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Pathogenic variants in LRBA, encoding the LPS Responsive Beige-Like Anchor (LRBA) protein, are responsible for recessive, early-onset hypogammaglobulinemia, severe multi-organ autoimmunity, and lymphoproliferation, with increased risk for malignancy. LRBA deficiency has a wide clinical spectrum with variable age of onset and disease severity. Three apparently unrelated patients with LRBA deficiency, of Georgian Jewish descent, were homozygous for LRBA c.6640C T, p.R2214*, leading to a stop upstream of the LRBA BEACH domain. Despite carrying the same LRBA genotype, the three patients differed in clinical course: the first patient was asymptomatic until age 25 years; the second presented with failure to thrive at age 3 months; and the third presented at age 7 years with immune cytopenias and severe infections. Two of the patients developed malignancies: the first patient was diagnosed with recurrent Hodgkin's disease at age 36 years, and the second patient developed aggressive gastric cancer at age 15 years. Among Georgian Jews, the carrier frequency of the LRBA p.R2214* allele was 1.6% (4 of 236 Georgian Jewish controls). The allele was absent from other populations. Haplotype analysis showed a shared origin of the mutation. These three patients revealed a pathogenic LRBA founder allele in the Georgian Jewish population, support the diverse and complex clinical spectrum of LRBA deficiency, and support the possibility that LRBA deficiency predisposes to malignancy.

Complex chromosomal rearrangements (CCRs), a class of structural variants (SVs) involving more than two chromosome breaks, were classically thought to be extremely rare. As advanced technologies become more available, it has become apparent that CCRs are more common than formerly thought, and are a substantial cause of genetic disorders. We attempted a novel approach for solving the mechanism of challenging CCRs, which involve repetitive sequences, by precisely identifying sequence-level changes and their order. Chromosomal microarray (CMA) and FISH analyses were used for interpretation of SVs detected by whole exome sequencing (WES). Breakpoint junctions were analyzed by Nanopore sequencing, a novel long-read whole genome sequencing tool. A large deletion identified by WES, encompassing the FOXF1 enhancer, was the cause of alveolar capillary dysplasia and respiratory insufficiency, resulting in perinatal death. CMA analysis of the newborn's mother revealed two duplications encompassing the deleted region in the proband, raising our hypothesis that the deletion resulted from the mother's CCR. Breakpoint junctions of complex SVs were determined at the nucleotide level using Nanopore long-read sequencing. According to sequencing results of breakpoint junctions, the CCR in the newborn was considered the consequence of at least one double-strand break during meiosis, and reassembly of DNA fragments by intra-chromosomal homologous recombination. Our comprehensive approach, combining cytogenetics and long-read sequencing, enabled delineation of the exact breakpoints in a challenging CCR, and proposal of a mechanism in which it arises. We suggest applying our integrative approach combining technologies for deciphering future challenging CCRs, enabling risk assessment in families.

ObjectiveTo determine the yield of genetic diagnoses using chromosomal microarray (CMA) and trio whole exome sequencing (WES), separately and combined, among patients with cryptogenic cerebral palsy (CP).MethodsTrio WES of patients with prior CMA analysis for cryptogenic CP, defined as disabling, non-progressive motor symptoms beginning before the age of 3 years without known cause.ResultsGiven both CMA analysis and trio WES, clinically significant genetic findings were identified for 58% of patients (26 of 45). Diagnoses were eight large CNVs detected by CMA and 18 point mutations detected by trio WES. None had more than one severe mutation. Approximately half of events (14 of 26) were de novo. Yield was significantly higher in patients with CP with comorbidities (69%, 22 of 32) than in those with pure motor CP (31%, 4 of 13; p=0.02). Among patients with genetic diagnoses, CNVs were more frequent than point mutations among patients with congenital anomalies (OR 7.8, 95% CI 1.2 to 52.4) or major dysmorphic features (OR 10.5, 95% CI 1.4 to 73.7). Clinically significant mutations were identified in 18 different genes: 14 with known involvement in CP-related disorders and 4 responsible for other neurodevelopmental conditions. Three possible new candidate genes for CP were ARGEF10, RTF1 and TAOK3.ConclusionsCryptogenic CP is genetically highly heterogeneous. Genomic analysis has a high yield and is warranted in all these patients. Trio WES has higher yield than CMA, except in patients with congenital anomalies or major dysmorphic features, but these methods are complementary. Patients with negative results with one approach should also be tested by the other.

