De novo variants in FRMD5 are associated with developmental delay, intellectual disability, ataxia, and abnormalities of eye movement.

Proteins containing the FERM (four-point-one, ezrin, radixin, and moesin) domain link the plasma membrane with cytoskeletal structures at specific cellular locations and have been implicated in the localization of cell-membrane-associated proteins and/or phosphoinositides. FERM domain-containing protein 5 (FRMD5) localizes at cell adherens junctions and stabilizes cell-cell contacts. To date, variants in FRMD5 have not been associated with a Mendelian disease in OMIM. Here, we describe eight probands with rare heterozygous missense variants in FRMD5 who present with developmental delay, intellectual disability, ataxia, seizures, and abnormalities of eye movement. The variants are de novo in all for whom parental testing was available (six out of eight probands), and human genetic datasets suggest that FRMD5 is intolerant to loss of function (LoF). We found that the fly ortholog of FRMD5, CG5022 (dFrmd), is expressed in the larval and adult central nervous systems where it is present in neurons but not in glia. dFrmd LoF mutant flies are viable but are extremely sensitive to heat shock, which induces severe seizures. The mutants also exhibit defective responses to light. The human FRMD5 reference (Ref) cDNA rescues the fly dFrmd LoF phenotypes. In contrast, all the FRMD5 variants tested in this study (c.340T>C, c.1051A>G, c.1053C>G, c.1054T>C, c.1045A>C, and c.1637A>G) behave as partial LoF variants. In addition, our results indicate that two variants that were tested have dominant-negative effects. In summary, the evidence supports that the observed variants in FRMD5 cause neurological symptoms in humans.

Recessive PRDM13 mutations cause fatal perinatal brainstem dysfunction with cerebellar hypoplasia and disrupt Purkinje cell differentiation.

Pontocerebellar hypoplasias (PCHs) are congenital disorders characterized by hypoplasia or early atrophy of the cerebellum and brainstem, leading to a very limited motor and cognitive development. Although over 20 genes have been shown to be mutated in PCHs, a large proportion of affected individuals remains undiagnosed. We describe four families with children presenting with severe neonatal brainstem dysfunction and pronounced deficits in cognitive and motor development associated with four different bi-allelic mutations in PRDM13, including homozygous truncating variants in the most severely affected individuals. Brain MRI and fetopathological examination revealed a PCH-like phenotype, associated with major hypoplasia of inferior olive nuclei and dysplasia of the dentate nucleus. Notably, histopathological examinations highlighted a sparse and disorganized Purkinje cell layer in the cerebellum. PRDM13 encodes a transcriptional repressor known to be critical for neuronal subtypes specification in the mouse retina and spinal cord but had not been implicated, so far, in hindbrain development. snRNA-seq data mining and in situ hybridization in humans show that PRDM13 is expressed at early stages in the progenitors of the cerebellar ventricular zone, which gives rise to cerebellar GABAergic neurons, including Purkinje cells. We also show that loss of function of prdm13 in zebrafish leads to a reduction in Purkinje cells numbers and a complete absence of the inferior olive nuclei. Altogether our data identified bi-allelic mutations in PRDM13 as causing a olivopontocerebellar hypoplasia syndrome and suggest that early deregulations of the transcriptional control of neuronal fate specification could contribute to a significant number of cases.

Diagnostic yield of rare skeletal dysplasia conditions in the radiogenomics era.

