Characterization of the Poly-T Variant in the TOMM40 Gene in Diverse Populations

We previously discovered that a polymorphic, deoxythymidine-homopolymer (poly-T, rs10524523) in intron 6 of the TOMM40 gene is associated with age-of-onset of Alzheimer's disease and with cognitive performance in elderly. Three allele groups were defined for rs10524523, hereafter ‘523’, based on the number of ‘T’-residues: ‘Short’ (S, T≤19), ‘Long’ (L, 20≤T≤29) and ‘Very Long’ (VL, T≥30). Homopolymers, particularly long homopolymers like ‘523’, are difficult to genotype because ‘slippage’ occurs during PCR-amplification. We initially genotyped this locus by PCR-amplification followed by Sanger-sequencing. However, we recognized the need to develop a higher-throughput genotyping method that is also accurate and reliable. Here we describe a new ‘523’ genotyping assay that is simple and inexpensive to perform in a standard molecular genetics laboratory. The assay is based on the detection of differences in PCR-fragment length using capillary electrophoresis. We discuss technical problems, solutions, and the steps taken for validation. We employed the novel assay to investigate the ‘523’ allele frequencies in different ethnicities. Whites and Hispanics have similar frequencies of S/L/VL alleles (0.45/0.11/0.44 and 0.43/0.09/0.48, respectively). In African-Americans, the frequency of the L-allele (0.10) is similar to Whites and Hispanics; however, the S-allele is more prevalent (0.65) and the VL-allele is concomitantly less frequent (0.25). The allele frequencies determined using the new methodology are compared to previous reports for Ghanaian, Japanese, Korean and Han Chinese cohorts. Finally, we studied the linkage pattern between TOMM40-‘523’ and APOE alleles. In Whites and Hispanics, consistent with previous reports, the L is primarily linked to ε4, while the majority of the VL and S are linked to ε3. Interestingly, in African-Americans, Ghanaians and Japanese, there is an increased frequency of the ‘523’S-APOEε4 haplotype. These data may be used as references for ‘523’ allele and ‘523’-APOE haplotype frequencies in diverse populations for the design of research studies and clinical trials

Satisfaction survey with DNA cards method to collect genetic samples for pharmacogenetics studies

BACKGROUND: Pharmacogenetic studies are essential in understanding the interindividual variability of drug responses. DNA sample collection for genotyping is a critical step in genetic studies. A method using dried blood samples from finger-puncture, collected on DNA-cards, has been described as an alternative to the usual venepuncture technique. The purpose of this study is to evaluate the implementation of the DNA cards method in a multicentre clinical trial, and to assess the degree of investigators' satisfaction and the acceptance of the patients perceived by the investigators. METHODS: Blood samples were collected on DNA-cards. The quality and quantity of DNA recovered were analyzed. Investigators were questioned regarding their general interest, previous experience, safety issues, preferences and perceived patient satisfaction. RESULTS: 151 patients' blood samples were collected. Genotyping of GST polymorphisms was achieved in all samples (100%). 28 investigators completed the survey. Investigators perceived patient satisfaction as very good (60.7%) or good (39.3%), without reluctance to finger puncture. Investigators preferred this method, which was considered safer and better than the usual methods. All investigators would recommend using it in future genetic studies. CONCLUSION: Within the clinical trial setting, the DNA-cards method was very well accepted by investigators and patients (in perception of investigators), and was preferred to conventional methods due to its ease of use and safety

A TOMM40 variable-length polymorphism predicts the age of late-onset Alzheimer's disease

The ɛ4 allele of the apolipoprotein E (APOE) gene is currently the strongest and most highly replicated genetic factor for risk and age of onset of late-onset Alzheimer's disease (LOAD). Using phylogenetic analysis, we have identified a polymorphic poly-T variant, rs10524523, in the translocase of outer mitochondrial membrane 40 homolog (TOMM40) gene that provides greatly increased precision in the estimation of age of LOAD onset for APOE ɛ3 carriers. In two independent clinical cohorts, longer lengths of rs10524523 are associated with a higher risk for LOAD. For APOE ɛ3/4 patients who developed LOAD after 60 years of age, individuals with long poly-T repeats linked to APOE ɛ3 develop LOAD on an average of 7 years earlier than individuals with shorter poly-T repeats linked to APOE ɛ3 (70.5±1.2 years versus 77.6±2.1 years, P=0.02, n=34). Independent mutation events at rs10524523 that occurred during Caucasian evolution have given rise to multiple categories of poly-T length variants at this locus. On replication, these results will have clinical utility for predictive risk estimates for LOAD and for enabling clinical disease prevention studies. In addition, these results show the effective use of a phylogenetic approach for analysis of haplotypes of polymorphisms, including structural polymorphisms, which contribute to complex diseases

TOM40 Mediates Mitochondrial Dysfunction Induced by α-Synuclein Accumulation in Parkinson's Disease.

