Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Simultaneous assessment of mitochondrial DNA copy number and nuclear epigenetic age towards predictive models of development and aging.

OBJECTIVE: Mitochondrial dysfunction and nuclear epigenetic alterations, two hallmarks of aging, are associated with aberrant development and complex disease risk. Here, we report a method for the simultaneous assessment of mitochondrial DNA copy number (mtDNA-CN) and DNA methylation age (DNAm age) from the same DNA extraction using quantitative polymerase chain reaction (qPCR) and array data, respectively. RESULT: We present methods for the concurrent estimation of mtDNA-CN and DNAm age from the same DNA samples. This includes qPCR to estimate mtDNA-CN, representing the number of circular mitochondrial genomes in a cell, and DNA methylation microarray data to estimate the epigenetic age of an individual. Further, we provide a method for the combination of these metrics into a shared metric termed 'mtEpiAge'. This approach provides a valuable tool for exploring the interplay between mitochondrial dysfunction and nuclear epigenetic alterations, and their associations with disease and aging.

Genome-wide association studies for economically important traits in mink using copy number variation.

Copy number variations (CNVs) are structural variants consisting of duplications and deletions of DNA segments, which are known to play important roles in the genetics of complex traits in livestock species. However, CNV-based genome-wide association studies (GWAS) have remained unexplored in American mink. Therefore, the purpose of the current study was to investigate the association between CNVs and complex traits in American mink. A CNV-based GWAS was performed with the ParseCNV2 software program using deregressed estimated breeding values of 27 traits as pseudophenotypes, categorized into traits of growth and feed efficiency, reproduction, pelt quality, and Aleutian disease tests. The study identified a total of 10,137 CNVs (6968 duplications and 3169 deletions) using the Affymetrix Mink 70K single nucleotide polymorphism (SNP) array in 2986 American mink. The association analyses identified 250 CNV regions (CNVRs) associated with at least one of the studied traits. These CNVRs overlapped with a total of 320 potential candidate genes, and among them, several genes have been known to be related to the traits such as ARID1B, APPL1, TOX, and GPC5 (growth and feed efficiency traits); GRM1, RNASE10, WNT3, WNT3A, and WNT9B (reproduction traits); MYO10, and LIMS1 (pelt quality traits); and IFNGR2, APEX1, UBE3A, and STX11 (Aleutian disease tests). Overall, the results of the study provide potential candidate genes that may regulate economically important traits and therefore may be used as genetic markers in mink genomic breeding programs.

Assessment of MYC and TERT copy number variations in lung cancer using digital PCR.

OBJECTIVE: Lung cancer is the second most frequent cancer type and the most common cause of cancer-related deaths worldwide. Alteration of gene copy numbers are associated with lung cancer and the determination of copy number variations (CNV) is appropriate for the discrimination between tumor and non-tumor tissue in lung cancer. As telomerase reverse transcriptase (TERT) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) play a role in lung cancer the aims of this study were the verification of our recent results analyzing MYC CNV in tumor and non-tumor tissue of lung cancer patients using an independent study group and the assessment of TERT CNV as an additional marker. RESULTS: TERT and MYC status was analyzed using digital PCR (dPCR) in tumor and adjacent non-tumor tissue samples of 114 lung cancer patients. The difference between tumor and non-tumor samples were statistically significant (p < 0.0001) for TERT and MYC. Using a predefined specificity of 99% a sensitivity of 41% and 51% was observed for TERT and MYC, respectively. For the combination of TERT and MYC the overall sensitivity increased to 60% at 99% specificity. We demonstrated that a combination of markers increases the performance in comparison to individual markers. Additionally, the determination of CNV using dPCR might be an appropriate tool in precision medicine.

Assessment of the bi-directional relationship between blood mitochondrial DNA copy number and type 2 diabetes mellitus: a multivariable-adjusted regression and Mendelian randomisation study.

