A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

Carotid artery disease: novel pathophysiological mechanisms identified by gene-expression profiling of peripheral blood.

OBJECT: The pathogenesis of carotid artery stenosis (CAS) as well as the mechanisms underlying the different localisation of the atherosclerotic lesions remains poorly understood. We used microarray technology to identify novel systemic mediators that could contribute to CAS pathogenesis. Moreover, we compared gene-expression profile of CAS with that of patients affected by abdominal aortic aneurysm (AAA), previously published by our group. METHODS AND RESULTS: By global gene-expression profiling in a pool of 10 CAS patients and 10 matched controls, we found 82 genes differentially expressed. Validation study in pools used for profiling and replication study in larger numbers of CAS patients (n = 40) and controls (n = 40) of 14 genes by real-time polymerase chain reaction (RT-PCR) confirmed microarray results. Fourteen out of 82 genes were similarly expressed in AAA patients. Gene ontology analysis identified a statistically significant enrichment in CAS of differentially expressed transcripts involved in immune response and oxygen transport. Whereas alteration of oxygen transport is a common tract of the two localisations, alteration of immune response in CAS and of lipid metabolic process in AAA represents distinctive tracts of the two atherosclerotic diseases. CONCLUSIONS: We describe the systemic gene-expression profile of CAS, which provides an extensive list of potential molecular markers.

Gene expression profiling of peripheral blood in patients with abdominal aortic aneurysm.

OBJECT: Abdominal aortic aneurysm (AAA) pathogenesis remains poorly understood. This study investigated the gene expression profile of peripheral blood from patients with AAA using microarray technology. METHODS AND RESULTS: We determined gene expression profiles in pooled RNA from 10 AAA patients and 10 matched controls with arrays representing 14,000 transcripts. Microarray data for selected genes were confirmed by real-time PCR in two different AAA (n=36) and control (n=36) populations and integrated with biochemical data. We identified 91 genes which were differentially expressed in AAA patients. Gene Ontology analysis indicated a significant alteration of oxygen transport (increased hemoglobin gene expression) and lipid metabolism [including monoglyceride lipase and low density lipoprotein receptor-related protein 5 (LRP5) gene]. LRP5 expression was associated inversely with serum lipoprotein(a) [Lp(a)] concentration. CONCLUSIONS: Increased expression of hemoglobin chain genes as well as of genes involved in erythrocyte mechanical stability were observed in the AAA RNA pools. The association between low levels of LRP5 gene expression and increased levels of Lp(a) in AAA patients suggests a potential role of LRP5 in Lp(a) catabolism. Our data underline the power of microarrays in identifying further molecular perturbations associated with AAA.

Genetic analysis of 56 polymorphisms in 17 genes involved in methionine metabolism in patients with abdominal aortic aneurysm.

BACKGROUND: Previous studies suggested an association between abdominal aortic aneurysm (AAA) and hyperhomocysteinaemia, a complex trait determined by genetic and environmental factors. Our hypothesis was that polymorphisms in genes directly or indirectly involved in methionine metabolism may contribute to AAA susceptibility. METHOD: We studied 56 polymorphisms in MTHFR, MTR, MTRR, CBS, MTHFD1, SLC19A1, NNMT, TCN2, AHCY, BHMT, BHMT2, FOLH1, TYMS, ENOSF1, SHMT1, PON1, PON2 genes according to their demonstrated/putative function, localisation in promoter or regulatory and coding regions and/or heterozygosity values >0.300. Polymorphisms were evaluated by using a primer extension based microarray technology in 423 AAA patients and 423 matched controls. RESULTS: All polymorphisms resulted in Hardy-Weinberg equilibrium in patients and controls. At the multiple logistic regression analysis adjusted for traditional cardiovascular risk factors (sex, age, hypertension, smoking habit, dyslipidaemia, diabetes) and chronic obstructive pulmonary disease (COPD), rs8003379 MTHFD1 (odds ratio (OR) 0.41, 95% confidence interval (CI) 0.26 to 0.65) and rs326118 MTRR (OR 0.47, 95% CI 0.29 to 0.77) polymorphisms resulted in independent susceptibility factor for AAA. CONCLUSIONS: After haplotype reconstruction, logistic regression analyses adjusted for traditional risk factors and COPD showed a significant association among AAA and AHCY, FOLH1, MTHFD1, MTR, NNMT, PON1 and TYMS haplotypes. Our findings offer new insights into the pathogenesis of AAA.

Dual-energy X-ray absorptiometry registers bone mineral in the liver.

