Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes.

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.

A novel homozygous SLC13A5 whole-gene deletion generated by Alu/Alu-mediated rearrangement in an Iraqi family with epileptic encephalopathy.

Biallelic loss-of-function (LoF) of SLC13A5 (solute carrier family 13, member 5) induced deficiency in sodium/citrate transporter (NaCT) causes autosomal recessive developmental epileptic encephalopathy 25 with hypoplastic amelogenesis imperfecta (DEE25; MIM #615905). Many pathogenic SLC13A5 single nucleotide variants (SNVs) and small indels have been described; however, no cases with copy number variants (CNVs) have been sufficiently investigated. We describe a consanguineous Iraqi family harboring an 88.5 kb homozygous deletion including SLC13A5 in Chr17p13.1. The three affected male siblings exhibit neonatal-onset epilepsy with fever-sensitivity, recurrent status epilepticus, global developmental delay/intellectual disability (GDD/ID), and other variable neurological findings as shared phenotypical features of DEE25. Two of the three affected subjects exhibit hypoplastic amelogenesis imperfecta (AI), while the proband shows no evidence of dental abnormalities or AI at 2 years of age with apparently unaffected primary dentition. Characterization of the genomic architecture at this locus revealed evidence for genomic instability generated by an Alu/Alu-mediated rearrangement; confirmed by break-point junction Sanger sequencing. This multiplex family from a distinct population elucidates the phenotypic consequence of complete LoF of SLC13A5 and illustrates the importance of read-depth-based CNV detection in comprehensive exome sequencing analysis to solve cases that otherwise remain molecularly unsolved.

REST Final-Exon-Truncating Mutations Cause Hereditary Gingival Fibromatosis.

Hereditary gingival fibromatosis (HGF) is the most common genetic form of gingival fibromatosis that develops as a slowly progressive, benign, localized or generalized enlargement of keratinized gingiva. HGF is a genetically heterogeneous disorder and can be transmitted either as an autosomal-dominant or autosomal-recessive trait or appear sporadically. To date, four loci (2p22.1, 2p23.3-p22.3, 5q13-q22, and 11p15) have been mapped to autosomes and one gene (SOS1) has been associated with the HGF trait observed to segregate in a dominant inheritance pattern. Here we report 11 individuals with HGF from three unrelated families. Whole-exome sequencing (WES) revealed three different truncating mutations including two frameshifts and one nonsense variant in RE1-silencing transcription factor (REST) in the probands from all families and further genetic and genomic analyses confirmed the WES-identified findings. REST is a transcriptional repressor that is expressed throughout the body; it has different roles in different cellular contexts, such as oncogenic and tumor-suppressor functions and hematopoietic and cardiac differentiation. Here we show the consequences of germline final-exon-truncating mutations in REST for organismal development and the association with the HGF phenotype.

Identification of likely pathogenic and known variants in TSPEAR, LAMB3, BCOR, and WNT10A in four Turkish families with tooth agenesis.

Tooth agenesis (TA), the failure of development of one or more permanent teeth, is a common craniofacial abnormality observed in different world populations. The genetic etiology of TA is heterogeneous; more than a dozen genes have been associated with isolated or nonsyndromic TA, and more than 80 genes with syndromic forms. In this study, we applied whole exome sequencing (WES) to identify candidate genes contributing to TA in four Turkish families. Likely pathogenic variants with a low allele frequency in the general population were identified in four disease-associated genes, including two distinct variants in TSPEAR, associated with syndromic and isolated TA in one family each; a variant in LAMB3 associated with syndromic TA in one family; and a variant in BCOR plus a disease-associated WNT10A variant in one family with syndromic TA. With the notable exception of WNT10A (Tooth agenesis, selective, 4, MIM #150400), the genotype-phenotype relationships described in the present cohort represent an expansion of the clinical spectrum associated with these genes: TSPEAR (Deafness, autosomal recessive 98, MIM #614861), LAMB3 (Amelogenesis imperfecta, type IA, MIM #104530; Epidermolysis bullosa, junctional, MIMs #226700 and #226650), and BCOR (Microphthalmia, syndromic 2, MIM #300166). We provide evidence supporting the candidacy of these genes with TA, and propose TSPEAR as a novel nonsyndromic TA gene. Our data also suggest potential multilocus genomic variation, or mutational burden, in a single family, involving the BCOR and WNT10A loci, underscoring the complexity of the genotype-phenotype relationship in the common complex trait of TA.

Identification of a pathogenic PMP2 variant in a multi-generational family with CMT type 1: Clinical gene panels versus genome-wide approaches to molecular diagnosis.

Charcot-Marie-Tooth (CMT) disease type 1 is an inherited peripheral neuropathy characterized by demyelination and reduced nerve conduction velocities. We present a multi-generational family with peripheral neuropathy in whom clinical CMT panel testing failed to conclude a molecular diagnosis. We found a PMP2 pathogenic variant c.155T > C, p.(Ile52Thr) that segregates with disease suggesting that PMP2 variants should be considered in patients with neuropathy and that it may be prudent to include in clinical CMT gene panels.

A biallelic ANTXR1 variant expands the anthrax toxin receptor associated phenotype to tooth agenesis.

