A rare missense mutation in CHRNA4 associates with smoking behavior and its consequences.

Using Icelandic whole-genome sequence data and an imputation approach we searched for rare sequence variants in CHRNA4 and tested them for association with nicotine dependence. We show that carriers of a rare missense variant (allele frequency=0.24%) within CHRNA4, encoding an R336C substitution, have greater risk of nicotine addiction than non-carriers as assessed by the Fagerstrom Test for Nicotine Dependence (P=1.2 × 10(-4)). The variant also confers risk of several serious smoking-related diseases previously shown to be associated with the D398N substitution in CHRNA5. We observed odds ratios (ORs) of 1.7-2.3 for lung cancer (LC; P=4.0 × 10(-4)), chronic obstructive pulmonary disease (COPD; P=9.3 × 10(-4)), peripheral artery disease (PAD; P=0.090) and abdominal aortic aneurysms (AAAs; P=0.12), and the variant associates strongly with the early-onset forms of LC (OR=4.49, P=2.2 × 10(-4)), COPD (OR=3.22, P=2.9 × 10(-4)), PAD (OR=3.47, P=9.2 × 10(-3)) and AAA (OR=6.44, P=6.3 × 10(-3)). Joint analysis of the four smoking-related diseases reveals significant association (P=6.8 × 10(-5)), particularly for early-onset cases (P=2.1 × 10(-7)). Our results are in agreement with functional studies showing that the human α4ß2 isoform of the channel containing R336C has less sensitivity for its agonists than the wild-type form following nicotine incubation.

Genetic profile of ischemic cerebrovascular disease and carotid stenosis.

OBJECTIVES: Carotid artery stenosis (CS) is a major risk factor for ischemic cerebrovascular disease (ICVD) and is therefore of interest in genetic investigating. Here we report the distribution of 100 polymorphisms in 47 suspected susceptibility genes for ICVD and its risk factors. MATERIALS AND METHODS: Previously published markers in suspected susceptibility genes were genotyped in ICVD patients and controls (928/602). Genotyping was performed using multiplex polymerase chain reaction (PCR) and linear immobilized probe array assays. ICVD cases were subtyped according to Trial of Org 10172 in Acute Stroke Treatment (TOAST) or subdivided into CS and non-CS patients by ultrasonography in a separate analysis. RESULTS: Three polymorphisms located in the lipoprotein lipase (LPL), angiotensinogen (AGT) and guanine nucleotide-binding protein beta-3 (GNB3) genes were significantly associated with ICVD after correction for age and gender. The strongest association was found for the protective LPL Ser447Term polymorphism. All the significant markers showed varying frequencies in different subphenotypes of ICVD. Factor VII, apolipoprotein E and two renin polymorphisms were differentially frequent in patients with evidence of CS compared with non-CS patients. CONCLUSIONS: We have found that some previously described susceptibility polymorphisms are weakly associated with ICVD and that subdivision of patients into CS and non-CS groups may help to identify new candidate polymorphisms.

BRCA2 and p53 mutations in primary breast cancer in relation to genetic instability.

The products of the BRCA breast cancer susceptibility genes have been implicated in cell cycle control and DNA repair. It has been suggested that mutations in the p53 gene are a necessary step in tumorigenesis in BRCA tumors. We tested samples from 402 breast cancer patients for germ-line BRCA2 and p53 mutations in tumors. p53 mutations are more frequent in BRCA2 mutation carriers than they are in controls. Tumors with mutations in either gene had multiple chromosomal abnormalities, as shown by cytogenetic analysis.

Replication error in colorectal carcinoma: association with loss of heterozygosity at mismatch repair loci and clinicopathological variables.

Instability of microsatellite DNA or replication error (RER) is characteristic of tumours caused by mismatch repair (MMR) deficiency. Germline mutations in MMR genes are associated with Hereditary non-polyposis colorectal carcinoma (HNPCC) and somatic mutations in these genes are also found in a substantial fraction of colorectal cancers (CRC). In this study we concurrently screened colorectal tumours for the RER phenotype and loss of heterozygosity (LOH) at MMR gene loci. The RER phenotype was evident in 47/197 (24%) tumours. RER was more commonly detected in young patients (< 50 years) and in tumours located in the proximal colon. RER was positively associated with LOH at the hMSH2/hMSH6 loci on chromosome 2p, where LOH was observed in 46% of the RER+ tumours. LOH at hMLH1 and hPMS1 loci was more frequent in the younger patients (< 50 years). RER was not associated with clinicopathological parameters, such as Duke's stage and tumour differentiation (grade). The RER phenotype was associated with better overall survival, but there was a trend towards significance when multivariate analysis was used. This indicates that loss of MMR genes generate a less aggressive phenotype, and raises the question about RER being a useful indicator of prognosis for CRC patients.