BACKGROUND: Skeletal dysplasia (SD) conditions are rare genetic diseases of the skeleton, encompassing a heterogeneous group of over 400 disorders, and represent approximately 5% of all congenital anomalies. Developments in genetic and treatment technologies are leading to unparalleled therapeutic advances; thus, it is more important than ever to molecularly confirm SD conditions. Data on 'rates-of-molecular yields' in SD conditions, through exome sequencing approaches, is limited. Figures of 39% and 52.5% have been reported in the USA (n = 54) and South Korea (n = 185) respectively. METHODS: We discuss a single-centre (in the UK) experience of whole-exome sequencing (WES) in a cohort of 15 paediatric patients (aged 5 months to 12 years) with SD disorders previously molecularly unconfirmed. Our cohort included patients with known clinical diagnoses and undiagnosed skeletal syndromes. Extensive phenotyping and expert radiological review by a panel of international SD radiology experts, coupled with a complex bioinformatics pipeline, allowed for both gene-targeted and gene-agnostic approaches. RESULTS: Significant variants leading to a likely or confirmed diagnosis were identified in 53.3% (n = 8/15) of patients; 46.7% (n = 7/15) having a definite molecular diagnosis and 6.7% (n = 1/15) having a likely molecular diagnosis. We discuss this in the context of a rare disease in general and specifically SD presentations. Of patients with known diagnoses pre-WES (n = 10), molecular confirmation occurred in 7/10 cases, as opposed to 1/5 where a diagnosis was unknown pre-test. Thus, diagnostic return is greatest where the diagnosis is known pre-test. For WGS (whole genome sequencing, the next iteration of WES), careful case selection (ideally of known diagnoses pre-test) will yield highest returns. CONCLUSIONS: Our results highlight the cost-effective use of WES-targeted bioinformatic analysis as a diagnostic tool for SD, particularly patients with presumed SD, where detailed phenotyping is essential. Thorough co-ordinated clinical evaluation between clinical, radiological, and molecular teams is essential for improved yield and clinical care. WES (and WGS) yields will increase with time, allowing faster diagnoses, avoiding needless investigations, ensuring individualised patient care and patient reassurance. Further diagnoses will lead to increased information on natural history/mechanistic details, and likely increased therapies and clinical trials.

High diagnostic yield in skeletal ciliopathies using massively parallel genome sequencing, structural variant screening and RNA analyses.

Skeletal ciliopathies are a heterogenous group of disorders with overlapping clinical and radiographic features including bone dysplasia and internal abnormalities. To date, pathogenic variants in at least 30 genes, coding for different structural cilia proteins, are reported to cause skeletal ciliopathies. Here, we summarize genetic and phenotypic features of 34 affected individuals from 29 families with skeletal ciliopathies. Molecular diagnostic testing was performed using massively parallel sequencing (MPS) in combination with copy number variant (CNV) analyses and in silico filtering for variants in known skeletal ciliopathy genes. We identified biallelic disease-causing variants in seven genes: DYNC2H1, KIAA0753, WDR19, C2CD3, TTC21B, EVC, and EVC2. Four variants located in non-canonical splice sites of DYNC2H1, EVC, and KIAA0753 led to aberrant splicing that was shown by sequencing of cDNA. Furthermore, CNV analyses showed an intragenic deletion of DYNC2H1 in one individual and a 6.7 Mb de novo deletion on chromosome 1q24q25 in another. In five unsolved cases, MPS was performed in family setting. In one proband we identified a de novo variant in PRKACA and in another we found a homozygous intragenic deletion of IFT74, removing the first coding exon and leading to expression of a shorter message predicted to result in loss of 40 amino acids at the N-terminus. These findings establish IFT74 as a new skeletal ciliopathy gene. In conclusion, combined single nucleotide variant, CNV and cDNA analyses lead to a high yield of genetic diagnoses (90%) in a cohort of patients with skeletal ciliopathies.

Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome.

CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signalling. Due to advances in next-generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic (BCD) syndrome albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 13 participants from nine families presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.

The CHD8 overgrowth syndrome: A detailed evaluation of an emerging overgrowth phenotype in 27 patients.

CHD8 has been reported as an autism susceptibility/intellectual disability gene but emerging evidence suggests that it additionally causes an overgrowth phenotype. This study reports 27 unrelated patients with pathogenic or likely pathogenic CHD8 variants (25 null variants, two missense variants) and a male:female ratio of 21:6 (3.5:1, p < .01). All patients presented with intellectual disability, with 85% in the mild or moderate range, and 85% had a height and/or head circumference ≥2 standard deviations above the mean, meeting our clinical criteria for overgrowth. Behavioral problems were reported in the majority of patients (78%), with over half (56%) either formally diagnosed with an autistic spectrum disorder or described as having autistic traits. Additional clinical features included neonatal hypotonia (33%), and less frequently seizures, pes planus, scoliosis, fifth finger clinodactyly, umbilical hernia, and glabellar hemangioma (≤15% each). These results suggest that, in addition to its established link with autism and intellectual disability, CHD8 causes an overgrowth phenotype, and should be considered in the differential diagnosis of patients presenting with increased height and/or head circumference in association with intellectual disability.

