Evaluation of an automated genome interpretation model for rare disease routinely used in a clinical genetic laboratory.

PURPOSE: The analysis of exome and genome sequencing data for the diagnosis of rare diseases is challenging and time-consuming. In this study, we evaluated an artificial intelligence model, based on machine learning for automating variant prioritization for diagnosing rare genetic diseases in the Baylor Genetics clinical laboratory. METHODS: The automated analysis model was developed using a supervised learning approach based on thousands of manually curated variants. The model was evaluated on 2 cohorts. The model accuracy was determined using a retrospective cohort comprising 180 randomly selected exome cases (57 singletons, 123 trios); all of which were previously diagnosed and solved through manual interpretation. Diagnostic yield with the modified workflow was estimated using a prospective "production" cohort of 334 consecutive clinical cases. RESULTS: The model accurately pinpointed all manually reported variants as candidates. The reported variants were ranked in top 10 candidate variants in 98.4% (121/123) of trio cases, in 93.0% (53/57) of single proband cases, and 96.7% (174/180) of all cases. The accuracy of the model was reduced in some cases because of incomplete variant calling (eg, copy number variants) or incomplete phenotypic description. CONCLUSION: The automated model for case analysis assists clinical genetic laboratories in prioritizing candidate variants effectively. The use of such technology may facilitate the interpretation of genomic data for a large number of patients in the era of precision medicine.

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.

Mutations in HOGA1 do Not Confer a Dominant Phenotype Manifesting as Kidney Stone Disease.

PURPOSE: The etiology of calcium-oxalate kidney stone formation remains elusive. Biallelic mutations in HOGA1 are responsible for primary hyperoxaluria type 3 and result in oxalate overproduction and kidney stone disease. Our previous study showed that carriers of HOGA1 mutations have elevated urinary levels of oxalate precursors. In this study we explored the possibility that mutations in HOGA1 confer a dominant phenotype in the form of kidney stone disease or hyperoxaluria. MATERIALS AND METHODS: An observational analytic case control study was designed to determine the prevalence of pathogenic HOGA1 mutations among adults with calcium-oxalate kidney stone disease. Given the high prevalence of HOGA1 mutations among Ashkenazi Jews, this group was evaluated separately. Carrier frequency of any of the 52 reported pathogenic mutations was compared to data derived from gnomAD for the corresponding ethnic group. Sanger sequencing of HOGA1 gene was performed on DNA samples from the following groups: 60 Ashkenazi Jews and 86 nonAshkenazi calcium-oxalate stone formers, 150 subjects with low and 150 with high urinary oxalate levels. RESULTS: The carrier prevalence of pathogenic mutations among the Ashkenazi Jews was 1.7% compared to 2.8% in the corresponding control group (p=0.9 OR=0.6 95% CI 0.01-3.51). We did not detect any mutation among the nonAshkenazi study group. No correlation was detected between hyperoxaluria and HOGA1 variants. CONCLUSIONS: This study shows that mutations in HOGA1 do not confer a dominant phenotype in the form of calcium-oxalate kidney stone disease or hyperoxaluria.

The Genomic History of the Bronze Age Southern Levant.

We report genome-wide DNA data for 73 individuals from five archaeological sites across the Bronze and Iron Ages Southern Levant. These individuals, who share the "Canaanite" material culture, can be modeled as descending from two sources: (1) earlier local Neolithic populations and (2) populations related to the Chalcolithic Zagros or the Bronze Age Caucasus. The non-local contribution increased over time, as evinced by three outliers who can be modeled as descendants of recent migrants. We show evidence that different "Canaanite" groups genetically resemble each other more than other populations. We find that Levant-related modern populations typically have substantial ancestry coming from populations related to the Chalcolithic Zagros and the Bronze Age Southern Levant. These groups also harbor ancestry from sources we cannot fully model with the available data, highlighting the critical role of post-Bronze-Age migrations into the region over the past 3,000 years.

