Unrelated Cases Research Articles

New insights into minor splicing-a transcriptomic analysis of cells derived from TALS patients.

Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in RNU4ATAC, transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi–Linder (TALS/MOPD1), Roifman (RFMN), and Lowry–Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning RNU4ATAC-mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients’ cells compared to controls.

Frequency of GJB2 mutations, GJB6-D13S1830 and GJB6-D13S1854 deletions among patients with non-syndromic hearing loss from the central region of Iran.

BackgroundIn the present study, we investigate the prevalence of the GJB2 gene mutations, and deletions in the GJB6 gene, namely del (GJB6‐D13S1830) and del (GJB6‐D13S1854), in patients with autosomal recessive non‐syndromic hearing loss (ARNSHL) from the central region of Iran.MethodsOne hundred and thirty‐one unrelated ARNSHL cases from the central part of Iran were recruited. Among them, 81% (106 cases) had at least two affected relatives. Coding and noncoding regions of the GJB2 gene were sequenced. Multiplex PCR was used for analysis of del (GJB6‐D13S1830) and del (GJB6‐D13S1854) deletions in GJB6.ResultsThe GJB2 variants were found in 16.79% (22/131) of the patients. The pathogenic variants were 21/131 (16.03%). The nonpathogenic variants were 1/131 (0. 07%). Allele frequency of the c.35delG as the pathogenic variant was the most common with 59.52% (25/42). The remaining pathogenic variants were c.235delC, p.T8M, p.R32H, p.R143Q, p.R143W, c‐23+1G>A. The only nonpathogenic variant was polymorphism p.V27I. Further segregation analysis showed that variant of p.R143Q might have incomplete penetrance. None of the patients had targeted deletions in the GJB6 gene.ConclusionIn comparison with reports from other areas of Iran, c.35delG demonstrates the highest frequency within the central region (accounting for 57.14% of cases), probably resulting from the founder effect and consanguineous marriage. The pathology of ARNSHL in such patients could be attributed to defects in Connexin 26 encoded by GJB2.

PB1874 STEREOTYPE ANTIGEN RECEPTORS IN B‐CELL LYMPHOPROLIFERATIVE DISEASES

Background:Studies over the last 20 years suggest a significant role of antigen stimulation in the pathogenesis of B‐cell lymphoproliferative diseases. Immunoglobulin gene heavy chain variable region (IGHV) repertoire narrowing is an evidence for the involvement of a limited set of antigens or superantigens in the development of lymphomas. For some diseases, e.g. chronic lymphocytic leukemia (CLL), the existence of more than 200 subgroups of tumor IGHV antigen‐binding sites, so called stereotypical antigen receptors (SAR), has been shown among unrelated cases. Most common subgroups (19 SARs) are responsible for more than 12% of all cases of CLL. For other lymphomas, the existence of SARs is also suggested.Aims:To compare the features of tumor IGHVs and possible SARs in CLL, mantle cell lymphoma (MCL) and in the splenic marginal zone lymphoma (SMZL).Methods:The study included samples of 680 patients with B‐CLL, 38 with SMZL and 40 with MCL directed to the National Research Center for Hematology for the genetic analysis between 2006 to 2019. The nucleotide sequences of the IGHV genes were determined according to ERIC protocol.Results:In CLL most common IGHV genes were IGHV1‐69, IGHV1‐2 (mostly unmutated) and IGHV3‐30, IGHV4‐34, IGHV3‐23 (mostly mutated) ‐ they occur in 40% of patients. The most common SARs were CLL#1 (genes of 1, 5 and 7 families, excluding IGHV1‐69), CLL#2 (IGHV3‐21), CLL#6,7,9 (IGHV1‐69). The most common SAR with mutated genes and, accordingly, with a favorable prognosis, was CLL # 4 (IGHV4‐34).In MCL the most common genes were IGHV4‐34, IGHV3‐21, IGHV3‐23, IGHV3‐30, IGHV4‐59 ‐ 57% of all patients. At the same time, unlike in CLL, the IGHV4‐34 gene was often unmutated. We found 3 pairs of MCL patients with sequences of CDR3 regions that formally meet SAR characteristics: IGHV4‐34/IGHD2‐2/IGHJ6 (MCL#1), IGHV3‐21/ IGHD3‐3/IGHJ6 (MCL#2), IGHV4‐59/IGHJ6 (MCL#3). CDR3 of one patient was highly homologous to the rearrangement IGHV4‐34/IGHD1‐26/IGHJ6 (MCL#4) described in the literature.In SMZL the most frequent gene was IGHV1‐2 ‐ 50% of all cases. Moreover, our sample of SMZL is characterized by a strict usage of IGHV1‐2∗04 allele, while in CLL this allele accounts for 25% of all cases only. Unlike in CLL, the frequencies of mutated and unmutated IGHV1‐2 cases were equal. Despite a strong narrowing of IGHV and IGHD repertoires in our SMZL sample (IGHD3‐3 and IGHD3‐10 genes are overrepresented), only 2 pairs of patients could have possible SARs (IGHV1‐2∗04/IGHD3‐3/IGHJ5 and IGHV1‐2∗04/IGHD3‐3/IGHJ5 rearrangements).When comparing the sequences of SARs between the diseases, it was found that in the vast majority of cases they are highly disease‐specific both at the level of nucleotide and amino acid sequences. The only exclusion were MCL#1 and CLL#64D SARs formed by the same set of genes, having similar amino acid motifs and differ only in length.Summary/Conclusion:Our results suggest that antigens crucial for the pathogenesis of B‐cell malignancies could be disease‐specific. More data obtained from the extended samples of non‐CLL patients are required to assess the role of SARs in pathogenesis of these diseases and may also contribute to the development of new diagnostic and prognostic markers.