Purpose We previously developed Haploseek, a method for comprehensive preimplantation genetic testing (PGT). However, some key features were missing, and the method has not yet been systematically validated. Methods We extended Haploseek to incorporate DNA from embryo grandparents and to allow testing of variants on chromosome X or in regions where parents share common haplotypes. We then validated Haploseek on 151 embryo biopsies from 27 clinical PGT cases. We sequenced all biopsies to low coverage (0.2×), and performed single-nucleotide polymorphism (SNP) microarray genotyping on the embryos' parents and siblings/grandparents. We used the extended Haploseek to predict chromosome copy-number variants (CNVs) and relevant variant-flanking haplotypes in each embryo. We validated haplotype predictions for each clinical sample against polymerase chain reaction (PCR)-based PGT case results, and CNV predictions against established commercial kits. Results For each of the 151 embryo biopsies, all Haploseek-derived haplotypes and CNVs were concordant with clinical PGT results. The cases included 17 autosomal dominant, 5 autosomal recessive, and 3 X-linked monogenic disorders. In addition, we evaluated 1 Robertsonian and 2 reciprocal translocations, and 17 cases of chromosome copy-number counting were performed. Conclusion Our results demonstrate that Haploseek is clinically accurate and fit for all standard clinical PGT applications.

PURPOSE: To review cases of couples presented to our PGT-unit with copy number variants (CNVs) classified as variants of uncertain significance (VUS) in order to better understand their needs. METHODS: Retrospective cohort study conducted in a tertiary medical-center, 2014–2019. We reviewed files of all couples applying for genetic counseling with CNVs classified as VUS. The main outcomes measured: number of VUS findings and their description, PGT-M procedures planned and performed, IVF cycles, clinical pregnancy, and live birth rates (LBR). VUS were classified according to the American-College of Medical-Genetics and Genomics classification at time of first consultation, and updated—December 2018. RESULTS: Twenty-four couples presented with a total of 30 VUS. Twelve couples (50%) had isolated VUS and 12 (50%) had VUS diagnosed in addition to a pathogenic mutation. Initially, nine findings (30%) were defined as VUS; eight (27%) as likely benign (b-VUS); and 13 (43%) as likely pathogenic (p-VUS). PGT-M was recommended for 17/30 CNVs (56.6%), 12 (70%) of which, isolated VUS. No couple had other indications for IVF. To date, nine couples performed PGT-M for isolated VUS; LBR per-couple—55.5%. Five couples performed PGT-M for both pathogenic findings and VUS, LBR—80%. After reviewing VUS classifications, 30% remained unchanged, 20% were more severely defined, and 50% less severely defined. CONCLUSION: The genomic era enables detection of VUS whose definition is subject to change as additional information becomes available. The uncertainty of variants’ clinical significance and changes in VUS definition over time complicates genetic counseling. Revised guidelines for VUS interpretation and reevaluation of patient counseling before each pregnancy must be practiced when counseling them regarding the justification of PGT-M for their diagnosed VUS.

Infantile Krabbe disease (OMIM 245200) is a severe, fatal autosomal recessive neurodegenerative disorder that is relatively frequent in two Muslims villages within Jerusalem. After the characterization of the founder mutation, a population carrier screening for Krabbe disease became a component of the Israeli program for the detection and the prevention of birth defects. Between 2010 and 2018, 3366 individuals were tested and among them 247 carriers for Krabbe disease were identified (7.3%). Most of the 21 carrier couples identified that had pregnancies after being informed that they were at risk used preventive measures including termination of pregnancies of affected fetuses. During the study period, eight children affected with Krabbe disease were born in the villages, four to couples not detected though the program. Twenty years after the beginning of the carrier screening program, Krabbe disease remained relatively frequent in the villages. The establishment of a genetic clinic in the villages may allow to improve the carrier screening program while giving individual counseling for the risk to the other genetic diseases existing in the villages.

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Deficiency of the endoplasmic reticulum transmembrane protein ARV1 leads to epileptic encephalopathy in humans and in mice. ARV1 is highly conserved, but its function in human cells is unknown. Studies of yeast arv1 null mutants indicate that it is involved in a number of biochemical processes including the synthesis of sphingolipids and glycosylphosphatidylinositol (GPI), a glycolipid anchor that is attached to the C-termini of many membrane bound proteins. GPI anchors are post-translational modifications, enabling proteins to travel from the endoplasmic reticulum (ER) through the Golgi and to attach to plasma membranes. We identified a homozygous pathogenic mutation in ARV1, p.Gly189Arg, in two brothers with infantile encephalopathy, and characterized the biochemical defect caused by this mutation. In addition to reduced expression of ARV1 transcript and protein in patients' fibroblasts, complementation tests in yeast showed that the ARV1 p.Gly189Arg mutation leads to deficient maturation of Gas1, a GPI-anchored protein, but does not affect sphingolipid synthesis. Our results suggest, that similar to mutations in other proteins in the GPI-anchoring pathway, including PIGM, PIGA, and PIGQ, ARV1 p.Gly189Arg causes a GPI anchoring defect and leads to early onset epileptic encephalopathy.

