Using multi-scale genomics to associate poorly annotated genes with rare diseases.

BACKGROUND: Next-generation sequencing (NGS) has significantly transformed the landscape of identifying disease-causing genes associated with genetic disorders. However, a substantial portion of sequenced patients remains undiagnosed. This may be attributed not only to the challenges posed by harder-to-detect variants, such as non-coding and structural variations but also to the existence of variants in genes not previously associated with the patient's clinical phenotype. This study introduces EvORanker, an algorithm that integrates unbiased data from 1,028 eukaryotic genomes to link mutated genes to clinical phenotypes. METHODS: EvORanker utilizes clinical data, multi-scale phylogenetic profiling, and other omics data to prioritize disease-associated genes. It was evaluated on solved exomes and simulated genomes, compared with existing methods, and applied to 6260 knockout genes with mouse phenotypes lacking human associations. Additionally, EvORanker was made accessible as a user-friendly web tool. RESULTS: In the analyzed exomic cohort, EvORanker accurately identified the "true" disease gene as the top candidate in 69% of cases and within the top 5 candidates in 95% of cases, consistent with results from the simulated dataset. Notably, EvORanker outperformed existing methods, particularly for poorly annotated genes. In the case of the 6260 knockout genes with mouse phenotypes, EvORanker linked 41% of these genes to observed human disease phenotypes. Furthermore, in two unsolved cases, EvORanker successfully identified DLGAP2 and LPCAT3 as disease candidates for previously uncharacterized genetic syndromes. CONCLUSIONS: We highlight clade-based phylogenetic profiling as a powerful systematic approach for prioritizing potential disease genes. Our study showcases the efficacy of EvORanker in associating poorly annotated genes to disease phenotypes observed in patients. The EvORanker server is freely available at https://ccanavati.shinyapps.io/EvORanker/ .

Autosomal dominant non-syndromic hearing loss maps to DFNA33 (13q34) and co-segregates with splice and frameshift variants in ATP11A, a phospholipid flippase gene.

Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3' UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.

The noncoding genome and hearing loss.

The age of sequencing has provided unprecedented insights into the human genome. The coding region of the genome comprises nearly 20,000 genes, of which approximately 4000 are associated with human disease. Beyond the protein-coding genome, which accounts for only 3% of the genome, lies a vast pool of regulatory elements in the form of promoters, enhancers, RNA species, and other intricate elements. These features undoubtably influence human health and disease, and as a result, a great deal of effort is currently being invested in deciphering their identity and mechanism. While a paucity of material has caused a lag in identifying these elements in the inner ear, the emergence of technologies for dealing with a minimal number of cells now has the field working overtime to catch up. Studies on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), methylation, histone modifications, and more are ongoing. A number of microRNAs and other noncoding elements are known to be associated with hearing impairment and there is promise that regulatory elements will serve as future tools and targets of therapeutics and diagnostics. This review covers the current state of the field and considers future directions for the noncoding genome and implications for hearing loss.

Helicase-inactivating BRIP1 mutation yields Fanconi anemia with microcephaly and other congenital abnormalities.

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.

Spectrum of genes for inherited hearing loss in the Israeli Jewish population, including the novel human deafness gene ATOH1.

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 show 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.

Genomic analysis of inherited hearing loss in the Palestinian population.

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.

Inclusion of hemimegalencephaly into the phenotypic spectrum of NPRL3 pathogenic variants in familial focal epilepsy with variable foci.

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.

The combination of whole-exome sequencing and clinical analysis allows better diagnosis of rare syndromic retinal dystrophies.

PURPOSE: To identify the accurate clinical diagnosis of rare syndromic inherited retinal diseases (IRDs) based on the combination of clinical and genetic analyses. METHODS: Four unrelated families with various autosomal recessive syndromic inherited retinal diseases were genetically investigated using whole-exome sequencing (WES). RESULTS: Two affected subjects in family MOL0760 presented with a distinctive combination of short stature, developmental delay, congenital mental retardation, microcephaly, facial dysmorphism and retinitis pigmentosa (RP). Subjects were clinically diagnosed with suspected Kabuki syndrome. WES revealed a homozygous nonsense mutation (c.5492dup, p.Asn1831Lysfs*8) in VPS13B that is known to cause Cohen syndrome. The index case of family MOL1514 presented with both RP and liver dysfunction, suspected initially to be related. WES identified a homozygous frameshift mutation (c.1787_1788del, p.His596Argfs*47) in AGBL5, associated with nonsyndromic RP. The MOL1592 family included three affected subjects with crystalline retinopathy, skin ichthyosis, short stature and congenital adrenal hypoplasia, and were found to harbour a homozygous nonsense mutation (c.682C>T, p.Arg228Cys) in ALDH3A2, reported to cause Sjögren-Larsson syndrome (SLS). In the fourth family, SJ002, two siblings presented with hypotony, psychomotor delay, dysmorphic facial features, pathologic myopia, progressive external ophthalmoplegia and diffuse retinal atrophy. Probands were suspected to have atypical Kearns-Sayre syndrome, but were diagnosed with combined oxidative phosphorylation deficiency-20 due to a novel suspected missense variant (c.1691C>T, p.Ala564Val) in VARS2. CONCLUSION: Our findings emphasize the important complement of WES and thorough clinical investigation in establishing precise clinical diagnosis. This approach constitutes the basis for personalized medicine in rare IRDs.