Tooth development is regulated by multiple genetic pathways, which ultimately drive the complex interactions between the oral epithelium and mesenchyme. Disruptions at any time point during this process may lead to failure of tooth development, also known as tooth agenesis (TA). TA is a common craniofacial abnormality in humans and represents the failure to develop one or more permanent teeth. Many genes and potentially subtle variants in these genes contribute to the TA phenotype. We report the clinical and genetic impact of a rare homozygous ANTXR1 variant (c.1312C>T), identified by whole exome sequencing (WES), in a consanguineous Turkish family with TA. Mutations in ANTXR1 have been associated with GAPO (growth retardation, alopecia, pseudoanodontia, and optic atrophy) syndrome and infantile hemangioma, however no clinical characteristics associated with these conditions were observed in our study family. We detected the expression of Antxr1 in oral and dental tissues of developing mouse embryos, further supporting a role for this gene in tooth development. Our findings implicate ANTXR1 as a candidate gene for isolated TA, suggest the involvement of specific hypomorphic alleles, and expand the previously known ANTXR1-associated phenotypes.

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

The role of combined SNV and CNV burden in patients with distal symmetric polyneuropathy.

PURPOSE: Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of genetic disorders of the peripheral nervous system. Copy-number variants (CNVs) contribute significantly to CMT, as duplication of PMP22 underlies the majority of CMT1 cases. We hypothesized that CNVs and/or single-nucleotide variants (SNVs) might exist in patients with CMT with an unknown molecular genetic etiology. METHODS: Two hundred patients with CMT, negative for both SNV mutations in several CMT genes and for CNVs involving PMP22, were screened for CNVs by high-resolution oligonucleotide array comparative genomic hybridization. Whole-exome sequencing was conducted on individuals with rare, potentially pathogenic CNVs. RESULTS: Putatively causative CNVs were identified in five subjects (~2.5%); four of the five map to known neuropathy genes. Breakpoint sequencing revealed Alu-Alu-mediated junctions as a predominant contributor. Exome sequencing identified MFN2 SNVs in two of the individuals. CONCLUSION: Neuropathy-associated CNV outside of the PMP22 locus is rare in CMT. Nevertheless, there is potential clinical utility in testing for CNVs and exome sequencing in CMT cases negative for the CMT1A duplication. These findings suggest that complex phenotypes including neuropathy can potentially be caused by a combination of SNVs and CNVs affecting more than one disease-associated locus and contributing to a mutational burden.Genet Med 18 5, 443-451.

Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis.

PURPOSE: We aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients. METHODS: WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. After deleterious mutations were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls. RESULTS: We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) in LRP6, respectively, in the patient with TA and OFC and in the patient with severe TA only. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo. CONCLUSION: Mutations in LRP6 cause TA in humans.Genet Med 18 11, 1158-1162.

Exome sequencing identifies a homozygous C5orf42 variant in a Turkish kindred with oral-facial-digital syndrome type VI.

Oral-facial-digital syndrome type VI (OFDVI) is a rare ciliopathy in the spectrum of Joubert syndrome (JS) and distinguished from other oral-facial-digital syndromes by metacarpal abnormalities with central polydactyly and by a molar tooth sign on cranial MRI. Additional characteristic features include short stature, micrognathia, posteriorly rotated low-set ears, hypertelorism, epicanthal folds, broad nasal tip, tongue hamartoma, upper lip notch, intraoral frenula, cleft lip/palate, and renal anomalies. Recently, novel mutations in C5orf42 were identified in 9 out of 11 OFDVI families. In a subsequent study C5orf42 was found to be mutated in only 2 out of 17 OFDVI probands while 28 patients with a pure JS phenotype also had pathogenic mutations of C5orf42. We report on two affected cousins diagnosed with OFDVI who were born from first degree cousin marriages. Whole exome sequencing (WES) identified a homozygous predicted damaging missense mutation (c.4034A > G; p.Gln1345Arg) in the C5orf42 gene. Our data contribute to the evidence that C5orf42 is one of the causative genes for OFDVI.

Whole exome sequencing identifies three novel mutations in ANTXR1 in families with GAPO syndrome.

GAPO syndrome (OMIM#230740) is the acronym for growth retardation, alopecia, pseudoanodontia, and optic atrophy. About 35 cases have been reported, making it among one of the rarest recessive conditions. Distinctive craniofacial features including alopecia, rarefaction of eyebrows and eyelashes, frontal bossing, high forehead, mid-facial hypoplasia, hypertelorism, and thickened eyelids and lips make GAPO syndrome a clinically recognizable phenotype. While this genomic study was in progress mutations in ANTXR1 were reported to cause GAPO syndrome. In our study we performed whole exome sequencing (WES) for five affected individuals from three Turkish kindreds segregating the GAPO trait. Exome sequencing analysis identified three novel homozygous mutations including; one frame-shift (c.1220_1221insT; p.Ala408Cysfs*2), one splice site (c.411A>G; p.Gln137Gln), and one non-synonymous (c.1150G>A; p.Gly384Ser) mutation in the ANTXR1 gene. Our studies expand the allelic spectrum in this rare condition and potentially provide insight into the role of ANTXR1 in the regulation of the extracellular matrix.

Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ∼ 45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy-associated genes in subjects versus controls, confirmed in a second ethnically discrete neuropathy cohort, suggesting that mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HPMVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.