Matrix metalloproteinase 9 gene haplotypes affect left ventricular hypertrophy in hypertensive patients

Background: Matrix metalloproteinases (MMPs) are involved in cardiac remodeling and are encoded by genes showing genetic polymorphisms that have functional implications. We examined whether MMP-9 genetic polymorphisms are associated with hypertension and with left ventricular (LV) remodeling in hypertensive patients. Methods: We studied 173 hypertensive patients and 137 age, race and gender matched healthy controls. Heart echocardiography was performed in all patients and the following MMP-9 genetic polymorphisms were analyzed: C-(1562)T (rs3918242). -90 (CA)(14-24) (rs2234681) and Q279R (rs17576). Haplo.stats analysis was used to assess whether MMP-9 haplotypes are associated with hypertension. Linear regression analysis was performed to assess whether MMP-9 haplotypes affect LV mass index (LVMI) and other echocardiography parameters. Results: MMP-9 90 (CA)14-24 ""HH"" genotype (H allele defined by number of CA repeats >= 21) was associated with hypertension (P = 0.0085; OR = 2.321, 95% confidence interval = 1.250 to 4.309). While one MMP-9 haplotype (""C. H, Q"") protects against LVMI and end-diastolic diameter increases due to remodeling (P = 0.0490 and P = 0.0367), another MMP-9 haplotype apparently has detrimental effects over both parameters in hypertensive patients (""T, H. Q"", P = 0.0015 and P = 0.0057. respectively). Conclusion: Genetic polymorphisms in MMP-9 gene may modify the susceptibility of hypertensive patients to LV remodeling. Further studies are necessary to examine whether these polymorphisms affect clinical events in hypertensive patients. (C) 2010 Elsevier B.V. All rights reserved.

Effect of beta-lactoglobulin polymorphism and seasonality on bovine milk composition

The objective was to evaluate the effect of beta-lactoglobulin (beta-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine beta-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of beta-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and beta-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein :true protein ratio were found.

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in Sao Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, STUB, GRA6, c22-8, c29-2, L358, PKI and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits. (C) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Genotyping of Brazilian Erysipelothrix spp. strains by amplified fragment length polymorphism

One hundred and fifty-one Erysipelothrix spp. isolates from diseased and carrier swine from Brazil were identified by PCR, submitted to serotyping and analyzed by amplified fragment length polymorphism with a single enzyme (AFLP). Reference strains from Australia and the United Kingdom were also examined. The 151 strains were classified into 18 different serotypes (1a, 1b, 2a, 2b, 4, 5, 6, 7, 8, 10, 11, 12, 15, 17, 19, 21, 24 and 25), being serotype 2b the most frequent (39.7%). By associating serotyping and PCR results, it was possible to identify 146 strains as E. rhusiopathiae and five strains as E. tonsillarum. Despite the fact that for this genus AFLP did not cluster all isolates according to serotype, origin, disease or isolation data, the execution of the technique was easy and fast, demonstrating high discriminatory power. The results produced by the AFLP analysis of Erysipelothrix spp. could also support its use as a discriminatory tool for E. rhusiopathiae and E. tonsillarum species. (C) 2010 Elsevier B.V. All rights reserved.

Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks

At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the Sao Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to the TCO and CEO vaccines. The two Group II isolates could not be distinguished from one of the CEO vaccines. The nucleotide and amino acid sequence analyses discriminated between the Brazilian and non-Brazilian isolates, as well as between the TCO and CEO vaccines. Sequence analysis of the TK gene enabled classification of the field isolates (Group I) as virulent and non-vaccine. This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain.

Insights from Studies with Oral Cleft Genes Suggest Associations between WNT-pathway Genes and Risk of Oral Cancer

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of the malignant neoplasms that arise in the mucosa of the upper aerodigestive tract. Recent studies of cleft lip/palate have shown the association of genes involved in cancer. WNT pathway genes have been associated with several types of cancer and recently with cleft lip/palate. To investigate if genes associated with cleft lip/palate were also associated with oral cancer, we genotyped 188 individuals with OSCC and 225 control individuals for markers in AXIN2, AXIN1, GSK3 beta, WNT3A, WNT5A, WNT8A, WNT11, WNT3, and WNT9B. Statistical analysis was performed with PLINK 1.06 software to test for differences in allele frequencies of each polymorphism between cases and controls. We found association of SNPs in GSK3B (p = 0.0008) and WNT11 (p = 0.03) with OSCC. We also found overtransmission of GSK3B haplotypes in OSCC cases. Expression analyses showed up-regulation of WNT3A, GSK3B, and AXIN1 and down-regulation of WNT11 in OSCC in comparison with control tissues (P < 0.001). Additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting and oral cancer.

