Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.

Genome-wide screening for genetic variants in polyadenylation signal (PAS) sites in mouse selection lines for fatness and leanness.

Alternative polyadenylation (APA) determines mRNA stability, localisation, translation and protein function. Several diseases, including obesity, have been linked to APA. Studies have shown that single nucleotide polymorphisms in polyadenylation signals (PAS-SNPs) can influence APA and affect phenotype and disease susceptibility. However, these studies focussed on associations between single PAS-SNP alleles with very large effects and phenotype. Therefore, we performed a genome-wide screening for PAS-SNPs in the polygenic mouse selection lines for fatness and leanness by whole-genome sequencing. The genetic variants identified in the two lines were overlapped with locations of PAS sites obtained from the PolyASite 2.0 database. Expression data for selected genes were extracted from the microarray expression experiment performed on multiple tissue samples. In total, 682 PAS-SNPs were identified within 583 genes involved in various biological processes, including transport, protein modifications and degradation, cell adhesion and immune response. Moreover, 63 of the 583 orthologous genes in human have been previously associated with human diseases, such as nervous system and physical disorders, and immune, endocrine, and metabolic diseases. In both lines, PAS-SNPs have also been identified in genes broadly involved in APA, such as Polr2c, Eif3e and Ints11. Five PAS-SNPs within 5 genes (Car, Col4a1, Itga7, Lat, Nmnat1) were prioritised as potential functional variants and could contribute to the phenotypic disparity between the two selection lines. The developed PAS-SNPs catalogue presents a key resource for planning functional studies to uncover the role of PAS-SNPs in APA, disease susceptibility and fat deposition.

Genetic variants of the hypoxia-inducible factor 3 alpha subunit (Hif3a) gene in the Fat and Lean mouse selection lines.

BACKGROUND: Adipose tissue hypoxia and members of the hypoxia-inducible factor alpha (HIFA) are involved in development of obesity. However, the mechanism and functions of HIF3A, one of three HIFA paralogs, in fat deposition have not been sufficiently studied. METHODS AND RESULTS: In the present study, we investigated whether Hif3a sequence variants are associated with divergent fat deposition in mouse selection lines for fatness and leanness. Sequencing and RFLP were used to analyse sequence variants within Hif3a. To identify candidate regulatory variants, we performed literature screening and used databases and bioinformatics tools like Ensembl, MethPrimer, TargetScanMouse, miRDB, PolyAsite, RISE, LncRRIsearch, RNAfold, PredictProtein, CAIcal, and switches.ELM Resource. There are 90 sequence variants in Hif3a between the two mouse lines. While most Fat line variants locate within intronic regions, Lean line variants are mainly in 3' UTR. We constructed a map of Hif3a potential regulatory regions and identified 39 regulatory variants by integrating data on constrained and regulatory elements, CpGs, and miRNAs and lncRNAs binding sites. Moreover, 3' UTR and two exonic variants may influence mRNA stability, translation rate and protein functionality. We propose as priority candidates for further functional studies a missense (rs37398126) and synonymous (rs37739792) variants, and intronic (rs47471302) variant that overlap conserved element in promoter region and predicted lncRNAs binding site. CONCLUSION: The results indicate a potential involvement of Hif3a in fat deposition. Additionally, approach used in the present study may serve as a general guideline for constructing an integrative gene map for prioritizing candidate gene variants with phenotypic effects.

The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia.

The typical response of the adult mammalian pulmonary circulation to a low oxygen environment is vasoconstriction and structural remodelling of pulmonary arterioles, leading to chronic elevation of pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. Some mammals, however, exhibit genetic resistance to hypoxia-induced pulmonary hypertension. We used a congenic breeding program and comparative genomics to exploit this variation in the rat and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodelling. Slc39a12 encodes the zinc transporter ZIP12. Here we report that ZIP12 expression is increased in many cell types, including endothelial, smooth muscle and interstitial cells, in the remodelled pulmonary arterioles of rats, cows and humans susceptible to hypoxia-induced pulmonary hypertension. We show that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia dependent and that targeted inhibition of ZIP12 inhibits the rise in intracellular labile zinc in hypoxia-exposed pulmonary vascular smooth muscle cells and their proliferation in culture. We demonstrate that genetic disruption of ZIP12 expression attenuates the development of pulmonary hypertension in rats housed in a hypoxic atmosphere. This new and unexpected insight into the fundamental role of a zinc transporter in mammalian pulmonary vascular homeostasis suggests a new drug target for the pharmacological management of pulmonary hypertension.

