Large Copy Number Variants Research Articles

Genomes of Leishmania parasites directly sequenced from patients with visceral leishmaniasis in the Indian subcontinent.

Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples

Targeted deep-intronic sequencing in a cohort of unexplained cases of suspected Lynch syndrome.

Lynch syndrome (LS) is caused by germline defects in DNA mismatch repair (MMR) pathway, resulting in microsatellite instability (MSI-H) and loss of immunohistochemical staining (IHC) of the respective protein in tumor tissue. However, not in all clinically suspected LS patients with MSI-H tumors and IHC-loss, causative germline alterations in the MMR genes can be detected. Here, we investigated 128 of these patients to possibly define new pathomechanisms. A search for large genomic rearrangements and deep-intronic regulatory variants was performed via targeted next-generation sequencing (NGS) of exonic, intronic, and chromosomal regions upstream and downstream of MLH1, MSH2, MSH6, PMS2, MLH3, MSH3, PMS1, and EPCAM. Within this cohort, two different large rearrangements causative for LS were detected in three cases, belonging to two families (2.3%). The sensitivity to detect large rearrangements or copy number variations (CNV) was evaluated to be 50%. In 9 of the 128 patients (7%), previously overlooked pathogenic single-nucleotide variants (SNV) and two variants of uncertain significance (VUS) were identified in MLH1, MSH2, and MSH6. Pathogenic aberrations were not found in MLH3, MSH3, and PMS1. A potential effect on regulation was exerted for 19% of deep-intronic SNVs, predominantly located in chromosomal regions where the modification of histone proteins suggests an enhancer function. In conclusion, conventional variation analysis of coding regions is missing rare genomic rearrangements, nevertheless they should be analyzed. Assessment of deep-intronic SNVs is so far non-conclusive for medical questioning.

RBV: Read balance validator, a tool for prioritising copy number variations in germline conditions

The popularisation and decreased cost of genome resequencing has resulted in an increased use in molecular diagnostics. While there are a number of established and high quality bioinfomatic tools for identifying small genetic variants including single nucleotide variants and indels, currently there is no established standard for the detection of copy number variants (CNVs) from sequence data. The requirement for CNV detection from high throughput sequencing has resulted in the development of a large number of software packages. These tools typically utilise the sequence data characteristics: read depth, split reads, read pairs, and assembly-based techniques. However, the additional source of information from read balance (defined as relative proportion of reads of each allele at each position) has been underutilised in the existing applications. Here we present Read Balance Validator (RBV), a bioinformatic tool that uses read balance for prioritisation and validation of putative CNVs. The software simultaneously interrogates nominated regions for the presence of deletions or multiplications, and can differentiate larger CNVs from diploid regions. Additionally, the utility of RBV to test for inheritance of CNVs is demonstrated in this report. RBV is a CNV validation and prioritisation bioinformatic tool for both genome and exome sequencing available as a python package from https://github.com/whitneywhitford/RBV.

