Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage.

Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes1-6. In this study, using single-cell RNA sequencing, we investigated genome editing outcomes in primary human T cells transfected with CRISPR-Cas9 and guide RNAs targeting genes for TCR chains and programmed cell death protein 1. Four days after transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells and a chromosome 14 gain in up to 1.4% of the cells. Chromosome 7, harboring the TCRß locus, was truncated in 9.9% of the cells. Aberrations were validated using fluorescence in situ hybridization and digital droplet PCR. Aneuploidy was associated with reduced proliferation, induced p53 activation and cell death. However, at 11 days after transfection, 0.9% of T cells still had a chromosome 14 loss. Aneuploidy and chromosomal truncations are, thus, frequent outcomes of CRISPR-Cas9 cleavage that should be monitored and minimized in clinical protocols.

Hyper IgE Syndrome in an Isolated Population in Israel.

Introduction: Hyper IgE syndromes (HIES) are a group of rare primary immunodeficiency characterized by high levels of serum IgE, cold abscesses, pulmonary infections, and eczema. ZNF341 deficiency was described in 2018 in 11 patients clinically diagnosed previously with HIES. Eight of those patients, all offspring of consanguineous couples, are from three families who live in a Muslim village in Israel which has approximately 15,000 residents. Objective: Our study aimed to evaluate the prevalence of ZNF341 mutation in the population of the village. Methods: Three hundred DNA samples of females were included in the study. The samples belong to females that were referred to the Meir Medical Center for prenatal genetic testing before pregnancy, during 2017-2019: 200 samples were from the village, and 100 samples of Muslim females were from other villages.All samples were tested by Sanger sequencing for the ZNF341 mutation (c.904C>T, NM_001282933.1). Results: Heterozygous nonsense mutation in ZNF341 was found in ten samples (5%) of the study group compared to zero in the control group (p<0.01). Conclusion: The carrier frequency of the mutation in ZNF341 in the studied village population is 1:20. This high frequency is probably due to founder mutation and consanguineous marriages.

Recurring pathogenic variants in the BRCA2 gene in the Ethiopian Jewish population. Founder mutations?

Mutations in the BRCA1 and BRCA2 genes increase the risk for various cancers including breast, ovarian, prostate, pancreas and melanoma. Identifying BRCA1/2 mutation carriers enables risk assessment, surveillance, early detection and risk reduction. In certain Israeli sub-populations recurring and founder mutations have been identified and for these, testing for founder mutations is simple, efficient and cost-effective. Founder mutations in the Jewish Ethiopian population have not been described. We report here the identification of a recurring BRCA2 mutation in the Ethiopian Jewish population; c.5159C>A; p.Ser1720Ter, which has only been described once before in this population. In addition, in another family of the same origin we found the BRCA2 c.7579delG; p.Val2527Ter mutation that has been previously described in two different Jewish Ethiopian families. In Israel genetic testing is performed in a sequential stepwise manner, first testing a panel of predominant mutations and if negative further testing by gene sequencing is offered. Recently it has been decided to expand the founder mutation panel to include mutations which have been found in two or more separate families. This new panel will include the BRCA2 c.7579delG; p.Val2527Ter mutation, and we recommend that the BRCA2 c.5159C>A; p.Ser1720Ter mutation should also be added to the new predominant mutation panel.

Prenatal diagnosis of a likely pathogenic variant in ZBTB18: Natural evolution of fetal phenotype including the long bones and corpus callosum.

Pathogenic variants in ZBTB18 gene have been described only postnatally with a variable phenotypic spectrum that includes intellectual disability, microcephaly, hypotonia, poor growth, corpus callosum abnormalities, seizures, and dysmorphic facial features. These features overlap with the phenotype of 1q43-q44 deletion syndrome (OMIM #612337). There are several genes within the 1q43-q44 deletion region, and ZBTB18 is of particular interest due to its known involvement in neuronal differentiation and migration. We describe here a fetus presenting with an intrauterine growth restriction, diminished long bones growth, single umbilical artery, and a short corpus callosum. On mid pregnancy ultrasound, all biometric parameters including the corpus callosum were relatively small but still within the normal range. Only a targeted follow-up during the third trimester, including neurosonographic and MRI exams, revealed the full extent of the malformation, leading to amniocentesis and a genetic workup that led to the identification of a de novo likely pathogenic variant in ZBTB18 gene. This is the first description of the evolving phenotype of a ZBTB18-related disorder in a fetus, which emphasizes the challenging diagnosis of subtle findings, that mandates a high level of clinical suspicion and a targeted follow-up throughout pregnancy.

