Abnormal phenotypes have played significant roles in the discovery of gene function, but organized collection of phenotype data has been overshadowed by developments in sequencing technology. In order to study phenotypes systematically, large-scale projects with standardized objective assessment across populations are considered necessary. The report of the 2006 Human Variome Project meeting recommended documentation of phenotypes through electronic means by collaborative groups of computational scientists and clinicians using standard, structured descriptions of disease-specific phenotypes. In this report, we describe progress over the past decade in 3D digital imaging and shape analysis of the face, and future prospects for large-scale facial phenotyping. Illustrative examples are given throughout using a collection of 1107 3D face images of healthy controls and individuals with a range of genetic conditions involving facial dysmorphism.

In 2005 the authors of this article formed an international working group to develop standardized definitions and terms to describe the physical variations used in human phenotypic analyses. This project, which came to be known as the Elements of Morphology, resulted in six articles proposing consensus definitions for almost 400 phenotypic variations of the head and face, periorbital region, ear, nose and philtrum, mouth and lips, and hands and feet. Every variation was accompanied by a representative figure depicting the feature. The papers were published in the January 2009 issue of the American Journal of Medical Genetics Part A and are available free access on both the Journal's web page and a National Institutes of Health-based site. The publication of the Elements definitions has spawned an ongoing dialogue about the proposed terms to describe phenotype. The working group considered the six articles as only the first step in the process, and four more papers on proposed terminology for the trunk, genital region, skin, and remainder of the limb terms are in preparation. The secondary outcome of the Elements project is the provision of a working methodology for the establishment of standardized terminology and definitions for phenotype analysis in general.

Whole genome analysis, now including whole genome sequencing, is moving rapidly into the clinical setting, leading to detection of human variation on a broader scale than ever before. Interpreting this information will depend on the availability of thorough and accurate phenotype information, and the ability to curate, store, and access data on genotype-phenotype relationships. This idea has already been demonstrated within the context of chromosome microarray (CMA) testing. The International Standards for Cytogenomic Arrays (ISCA) Consortium promotes standardization of variant interpretation for this technology through its initiatives, including the formation of a publicly available database housing clinical CMA data. Recognizing that phenotypic data is essential for the interpretation of genomic variants, the ISCA Consortium has developed tools to facilitate the collection of this data and its deposition in a standardized, structured format within the ISCA Consortium database. This rich source of phenotypic data can also be used within broader applications, such as developing phenotypic profiles of emerging genomic disorders, the identification of candidate regions for particular phenotypes, or the creation of tools for use in clinical practice. We summarize the ISCA experience as a model for ongoing efforts incorporating phenotype data with genotype data to improve the quality of research and clinical care in human genetics.

Mouse phenotype data represents a valuable resource for the identification of disease-associated genes, especially where the molecular basis is unknown and there is no clue to the candidate gene's function, pathway involvement or expression pattern. However, until recently these data have not been systematically used due to difficulties in mapping between clinical features observed in humans and mouse phenotype annotations. Here, we describe a semantic approach to solve this problem and demonstrate highly significant recall of known disease-gene associations and orthology relationships. A web application (MouseFinder; www.mousemodels.org) has been developed to allow users to search the results of our whole-phenome comparison of human and mouse. We demonstrate its use in identifying ARTN as a strong candidate gene within the 1p34.1-p32 mapped locus for a hereditary form of ptosis.

Genome-wide arrays are rapidly replacing conventional karyotyping in postnatal cytogenetic diagnostics and there is a growing request for arrays in the prenatal setting. Several studies have documented 1-3% additional abnormal findings in prenatal diagnosis with arrays compared to conventional karyotyping. A recent meta-analysis demonstrated that 5.2% extra diagnoses can be expected in foetuses with ultrasound abnormalities. However, no consensus exists as to whether the use of genome-wide arrays should be restricted to pregnancies with ultrasound abnormalities, performed in all women undergoing invasive prenatal testing or offered to all pregnant women. Moreover, the interpretation of array results in the prenatal situation is challenging due to the large numbers of copy number variants with no major phenotypic effect. This also raises the question of what, or what not to report, i.e. how to deal with unsolicited findings. These issues were discussed at a working group meeting that preceded the European Society of Human Genetics 2011 Conference in Amsterdam. This paper is the result of this meeting and explores the introduction of genome-wide arrays into routine prenatal diagnosis. We aim to give some general recommendations on how to develop practical guidelines that can be implemented in the local setting and that are consistent with the emerging international consensus.

Next-Generation Sequencing (NGS) is the most powerful diagnostic tool since the roentgenogram. NGS will facilitate diagnosis on a massive scale -allowing interrogation of all genes in a single assay. It has been suggested that NGS will decrease the need for phenotyping in general, and medical geneticists in particular. We argue that NGS will shift focus and approach of phenotyping. We predict that NGS performed for diagnostic purposes will yield variants in several genes, and consequences of these variants will need to be analyzed and integrated with clinical findings to make a diagnosis. Diagnostic skills of medical specialists will shift from a pre-NGS-test differential diagnostic mode to a post-NGS-test diagnostic assessment mode. In research phenotyping and medical genetic assessments will remain essential as well. NGS can identify primary causative variants in phenotypes inherited in a Mendelian pattern, but biology is much more complex. Phenotypes are caused by the actions of several genes, and epigenetic and environmental influences. Dissecting all influences necessitates ongoing and detailed phenotyping, refinement of clinical diagnostic assignments, and iterative analyses of NGS data. We conclude that there will be a critical need for phenotyping and clinical analysis and that medical geneticists are uniquely positioned to address this need.

Neurofibromatosis type-1 (NF1) is associated with the growth of benign and malignant tumours. ∼15% of NF1 patients develop malignant peripheral nerve sheath tumours (MPNSTs), underlining the need to identify specific diagnostic/prognostic biomarkers associated with MPNST development. The Affymetrix Genome-wide Human SNP-Array-6.0 was used to perform single nucleotide polymorphism genotyping and copy number alteration (CNA), loss-of-heterozygosity (LOH) and copy number neutral-LOH (CNN-LOH) analyses of DNA isolated from 15 MPNSTs, 5 benign plexiform neurofibromas (PNFs) and patient-matched lymphocyte DNAs. MPNSTs exhibited high-level LOH, with recurrent changes occurring in MPNSTs but not PNFs. CNN-LOH was evident in MPNSTs but occurred less frequently than genomic deletions. CNAs involving the ITGB8, PDGFA,RAC1 (7p21-p22), PDGFRL (8p22-p21.3) and MMP12 (11q22.3) genes were specific to MPNSTs. Pathway analysis revealed the MPNST-specific amplification of seven Rho-GTPase pathway genes and several cytoskeletal remodelling/cell adhesion genes. In knockdown experiments employing short-hairpin RAC1,ROCK2, PTK2 and LIMK1 RNAs to transfect both control and MPNST-derived cell lines, cell adhesion was significantly increased in the MPNST cell lines whereas wound healing, cell migration and invasiveness were reduced, consistent with a role for these Rho-GTPase pathway genes in MPNST development and metastasis. These results suggest new targets for therapeutic intervention in relation to MPNSTs.

Familial adenomatous polyposis (FAP) is an autosomal dominant condition mainly due to a mutation of the adenomatous polyposis coli (APC) gene. The present study reports evidence of a technical issue occurring during the mutational analysis of APC exon 4. Genetic conventional direct sequence analysis of a repetitive AT-rich region in the splice acceptor site of APC intron 3 could be misinterpreted as a pathogenetic frameshift result. However, this potential bias may be bypassed adopting a method for random mutagenesis of DNA based on the use of a triphosphates nucleoside analogues mixture. Using this method as a second level analysis we also demonstrated the non-pathogenic nature of the variant in the poly A trait in APC exon 4 region (c.423-4delA) that do not result in aberrant splicing of APC exons 3-4, conversely we did not find a previously reported T deletion/insertion polymorphism.