A genetic polymorphism of matrix metalloproteinase 9 (MMP-9) affects the changes in circulating MMP-9 levels induced by highly active antiretroviral therapy in HIV patients

We examined whether two functional polymorphisms (g.-1562C>T and g.-90(CA)14-24) in the matrix metalloproteinase (MMP)-9 gene or MMP-9 haplotypes affect the circulating levels of pro-MMP-9 and pro-MMP-9/TIMP-1 (tissue inhibitor of metalloproteinase-1) ratios in AIDS patients, and modulate alterations in these biomarkers after highly active antiretroviral therapy (HAART). We studied 82 patients commencing HAART. Higher pro-MMP-9 concentrations and pro-MMP-9/TIMP-1 ratios were found in CT/TT patients compared with CC patients. HAART decreased pro-MMP-9 levels and pro-MMP-9/TIMP-1 ratios in CT/TT patients, it did not modify pro-MMP-9 levels and it increased pro-MMP-9/TIMP-1 ratios in CC patients. The g.-90(CA)14-24 polymorphism, however, produced no significant effects. Moreover, we found no significant differences in HAART-induced changes in plasma pro-MMP-9, TIMP-1 and pro-MMP-9/TIMP-1 ratios when different MMP-9 haplotypes were compared. These findings suggest that the g.-1562C>T polymorphism affects pro-MMP-9 levels in patients with AIDS and modulates the alterations in pro-MMP-9 levels caused by HAART, thus possibly affecting the risk of cardiovascular complications. The Pharmacogenomics Journal (2009) 9, 265-273; doi: 10.1038/tpj.2009.13; published online 21 April 2009

Polymorphisms of the renin-angiotensin system in patients with multifocal renal arterial fibromuscular dysplasia

Fibromuscular dysplasia (FMD) is an important cause of renal artery stenosis, particularly in young females. Polymorphisms of the renin-angiotensin (RA) system have been implicated in the pathogenesis of hypertension and atherosclerotic vascular disease, and may play a role in the development of FMD. Examination of polymorphisms by PCR for angiotensin-converting enzyme (ACE) I/D, angiotensin II type 1 receptor (AT(1)R) A1166C and angiotensinogen (AGT) M235T and T174M was undertaken in 43 patients with typical multifocal renal arterial FMD (MF-FMD) and in 89 controls. The age of NIF-FMD patients at the time of diagnosis of hypertension did not differ (38.6 + 11.1 years vs 35.5 +/- 10.3 years, P = 0.12) from controls and the proportion (95% vs 86%, P = 0.14) of females was similar. Allele frequencies did not differ significantly between groups, except that MF-FMD patients had a significantly higher frequency of the ACE I allele than control subjects (0.62 vs 0.47, P = 0.026). Since the ACE I allele is associated with lower circulating ACE levels and possibly lower tissue levels of angiotensin II (Ang II), and since Ang II modulates vascular smooth muscle cell growth and synthetic activity, the I allele might predispose to defective remodelling of the arterial media, and thus to the development of MF-FMD. This contrasts with atherosclerotic renal artery stenosis, coronary stent restenosis and carotid intimal thickening, which are diseases affecting the arterial intima, and which are associated with increased frequency of the D allele.

Identification of an apolipoprotein(e) variant associated with type III hyperlipoproteinaemia in an indigenous Australian

As a result of testing for lipid and apolipoprotein(e) (apo E) phenotype status of an indigenous Australian community, an apo E variant associated with type III hyperlipoproteinaemia has been identified. Apo E phenotype was determined by analysis of VLDL by isoelectric focusing, and genotype on DNA amplified by polymerase chain reaction, using two different restriction enzyme isotyping assays. Phenotypes and genotypes were discordant in samples from two subjects and an abnormal-sized restriction fragment was also observed in their genotyping gel patterns. DNA sequencing studies revealed this was due to a single nucleotide deletion. 3817delC, at amino acid 136 on apo E. This resulted in a new reading frame and the premature termination of the apo E protein due to a stop codon (TGA) at nucleotide 4105. The variant apo E null allele showed a recessive mode of inheritance and, in combination with the E2 allele, resulted in the type III hyperlipoproteinaemic phenotype but when inherited with the E4 allele had no marked effect on plasma lipids.

