RESUMEN
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.
Asunto(s)
Variaciones en el Número de Copia de ADN , ADN Mitocondrial/genética , Estudio de Asociación del Genoma Completo/métodos , Adolescente , Adulto , Niño , Estudios de Cohortes , Femenino , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
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.
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Enfermedades Cardiovasculares/genética , Variaciones en el Número de Copia de ADN , ADN Mitocondrial/análisis , Enfermedades Metabólicas/genética , Adulto , Anciano , Enfermedades Cardiovasculares/epidemiología , Femenino , Humanos , Enfermedades Metabólicas/epidemiología , Persona de Mediana Edad , Reino Unido/epidemiologíaRESUMEN
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.
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Catepsina C/genética , Mutación Missense , Enfermedad de Papillon-Lefevre/diagnóstico , Análisis de Secuencia de ADN/métodos , Árabes/legislación & jurisprudencia , Consanguinidad , Exoma , Femenino , Humanos , Masculino , Enfermedad de Papillon-Lefevre/genética , Linaje , Arabia SauditaRESUMEN
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.
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Ácido Ascórbico/sangre , Haptoglobinas/genética , Hemoglobinas/metabolismo , Femenino , Dosificación de Gen , Duplicación de Gen , Frecuencia de los Genes , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Caracteres Sexuales , GalesRESUMEN
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.
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Proteínas Portadoras/genética , Codón sin Sentido , Predisposición Genética a la Enfermedad , Síndrome de Kartagener/genética , HumanosRESUMEN
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.
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Eliminación de Gen , Calicreínas/genética , Antígeno Prostático Específico/sangre , Exones , Humanos , Masculino , Mutación , Reacción en Cadena de la Polimerasa , Antígeno Prostático Específico/genéticaRESUMEN
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.
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Variaciones en el Número de Copia de ADN , Haptoglobinas/genética , Polimorfismo de Nucleótido Simple , Selección Genética , Duplicación de Gen , Haplotipos , Humanos , Desequilibrio de Ligamiento , Reino Unido , Población Blanca/genéticaRESUMEN
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.
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Antígenos de Neoplasias/genética , Colesterol/genética , Variación Genética/fisiología , Haptoglobinas/genética , Hemoglobinas/genética , Sitios de Carácter Cuantitativo/genética , Anciano , Colesterol/metabolismo , Estudios de Cohortes , Epistasis Genética , Femenino , Interacción Gen-Ambiente , Ligamiento Genético , Genotipo , Humanos , Persona de Mediana Edad , Modelos Biológicos , Polimorfismo de Nucleótido SimpleRESUMEN
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.
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Proteínas del Citoesqueleto/genética , Síndrome de Kartagener/genética , Análisis de Secuencia de ADN , Consanguinidad , Análisis Mutacional de ADN , Exoma , Humanos , Mutación , Arabia SauditaRESUMEN
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.
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Dosificación de Gen , Reacción en Cadena de la Polimerasa/métodos , Emparejamiento Base , Quimiocinas CC/genética , Variaciones en el Número de Copia de ADN , Cartilla de ADN/química , Genotipo , Haptoglobinas/genética , Desnaturalización de Ácido NucleicoRESUMEN
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.
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Apolipoproteínas E/genética , Escolaridad , Inteligencia/genética , Lípidos/sangre , Lípidos/genética , Memoria/fisiología , Niño , Colesterol/sangre , Colesterol/genética , Cognición/fisiología , Educación , Femenino , Frecuencia de los Genes , Humanos , Lipoproteínas/sangre , Lipoproteínas/genética , Masculino , Pruebas Neuropsicológicas , Embarazo , Encuestas y CuestionariosRESUMEN
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.
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Algoritmos , Alelos , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN/genética , Genética de Población , Mutación/genética , Arilsulfotransferasa/genética , Humanos , alfa-Amilasas Salivales/genéticaRESUMEN
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.