Rare Deletions Research Articles

Comprehensive genetic analysis of 961 unrelated Duchenne Muscular Dystrophy patients: Focus on diagnosis, prevention and therapeutic possibilities.

Recently DNA sequencing analysis has played a vital role in the unambiguous diagnosis of clinically suspected patients with Duchenne Muscular Dystrophy (DMD). DMD is a monogenic, X-linked, recessive, degenerative pediatric neuromuscular disorder affecting males, invariably leading to fatal cardiopulmonary failure. Early and precise diagnosis of the disease is an essential part of an effective disease management strategy as care guidelines and prevention through counseling need to be initiated at the earliest particularly since therapies are now available for a subset of patients. In this manuscript we report the DMD gene mutational profiles of 961 clinically suspected male DMD patients, 99% of whom were unrelated. We utilized a molecular diagnostic approach which is cost-effective for most patients and follows a systematic process that sequentially involves identification of hotspot deletions using mPCR, large deletions and duplications using MLPA and small insertions/ deletions and point mutations using an NGS muscular dystrophy gene panel. Pathogenic DMD gene mutations were identified in 84% of patients. Our data compared well with the frequencies and distribution of deletions and duplications reported in the DMD gene in other published studies. We also describe a number of rare in-frame mutations, which appeared to be enriched in the 5’ proximal hotspot region of the DMD gene. Furthermore, we identified a family with a rare non-contiguous deletion mutation in the DMD gene where three males were affected and two females were deemed carriers. A subset of patients with mutations in the DMD gene who are likely to benefit therapeutically from new FDA and EMA approved drugs were found in our cohort. Given that the burden of care for DMD patients invariably falls on the mothers, particularly in rural India, effective genetic counseling followed by carrier screening is crucial for prevention of this disorder. We analyzed the carrier status of consented female relatives of 463 probands to gauge the percentage of patients with familial disease. Our analysis revealed 43.7% of mothers with DMD gene mutations. Our comprehensive efforts, involving complete genetic testing coupled with compassionate genetic counseling provided to DMD patients and their families, are intended to improve the quality of life of DMD patients and to empower carrier females to make informed reproductive choices to impede the propagation of this deadly disease.

A 24-base pair deletion in the ABO gene causes a hereditary splice site defect: a novel mechanism underlying ABO blood group O.

BACKGROUNDBlood group A and B antigens are synthesized by glycosyltransferases regulated by a complex molecular genetic background. A multibase deletion in the ABO gene was identified in two related blood donors. To define its hereditary character and to evaluate genotype–phenotype associations, a detailed study including 30 family members was conducted.METHODS AND MATERIALSABO phenotyping was performed with agglutination techniques and adsorption‐elution tests. The secretor status was determined. Allele‐specific sequencing of ABO and genotyping of family members by a mutation‐specific polymerase chain reaction were carried out. Functional analysis included cloning of complementary DNA and transfection experiments in HeLa cells. The antigen expression was investigated by flow cytometry and adsorption‐elution method.RESULTSSequencing analysis revealed a 24‐bp deletion in Exon 5 and the adjacent intronic region of ABO. The alteration was inherited by 16 family members. Nine of them being heterozygous for the mutated allele failed to express A antigen on their erythrocytes as found by routine typing. In particular samples, however, adsorption‐elution studies indicated inconclusive results. HeLa cells transfected with aberrant gene transcripts did not express blood group antigen A.CONCLUSIONThe variation causes defects in messenger RNA splicing, most likely inactivating the transferase as observed by serological typing and in vitro expression analysis. These data suggest a novel mechanism associated with blood group O and extend the knowledge of exceptionally rare ABO splice site mutations and deletions. With increased understanding of the molecular bases of ABO, the diagnostics may be further enhanced to ensure the safest possible use of the blood supply.

Compound craniosynostosis, intellectual disability, and Noonan-like facial dysmorphism associated with 7q32.3-q35 deletion.