Mutations in the gene HSD17B3 encoding the 17-beta hydroxysteroid dehydrogenase 3 enzyme cause testosterone insufficiency leading to XY disorders of sex development. In this study the clinical and molecular characteristics of three patients from consanguineous families are elucidated. We identified three patients from two unrelated families with XY DSD and a novel homozygous HSD17B3:c. 673G>A mutation. The effect of the mutation on splicing was determined in RNA extracted from the testis of one patient. Three patients presented at ages 0.1, 8 and 0.7 years with ambiguous genitalia and an XY Karyotype. Endocrine workup showed normal cortisol and mineralocorticoid levels with a low testosterone/androstenedione ratio. Whole-exome sequencing, carried out in the first family, revealed a homozygous novel mutation in the HSD17B3 gene: c. 673G>A, p. V225M. The same mutation was found by Sanger sequencing in the third unrelated patient. Haplotype analysis of a 4 Mb region surrounding the HSD17B3 gene on chromosome 9 revealed that the mutation resides on the same allele in all three patients. The mutation, being the first nucleic acid on exon 10, affects splicing and causes exon 10 skipping in one of our patients’ testes. The novel homozygous c. 673G>A, p. V225M mutation in the 17HSDB3 gene is likely a founder mutation and causes severe XY-DSD. It changes a conserved amino acid residue, and also alters 17HSDB3 gene transcription by causing skipping of exon 10, thereby contributing to an imbalance in the relevant protein isoforms and consequently, significant decreased 17HDSB3 enzymatic activity.

To develop an economical, user-friendly, and accurate all-in-one next-generation sequencing (NGS)-based workflow for single-cell gene variant detection combined with comprehensive chromosome screening in a 24-hour workflow protocol. We subjected single lymphoblast cells or blastomere/blastocyst biopsies from four different families to low coverage (0.3×–1.4×) genome sequencing. We combined copy-number variant (CNV) detection and whole-genome haplotype phase prediction via Haploseek, a novel, user-friendly analysis pipeline. We validated haplotype predictions for each sample by comparing with clinical preimplantation genetic diagnosis (PGD) case results or by single-nucleotide polymorphism (SNP) microarray analysis of bulk DNA from each respective lymphoblast culture donor. CNV predictions were validated by established commercial kits for single-cell CNV prediction. Haplotype phasing of the single lymphoblast/embryo biopsy sequencing data was highly concordant with relevant ground truth haplotypes in all samples/biopsies from all four families. In addition, whole-genome copy-number assessments were concordant with the results of a commercial kit. Our results demonstrate the establishment of a reliable method for all-in-one molecular and chromosomal diagnosis of single cells. Important features of the Haploseek pipeline include rapid sample processing, rapid sequencing, streamlined analysis, and user-friendly reporting, so as to expedite clinical PGD implementation.

