Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes.

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.

A de novo microdeletion of SEMA5A in a boy with autism spectrum disorder and intellectual disability.

Semaphorins are a large family of secreted and membrane-associated proteins necessary for wiring of the brain. Semaphorin 5A (SEMA5A) acts as a bifunctional guidance cue, exerting both attractive and inhibitory effects on developing axons. Previous studies have suggested that SEMA5A could be a susceptibility gene for autism spectrum disorders (ASDs). We first identified a de novo translocation t(5;22)(p15.3;q11.21) in a patient with ASD and intellectual disability (ID). At the translocation breakpoint on chromosome 5, we observed a 861-kb deletion encompassing the end of the SEMA5A gene. We delineated the breakpoint by NGS and observed that no gene was disrupted on chromosome 22. We then used Sanger sequencing to search for deleterious variants affecting SEMA5A in 142 patients with ASD. We also identified two independent heterozygous variants located in a conserved functional domain of the protein. Both variants were maternally inherited and predicted as deleterious. Our genetic screens identified the first case of a de novo SEMA5A microdeletion in a patient with ASD and ID. Although our study alone cannot formally associate SEMA5A with susceptibility to ASD, it provides additional evidence that Semaphorin dysfunction could lead to ASD and ID. Further studies on Semaphorins are warranted to better understand the role of this family of genes in susceptibility to neurodevelopmental disorders.

Compound heterozygous PKHD1 variants cause a wide spectrum of ductal plate malformations.

Ductal plate malformations (DPM) present with a wide phenotypic spectrum comprising Von Meyenburg complexes (VMC), Caroli disease (CD), Caroli syndrome (CS), and autosomal recessive polycystic kidney disease (ARPKD). Variants in PKHD1 are responsible for ARPKD and CS with a high inter- and intra-familial phenotypic variability. Rare familial cases of CD had been reported and exceptional cases of CD are associated with PKHD1 variants. In a family of three siblings presenting with a wide spectrum of severity of DPM, we performed whole exome sequencing and identified two PKHD1 compound heterozygous variants (c.10444G>A; p.Arg3482Cys and c.5521C>T; p.Glu1841Lys), segregating with the symptoms. Two compound heterozygous PKHD1 variants, including one hypomorphic variant, were identified in two other familial cases of DPM with at least one patient presenting with CD. This report widens the phenotypic variability of PKHD1 variants to VMC, and others hepatic bile ducts malformations with inconstant renal phenotype in adults and highlights the important intra-familial phenotypic variability. It also showed that PKHD1 might be a major gene for CD. This work adds an example of the contribution of exome sequencing, not only in the discovery of new genes but also in expanding the phenotypic spectrum of well-known disease-associated genes, using reverse phenotyping.

Severe X-linked chondrodysplasia punctata in nine new female fetuses.

OBJECTIVES: Conradi-Hünermann-Happle [X-linked dominant chondrodysplasia punctata 2 (CDPX2)] syndrome is a rare X-linked dominant skeletal dysplasia usually lethal in men while affected women show wide clinical heterogeneity. Different EBP mutations have been reported. Severe female cases have rarely been reported, with only six antenatal presentations. METHODS: To better characterize the phenotype in female fetuses, we included nine antenatally diagnosed cases of women with EBP mutations. All cases were de novo except for two fetuses with an affected mother and one case of germinal mosaicism. RESULTS: The mean age at diagnosis was 22 weeks of gestation. The ultrasound features mainly included bone abnormalities: shortening (8/9 cases) and bowing of the long bones (5/9), punctuate epiphysis (7/9) and an irregular aspect of the spine (5/9). Postnatal X-rays and examination showed ichthyosis (8/9) and epiphyseal stippling (9/9), with frequent asymmetric short and bowed long bones. The X-inactivation pattern of the familial case revealed skewed X-inactivation in the mildly symptomatic mother and random X-inactivation in the severe fetal case. Differently affected skin samples of the same fetus revealed different patterns of X-inactivation. CONCLUSION: Prenatal detection of asymmetric shortening and bowing of the long bones and cartilage stippling should raise the possibility of CPDX2 in female fetuses, especially because the majority of such cases involve de novo mutations.

Autosomal-recessive SASH1 variants associated with a new genodermatosis with pigmentation defects, palmoplantar keratoderma and skin carcinoma.

