Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT

Mutations in ABCA1, APOA1, and LCAT reduce HDL cholesterol (HDLc) in humans. However, the prevalence of these mutations and their relative effects on HDLc reduction and risk of coronary artery disease (CAD) are less clear. Here we searched for ABCA1, APOA1, and LCAT mutations in 178 unrelated probands with HDLc < 10th percentile but no other major lipid abnormalities, including 89 with ≥ 1 first-degree relative with low HDLc (familial probands) and 89 where familial status of low HDLc is uncertain (unknown probands). Mutations were most frequent in LCAT (15.7%), followed by ABCA1 (9.0%) and APOA1 (4.5%), and were found in 42.7% of familial but only 14.6% of unknown probands (p = 2.44 ∗ 10^− 5). Interestingly, only 16 of 24 (66.7%) mutations assessed in families conferred an average HDLc < 10th percentile. Furthermore, only mutation carriers with HDLc < 5th percentile had elevated risk of CAD (odds ratio (OR) = 2.26 for 34 ABCA1 mutation carriers vs. 149 total first-degree relative controls, p = 0.05; OR = 2.50 for 26 APOA1 mutation carriers, p = 0.04; OR = 3.44 for 38 LCAT mutation carriers, p = 1.1 ∗ 10^− 3). These observations show that mutations in ABCA1, APOA1, and LCAT are sufficient to explain > 40% of familial hypoalphalipoproteinemia in this cohort. Moreover, individuals with mutations and large reductions in HDLc have increased risk of CAD. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Segregation of LIPG, CETP, and GALNT2 Mutations in Caucasian Families with Extremely High HDL Cholesterol

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.     

Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband’s family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.     

A novel splicing mutation in the ABCA1 gene,causing Tangier disease and familial HDL deficiency in a large family

Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene.ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G?>?A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86?±?0.16?mmol/L and 92.2?±?10.9?mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G?>?A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency.Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided.     

ABCA1 gene promoter DNA methylation is associated with HDL particle profile and coronary artery disease in familial hypercholesterolemia

High-density lipoproteins cholesterol (HDL-C) level, a strong coronary artery disease (CAD) clinical biomarker, shows significant interindividual variability. However, the molecular mechanisms involved remain mostly unknown. ATP-binding cassette A1 (ABCA1) catalyzes the cholesterol transfer from peripheral cells to nascent HDL particles. Recently, a differentially methylation region was identified in ABCA1 gene promoter locus, near the first exon. Therefore, we hypothesized that DNA methylation changes at ABCA1 gene locus is one of the molecular mechanisms involved in HDL-C interindividual variability. The study was conducted in familial hypercholesterolemia (FH), a monogenic disorder associated with a high risk of CAD . Ninety-seven FH patients (all p.W66G for the LDLR gene mutation and not under lipid-lowering treatment) were recruited and finely phenotyped for DNA methylation analyses at ABCA1 gene locus. ABCA1 DNA methylation levels were found negatively correlated with circulating HDL-C (r = -0.20; p = 0.05), HDL2-phospholipid levels (r = -0.43; p = 0.04), and with a trend for association with HDL peak particle size (r = -0.38; p = 0.08). ABCA1 DNA methylation levels were also found associated with prior history of CAD (CAD = 40.2% vs. without CAD = 34.3%; p = 0.003). These results suggest that epigenetic changes within the ABCA1 gene promoter contribute to the interindividual variability in plasma HDL-C concentrations and are associated with CAD expression. These findings could change our understanding of the molecular mechanisms involved in the pathophysiological processes leading to CAD.     

