Cardiac valvular Ehlers‐Danlos syndrome is a well‐defined condition due to recessive null variants in COL1A2

Cardiac valvular Ehlers‐Danlos syndrome (EDS) is a rare EDS subtype, caused by specific recessive variants in the gene encoding pro‐α2‐chain of type I collagen (COL1A2). Cardiac valvular EDS is mainly characterized by generalized/peripheral joint hypermobility, moderate–severe cardiac valvular disease, skin hyperextensibility and other minor soft tissues features. Only five molecularly confirmed patients have been reported to date. Here, we describe two additional affected sisters, who share the homozygous c.3601G>T nonsense variant in COL1A2. Clinical data and literature review allowed to better define the clinical spectrum of cardiac valvular EDS which now emerges as a more recognizable EDS variant with progressive heart valve disease firstly affecting the mitral valve. Possibly distinguishing features include bilateral flatfeet with hindfoot pronation, lower eyelid ptosis and hypoplasia of the interphalangeal creases. The absence of bone fragility in our patients indicates that cardiac valvular EDS is also separated from patients with autosomal recessive osteogenesis imperfecta and variants in COL1A2, as well as from individuals with autosomal dominant osteogenesis imperfecta and severe cardiac valvular disease.     

TOPORS as a novel causal gene for Joubert syndrome

Joubert syndrome (JBTS) is a Mendelian disorder of the primary cilium defined by the clinical triad of hypotonia, developmental delay, and a distinct cerebellar malformation called the molar tooth sign. JBTS is inherited in an autosomal recessive, autosomal dominant, or X-linked recessive manner. Though over 40 genes have been identified as causal for JBTS, molecular diagnosis is not made in 30%–40% of individuals who meet clinical criteria. TOPORS encodes topoisomerase I-binding arginine/serine-rich protein, and homozygosity for a TOPORS missense variant (c.29C > A; p.(Pro10Gln)) was identified in individuals with the ciliopathy oral-facial-digital syndrome in two families of Dominican descent. Here, we report an additional proband of Dominican ancestry with JBTS found by exome sequencing to be homozygous for the identical p.(Pro10Gln) TOPORS missense variant. Query of the Mount Sinai BioMe biobank, which includes 1880 individuals of Dominican ancestry, supports a high carrier frequency of the TOPORS p.(Pro10Gln) variant in individuals of Dominican descent. Our data nominates TOPORS as a novel causal gene for JBTS and suggests that TOPORS variants should be considered in the differential of ciliopathy-spectrum disease in individuals of Dominican ancestry.     

Diagnostic Exome Sequencing to Elucidate the Genetic Basis of Likely Recessive Disorders in Consanguineous Families

Rare, atypical, and undiagnosed autosomal‐recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal‐recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal‐recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High‐confidence pathogenic variants were found in homozygosity in known disease‐causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.     

Identification of C12orf4 as a gene for autosomal recessive intellectual disability

Intellectual disability (ID) is a major health problem in our society. Genetic causes of ID remain unknown because of its vast heterogeneity. Here we report two Finnish families and one Dutch family with affected individuals presenting with mild to moderate ID, neuropsychiatric symptoms and delayed speech development. By utilizing whole exome sequencing (WES), we identified a founder missense variant c.983T>C (p.Leu328Pro) in seven affected individuals from two Finnish consanguineous families and a deletion c.799_1034‐429delinsTTATGA (p.Gln267fs) in one affected individual from a consanguineous Dutch family in the C12orf4 gene on chromosome 12. Both the variants co‐segregated in the respective families as an autosomal recessive trait. Screening of the p.Leu328Pro variant showed enrichment in the North Eastern sub‐isolate of Finland among anonymous local blood donors with a carrier frequency of 1:53, similar to other disease mutations with a founder effect in that region. To date, only one Arab family with a three affected individuals with a frameshift insertion variant in C12orf4 has been reported. In summary, we expand and establish the clinical and mutational spectrum of C12orf4 variants. Our findings implicate C12orf4 as a causative gene for autosomal recessive ID.     

