A genome-wide association study of mitochondrial DNA copy number in two population-based cohorts.

BACKGROUND: Mitochondrial DNA copy number (mtDNA CN) exhibits interindividual and intercellular variation, but few genome-wide association studies (GWAS) of directly assayed mtDNA CN exist. We undertook a GWAS of qPCR-assayed mtDNA CN in the Avon Longitudinal Study of Parents and Children (ALSPAC) and the UK Blood Service (UKBS) cohort. After validating and harmonising data, 5461 ALSPAC mothers (16-43 years at mtDNA CN assay) and 1338 UKBS females (17-69 years) were included in a meta-analysis. Sensitivity analyses restricted to females with white cell-extracted DNA and adjusted for estimated or assayed cell proportions. Associations were also explored in ALSPAC children and UKBS males. RESULTS: A neutrophil-associated locus approached genome-wide significance (rs709591 [MED24], ß (change in SD units of mtDNA CN per allele) [SE] - 0.084 [0.016], p = 1.54e-07) in the main meta-analysis of adult females. This association was concordant in magnitude and direction in UKBS males and ALSPAC neonates. SNPs in and around ABHD8 were associated with mtDNA CN in ALSPAC neonates (rs10424198, ß [SE] 0.262 [0.034], p = 1.40e-14), but not other study groups. In a meta-analysis of unrelated individuals (N = 11,253), we replicated a published association in TFAM (ß [SE] 0.046 [0.017], p = 0.006), with an effect size much smaller than that observed in the replication analysis of a previous in silico GWAS. CONCLUSIONS: In a hypothesis-generating GWAS, we confirm an association between TFAM and mtDNA CN and present putative loci requiring replication in much larger samples. We discuss the limitations of our work, in terms of measurement error and cellular heterogeneity, and highlight the need for larger studies to better understand nuclear genomic control of mtDNA copy number.

Cardiometabolic phenotypes and mitochondrial DNA copy number in two cohorts of UK women.

The mitochondrial genome is present at variable copy number between individuals. Mitochondria are vulnerable to oxidative stress, and their dysfunction may be associated with cardiovascular disease. The association of mitochondrial DNA copy number with cardiometabolic risk factors (lipids, glycaemic traits, inflammatory markers, anthropometry and blood pressure) was assessed in two independent cohorts of European origin women, one in whom outcomes were measured at mean (SD) age 30 (4.3) years (N=2278) and the second at 69.4 (5.5) years (N=2872). Mitochondrial DNA copy number was assayed by quantitative polymerase chain reaction. Associations were adjusted for smoking, sociodemographic status, laboratory factors and white cell traits. Out of a total of 12 outcomes assessed in both cohorts, mitochondrial DNA copy number showed little or no association with the majority (point estimates were close to zero and nearly all p-values were >0.01). The strongest evidence was for an inverse association in the older cohort with insulin (standardised beta [95%CI]: -0.06, [-0.098, -0.022], p=0.002), but this association did not replicate in the younger cohort. Our findings do not provide support for variation in mitochondrial DNA copy number having an important impact on cardio-metabolic risk factors in European origin women.

Frequency of KLK3 gene deletions in the general population.

Background One of the kallikrein genes ( KLK3) encodes prostate-specific antigen, a key biomarker for prostate cancer. A number of factors, both genetic and non-genetic, determine variation of serum prostate-specific antigen concentrations in the population. We have recently found three KLK3 deletions in individuals with very low prostate-specific antigen concentrations, suggesting a link between abnormally reduced KLK3 expression and deletions of KLK3. Here, we aim to determine the frequency of kallikrein gene 3 deletions in the general population. Methods The frequency of KLK3 deletions in the general population was estimated from the 1958 Birth Cohort sample ( n = 3815) using amplification ratiometry control system. In silico analyses using PennCNV were carried out in the same cohort and in NBS-WTCCC2 in order to provide an independent estimation of the frequency of KLK3 deletions in the general population. Results Amplification ratiometry control system results from the 1958 cohort indicated a frequency of KLK3 deletions of 0.81% (3.98% following a less stringent calling criterion). From in silico analyses, we found that potential deletions harbouring the KLK3 gene occurred at rates of 2.13% (1958 Cohort, n = 2867) and 0.99% (NBS-WTCCC2, n = 2737), respectively. These results are in good agreement with our in vitro experiments. All deletions found were in heterozygosis. Conclusions We conclude that a number of individuals from the general population present KLK3 deletions in heterozygosis. Further studies are required in order to know if interpretation of low serum prostate-specific antigen concentrations in individuals with KLK3 deletions may offer false-negative assurances with consequences for prostate cancer screening, diagnosis and monitoring.