Association between the gene encoding 5-lipoxygenase-activating protein and stroke replicated in a Scottish population.

Cardiovascular diseases, including myocardial infarction (MI) and stroke, most often occur on the background of atherosclerosis, a condition attributed to the interactions between multiple genetic and environmental risk factors. We recently reported a linkage and association study of MI and stroke that yielded a genetic variant, HapA, in the gene encoding 5-lipoxygenase-activating protein (ALOX5AP), that associates with both diseases in Iceland. We also described another ALOX5AP variant, HapB, that associates with MI in England. To further assess the contribution of the ALOX5AP variants to cardiovascular diseases in a population outside Iceland, we genotyped seven single-nucleotide polymorphisms that define both HapA and HapB from 450 patients with ischemic stroke and 710 controls from Aberdeenshire, Scotland. The Icelandic at-risk haplotype, HapA, had significantly greater frequency in Scottish patients than in controls. The carrier frequency in patients and controls was 33.4% and 26.4%, respectively, which resulted in a relative risk of 1.36, under the assumption of a multiplicative model (P=.007). We did not detect association between HapB and ischemic stroke in the Scottish cohort. However, we observed that HapB was overrepresented in male patients. This replication of haplotype association with stroke in a population outside Iceland further supports a role for ALOX5AP in cardiovascular diseases.

Molecular studies on two variant repeat types of the common cetacean DNA satellite of the sperm whale, and the relationship between Physeteridae (sperm whales) and Ziphiidae (beaked whales).

In the sperm whale (Physeter macrocephalus) two different repeat types (A and B) of the common cetacean DNA satellite were identified. The evolution of each group of repeats appears to be independent from that of the other. The sequence similarity between the two groups is less than the similarity between group A and repeats of the satellite in related whale species. The systematic relationship within and between the families Physeteridae (sperm whales) and Ziphiidae (beaked whales) was addressed by both sequence analysis of the satellite and comparisons with the families Delphinidae and Phocoenidae. The mysticete blue whale (Balaenoptera musculus) was used as an outgroup in the comparisons. The molecular phylogeny, when maximum-parsimony analysis and the neighbor-joining method were used, grouped together species of each family. At the family level the ziphiids grouped closet to the families Phocoenidae and Delphinidae. The similarities between the common cetacean satellite of the blue whale and the sperm whale were greater than those between the blue whale and the other odontocetes included, suggesting that the evolution of the satellite is slower in the sperm whale than in the other odontocetes.

Evolution of the common cetacean highly repetitive DNA component and the systematic position of Orcaella brevirostris.

The common cetacean highly repetitive DNA component was analyzed with respect to its evolution and value for establishing phylogenetic relationships. The repeat length of the component, which is tandemly organized, is approximately 1750 bp in all cetaceans except the delphinids, in which the repeat length is approximately 1580 bp. The evolution of the component was studied after sequencing the component in different odontocetes representing the Delphinidae (delphinids), Monodontidae (narwhals), and Ziphiidae (beaked whales). The evolution of this component is very slow, and comparisons showed that sequence divergence among species corresponds closely to their generally accepted phylogenetic relationships and that the component evolves in a concerted manner. The phylogenetic information obtained in this study identified the Irrawaddy dolphin (Orcaella brevirostris) as a delphinid and did not support a close relationship of this species with the Monodontidae.

Mysticete (baleen whale) relationships based upon the sequence of the common cetacean DNA satellite.

The genomes of all extant cetaceans are characterized by the presence of the so-called common cetacean DNA satellite. In the mysticetes (whalebone whales) the repeat length of the satellite is 1,760 bp. In the odontocetes (toothed whales), other than the family Delphinidae, the repeat length is usually approximately 1,740 bp. The Delphinidae are characterized by a repeat length of approximately 1,580 bp. It has been shown in odontocetes that the satellite evolves in concert and that differences between species, with respect to the sequence of the satellite, correspond reasonably well to their evolutionary distances. In the present study the sequence of the satellite was determined in three repeats in each of seven mysticete species, and a consensus for each species established. Parsimony and neighbor-joining analyses based upon sequences of all repeats showed that the primary evolutionary distinction among the mysticetes is between the Balaenidae sensu stricto (i.e., the bowhead whale and the right whale) and all remaining species, including the pygmy right whale, a species that usually has been included in the Balaenidae. The comparisons also showed that the humpback whale and the gray whale were approximately equidistant from the blue whale and the fin whale (genus Balaenoptera). Concerted evolution of the satellite was also demonstrated among the mysticetes, but it appeared to evolve more slowly in the mysticetes than in the odontocetes.

