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PubMed2003

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay.

BACKGROUND: Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. RESULTS: A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. CONCLUSIONS: Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

Chromosomes, Human, Pair 6↗

PubMed2007

TAMGeS: a Three-array Method for Genotyping of SNPs by a dual-colour approach.

BACKGROUND: Many of the most effective high-throughput protocols for SNP genotyping employ microarrays. Genotypes are assessed by comparing the signal intensities that derive from the hybridization of different allele-specific probes labelled either by using four fluorescent dyes, one for each base, or by using only two dyes and investigating the polymorphic alleles two by two on separate arrays. The employment of only two dyes makes it possible to use a dual-laser scanner, which has the advantage of being present in every microarray laboratory. However, this protocol may present some drawbacks. To infer all the six possible genotypes it is necessary to compare signals from two arrays, but this comparison not always is successful. A number of systematic errors in the experimental protocol, in fact, may differently affect signal intensities on separate arrays. Here we present TAMGeS (Three-Array Method for Genotyping of SNPs), an exhaustive method for SNP genotyping through SBE (Single Base Extension) and dual-colour microarrays, which makes the comparison of signals on distinct arrays reliable by using a third array and a data handling method for signal normalization based on bilinear regression theory. RESULTS: We tested the effectiveness of the proposed method by evaluating the results obtained from the direct comparison of the two arrays or by applying TAMGeS, both on experimental and synthetic data. With synthetic data, TAMGeS reduced the frequency of errors by an order of magnitude, when the incidence of systematic errors was not negligible. With the experimental data, produced by genotyping 25 SNPs in 437 subjects, TAMGeS reduced the percentage of missing genotypes from 54% (Two-Array Method) to 14.5%. Allelic and genotypic call rates were 99.3% and 99.5%, respectively. The normalization procedure takes into account also systematic errors, which can be generated by a time-delayed assay, thus making the protocol more flexible. CONCLUSION: TAMGeS represents an innovative method, which proved to be very effective in producing reliable SNP genotyping data by dual-colour microarrays. The requirement of a third array is well balanced by the strong enhancement in data quality and by the greater flexibility of the experimental protocol.

Base Sequence↗

PubMed2006

Human papillomavirus infections in women seeking cervical Papanicolaou cytology of Durango, Mexico: prevalence and genotypes.

BACKGROUND: HPV infection in women from developing countries is an important public health problem. Therefore, we sought to determine the prevalences of HPV infection and HPV genotypes in a female population of Durango City, Mexico. Also to determine whether any socio-demographic characteristic from the women associated with HPV infection exists. METHODS: Four hundred and ninety eight women seeking cervical Papanicolaou examination in three public Health Centers were examined for HPV infection. All women were tested for HPV DNA PCR by using HPV universal primers. In addition, all positive HPV DNA PCR samples were further analyzed for genotyping of HPV genotype 16, 18 and 33. Socio-demographic characteristics from each participant were also obtained. RESULTS: Twenty-four out of four hundred and ninety-eight (4.8%) women were found infected by HPV. HPV genotype 16 was found in 18 out of the 24 (75%) infected women. Two of them were also coinfected by HPV genotype 18 (8.3%). In the rest 6 PCR positive women, genotyping for HPV genotypes 16, 18 and 33 were negative. CONCLUSION: The prevalence of HPV in women of Durango City is low; however, most infected women have high risk HPV genotype. The women who were studied showed low frequency of risk factors for HPV infection and this may explain the low prevalence of HPV infection. The high frequency of high risk HPV genotypes observed might explain the high rate of mortality for cervical cancer in our region.

Adult↗

PubMed2006

T null and M null genotypes of the glutathione S-transferase gene are risk factor for CAD independent of smoking.