Next-Generation Optical Mapping Reveals Numerous Previously Unrecognizable Structural Variants in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in western countries. Although a number of genetic aberrations (complex karyotype, del(17p), del(11q), unmutated IGVH status, TP53 mutations) having predictive and prognostic significance in CLL was reported, there is still a lack of information about large structural variants, repetitive regions, copy number variations, and inversions in the CLL genome due to limitations of current methods (NGS, cytogenetics). Aim: Using novel next-generation optical mapping technology, we analyzed leukemic cells from peripheral blood of CLL patients at different stages of the disease. Methods and Patients: High-molecular weight DNA was isolated from peripheral blood (>90% CD5+/CD19+/CD20+/CD23+ B-cells) from two CLL patients (P1: 70-y-old male, diagnosis of CLL in 2018, Binet Stage B, unmutated IGVH, untreated; P2: 59-y-old male, diagnosis of CLL in 2005, Binet Stage A, unmutated IGVH, on ibrutinib (2nd line) since 4/2018). For optical mapping, DNA was labeled by DLS chemistry and visualized on Saphyr system (BioNano Genomics). The optical genome maps (480 Gbp, coverage 124X) were aligned to a human reference genome (GRCh38) and the detected structural variants (SVs) were compared against the database of healthy controls; only rare de novo variants were analyzed. In both patients, cytogenetic analysis (karyotype, FISH, and arrayCGH) and mutational analysis by NGS (TP53, ATM, BIRC3, NOTCH1, SF3B1, POT1 genes) were available. Results: For both CLL patients, the optical mapping method identified 97% of chromosomal abnormalities previously reported by standard cytogenetic methods such as deletions on 6q, 13q, 11q, 17p and trisomy 12. In both patients, same large deletions of 17.5 kbp in 9q21 and 7.1 kbp in 14q21 chromosome region were detected, undetectable by standard methods. Both patients also carried intra-chromosomal translocation t(13;13) located in 13q14 region. Additionally, P1 genome carried 2 kbp deletion in oncogene EGFR and 500 bp insertion affecting tumor suppressor TCF4, both genes previously associated with CLL. Moreover, numerous additional copy number variations and large somatic SVs, including on average tens of deletions, insertions, inversions, duplications, and translocations were detected in both patients. Conclusion: Our study revealed numerous novel genomic rearrangements in CLL genome, unrecognizable by standard methods. These data highlights a high potential of optical maps for refinement of genomic variability in CLL, mainly for detection of large structural variations and copy number variants. The study on larger patient cohort and longer follow-up of patients may identify genetic aberrations associated with the clinical course and therapy response. Support: MZ ČR VES16-32339A, MZ ČR - RVO (FNOl, 00098892), IGA UP_2019_014, IGA UP_2019_01 Disclosures Minarik: Celgene: Consultancy, Honoraria, Research Funding; Amgen, BMS, Janssen-Cilag, Takeda: Consultancy, Honoraria.

Differentiating between Hyperdiploidy and Pseudo-Hyperdiploidy in B-Lymphoblastic Leukemia Utilizing Low-Coverage Mate-Pair Sequencing

Purpose: B-cell acute lymphoblastic leukemia/lymphoma (B-ALL/LBL) represents the most common childhood malignancy. Classification of recurrent genetic abnormalities in B-ALL/LBL is essential for risk stratification and patient management. Observed in approximately 5% of B-ALL/LBL cases, hypodiploidy is considered a poor prognostic finding and can be further characterized as near-haploidy (24-31 chromosomes), low hypodiploidy (32-39 chromosomes) and high hypodiploidy (40-43 chromosomes). Importantly, masked hypodiploid clones that have undergone endoreduplication can be mischaracterized as hyperdiploidy, a favorable prognostic finding in B-ALL/LBL. These "doubled" hypodiploid clones are termed pseudo-hyperdiploid and their accurate detection is critical. Mate-pair sequencing (MPseq) is a next-generation sequencing (NGS) technology designed to sequence larger DNA fragments (2000-5000bp) than paired-end sequencing. This allows for the detection of large genetic rearrangements (e.g. translocations, inversions, etc.) and copy number variants (CNVs) at a high resolution with low base coverage. It has demonstrated particular clinical utility for genetic characterization of hematologic malignancies. To expand the role of MPseq in the evaluation of hematologic neoplasms, we aimed to improve the analysis of MPseq results to allow for the detection of copy-neutral loss of heterozygosity (cnLOH) that would enable the differentiation between hyperdiploid and pseudo-hyperdiploid clones. Methods: A new algorithm was applied that statistically analyzes common SNP locations in MPseq data in order to detect both cnLOH regions, and provides global heterozygosity levels at a resolution that allows for the detection of hypodiploidy. This method accounts for the low base-coverage of MPseq data (<10x) in order to provide the highest possible accuracy and precision without increasing cost by requiring changes to the input or sequencing requirements. The algorithm was applied to 17 B-ALL samples classified by conventional chromosome analysis, FISH, and/or chromosomal microarray as either pseudo-hyperdiploidy (n=8) or hyperdiploidy (n=9). The cases were sequenced using MPseq with 101bp read lengths across a range of base coverages (1-10x; 30-135 million fragments) and tumor percentages (50-100%). Results: We demonstrate the method's ability to differentiate between hyperdiploidy and pseudo-hyperdiploidy in all cases without any changes to the MPseq input or sequencing requirements. We detected an average of 11.5 whole-chromosome cnLOH (range 8-20) in the pseudo-hyperdiploidy cases, compared to an average of 0.67 whole-chromosome cnLOH (range 0-2) in the hyperdiploidy cases. Additionally, the method allows for the detection of cnLOH regions across the genome. For example, we observed a recurrent cnLOH of chromosome 9 in the hyperdiploid subtype (3/9 hyperdiploid cases), which is a recurrent abnormality in the disease. The method provides an easy to read visual output that compares favorably to the output of chromosomal microarray, increasing its utility for clinical application. Conclusions: This advancement in the analysis of low-coverage sequencing data makes MPseq available for the genetic characterization of B-ALL/LBL, where hypodiploidy is a primary cytogenetic abnormality and is sometimes missed by conventional cytogenetic techniques. Additionally, the algorithm allows for the detection of cnLOH regions across the genome, which is important across many disease types for diagnosis, prognosis, and the detection of potential therapeutic targets. The addition of the algorithm to the MPseq analysis pipeline makes it a more complete genetic characterization tool and brings it closer to its promise as a potential single assay replacement for conventional cytogenetic techniques. Disclosures No relevant conflicts of interest to declare.

