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Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

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

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Maroteaux-Lamy syndrome (MPS-VI) is a rare autosomal-recessive disorder with a wide spectrum of clinical manifestations, ranging from an attenuated to a rapidly progressive disease. It is caused by variants in ARSB, which encodes the lysosomal arylsulfatase B (ARSB) enzyme, part of the degradation process of glycosaminoglycans in lysosomes. Over 220 variants have been reported so far, with a majority of missense variants. We hereby report two siblings of Bedouin origin with a diagnosis of MPS-VI. Western blots in patient fibroblasts revealed total absence of ARSB protein production. Complete sequencing of the coding region of ARSB did not identify a candidate disease-associated variant. However, deep sequencing of the noncoding region of ARSB by whole genome sequencing (WGS) revealed a c.1142+581A to G variant. The variant is located within intron 5 and fully segregated with the disease in the family. Determination of the genetic cause for these patients enabled targeted treatment by enzyme replacement therapy, along with appropriate genetic counseling and prenatal diagnosis for the family. These results highlight the advantage of WGS as a powerful tool, for improving the diagnostic rate of rare disease-causing variants, and emphasize the importance of studying deep intronic sequence variation as a cause of monogenic disorders.

BackgroundThe molecular basis of heterotaxy and congenital heart malformations associated with disruption of left–right asymmetry is broad and heterogenous, with over 25 genes implicated in its pathogenesis thus far.ObjectiveWe sought to elucidate the molecular basis of laterality disorders and associated congenital heart defects in a cohort of 30 unrelated probands of Arab–Muslim descent, using next-generation sequencing techniques.MethodsDetailed clinical phenotyping followed by whole-exome sequencing (WES) was pursued for each of the probands and their parents (when available). Sanger sequencing was used for segregation analysis of disease-causing mutations in the families.ResultsUsing WES, we reached a molecular diagnosis for 17 of the 30 probands (56.7%). Genes known to be associated with heterotaxy and/or primary ciliary dyskinesia, in which homozygous pathogenic or likely pathogenic variants were detected, included CFAP53 (CCDC11), CFAP298 (C21orf59), CFAP300, LRRC6, GDF1, DNAAF1, DNAH5, CCDC39, CCDC40, PKD1L1 and TTC25. Additionally, we detected a homozygous disease causing mutation in DAND5, as a novel recessive monogenic cause for heterotaxy in humans. Three additional probands were found to harbour variants of uncertain significance. These included variants in DNAH6, HYDIN, CELSR1 and CFAP46.ConclusionsOur findings contribute to the current knowledge regarding monogenic causes of heterotaxy and its associated congenital heart defects and underscore the role of next-generation sequencing techniques in the diagnostic workup of such patients, and especially among consanguineous families.

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that regulates numerous biological processes. PPP2R1A encodes the scaffolding "Aα" subunit of PP2A. To date, nearly 40 patients have been previously reported with 19 different pathogenic PPP2R1A variants, with phenotypes including intellectual disability, developmental delay, epilepsy, infant agenesis/dysgenesis of the corpus callosum, and dysmorphic features. Apart from a single case, severe congenital heart defects (CHD) have not been described. We report four new unrelated individuals with pathogenic heterozygous PPP2R1A variants and CHD and model the crystal structure of several variants to investigate mechanisms of phenotype disparity. Individuals 1 and 2 have a previously described variant (c.548GA, p.R183Q) and similar phenotypes with severe ventriculomegaly, agenesis/dysgenesis of the corpus callosum, and severe CHD. Individual 3 also has a recurrent variant (c.544CT, p.R182W) and presented with agenesis of corpus callosum, ventriculomegaly, mild pulmonic stenosis, and small patent foramen ovale. Individual 4 has a novel variant (c.536CA, p.P179H), ventriculomegaly, and atrial septal defect. To conclude, we propose expansion of the phenotype of PPP2R1A neurodevelopmental disorder to include CHD. Further, the R183Q variant has now been described in three individuals, all with severe neurologic abnormalities, severe CHD, and early death suggesting that this variant may be particularly deleterious.