BACKGROUND: The association of the deletion in GSTT1 and GSTM1 genes with coronary artery disease (CAD) among smokers is controversial. In addition, no such investigation has previously been conducted among Arabs. METHODS: We genotyped 1054 CAD patients and 762 controls for GSTT1 and GSTM1 deletion by multiplex polymerase chain reaction. Both CAD and controls were Saudi Arabs. RESULTS: In the control group (n = 762), 82.3% had the T wild M wildgenotype, 9% had the Twild M null, 2.4% had the Tnull M wild and 6.3% had the Tnull M null genotype. Among the CAD group (n = 1054), 29.5% had the Twild M wild genotype, 26.6% (p < .001) had the Twild M null, 8.3% (p < .001) had the Tnull M wild and 35.6% (p < .001) had the Tnull M null genotype, indicating a significant association of the Twild M null, Tnull M wild and Tnull M null genotypes with CAD. Univariate analysis also showed that smoking, age, hypercholesterolemia and hypertriglyceridemia, diabetes mellitus, family history of CAD, hypertension and obesity are all associated with CAD, whereas gender and myocardial infarction are not. Binary logistic regression for smoking and genotypes indicated that only M null and Tnullare interacting with smoking. However, further subgroup analysis stratifying the data by smoking status suggested that genotype-smoking interactions have no effect on the development of CAD. CONCLUSION: GSTT1 and GSTM1 null-genotypes are risk factor for CAD independent of genotype-smoking interaction.

Arabs↗

PubMed2006

High prevalence of ACE DD genotype among north Indian end stage renal disease patients.

BACKGROUND: The Renin-Angiotensin system (RAS) is a key regulator of both blood pressure and kidney functions and their interaction. In such a situation, genetic variability in the genes of different components of RAS is likely to contribute for its heterogeneous association in the renal disease patients. Angiotensin converting enzyme-1 (ACE-1) is an important component of RAS which determines the vasoactive peptide Angiotensin-II. METHODS: In the present study, we have investigated 127 ESRD patients and 150 normal healthy controls from north India to deduce the association between ACE gene polymorphism and ESRD. The inclusion criteria for patients included a constantly elevated serum creatinine level above normal range (ranging from 3.4 to 15.8) and further the patients were recommended for renal transplantation. A total of 150 normal healthy controls were also genotyped for ACE I/D polymorphism. The criterion of defining control sample as normal was totally based on the absence of any kidney disease determined from the serum creatinin level. Genotyping of ACE I/D were assayed by polymerase chain reaction (PCR) based DNA amplification using specific flanking primers Based on the method described elsewhere. RESULTS: The difference of DD and II genotypes was found highly significant among the two groups (p = 0.025; OR = 3.524; 95% CI = 1.54-8.07). The combined genotype DD v/s ID+II comparison validated that DD genotype is a high risk genotype for ESRD (p = 0.001; OR = 5.74; 95% CI limit = 3.4-8.5). However, no correlation was obtained for different biochemical parameters of lipid profile and renal function among DD and non DD genotype. Interestingly, approximately 87% of the DD ESRD patients were found hypertensive in comparison to the 65% patients of non DD genotype CONCLUSION: Based on these observations we conclude that ACE DD genotype implicate a strong possible role in the hypertensive state and in renal damage among north Indians. The study will help in predetermining the timing, type and doses of anti-hypertensive therapy for ESRD patients.

Adult↗

PubMed2004

Modulating effects of mannose binding lectin genotype on arterial stiffness in children after Kawasaki disease.

Systemic arterial stiffness is increased in patients after Kawasaki disease (KD). Recently, associations between mannose-binding lectin (MBL) gene mutation and coronary complications in infants with KD and atherosclerosis in adults have been reported. We tested the hypothesis that MBL genotype modulates arterial stiffness in children after KD. Seventy-one KD patients (42 with and 29 without coronary aneurysms), aged 9.5 +/- 3.7 y, and 41 age-matched controls were studied. We determined and compared their blood pressure, brachioradial arterial stiffness as determined by pulse wave velocity (PWV), fasting total cholesterol, serum MBL level, and MBL genotype. Additionally, the modulating effects of different MBL expression genotypes [high level (HL) versus intermediate or low level (IL/LL)] on arterial stiffness in different groups were assessed. The MBL genotype distributions did not differ between patients and controls (p = 0.41) or between patients with and without coronary aneurysms (p = 0.42). Patients with IL/LL expression genotypes had significantly faster PWV than those with HL expression genotypes (7.93 +/- 1.38 m/s versus 6.67 +/- 2.28 m/s, p = 0.027). This genotype-modulating effect is more pronounced in patients without (HL 8.86 +/- 0.77 m/s versus IL/LL 6.48 +/- 2.32 m/s, p = 0.02) than those with (HL 7.50 +/- 1.41 m/s versus IL/LL 6.80 +/- 2.28 m/s, p = 0.32) coronary aneurysms. Multiple linear regression analysis identified age (beta = 0.26, p = 0.012), being a Kawasaki patient (beta = 0.22, p = 0.015), and MBL IL/LL genotype subgroup (beta = 0.20, p = 0.03) as significant determinants of arterial stiffness in the entire cohort. In conclusion, MBL genotype modulates arterial stiffness, an important cardiovascular risk factor, in children after KD.