Biallelic sequence and structural variants in RAX2 are a novel cause for autosomal recessive inherited retinal disease

PurposeRAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease.MethodsExome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members.ResultsBiallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry.ConclusionThis study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases.

Phenotypic spectrum and genetic heterogeneity of cystic fibrosis in Sri Lanka

BackgroundCystic fibrosis has been largely under-diagnosed and thus, limited data is available on the incidence of cystic fibrosis in Sri Lanka. Our aim is to describe the phenotypic and genotypic spectrum of children with cystic fibrosis in Sri Lanka.Case presentationThis report describes 10 unrelated cystic fibrosis cases with phenotypic features of cystic fibrosis and abnormal or intermediate sweat tests. The most common phenotypic features in this sample of symptomatic patients were persistent or recurrent lower respiratory tract infections, failure to thrive and Pseudo-Bartter syndrome. Altogether 7 cystic fibrosis causing mutations were identified in 10 patients. Except delta F508 which is the commonest mutation worldwide all the other mutations detected in Sri Lankan patients are rare mutations. 1161delC and V456A detected in our patients are South Asian mutations. The other mutations such as [C.1282C > G; C.2738A > G], C.53 + 1G > C, 2184insA and a deletion encompassing exons 4 to 11 have been reported previously from European patients with cystic fibrosis.ConclusionThese cases highlight the importance of considering the diagnosis of cystic fibrosis in children and young adults presenting with persistent respiratory tract infections associated with severe malnutrition and Pseudo-Bartter syndrome, especially in low income countries where newborn screening for cystic fibrosis is not available. The spectrum of CFTR mutations in Sri Lanka is heterogeneous and possibly linked to genetic flow from Indian subcontinent and Europe. The common mutations should be identified by sequencing the entire CFTR gene in adequate number of cystic fibrosis patients in order to design a mutation panel for common regional mutations.