The yield of chromosomal microarray analysis among pregnancies terminated due to fetal malformations.

BACKGROUND: Chromosomal microarray analysis (CMA) is preferred for genetic work-up when fetal malformations are detected prenatally. OBJECTIVES: To assess the detection rate of CMA after pregnancy termination due to abnormal ultrasound findings. METHODS: CMA was successfully performed in 71 pregnancies using fetal DNA (mainly from skin) or placenta. Data regarding clinical background, pregnancy work-up, and CMA were analyzed. RESULTS: Findings were abnormal in 17 cases (23.9%), of which 13 were detectable by karyotype. The incremental yield of CMA was 4/71 (5.6%); 1/32 (3.1%) for cases with an isolated anomaly and 3/39 (7.7%) for cases with nonisolated anomalies. CONCLUSIONS: CMA yield from terminated pregnancies was 23.9%. Although most chromosomal abnormalities are detectable by karyotype, CMA does not require viable dividing cells; hence, it is more practical for work-up after termination. In most cases, the diagnosis was followed by consultation regarding the risk of recurrence and recommendations for testing in subsequent pregnancies.

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.

Diagnostic yield of multigene panel testing in an Israeli cohort: enrichment of low-penetrance variants.

BACKGROUND: Carriers of pathogenic variants (PVs) in moderate-high-penetrance cancer susceptibility genes are offered tailored surveillance schemes for early cancer diagnosis. The clinical implications of low-penetrance variant carriers are less clear. METHODS: Clinical and demographic data were retrieved for a cohort of Israeli individuals who underwent oncogenetic testing by the 30-gene cancer panel at Color Genomics laboratory, between 04/2013 and 12/2018. RESULTS: Of 758 genotyped individuals, 504 had been diagnosed with cancer prior to testing: 283 (56%) had breast cancer and 106 (21%) colorectal cancer. Pathogenic or likely pathogenic (P/LP) variants were detected in 123 (16%) individuals. Overall, 44 different P/LP variants were detected in 18/30 cancer susceptibility genes; 20 of them were founder/recurrent mutations. Of the carriers, 39 (32%), 10 (8%), and 74 (60%) carried high-, moderate-, or low-penetrance variants, respectively. After excluding low-penetrance variants, 7% (33/504) of all cancer patients, 6% of breast or ovarian cancer patients were found to be carriers, as well as 7% (14/203) of individuals with colonic polyps, and 4% (11/254) of cancer-free individuals. CONCLUSIONS: The diagnostic yield of moderate- and high-penetrance PVs using multigene panel testing was 6%, with 3.7% carriers of non-recurrent PVs. This yield should be discussed during pre-test counseling, and emphasizes the need for harmonized recommendations regarding clinical implications of low-penetrance variants.

NFIB Haploinsufficiency Is Associated with Intellectual Disability and Macrocephaly.

The nuclear factor I (NFI) family of transcription factors play an important role in normal development of multiple organs. Three NFI family members are highly expressed in the brain, and deletions or sequence variants in two of these, NFIA and NFIX, have been associated with intellectual disability (ID) and brain malformations. NFIB, however, has not previously been implicated in human disease. Here, we present a cohort of 18 individuals with mild ID and behavioral issues who are haploinsufficient for NFIB. Ten individuals harbored overlapping microdeletions of the chromosomal 9p23-p22.2 region, ranging in size from 225 kb to 4.3 Mb. Five additional subjects had point sequence variations creating a premature termination codon, and three subjects harbored single-nucleotide variations resulting in an inactive protein as determined using an in vitro reporter assay. All individuals presented with additional variable neurodevelopmental phenotypes, including muscular hypotonia, motor and speech delay, attention deficit disorder, autism spectrum disorder, and behavioral abnormalities. While structural brain anomalies, including dysgenesis of corpus callosum, were variable, individuals most frequently presented with macrocephaly. To determine whether macrocephaly could be a functional consequence of NFIB disruption, we analyzed a cortex-specific Nfib conditional knockout mouse model, which is postnatally viable. Utilizing magnetic resonance imaging and histology, we demonstrate that Nfib conditional knockout mice have enlargement of the cerebral cortex but preservation of overall brain structure and interhemispheric connectivity. Based on our findings, we propose that haploinsufficiency of NFIB causes ID with macrocephaly.

