Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease.

Alzheimer's disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals-16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-ß precursor protein processing, amyloid-ß aggregation, lipid metabolism and microglial function in AD.

A blood miRNA signature associates with sporadic Creutzfeldt-Jakob disease diagnosis.

Sporadic Creutzfeldt-Jakob disease (sCJD) presents as a rapidly progressive dementia which is usually fatal within six months. No clinical blood tests are available for diagnosis or disease monitoring. Here, we profile blood microRNA (miRNA) expression in sCJD. Sequencing of 57 sCJD patients, and healthy controls reveals differential expression of hsa-let-7i-5p, hsa-miR-16-5p, hsa-miR-93-5p and hsa-miR-106b-3p. Downregulation of hsa-let-7i-5p, hsa-miR-16-5p and hsa-miR-93-5p replicates in an independent cohort using quantitative PCR, with concomitant upregulation of four mRNA targets. Absence of correlation in cross-sectional analysis with clinical phenotypes parallels the lack of association between rate of decline in miRNA expression, and rate of disease progression in a longitudinal cohort of samples from 21 patients. Finally, the miRNA signature shows a high level of accuracy in discriminating sCJD from Alzheimer's disease. These findings highlight molecular alterations in the periphery in sCJD which provide information about differential diagnosis and improve mechanistic understanding of human prion diseases.

Genetic, physiological and comparative genomic studies of hypertension and insulin resistance in the spontaneously hypertensive rat.

We previously mapped hypertension-related insulin resistance quantitative trait loci (QTLs) to rat chromosomes 4, 12 and 16 using adipocytes from F2 crosses between spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats, and subsequently identified Cd36 as the gene underlying the chromosome 4 locus. The identity of the chromosome 12 and 16 genes remains unknown. To identify whole-body phenotypes associated with the chromosome 12 and 16 linkage regions, we generated and characterised new congenic strains, with WKY donor segments introgressed onto an SHR genetic background, for the chromosome 12 and 16 linkage regions. We found a >50% increase in insulin sensitivity in both the chromosome 12 and 16 strains. Blood pressure and left ventricular mass were reduced in the two congenic strains consistent with the congenic segments harbouring SHR genes for insulin resistance, hypertension and cardiac hypertrophy. Integrated genomic analysis, using physiological and whole-genome sequence data across 42 rat strains, identified variants within the congenic regions in Upk3bl, RGD1565131 and AABR06087018.1 that were associated with blood pressure, cardiac mass and insulin sensitivity. Quantitative trait transcript analysis across 29 recombinant inbred strains showed correlation between expression of Hspb1, Zkscan5 and Pdgfrl with adipocyte volume, systolic blood pressure and cardiac mass, respectively. Comparative genome analysis showed a marked enrichment of orthologues for human GWAS-associated genes for insulin resistance within the syntenic regions of both the chromosome 12 and 16 congenic intervals. Our study defines whole-body phenotypes associated with the SHR chromosome 12 and 16 insulin-resistance QTLs, identifies candidate genes for these SHR QTLs and finds human orthologues of rat genes in these regions that associate with related human traits. Further study of these genes in the congenic strains will lead to robust identification of the underlying genes and cellular mechanisms.

Genetic analysis of the cardiac methylome at single nucleotide resolution in a model of human cardiovascular disease.

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.

Targeted genetic testing for familial hypercholesterolaemia using next generation sequencing: a population-based study.

BACKGROUND: Familial hypercholesterolaemia (FH) is a common Mendelian condition which, untreated, results in premature coronary heart disease. An estimated 88% of FH cases are undiagnosed in the UK. We previously validated a method for FH mutation detection in a lipid clinic population using next generation sequencing (NGS), but this did not address the challenge of identifying index cases in primary care where most undiagnosed patients receive healthcare. Here, we evaluate the targeted use of NGS as a potential route to diagnosis of FH in a primary care population subset selected for hypercholesterolaemia. METHODS: We used microfluidics-based PCR amplification coupled with NGS and multiplex ligation-dependent probe amplification (MLPA) to detect mutations in LDLR, APOB and PCSK9 in three phenotypic groups within the Generation Scotland: Scottish Family Health Study including 193 individuals with high total cholesterol, 232 with moderately high total cholesterol despite cholesterol-lowering therapy, and 192 normocholesterolaemic controls. RESULTS: Pathogenic mutations were found in 2.1% of hypercholesterolaemic individuals, in 2.2% of subjects on cholesterol-lowering therapy and in 42% of their available first-degree relatives. In addition, variants of uncertain clinical significance (VUCS) were detected in 1.4% of the hypercholesterolaemic and cholesterol-lowering therapy groups. No pathogenic variants or VUCS were detected in controls. CONCLUSIONS: We demonstrated that population-based genetic testing using these protocols is able to deliver definitive molecular diagnoses of FH in individuals with high cholesterol or on cholesterol-lowering therapy. The lower cost and labour associated with NGS-based testing may increase the attractiveness of a population-based approach to FH detection compared to genetic testing with conventional sequencing. This could provide one route to increasing the present low percentage of FH cases with a genetic diagnosis.

The use of next-generation sequencing in clinical diagnosis of familial hypercholesterolemia.