AIMS/HYPOTHESIS: Mitochondrial dysfunction, which can be approximated by blood mitochondrial DNA copy number (mtDNA-CN), has been implicated in the pathogenesis of type 2 diabetes mellitus. Thus far, however, insights from prospective cohort studies and Mendelian randomisation (MR) analyses on this relationship are limited. We assessed the association between blood mtDNA-CN and incident type 2 diabetes using multivariable-adjusted regression analyses, and the associations between blood mtDNA-CN and type 2 diabetes and BMI using bi-directional MR. METHODS: Multivariable-adjusted Cox proportional hazard models were used to estimate the association between blood mtDNA-CN and incident type 2 diabetes in 285,967 unrelated European individuals from UK Biobank free of type 2 diabetes at baseline. Additionally, a cross-sectional analysis was performed to investigate the association between blood mtDNA-CN and BMI. We also assessed the potentially causal relationship between blood mtDNA-CN and type 2 diabetes (N=898,130 from DIAGRAM, N=215,654 from FinnGen) and BMI (N=681,275 from GIANT) using bi-directional two-sample MR. RESULTS: During a median follow-up of 11.87 years, 15,111 participants developed type 2 diabetes. Participants with a higher level of blood mtDNA-CN are at lower risk of developing type 2 diabetes (HR 0.90 [95% CI 0.89, 0.92]). After additional adjustment for BMI and other confounders, these results attenuated moderately and remained present. The multivariable-adjusted cross-sectional analyses showed that higher blood mtDNA-CN was associated with lower BMI (-0.12 [95% CI -0.14, -0.10]) kg/m2. In the bi-directional MR analyses, we found no evidence for causal associations between blood mtDNA-CN and type 2 diabetes, and blood mtDNA-CN and BMI in either direction. CONCLUSIONS/INTERPRETATION: The results from the present study indicate that the observed association between low blood mtDNA-CN and higher risk of type 2 diabetes is likely not causal.

Assessment of burden and segregation profiles of CNVs in patients with epilepsy.

OBJECTIVE: Microdeletions are associated with different forms of epilepsy but show incomplete penetrance, which is not well understood. We aimed to assess whether unmasked variants or double CNVs could explain incomplete penetrance. METHODS: We analyzed copy number variants (CNVs) in 603 patients with four different subgroups of epilepsy and 945 controls. CNVs were called from genotypes and validated on whole-genome (WGS) or whole-exome sequences (WES). CNV burden difference between patients and controls was obtained by fitting a logistic regression. CNV burden was assessed for small and large (>1 Mb) deletions and duplications and for deletions overlapping different gene sets. RESULTS: Large deletions were enriched in genetic generalized epilepsies (GGE) compared to controls. We also found enrichment of deletions in epilepsy genes and hotspots for GGE. We did not find truncating or functional variants that could have been unmasked by the deletions. We observed a double CNV hit in two patients. One patient also carried a de novo deletion in the 22q11.2 hotspot. INTERPRETATION: We could corroborate previous findings of an enrichment of large microdeletions and deletions in epilepsy genes in GGE. We could also replicate that microdeletions show incomplete penetrance. However, we could not validate the hypothesis of unmasked variants nor the hypothesis of double CNVs to explain the incomplete penetrance. We found a de novo CNV on 22q11.2 that could be of interest. We also observed GGE families carrying a deletion on 15q13.3 hotspot that could be investigated in the Quebec founder population.

Assessment of Multiple Annealing and Looping-Based Amplification Cycle-Based Whole-Genome Amplification for Short Tandem Repeat Genotyping of Low Copy Number-DNA.

Aim: A common problem in forensic practice is the lack of sufficient amounts of good quality genomic DNA. A possible solution is the amplification of the available genomic DNA before locus-specific polymerase chain reaction (PCR) analysis. The aim of this study was to evaluate multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) for short tandem repeat (STR) genotyping of low copy number DNA (LCN-DNA). Materials and Methods: DNA isolated from five blood samples was quantified and diluted to 250, 150, 100, 50, 25, and 5 pg/µL. After preamplification with MALBAC, WGA products were quantified. PCR-STR genotyping was performed in triplicate using dilution or purification-treated WGA products for each level of DNA. STR profiles were analyzed and compared with that from non-WGA DNA. Results: The purification treatment performed better than dilution of the MALBAC-based WGA products. Compared with the non-WGA DNA, both the average number and peak heights of correct alleles were significantly improved after preamplification with the MALBAC-based WGA at DNA inputs of ≤50 pg. Like other WGA methods, allele dropout and allele drop-in were observed in the profiling results for many samples. Conclusions: MALBAC shows great potential in LCN-DNA analysis and could find broader application in the fields of forensics and genetics.

Copy number assessment of SMN1 based on real-time PCR with high-resolution melting: fast and highly reliable testing.

