The rs2516839 variation of USF1 gene is associated with 4-year mortality of nonagenarian women: The Vitality 90+ study.

Upstream transcription factor 1 (USF1) regulates the transcription of many genes related to cell and organism survival processes such as stress and immune response, regulation of cellular senesce, and carcinogenesis. In this study, our aim was to investigate the effect of USF1 single nucleotide variations (SNVs) on longevity in the Vitality 90+ study, a population-based study of nonagenarians (90 ±1 years of age) living in the area of Tampere municipality, Finland. Altogether 509 voluntary nonagenarians (115 males, 394 females) were genotyped using the 5'-nuclease assay for rs2774279G > A, rs2516839T > C, and rs2073658C > T SNVs. During the 4 years of follow-up, the total mortality rate was 64.2%. In the study, we found that the frequency of C-allele of rs2516839 among nonsurviving nonagenarians (52.5%) was higher than those who survived (41.2%; P = 0.0006, odds ratio = 1.575, 95% confidence interval [CI]: 1.215-2.041). Furthermore, carriage of this variation and its haplotypes had a significant gender by genotype interaction (P < 0.05) on mortality. Kaplan-Meier log-rank test during 4-years of follow-up showed significantly higher mortality rate in the case of CC genotype carriage than other genotype carriages in nonagenarian women (P < 0.0001). In addition, after adjusting for age in Cox regression analysis, cardiovascular disease, diabetes, infectious disease, dementia, and living place (nursing home or home), CC genotype of rs2516839T > C was found to be associated with shorter life expectancy in nonagenarian women (hazard ratio = 2.27; 95% CI, 1.34-3.85 P = 0.002). In conclusion, rs2516839 variation and related haplotypes of the USF1 gene are strongly related to all-cause mortality in Finnish nonagenarians, especially among women.

Low "quotient" Lp(a) concentration mediates autoimmune activation and independently predicts cardiometabolic risk.

OBJECTIVE: We determined whether U-shaped relationships exist between serum lipoprotein[Lp](a) and cardiometabolic risk. METHODS: In population-based nondiabetic and diabetic middle-aged adults (n=1 428 and 241, respectively) who had been genotyped for the LPA rs10455872 A>G polymorphism, we adjusted the Lp(a) concentration for the effects of genotype and other covariates. Via sex-specific equations we estimated expected Lp(a) concentration in each participant, and the quotient between observed to expected Lp(a) values was determined. Lp(a) and Lp(a) quotient tertiles served to identify non-linear associations with outcomes. RESULTS: Incident 81 cases of diabetes and 128 of coronary heart disease (CHD) developed at 5.1 years' follow-up. Lp(a) concentration was linearly associated with the LPA genotype, gender, total cholesterol, (inversely) fasting insulin, which together with age formed the variables to derive the equations. In logistic regression for incident diabetes, the low Lp(a) quotient tertile was a predictor (RR 1.95 [95%CI 1.10; 3.47]) alike the low Lp(a) tertile, additively to major confounders. Cox regression models comprising sex, age, LPA genotype, smoking status, systolic pressure and serum HDL-cholesterol disclosed that, compared with the mid-tertile, both low (HR 1.77) and high Lp(a) quotient tertiles significantly predicted incident CHD, especially in women. CONCLUSION: Elevated cardiometabolic risk is conferred by apparently reduced circulating Lp(a) assays supporting the notion that "low" serum Lp(a), mediating autoimmune activation, is a major determinant of cardiometabolic risk.

Genetic bases and phenotypes of autosomal recessive Parkinson disease in a Turkish population.

BACKGROUND AND PURPOSE: To evaluate the phenotype and the frequencies of mutations in PRKN, DJ1 and PINK1 genes in patients with Parkinson disease (PD) in Turkey. METHODS: Eighty-six patients from 77 PD families participated in the study. Seventy-four families were originating from Turkey, two families from Greece and one family from Bulgaria. All patients underwent detailed neurological examination. PRKN, PINK1 and DJ1 genes were sequenced, and dosage analysis was performed by multiplex ligation-dependent probe amplification. RESULTS: Sixteen patients with PD were found to carry homozygous (n = 14) or compound heterozygous (n = 2) PRKN mutations. We identified exon rearrangements, three point mutations and one new point mutation in exon 2 (p.K27del). In two families, two new PINK1 point mutations (L31X and P416L) were identified. No pathogenic mutations were found in DJ1 gene. Clinical phenotypes of PRKN patients were comparable to previously described features, but only in four of 13 families, the pedigree structure was clearly consistent with an autosomal recessive (AR) mode of inheritance in comparison with nine families where also different pattern of transmission could have been possible. CONCLUSIONS: Our data suggest that the PRKN gene mutation is the most frequent form of ARPD in Turkey. The proportion of mutations with regard to the age of onset in our population is in the range of those previously described, but our pedigrees are characterized by high rate of consanguinity, which might explain the high proportion of families with homozygous mutations and of patients in more than one generation. Pathogenic DJ1 mutations do not seem to play a major role in Turkey.