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.

Genomic structure of nucleotide diversity among Lyon rat models of metabolic syndrome.

BACKGROUND: The metabolic syndrome (MetS), a complex disorder involving hypertension, obesity, dyslipidemia and insulin resistance, is a major risk factor for heart disease, stroke, and diabetes. The Lyon Hypertensive (LH), Lyon Normotensive (LN) and Lyon Low-pressure (LL) rats are inbred strains simultaneously derived from a common outbred Sprague Dawley colony by selection for high, normal, and low blood pressure, respectively. Further studies found that LH is a MetS susceptible strain, while LN is resistant and LL has an intermediate phenotype. Whole genome sequencing determined that, while the strains are phenotypically divergent, they are nearly 98% similar at the nucleotide level. Using the sequence of the three strains, we applied an approach that harnesses the distribution of Observed Strain Differences (OSD), or nucleotide diversity, to distinguish genomic regions of identity-by-descent (IBD) from those with divergent ancestry between the three strains. This information was then used to fine-map QTL identified in a cross between LH and LN rats in order to identify candidate genes causing the phenotypes. RESULTS: We identified haplotypes that, in total, contain at least 95% of the identifiable polymorphisms between the Lyon strains that are likely of differing ancestral origin. By intersecting the identified haplotype blocks with Quantitative Trait Loci (QTL) previously identified in a cross between LH and LN strains, the candidate QTL regions have been narrowed by 78%. Because the genome sequence has been determined, we were further able to identify putative functional variants in genes that are candidates for causing the QTL. CONCLUSIONS: Whole genome sequence analysis between the LH, LN, and LL strains identified the haplotype structure of these three strains and identified candidate genes with sequence variants predicted to affect gene function. This approach, merged with additional integrative genetics approaches, will likely lead to novel mechanisms underlying complex disease and provide new drug targets and therapies.

Whole genome sequencing of mouse lines divergently selected for fatness (FLI) and leanness (FHI) revealed several genetic variants as candidates for novel obesity genes.

BACKGROUND: Analysing genomes of animal model organisms is widely used for understanding the genetic basis of complex traits and diseases, such as obesity, for which only a few mouse models exist, however, without their lean counterparts. OBJECTIVE: To analyse genetic differences in the unique mouse models of polygenic obesity (Fat line) and leanness (Lean line) originating from the same base population and established by divergent selection over more than 60 generations. METHODS: Genetic variability was analysed using WGS. Variants were identified with GATK and annotated with Ensembl VEP. g.Profiler, WebGestalt, and KEGG were used for GO and pathway enrichment analysis. miRNA seed regions were obtained with miRPathDB 2.0, LncRRIsearch was used to predict targets of identified lncRNAs, and genes influencing adipose tissue amount were searched using the IMPC database. RESULTS: WGS analysis revealed 6.3 million SNPs, 1.3 million were new. Thousands of potentially impactful SNPs were identified, including within 24 genes related to adipose tissue amount. SNP density was highest in pseudogenes and regulatory RNAs. The Lean line carries SNP rs248726381 in the seed region of mmu-miR-3086-3p, which may affect fatty acid metabolism. KEGG analysis showed deleterious missense variants in immune response and diabetes genes, with food perception pathways being most enriched. Gene prioritisation considering SNP GERP scores, variant consequences, and allele comparison with other mouse lines identified seven novel obesity candidate genes: 4930441H08Rik, Aff3, Fam237b, Gm36633, Pced1a, Tecrl, and Zfp536. CONCLUSION: WGS revealed many genetic differences between the lines that accumulated over the selection period, including variants with potential negative impacts on gene function. Given the increasing availability of mouse strains and genetic polymorphism catalogues, the study is a valuable resource for researchers to study obesity.