The island of Gran Canaria: A genetic isolate for familial hypercholesterolemia

Genetic diagnosis of familial hypercholesterolemia (FH) has not been universally performed in the Canary Islands (Spain). This study aimed to genetically characterize a cohort of patients with FH in the island of Gran Canaria. Study subjects were 70 unrelated index cases attending a tertiary hospital in Gran Canaria, with a clinical diagnosis of FH, according to the criteria of the Dutch Lipid Clinic Network. Given that 7 of the first 10 cases with positive genetic study were carriers of a single mutation in the LDLR gene [p.(Tyr400_Phe402del)], a specific polymerase chain reaction-based assay was developed for the detection of this variant as a first screening step on the remaining subjects. In those without this mutation, molecular diagnosis was completed using a next-generation sequencing panel including LDLR, APOB, PCSK9, LDLRAP1, APOE, STAP1, and LIPA genes and incorporating copy number variation detection in LDLR. On the whole, 44 subjects (62%) had a positive genetic study, of whom 30 (68%) were heterozygous carriers of the p.(Tyr400_Phe402del) variant. Eleven subjects carried other mutations in LDLR, including the novel mutation NM_000527.4: c.877dupG; NP_000518.1: p.(Asp293Glyfs*8). An unclassified PCSK9 gene variant was found in one subject [(NM_174936.3:c.1496G>A; NP_777596.2: p.(Arg499His)]. Other single patients had mutations in APOB (heterozygous) and in LIPA (homozygous). All identified variants co-segregated with the disease phenotype. These findings suggest a founder effect for the p.(Tyr400_Phe402del) LDLR mutation in Gran Canaria. A cost-effective local screening strategy for genetic diagnosis of FH could be implemented in this region.

Novel Usher syndrome pathogenic variants identified in cases with hearing and vision loss

BackgroundUsher syndrome, the most common form of inherited deaf-blindness, is unlike many other forms of syndromic hereditary hearing loss in that the extra aural clinical manifestations are also detrimental to communication. Usher syndrome patients with early onset deafness also experience vision loss due to progressive retinitis pigmentosa that can lead to legal blindness in their third or fourth decade.MethodsUsing a multi-omic approach, we identified three novel pathogenic variants in two Usher syndrome genes (USH2A and ADGRV1) in cases initially referred for isolated vision or hearing loss.ResultsIn a multiplex hearing loss family, two affected sisters, the product of a second cousin union, are homozygous for a novel nonsense pathogenic variant in ADGRV1 (c.17062C > T, p.Arg5688*), predicted to create a premature stop codon near the N-terminus of ADGRV1. Ophthalmological examination of the sisters confirmed typical retinitis pigmentosa and prompted a corrected Usher syndrome diagnosis. In an unrelated clinical case, a child with hearing loss tested positive for two novel USH2A splicing variants (c.5777-1G > A, p. Glu1926_Ala1952del and c.10388-2A > G, p.Asp3463Alafs*6) and RNA studies confirmed that both pathogenic variants cause splicing errors. Interestingly, these same USH2A variants are also identified in another family with vision loss where subsequent clinical follow-up confirmed pre-existing hearing loss since early childhood, eventually resulting in a reassigned diagnosis of Usher syndrome.ConclusionThese findings provide empirical evidence to increase Usher syndrome surveillance of at-risk children. Given that novel antisense oligonucleotide therapies have been shown to rescue retinal degeneration caused by USH2A splicing pathogenic variants, these solved USH2A patients may now be eligible to be enrolled in therapeutic trials.

Dominant PAX2 mutations may cause steroid-resistant nephrotic syndrome and FSGS in children

Heterozygous PAX2 mutations cause renal coloboma syndrome (RCS) [OMIM no. 120330]. RCS is a renal syndromic disease encompassing retinal coloboma and sensorineural hearing loss. Recently, a causative role for PAX2 was reported in adult-onset nephrotic syndrome secondary to focal segmental glomerulosclerosis (FSGS). However, the prevalence of PAX2 mutations among large cohort of children with steroid-resistant nephrotic syndrome (SRNS) and FSGS has not been systematically studied. We employed whole-exome sequencing (WES) to identify the percentage of SRNS cases explained by monogenic mutations in known genes of SRNS/FSGS. As PAX2 mutations are not an established cause of childhood FSGS, we evaluated a cohort of 215 unrelated families with SRNS, in whom no underlying genetic etiology had been previously established. Using WES, we identified 3 novel causative heterozygous PAX2 mutations in 3 out of the 215 unrelated index cases studied (1.3%). All three cases were detected in individuals from families with more than one affected and compatible with an autosomal dominant mode of inheritance (3/57 familial cases studied (5.2%)). The clinical diagnosis in three out of four pediatric index patients was done during routine medical evaluation. Our findings demonstrate high frequency of PAX2 mutations in familial form of SRNS (5.2%) and further expand the phenotypic spectrum of PAX2 heterozygous mutations to include autosomal dominant childhood-onset FSGS. These results highlight the importance of including PAX2 in the list of genes known to cause FSGS in children.