Endothelial function and endothelial nitric oxide synthase intron 4a/b polymorphism in primary hyperparathyroidism.

BACKGROUND AND AIM: Patients with symptomatic primary hyperparathyroidism (pHT) have increased cardiovascular morbidity and mortality. Endothelial nitric oxide synthase (eNOS) intron 4a/b polymorphism is associated with coronary artery disease and hypertension in various populations. Our aim is to evaluate endothelial function in patients with pHT during pre-operative hypercalcemic and post-operative normocalcemic periods and to determine whether intron 4a/b polymorphism of eNOS gene influences endothelial function. SUBJECTS AND METHODS: Forty patients with pHT (age 48.48+/-11.64 yr) were examined pre-operatively and reexamined 5.8+/-1.9 months after parathyroidectomy. Forty-three healthy subjects (age 47.13+/-8.14 yr) were served as control group. Endothelial function was determined by flow-mediated dilation of brachial artery (FMD). eNOS4a/b polymorphism was detected by polymerase chain reaction. RESULTS: FMD was significantly lower in patients pre-operatively compared with controls (8.48+/-1.78% vs 19.49+/-2.34%, p<0.001). FMD improved significantly after parathyroidectomy (16.19+/-2.16%, p<0.001 compared with pre-operative measurements), but was still significantly lower than controls (p<0.001). The distribution of eNOS4a/b genotype frequencies was not significantly different between patients and controls. Logistic regression analysis showed that increased serum calcium (>2.47 mmol/l) and PTH concentrations (>7.75 pmol/l) were significant independent predictors of lower FMD (<16.7%). ENOS4a/b polymorphism did not enter in this model. CONCLUSION: Impaired endothelial function in patients with pHT improves after successful parathyroid surgery. No compelling data are evident to suggest that eNOS4a/b polymorphism modifies the endothelial function in patients with pHT.

Association of C-reactive protein (CRP) gene allelic variants with serum CRP levels and hypertension in Turkish adults.

OBJECTIVE: Serum C-reactive protein (CRP) is an independent risk factor for cardiovascular disease and metabolic syndrome (MetS). The aim of this study was to analyze the CRP gene allelic variations in the Turkish adult risk factor (TARF) study and relate them with serum CRP levels as well as MetS and its components. METHODS: We analyzed CRP gene polymorphisms (-286C>T>A [rs3091244], +1444C>T [rs1130864], +1059G>C [rs1800947], and +1846G>A [rs1205]) as well as their haplotypes, in addition to measuring CRP levels (n=1138) and collecting risk factor data from 1987 adults (mean age 54.3+/-11.9 years, 51.3% women) participating in the TARF Study. MetS was defined by using the criteria of the National Cholesterol Education Program modified for pre-diabetes and in men for abdominal obesity. RESULTS: After adjustment for the major cardiovascular risk factors, four CRP SNPs (-286C>T>A, +1059G>C, +1444C>T, and +1846G>A) were significantly associated with serum CRP levels in women (p<0.05), whereas the -286C>T>A and +1444C>T polymorphisms were associated with CRP levels in men (p<0.05). The haplotype analyses revealed four common CRP haplotypes. The haplotype 1 (CGCA) in women and the haplotype 3 (TGTG) in men were associated with serum CRP levels and hypertension (p<0.05). However, no haplotype association was observed for MetS or its components. CONCLUSION: CRP gene allelic variation is associated with serum CRP levels as well as hypertension in Turkish adults.

A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

Downregulation of N1 gene expression inhibits the initial heartbeating and heart development in axolotls.

