38th Annual David W. Smith Workshop on Malformations and Morphogenesis: Abstracts of the 2017 Annual Meeting

The 38th Annual David W. Smith Workshop on Malformations and Morphogenesis occurred on August 26th – 29th, 2017 at the Stoweflake Resort and Conference Center in Stowe, VT. The Workshop, which honors the legacy of David W Smith, brought together clinicians and researchers interested in congenital malformations and their underlying mechanisms of morphogenesis. The Workshop highlighted four themes besides mechanisms of morphogenesis and new syndromes: Disorders of Transcriptional Regulation, Dysmorphology (Syndromes and Malformations) in Minority and Unique Populations, Syndromes and Isolated Birth Defects Involving Malformations of the Developing Foregut, and the Natural History of Syndromes. This Conference Report includes the abstracts presented at the 2017 Workshop.

The roles of many of these proteins and genes in transcription and development have been intensively studied in Drosophila melanogaster. With strong genetic tools, and a significantly smaller genome, studies in Drosophila often provide unique and important insights that are more difficult to obtain in mammalian models.
As an example, much is known about how the proteins affected in

DISORDERS OF TRANSCRIPTIONAL REGULATION (DTRS) -A GROWING GROUP OF DISORDERS WITH
PHENOTYPIC OVERLAP WITH CORNELIA DE LANGE SYNDROME (e.g. the Cornelia de Lange, Rubinstein-Taybi, Coffin-Siris, Bohring-Opitz, CHOPS syndromes and others). Both reverse and forward genetic approaches have led to both insights into transcriptional regulation as well as the identification of novel disease genes. An overview of these disorders and their molecular and clinical interrelatedness will be discussed.

DIAS-LOGAN SYNDROME: DELINEATING A NEWLY RECOGNIZED DISORDER OF TRANSCRIPTIONAL REGULATION
Dias-Logan syndrome is a recently described condition characterized by intellectual disability (ID) and persistence of fetal hemoglobin (HbF).
It is caused by haploinsufficiency of BCL11A, on 2p16.1, encoding a transcription factor belonging to the SWI/SNF chromatin remodeling complex (Dias et al., 2016). Here we describe a newly diagnosed patient, and discuss additional clinical features and pathogenesis.
Our patient presented with severe ID, early-onset seizures, postnatal deceleration of head circumference resulting in microcephaly (24 SD), short stature, scoliosis, small testes, and cutis marmorata. His distinctive facial gestalt included short forehead, deep-set eyes with strabismus and long palpebral fissures, wide mouth with tented vermillion of the upper lip and full and everted lower lip, and widely spaced teeth.
A possible diagnosis of Alpha-Thalassemia X-Linked Intellectual Disability syndrome (ATRX) had previously been proposed based on clinical features and facial gestalt, but molecular analysis of ATRX failed to identify a mutation and there were no erythrocyte HbH inclusion bodies. Of note, sequential hemoglobin (Hb) electrophoresis showed low HbA1 (72%, normal 95-98%) and normal HbA2 (2%) but persistence of fetal hemoglobin (26%; normal <2.1%). Exome sequencing identified a novel de novo pathogenic variant in BCL11A (c.1078dupC; p.Leu360fs), causative for Dias-Logan syndrome and consistent with his phenotype. However, our patient was more severely affected than previously reported individuals.
A total of 11 patients with point mutations have been described (Dias et al., 2016) and, by review of the literature, we found 19 additional individuals with 2p15p16.1 microdeletions involving BCL11A: only one of them had seizures, with onset at age 12 weeks (Hucthagowder et al., 2012). Given a positive family history of seizures (maternal uncle) and the paucity of patients with this syndrome and epilepsy in the literature, we reviewed the exome results looking for possible mutations in over 100 epilepsy genes. No pathogenic or likely pathogenic mutations were identified, suggesting that early onset seizures could represent an additional manifestation of Dias-Logan syndrome.
It is notable that the facial gestalt of our patient resembled ATRX more than Dias-Logan syndrome and, by review of available photos, 38% of previously described individuals had a facial gestalt consistent with the ATRX phenotype. BCL11A acts as a transcriptional repressor of fetal hemoglobin, and ATRX encodes a SWI/SNF-like protein that regulates the alpha-globin locus. Of note, STRING network analysis identified HDAC1 to play an important role in transcription regulation, as functionally associated with both ATRX and BCL11A proteins.
Interestingly, both gene products cause hemoglobin changes and two overlapping syndromes with ID, microcephaly and similar facial features, warranting further studies on their respective roles in chromatin remodeling in early development.
In conclusion, our report expands the phenotype of BCL11A mutations to include early onset seizures, and the overlapping manifestations between Dias-Logan syndrome and ATRX syndrome suggest convergence on a common pathway of transcription regulation of hemoglobin genes.

MUTATIONS IN H3F3A AND H3F3B ENCODING HISTONE 3.3 CAUSE THE FIRST REPORTED GERMLINE HISTONE SYNDROME: REPORT OF 23 PATIENTS WITH NEURODEVELOPMENTAL AND CONGENITAL MANIFESTATIONS
INSERM, UMR-S 957, Nantes, France; 23 These authors contributed equally to this work.
From a GeneMatcher-enabled international collaboration, we identified ten individuals affected by intellectual disability, speech delay, ataxia, and facial dysmorphism and carrying a deleterious EBF3 variant detected by whole-exome sequencing. One 9-bp duplication and one splice-site, five missense, and two nonsense variants in EBF3 were found; the mutations occurred de novo in eight individuals, and the missense variant c.625C>T (p.Arg209Trp) was inherited by two affected siblings from their healthy mother, who is mosaic. EBF3 belongs to the early B cell factor family (also known as Olf, COE, or O/ E) and is a transcription factor involved in neuronal differentiation and maturation. Structural assessment predicted that the five amino acid substitutions have damaging effects on DNA binding of EBF3. Transient expression of EBF3 mutant proteins in HEK293T cells revealed mislocalization of all but one mutant in the cytoplasm, as well as nuclear localization. By transactivation assays, all EBF3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene CDKN1A, and in situ subcellular fractionation experiments demonstrated that EBF3 mutant proteins were less tightly associated with chromatin. Finally, in RNA-seq and ChIP-seq experiments, EBF3 acted as a transcriptional regulator, and mutant EBF3 had reduced genome-wide DNA binding and gene-regulatory activity. Our findings demonstrate that variants disrupting EBF3-mediated transcriptional regulation cause intellectual disability and developmental delay and are present in 0.1% of individuals with unexplained neurodevelopmental disorders.

NSD1 OVEREXPRESSION ALTERS THE LEVELS OF INSULIN RECEPTOR (INR) AND TSC2
contrasting features that occur in microdeletion-duplication syndromes.
Our study documents the long term prognosis and penetrance of this recently delineated microduplication syndrome, and suggests the possibility of a differential diagnosis for children with delayed bone age and short stature. Focusing specifically on the growth phenotype, we have modeled the effects of NSD1 overexpression in Drosophila melanogaster. Overexpression of the fly homologue of NSD1 in the developing wing causes undergrowth associated with increased H3K36 methylation and increased apoptosis.
Altering the levels of insulin receptor (InR) and TSC2, a negative regulator of mTor signaling, rescues the apoptosis and the wing undergrowth phenotype, demonstrating alterations in PI3K/mTOR pathway signaling. Most interestingly, supplementing the diet with leucine also rescues the cell death, suggesting that this may represent a potential intervention for treating the growth phenotype. We report a patient with homozygous splice site variants in another TFIID component, TAF8, a gene not previously reported in human disease. First seen at age one with severe delay and chronic aspiration, she was born to non-consanguineous teenaged Hispanic parents. An extensive metabolic and genetic evaluation, including a normal SNP array, was negative. MRI revealed reduced myelination, small vermis and partial absence of the corpus callosum. Currently at age 4, she is G tube fed, has mild microcephaly (22 2 3 SD), brachycephaly, dysmorphic features with a vague resemblance to CDLS and a picture of evolving lower extremity spasticity. She rolls and smiles but has no other developmental achievements. Exome sequencing revealed a c.781-1G>A homozygous splice site variant carried by her parents.
Mice with homozygous germline deletions in the Taf8 gene die at the blastocyst stage of embryonic development (embryonic Day 4) (Voss et al., 2000). Deletion of Taf8 using the Nestin-Cre transgene, which is activated mid-gestation, results in embryos with massive cell death in the cerebral cortex, and reduced brain size as compared to control littermates. Embryos are born but do not survive beyond postnatal Day 2 because they fail to suckle (El Saafin unpubl).
Studies on this child's fibroblasts, performed by the French investigators, reveal that the TAF8 c.781-1G>A intron 7 mutation results in malformations, the female genital tract is evaluated. Following ultrasounds of 24 females with CdLS, ages 12 to 54 years, notably six (25%) were found to have a bicornuate uterus. Some uteri measured small, but most ovaries were normal in appearance, and no other pathology was noted, including no ambiguous or intersex genitalia. Few genetic syndromes present with Mullerian fusion anomalies such as bicornuate uteri, which typically do not cause medical complications other than pregnancy difficulties. Bicornuate uterus has been seen as a distinct malformation in Fryns syndrome (Slavotinek, 2004), Bardet-Biedl syndrome (Stoler et al., 1995) and, interestingly, in Roberts syndrome (Freeman et al., 1974), another disorder of the cohesin protein complex. In addition, the Hand-Foot-Genital syndrome (HFGS) presents with genital malformations in both males and females, including Mullerian fusion defects. Specific extremity findings include small hands and feet, short first metacarpals/metatarsals, brachyclinodactyly of 5th fingers, and short great toes. Facial features have been infrequently reported. A recent female patient was found to have speech delay (Pezzani et al., 2015). Of note, one report includes a family with typical hand and foot findings, in which the mother has bicornuate uterus, and her son has feeding problems, poor weight gain, genital abnormalities, gastroesophageal reflux, obstructive sleep apnea, peripheral pulmonic stenosis, diaphragmatic hernia, and scoliosis (Tas et al., 2016). Many of the HFGS findings can also be seen in CdLS. Half of the females in our cohort with CdLS and bicornuate uteri have short great toes, and half have brachyclinodactyly of the 5th fingers. HFGS is due to mutations or deletions in HOXA13 in the HOXA gene cluster on 7p15. Cohesin is known to coordinate HOXA chromatin structure and gene expression (Wang et al., 2015) and maintain associated domains at HOXA (Nwigwe et al., 2015) during development. We hypothesize that chromatin-coordinating role of cohesin is related to proper expression of the HOXA cluster. If this activity is disrupted, this could lead to uterine fusion anomalies and limb features, as well as other features overlapping with those seen in CdLS.