Plasma levels of lecithin:cholesterol acyltransferase and risk of future coronary artery disease in apparently healthy men and women: a prospective case-control analysis nested in the EPIC-Norfolk population study

LCAT plays a key role in the maturation of HDL, as evidenced by low HDL-cholesterol levels in carriers of deleterious mutations in LCAT. However, the role of LCAT in atherosclerosis is unclear. We set out to study this in a prospective study. Plasma LCAT levels, which strongly correlate with LCAT activity, were measured in baseline nonfasting samples of 933 apparently healthy men and women who developed coronary artery disease (CAD) and 1,852 matched controls who remained free of CAD during 6 year follow-up. LCAT levels did not differ between cases and controls but were higher in women than men. Stratification into LCAT quartiles revealed a positive association with plasma LDL-cholesterol and triglyceride levels in the unexpected absence of an association with HDL-cholesterol. In mixed-gender analyses, the odds ratio (OR) for future CAD in the highest LCAT quartile versus the lowest was 1.00 [confidence interval (CI): 0.76–1.29, P for linearity = 0.902], although opposite trends were observed in men and women. In fact, high LCAT levels were associated with an increased CAD risk in women (unadjusted OR 1.45, CI: 0.94–2.22, P for linearity = 0.036). In contrast to our studies in carriers of LCAT mutations, the current data show that low LCAT plasma levels are not associated with increased atherosclerosis in the general population.     

Mosaic SCN1A mutations in familial partial epilepsy with antecedent febrile seizures

SCN1A is the most relevant epilepsy gene. Mutations of SCN1A generate phenotypes ranging from the extremely severe form of Dravet syndrome (DS) to a mild form of generalized epilepsy with febrile seizures plus (GEFS+). Mosaic SCN1A mutations have been identified in rare familial DS. It is suspected that mosaic mutations of SCN1A may cause other types of familial epilepsies with febrile seizures (FS), which are more common clinically. Thus, we screened SCN1A mutations in 13 families with partial epilepsy with antecedent febrile seizures (PEFS+) using denaturing high-performance liquid chromatography and sequencing. The level of mosaicism was further quantified by pyrosequencing. Two missense SCN1A mutations with mosaic origin were identified in two unrelated families, accounting for 15.4% (2/13) of the PEFS+ families tested. One of the mosaic carriers with ~25.0% mutation of c.5768A>G/p.Q1923R had experienced simple FS; another with ~12.5% mutation of c.4847T>C/p.I1616T was asymptomatic. Their heterozygous children had PEFS+. Recurrent transmission occurred in both families, as noted in most of the families with germline mosaicism reported previously. The two mosaic mutations identified in this study are less destructive missense, compared with the more destructive truncating and splice-site mutations identified in the majority of previous studies. This is the first report of mosaic SCN1A mutations in families with probands that do not exhibit DS, but manifest only a milder phenotype. Therefore, such families with mild cases should be approached with caution in genetic counseling and the possibility of mosaicism origin associated with high recurrence risk should be excluded.     

Inherited disorders of HDL metabolism and atherosclerosis

PURPOSE OF REVIEW: Genetic disorders of HDL metabolism are rare and, as a result, the assessment of atherosclerosis risk in individuals suffering from these disorders has been difficult. Ultrasound imaging of carotid arteries has provided a tool to assess the risk in hereditary hypo and hyperalphalipoproteinemia. This review gives a comprehensive summary. RECENT FINDINGS: Epidemiological studies have unequivocally shown that HDL cholesterol levels are inversely related to coronary artery disease risk, but the literature concerning genetic disorders of HDL metabolism provides less convincing information. Fortuitously, we were able to directly compare carotid intima media thickness data of substantial numbers of individuals with mutations in either apolipoprotein A-I (apoA-I), ATP binding cassette AI (ABCA1), lecithin: cholesterol acyltransferase (LCAT) or cholesteryl ester transfer protein. These data show that carriers of an apoA-I mutation exhibit the most pronounced accelerated atherosclerosis compared with those carrying mutations in ABCA1 and LCAT. Heterozygosity for a non-sense mutation in cholesteryl ester transfer protein did, by contrast, not distinguish carriers from controls in terms of intima media thickness progression. We will discuss these results in the context of the current literature. SUMMARY: Intima media thickness studies have provided evidence that hypoalphalipoproteinemia due to mutations in apoA-I, ABCA1, and LCAT is associated with increased progression of atherosclerosis. In contrast, hyperalphalipoproteinemia as a result of loss of cholesteryl ester transfer protein function is associated with unaltered atherosclerosis progression compared with family controls. This insight is of interest, since it can assist in the prioritizing of antiatherogenic therapy by increasing HDL cholesterol levels.     

Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer

A population-based series of 649 unselected incident cases of ovarian cancer diagnosed in Ontario, Canada, during 1995-96 was screened for germline mutations in BRCA1 and BRCA2. We specifically tested for 11 of the most commonly reported mutations in the two genes. Then, cases were assessed with the protein-truncation test (PTT) for exon 11 of BRCA1, with denaturing gradient gel electrophoresis for the remainder of BRCA1, and with PTT for exons 10 and 11 of BRCA2. No mutations were found in all 134 women with tumors of borderline histology. Among the 515 women with invasive cancers, we identified 60 mutations, 39 in BRCA1 and 21 in BRCA2. The total mutation frequency among women with invasive cancers, 11.7% (95% confidence interval [95%CI] 9.2%-14.8%), is higher than previous estimates. Hereditary ovarian cancers diagnosed at age <50 years were mostly (83%) due to BRCA1, whereas the majority (60%) of those diagnosed at age >60 years were due to BRCA2. Mutations were found in 19% of women reporting first-degree relatives with breast or ovarian cancer and in 6.5% of women with no affected first-degree relatives. Risks of ovarian, breast, and stomach cancers and leukemias/lymphomas were increased nine-, five-, six- and threefold, respectively, among first-degree relatives of cases carrying BRCA1 mutations, compared with relatives of noncarriers, and risk of colorectal cancer was increased threefold for relatives of cases carrying BRCA2 mutations. For carriers of BRCA1 mutations, the estimated penetrance by age 80 years was 36% for ovarian cancer and 68% for breast cancer. In breast-cancer risk for first-degree relatives, there was a strong trend according to mutation location along the coding sequence of BRCA1, with little evidence of increased risk for mutations in the 5' fifth, but 8.8-fold increased risk for mutations in the 3' fifth (95%CI 3.6-22.0), corresponding to a carrier penetrance of essentially 100%. Ovarian, colorectal, stomach, pancreatic, and prostate cancer occurred among first-degree relatives of carriers of BRCA2 mutations only when mutations were in the ovarian cancer-cluster region (OCCR) of exon 11, whereas an excess of breast cancer was seen when mutations were outside the OCCR. For cancers of all sites combined, the estimated penetrance of BRCA2 mutations was greater for males than for females, 53% versus 38%. Past studies may have underestimated the contribution of BRCA2 to ovarian cancer, because mutations in this gene cause predominantly late-onset cancer, and previous work has focused more on early-onset disease. If confirmed in future studies, the trend in breast-cancer penetrance, according to mutation location along the BRCA1 coding sequence, may have significant impact on treatment decisions for carriers of BRCA1-mutations. As well, BRCA2 mutations may prove to be a greater cause of cancer in male carriers than previously has been thought.     

Average age-specific cumulative risk of breast cancer according to type and site of germline mutations in BRCA1 and BRCA2 estimated from multiple-case breast cancer families attending Australian family cancer clinics