Exome sequencing reveals a de novo POLD1 mutation causing phenotypic variability in mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL).

BACKGROUND: Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL) is an autosomal dominant systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This syndrome is caused by heterozygous de novo mutations in the POLD1 gene. To date, 19 patients with MDPL have been reported in the literature and among them 14 patients have been characterized at the molecular level. Twelve unrelated patients carried a recurrent in-frame deletion of a single codon (p.Ser605del) and two other patients carried a novel heterozygous mutation in exon 13 (p.Arg507Cys). Additionally and interestingly, germline mutations of the same gene have been involved in familial polyposis and colorectal cancer (CRC) predisposition. PATIENTS AND METHODS: We describe a male and a female patient with MDPL respectively affected with mild and severe phenotypes. Both of them showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, a small mouth, growth retardation, muscle and skin atrophy, but the female patient showed such a severe and early phenotype that a first working diagnosis of Hutchinson-Gilford Progeria was made. The exploration was performed by direct sequencing of POLD1 gene exon 15 in the male patient with a classical MDPL phenotype and by whole exome sequencing in the female patient and her unaffected parents. RESULTS: Exome sequencing identified in the latter patient a de novo heterozygous undescribed mutation in the POLD1 gene (NM_002691.3: c.3209T>A), predicted to cause the missense change p.Ile1070Asn in the ZnF2 (Zinc Finger 2) domain of the protein. This mutation was not reported in the 1000 Genome Project, dbSNP and Exome sequencing databases. Furthermore, the Isoleucine1070 residue of POLD1 is highly conserved among various species, suggesting that this substitution may cause a major impairment of POLD1 activity. For the second patient, affected with a typical MDPL phenotype, direct sequencing of POLD1 exon 15 revealed the recurrent in-frame deletion (c.1812_1814del, p.S605del). CONCLUSION: Our work highlights that mutations in different POLD1 domains can lead to phenotypic variability, ranging from dominantly inherited cancer predisposition syndromes, to mild MDPL phenotypes without lifespan reduction, to very severe MDPL syndromes with major premature aging features. These results also suggest that POLD1 gene testing should be considered in patients presenting with severe progeroid features.

Oculo-auriculo-vertebral spectrum: clinical and molecular analysis of 51 patients.