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by the absence of both copies of the survival motor neuron 1 (SMN1) gene. Multiple regions recommended population-wide SMA screening to quantify the copy number of SMN1. SMN1 diagnostic assays for the simplified procedure, high sensitivity, and throughput continue to be needed. METHODS: Real-time PCR with high-resolution melting for the quantifying of the SMN1 gene exon 7 copies and exon 8 copies were established and confirmed by multiplex ligation-dependent probe amplification (MLPA). The diagnosis of 2563 individuals, including SMA patients, suspected cases, and the general population, was tested by real-time PCR. The results were compared with the gold standard test MLPA. RESULTS: In this study, the homozygous and heterozygous deletions were detected by real-time PCR with a high-resolution melting method with an incidence of 10.18% and 2.26%, respectively. In addition, the R-value distribution (P > 0.05) among 8 replicates and the coefficient of variation (CV < 0.003) suggested that the real-time PCR screening test had high reproducibility. High concordance was obtained between real-time PCR with high-resolution melting and MLPA. CONCLUSIONS: The real-time PCR based on high-resolution melting provides a sensitive and high-throughput approach to large-scale SMA carrier screening with low cost and labor.

Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis.

Oral leukoplakia is the most common type of oral potentially malignant disorders and considered a precursor lesion to oral squamous cell carcinoma. However, a predictor of oral leukoplakia prognosis has not yet been identified. We investigated whether copy number alteration patterns may effectively predict the prognostic outcomes of oral leukoplakia using routinely processed paraffin sections. Comparison of copy number alteration patterns between oral leukoplakia with hyperplasia (HOL, n=22) and dysplasia (DOL, n=21) showed that oral leukoplakia with dysplasia had a higher copy number alteration rate (86%) than oral leukoplakia with hyperplasia (46%). Oral leukoplakia with dysplasia exhibited a wider range of genomic variations across all chromosomes compared with oral leukoplakia with hyperplasia. We also examined a retrospective cohort of 477 patients with oral leukoplakia with hyperplasia with detailed follow-up information. The malignant transformation (MT, n=19) and leukoplakia recurrence (LR, n=253) groups had higher frequencies of aneuploidy events and copy number loss rate than the free of disease (FD, n=205) group. Together, our results revealed the association between the degree of copy number alterations and the histological grade of oral leukoplakia and demonstrated that copy number alteration may be effective for prognosis prediction in oral leukoplakia patients with hyperplasia.

Mitochondrial DNA Copy Number Variations and Serum Pepsinogen Levels for Risk Assessment in Gastric Cancer

Background: Variations in mitochondrial DNA copy number (mtDNA-CN) of peripheral blood leukocytes (PBLs), as a potential biomarker for gastric cancer (GC) screening has currently been subject to controversy. Herein, we have assessed its efficiency in GC screening, in parallel and in combination with serum pepsinogen (sPG) I/II ratio, as an established indicator of gastric atrophy. Methods: The study population included GC (n = 53) and non-GC (n = 207) dyspeptic patients. The non-GC group was histologically categorized into CG (n = 104) and NM (n = 103) subgroups. The MtDNA-CN of PBLs was measured by quantitative real-time PCR. The sPG I and II levels and anti-H. pylori serum IgG were measured by ELISA. Results: The mtDNA-CN was found significantly higher in GC vs. non-GC (OR = 3.0; 95% CI = 1.4, 6.4) subjects. Conversely, GC patients had significantly lower sPG I/II ratio than the non-GC (OR = 3.2; CI = 1.4, 7.2) subjects. The combination of these two biomarkers yielded a dramatic amplification of the odds of GC risk in double-positive (high mtDNA-CN-low sPGI/II) subjects, in reference to double-negatives (low mtDNA-CN-high sPGI/II), when assessed against non-GC (OR = 27.1; CI = 5.0, 147.3), CG (OR = 13.1; CI = 2.4, 72.6), or NM (OR = 49.5; CI = 7.9, 311.6) groups. Conclusion: The combination of these two biomarkers, namely mtDNA-CN in PBLs and serum PG I/II ratio, drastically enhanced the efficiency of GC risk assessment, which calls for further validations.

Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases.

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

Clinical pharmacogenomics in action: design, assessment and implementation of a novel pharmacogenetic panel supporting drug selection for diseases of the central nervous system (CNS).