If the risk of disease is not the same for all germline mutations in a given gene, or if there are other familial modifiers of risk in carriers, then family-history-based estimates of average risk for detected mutations in that gene will depend on how carriers are sampled. Risk may also depend on the site or type of mutation. We studied 51 families with strong histories of breast cancer who attended Australian family cancer clinics and in which a germline mutation in BRCA1 or BRCA2 had been identified (28 and 23 families, respectively). Breast cancer risk in carriers was estimated under maximum likelihood theory, using information from all family members including those not tested, with adjustment for ascertainment by conditioning on genotype of the proband and family phenotype. The average cumulative risk of breast cancer for mutations in either BRCA1 or BRCA2 was 27% (95% confidence interval 16-43%) to age 50 and 64% (44-83%) to age 70. When grouped, the incidence in carriers was on average 17 (10-30) times that in non-carriers, independent of gene or mutation type (hazard ratios: 11 (4-29) for BRCA1, 23 (12-43) for BRCA2 (P for difference = 0.23); 13 (6-29) for protein-truncating mutations, 30 (9-104) for missense mutations and 30 (10-90) for splice-site mutations). For missense mutations, this was equivalent to a cumulative risk to age 70 of 83% (40-100%) and was due in part, but not totally, to the missense mutations 300 T>G in BRCA1 and 4486 G>T in BRCA2, which were individually found to be associated with high risk (P<0.001). Mutations in the central region of BRCA1 may be associated with a lower risk. The issue of the pathogenicity of specific variants may be addressed analytically providing there are one or more suitably informative families with that mutation.     

Contribution of BRCA1 and BRCA2 mutations to breast and ovarian cancer in Pakistan

The population of Pakistan has been reported to have the highest rate of breast cancer of any Asian population (excluding Jews in Israel) and one of the highest rates of ovarian cancer worldwide. To explore the contribution that genetic factors make to these high rates, we have conducted a case-control study of 341 case subjects with breast cancer, 120 case subjects with ovarian cancer, and 200 female control subjects from two major cities of Pakistan (Karachi and Lahore). The prevalence of BRCA1 or BRCA2 mutations among case subjects with breast cancer was 6.7% (95% confidence interval [CI] 4.1%-9.4%), and that among case subjects with ovarian cancer was 15.8% (95% CI 9.2%-22.4%). Mutations of the BRCA1 gene accounted for 84% of the mutations among case subjects with ovarian cancer and 65% of mutations among case subjects with breast cancer. The majority of detected mutations are unique to Pakistan. Five BRCA1 mutations (2080insA, 3889delAG, 4184del4, 4284delAG, and IVS14-1A-->G) and one BRCA2 mutation (3337C-->T) were found in multiple case subjects and represent candidate founder mutations. The penetrance of deleterious mutations in BRCA1 and BRCA2 is comparable to that of Western populations. The cumulative risk of cancer to age 85 years in female first-degree relatives of BRCA1-mutation-positive case subjects was 48% and was 37% for first-degree relatives of the BRCA2-mutation-positive case subjects. A higher proportion of case subjects with breast cancer than of control subjects were the progeny of first-cousin marriages (odds ratio [OR] 2.1; 95% CI 1.4-3.3; P=.001). The effects of consanguinity were significant for case subjects with early-onset breast cancer (age <40 years) (OR=2.7; 95% CI 1.5-4.9; P=.0008) and case subjects with ovarian cancer (OR=2.4; 95% CI 1.4-4.2; P=.002). These results suggest that recessively inherited genes may contribute to breast and ovarian cancer risk in Pakistan.     