Germline variant in REXO2 is a novel candidate gene in familial pheochromocytoma.

Pheochromocytoma (PCC) is a rare, mostly benign tumour of the adrenal medulla. Hereditary PCC accounts for ~35% of cases and has been associated with germline mutations in several cancer susceptibility genes (e.g., KIF1B, SDHB, VHL, SDHD, RET). We performed whole-exome sequencing in a family with four PCC-affected patients in two consecutive generations and identified a potential novel candidate pathogenic variant in the REXO2 gene that affects splicing (c.531-1G>T (NM 015523.3)), which co-segregated with the phenotype in the family. REXO2 encodes for RNA exonuclease 2 protein and localizes to 11q23, a chromosomal region displaying allelic imbalance in PCC. REXO2 protein has been associated with DNA repair, replication and recombination processes and thus its inactivation may contribute to tumorigenesis. While the study suggests that this novel REXO2 gene variant underlies PCC in this family, additional functional studies are required in order to establish the putative role of the REXO2 gene in PCC predisposition.

Lysine acetyltransferase 8 is involved in cerebral development and syndromic intellectual disability.

Epigenetic integrity is critical for many eukaryotic cellular processes. An important question is how different epigenetic regulators control development and influence disease. Lysine acetyltransferase 8 (KAT8) is critical for acetylation of histone H4 at lysine 16 (H4K16), an evolutionarily conserved epigenetic mark. It is unclear what roles KAT8 plays in cerebral development and human disease. Here, we report that cerebrum-specific knockout mice displayed cerebral hypoplasia in the neocortex and hippocampus, along with improper neural stem and progenitor cell (NSPC) development. Mutant cerebrocortical neuroepithelia exhibited faulty proliferation, aberrant neurogenesis, massive apoptosis, and scant H4K16 propionylation. Mutant NSPCs formed poor neurospheres, and pharmacological KAT8 inhibition abolished neurosphere formation. Moreover, we describe KAT8 variants in 9 patients with intellectual disability, seizures, autism, dysmorphisms, and other anomalies. The variants altered chromobarrel and catalytic domains of KAT8, thereby impairing nucleosomal H4K16 acetylation. Valproate was effective for treating epilepsy in at least 2 of the individuals. This study uncovers a critical role of KAT8 in cerebral and NSPC development, identifies 9 individuals with KAT8 variants, and links deficient H4K16 acylation directly to intellectual disability, epilepsy, and other developmental anomalies.

Primary Coenzyme Q deficiency Due to Novel ADCK3 Variants, Studies in Fibroblasts and Review of Literature.

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II + III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient's cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.

Gain-of-function mutation of microRNA-140 in human skeletal dysplasia.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1-5, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA-target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs7,8. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs.

Biallelic mutations in EXOC3L2 cause a novel syndrome that affects the brain, kidney and blood.

BACKGROUND: Dandy-Walker malformation features agenesis/hypoplasia of the cerebellar vermis, cystic dilatation of the fourth ventricle and enlargement of posterior fossa. Although Dandy-Walker malformation is relatively common and several genes were linked to the syndrome, the genetic cause in the majority of cases is unknown. OBJECTIVE: To identify the mutated gene responsible for Dandy-Walker malformation, kidney disease and bone marrow failure in four patients from two unrelated families. METHODS: Medical assessment, sonographic, MRI and pathological studies were used to define phenotype. Chromosomal microarray analysis and whole-exome sequence were performed to unravel the genotype. RESULTS: We report four subjects from two unrelated families with homozygous mutations in the Exocyst Complex Component 3-Like-2 gene (EXOC3L2).EXOC3L2 functions in trafficking of post-Golgi vesicles to the plasma membrane. In the first family a missense mutation in a highly conserved amino acid, p.Leu41Gln, was found in three fetuses; all had severe forms of Dandy-Walker malformation that was detectable by prenatal ultrasonography and confirmed by autopsy. In the second family, the affected child carried a nonsense mutation, p.Arg72*, and no detected protein. He had peritrigonal and cerebellar white matter abnormalities with enlargement of the ventricular trigones, developmental delay, pituitary hypoplasia, severe renal dysplasia and bone marrow failure. CONCLUSION: We propose that biallelic EXOC3L2 mutations lead to a novel syndrome that affects hindbrain development, kidney and possibly the bone marrow.