Sequencing as a first-line methodology for cystic fibrosis carrier screening

PurposeMedical society guidelines recommend offering genotyping-based cystic fibrosis (CF) carrier screening to pregnant women or women considering pregnancy. We assessed the performance of sequencing-based CF screening relative to genotyping, in terms of analytical validity, clinical validity, clinical impact, and clinical utility. MethodsAnalytical validity was assessed using orthogonal confirmation and reference samples. Clinical validity was evaluated using the CFTR2 database. Clinical impact was assessed using ~100,000 screened patients. Three screening strategies were compared: genotyping 23 guideline-recommended variants (“CF23”), sequencing all coding bases in CFTR (“NGS”), and sequencing with large copy-number variant (CNV) identification(“NGS + CNV”). Clinical utility was determined via self-reported actions of at-risk couples (ARCs). ResultsAnalytical accuracy of NGS + CNV was 100% for SNVs, indels, and CNVs; interpretive clinical specificity relative to CFTR2 was 99.5%. NGS + CNV detected 58 ARCs, 18 of whom would have gone undetected with CF23 alone. Most ARCs (89% screened preconceptionally, 56% prenatally) altered pregnancy management, and no significant differences were observed between ARCs with or without at least one non-CF23 variant. ConclusionModern NGS and variant interpretation enable accurate sequencing-based CF screening. Limiting screening to 23 variants does not improve analytical validity, clinical validity, or clinical utility, but does fail to detect approximately 30% (18/58) of ARCs.

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes.

Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.

Polygenic risk scores in schizophrenia with clinically significant copy number variants.

AimsRecent studies have revealed that the interplay between polygenic risk scores (PRS) and large copy number variants (CNV; >500kb) is essential for the etiology of schizophrenia (SCZ). To replicate previous findings, including those for smaller CNV (>10kb), the PRS between SCZ patients with and without CNV were compared.MethodsThe PRS were calculated for 724 patients with SCZ and 1178 healthy controls (HC), genotyped using array‐based comparative genomic hybridization and single nucleotide polymorphisms chips, and comparisons were made between cases and HC, or between subjects with and without ‘clinically significant’ CNV.ResultsFirst, we replicated the higher PRS in patients with SCZ compared to that in HC (without taking into account the CNV). For clinically significant CNV, as defined by the American College of Medical Genetics (‘pathogenic’ and ‘uncertain clinical significance, likely pathogenic’ CNV), 66 patients with SCZ carried clinically significant CNV, whereas 658 SCZ patients had no such CNV. In the comparison of PRS between cases with/without the CNV, despite no significant difference in PRS, significant enrichment of the well‐established risk CNV (22q11.2 deletion and 47,XXY/47,XXX) was observed in the lowest decile of PRS in SCZ patients with the CNV.ConclusionAlthough the present study failed to replicate the significant difference in PRS between SCZ patients with and without clinically significant CNV, SCZ patients with well‐established risk CNV tended to have a lower PRS. Therefore, we speculate that the CNV in SCZ patients with lower PRS may contain ‘genuine’ risk; PRS is a possible tool for prioritizing clinically significant CNV because the power of the CNV association analysis is limited due to their rarity.