Recessive mutant gene c in the axolotl results in a failure of affected embryos to develop contracting hearts. This abnormality can be corrected by treating the mutant heart with RNA isolated from normal anterior endoderm or from endoderm conditioned medium. A cDNA library was constructed from the total conditioned medium RNA using a random priming technique in a pcDNAII vector. We have previously identified a clone (designated as N1) from the constructed axolotl cDNA library, which has a unique nucleotide sequence. We have also discovered that the N1 gene product is related to heart development in the Mexican axolotl [Cell Mol. Biol. Res. 41 (1995) 117]. In the present studies, we further investigate the role of N1 on heartbeating and heart development in axolotls. N1 mRNA expression has been determined by using semi-quantitative RT-PCR with specifically designed primers. Normal embryonic hearts (at stages 30-31) have been transfected with anti-sense oligonucleotides against N1 to determine if downregulation of N1 gene expression has any effect on normal heart development. Our results show that cardiac N1 mRNA expression is partially blocked in the hearts transfected with anti-sense nucleotides and the downregulation of N1 gene expression results in a decrease of heartbeating in normal embryos, although the hearts remain alive as indicated by calcium spike movement throughout the hearts. Confocal microscopy data indicate some myofibril disorganization in the hearts transfected with the anti-sense N1 oligonucleotides. Interestingly, we also find that N1 gene expression is significantly decreased in the mutant axolotl hearts. Our results suggest that N1 is a novel gene in Mexican axolotls and it probably plays an important role in myofibrillogenesis and in the initiation of heartbeating during heart development.

Clinical characteristics and genetic screening of an extended family with MEN2A.

MEN-2A is characterized by medullary thyroid carcinoma (MTC) with pheochromocytoma and sometimes parathyroid adenoma. In affected members of the family, the risk of MTC is about 100%. Biochemical screening allows tumors to be detected early but even at this stage treatment is not always curative. Missense mutations in exon 10 and 11 of the RET proto-oncogene are associated with MEN-2A. Early detection of this mutation by DNA analysis allows the identification of the carriers of the gene. We performed genetic screening in 88 members of an extended family with MEN-2A and found 18 members positive for RET mutation (Cys634Gly). Only three of these 18 RET positive cases had a previous diagnosis of medullary cancer and/or pheochromocytoma. Up to now, 12 of the RET positive cases have undergone thyroidectomy. There was extended disease with cervical lymph node metastasis in 6 of them, bilateral medullary microcancer in 3 and c-cell hyperplasia in the remaining 3. Three of the 18 RET positive patients had also pheochromocytoma. Primary hyperparathyroidism was present in only one patient. The mean age of diagnosis of medullary cancer was between 25-50 yr and mean age of death was between 35-95 yr in affected members of the family. The family had many other affected members in other cities in Turkey and in other countries throughout the world from Australia to the Netherlands. So this family is perhaps one of the most extended families with MEN-2A.

Association of a polymorphism of the ecNOS gene with myocardial infarction in a subgroup of Turkish MI patients.

In this study we examined a possible association between a 27 base pair (bp)-repeat polymorphism in intron 4 of the ecNOS gene and myocardial infarction (MI) in a subgroup of the Turkish population. We compared MI and control groups for the frequencies of the ecNOS alleles and their genotypes. The frequency of the ecNOS 4a/a and 4a/b genotypes was found to be significantly higher in the MI group than in the control group. Interestingly, the frequency of the ecNOS 4a/b polymorphism was found to be significantly higher in the selected MI group (patients with no known secondary risk factors) than in the control and non-selected MI group. We found that the patients with MI had the frequency of the a/a genotype 4.3%, of the a/b genotype 26.6% and the b/b genotype 69.1%. The controls, however, showed only 0.6% for a/a, 18.0% for a/b and 81.4% for the b/b genotype (P < 0.001; chi2 = 13.626). In this study, we show that myocardial infarction is associated with one subtype of ecNOS gene polymorphism.

HLA-DQ alleles in patients with celiac disease in Turkey.

The prevalence of the HLA-DQA1 and DQB1 alleles in 55 Turkish children with celiac disease and 50 control subjects was investigated by using an allele-specific DNA-based polymerase chain reaction-sequence-specific primer (PCR-SSP) method. The frequency of the DQA1*0501 and DQB1*02 alleles was higher in celiac patients than in controls. The DQA1B1 (*0501; *02) haplotype was present in 46 (83.6%) patients and only in 12 (24%) controls. The remaining 9 celiac patients which were negative for DQA1B1 (*0501;*02) carried the DQA1B1 (*03;*0302) haplotype. We found an excess homozygosity of the DQB1*02 allele and the DQA1B1 (*0501;*02) haplotype in the patients. No statistically significant correlation was found between the homozygosity of this haplotype or the DQB1*02 allele and an earlier onset of the disease.