Wo r ksh o p S ess i on 3 D I SOR DE RS OF T RA N SCR IP T I ONA L R EG U LA TI ON I I I
Kabuki syndrome (KS, OMIM #147920) is a rare congenital disorder characterized by distinct dysmorphic facial features, intellectual disabilities, postnatal growth retardation, skeletal malformations and dermatoglyphic abnormalities. Up to 80% of cases described in the literature have de novo mutations. However, familial occurrence with autosomal dominant inheritance has also been reported. The major genetic cause of KS are mutations in the KMT2D (lysine-specific methyltransferase 2D; MIM #602113) gene, also known as MLL2,and,in up to 10% of the cases, mutations in the KDM6A (lysine-specific demethylase 6A; MIM #30012) gene. Most identified pathogenic mutations are truncating, likely leading to haploinsufficiency of the KMT2D protein.
KMT2D is required for H3K4 di-and trimethylation, a hallmark of transcriptional activation. We hypothesized that the truncating mutations of KMT2D seen in KS could cause differential methylation, and that these differences may provide insight into the pathogenesis of this disorder, in addition to providing a tool for biomarker development and Oculo-Auriculo-Vertebral Spectrum (OAVS) affects 1:5,600 live births and is characterized by a variable array of features that includes microtia, maxillary and mandibular hypoplasia, pre-auricular or lateral facial tags, ear canal atresia with associated hearing deficits, and cervical vertebral anomalies. OAVS is also particularly noted for its frequent asymmetric presentation. Despite rare examples of familial inheritance, few genes have been definitively implicated. However, an increased risk has been associated with elevated Vitamin A exposure during pregnancy.
Here we report a unique mutant mouse line that presents almost all of the classic OAVS features, including the marked phenotypic variability and asymmetry. Using low-pass whole genome sequencing we identified a 20kb inversion within the mapping interval that physically disrupted only a single gene. This gene encodes a zinc-finger CONFERENCE REPORT | 1469 transcription factor, the expression of which was decreased by >90% in mutant tissue. However, complementation experiments demonstrated that loss of this gene is not responsible for the classic OAVS features.
Re-evaluation of the chromosomal anomaly revealed that a small evolutionarily conserved intronic sequence was also deleted with the inversion. This deletion removes a validated chromatin insulator element, suggesting it could be disrupting local gene expression. Consistent with this, we demonstrate that the expression levels of two genes immediately adjacent to the transcription factor-encoding gene, which themselves are not physically disrupted, are upregulated more than 8 fold in mutant branchial arch tissue. Notably, these two genes are paralogs that encode relatively uncharacterized retinol dehydrogenase enzymes.
Our data point to elevated retinoid signaling as a likely mechanism in this OAVS mouse model, providing the first direct evidence that genetic mutations disrupting retinoid signaling could underlie OAVS in patients.
This work was funded in part by the Laurel Foundation Endowment for Craniofacial Research and NIDCR grants R01-DE022561 (TCC) and T90-DE021984 (EDC). Despite significant advances in genomic technologies, it is estimated that more than half of the genes that cause rare diseases remain undiscovered. Using large-scale human genomics of healthy individuals, we can identify genes that are depleted for loss-of-function or missense variation, known as constrained genes. The Exome Aggregation Database (ExAC) is a large-scale reference database with high quality, jointly processed exome data from 60,706 individuals. Leveraging the ExAC dataset, we have identified 3,300 genes that are constrained for loss-of-function variation. Over two-thirds of these candidate haploinsufficient genes are not yet associated with any human disease phenotype. KMT2E (MLL5) is one such candidate haploinsufficient gene in which there are no high quality nonsense or canonical splice site mutations seen in ExAC, while 52 are expected based on the gene length and sequence. KMT2E has previously been implicated (but not definitely proven) as a candidate autism disease gene in exome sequencing studies but no further phenotype information is available.
Loss of function in KMT2E is predicted to cause a disorder of transcriptional regulation. KMT2E encodes a histone methyltransferase epigenetic protein, a transcriptional regulator reported to play key roles in diverse biological processes, including cell cycle progression, genomic stability maintenance, adult hematopoiesis, and spermatogenesis.
We report here on the phenotype of heterozygous KMT2E predicted loss-of-function variants. Clinical exome sequencing performed on a 2-year-old male with macrocephaly, infantile epilepsy, and developmental regression identified a de novo Y284H missense variant in KMT2E. The proband had normal development until 6 months of age.
He developed medically refractory epilepsy at 6 months, associated with developmental stagnation. At 14 months of age, a ketogenic diet was initiated and maintained for the subsequent 6 months, during which time he had severe developmental regression. Since the discontinuation of the ketogenic diet, he has started to make slow developmental gains but has not returned to his prior baseline.
Recent studies have shown that beta-hydroxybutyrate (ketone bodies) are natural inhibitors of histone deacetylases, leading to an altered brain epigenetic landscape. It is intriguing to hypothesize that the abnormal brain epigenetic landscape of individuals with loss of KMT2E function could have become detrimentally more abnormal as a result of the ketogenic diet.
Through GeneMatcher, we have identified eleven additional probands (ages 1-12 years) with de novo protein truncating variants in KMT2E to further delineate the phenotype. All of these children have developmental delay and intellectual disability, while 80% have hypotonia.
Three have a formal diagnosis of autism. Two-thirds have relative macrocephaly. Several have a short nose and tented vermilion of the upper lip.
Only one additional subject has childhood onset epilepsy. This case series supports KMT2E as a novel intellectual disability and autism disease gene.

SMC1A MUTATIONS CAUSE MECHANISTICALLY SEPARABLE ALLELIC DISORDERS: ATYPICAL CORNELIA DE LANGE SYNDROME AND A RETT-LIKE EPILEPTIC ENCEPHALOPATHY
Mutations in SMC1A were first identified in 2006 in males with a variant presentation of Cornelia de Lange syndrome (CdLS;Musio et al., 2006). Subsequent efforts (Deardorff et al., 2007;Huisman et al., 2017) have identified a substantial number of males and females with CdLS-like presentations caused by mutations in SMC1A. These subjects demonstrate features that vary from classical CdLS that include broad full brows, more normal growth parameters and less frequent congenital malformations, although their intellectual disability can be as severe.
SMC1A mutations that cause CdLS-like features are missense mutations. Notably, SMC1A is on the X chromosome but not inactivated and expressed biallelically in females. The findings that females can be as severely affected as males suggests that mutations in SMC1A that cause a CdLS-like presentation cause a dominant-negative effect, and potentially disrupt a specific function of the protein.
Recently, an allelic disorder has been emerging caused by SMC1A loss of function mutations. All individuals are girls who present, not with CdLS features, but with an early onset epileptic encephalopathy.
We have identified over 15 girls with this clinical picture, some of whom have no dysmorphic features of CdLS, but are often described as "Rett-like". Their seizures typically have an onset in the first year of life, are generalized tonic-clonic, can be difficult to manage, but have resolved in a small number of cases.
SMC1A is a core component of the cohesin complex that forms a dimer with SMC3. Together SMC1A and SMC3 undergo a complex folding process to form an antiparallel coiled-coil dimer, attaching at their midportions to generate a bracelet-like structure. Cohesin is loaded onto chromatin and re-positioned to generally regulate chromatin dynamics and transcription. This process is regulated by a series of proteins, including NIPBL, the gene in which loss of function mutations have been identified in CdLS.
These phenotypic data, suggest that SMC1A mutations that cause CdLS-like features lead to disrupted loading, repositioning or chromatin regulation, in a manner similar to that noted for loss of NIPBL function.
In contrast, loss of function mutations in SMC1A that result in X-linked female epileptic encephalopathy likely cause a haploinsufficiency of SMC1A, leading to specifically to reduced cohesin function, rather than altered cohesin function. Consistent with the clinical neurologic presentation, Drosophila and mouse models have also noted that neural cells, a post-mitotic cell type, are more sensitive to a loss of cohesin function than other cell types. These observations strongly suggest a dosagedependent post-replicative role for cohesin in neural development.
Elucidation of the specific mechanism of these likely chromatin and transcriptional-regulatory deficiencies will serve to provide insight into better understanding and management of these allelic clinical disorders.

NEUROLOGIC, NEUROMUSCULAR AND CRANIOFACIAL SYNDROMES THE GENETIC LANDSCAPE OF CEREBELLAR MALFORMATIONS
The cerebellum is a complex part of the brain that in humans accounts for only 10% of total brain volume, but is densely packed and contains more than 80% of all neurons. It is best known for its function in motor coordination, but appears to coordinate cognition, emotion and attention as well. Malformations of the cerebellum are collectively common and difficult to classify birth defects that are associated with epilepsy, autism, intellectual disability, mood dysregulation and attention deficit disorder. The genetic basis of these developmental brain disorders is poorly understood, and few patients receive a genetic diagnosis.
We selected 104 children from 98 families diagnosed with cerebellar malformations from a large cohort of developmental brain disorder patients recruited between 1983 and 2012 with DNA available but without a known genetic diagnosis. The cohort includes common types of diffuse cerebellar (CBLH) and cerebellar vermis (CBVH) hypoplasia as well as Dandy-Walker malformation (DWM), but excluded several rare and specific malformations such as (near) complete cerebellar agenesis, giant tectum/absent vermis, and rhombencephalosynapsis.
We performed exome sequencing in 98 families in which one or more children had CBLH-CBVH or DWM, using an in house analysis pipeline that calls sequence variants detected by both GATK and FreeBayes with depth of coverage 10, AAF 0.1%, and a functional change with CADD score 10 if applicable. Our final interpretation of variants often required searches of clinical and research variant databases plus recent literature, as well as the GeneMatcher website. We have so far found a genetic diagnosis in 40 of 98 (41%) families. Unexpectedly, we were able to solve 28 of 54 (52%) children with CBLH-CBVH, but only 11 of 44 (25%) with DWM. The difference was statistically significant (p 5 .007), which suggests greater causative complexity for DWM than for isolated CBLH-CBVH. Notably, several prenatal extrinsic factors are known to cause cerebellar hypoplasia, including prenatal hemorrhage, hypoxia-ischemia (especially in extremely low gestational age newborns), infections (i.e. Zika virus), and teratogens such as retinoid acid.
We identified single gene mutations in 32 different genes including 27 previously associated with developmental brain disorders, and 5 novel genes (for example, FZD3). For a majority of the known genes, cerebellar malformations had not been reported as part of the phenotype. The mutations were de novo heterozygous for 19, homozygous or compound heterozygous for 10, and X-linked for 3 genes. We found mutations in more than one child for 6 of the 32 genes (CASK, BCL11A, DDX3X, FOXP1, STXBP1 and TUBA1A), but only CASK was seen in more than two children. All of the genes were expressed in fetal or adult cerebellum. Our results show that cerebellar malformations occur as a co-morbidity with numerous different developmental brain disorders, and that the yield of testing is higher for CBLH-CBVH that for classic DWM. Our results suggest that CASK may be the only "common" CBLH gene and that non-genetic causes are an important cause of cerebellar malformations.
TUBA1A: OUTCOME OF MOSAICISM AND PHENOTYPIC ANALYSIS pattern peaking in fetal brain and decreasing in the post-natal period into adulthood. Abnormal TUBA1A protein is proposed to interfere with microtubule function, thus impairing neuronal migration. Pathogenic variants (PV) in TUBA1A cause a spectrum of brain malformations associated with disrupted neuronal migration including classic lissencephaly, lissencephaly with cerebellar hypoplasia and agenesis of the corpus callosum, cerebellar hypoplasia without lissencephaly, perisylvian asymmetrical polymicrogyria, and polymicrogyria-like cortical dysplasia among others.
Somatic mosaicism of PV is known to alter the phenotype in several genetic conditions. This article presents phenotypic findings in four individuals mosaic for PV in TUBA1A.
Case 1: A two-year-old girl presented with developmental delay, microcephaly, stereotypies and MR with decreased cortical volume but no structural abnormalities. Whole Genome Sequencing identified a de novo variant in the TUBA1A gene, c.167C>T (p.Thr56Met), which has been previously reported in three unrelated individuals associated with lissencephaly with cerebellar hypoplasia. The variant was present in only 16% of cells (confirmed by qPCR) and was not present in either parent.
Case 2: 2 siblings presented with cognitive impairment and structural brain anomalies consistent with a neuronal migration abnormality.
The mother carries this PV in 5.6% of her fibroblasts. MR of the mother, who was described as asymptomatic, showed a thin corpus callosum, hypoplasia of the superior vermis, and a thin medulla.
Case 3: 2 siblings were referred for evaluation of developmental delay, microcephaly and structural brain anomalies. Whole exome sequencing in one sibling identified a heterozygous PV in the TUBA1A gene, c.1091C>G (p.P364R), which was confirmed in the sibling by single site testing. The mother has mosaicism for this PV in blood. She has mild cognitive impairment.
Case 4: A fetus was evaluated with MR at 30 weeks EGA due to intertwin discordant cephalic measurements. Multiple brain anomalies consistent with neuronal migration abnormality were found and subsequently confirmed postnatally. Infantile spasms and profound developmental delay were present. A next-generation brain malformation panel identified a heterozygous c.790C>G (p.R264G) PV in TUBA1A.
The father was mosaic in blood for this PV. He had normal intellect, no history of seizures, and normal brain MRI.
Somatic mosaicism for PV in TUBA1A should be considered in the evaluation of children with developmental delay and intellectual disability particularly if structural brain anomalies are present and in parents of affected siblings. Parental testing should also be offered for recurrence counseling in isolated cases. Genetic and genomic testing should include assessment of mosaicism as part of the analytic pipeline.