Digital clubbing is usually secondary to different acquired diseases. Primary hypertrophic osteoarthropathy (PHO) is a rare hereditary disorder with variable digital clubbing as the most prominent feature, subperiosteal new bone formation, and arthropathy. Recently, mutations in the 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) encoding gene HPGD were found to cause PHO. Here, we identified three unrelated families with different mutations in the prostaglandin transporter (PGT) encoding gene SLCO2A1 which presumably result in reduced metabolic clearance by 15-PGDH due to diminished cellular uptake of prostaglandin E2 (PGE2) by mutant PGT. In two consanguineous families, homozygous mutations, an intragenic deletion that results in frameshift and a missense mutation, are associated with a severe PHO phenotype. In a third family, a heterozygous carrier of a stop mutation presents with isolated digital clubbing. Thus, our study further supports the importance of PGE2 metabolism in the pathogenesis of digital clubbing and PHO.

The number of known disease-causing mutations has increased dramatically. However, there have been few organized mutation databases developed that are available to the public or not-for-profit entities. Thus, clinicians and diagnostic laboratories had to spend time searching many publications and databases to determine whether a mutation has been previously reported. To assist in genetic diagnoses, the systematic collection and curation of mutations is necessary. The Korean Mutation Database (KMD, http://kmd.cdc.go.kr) is a country-specific database of human gene mutations that was established in September, 2009. The KMD is a database consolidating mutations of genes related to diseases in Korea; it now contains more than 1,600 mutations from 245 genes. We collected mutation data from diagnostic laboratories and published journals over recent decades in Korea. KMD has been open to the public for searches and registration of mutation data without charge. Our aim is to provide organized information for clinicians and researchers who are interested in genetic diseases. It will be useful not only for researchers in Korea but also for those in countries with similar ethnic backgrounds. Ultimately, KMD will be an essential base to improve researches in genetic diseases, developments of diagnostics, and therapeutic optimization.

In this study we assess exome sequencing (ES) as a diagnostic alternative for genetically heterogeneous disorders. Since ES readily identified a previously reported homozygous mutation in the CAPN3 gene for an individual with an undiagnosed limb girdle muscular dystrophy, we evaluated ES as a generalizable clinical diagnostic tool by assessing the targeting efficiency and sequencing-coverage of 88 genes associated with muscle disease (MD) and spastic paraplegia (SPG). We used three exome-capture kits on 125 individuals. Exons constituting each gene were defined using the UCSC and CCDS databases. The three exome-capture kits targeted 47-92% of bases within the UCSC-defined exons, and 97%-99% of bases within the CCDS-defined exons. An average of 61.2-99.5% and 19.1-99.5% of targeted bases per gene were sequenced to 20X coverage within the CCDS-defined MD and SPG coding exons, respectively. Greater than 95-99% of targeted known mutation positions were sequenced to =1X coverage and 55-87% to =20X coverage in every exome. We conclude therefore that ES is a rapid and efficient first tier method to screen for mutations, particularly within the CCDS annotated exons, although its application requires disclosure of the extent of coverage for each targeted gene and supplementation with second tier Sanger sequencing for full coverage.

Disease gene discovery has been transformed by affordable sequencing of exomes and genomes. Identification of disease-causing mutations requires sifting through a large number of sequence variants. A subset of the variants are unlikely to be good candidates for disease causation based on one or more of the following criteria: (1) being located in genomic regions known to be highly polymorphic, (2) having characteristics suggesting assembly misalignment, and/or (3) being labeled as variants based on misleading reference genome information. We analyzed exome sequence data from 118 individuals in 29 families seen in the NIH Undiagnosed Diseases Program (UDP) to create lists of variants and genes with these characteristics. Specifically, we identified several groups of genes that are candidates for provisional exclusion during exome analysis; 23,389 positions with excess heterozygosity suggestive of alignment errors; and 1,009 positions in which the hg18 human genome reference sequence appeared to contain a minor allele. Exclusion of such variants, which we provide in supplemental lists, will likely enhance identification of disease-causing mutations using exome sequence data.

The multidrug resistance protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel “Screen and Insert” (ScIn) technology to achieve stable and highly reproducible expression of thirteen ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared to wild-type (100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein and gluthatione-monochlorobimane by 80% and 50%, respectively, while R1181L and P1291L reduced only gluthatione-monochlorobimane transport by 50% as compared to wild-type. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for glutathione-methylfluorescein and gluthatione-monochlorobimane by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L and double V1188E/C1515Y have been identified as most promising for further clinical evaluation.

Genome-wide associations with glycosylated hemoglobin, which reflects the long-term glycemia, were examined using two independent cohorts of the Korea Association Resource (KARE) consortium. We first identified sequence variants within a linkage disequilibrium block (r² > 0.98) in the intron 5 of cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1) gene using 4,275 normoglycemic subjects of Cohort 1 (P < 2.5×10⁻⁸). The association was replicated in 3,782 normoglycemic subjects of Cohort 2. Furthermore, the sequence variants were also associated with glucose levels after oral glucose tolerance test. Especially, a strong association with 1 h glucose (P = 1.3×10⁻¹¹) led us to interpreting that CDKAL1 might influence the level of glycosylated hemoglobin by affecting 1 h glucose level. Ultimately, accumulated effects on the glycosylated hemoglobin level by the genetic variation of CDKAL1 might affect susceptibility to type 2 diabetes mellitus.

Numerous mismatch repair (MMR) gene variants have been identified in Lynch syndrome and other cancer patients, but knowledge about their pathogenicity is frequently missing. The diagnosis and treatment of patients would benefit from knowing which variants are disease-related. Bioinformatic approaches are well suited to the problem and can handle large numbers of cases. Functional effects were revealed based on literature for 168 MMR missense variants. Performance of numerous prediction methods was tested with this dataset. Among the tested tools only the results of tolerance prediction methods correlated to functional information, however with poor performance. Therefore a novel consensus based predictor was developed. The novel prediction method, PON-MMR, achieved accuracy of 0.87 and Matthews correlation coefficient of 0.77 on the experimentally verified variants. When applied to 616 MMR cases with unknown effects, 81 missense variants were predicted to be pathogenic and 167 neutral. With PON-MMR the number of MMR missense variants with unknown effect was reduced by classifying a large number of cases as likely pathogenic or benign. The results can be used e.g. to prioritize cases for experimental studies and assist in the classification of cases.

SOX5 encodes a transcription factor involved in the regulation of chondrogenesis and the development of the nervous system. Despite its important developmental roles, SOX5 disruption has yet to be associated with human disease. We report one individual with a reciprocal translocation breakpoint within SOX5, eight individuals with intragenic SOX5 deletions (four are apparently de novo and one inherited from an affected parent), and seven individuals with larger 12p12 deletions encompassing SOX5. Common features in these subjects include prominent speech delay, intellectual disability, behavior abnormalities, and dysmorphic features. The phenotypic impact of the deletions may depend on the location of the deletion and consequently which of the three major SOX5 protein isoforms are affected. One intragenic deletion involving only untranslated exons was present in a more mildly affected subject, was inherited from a healthy parent and grandparent, and is similar to a deletion found in a control cohort. Therefore, some intragenic SOX5 deletions may have minimal phenotypic effect. Based on the location of the deletions in the subjects compared to the controls, the de novo nature of most of these deletions, and the phenotypic similarities among cases, SOX5 appears to be a dosage-sensitive, developmentally important gene.

The analysis of variants generated by exome sequencing of families with rare Mendelian diseases is a time-consuming, manual process that represents one barrier to applying the technology routinely. To address this issue, we have developed a software tool, VAR-MD (http://research.nhgri.nih.gov/software/var-md/), for analyzing the DNA sequence variants produced by human exome sequencing. VAR-MD generates a ranked list of variants using predicted pathogenicity, Mendelian inheritance models, genotype quality and population variant frequency data. VAR-MD was tested using two previously solved data sets and one unsolved data set. In the solved cases, the correct variant was listed at the top of VAR-MD's variant ranking. In the unsolved case, the correct variant was highly ranked allowing for subsequent identification and validation. We conclude that VAR-MD has the potential to enhance mutation identification using family-based, annotated next generation sequencing data. Moreover, we predict an incremental advancement in software performance as the reference databases, such as dbSNP and HGMD, continue to improve.