INTRODUCTION: Oculo-auriculo-vertebral spectrum (OAVS OMIM 164210) is a craniofacial developmental disorder affecting the development of the structures derived from the 1st and the 2nd branchial arches during embryogenesis, with consequential maxillary, mandibular, and ear abnormalities. The phenotype in OAVS is variable and associated clinical features can involve the cardiac, renal, skeletal, and central nervous systems. Its aetiology is still poorly understood. METHODS: We have evaluated the clinical phenotypes of 51 previously unpublished patients with OAVS and their parents, and performed comparative genomic hybridization microarray studies to identify potential causative loci. RESULTS: Of all 51 patients, 16 (31%) had a family history of OAVS. Most had no relevant pre-natal history and only 5 (10%) cases had a history of environmental exposures that have previously been described as risk factors for OAVS. In 28 (55%) cases, the malformations were unilateral. When the involvement was bilateral, it was asymmetric. Ear abnormalities were present in 47 (92%) patients (unilateral in 24; and bilateral in 23). Hearing loss was common (85%), mostly conductive, but also sensorineural, or a combination of both. Hemifacial microsomia was present in 46 (90%) patients (17 also presented facial nerve palsy). Ocular anomalies were present in 15 (29%) patients. Vertebral anomalies were confirmed in 10 (20%) cases; 50% of those had additional heart, brain and/or other organ abnormalities. Brain abnormalities were present in 5 (10%) patients; developmental delay was more common among these patients. Limb abnormalities were found in 6 (12%) patients, and urogenital anomalies in 5 (10%). Array-CGH analysis identified 22q11 dosage anomalies in 10 out of 22 index cases screened. DISCUSSION: In this study we carried out in-depth phenotyping of OAVS in a large, multicentre cohort. Clinical characteristics are in line with those reported previously, however, we observed a higher incidence of hemifacial microsomia and lower incidence of ocular anomalies. Furthermore our data suggests that OAVS patients with vertebral anomalies or congenital heart defects have a higher frequency of additional brain, limb or other malformations. We had a higher rate of familial cases in our cohort in comparison with previous reports, possibly because these cases were referred preferentially to our genetic clinic where family members underwent examination. We propose that familial OAVS cases show phenotypic variability, hence, affected relatives might have been misclassified in previous reports. Moreover, in view of its phenotypic variability, OAVS is potentially a spectrum of conditions, which overlap with other conditions, such as mandibulofacial dysostosis. Array CGH in our cohort identified recurrent dosage anomalies on 22q11, which may contribute to, or increase the risk of OAVS. We hypothesize that although the 22q11 locus may harbour gene(s) or regulatory elements that play a role in the regulation of craniofacial symmetry and 1st and 2nd branchial arch development, OAVS is a heterogeneous condition and many cases have a multifactorial aetiology or are caused by mutations in as yet unidentified gene(s).

Oculo-auriculo-vertebral spectrum: a review of the literature and genetic update.

Oculo-auriculo-vertebral spectrum (OAVS, OMIM 164 210) is a developmental disorder primarily involving structures derived from the first and second pharyngeal arches during embryogenesis. The phenotype is clinically heterogeneous and is typically characterised by abnormal development of the ear, mandible anomalies and defects of the vertebral column. OAVS may occur as a multiple congenital abnormality, and associated findings include anomalies of the eye, brain, heart, kidneys and other organs and systems. Both genetic and environmental factors are thought to contribute to this craniofacial condition, however, the mechanisms are still poorly understood. Here, we present a review of the literature on OAVS, discussing what is known about the aetiology, candidate loci, possible mechanisms and the range of clinical features that characterise this condition. We also comment on some important aspects of recurrence risk counselling to aid clinical management.

Risk factors for hypospadias in the estrogen receptor 2 gene.

CONTEXT: Hypospadias is a common inborn error of the male genitalia of complex, and still elusive, etiology. The presence of active estrogen receptors (ESRs) in the developing male urethra, predominantly the ESR2, has suggested a role of estrogens in the otherwise androgen-dependent male genital differetiation. Moreover, imbalances between these two steroid hormones have been suggested to disturb the external genital development. This has been supported by the association between longer (CA)n variants in the ESR2 gene with lower androgen levels as well as with hypospadias. OBJECTIVE: The aim of this study was to analyze the effect of ESR2 gene variants on the risk to hypospadias. DESIGN, PARTICIPANTS, AND METHODS: Four haplotype-tagging single nucleotide polymorphisms (rs2987983, rs1887994, rs1256040, and rs1256062), the (CA)n polymorphism, and two additional promoter single nucleotide polymorphisms (rs10483774 and rs1271572), mapping to a transcription factor binding region, were typed and analyzed in a Swedish cohort of 354 boys with nonsyndromic hypospadias and 380 healthy controls. RESULTS: Association was identified with longer variants of the (CA)n polymorphism in intron 6 and with a region of intense transcription factor binding, in the putative promoter region, mapping to rs2987983 and rs10483774. The two regions are in low-linkage disequilibrium, meaning that they are not necessarily inherited together as a haplotype; logistic regression analysis indicates that these two risk effects are not independent. CONCLUSIONS: The present study evidences two nonindependent risk factors for hypospadias in the ESR2 gene. We discuss possible mechanisms that explain how these variants may affect male urethral development.