PHENOTYPIC SPECTRUM OF FEMALES CARRYING MUTATIONS IN ZC4H2
Arthrogryposis is a highly heterogeneous group of conditions defined as non-progressive congenital joint contractures of two or more different body areas. Arthrogryposis is a clinical feature of more than 500 rare conditions and can be caused by abnormalities of the central or peripheral nervous system, neuromuscular junction, or muscle. The distal arthrogryposes (DA) are a phenotypically and genetically heterogeneous subset of arthrogryposis conditions putatively caused by mutations in 15 genes. Distinguishing among the different DA early in life can difficult.
Analysis of exome sequence data from a large cohort of families putatively diagnosed with DA identified predicted loss-of-function (LOF) mutations in ZC4H2 in six females, each with a different mutationfive of which were confirmed to be de novo. Three females with deletions of ZC4H2 have been reported bringing the total number of females with putative LOF variants to nine.
Hemizygous mutations in ZC4H2 cause Wieacker-Wolff syndrome, a X-linked recessive condition characterized by arthrogryposis, hypotonia, motor and developmental delay, and sometimes early death. Some carrier females are unaffected, while others are reportedly "mildly affected" with mild intellectual disability, camptodactyly, and clubfoot.
In contrast, clinical characteristics of females with LOF variants in ZC4H2 included moderate to severe intellectual disability, seizures, poor growth, scoliosis, and severe contractures.
Overall, females with predicted LOF mutations ZC4H2 appear to be comparatively more severely affected than either females or males with missense mutations in ZC4H2. This suggests that LOF mutations in females leads to a phenotype than is more severe than missense mutations in ZC4H2 in either males or females. LOF variants in ZC4H2 have not been reported in males.
We hypothesize that most ZC4H2 missense variants are hypomorphic whereas LOF variants result in haploinsufficiency and that haploinsufficiency in males is embryonic lethal and in females results in severe disease. The spectrum of disease severity in females with LOF variants in ZC4H2 may be associated with the level of X-inactivation of the mutant allele.

UAB's Undiagnosed Disease Program and HudsonAlpha's Clinical
Sequencing Exploratory Research program have identified 5 cases of KBG syndrome due to ANKRD11 variants. Each case has a unique phenotype that may explain why patients were not previously diagnosed and referred to these "last resort" programs.
Case 2: 10yo male with seizures, autism, speech delay, and moderate intellectual disability. He had a history of tremors, failure to thrive, gastroesophageal reflux, and dysmorphic facial features. He was found to have de novo ANKRD11 p.Glu2158X.
Case 3: 12yo female with seizures, hearing impairment, myalgia, moderate intellectual disability, and dysmorphic features. Prior diagnoses considered included a mitochondrial disorder, as she had atypical Complex I, III, V activity and two mitochondrial variants of uncertain significance. WGS revealed de novo ANKRD11 p.Ala2265fs. It is unclear if there is a second, undetected condition, or if the KBG phenotype could include myalgia or hearing loss.
Case 4: 12yo female with complex congenital heart defect (single ventricle, mitral atresia, restrictive ASD), esophageal varices, portal hypertension, short stature, hearing impairment, moderate intellectual disability, macrocephaly and dysmorphic facial features. She harbors a pathogenic ANKRD11 variant (p.Lys803fs), inheritance unknown as the egg donor was unavailable for testing.
Case 5: 6yo male with seizures, developmental delay, and moderate intellectual disability. A nonsense variant in ANKRD11 (p.R733X) was inherited from his mother with seizures, and testing is pending in a similarly affected brother.
Three of our cases have hearing impairment (one attributed to a blended phenotype), 1 with cardiac manifestations and 1 with myalgias.
These may represent phenotypic expansion or unrelated conditions. An additional patient is presented with a de novo, nonsense mutation (c.2872C>T, p.Gln958Ter) in exome 15 of ARID2 detected on whole exome sequencing. This mutation has not previously been reported but is predicted to cause loss of normal protein function. The patient was 26 months of age at the time of testing, had minimal words, and did not walk independently. The patient's delivery was complicated by nuchal cord times two, and there was resultant concern for a hypoxic event leading the patient to be diagnosed with cerebral palsy prior to genetic testing.x Health concerns for this young man include difficulty gaining weight (weight less than 1%ile), shorter stature (height 3%ile), laryngotracheomalacia, hypotonia, and white matter loss on brain MRI. Physical features include: coarse face with prominent forehead, protruding and posteriorly rotated ears, thin upper lip, wide nasal bridge, as well as hypoplastic 5 th digit nails of both fingers and toes. The patient is very jovial and often smiling broadly. Many of this patient's features are shared by those previously reported with ARID2 mutations as well as those with Coffin-Siris syndrome.
The BAF subclass of the SWI-SNF complex contains the majority of the genes already associated with CSS. While BAF and PBAF are distinct entities, they share many core components and are major parts of SWI-SNF. ARID2 is exclusively found in PBAF, yet it interacts with BAF during some developmental stages. It is expressed in all tissues and appears integral for tissue and cell-specific gene expression and maintenance. ARID2 mutations likely cause haploinsufficiency, which is similar to ARID1A and ARID1B.
Though several genes are known to cause CSS, nearly 40% of clinical cases elude genetic diagnosis. With the similarity in clinical concerns and physical features, as well as the relationship between ARID2 to the complex indicted in CSS, consideration should be made to including ARID2 as a causative gene for CSS or at least a CSS-like condition. There is considerable clinical variability in CSS, and most of the ARID2 patients seem to share the more common features. This may represent some ascertainment bias regarding ARID2. However, for the time being in clinical situations, if other genetic etiologies for CSS are not found, ARID2 should be considered.

DE NOVO MISSENSE VARIANT IN UPF1, A KEY REGULATORY COMPONENT OF NONSENSE-MEDIATED
The nonsense-mediated decay (NMD) pathway is best known for its selective degradation of mRNA transcripts harboring premature termination codons (PTC). UPF1 (up-frameshift-1) is one of 12 known NMD factors, and is the key central component in PTC-containing mRNA recognition and degradation. When recruited to a translating ribosome by a PTC in an mRNA transcript and complexed with UPF2 and UPF3, UPF1 becomes phosphorylated and initiates enzymatic degradation of the transcript. Despite its central role in NMD (a highly evolutionary conserved process which has been in yeast through mammals), variants in UPF1 have not been associated with human Mendelian conditions. However, pathogenic variants in UPF3B (an X-linked paralogue of UPF3) are associated with a distinct facial gestalt with intellectual disability (Tarpey et al., 2007;Laumonnier et al., 2010). We recently identified a de novo missense variant in UPF1 in a toddler with global developmental delay and a distinctive facial gestalt. The proband was initially evaluated at 2 years and 3 months old for global developmental delay and non-familial facial features. He is the second child of nonconsanguineous parents and there is no family history of X-linked intellectual disability. Pregnancy was uncomplicated and he was AGA at 37 weeks. He was noted to have "crunched up ears" at birth. He had poor weight gain on breast milk and skin sensitivity improved with switch to formula. He had gastric dysmotility and mild hydronephrosis. He had delayed head control and showed little developmental progress between 12 and 24 months. He sat independently at 12-15 months, crawled at 24 months, pulled to stand at 27 months, and babbles at 24 months with just a couple of word-like vocalizations. He is curious and described as "laid back". He had normal cytogenomic SNP microarray and brain MRI. On physical exam he is hesitant to stand and has poor balance. His length is 25th centile, weight is 10th centile, and OFC is 2nd centile. He has a long face with a high forehead, cupped ears with prominent lobes, myopathic face with intermittent ptosis, underdeveloped calves and puffy dorsum of the feet. Whole exome sequence analysis identified a de novo sequence variant of uncertain significance in UPF1 (c.1762T>C; p.Ser588Pro). Located in the RecA1 domain (RNA binding domain) of UPF1, the p.Ser588Pro variant is not listed in population databases, and serine 588 is highly conserved, including yeast and Drosophila. It is an exposed amino acid at the juncture of two alpha helix domains, but it is not a commonly phosphorylated serine residue. Lymphoblastoid cell lines have been generated from the proband and his parents to determine the efficiency of NMD (Tarpey et al., 2007). We hypothesize that this novel amino acid change in UPF1, the key regulatory component of nonsense-mediated mRNA decay, leads to less efficient NMD. We further speculate that similar to pathogenic variants in UPF3B, attenuated NMD due to this UPF1 variant leads to deregulation of neuronal-specific transcripts important in normal intellectual development. were reported with severe-profound developmental delay, hypotonia, choreiform movements, and cerebellar atrophy in one sibling (Hirabayashi et al., 2016). We report a 6 year-old girl of mixed European, Pacific Islander, and Native American ancestry with severe developmental delays, Duane anomaly, hematopoietic myelodysplasia, and cerebellar hypoplasia found to have compound heterozygous mutations in