Data-adaptive multi-locus association testing in subjects with arbitrary genealogical relationships.

Genome-wide sequencing enables evaluation of associations between traits and combinations of variants in genes and pathways. But such evaluation requires multi-locus association tests with good power, regardless of the variant and trait characteristics. And since analyzing families may yield more power than analyzing unrelated individuals, we need multi-locus tests applicable to both related and unrelated individuals. Here we describe such tests, and we introduce SKAT-X, a new test statistic that uses genome-wide data obtained from related or unrelated subjects to optimize power for the specific data at hand. Simulations show that: a) SKAT-X performs well regardless of variant and trait characteristics; and b) for binary traits, analyzing affected relatives brings more power than analyzing unrelated individuals, consistent with previous findings for single-locus tests. We illustrate the methods by application to rare unclassified missense variants in the tumor suppressor gene BRCA2, as applied to combined data from prostate cancer families and unrelated prostate cancer cases and controls in the Multi-ethnic Cohort (MEC). The methods can be implemented using open-source code for public use as the R-package GATARS (Genetic Association Tests for Arbitrarily Related Subjects) <https://gailg.github.io/gatars/>.

Retreating to the History of Judicial Review?

Less than a decade ago, the High Court said, in effect, that State Supreme Courts have substantially the same entrenched jurisdiction to issue relief in the nature of the prerogative writs as the Constitution confers on the High Court. The New South Wales Court of Appeal reads this narrowly, holding that only three specific remedies are protected, together with the rules and limitations that existed at federation. If correct, we might see the emergence of two bodies of doctrine, the old law of remedies and the new law of ‘judicial review’ (a concept that emerged only after federation). In an unrelated case, six High Court judges implied a limited privative clause ousting non-jurisdictional certiorari. The seventh judge said that this was unnecessary, because in his view, that branch of certiorari conflicts with judicial review fundamentals, is historically misconceived, and should apply only where the reviewing court can try the matter afresh. This article reviews these developments, discusses their rationales and questions both the history and its importance.

Genomic Landscape of Sporadic Retinitis Pigmentosa: Findings from 877 Spanish Cases

We aimed to unravel the molecular basis of sporadic retinitis pigmentosa (sRP) in the largest cohort reported to date. Case series. A cohort of 877 unrelated Spanish sporadic cases with a clinical diagnosis of retinitis pigmentosa (RP) and negative family history. The cohort was studied by classic genotyping or targeted next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization were performed to confirm copy number variations detected by NGS. Quantitative fluorescent polymerase chain reaction was assessed in sRP cases carrying de novo variants to confirm paternity. The study of the sRP cohort showed a high proportion of causal autosomal dominant (AD) and X-linked (XL) variants, most of them being de novo. Causative variants were identified in 38% of the patients studied, segregating recessively in 84.5% of the solved cases. Biallelic variants detected in only 6 different autosomal recessive genes explained 50% of the cases characterized. Causal AD and XL variants were found in 7.6% and 7.9% of cases, respectively. Remarkably, 20 de novo variants were confirmed after trio analysis, explaining 6% of the cases. In addition, 17% of the solved sRP cases were reclassified to a different retinopathy phenotype. This study highlights the clinical utility of NGS testing for sRP cases, expands the mutational spectrum, and provides accurate prevalence of mutated genes. Our findings evidence the underestimated role of de novo variants in the etiology of RP, emphasizing the importance of segregation analysis as well as comprehensive screening of genes carrying XL and AD variants in sporadic cases. Such in-depth study is essential for accurate family counseling and future enrollment in gene therapy-based treatments.

