Deep whole-genome sequencing of 100 southeast Asian Malays.

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.

One CNV Discordance in NRXN1 Observed Upon Genome-wide Screening in 38 Pairs of Adult Healthy Monozygotic Twins.

Monozygotic (MZ) twins stem from the same single fertilized egg and therefore share all their inherited genetic variation. This is one of the unequivocal facts on which genetic epidemiology and twin studies are based. To what extent this also implies that MZ twins share genotypes in adult tissues is not precisely established, but a common pragmatic assumption is that MZ twins are 100% genetically identical also in adult tissues. During the past decade, this view has been challenged by several reports, with observations of differences in post-zygotic copy number variations (CNVs) between members of the same MZ pair. In this study, we performed a systematic search for differences of CNVs within 38 adult MZ pairs who had been misclassified as dizygotic (DZ) twins by questionnaire-based assessment. Initial scoring by PennCNV suggested a total of 967 CNV discordances. The within-pair correlation in number of CNVs detected was strongly dependent on confidence score filtering and reached a plateau of r = 0.8 when restricting to CNVs detected with confidence score larger than 50. The top-ranked discordances were subsequently selected for validation by quantitative polymerase chain reaction (qPCR), from which one single ~120kb deletion in NRXN1 on chromosome 2 (bp 51017111-51136802) was validated. Despite involving an exon, no sign of cognitive/mental consequences was apparent in the affected twin pair, potentially reflecting limited or lack of expression of the transcripts containing this exon in nerve/brain.

Regions of homozygosity in three Southeast Asian populations.

The genomes of outbred populations were first shown in 2006 to contain regions of homozygosity (ROHs) of several megabases. Further studies have also investigated the characteristics of ROHs in healthy individuals in various populations but there are no studies on Singapore populations to date. This study aims to identify and investigate the characteristics of ROHs in three Singapore populations. A total of 268 samples (96 Chinese, 89 Malays and 83 Indians) are genotyped on Illumina Human 1 M Beadchip and Affymetrix Genome-Wide Human SNP Array 6.0. We use the PennCNV algorithm to detect ROHs. We report an abundance of ROHs (≥500 kb), with an average of more than one hundred regions per individual. On average, the Indian population has the lowest number of ROHs and smallest total length of ROHs per individual compared with the Chinese and Malay populations. We further investigate the relationship between the occurrence of ROHs and haplotype frequency, regional linkage disequilibrium (LD) and positive selection. Based on the results of this data set, we find that the frequency of occurrence of ROHs is positively associated with haplotype frequency and regional LD. The majority of regions detected for recent positive selection and regions with differential LD between populations overlap with the ROH loci. When we consider both the location of the ROHs and the allelic form of the ROHs, we are able to separate the populations by principal component analysis, demonstrating that ROHs contain information on population structure and the demographic history of a population.

A population-based study of copy number variants and regions of homozygosity in healthy Swedish individuals.

The abundance of copy number variants (CNVs) and regions of homozygosity (ROHs) have been well documented in previous studies. In addition, their roles in complex diseases and traits have since been increasingly appreciated. However, only a limited amount of CNV and ROH data is currently available for the Swedish population. We conducted a population-based study to detect and characterize CNVs and ROHs in 87 randomly selected healthy Swedish individuals using the Affymetrix SNP Array 6.0. More than 600 CNV loci were detected in the population using two different CNV-detection algorithms (PennCNV and Birdsuite). A total of 196 loci were consistently identified by both algorithms, suggesting their reliability. Numerous disease-associated and pharmacogenetics-related genes were found to be overlapping with common CNV loci such as CFHR1/R3, LCE3B/3C, UGT2B17 and GSTT1. Correlation analysis between copy number polymorphisms (CNPs) and genome-wide association studies-identified single-nucleotide polymorphisms also indicates the potential roles of several CNPs as causal variants for diseases and traits such as body mass index, Crohn's disease and multiple sclerosis. In addition, we also identified a total of 14 815 ROHs 500 kb or 2814 ROHs 1M in the Swedish individuals with an average of 170 and 32 regions detected per individual respectively. Approximately 141 Mb or 4.92% of the genome is homozygous in each individual of the Swedish population. This is the first population-based study to investigate the population characteristics of CNVs and ROHs in the Swedish population. This study found many CNV loci that warrant further investigation, and also highlighted the abundance and importance of investigating ROHs for their associations with complex diseases and traits.

Copy number polymorphisms in new HapMap III and Singapore populations.

