The correlation between CpG methylation and gene expression is driven by sequence variants.

Gene promoter and enhancer sequences are bound by transcription factors and are depleted of methylated CpG sites (cytosines preceding guanines in DNA). The absence of methylated CpGs in these sequences typically correlates with increased gene expression, indicating a regulatory role for methylation. We used nanopore sequencing to determine haplotype-specific methylation rates of 15.3 million CpG units in 7,179 whole-blood genomes. We identified 189,178 methylation depleted sequences where three or more proximal CpGs were unmethylated on at least one haplotype. A total of 77,789 methylation depleted sequences (~41%) associated with 80,503 cis-acting sequence variants, which we termed allele-specific methylation quantitative trait loci (ASM-QTLs). RNA sequencing of 896 samples from the same blood draws used to perform nanopore sequencing showed that the ASM-QTL, that is, DNA sequence variability, drives most of the correlation found between gene expression and CpG methylation. ASM-QTLs were enriched 40.2-fold (95% confidence interval 32.2, 49.9) among sequence variants associating with hematological traits, demonstrating that ASM-QTLs are important functional units in the noncoding genome.

A population-based survey of FBN1 variants in Iceland reveals underdiagnosis of Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant condition characterized by aortic aneurysm, skeletal abnormalities, and lens dislocation, and is caused by variants in the FBN1 gene. To explore causes of MFS and the prevalence of the disease in Iceland we collected information from all living individuals with a clinical diagnosis of MFS in Iceland (n = 32) and performed whole-genome sequencing of those who did not have a confirmed genetic diagnosis (27/32). Moreover, to assess a potential underdiagnosis of MFS in Iceland we attempted a genotype-based approach to identify individuals with MFS. We interrogated deCODE genetics' database of 35,712 whole-genome sequenced individuals to search for rare sequence variants in FBN1. Overall, we identified 15 pathogenic or likely pathogenic variants in FBN1 in 44 individuals, only 22 of whom were previously diagnosed with MFS. The most common of these variants, NM_000138.4:c.8038 C > T p.(Arg2680Cys), is present in a multi-generational pedigree, and was found to stem from a single forefather born around 1840. The p.(Arg2680Cys) variant associates with a form of MFS that seems to have an enrichment of abdominal aortic aneurysm, suggesting that this may be a particularly common feature of p.(Arg2680Cys)-associated MFS. Based on these combined genetic and clinical data, we show that MFS prevalence in Iceland could be as high as 1/6,600 in Iceland, compared to 1/10,000 based on clinical diagnosis alone, which indicates underdiagnosis of this actionable genetic disorder.

Multiple transmissions of de novo mutations in families.

De novo mutations (DNMs) cause a large proportion of severe rare diseases of childhood. DNMs that occur early may result in mosaicism of both somatic and germ cells. Such early mutations can cause recurrence of disease. We scanned 1,007 sibling pairs from 251 families and identified 878 DNMs shared by siblings (ssDNMs) at 448 genomic sites. We estimated DNM recurrence probability based on parental mosaicism, sharing of DNMs among siblings, parent-of-origin, mutation type and genomic position. We detected 57.2% of ssDNMs in the parental blood. The recurrence probability of a DNM decreases by 2.27% per year for paternal DNMs and 1.78% per year for maternal DNMs. Maternal ssDNMs are more likely to be T>C mutations than paternal ssDNMs, and less likely to be C>T mutations. Depending on the properties of the DNM, the recurrence probability ranges from 0.011% to 28.5%. We have launched an online calculator to allow estimation of DNM recurrence probability for research purposes.

Whole genome characterization of sequence diversity of 15,220 Icelanders.

Understanding of sequence diversity is the cornerstone of analysis of genetic disorders, population genetics, and evolutionary biology. Here, we present an update of our sequencing set to 15,220 Icelanders who we sequenced to an average genome-wide coverage of 34X. We identified 39,020,168 autosomal variants passing GATK filters: 31,079,378 SNPs and 7,940,790 indels. Calling de novo mutations (DNMs) is a formidable challenge given the high false positive rate in sequencing datasets relative to the mutation rate. Here we addressed this issue by using segregation of alleles in three-generation families. Using this transmission assay, we controlled the false positive rate and identified 108,778 high quality DNMs. Furthermore, we used our extended family structure and read pair tracing of DNMs to a panel of phased SNPs, to determine the parent of origin of 42,961 DNMs.

Parental influence on human germline de novo mutations in 1,548 trios from Iceland.