Genetic diagnostic tools including whole-exome sequencing (WES) have advanced our understanding in human diseases and become common practice in diagnosing patients with suspected primary immune deficiencies. Establishing a genetic diagnosis is of paramount importance for tailoring adequate therapeutic regimens, including identifying the need for hematopoietic stem cell transplantation (HSCT) and genetic-based therapies. Here, we genetically studied two adult patients who were clinically diagnosed during infancy with severe combined immune deficiency (SCID). Two unrelated patients, both of consanguineous kindred, underwent WES in adulthood, 2 decades after their initial clinical manifestations. Upon clinical presentation, immunological workup was performed, which led to a diagnosis of SCID. The patients presented during infancy with failure to thrive, generalized erythematous rash, and recurrent gastrointestinal and respiratory tract infections, including episodes of Pneumocystis pneumonia infection and Candida albicans fungemia. Hypogammaglobulinemia and T-cell lymphopenia were detected. Both patients were treated with a 10/10 HLA matched sibling donor unconditioned HSCT. Retrospective genetic workup revealed homozygous bi-allelic mutations in IL7RA in one patient and in RAG2 in the other. Our study exemplifies the impact of retrospectively establishing a genetic diagnosis. Pinpointing the genetic cause raises several issues including optimized surveillance and treatment, understanding disease mechanisms and outcomes, future family planning, and social and psychological considerations.

The genetic basis of congenital heart malformations associated with disruption of left-right (L-R) asymmetry is broad and heterogenous, with variants in over 25 genes implicated thus far. Of these, deleterious variants in the Growth/Differentiation Factor 1 (GDF1) gene have been shown to cause heterotaxy with varied complex heart malformations of left-right patterning, in 23 individuals reported to date, either in monoallelic or biallelic state. We report three unrelated individuals exhibiting right isomerism with congenital heart defects, each originating from a consanguineous kindred of Arab-Muslim descent. Using whole exome sequencing, a shared novel homozygous truncating c.608G > A (p.W203*) variant in the GDF1 gene was revealed as the molecular basis of their disease. Subsequently, targeted sequencing of this variant showed full segregation with the disease in these families, with a total of over 15 reportedly affected individuals, enabling genetic counseling, prenatal diagnosis, and planning of future pregnancies. Our findings further confirm the association of biallelic GDF1 variants, heterotaxy and congenital heart defects of left-right patterning, and expand the previously described phenotypic spectrum and mutational profile. Moreover, we suggest targeted screening for the p.W203* variant in relevant clinical circumstances.

BACKGROUND AND AIMS Genetic etiologies are estimated to affect ∼10% of adults with advanced CKD. However, significant population disparities in genetic kidney disease exist and population-based screening are lacking especially among minority groups. The Druze population is a middle-eastern minority with high rates of end-stage renal disease (ESRD) and consanguinity, which suggests high rates of population-specific monogenic CKD etiologies. We therefore hypothesized that by exome sequencing we will identify a unique distribution of monogenic ESRD causes as compared with prior studies and that important ramifications for clinical practice may ensue. METHOD We initiated a national multicenter study of all Israeli dialysis units in order to provide comprehensive evidence for ESRD's genetic basis (Israeli ESRD Genetic Consortium Cohort). Specifically, during 2020, we recruited 97% (n = 94) of Israeli Druze individuals on dialysis from 12 different hospitals and community-based units. We conducted exome sequencing and diagnostic analysis for all patients. We assessed the diagnostic yield of genetic analysis and its relation to baseline clinical phenotypes. RESULTS Overall, the cohort consists of 94 individuals from 91 different families with first-degree consanguinity rate of 28%. Participants mean age was 62 years (ranging from 18 to 88 years). Most common primary clinical diagnoses were diabetic kidney disease, nephropathy of unknown origin, glomerular or cystic kidney disease, together encompassing 90% of all cases. None had previous molecular diagnosis. Using exome sequencing, we identified a genetic etiology in 27 out of 94 (28.7%) participants. We identified WDR19 (NPHP13) homozygous mutation c.878G > A (p.C293Y) as the most common genetic diagnosis (5.3%). Follow-up clinical characterization revealed that all affected individuals exhibited non-syndromic adulthood-onset ESRD, supporting a profound mutation-dependent phenotypic heterogeneity and weak pre-exome phenotypic specificity. Other prevalent genetic diagnoses included type IV collagen, PKD1, PKD2 and UMOD mutations as well as monogenic forms of diabetes and hyperlipidemia. Molecular diagnosis corresponded to the pre-exome clinical diagnosis in only one-third of the participants. CONCLUSION Exome sequencing in Druze with ESRD yields a genetic diagnosis in just