Identification of Two Homozygous Sequence Variants in the COL7A1 Gene Underlying Dystrophic Epidermolysis Bullosa by Whole‐Exome Analysis in a Consanguineous Family

Dystrophic epidermolysis bullosa (DEB) is an inherited skin disorder with variable severity and heterogeneous genetic involvement. Diagnostic approaches for this condition include clinical evaluations and electron microscopy of patients’ skin biopsies, followed by Sanger sequencing (SS) of a large gene (118 exons) that encodes the alpha chain of type VII collagen (COL7A1) located on Chromosome 3p21.1. However, the use of SS may hinder diagnostic efficiency and lead to delays because it is costly and time‐consuming. We evaluated a 5‐generation consanguineous family with 3 affected individuals presenting the severe generalised DEB phenotype. Human whole‐exome sequencing (WES) revealed 2 homozygous sequence variants: the previously reported variant p.Arg578* in exon 13 and a novel variant p.Arg2063Gln in exon 74 of the COL7A1 gene. Validation by SS, performed on all family members, confirmed the cosegregation of the 2 variants with the disease phenotype. To the best of our knowledge, 2 homozygous COL7A1 variants have never been simultaneously reported in DEB patients; however, the upstream protein truncation variant is more likely to be disease‐causing than the novel missense variant. WES can be used as an efficient molecular diagnostic tool for evaluating autosomal recessive forms of DEB.     

Exome sequencing revealed a novel splice site variant in the ALX1 gene underlying frontonasal dysplasia

Frontonasal dysplasia (FND) is a heterogeneous group of disorders characterized by hypertelorism, telecanthus, broad nasal root, wide prominent nasal bridge, short and wide nasal ridge, broad columella and smooth philtrum. To date one X‐linked and three autosomal recessive forms of FND have been reported in different ethnic groups. We sought to identify the gene responsible for FND in a consanguineous Pakistani family segregating the disorder in autosomal recessive pattern. Genome‐wide homozygosity mapping using 250KNsp array revealed five homozygous regions in the selected affected individuals. Exome sequencing found a novel splice acceptor site variant (c.661‐1G>C: NM_006982.2) in ALX1. Sanger sequencing confirmed the correct segregation of the pathogenic variant in the whole family. Our study concludes that the splice site variant identified in the ALX1 gene causes mild form of FND.     

Homozygous FIBP nonsense variant responsible of syndromic overgrowth,with overgrowth,macrocephaly, retinal coloboma and learning disabilities

The acidic fibroblast growth factor (FGF) intracellular binding protein (FIBP) interacts directly with the fibroblast growth factor FGF1. Although FIBP is known to be implicated in the FGF signaling pathway, its precise function remains unclear. Gain‐of‐function variants in several FGF receptors (FGFRs) are implicated in a wide spectrum of growth disorders from achondroplasia to overgrowth syndromes. In a unique case from a consanguineous union presenting with overgrowth, macrocephaly, retinal coloboma, large thumbs, severe varicose veins and learning disabilities, exome sequencing identified a homozygous nonsense FIBP variant. The patient's fibroblasts exhibit FIBP cDNA degradation and an increased proliferation capacity compared with controls. The phenotype defines a new multiple congenital abnormalities (MCA) syndrome, overlapping with the heterogeneous group of overgrowth syndromes with macrocephaly. The different clinical features can be explained by the alteration of the FGFR pathway. Taken together, these results suggest the implication of FIBP in a new autosomal recessive MCA.     

RP1L1 Variants are Associated with a Spectrum of Inherited Retinal Diseases Including Retinitis Pigmentosa and Occult Macular Dystrophy

In one consanguineous family with retinitis pigmentosa (RP), a condition characterized by progressive visual loss due to retinal degeneration, homozygosity mapping, and candidate gene sequencing suggested a novel locus. Exome sequencing identified a homozygous frameshifting mutation, c.601delG, p.Lys203Argfs*28, in RP1L1 encoding RP 1‐like1, a photoreceptor‐specific protein. A screen of a further 285 unrelated individuals with autosomal recessive RP identified an additional proband, homozygous for a missense variant, c.1637G>C, p.Ser546Thr, in RP1L1. A distinct retinal disorder, occult macular dystrophy (OCMD) solely affects the central retinal cone photoreceptors and has previously been reported to be associated with variants in the same gene. The association between mutations in RP1L1 and the disorder OCMD was explored by screening a cohort of 28 unrelated individuals with the condition; 10 were found to harbor rare (minor allele frequency ≤0.5% in the 1,000 genomes dataset) heterozygous RP1L1 missense variants. Analysis of family members revealed many unaffected relatives harboring the same variant. Linkage analysis excluded the possibility of a recessive mode of inheritance, and sequencing of RP1, a photoreceptor protein that interacts with RP1L1, excluded a digenic mechanism involving this gene. These findings imply an important and diverse role for RP1L1 in human retinal physiology and disease.     