Proxy molecular diagnosis from whole-exome sequencing reveals Papillon-Lefevre syndrome caused by a missense mutation in CTSC.

Papillon-Lefevre syndrome (PLS) is an autosomal recessive disorder characterised by severe early onset periodontitis and palmoplantar hyperkeratosis. A previously reported missense mutation in the CTSC gene (NM_001814.4:c.899G>A:p.(G300D)) was identified in a homozygous state in two siblings diagnosed with PLS in a consanguineous family of Arabic ancestry. The variant was initially identified in a heterozygous state in a PLS unaffected sibling whose whole exome had been sequenced as part of a previous Primary ciliary dyskinesia study. Using this information, a proxy molecular diagnosis was made on the PLS affected siblings after consent was given to study this second disorder found to be segregating within the family. The prevalence of the mutation was then assayed in the local population using a representative sample of 256 unrelated individuals. The variant was absent in all subjects indicating that the variant is rare in Saudi Arabia. This family study illustrates how whole-exome sequencing can generate findings and inferences beyond its primary goal.

Haptoglobin duplicon, hemoglobin, and vitamin C: analyses in the british women's heart and health study and Caerphilly prospective study.

BACKGROUND: Haptoglobin acts as an antioxidant by limiting peroxidative tissue damage by free hemoglobin. The haptoglobin gene allele Hp2 comprises a 1.7 kb partial duplication. Relative to allele Hp1, Hp2 carriers form protein multimers, suboptimal for hemoglobin scavenging. OBJECTIVE: To examine the association of haptoglobin genotype with a range of phenotypes, with emphasis on vitamin C and hemoglobin levels. METHODS: We applied a quantitative PCR assay for the duplication junction to two population cohorts including 2747 British women and 1198 British men. We examined the association of haptoglobin duplicon copy number with hemoglobin and vitamin C and used the copy number to complete a phenome scan. RESULTS: Hemoglobin concentrations were greater in those with Hp2,2 genotype, in women only (Hp1,1 13.45 g/dL, Hp1,2 13.49 g/dL, Hp2,2 13.61 g/dL; P = 0.002), though statistically there was no evidence of a difference between the sexes (z value = 1.2, P = 0.24). Haptoglobin genotype was not associated with vitamin C or any other phenotype in either cohort. CONCLUSIONS: Our results do not support association of haptoglobin genotype with vitamin C or with other phenotypes measured in two population cohorts. The apparent association between haptoglobin genotype and hemoglobin in the women's cohort merits further investigation.

Nonsense mutation in coiled-coil domain containing 151 gene (CCDC151) causes primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder characterized by impaired ciliary function that leads to subsequent clinical phenotypes such as chronic sinopulmonary disease. PCD is also a genetically heterogeneous disorder with many single gene mutations leading to similar clinical phenotypes. Here, we present a novel PCD causal gene, coiled-coil domain containing 151 (CCDC151), which has been shown to be essential in motile cilia of many animals and other vertebrates but its effects in humans was not observed until currently. We observed a novel nonsense mutation in a homozygous state in the CCDC151 gene (NM_145045.4:c.925G>T:p.[E309*]) in a clinically diagnosed PCD patient from a consanguineous family of Arabic ancestry. The variant was absent in 238 randomly selected individuals indicating that the variant is rare and likely not to be a founder mutation. Our finding also shows that given prior knowledge from model organisms, even a single whole-exome sequence can be sufficient to discover a novel causal gene.

Very low PSA concentrations and deletions of the KLK3 gene.

BACKGROUND: Prostate-specific antigen (PSA), a widely used biomarker for prostate cancer (PCa), is encoded by a kallikrein gene (KLK3, kallikrein-related peptidase 3). Serum PSA concentrations vary in the population, with PCa patients generally showing higher PSA concentrations than control individuals, although a small proportion of individuals in the population display very low PSA concentrations. We hypothesized that very low PSA concentrations might reflect gene-inactivating mutations in KLK3 that lead to abnormally reduced gene expression. METHODS: We have sequenced all KLK3 exons and the promoter and searched for gross deletions or duplications in KLK3 in the 30 individuals with the lowest observed PSA concentrations in a sample of approximately 85 000 men from the Prostate Testing for Cancer and Treatment (ProtecT) study. The ProtecT study examines a community-based population of men from across the UK with little prior PSA testing. RESULTS: We observed no stop codons or frameshift mutations, but we did find 30 single-base genetic variants, including 3 variants not described previously. These variants included missense variants that could be functionally inactivating and splicing variants. At this stage, however, we cannot confidently conclude whether these variants markedly lower PSA concentration or activity. More importantly, we identified 3 individuals with different large heterozygous deletions that encompass all KLK3 exons. The absence of a functional copy of KLK3 in these individuals is consistent with their reduced serum PSA concentrations. CONCLUSIONS: The clinical interpretation of the PSA test for individuals with KLK3 gene inactivation could lead to false-negative PSA findings used for screening, diagnosis, or monitoring of PCa.