Target enrichment strategies are a very common approach to sequence a predefined part of an individual's genome using second-generation sequencing technologies. While highly dependent on the technology and the target sequences selected, the performance of the various assays is variable also between samples and influenced by the way how the libraries are handled in the laboratory. Here, we show how we find detailed information about the enrichment performance using a novel software package called NGSrich which we developed as part of a whole-exome resequencing pipeline in a medium-sized genomics center. Our software is suitable for high-throughput use and the results can be shared using HTML and a webserver. Finally, we have sequenced exome-enriched DNA libraries of 18 human individuals using 3 different enrichment products and used our new software for a comparative analysis of their performance.

The FCGR locus encoding the low-affinity Fcγ receptors for IgG has largely been missed by genome-wide association studies due to complications with structural variation and segmental duplication. Recently identified CNVs affecting FCGR3A and FCGR3B have been linked to a number of autoimmune disorders. We have developed and validated a novel quantitative sequence variant (QSV) assay in combination with an adapted paralogue ratio test to examine independent CNVs carrying FCGR3A and FCGR3B in rheumatoid arthritis (RA) compared with healthy controls (n=1115 and 654, respectively). Implementation of a robust statistical analysis framework (CNVtools) allowed for systematic batch effects and for the inherent uncertainty of copy number assignment, thus avoiding two major sources of false positive results. Evidence for association with neither duplications nor deletions of FCGR3A was found, however, in line with previous studies there was evidence of overrepresentation of FCGR3B deletions in RA (OR 1.50, P=0.028), which was more apparent in rheumatoid factor positive disease (OR 1.61, P=0.011). The level of FcγRIIIb, encoded by FCGR3B, expression on neutrophils was shown to correlate with gene copy number. Thus, our results may highlight an important role for neutrophils in the pathogenesis of RA, potentially through reduced FcγRIIIb-mediated immune-complex clearance.

The Undiagnosed Diseases Program at the National Institutes of Health uses High Throughput Sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The paper is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects.

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation (SEPT12^T89M) significantly reduced GTP hydrolytic activity, and the p.Asp197Asn mutation (SEPT12^D197N) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12^D197N presented with oligo-asthenozoospermia while the SEPT12^T89M patient had astheno-teratozoospermia. The characteristic sperm pathology of the SEPT12^D197N patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation.

We evaluated an autopsy case with severe neonatal respiratory distress, hypoplasia of thymus, thyroid gland and cerebellum, and agenesis of the corpus callosum displaying striking phenotypic similarity to the CrebA knockout mouse. On the assumption that comparable genetic alterations must be present, we checked the whole genomic DNA sequence of CREB1, the human counterpart of mouse CrebA, and found a missense c.347A>G mutation corresponding to p.D116G within the kinase inducible domain (KID) of CREB1. When transcribed in vitro, while Ser133-phosphorylation of KID was maintained upon forskolin treatment, mutated CREB1 protein failed to associate with the KIX domain of coactivator CREBBP/EP300, and thereby, interrupted cAMP-dependent protein kinase (PKA) signal transduction as the dominant-negative form. This is the first report of a sporadic CREB1-related multiple malformation syndrome that, in light of accumulated knowledge of phenotypic features in gene-targeted animals, clearly emphasizes the importance of cross-species translational research.

PCDH19 encodes protocadherin 19 on chromosome Xq22.3. This 1148 amino-acid protein, highly expressed during brain development, could play significant roles in neuronal migration or establishment of synaptic connections. PCDH19 is composed of 6 exons, with a large first exon encoding the entire extracellular domain of the protein. Heterozygous PCDH19 mutations were initially identified in epilepsy and mental retardation limited to females, a familial disorder with a singular mode of inheritance since only heterozygous females are affected whereas hemizygous males are asymptomatic. Yet, mosaic males can also be affected, supporting cellular interference as the pathogenic mechanism. Recently, mutations in PCDH19, mostly occurring de novo, were shown to be a frequent cause of sporadic infantile-onset epileptic encephalopathy in females. PCDH19 mutations were also identified in epileptic females without cognitive impairment. Typical features of this new epileptic syndrome include generalized or focal seizures highly sensitive to fever, and brief seizures occurring in clusters, repeating during several days. Here we present a review of the published mutations in the PCDH19 gene to date and report on new mutations. PCDH19 has become the second most relevant gene in epilepsy after SCN1A.

Congenital cardiovascular malformation (CVM) exhibits familial predisposition but most of the specific genetic factors involved are unknown. Postulating that rare variants in genes in critical cardiac developmental pathways predispose to CVM, we systematically surveyed three genes of the bone morphogenetic protein (BMP) signalling pathway for novel variants. Exonic, splice site and untranslated regions of BMPR1A, BMPR2 and SMAD6 genes were sequenced in 90 unrelated sporadic cases of CVM. One non-synonymous variant (p.C484F) with predicted functional impact was found in the MAD homology 2 (MH2) domain of SMAD6, an intracellular inhibitor of BMP signalling. Sequencing this domain in an additional 346 cases of CVM yielded two further non-synonymous variants (p.P415L and p.A325T). Functional effects of all three SMAD6 mutations were investigated using BMP signalling assays in vitro. Two SMAD6 variants (p.C484F and p.P415L) had significantly (p<0.05) lower activity than wild-type SMAD6 in inhibiting BMP signalling in a transcriptional reporter assay. In addition, the p.C484F variant had a significantly (p<0.05) lower capacity to inhibit an osteogenic response to BMP signalling. We conclude that low-frequency deleterious variants in SMAD6 predispose to CVM. This is the first report of a human disease phenotype related to genetic variation in SMAD6..

Noonan syndrome (NS) is among the most common non-chromosomal disorders affecting development and growth. NS is genetically heterogeneous, being caused by germline mutations affecting various genes implicated in the RAS signaling network. This network transduces extracellular signals into intracellular biochemical and transcriptional responses controlling cell proliferation, differentiation, metabolism, and senescence. To explore the transcriptional consequences of NS-causing mutations, we performed global mRNA expression profiling on peripheral blood mononuclear cells obtained from 23 NS patients carrying heterozygous mutations in PTPN11 or SOS1. Gene expression profiling was also resolved in 5 subjects with Noonan-like syndrome with loose anagen hair (NS/LAH), a condition clinically related to NS and caused by an invariant mutation in SHOC2. Robust transcriptional signatures were found to specifically discriminate each of the three mutation groups from 21 age- and sex-matched controls. Despite the only partial overlap in terms of gene composition, the three signatures showed a notable concordance in terms of biological processes and regulatory circuits affected. These data establish expression profiling of peripheral blood mononuclear cells as a powerful tool to appreciate differential perturbations driven by germline mutations of transducers involved in RAS signaling, and to dissect molecular mechanisms underlying NS and other RASopathies.

The fourth Personal Genomes meeting was held at Cold Spring Harbor Laboratory, New York, from 30 September to 2 October and provided an exciting collection of science built on recent significant milestones in individual human genome sequencing, from the first personal genomes to thousands of human genomes sequenced. As ultra-high throughput sequencing platforms enable the production of more and more individual genomes, a growing number of scientists, physicians and clinical geneticists are actively exploring the promise and the implications of these new data. Personal Genomes brought many of these pioneers together with nearly 200 scientists, physicians, ethicists and others to discuss the progress and opportunities around the sequencing and medical interpretation of individual genome sequences.