Comparison of competitively primed and conventional allele-specific nucleic acid amplification

A simulation of competitively primed allele-specific DNA amplification has been constructed and its behavior examined, This has shown that when the ratio of the amount of homoduplex misprime product to the total amount of amplimer is low, it increases by approximately one-fourth of the mispriming frequency with each doubling of the total amount of amplimer, When the ratio is high acid reverse mispriming becomes significant, it asymptotes toward a value

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 90 involved in the development of melanoma, Although LOH at 90 has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 90. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations hy single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele, Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748, the markers closest to CDKN2A. Of the remaining 11 tumors with LOH, 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion, This report supports the conclusions of previous studies that at least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

The microsomal epoxide hydrolase Tyr113His polymorphism: Association with risk of ovarian cancer

Functional significance has been demonstrated in vitro for the exon 3 T-->C Tyr113His amino acid substitution polymorphism of the microsomal epoxide hydrolase (EPHX) gene. The higher activity or fast TT genotype was previously reported to be associated with an increased risk of ovarian cancer, and this association may reflect enhanced activation of endogenous or exogenous substrates to more reactive and mutagenic derivatives. Components of cigarette smoke are examples of exogenous substrates subject to such bioactivation, and smoking exposure may thus modify the risk associated with the EPHX polymorphism. We examined 545 cases of epithelial ovarian cancer and 287 unaffected controls for this EPHXT-C genetic variant to investigate whether, in the Australian population, the TT genotype was associated with (i) specific ovarian tumor characteristics; (ii) risk of ovarian cancer, overall or for specific subgroups; and (iii) risk of ovarian cancer in smokers specifically. Genotyping was carried out using the Perkin-Elmer ABI Prism 7700 Sequence Detection System for fluorogenic polymerase chain reaction allelic discrimination. Stratification of the ovarian cancer cases according to tumor behavior (low malignant potential or invasive), grade, stage, and p53 immunohistochemical status failed to show any heterogeneity with respect to the genotype defined by the EPHX polymorphism. There was a suggestion of heterogeneity with respect to histologic subtype (P= 0.03), largely due to a decreased frequency of the TT genotype in endometrioid tumors. EPHX genotype distribution did not differ significantly between unaffected controls and ovarian cancer cases (overall, low malignant potential, or invasive) either overall or after stratification by smoking status. However, the TT genotype was associated with a decreased risk of invasive ovarian cancer of the endometrioid subtype specifically (age-adjusted odds ratio = 0.38, 95% confidence interval=0.17-0.87). The results suggest that the proposed EPHX-mediated bioactivation of components of cigarette smoke to mutagenic forms is unlikely to be involved in the etiology of ovarian cancer in general but that a greater rate of EPHX-mediated detoxification may decrease the risk of endometrioid ovarian cancer. (C) 2001 Wiley-Liss, Inc.

A longitudinal study of interleukin-1 gene polymorphisms and periodontal disease in a general adult population

Background: Cross-sectional studies have demonstrated that a specific polymorphism (allele 2 of both IL-1A +4845 and IL-1B +3954) in the IL-1 gene cluster has been associated with an increased susceptibility to severe periodontal disease and to an increased bleeding tendency during periodontal maintenance. The aim of the present study was to investigate the relationship between IL-1 genotype and periodontitis in a prospective longitudinal study in an adult population of essentially European heritage. Methods: From an ongoing study of the Oral Care Research Programme of The University of Queensland, 295 subjects consented to genotyping for IL-1 allele 2 polymorphisms. Probing depths and relative attachment levels were recorded at baseline, 6, 12, 24, 36, 48 and 60 months using the Florida probe. Periodontitis progression at a given site was defined as attachment loss greater than or equal to2 mm at any observation period during the 5 years of the study and the extent of disease progression determined by the number of sites showing attachment loss. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia were detected using ELISA. Results: 38.9% of the subjects were positive for the composite IL-1 genotype. A relationship between the IL-1 positive genotype and increased mean probing pocket depth in non-smokers greater than 50 years of age was found. Further, IL-1 genotype positive smokers and genotype positive subjects with P. gingivalis in their plaque had an increase in the number of probing depths greater than or equal to3.5 mm, There was a consistent trend for IL-1 genotype positive subjects to experience attachment loss when compared with IL-1 genotype negative subjects. Conclusion: The results of this study have shown an interaction of the IL-1 positive genotype with age, smoking and P. gingivalis which suggests that IL-1 genotype is a contributory but non-essential risk factor for periodontal disease progression in this population.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio=3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information gained through observation of pigmentation phenotype.