BACKGROUND: Pharmacogenomics describes the link between gene variations (polymorphisms) and drug responses. In view of the implementation of precision medicine in personalized healthcare, pharmacogenetic tests have recently been introduced in the clinical practice. However, the translational aspects of such tests have been limited due to the lack of robust population-based evidence. MATERIALS: In this paper we present a novel pharmacogenetic panel (iDNA Genomics-PGx-CNS or PGx-CNS), consisting of 24 single nucleotide polymorphisms (SNPs) on 13 genes involved in the signaling or/and the metabolism of 28 approved drugs currently administered to treat diseases of the Central Nervous System (CNS). We have tested the PGx-CNS panel on 501 patient-derived DNA samples from a southeastern European population and applied biostatistical analyses on the pharmacogenetic associations involving drug selection, dosing and the risk of adverse drug events (ADEs). RESULTS: Results reveal the occurrences of each SNP in the sample and a strong correlation with the European population. Nonlinear principal component analysis strongly indicates co-occurrences of certain variants. The metabolization efficiency (poor, intermediate, extensive, ultra-rapid) and the frequency of clinical useful pharmacogenetic, associations in the population (drug relevance), are also described, along with four exemplar clinical cases illustrating the strong potential of the PGx-CNS panel, as a companion diagnostic assay. It is noted that pharmacogenetic associations involving copy number variations (CNVs) or the HLA gene were not included in this analysis. CONCLUSIONS: Overall, results illustrate that the PGx-CNS panel is a valuable tool supporting therapeutic medical decisions, urging its broad clinical implementation.

The phenotype of 15 cases with rare 8q24.13-q24.3 deletions-A new syndrome or still an enigma?

Diagnosis of rare copy number variants (CNVs) with scarce literature evidence poses a major challenge for interpretation of the clinical significance of chromosomal microarray analysis (CMA) results, especially in the prenatal setting. Bioinformatic tools can be used to assist in this issue; however, this prediction can be imprecise. Our objective was to describe the phenotype of the rare copy number losses encompassing the 8q24.13-q24.3 locus, and to find common features in terms of genomic coordinates, gene content, and clinical phenotypic characteristics. Appropriate cases were retrieved using local databases of two largest Israeli centers performing CMA analysis. In addition, literature and public databases search was performed. Local database search yielded seven new patients with del (8)(q24.13q24.3) (one of these with an additional copy number variant). Literature and public databases search yielded eight additional patients. The cases showed high phenotypic variability, ranging from asymptomatic adults and fetuses with normal ultrasound to patients with autism/developmental delay (6/11 postnatal cases, 54.5%). No clear association was noted between the specific disease-causing/high-pLI gene content of the described del (8)(q24.13q24.3) to neurodevelopmental disorders, except for a possibly relevant locus encompassing the KCNQ3 gene. We present the challenges in classification of rare variants with limited clinical information. In such cases, genotype-phenotype correlation must be assessed with extra-caution and possibly using additional methods to assist the classification, especially in the prenatal setting.

Post-selection inference for changepoint detection algorithms with application to copy number variation data.

Changepoint detection methods are used in many areas of science and engineering, for example, in the analysis of copy number variation data to detect abnormalities in copy numbers along the genome. Despite the broad array of available tools, methodology for quantifying our uncertainty in the strength (or the presence) of given changepoints post-selection are lacking. Post-selection inference offers a framework to fill this gap, but the most straightforward application of these methods results in low-powered hypothesis tests and leaves open several important questions about practical usability. In this work, we carefully tailor post-selection inference methods toward changepoint detection, focusing on copy number variation data. To accomplish this, we study commonly used changepoint algorithms: binary segmentation, as well as two of its most popular variants, wild and circular, and the fused lasso. We implement some of the latest developments in post-selection inference theory, mainly auxiliary randomization. This improves the power, which requires implementations of Markov chain Monte Carlo algorithms (importance sampling and hit-and-run sampling) to carry out our tests. We also provide recommendations for improving practical useability, detailed simulations, and example analyses on array comparative genomic hybridization as well as sequencing data.

A Comprehensive Assessment of Genetic and Epigenetic Alterations Identifies Frequent Variations Impacting Six Prototypic SCF Complex Members.