The characterization of mutational processes that generate sequence diversity in the human genome is of paramount importance both to medical genetics and to evolutionary studies. To understand how the age and sex of transmitting parents affect de novo mutations, here we sequence 1,548 Icelanders, their parents, and, for a subset of 225, at least one child, to 35× genome-wide coverage. We find 108,778 de novo mutations, both single nucleotide polymorphisms and indels, and determine the parent of origin of 42,961. The number of de novo mutations from mothers increases by 0.37 per year of age (95% CI 0.32-0.43), a quarter of the 1.51 per year from fathers (95% CI 1.45-1.57). The number of clustered mutations increases faster with the mother's age than with the father's, and the genomic span of maternal de novo mutation clusters is greater than that of paternal ones. The types of de novo mutation from mothers change substantially with age, with a 0.26% (95% CI 0.19-0.33%) decrease in cytosine-phosphate-guanine to thymine-phosphate-guanine (CpG>TpG) de novo mutations and a 0.33% (95% CI 0.28-0.38%) increase in C>G de novo mutations per year, respectively. Remarkably, these age-related changes are not distributed uniformly across the genome. A striking example is a 20 megabase region on chromosome 8p, with a maternal C>G mutation rate that is up to 50-fold greater than the rest of the genome. The age-related accumulation of maternal non-crossover gene conversions also mostly occurs within these regions. Increased sequence diversity and linkage disequilibrium of C>G variants within regions affected by excess maternal mutations indicate that the underlying mutational process has persisted in humans for thousands of years. Moreover, the regional excess of C>G variation in humans is largely shared by chimpanzees, less by gorillas, and is almost absent from orangutans. This demonstrates that sequence diversity in humans results from evolving interactions between age, sex, mutation type, and genomic location.

HLA class II sequence variants influence tuberculosis risk in populations of European ancestry.

Mycobacterium tuberculosis infections cause 9 million new tuberculosis cases and 1.5 million deaths annually. To identify variants conferring risk of tuberculosis, we tested 28.3 million variants identified through whole-genome sequencing of 2,636 Icelanders for association with tuberculosis (8,162 cases and 277,643 controls), pulmonary tuberculosis (PTB) and M. tuberculosis infection. We found association of three variants in the region harboring genes encoding the class II human leukocyte antigens (HLAs): rs557011[T] (minor allele frequency (MAF) = 40.2%), associated with M. tuberculosis infection (odds ratio (OR) = 1.14, P = 3.1 × 10(-13)) and PTB (OR = 1.25, P = 5.8 × 10(-12)), and rs9271378[G] (MAF = 32.5%), associated with PTB (OR = 0.78, P = 2.5 × 10(-12))--both located between HLA-DQA1 and HLA-DRB1--and a missense variant encoding p.Ala210Thr in HLA-DQA1 (MAF = 19.1%, rs9272785), associated with M. tuberculosis infection (P = 9.3 × 10(-9), OR = 1.14). We replicated association of these variants with PTB in samples of European ancestry from Russia and Croatia (P < 5.9 × 10(-4)). These findings show that the HLA class II region contributes to genetic risk of tuberculosis, possibly through reduced presentation of protective M. tuberculosis antigens to T cells.

The effect of SNPs on expression levels in Nimblegen RNA expression microarrays.

In this paper the effect of SNPs on expression levels in Nimblegen RNA expression microarrays is investigated. A vast number of replicates of probe pairs representing both alleles of SNPs on 14 loci allows accurate estimation of the difference in signal intensities both within and between probe pairs. The majority of probe-pairs with sufficiently high expression have significant differences in expression levels within the pair and the difference shows concordance with the genotype of the samples. With two or more replicates of each probe, the allele-to-allele variance dominates the error in estimating the difference within the probe-pair, ten replicates are needed for adequate power in calling a true difference within a single probe-pair. Using the expression level of the probe within the probe-pair that has the higher value gives more accurate estimates. When using probes at loci containing known SNP's one should use probes containing both alleles of the SNP.

A method for detecting long non-coding RNAs with tiled RNA expression microarrays.

Long non-coding ribonucleic acids (lncRNAs) have been proposed as biomarkers in prostate cancer. This paper proposes a selection method which uses data from tiled microarrays to identify relatively long regions of moderate expression independent of the microarray platform and probe design. The method is used to search for candidate long non-coding ribonucleic acids (lncRNAs) at locus 8q24 and is run on three independent experiments which all use samples from prostate cancer patients. The robustness of the method is tested by utilizing repeated copies of tiled probes. The method shows high consistency between experiments that used the same samples, but different probe layout. There also is statistically significant consistency when comparing experiments with different samples. The method selected the long non-coding ribonucleic acid PCNCR1 in all three experiments.