TCIRG1 and SNX10 gene mutations in the patients with autosomal recessive osteopetrosis

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease characterized by dense and fragile bone, caused by a defect in osteoclasts responsible for the bone destruction. In this study, we aimed to investigate the mutations in TCIRG1 and SNX10 that are responsible for 50% and 4% of the cases, respectively. All amplicons were sequenced by Sanger sequencing following PCR amplification. As a result, six different mutations of the TCIRG1 gene were found in five of the twelve unrelated cases. These include two novel mutations, namely c.630 + 1G > T mutation and c.1778_1779delTG mutation of the gene which are identified as homozygous. A compound heterozygosity of known mutations c.649_674del26 and c.1372G > A and homozygous presence of the known c.2235 + 1G > A mutation were also observed in different patients. In addition, as a result of the prenatal testing in a family with osteopetrosis infant, the c.1674-1G > A mutation was detected as homozygous for the fetus. In TCIRG1, c.166C > T change, which is indicated as likely benign according to ClinVar database, was heterozygous. Several known polymorphisms; c.117 + 83 T > C, c.417 + 11A > G and c.714-19C > A in TCIRG1 gene; c.24 + 36 T > A and c.112-84G > A in SNX10 gene were also detected. In conclusion, our study revealed that five of the twelve cases carry at least one mutation of TCIRG1 gene. Further studies with more patients and other genes would help better understanding of genetic etiology of the disease.

Mutational spectrum and identification of five novel mutations in G6PC1 gene from a cohort of Glycogen Storage Disease Type 1a

BackgroundGlycogen storage disease type-1a is an inherited, autosomal recessive disorder caused by mutations in G6PC1 gene leading to deficiency of glucose-6-phosphatase-α specifically in the liver/kidney/intestine. Patients and methodsDNA of six unrelated Indian GSD-1a patients were screened for mutations in the entire coding region of G6PC1 gene followed by direct DNA sequencing and functional was tested using glucose-6-phosphatase assay. ResultsMutational screening of GSD-1a patients identified five novel mutations, viz., 1) p.V99Cfs*3, 2) p.G125R, 3) IVS1-2A > T, 4) IVS3 + 39G > A and 5) IVS3 + 42G > A along with three previously reported mutations p.G118D, p.R149Q and p.A331V. Interestingly, each of the p.V99Cfs*3, IVS1-2A > T and p.G118D mutations are identified in two unrelated GSD-1a cases. Further allelic distribution of p.V99Cfs*3 and p.A331V mutations were confirmed by RFLP analysis, consistent with autosomal recessive inheritance. Functional characterization revealed that glucose-6-phosphatase activity was completely abrogated with the mutant proteins p.G125R, p.R149Q, p.G118D, p.A331V and p.V99Cfs*3 than wild-type. However, no significant changes were observed in the expression of mutant constructs at transcription and translation level. ConclusionFive novel mutations, p.V99Cfs*3, p.G125R, IVS1-2A > T, IVS3 + 39G > A and IVS3 + 42G > A are reported first time to cause GSD-1a among Indian ethnicity and are not yet reported elsewhere, suggesting separate ethnic founder effects for some mutations among Indian ethnicity.

Molecular analysis of clonally related Salmonella Typhi recovered from epidemiologically unrelated cases of typhoid fever, Brazil

BackgroundThe primary method of molecular subtyping for the identification and investigation of outbreaks has been pulsed-field gel electrophoresis (PFGE). In some cases, this technique has not been able to show discrimination between the unrelated strains that can be achieved by whole genome sequencing (WGS). MethodsThe aim of this study was to determine the strengths and drawbacks of WGS using different analytic approaches compared to traditional typing method, PFGE, for retrospectively typing clusters cases of 28 S. Typhi. ResultsWe evaluated three analytical approaches on the WGS data set (Nucleotide Difference (ND), (SNPs) and Whole genome multi locus sequence typing (wgMLST) that identically classified the clusters-related strains into two clusters, cluster A (with strains from 2017), and Cluster B (with strains from 2007). ConclusionsIn this study WGS based typing, was able to compete with PFGE for differentiation of the clusters of S. Typhi strains.

A Follow-Up from Infancy to Puberty in a Japanese Male with SRY-Negative 46,XX Testicular Disorder of Sex Development Carrying a p.Arg92Trp Mutation in NR5A1

SRY-negative 46,XX testicular disorders of sex development (DSD) are very rare conditions. Recently, we identified a novel heterozygous NR5A1 mutation, p.Arg92Trp (c.274C>T, p.R92W), in 2 unrelated cases of 46,XX testicular/ovotesticular DSD. We report the clinical course from infancy to puberty in a Japanese male with SRY-negative 46,XX testicular DSD, carrying this p.Arg92Trp mutation in NR5A1. The patient naturally acquired the development of a penis and pubic hair during puberty. However, hypergonadotropic hypogonadism subsequently developed. More clinical cases will be needed to fully understand the effects of the p.Arg92Trp mutation on the ability to maintain testosterone secretion in 46,XX testicular DSD.

