Responsible for the metabolism of ~21% of clinically used drugs, CYP2D6 is a critical component of personalized medicine initiatives. Genotyping CYP2D6 is challenging due to sequence similarity with ...its pseudogene paralog CYP2D7 and a high number and variety of common structural variants (SVs). Here we describe a novel bioinformatics method, Cyrius, that accurately genotypes CYP2D6 using whole-genome sequencing (WGS) data. We show that Cyrius has superior performance (96.5% concordance with truth genotypes) compared to existing methods (84-86.8%). After implementing the improvements identified from the comparison against the truth data, Cyrius's accuracy has since been improved to 99.3%. Using Cyrius, we built a haplotype frequency database from 2504 ethnically diverse samples and estimate that SV-containing star alleles are more frequent than previously reported. Cyrius will be an important tool to incorporate pharmacogenomics in WGS-based precision medicine initiatives.
Identification of the genetic polymorphisms that contribute to susceptibility for common diseases such as type 2 diabetes and schizophrenia will aid in the development of diagnostics and ...therapeutics. Previous studies have focused on the technique of genetic linkage, but new technologies and experimental resources make whole-genome association studies more feasible. Association studies of this type have good prospects for dissecting the genetics of common disease, but they currently face a number of challenges, including problems with multiple testing and study design, definition of intermediate phenotypes and interaction between polymorphisms.
Tandem DNA repeats vary in the size and sequence of each unit (motif). When expanded, these tandem DNA repeats have been associated with more than 40 monogenic disorders
. Their involvement in ...disorders with complex genetics is largely unknown, as is the extent of their heterogeneity. Here we investigated the genome-wide characteristics of tandem repeats that had motifs with a length of 2-20 base pairs in 17,231 genomes of families containing individuals with autism spectrum disorder (ASD)
and population control individuals
. We found extensive polymorphism in the size and sequence of motifs. Many of the tandem repeat loci that we detected correlated with cytogenetic fragile sites. At 2,588 loci, gene-associated expansions of tandem repeats that were rare among population control individuals were significantly more prevalent among individuals with ASD than their siblings without ASD, particularly in exons and near splice junctions, and in genes related to the development of the nervous system and cardiovascular system or muscle. Rare tandem repeat expansions had a prevalence of 23.3% in children with ASD compared with 20.7% in children without ASD, which suggests that tandem repeat expansions make a collective contribution to the risk of ASD of 2.6%. These rare tandem repeat expansions included previously undescribed ASD-linked expansions in DMPK and FXN, which are associated with neuromuscular conditions, and in previously unknown loci such as FGF14 and CACNB1. Rare tandem repeat expansions were associated with lower IQ and adaptive ability. Our results show that tandem DNA repeat expansions contribute strongly to the genetic aetiology and phenotypic complexity of ASD.
Adult-onset cerebellar ataxias are a group of neurodegenerative conditions that challenge both genetic discovery and molecular diagnosis. In this study, we identified an intronic (GAA) repeat ...expansion in fibroblast growth factor 14 (FGF14). Genetic analysis of 95 Australian individuals with adult-onset ataxia identified four (4.2%) with (GAA)
and a further nine individuals with (GAA)
. PCR and long-read sequence analysis revealed these were pure (GAA) repeats. In comparison, no control subjects had (GAA)
and only 2/311 control individuals (0.6%) had a pure (GAA)
. In a German validation cohort, 9/104 (8.7%) of affected individuals had (GAA)
and a further six had (GAA)
, whereas 10/190 (5.3%) control subjects had (GAA)
but none were (GAA)
. The combined data suggest (GAA)
are disease causing and fully penetrant (p = 6.0 × 10
, OR = 72 95% CI = 4.3-1,227), while (GAA)
is likely pathogenic with reduced penetrance. Affected individuals had an adult-onset, slowly progressive cerebellar ataxia with variable features including vestibular impairment, hyper-reflexia, and autonomic dysfunction. A negative correlation between age at onset and repeat length was observed (R
= 0.44, p = 0.00045, slope = -0.12) and identification of a shared haplotype in a minority of individuals suggests that the expansion can be inherited or generated de novo during meiotic division. This study demonstrates the power of genome sequencing and advanced bioinformatic tools to identify novel repeat expansions via model-free, genome-wide analysis and identifies SCA50/ATX-FGF14 as a frequent cause of adult-onset ataxia.