Adolescent↗

PubMed2000

CYP2D6 genotyping as an alternative to phenotyping for determination of metabolic status in a clinical trial setting.

The emerging application of pharmacogenomics in the clinical trial setting requires careful comparison with more traditional phenotyping methodologies, particularly in the drug metabolism area where phenotyping is used extensively. The research objectives of this study were 1) to assess the utility of cytochrome P450 2D6 (CYP2D6) genotyping as an alternative to traditional phenotyping as a predictor of poor metabolizer status; 2) to identify issues for consideration when implementing CYP2D6 genotyping in clinical trials; and 3) to outline the advantages and disadvantages of CYP2D6 genotyping compared with phenotyping. DNA samples obtained from 558 previously phenotyped individuals were blindly genotyped at the CYP2D6 locus, and the genotype-phenotype correlation was then determined. The CYP2D6 genotyping methodology successfully predicted all but 1 of the 46 poor metabolizer subjects, and it was determined that this 1 individual had a novel (presumably inactive) mutation within the coding region. In addition, we identified 2 subjects with CYP2D6 genotypes indicative of poor metabolizers who had extensive metabolizer phenotypes as determined by dextromethorphan/dextrorphan ratios. This finding suggests that traditional phenotyping methods do not always offer 100% specificity. Our results suggest that CYP2D6 genotyping is a valid alternative to traditional phenotyping in a clinical trial setting, and in some cases may be better. We also discuss some of the issues and considerations related to the use of genotyping in clinical trials and medical practice.

Cytochrome P-450 CYP2D6↗

PubMed2004

Relationship of estrogen receptor genotypes to bone mineral density and to rates of bone loss in men.

Estrogen is now known to play an important role in bone metabolism in men. Thus, we examined possible relationships between polymorphisms of the estrogen receptor (ER)-alpha and -beta genes, bone mineral density (BMD), and rates of bone loss in an age-stratified random sample of 283 Rochester, Minnesota, men aged 22-90 yr. DNA was analyzed for the XbaI and PvuII ER-alpha and AluI ER-beta polymorphisms. The X/P and x/p alleles of the ER-alpha gene were in strong linkage disequilibrium. BMD at multiple sites did not differ as a function of either the ER-alpha or -beta genotype. However, the ER-alpha (but not ER-beta) genotypes did modulate the previously described relationships between BMD or rates of bone loss and bioavailable estradiol (E(2)) levels in these men. At the femoral neck, BMD was associated with bioavailable E(2) levels in men with the XX (R = 0.66) or PP (R = 0.51) genotypes (P < 0.001 for both) but not in men with the xx (R = 0.15; P = 0.188) or pp (R = 0.12; P = 0.356) genotypes. The interactions between bioavailable E(2) levels and the XbaI and PvuII genotypes were significant at the P < 0.001 and P < 0.009 levels, respectively. Moreover, rates of bone loss at the midradius in men aged 60-90 yr were modestly correlated with serum bioavailable E(2) levels in subjects with the X (R = 0.47) or P (R = 0.42) alleles (P < 0.001 for both) but not in those with the xx (R = 0.15; P = 0.430) or pp (R = 0.21; P = 0.372) genotypes. The overall effect of genotype on midradius rate of bone loss was clearly significant for the XbaI polymorphism (P = 0.009) when bioavailable E(2) levels were low (<40 pmol/liter) but not for the PvuII polymorphism. These data thus indicate that the ER-alpha genotype may modulate the relationship between BMD or rates of bone loss and estrogen levels in men and that bone mass in men with the X or P alleles may be more susceptible to the consequences of estrogen deficiency (and conversely, benefit most from estrogen sufficiency) than in men with the xx or pp genotypes.

Adult↗

PubMed2004

Ala12Ala genotype of the peroxisome proliferator-activated receptor gamma2 protects against atherosclerosis.