BACKGROUND Chronic kidney disease in children is estimated to be secondary to a monogenic etiology in ∼20% of patients and can arise from mutations in a multitude of different single-gene causes. Still, data are lacking on the true prevalence of genetic etiologies dates from a large scale unbiased population screen of children with advanced kidney disease. METHOD In order to provide comprehensive real-world evidence for monogenic etiologies of childhood end-stage kidney disease—on a national level—we initiated a nation-wide multicenter study of all pediatric Israeli dialysis units. Specifically, between 2020 and 2021, we recruited ∼90% (n = 66) of children on dialysis from all six dialysis units treating children in Israel. We conducted exome sequencing and diagnostic analysis for all patients. We assessed the diagnostic yield of genetic analysis and its relation to baseline clinical phenotypes. RESULTS Overall, the cohort comprises 66 individuals from different families with a first-degree consanguinity rate of 47%. Participants' mean age at renal replacement therapy initiation was 8.1 years (range 1-month to 20-years). Using exome sequencing we identified a genetic etiology in 29 out of 66 (44%) participants. The most common subgroup of diagnostic variants was in genes causing renal cystic ciliopathies (e.g. NPHP1, NPHP4, PKHD1 and BBS9), which together explain 31% of all monogenic etiologies. This was followed by mutations in genes causing CAKUT (e.g. EYA1, HNF1B, PAX2, COL4A1 and GREB1L), steroid-resistant nephrotic syndrome (e.g. LAGE3, NPHS1, NPHS2, LMX1B, SMARCAL1 and CRB2) and tubulopathies (e.g. CTNS, AQP2), which explain 21%, 21% and 17% of all genetic etiologies, respectively. The yield of exome sequencing was higher among non-Jewish compared with Jewish individuals (52% versus 29%) and in children from consanguineous families compared with non-consanguineous families (56% versus 31%). The final molecular diagnosis did not correspond with the pre-exome clinical diagnosis in 17% of cases. CONCLUSION Exome sequencing in an unbiased pediatric cohort with end-stage kidney disease yields a genetic diagnosis in 44% of cases and reveals many underappreciated monogenic etiologies. Surprisingly, renal cystic ciliopathies causing-genes were more common than CAKUT genes in our cohort. These results emphasize the importance of genetic testing among children with advanced chronic kidney disease and validate the role of exome sequencing as a standard routine diagnostic tool.

Purpose: Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important regulator of necroptosis and inflammatory responses. We present the clinical features, genetic analysis and immune work-up of two patients with infantile-onset inflammatory bowel disease (IBD) resulting from novel RIPK1 mutations.Methods: Whole exome and Sanger sequencing was performed in two IBD patients. Mass cytometry time of flight (CyTOF) was conducted for in-depth immunophenotyping on one of the patient’s peripheral blood mononuclear cells, and compared to control subjects and patients with Crohn’s disease.Results: The patients presented with severe colitis and perianal fistulas in the first months of life, without severe/atypical infections. Genetic studies identified novel and pathogenic genetic variants in RIPK1. Using CyTOF unbiased clustering analysis, we identified peripheral immune dysregulation in one of these patients, characterized by an increase in IFNγ CD8+ T cells along with a decrease in monocytes, dendritic cells and B cells. Moreover, RIPK1-deficient patient’s immune cells exhibited decreased IL-6 production in response to LPS across multiple cell types including T cells, B cells and innate immune cells. Summary: Mutations in RIPK1 should be considered in very young patients presenting with colitis and perianal fistulas. Given RIPK1’s role in inflammasome activation, but also in epithelial cells, it is unclear whether IL1 blockade or allogeneic hematopoietic stem cell transplantation can suppress or cure the hyper-inflammatory response in these patients. Additional studies in humans are required to better define the role of RIPK1 in regulating intestinal immune responses, and how treatment can be optimized for patients with RIPK1 deficiency.