Craniosynostosis (CS) is the premature fusion of the cranial sutures, occurring either in isolated or syndromic form. Syndromic CS, which was described in over 180 genetic syndromes, accounts for 15-30% of all CS cases and usually originates from mutations within the FGFR1, FGFR2, FGFR3, and TWIST1 genes. However, causative alterations in other genes, or rarely copy number variations (CNVs) were also reported. In this article, we describe a patient with Noonan-like facial dysmorphism accompanied by intellectual disability and compound CS, involving coronal, sagittal, and squamous sutures. We applied karyotyping, copy number variations analysis using array comparative genomic hybridization, and microarray-based genes expresion analysis. We have shown that the index carried a large and rare heterozygous deletion, which encompassed 12.782 Mb and mapped to a chromosomal region of 7q32.3-q35 (HG38 - chr7:131837067-144607071). The aberration comprised 109 protein-coding genes, including BRAF, that encodes serine/threonine-protein kinase B-Raf, being a part of the RAS/MAPK signaling pathway. The RAS/MAPK pathway plays an essential role in human development; hence, its dysregulation not surprisingly results in severe congenital anomalies, such as phenotypically overlapping syndromes termed RASopathies. To our best knowledge, we report here the first CNV causing haploinsufficiency of BRAF, resulting in dysregulation of the RAS/MAPK cascade, and consequently, in the phenotype observed in our patient. To conclude, with this report, we have pointed to the involvement of the RAS/MAPK signaling pathway in CS development. Moreover, we have shown that the molecular analysis based on both DNA and RNA profiling, undoubtedly constitutes a comprehensive diagnostic and research strategy for elucidating a cause of genetic diseases.

SUN-089 Novel Genetic and Biochemical Findings of DLK1 Deficiency in Children with Central Precocious Puberty - a Collaborative Brazilian-Spanish Study

Background: Delta-like 1 homolog (DLK1), also known as pre-adipocyte factor 1 (Pref-1), is part of the Notch signaling pathway that controls many developmental processes. Loss-of-function mutations of DLK1 have been identified in children with central precocious puberty (CPP), as well as in women who had precocious menarche (≤ 9 years) with an unfavorable metabolic profile. Objective: To investigate genetic and biochemical aspects of DLK1 in a cohort of children with CPP. Patients: A large cohort of Spanish children with idiopathic CPP (Spanish PUBERE Registry) was studied. Genomic DNA was obtained from 444 individuals, including 168 index cases with CPP and their close relatives. Automatic sequencing of the coding region (5 exons) of DLK1 was performed in all index cases. Serum DLK1 levels were measured by using a soluble DLK1 enzyme-linked immunosorbent assay (ELISA). Results: A rare allelic deletion (c.401_404 + 8del) of a splice site junction of DLK1 was identified in a girl with sporadic CPP. Pubertal signs appeared at 5.7 years of age with progressive puberty (basal LH: 1.7 mIU/mL, peak LH: 32.77 mIU/mL; basal FSH: 6.32 mIU/mL, peak FSH: 19.89 mIU/mL), BA/CA 1.7 years; normal cranial MRI). She received LHRH analogues (6.3 - 10.1 years of CA) with no side effects. At 14.9 years of age height and BMI are 152.9 cms and 18 kg/m2, respectively, presenting regular menses. Familial segregation analysis showed that the affected child was the only carrier of this deletion characterizing a de novo mutation (biological paternity and maternity were confirmed by microsatellite analysis). Serum DLK1 levels were undetectable (<0.4 ng/mL) in this girl, supporting that the deletion lead to complete lack of DLK1 production. Her father, mother and sister had normal serum DLK1 levels (ranged 6.36 ng/mL to 8.98 ng/mL). Two rare consecutive nucleotide changes in the promoter region of the DLK1 gene (c.-222 C>A and c.-223 G>A) were also identified in an adopted child with CPP. They are located in a transcription factor binding site for SP1 (a zinc finger transcription factor). Pubertal signs appeared at 6.7 years of age with progressive puberty (Basal LH: 0.5 mIU/mL, peak LH: 15.9 mIU/mL, basal FSH: 1.52 mIU/mL, peak FSH: 6.56 mIU/mL, BA/CA 1.4 years; normal cranial MRI). She is under therapy with LHRH analogues with no side effects. Conclusion: Novel DLK1 findings were identified in the Spanish cohort of children with CPP, reinforcing a significant role of this factor in human pubertal timing.

