Expanding the genetic landscape of oral‐facial‐digital syndrome with two novel genes

Abstract Oral‐facial‐digital syndromes (OFDS) are a heterogeneous and rare group of Mendelian disorders characterized by developmental abnormalities of the oral cavity, face, and digits caused by dysfunction of the primary cilium, a mechanosensory organelle that exists atop most cell types that facilitates organ patterning and growth. OFDS is inherited both in an X‐linked dominant, X‐linked recessive, and autosomal recessive manner. Importantly, though many of the causal genes for OFDS have been identified, up to 40% of OFD syndromes are of unknown genetic basis. Here we describe three children with classical presentations of OFDS including lingual hamartomas, polydactyly, and characteristic facial features found by exome sequencing to harbor variants in causal genes not previously associated with OFDS. We describe a female with hypothalamic hamartoma, urogenital sinus, polysyndactyly, and multiple lingual hamartomas consistent with OFDVI with biallelic pathogenic variants in CEP164, a gene associated with ciliopathy‐spectrum disease, but never before with OFDS. We additionally describe two unrelated probands with postaxial polydactyly, multiple lingual hamartomas, and dysmorphic features both found to be homozygous for an identical TOPORS missense variant, c.29 C>A; (p.Pro10Gln). Heterozygous TOPORS pathogenic gene variants are associated with autosomal dominant retinitis pigmentosa, but never before with syndromic ciliopathy. Of note, both probands are of Dominican ancestry, suggesting a possible founder allele.

Importantly, features of OFDS can be seen in other ciliopathy syndromes, most commonly Joubert syndrome (JS), which is defined by the clinical triad of hypotonia, developmental delays, and a pathognomonic cerebellar and brain stem malformation referred to as a "molar tooth sign" (MTS) (Parisi & Glass, 2003).
Since its initial description in 1941 (Mohr, 1941), multiple OFDS types have been described with significant phenotypic overlap but with distinct patterns of organ system involvement and malformations (Bruel et al., 2017;Franco & Thauvin-Robinet, 2016).
OFD is inherited in an X-linked dominant, X-linked recessive, and autosomal recessive manner. Causal genes have not been identified for approximately 40% of OFD types (Bruel et al., 2017;Franco & Thauvin-Robinet, 2016).
Here we describe three probands with a clinical diagnosis of OFDS VI. One patient was compound heterozygous for nonsense and frameshift CEP164 variants, and two unrelated probands of Dominican Republic descent were homozygous for a c.29 C>A; p.(Pro10Gln)) missense variant in TOPORS. We propose that CEP164 and TOPORS are novel causal genes for OFD VI, and suggest that the c.29 C>A allele is may represent a founder allele in the Dominican Republic population.

| Editorial policies and ethical considerations
All individuals and families agreed to participate in this study and signed appropriate consent forms. Permission for clinical photographs was given separately. This study was approved by the institutional IRB (Protocol # 16-013278). Patient 1 was the product of a naturally-conceived pregnancy to a then 40-year-old G5P3à4 mother. Family history was notable for a 12-year-old brother with Trisomy 21 (conceived at 27 years of age) and a 14-year-old sister with suspected autoimmune aplastic anemia status post bone marrow transplant. There were no medications or exposures during the pregnancy. Noninvasive prenatal testing was done due to maternal age and was normal.

| Genetic testing methodology
Twenty-week ultrasound was also normal. An additional ultrasound was done at 29 weeks due to loss of the cervical mucus plug, and was notable for multiple congenital anomalies, prompting fetal MRI and echocardiogram, which were notable for polydactyly, brain cyst, absent vagina, and hydronephrosis. Patient was ultimately born via caesarian section at 33 weeks gestational age due to fetal tachycardia and worsening hydronephrosis. Birth weight was 3.115 kg (>97%; 50% for 36 weeks gestational age) and birth length was 43 cm (25-50%). She was admitted to the NICU for management. She was noted to have hypotonia, fourextremity polydactyly, syndactyly of multiple digits, multiple tongue hamartomas, and urogenital sinus. A suprapubic catheter was placed with improvement in her hydronephrosis and hydrometrocolpos. Echocardiogram and ophthalmology examination were normal. Head ultrasound showed a prominent temporal horn of the left lateral ventricle. Brain MRI showed hypothalamic hamartoma and asymmetric ventriculomegaly.
She was evaluated by Genetics at 3 months of age. Growth parameters were notable for a weight of 3.65 kg (10-25% corrected), height of 48.3 cm (<3%; 50% for newborn), and head circumference of 36.5 cm (75% corrected). Physical examination was notable for hypotonia, relative macrocephaly, down-slanting palpebral fissures, hypertelorism, broad and flat nasal bridge, upper alveolar ridge notch, multiple lingual hamartomas, accessory frenula, high palate, mild micrognathia, and linear nevus simplex extending from the forehead to the upper lip (Figure 1a Presence of hypothalamic hamartoma, lingual hamartomas, polydactyly, and syndactyly was concerning for OFD VI (Poretti et al., 2012). Kidney ultrasound was performed, which showed moderate, symmetric hydronephrosis with no kidney cysts. An EEG was performed, which showed intermittent focal slowing in the bifrontal region and rare epileptiform discharges in the left temporal and right occipital regions. She underwent surgical removal of her lingual hamartomas and accessory frenula, and was admitted postoperatively due to concern for silent aspiration, later confirmed on video swallow study. She is currently NPO. Patient is now 6 months of age, gestationally corrected to 4 months of age. Motor milestones are delayed with minimal head control. Social skills are normal; patient fixes and follows, smiles, and is extremely interactive.

