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Mutations in more than 150 genes are responsible for inherited hearing loss, with thousands of different, severe causal alleles that vary among populations. The Israeli Jewish population includes communities of diverse geographic origins, revealing a wide range of deafness-associated variants and enabling clinical characterization of the associated phenotypes. Our goal was to identify the genetic causes of inherited hearing loss in this population, and to determine relationships among genotype, phenotype, and ethnicity. Genomic DNA samples from informative relatives of 88 multiplex families, all of self-identified Jewish ancestry, with either non-syndromic or syndromic hearing loss, were sequenced for known and candidate deafness genes using the HEar-Seq gene panel. The genetic causes of hearing loss were identified for 60% of the families. One gene was encountered for the first time in human hearing loss: ATOH1 (Atonal), a basic helix-loop-helix transcription factor responsible for autosomal dominant progressive hearing loss in a five-generation family. Our results demonstrate that genomic sequencing with a gene panel dedicated to hearing loss is effective for genetic diagnoses in a diverse population. Comprehensive sequencing enables well-informed genetic counseling and clinical management by medical geneticists, otolaryngologists, audiologists, and speech therapists and can be integrated into newborn screening for deafness.

The genetic characterization of a common phenotype for an entire population reveals both the causes of that phenotype for that place and the power of family-based, population-wide genomic analysis for gene and mutation discovery. We characterized the genetics of hearing loss throughout the Palestinian population, enrolling 2,198 participants from 491 families from all parts of the West Bank and Gaza. In Palestinian families with no prior history of hearing loss, we estimate that 56% of hearing loss is genetic and 44% is not genetic. For the great majority (87%) of families with inherited hearing loss, panel-based genomic DNA sequencing, followed by segregation analysis of large kindreds and transcriptional analysis of participant RNA, enabled identification of the causal genes and mutations, including at distant noncoding sites. Genetic heterogeneity of hearing loss was striking with respect to both genes and alleles: The 337 solved families harbored 143 different mutations in 48 different genes. For one in four solved families, a transcription-altering mutation was the responsible allele. Many of these mutations were cryptic, either exonic alterations of splice enhancers or silencers or deeply intronic events. Experimentally calibrated in silico analysis of transcriptional effects yielded inferences of high confidence for effects on splicing even of mutations in genes not expressed in accessible tissue. Most (58%) of all hearing loss in the population was attributable to consanguinity. Given the ongoing decline in consanguineous marriage, inherited hearing loss will likely be much rarer in the next generation.

Mutations in more than 150 genes are responsible for inherited hearing loss, with thousands of different, severe causal alleles that vary among populations. The Israeli Jewish population includes communities of diverse geographic origins, revealing a wide range of deafness-associated variants and enabling clinical characterization of the associated phenotypes. Our goal was to identify the genetic causes of inherited hearing loss in this population, and to determine relationships among genotype, phenotype, and ethnicity. Genomic DNA samples from informative relatives of 88 multiplex families, all of self-identified Jewish ancestry, with either non-syndromic or syndromic hearing loss, were sequenced for known and candidate deafness genes using the HEar-Seq gene panel. The genetic causes of hearing loss were identified for 60% of the families. One gene was encountered for the first time in human hearing loss:ATOH1(Atonal), a basic helix-loop-helix transcription factor responsible for autosomal dominant progressive hearing loss in a five-generation family. Our results demonstrate that genomic sequencing with a gene panel dedicated to hearing loss is effective for genetic diagnoses in a diverse population. Comprehensive sequencing enables well-informed genetic counseling and clinical management by medical geneticists, otolaryngologists, audiologists, and speech therapists and can be integrated into newborn screening for deafness.

Fanconi anemia is a genetically and phenotypically heterogeneous disorder characterized by congenital anomalies, bone marrow failure, cancer, and sensitivity of chromosomes to DNA cross-linking agents. One of the 22 genes responsible for Fanconi anemia is BRIP1, in which biallelic truncating mutations lead to Fanconi anemia group J and monoallelic truncating mutations predispose to certain cancers. However, of the more than 1000 reported missense mutations in BRIP1, very few have been functionally characterized. We evaluated the functional consequence of BRIP1 p.R848H (c.2543G > A), which was homozygous in two cousins with low birth weight, microcephaly, upper limb abnormalities, and imperforate anus and for whom chromosome breakage analysis of patient cells revealed increased mitomycin C sensitivity. BRIP1 p.R848H alters a highly conserved residue in the catalytic DNA helicase domain. We show that BRIP1 p.R848H leads to a defect in helicase activity. Heterozygosity at this missense has been reported in multiple cancer patients but, in the absence of functional studies, classified as of unknown significance. Our results support that this mutation is pathogenic for Fanconi anemia in homozygotes and for increased cancer susceptibility in heterozygous carriers.