MED17.
The patient was born at term following an uncomplicated pregnancy and delivery.
Developmental delays were noted in the first 6 months. At 12 months she was diagnosed with bilateral Duane anomaly. At age 4 years she developed thrombocytopenia and bone marrow biopsy revealed myelodysplasia. She has remained severely delayed but with no developmental regression. She sat independently at 3 years and now rolls and scoots. She remains nonverbal without any specific communicative signing. She does finger feed. She has a friendly demeanor and frequently laughs without provocation.
Her exam is notable for normal growth with head circumference at the 15 th percentile, diffuse hypotonia, bilateral intention tremors, thick eyebrows with mild synophyrys, and a right preauricular tag.
Brain MRI at 17 months revealed moderate cerebellar hypoplasia involving the vermis more than the hemispheres, mild pons hypoplasia, and mild enlargement of the frontal horns. Clinical whole exome sequencing revealed compound heterozygous variants in MED17: maternally-inherited c.1299_1302del (p.Ala435SerfsTer5) and paternally-inherited c.1183C>G (p.Pro395Ala). The p.Ala435fs variant is expected to lead to non-sense mediated decay and has an allele frequency in the Exome Aggregation Consortium (ExAC) of 0.0045% in Europeans. The p.Pro395Ala variant is predicted to be damaging by computational analysis programs and is not listed in population frequency databases.
Mediator is a multi-protein variable complex that plays an essential role in eukaryotic transcriptional regulation through its control of RNA polymerase II activity. The human mediator complex is composed of up to 26 proteins and mutations in five of these genes have been associated with severe intellectual disability syndromes with variable brain malformations and neurologic findings. We believe that our patient's neurodevelopmental disabilities and cerebellar dysgenesis are a consequence of her biallelic MED17 mutations and that mediator complex abnormalities may represent a particular category of transcriptional regulation disorders. A few individuals with more N-terminal or copy number KAT6B variants, and a non-specific developmental or "atypical Ohdo" phenotype have been described 2 . Given the rarity of such reports, the pathogenicity of non-exon 18 variants may be under called. We hypothesize that reverse phenotyping in instances of non-exon 18 variants is improved by thinking beyond the features seen in genitopatellar and Ohdo syndromes, and assessing for the features seen across the lysine acetyl transferases syndromes.
We describe a normocephalic, well grown 39 month boy with a left partial cleft lip, distinct from common clefts in that it is associated with more significant deficiency of regional tissues. He has shortening of the proximal and distal segments of his right leg. His right foot has four toes.
He has history of a small PFO and ASD. His facial features are neither genitopatellar nor Ohdo like. Further, his knees are normal without contracture, and the patellae are normal to palpate. The genitalia are normal. His development was normal until 30 months when, over the course of a week, he lost his words and toilet training, behaviours changed. He has subsequently been formally diagnosed with autism.
Exome sequencing identified one variant of interest, a de novo missense variant (c.458G>A; p.Arg153Gln), which has a CADD score of 25, and is not found in gene specific data bases or the population databases.
In comparing this boy to published and other cases we have cared for with lysine acetyl transferases syndromes, this boy's cleft, hypomelia, and foot malformation are similar to those of Roberts syndrome.
Further, the acute loss of skills and development of autism is similar to that which we observed in an individual with EP300 syndrome. Careful description of this boy's features will support reverse phenotyping in other more subtly affected individuals where variants in lysine acetyl transferases are identified.
The spliceosome is a dynamic and flexible biological complex fundamentally involved in cell differentiation, homeostasis, and disease (Papasaikas and Valcarcel, 2016). Since the first draft of the human genome was published, over a dozen genes involved in the spliceosomal machinery have been reported in relation to the etiology of Mendelian diseases.
Nager syndrome (NS), the prototype for mandibulofacial dysostoses with preaxial limb defects, is one of such disorders due to mutations in SF3B4, a component of the U2 complex of the spliceosome (Bernier et al., 2012). SF3B4 encodes the spliceosome associated protein 49 (SAP49), a highly conserved protein with two RNA-recognition motifs (RRMs) in its N-terminal followed by a proline-glycine rich region domain in its C-terminal. During the pre-spliceosomal assembly of the U2 complex, SAP49 and SAP145 form a complex in which the two RRMs of SAP49 are required in the interaction (Champion-Arnaud and Reed, 1994). In Nager syndrome, approximately 60% of the patients have a heterozygous loss of function of SAP49 as a result of truncating mutations in SF3B4 (Bernier et al., 2012;Czeschik et al., 2013;Macpherson et al., 2014;Petit et al., 2014). We are reporting on a patient with a clinical diagnosis of NS who was found to have a novel missense substitution in exon 3 of SF3B4, in the RRM1 domain.
The patient has a recognizable gestalt, normal growth parameters, repair for duodenal atresia, hearing loss, mild limb anomalies, and a neurobehavioral phenotype. The missense mutation, c.251T>G (I84R) is deemed pathogenic by all in silico analyses performed.
Further, in order to test the effect of this mutation on viability, a plasmid shuffle in a yeast strain for I84R was performed and no viable transformants were identified. Following this results, we have surveyed the ExAC database for missense mutations in the SF3B4 gene and applied the same in vitro yeast model to test their pathogenicity. To date, we have identified one additional SF3B4 missense substitution L28P for which the yeast construct is lethal.
In this article, we bring evidence to support that pathogenicity of mutations in SF3B4 are not only related to the type of mutation but more importantly to the domain of the protein involved in specific spliceosomal function. We propose a rapid functional in vitro model that could be used particularly when studying the significance of mutations involved in spliceosomopathies and raise attention for potential pathogenic mutations reported in the ExAC database. This latter finding has particular implications for the counseling of patients in regards to disorders known to be underlined by incomplete penetrance including mandibulofacial dysostoses. Mutations in MECP2 are associated with Rett syndrome characterized by initially normal progress followed by a period of regression, loss of functional use of the hands, feeding problems, seizures and severe intellectual disability in females. Affected males are rare with a high rate of early loss in male pregnancies. The function of MECP2 protein is incompletely understood. It binds to methylated DNA, and can then promote or repress transcription. We report a family with a mutation at c.925C>T (p.Arg309Trp) with multiple affected male and female family members. The initial presentation was a male infant with an apparently syndromic developmental disability. The proband was delivered to a G1P0 female at term; As a newborn he was noted to have hypotonia, macroglossia, macrocephaly, and mildly dysmorphic features.
Further evaluation identified congenital hypothyroidism, mild hepatomegaly, dilated aortic root (Z score 2.4 but has improved to 1.6 with growth), and severe developmental disability. He is nonverbal at age 9.
He is able to walk and can follow a few one step commands. The family has had several additional children including 2 additional boys with presentation similar to the proband current ages 7 years and 22 months.
The second boy also had aortic root dilation (z score 4.5 at age 7), and seizures. The oldest sister has developmental disability. She is verbal and ambulatory, but has not had movement disorder or regression. She has continued to gain skills.
The second sister has had normal development. The mother had a mild intellectual disability, but was able to communicate effectively and care for the children. She has a sister with similar disability who has 2 children with more severe developmental disability who reportedly are very similar to our proband. to successfully identify causal mutations in known and novel Mendelian genes for 31.5% of kindreds. The identified mutations were mostly de novo dominant (n 5 51, 81%), consistent with the majority of cases of the study cohort arise sporadically without familial occurrence, but also X-linked or autosomal recessive (n 5 12, 19%). Among them, we have identified and characterized a number of known and novel diseasecausing genes in transcriptional processes and related epigenetic modification pathway important for human developmental disorders in 15% of patients in this cohort, such as H3F3A, KMT5B, SMARCE1, U2AF2,   STAG2, MED12, SMC1A, MAF, SATB2, ADNP, HIVEP2, HNPNPK, TP63,   DDX3X, SF3B4, LEUTX, EFTUD2, FOXP1, FOXC1, MED13L, TFAP2A, and EYA1. WES also identified likely pathogenic variants in about a dozen genes not previously implicated in rare multiple congenital anomalies, including but not limited to DDB1, BMP2, MTOR, RICTOR, TUBA1B, SMG5, and SPECC1. We will present an overview of our recent novel discoveries in MCA, discuss options to partner with laboratories that can provide follow up functional measurements, and explore novel mechanisms as therapeutic targets for part of symptoms representing in MCA.  (1). The series suggested association with features such as scoliosis and identified a distinctive facial appearance characterized by a round face, heavy horizontal eyebrows and narrow palpebral fissures (1,2). The purpose of this report is to expand the phenotype of TBR syndrome and highlight it as a diagnostic consideration in the differential diagnosis of Loeys Dietz and Marfan syndromes. We report a 12 year old female referred for query connective tissue disorder. She had tall stature 169cm (> 99th percentile), mitral valve prolapse, arachnodactyly, thoracic lordosis and scoliosis, straight heavy eyebrows, short palpebral fissures (<3rd centile), high arched palate, bifid uvula, droplet cortical lens opacities, failure and retention of some primary dentition and hirsutism. There was mild ID and a history of three nocturnal seizures. A de novo pathogenic missense mutation in DNMT3A was identified by whole exome sequencing.
The presented case has some hallmarks of TBR syndrome such as the mild ID, distinctive facies, tall stature and scoliosis. Other features such as bifid uvula and droplet lens opacities have not been previously reported, but high arched palate, mitral valve prolapse, scoliosis each do appear > of the previously described cases. This case expands the TBR syndrome phenotype and suggests it should be considered a differential diagnosis of patients being assessed for Marfan (like) conditions. The novel mutation identified in this case is

THE MANY FACES OF PEROXISOMAL DISORDERS: LESSONS FROM A LARGE COHORT
Peroxisomes are single-membrane organelles that enclose various enzymes required for many important functions in human metabolism.
Defects in the biogenesis and/or function of peroxisomes result in such phenotypes as Zellweger-spectrum, rhizomelic chondrodysplasia punctate, isolated fatty acid beta-oxidation deficiency, as well as less known phenotypes that were only recently described. In this study, we describe the largest cohort to date (75 families) of clinically, biochemically and molecularly characterized patients with peroxisomal disorders.
At the molecular level, we identified 41 disease-causing variants, more than half of which (22) are novel. Missense variants were the predominant class of variants accounting for 34% (14/41), followed by small indels 24% (10/41), nonsense 20% (8/41), splicing 12%, and large rearrangements in 10%. The founder nature of many of the variants identified in this study allowed us to make a minimum estimate of disease incidence of around 1 per 20,000, much higher than previous estimates in other populations.
Clinically, we found a strong genotype/phenotype correlation that is primarily driven by individual alleles rather than individual genes or mutations classes. Although Zellweger spectrum disorder is known for its severity that results in death in infancy, 39% (16/40) of the patients have documented survival at the time of reporting the study above one year of life.
Most unusual among the long-term survivors was a multiplex family in which the affected members had minimum to no elevation of VLCFA and presented as adults with nonspecific intellectual disability.
Other unusual presentations include Usher syndrome-like phenotype, as well as the very recently described Peroxisomal fatty acyl-CoA reductase 1 disorder as well as CRSPW syndrome (cone-rod dystrophy, developmental delay, spastic paraparesis, and white matter disease).
We conclude that peroxisomal disorders are more heterogeneous in their clinical presentation than previously thought and should be considered in the differential diagnosis of a wide array of clinical presentations. Our data also demonstrate that milder forms cannot be ruled out by the "gold standard" VLCFA assay, which highlights the value of a genomics-first approach in these cases.

Isabel Filges
Medical Genetics, University Hospital Basel, Basel, Switzerland The clinical presentation of fetal malformations or malformation syndromes, particularly if lethal in utero or perinatally, and their genetic causes often remain undefined for various reasons. Intestinal atresias have long been hypothesized to result from either failure of recanalization of the intestinal lumen or in utero vascular accidents, jejunal atresia being the most common in newborns. However, some animal models implicate disruptions in molecular pathways in atresia formation. Clinically, few familial hereditary forms have been reported. Apple peel atresia is most likely due to agenesis or maldevelopment of the superior mesenteric artery and its branches.
We have recently described autosomal recessive mutations in CENPF to be causal for Strømme syndrome in the original sibs presenting with the triad of apple peel intestinal atresia, ocular malformations and microcephaly and a novel family with two fetuses with duodenal and multiple jejunal atresias, hyperrotation of the midgut, histopathological signs of intestinal and cardiac myopathy and overrepresentation of bi-nucleated cells, additional organ anomalies including eye anomalies and preaxial polydactylyphenotypic variability compatible with the CENPF regulatory role in cell proliferation and ciliary function. In addition, autosomal recessive multiple intestinal atresia with or without immunodeficiency is due to mutations in TTC7A, recessive mutations in RFX6 have been described in the clinically overlapping Mitchell-Riley and Martinez-Frias syndromes. Interestingly, autosomal dominant mutations in ACTG2 were shown to cause familial visceral myopathy presenting with functional gastrointestinal obstruction even leading to death due to impaired function of enteric smooth muscle cells. Single case reports mention the association of jejunal atresia and polydactyly, biliary atresia or heterotaxy.
Since we systematically use exome sequencing for discovery of novel autosomal recessive conditions in non-consanguineous families with recurrent lethal fetal phenotypes after autopsy, we now report on a family with a first girl presenting with duodenal atresia and heterotaxy, the intrauterine death of the second boy at 21 gestational weeks with IUGR, anal atresia, malrotation, and cystic dysplastic kidneys, and a boy born and deceased at 32 weeks with arthrogryposis, hypotrophic skeletal and smooth muscle, intestinal string of pearls stenosis and hypoplastic genitalia. We hypothesize an autosomal recessive disorder due to mutations in a gene implied in an embryonic pathway affecting cell cycle and ciliary function leading to a variably expressed phenotype linking all three phenotypic findings -atresia, muscle impairment and clinical signs of ciliopathies. We will present data of the currently ongoing analysis.
Atresia phenotypes show great overlap, but also important even intrafamilial variabilityin severity but also type of atresia as well as associated anomalies. Therefore, they likely represent different degrees of malformations of the same or linked embryologic process. Autosomal recessive inheritance may play a significant role. We discuss the degrees of phenotypes and linked pathways as well as the notion that genes involved in cell cycle control and division as well as ciliogenesis and -function may be considered important candidates when elucidating underlying pathways in intestinal atresia. Establishment of the various body axes, including left-right asymmetry, begins just before and during gastrulation with initiation of the primitive streak by NODAL, and results in a complex pattern of spatial and temporal gene expression to allow the development of both symmetric and asymmetric structures. Midline defects are reported in heterotaxy disorders, although foregut defects receive less attention than the asymmetric midgut. The purpose of this report is to highlight foregut malformation in two malformed fetuses with laterality defects and review the pertinent embryology of the trachea/esophagus. Case 1 is a stillborn male delivered to a 26 yo G2P1sAb1 healthy mother at 33 wk gestation. Autopsy showed occipital encephalocele, cleft palate, right arm phocomelia with 2 digits, absent left thumb, dextrocardia, complex heart defect, abnormal lung lobation, complete tracheal rings throughout, accessory spleens, GI malrotation, multicystic right and absent left kidney, abnormal ribs and scoliosis. Karyotype and array CGH normal. WES revealed 2 inherited heterozygous variants considered likely pathogenic; a NODAL missense variant (mat) and a frameshift in DYNC2H1 (pat), associated with Short Rib Thoracic Dysplasia 3. In addition were 2 more VUS, considered possibly related to the phenotype. Case 2 is a male delivered to a 21 yo G1P1 mother at 26 wk gestation. Autopsy showed alobar holoprosencephaly, bilateral microphthalmia, cleft lip, dysgnathia, microglossia,/glossoptosis, dextrocardia, marked LV dilation, bilateral unsegmented lungs and esophageal atresia with tracheoesophageal (TE) fistula. Array CGH was normal.
The developmental biology of the foregut derivatives, including patterning of the tracheal cartilaginous rings has just recently been studied in detail. Shh, a familiar gene in left-right signaling and patterning the ventral neural tube, has also been shown in a mouse model to be critical in foregut development. Shh signaling is required for the for-