Late detection, compromised immune system, and chemotherapy resistance underlie the poor patient prognosis for pancreatic ductal adenocarcinoma (PDAC) patients, making it the 3rd leading cause of ...cancer-related deaths in the United States. Cooperation between the tumor cells and the immune system leads to the immune escape and eventual establishment of the tumor. For more than 20 years, sincere efforts have been made to intercept the tumor-immune crosstalk and identify the probable therapeutic targets for breaking self-tolerance toward tumor antigens. However, the success of these studies depends on detailed examination and understanding of tumor-immune cell interactions, not only in the primary tumor but also at distant systemic niches. Innate and adaptive arms of the immune system sculpt tumor immunogenicity, where they not only aid in providing an amenable environment for their survival but also act as a driver for tumor relapse at primary or distant organ sites. This review article highlights the key events associated with tumor-immune communication and associated immunosuppression at both local and systemic microenvironments in PDAC. Furthermore, we discuss the approaches and benefits of targeting both local and systemic immunosuppression for PDAC patients. The present articles integrate data from clinical and genetic mouse model studies to provide a widespread consensus on the role of local and systemic immunosuppression in undermining the anti-tumor immune responses against PDAC.
Spinal muscular atrophy (SMA), caused by loss of the SMN1 gene, is a leading cause of early childhood death. Due to the near identical sequences of SMN1 and SMN2, analysis of this region is ...challenging. Population-wide SMA screening to quantify the SMN1 copy number (CN) is recommended by the American College of Medical Genetics and Genomics.
We developed a method that accurately identifies the CN of SMN1 and SMN2 using genome sequencing (GS) data by analyzing read depth and eight informative reference genome differences between SMN1/2.
We characterized SMN1/2 in 12,747 genomes, identified 1568 samples with SMN1 gains or losses and 6615 samples with SMN2 gains or losses, and calculated a pan-ethnic carrier frequency of 2%, consistent with previous studies. Additionally, 99.8% of our SMN1 and 99.7% of SMN2 CN calls agreed with orthogonal methods, with a recall of 100% for SMA and 97.8% for carriers, and a precision of 100% for both SMA and carriers.
This SMN copy-number caller can be used to identify both carrier and affected status of SMA, enabling SMA testing to be offered as a comprehensive test in neonatal care and an accurate carrier screening tool in GS sequencing projects.
Common genetic polymorphisms may explain a portion of the heritable risk for common diseases. Within candidate genes, the number of common polymorphisms is finite, but direct assay of all existing ...common polymorphism is inefficient, because genotypes at many of these sites are strongly correlated. Thus, it is not necessary to assay all common variants if the patterns of allelic association between common variants can be described. We have developed an algorithm to select the maximally informative set of common single-nucleotide polymorphisms (tagSNPs) to assay in candidate-gene association studies, such that all known common polymorphisms either are directly assayed or exceed a threshold level of association with a tagSNP. The algorithm is based on the
r
2 linkage disequilibrium (LD) statistic, because
r
2 is directly related to statistical power to detect disease associations with unassayed sites. We show that, at a relatively stringent
r
2 threshold (
r
2>0.8), the LD-selected tagSNPs resolve >80% of all haplotypes across a set of 100 candidate genes, regardless of recombination, and tag specific haplotypes and clades of related haplotypes in nonrecombinant regions. Thus, if the patterns of common variation are described for a candidate gene, analysis of the tagSNP set can comprehensively interrogate for main effects from common functional variation. We demonstrate that, although common variation tends to be shared between populations, tagSNPs should be selected separately for populations with different ancestries.