Breast cancer among Palestinian women has lower incidence than in Europe or North America, yet is very frequently familial. We studied genetic causes of this familial clustering in a consecutive hospital-based series of 875 Palestinian patients with invasive breast cancer, including 453 women with diagnosis by age 40, or with breast or ovarian cancer in a mother, sister, grandmother, or aunt (“discovery series”); and 422 women diagnosed after age 40 and with negative family history (“older-onset sporadic patient series”). Genomic DNA from women in the discovery series was sequenced for all known breast cancer genes, revealing a pathogenic mutation in 13% (61/453) of patients. These mutations were screened in all patients and in 300 Palestinian female controls, revealing 1.0% (4/422) carriers among older, non-familial patients and two carriers among controls. The mutational spectrum was highly heterogeneous, including pathogenic mutations in eleven different genes: BRCA1, BRCA2, TP53, ATM, CHEK2, BARD1, BRIP1, PALB2, MRE11A, PTEN, and XRCC2. BRCA1 carriers were significantly more likely than other patients to have triple negative tumors (P = 0.03). The single most frequent mutation was TP53 p.R181C, which was significantly enriched in the discovery series compared to controls (P = 0.01) and was responsible for 15% of breast cancers among young onset or familial patients. TP53 p.R181C predisposed specifically to breast cancer with incomplete penetrance, and not to other Li-Fraumeni cancers. Palestinian women with young onset or familial breast cancer and their families would benefit from genetic analysis and counseling. This article is protected by copyright. All rights reserved.

Despite tremendous progress through next generation sequencing technologies, familial focal epilepsies are insufficiently understood. We sought to identify the genetic basis in multiplex Palestinian families with familial focal epilepsy with variable foci (FFEVF). Family I with 10 affected individuals and Family II with five affected individuals underwent detailed phenotyping over three generations. The phenotypic spectrum of the two families varied from nonlesional focal epilepsy including nocturnal frontal lobe epilepsy to severe structural epilepsy due to hemimegalencephaly. Whole-exome sequencing and single nucleotide polymorphism array analysis revealed pathogenic variants in NPRL3 in each family, a partial ~38-kb deletion encompassing eight exons (exons 8-15) and the 3'-untranslated region of the NPRL3 gene in Family I, and a de novo nonsense variant c.1063C>T, p.Gln355* in Family II. Furthermore, we identified a truncating variant in the PDCD10 gene in addition to the NPRL3 variant in a patient with focal epilepsy from Family I. The individual also had developmental delay and multiple cerebral cavernomas, possibly demonstrating a digenic contribution to the individual's phenotype. Our results implicate the association of NPRL3 with hemimegalencephaly, expanding the phenotypic spectrum of NPRL3 in FFEVF and underlining that partial deletions are part of the genotypic spectrum of NPRL3 variants.

Tooth development is controlled by the same processes that regulate formation of other ectodermal structures. Mutations in the genes underlying these processes may cause ectodermal dysplasia, including severe absence of primary or permanent teeth. Four consanguineous Palestinian families presented with oligodontia and hair and skin features of ectodermal dysplasia. Appearance of ectodermal dysplasia was consistent with autosomal recessive inheritance. Exome sequencing followed by genotyping of 56 informative relatives in the 4 families suggests that the phenotype is due to homozygosity for KREMEN1 p.F209S (c.626 T>C) on chromosome 22 at g.29,521,399 (hg19). The variant occurs in the highly conserved extracellular WSC domain of KREMEN1, which is known to be a high affinity receptor of Dickkopf-1, a component of the Dickkopf–Kremen–LRP6 complex, and a potent regulator of Wnt signaling. The Wnt signaling pathway is critical to development of ectodermal structures. Mutations in WNT10A, LRP6, EDA, and other genes in this pathway lead to tooth agenesis with or without other ectodermal anomalies. Our results implicate KREMEN1 for the first time in a human disorder and provide additional details on the role of the Wnt signaling in ectodermal and dental development.

Background Primary gonadal failure is characterised by primary amenorrhoea or early menopause in females, and oligospermia or azoospermia in males. Variants of the minichromosome maintenance complex component 8 gene (MCM8) have recently been shown to be significantly associated with women’s menopausal age in genome-wide association studies. Furthermore, MCM8-knockout mice are sterile. The objective of this study was to elucidate the genetic aetiology of gonadal failure in two consanguineous families presenting as primary amenorrhoea in the females and as small testes and azoospermia in a male. Methods and results Using whole exome sequencing, we identified two novel homozygous mutations in the MCM8 gene: a splice (c.1954-1G>A) and a frameshift (c.1469-1470insTA). In each consanguineous family the mutation segregated with the disease and both mutations were absent in 100 ethnically matched controls. The splice mutation led to lack of the wild-type transcript and three different aberrant transcripts predicted to result in either truncated or significantly shorter proteins. Quantitative analysis of the aberrantly spliced transcripts showed a significant decrease in total MCM8 message in affected homozygotes for the mutation, and an intermediate decrease in heterozygous family members. Chromosomal breakage following exposure to mitomcyin C was significantly increased in cells from homozygous individuals for c.1954-1G>A, as well as c.1469-1470insTA. Conclusions MCM8, a component of the pre-replication complex, is crucial for gonadal development and maintenance in humans—both males and females. These findings provide new insights into the genetic disorders of infertility and premature menopause in women.