During the process of generating diverse T and B cell receptor (TCR and BCR, respectively) repertoires, double-strand DNA breaks are produced. Subsequently, these breaks are corrected by a complex system led by the non-homologous end-joining (NHEJ). Pathogenic variants in genes involved in this process, such as theTo provide new clinical and immunological insights on NHEJ1 deficiency arising from a newly diagnosed patient with severe immunodeficiency.A male infant, born to consanguineous parents, suspected of having primary immunodeficiency underwent immunological and genetic workup. This included a thorough assessment of T cell phenotyping and lymphocyte activation by mitogen stimulation tests, whole-exome sequencing (WES), TCR repertoire Vβ repertoireClinical findings included microcephaly, recurrent pneumonia, and failure to thrive. An immune workup revealed lymphopenia, reduced T cell function, and hypogammaglobulinemia. Skewed TCR Vβ repertoire, TCR gamma (TRG) repertoire, and BCR repertoire were determined in the patient. Genetic analysis identified a novel homozygous missense pathogenic variant inA novel

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

The role of lysine methyltransferases (KMTs) and demethylases (KDMs) in the regulation of chromatin modification is well-established. Recently, deleterious heterozygous variants in

Purpose: The clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis. Methods: Clinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants. Results: In this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments. Conclusion: Our data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.

Exome sequencing (ES) is an important diagnostic tool for individuals with neurodevelopmental disorders (NDD) and/or multiple congenital anomalies (MCA). However, the cost of ES limits the test's accessibility for many patients. We evaluated the yield of publicly funded clinical ES, performed at a tertiary center in Israel, over a 3-year period (2018–2020). Probands presented with (1) moderate-to-profound global developmental delay (GDD)/intellectual disability (ID); or (2) mild GDD/ID with epilepsy or congenital anomaly; and/or (3) MCA. Subjects with normal chromosomal microarray analysis who met inclusion criteria were included, totaling 280 consecutive cases. Trio ES (proband and parents) was the default option. In 252 cases (90.0%), indication of NDD was noted. Most probands were males (62.9%), and their mean age at ES submission was 9.3 years (range 1 month to 51 years). Molecular diagnosis was reached in 109 probands (38.9%), mainly due to de novo variants (91/109, 83.5%). Disease-causing variants were identified in 92 genes, 15 of which were implicated in more than a single case. Male sex, families with multiple-affected members and premature birth were significantly associated with lower ES yield (p

Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.

Genetic kidney diseases contribute a significant portion of kidney diseases in children and young adults. Nephrogenetics is a rapidly evolving subspecialty; however, in the clinical setting, increased use of genetic testing poses implementation challenges. Consequently, we established a national nephrogenetics clinic to apply a multidisciplinary model.Patients were referred from different pediatric or adult nephrology units across the country if their primary nephrologist suspected an undiagnosed genetic kidney disease. We determined the diagnostic rate and observed the effect of diagnosis on medical care. We also discuss the requirements of a nephrogenetics clinic in terms of logistics, recommended indications for referral, and building a multidisciplinary team.Over 24 months, genetic evaluation was completed for a total of 74 unrelated probands, with an age range of 10 days to 72 years. The most common phenotypes included congenital anomalies of the kidneys and urinary tract, nephrotic syndrome or unexplained proteinuria, nephrocalcinosis/nephrolithiasis, tubulopathies, and unexplained kidney failure. Over 80% of patients were referred due to clinical suspicion of an undetermined underlying genetic diagnosis. A molecular diagnosis was reached in 42/74 probands, yielding a diagnostic rate of 57%. Of these, over 71% of diagnoses were made via next generation sequencing (gene panel or exome sequencing).We identified a substantial fraction of genetic kidney etiologies among previously undiagnosed individuals which influenced subsequent clinical management. Our results support that nephrogenetics, a rapidly evolving field, may benefit from well-defined multidisciplinary co-management administered by a designated team of nephrologist, geneticist, and bioinformatician. A higher resolution version of the Graphical abstract is available as Supplementary information.