Variant in SCYL1 gene causes aberrant splicing in a family with cerebellar ataxia, recurrent episodes of liver failure, and growth retardation.

Herein, we describe two members of one family who presented with recurrent episodes of hepatic failure, cerebellar ataxia, peripheral neuropathy, and short stature. Liver transplantation was considered. Whole-exome sequencing (Trio) revealed a synonymous variant in exon 4 of SCYL1:c.459C>T p. (Gly153Gly), which did not appear to affect the protein sequence. Computational prediction analysis suggested that this modification could alter the SCYL1 mRNA splicing processing to create a premature termination codon. The SCYL1 mRNAs in our patient's lymphocytes were analyzed and aberrant splicing was found. Molecular analysis of family members identified the parents as heterozygous recessive carriers and the proband as well as an affected aunt as homozygous. Evidently, harmless synonymous variants in the SCYL1 gene can damage gene splicing and hence the expression. We confirmed that the pathogenicity of this variant in the SCYL1 gene was associated with spinocerebellar ataxia, autosomal recessive 21 (SCAR21). Other reported cases (accept one) of liver failure found in the SCYL1 variants resolved during childhood, therefore orthotropic liver transplantation was no longer appropriate.

Big data analysis of human mitochondrial DNA substitution models: a regression approach.

BACKGROUND: We study Phylotree, a comprehensive representation of the phylogeny of global human mitochondrial DNA (mtDNA) variations, to better understand the mtDNA substitution mechanism and its most influential factors. We consider a substitution model, where a set of genetic features may predict the rate at which mtDNA substitutions occur. To find an appropriate model, an exhaustive analysis on the effect of multiple factors on the substitution rate is performed through Negative Binomial and Poisson regressions. We examine three different inclusion options for each categorical factor: omission, inclusion as an explanatory variable, and by-value partitioning. The examined factors include genes, codon position, a CpG indicator, directionality, nucleotide, amino acid, codon, and context (neighboring nucleotides), in addition to other site based factors. Partitioning a model by a factor's value results in several sub-models (one for each value), where the likelihoods of the sub-models can be combined to form a score for the entire model. Eventually, the leading models are considered as viable candidates for explaining mtDNA substitution rates. RESULTS: Initially, we introduce a novel clustering technique on genes, based on three similarity tests between pairs of genes, supporting previous results regarding gene functionalities in the mtDNA. These clusters are then used as a factor in our models. We present leading models for the protein coding genes, rRNA and tRNA genes and the control region, showing it is disadvantageous to separate the models of transitions/transversions, or synonymous/non-synonymous substitutions. We identify a context effect that cannot be attributed solely to protein level constraints or CpG pairs. For protein-coding genes, we show that the substitution model should be partitioned into sub-models according to the codon position and input codon; additionally we confirm that gene identity and cluster have no significant effect once the above factors are accounted for. CONCLUSIONS: We leverage the large, high-confidence Phylotree mtDNA phylogeny to develop a new statistical approach. We model the substitution rates using regressions, allowing consideration of many factors simultaneously. This admits the use of model selection tools helping to identify the set of factors best explaining the mutational dynamics when considered in tandem.

A de novo GABRA2 missense mutation in severe early-onset epileptic encephalopathy with a choreiform movement disorder.