Some cases of maternally inherited isolated deafness are caused by mtDNA mutations, frequently following an exposure to aminoglycosides. Two mitochondrial genes have been clearly described as being affected by mutations responsible for this pathology: the ribosomal RNA 12S gene and the transfer RNA Serine (UCN) gene. A previous study identified several candidate novel mtDNA mutations, localized in a variety of mitochondrial genes, found in patients with no previous treatment with aminoglycosides (Leveque, et al., 2007). Five of these candidate mutations are characterized in the present study. These mutations are localized in subunit ND1 of complex I of the respiratory chain (m.3388C>A (p.MT-ND1:Leu28Met)), the tRNA for Isoleucine (m.4295A>G), subunit COII of complex IV (m.8078G>A (p.MT-CO2:Val165Ile)), the tRNA of Serine 2 (AGU/C) (m.12236G>A) and Cytochrome B, subunit of complex III (m.15077G>A (p.MT-CYB:Glu111Lys)). Cybrid cell lines have been constructed for each of the studied mtDNA mutations and functional studies have been performed to assess the possible consequences of these mutations on mitochondrial bioenergetics. This study shows that a variety of mitochondrial genes, including protein coding genes, can be responsible for non-syndromic deafness, and that exposure to aminoglycosides is not required to develop the disease, giving new insights on the molecular bases of this pathology.

One of the most serious complications faced by inflammatory bowel disease (IBD) is the potential development of colorectal cancer (CRC). There is a compelling need to enhance the accuracy of cancer screening of IBD patients. MicroRNAs (miRNAs) are small non-protein-coding RNAs that play important roles in CRC oncogenesis. In this study, we report differential miRNA expression in IBD patients with associated CRC, from non-neoplastic tissue to dysplasia and eventually cancer. In addition, we identify and examine the role of dysregulated miRNAs in the TP53 pathway. In our CD patients, six miRNAs were up-regulated from non-neoplastic tissue to dysplasia, but down-regulated from dysplasia to cancer (miR-122, miR-181a, miR-146b-5p, let-7e, miR-17, miR-143) (p < 0.001). Six differentially expressed miRNAs affected the TP53 pathway (miR-122, miR-214, miR-372, miR-15b, let-7e, miR-17) (p < 0.001). Using two human colon cancer cell lines (HT-29 and HCT-116), E2F1, an upstream regulator of TP53, was down-regulated in both cell lines transfected with let-7e (p < 0.05) as well as in HCT-116 cells transfected with miR-17 (p < 0.05). Additionally, cyclin G, a cell-cycle regulator miR-122 target was down-regulated in both cell lines (p < 0.05). Unique differentially expressed miRNAs were observed in CD-associated CRC progression. Six of these miRNAs had a tumorigenic effect on the TP53 pathway; the effect of three of which was studied using cell lines.

Renal coloboma syndrome, also known as papillorenal syndrome is an autosomal-dominant disorder characterized by ocular and renal malformations. Mutations in the paired-box gene, PAX2, have been identified in approximately half of individuals with classic findings of renal hypoplasia/dysplasia and abnormalities of the optic nerve. Prior to 2011, there was no actively maintained locus-specific database (LSDB) cataloguing the extent of genetic variation in the PAX2 gene and phenotypic variation in individuals with renal coloboma syndrome. Review of published cases and the collective diagnostic experience of three laboratories in the United States, France, and New Zealand identified 55 unique mutations in 173 individuals from 86 families. The three clinical laboratories participating in this collaboration contributed 28 novel variations in 68 individuals in 33 families, which represent a 50% increase in the number of variations, patients, and families published in the medical literature. An LSDB was created using the Leiden Open Variation Database platform: www.lovd.nl/PAX2. The most common findings reported in this series were abnormal renal structure or function (92% of individuals), ophthalmological abnormalities (77% of individuals), and hearing loss (7% of individuals). Additional clinical findings and genetic counseling implications are discussed.

Congenital disorders of glycosylation (CDG) comprise a clinically and biochemically heterogeneous group of monogenetic-inherited, multisystemic diseases that affect the biosynthesis of N- and/or O-glycans linked to glycoconjugates. Recently, we identified the first patient with a defect in the cytosolic-orientated GDP-mannose:Man_3-4GlcNAc₂-PP-dolichol alpha-1,2-mannosyltransferase (ALG11), who presented an accumulation of shortened dolichol-linked oligosaccharides leading to CDG-Ip (ALG11-CDG). Here we describe an improved metabolic labeling method that allowed the identification of three new CDG-Ip cases that were missed so far in routine diagnostics. Although all CDG-Ip patients carry different mutations in the ALG11 gene, they share a variety of clinical syndromes like an unremarkable prenatal period followed by developmental delay, psychomotor, and mental retardation, strabismus convergens and seizures occurring in the first year of life.

The 2011 annual scientific meeting of the Human Genome Variation Society (HGVS) was held on the 11th of October, in Montreal, Canada. The theme of this meeting was “Exome and Genome Analysis as a Tool for Disease Identification and Treatment.” In the last few years, there has been a substantial increase in the use of next-generation sequencing in identifying variants associated with both single-gene disorders and complex diseases. The advent of exome sequencing with the subsequent transition to whole genome sequencing will require methods to identify candidate causal variants both in coding and regulatory regions. As this technology slowly moves into the clinical diagnostic laboratory, the need to accurately predict the functional consequences of variants becomes more critical for the both the diagnosis and treatment of disease. This year's annual meeting focused on these issues.

Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan^® low-density array technology and is able to define the full-exon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies.

Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with multisystem involvement and cancer predisposition, caused by mutations in the A-T mutated (ATM) gene. To study genotype–phenotype correlations, we evaluated the clinical and laboratory data of 51 genetically proven A-T patients, and additionally measured ATM protein expression and kinase activity. Patients without ATM kinase activity showed the classical phenotype. The presence of ATM protein, correlated with slightly better immunological function. Residual kinase activity correlated with a milder and essentially different neurological phenotype, absence of telangiectasia, normal endocrine and pulmonary function, normal immunoglobulins, significantly lower X-ray hypersensitivity in lymphocytes, and extended lifespan. In these patients, cancer occurred later in life and generally consisted of solid instead of lymphoid malignancies. The genotypes of severely affected patients generally included truncating mutations resulting in total absence of ATM kinase activity, while patients with milder phenotypes harbored at least one missense or splice site mutation resulting in expression of ATM with some kinase activity. Overall, the phenotypic manifestations in A-T show a continuous spectrum from severe classical childhood-onset A-T to a relatively mild adult-onset disorder, depending on the presence of ATM protein and kinase activity. Each patient is left with a tremendously increased cancer risk.

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/−) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94–99%) of del(+/−) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/−) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/−) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/−) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/−) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/−) cells in peripheral blood from the affected patients.

Dominant missense mutations in FLNB, encoding the actin-cross linking protein filamin B (FLNB), cause a broad range of skeletal dysplasias with varying severity by an unknown mechanism. Here these FLNB mutations are shown to cluster in exons encoding the actin-binding domain (ABD) and filamin repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin-containing cytoplasmic focal accumulations of the substituted protein to a degree that was correlated with the severity of the associated phenotypes. In contrast, study of mutations leading to substitutions in the FLNB rod domain that result in the same phenotypes as ABD mutations demonstrated that with only one exception disease-associated substitutions, surrounding hinge 1 demonstrated no tendency to form actin-filamin foci. The exception, a substitution in filamin repeat 6, lies within a region previously implicated in filamin-actin binding. These data are consistent with mutations in the ABD conferring enhanced actin-binding activity but suggest that substitutions affecting repeats near the flexible hinge region of FLNB precipitate the same phenotypes through a different mechanism.