Objective To explore the indications for and diagnostic outcomes of fetal exome sequencing in a tertiary referral center. Methods Between 2012 and 2017, 77 unrelated fetal samples from pregnancies referred to our center underwent exome sequencing. The cohort included 37 fetuses, 36 products of conception (from cases of pregnancy termination or intrauterine fetal death), one case with DNA from both the fetus and a previous termination of pregnancy, and three cases with DNA of unknown origin. Exome sequencing was performed on DNA extracted from amniocytes or fetal tissue and, in some cases, from parental peripheral blood. Indications, turnaround time, diagnostic rates and pregnancy outcomes were investigated. Diagnostic yield was analyzed according to consanguinity (yes or no), sample type (proband only, or trio or other) and referral indication (malformation or isolated nuchal translucency (NT)). Results The most common indication for fetal exome sequencing was multiple malformations (21/77, 27%), followed by isolated brain malformation (15/77, 19%). Twelve (16%) fetuses were referred for isolated increased NT. Exome analysis was diagnostic for 16 fetuses (21%); when subclassified into fetal malformations vs isolated increased NT it became clear that exome analysis did not reveal any known or probable pathogenic variants in cases referred for isolated increased NT, whereas, among the remaining fetuses, a molecular diagnosis was reached in 16/65 (25%). Proband-only cases received a diagnosis more often than did cases that had trio exome sequencing. Conclusions Exome sequencing has the potential to provide molecular diagnoses in cases in which conventional prenatal cytogenetic testing is negative. Referral bias of consanguineous cases could account for the high diagnostic rate of proband-only sequencing. Syndrome-specific prognostic information enables parents to make informed decisions, whereas challenges include time limitations and variant interpretation in the setting of non-specific fetal findings. As we report only established disease-gene associations, further segregation and functional studies in a research setting are expected to increase significantly the diagnostic yield. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Objective To examine chromosomal microarray analysis results in pregnancies with various ultrasonographic anomalies and to characterize the copy number variants in diverse fetal phenotypes. Methods We retrospectively examined chromosomal microarray analyses of amniocenteses performed nationwide as a result of fetal ultrasonographic anomalies (structural defects, fetal growth restriction, and polyhydramnios) between January 2013 and September 2017. The rate of abnormal chromosomal microarray findings was compared between the different phenotypes and with a previously described control population of 15,225 pregnancies with normal ultrasonographic findings. Results Clinically significant chromosomal microarray aberrations were detected in 272 of 5,750 pregnancies (4.7%): 115 (2%) karyotype-detectable and 157 (2.7%) submicroscopic. Most commonly detected copy number variants were 22q11.21 deletions (0.4%) followed by 22q11.21 gain of copy number (0.2%). Specific copy number variants detected among pregnancies with abnormal ultrasonographic findings were up to 20-fold more prevalent compared with low-risk pregnancies. Some variants were associated with specific phenotypes (eg, 22q11.21 microdeletions with cardiovascular and 17q12 microdeletions with genitourinary defects). Conclusion The rate of abnormal amniotic chromosomal microarray analysis results is twice that of karyotypic abnormalities in pregnancies with various abnormal ultrasonographic findings.

BackgroundCopy number variants (CNVs) associated with late-onset medical conditions are rare but important secondary findings in chromosomal microarray analysis (CMA) performed during pregnancy. Here, we critically review the cases at two tertiary centres to assess the criteria which guide the disclosure of such findings and develop a disclosure decision tool (DDT) aimed at facilitating disclosure decision. Parental decisions on receiving CNVs associated with risks for late-onset conditions were also recorded.MethodsPrenatal CMAs in Hadassah and Shaare Zedek Medical Centers from November 2013 to October 2021 were reviewed for CNVs associated with late-onset conditions. The DDT proposed uses a five-parameter scoring system, which considers the severity, median age of onset, penetrance, understanding of genotype-phenotype correlation and actionability of the finding.ResultsOut of 16 238 prenatal CMAs, 16 (0.1%) harboured CNVs associated with late-onset conditions, 15 of which were disclosed. Outcome information was available on 13 of the 16 pregnancies, all of which continued to delivery.ConclusionsOur suggested DDT will help clinicians to quantitatively weigh the variables associated with CNVs of this type and arrive at a well thought out clinical decision regarding disclosure. Although the prevalence of late-onset conditions as a major finding in the prenatal setup is low, it is expected to rise with the increasing use of non-invasive CMA testing and whole exome and genome sequencing.

Background Evidence comparing the yield of chromosomal microarray analysis to noninvasive prenatal screening in pregnancies with congenital heart anomalies is currently limited. Objective This study aimed to examine the residual risk of clinically significant chromosomal microarray analysis results in fetuses with congenital heart defects by its various subtypes following a normal noninvasive prenatal screening. Study Design Using a population-based, countrywide computerized database, we retrieved the reports of all pregnancies undergoing chromosomal microarray analysis because of congenital heart defects through the years 2013–2019. We examined the risk of clinically significant (pathogenic and likely pathogenic) chromosomal microarray analysis results and compared it with the results of a local cohort of low-risk pregnancies. Of 5541 fetuses, 78 (1.4%) showed abnormal results. The residual risk of abnormal chromosomal microarray analysis results was calculated using several options—trisomies 21, 18, and 13; sex chromosome aneuploidies; 22q11.2 deletion, and deletions and duplications of at least 10 MB in size (genome-wide noninvasive prenatal screening)—following the exclusion of theoretically detectable noninvasive prenatal screening anomalies. Results Of the 1728 fetuses with congenital heart defects, 93 (5.4%) showed clinically significant chromosomal microarray analysis results (relative risk, 2.7; 95% confidence interval, 2.3–3.1). The result of pregnancies with fetuses with congenital heart defects was compared with the results of the control population. Unique variants were found in 15 pregnancies (16.1%). The detection rate of noninvasive prenatal screening in isolated congenital heart defects varied from 1.0% (aimed at 3 common trisomies) to 2.2% (aimed at 5 common aneuploidies and 22q11.2 deletion) using noninvasive prenatal screening. In nonisolated congenital heart defects, the noninvasive prenatal screening detection rates ranged from 7.8% (aimed at common autosomal trisomies) to 9.2% using genome-wide noninvasive prenatal screening. The residual risk of clinically significant chromosomal microarray analysis results following normal noninvasive prenatal screening ranged from 2.0% to 2.8% in isolated congenital heart defects and 4.5% to 5.9% in nonisolated cases and was significantly higher than those of the control cohort in all noninvasive prenatal screening options. In addition, the residual risk following noninvasive prenatal screening aimed at chromosomes 13, 18, 21, X, and Y was significantly higher than those of the control cohort for most specific congenital heart defect subtypes, except for ventricular septal defects and aberrant right subclavian artery. Conclusion The residual risk of clinically significant chromosomal microarray analysis results in pregnancies with fetuses with congenital heart defects following normal noninvasive prenatal screening was higher than those in pregnancies with normal ultrasound in most isolated and nonisolated congenital heart defect subtypes. This information should be taken into account by obstetricians and genetic counselors when considering the option of diagnostic testing.