BACKGROUND: Early-onset epileptic encephalopathy (EOEE) is a severe convulsive disorder with a poor developmental prognosis. Although it has been associated with mutations in a number of genes, the fact that there is a large proportion of patients who remain undiagnosed suggests that there are many more still-unknown genetic causes of EOEE. Achieving a genetic diagnosis is important for understanding the biological basis of the disease, with its implications for treatment and family planning. METHODS: Whole-exome sequencing was performed in a family of Ashkenazi Jewish origin in which a male infant was diagnosed with EOEE. There was no family history of a similar neurologic disease. The patient had extreme hypotonia, neonatal hypothermia, choreiform movements, and vision impairment in addition to the convulsive disorder. RESULTS: A de novo heterozygous missense mutation, c.1003A > C, p.Asn335His, was identified in a conserved domain of GABRA2. GABRA2 encodes the α2 subunit of the GABAA receptor. CONCLUSIONS: In the context of previous reports of an association of de novo mutations in genes encoding different subunits of the GABAA receptor (GABRB1, GABRA1, GABRG2, GABRB3) with autosomal dominant epileptic disorders, we conclude that a de novo mutation in GABRA2 is likely to cause autosomal dominant EOEE accompanied by a movement disorder and vision impairment.

A null variant in the apolipoprotein L3 gene is associated with non-diabetic nephropathy.

Background: Inheritance of apolipoprotein L1 gene (APOL1) renal-risk variants in a recessive pattern strongly associates with non-diabetic end-stage kidney disease (ESKD). Further evidence supports risk modifiers in APOL1-associated nephropathy; some studies demonstrate that heterozygotes possess excess risk for ESKD or show earlier age at ESKD, relative to those with zero risk alleles. Nearby loci are also associated with ESKD in non-African Americans. Methods: We assessed the role of the APOL3 null allele rs11089781 on risk of non-diabetic ESKD. Four cohorts containing 2781 ESKD cases and 2474 controls were analyzed. Results: Stratifying by APOL1 risk genotype (recessive) and adjusting for African ancestry identified a significant additive association between rs11089781 and ESKD in each stratum and in a meta-analysis [meta-analysis P = 0.0070; odds ratio (OR) = 1.29]; ORs were consistent across APOL1 risk strata. The biological significance of this association is supported by the finding that the APOL3 gene is co-regulated with APOL1, and that APOL3 protein was able to bind to APOL1 protein. Conclusions: Taken together, the genetic and biological data support the concept that other APOL proteins besides APOL1 may also influence the risk of non-diabetic ESKD.

Whole-exome sequencing reveals POC5 as a novel gene associated with autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is associated with different groups of genes, including those encoding proteins involved in centriole and cilium biogenesis. Exome sequencing revealed a homozygous nonsense mutation [c.304_305delGA (p. D102*)] in POC5, encoding the Proteome Of Centriole 5 protein, in a patient with RP, short stature, microcephaly and recurrent glomerulonephritis. The POC5 gene is ubiquitously expressed, and immunohistochemistry revealed a distinct POC5 localization at the photoreceptor connecting cilium. Morpholino-oligonucleotide-induced knockdown of poc5 translation in zebrafish resulted in decreased length of photoreceptor outer segments and a decreased visual motor response, a measurement of retinal function. These phenotypes could be rescued by wild-type human POC5 mRNA. These findings demonstrate that Poc5 is important for normal retinal development and function. Altogether, this study presents POC5 as a novel gene involved autosomal recessively inherited RP, and strengthens the hypothesis that mutations in centriolar proteins are important cause of retinal dystrophies.

The genetic variation in the R1a clade among the Ashkenazi Levites' Y chromosome.

Approximately 300,000 men around the globe self-identify as Ashkenazi Levites, of whom two thirds were previously shown to descend from a single male. The paucity of whole Y-chromosome sequences precluded conclusive identification of this ancestor's age, geographic origin and migration patterns. Here, we report the variation of 486 Y-chromosomes within the Ashkenazi and non-Ashkenazi Levite R1a clade, other Ashkenazi Jewish paternal lineages, as well as non-Levite Jewish and non-Jewish R1a samples. Cumulatively, the emerging profile is of a Middle Eastern ancestor, self-affiliating as Levite, and carrying the highly resolved R1a-Y2619 lineage, which was likely a minor haplogroup among the Hebrews. A star-like phylogeny, coalescing similarly to other Ashkenazi paternal lineages, ~1,743 ybp, suggests it to be one of the Ashkenazi paternal founders; to have expanded as part of the overall Ashkenazi demographic expansion, without special relation to the Levite affiliation; and to have subsequently spread to non-Ashkenazi Levites.