Measuring and Estimating the Effect Size of Rare Non-Recurrent Deletions and Duplications on General Intelligence

Measuring and Estimating the Effect Size of Rare Non-Recurrent Deletions and Duplications on General Intelligence

A single nucleotide deletion resulting in a frameshift in exon 4 of TAB2 is associated with a polyvalular syndrome

Polyvalvularmyxomatous degeneration is a rare clinical condition. A 51-year-old male patient presented at our centre with all four heart valves with myxomatous degeneration and severe mitral and aortic regurgitation due to leaflet prolapse. The patient referred five further family members with valvular heart disease at different stages of presentation. The aim of this study was to investigate the genetic basis of this familial polyvalvularmyxomatous degeneration which was associated with mild dysmorphic facial anomalies and short stature. A detailed family history was recorded. Nine members of the family, affected or not by valvular heart disease, were studied clinically, echocardiographically and by detailed genetic analyses. Six of the nine family members had echocardiographic features of different degrees of degenerative heart valve disease. In addition, the affected subjects shared similar mild dysmorphic facial anomalies and short stature. Exome sequencing identified a rare heterozygous single nucleotide deletion in the TAB2 gene in all affected family members, which was absent in the unaffected members. A variant in the TAB2 gene is proposed as the cause of syndromic congenital heart disease, displaying congenital myxomatous degenerative heart valve disease, mild dysmorphic fascial anomalies and short stature in this family.

Characterization of a schizophrenia patient with a rare RELN deletion by combining genomic and patient-derived cell analyses

Genetic studies have identified rare RELN variants as risk factors for psychiatric disorders. However, additional genetic factors appear to be necessary for disease onset. Detailed genetic information and the use of patient-derived neuronal cells may thus enable to discover these disease-related additional factors. Here, we performed whole-genome sequencing of a schizophrenia patient with a rare RELN deletion and his healthy mother, and examined the phenotypes of 3D-cultured neuronal cells derived from induced pluripotent stem cells of this patient. Our results revealed that, along with the RELN deletion, neuronal death was promoted in this patient; thus, neuronal death may be a vulnerable factor for schizophrenia.

Revealing the Impact of Recurrent and Rare Structural Variations in Multiple Myeloma

Whole genome sequencing (WGS) studies have started to reveal the critical role of structural variants (SVs) in multiple myeloma (MM) pathogenesis and evolution. We have recently revealed the existence of three main classes of complex events in 30 MM patients: chromothripsis, chromoplexy and templated insertions (Maura F et al, Nat Comm, 2019). Here, drawing on a large cohort of 768 MM patients enrolled in the MMRF CoMMpass study (NCT01454297), we comprehensively characterized the landscape of SVs and their functional implications. Low coverage long-insert WGS (median 4-8X) was available from all patients, of whom 591 also had RNAseq data. Overall, we identified a median of 15 total SVs (range 1-253). Fifty-one percent of SVs (n = 8766) were defined as part of complex events, with a median of one per patient (range 0-14). Chromothripsis, chromoplexy and templated insertions involving &amp;gt;2 chromosomes were observed in 21%, 11% and 21 %, respectively. Chromothripsis was the only SV class with clear prognostic implications after adjustment for molecular and clinical features, resulting in adverse PFS (adjusted HR = 1.57; 95% CI 1.13-2.22; p = 0.008) and OS (adjusted HR = 2.4; 95% CI 1.5-3.83; p &amp;lt; 0.001). Templated insertions emerged as the cause of CCND1-IGH and MYC translocations in 34 % and 73 % of cases, respectively. This is particularly important given the capability of templated insertions to connect and amplify multiple regions of the genome, involving several oncogenes and regulatory regions (e.g. super enhancers). Twenty-four patients (3.1 %) had translocation between an immunoglobulin locus and a non-canonical driver gene (e.g. PAX5, CD40 and MAP3K14), showing outlier expression by RNAseq where available. SV hotspot analysis was carried out using the Piecewise Constant Fitting algorithm, comparing the local SV breakpoint density to an empirical background model (Glodzik et al, Nat Genet, 2017). To identify functionally important hotspots, we integrated: 1) local cumulative copy number data, 2) amplification and deletion peaks identified by GISTIC v2 (q &amp;lt; 0.1), 3) gene fusion data and 4) differential expression analysis with adjustment for main molecular subgroups (limma; Bonferroni-Holm adjusted p-values &amp;lt; 0.01). Ninety-eight hotspots were identified (Figure 1), of which 71 (72%) have not previously been reported. Among these novel hotspots, 23 (33 %) contained a known or suspected driver gene, including TNFRSF17 (encoding CAR-T target BCMA), SYK (BCR signal transduction) and KLF2 (key myeloma transcription factor and germline predisposition locus). Active enhancer regions were present in 29 of the novel hotspots (41 %), including 65 % of those with a concurrent putative driver gene involved. For 34 hotspots (48 %) no clear target gene or regulatory region emerged. SV hotspots and GISTIC peaks covered 13 % of the genome. Overall 38 % of simple and complex SVs had at least one breakpoint falling within a recurrently involved region. The majority of chromoplexy, chromothripsis and templated insertions involved recurrent regions (64, 76 and 86 %, respectively). Simple events were most commonly rare, ranging from 74 % of deletions to 45 % for translocations. Quantifying the global functional impact of the remaining 72 % of non-recurrent or rare SVs, we observed that genes involved by a rare SV were significantly enriched for outlier expression (z-score +/- 2) compared to a permutation background model. Rare deletions and duplications exerted their effects within 10 Kb of the gene body. Translocations and templated insertions were associated with overexpression up to 1 Mb from the gene, but had no effect when involving the gene body, consistent with a major enhancer hijacking mechanism. Finally, we sought to understand the role of recurrent and rare SVs in evolutionary dynamics, analyzing 27 patients that progressed with branching evolution. Seventy-two acquired SVs involved a hotspot region (42 driver and/or enhancer; 48 unknown), while 328 were rare. In conclusion, the SV landscape in multiple myeloma is characterized by multiple recurrently involved genes and regulatory regions. These regions account for the majority of complex SVs, indicating strong positive selection of these events. Nonetheless, the majority of SVs remain unaccounted for. Rare SVs were associated with outlier gene expression and may contribute to the tumor evolutionary trajectory of individual patients. Disclosures Papaemmanuil: Celgene: Research Funding. Landgren:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Genetic Analysis, Prenatal Diagnosis and Preimplantation Genetic Diagnosis of Taiwanese Deletion β-Thalassemia