Glycosylphosphatidylinositol (GPI) is synthesized in the endoplasmic reticulum (ER) and added onto proteins to form GPI-anchored proteins. Among the many proteins involved in this process, ACAT-related enzyme-2 required for viability 1 (Arv1) is a candidate, functioning as a flippase that translocates GPI intermediates from the cytoplasmic side into the luminal side of the ER membranes. Here, we show that the deletion of the ARV1 gene in yeast leads to cold-sensitive defects in cell growth and GPI anchor synthesis. Furthermore, complementation assays show that the overexpression of a missense human ARV1-G189R mutant does not completely restore the cold-sensitive phenotypes of the yeast arv1 mutant. Our results support the proposed role of Arv1 in GPI anchor synthesis and suggest that ARV1-linked human diseases result from defective GPI anchor synthesis.

Mutations in the gene HSD17B3 encoding the 17-beta hydroxysteroid dehydrogenase 3 enzyme cause testosterone insufficiency leading to XY disorders of sex development. In this study the clinical and molecular characteristics of three patients from consanguineous families are elucidated.We identified three patients from two unrelated families with XY DSD and a novel homozygous HSD17B3:c. 673GA mutation. The effect of the mutation on splicing was determined in RNA extracted from the testis of one patient.Three patients presented at ages 0.1, 8 and 0.7 years with ambiguous genitalia and an XY Karyotype. Endocrine workup showed normal cortisol and mineralocorticoid levels with a low testosterone/androstenedione ratio. Whole-exome sequencing, carried out in the first family, revealed a homozygous novel mutation in the HSD17B3 gene: c. 673GA, p. V225M. The same mutation was found by Sanger sequencing in the third unrelated patient. Haplotype analysis of a 4 Mb region surrounding the HSD17B3 gene on chromosome 9 revealed that the mutation resides on the same allele in all three patients. The mutation, being the first nucleic acid on exon 10, affects splicing and causes exon 10 skipping in one of our patients' testes.The novel homozygous c. 673GA, p. V225M mutation in the 17HSDB3 gene is likely a founder mutation and causes severe XY-DSD. It changes a conserved amino acid residue, and also alters 17HSDB3 gene transcription by causing skipping of exon 10, thereby contributing to an imbalance in the relevant protein isoforms and consequently, significant decreased 17HDSB3 enzymatic activity.

ObjectiveTo examine the risk for abnormal chromosomal microarray analysis (CMA) results among fetuses with an apparently isolated pelvic kidney.MethodsData from all CMA analyses performed due to an isolated pelvic kidney reported to the Israeli Ministry of Health between January 2013 and September 2016 were retrospectively obtained. Risk estimation was performed comparing the rate of abnormal observed CMA findings to the general population risk, based on a systematic review encompassing 9272 cases and on local data of 5541 cases.ResultsOf 120 pregnancies with an isolated pelvic kidney, two gain-of-copy number variants suggesting microduplication syndromes were demonstrated (1.67%). In addition, three variants of unknown significance were detected (2.5%).ConclusionThe risk for clinically significant CMA findings among pregnancies with an isolated single pelvic kidney was not significantly different compared to both control populations. The results of our study question the practice of routine CMA analysis in fetuses with an isolated pelvic kidney.