Clinical Heterogeneity and Phenotypic Expansion of NaPi-IIa-Associated Disease.

Context: NaPi-IIa, encoded by SLC34A1, is a key phosphate transporter in the mammalian proximal tubule and plays a cardinal role in renal phosphate handling. NaPi-IIa impairment has been linked to various overlapping clinical syndromes, including hypophosphatemic nephrolithiasis with osteoporosis, renal Fanconi syndrome with chronic kidney disease, and, most recently, idiopathic infantile hypercalcemia and nephrocalcinosis. Objectives: We studied the molecular basis of idiopathic infantile hypercalcemia with partial proximal tubulopathy in two apparently unrelated patients of Israeli and Turkish descent. Design: Genetic analysis in two affected children and their close relatives was performed using whole-exome sequencing, followed by in vitro localization and trafficking analysis of mutant NaPi-IIa. Results: Mutation and haplotype analyses in both patients revealed a previously described homozygous loss-of-function inserted duplication (p.I154_V160dup) in NaPi-IIa, which is inherited identical-by-descent from a common ancestor. The shared mutation was originally reported by our team in two adult siblings with renal Fanconi syndrome, hypophosphatemic bone disease, and progressive renal failure who are family members of one of the infants reported herein. In vitro localization assays and biochemical analysis of p.I154_V160dup and of additional NaPi-IIa mutants harboring a trafficking defect indicate aberrant retention at the endoplasmic reticulum in an immature and underglycosylated state, leading to premature proteasomal degradation. Conclusions: Our findings expand the phenotypic spectrum of NaPi-IIa disruption, reinforce its link with proximal tubular impairment, enable longitudinal study of the natural history of the disease, and shed light on cellular pathways associated with loss of function and impaired trafficking of NaPi-IIa mutants.

[UTILIZATION OF WHOLE EXOME SEQUENCING IN DIAGNOSTICS OF GENETIC DISEASE: RABIN MEDICAL CENTER'S EXPERIENCE].

INTRODUCTION: Whole exome sequencing is a diagnostic approach for the identification of molecular etiology in patients with suspected monogenic diseases. In this article we report on our experience with whole-exome sequencing (WES) of DNA samples taken from patients referred for genetic evaluation due to suspected undiagnosed genetic conditions. METHODS: Exome enrichment was achieved by Nextera Rapid Capture Expanded Exome Kit. Whole-exome sequencing was performed on Illumina HiSeq 2500. Potentially damaging rare variants were selected for familial cosegregation analysis. RESULTS: A total of 39 patients presenting a wide range of phenotypes suspected to have a genetic cause were sent to WES. Approximately 80% were children with neurological phenotypes. Variations having a high probability of being causative were identified in 20 families, achieving a 51.3% molecular diagnostic rate. Among these, 7 exhibited autosomal dominant disease, 12 autosomal recessive diseases and one X-linked disease; 28% of the patients (11/39) were found to carry a novel mutation located in previously reported genes. Novel mutations located in genes not known to be associated with genetic disease were identified in 23% of the patients (9/39). CONCLUSIONS: Whole exome sequencing identified the underlying genetic cause in more than half of the patients referred for evaluation in the genetics clinic at the tertiary hospital. These data demonstrate the utility of WES as a powerful tool for effective diagnostics of monogenic genetic diseases.

Deficiency of the sphingosine-1-phosphate lyase SGPL1 is associated with congenital nephrotic syndrome and congenital adrenal calcifications.