Inflammation and not cardiovascular risk factors is associated with short leukocyte telomere length in 13- to 16-year-old adolescents.

OBJECTIVE: Short leukocyte telomere length (LTL) is associated with cardiovascular (CV) disease in adulthood. However, the biological basis of this association remains unclear. We sought to define early determinants of the association between CV disease and LTL in an adolescent population. METHODS AND RESULTS: One thousand eighty adolescents, aged 13 to 16 years and participating in the Ten Towns Heart Health Study, provided blood samples for DNA extraction and measurement of a range of CV risk factors. LTL was measured by real-time polymerase chain reaction. LTL was inversely associated with age (P=0.04), longer in females than in males (P=0.03), and longer in South Asians than in white Europeans (P=0.01). No associations were found between LTL and traditional CV risk factors. There was a significant and inverse association between LTL and inflammatory markers, including C-reactive protein (P<0.001) and fibrinogen (P=0.001). The associations between LTL and inflammatory markers were not affected by multiple adjustments for behavioral and metabolic factors. CONCLUSIONS: High levels of inflammation are associated with shorter LTL from early adolescence; traditional CV risk factors have little association with LTL in adolescence. Inflammation in early life may play a causal role in the adult association between short LTL and CV disease.

Molecular and population analysis of natural selection on the human haptoglobin duplication.

Haptoglobin binds free haemoglobin that prevents oxidative damage produced by haemolysis. There is a copy number variant (CNV) in the haptoglobin gene (HP) consisting of two alleles, Hp1 (no duplication), and Hp2 (1.7kb duplication involving two exons). The spread of the Hp2 allele is believed to have taken place under selective pressures conferred by malaria resistance. However, molecular evidence is lacking and Hp did not emerge in genomewide SNPs surveys for evidence of selection. In Europe, there is geographical constancy of Hp2 frequency, indicative of absence of clinal pressures and that modern day European alleles represent a "snapshot" of their out-of-Africa migrations. In this work we test for signatures of natural selection acting on the Hp CNV in a sample from the UK population (Avon Longitudinal Study of Parents and Children, ALSPAC). We present here heterozygosity decay, pairwise F(ST) values observed between ALSPAC and 301 populations from all five populated continents, extended haplotype homozygosity analyses involving the CNV and 80 SNPs surrounding the CNV ~500kb in each direction, and linkage disequilibrium and pairwise haplotypic analyses involving 160 SNPs on chromosome 16q22.1. Taken together, our results represent the first molecular analysis of natural selection in the Hp CNV genetic region.

Complexity of a complex trait locus: HP, HPR, haemoglobin and cholesterol.

HP and HPR are related and contiguous genes in strong linkage disequilibrium (LD), encoding haptoglobin and haptoglobin-related protein. These bind and chaperone free Hb for recycling, protecting against oxidation. A copy number variation (CNV) within HP (Hp1/Hp2) results in different possible haptoglobin complexes which have differing properties. HPR rs2000999 (G/A), identified in meta-GWAS, influences total cholesterol (TC) and LDL-cholesterol (LDL-C). We examined the relationship between HP CNV, HPR rs2000999, Hb, red cell count (RCC), LDL-C and TC in the British Women's Heart and Health Study (n=2779 for samples having CNV, rs2000999, and phenotypes). Analysing single markers by linear regression, rs2000999 was associated with LDL-C (ß=0.040 mmol/L, p=0.023), TC (ß=-0.040 mmol/L, p=0.019), Hb (ß=-0.044 g/dL, p=0.028) and borderline with RCC (ß=-0.032 × 10(12)/L, p=0.066). Analysis of CNV by linear regression revealed an association with Hb (Hp1 vs Hp2, ß=0.057 g/dL, p=0.004), RCC (ß=0.045 × 10(12)/L, p=0.014), and showed a trend with LDL-C and TC. There were 3 principal haplotypes (Hp1-G 36%; Hp2-G 45%; Hp2-A 18%). Haplotype comparisons showed that LDL-C and TC associations were from rs2000999; Hb and RCC associations derived largely from the CNV. Distinct genotype-phenotype effects are evident at the genetic epidemiological level once LD has been analysed, perhaps reflecting HP-HPR functional biology and evolutionary history. The derived Hp2 allele of the HP gene has apparently been subject to malaria-driven positive selection. Haptoglobin-related protein binds Hb and apolipoprotein-L, i.e. linking HPR to the cholesterol system; and the HPR/apo-L complex is specifically trypanolytic. Our analysis illustrates the complex interplay between functions and haplotypes of adjacent genes, environmental context and natural selection, and offers insights into potential use of haptoglobin or haptoglobin-related protein as therapeutic agents.