SASH1 (SAM and SH3 domain-containing protein 1) is a tumor suppressor gene involved in the tumorigenesis of a spectrum of solid cancers. Heterozygous SASH1 variants are known to cause autosomal-dominant dyschromatosis. Homozygosity mapping and whole-exome sequencing were performed in a consanguineous Moroccan family with two affected siblings presenting an unclassified phenotype associating an abnormal pigmentation pattern (hypo- and hyperpigmented macules of the trunk and face and areas of reticular hypo- and hyperpigmentation of the extremities), alopecia, palmoplantar keratoderma, ungueal dystrophy and recurrent spinocellular carcinoma. We identified a homozygous variant in SASH1 (c.1849G>A; p.Glu617Lys) in both affected individuals. Wound-healing assay showed that the patient's fibroblasts were better able than control fibroblasts to migrate. Following the identification of SASH1 heterozygous variants in dyschromatosis, we used reverse phenotyping to show that autosomal-recessive variants of this gene could be responsible for an overlapping but more complex phenotype that affected skin appendages. SASH1 should be added to the list of genes responsible for autosomal-dominant and -recessive genodermatosis, with no phenotype in heterozygous patients in the recessive form, and to the list of genes responsible for a predisposition to skin cancer.

The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation.

Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

Cohen syndrome is associated with major glycosylation defects.

Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane protein involved in Golgi integrity. As the Golgi complex is the place where glycosylation of newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of the main clinical manifestations of CS. The glycosylation status of CS serum proteins showed a very unusual pattern of glycosylation characterized by a significant accumulation of agalactosylated fucosylated structures as well as asialylated fucosylated structures demonstrating a major defect of glycan maturation in CS. However, CS transferrin and α1-AT profiles, two liver-derived proteins, were normal. We also showed that intercellular cell adhesion molecule 1 and LAMP-2, two highly glycosylated cellular proteins, presented an altered migration profile on SDS-PAGE in peripheral blood mononuclear cells from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore, early endosomes were almost absent in these cells and lysosomes were abnormally enlarged, suggesting a crucial role of VPS13B in endosomal-lysosomal trafficking. Our work provides evidence that CS is associated to a tissue-specific major defect of glycosylation and endosomal-lysosomal trafficking defect, suggesting that this could be a new key element to decipher the mechanisms of CS physiopathology.

C5orf42 is the major gene responsible for OFD syndrome type VI.

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the "Joubert syndrome related disorders". Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.

Congenital neutropenia with retinopathy, a new phenotype without intellectual deficiency or obesity secondary to VPS13B mutations.

Over one hundred VPS13B mutations are reported in Cohen syndrome (CS). Most cases exhibit a homogeneous phenotype that includes intellectual deficiency (ID), microcephaly, facial dysmorphism, slender extremities, truncal obesity, progressive chorioretinal dystrophy, and neutropenia. We report on a patient carrying two VPS13B splicing mutations with an atypical phenotype that included microcephaly, retinopathy, and congenital neutropenia, but neither obesity nor ID. RNA analysis of the IVS34+2T_+3AinsT mutation did not reveal any abnormal splice fragments but mRNA quantification showed a significant decrease in VPS13B expression. RNA sequencing analysis up- and downstream from the IVS57+2T>C mutation showed abnormal splice isoforms. In contrast to patients with typical CS, who express only abnormal VPS13B mRNA and truncated protein, a dose effect of residual normal VPS13B protein possibly explains the incomplete phenotype in the patient. This observation emphasizes that VPS13B analysis should be performed in cases of congenital neutropenia associated with retinopathy, even in the absence of ID, therefore extending the VPS13B phenotype spectrum.

Detailed clinical, genetic and neuroimaging characterization of OFD VI syndrome.

Oral-facial-digital syndrome type VI (OFD VI) is characterized by the association of malformations of the face, oral cavity and extremities, distinguished from the 12 other OFD syndromes by cerebellar and metacarpal abnormalities. Cerebellar malformations in OFD VI have been described as a molar tooth sign (MTS), thus, including OFD VI among the "Joubert syndrome related disorders" (JSRD). OFD VI diagnostic criteria have recently been suggested: MTS and one or more of the following: 1) tongue hamartoma(s) and/or additional frenula and/or upper lip notch; 2) mesoaxial polydactyly of hands or feet; 3) hypothalamic hamartoma. In order to further delineate this rare entity, we present the neurological and radiological data of 6 additional OFD VI patients. All patients presented oral malformations, facial dysmorphism and distal abnormalities including frequent polydactyly (66%), as well as neurological symptoms with moderate to severe mental retardation. Contrary to historically reported patients, mesoaxial polydactyly did not appear to be a predominant clinical feature in OFD VI. Sequencing analyzes of the 14 genes implicated in JSRD up to 2011 revealed only an OFD1 frameshift mutation in one female OFD VI patient, strengthening the link between these two oral-facial-digital syndromes and JSRD.

