Rare variants in the notch signaling pathway describe a novel type of autosomal recessive Klippel-Feil syndrome.

Klippel-Feil syndrome is a rare disorder represented by a subgroup of segmentation defects of the vertebrae and characterized by fusion of the cervical vertebrae, low posterior hairline, and short neck with limited motion. Both autosomal dominant and recessive inheritance patterns were reported in families with Klippel-Feil. Mutated genes for both dominant (GDF6 and GDF3) and recessive (MEOX1) forms of Klippel-Feil syndrome have been shown to be involved in somite development via transcription regulation and signaling pathways. Heterotaxy arises from defects in proteins that function in the development of left-right asymmetry of the developing embryo. We describe a consanguineous family with a male proband who presents with classical Klippel-Feil syndrome together with heterotaxy (situs inversus totalis). The present patient also had Sprengel's deformity, deformity of the sternum, and a solitary kidney. Using exome sequencing, we identified a homozygous frameshift mutation (c.299delT; p.L100fs) in RIPPLY2, a gene shown to play a crucial role in somitogenesis and participate in the Notch signaling pathway via negatively regulating Tbx6. Our data confirm RIPPLY2 as a novel gene for autosomal recessive Klippel-Feil syndrome, and in addition-from a mechanistic standpoint-suggest the possibility that mutations in RIPPLY2 could also lead to heterotaxy. © 2015 Wiley Periodicals, Inc.

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.

Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D.

PURPOSE: Copy-number variations as a mutational mechanism contribute significantly to human disease. Approximately one-half of the patients with Charcot-Marie-Tooth (CMT) disease have a 1.4 Mb duplication copy-number variation as the cause of their neuropathy. However, non-CMT1A neuropathy patients rarely have causative copy-number variations, and to date, autosomal-recessive disease has not been associated with copy-number variation as a mutational mechanism. METHODS: We performed Agilent 8 × 60 K array comparative genomic hybridization on DNA from 12 recessive Turkish families with CMT disease. Additional molecular studies were conducted to detect breakpoint junctions and to evaluate gene expression levels in a family in which we detected an intragenic duplication copy-number variation. RESULTS: We detected an ~6.25 kb homozygous intragenic duplication in NDRG1, a gene known to be causative for recessive HMSNL/CMT4D, in three individuals from a Turkish family with CMT neuropathy. Further studies showed that this intragenic copy-number variation resulted in a homozygous duplication of exons 6-8 that caused decreased mRNA expression of NDRG1. CONCLUSION: Exon-focused high-resolution array comparative genomic hybridization enables the detection of copy-number variation carrier states in recessive genes, particularly small copy-number variations encompassing or disrupting single genes. In families for whom a molecular diagnosis has not been elucidated by conventional clinical assays, an assessment for copy-number variations in known CMT genes might be considered.

Exome sequencing in mostly consanguineous Arab families with neurologic disease provides a high potential molecular diagnosis rate.

BACKGROUND: Neurodevelopment is orchestrated by a wide range of genes, and the genetic causes of neurodevelopmental disorders are thus heterogeneous. We applied whole exome sequencing (WES) for molecular diagnosis and in silico analysis to identify novel disease gene candidates in a cohort from Saudi Arabia with primarily Mendelian neurologic diseases. METHODS: We performed WES in 31 mostly consanguineous Arab families and analyzed both single nucleotide and copy number variants (CNVs) from WES data. Interaction/expression network and pathway analyses, as well as paralog studies were utilized to investigate potential pathogenicity and disease association of novel candidate genes. Additional cases for candidate genes were identified through the clinical WES database at Baylor Miraca Genetics Laboratories and GeneMatcher. RESULTS: We found known pathogenic or novel variants in known disease genes with phenotypic expansion in 6 families, disease-associated CNVs in 2 families, and 12 novel disease gene candidates in 11 families, including KIF5B, GRM7, FOXP4, MLLT1, and KDM2B. Overall, a potential molecular diagnosis was provided by variants in known disease genes in 17 families (54.8 %) and by novel candidate disease genes in an additional 11 families, making the potential molecular diagnostic rate ~90 %. CONCLUSIONS: Molecular diagnostic rate from WES is improved by exome-predicted CNVs. Novel candidate disease gene discovery is facilitated by paralog studies and through the use of informatics tools and available databases to identify additional evidence for pathogenicity. TRIAL REGISTRATION: Not applicable.