Rhabdomyolysis is a clinical emergency that may cause acute kidney injury (AKI). It can be acquired or due to monogenic mutations. Around 60 different rare monogenic forms of rhabdomyolysis have been reported to date. In the clinical setting, identifying the underlying molecular diagnosis is challenging due to nonspecific presentation, the high number of causative genes, and current lack of data on the prevalence of monogenic forms. We employed whole exome sequencing (WES) to reveal the percentage of rhabdomyolysis cases explained by single-gene (monogenic) mutations in one of 58 candidate genes. We investigated a cohort of 21 unrelated families with rhabdomyolysis, in whom no underlying etiology had been previously established. Using WES, we identified causative mutations in candidate genes in nine of the 21 families (43%). We detected disease-causing mutations in eight of 58 candidate genes, grouped into the following categories: (1) disorders of fatty acid metabolism (CPT2), (2) disorders of glycogen metabolism (PFKM and PGAM2), (3) disorders of abnormal skeletal muscle relaxation and contraction (CACNA1S, MYH3, RYR1 and SCN4A), and (4) disorders of purine metabolism (AHCY). Our findings demonstrate a very high detection rate for monogenic etiologies using WES and reveal broad genetic heterogeneity for rhabdomyolysis. These results highlight the importance of molecular genetic diagnostics for establishing an etiologic diagnosis. Because these patients are at risk for recurrent episodes of rhabdomyolysis and subsequent risk for AKI, WES allows adequate prophylaxis and treatment for these patients and their family members and enables a personalized medicine approach.

The causes of ovarian dysgenesis remain incompletely understood. Two sisters with XX ovarian dysgenesis carried compound heterozygous truncating mutations in the BRCA2 gene that led to reduced BRCA2 protein levels and an impaired response to DNA damage, which resulted in chromosomal breakage and the failure of RAD51 to be recruited to double-stranded DNA breaks. The sisters also had microcephaly, and one sister was in long-term remission from leukemia, which had been diagnosed when she was 5 years old. Drosophila mutants that were null for an orthologue of BRCA2 were sterile, and gonadal dysgenesis was present in both sexes. These results revealed a new role for BRCA2 and highlight the importance to ovarian development of genes that are critical for recombination during meiosis. (Funded by the Israel Science Foundation and others.).

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive autoinflammatory disease resulting from mutations in ADA2 (formerly named CECR1). Initially described by two groups in 2014, additional reports have documented that the phenotypic expression is quite broad. Although commonly presenting in childhood, patients can present throughout their lives with varying forms of inflammatory, neurologic, hematologic, and immunologic phenotypes. Frequently reported disease manifestations include lacunar strokes, non-cirrhotic portal hypertension, immunodeficiencies, and bone marrow involvement that can include an overt pancytopenia ranging to cell-specific immune destruction. The role of adenosine deaminase 2 (ADA2) remains incompletely understood. The lack of ADA2 in patients with DADA2 results in endothelial cell fragility and a skewing of macrophage development toward the inflammatory, M1 macrophage. Current treatment options are tailored toward the individual clinical presentations but the utilization of anti-tumor necrosis factor (TNF) medications has been highly effective at reducing the risk for stroke. Hematopoietic stem cell transplant offers a potential cure for the disease.

Recessive CRB2 mutations were recently reported to cause both steroid resistant nephrotic syndrome and prenatal onset ventriculomegaly with kidney disease. We report two Ashkenazi Jewish siblings clinically diagnosed with ciliopathy. Both presented with severe congenital hydrocephalus and mild urinary tract anomalies. One affected sibling also has lung hypoplasia and heart defects. Exome sequencing and further CRB2 analysis revealed that both siblings are compound heterozygotes for CRB2 mutations p.N800K and p.Gly1036Alafs*43, and heterozygous for a deleterious splice variant in the ciliopathy gene TTCB21. CRB2 is a polarity protein which plays a role in ciliogenesis and ciliary function. Biallelic CRB2 mutations in animal models result in phenotypes consistent with ciliopathy. This report expands the phenotype of CRB2 mutations to include lung hypoplasia and uretero-pelvic renal anomalies, and confirms cardiac malformation as a feature. We suggest that CRB2-associated disease is a new ciliopathy syndrome with possible digenic/triallelic inheritance, as observed in other ciliopathies. Clinically, CRB2 should be assessed when ciliopathy is suspected, especially in Ashkenazi Jews, where we found that p.N800K carrier frequency is 1 of 64. Patients harboring CRB2 mutations should be tested for the complete range of ciliopathy manifestations.