We identified two unrelated consanguineous families with three children affected by the rare association of congenital nephrotic syndrome (CNS) diagnosed in the first days of life, of hypogonadism, and of prenatally detected adrenal calcifications, associated with congenital adrenal insufficiency in one case. Using exome sequencing and targeted Sanger sequencing, two homozygous truncating mutations, c.1513C>T (p.Arg505*) and c.934delC (p.Leu312Phefs*30), were identified in SGPL1-encoding sphingosine-1-phosphate (S1P) lyase 1. SGPL1 catalyzes the irreversible degradation of endogenous and dietary S1P, the final step of sphingolipid catabolism, and of other phosphorylated long-chain bases. S1P is an intracellular and extracellular signaling molecule involved in angiogenesis, vascular maturation, and immunity. The levels of SGPL1 substrates, S1P, and sphingosine were markedly increased in the patients' blood and fibroblasts, as determined by liquid chromatography-tandem mass spectrometry. Vascular alterations were present in a patient's renal biopsy, in line with changes seen in Sgpl1 knockout mice that are compatible with a developmental defect in vascular maturation. In conclusion, loss of SGPL1 function is associated with CNS, adrenal calcifications, and hypogonadism.

Strictly conserved tri-nucleotide motif "CAT" is associated with TAS DNA protein-binding sites in human mitochondrial DNA control region.

The mitochondrial DNA (mtDNA) control region is a highly variable segment that contains functional elements that control mtDNA transcription and replication. By analysis of the polymorphic nucleotide spectrum of that segment, we aimed to identify the most conserved sites that should be associated with these elements. For that aim, we analyzed 50 033 human mtDNA control region sequences (mtDNA positions 16 066-16 374). We identified 10 conserved tri-nucleotides, one conserved tetra-nucleotide, and one conserved penta-nucleotide, containing six repetitions of the motif CAT, and two of its complement motif ATG (p value < 2 × 10 - 4). Three other appearances of the tri-nucleotide CAT were almost perfectly preserved. The positions of the preserved CAT elements are associated with the location of previously identified termination-associated sequences (TAS) which are the binding locations for proteins involved in mtDNA replication. We, therefore, hypothesize that the CAT tri-nucleotide elements within the control region may be the binding sites for TAS proteins and are directly involved in mtDNA transcription and replication.

A Biallelic Mutation in the Homologous Recombination Repair Gene SPIDR Is Associated With Human Gonadal Dysgenesis.

Context: Primary ovarian insufficiency (POI) is caused by ovarian follicle depletion or follicle dysfunction, characterized by amenorrhea with elevated gonadotropin levels. The disorder presents as absence of normal progression of puberty. Objective: To elucidate the cause of ovarian dysfunction in a family with POI. Design: We performed whole-exome sequencing in 2 affected individuals. To evaluate whether DNA double-strand break (DSB) repair activities are altered in biallelic mutation carriers, we applied an enhanced green fluorescent protein-based assay for the detection of specific DSB repair pathways in blood-derived cells. Setting: Diagnoses were made at the Pediatric Endocrine Clinic, Clalit Health Services, Sharon-Shomron District, Israel. Genetic counseling and sample collection were performed at the Pediatric Genetics Unit, Schneider Children's Medical Center Israel, Petah Tikva, Israel. Patients and Intervention: Two sisters born to consanguineous parents of Israeli Muslim Arab ancestry presented with a lack of normal progression of puberty, high gonadotropin levels, and hypoplastic or absent ovaries on ultrasound. Blood samples for DNA extraction were obtained from all family members. Main Outcome Measure: Exome analysis to elucidate the cause of POI in 2 affected sisters. Results: Analysis revealed a stop-gain homozygous mutation in the SPIDR gene (KIAA0146) c.839G>A, p.W280*. This mutation altered SPIDR activity in homologous recombination, resulting in the accumulation of 53BP1-labeled DSBs postionizing radiation and γH2AX-labeled damage during unperturbed growth. Conclusions: SPIDR is important for ovarian function in humans. A biallelic mutation in this gene may be associated with ovarian dysgenesis in cases of autosomal recessive inheritance.