Molecular etiology of arthrogryposis in multiple families of mostly Turkish origin.

BACKGROUND: Arthrogryposis, defined as congenital joint contractures in 2 or more body areas, is a clinical sign rather than a specific disease diagnosis. To date, more than 400 different disorders have been described that present with arthrogryposis, and variants of more than 220 genes have been associated with these disorders; however, the underlying molecular etiology remains unknown in the considerable majority of these cases. METHODS: We performed whole exome sequencing (WES) of 52 patients with clinical presentation of arthrogryposis from 48 different families. RESULTS: Affected individuals from 17 families (35.4%) had variants in known arthrogryposis-associated genes, including homozygous variants of cholinergic γ nicotinic receptor (CHRNG, 6 subjects) and endothelin converting enzyme-like 1 (ECEL1, 4 subjects). Deleterious variants in candidate arthrogryposis-causing genes (fibrillin 3 [FBN3], myosin IXA [MYO9A], and pleckstrin and Sec7 domain containing 3 [PSD3]) were identified in 3 families (6.2%). Moreover, in 8 families with a homozygous mutation in an arthrogryposis-associated gene, we identified a second locus with either a homozygous or compound heterozygous variant in a candidate gene (myosin binding protein C, fast type [MYBPC2] and vacuolar protein sorting 8 [VPS8], 2 families, 4.2%) or in another disease-associated genes (6 families, 12.5%), indicating a potential mutational burden contributing to disease expression. CONCLUSION: In 58.3% of families, the arthrogryposis manifestation could be explained by a molecular diagnosis; however, the molecular etiology in subjects from 20 families remained unsolved by WES. Only 5 of these 20 unrelated subjects had a clinical presentation consistent with amyoplasia; a phenotype not thought to be of genetic origin. Our results indicate that increased use of genome-wide technologies will provide opportunities to better understand genetic models for diseases and molecular mechanisms of genetically heterogeneous disorders, such as arthrogryposis. FUNDING: This work was supported in part by US National Human Genome Research Institute (NHGRI)/National Heart, Lung, and Blood Institute (NHLBI) grant U54HG006542 to the Baylor-Hopkins Center for Mendelian Genomics, and US National Institute of Neurological Disorders and Stroke (NINDS) grant R01NS058529 to J.R. Lupski.

Whole-exome sequencing identifies homozygous GPR161 mutation in a family with pituitary stalk interruption syndrome.

CONTEXT: Pituitary stalk interruption syndrome (PSIS) is a rare, congenital anomaly of the pituitary gland characterized by pituitary gland insufficiency, thin or discontinuous pituitary stalk, anterior pituitary hypoplasia, and ectopic positioning of the posterior pituitary gland (neurohypophysis). The clinical presentation of patients with PSIS varies from isolated growth hormone (GH) deficiency to combined pituitary insufficiency and accompanying extrapituitary findings. Mutations in HESX1, LHX4, OTX2, SOX3, and PROKR2 have been associated with PSIS in less than 5% of cases; thus, the underlying genetic etiology for the vast majority of cases remains to be determined. OBJECTIVE: We applied whole-exome sequencing (WES) to a consanguineous family with two affected siblings who have pituitary gland insufficiency and radiographic findings of hypoplastic (thin) pituitary gland, empty sella, ectopic neurohypophysis, and interrupted pitiutary stalk-characteristic clinical diagnostic findings of PSIS. DESIGN AND PARTICIPANTS: WES was applied to two affected and one unaffected siblings. RESULTS: WES of two affected and one unaffected sibling revealed a unique homozygous missense mutation in GPR161, which encodes the orphan G protein-coupled receptor 161, a protein responsible for transducing extracellular signals across the plasma membrane into the cell. CONCLUSION: Mutations of GPR161 may be implicated as a potential novel cause of PSIS.