OBJECTIVE: To analyse the difference in bone mineral content (BMC) between the left and right trunk generally obtained by dual-energy X-ray absorptiometry (DXA) by creating identical images over the liver region and the contralateral side. PATIENTS AND DESIGN: Fifty-four patients were selected at random from 1,722 subjects examined by DXA because of osteoporosis. Another five patients were selected who had been followed for osteoporosis by repeated DXA two to five times at intervals from 2 to 36 months. One healthy volunteer was followed for one day by means of DXA total body measurements. All protocols were analysed with respect to BMC, fat mass (FM) and lean tissue mass (LTM) of the imaged trunk and liver. RESULTS: BMC of the right trunk exceeded that of the left trunk in 78% of the investigated subjects. The right (liver) image dominated in all 81 investigations calculated from 60 subjects. There were intraindividual short- and longterm variations between repeated DXA examinations. The amounts of FM and LTM were distributed symmetrically between the right and left trunk. CONCLUSIONS: DXA registers BMC in the liver, which explains the general dominance of the right trunk. The absorption over the liver region varies in the same individual in repeated measurements at intervals of hours to months.

Homocysteine status in former top-level male athletes: possible effect of physical activity and physical fitness.

The importance of elevated homocysteine (Hcy) as a risk marker for cardiovascular disease is continuously under debate. Lifestyle factors may increase the total Hcy (tHcy) level of the plasma, but there are no consistent findings relating to Hcy, physical activity, and cardiorespiratory fitness. Cross-sectional measurement from an ongoing follow-up study was performed on 77 former male athletes and 33 sedentary controls (age range 35-62 years). Lifestyle parameters (current physical activity patterns, smoking, etc.), anthropometric and blood pressure data, and data about tHcy, reduced, and oxidized glutathione (GSH, GSSG, respectively) in blood, lipoproteins, and maximal oxygen consumption (VO(2max)) were collected. Our study results showed that the subgroup of physically active ex-athletes (n=52) had a significantly lower tHcy level and glutathione redox ratio (GSSG:GSH) in comparison with the subgroup of sedentary ex-athletes (n=25). tHcy level was inversely related to cardiorespiratory fitness (VO(2max)/kg). Dietary and smoking habits were not significantly associated with the tHcy level in our study group. In conclusion, the research findings indicate that both current physical activity and cardiorespiratory fitness are significantly inversely associated with an elevated homocysteine level in middle-aged former athletes.

Atherogenic inflammatory and oxidative stress markers in relation to overweight values in male former athletes.

OBJECTIVE: To evaluate inflammation- and oxidative stress-related (OxS) background in former athletes in relation to overweight and abdominal obesity status. DESIGN: Cross-sectional data from ongoing follow-up study. SUBJECTS: A total of 60 middle-aged former athletes (46.6+/-7.5 years; 181.1+/-7.2 cm; 88.1 +/- 12.9 kg) and 54 age-matched controls (48.1+/-7.3 years; 181.4 +/- 6.2 cm; 89.7 +/- 14.4 kg). MEASUREMENTS: Anthropometric characteristics, serum lipoproteins (CHOL, HDL-C, LDL-C, TG), oxidized LDL (oxLDL), diene conjugates (DC) and high-sensitive C-reactive protein (hsCRP). Information about the physical activity and other lifestyle variables were collected by the questionnaire. RESULTS: Ex-athletes were characterized by significantly higher physical activity characteristics and lower CHOL and oxLDL in comparison with controls. Correlation analysis among ex-athletes revealed negative associations between all measured overweight data (body mass index, fat percentage, waist to hip circumferences and waist circumference (WC)), and current physical activity. Current physical activity was significantly related to OxS and inflammatory characteristics (oxLDL, DC and hsCRP) among the ex-athletes, but not among the control group. The most expressed positive correlations were found between WC, hsCRP, triglycerides (TG), DC and oxLDL in both study groups. CONCLUSION: Our study results suggest that there exists an independent (adjusted for potential confounders) association between overweight, abdominal obesity, and atherogenic inflammatory and oxidative stress markers in ex-athletes as well as in age-matched controls. Major findings of our study show that WC is the best correlate of hsCRP, oxLDL, DC and TG levels.

Psychological factors related to health, back pain, and dysfunction.

Psychosocial variables may be important determinants of experienced back pain as well as dysfunction. This paper reports on differences on a battery of psychosocial variables between women, from the same work place, off work because of back pain, having only back pain (not off work), and those without back pain. The groups suffering pain had similar levels of pain intensity and frequency and the covariates of age and work load were used in MANCOVA analyses. The results showed significant overall differences on the Coping Strategies Questionnaire, Handicap Index, Duke Health Profile, as well as items concerning family support and the relation of pain to work. Several variables differed between the Healthy group on the one hand and the two groups suffering pain on the other hand. However, coping strategies and perceived health produced significant differences between all three groups in univariate analyses. Unlike other studies the Work APGAR produced no significant results. These data suggest that work status is not directly related to pain intensity, but rather to an interaction between psychosocial factors and the pain experience. Future research should delineate which variables may be used in screening.