| Patient 2
Patient 2 was born to a 31-year-old G4P3à4 mother. Family history was noncontributory. Both parents were from the Dominican Republic. Pregnancy was complicated by gestational diabetes treated with metformin. Prenatal ultrasounds and follow up fetal MRI were notable for four extremity polydactyly, severely hypoplastic/absent cerebellar vermis, mildly enlarged posterior fossa, and micropenis. Patient was born via repeat caesarian section at 38 weeks gestational age. APGARS were 9 and 9 at 1 and 5 min of life, respectively. Birth weight was 3.73 kg (90%), birth length was 50.5 cm (75-90%), and head circumference was 37 cm (>97%). fix or follow. Kidney ultrasound was normal, and ophthalmology examination was performed, but the results are unknown. Patient is currently 10 months of age. Clinical course is complicated by severe developmental delay.

| Patient 3
Patient 3 was the product of a naturally-conceived pregnancy to a then 21-year-old G2P0à1 mother. Both parents were from the Dominican Republic. Family history was notable for consanguinity with the mother's maternal grandfather and the father's paternal grandmother being siblings (second-cousin union) and for a prior pregnancy terminated for multiple congenital anomalies, including lemonshaped head with frontal bossing, posterior fossa cyst, encephalocele, and arthrogryposis with bilateral talipes equinovarus. Microarray and exome sequencing were nondiagnostic.
Pregnancy was complicated by prenatal ultrasound concerning for multiple congenital anomalies, prompting fetal MRI, which demonstrated bilateral ventriculomegaly, dandy-walker malformation, occipital meningocele, ambiguous genitalia, bilateral talipes equinovarus, and shortened long bones. Patient was born at 36 weeks gestational age via caesarian section for fetal macrocephaly. APGARs were 2 and 8 and 1 and 5-min of life, respectively. Birth weight was 3.17 kg (75%), birth length was 45 cm (20%), and head circumference was 42 cm (>97%). He was diagnosed postnatally with cleft palate and four-extremity polydactyly. He developed respiratory distress in the delivery room requiring positive pressure ventilation, and was intubated shortly thereafter for persistent apneas and desaturations.
He was evaluated by Genetics on day of life 3. Physical examination was notable for macrocephaly with prominent forehead and occiput, down-slanting palpebral fissures, hypertelorism, nasal milia, bifid tongue, lingual hamartomas, micrognathia, fourextremity postaxial polydactyly, micropenis and hypotonia. Due to concern for OFDS, an abdominal ultrasound, echocardiogram, brain MRI and ophthalmology examination were requested. Abdominal ultrasound showed structurally normal liver and kidneys, echocardiogram showed a structurally normal heart, ophthalmology examination showed bilateral optic nerve colobomas, and brain MRI