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.Identification of the responsible gene by whole exome sequencing and homozygosity mapping.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.2515CT, p.R839X, to be homozygous in the proband and to cosegregate with the condition in her family. The allele frequency of PCDH12 p.R839X is0.00001 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% (p2.4 × 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.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.

ObjectiveTo identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy.MethodsIdentification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients’ cells and in yeast.ResultsTwo brothers from a consanguineous Palestinian family presented with progressive spinocerebellar ataxia, mental retardation and psychotic episodes. Serial brain imaging showed severe progressive cerebellar atrophy. Whole exome sequencing revealed a novel mutation: pitrilysin metallopeptidase 1 (PITRM1) c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in PITRM1 protein. Whole genome sequencing revealed a chromosome X structural rearrangement that also segregated with the disease. Independently, two siblings from a second Palestinian family presented with similar, somewhat milder symptoms and the same PITRM1 mutation on a shared haplotype. PITRM1T931M carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. PITRM1 is a mitochondrial matrix enzyme that degrades 10–65 amino acid oligopeptides, including the mitochondrial fraction of amyloid-beta peptide. Analysis of peptide cleavage activity by the PITRM1T931M protein revealed a significant decrease in the degradation capacity specifically of peptides ≥40 amino acids.ConclusionPITRM1T931M results in childhood-onset recessive cerebellar pathology. Severity of PITRM1-related disease may be affected by the degree of impairment in cleavage of mitochondrial long peptides. Disruption and deletion of X linked regulatory segments may also contribute to severity.

Background: Tuberous sclerosis complex (TSC) is a multisystem disorder diagnosed by clinical criteria and/or genetic testing. Genetic testing reveals atypical phenotypes that have not met clinical criteria, with practical implications. Methods: We describe 4 family members with pathogenic partial deletion in TSC1 who individually did not meet tuberous sclerosis complex clinical criteria. Results: Family members had different and atypical findings of tuberous sclerosis complex. Although none of the family members fulfilled the clinical criteria for tuberous sclerosis complex, they all carried the same genomic deletion (9q34.13q34.2) that included part of the TSC1 gene. One member had ganglioglioma and intractable seizures, one sibling presented with seizures, developmental delay, and displayed white matter abnormalities; another sibling had no clinical manifestations but has cortical tuber. Their mother has facial angiofibroma, cortical tuber, and seizures during infancy. Conclusions: Ganglioglioma may be a phenotypic expression of TSC1. Genetic testing is recommended for infants with brain tumors, especially those with an abnormal familial history.

Purpose: To investigate immune tolerance induction with transient low-dose methotrexate (TLD-MTX) initiated with recombinant human acid α-glucosidase (rhGAA), in treatment-naïve cross-reactive immunologic material (CRIM)-positive infantile-onset Pompe disease (IOPD) patients. Methods: Newly-diagnosed IOPD patients received subcutaneous or oral 0.4 mg/kg TLD-MTX for 3 cycles (3 doses/cycle) with the first 3 rhGAA infusions. Anti-rhGAA IgG titers, classified as high-sustained (HSAT; ≥51,200, ≥2 times after 6 months), sustained intermediate (SIT; ≥12,800 and

The objective of this study is to examine the frequency of abnormal Chromosomal Microarray (CMA) analyses among fetuses with isolated non-visualization of fetal gallbladder.Data from CMA analyses performed due to isolated non-visualization of fetal gallbladder between January 2013 and September 2016 were retrospectively acquired from a computerized database of the Israeli Ministry of Health. The results were compared with the rate for clinically significant CMA findings in general population, based on a large cohort of 5541 pregnancies undergoing CMA due to maternal request, and a systematic review of 9272 cases with normal ultrasound.Of 45 pregnancies with isolated non-visualization of fetal gallbladder, CMA testing yielded one (2.22%) gain-of-copy-number variant at 16p11.2, categorized as "pathogenic". In addition, one finding of unknown significance was demonstrated. The risk for clinically meaningful CMA findings among pregnancies with isolated absent gallbladder was not significantly increased compared to control population.To the best of our knowledge, this study is the first report describing the rate of pathogenic CMA results in fetuses with isolated non-visualization of fetal gallbladder. The results, in conjunction with previous studies, show that the risk for abnormal CMA results in pregnancies diagnosed with non-visualized gallbladder is not significantly different from pregnancies with normal ultrasound.