Schizophrenia and Nail Patella Syndrome: The Dopamine Connection.

BACKGROUND: Nail-patella syndrome (NPS) is characterized by changes in the nails, knees, and elbows, as well as the presence of iliac horns detected by X-ray of the pelvis. A higher occurrence of psychiatric disorders has also been suggested in NPS. Heterozygous mutations in the gene encoding the LIM-homeodomain transcription factor (LMX1B) are identified in most patients with typical clinical findings of NPS. OBJECTIVES: To report on the association between NPS and schizophrenia. METHODS: Genomic DNA were isolated from a patient's venous blood and collected on ethylenediaminetetraacetic 5% with the Gentra Puregene Blood Kit. All exons and flanking regions of the LMX1B gene (LMX1B: NM_001174146.1) were amplified by standard polymerase chain reaction and analyzed by direct DNA sequencing with BigDye Terminators on an ABI 3100 sequencer. Sequence chromatograms were analyzed using SeqScape software version 1.1. Mutation analysis and characterization of variants was performed with the Alamut Software Version 2.1. RESULTS: We report a patient presenting to the psychiatry department with schizophrenia. Clinical examination revealed characteristic findings consistent with NPS. Since NPS was suspected, based on clinical findings, sequencing of all coding exons of LMX1B gene was completed. Results revealed a novel heterozygous mutation in the proband: c.546_547insACCG(het); p.Glu183Thrfs*11. CONCLUSIONS: Based on LMX1B expression in brain regions that are implicated in neuropsychiatric illness, and especially in the development of dopaminergic neurons, we hypothesize that schizophrenia may be part of the clinical spectrum of NPS.

The association between maternal serum first trimester free ßhCG, second trimester intact hCG levels and foetal growth restriction and preeclampsia.

The purpose of this study was to analyse the association between free beta hCG (fßhCG) increased levels and pregnancy complications (PC), foetal growth restriction (FGR) and preeclampsia (PE). This connection was evaluated in two stages (i) investigating the association between those PC with first trimester fßhCG and second trimester intact hCG (ihCG), and (ii) studying the association between these two analytes in the same pregnancy. This was a retrospective study in two settings: medical centre that provided data on fßhCG and ihCG levels in pregnancies with FGR and PE, and central laboratory that provided fßhCG and ihCG levels that were compared in the same pregnancy. No association was found between those PC and the hCG analytes, except for elevated ihCG levels and FGR. Elevated fßhCG (>3.00 MoM) was found in 570/16,849 (3.4%) women. However, only 14% of whom had elevated second trimester ihCG. A positive correlation was found between the magnitude of first trimester fßhCG levels and the percentage of women who had elevated second trimester ihCG. This association was determined by the magnitude of the elevation of fßhCG levels. Impact statement What is already known on this subject: The two analytes, first trimester fßhCG and second trimester ihCG, are independently produced and parameters of the biochemical screening during pregnancy. What the results of this study add: Referring to 3.00 MoM as cut-off levels, most pregnancies with elevated levels of first trimester fßhCG will have normal ihCG second trimester levels. What the implications are of these findings for clinical practice and/or further research: The risk of developing pregnancy complications, FGR and PE should be associated with second trimester ihCG levels. About 3.5% of women had high fßhCG levels during the first trimester. However, only 14% also had increased ihCG levels, defined as >3.00 MoM; additional studies are needed to explore the association between increased first trimester fßhCG levels and the risk of developing pregnancy complications, independent of ihCG levels in the second trimester.