| DISCUSSION
Oral facial digital syndromes (OFDS) are a group of rare ciliopathy syndromes characterized by the core features of oral cavity malformations, facial dysmorphisms, and digit anomalies (Bruel et al., 2017;Franco & Thauvin-Robinet, 2016). OFDS subtypes are classified based on pattern of associated features such as structural brain differences, intellectual disability, skeletal differences, congenital heart disease and kidney disease, with over 18 subtypes described to date (Table 1).
Over 16 genes have now been identified as causal for OFDS (Bruel et al., 2017). These genes encode components of the primary cilium, a mechanosensory organelle that facilitates organ growth and patterning (Berbari et al., 2009;Fry et al., 2014). OFDS genes localize to virtually every ciliary subsegment, including the basal body, centriole, transition zone, and axoneme ( Figure 2). The exact mechanism by which ciliary dysfunction causes the clinical features of OFDS is unknown.
Though initially described 80 years ago, up to 40% of the described OFDS subtypes do not have an associated gene and many patients with a clinical diagnosis of OFDS have negative molecular testing, highlighting our incomplete understanding of the genetic landscape of these conditions (Bruel et al., 2017;Franco & Thauvin-Robinet, 2016). Additionally, many genes associated with OFDS are also associated with other ciliopathy syndromes, most commonly JS; however, genotype-phenotype correlation remains elusive, and it is difficult to use mutation type to predict which ciliopathy phenotype a Here we describe two novel genes identified in patients with clinical features consistent with OFD VI: CEP164, previously identified as causal for the ciliopathies nephronophthisis, JS and Bardet Biedl syndrome, and TOPORS, a gene associated with the autosomal dominant ciliopathy RP, but never before with syndromic ciliopathy.
CEP164 maps to 11q23.3 and encodes centrosomal protein of 164 kDa, a component of the centriolar distal appendage. CEP164 is required for vesicular docking at the centriole, thereby enabling ciliogenesis (Graser et al., 2007). CEP164 has also been associated with coordination of the DNA damage response (Pan & Lee, 2009;Sivasubramaniam et al., 2008); however, this has recently been challenged (Daly et al., 2016). Morpholino-mediated cep164 knockdown in zebrafish disrupts the DNA damage pathway and causes nephronophthisis-spectrum disease and hydrocephalus (Chaki et al., 2012). Total body cep164 deficiency is embryonic lethal in mice, associated with holoprosencephaly, abnormal heart development, and gross patterning defects, and conditional cep164 deficiency in multiciliated cells causes hydrocephalus, lung disease, and infertility, supporting a role for CEP164 in ciliary function (Siller et al., 2017).  variants are believed to cause ciliopathy-spectrum disease by disrupting ciliogenesis and possibly also through modulation of the DNA damage response and cell cycle control (Chaki et al., 2012;Graser et al., 2007;Pan & Lee, 2009;Sivasubramaniam et al., 2008). We hypothesize that Patient 1 presented with severe ciliopathy-spectrum disease because her nonsense and frameshift CEP164 variants more significantly impair CEP164 function. Indeed, patients with biallelic nonsense CEP164 variants presented with Bardet-Biedl and JS phenotypes, as opposed to patients harboring missense variants, who presented with nephronophthisis and retinal disease (Chaki et al., 2012;Shamseldin et al., 2020).
The mechanism by which heterozygous TOPORS variants cause disease is unknown. Biallelic TOPORS variants have not been previously reported. Given the localization of TOPORS at the ciliary base and in the nucleus, loss of TOPORS function could cause ciliopathy-spectrum disease by disrupting ciliary homeostasis directly or through dysregulated gene expression. We hypothesize that loss of TOPORS function disrupts ciliogenesis and ciliary function by interfering with the proper ubiquitination and turnover of ciliary proteins and morphogens ( Figure 3). Disruption of the ciliary ubiquitin-proteasome system disrupts ciliogenesis, and biallelic variants in genes involved in regulation of the ubiquitin-proteasome system have been identified in ciliopathy patients (Gerhardt et al., 2015;Izawa et al., 2015;Kasahara et al., 2014).  (Bruel et al., 2017;Coene et al., 2009;Franco & Thauvin-Robinet, 2016). We propose that TOPORS also bridges OFDS and JS. It is unknown at this time whether this TOPORS phenotype is specific to the Pro10Gln variant identified in our probands, or whether it can be generalized to other gene variants. It is also unclear why Patient 3 and his affected sibling presented with neural tube defects while Patient 2 presented with MTS. Of note, both families trace their ancestry to the Dominican Republic, suggesting a possible founder allele.
In conclusion, we report on two novel genes associated with OFD VI, TOPORS and CEP164, further expanding the genetic differential of OFDS. Our cases highlight the phenotypic and genetic diversity of this family of diseases, and again implicate the ubiquitin-proteasome system in ciliary disease.

CONFLICT OF INTEREST
None.

AUTHOR CONTRIBUTIONS
Alanna Strong conceptualized and designed the study, evaluated contributed to data analysis, critically reviewed and edited the manuscript, and provided funding for the work. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.