PURPOSE: Familial hypercholesterolemia is a common Mendelian disorder associated with early-onset coronary heart disease that can be treated by cholesterol-lowering drugs. The majority of cases in the United Kingdom are currently without a molecular diagnosis, which is partly due to the cost and time associated with standard screening techniques. The main purpose of this study was to test the sensitivity and specificity of two next-generation sequencing protocols for genetic diagnosis of familial hypercholesterolemia. METHODS: Libraries were prepared for next-generation sequencing by two target enrichment protocols; one using the SureSelect Target Enrichment System and the other using the PCR-based Access Array platform. RESULTS: In the validation cohort, both protocols showed 100% specificity, whereas the sensitivity for short variant detection was 100% for the SureSelect Target Enrichment and 98% for the Access Array protocol. Large deletions/duplications were only detected using the SureSelect Target Enrichment protocol. In the prospective cohort, the mutation detection rate using the Access Array was highest in patients with clinically definite familial hypercholesterolemia (67%), followed by patients with possible familial hypercholesterolemia (26%). CONCLUSION: We have shown the potential of target enrichment methods combined with next-generation sequencing for molecular diagnosis of familial hypercholesterolemia. Adopting these assays for patients with suspected familial hypercholesterolemia could improve cost-effectiveness and increase the overall number of patients with a molecular diagnosis.

Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia.

Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms.

FCGR3B copy number variation is associated with susceptibility to systemic, but not organ-specific, autoimmunity.

Naturally occurring variation in gene copy number is increasingly recognized as a heritable source of susceptibility to genetically complex diseases. Here we report strong association between FCGR3B copy number and risk of systemic lupus erythematosus (P = 2.7 x 10(-8)), microscopic polyangiitis (P = 2.9 x 10(-4)) and Wegener's granulomatosis in two independent cohorts from the UK (P = 3 x 10(-3)) and France (P = 1.1 x 10(-4)). We did not observe this association in the organ-specific Graves' disease or Addison's disease. Our findings suggest that low FCGR3B copy number, and in particular complete FCGR3B deficiency, has a key role in the development of systemic autoimmunity.

Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans.

Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.

Segregation of experimental autoimmune glomerulonephritis as a complex genetic trait and exclusion of Col4a3 as a candidate gene.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar-Kyoto (WKY) rats (RT1-l) by immunization with rat glomerular basement membrane (GBM) in adjuvant. The model in this rat strain is characterized by anti-GBM antibody production accompanied by focal necrotizing glomerulonephritis with crescent formation. The main autoantigen in humans and rats has been identified as the non-collagenous domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). By contrast, Lewis (LEW) rats with the same MHC background (RT1-l), immunized with the same antigen, develop similar levels of circulating anti-GBM antibodies, but no histological evidence of nephritis. In order to investigate the genetic basis of susceptibility to EAG, we examined the response of both F1 (WKY x LEW) and backcross (BC1; WKY x F1) rats to immunization with rat GBM. F1 animals were completely resistant to the development of EAG, while BC1 animals showed a range of responses from severe crescentic glomerulonephritis to no histological evidence of disease. The results indicate that EAG is inherited as a complex trait under the control of WKY genes unlinked to the MHC. cDNA sequence analysis of alpha3(IV)NC1 in the two parental strains was identical, indicating no predicted amino acid sequence variation in the alpha3(IV)NC1 domain between these strains. Radiation hybrid mapping, using two separate PCR amplicons from rat alpha3(IV)NC1, localized rat Col4a3 to a region of chromosome 9. Since Col4a3 (encoding the autoantigen) is a candidate for susceptibility to EAG, we screened the region of rat chromosome 9 where Col4a3 is localized, using polymorphic microsatellite markers in segregating BC1 progeny. No significant linkage was detected. These results exclude Col4a3 as a recessive susceptibility gene for EAG in the BC1 progeny.

Radiation hybrid mapping of 70 rat genes from a data set of differentially expressed genes.

The spontaneously hypertensive rat (SHR) is a model of human essential hypertension. Increased blood pressure in SHR is associated with other risk factors associated with cardiovascular disease, including insulin resistance and dyslipidemia. DNA microarray studies identified over 200 differentially expressed genes and ESTs between SHR and normotensive control rats. These clones represent candidate genes that may underlie previously detected QTLs in SHR. This study made use of the publication of two whole-genome maps to identify positional QTL candidates. Radiation hybrid (RH) mapping was used to determine the chromosomal locations of 70 rat genes and ESTs from this dataset. Most of the locations are novel, but in five cases we identified a definitive map location for genes previously mapped by somatic cell hybrids and/or linkage analysis. Genes for which the mouse genome map location was already determined mapped to syntenic segments in the rat genome map, except for two rat genes whose map locations confirmed previous findings. Where synteny comparisons could be made only with the human, 74% of the genes mapped in this study lay in a conserved syntenic segment. Chromosomal localisation of these mouse and human orthologs to syntenic segments produces a high level of confidence in the data presented in this study. The data provide new map locations for rat genes and will aid efforts to advance the rat genome map. The data may also be used to prioritize candidate QTL genes in SHR and other rat strains on the basis of their map location.