Homozygous loss-of-function mutations in SOHLH1 in patients with nonsyndromic hypergonadotropic hypogonadism.

CONTEXT: Hypergonadotropic hypogonadism presents in females with delayed or arrested puberty, primary or secondary amenorrhea due to gonadal dysfunction, and is further characterized by elevated gonadotropins and low sex steroids. Chromosomal aberrations and various specific gene defects can lead to hypergonadotropic hypogonadism. Responsible genes include those with roles in gonadal development or maintenance, sex steroid synthesis, or end-organ resistance to gonadotropins. Identification of novel causative genes in this disorder will contribute to our understanding of the regulation of human reproductive function. OBJECTIVES: The aim of this study was to identify and report the gene responsible for autosomal-recessive hypergonadotropic hypogonadism in two unrelated families. DESIGN AND PARTICIPANTS: Clinical evaluation and whole-exome sequencing were performed in two pairs of sisters with nonsyndromic hypergonadotropic hypogonadism from two unrelated families. RESULTS: Exome sequencing analysis revealed two different truncating mutations in the same gene: SOHLH1 c.705delT (p.Pro235fs*4) and SOHLH1 c.27C>G (p.Tyr9stop). Both mutations were unique to the families and segregation was consistent with Mendelian expectations for an autosomal-recessive mode of inheritance. CONCLUSIONS: Sohlh1 was known from previous mouse studies to be a transcriptional regulator that functions in the maintenance and survival of primordial ovarian follicles, but loss-of-function mutations in human females have not been reported. Our results provide evidence that homozygous-truncating mutations in SOHLH1 cause female nonsyndromic hypergonadotropic hypogonadism.

Global transcriptional disturbances underlie Cornelia de Lange syndrome and related phenotypes.

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder that presents with extensive phenotypic variability, including facial dysmorphism, developmental delay/intellectual disability (DD/ID), abnormal extremities, and hirsutism. About 65% of patients harbor mutations in genes that encode subunits or regulators of the cohesin complex, including NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Wiedemann-Steiner syndrome (WDSTS), which shares CdLS phenotypic features, is caused by mutations in lysine-specific methyltransferase 2A (KMT2A). Here, we performed whole-exome sequencing (WES) of 2 male siblings clinically diagnosed with WDSTS; this revealed a hemizygous, missense mutation in SMC1A that was predicted to be deleterious. Extensive clinical evaluation and WES of 32 Turkish patients clinically diagnosed with CdLS revealed the presence of a de novo heterozygous nonsense KMT2A mutation in 1 patient without characteristic WDSTS features. We also identified de novo heterozygous mutations in SMC3 or SMC1A that affected RNA splicing in 2 independent patients with combined CdLS and WDSTS features. Furthermore, in families from 2 separate world populations segregating an autosomal-recessive disorder with CdLS-like features, we identified homozygous mutations in TAF6, which encodes a core transcriptional regulatory pathway component. Together, our data, along with recent transcriptome studies, suggest that CdLS and related phenotypes may be "transcriptomopathies" rather than cohesinopathies.

Genes that Affect Brain Structure and Function Identified by Rare Variant Analyses of Mendelian Neurologic Disease.

Development of the human nervous system involves complex interactions among fundamental cellular processes and requires a multitude of genes, many of which remain to be associated with human disease. We applied whole exome sequencing to 128 mostly consanguineous families with neurogenetic disorders that often included brain malformations. Rare variant analyses for both single nucleotide variant (SNV) and copy number variant (CNV) alleles allowed for identification of 45 novel variants in 43 known disease genes, 41 candidate genes, and CNVs in 10 families, with an overall potential molecular cause identified in >85% of families studied. Among the candidate genes identified, we found PRUNE, VARS, and DHX37 in multiple families and homozygous loss-of-function variants in AGBL2, SLC18A2, SMARCA1, UBQLN1, and CPLX1. Neuroimaging and in silico analysis of functional and expression proximity between candidate and known disease genes allowed for further understanding of genetic networks underlying specific types of brain malformations.