Introduction The new chromosomal microarray CMA technology, allows the identification of genetic defects in many disorders that would previously have escaped detection. Alongside the benefits, it exposes us to copy number variants (CNV) whose pathogenic significance is unclear "Variants of unknown significance" (VUS). In our cohort we reviewed all files of couples that applied for PGT-M at our clinic with a finding of VUS. We aimed to characterize these VUS and the cases in which PGT-M was decided upon. Materials and methods A retrospective cohort study in the Zohar Unit for PGT-M, Shaare Zedek medical center, 2014-2019. Files of all couples with a finding of VUS among those who applied for PGT-M with any CMA findings were reviewed. VUS were classified (likely pathogenic, VUS, likely benign) according to the American collage of medical genetics and Genomics classification at time of first consultation and to date (December 2018). Pathogenic variants were not included in the study. PGT-M was performed on blastromeres of throphoectoderm biopsy by PCR using surrounding polymorphic markers. For all microduplications, FISH was performed prior to the PGT-M in order to confirm the tandem location. Results Of 45 couples requesting PGT-M for CMA findings, 24 (53%) presented with VUS. VUS was an isolated finding in 54% of cases and as an additional finding to a known pathogenic mutation in 46%. The mean age of women was 29.8. Half the couples had children at the time of PGT-M consultation. 11 couples (46%) had a termination of pregnancy (TOP) due to VUS; three solely for the VUS and eight for VUS accompanying a sonographic finding in a prenatal screening. Indications for detecting the VUS were as follows- 33% amniocentesis for maternal request not otherwise indicated, 50% amniocentesis for a prenatal ultrasound finding, 25% had a CMA testing due to an affected child and another 12.5% for a CMA finding on a previously aborted fetus. Out of 30 VUS detected, 33% were defined VUS, 26% likely benign VUS and 40% likely pathogenic VUS. 19 VUS (63%) were microduplications (size ranging 31-2800Kbp) and 11 (37%) were microdeletions (size ranging 57-1200Kbp). Reviewing VUS classifications up to date- 33% remained unchanged, 20% were more severely defined and 46% were less severely defined. Based on genetic counseling, PGT-M was assigned for 17/30 CNVs (56%), 13(76.4%) of which were isolated VUS. 11/13 cases of isolated VUS were performed to date with a mean number of: 1.5 IVF cycles per couple (1-3), 8.3 embryos biopsied (1-15) and a mean of 2.9 wild type embryos per couple (0-6). Of 6 couples who had an embryo transfer at our institution there were 4 clinical pregnancies. Conclusions The genomic era allows the detection of varies VUS whose definition is submitted to changes as more information is gathered. Couples are already performing TOP and requesting PGT-M for VUS including likely benign VUS. It is thus of great importance to carefully use guidelines for the interpretation of these findings while counseling couples and determining the justification to practice PGT-M for their VUS finding.

Item does not contain fulltext Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.

Objective:To identify the genetic basis of a recessive congenital neurologic syndrome characterized by severe hypotonia, arthrogryposis, and respiratory failure.Methods:Identification of the responsible gene by exome sequencing and assessment of the effect of the mutation on protein stability in transfected rat neuronal-like PC12A123.7 cells.Results:Two brothers from a nonconsanguineous Yemeni Jewish family manifested at birth with severe hypotonia and arthrogryposis. The older brother died of respiratory failure at 5 days of age. The proband, now 4.5 years old, has been mechanically ventilated since birth with virtually no milestones achievement. Whole exome sequencing revealed homozygosity of SLC18A3 c.1078G>C, p.Gly360Arg in the affected brothers but not in other family members. SLC18A3 p.Gly360Arg is not reported in world populations but is present at a carrier frequency of 1:30 in healthy Yemeni Jews. SLC18A3 encodes the vesicular acetylcholine transporter (VAChT), which loads newly synthesized acetylcholine from the neuronal cytoplasm into synaptic vesicles. Mice that are VAChT-null have been shown to die at birth of respiratory failure. In human VAChT, residue 360 is located in a conserved region and substitution of arginine for glycine is predicted to disrupt proper protein folding and membrane embedding. Stable transfection of wild-type and mutant human VAChT into neuronal-like PC12A123.7 cells revealed similar mRNA levels, but undetectable levels of the mutant protein, suggesting post-translational degradation of mutant VAChT.Conclusion:Loss of function of VAChT underlies severe arthrogryposis and respiratory failure. While most congenital myasthenic syndromes are caused by defects in postsynaptic proteins, VAChT deficiency is a presynaptic myasthenic syndrome.

Nat. Genet. 49, 223–237 (2017); published online 19 December 2016; corrected after print 20 April 2017 Following publication of this article, the authors were asked to remove a clinical image and some video footage of one of the affected individuals. Although consent was obtained, in keeping with their ethical consent framework, the authors allow for withdrawal of consent and are carrying out the wishes of the research subjects under their consent process.