LIKELIHOOD OF VATER/VACTERL IN PATIENTS WITH ESOPHAGEAL ATRESIA
The purpose of our study was to evaluate cases of EA1/-TEF within the context of two standardized definitions of VATER/VACTERL to investigate how many cases meet formal criteria for diagnosis.
In our study we reviewed all the cases of EA1/-TEF as recorded into Texas Birth Defects Registry (TxBDR) from 1999 to 2014. We defined VATER as described by Quan and Smith in 1973, and VAC-TERL based on the 2011 review by Solomon. Cases were classified into five groups using National Birth Defects Prevention Study criteria -Isolated, EA1/-TEF with malformations meeting defined criteria for VATER and/or VACTERL, EA1/-TEF associated with multiple malformations not meeting the VATER/VACTERL definitions (unknown etiology), and those with known chromosomal or single gene disorders. However, whether matUPD16 causes a recognizable syndrome is controversial. One complication is that matUPD16 is almost always accompanied by mosaic trisomy 16, either confined to the placenta or also present mosaically in the fetus. The challenge lies in determining whether mosaic trisomy 16 or matUPD16 (or both) are responsible for the subsequent phenotypic effects in the infant. Identification of pregnancies affected by trisomy 16 is becoming more common as select laboratories report on trisomy 16 in addition to trisomy 13, 18, and 21.
We report the prenatal, perinatal, and postnatal findings for seven maternal and fetal/infant pairs with trisomy 16 detected during pregnancy. Two mothers with elevated hCG levels on a quad screen were labeled with a risk for trisomy 21 that was >10 times their age-related risk. Average maternal age was 32 years (range 25-37). Maternal preeclampsia leading to eclampsia or HELPP syndrome was common, with six infants delivered by C-section between 26-35 weeks gestation.
One pregnancy was electively terminated at 24 weeks. A two-vessel cord was noted in two cases. Echogenic bowel was noted in three fetuses prenatally. In almost all of the pregnancies, slowing of fetal growth was a concern. Although these fetuses were said to have IUGR (intrauterine growth restriction) during the pregnancies, values for birth weight, length and OFC were 2 nd -60 th centile for all newborns.
In three cases, the cell-free DNA (cfDNA) screen identified an increased risk of trisomy 16. Three mothers had normal cfDNA screening performed by laboratories that did not report trisomy 16. Homozygosity on chromosome 16, suggestive but not diagnostic of uniparental disomy, were detected in two cases by SNP microarray. Myhre syndrome (OMIM #139210) is due to specific SMAD4 mutations, which cause short stature and a striking multisystem phenotype.  Angelman syndrome (AS) results from the loss of expression of the maternally-inherited copy of UBE3A in the brain due to one of four mechanisms, viz. a deletion on maternal chromosome 15q11q13, paternal uniparental disomy (UPD) of chromosome 15, an imprinting defect, or a mutation in the maternally-inherited copy of UBE3A. Although AS is a neurodevelopmental disorder that was described over 50 years ago, the ages at which these individuals achieve various developmental milestones have not been systematically studied.
The AS Natural History Study initiated in 2006 is a longitudinal study in which each participant is evaluated at a study site at regular, typically annual, intervals. At each study visit, we ask parents the ages at which the participants achieved specific motor and language skills.
We have enrolled 302 individuals with a cytogenetic or molecular confirmation of AS (age range: 5 months to 40 1 =2 years old). While the latter remains possible, our family challenges the concept that mutations in PNPT1 cause a pure sensory phenotype. Identification of further families will be required to fully understand the natural history associated with mutations in PTPN1. However, given the large window of time (four decades) with only a single symptom, there may be a future therapeutic opportunity for similar families.
It is likely that other rare disorders presenting in childhood with single system involvement may ultimately be associated with other complications. Reporting such families, studying more undiagnosed adult patients, creating gene or disease-specific databases with natural history information, and working in partnership with patients and their families will be necessary to further our understanding of such rare disorders. PMM2-CDG patients, who ranged in age from 2 months to 67 years.
In contrast to the reported mortality rate of 20% with the infantile multisystem presentation, we observed a mortality rate of 55% (5/9) in patients presenting in the first year of life. One patient with the childhood ataxia-intellectual disability presentation is now 21 years old and exhibits seizure disorder, retinitis pigmentosa, and progressive kyphoscoliosis. Another patient was diagnosed at age 67 years after evaluation of progressive ataxia and cerebellar atrophy. This patient had been thought to have cerebral palsy due to abnormal childhood gait.  The core entity of TCS has been difficult to settle upon for a variety of reasons encompassing, the inherent variability of TCS itself, coupled with phenocopy admixture including unbalanced chromosome rearrangements. TCS, being such a fraught clinical assignment has unsurprisingly eluded gene identification so far.
A severely disabled female now aged 12 years has been followed for a decade for a diagnostically elusive combination of severe cognitive disability, postnatal growth delays, CNS dysgenesis with partial ACC and cerebellar hypoplasia with hypotonia and a progressive severe neuromuscular scoliosis, with distinctive external features, including facial asymmetry, a wide forehead, squared fleshy ears, telecanthus, tear-shaped philtrum and thin upper lip and unilateral 2/3 toe syndactyly. A prior epic workup including biochemical testing, array CGH, and de Lange gene sequencing was unrevealing.
Over the past 2 years, distinctive hyperkeratosis of the dorsum of both thumbs has appeared. As hyperkeratosis has been described in the LDDB as a feature of TCS this prompted re-consideration of TCS  Of the critical genotypes identified in the 13 resolved cases, 5 involve de novo mutations, 2 are compound heterozygous, 1 is heterozygous and inherited from an affected parent, 1 is X-linked, and 4 are homozygous. Only one family was aware of even distant relationship between the parents. A particularly interesting patient has Crouzon syndrome with an identified FGFR2 missense mutation, but more severe cognitive disabilities than described in his father and sister who share the Crouzon syndrome diagnosis. It may be that mutations in a second gene contribute to this patient's phenotype. Another interesting patient has both Robinow syndrome and G6PD deficiency, resolved by a de novo deletion in DVL1 and a point mutation in G6PD.
Among the many benefits from this collaboration are the ability to provide diagnoses for patients and families, including the possibility of and Brazil, with no specific mention of ethnic diversity. We hypothesized that in our clinical cohort, there would be a higher percentage of underrepresented minority populations in the more severe subgroup than in the more mildly affected individuals, and that there would be specific features that would be distinctive in some of the populations.
We reviewed 136 patients seen in our biannual multidisciplinary CdLS aging clinic, and 30 outpatients with CdLS seen in our pediatric genetics clinic, for a total of 166. Of these, 32 (19%) were found to be either Black (13, or 8%), Latino (11, or 7%), Asian (4, or 2.4%), or Pacific Islander (4, 2.4%). Some were mixed racial (7, or 5%), and no patients BBS is an autosomal recessive ciliopathy currently associated with 19 genes. We reviewed our PKS patient database for individuals with con- to the phenotypic variability noted in our cohorts.
In conclusion, a small catalog of minority PKS patients have been identified and characterized. While the numbers in our study limit statistical significance in this rare syndrome, the differences and similarities across several ancestries will be presented. Multiplex families with non-syndromic microtia have been clinically described, but causative mutations have been only identified for two families with HOXA2 mutations. We performed whole exome sequencing (WES) on a family from Colombia, presenting with isolated cupshaped microtia in an autosomal dominant mode of inheritance with incomplete penetrance, where 5 individuals were affected and two individuals were unaffected, but transmitted the mutation (carriers).

FGFR2 MUTATION IN MULTIPLEX FAMILY WITH CUP-SHAPED EARS
WES revealed that affected individuals and mutation carriers shared 7 conserved and rare variants (GERP>4, frequency < 0.0001), of those 2 were expressed in the mouse 2 nd branchial arch (E10.5 and E11.5) and in the human embryo developing ear (at 54 days). One of these two genes is FGFR2, an heterozygous TTC insertion was identified in this gene resulting in an insertion of a serine. This mutation has not been described in ExAC and in 40 trios with non-syndromic microtia from the same region. It is predicted to be a frameshift disruptive mutation,  patient was born to non-consanguineous German parents. Her similarly affected brother died in infancy. At birth, she was too short (-2.8 SD) and mildly microcephalic (-2.3 SD). She developed intractable seizures within the first hour of life. Her growth continued to be mildly retarded (-2.8 SD at the age of 9 years) and microcephaly was progressive (-6.5 SD at the age of 9 years). She did not achieve any of the motor or cognitive developmental milestones, she did not have eye contact, and the only interaction with her surrounding was a mild reaction of being touched. The third patient was initially evaluated at 11 days of age when she exhibited myoclonic seizures, intrauterine growth retardation, microcephaly, and elevated lactic acid. At birth, she was microcephalic (-2.9 SD) and microcephaly was progressive (-5.4 SD at the age of 19 months). Later she was found to have nystagmus, but this appeared to be secondary to therapy with valproic acid. She has required a gastrostomy feeding tube. The fourth patient was born to non-consanguineous German parents. At birth, she was also too short (-2.7 SD) and microcephalic (-3.9 SD). At 2 years of age, she still showed short stature (-4.7 SD) and progressive microcephaly (-9.7 SD). She developed pharmacoresistant epilepsy, beginning 3 hr after birth. Initially, she showed hypotonia, and presented with severe spasticity at follow up. She shows profound delay with no visual reactions. We performed detailed evaluation of cranial MRI in the patients. Characteristic neuroimaging features include delayed myelination, hypoplasia or aplasia of corpus callosum, cortical anomalies and enlarged cerebral ventricles.
QARS variants may likely be diagnosed more frequently in the future due to the wide and increasing application of NGS technologies.
NGS panels of genes associated with microcephaly, epilepsy and brain anomalies should include QARS. These new patients and additional cases will allow for further delineation of the phenotype of this rare syndrome. gene, but is associated with overgrowth without macrocephaly and includes long fingers, large feet, and a facial phenotype without hypertelorism. The proband was a girl seen in the neurology clinic at the age of 2 for developmental delay. On physical examination, she was noted to have tall stature, prominent eyes, a somewhat bulbous nose, and a head circumference at the 5 th % ile. She was also far-sighted and wore glasses at the age of 2 years. Her family history was positive for learning problems in both parents. A subsequent microarray revealed a 464 kb deletion at 17q11.2 that was felt to be of unknown significance.