Isolation and high-throughput sequencing of two closely linked epistatic hypertension susceptibility loci with a panel of bicongenic strains.

Interactions or epistasis between genetic factors may contribute to "missing heritability." While linkage analyses detect epistasis, defining the limits of the interacting segments poses a significant challenge especially when the interactions are between loci in close proximity. The goal of the present study was to isolate two such epistatic blood pressure (BP) loci on rat chromosome 5. A panel of S.LEW bicongenic strains along with the corresponding monocongenic strains was constructed. BP of each set comprising of one bicongenic and two corresponding monocongenic strains were determined along with the parental Salt-sensitive (S) strain. Epistasis was observed in one out of four sets of congenic strains, wherein systolic blood pressures (SBP) of the two monocongenic strains S.LEW(5)x6Bx9x5a and S.LEW(5)x6Bx9x5b were comparable to that of S, but the SBP of the bicongenic strain S.LEW(5)x6Bx9x5 (157 ± 4.3 mmHg) was significantly lower than that of S (196 ± 6.8 mmHg, P < 0.001). A two-way ANOVA indicated significant interactions between the LEW alleles at the two loci. The interacting loci were 2.02 Mb apart and located within genomic segments spanning 7.77 and 4.18 Mb containing 7,360 and 2,753 candidate variants, respectively. The current study demonstrates definitive evidence for epistasis and provides genetic tools for further dissection of the isolated epistatic BP loci.

Combined ChIP-Seq and transcriptome analysis identifies AP-1/JunD as a primary regulator of oxidative stress and IL-1ß synthesis in macrophages.

BACKGROUND: The oxidative burst is one of the major antimicrobial mechanisms adopted by macrophages. The WKY rat strain is uniquely susceptible to experimentally induced macrophage-dependent crescentic glomerulonephritis (Crgn). We previously identified the AP-1 transcription factor JunD as a determinant of macrophage activation in WKY bone marrow-derived macrophages (BMDMs). JunD is over-expressed in WKY BMDMs and its silencing reduces Fc receptor-mediated oxidative burst in these cells. RESULTS: Here we combined Jund RNA interference with microarray analyses alongside ChIP-sequencing (ChIP-Seq) analyses in WKY BMDMs to investigate JunD-mediated control of macrophage activation in basal and lipopolysaccharide (LPS) stimulated cells. Microarray analysis following Jund silencing showed that Jund activates and represses gene expression with marked differential expression (>3 fold) for genes linked with oxidative stress and IL-1ß expression. These results were complemented by comparing whole genome expression in WKY BMDMs with Jund congenic strain (WKY.LCrgn2) BMDMs which express lower levels of JunD. ChIP-Seq analyses demonstrated that the increased expression of JunD resulted in an increased number of binding events in WKY BMDMs compared to WKY.LCrgn2 BMDMs. Combined ChIP-Seq and microarray analysis revealed a set of primary JunD-targets through which JunD exerts its effect on oxidative stress and IL-1ß synthesis in basal and LPS-stimulated macrophages. CONCLUSIONS: These findings demonstrate how genetically determined levels of a transcription factor affect its binding sites in primary cells and identify JunD as a key regulator of oxidative stress and IL-1ß synthesis in primary macrophages, which may play a role in susceptibility to Crgn.

The genome sequence of the spontaneously hypertensive rat: Analysis and functional significance.

The spontaneously hypertensive rat (SHR) is the most widely studied animal model of hypertension. Scores of SHR quantitative loci (QTLs) have been mapped for hypertension and other phenotypes. We have sequenced the SHR/OlaIpcv genome at 10.7-fold coverage by paired-end sequencing on the Illumina platform. We identified 3.6 million high-quality single nucleotide polymorphisms (SNPs) between the SHR/OlaIpcv and Brown Norway (BN) reference genome, with a high rate of validation (sensitivity 96.3%-98.0% and specificity 99%-100%). We also identified 343,243 short indels between the SHR/OlaIpcv and reference genomes. These SNPs and indels resulted in 161 gain or loss of stop codons and 629 frameshifts compared with the BN reference sequence. We also identified 13,438 larger deletions that result in complete or partial absence of 107 genes in the SHR/OlaIpcv genome compared with the BN reference and 588 copy number variants (CNVs) that overlap with the gene regions of 688 genes. Genomic regions containing genes whose expression had been previously mapped as cis-regulated expression quantitative trait loci (eQTLs) were significantly enriched with SNPs, short indels, and larger deletions, suggesting that some of these variants have functional effects on gene expression. Genes that were affected by major alterations in their coding sequence were highly enriched for genes related to ion transport, transport, and plasma membrane localization, providing insights into the likely molecular and cellular basis of hypertension and other phenotypes specific to the SHR strain. This near complete catalog of genomic differences between two extensively studied rat strains provides the starting point for complete elucidation, at the molecular level, of the physiological and pathophysiological phenotypic differences between individuals from these strains.