In vivo tissue cholesterol efflux is reduced in carriers of a mutation in APOA1

Atheroprotection by high density lipoprotein (HDL) is considered to be mediated through reverse cholesterol transport (RCT) from peripheral tissues. We investigated in vivo cholesterol fluxes through the RCT pathway in patients with low plasma high density lipoprotein cholesterol (HDL-c) due to mutations in APOA1. Seven carriers of the L202P mutation in APOA1 (mean HDL-c: 20 ± 19 mg/dl) and seven unaffected controls (mean HDL-c: 54 ± 11 mg/dl, P < 0.0001) received a 20 h infusion of ¹³C₂-cholesterol (¹³C-C). Enrichment of plasma and erythrocyte free cholesterol and plasma cholesterol esters was measured. With a three-compartment SAAM-II model, tissue cholesterol efflux (TCE) was calculated. TCE was reduced by 19% in carriers (4.6 ± 0.8 mg/kg/h versus 5.7 ± 0.7 mg/kg/h in controls, P = 0.02). Fecal ¹³C recovery and sterol excretion 7 days postinfusion did not differ significantly between carriers and controls: 21.3 ± 20% versus 13.3 ± 6.3% (P = 0.33), and 2,015 ± 1,431 mg/day versus 1456 ± 404 mg/day (P = 0.43), respectively. TCE is reduced in carriers of mutations in APOA1, suggesting that HDL contributes to efflux of tissue cholesterol in humans. The residual TCE and unaffected fecal sterol excretion in our severely affected carriers suggest, however, that non-HDL pathways contribute to RCT significantly.     

Spectrum of the GJB2 mutations in Belarussian patients with hearing loss. Findings of pilot genetic screening of hearing impairment in newborns

A total of 111 unrelated probands and their 8 sibs from Grodno oblast (Belarus) with bilateral isolated sensorineural hearing impairment were studied for the presence of mutations in the connexin 26 (GJB2) gene. Mutations were detected in 51 probands (46% of the sample). A significantly higher frequency of the GJB2 gene mutations was observed in familial cases of the disease with the autosomal recessive mode of inheritance (in 78% of families). Detected characteristics of the GJB2 gene mutation spectrum demonstrated that the using the algorithm, which was designed for Russian patients, is optimal for the molecular study of patients from Belarus. In the sample of patients with hearing loss, the highest (among other similar samples studied in the world) allele frequency of c.313_326del14 mutation (7% of all pathological GJB2 alleles) was registered; Polish origin of this deletion was suggested. It was demonstrated that detection of the GJB2 gene mutation on one patient’s chromosome only is insufficient to confirm a molecular genetic diagnosis of hearing loss of the DFNB1 genetic type (autosomal recessive hearing loss caused by the GJB2 gene mutations). Pilot screening for the GJB2 gene mutations in newborns from Grodno oblast was performed. The material from 235 children was studied during the screening; nine heterozygous carriers of the mutation were found. The c.35delG mutation was detected in a homozygous state in a single newborn (hearing loss of moderate severity was subsequently audiologically confirmed in this child).     

Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

Breast cancer is strongly influenced by hereditary risk factors, a majority of which still remain unknown. Here, we performed a targeted next-generation sequencing of 796 genes implicated in DNA repair in 189 Finnish breast cancer cases with indication of hereditary disease susceptibility and focused the analysis on protein truncating mutations. A recurrent heterozygous mutation (c.904_916del, p.Arg304ValfsTer3) was identified in early DNA damage response gene, MCPH1, significantly associating with breast cancer susceptibility both in familial (5/145, 3.4%, P = 0.003, OR 8.3) and unselected cases (16/1150, 1.4%, P = 0.016, OR 3.3). A total of 21 mutation positive families were identified, of which one-third exhibited also brain tumors and/or sarcomas (P = 0.0007). Mutation carriers exhibited significant increase in genomic instability assessed by cytogenetic analysis for spontaneous chromosomal rearrangements in peripheral blood lymphocytes (P = 0.0007), suggesting an effect for MCPH1 haploinsufficiency on cancer susceptibility. Furthermore, 40% of the mutation carrier tumors exhibited loss of the wild-type allele. These findings collectively provide strong evidence for MCHP1 being a novel breast cancer susceptibility gene, which warrants further investigations in other populations.     

High prevalence of mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands.