The SKP1, CUL1, F-box protein (SCF) complex represents a family of 69 E3 ubiquitin ligases that poly-ubiquitinate protein substrates marking them for proteolytic degradation via the 26S proteasome. Established SCF complex targets include transcription factors, oncoproteins and tumor suppressors that modulate cell cycle activity and mitotic fidelity. Accordingly, genetic and epigenetic alterations involving SCF complex member genes are expected to adversely impact target regulation and contribute to disease etiology. To gain novel insight into cancer pathogenesis, we determined the prevalence of genetic and epigenetic alterations in six prototypic SCF complex member genes (SKP1, CUL1, RBX1, SKP2, FBXW7 and FBXO5) from patient datasets extracted from The Cancer Genome Atlas (TCGA). Collectively, ~45% of observed SCF complex member mutations are predicted to impact complex structure and/or function in 10 solid tumor types. In addition, the distribution of encoded alterations suggest SCF complex members may exhibit either tumor suppressor or oncogenic mutational profiles in a cancer type dependent manner. Further bioinformatic analyses reveal the potential functional implications of encoded alterations arising from missense mutations by examining predicted deleterious mutations with available crystal structures. The SCF complex also exhibits frequent copy number alterations in a variety of cancer types that generally correspond with mRNA expression levels. Finally, we note that SCF complex member genes are differentially methylated across cancer types, which may effectively phenocopy gene copy number alterations. Collectively, these data show that SCF complex member genes are frequently altered at the genetic and epigenetic levels in many cancer types, which will adversely impact the normal targeting and timely destruction of protein substrates, which may contribute to the development and progression of an extensive array of cancer types.

Characterizing Intra-Tumor Heterogeneity From Somatic Mutations Without Copy-Neutral Assumption.

Bulk samples of the same patient are heterogeneous in nature, comprising of different subpopulations (subclones) of cancer cells. Cells in a tumor subclone are characterized by unique mutational genotype profile. Resolving tumor heterogeneity by estimating the genotypes, cellular proportions and the number of subclones present in the tumor can help in understanding cancer progression and treatment. We present a novel method, ChaClone2, to efficiently deconvolve the observed variant allele fractions (VAFs), with consideration for possible effects from copy number aberrations at the mutation loci. Our method describes a state-space formulation of the feature allocation model, deconvolving the observed VAFs from samples of the same patient into three matrices: subclonal total and variant copy numbers for mutated genes, and proportions of subclones in each sample. We describe an efficient sequential Monte Carlo (SMC) algorithm to estimate these matrices. Extensive simulation shows that the ChaClone2 yields better accuracy when compared with other state-of-the-art methods for addressing similar problem and it offers scalability to large datasets. Also, ChaClone2 features that the model parameter estimates can be refined whenever new mutation data of freshly sequenced genomic locations are available. MATLAB code and datasets are available to download at: https://github.com/moyanre/method2.

Noninvasive Detection of Urothelial Carcinoma by Cost-effective Low-coverage Whole-genome Sequencing from Urine-Exfoliated Cell DNA.

PURPOSE: Urothelial carcinoma is a malignant cancer with frequent chromosomal aberrations. Here, we investigated the application of a cost-effective, low-coverage whole-genome sequencing technology in detecting all chromosomal aberrations. EXPERIMENTAL DESIGN: Patients with urothelial carcinomas and nontumor controls were prospectively recruited in clinical trial NCT03998371. Urine-exfoliated cell DNA was analyzed by Illumina HiSeq XTen, followed by genotyping with a customized bioinformatics workflow named Urine Exfoliated Cells Copy Number Aberration Detector (UroCAD). RESULTS: In the discovery phase, urine samples from 126 patients with urothelial carcinomas and 64 nontumor disease samples were analyzed. Frequent chromosome copy-number changes were found in patients with tumor as compared with nontumor controls. A novel diagnosis model, UroCAD, was built by incorporating all the autosomal chromosomal changes. The model reached performance of AUC = 0.92 (95% confidence interval, 89.4%-97.3%). At the optimal cutoff, |Z| ≥ 3.21, the sensitivity, specificity, and accuracy were 82.5%, 96.9%, and 89.0%, respectively. The prediction positivity was found correlated with tumor grade (P = 0.01). In the external validation cohort of 95 participants, the UroCAD assay identified urothelial carcinomas with an overall sensitivity of 80.4%, specificity of 94.9%, and AUC of 0.91. Meanwhile, UroCAD assay outperformed cytology tests with significantly improved sensitivity (80.4% vs. 33.9%; P < 0.001) and comparable specificity (94.9% vs. 100%; P = 0.49). CONCLUSIONS: UroCAD could be a robust urothelial carcinoma diagnostic method with improved sensitivity and similar specificity as compared with cytology tests. It may be used as a noninvasive approach for diagnosis and recurrence surveillance in urothelial carcinoma prior to the use of cystoscopy, which would largely reduce the burden on patients.

Recurrent Rare Copy Number Variants Increase Risk for Esotropia.