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.

Regulatory de novo mutations underlying intellectual disability.

The genetic aetiology of a major fraction of patients with intellectual disability (ID) remains unknown. De novo mutations (DNMs) in protein-coding genes explain up to 40% of cases, but the potential role of regulatory DNMs is still poorly understood. We sequenced 63 whole genomes from 21 ID probands and their unaffected parents. In addition, we analysed 30 previously sequenced genomes from exome-negative ID probands. We found that regulatory DNMs were selectively enriched in fetal brain-specific enhancers as compared with adult brain enhancers. DNM-containing enhancers were associated with genes that show preferential expression in the prefrontal cortex. Furthermore, we identified recurrently mutated enhancer clusters that regulate genes involved in nervous system development (CSMD1, OLFM1, and POU3F3). Most of the DNMs from ID probands showed allele-specific enhancer activity when tested using luciferase assay. Using CRISPR-mediated mutation and editing of epigenomic marks, we show that DNMs at regulatory elements affect the expression of putative target genes. Our results, therefore, provide new evidence to indicate that DNMs in fetal brain-specific enhancers play an essential role in the aetiology of ID.

Cap analysis of gene expression reveals alternative promoter usage in a rat model of hypertension.

The role of alternative promoter usage in tissue-specific gene expression has been well established; however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) sequencing from the left ventricle of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, Brown Norway to understand the role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites in the rat left ventricle, including 1,970 novel cardiac transcription start sites. We identified 28 genes with alternative promoter usage between SHR and Brown Norway, which could lead to protein isoforms differing at the amino terminus between two strains and 475 promoter switching events altering the length of the 5' UTR. We found that the shift in Insr promoter usage was significantly associated with insulin levels and blood pressure within a panel of HXB/BXH recombinant inbred rat strains, suggesting that hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.

Cardiorenal Tissues Express SARS-CoV-2 Entry Genes and Basigin (BSG/CD147) Increases With Age in Endothelial Cells.

Vascular and cardiovascular inflammation and thrombosis occur in patients with severe coronavirus disease-2019 (COVID-19). Advancing age is the most significant risk factor for severe COVID-19. Using transcriptomic databases, the authors found that: 1) cardiovascular tissues and endothelial cells express putative genes for severe acute respiratory syndrome coronavirus-2 infection, including angiotensin-converting enzyme 2 (ACE2) and basigin (BSG); 2) severe acute respiratory syndrome coronavirus-2 receptor pathways ACE2/transmembrane serine protease 2 and BSG/peptidylprolyl isomerase B(A) polarize to lung/epithelium and vessel/endothelium, respectively; 3) expression of host genes is relatively stable with age; and 4) notable exceptions are ACE2, which decreases with age in some tissues, and BSG, which increases with age in endothelial cells, suggesting that BSG expression in the vasculature may explain the heightened risk for severe disease with age.

Impulsivity is a heritable trait in rodents and associated with a novel quantitative trait locus on chromosome 1.