[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.

The 2017 international classification of the Ehlers-Danlos syndromes.

The Ehlers-Danlos syndromes (EDS) are a clinically and genetically heterogeneous group of heritable connective tissue disorders (HCTDs) characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Over the past two decades, the Villefranche Nosology, which delineated six subtypes, has been widely used as the standard for clinical diagnosis of EDS. For most of these subtypes, mutations had been identified in collagen-encoding genes, or in genes encoding collagen-modifying enzymes. Since its publication in 1998, a whole spectrum of novel EDS subtypes has been described, and mutations have been identified in an array of novel genes. The International EDS Consortium proposes a revised EDS classification, which recognizes 13 subtypes. For each of the subtypes, we propose a set of clinical criteria that are suggestive for the diagnosis. However, in view of the vast genetic heterogeneity and phenotypic variability of the EDS subtypes, and the clinical overlap between EDS subtypes, but also with other HCTDs, the definite diagnosis of all EDS subtypes, except for the hypermobile type, relies on molecular confirmation with identification of (a) causative genetic variant(s). We also revised the clinical criteria for hypermobile EDS in order to allow for a better distinction from other joint hypermobility disorders. To satisfy research needs, we also propose a pathogenetic scheme, that regroups EDS subtypes for which the causative proteins function within the same pathway. We hope that the revised International EDS Classification will serve as a new standard for the diagnosis of EDS and will provide a framework for future research purposes. © 2017 Wiley Periodicals, Inc.

Familial Occurrence of Atrioventricular Nodal Reentrant Tachycardia.

BACKGROUND: Atrioventricular nodal reentrant tachycardia (AVNRT) is considered a sporadic disease occurring in ≈22.5 cases per 10 000 in the general population. We define the prevalence and characteristics of familial AVNRT among patients who underwent radiofrequency ablation. METHODS AND RESULTS: Ablation reports of all patients with familial AVNRT (at least 2 first-degree family members) who underwent radiofrequency ablation in our institution and in another hospital were reviewed. There were 1587 patients from our institution, of whom 20 had ≥1 first-degree relatives with AVNRT. This indicates a familial AVNRT prevalence of 127 cases per 10 000 (95% confidence interval, 82-196/10 000). First-degree relatives of patients with AVNRT presented a hazard ratio of at least 3.6 for exhibiting AVNRT compared with the general population. After inclusion of 4 families with familial AVNRT who underwent ablation at another hospital our population study comprised a total of 24 families (50 patients) with AVNRT. Patients at ablation were younger in the familial AVNRT group when compared with the sporadic AVNRT group (44.2±19 versus 54.8±18 years old, P=0.0001). The male/female ratio was similar, with female predominance. The supraventricular tachycardia mechanism was typical slow/fast reentry in most cases in both groups. The most common familial relationship in our 24 families included a parent and a child in 67% of cases and less often 2 siblings (29%). CONCLUSIONS: Familial AVNRT prevalence is higher than previously believed suggesting that this arrhythmia may have a genetic component. Autosomal dominance with incomplete penetrance is the most likely mode of inheritance.

Periodontal Ehlers-Danlos Syndrome Is Caused by Mutations in C1R and C1S, which Encode Subcomponents C1r and C1s of Complement.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.

Congenital dilated cardiomyopathy caused by biallelic mutations in Filamin C.

In the vast majority of pediatric patients with dilated cardiomyopathy, the specific etiology is unknown. Studies on families with dilated cardiomyopathy have exemplified the role of genetic factors in cardiomyopathy etiology. In this study, we applied whole-exome sequencing to members of a non-consanguineous family affected by a previously unreported congenital dilated cardiomyopathy syndrome necessitating early-onset heart transplant. Exome analysis identified compound heterozygous variants in the FLNC gene. Histological analysis of the cardiac muscle demonstrated marked sarcomeric and myofibrillar abnormalities, and immunohistochemical staining demonstrated the presence of Filamin C aggregates in cardiac myocytes. We conclude that biallelic variants in FLNC can cause congenital dilated cardiomyopathy. As the associated clinical features of affected patients are mild, and can be easily overlooked, testing for FLNC should be considered in children presenting with dilated cardiomyopathy.