To perform genetic analysis, prenatal diagnosis and preimplantation genetic diagnosis (PGD) in a family with a rare deletional β- thalassemia. Hematological parameters of the peripheral blood collected from all the family members were analyzed by whole blood cell analysis and capillary zone electrophoresis (CZE). Polymerase chain reaction-reverse dot blot (PCR-RDB) was used to identify 17 common β- thalassemia gene mutations, the multiplex ligation-dependent probe amplification (MLPA) and gap-polymerase chain reaction (gap-PCR) were used to identify β- globin gene cluster deletions. Chorionic villus sample or umbilical cord blood was obtained for prenatal diagnosis. Oligo-cells from blastocyst biopsy were collected for preimplantation genetic diagnosis by whole genome amplification and next generation sequencing. The proband was a carrier of Taiwanese deletion β- thalassemia, two fetuses were both thalassemia majors. The PGD results showed that 6 of 11 tested embryos could be choose for transplantation. The Taiwanese deletion is a rare type deletion of β- globin gene cluster, and it can lead to thalassemia intermedia or thalassemia major when compounded with other β- globin gene mutation. PGD is another choice for thalassemia couples.

NRXN1 is associated with enlargement of the temporal horns of the lateral ventricles in psychosis

Schizophrenia, Schizoaffective, and Bipolar disorders share behavioral and phenomenological traits, intermediate phenotypes, and some associated genetic loci with pleiotropic effects. Volumetric abnormalities in brain structures are among the intermediate phenotypes consistently reported associated with these disorders. In order to examine the genetic underpinnings of these structural brain modifications, we performed genome-wide association analyses (GWAS) on 60 quantitative structural brain MRI phenotypes in a sample of 777 subjects (483 cases and 294 controls pooled together). Genotyping was performed with the Illumina PsychChip microarray, followed by imputation to the 1000 genomes multiethnic reference panel. Enlargement of the Temporal Horns of Lateral Ventricles (THLV) is associated with an intronic SNP of the gene NRXN1 (rs12467877, P = 6.76E–10), which accounts for 4.5% of the variance in size. Enlarged THLV is associated with psychosis in this sample, and with reduction of the hippocampus and enlargement of the choroid plexus and caudate. Eight other suggestively significant associations (P < 5.5E–8) were identified with THLV and 5 other brain structures. Although rare deletions of NRXN1 have been previously associated with psychosis, this is the first report of a common SNP variant of NRXN1 associated with enlargement of the THLV in psychosis.