TBC1D24 Mutation Causes Autosomal‐Dominant Nonsyndromic Hearing Loss

Hereditary hearing loss is extremely heterogeneous. Over 70 genes have been identified to date, and with the advent of massively parallel sequencing, the pace of novel gene discovery has accelerated. In a family segregating progressive autosomal‐dominant nonsyndromic hearing loss (NSHL), we used OtoSCOPE® to exclude mutations in known deafness genes and then performed segregation mapping and whole‐exome sequencing to identify a unique variant, p.Ser178Leu, in TBC1D24 that segregates with the hearing loss phenotype. TBC1D24 encodes a GTPase‐activating protein expressed in the cochlea. Ser178 is highly conserved across vertebrates and its change is predicted to be damaging. Other variants in TBC1D24 have been associated with a panoply of clinical symptoms including autosomal recessive NSHL, syndromic hearing impairment associated with onychodystrophy, osteodystrophy, mental retardation, and seizures (DOORS syndrome), and a wide range of epileptic disorders.     

Prenatal exome sequencing in anomalous fetuses: new opportunities and challenges

PurposeWe investigated the diagnostic and clinical performance of exome sequencing in fetuses with sonographic abnormalities with normal karyotype and microarray and, in some cases, normal gene-specific sequencing.MethodsExome sequencing was performed on DNA from 15 anomalous fetuses and from the peripheral blood of their parents. Parents provided consent to be informed of diagnostic results in the fetus, medically actionable findings in the parents, and their identification as carrier couples for significant autosomal recessive conditions. We assessed the perceptions and understanding of exome sequencing using mixed methods in 15 mother−father dyads.ResultsIn seven (47%) of 15 fetuses, exome sequencing provided a diagnosis or possible diagnosis with identification of variants in the following genes: COL1A1, MUSK, KCTD1, RTTN, TMEM67, PIEZO1 and DYNC2H1. One additional case revealed a de novo nonsense mutation in a novel candidate gene (MAP4K4). The perceived likelihood that exome sequencing would explain the results (5.2 on a 10-point scale) was higher than the approximately 30% diagnostic yield discussed in pretest counseling.ConclusionExome sequencing had diagnostic utility in a highly select population of fetuses where a genetic diagnosis was highly suspected. Challenges related to genetics literacy and variant interpretation must be addressed by highly tailored pre- and posttest genetic counseling.     

Genetic basis of neurodevelopmental disorders in 103 Jordanian families

We recruited 103 families from Jordan with neurodevelopmental disorders (NDD) and patterns of inheritance mostly suggestive of autosomal recessive inheritance. In each family, we investigated at least one affected individual using exome sequencing and an in-house diagnostic variant interpretation pipeline including a search for copy number variation. This approach led us to identify the likely molecular defect in established disease genes in 37 families. We could identify 25 pathogenic nonsense and 11 missense variants as well as 3 pathogenic copy number variants and 1 repeat expansion. Notably, 11 of the disease-causal variants occurred de novo. In addition, we prioritized a homozygous frameshift variant in PUS3 in two sisters with intellectual disability. To our knowledge, PUS3 has been postulated only recently as a candidate disease gene for intellectual disability in a single family with three affected siblings. Our findings provide additional evidence to establish loss of PUS3 function as a cause of intellectual disability.     

Expanding the MYBPC1 phenotypic spectrum: a novel homozygous mutation causes arthrogryposis multiplex congenita

Arthrogryposis multiplex congenita (AMC) is characterized by heterogeneous nonprogressive multiple joint contractures appearing at birth. We present a consanguineous Israeli‐Druze family with several members presenting with AMC. A variable intra‐familial phenotype and pected autosomal recessive inheritance prompted molecular diagnosis by whole‐exome sequencing. Variant analysis focused on rare homozygous changes, revealed a missense variant in MYBPC1, NM_002465:c.556G>A (p.E286K), affecting the last nucleotide of Exon 8. This novel variant was not observed in the common variant databases and co‐segregated as expected within the extended family. MYBPC1 encodes a slow skeletal muscle isoform, essential for muscle contraction. Heterozygous mutations in this gene are associated with distal arthrogryposis types 1b and 2, whereas a homozygous nonsense mutation is implicated in one family with lethal congenital contractural syndrome 4. We present a novel milder MYBPC1 homozygous phenotype.     