Mutations in microtubule-associated protein tau recently have been identified in familial cases of frontotemporal dementia (FTD). We report the frequency of tau mutations in a large population-based study of FTD carried out in the Netherlands from January 1994 to June 1998. Thirty-seven patients had >/=1 first-degree relative with dementia. A mutation in the tau gene was found in 17.8% of the group of patients with FTD and in 43% of patients with FTD who also had a positive family history of FTD. Three distinct missense mutations (G272V, P301L, R406W) accounted for 15.6% of the mutations. These three missense mutations, and a single amino acid deletion (DeltaK280) that was detected in one patient, strongly reduce the ability of tau to promote microtubule assembly. We also found an intronic mutation at position +33 after exon 9, which is likely to affect the alternative splicing of tau. Tau mutations are responsible for a large proportion of familial FTD cases; however, there are also families with FTD in which no mutations in tau have been found, which indicates locus and/or allelic heterogeneity. The different tau mutations may result in disturbances in the interactions of the protein tau with microtubules, resulting in hyperphosphorylation of tau protein, assembly into filaments, and subsequent cell death.     

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from Apulia, Italy

INTRODUCTION: Hereditary breast cancer has been partly attributed to germline mutations in the BRCA1 gene that are deleterious for BRCA1 protein activity. This paper analyzes the incidence and characteristics of detectable BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from southern Italy to investigate the incidence and the association of these molecular alterations with breast cancer biology and family history. METHODS: One hundred cases with familial characteristics were selected from a consecutive series of 511 patients with a first diagnosis of breast cancer. DNA from peripheral blood was screened for whole BRCA1 gene mutations utilizing dHPLC as a pre-screening analysis and automatic DNA sequencing for the identification of specific alterations. RESULTS: In the overall series of 511 patients, 100 had a family history of breast cancer and were investigated for BRCA1 mutations. Two types of BRCA1 mutations were identified, 5382insC in six cases and 4566delA in one case. The 5382insC mutation was present in two out of six cases with ovarian cancer while 4566delA in one case of male cancer. The most frequent missense polymorphisms were E1038G, P871L, K1183R in exon 11, S1613G, M1652I in exon 16 and D1778G in exon 22. Confirming what found in previous studies, patients in whom pathological BRCA1 mutations were detected had early-onset breast cancer (p=0.05), positive nodal status (p=0.05), lower ER (p=0.02) and PgR (p=0.01) content. Interestingly, the K1183R polymorphism and, less strongly, S1613G polymorphism were associated to mutational risk (K1183R: OR 0.1 p=0.03; S1613G: OR 2.7 p=0.08). CONCLUSION: Mutations in the BRCA1 gene are frequent also in our consecutive series of patients from southern Italy. An association between two detected single nucleotide polymorphisms (SNPs) and BRCA1 mutational risk was ascertained. Finally, we confirm the fact that peculiar clinical-pathological features seem to characterize patients with a family history of breast cancer and BRCA1 alterations.     

Recurrent and Founder Mutations in the Netherlands: the Long-QT Syndrome

Background and objective

The long-QT syndrome (LQTS) is associated with premature sudden cardiac deaths affecting whole families and is caused by mutations in genes encoding for cardiac proteins. When the same mutation is found in different families (recurrent mutations), this may imply either a common ancestor (founder) or multiple de novo mutations. We aimed to review recurrent mutations in patients with LQTS.

Methods

By use of our databases, we investigated the number of mutations that were found recurrently (at least three times) in LQT type 1–3 patients in the Netherlands. We studied familial links in the apparently unrelated probands, and we visualised the geographical distribution of these probands. Our results were compared with published literature of founder effects in LQTS outside the Netherlands.

Results

We counted 14 recurrent LQT mutations in the Netherlands. There are 326 identified carriers of one of these mutations. For three of these mutations, familial links were found between apparently unrelated probands.

Conclusion

Whereas true LQT founder mutations are described elsewhere in the world, we cannot yet demonstrate a real founder effect of these recurrent mutations in the Netherlands. Further studies on the prevalence of these mutations are indicated, and haplotype-sharing of the mutation carriers is pertinent to provide more evidence for founder mutation-based LQTS pathology in our country.     

An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations

The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.