Alpha‐globin gene mutation spectrum in patients with microcytic hypochromic anemia from Mazandaran Province, Iran

BackgroundIt is estimated about 7% of the world population is carriers of hemoglobin diseases. Alpha‐thalassemia is one of the most common hereditary hemoglobin disorders in the world. This study investigated alpha‐globin mutations in potential carriers with hypochromic and microcytic anemia from Mazandaran, in northern Iran.MethodsA total of 859 subjects were selected; genomic DNA was extracted and examined for the presence of mutations in the alpha‐globin genes.ResultsMutation analysis of alpha‐globin genes revealed 27 different mutations. Seven variants were seen in 91.45% of all alpha‐1 and alpha‐2 mutations among patients in this study. The 3.7 kb deletion is the most frequent mutation with a frequency of 49.53%, followed by PolyA2 (15.19%), −4.2 deletion (8.76%), ‐‐MED (5.84%), IVSI‐5nt deletion (5.49%), Hb constant spring (3.62%), and Cd 19 (−G; 3.04%), respectively. There are also seven new variants which were reported for the first time either in alpha‐1 or alpha‐2 genes, including codon 9 (C > A; α2), deletion of codon 60 (AAG deletion; α2), duplication of codon 94‐100 plus 3 base pairs of intron 2 (IVSII + 3; α1), codon 99 (C > A; α2), codon 108 (A > G; α2), codon 128 (A > T; α2), and codon 129 (T > G; α2), respectively. The MLPA method also revealed three rare and novel deletions in alpha‐cluster region with about 30 kilobases long.ConclusionThis study showed an efficient identification of α‐thalassemia can be achieved using standard hematological indices in our population. The details of these variations will help local genetic services for diagnostic and prenatal diagnosis services.

The Spectrum of Genetic Defects in Congenital Adrenal Hyperplasia in the Population of Cyprus: A Retrospective Analysis.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is caused by mutations in the CYP21A2 gene. The study refers to CAH patients of Greek-Cypriot ancestry between years 2007 and 2018. One hundred and twenty patients with various degrees of CAH were categorized and genotyped. The patients were categorized in 4 mutation groups based on their clinical and biochemical findings. The majority of patients (85.0%) belonged to the non-classic (NC)-CAH form and the disorder was more often diagnosed in females (71.7%). The most severe classic salt-wasting (SW) form was identified in 11 neonates (9.2%). Seven (5.8%) children were also identified with the simple virilizing (SV) form and a median presentation age of 5 years [interquartile range (IQR) 3.2-6.5]. In the 240 nonrelated alleles, the most frequent mutation was p.Val281Leu (60.0%) followed by c.655 A/C>G (IVS2-13A/C>G) (8.8%), p.Pro453Ser (5.8%), DelEx1-3 (4.6%), p.Val304Met (4.6%), and p.Gln318stop (4.2%). Other less frequent mutations including rare deletions were also identified. Following our recent report that the true carrier frequency of CYP21A2 in Greek-Cypriots is 1:10, this study reports that the CAH prevalence is predicted around 1.7 cases per 10 000 people. Therefore, the up-to-date 120 CAH patients identified by our group make only the 6.9% of the ones estimated (approximately 1750) to exist in the Greek Cypriot population. The compiled data from a coherent population such as the Greek-Cypriot could be valuable for the antenatal diagnosis, management and genetic counselling of the existing and prospect families with CAH.

Genome-wide analysis identifies rare copy number variations associated with inflammatory bowel disease.

BackgroundInflammatory bowel disease (IBD) is an idiopathic, chronic disorder of unclear etiology with an underlying genetic predisposition. Recent genome-wide association studies have identified more than 200 IBD susceptibility loci, but the causes of IBD remain poorly defined. We hypothesized that rare (<0.1% population frequency) gene copy number variations (CNVs) could play an important mechanism for risk of IBD. We aimed to examine changes in DNA copy number in a population-based cohort of patients with IBD and search for novel genetic risk factors for IBD.MethodsDNA samples from 243 individuals with IBD from the Manitoba IBD Cohort Study and 2988 healthy controls were analyzed using genome-wide SNP microarray technology. Three CNV calling algorithms were applied to maximize sensitivity and specificity of CNV detection. We identified IBD-associated genes affected by rare CNV from comparing the number of overlapping CNVs in IBD samples with the number of overlapping CNVs in controls for each gene.Results4,402 CNVs detected by two or three algorithms intersected 7,061 genes, in at least one analyzed sample. Four genes (e.g. DUSP22 and IP6K3) intersected by rare deletions and fourteen genes (e.g. SLC25A10, PSPN, GTF2F1) intersected by rare duplications demonstrated significant association with IBD (FDR-adjusted p-value < 0.01). Of these, ten genes were functionally related to immune response and intracellular signalling pathways. Some of these genes were also identified in other IBD related genome-wide association studies. These suggested that the identified genes may play a role in the risk of IBD.ConclusionOur results revealed new genomic loci associated with IBD, which suggested the role of rare CNVs in IBD risk.