THE GENETIC LANDSCAPE OF FAMILIAL CONGENITAL HYDROCEPHALUS
The family was referred to genetics when a subsequent maternal sample identified the same deletion.
This began a long relationship in the genetics clinic involving subsequent children and the previous generation. The proband's mother, maternal grandmother, and maternal half-sister were found to have the microdeletion and share the characteristics of tall stature, normal head circumference, long fingers and large feet and facial features that are more recognizable as younger children, but include prominent eyes, a pointed chin and a triangular face. All affected individuals have developmental delay or intellectual disability and some degree of vision and hearing problems. The proband also has a full brother and a maternal half-sister who do not have the deletion, nor do they have the same phenotype.
The deleted interval involves six known genes.  then became a chimera with an affected lost triplet, or; 4) the abnormal interphase FISH nuclei in the maternal blood were persistent fetal cells resulting in a fetal microchimerism. The last of these seems most likely: that the mother's germline is normal and the interphase FISH result is acquired. This interpretation is also consistent with the normal female karyotype present in 30 cells since a 30 cell evaluation excludes a 10% level of mosaicism at a 95% confidence interval.
In a research setting, women with previous pregnancies have been Although peroxisomes are evolutionarily unrelated organelles, DRP1 also plays a role in promoting peroxisomal proliferation through a similar cleavage mechanism. DRP1 is produced in the cytosol and contains Decades of public health research have documented that active smoking in pregnancy poses significant health risks to both mother and child.
More recent studies have shown that even passive maternal exposure to secondhand smoke is associated with negative birth outcomes (1,2).
However, the mechanisms linking exposure to outcomes have The 8 year-old had mild speech delay and could get by in school with minimal help. Four of the affected had brain MRI, and no structural malformation was noted in any. On physical exam, they were all of normal stature (50-75th%) and head circumference (50-98th%) and not overtly dysmorphic. The younger boys had normal male genitalia. The adult males had normal sexual development and secondary sexual features (exam declined). None of the affected has movement disorders or spasticity. The 21 year-old has significant issues with social sexual boundaries and the mother is very worried that he might be arrested for sexually inappropriate behaviours. The ARX gene belongs to homeodomain transcript factor family that is involved in cerebral genesis and patterning. ARX-related phenotype ranges from mild delay to lethal brain malformation. While truncating and null mutations tend to give rise to severe phenotype, missense and duplications are often associated with variable expressivity. In the latter case, intracellular mislocalization of the transcription factor may be the underlying mechanism for the proteins dysfunction and the resultant clinical phenotype. reported foregut malformations were pyloric stenosis (incidence 1/ 700, as compared to reported incidence of 1/250 21/500); esophageal atresia with or without tracheoesophageal fistula (incidence 1/4500, as compared to reported incidence of 1/3500); duodenal atresia (incidence 1/7000, as compared to reported incidence of 1/6000); biliary atresia (incidence 1/13,000, as compared to reported incidence of 1/ 18,000); and annular pancreas (incidence 1/50,000, as compared to reported incidence of 1/20,000 to 1/7000).

MUTATIONS IN FIBRONECTIN CAUSE A SUBTYPE OF SPONDYLOMETAPHYSEAL DYSPLASIA WITH "CORNER FRACTURES"
Fibronectin is a master organizer of extracellular matrices (ECM), promoting assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its expression in osteoblasts, chondrocytes and mesenchymal cells. Spondylometaphyseal dysplasias (SMD) comprise a diverse group of skeletal dysplasias often presenting with short stature, growth plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of "corner fractures" at metaphyses, we identified three individuals with novel variants in the fibronectin gene (FN1) affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotye.
The severe scoliosis in most individuals and rare developmental coxa vara distinguishes individuals with FN1 mutations from those with classical Sutcliffe type SMD. To study functional consequences of these Other anomalies of the GI tract are less commonly reported and there has only been one prior report of an anorectal malformation and Noonan syndrome (rectal atresia).
We report three additional patients with Noonan syndrome and anorectal anomalies. The first was evaluated at 3 months of age and found to have a patent, but significantly anteriorly displaced anus. She had a de novo c.770C>T (p.Ser257Leu) variant in RAF1. Our second patient was a 12-day old male with anteriorly displaced anus versus anal stricture. He was found to have the same c.770C>T (p.Ser257Leu) variant in RAF1 that was identified in our first patient. Both patients required anal dilation. Our third patient was evaluated at 4 days of age.
He was found to have a prolapsed rectum. He had a c.236A>G (p.

Gln79Arg) variant in PTPN11.
Based on the anorectal findings of these cases, we retrospectively reviewed 53 cases of Noonan syndrome from a research cohort to further explore the relative frequency of associated GI concerns. Of the 53 individuals with Noonan syndrome, 29 had known mutations in causative genes, (79% were in PTPN11 and 7% were in RAF1). A GI complication was identified in 19 individuals (36%) within this research cohort. These complications ranged from feeding issues (i.e. swallowing difficulties, oral aversion, NG tube feeds) in 5 individuals (9%), to structural anomalies (i.e. malrotation) in 2 individuals (4%). A gastrostomy tube was present in 7 individuals (13%) within this cohort, though the indication was not specified. Anorectal anomalies were not reported for any of these patients. types. These include rare clinical presentations that have been described in <10 kindreds (n 5 47, 29.2%, e.g., Wahab syndrome), more recognizable syndromes that remain without a confirmed etiology (n 5 1, 0.6%, e.g., Dubowitz syndrome), mapped loci for a disease known to be genetically heterogeneous (n 5 33, 20.5%, e.g. retinitis pigmentosa), relatively common multifactorial entities (n 5 1, 0.6%, e.g., orofacial cleft) and descriptions of minor traits (n 5 9, 5.6%, e.g., Darwinian tubercle of the ear, musical perfect pitch). The remaining 70 entries (43.5%) describe loci associated with either quantitative traits (e.g., ALT levels) or susceptibility to common disorders (e.g., glaucoma) or traits (e.g., myopia, nocturnal enuresis).
We also considered how these phenotypic entries were being reclassified from one year to the next. Twenty-two entries denoted by a % sign in 2016 (May 2016, n 5 1622) had been reclassified by 2017.
Of these, 17 phenotypes were now associated with pathogenic variants in a specific gene (e.g., uncombable hair syndrome: PADI3). The remaining 5 were incorporated into other OMIM entries. For example, Miles-Carpenter syndrome (previously 309605), which was suspected to be an X-linked mental retardation syndrome, has now been incorporated into Wieacker-Wolff syndrome (314580), with the gene identified to be ZC4H2 on chromosome X. Interestingly, of the 7 new % phenotypes in 2017, some (2/7) appear to be reclassified due to downgraded genetic evidence for causality based on new control databases.
We will present our review of the 1600 unsolved clinical presentations with a focus on those associated with multiple malformations and will discuss the contribution of these unsolved syndromes to current practices of dysmorphology.

Maria Haanpää, MD, PhD
Stanford University School of Medicine, Stanford, California Nance-Horan syndrome (NHS: OMIM 302350) is a rare X-linked disorder characterized by congenital cataract, specific dental anomalies, and dysmorphic features. Mental retardation is present in about 30% of the cases. It is caused by mutations in the NHS action remodelling regulator gene on the chromosome Xp22.13. The encoded protein functions in eye, tooth, craniofacial and brain development. NHS syndrome is also known as cataract-oto-dental syndrome.
Ophthalmological findings in affected males are severe bilateral congenital dense nuclear cataracts which lead to profound vision loss and usually require surgery at an early age. Microcornea, microphthalmia and nystagmus have also been reported. Characteristic facial features such as large anteverted and simple pinnaes, long narrow face, prominent nose and nasal bridge, and distinctive dental anomalies such as supernumerary incisors (mesiodens), diastema between the teeth and screwdriver blade-shaped incisors are seen in affected males. Also, several families have been reported to have lateral brachymetacarpalia.
The disorder appears to be inherited in a semi-dominant fashion, with heterozygous females often manifesting similar but less severe features.
To date the number of cases reported is very small and the spectrum of phenotypic features especially in females, is not well defined.
Here we report the clinical and molecular findings in a three generation In conclusion, we report a novel nonsense mutation in NHS gene.
The spectrum of clinical features in Nance-Horan syndrome is variable.
In particular, for female patients, it can be very subdued. Our findings broaden the spectrum of NHS mutations causing Nance-Horan syndrome and describe detailed clinical findings of phenotypic spectrum in all three generations.    Importantly, some of these cellular and molecular processes are also essential or proper alveolar regeneration after acute lung injury. These ongoing studies provide critical insight into reinitiating lung growth in developmental lung disease and regenerating lung epithelium following injury. R255Q), suggesting locus heterogeneity. Here we report the second case of congenital acinar dysplasia due to a de novo mutation in TBX4.