Immunochemotherapy for Richter syndrome: current insights.

Richter syndrome (RS) is recognized as the development of a secondary and aggressive lymphoma during the clinical course of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Most of such histological transformations are from RS to diffuse large B-cell lymphoma (DLBCL-RS, 90%) and Hodgkin’s lymphoma (HL-RS, 10%). Histopathological examination is a prerequisite for diagnosis. It is crucial to assess the relationship between the RS clone and the underlying CLL/SLL because clonally related DLBCL-RS has a poor outcome, while clonally unrelated cases have a prognosis similar to de novo DLBCL. An anti-CD20 antibody-based immunochemotherapy is hitherto the frontline treatment of choice for DLBCL-RS; nonetheless, the results are unsatisfactory. Allogeneic stem cell transplantation should be offered to younger and fit patients as a consolidative treatment; however, the majority of the patients may not be qualified for this procedure. The HL-RS transformation has better outcomes than those of DLBCL-RS and can effectively be treated by the adriamycin, bleomycin, vinblastine, and dacarbazine regimen. Although novel agents are currently being investigated for RS, immunochemotherapy nevertheless remains a standard treatment for DLBCL-RS.

NR2F2 loss‑of‑function mutation is responsible for congenital bicuspid aortic valve.

Congenital bicuspid aortic valve (BAV) represents the most common type of cardiac birth defect affecting 0.4‑2% of the general population, and accounts for a markedly increased incidence of life‑threatening complications, including valvulopathy and aortopathy. Accumulating evidence has demonstrated the genetic basis of BAV. However, the genetic basis for BAV in the majority of cases remains to be elucidated. In the present study, the coding regions and splicing donors/acceptors of the nuclear receptor subfamily 2 group F member 2 (NR2F2) gene, which encodes a transcription factor essential for proper cardiovascular development, were sequenced in 176 unrelated cases of congenital BAV. The available family members of the proband carrying an identified NR2F2 mutation and 280 unrelated, sex‑ and ethnicity‑matched healthy individuals as controls were additionally genotyped for NR2F2. The functional effect of the mutation was characterized using a dual‑luciferase reporter assay system. As a result, a novel heterozygous NR2F2 mutation, NM_021005.3: c.288C>A; p.(Cys96*), was identified in a family with BAV, which was transmitted in an autosomal dominant mode with complete penetrance. The nonsense mutation was absent from the 560 control chromosomes. Functional analysis identified that the mutant NR2F2 protein had no transcriptional activity. Furthermore, the mutation disrupted the synergistic transcriptional activation between NR2F2 and transcription factor GATA‑4, another transcription factor that is associated with BAV. These findings suggested NR2F2 as a novel susceptibility gene of human BAV, which reveals a novel molecular pathogenesis underpinning BAV.

Genetic architecture of laterality defects revealed by whole exome sequencing.

Aberrant left-right patterning in the developing human embryo can lead to a broad spectrum of congenital malformations. The causes of most laterality defects are not known, with variants in established genes accounting for <20% of cases. We sought to characterize the genetic spectrum of these conditions by performing whole-exome sequencing of 323 unrelated laterality cases. We investigated the role of rare, predicted-damaging variation in 1726 putative laterality candidate genes derived from model organisms, pathway analyses, and human phenotypes. We also evaluated the contribution of homo/hemizygous exon deletions and gene-based burden of rare variation. A total of 28 candidate variants (26 rare predicted-damaging variants and 2 hemizygous deletions) were identified, including variants in genes known to cause heterotaxy and primary ciliary dyskinesia (ACVR2B, NODAL, ZIC3, DNAI1, DNAH5, HYDIN, MMP21), and genes without a human phenotype association, but with prior evidence for a role in embryonic laterality or cardiac development. Sanger validation of the latter variants in probands and their parents revealed no de novo variants, but apparent transmitted heterozygous (ROCK2, ISL1, SMAD2), and hemizygous (RAI2, RIPPLY1) variant patterns. Collectively, these variants account for 7.1% of our study subjects. We also observe evidence for an excess burden of rare, predicted loss-of-function variation in PXDNL and BMS1- two genes relevant to the broader laterality phenotype. These findings highlight potential new genes in the development of laterality defects, and suggest extensive locus heterogeneity and complex genetic models in this class of birth defects.