Mutation of a histidine-rich calcium-binding-protein gene in wheat confers resistance to Fusarium head blight.

Head or ear blight, mainly caused by Fusarium species, can devastate almost all staple cereal crops (particularly wheat), resulting in great economic loss and imposing health threats on both human beings and livestock1-3. However, achievement in breeding for highly resistant cultivars is still not satisfactory. Here, we isolated the major-effect wheat quantitative trait locus, Qfhs.njau-3B, which confers head blight resistance, and showed that it is the same as the previously designated Fhb1. Fhb1 results from a rare deletion involving the 3' exon of the histidine-rich calcium-binding-protein gene on chromosome 3BS. Both wheat and Arabidopsis transformed with the Fhb1 sequence showed enhanced resistance to Fusarium graminearum spread. The translation products of this gene's homologs among plants are well conserved and might be essential for plant growth and development. Fhb1 could be useful not only for curbing Fusarium head blight in grain crops but also for improving other plants vulnerable to Fusarium species.

Disruption of the exocyst induces podocyte loss and dysfunction

Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-β pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.

Systematic analysis of dark and camouflaged genes reveals disease-relevant genes hiding in plain sight

BackgroundThe human genome contains “dark” gene regions that cannot be adequately assembled or aligned using standard short-read sequencing technologies, preventing researchers from identifying mutations within these gene regions that may be relevant to human disease. Here, we identify regions with few mappable reads that we call dark by depth, and others that have ambiguous alignment, called camouflaged. We assess how well long-read or linked-read technologies resolve these regions.ResultsBased on standard whole-genome Illumina sequencing data, we identify 36,794 dark regions in 6054 gene bodies from pathways important to human health, development, and reproduction. Of these gene bodies, 8.7% are completely dark and 35.2% are ≥ 5% dark. We identify dark regions that are present in protein-coding exons across 748 genes. Linked-read or long-read sequencing technologies from 10x Genomics, PacBio, and Oxford Nanopore Technologies reduce dark protein-coding regions to approximately 50.5%, 35.6%, and 9.6%, respectively. We present an algorithm to resolve most camouflaged regions and apply it to the Alzheimer’s Disease Sequencing Project. We rescue a rare ten-nucleotide frameshift deletion in CR1, a top Alzheimer’s disease gene, found in disease cases but not in controls.ConclusionsWhile we could not formally assess the association of the CR1 frameshift mutation with Alzheimer’s disease due to insufficient sample-size, we believe it merits investigating in a larger cohort. There remain thousands of potentially important genomic regions overlooked by short-read sequencing that are largely resolved by long-read technologies.

A new microdeletion syndrome involving TBC1D24, ATP6V0C, and PDPK1 causes epilepsy, microcephaly, and developmental delay

PurposeContiguous gene deletions are known to cause several neurodevelopmental syndromes, many of which are caused by recurrent events on chromosome 16. However, chromosomal microarray studies (CMA) still yield copy-number variants (CNVs) of unknown clinical significance. We sought to characterize eight individuals with overlapping 205-kb to 504-kb 16p13.3 microdeletions that are distinct from previously published deletion syndromes. MethodsClinical information on the patients and bioinformatic scores for the deleted genes were analyzed. ResultsAll individuals in our cohort displayed developmental delay, intellectual disability, and various forms of seizures. Six individuals were microcephalic and two had strabismus. The deletion was absent in all 13 parents who were available for testing. The area of overlap encompasses seven genes including TBC1D24, ATP6V0C, and PDPK1 (also known as PDK1). Bi-allelic TBC1D24 pathogenic variants are known to cause nonsyndromic deafness, epileptic disorders, or DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures). Sanger sequencing of the nondeleted TBC1D24 allele did not yield any additional pathogenic variants. ConclusionsWe propose that 16p13.3 microdeletions resulting in simultaneous haploinsufficiencies of TBC1D24, ATP6V0C, and PDPK1 cause a novel rare contiguous gene deletion syndrome of microcephaly, developmental delay, intellectual disability, and epilepsy.