A mutation in the peroxisome proliferator-activated receptor gamma2 (PPARgamma2) gene with a cytosine to guanine substitution results in an exchange of proline (Pro) with alanine (Ala) in exon B (codon 12) of this gene. This polymorphism has been associated with high insulin sensitivity and low body weight, but no data have been published to date about its effect on early atherosclerosis. We investigated the relationship of the Pro(12)Ala polymorphism to early atherosclerosis, measured by the intima-media thickness (IMT). A total of 622 subjects were included, aged 40-70 yr, who were participants of the RIAD (Risk factors in Impaired glucose tolerance for Atherosclerosis and Diabetes) study and were at risk of developing type 2 diabetes. Altogether, 449 of the subjects had the common genotype (Pro(12)Pro), 162 had the Pro(12)Ala genotype, and 11 the Ala(12)Ala genotype. IMT was significantly decreased in subjects with the Ala(12)Ala genotype compared with subjects with the other two genotypes. Body mass index, free fatty acid levels, and leukocyte count were lower in subjects with the Ala(12)Ala genotype compared with subjects with the Pro(12)Pro or Pro(12)Ala genotypes. In multivariate analysis, the Ala(12)Ala genotype was a significant independent determinant of IMT. Furthermore, we demonstrated specific expression of the PPARgamma2 gene in human atherosclerotic lesions as well as in cultured primary macrophages and foam cells. In conclusion, our data suggest that the Ala(12)Ala genotype of the PPARgamma2 gene may protect from early atherosclerosis in subjects at risk for diabetes.

Adult↗

PubMed1995

Lack of a high prevalence of the BB vitamin D receptor genotype in severely osteoporotic women.

Studies of twins strongly suggest that more than 50% of the peak spinal bone density is determined by genetics. It was reported recently that this genetic effect is primarily determined by vitamin D receptor (VDR) alleles; specifically, a VDR genotype termed BB has been highly associated with low peak bone density. Homozygotes for the second VDR allele, bb, are associated with high peak bone density. If peak bone density is an important determinant of osteoporosis and if the VDR genotype is an important determinant of peak bone density, then patients with severe osteoporosis should have a high prevalence of the BB VDR genotype compared with that of control subjects. To test this hypothesis, we used Southern blot analysis to determine the VDR genotype of 41 Caucasian patients (72 +/- 14 yr) with severe osteoporosis (27 women with spinal bone densities below 50 mg/cm3 as determined by quantitative computed tomography; 14 women with spinal bone densities below 0.75 g/cm2 as determined by dual energy x-ray absorptiometry) and 23 Caucasian control subjects (68 +/- 7 yr) without osteoporosis (quantitative computed tomography values at or above the fracture threshold of 100 mg/cm3). Only 6 of the 41 individuals in the group with severe osteoporosis had the BB genotype, whereas 16 had the bb genotype. In the control group comprising 23 individuals, 7 had the BB genotype and only 6 had the bb genotype. We conclude that the BB VDR genotype is not a good predictor of risk for developing severe osteoporosis in our population.

Age Factors↗

PubMed2000

Association of estrogen receptor-alpha genotypes with body mass index in normal healthy postmenopausal Caucasian women.

Several lines of evidence suggest the importance of the estrogen receptor (ER) in determining body mass index (BMI). Our purpose was to investigate whether genetic polymorphisms at the restriction enzyme PvuII site of the ER-alpha gene locus are associated with BMI variation. Data on BMI, age, and ER-alpha genotypes were obtained from 108 healthy midwestern U.S. postmenopausal Caucasian women. The study subjects were unrelated and aged 65 yr and over (mean age +/- SD, 73.4 +/- 5.1 yr), with an average BMI of 25.25 (SD, 4.04). The ER-alpha genotypes were obtained by PCR followed by restriction enzyme PvuII digestion. We found that in our study subjects the ER-alpha genotypes are significantly associated with BMI (by ANOVA, P = 0.04), explaining about 6.2% of the BMI variation in our study sample. The allelic effects of this locus on BMI are approximately additive. In our sample, individuals of the PP and Pp genotypes have, respectively, 11.4% and 4.8% higher BMI than individuals of the pp genotype. There is a significant ER-alpha genotype by age interaction, so that in our sample PP individuals tend to gain weight with age, whereas Pp and pp individuals tend to lose weight with age. Therefore, the ER-alpha polymorphisms are associated with BMI variation in healthy postmenopausal Caucasian women aged 65 yr and over. Our result is consistent with some recent findings suggesting the potential effects of the ER on BMI. The importance of the ER-alpha genotypes in other populations and other age groups needs to be demonstrated. Although the results of the ER-alpha genotype by age interaction are obtained here from cross-sectional data, direct confirmation may come from longitudinal studies in which individuals are measured multiple times over several years. The importance of the ER-alpha genotypes on BMI should be confirmed by further studies using methods robust to the potential problem of population substructuring that may confound the conclusions of population association studies.