Changing facial phenotype in Cohen syndrome: towards clues for an earlier diagnosis.

Cohen syndrome (CS) is a rare autosomal recessive condition caused by mutations and/or large rearrangements in the VPS13B gene. CS clinical features, including developmental delay, the typical facial gestalt, chorioretinal dystrophy (CRD) and neutropenia, are well described. CS diagnosis is generally raised after school age, when visual disturbances lead to CRD diagnosis and to VPS13B gene testing. This relatively late diagnosis precludes accurate genetic counselling. The aim of this study was to analyse the evolution of CS facial features in the early period of life, particularly before school age (6 years), to find clues for an earlier diagnosis. Photographs of 17 patients with molecularly confirmed CS were analysed, from birth to preschool age. By comparing their facial phenotype when growing, we show that there are no special facial characteristics before 1 year. However, between 2 and 6 years, CS children already share common facial features such as a short neck, a square face with micrognathia and full cheeks, a hypotonic facial appearance, epicanthic folds, long ears with an everted upper part of the auricle and/or a prominent lobe, a relatively short philtrum, a small and open mouth with downturned corners, a thick lower lip and abnormal eye shapes. These early transient facial features evolve to typical CS facial features with aging. These observations emphasize the importance of ophthalmological tests and neutrophil count in children in preschool age presenting with developmental delay, hypotonia and the facial features we described here, for an earlier CS diagnosis.

In-frame mutations in exon 1 of SKI cause dominant Shprintzen-Goldberg syndrome.

Shprintzen-Goldberg syndrome (SGS) is characterized by severe marfanoid habitus, intellectual disability, camptodactyly, typical facial dysmorphism, and craniosynostosis. Using family-based exome sequencing, we identified a dominantly inherited heterozygous in-frame deletion in exon 1 of SKI. Direct sequencing of SKI further identified one overlapping heterozygous in-frame deletion and ten heterozygous missense mutations affecting recurrent residues in 18 of the 19 individuals screened for SGS; these individuals included one family affected by somatic mosaicism. All mutations were located in a restricted area of exon 1, within the R-SMAD binding domain of SKI. No mutation was found in a cohort of 11 individuals with other marfanoid-craniosynostosis phenotypes. The interaction between SKI and Smad2/3 and Smad 4 regulates TGF-ß signaling, and the pattern of anomalies in Ski-deficient mice corresponds to the clinical manifestations of SGS. These findings define SGS as a member of the family of diseases associated with the TGF-ß-signaling pathway.

The DYRK1A gene is a cause of syndromic intellectual disability with severe microcephaly and epilepsy.

BACKGROUND: DYRK1A plays different functions during development, with an important role in controlling brain growth through neuronal proliferation and neurogenesis. It is expressed in a gene dosage dependent manner since dyrk1a haploinsufficiency induces a reduced brain size in mice, and DYRK1A overexpression is the candidate gene for intellectual disability (ID) and microcephaly in Down syndrome. We have identified a 69 kb deletion including the 5' region of the DYRK1A gene in a patient with growth retardation, primary microcephaly, facial dysmorphism, seizures, ataxic gait, absent speech and ID. Because four patients previously reported with intragenic DYRK1A rearrangements or 21q22 microdeletions including only DYRK1A presented with overlapping phenotypes, we hypothesised that DYRK1A mutations could be responsible for syndromic ID with severe microcephaly and epilepsy. METHODS: The DYRK1A gene was studied by direct sequencing and quantitative PCR in a cohort of 105 patients with ID and at least two symptoms from the Angelman syndrome spectrum (microcephaly < -2.5 SD, ataxic gait, seizures and speech delay). RESULTS: We identified a de novo frameshift mutation (c.290_291delCT; p.Ser97Cysfs*98) in a patient with growth retardation, primary severe microcephaly, delayed language, ID, and seizures. CONCLUSION: The identification of a truncating mutation in a patient with ID, severe microcephaly, epilepsy, and growth retardation, combined with its dual function in regulating the neural proliferation/neuronal differentiation, adds DYRK1A to the list of genes responsible for such a phenotype. ID, microcephaly, epilepsy, and language delay are the more specific features associated with DYRK1A abnormalities. DYRK1A studies should be discussed in patients presenting such a phenotype.