ACINAR DYSPLASIA, TBX4 AND LUNG BRANCHING MORPHOGENESIS
The proband was the product of a natural conception to a 26 yo G1P0->1 mother. Pregnancy was uncomplicated. She was delivered at 41 1/7 weeks gestation via c-section due to failure to progress. Labor  findings thought to be consistent with "cat's paw" pigmentation. She was hypotonic, but now at age 2 years has normal development. We review a series of cases plus an MCA family with recurrent malrotation. Additionally, a tabulation of literature MCA syndromes associated with malrotation will be presented. ACDMPV. We present a female patient born to a 24 yo G1P0 white female with hypertension and obesity, who was noted at birth to have a small omphalocele. She quickly went into respiratory distress requiring intubation with 100% FIO2 and nitrous oxide. Echocardiogram revealed pulmonary hypertension, and a hypoplastic left heart variant with ASD, VSD, PDAs, bilateral SVCs, and coarctation of the aorta. Pulmonary hypertension continued to worsen, and the patient was placed on ECMO. A lung biopsy was performed which showed evidence of alveolar capillary dysplasia with misalignment of pulmonary veins. Given worsening cardiopulmonary status and biopsy results, the family decided to withdraw ECMO and the patient died at 14 days of age. A chromosomal microarray was negative. Sequence analysis of the FOXF1 gene found a pathogenic deletion of two nucleotides (c.236_237delAG, p.Ser80ProfsX293). This is a previously unreported de novo mutation. This is only the second known case of ACDMPV with omphalocele. We will review the clinical and histopathologic findings of ACDMPV. We will also review all of the reported foregut derivative abnormalities seen in patients with FOXF1 mutations and deletions. Finally, we will review the interaction between mesenchymal, to 30% of EA/TEF patients. In 6-10% of all EA/TEF cases, a defined genetic syndrome can be diagnosed, but in the remainder, the etiology is unknown. Rady Children's Hospital San Diego (RCHSD) is the largest surgical pediatric referral center in San Diego county. We performed a retrospective chart review of all the EA/TEF patients seen at this center from 2012-2016. The great majority of patients were managed at RCHSD neonatal critical care unit, but patients seen at the outpatient clinics or emergency department were also included if adequate records were available. A query to the electronic record system resulted in 67 cases. Eight were excluded because there was no EA or TEF, and nine were excluded because of incomplete records. This left us with 50 patients to review. As expected, the most common subtype was EA Gross Type C (78%), followed by EA without Fistula (Gross Type A, 10%) and type H TEF without EA (Gross Type E, 6%). About half (54%) of all the EA/TEF cases were evaluated by a geneticist. In 78% of the cases, there were other congenital anomalies present, most commonly cardiac (38%), vertebral (24%) and renal (22%). VATER association was diagnosed in 26% of cases. There were no cases clearly associated with teratogenic exposure. Gestational diabetes was present in two mothers (4%) but both cases were detected in the third trimester and were well controlled with diet. There was one case of maternal hyperthyroidism with third trimester methimazole exposure. Eleven of the 50 cases (22%) had an identified syndrome (See Table), most confirmed with laboratory testing. This number is higher than reported in the literature, which may reflect the increased likelihood of referral of complex cases. The pathologic mechanisms leading to EA/TEF remain unknown. Associated anomalies identified after birth included: airway/pulmonary (77%), cardiac (75%), spine (58%), genitourinary (42%), other gastrointestinal (38%), neurological (34%), limb (29%), and ophthalmic (27%) malformations. Three (6%) had isolated EA with or without tracheoesophageal fistula. Twenty-one (45%) of these cases were born prematurely before 37 weeks' gestation. More than half of (21/37 (57%)) children did not have any documented developmental delay.
Three cases were born in an outside hospital and were excluded from further analysis due to no access to their postnatal information.
Twenty-one out of 46 (45%) of the patients were evaluated by a clinical geneticist after birth, and genetic testing was done in 23/46 (50%) of patients postnatally, including some who have had prenatal testing.
Out of those patients seen by a geneticist prenatally, 59% were seen in follow up by a geneticist postnatally.
Twenty-six of 46 patients (57%) were given a genetic diagnosis.
VACTERL association was the most common clinical diagnosis (21/26 (80%)), followed by Down syndrome in 3/26 (11%), Feingold syndrome in one patient and CHARGE syndrome in one patient. For 20/46 of patients (43%), no genetic diagnosis was made. While many of the diagnoses were made by clinical geneticists (9/21 (42%)), nine VAC-TERL patients and two Down syndrome patients were diagnosed by surgeons and neonatologists. Patients were followed up for an average of 6.7 years (median 5 6.2, range 5 0 -14.9). Based on our current data, 75% of the patients were still alive, 23% were deceased, and 2% were lost to follow-up.
Our data suggest that many patients with EA have a unifying genetic diagnosis and would benefit from a clinical genetics consultation, which the majority of patients have not had. These data will help improve our counseling of women who are carrying a fetus with EA and provide the first insights into the natural history of EA. Random mating in the general population tends to limit the occurrence of homozygous and compound heterozygous forms of dominant hereditary disorders. Certain phenotypes, the most recognized being dwarfism/short stature, lead to cultural interaction and assortative mating.

BIALLELIC MUTATIONS IN WAARDENBURG SYNDROME GENES CAUSE RECOGNIZABLE ARTHROGRYPOSIS SYNDROMES
To this well-known example, we add deafness which brings together individuals with a variety of deafness genotypes, some being dominant.
Waardenburg syndrome is one such autosomal dominant disorder in which affected individuals may interact culturally because of deafness. tive Shh signaling and HPE. Much of ethanol's toxicity is ascribed to its oxidative metabolism by alcohol and aldehyde dehydrogenase. We find that treatment of Cdon mutant mice with t-butyl alcohol, which is not subject to such oxidative metabolism, induces HPE and defects in Shh signaling in Cdon -/embryos. We propose a model wherein ethanol itself, not a consequence of its metabolism, is an HPE-inducing teratogen and that it acts to modify Nodal signaling in Cdon -/embryos. Several years ago at this meeting we presented preliminary results of a project to collate data regarding critical periods during gestation when interference with normal development causes specific malformations.

UPDATE ON A TIMELINE OF CRITICAL DEVELOPMENTAL STAGES FOR THE TERATOGENIC CAUSATION OF BIRTH DEFECTS
The purpose of this presentation is to update progress on this project to identify and catalogue windows of teratogenic exposure for common birth defects using published data from human observations as well as animal studies (mouse, rat, golden hamster, and chick), translating non-human developmental stages to the corresponding human developmental days. Such an integrated timeline may be useful for epidemiologic studies involving multiple birth defects, in understanding gene-environment interactions, and for teaching normal embryologic development.
Human epidemiologic studies of birth defects may include multiple phenotypes and exposures to maximize resources. Statistical analyses of small groups can lead to spurious, misleading conclusions with low power and inflated false positive rates. Grouping defects can increase statistical power, but may cause a loss of biologic significance unless the groupings are based on common causal and/or pathogenetic factors. Our strategy is to consider clustering birth defects caused at the same stage in development. We propose that such a grouping can be combined with advanced statistical methods to find a compromise between lumping and splitting in studies designed to investigate numerous birth defects and potential risk factors.
In our previous, preliminary report at this meeting, we noted that a group of malformations appear to be caused early during gastrulation and neurogenesis, while there seemed to be another cluster during subsequent formation of the heart, sensory placodes, and pharyngeal arches. In addition, we found that some malformations appear to result from teratogenic exposures at more than one specific developmental stage, suggesting multiple mechanisms of causation. This presentation will provide an update of progress on this timeline, with the annotation of more than 30 clinically important structural anomalies. Approximately 1-2% of pregnancies are complicated by pre-gestational diabetes, either type 1 or type 2. The risk of congenital anomalies in infants born to diabetic mothers is 2-8 times higher than the background risk. These anomalies most frequently involve the craniofacial region, CNS, heart, GI tract, urogenital tract, and skeleton. This report describes an infant born to a diabetic mother with unilateral abdominal wall hypoplasia, preaxial hallucal polydactyly, and other anomalies and reviews the evidence that abdominal wall hypoplasia may be due to diabetic embryopathy.
A female infant was born to an insulin-dependent diabetic mother at 37 weeks gestation; HgbA1C levels were significantly elevated in early pregnancy. At birth she had unilateral preaxial hallucal polydactyly, absent left kidney, multiple thoracic, lumbar, and sacral segmenta- EMG findings in all three suggested moderate chronic involvement of peripheral motor neurons; compound muscle action potential of the phrenic nerve was reduced unilaterally in two infants and bilaterally in one. The spinal cord in one infant was thinner than normal in the thoracic area, predominantly in the ventral aspect reducing the ventral roots.
All mammals have movement of the diaphragm in utero preparing the newborn to breathe effectively upon delivery. In experimental animals, control of this process has been shown to be the result of medullary centers that generate the rhythmic bursts and interaction between the phrenic motor neuron and the muscles of the diaphragm. The phrenic nerve arises from the neck at C3-C5 and receives innervation from both the cervical and brachial plexus. One hypothesis suggests that the muscle of the diaphragm may arise from forelimb muscle cells.
We postulate that one clinical phenotype associated with congeni- In mice, VPA increases the risk of exencephaly, an anterior NTD, and the degree of susceptibility depends on the genetic background of the mouse strain exposed. Since the early 1980s, we have developed two mice strains who respond differently to maternal VPA treatment.
One strain (C57BL/6J) is resistant to VPA, with only a modest portion (10%) of the exposed embryos present with NTDs. While the SWV/ Fnn inbred mouse strain is highly sensitive to in utero VPA exposure, with no less than 80% of the embryos having NTDs after the dam exposed to VPA at E8. Using these two strains in a series of backcrossing linkage studies, we were able to map purported sensitivity genes to a 6Mb chromosomal region located on chromosome 7, which are responsible for VPA interactions in the SWV/Fnn embryos. To further this initial investigation, we performed whole genome sequencing (WGS) in VPA-exposed embryos from these two strains, and called genetic variants including both single nucleotide variants (SNVs) and copy number variants (CNVs) that differed between these two strains. We report two adult sisters followed since birth. Both share a remarkably similar phenotype with congenital cataracts, severe (   Using a facial analysis algorithm, we showed that when applied to individuals with Down syndrome from around the world, we found the sensitivity to be 96.1% and specificity to be 92.4%. Importantly, the accuracy of the technology increased significantly when applied to distinct population groups (African, Asian, and Caucasian). When using facial analysis technology in 22q11.2 deletion syndrome, we also found differences between population groups. Interestingly, all four groups (Caucasian, African, Asian, and Latin American) only shared two common geographic facial analysis features: telecanthus and narrow palpebral fissures.
In summary, genetic syndromes in diverse populations are underrepresented in the medical literature and underdiagnosed in the clinics.
We have shown that both subjective clinical examination and objective facial analysis technology have found differences in diverse population groups with selected genetic syndrome. And most importantly, we know that facial analysis technology is both sensitive and specific. We believe in both developed countries and in parts of the world where molecular testing is not available, facial analysis technology will be a valuable tool that will assist in earlier diagnoses and earlier treatment of patients with genetic syndromes. with clinical diagnosis of Carpenter syndrome due to his mild but typical facial configuration was found to have Primrose syndrome many years later after whole exome sequencing analysis. These cases are just part of the multiple unusual presentations seen in our cohort.
We conclude that initial genetics evaluation, of patients considered being part of the "minority" populations, needs to be carefully performed due to their atypical presentations and numerous limitations to receive a comprehensive genetic evaluation. We believe that some of these unusual findings can be part of a different presentation of the clinical spectrum of the genetic syndromes and some may be due the ethnic related physical traits. It is also possible that there are other unknown confounding genetic factors or modifiers that need to be studied further. Genetic, environmental and epigenetic factors contribute to gene expression differences during development, and these same factors likely play a role in modifying facial features as significantly as a strong

EMBRYONIC LETHAL MENDELIAN PHENOTYPES: A LARGE COHORT FROM A CONSANGUINEOUS POPULATION
Mendelian mutation. It is also clear that these modifiers play a key role in phenotypic variability between different ancestry groups. Of note, there are higher proportions of Caucasians in the databases used for this analysis. However, our approach to use the objectivity of computer software facial anthropometric analysis will allow us to accurately identify differences across underrepresented minorities (URM), and further assess its utility as a tool to help the clinician in the diagnosis of CdLS across diverse ancestral groups.
We have evaluated the dysmorphologic features in a large cohort of individuals who were self-or family-identified as a URM with CdLS.
All subjects had previously been enrolled in an IRB-approved protocol of informed consent. Our CdLS subject database was reviewed for all individuals with a clinical diagnosis. This identified 3078 overall subjects of whom 147 were identified as a URM belonging to one of the following ancestry cohorts -African American, Asian, Native American, Middle Eastern, and Hispanic/Latino. Of these clinically diagnosed individuals, we refined our analysis to patients with clinical images available, and identified 40 URM individuals and 180 matched Caucasians who also had confirmed molecular diagnoses. We restricted subsequent analyses to these 220 subjects.
Using facial recognition technology, we compared frontal photos between the URM and Caucasian cohorts. We assessed average scores between individuals grouped by mutated gene. We subsequently com- genes not known to be associated with CHD, variants not present in the ExAC database, and CADD scores greater than 15. Sixteen genes met this criteria and zebrafish evaluation is currently underway to validate pathogenicity. We have initiated the largest CHD study in a non-European cohort using next generation sequencing project to search for novel genetic associations with CHD. With 2/3 of next generation sequencing complete on our cohort, we are now finding novel genes that explain the CHD phenotype. Similar to European populations, we found that CHD in diverse populations is enriched with genes associated with genetic syndromes in which CHD comprises a major part of the phenotype. More interesting is our new gene discovery that will increase our understanding of the genetic basis of CHD. Additionally, we are conducting mouse model experiments on unique vitamin A pathway variants from our cohort that will contribute to our und rstanding of gene-environmental interactions.