Aged↗

PubMed2005

Evaluating whole genome amplification via multiply-primed rolling circle amplification for SNP genotyping of samples with low DNA yield.

The amount of available DNA is often a limiting factor in pursuing genetic analyses of large-scale population cohorts. An association between higher DNA yield from blood and several phenotypes associated with inflammatory states has recently been demonstrated, suggesting that exclusion of samples with very low DNA yield may lead to biased results in statistical analyses. Whole genome amplification (WGA) could present a solution to the DNA concentration-dependent sample selection. The aim was to thoroughly assess WGA for samples with low DNA yield, using the multiply-primed rolling circle amplification method. Fifty-nine samples were selected with the lowest DNA yield (less than 7.5 microg) among 799 samples obtained for one population cohort. The genotypes obtained from two replicate WGA samples and the original genomic DNA were compared by typing 24 single nucleotide polymorphisms (SNPs). Multiple genotype discrepancies were identified for 13 of the 59 samples. The largest portion of discrepancies was due to allele dropout in heterozygous genotypes in WGA samples. Pooling the WGA DNA replicates prior to genotyping markedly improved genotyping reproducibility for the samples, with only 7 discrepancies identified in 4 samples. The nature of discrepancies was mostly homozygote genotypes in the genomic DNA and heterozygote genotypes in the WGA sample, suggesting possible allele dropout in the genomic DNA sample due to very low amounts of DNA template. Thus, WGA is applicable for low DNA yield samples, especially if using pooled WGA samples. A higher rate of genotyping errors requires that increased attention be paid to genotyping quality control, and caution when interpreting results.

DNA↗

PubMed1998

Infant TGF-alpha genotype, orofacial clefts, and maternal periconceptional multivitamin use.

OBJECTIVE: We previously demonstrated a strong association between periconceptional maternal cigarette smoking, infant transforming growth factor-alpha (TGFa) genotype, and risk of orofacial clefts. Because serum folate may be decreased by cigarette smoking and because maternal periconceptional use of multivitamins containing folic acid has been associated with a reduced risk of clefting, we explored whether a potential relation existed between infant TGFa genotype, maternal multivitamin use, and risk of orofacial cleft phenotypes. DESIGN: Data were derived from a population-based case-control study of fetuses and live-born infants among a cohort of 1987 to 1989 California births (n = 548,844). Information concerning periconceptional multivitamin use was obtained via telephone interviews with mothers of 731 (84.7% of eligible) orofacial cleft case infants, and of 734 (78.2%) nonmalformed control infants. DNA was obtained from newborn screening bloodspots and genotyped for the Taql polymorphism of TGFa. Among infants of interviewed mothers, genotypes were available for 571 (78.1%) case infants and 640 (87.2%) control infants. SETTING: The study encompassed all hospitals in selected California counties. MAIN OUTCOME MEASURE: The main outcome measures were the risks of specific cleft phenotypes among infants with uncommon TGFa genotypes and whose mothers did not use multivitamins periconceptionally. RESULTS: Compared with infants homozygous for the common TGFa genotype and whose mothers used multivitamins, increased clefting risks were observed for infants with the A2 genotype (homozygous or heterozygous) and whose mothers did not use multivitamins. Risk estimates were 3.0 (1.4-6.6 [95% confidence interval]) for isolated cleft lip with or without cleft palate (CLP), 2.4 (0.69-11.6) for multiple CLP, 2.6 (0.97-7.7) for isolated cleft palate (CP), 4.2 (1.3-16.2) for multiple CP, and 8.1 (2.6-27.7) for "known-syndrome" clefts. Clefting risks for infants with the A2 genotype and whose mothers used multivitamins were substantially smaller, as were the risks for infants with the A1 genotype whose mothers did not use multivitamins. CONCLUSION: These data provide preliminary evidence for a gene-nutrient interaction in risk of clefting.

Adult↗

PubMed2003

The LightTyper: high-throughput genotyping using fluorescent melting curve analysis.