Intellectual disability and non-compaction cardiomyopathy with a de novo NONO mutation identified by exome sequencing.

Pathogenic variants in the NONO gene have been most recently implicated in X-linked intellectual disability syndrome. This observation has been supported by studies of NONO-deficient mice showing that NONO has an important role in regulating inhibitory synaptic activity. Thus far, the phenotypic spectrum of affected patients remains limited. We applied whole exome sequencing to members of a family in which the proband was presented with a complex phenotype consisting of developmental delay, dysmorphism, and non-compaction cardiomyopathy. Exome analysis identified a novel de novo splice-site variant c.1171+1G>T in exon 11 of NONO gene that is suspected to abolish the donor splicing site. Thus, we propose that the phenotypic spectrum of NONO-related disorder is much broader than described and that pathogenic variants in NONO cause a recognizable phenotype.

Exome sequencing identified a novel de novo OPA1 mutation in a consanguineous family presenting with optic atrophy.

Inherited optic neuropathies are a heterogeneous group of disorders characterized by mild to severe visual loss, colour vision deficit, central or paracentral visual field defects and optic disc pallor. Optic atrophies can be classified into isolated or non-syndromic and syndromic forms. While multiple modes of inheritance have been reported, autosomal dominant optic atrophy and mitochondrial inherited Leber's hereditary optic neuropathy are the most common forms. Optic atrophy type 1, caused by mutations in the OPA1 gene is believed to be the most common hereditary optic neuropathy, and most patients inherit a mutation from an affected parent. In this study we used whole-exome sequencing to investigate the genetic aetiology in a patient affected with isolated optic atrophy. Since the proband was the only affected individual in his extended family, and was a product of consanguineous marriage, homozygosity mapping followed by whole-exome sequencing were pursued. Exome results identified a novel de novo OPA1 mutation in the proband. We conclude, that though de novo OPA1 mutations are uncommon, testing of common optic atrophy-associated genes such as mitochondrial mutations and OPA1 gene sequencing should be performed first in single individuals presenting with optic neuropathy, even when dominant inheritance is not apparent.

Exome sequencing identified mutations in CASK and MYBPC3 as the cause of a complex dilated cardiomyopathy phenotype.

Whole-exome sequencing for clinical applications is now an integral part of medical genetics practice. Though most studies are performed in order to establish diagnoses in individuals with rare and clinically unrecognizable disorders, due to the constantly decreasing costs and commercial availability, whole-exome sequencing has gradually become the initial tool to study patients with clinically recognized disorders when more than one gene is responsible for the phenotype or in complex phenotypes, when variants in more than one gene can be the cause for the disease. Here we report a patient presenting with a complex phenotype consisting of severe, adult-onset, dilated cardiomyopathy, hearing loss and developmental delay, in which exome sequencing revealed two genetic variants that are inherited from a healthy mother: a novel missense variant in the CASK gene, mutations in which cause a spectrum of neurocognitive manifestations, and a second variant, in MYBPC3, that is associated with hereditary cardiomyopathy. We conclude that although the potential for co-occurrence of rare diseases is higher when analyzing undefined phenotypes in consanguineous families, it should also be given consideration in the genetic evaluation of complex phenotypes in non-consanguineous families.

Terminal microdeletions of 13q34 chromosome region in patients with intellectual disability: Delineation of an emerging new microdeletion syndrome.

The increasing use of chromosomal microarray studies in patients with intellectual disability has led to the description of new microdeletion and microduplication syndromes. We report terminal microdeletions in 13q34 chromosome region in 5 adult patients of two unrelated families. Patients harboring 13q34 microdeletions display common clinical features, including intellectual disability, obesity, and mild facial dysmorphism. These individuals can become fairly self-sufficient, however they do not live independently, and require community and social support. Further systematic analysis of the genes comprised in the deleted region will allow the identification of genes whose haploinsufficiency is expected to lead to disease manifestations, in particular intellectual disability.