Genomic analysis of inherited breast cancer among Palestinian women: Genetic heterogeneity and a founder mutation in TP53.

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, nonfamilial patients and two carriers among controls. The mutational spectrum was highly heterogeneous, including pathogenic mutations in 11 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.

Loss of function of PCDH12 underlies recessive microcephaly mimicking intrauterine infection.

OBJECTIVE: To identify the genetic basis of a recessive syndrome characterized by prenatal hyperechogenic brain foci, congenital microcephaly, hypothalamic midbrain dysplasia, epilepsy, and profound global developmental disability. METHODS: Identification of the responsible gene by whole exome sequencing and homozygosity mapping. RESULTS: Ten patients from 4 consanguineous Palestinian families manifested in utero with hyperechogenic brain foci, microcephaly, and intrauterine growth retardation. Postnatally, patients had progressive severe microcephaly, neonatal seizures, and virtually no developmental milestones. Brain imaging revealed dysplastic elongated masses in the midbrain-hypothalamus-optic tract area. Whole exome sequencing of one affected child revealed only PCDH12 c.2515C>T, p.R839X, to be homozygous in the proband and to cosegregate with the condition in her family. The allele frequency of PCDH12 p.R839X is <0.00001 worldwide. Genotyping PCDH12 p.R839X in 3 other families with affected children yielded perfect cosegregation with the phenotype (probability by chance is 2.0 × 10(-12)). Homozygosity mapping revealed that PCDH12 p.R839X lies in the largest homozygous region (11.7 MB) shared by all affected patients. The mutation reduces transcript expression by 84% (p < 2.4 × 10(-13)). PCDH12 is a vascular endothelial protocadherin that promotes cellular adhesion. Endothelial adhesion disruptions due to mutations in OCLN or JAM3 also cause congenital microcephaly, intracranial calcifications, and profound psychomotor disability. CONCLUSIONS: Loss of function of PCDH12 leads to recessive congenital microcephaly with profound developmental disability. The phenotype resembles Aicardi-Goutières syndrome and in utero infections. In cases with similar manifestations but no evidence of infection, our results suggest consideration of an additional, albeit rare, cause of congenital microcephaly.

Mutation of KREMEN1, a modulator of Wnt signaling, is responsible for ectodermal dysplasia including oligodontia in Palestinian families.

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.

Combined mineralocorticoid and glucocorticoid deficiency is caused by a novel founder nicotinamide nucleotide transhydrogenase mutation that alters mitochondrial morphology and increases oxidative stress.

BACKGROUND: Familial glucocorticoid deficiency (FGD) reflects specific failure of adrenocortical glucocorticoid production in response to adrenocorticotropic hormone (ACTH). Most cases are caused by mutations encoding ACTH-receptor components (MC2R, MRAP) or the general steroidogenesis protein (StAR). Recently, nicotinamide nucleotide transhydrogenase (NNT) mutations were found to cause FGD through a postulated mechanism resulting from decreased detoxification of reactive oxygen species (ROS) in adrenocortical cells. METHODS AND RESULTS: In a consanguineous Palestinian family with combined mineralocorticoid and glucocorticoid deficiency, whole-exome sequencing revealed a novel homozygous NNT_c.598 G>A, p.G200S, mutation. Another affected, unrelated Palestinian child was also homozygous for NNT_p.G200S. Haplotype analysis showed this mutation is ancestral; carrier frequency in ethnically matched controls is 1/200. Assessment of patient fibroblasts for ROS production, ATP content and mitochondrial morphology showed that biallelic NNT mutations result in increased levels of ROS, lower ATP content and morphological mitochondrial defects. CONCLUSIONS: This report of a novel NNT mutation, p.G200S, expands the phenotype of NNT mutations to include mineralocorticoid deficiency. We provide the first patient-based evidence that NNT mutations can cause oxidative stress and both phenotypic and functional mitochondrial defects. These results directly demonstrate the importance of NNT to mitochondrial function in the setting of adrenocortical insufficiency.