Instrumentation, chemistry, and software for high-throughput genotyping using fluorescent melting curves are described. The LightTyper system provides post-amplification genotyping within 10 min using samples in 96- or 384-well microplate formats. The system is homogenous because all reagents are added at the beginning of the reaction and there is no sample manipulation between amplification and genotyping. High-resolution melting curves are achieved by slow and steady heating. As samples are heated, panels of blue light-emitting diodes excite the probes, and fluorescence emission is acquired with a cooled charge-coupled device camera. A variety of probe chemistries are compatible for genotyping on the LightTyper, including dsDNA dyes, single-labeled probes, and fluorescence resonance energy transfer systems. Genotyping is performed automatically, and each sample is given a score reflecting the similarity of the genotype to the standards provided. Standard genotypes can be selected from within the run or imported from other files. Samples and genotypes can be grouped to allow multiple-allele detection on one or many samples. The utility of the LightTyper is illustrated by genotyping samples for the Factor V Leiden mutation and for mutations in the CFTR gene.

Base Sequence↗

PubMed2005

The role of genotyping in diagnosing cardiac channelopathies : progress to date.

The role of genotyping for diagnosis of the cardiac ion channelopathies is a work in progress. No formal guidelines or other publications discussing current recommendations for genotyping exist, particularly for clinical/commercial genotyping. Further, the field is changing rapidly, opinions vary and, additionally, circumstances inside the US are different from outside. The following considerations are a current summary based on a review of the literature, discussions with experts in the field, and our own opinions and also include a brief discussion about genotyping for therapeutic decision making. Research-based genotyping is very important for continued understanding of the details of pathophysiology and the complex regulatory processes in these diseases. Clinical/commercial genotyping for diagnosis is important for identifying patients with reduced penetrance of the phenotype since effective therapies to prevent sudden death exist. Clinical genotyping for therapeutic advantage has limited application at present but will become much more important if and when genotype-/mutation-type specific therapies are shown to be effective. The recommendations will progressively change as new research findings and new genotyping technologies appear.

Genotype↗

PubMed2006

Development and clinical evaluation of oligonucleotide microarray for HLA-AB genotyping.

BACKGROUND: The antigens encoded by human leukocyte antigen (HLA) genes are primary antigens in immunological response of transplantation, and genotypes of HLA-A, B and DRB1 must be determined on donors and recipients before the transplantation is carried out. In this study, oligonucleotide microarray for HLA-AB genotyping was prepared and evaluated. METHODS: Oligonucleotide probes were designed based on the sequence of the different genotypes of HLA-A and B and were fixed on silylated glass slides to form a microarray. Fluorescence-labeled target DNA was obtained by asymmetric polymerase chain reaction amplification of exon 2 and exon 3 of HLA-A and -B genes and hybridized to the microarray. The hybridized microarray was subsequently scanned and the result was analyzed by software in order to determine the genotypes of the tested sample. RESULTS: The sensitivity of the microarray for HLA-AB genotyping was 10 ng/microl, with a coincidence rate of 100% with international reference, and a combined variation value of detected signal below 15%. Analysis of genotyping of 1295 cases of clinical samples showed that the general coincidence rate between the microarray method and conventional method (polymerase chain reaction-sequence-specific primer and flow cytometry reverse polymerase chain reaction sequence-specific oligonucleotide) was HLA-A: 99.61% and HLA-B: 98.30%, respectively. A total of five out of seven samples that had conflicting results of genotypes were proved to be microarray-assay reliable by DNA sequencing, suggesting a higher accuracy of the microarray method. CONCLUSION: The microarray for HLA-AB genotyping is satisfactory for clinical use in HLA-AB genotyping with its good specificity, sensitivity and reproducibility.

DNA Probes↗

PubMed1997

Effects of somatotropin and salbutamol in three genotypes of finishing barrows: growth, carcass, and calorimeter criteria.

