Jennifer A. Collins

Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

Mutations in the ABC1 gene in familial HDL deficiency with defective cholesterol efflux

BACKGROUND A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients with premature atherosclerosis. We have shown that Tangier disease, a rare and severe form of HDL deficiency characterised by a biochemical defect in cellular cholesterol efflux, is caused by mutations in the ATP-binding-cassette (ABC1) gene. This gene codes for the cholesterol-efflux regulatory protein (CERP). We investigated the presence of mutations in this gene in patients with familial HDL deficiency. METHODS Three French-Canadian families and one Dutch family with familial HDL deficiency were studied. Fibroblasts from the proband of each family were defective in cellular cholesterol efflux. Genomic DNA of each proband was used for mutation detection with primers flanking each exon of the ABC1 gene, and for sequencing of the entire coding region of the gene. PCR and restriction-fragment length polymorphism assays specific to each mutation were used to investigate segregation of the mutation in each family, and to test for absence of the mutation in DNA from normal controls. FINDINGS A different mutation was detected in ABC1 in each family studied. Each mutation either created a stop codon predicted to result in truncation of CERP, or altered a conserved aminoacid residue. Each mutation segregated with low concentrations of HDL-cholesterol in the family, and was not observed in more than 500 control chromosomes tested. INTERPRETATION These data show that mutations in ABC1 are the major cause of familial HDL deficiency associated with defective cholesterol efflux, and that CERP has an essential role in the formation of HDL. Our findings highlight the potential of modulation of ABC1 as a new route for increasing HDL concentrations.

Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes

We and others have recently identified mutations in the ABCA1 gene as the underlying cause of Tangier disease (TD) and of a dominantly inherited form of familial hypoalphalipoproteinemia (FHA) associated with reduced cholesterol efflux. We have now identified 13 ABCA1 mutations in 11 families (five TD, six FHA) and have examined the phenotypes of 77 individuals heterozygous for mutations in the ABCA1 gene. ABCA1 heterozygotes have decreased HDL cholesterol (HDL-C) and increased triglycerides. Age is an important modifier of the phenotype in heterozygotes, with a higher proportion of heterozygotes aged 30-70 years having HDL-C greater than the fifth percentile for age and sex compared with carriers less than 30 years of age. Levels of cholesterol efflux are highly correlated with HDL-C levels, accounting for 82% of its variation. Each 8% change in ABCA1-mediated efflux is predicted to be associated with a 0.1 mmol/l change in HDL-C. ABCA1 heterozygotes display a greater than threefold increase in the frequency of coronary artery disease (CAD), with earlier onset than unaffected family members. CAD is more frequent in those heterozygotes with lower cholesterol efflux values. These data provide direct evidence that impairment of cholesterol efflux and consequently reverse cholesterol transport is associated with reduced plasma HDL-C levels and increased risk of CAD.

CAG Expansion in the Huntington Disease Gene Is Associated with a Specific and Targetable Predisposing Haplogroup

Huntington disease (HD) is an autosomal-dominant disorder that results from >or=36 CAG repeats in the HD gene (HTT). Approximately 10% of patients inherit a chromosome that underwent CAG expansion from an unaffected parent with <36 CAG repeats. This study is a comprehensive analysis of genetic diversity in HTT and reveals that HD patients of European origin (n = 65) have a significant enrichment (95%) of a specific set of 22 tagging single nucleotide polymorphisms (SNPs) that constitute a single haplogroup. The disease association of many SNPs is much stronger than any previously reported polymorphism and was confirmed in a replication cohort (n = 203). Importantly, the same haplogroup is also significantly enriched (83%) in individuals with 27-35 CAG repeats (intermediate alleles, n = 66), who are unaffected by the disease, but have increased CAG tract sizes relative to the general population (n = 116). These data support a stepwise model for CAG expansion into the affected range (>or=36 CAG) and identifies specific haplogroup variants in the general population associated with this instability. The specific variants at risk for CAG expansion are not present in the general population in China, Japan, and Nigeria where the prevalence of HD is much lower. The current data argue that cis-elements have a major predisposing influence on CAG instability in HTT. The strong association between specific SNP alleles and CAG expansion also provides an opportunity of personalized therapeutics in HD where the clinical development of only a small number of allele-specific targets may be sufficient to treat up to 88% of the HD patient population.

HTT haplotypes contribute to differences in Huntington disease prevalence between Europe and East Asia.

Huntington disease (HD) results from CAG expansion in the huntingtin (HTT) gene. Although HD occurs worldwide, there are large geographic differences in its prevalence. The prevalence in populations derived from Europe is 10–100 times greater than in East Asia. The European general population chromosomes can be grouped into three major haplogroups (group of similar haplotypes): A, B and C. The majority of HD chromosomes in Europe are found on haplogroup A. However, in the East-Asian populations of China and Japan, we find the majority of HD chromosomes are associated with haplogroup C. The highest risk HD haplotypes (A1 and A2), are absent from the general and HD populations of China and Japan, and therefore provide an explanation for why HD prevalence is low in East Asia. Interestingly, both East-Asian and European populations share a similar low level of HD on haplogroup C. Our data are consistent with the hypothesis that different HTT haplotypes have different mutation rates, and geographic differences in HTT haplotypes explain the difference in HD prevalence. Further, the bias for expansion on haplogroup C in the East-Asian population cannot be explained by a higher average CAG size, as haplogroup C has a lower average CAG size in the general East-Asian population compared with other haplogroups. This finding suggests that CAG-tract size is not the only factor important for CAG instability. Instead, the expansion bias may be because of genetic cis-elements within the haplotype that influence CAG instability in HTT, possibly through different mutational mechanisms for the different haplogroups.