Genome-wide association studies (GWAS) that allow for allelic heterogeneity may facilitate the discovery of novel genes not detectable by models that require replication of a single variant site. One strategy to accomplish this is to focus on genes rather than markers as units of association, and so potentially capture a spectrum of causal alleles that differ across populations. Here, we conducted a GWAS of Alzheimer disease (AD) in 2,586 Swedes and performed gene-based meta-analysis with three additional studies from France, Canada, and the United States, in total encompassing 4,259 cases and 8,284 controls. Implementing a newly designed gene-based algorithm, we identified two loci apart from the region around APOE that achieved study-wide significance in combined samples, the strongest finding being for FRMD6 on chromosome 14q (P = 2.6 × 10⁻¹⁴) and a weaker signal for NARS2 that is immediately adjacent to GAB2 on chromosome 11q (P = 7.8 × 10⁻⁹). Ontology-based pathway analyses revealed significant enrichment of genes involved in glycosylation. Results suggest that gene-based approaches that accommodate allelic heterogeneity in GWAS can provide a complementary avenue for gene discovery and may help to explain a portion of the missing heritability not detectable with single nucleotide polymorphisms (SNPs) derived from marker-specific meta-analysis.

The lysosomal storage disorder alpha-mannosidosis is caused by deficiency of the enzyme lysosomal alpha-mannosidase (MAN2B1). In this study, 96 disease-associated sequence variants were identified in 130 unrelated alpha-mannosidosis patients from 30 countries. Eighty-three novel variants were detected, extending the mutation spectrum from 42 to 125. In total, 256 of the 260 mutant alleles (98.5%) were identified. Most of the variants were unique to each family, however, c.2248C>T (p.Arg750Trp) was detected in 50 patients from 16 countries, and accounted for 27.3% of the disease alleles. Haplotype analysis revealed that the c.2248T variant was present on four MAN2B1 haplotype backgrounds, where one major haplotype accounted for 95% of the alleles. The distribution of the c.2248T-associated haplotypes differed remarkably from those of the control populations, suggesting that c.2248C>T has occurred on a few ancestral haplotypes where the major haplotype subsequently has spread by founder effects. The disease-associated missense mutations were introduced into the human MAN2B1 cDNA, expressed in cell culture and assayed for MAN2B1 activity. The majority of the variants were inactive, however, ten showed MAN2B1 activity above background, and more detailed studies are necessary to further evaluate the pathogenicity of these variants.

Inhibitory costimulatory molecule CD274 expresses in various cancers and contributes to cancer immune evasion by inhibiting T cell activation and proliferation, yet the regulatory mechanisms for CD274 overexpression in cancers are poorly understood. In this study, we discovered a novel mechanism of CD274 expression regulated by miR-570. A guanine-to-cytosine mutation at the 3′-UTR of CD274 mRNA led to CD274 overexpression by disrupting the miR-570 binding. The mutations were widely observed in cancers by sequencing of 276 gastrointestinal cancers (esophageal, gastric, colorectal, hepatocellular, and pancreatic cancers). This mutation was significantly associated with CD274 overexpression in gastric cancer (P = 1.44×10⁻¹⁰) and with the pathological features including differentiation grade, depth of tumor invasion, lymph node metastasis, and tumor-node-metastases (TNM) stage. These findings suggest a novel regulatory mechanism for CD274 overexpression in gastric cancer mediated by miR-570 and a somatic mutation in CD274 3′-UTR, and provide a new insight to gastric carcinogenesis.

Familial dysautonomia (FD) is a rare inherited neurodegenerative disorder. The most common mutation is a c.2204+6T>C transition in the 5′ splice site (5′ss) of IKBKAP intron 20, which causes a tissue-specific skipping of exon 20, resulting in lower synthesis of IKAP/hELP1 protein. To better understand the specificity of neuron loss in FD, we modeled the molecular mechanisms of IKBKAP mRNA splicing by studying human olfactory ecto-mesenchymal stem cells (hOE-MSCs) derived from FD patient nasal biopsies. We explored how the modulation of IKBKAP mRNA alternative splicing impacts the transcriptome at the genome-wide level. We found that the FD transcriptional signature was highly associated with biological functions related to the development of the nervous system. In addition, we identified target genes of kinetin, a plant cytokinin that corrects IKBKAP mRNA splicing and increases the expression of IKAP/hELP1. We identified this compound as a putative regulator of splicing factors and added new evidence for a sequence-specific correction of splicing. In conclusion, hOE-MSCs isolated from FD patients represent a promising avenue for modeling the altered genetic expression of FD, demonstrating a methodology that can be applied to a host of other genetic disorders to test the therapeutic potential of candidate molecules.

The 12th International Meeting on Human Genome Variation and Complex Genome Analysis (HGV2011: Berkeley, California, USA, 8th–10th September 2011) was a stimulating workshop where researchers from academia and industry explored the latest progress, challenges, and opportunities in genome variation research. Key themes included progress beyond GWAS, variation in human populations, use of sequence data in medical settings, large-scale sequencing data analysis, and bioinformatics approaches to large datasets.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Previously, we demonstrated that pathogenic amino acid substitutions in the N-terminal domain of TSC1 (amino acids 50–224) are destabilizing. Here we investigate an additional 21 unclassified TSC1 variants. Our functional assessment identified four substitutions (p.L61R, p.G132D, p.F158S, and p.R204P) between amino acids 50 and 224 that reduced TSC1 stability and prevented the TSC1-TSC2-dependent inhibition of TORC1. In four cases (20%), our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our new data confirm our previous finding that the N-terminal region of TSC1 is essential for TSC1 function.

We have systematically analyzed the two known minor genes involved in Usher syndrome type 2, DFNB31 and GPR98, for mutations in a cohort of 31 patients not linked to USH2A. PDZD7, an Usher syndrome type 2 (USH2) related gene, was analyzed when indicated. We found that mutations in GPR98 contribute significantly to USH2. We report 17 mutations in 10 individuals, doubling the number of GPR98 mutations reported to date. In contrast to mutations in usherin, the mutational spectrum of GPR98 predominantly results in a truncated protein product. This is true even when the mutation affects splicing, and we have incorporated a splicing reporter minigene assay to show this, where appropriate. Only two mutations were found which we believe to be genuine missense changes. Discrepancy in the mutational spectrum between GPR98 and USH2A is discussed. Only two patients were found with mutations in DFNB31, showing that mutations of this gene contribute to only a very small extent to USH2. Close examination of the clinical details, where available, for patients in whom no mutation was found in USH2A, GPR98, or DFNB31, showed that most of them had atypical features. In effect, these three genes account for the vast majority of USH2 patients and their analysis provide a robust pathway for routine molecular diagnosis.

Chronic granulomatous disease (CGD) is mainly caused by mutations in X-linked CYBB that encodes gp91. We have identified two novel mutations in CYBB resulting in the rare X91⁺-CGD variant, c.1500T>G (p.Asp500Glu) in two male siblings and c.1463C>A (p.Ala488Asp) in an unrelated male. Zymosan and/or PMA (Phorbol 12-myristate 13-acetate)-induced recruitment of p47^phox and p67^phox to the membrane fraction was normal for both mutants. Cell-free assays using recombinant wild-type and the mutant proteins revealed that these mutants were not activated by NADPH (nicotinamide adenine dinucleotide phosphate). Interestingly, the Ala488Asp mutant was activated by NADPH in the presence of glutathione. These data suggest that the mutations prevented NADPH from binding to gp91^phox and the requirement of a negative charge at residue 500 in gp91^phox for NADPH oxidase assembly, in contrast to a previously described Asp500Gly change. These mutations and the effect of glutathione provide a unique insight into disease pathogenesis and potential therapy in variant X91⁺-CGD.

Mutations in the dysferlin gene (DYSF) lead to a complete or partial absence of the dysferlin protein in skeletal muscles and are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. As a step towards a better understanding of the DYSF mutational spectrum, and towards possible inclusion of patients in future therapeutic clinical trials, we set up the Universal Mutation Database for Dysferlin (UMD-DYSF), a Locus-Specific Database developed with the UMD® software. The main objective of UMD-DYSF is to provide an updated compilation of mutational data and relevant interactive tools for the analysis of DYSF sequence variants, for diagnostic and research purposes. In particular, specific algorithms can facilitate the interpretation of newly identified intronic, missense- or isosemantic-exonic sequence variants, a problem encountered recurrently during genetic diagnosis in dysferlinopathies. UMD-DYSF v1.0 is freely accessible at www.umd.be/DYSF/. It contains a total of 742 mutational entries corresponding to 266 different disease-causing mutations identified in 558 patients worldwide diagnosed with dysferlinopathy. This article presents for the first time a comprehensive analysis of the dysferlin mutational spectrum based on all compiled DYSF disease-causing mutations reported in the literature to date, and using the main bioinformatics tools offered in UMD-DYSF. ©2011 Wiley-Liss, Inc.