Distribution of the M129V polymorphism of the prion protein gene in a Turkish population suggests a high risk for Creutzfeldt-Jakob disease.

A polymorphism (M129V) at codon 129 of the prion protein gene (PRNP) results in either a methionine residue (Met) or a valine residue (Val) and is known to determine susceptibility for the development of sporadic or acquired Creutzfeldt-Jakob disease (CJD). The distributions of M129V genotypes and alleles in various general populations have been reported and there are clear differences between Western Europeans and East Asians. We analysed the coding sequence of the PRNP gene in 100 healthy Turkish subjects to determine whether the distributions of the M129V genotypes and alleles or other PRNP gene variants in the Turkish population differ from those in other normal populations. Three known polymorphisms but no other gene variants were detected in the PRNP coding sequence of the Turkish individuals. Genotype frequencies at codon 129 were 57% Met/Met, 34% Met/Val and 9% Val/Val, with an allele frequency of 0.740:0.260 Met:Val. These distributions are considerably different from those reported for other normal populations residing in Western Europe and East Asia, except in Crete. The higher frequency of 129 Met-homozygotes in Turkey than in Western Europe suggests that the Turkish are at greater risk of developing CJD.

Genomic organization and expression of KCNJ8/Kir6.1, a gene encoding a subunit of an ATP-sensitive potassium channel.

ATP-sensitive K+ (KATP) channels are implicated in the coupling of metabolic energy to membrane potential, thereby regulating many essential cell functions. Here, we demonstrate that a subunit of human KATP channel, KCNJ8/Kir6.1, is expressed preferentially in the human heart. Somatic cell-hybrid mapping and fluorescence in-situ hybridization (FISH) localize human KCNJ8 to the short arm of human chromosome 12, at 12p12. Partial characterization of the human Kir6. 1 gene demonstrates that there is one large intron in the coding region and at least two additional introns in the 5' untranslated region resulting in transcripts that have differential expression in human tissues examined. Our studies provide information on the complexity of the Kir6.1 transcript in the 5' UTR that may be useful for future investigations on the tissue-specific regulation and function of this KATP channel gene.

A specific synthetic RNA promotes cardiac myofibrillogenesis in the Mexican axolotl.

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. However, the defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or RNA from endoderm mesoderm-conditioned medium. We constructed a cDNA library from total conditioned medium RNA in a pcDNAII expression vector. We screened the cDNA library by an organ culture bioassay and isolated a single clone (Cl#4), the synthetic RNA from which corrects the heart defect by promoting myofibrillogenesis. The insert size of the active clone is 166 nt in length with a unique nucleotide sequence. The anti-sense RNA from Cl#4 using SP6 RNA polymerase failed to rescue mutant hearts. The ability of this small RNA to correct the mutant heart defect suggests that the RNA probably does not act as an mRNA. While the precise mechanism of action is not yet known, on the basis of our studies to date it is very clear that the sense strand of Cl#4 RNA has the ability to promote myofibrillogenesis and rescue the mutant hearts both in vitro and in vivo.

Expression of myosin heavy chain transcripts in normal and cardiac mutant Mexican axolotls.

In this study, we have cloned a 1.0 kb myosin heavy chain (MHC) cDNA by screening an axolotl heart cDNA library with the monoclonal antibody MF20 against a light meromyosin (LMM) region of MHC. The nucleotide sequence analysis shows 85-86% homology at the amino acid and 78-81% homology at the nucleic acid level with MHC from other vertebrates. Phylogenetic analyses suggest that axolotl beta-MHC forms a cluster with the myosin II group of vertebrate striated muscles. Within the myosin II cluster, axolotl beta-MHC forms a distinct subclade from avian MHC and is instead closer to mammalian MHC. RT-PCR analyses show that transcripts of beta-MHC are present at stage 2 and the onset of the MHC gene expression is at stage 8-10 (gastrulation). Expression increases with embryonic development and reaches a maximum at stage 20. Beyond stage 35, the heart-beat initiation stage, the expression level of beta-MHC is higher in cardiac muscle than in skeletal muscle. We could not detect significant differences in the levels of expression of MHC transcripts in normal and cardiac lethal mutant (c/c) axolotls (Ambystoma mexicanum).