P.160The spectrum of disease-causing and normal variation in the nebulin gene

The nebulin gene (NEB) with 183 exons is one of the largest genes in human. More than 240 different disease-causing variants in NEB have been identified in some 220 families. Around 50% of nemaline myopathies (NM) are caused by mutations in NEB. Mutations in NEB also cause core-rod myopathy, distal forms of NM, distal myopathy without nemaline rods, and multiple pterygium syndrome. The majority (95%) of the disease-causing variants in NEB are recessive point mutations, mainly nonsense and splice site mutations, and small indels, causing frameshifts. The remaining 5% of mutations are large copy number variations (CNVs). In addition to the recurrent CNVs in the triplicate region and the founder mutation with world-wide occurrence, deletion of exon 55, 11 different large pathogenic duplications and deletions have been identified in NEB. Until recently, all identified mutations in NEB have been recessive. Now it is known that large in-frame deletions in NEB, leading to the expression of substantially smaller proteins, can have a dominant-negative effect, and cause dominant distal forms of NM. The genome aggregation database (gnomAD) contains exome and genome data from 141,456 unrelated individuals, excluding patients with severe pediatric disorders. The total number of different NEB variants listed in gnomAD is 10,634. Most of the variants (96%) are very rare, only present in a few heterozygotes. Intronic variants comprise 43%, missense variants 40%, synonymous variants 14%, loss-of-function variants 3%, and UTR variants 0.2% of the total variation. It is noteworthy that 12.5% of the intronic variants are in the splice regions (excluding splice donor and acceptor sites), and may affect splicing. Rare variants are the most common variant type in NEB, complicating the interpretation of pathogenicity. Therefore, the pathogenicity of especially missense and splice site variants in NEB requires verification by functional analyses at the protein and mRNA level.

Detecting Large Chromosomal Modifications Using Short Read Data From Genotyping-by-Sequencing.

Markers linked to agronomic traits are of the prerequisite for molecular breeding. Genotyping-by-sequencing (GBS) data enables to detect small polymorphisms including single nucleotide polymorphisms (SNPs) and short insertions or deletions (InDels) that can be used, for instance, for marker-assisted selection, population genetics, and genome-wide association studies (GWAS). Here, we aim at detecting large chromosomal modifications in barley and wheat based on GBS data. These modifications could be duplications, deletions, substitutions including introgressions as well as alterations of DNA methylation. We demonstrate that GBS coverage analysis is capable to detect Hordeum vulgare/Hordeum bulbosum introgression lines. Furthermore, we identify large chromosomal modifications in barley and wheat collections. Hence, large chromosomal modifications, including introgressions and copy number variations (CNV), can be detected easily and can be used as markers in research and breeding without additional wet-lab experiments.