HOXA2 Haploinsufficiency in Dominant Bilateral Microtia and Hearing Loss

Microtia is a rare, congenital malformation of the external ear that in some cases has a genetic etiology. We ascertained a three‐generation family with bilateral microtia and hearing loss segregating as an autosomal dominant trait. Exome sequencing of affected family members detected only seven shared, rare, heterozygous, nonsynonymous variants, including one protein truncating variant, a HOXA2 nonsense change (c.703C>T, p.Q235*). The HOXA2 variant was segregated with microtia and hearing loss in the family and was not seen in 6,500 individuals sequenced by the NHLBI Exome Sequencing Project or in 218 control individuals sequenced in this study. HOXA2 has been shown to be critical for outer and middle ear development through mouse models and has previously been associated with autosomal recessive bilateral microtia. Our data extend these conclusions and define HOXA2 haploinsufficiency as the first genetic cause for autosomal‐dominant nonsyndromic microtia.     

Homozygous variants in AMPD2 and COL11A1 lead to a complex phenotype of pontocerebellar hypoplasia type 9 and Stickler syndrome type 2

Pontocerebellar hypoplasia type 9 (PCH9) is an autosomal recessive neurodevelopmental disorder caused by pathogenic variants in the AMPD2 gene. We evaluated the son of a consanguineous couple who presented with profound hypotonia and global developmental delay. Other features included sensorineural hearing loss, asymmetric astigmatism, and high myopia. Clinical whole‐exome sequence analysis identified a homozygous missense variant in AMPD2 (NM_001257360.1:c.2201C > T, p.[Pro734Leu]) that has not been previously reported. Given the strong phenotypic overlap with PCH9, including the identification of the typical “Figure 8” appearance of the brainstem on neuroimaging, we suspect this variant was causative of the neurodevelopmental disability in this individual. An additional homozygous nonsense variant in COL11A1 (NM_001854.4:c.1168G > T, p.[Glu390Ter]) was identified. Variants in this alternatively spliced region of COL11A1 have been identified to cause an autosomal recessive form of Stickler syndrome type 2 characterized by sensorineural hearing loss and eye abnormalities, but without musculoskeletal abnormalities. The COL11A1 variant likely also contributed to the individual's phenotype, suggesting two potentially relevant genetic findings. This challenging case highlights the importance of detailed phenotypic characterization when interpreting whole exome data.     

Bohring‐Opitz syndrome caused by an ASXL1 mutation inherited from a germline mosaic mother

Bohring‐Opitz syndrome (BOS) is characterized clinically by severe developmental delays, microcephaly, failure to thrive, and characteristic facial features (prominent eyes, facial nevus simplex [flammeus], and others). Most patients meeting the clinical criteria for BOS (MIM: 605039) have a de novo nonsense or frameshift variant in ASXL1. We report a case of BOS caused by a pathogenic ASXL1 variant inherited from a germline mosaic mother. The ASXL1 mutation was detected via trio exome sequencing. The sequencing data demonstrated that the variant was inherited maternally but that the maternal variant was underrepresented in comparison to the normal allele. These results suggested maternal mosaicism for the variant. Additional testing on the mother was performed on buccal cell DNA, which was also consistent with mosaicism. The mother had been reported to be healthy and the family history is unremarkable. This is the first report of BOS caused by a mutation inherited from an unaffected, presumed germline mosaic parent. This phenomenon has been reported for other traditionally de novo dominant disorders like CHARGE syndrome and Cornelia de Lange syndrome. This case emphasizes the need for accurate low but non‐negative recurrence risk counseling for families with children with BOS and it impacts exome interpretation strategy.     

Diagnostic exome sequencing in early‐onset Parkinson's disease confirms VPS13C as a rare cause of autosomal‐recessive Parkinson's disease

Parkinson's disease (PD) is a genetically heterogeneous disorder and new putative disease genes are discovered constantly. Therefore, whole‐exome sequencing could be an efficient approach to genetic testing in PD. To evaluate its performance in early‐onset sporadic PD, we performed diagnostic exome sequencing in 80 individuals with manifestation of PD symptoms at age 40 or earlier and a negative family history of PD. Variants in validated and candidate disease genes and risk factors for PD and atypical Parkinson syndromes were annotated, followed by further analysis for selected variants. We detected pathogenic variants in Mendelian genes in 6.25% of cases and high‐impact risk factor variants in GBA in 5% of cases, resulting in overall maximum diagnostic yield of 11.25%. One individual was compound heterozygous for variants affecting canonical splice sites in VPS13C, confirming the causal role of protein‐truncating variants in this gene linked to autosomal‐recessive early‐onset PD. Despite the low diagnostic yield of exome sequencing in sporadic early‐onset PD, the confirmation of the recently discovered VPS13C gene highlights its advantage over using predefined gene panels.     

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1–4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA‐binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole‐exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense‐mediated mRNA decay (NMD), and one canonical splice site variant, c.272‐1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272‐1G>C, were clustered within 20 amino acid residues of the C‐terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.