Introduction: Hemophilia A (HA) is an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency or dysfunction due to F8 gene mutations. HA carriers are usually asymptomatic because their FVIII levels correspond to approximately half of the concentration found in healthy individuals. However, in rare cases, a carrier may exhibit symptoms of moderate to severe HA primarily due to skewed inactivation of her non-hemophilic X chromosome. Aim: The aim of the study was to investigate X-chromosome inactivation (XCI) patterns in HA carriers, with special emphasis on three karyotypically normal HA carriers presenting with moderate to severe HA phenotype due to skewed XCI, in an attempt to elucidate the molecular mechanism underlying skewed XCI in these symptomatic HA carriers. The study was based on the hypothesis that the presence of a pathogenic mutation on the non-hemophilic X chromosome is the cause of extreme inactivation of that X chromosome. Methods: XCI patterns were studied by PCR analysis of the CAG repeat region in the HUMARA gene. HA carriers that demonstrated skewed XCI were further studied by whole-exome sequencing (WES) followed by X chromosome-targeted bioinformatic analysis. Results: All three HA carriers presenting with the moderate to severe HA phenotype due to skewed XCI were found to carry pathogenic mutations on their non-hemophilic X chromosomes. Patient 1 was diagnosed with a frameshift mutation in the PGK1 gene that was associated with familial XCI skewing in three generations. Patient 2 was diagnosed with a missense mutation in the SYTL4 gene that was associated with familial XCI skewing in two generations. Patient 3 was diagnosed with a nonsense mutation in the NKAP gene that was associated with familial XCI skewing in two generations. Conclusion: Our results indicate that the main reason for skewed XCI in our female HA patients was negative selection against cells with a disadvantage caused by an additional deleterious mutation on the silenced X chromosome, thus complicating the phenotype of a monogenic X-linked disease. Based on our study, we are currently offering the X inactivation test to symptomatic hemophilia carriers and plan to expand this approach to symptomatic carriers of other X-linked diseases, which can be further used in pregnancy planning.

Avoidance of fasting and regular ingestion of uncooked-cornstarch have long been the mainstay dietary treatment of Glycogen Storage Disease type Ia (GSD-Ia). However, GSD-Ia patients who despite optimal dietary treatment show poor glycemic control and are intolerant to cornstarch, present a complex clinical challenge. We pursued Whole Exome Sequencing (WES) in three such unrelated patients, to both confirm a molecular diagnosis of GSD-Ia, and seek additional variants in other genes (e.g. genes associated with amylase production) which may explain their persistent symptoms. WES confirmed the GSD-Ia diagnosis, with all three probands harboring the homozygous p.R83C variant in G6PC. While no other significant variants were identified for patients A and B, a homozygous p.G276V variant in the SI gene was detected in patient C, establishing the dual-diagnosis of GSD-Ia and Sucrase-Isomaltase Deficiency. To conclude, we suggest that WES should be considered in GSD-Ia patients who show persistent symptoms despite optimal dietary management.

Whole genome approaches are superior for identifying recessive genes, however discovery of dominant genes including deafness genes (DFNA) remains challenging. Herein we report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Targeted screening of DFNA genes based on audioprofiles was unsuccessful. Genome-wide SNP genotyping linked SNHL to DFNA33 (Lod = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL in 2009. WGS identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 Kb, excluding all but one variant. ATP11A (NC_000013.11: g.190616G > A) is a novel point mutation predicted to be a cryptic donor splice site. RNA studies in patient-derived tissues verified in silico predictions, revealing the retention of 153bp of intron in the 3’ UTR of several ATP11A isoforms. A second, unresolved family from Israel with a similar, variable form of SNHL and a novel duplication in exon 28 of ATP11A that occurs within the splice donor sequence (intron 28). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.

Signal transducer and activator of transcription (STAT)1 heterozygous gain-of-function (GOF) mutations are known to induce immune dysregulation and chronic mucocutaneous candidiasis (CMCC). Previous reports suggest an association between demodicosis and STAT1 GOF. However, immune characterization of these patients is lacking. Here, we present a retrospective analysis of patients with immune dysregulation and STAT1 GOF who presented with facial and ocular demodicosis. In-depth immune phenotyping and functional studies were used to characterize the patients. We identified five patients (three males) from two non-consanguineous Jewish families. The mean age at presentation was 11.11 (range = 0.58–24) years. Clinical presentation included CMCC, chronic demodicosis and immune dysregulation in all patients. Whole-exome and Sanger sequencing revealed a novel heterozygous c.1386C>A; p.S462R STAT1 GOF mutation in four of the five patients. Immunophenotyping demonstrated increased phosphorylated signal transducer and activator of transcription in response to interferon-α stimuli in all patients. The patients also exhibited decreased T cell proliferation capacity and low counts of interleukin-17-producing T cells, as well as low forkhead box protein 3+ regulatory T cells. Specific antibody deficiency was noted in one patient. Treatment for demodicosis included topical ivermectin and metronidazole. Demodicosis may indicate an underlying primary immune deficiency and can be found in patients with STAT1 GOF. Thus, the management of patients with chronic demodicosis should include an immunogenetic evaluation.