Five novel somatic CDKN2/p16 mutations identified in melanoma, glioma and carcinoma of the pancreas. Mutations in brief no. 170. Online.

Germ-line mutations in the p16/CDKN2 gene are known to predispose to melanoma. This gene belongs to a family of cyclin-dependent kinase inhibitors and blocks G1-S progression. The occurrence of p16/CDKN2 germline mutations in 12 Icelandic melanoma kindreds (kindreds with two or more cases of melanoma or melanoma, pancreas and/or glioma cases) was examined. No germ-line mutation was found, however five mutations not previously discribed in solid tumours were identified, Pro48Leu, Ala57Val, Gly89Asp, Leu117Met, Tyr129Stop.

TP53 abnormalities and genetic instability in breast cancer.

TP53 abnormalities in breast carcinomas and inherited TP53 changes in breast cancer patients and in Li-Fraumeni-like families were looked for. Tumours were screened for mutations in the TP53 gene by means of the PCR-CDGE method followed by PCR and direct sequencing. Allelic loss was determined by polymorphic markers, by comparing normal and tumour DNA. Abnormal protein expression was examined by immunohistochemical staining. TP53 abnormalities in the tumours were examined in relation to genetic instability, clinical data and family history. Genetic instability was studied by detection of oncogene amplification, allelic loss, karyotype analysis and fluorescent in situ hybridization, FISH. Our studies showed that TP53 abnormalities were significantly associated with amplification of the erbB2 oncogene and allelic loss on chromosome 17. Chromosomal abnormalities were also significantly more common in tumours with TP53 abnormalities. Looking at clinical data we found significant association between TP53 abnormalities and poor prognosis.

The mitochondrial genome of the sperm whale and a new molecular reference for estimating eutherian divergence dates.

Extant cetaceans are systematically divided into two suborders: Mysticeti (baleen whales) and Odontoceti (toothed whales). In this study, we have sequenced the complete mitochondrial (mt) genome of an odontocete, the sperm whale (Physeter macrocephalus), and included it in phylogenetic analyses together with the previously sequenced complete mtDNAs of two mysticetes (the fin and blue whales) and a number of other mammals, including five artiodactyls (the hippopotamus, cow, sheep, alpaca, and pig). The most strongly supported cetartiodactyl relationship was: outgroup,((pig, alpaca), ((cow, sheep),(hippopotamus,(sperm whale,(baleen whales))))). As in previous analyses of complete mtDNAs, the sister-group relationship between the hippopotamus and the whales received strong support, making both Artiodactyla and Suiformes (pigs, peccaries, and hippopotamuses) paraphyletic. In addition, the analyses identified a sister-group relationship between Suina (the pig) and Tylopoda (the alpaca), although this relationship was not strongly supported. The paleontological records of both mysticetes and odontocetes extend into the Oligocene, suggesting that the mysticete and odontocete lineages diverged 32-34 million years before present (MYBP). Use of this divergence date and the complete mtDNAs of the sperm whale and the two baleen whales allowed the establishment of a new molecular reference, O/M-33, for dating other eutherian divergences. There was a general consistency between O/M-33 and the two previously established eutherian references, A/C-60 and E/R-50. Cetacean (whale) origin, i.e., the divergence between the hippopotamus and the cetaceans, was dated to approximately 55 MYBP, while basal artiodactyl divergences were dated to >/=65 MYBP. Molecular estimates of Tertiary eutherian divergences were consistent with the fossil record.

TP53 mutation analyses on breast carcinomas: a study of paraffin-embedded archival material.

The aim of this investigation was to examine the possibility of analysing TP53 mutations in archival paraffin-embedded material with the constant denaturant gel electrophoresis (CDGE) method. We extracted DNA from 193 archival primary breast carcinoma samples, diagnosed in 1981-83; further analysis was possible for 186 of these. TP53 mutations in exons 5-8 were detected with CDGE in 30 samples (16.1%) and 17 of these mutations were confirmed by sequencing. Immunohistochemistry demonstrated TP53 nuclear accumulation in 58 tumours (31%). A strong association between the presence of TP53 mutations and TP53 immunostaining was observed (P < 0.001). Our mutation and immunohistochemistry results are in agreement with other findings based on fresh tumour tissue. TP53 abnormalities were significantly related to high S-phase fraction, low oestrogen receptor (ER) content and high tumour grade. Survival of patients with TP53 abnormalities, in the group as a whole, did not differ from patients with normal TP53. Our study did, however, show that patients with abnormal TP53 had a significantly shorter post-recurrence survival (P = 0.005) than patients with normal TP53.