Purpose: To determine whether rare copy number variants (CNVs) increase risk for comitant esotropia. Methods: CNVs were identified in 1614 Caucasian individuals with comitant esotropia and 3922 Caucasian controls from Illumina SNP genotyping using two Hidden Markov model (HMM) algorithms, PennCNV and QuantiSNP, which call CNVs based on logR ratio and B allele frequency. Deletions and duplications greater than 10 kb were included. Common CNVs were excluded. Association testing was performed with 1 million permutations in PLINK. Significant CNVs were confirmed with digital droplet polymerase chain reaction (ddPCR). Whole genome sequencing was performed to determine insertion location and breakpoints. Results: Esotropia patients have similar rates and proportions of CNVs compared with controls but greater total length and average size of both deletions and duplications. Three recurrent rare duplications significantly (P = 1 × 10-6) increase the risk of esotropia: chromosome 2p11.2 (hg19, 2:87428677-87965359), spanning one long noncoding RNA (lncRNA) and two microRNAs (OR 14.16; 95% confidence interval [CI] 5.4-38.1); chromosome 4p15.2 (hg19, 4:25554332-25577184), spanning one lncRNA (OR 11.1; 95% CI 4.6-25.2); chromosome 10q11.22 (hg19, 10:47049547-47703870) spanning seven protein-coding genes, one lncRNA, and four pseudogenes (OR 8.96; 95% CI 5.4-14.9). Overall, 114 cases (7%) and only 28 controls (0.7%) had one of the three rare duplications. No case nor control had more than one of these three duplications. Conclusions: Rare CNVs are a source of genetic variation that contribute to the genetic risk for comitant esotropia, which is likely polygenic. Future research into the functional consequences of these recurrent duplications may shed light on the pathophysiology of esotropia.

CONY: A Bayesian procedure for detecting copy number variations from sequencing read depths.

Copy number variations (CNVs) are genomic structural mutations consisting of abnormal numbers of fragment copies. Next-generation sequencing of read-depth signals mirrors these variants. Some tools used to predict CNVs by depth have been published, but most of these tools can be applied to only a specific data type due to modeling limitations. We develop a tool for copy number variation detection by a Bayesian procedure, i.e., CONY, that adopts a Bayesian hierarchical model and an efficient reversible-jump Markov chain Monte Carlo inference algorithm for whole genome sequencing of read-depth data. CONY can be applied not only to individual samples for estimating the absolute number of copies but also to case-control pairs for detecting patient-specific variations. We evaluate the performance of CONY and compare CONY with competing approaches through simulations and by using experimental data from the 1000 Genomes Project. CONY outperforms the other methods in terms of accuracy in both single-sample and paired-samples analyses. In addition, CONY performs well regardless of whether the data coverage is high or low. CONY is useful for detecting both absolute and relative CNVs from read-depth data sequences. The package is available at https://github.com/weiyuchung/CONY.

Identifying schizophrenia patients who carry pathogenic genetic copy number variants using standard clinical assessment: retrospective cohort study.

BACKGROUND: Copy number variants (CNVs) play a significant role in disease pathogenesis in a small subset of individuals with schizophrenia (~2.5%). Chromosomal microarray testing is a first-tier genetic test for many neurodevelopmental disorders. Similar testing could be useful in schizophrenia. AIMS: To determine whether clinically identifiable phenotypic features could be used to successfully model schizophrenia-associated (SCZ-associated) CNV carrier status in a large schizophrenia cohort. METHOD: Logistic regression and receiver operating characteristic (ROC) curves tested the accuracy of readily identifiable phenotypic features in modelling SCZ-associated CNV status in a discovery data-set of 1215 individuals with psychosis. A replication analysis was undertaken in a second psychosis data-set (n = 479). RESULTS: In the discovery cohort, specific learning disorder (OR = 8.12; 95% CI 1.16-34.88, P = 0.012), developmental delay (OR = 5.19; 95% CI 1.58-14.76, P = 0.003) and comorbid neurodevelopmental disorder (OR = 5.87; 95% CI 1.28-19.69, P = 0.009) were significant independent variables in modelling positive carrier status for a SCZ-associated CNV, with an area under the ROC (AUROC) of 74.2% (95% CI 61.9-86.4%). A model constructed from the discovery cohort including developmental delay and comorbid neurodevelopmental disorder variables resulted in an AUROC of 83% (95% CI 52.0-100.0%) for the replication cohort. CONCLUSIONS: These findings suggest that careful clinical history taking to document specific neurodevelopmental features may be informative in screening for individuals with schizophrenia who are at higher risk of carrying known SCZ-associated CNVs. Identification of genomic disorders in these individuals is likely to have clinical benefits similar to those demonstrated for other neurodevelopmental disorders.