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson's disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery -TOM40- might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies

Phenotype Prediction Using Regularized Regression on Genetic Data in the DREAM5 Systems Genetics B Challenge

A major goal of large-scale genomics projects is to enable the use of data from high-throughput experimental methods to predict complex phenotypes such as disease susceptibility. The DREAM5 Systems Genetics B Challenge solicited algorithms to predict soybean plant resistance to the pathogen Phytophthora sojae from training sets including phenotype, genotype, and gene expression data. The challenge test set was divided into three subcategories, one requiring prediction based on only genotype data, another on only gene expression data, and the third on both genotype and gene expression data. Here we present our approach, primarily using regularized regression, which received the best-performer award for subchallenge B2 (gene expression only). We found that despite the availability of 941 genotype markers and 28,395 gene expression features, optimal models determined by cross-validation experiments typically used fewer than ten predictors, underscoring the importance of strong regularization in noisy datasets with far more features than samples. We also present substantial analysis of the training and test setup of the challenge, identifying high variance in performance on the gold standard test sets.National Science Foundation (U.S.). Graduate Research Fellowship ProgramNational Defense Science and Engineering Graduate Fellowshi

Pharmacogenetics of telatinib, a VEGFR-2 and VEGFR-3 tyrosine kinase inhibitor, used in patients with solid tumors

Purpose Telatinib is an orally active small-molecule tyrosine kinase inhibitor of kinase insert domain receptor (KDR; VEGFR-2) and fms-related tyrosine kinase 4 (FLT4; VEGFR-3). This study aims at the identification of relationships between single nucleotide polymorphisms (SNPs) in genes encoding for transporter proteins and pharmacokinetic parameters in order to clarify the significant interpatient variability in drug exposure. In addition, the potential relationship between target receptor polymorphisms and toxicity of telatinib is explored. Methods Blood samples from 33 patients enrolled in a phase I dose-escalation study of telatinib were analyzed. For correlation with dose normalized AUC(0–12), ATP-binding cassette (ABC) B1 (ABCB1), ABCC1, and ABCG2 were the genes selected. For correlation with telatinib toxicity, selected genes were the drug target genes KDR and FLT4. Results No association between dose normalized AUC(0–12) and drug transporter protein polymorphisms was observed. In addition, no association between toxicity and KDR or FLT4 genotype or haplotype was seen. Conclusions Our pharmacogenetic analysis could not reveal a correlation between relevant gene polymorphisms and clinical and pharmacokinetic observations of telatinib

APOE/TOMM40 genetic loci, white matter hyperintensities and cerebral microbleeds

Background: Two markers of cerebral small vessel disease are white matter hyperintensities and cerebral microbleeds, which commonly occur in people with Alzheimer's disease. Aim and/or hypothesis: To test for independent associations between two Alzheimer's disease-susceptibility gene loci – APOE ε and the TOMM40 ‘523’ poly-T repeat – and white matter hyperintensities/cerebral microbleed burden in community-dwelling older adults. Methods: Participants in the Lothian Birth Cohort 1936 underwent genotyping for APOE ε and TOMM40 523, and detailed structural brain magnetic resonance imaging at a mean age of 72·70 years (standard deviation = 0·7; range = 71–74). Results: No significant effects of APOE ε or TOMM40 523 genotypes on white matter hyperintensities or cerebral microbleed burden were found amongst 624 participants. Conclusions: Lack of association between two Alzheimer's disease susceptibility gene loci and markers of cerebral small vessel disease may reflect the relative health of this population compared with those in other studies in the literature