High frequencies of Non Allelic Homologous Recombination (NAHR) events at the AZF loci and male infertility risk in Indian men

Deletions in the AZoospermia Factor (AZF) regions (spermatogenesis loci) on the human Y chromosome are reported as one of the most common causes of severe testiculopathy and spermatogenic defects leading to male infertility, yet not much data is available for Indian infertile men. Therefore, we screened for AZF region deletions in 973 infertile men consisting of 771 azoospermia, 105 oligozoospermia and 97 oligoteratozoospermia cases, along with 587 fertile normozoospermic men. The deletion screening was carried out using AZF-specific markers: STSs (Sequence Tagged Sites), SNVs (Single Nucleotide Variations), PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) analysis of STS amplicons, DNA sequencing and Southern hybridization techniques. Our study revealed deletion events in a total of 29.4% of infertile Indian men. Of these, non-allelic homologous recombination (NAHR) events accounted for 25.8%, which included 3.5% AZFb deletions, 2.3% AZFbc deletions, 6.9% complete AZFc deletions, and 13.1% partial AZFc deletions. We observed 3.2% AZFa deletions and a rare long AZFabc region deletion in 0.5% azoospermic men. This study illustrates how the ethnicity, endogamy and long-time geographical isolation of Indian populations might have played a major role in the high frequencies of deletion events.

Genome-wide Burden of Rare Short Deletions Is Enriched in Major Depressive Disorder in Four Cohorts

BackgroundMajor depressive disorder (MDD) is moderately heritable, with a high prevalence and a presumed high heterogeneity. Copy number variants (CNVs) could contribute to the heritable component of risk, but the two previous genome-wide association studies of rare CNVs did not report significant findings. MethodsIn this meta-analysis of four cohorts (5780 patients and 6626 control subjects), we analyzed the association of MDD to 1) genome-wide burden of rare deletions and duplications, partitioned by length (<100 kb or >100 kb) and other characteristics, and 2) individual rare exonic CNVs and CNV regions. ResultsPatients with MDD carried significantly more short deletions than control subjects (p = .0059) but not long deletions or short or long duplications. The confidence interval for long deletions overlapped with that for short deletions, but long deletions were 70% less frequent genome-wide, reducing the power to detect increased burden. The increased burden of short deletions was primarily in intergenic regions. Short deletions in cases were also modestly enriched for high-confidence enhancer regions. No individual CNV achieved thresholds for suggestive or significant association after genome-wide correction. p values < .01 were observed for 15q11.2 duplications (TUBGCP5, CYFIP1, NIPA1, and NIPA2), deletions in or near PRKN or MSR1, and exonic duplications of ATG5. ConclusionsThe increased burden of short deletions in patients with MDD suggests that rare CNVs increase the risk of MDD by disrupting regulatory regions. Results for longer deletions were less clear, but no large effects were observed for long multigenic CNVs (as seen in schizophrenia and autism). Further studies with larger sample sizes are warranted.

Targeted next generation sequencing of pancreatic solid pseudopapillary neoplasms show mutations in Wnt signaling pathway genes.

Solid pseudopapillary neoplasms of the pancreas are rare neoplasms that have been shown to harbor recurrent somatic pathogenic variants in the beta-catenin gene, CTNNB1. Here, we used targeted next generation sequencing to analyze these tumors for other associated mutations. Six cases of solid pseudopapillary neoplasms were studied. DNA extracted from formalin-fixed paraffin embedded tissue blocks was analyzed using the Ion Torrent platform, with the 50-gene Ampliseq Cancer Hotspot Panel v2 (CHPv2), with further variant validation performed by Sanger sequencing. Four tumors (67%) were confirmed to harbor mutations within CTNNB1, two with c.109T > G p.(Ser37Ala) and two with c.94G > A p.(Asp32Asn). One case showed a frameshift deletion in the Adenomatous Polyposis Coli gene, APC c.3964delG p.(Glu1322Lysfs*93) with a variant allele frequency of 42.6%. Sanger sequencing on non-tumoral tissue confirmed the variant was somatic. The patient with the APC mutation developed metastasis and died. In addition to the four cases harboring CTNNB1 variants, we found a case characterized by poor outcome, showing a rare frameshift deletion in the APC gene. Since the APC product interacts with beta-catenin, APC variants may, in addition to CTNNB1, contribute to the pathogenesis of solid pseudopapillary neoplasms via the Wnt signaling pathway.