Systematic search for neutropenia should be part of the first screening in patients with poikiloderma.

Poikiloderma occurs in a number of hereditary syndromes, the best known of which is Rothmund-Thomson syndrome (RTS). Differential diagnoses include Dyskeratosis Congenita (DC) with high genetic heterogeneity and Clericuzio-type Poikiloderma with Neutropenia (CPN) due to mutations in the C16orf57 gene. Mutations in the RECQL4 gene are only observed in two thirds of RTS patients. In this study, 10 patients referred for syndromic poikiloderma and negative for RECQL4 sequencing analysis were investigated for C16orf57 mutations. Two C16orf57 heterozygous nonsense mutations (p.W81X and p.Y89X) were identified in a 5-year-old female child presenting with generalized poikiloderma, dental dysplasia, gingivitis, nail dystrophy, palmoplantar keratoderma and pachyonychia of the great toenails. Previously undetected and silent neutropenia was evidenced after C16orf57 molecular analysis. Neutropenia was absent in the C16orf57-negative patients. This report confirms that neutrophil count should be performed in all patients with poikiloderma to target the C16orf57 gene sequencing analysis, prior to RECQL4 analysis.

Search for the best indicators for the presence of a VPS13B gene mutation and confirmation of diagnostic criteria in a series of 34 patients genotyped for suspected Cohen syndrome.

BACKGROUND: Cohen syndrome is a rare autosomal recessive inherited disorder that results from mutations of the VPS13B gene. Clinical features consist of a combination of mental retardation, facial dysmorphism, postnatal microcephaly, truncal obesity, slender extremities, joint hyperextensibility, myopia, progressive chorioretinal dystrophy, and intermittent neutropenia. PATIENTS AND METHODS: The aim of the study was to determine which of the above clinical features were the best indicators for the presence of VPS13B gene mutations in a series of 34 patients with suspected Cohen syndrome referred for molecular analysis of VPS13B. RESULTS: 14 VPS13B gene mutations were identified in 12 patients, and no mutation was found in 22 patients. The presence of chorioretinal dystrophy (92% vs 32%, p=0.0023), intermittent neutropenia (92% vs 5%, p<0.001), and postnatal microcephaly (100% vs 48%, p=0.0045) was significantly higher in the group of patients with a VPS13B gene mutation compared to the group of patients without a mutation. All patients with VPS13B mutations had chorioretinal dystrophy and/or intermittent neutropenia. The Kolehmainen diagnostic criteria provided 100% sensibility and 77% specificity when applied to this series. CONCLUSION: From this study and a review of more than 160 genotyped cases from the literature, it is concluded that, given the large size of the gene, VPS13B screening is not indicated in the absence of chorioretinal dystrophy or neutropenia in patients aged over 5 years. The follow-up of young patients could be a satisfactory alternative unless there are some reproductive issues.

Genomic deletions of OFD1 account for 23% of oral-facial-digital type 1 syndrome after negative DNA sequencing.

Oral-facial-digital type I syndrome (OFDI) is characterised by an X-linked dominant mode of inheritance with lethality in males. Clinical features include facial dysmorphism with oral, dental and distal abnormalities, polycystic kidney disease and central nervous system malformations. Considerable allelic heterogeneity has been reported within the OFD1 gene, but DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene remains negative in more than 20% of cases. We hypothesized that genomic rearrangements could account for the majority of the remaining undiagnosed cases. Thus, we took advantage of two independent available series of patients with OFDI syndrome and negative DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene from two different European labs: 13/36 cases from the French lab; 13/95 from the Italian lab. All patients were screened by a semiquantitative fluorescent multiplex method (QFMPSF) and relative quantification by real-time PCR (qPCR). Six OFD1 genomic deletions (exon 5, exons 1-8, exons 1-14, exons 10-11, exons 13-23 and exon 17) were identified, accounting for 5% of OFDI patients and for 23% of patients with negative mutation screening by DNA sequencing. The association of DNA direct sequencing, QFMPSF and qPCR detects OFD1 alteration in up to 85% of patients with a phenotype suggestive of OFDI syndrome. Given the average percentage of large genomic rearrangements (5%), we suggest that dosage methods should be performed in addition to DNA direct sequencing analysis to exclude the involvement of the OFD1 transcript when there are genetic counselling issues.