We evaluated the combined use of 0 or 4 mg/d of recombinant porcine somatotropin (pST) and 0 or 2.75 ppm salbutamol in three genotypes of growing barrows (139 d old) differing in lean and lipid accretion potential. Treatments were in a 2 x 2 x 3 factorial arrangement, and the three genotypes tested were Meishan (M, n = 32, 49 kg), Duroc x White composite (D x Wc, n = 31, 62 kg), and Meishan x White composite (M x Wc, n = 31, 64 kg) pigs. Growth performance was evaluated over 28 d for individual pigs, and 20-h feed-deprived heat production was measured before slaughter (d 34). Daily pST injection increased ADG (+70 g/d) and reduced ADFI (-.61 kg/d) across genotypes (P < .05). Salbutamol increased (P < .05) ADG in M x Wc pigs (+146 g/d) but not in M pigs (-60 g/d) or D x Wc (+80 g/d) pigs. However, M pigs had the lowest ADG and ADFI, and M x Wc pigs not treated with salbutamol grew slower than D x Wc pigs (P < .05). Carcass protein and moisture accretion were additively (P < .05) increased by pST and salbutamol for D x Wc and M x Wc pigs. Meishan pigs had increased carcass protein and moisture accretion from pST, whereas only moisture accretion was increased by salbutamol (P < .05). The longissimus muscle area and semitendinosus weight increased as the percentage of M in the genotype decreased (P < .05), and both were increased by pST or salbutamol treatment (P < .001). Leaf fat was decreased more (P < .05) in M pigs than in D x Wc or M x Wc pigs with pST injection. The similar magnitude of leaf fat reduction between D x Wc and M x Wc pigs resulted in a mean genotype difference (P < .05), and salbutamol decreased leaf fat across genotypes. Oxygen consumption and heat production were increased by pST in M pigs more than in the crossbred genotypes, but CO2 production was reduced by similar magnitudes across genotypes, and salbutamol only tended to reduce CO2 production in D x Wc pigs. In general, these data indicate that pST and salbutamol result in additive increases in carcass lean composition; however, growth rate, carcass accretion, and various organ weights may vary among genotypes with salbutamol and pST treatment.

Albuterol↗

PubMed2001

The effects of Piedmontese inheritance and myostatin genotype on the palatability of longissimus thoracis, gluteus medius, semimembranosus, and biceps femoris.

The objective of this study was to determine the relative contributions of Piedmontese inheritance (0, 25, 50, or 75%) and myostatin genotype (+/+, mh/+, and mh/mh) to tenderness of four major muscles. Matings were made to produce animals with 0 (+/+), 1 (mh/+), or 2 (mh/mh) inactive myostatin alleles that were known to result in normal muscling, heavy muscling, and extremely heavy muscling, respectively. Over a 4-yr period, 395 steers and heifers (14 to 17 mo of age) were humanely slaughtered and the carcasses were chilled 48 h at 0 degrees C. An eight-member trained descriptive attribute panel evaluated tenderness, ease of fragmentation, connective tissue amount, juiciness, and beef flavor intensity of longissimus thoracis (LD), gluteus medius (GM), semimembranosus (SM), and biceps femoris (BF) steaks at 14 d postmortem. Data were analyzed for the main effects of group (eight combinations of myostatin genotype and percentage Piedmontese; [+/+]/0%, [+/+]/25%, [+/+]/50%, [mh/+]/25%, [mh/+]/50%, [mh/+]/75%, [mh/mh]/50%, [mh/mh]/75%) and muscle. Muscle x group interactions were not significant (P > 0.05). Within myostatin genotypes, contrasts to test the effect of percentage Piedmontese were not significant (P > 0.05). Data were reanalyzed for the main effects of myostatin genotype and muscle. Tenderness, ease of fragmentation, and amount of connective tissue ratings were higher (P < 0.05) for the mh/+ and mh/mh genotypes relative to +/+ in all muscles. In biceps femoris, mh/mh had higher (P < 0.05) tenderness, ease of fragmentation, and amount of connective tissue ratings than the mh/+ genotype. Juiciness ratings were lower (P < 0.05) for mh/mh than for mh/+ in all muscles and were lower for mh/mh than for +/+ in all muscles except gluteus medius. Beef flavor intensity ratings were lower (P < 0.05) for mh/mh than for +/+ in all muscles. Muscle ranks for tenderness within myostatin genotype were LD > GM > SM > BF, LD > GM > SM > BF, LD > GM > BF > SM, for +/+, mh/+, and mh/mh genotypes, respectively. The effects of Piedmontese inheritance on meat tenderness were all due to myostatin genotype. Piedmontese mh/mh bulls could be used as terminal sires to produce mh/+ progeny with improved carcass value due to improved tenderness in the four muscles studied.

Animals↗