Copy number variants in lipid metabolism genes are associated with gallstones disease in men

Gallstones Disease (GSD) is one of the most common digestive diseases requiring hospitalization and surgical procedures in the world. GSD has a high prevalence in populations with European or Amerindian ancestry (10–20%) and the influence of genetic factors is broadly acknowledged. However, known genetic variants do not entirely explain the disease heritability suggesting that additional genetic variants remain to be identified. Here, we examined the association of copy number variants (CNVs) with GSD in a sample of 4778 individuals (1929 GSD cases and 2849 controls) including two European cohorts from Germany (n = 3702) and one admixed Latin American cohort from Chile (n = 1076). We detected 2936 large and rare CNVs events (size > 100 kb, frequency < 1%). Case-control burden analysis and generalized linear regression models revealed significant association of CNVs with GSD in men, with the strongest effect observed with CNVs overlapping lipid metabolism genes (p-value = 6.54 × 10–4; OR = 2.76; CI 95% = 1.53–4.89). Our results indicate a clear link between CNVs and GSD in men and provides additional evidence that the genetic components of risk for GSD are complex, can be sex specific and include CNVs affecting genes involved in lipid metabolism.

A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations.

Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods. The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

DNA copy number variations in children with vesicoureteral reflux and urinary tract infections.

Vesicoureteral reflux (VUR) is a complex, heritable disorder. Genome-wide linkage analyses of families affected by VUR have revealed multiple genomic loci linked to VUR. These loci normally harbor a number of genes whose biologically functional variant is yet to be identified. DNA copy number variations (CNVs) have not been extensively studied at high resolution in VUR patients. In this study, we performed array comparative genomic hybridization (aCGH) on a cohort of patients with a history of both VUR and urinary tract infection (UTI) with the objective of identifying genetic variations responsible for VUR and/or UTI susceptibility. UTI/VUR-associated CNVs were identified by aCGH results from the 192 Randomized Intervention for Children With Vesicoureteral Reflux (RIVUR) patients compared to 683 controls. Rare, large CNVs that are likely pathogenic and lead to VUR development were identified using stringent analysis criteria. Because UTI is a common affliction with multiple risk factors, we utilized standard analysis to identify potential disease-modifying CNVs that can contribute to UTI risk. Gene ontology analysis identified that CNVs in innate immunity and development genes were enriched in RIVUR patients. CNVs affecting innate immune genes may contribute to UTI susceptibility in VUR patients and may provide the first step in assisting clinical medicine in determining adverse outcome risk in children with VUR.

Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks.

We performed a comprehensive assessment of rareinherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extensionof previousfindings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

50. ASSESSMENT OF MICROBIAL CONTAMINATION TO ELIMINATE ARTIFACTS IN SEQUENCING-BASED PGT-A/PGT-SR

Introduction Low pass high throughput sequencing has become a choice method for obtaining chromosome copy number information from biopsies of pre-implantation embryos. In addition to large copy number variants (CNVs) in human embryo samples, data sets from these routine procedures also provide information regarding the microbial content of each specimen and it's derived sequencing library. The aim of this study was to probe raw sequencing data from clinical PGT-A/PGT-SR cycles for microbial contamination which might explain artifactual or difficult to interpret CNV results in contaminated samples. Materials & Methods Between January 2018 and July 2018, PGT-A/PGT-SR was performed on a total of 448 blastomere/blastocyst biopsies at Shaare Zedek Medical Center, Jerusalem, Israel. Library prep and sequencing was performed using the VeriSeq kit (Illumina) according to the manufacturer's protocol. Derivative log ratio (DLR) noise measurements as well as whole chromosomal and segmental CNVs were identified and reported using BlueFuse software. To assess microbial contamination, raw fastq files from each sequencing library were input to PathoScope, a metagenomics suite, for non-human read parsing and microbe identification. Both bacterial and DNA viral taxonomes were fed to PathoScope for non-human read mapping. Results An average of 0.18%+/-0.76% of total reads per sequencing data set, mapped to bacterial/viral genomes, and a positive correlation was observed between qualitative copy number noise measurement (DLR) and microbial sequence contamination. Notably, 6 of the most heavily contaminated samples (>0.9% non-human read fraction) presented with over-representation of sequencing reads mapping to the Streptococcus suis genome. In depth post-hoc analysis indicated that sequencing libraries from all 6 samples were prepared from the same index PCR primer which had likely been contaminated by the manufacturer during or shortly after oligo synthesis. Conclusions Microbial contamination is a genuine threat to the quality of molecular analyses such as PGT-A and PGT-SR. This study draws attention to the prospect that clinical labs, conducting sequencing-based PGT-A/PGT-SR, consider quality control measures to control for artifactual results that may arise from data sets that are contaminated by genomes of foreign organisms.