Copy number variations can be identified using newer genotyping arrays with higher single nucleotide polymorphisms (SNPs) density and copy number probes accompanied by newer algorithms. McCarroll et al. (2008) applied these to the HapMap II samples and identified 1316 copy number polymorphisms (CNPs). In our study, we applied the same approach to 859 samples from three Singapore populations and seven HapMap III populations. Approximately 50% of the 1291 autosomal CNPs were found to be polymorphic only in populations of non-African ancestry. Pairwise comparisons among the 10 populations showed substantial differences in the CNPs frequencies. Additionally, 698 CNPs showed significant differences with false discovery rate (FDR)<0.01 among the 10 populations and these loci overlap with known disease-associated or pharmacogenetic-related genes such as CFHR3 and CFHR1 (age related macular degeneration), GSTTI (metabolism of various carcinogenic compounds and cancers) and UGT2B17 (prostate cancer and graft-versus-host disease). The correlations between CNPs and genome-wide association studies-SNPs were investigated and several loci, which were previously unreported, that may potentially be implicated in complex diseases and traits were found; for example, childhood acute lymphoblastic leukaemia, age-related macular degeneration, breast cancer, response to antipsychotic treatment, rheumatoid arthritis and type-1 diabetes. Additionally, we also found 5014 novel copy number loci that have not been reported previously by McCarroll et al. (2008) in the 10 populations.

Luminal microbiota related to Crohn's disease recurrence after surgery.

BACKGROUND: Microbial factors are likely to be involved in the recurrence of Crohn's disease (CD) after bowel resection. We investigated the luminal microbiota before and longitudinally after surgery, in relation to disease recurrence, using 16S metagenomic techniques. METHODS: In the prospective Post-Operative Crohn's Endoscopic Recurrence (POCER) study, fecal samples were obtained before surgery and 6, 12, and 18 months after surgery from 130 CD patients. Endoscopy was undertaken to detect disease recurrence, defined as Rutgeerts score ≥i2, at 6 months in two-thirds of patients and all patients at 18 months after surgery. The V2 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Cluster analysis was performed at family level, assessing microbiome community differences between patients with and without recurrence. RESULTS: Six microbial cluster groups were identified. The cluster associated with maintenance of remission was enriched for the Lachnospiraceae family [adjusted OR 0.47 (0.27-0.82), P = .007]. The OTU diversity of Lachnospiraceae within this cluster was significantly greater than in all other clusters. The cluster enriched for Enterobacteriaceae was associated with an increased risk of disease recurrence [adjusted OR 6.35 (1.24-32.44), P = .026]. OTU diversity of Enterobacteriaceae within this cluster was significantly greater than in other clusters. CONCLUSIONS: Luminal bacterial communities are associated with protection from, and the occurrence of, Crohn's disease recurrence after surgery. Recurrence may relate to a higher abundance of facultatively anaerobic pathobionts from the Enterobacteriaceae family. The ecologic change of depleted Lachnospiraceae, a genus of butyrate-producing bacteria, may permit expansion of Enterobacteriaceae through luminal environmental perturbation.

Microbial Factors Associated with Postoperative Crohn's Disease Recurrence.

BACKGROUND AND AIMS: The intestinal microbiota is a key antigenic driver in Crohn's disease [CD]. We aimed to identify changes in the gut microbiome associated with, and predictive of, disease recurrence and remission. METHODS: A total of 141 mucosal biopsy samples from 34 CD patients were obtained at surgical resection and at colonoscopy 6 and/or 18 months postoperatively; 28 control samples were obtained: 12 from healthy patients [healthy controls] and 16 from hemicolectomy patients [surgical controls]. Bacterial 16S ribosomal profiling was performed using the Illumina MiSeq platform. RESULTS: CD was associated with reduced alpha diversity when compared with healthy controls but not surgical controls [p < 0.001 and p = 0.666, respectively]. Beta diversity [composition] differed significantly between CD and both healthy [p < 0.001] and surgical [p = 0.022] controls, but did not differ significantly between those with and without endoscopic recurrence. There were significant taxonomic differences between recurrence and remission. Patients experiencing recurrence demonstrated elevated Proteus genera [p = 0.008] and reduced Faecalibacterium [p< 0.001]. Active smoking was associated with elevated levels of Proteus [p = 0.013] postoperatively. Low abundance of Faecalibacterium [< 0.1%] and detectable Proteus in the postoperative ileal mucosa was associated with a higher risk of recurrence (odds ratio [OR] 14 [1.7-110], p = 0.013 and 13 [1.1-150], p = 0.039, respectively) when corrected for smoking. A model of recurrence comprising the presence of Proteus, abundance of Faecalibacterium, and smoking status showed moderate accuracy (area under the curve [AUC] 0.740, 95% confidence interval [CI] [0.69-0.79]). CONCLUSIONS: CD is associated with a microbial signature distinct from health. Microbial factors and smoking independently influence postoperative CD recurrence. The genus Proteus may play a role in the development of CD.