Cloning, sequencing and expression of an isoform of cardiac C-protein from the Mexican axolotl (Ambystoma mexicanum).

C-protein, a myosin binding protein, is thought to regulate and stabilize thick filaments during assembly of sarcomeric A-bands. Multiple isoforms of C-protein have been characterized in avian and mammalian systems. We now report the isolation and the nucleic acid sequence of a partial C-protein cDNA clone from an axolotl heart cDNA expression library in lambda gt11. The clone was isolated by screening the library with a heterologous monoclonal anti-C-protein antibody (MF1). Sequence comparison revealed that CPROAxocard1 has an average sequence identity of 62-68% at the nuclei acid and 72-78% at the amino acid levels respectively to human and chicken sequences. We could not detect any significant differences at the levels of expression of the cardiac isoform of C-protein (CPROAxocard1) in normal and non-beating heart tissues of the double-recessive cardiac lethal mutant (c/c) axolotl, Ambystoma mexicanum. This is the first report of a C-protein sequence from an amphibian species.

The cardiac mutant gene c in axolotls: cellular, developmental, and molecular studies.

The cardiac mutant axolotl is an interesting model for studying heart development. The mutant gene results in a failure of heart cells to form organized myofibrils and as a consequence the heart fails to beat. Experiments have shown that mutant hearts can be "rescued" (i.e., turned into normally contracting organs) by the addition of RNA purified from conditioned media produced by normal embryonic anterior endoderm-mesoderm cultures. These corrected hearts form myofibrils of normal morphology. New advances in recombinant DNA technology applied to this system should provide significant insights into the regulatory mechanisms of myofibrillogenesis as well as the inductive processes related to the control of gene expression during embryonic heart development. In a broader biological sense, the use of gene c in axolotls is potentially capable of helping to solve major unanswered questions in modern biology related to the genetic regulation of differentiation in vertebrates.

In vivo protein synthesis in developing hearts of normal and cardiac mutant axolotls (Ambystoma mexicanum).

Recessive mutant gene c in axolotls causes a failure of the hearts of affected embryos to function. The mutant hearts (c/c) lack organized sarcomeric myofibrils. The present study was undertaken to determine the overall pattern of in vivo protein synthesis and subsequent accumulation of the newly synthesized proteins for a 24-h period in normal (+/+ or +/c) and cardiac mutant (c/c) axolotl hearts at various stages of development. Additionally, selected cytoskeletal/myofibrillar proteins were analyzed in detail for their synthesis during heart development. For such analyses, the hearts were radiolabeled with 35S-methionine for 24 h and subjected to SDS-PAGE and autoradiography. Quantitative densitometric analyses of the bands show that, even though the overall protein pattern is similar in normal and mutant heart tissues, a general reduction in the synthesis of the proteins in mutant hearts is observed even at the earlier stages of development (stages 35-36 and 37-38). Synthesis and accumulation of most of the proteins is significantly inhibited in mutant hearts at later stages (stages 41-42). Tropomyosin synthesis in mutant hearts is at a level of only 72.6% of that in normal embryonic hearts at stage 35. The synthesis and the accumulation of the tropomyosin in mutant hearts decreases further with increasing age until the protein essentially stops being synthesized by stage 41.

Confocal microscopy of a newly identified protein associated with heart development in the Mexican axolotl.

Recessive mutant gene c for "'cardiac nonfunction" in the mexican axolotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 weeks after fertilization, but eventually die from a lack of circulation. Morphological studies show that mutant hearts lack organized sarcomeric myofibrils. This abnormality can be corrected by co-culturing early mutant hearts with normal anterior endoderm/mesoderm tissues, by culturing them in a medium "conditioned" by this normal tissue, or by RNA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is constructed using this RNA. On the basis of sequence analyses on this cDNA library, it was estimated that 56% of the total RNA present in the conditioned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribosomal RNAs that showed no significant homology with other known sequences in the Genebank was examined further. An RT-PCR analysis showed that this RNA (designated "N1") is expressed in juvenile skeletal muscle, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide antibodies were produced against the most antigenic portion of the polypeptide which was deduced from this RNA. Western blot analyses of adult heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analyzed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investigated to determine its localization in normal isolated hearts at embryonic stages 35, 38, and 41 and on cross-sections through the heart regions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 using immunohistochemical techniques and confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.