From a single whole exome read to notions of clinical screening: primary ciliary dyskinesia and RSPH9 p.Lys268del in the Arabian Peninsula.

Primary ciliary dyskinesia (PCD) is a genetic disorder, usually autosomal recessive, causing early respiratory disease and later subfertility. Whole exome sequencing may enable efficient analysis for locus heterogeneous disorders such as PCD. We whole-exome-sequenced one consanguineous Saudi Arabian with clinically diagnosed PCD and normal laterality, to attempt ab initio molecular diagnosis. We reviewed 13 known PCD genes and potentially autozygous regions (extended homozygosity) for homozygous exon deletions, non-dbSNP codon, splice-site base variants or small indels. Homozygous non-dbSNP changes were also reviewed exome-wide. One single molecular read representing RSPH9 p.Lys268del was observed, with no wild-type reads, and a notable deficiency of mapped reads at this location. Among all observations, RSPH9 was the strongest candidate for causality. Searching unmapped reads revealed seven more mutant reads. Direct assay for p.Lys268del (MboII digest) confirmed homozygosity in the affected individual, then confirmed homozygosity in three siblings with bronchiectasis. Our finding in southwest Saudi Arabia indicates that p.Lys268del, previously observed in two Bedouin families (Israel, UAE), is geographically widespread in the Arabian Peninsula. Analogous with cystic fibrosis CFTR p.Phe508del, screening for RSPH9 p.Lys268del (which lacks sentinel dextrocardia) in those at risk would help in early diagnosis, tailored clinical management, genetic counselling and primary prevention.

Amplification ratio control system for copy number variation genotyping.

We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP') in each strand form a hairpin. The bases immediately beyond the 3'-end of the UP and 5' of UP' are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3'-ends of amplicon strands. The resultant 'amplification ratio control system' (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and 'chemokine (C-C motif) ligand 3-like 1' gene.

IQ, educational attainment, memory and plasma lipids: associations with apolipoprotein E genotype in 5995 children.

BACKGROUND: Apolipoprotein E (APOE) genotype (ε2/ε3/ε4: rs429358 ε4 allele; rs7412 ε2 allele) is strongly associated with both lipid levels and Alzheimer's disease. Although there is also evidence of milder cognitive impairment in later life in carriers of the APOE ε4 allele, there have been few studies investigating the impact of APOE genotype on cognitive function in children. METHODS: We determined APOE genotype in 5995 children from the Avon Longitudinal Study of Parents and Children and investigated associations between APOE genotype and plasma lipids (at age 9), IQ (at age 8), memory (at ages 8 and 10), and performance in school attainment tests (at ages 7, 11, and 14). RESULTS: Observed genotype group counts were consistent with Hardy-Weinberg equilibrium (χ(2)p value = .84). There were strong relationships between APOE genotype and low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides, which follow the same patterns as in adults. There was no strong evidence to suggest that APOE genotype was associated with IQ (all p values ≥ .46), memory function (p ≥ .35), or school attainment test results (p ≥ .28). CONCLUSION: Although APOE genotype does have strong associations with lipid levels in childhood, there does not seem to be meaningful effects on cognitive performance, suggesting that any detrimental effects of the ε4 allele on cognitive function are not important until later life.

MeltMADGE for mutation scanning of specific genes in population studies.