SU68 - COMMON VARIANTS OF NRXN1, LRP1B AND RORA ARE ASSOCIATED WITH INCREASED VENTRICULAR VOLUMES IN SCHIZOPHRENIA AND BIPOLAR DISORDER

Background Schizophrenia, schizoaffective and bipolar disorder probands share many behavioral and biological traits including brain morphology features, as well as a partially overlapping complex polygenic basis. Ventricular volume enlargement is one of the most enduring anatomical findings in these disorders, representing a quantitative trait associated with severity. Methods We performed GWAS on a multi-ethnic sample from the Bipolar and Schizophrenia Network for Intermediate Phenotypes study (B-SNIP1) containing 1,115 unrelated cases and controls. FreeSurfer-processed volumes from structural MRI were used as quantitative phenotypes in GWAS with PsychChip genotypes imputed to the 1000 Genomes reference panel. Results Genome-wide significance was reached in associations with two regional volumes within the ventricular system: (1) The volume of the Temporal Horn of the Left Lateral Ventricle was associated with a SNP marker of NRXN1 (P= 1.156E-11), and (2) The volume of the Cavum Septum Pellucidum was associated with markers in LRP1B (P=1.661E-11) and RORA (P=1.72E-10). Discussion NRXN1 rare structural variants have been already associated with psychosis and cognition, but this is the first report of common SNP variants associated with increased ventricular volume. Presumably, the observed NRNX1 effect on temporal horn volume reflects shared regulation of morphometric characteristics of the surrounding limbic structures of the medial temporal lobe, not evident when these structures are considered separately. Previous research associated Cavum Septum Pellucidum persistence with schizophrenia, and this is the first report of a common genetic variant associated with its persistence related to neuropsychiatric disease. Co-expression analysis of NRXN1 and LRP1B identified genes involved in cell adhesion and neurotransmission. Our findings represent novel genetic associations implicated in the molecular basis of neuropsychiatric disorders at the stage of brain development.

A novel SLC40A1 p.Y333H mutation with gain of function of ferroportin: A recurrent cause of haemochromatosis in China.

Haemochromatosis type 4, also known as ferroportin disease, is an autosomal dominant genetic disorder caused by pathogenic mutations in the SLC40A1 gene, which encodes ferroportin 1 (FPN1). We have identified a novel SLC40A1 p.Y333H mutation in our previous study. In the present study, we tried to investigate the frequency and pathogenicity of the SLC40A1 p.Y333H mutation in haemochromatosis in China. Patients were analysed for SLC40A1 p.Y333H as well as mutations in the other classic haemochromatosis-related genes by Sanger sequencing. To analyse iron export capacity of the SLC40A1 p.Y333H mutant, the 293T cells were transfected with the SLC40A1 p.Y333H construct and then treated with hepcidin after exposure to ferric ammonium citrate. Cellular localization of mutant FPN1, expression of FPN1 and intracellular ferritin were analysed by immunofluorescence and Western blotting. Of 22 unrelated cases with primary iron overload, three cases (3/22, 13.6%) harboured the SLC40A1 p.Y333H, with no missense mutations identified in any other classical haemochromatosis-related genes including HFE, HJV, HAMP and TFR2. Pedigree analysis showed that three probands and the son of one proband had haemochromatosis of stage 3, while the son of another proband with age of 16 showed elevated transferrin saturation but normal serum ferritin level. In vitro studies showed the mutant p.Y333H ferroportin was resistant to hepcidin, affecting the subsequent internalization and degradation of FPN1, and was associated with ferroportin gain of function. The SLC40A1 p.Y333H mutation is associated with gain of function of ferroportin, representing one of the major aetiological factors of haemochromatosis in China.