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulfur cluster assembly gene ISCU, which leads to the activation of cryptic splice sites and the retention of part of intron 4. This incorrect splicing is more pronounced in muscle than in other tissues, resulting in a muscle-specific phenotype. In this study, we identified five nuclear factors that interact with the sequence harboring the mutation and analyzed their effect on the splicing of the ISCU gene. The identification revealed three splicing factors, SFRS14, RBM39, and PTBP1, and two additional RNA binding factors, matrin 3 (MATR3) and IGF2BP1. IGF2BP1 showed a preference for the mutant sequence, whereas the other factors showed similar affinity for both sequences. PTBP1 was found to repress the defective splicing of ISCU, resulting in a drastic loss of mutant transcripts. In contrast, IGF2BP1 and RBM39 shifted the splicing ratio toward the incorrect splice form. Hum Mutat 00:1–4, 2011. © 2011 Wiley Periodicals, Inc.

Primary ciliary dyskinesia (PCD) is an inherited disorder causing significant upper and lower respiratory tract morbidity and impaired fertility. Half of PCD patients show abnormal situs. Human disease loci have been identified but a mouse model without additional deleterious defects is elusive. The inversus viscerum mouse, mutated at the outer arm dynein heavy chain 11 locus (Dnahc11) is a known model of heterotaxy. We demonstrated immotile tracheal cilia with normal ultrastructure and reduced sperm motility in the Dnahc11^iv mouse. This is accompanied by gross rhinitis, sinusitis, and otitis media, all indicators of human PCD. Strikingly, age-related progression of the disease is evident. The Dnahc11^iv mouse is robust, lacks secondary defects, and requires no intervention to precipitate the phenotype. Together these findings show the Dnahc11^iv mouse to be an excellent model of many aspects of human PCD. Mutation of the homologous human locus has previously been associated with hyperkinetic tracheal cilia in PCD. Two PCD patients with normal ciliary ultrastructure, one with immotile and one with hyperkinetic cilia were found to carry DNAH11 mutations. Three novel DNAH11 mutations were detected indicating that this gene should be investigated in patients with normal ciliary ultrastructure and static, as well as hyperkinetic cilia. Hum Mutat 00:1–9, 2011. © 2011 Wiley Periodicals, Inc.

Lynch syndrome (LS) is an autosomal dominant disorder that predisposes to colon, endometrial, and other cancers. LS is caused by a heterozygous germline mutation in one of the DNA mismatch repair (MMR) genes. A significant proportion of all mutations found in suspected LS patients comprises single amino acid alterations. The pathogenicity of these variants of uncertain significance (VUS) is difficult to assess, precluding diagnosis of carriers and their relatives. Here we present a rapid cell-free assay to investigate MMR activity of MSH2 or MSH6 VUS. We used this assay to analyze a series of MSH2 and MSH6 VUS, selected from the Leiden Open Variation Database. Whereas a significant fraction of the MSH2 VUS has lost MMR activity, suggesting pathogenicity, the large majority of the MSH6 VUS appears MMR proficient. We anticipate that this assay will be an important tool in the development of a comprehensive and widely applicable diagnostic procedure for LS-associated VUS. Hum Mutat 00:1–7, 2011. © 2011 Wiley Periodicals, Inc.

Gene variant databases or Locus-Specific DataBases (LSDBs) are used to collect and display information on sequence variants on a gene-by-gene basis. Their most frequent use is in relation to DNA-based diagnostics, giving clinicians and scientists easy access to an up-to-date overview of all gene variants identified worldwide and whether they influence the function of the gene (“pathogenic or not”). While literature on gene variant databases is extensive, little has been published on the process of database curation itself. Based on our extensive experience as LSDB curators and our contributions to database curation courses, we discuss the subject of database curation. We describe the tasks involved, the steps to take, and the issues that might occur. Our overview is a first step toward establishing overall guidelines for database curation and ultimately covers one aspect of establishing quality-assured gene variant databases. Hum Mutat 33:291–297, 2012. © 2011 Wiley Periodicals, Inc.

Information about genetic variation has been collected for some 20 years into registries, known as locus specific databases (LSDBs), which nowadays often contain information in addition to the actual genetic variation. Several issues have to be taken into account when considering establishing and maintaining LSDBs and these have been discussed previously in a number of articles describing guidelines and recommendations. This information is widely scattered and, for a newcomer, it would be difficult to obtain the latest information and guidance. Here, a sequence of steps essential for establishing an LSDB is discussed together with guidelines for each step. Curators need to collect information from various sources, code it in systematic way, and distribute to the research and clinical communities. In doing this, ethical issues have to be taken into account. To facilitate integration of information to, for example, analyze genotype–phenotype correlations, systematic data representation using established nomenclatures, data models, and ontologies is essential. LSDB curation and maintenance comprises a number of tasks that can be managed by following logical steps. These resources are becoming ever more important and new curators are essential to ensure that we will have expertly curated databases for all disease-related genes in the near future. Hum Mutat 33:298–305, 2012. © 2011 Wiley Periodicals, Inc.

Mutations in the CRB1 gene are associated with variable phenotypes of severe retinal dystrophies, ranging from leber congenital amaurosis (LCA) to rod–cone dystrophy, also called retinitis pigmentosa (RP). Moreover, retinal dystrophies resulting from CRB1 mutations may be accompanied by specific fundus features: preservation of the para-arteriolar retinal pigment epithelium (PPRPE) and retinal telangiectasia with exudation (also referred to as Coats-like vasculopathy). In this publication, we report seven novel mutations and classify over 150 reported CRB1 sequence variants that were found in more that 240 patients. The data from previous reports were used to analyze a potential correlation between CRB1 variants and the clinical features of respective patients. This meta-analysis suggests that the differential phenotype of patients with CRB1 mutations is due to additional modifying factors rather than particular mutant allele combination. Hum Mutat 33:306–315, 2012. © 2011 Wiley Periodicals, Inc.

Autosomal recessive renal tubular dysgenesis (RTD) is a severe disorder of renal tubular development characterized by early onset and persistent fetal anuria leading to oligohydramnios and the Potter sequence, associated with skull ossification defects. Early death occurs in most cases from anuria, pulmonary hypoplasia, and refractory arterial hypotension. The disease is linked to mutations in the genes encoding several components of the renin–angiotensin system (RAS): AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), and AGTR1 (angiotensin II receptor type 1). Here, we review the series of 54 distinct mutations identified in 48 unrelated families. Most of them are novel and ACE mutations are the most frequent, observed in two-thirds of families (64.6%). The severity of the clinical course was similar whatever the mutated gene, which underlines the importance of a functional RAS in the maintenance of blood pressure and renal blood flow during the life of a human fetus. Renal hypoperfusion, whether genetic or secondary to a variety of diseases, precludes the normal development/ differentiation of proximal tubules. The identification of the disease on the basis of precise clinical and histological analyses and the characterization of the genetic defects allow genetic counseling and early prenatal diagnosis. Hum Mutat 33:316–326, 2012. © 2011 Wiley Periodicals, Inc.

Dystrophic Epidermolysis Bullosa (DEB) is a genetic disease caused by mutations in the COL7A1 gene that is inherited in the autosomal dominant or recessive mode. We have developed a curated, freely accessible COL7A1 specific database (http://www.col7.info), which contains more than 730 reported and unpublished sequence variants of the gene. Molecular defects are reported according to HGVS recommendation. The clinical description module is provided with an advanced search tool together with a CSV (comm. separated values) data format download option. This compilation of COL7A1 data and nomenclature is aimed at assisting molecular and clinical geneticists to enhance the collaboration between researchers worldwide. Hum Mutat 33:327–331, 2012. © 2011 Wiley Periodicals, Inc.