The LPL S447X cSNP is associated with decreased blood pressure and plasma triglycerides, and reduced risk of coronary artery disease

Linkage of the lipoprotein lipase (LPL) gene to blood pressure levels has been reported. The LPL S447X single nucleotide polymorphism (cSNP) has been associated with decreased triglycerides (TG), increased high density lipoprotein cholesterol, and a decreased risk of coronary artery disease (CAD), which may occur independently of its beneficial lipid changes. To investigate the relationship between LPL S447X cSNP and these parameters, we studied a cohort of individuals with familial hypercholesterolemia in whom blood pressures and information regarding the use of blood pressure lowering medications were available. Carriers of the S447X variant had decreased TG (1.21±0.47 vs. 1.52±0.67, p<0.001) and a trend towards decreased vascular disease (12.7 vs. 19.5%) compared to non‐carriers. More interestingly, however, carriers of this cSNP had decreased diastolic blood pressure compared to non‐carriers (78±10 vs. 82±11, p=0.002), evident in both men and women, youths and adults, with similar trends for systolic blood pressure. Furthermore, the decrease in blood pressure appeared independent of the decrease in TG (p=0.02), suggesting that the LPL protein may have a direct influence on the vascular wall. This suggests an additional mechanism whereby this variant may have protective effects, independent of changes in plasma lipid levels.

A fully humanized transgenic mouse model of Huntington disease

Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh(-/-) background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT.

Genetic Variation in Healthy Oldest-Old

Individuals who live to 85 and beyond without developing major age-related diseases may achieve this, in part, by lacking disease susceptibility factors, or by possessing resistance factors that enhance their ability to avoid disease and prolong lifespan. Healthy aging is a complex phenotype likely to be affected by both genetic and environmental factors. We sequenced 24 candidate healthy aging genes in DNA samples from 47 healthy individuals aged eighty-five years or older (the ‘oldest-old’), to characterize genetic variation that is present in this exceptional group. These healthy seniors were never diagnosed with cancer, cardiovascular disease, pulmonary disease, diabetes, or Alzheimer disease. We re-sequenced all exons, intron-exon boundaries and selected conserved non-coding sequences of candidate genes involved in aging-related processes, including dietary restriction (PPARG, PPARGC1A, SIRT1, SIRT3, UCP2, UCP3), metabolism (IGF1R, APOB, SCD), autophagy (BECN1, FRAP1), stem cell activation (NOTCH1, DLL1), tumor suppression (TP53, CDKN2A, ING1), DNA methylation (TRDMT1, DNMT3A, DNMT3B) Progeria syndromes (LMNA, ZMPSTE24, KL) and stress response (CRYAB, HSPB2). We detected 935 variants, including 848 single nucleotide polymorphisms (SNPs) and 87 insertion or deletions; 41% (385) were not recorded in dbSNP. This study is the first to present a comprehensive analysis of genetic variation in aging-related candidate genes in healthy oldest-old. These variants and especially our novel polymorphisms are valuable resources to test for genetic association in models of disease susceptibility or resistance. In addition, we propose an innovative tagSNP selection strategy that combines variants identified through gene re-sequencing- and HapMap-derived SNPs.

A SNP in the HTT promoter alters NF-[kappa]B binding and is a bidirectional genetic modifier of Huntington disease

Cis-regulatory variants that alter gene expression can modify disease expressivity, but none have previously been identified in Huntington disease (HD). Here we provide in vivo evidence in HD patients that cis-regulatory variants in the HTT promoter are bidirectional modifiers of HD age of onset. HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A) variant on the HD disease allele was associated with delayed age of onset in familial cases, whereas the presence of the rs13102260 (A) variant on the wild-type HTT allele was associated with earlier age of onset in HD patients in an extreme case–based cohort. Our findings suggest a previously unknown mechanism linking allele-specific effects of rs13102260 on HTT expression to HD age of onset and have implications for HTT silencing treatments that are currently in development.

Unstable familial transmissions of Huntington disease alleles with 27–35 CAG repeats (intermediate alleles)†

There are inconsistent reports regarding the likelihood of repeat instability for alleles with 27–35 CAG repeats in the Huntington disease (HD) gene. We have examined the intergenerational stability of such intermediate alleles in 51 families from the University of British Columbias DNA and Tissue Bank for Huntington Disease Research (UBC‐HD Databank). A total of 181 transmissions were identified, with 30% (n = 54/181) of the alleles being unstable upon transmission. The unstable transmissions included both expansions (n = 37) and contractions (n = 17) of CAG size. Of the expanded alleles, 68% (n = 25/37) expanded into the HD range (>36 CAG). Therefore, 14% (n = 25/181) of the 27–35 CAG allele transmissions examined expanded into the disease‐associated range resulting in a new mutation for HD. Significantly, of these new mutations, 40% (n = 10/25) originated from an allele with 35 CAG repeats with CAG repeat expansions ranging from +1 CAG to +23 CAG. The proportion of new mutations in the UBC‐HD Databank is consistent with the most recent new mutation rate for HD, estimated to be at least 10%. The observed difference in the stability of HD intermediate allele transmissions in this data set and in other studies may be a reflection of a small sample size. Alternately, these inconsistencies may indicate an underlying difference in genetic factors which influence repeat instability between the different populations examined. Additional studies determining the frequency and magnitude of repeat instability in this CAG repeat range and factors that influence instability are urgently needed. Until we understand the clinical implications of HD alleles with 27–35 CAG repeats and establish reliable risks of instability, we should exercise caution when translating these results to the clinic.