Association between Copy Number Variation and Response to Social Skills Training in Autism Spectrum Disorder

Challenges in social communication and interaction are core features of autism spectrum disorder (ASD) for which social skills group training (SSGT) is a commonly used intervention. SSGT has shown modest and heterogeneous effects. One of the major genetic risk factors in ASD is rare copy number variation (CNV). However, limited information exists whether CNV profiles could be used to aid intervention decisions. Here, we analyzed the rare genic CNV carrier status for 207 children, of which 105 received SSGT and 102 standard care as part of a randomized clinical trial for SSGT. We found that being a carrier of rare genic CNV did not have an impact on the SSGT outcome measured by the parent-report Social Responsiveness Scale (SRS). However, when stratifying by pathogenicity and size of the CNVs, we identified that carriers of clinically significant and large genic CNVs (>500 kb) showed inferior SRS outcomes at post-intervention (P = 0.047 and P = 0.036, respectively) and follow-up (P = 0.008 and P = 0.072, respectively) when adjusting for standard care effects. Our study provides preliminary evidence that carriers of clinically significant and large genic CNVs might not benefit as much from SSGT as non-carriers. Our results indicate that genetic information might help guide the modifications of interventions in ASD.

Clinical indicators of treatment-resistant psychosis.

Around 30% of individuals with schizophrenia remain symptomatic and significantly impaired despite antipsychotic treatment and are considered to be treatment resistant. Clinicians are currently unable to predict which patients are at higher risk of treatment resistance. To determine whether genetic liability for schizophrenia and/or clinical characteristics measurable at illness onset can prospectively indicate a higher risk of treatment-resistant psychosis (TRP). In 1070 individuals with schizophrenia or related psychotic disorders, schizophrenia polygenic risk scores (PRS) and large copy number variations (CNVs) were assessed for enrichment in TRP. Regression and machine-learning approaches were used to investigate the association of phenotypes related to demographics, family history, premorbid factors and illness onset with TRP. Younger age at onset (odds ratio 0.94, P = 7.79 × 10-13) and poor premorbid social adjustment (odds ratio 1.64, P = 2.41 × 10-4) increased risk of TRP in univariate regression analyses. These factors remained associated in multivariate regression analyses, which also found lower premorbid IQ (odds ratio 0.98, P = 7.76 × 10-3), younger father's age at birth (odds ratio 0.97, P = 0.015) and cannabis use (odds ratio 1.60, P = 0.025) increased the risk of TRP. Machine-learning approaches found age at onset to be the most important predictor and also identified premorbid IQ and poor social adjustment as predictors of TRP, mirroring findings from regression analyses. Genetic liability for schizophrenia was not associated with TRP. People with an earlier age at onset of psychosis and poor premorbid functioning are more likely to be treatment resistant. The genetic architecture of susceptibility to schizophrenia may be distinct from that of treatment outcomes.

Understand the genomic diversity and evolution of fungal pathogen Candida glabrata by genome-wide analysis of genetic variations

The yeast Candida glabrata, an opportunistic human fungal pathogen, is the second most prevalent cause of candidiasis worldwide, with an infection incidence that has been increasing in the past decades. The completion of the C. glabrata reference genome made fundamental contributions to the understanding of the molecular basis of its pathogenic phenotypes. However, knowledge of genome-wide genetic variations among C. glabrata strains is limited. In this study, we present a population genomic study of C. glabrata based on whole genome re-sequencing of 47 clinical strains to an average coverage of ∼63×. Abundant genetic variations were identified in these strains, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels) and copy number variations (CNVs). The observed patterns of variations revealed clear population structure of these strains. Using population genetic tests, we detected fast evolution of several genes involved in C. glabrata adherence ability, such as EPA9 and EPA10. We also located genome structural variations, including aneuploidies and large fragment CNVs, in regions that are functionally related to virulence. Subtelometric regions were hotspots of CNVs, which may contribute to variation in expression of adhesin genes that are important for virulence. We further conducted a genome-wide association study that identified two SNPs in the 5'UTR region of CST6 that were associated with fluconazole susceptibility. These observations provide convincing evidence for the highly dynamic nature of the C. glabrata genome with potential adaptive evolution to clinical environments, and offer valuable resources for investigating the mechanisms underlying drug resistance and virulence in this fungal pathogen. (249 words).