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn (ΔC) mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn (ΔC) allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.

Ovarian development and maintenance are poorly understood; however, diseases that affect these processes can offer insights into the underlying mechanisms. XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder that is characterized by underdeveloped, dysfunctional ovaries, with subsequent lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism. Here, we report an extended consanguineous family of Palestinian origin, in which 4 females exhibited XX-GD. Using homozygosity mapping and whole-exome sequencing, we identified a recessive missense mutation in nucleoporin-107 (NUP107, c.1339G>A, p.D447N). This mutation segregated with the XX-GD phenotype and was not present in available databases or in 150 healthy ethnically matched controls. NUP107 is a component of the nuclear pore complex, and the NUP107-associated protein SEH1 is required for oogenesis in Drosophila. In Drosophila, Nup107 knockdown in somatic gonadal cells resulted in female sterility, whereas males were fully fertile. Transgenic rescue of Drosophila females bearing the Nup107D364N mutation, which corresponds to the human NUP107 (p.D447N), resulted in almost complete sterility, with a marked reduction in progeny, morphologically aberrant eggshells, and disintegrating egg chambers, indicating defective oogenesis. These results indicate a pivotal role for NUP107 in ovarian development and suggest that nucleoporin defects may play a role in milder and more common conditions such as premature ovarian failure.

To determine antimicrobial drug resistance of Streptococcus pneumoniae serotypes, we analyzed isolates from blood cultures of sick children residing in the West Bank before initiation of pneumococcal vaccination. Of 120 serotypes isolated, 50.8%, 73.3%, and 80.8% of the bacteremia cases could have been prevented by pneumococcal conjugate vaccines. Serotype 14 was the most drug-resistant serotype isolated.

Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss.

In communities with high rates of consanguinity and consequently high prevalence of recessive phenotypes, homozygosity mapping with SNP arrays is an effective approach for gene discovery. In 20 Palestinian kindreds with prelingual nonsyndromic hearing loss, we generated homozygosity profiles reflecting linkage to the phenotype. Family sizes ranged from small nuclear families with two affected children, one unaffected sibling, and parents to multigenerational kindreds with 12 affected relatives. By including unaffected parents and siblings and screening 250 K SNP arrays, even small nuclear families yielded informative profiles. In 14 families, we identified the allele responsible for hearing loss by screening a single candidate gene in the longest homozygous region. Novel alleles included missense, nonsense, and splice site mutations of CDH23, MYO7A, MYO15A, OTOF, PJVK, Pendrin/SLC26A4, TECTA, TMHS, and TMPRSS3, and a large genomic deletion of Otoancorin (OTOA). All point mutations were rare in the Palestinian population (zero carriers in 288 unrelated controls); the carrier frequency of the OTOA genomic deletion was 1%. In six families, we identified five genomic regions likely to harbor novel genes for human hearing loss on chromosomes 1p13.3 (DFNB82), 9p23–p21.2/p13.3–q21.13 (DFNB83), 12q14.3–q21.2 (DFNB84; two families), 14q23.1–q31.1, and 17p12–q11.2 (DFNB85).

Massively parallel sequencing of targeted regions, exomes, and complete genomes has begun to dramatically increase the pace of discovery of genes responsible for human disorders. Here we describe how exome sequencing in conjunction with homozygosity mapping led to rapid identification of the causative allele for nonsyndromic hearing loss DFNB82 in a consanguineous Palestinian family. After filtering out worldwide and population-specific polymorphisms from the whole exome sequence, only a single deleterious mutation remained in the homozygous region linked to DFNB82. The nonsense mutation leads to an early truncation of the G protein signaling modulator GPSM2, a protein that is essential for maintenance of cell polarity and spindle orientation. In the mouse inner ear, GPSM2 is localized to apical surfaces of hair cells and supporting cells and is most highly expressed during embryonic development. Identification of GPSM2 as essential to the development of normal hearing suggests dysregulation of cell polarity as a mechanism underlying hearing loss.

Background: Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity. Results: A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands cosegregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p. S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population. Conclusions: Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.