Impulsivity describes the tendency to act prematurely without appropriate foresight and is symptomatic of a number of neuropsychiatric disorders. Although a number of genes for impulsivity have been identified, no study to date has carried out an unbiased, genome-wide approach to identify genetic markers associated with impulsivity in experimental animals. Herein we report a linkage study of a six-generational pedigree of adult rats phenotyped for one dimension of impulsivity, namely premature responding on the five-choice serial reaction time task, combined with genome wide sequencing and transcriptome analysis to identify candidate genes associated with the expression of the impulsivity trait. Premature responding was found to be heritable (h2 = 13-16%), with significant linkage (LOD 5.2) identified on chromosome 1. Fine mapping of this locus identified a number of polymorphic candidate genes, however only one, beta haemoglobin, was differentially expressed in both the founder strain and F6 generation. These findings provide novel insights into the genetic substrates and putative neurobiological mechanisms of impulsivity with broader translational relevance for impulsivity-related disorders in humans.

Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.

Adult cardiac tissue undergoes a rapid process of dedifferentiation when cultured outside the body. The in vivo environment, particularly constant electromechanical stimulation, is fundamental to the regulation of cardiac structure and function. We investigated the role of electromechanical stimulation in preventing culture-induced dedifferentiation of adult cardiac tissue using rat, rabbit and human heart failure myocardial slices. Here we report that the application of a preload equivalent to sarcomere length (SL) = 2.2 µm is optimal for the maintenance of rat myocardial slice structural, functional and transcriptional properties at 24 h. Gene sets associated with the preservation of structure and function are activated, while gene sets involved in dedifferentiation are suppressed. The maximum contractility of human heart failure myocardial slices at 24 h is also optimally maintained at SL = 2.2 µm. Rabbit myocardial slices cultured at SL = 2.2 µm remain stable for 5 days. This approach substantially prolongs the culture of adult cardiac tissue in vitro.

Reversal of endothelial dysfunction reduces white matter vulnerability in cerebral small vessel disease in rats.

Dementia is a major social and economic problem for our aging population. One of the most common of dementia in the elderly is cerebral small vessel disease (SVD). Magnetic resonance scans of SVD patients typically show white matter abnormalities, but we do not understand the mechanistic pathological link between blood vessels and white matter myelin damage. Hypertension is suggested as the cause of sporadic SVD, but a recent alternative hypothesis invokes dysfunction of the blood-brain barrier as the primary cause. In a rat model of SVD, we show that endothelial cell (EC) dysfunction is the first change in development of the disease. Dysfunctional ECs secrete heat shock protein 90α, which blocks oligodendroglial differentiation, contributing to impaired myelination. Treatment with EC-stabilizing drugs reversed these EC and oligodendroglial pathologies in the rat model. EC and oligodendroglial dysfunction were also observed in humans with early, asymptomatic SVD pathology. We identified a loss-of-function mutation in ATPase11B, which caused the EC dysfunction in the rat SVD model, and a single-nucleotide polymorphism in ATPase11B that was associated with white matter abnormalities in humans with SVD. We show that EC dysfunction is a cause of SVD white matter vulnerability and provide a therapeutic strategy to treat and reverse SVD in the rat model, which may also be of relevance to human SVD.

Systems biology with high-throughput sequencing reveals genetic mechanisms underlying the metabolic syndrome in the Lyon hypertensive rat.

BACKGROUND: The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including obesity, hypertension, dyslipidemia, and insulin resistance. The Lyon hypertensive and Lyon normotensive rats are models of MetS sensitivity and resistance, respectively. To identify genetic determinants and mechanisms underlying MetS, an F2 intercross between Lyon hypertensive and Lyon normotensive was comprehensively studied. METHODS AND RESULTS: Multidimensional data were obtained including genotypes of 1536 single-nucleotide polymorphisms, 23 physiological traits, and >150 billion nucleotides of RNA-seq reads from the livers of F2 intercross offspring and parental rats. Phenotypic and expression quantitative trait loci (eQTL) were mapped. Application of systems biology methods identified 17 candidate MetS genes. Several putative causal cis-eQTL were identified corresponding with phenotypic QTL loci. We found an eQTL hotspot on rat chromosome 17 that is causally associated with multiple MetS-related traits and found RGD1562963, a gene regulated in cis by this eQTL hotspot, as the most likely eQTL driver gene directly affected by genetic variation between Lyon hypertensive and Lyon normotensive rats. CONCLUSIONS: Our study sheds light on the intricate pathogenesis of MetS and demonstrates that systems biology with high-throughput sequencing is a powerful method to study the pathogenesis of complex genetic diseases.

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.