Variations are mostly due to nonsynonymous single nucleotide polymorphisms (nsSNPs), some of which are associated with certain diseases. Phenotypic effects of a large number of nsSNPs have not been characterized. Although several methods have been developed to predict the effects of nsSNPs as “disease” or “neutral,” there is still a need for development of methods with improved prediction accuracies. We, therefore, developed a support vector machine (SVM) based method named Hansa which uses a novel set of discriminatory features to classify nsSNPs into disease (pathogenic) and benign (neutral) types. Validation studies on a benchmark dataset and further on an independent dataset of well-characterized known disease and neutral mutations show that Hansa outperforms the other known methods. For example, fivefold cross-validation studies using the benchmark HumVar dataset reveal that at the false positive rate (FPR) of 20% Hansa yields a true positive rate (TPR) of 82% that is about 10% higher than the best-known method. Hansa is available in the form of a web server at http://hansa.cdfd.org.in:8080. Hum Mutat 33:332–337, 2012. © 2011 Wiley Periodicals, Inc.

Autozygosity mapping has been a powerful method for the identification of autosomal recessive disease genes. However, the approach is limited by the availability of suitable consanguineous pedigrees. While rare autosomal recessive diseases are overrepresented in consanguineous families, a significant proportion of affected patients nonetheless originate in families where the parents are apparently unrelated. However, due to their relative rarity and the heterogeneity of disease alleles, it has proved difficult to use these patients to identify disease loci. Therefore, we developed “Phaser,” a computer application that is able to infer the phase of SNP alleles and so haplotype entire chromosomes in small nuclear families (http://dna.leeds.ac.uk/Phaser). Once the index case's chromosomes have been haplotyped, it is then possible to deduce those of the parents and subsequently identify the parental origin of all the siblings' DNA. By combining information from a small number of nuclear families, it may then be possible to identify linkage to the recessive disease locus, in both in-bred and out-bred families. We have illustrated the program's utility by using it to correctly identify both the cystic fibrosis locus (using two unrelated compound heterozygous CEPH families) and a new gene mutated in early-onset myopathy with respiratory distress and dysphagia locus in a single consanguineous pedigree. Hum Mutat 33:338–342, 2012. © 2011 Wiley Periodicals, Inc.

Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of proα1(I) in the culture media being C-terminally processed, proα1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR-OI. Hum Mutat 33:343–350, 2012. © 2011 Wiley Periodicals, Inc.

Autosomal recessive ataxias are heterogeneous group of disorders characterized by cerebellar atrophy and peripheral sensorimotor neuropathy. Molecular characterization of this group of disorders identified a number of genes contributing to these overlapping phenotypes. Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive form of ataxia caused by mutations in the SETX gene. We report on a consanguineous family with autosomal recessive inheritance and clinical characteristics of AOA2, and no mutations in the SETX gene. We mapped the AOA locus in this family to chromosome 17p12-p13. Sequencing of all genes in the refined region identified a homozygous missense mutation in PIK3R5 that was absent in 477 normal controls. Our characterization of the PIK3R5 protein and findings suggest that it may play a role in the development of the cerebellum and vermis. Hum Mutat 33:351–354, 2012. © 2011 Wiley Periodicals, Inc.

GRIA3 encodes glutamate receptor ionotropic AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) subunit 3 and has been previously involved in X-linked intellectual disability (ID). We report on a male proband with ID and epilepsy associated with a duplication mapping within a gene desert, 874-kb upstream of the GRIA3 gene. This 970-kb duplication is maternally inherited. The proband's mother has a skewed X chromosome-inactivation pattern in agreement with her normal cognitive function. Quantitative polymerase chain reaction analysis indicates absence of GRIA3 mRNA in the proband lymphocytes relative to a wild-type control. Centromeric to the duplicated region, comparative genomic analysis showed a 2268-bp evolutionarily conserved region that could be a critical transcription factor binding-site for GRIA3 expression. The repositioning of distant-acting sequences, rather a missense/nonsense mutation, is considered to be causative for GRIA3-linked ID. This study illustrates the importance of high-resolution array-Comparative Genomic Hybridization analysis in exploring the potential role of disease-causing mutation in functional noncoding sequences. Hum Mutat 33:355–358, 2012. © 2011 Wiley Periodicals, Inc.

Many nonsynonymous single nucleotide polymorphisms (nsSNPs) are disease causing due to effects at protein-protein interfaces. We have integrated a database of the three-dimensional (3D) structures of human protein/protein complexes and the humsavar database of nsSNPs. We analyzed the location of nsSNPS in terms of their location in the protein core, at protein-protein interfaces, and on the surface when not at an interface. Disease-causing nsSNPs that do not occur in the protein core are preferentially located at protein-protein interfaces rather than surface noninterface regions when compared to random segregation. The disruption of the protein-protein interaction can be explained by a range of structural effects including the loss of an electrostatic salt bridge, the destabilization due to reduction of the hydrophobic effect, the formation of a steric clash, and the introduction of a proline altering the main-chain conformation. Hum Mutat 33:359–363, 2012. © 2011 Wiley Periodicals, Inc.

Vax1 and Vax2 have been implicated in eye development and the closure of the choroid fissure in mice and zebrafish. We sequenced the coding exons of VAX1 and VAX2 in 70 patients with anophthalmia/microphthalmia (A/M). In VAX1, we observed homozygosity for two successive nucleotide substitutions c.453G>A and c.454C>A, predicting p.Arg152Ser, in a proband of Egyptian origin with microphthalmia, small optic nerves, cleft lip/palate, and corpus callosum agenesis. This mutation affects an invariant residue in the homeodomain of VAX1 and was absent from 96 Egyptian controls. It is likely that the mutation results in a loss of function, as the mutation results in a phenotype similar to the Vax1 homozygous null mouse. We did not identify any mutations in VAX2. This is the first description of a phenotype associated with a VAX1 mutation in humans and establishes VAX1 as a new causative gene for A/M. Hum Mutat 33:364–368, 2012. © 2011 Wiley Periodicals, Inc.

Long INterspersed Element-1 (LINE-1) retrotransposons comprise 17% of the human genome, and move by a potentially mutagenic “copy and paste” mechanism via an RNA intermediate. Recently, the retrotransposition-mediated insertion of a new transcript was described as a novel cause of genetic disease, Duchenne muscular dystrophy, in a Japanese male. The inserted sequence was presumed to derive from a single-copy, noncoding RNA transcribed from chromosome 11q22.3 that retrotransposed into the dystrophin gene. Here, we demonstrate that a nonreference full-length LINE-1 is situated in the proband and maternal genome at chromosome 11q22.3, directly upstream of the sequence, whose copy was inserted into the dystrophin gene. This LINE-1 is highly active in a cell culture assay. LINE-1 insertions are often associated with 3′ transduction of adjacent genomic sequences. Thus, the likely explanation for the mutagenic insertion is a LINE-1-mediated 3′ transduction with severe 5′ truncation. This is the first example of LINE-1-induced human disease caused by an “orphan” 3′ transduction. Hum Mutat 33:369–371, 2012. © 2011 Wiley Periodicals, Inc.

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties. Hum Mutat 33:372–383, 2012. © 2011 Wiley Periodicals, Inc.

Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double Fzd3/Fzd6^−/− mutant mice, we investigated the role of the human orthologues, FZD3 and FZD6, by resequencing a cohort of 473 NTDs patients and 639 ethnically matched controls. While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes (p.Arg405Gln, p.Arg511Cys p.Arg511His), and one substitution (c.^*20C>T) affecting the 3′-untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1-fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs. Hum Mutat 33:384–390, 2012. © 2011 Wiley Periodicals, Inc.

Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G<T mutation in CRYGC that resulted in the substitution of a highly conserved glycine by cysteine at codon 129 (p.Gly129Cys) in a three-generation Chinese family with autosomal dominant congenital nuclear cataract by sequencing candidate genes. Using zebrafish as a model, we demonstrated that γC-crystallin p.Gly129Cys mutant caused the vacuole and the incomplete denucleation of lens, recapitulating the cataract phenotype in human beings. Molecular modeling and spectroscopic studies indicated that the mutation impaired the tertiary structure of the protein by modifying the H-bonding network in the C-terminal domain. The mutation led to a dramatic decrease in the thermal stability of γC-crystallin, and a significant increase in the propensity of aggregation when subject to storage at high concentrations, heat, and UV- irradiation stresses. Taken together, these results indicate that a novel γC-crystallin p.Gly129Cys mutation impaired the tertiary structure of the protein and caused cataract formation, which provides a new insight into how the mutation may affect the γC-crystallin structure, stability, and function. Our study also highlighted zebrafish as a valuable model tool for studying congenital inherited cataract. Hum Mutat 33:391–401, 2012. © 2011 Wiley Periodicals, Inc.

Adiponectin is most abundantly expressed in adipose tissue and well known to play an important role in metabolic regulation. Several studies have attempted to identify the genetic determinants of metabolic syndrome (MetS), though no study has revealed a cis- or trans-single nucleotide polymorphism (SNP) that affects plasma adiponectin levels, except the adiponectin structure gene and genes encoding adiponectin-regulatory proteins. We performed a genome-wide association study in regards to plasma adiponectin concentrations in 3,310 Japanese subjects. We identified the strongest statistically associated SNP (rs4783244) with adiponectin levels (P = 3.8 × 10⁻¹⁹) in the first intron of CDH13 (T-cadherin) gene in a 30-kb haplotype block covering the promoter region to first intron. In addition, rs12051272 SNP genotypes in linkage disequilibrium with rs4783244 were found to be more significantly associated with adiponectin levels (P = 9.5×10⁻²⁰) and specifically with the levels of high-molecular weight (HMW) adiponectin, a subtype form associated with parameters related to glucose metabolism. Our results did show more significant association with adiponectin levels than rs12444338 (in CDH13) SNP genotypes reported recently. We suggest that the phenotype-affecting haplotype tagged by rs12051272 SNP would affect the plasma adiponectin levels and that we have to take the CDH13 genotype into account before considering the functional relevance of the adiponectin level. Hum Mutat 33:402–410, 2012. © 2011 Wiley Periodicals, Inc.

Next-generation sequencing (NGS) is transitioning from being a research tool to being used in routine genetic diagnostics, where a major challenge is distinguishing which of many sequence variants in an individual are truly pathogenic. We describe some limitations of in silico analyses of NGS data that emphasize the need for experimental confirmation. Using NGS, we recently identified an apparently homozygous missense mutation in NUBPL in a patient with mitochondrial complex I deficiency. Causality was established via lentiviral correction studies with wild-type NUBPL cDNA. NGS data, however, provided an incomplete understanding of the genetic abnormality. We show that the maternal allele carries an unbalanced inversion, while the paternal allele carries a branch-site mutation in addition to the missense mutation. We demonstrate that the branch-site mutation, which is present in approximately one of 120 control chromosomes, likely contributes to pathogenicity and may be one of the most common autosomal mutations causing mitochondrial dysfunction. Had these analyses not been performed following NGS, the original missense mutation may be incorrectly annotated as pathogenic and a potentially common pathogenic variant not detected. It is important that locus-specific databases contain accurate information on pathogenic variation. NGS data, therefore, require rigorous experimental follow-up to confirm mutation pathogenicity. Hum Mutat 33:411–418, 2012. © 2011 Wiley Periodicals, Inc.

Mutations in the SLC4A11 gene, which encodes a plasma membrane borate transporter, cause recessive congenital hereditary endothelial corneal dystrophy type 2 (CHED2), corneal dystrophy and perceptive deafness (Harboyan syndrome), and dominant late-onset Fuchs endothelial corneal dystrophy (FECD). We analyzed missense SLC4A11 mutations identified in FECD and CHED2 patients and expressed in transfected HEK 293 cells. Chemical cross-linking and migration in nondenaturing gels showed that SLC4A11 exists as a dimer. Furthermore, co-immunoprecipitation of epitope-tagged proteins revealed heteromeric interactions between wild-type (WT) and mutant SLC4A11 proteins. When expressed alone, FECD- and CHED2-causing mutant SLC4A11 proteins are primarily retained intracellularly. Co-expression with WT SLC4A11 partially rescued the cell surface trafficking of CHED2 mutants, but not FECD mutants. CHED2 alleles of SLC4A11 did not affect cell surface processing of WT SLC4A11. In contrast, FECD mutants reduced WT cell surface processing efficiency, consistent with dominant inheritance of FECD. The reduction in movement of WT protein to the cell surface caused by FECD SLC4A11 helps to explain the dominant inheritance of this disorder. Similarly, the failure of CHED2 mutant SLC4A11 to affect the processing of WT protein, explains the lack of symptoms found in CHED2 carriers and the recessive inheritance of the disorder. Hum Mutat 33:419–428, 2012. © 2011 Wiley Periodicals, Inc.

Sarcoglycanopathies (SGP) are a group of autosomal recessive muscle disorders caused by primary mutations in one of the four sarcoglycan genes. The sarcoglycans (α-, β-, γ-, and δ-sarcoglycan) form a tetrameric complex at the muscle membrane that is part of the dystrophin-glycoprotein complex and plays an essential role for membrane integrity during muscle contractions. We previously showed that the most frequent missense mutation in α-sarcoglycan (p.R77C) leads to the absence of the protein at the cell membrane due to its blockade by the endoplasmic reticulum (ER) quality control. Moreover, we demonstrated that inhibition of the ER α-mannosidase I activity using kifunensine could rescue the mutant protein localization at the cell membrane. Here, we investigate 25 additional disease-causing missense mutations in the sarcoglycan genes with respect to intracellular fate and localization rescue of the mutated proteins by kifunensine. Our studies demonstrate that, similarly to p.R77C, 22 of 25 of the selected mutations lead to defective intracellular trafficking of the SGs proteins. Six of these were saved from ER retention upon kifunensine treatment. The trafficking of SGs mutants rescued by kifunensine was associated with mutations that have moderate structural impact on the protein. Hum Mutat 33:429–439, 2012. © 2011 Wiley Periodicals, Inc.

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein–protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism. Hum Mutat 33:440–447, 2012. © 2011 Wiley Periodicals, Inc.

Autosomal Dominant Hypercholesterolemia (ADH) is caused by LDLR and APOB mutations. However, genetically diagnosed ADH patients do not always exhibit the expected hypercholesterolemic phenotype. Of 4,669 genetically diagnosed ADH patients, identified through the national identification screening program for ADH, 75 patients (1.6%) had LDL-cholesterol (LDL-C) levels below the 50th percentile for age and gender prior to lipid-lowering therapy. The genes encoding APOB, PCSK9, and ANGPTL3 were sequenced in these subjects to address whether monogenic dominant loss-of-function mutations underlie this paradoxical phenotype. APOB mutations, resulting in truncated APOB, were found in five (6.7%) probands, reducing LDL-C by 56%. Rare variants in PCSK9, and ANGPTL3 completely correcting the hypercholesterolemic phenotype were not found. The common variants p.N902N, c.3842+82T>A, p.D2312D, and p.E4181K in APOB, and c.1863+94A>G in PCSK9 were significantly more prevalent in our cohort compared to the general European population. Interestingly, 40% of our probands carried at least one minor allele for all four common APOB variants compared to 1.5% in the general European population. While we found a low prevalence of rare variants in our cohort, our data suggest that regions in proximity of the analyzed loci, and linked to specific common haplotypes, might harbor additional variants that correct an ADH phenotype. Hum Mutat 33:448–455, 2012. © 2011 Wiley Periodicals, Inc.