Molecular Genetic Anatomy and Risk Profile of Hirschsprung’s Disease

BackgroundHirschsprung’s disease, or congenital aganglionosis, is a developmental disorder of the enteric nervous system and is the most common cause of intestinal obstruction in neonates and infants. The disease has more than 80% heritability, including significant associations with rare and common sequence variants in genes related to the enteric nervous system, as well as with monogenic and chromosomal syndromes.MethodsWe genotyped and exome-sequenced samples from 190 patients with Hirschsprung’s disease to quantify the genetic burden in patients with this condition. DNA sequence variants, large copy-number variants, and karyotype variants in probands were considered to be pathogenic when they were significantly associated with Hirschsprung’s disease or another neurodevelopmental disorder. Novel genes were confirmed by functional studies in the mouse and human embryonic gut and in zebrafish embryos.ResultsThe presence of five or more variants in four noncoding elements defined a widespread risk of Hirschsprung’s disease (48.4% of patients and 17.1% of controls; odds ratio, 4.54; 95% confidence interval [CI], 3.19 to 6.46). Rare coding variants in 24 genes that play roles in enteric neural-crest cell fate, 7 of which were novel, were also common (34.7% of patients and 5.0% of controls) and conferred a much greater risk than noncoding variants (odds ratio, 10.02; 95% CI, 6.45 to 15.58). Large copy-number variants, which were present in fewer patients (11.4%, as compared with 0.2% of controls), conferred the highest risk (odds ratio, 63.07; 95% CI, 36.75 to 108.25). At least one identifiable genetic risk factor was found in 72.1% of the patients, and at least 48.4% of patients had a structural or regulatory deficiency in the gene encoding receptor tyrosine kinase (RET). For individual patients, the estimated risk of Hirschsprung’s disease ranged from 5.33 cases per 100,000 live births (approximately 1 per 18,800) to 8.38 per 1000 live births (approximately 1 per 120).ConclusionsAmong the patients in our study, Hirschsprung’s disease arose from common noncoding variants, rare coding variants, and copy-number variants affecting genes involved in enteric neural-crest cell fate that exacerbate the widespread genetic susceptibility associated with RET. For individual patients, the genotype-specific odds ratios varied by a factor of approximately 67, which provides a basis for risk stratification and genetic counseling. (Funded by the National Institutes of Health.)

Population-based identity-by-descent mapping combined with exome sequencing to detect rare risk variants for schizophrenia.

Genome-wide association studies (GWASs) are highly effective at identifying common risk variants for schizophrenia. Rare risk variants are also important contributors to schizophrenia etiology but, with the exception of large copy number variants, are difficult to detect with GWAS. Exome and genome sequencing, which have accelerated the study of rare variants, are expensive so alternative methods are needed to aid detection of rare variants. Here we re-analyze an Irish schizophrenia GWAS dataset (n = 3,473) by performing identity-by-descent (IBD) mapping followed by exome sequencing of individuals identified as sharing risk haplotypes to search for rare risk variants in coding regions. We identified 45 rare haplotypes (>1 cM) that were significantly more common in cases than controls. By exome sequencing 105 haplotype carriers, we investigated these haplotypes for functional coding variants that could be tested for association in independent GWAS samples. We identified one rare missense variant in PCNT but did not find statistical support for an association with schizophrenia in a replication analysis. However, IBD mapping can prioritize both individual samples and genomic regions for follow-up analysis but genome rather than exome sequencing may be more effective at detecting risk variants on rare haplotypes.