MeltMADGE reconfigures the mutation scanning process of denaturing gradient gel electrophoresis so that the independent variable is time rather than space and the dependent (denaturing) variable is temperature rather than concentration of chemical denaturant. Use of a thermal ramp enables the use of a homogeneous gel and therefore of high-density arrays of wells such as those of microplate array diagonal gel electrophoresis (MADGE). In this configuration, electrophoresis of products on 10-12 96-well meltMADGE gels can be conducted in a 1- to 2-liter tank in a 1- to 2-h run, enabling the scanning of a target amplicon in over 1,000 subjects simultaneously. Gels are read by imaging the fluorescence of UV-excited ethidium bromide, giving a simple, economical system for identifying rarer sequence variants in target genes; it is suitable for large-scale case-control or population studies and other comparable applications. Different amplicons with similar melting characteristics can also be combined in the same run.

An expectation-maximization program for determining allelic spectrum from CNV data (CoNVEM): insights into population allelic architecture and its mutational history.

Copy number variations (CNVs) are a common form of genetic variation in which the allelic population contains a distribution of copy numbers of a particular gene (or other large sequence/region). The simplest forms describe deletion (0 vs. 1 copy) or duplication (1 vs. 2) events. However, some CNV loci contain a much wider range of copy numbers, such as that seen for the CCL3L1 locus. CNV classification methods typically only describe the total (diploid) copy number, leaving the underlying genotypic and allelic frequency distribution unknown. We have developed an expectation-maximization approach for the analysis of data from tandem CNVs that enables estimation of both the allelic copy number frequency distribution and the expected copy number genotype and class distribution under the Hardy-Weinberg equilibrium (HWE). The CNV expectation-maximization algorithm is available in a Web-tool (CoNVEM, http://apps.biocompute.org.uk/convem/), which graphically and numerically presents CNV allele and genotype distributions. We have applied this approach to the analysis of salivary amylase (AMY1A, B, and C), CCL3L1, and SULT1A1 CNVs using published data, and present inferences about the evolutionary history of these loci based on CoNVEM results.

Homogeneous assay of rs4343, an ACE I/D proxy, and an analysis in the British Women's Heart and Health Study (BWHHS).

Current literature suggests that ACE SNP rs4343, ACE 2350A>G in exon 17, T202T, may be the best proxy for the ACE Alu I/D whereas rs4363 and rs4362 may be slightly stronger predictors of ACE levels. Considering reported difficulties in genotyping ACE I/D and stronger associations of rs4343 than ACE I/D with plasma ACE levels in Africans, and suitability of rs4343 for allelic mRNA (cDNA) studies, we developed and validated a liquid phase assay for rs4343, which has advantage on both functional and technical grounds. We confirmed that rs4343, is in near perfect linkage disequilibrium (D'=1, r2=0.88, n=64) with ACE I/D in Europeans (A and G alleles of rs4343 marking insertion and deletion alleles of ACE I/D respectively). We then studied its association with metabolic and cardiovascular traits in 3253 British women (60-79 years old). Apart from a nominal trend of association with diastolic blood pressure (p anova=0.08; p trend=0.05), no other associations were observed. A post-hoc vascular and general phenome scan revealed no further associations. We conclude that ACE I/D is not a major determinant of metabolic and cardiovascular traits in this population. Liquid phase genotyping of SNP rs4343 may be preferable to gel based ACE I/D genotyping both for technical and functional reasons.

Integrated single-label liquid-phase assay of APOE codons 112 and 158 and a lipoprotein study in British women.

BACKGROUND: Apolipoprotein E (APOE) is an important element of lipid metabolism and, hence, cardiovascular disorders. APOE has 3 main allelic variants: epsilon3, epsilon4, and epsilon2. Of these, epsilon3 is the most common, followed by epsilon4 and epsilon2. The associations of these isoforms with cardiovascular disorders and Alzheimer disease have been widely studied in different populations. Most of the genotyping in these studies has been performed with gel-based methods, which have important limitations, particularly for large epidemiologic studies. We therefore developed an integrated "one-tube" liquid-phase assay. METHODS: To measure APOE isoforms, we developed an integrated single-label liquid-phase fluorescence assay containing 2 PCR primers, 2 probes, and 2 quencher oligonucleotides. We used a 384-well LightTyper, but the assay would be generically applicable for use with any fluorescence detector with thermal ramp control. We validated this method and applied it in the British Women's Heart and Health Study. RESULTS: There were 4 melting peaks, at 41, 56, 61, and 69 degrees C, which generated 6 distinctive patterns representing genotypic combinations of epsilon3, epsilon4, and epsilon2. The magnitude and direction of the associations found with total cholesterol, HDL-cholesterol, triglycerides, and estimated LDL-cholesterol were consistent with previous reports. CONCLUSION: The one-tube LightTyper assay presented here enables accurate, convenient, and economical genotyping of APOE and can be used for large epidemiologic studies.