RITSCHER-SCHINZEL/3-C SYNDROME -FURTHER DELINEATION OF A FIRST NATIONS COHORT AND IMPLICATIONS IN CHOLESTEROL HOMEOSTASIS
proposed in numerous patients with findings involving these three sys-  A number of disorders well known to this audience provide prototypes for the study of natural history; these include NF1, achondroplasia, and Prenatal findings of NBCCS in our cohort were ventriculomegaly (n 5 6), including two cases of unilateral ventriculomegaly, and cleft lip and palate (n 5 1). Features identified in the neonatal period were macrocephaly (n 5 4), cardiac fibroma (n 5 2), and metopic craniosynostosis (n 5 2). Rhabdomyoma, facial myofibroma, lingual cyst, and natal tooth were each observed in one individual. Manifestations in the three years after the neonatal period were medulloblastoma (n 5 3), nasal dermoid cyst (n 5 1), and cardiac fibroma (n 5 1).
No individual developed jaw keratocysts or basal cell carcinomas (BCC) prior to three years of age.
The most common manifestation in middle childhood and adolescence was jaw keratocysts (n 5 7), which developed at a mean age of 9 years. Only 3 patients had BCC. One developed thousands of BCC at age 3, following radiation therapy at age 7 months for medulloblastoma; the other two individuals had BCC by 6 and 13 years of age. One patient developed an osteochondroma of the tibia at 17 years.
Almost all patients had either neurologic or ophthalmologic mani- We discuss these findings here to reflect the broadening phenotype of CSS, in order to encourage clinicians to consider the diagnosis even when previously reported "key features" may be absent. Seven additional NAA10 mutations have been identified in nine males and sixteen manifesting females (the vast majority of which, were proven to be de novo). As one might expect, females generally had a milder and less characteristic clinical presentation than the males, but commonalities included moderate to severe neurocognitive impairments, postnatal growth failure commonly occurred, also resulting in severe microcephaly. Skeletal, brain, and organ anomalies were frequent, but without any apparent specific pattern.
Mutations in NAA10 are associated with reduced enzyme activity of N-Acetyltransferase (NatA  Among the first 20 patients we reviewed in detail, all of them had some degree of retinal degeneration, visual field constriction, and night blindness starting between ages 3 to 8 years of age. Seventy five percent had obesity (BMI greater than 30 kg/m 2 in adults or 95 th percentile for ages 2-20 years), with onset between ages 5 to 22 years old. Fifty percent of the patients had intellectual disability or developmental delay. Postaxial polydactyly was present in sixty percent. Renal function was normal for all 20, but renal ultrasound abnormalities were present in 5 patients (calyceal blunting, pelviectasis, hydronephrosis, ureteropelvic junction obstruction, and renal hypoplasia). Hypogenitalism including small penile shaft, reduced volume of the testes, and cryptorchidism was present in 5 patients. We will present data on the prevalence and age of onset of all primary and secondary findings of BBS in all 47 molecularly confirmed cases evaluated at our center.
The presence of both obesity and retinal disease are the most consistent findings in BBS in childhood, and these findings should prompt diagnostic workup for BBS even without other clinical findings to allow appropriate anticipatory management. Better phenotypic and natural history information will be critical for better management of this complex multisystem condition. decrease mobility. This is especially important in a population that seems to have increased risk for osteopenia. Also of note is the lack of cancer diagnoses in adulthood in this group, although the cohort is too small to draw any definitive conclusions about cancer risk in adults with CS. We plan ongoing follow-up of the current cohort of adults with CS in order to better understand progressive medical and physical problems, which is essential for providing targeted management recommendations and anticipatory guidance to families. associated with HHT which was negative. Further clinical details will be presented.

RASA1-RELATED DISORDERS
Our cohort confirms that the phenotype of individuals with germline RASA1 variants has significant variable expressivity. Although the majority of individuals had multifocal CMs, approximately 10% had a solitary CM. In addition, the AVMs were located in multiple different body regions beyond just the brain. Based on the relatively random locations and variation in the number of vascular malformations between individuals, we hypothesized that stochastic events were key drivers of the phenotypic expression. We previously documented a somatic second hit in one capillary malformation of an individual with CM-AVM syndrome (MacMurdo et al., 2016), and to further support this hypothesis, we obtained blood and tissue from the hypertrophic affected limb of an individual with the clinical phenotype of Parkes Weber syndrome. A RASA1 germline (c.1981_1985del, p. Thr662Glufs*6) and somatic (c.463G>T, p.Glu155*) mutation (6%) were identified, further supporting the role of "second hits" in the etiology of the vascular malformations in RASA1-related disorders. Although a somatic RASA1 mutation was documented in the biopsied tissue, the low level of mosacism suggests a mixed cellular origin. We hypothesize that the cells of origin are the endothelial cells and future studies of specific cell types are warranted. Our findings also show that RASA1related disorders have a high degree of variable expressivity, which is likely secondary to stochastic events such as a" second hit". The following observations emerged:

MALADAPTIVE BEHAVIORS IN CHILDREN WITH ANGELMAN SYNDROME
At baseline, there were statistically significant differences in the domains of irritability, lethargy, stereotypy and hyperactivity based on molecular subtype and level of developmental functioning.
At baseline, AS females were more lethargic than males Stereotypy did not change over time As individuals with AS mature, those with UBE3A mutations had greater increases in irritability, lethargy and stereotypy than those with Deletion or UPD/ID.
Hyperactivity increases with age for all molecular subclasses.
Correlations between severity of behavior problems with caregiver stress and family quality of life will be discussed. At birth Patient 1 had constricted pupils, enophthalmos, iridodonesis, dolichostenomelia with arachnodactyly and a murmur. At 2 months of age she had mitral valve prolapse, moderate aortic root dilation with a Z-score of 3.8 and bilateral ectopia lentis. At age 2.5 years she had her mitral valve replaced. At age 3 her aorta had enlarged to a Z-score of 11.6. She died at age 4.5 years following kyphoscoliosis surgery. She had a c.3218G>A mutation in exon 26 of the FBN1. Patient 2 presented at 1 month with mitral valve prolapse and regurgitation, a long face and trunk, deep-set eyes, arachnodactyly and long feet. Her aorta had become enlarged by age 18 months and at ages 5 and 9 years, her aortic Z-scores were 4.1 and 5.8, respectively. She has not had aorta replacement. As an infant, she also had a dislocated right lens, which was removed and replaced at age 18 months. At age 2 years she had rod-placement back surgery. At age 11 she was stable. She has an IVS30 1 5G>A mutation in FBN1. Patient 3 had cardiovascular involvement as an infant and at 10 years had her aortic valve and aortic root replaced. At the age of 20 she had aorta replacement, and at age 25, a mitral valve replacement. On physical exam at age 32 she had down slanting palpebral fissures, enophthalmos, abnormal ears, pectus excavatum, severe scoliosis, dolichostenomelia, arachnodactyly and decreased muscle mass. She has a c.3130T>G mutation in exon 25 of

FBN1.
We also report on 105 patients from the literature who we Twenty children were alive at the time of being reported and their ages ranged from 12 days to 11 years with a mean age of 2.7 years.
For both groups, there was also a large variety of other defects reported occurring at lesser frequencies.
At the present time there is no agreed on criteria for nMFS. We propose that the criteria include the presence of one or more systemic features of classic Marfan syndrome, aortic root dilation and major involvement of the mitral and/or tricuspid valves all before age 1 year, and an FBN1 mutation within exons 24-32. While we are not completely satisfied with the name nMFS, since not all children are diagnosed with the condition in the neonatal period, we do not have a better term and recommend continuous use of this name. A genotypephenotype correlation has not been made for mutations in exons 24-32. Such a correlation could account for the longer survival in some children. reported patients with PS, and to review the mechanisms, in addition to AKT1 mutation, which may lead to development of malignancy in these tumors.
We postulate that in addition to the risk for CHF and other complications of circulatory overload in the recipient, monozygotic twins are at increased risk for a spectrum of structural cardiac malformations which reflect a smaller number of cells affecting not only the cardiac primordium, but also overall fetal and placental growth. In severe cases the affected twin has a poorly developed heart and inadequate circulation, eventually becoming acardiac, while in less severe cases, the smaller infant has not only deficient septal growth sometimes resulting in VSD, but also less angiogenesis compared to the co-twin and becomes the "donor". Our observations of low concordance for cardiac defects and greater weight differential for pairs with structural heart disease among both liveborn and stillborn pairs with TTT relative to like sex controls without TTT support this hypothesis. Pleiotropy is the phenomenon by which the same gene can result in multiple phenotypes. Although this occurrence has been known for over 100 years, its contribution to rare genetic diseases has only really been appreciated since the boom in genetic discoveries precipitated by the advent of next-generation sequencing strategies. In the past year alone, the percentage of genes with phenotype-causing mutations associated with >1 phenotype in OMIM has increased from 25% to 32%. Pleiotropic proteins are therefore emerging as an important contributor to both rare and common disorders, but little is known of the mechanisms underlying pleiotropy, particularly with how they relate to congenital malformation syndromes.

LANDSCAPE OF PLEIOTROPIC PROTEINS CAUSING HUMAN MALFORMATION SYNDROMES
To examine the contribution of this phenomenon to congenital malformation syndromes we referred to the 7th edition of Smith's Recognizable Patterns of Human Malformations and compared it to OMIM data accessed in April 2017. Each listed syndrome was queried within OMIM and PubMed to identify the currently understood molecular etiologies (all genes in which pathogenic variants have been found to cause the syndrome). Syndromes associated with chromosomal abnormalities and disorders caused by contiguous gene deletions were excluded. In total, 231 syndromes were examined. Of these, 10 remained 'unsolved' (defined as molecular cause unknown).
In total, 402 unique genes had been implicated in Smith malformation syndromes; 72 of the 'solved' 221 syndromes (32%) were associated with pathogenic variants in more than one gene (genetic heterogeneity). Of the 402 genes, 271 (67%) were associated with more than one phenotype within OMIM; a rate that is twice as high as observed for all genes listed in the OMIM dataset (32%). Forty-eight of the 270 genes (18%) were associated with more than one distinct malformation syndrome within Smith's. Not surprisingly, the rates differed amongst categories of syndrome; being highest in neurological phenotypes (75%) and lowest amongst storage disorders (40%). The mechanisms of pleiotropy also varied tremendously at the protein level and could be sorted into four different categories: (1) distinct location of the mutations in different domains of the protein (e.g., FGFR3), (2) quantitative differences in the level of protein (e.g., POMT1) (3) qualitative effects on the protein (e.g., GOF vs LOF; FLNA), and (4) other influences on the protein (i.e., same mutation but different phenotypes due to unknown contributions; LMNA).
These results provide a better understanding of the pleiotropic proteins related to congenital malformations. They further support evidence that pleiotropic proteins are more likely to be 'essential' in human development and will be used to guide future discovery efforts for unsolved malformation syndromes. failure to thrive, with only 50% surviving to weaning. We also generated embryos mosaic for humanized Fzd2 W553* knock-in; a "F0-CRISPR" approach of harvesting injected embryos at E17.5 revealed five edited embryos with the Fzd2 W553* mutation. All F0 embryos exhibited cleft palate, smaller weights, and shortened limbs, consistent with FZD2 W548* patients. This in-vivo replication of clinical features seen in patients represents an approach which may be used to further investigate the mechanism of the autosomal dominant omodysplasia phenotypes and validates the utility of CRISPR knock-in mice as a tool for demonstrating pathogenicity of human genetic variants. Ongoing studies are focused on clarifying the mechanism of dominant inheritance via the role of DVL-FZD2 binding mediating canonical Wnt signaling in the face, as well as the effects of genetic background on the observed phenotypes.