A Genome-Wide Investigation of SNPs and CNVs in Schizophrenia

We report a genome-wide assessment of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) in schizophrenia. We investigated SNPs using 871 patients and 863 controls, following up the top hits in four independent cohorts comprising 1,460 patients and 12,995 controls, all of European origin. We found no genome-wide significant associations, nor could we provide support for any previously reported candidate gene or genome-wide associations. We went on to examine CNVs using a subset of 1,013 cases and 1,084 controls of European ancestry, and a further set of 60 cases and 64 controls of African ancestry. We found that eight cases and zero controls carried deletions greater than 2 Mb, of which two, at 8p22 and 16p13.11-p12.4, are newly reported here. A further evaluation of 1,378 controls identified no deletions greater than 2 Mb, suggesting a high prior probability of disease involvement when such deletions are observed in cases. We also provide further evidence for some smaller, previously reported, schizophrenia-associated CNVs, such as those in NRXN1 and APBA2. We could not provide strong support for the hypothesis that schizophrenia patients have a significantly greater “load” of large (>100 kb), rare CNVs, nor could we find common CNVs that associate with schizophrenia. Finally, we did not provide support for the suggestion that schizophrenia-associated CNVs may preferentially disrupt genes in neurodevelopmental pathways. Collectively, these analyses provide the first integrated study of SNPs and CNVs in schizophrenia and support the emerging view that rare deleterious variants may be more important in schizophrenia predisposition than common polymorphisms. While our analyses do not suggest that implicated CNVs impinge on particular key pathways, we do support the contribution of specific genomic regions in schizophrenia, presumably due to recurrent mutation. On balance, these data suggest that very few schizophrenia patients share identical genomic causation, potentially complicating efforts to personalize treatment regimens

The value of mastectomy flap fixation in reducing fluid drainage and seroma formation in breast cancer patients

<p>Abstract</p> <p>Background</p> <p>Prolonged and excessive drainage of serous fluid and seroma formation constitute the most common complications after mastectomy for breast carcinoma. Seroma formation delays wound healing, increases susceptibility to infection, skin flap necrosis, persistent pain and prolongs convalescence. For this, several techniques have been investigated to improve primary healing and minimize seroma formation.</p> <p>Materials and methods</p> <p>Between June 2009 and July 2010 forty patients with breast carcinoma, scheduled for modified radical mastectomy, were randomly divided into 2 groups, the study group (20) and the control group (20). In the study group; the mastectomy flaps were fixed to the underlying muscles in raws, at various parts of the flap and at the wound edge using fine absorbable sutures. In the control group; the wound was closed in the conventional method at the edges. Closed suction drains were used in both groups. Patients, tumor characteristics and operative related factors were recorded. The amount and color of drained fluid were recorded daily. The drains were removed when the amount become less than 50 cc. The total amount and duration of drained fluid and the formation of seroma were recorded and the results were compared between the two groups.</p> <p>Results</p> <p>In the flap fixation group, the drain was removed in significantly shorter time compared to the control group (p < 0.001). Also, the total amount of fluid drained was significantly lower in the flap fixation group (p < 0.001). The flap fixation group showed a significantly lower frequency of seroma formation compared to the control group, both clinically (p = 0.028) and ultrasonographically (p = 0.047).</p> <p>Conclusions</p> <p>The mastectomy flap fixation technique is a valuable procedure that significantly decreases the incidence of seroma formation, and reduces the duration and amount of drained fluid. However, it should be tried on a much wider scale to prove its validity.</p

Genotypes at the APOE and SCA2 loci do not predict the course of multiple sclerosis in patients of Portuguese origin

Prova tipográfica (In Press)Multiple sclerosis (MS) is a demyelinating disease that affects about one in 500 young Europeans. In order to test the previously proposed influence of the APOE and SCA2 loci on susceptibility to MS, we studied these loci in 243 Portuguese patients and 192 healthy controls and both parents of 92 patients. We did not detect any significant difference when APOE and SCA2 allele frequencies of cases and controls were compared, or when we compared cases with different forms of the disease. Disequilibrium of transmission was tested for both loci in the 92 trios, and we did not observe segregation distortion. To test the influence of the APOE o4 and SCA2 22 CAGs alleles on severity of disease, we compared age at onset and progression rate between groups with and without those alleles. We did not observe an association of the o4 or the 22 CAGs alleles with rate of progression in our total patient population; allele o4 was associated with increased rate of progression of MS in a subset of patients with less than 10 years of the disease. However, globally in the Portuguese population, the APOE and SCA2 genes do not seem to be useful in the clinical context as prognostic markers of this disorder.Fundação para a Ciência e a Tecnologia (FCT) - grant SFRH/BD/9111/2002.Serono Portugal