Screening for short tandem repeat (STR) expansions in next-generation sequencing data can enable diagnosis, optimal clinical management/treatment, and accurate genetic counseling of patients with ...repeat expansion disorders. We aimed to develop an efficient computational workflow for reliable detection of STR expansions in next-generation sequencing data and demonstrate its clinical utility.
We characterized the performance of eight STR analysis methods (lobSTR, HipSTR, RepeatSeq, ExpansionHunter, TREDPARSE, GangSTR, STRetch, and exSTRa) on next-generation sequencing datasets of samples with known disease-causing full-mutation STR expansions and genomes simulated to harbor repeat expansions at selected loci and optimized their sensitivity. We then used a machine learning decision tree classifier to identify an optimal combination of methods for full-mutation detection. In Burrows-Wheeler Aligner (BWA)-aligned genomes, the ensemble approach of using ExpansionHunter, STRetch, and exSTRa performed the best (precision = 82%, recall = 100%, F1-score = 90%). We applied this pipeline to screen 301 families of children with suspected genetic disorders.
We identified 10 individuals with full-mutations in the AR, ATXN1, ATXN8, DMPK, FXN, or HTT disease STR locus in the analyzed families. Additional candidates identified in our analysis include two probands with borderline ATXN2 expansions between the established repeat size range for reduced-penetrance and full-penetrance full-mutation and seven individuals with FMR1 CGG repeats in the intermediate/premutation repeat size range. In 67 probands with a prior negative clinical PCR test for the FMR1, FXN, or DMPK disease STR locus, or the spinocerebellar ataxia disease STR panel, our pipeline did not falsely identify aberrant expansion. We performed clinical PCR tests on seven (out of 10) full-mutation samples identified by our pipeline and confirmed the expansion status in all, showing absolute concordance between our bioinformatics and molecular findings.
We have successfully demonstrated the application of a well-optimized bioinformatics pipeline that promotes the utility of genome-wide sequencing as a first-tier screening test to detect expansions of known disease STRs. Interrogating clinical next-generation sequencing data for pathogenic STR expansions using our ensemble pipeline can improve diagnostic yield and enhance clinical outcomes for patients with repeat expansion disorders.
Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases
. However, repeat expansions are often not explored beyond neurological and ...neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer
. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.
Standard whole-genome genotyping technologies are unable to determine haplotypes. Here we describe a method for rapid and cost-effective long-range haplotyping. Genomic DNA is diluted and distributed ...into multiple aliquots such that each aliquot receives a fraction of a haploid copy. The DNA template in each aliquot is amplified by multiple displacement amplification, converted into barcoded sequencing libraries using Nextera technology, and sequenced in multiplexed pools. To assess the performance of our method, we combined two male genomic DNA samples at equal ratios, resulting in a sample with diploid X chromosomes with known haplotypes. Pools of the multiplexed sequencing libraries were subjected to targeted pull-down of a 1-Mb contiguous region of the X-chromosome Duchenne muscular dystrophy gene. We were able to phase the Duchenne muscular dystrophy region into two contiguous haplotype blocks with a mean length of 494 kb. The haplotypes showed 99% agreement with the consensus base calls made by sequencing the individual DNAs. We subsequently used the strategy to haplotype two human genomes. Standard genomic sequencing to identify all heterozygous SNPs in the sample was combined with dilution-amplification–based sequencing data to resolve the phase of identified heterozygous SNPs. Using this procedure, we were able to phase >95% of the heterozygous SNPs from the diploid sequence data. The N50 for a Yoruba male DNA was 702 kb whereas the N50 for a European female DNA was 358 kb. Therefore, the strategy described here is suitable for haplotyping of a set of targeted regions as well as of the entire genome.
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