Background KCNC2 encodes a member of the shaw-related voltage-gated potassium channel family (KV3.2), which are important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain.Methods Individuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic and functional analysis. The cases were referred through clinical and research collaborations in our study. Four de novo variants were examined electrophysiologically in Xenopus laevis oocytes.Results We identified novel KCNC2 variants in 27 patients with various forms of epilepsy. Functional analysis demonstrated gain-of-function in severe and loss-of-function in milder phenotypes as the underlying pathomechanisms with specific response to valproic acid.Conclusion These findings implicate KCNC2 as a novel causative gene for epilepsy emphasizing the critical role of KV3.2 in the regulation of brain excitability with an interesting genotype-phenotype correlation and a potential concept for precision medicine.

Diagnosis of primary complement deficiencies requires a high index of suspicion. Thus, susceptible patients are often underdiagnosed and untreated. Here, we present a multi-center experience with three novel inborn errors of the classical complement system. This is a retrospective multicenter analysis of computerized medical records of children (> 18 years) admitted in the period between 2003 and 2018 at Shaare Zedek Medical Center in Jerusalem and Edmond and Lily Safra Children's Hospital, Tel-Hashomer Medical Center, in Ramat Gan, Israel. Patients were genetically diagnosed by a complimentary immune work-up. We identified 5 patients (3 males) from four different consanguineous families harboring three novel mutations in the complement components C6-C8. Genetic mutations were identified by whole exome sequencing. Clinical manifestations consisted of meningitis with or without meningococcemia. The immune work-up demonstrated nearly absent levels of CH50, compatible with a complement pathway defect. The mean diagnosis delay was 10.56 (0–30) years. Conclusion: Invasive meningococcal infections may be life-threatening and cause severe neurological sequela. Awareness of risk factors for primary complement deficiencies, even at the firs infectious episode, should facilitate prompt immune and genetic investigations, commencing diagnosis and proper treatment.

Background: During the process of generating diverse T and B cell receptor (TCR and BCR, respectively) repertoires, double strand DNA breaks are produced. Subsequently, these breaks are corrected by a complexed system led mainly by the non-homologous end-joining (NHEJ). Mutations in proteins involved in this process, including the XLF/ Cernunnos gene, cause severe combined immunodeficiency syndrome (SCID) along with neurodevelopmental disease and susseptability to inoizing radiation. Objective: To provide new clinical and immunological insights on XLF/Cernunnos deficiency, arising from a newly diagnosed patient with severe immunodeficiency. Methods: A male infant, born to consanguineous parents, suspected of having primary immunodeficiency underwent immunological and genetic work up. This included a thorough assessment of T cell phenotyping and lymphocyte activation by mitogen stimulation tests, whole exome sequencing (WES), TCR repertoire Vβ repertoire via flow cytometry analysis and TCR and BCR via next generation sequencing (NGS). Results: Clinical findings included microcephaly, recurrent bacterial viral pneumonia and failure to thrive. Immune workup revealed lymphopenia, reduced T cell function and hypogammaglubolinemia. A skewed TCR Vβ repertoire, TCR gamma (TRG) repertoire and BCR repertoire were determined in the patient. Genetic analysis identified a novel autosomal recessive homozygous missense mutation in XLF/Cernunnos c. A580Ins.T; p. M194fs. The patient underwent a successful hematopoietic stem cell transplantation (HSCT). Conclusions: A novel XLF/Cernunnos mutation is reported in a patient presented with SCID phenotype that displayed clonally expanded T and B cells. An adjusted HSCT was safe to ensure full T cell immune reconstitution.

Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit-encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.

Introduction Glanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. Aim We aimed to study the genetic cause of IPFD mimicking GT. Methods During 2017–2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. Results Using flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbβ3 and αvβ3, as well as normal integrin αIIbβ3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. Discussion Both RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. Conclusion Patients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.