An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype.

BACKGROUND: Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. METHODS: A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. RESULTS: Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. CONCLUSIONS: The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies.

Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene.

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder linked to a region on chromosome 10 that includes PLAU, the urokinase plasminogen activator gene. QPD increases urokinase plasminogen activator mRNA levels, particularly during megakaryocyte differentiation, without altering expression of flanking genes. Because PLAU sequence changes were excluded as the cause of this bleeding disorder, we investigated whether the QPD mutation involved PLAU copy number variation. All 38 subjects with QPD had a direct tandem duplication of a 78-kb genomic segment that includes PLAU. This mutation was specific to QPD as it was not present in any unaffected family members (n = 114), unrelated French Canadians (n = 221), or other persons tested (n = 90). This new information on the genetic mutation will facilitate diagnostic testing for QPD and studies of its pathogenesis and prevalence. QPD is the first bleeding disorder to be associated with a gene duplication event and a PLAU mutation.

Genome-wide association identifies the ABO blood group as a major locus associated with serum levels of soluble E-selectin.

BACKGROUND: Elevated serum soluble E-selectin levels have been associated with a number of diseases. Although E-selectin levels are heritable, little is known about the specific genetic factors involved. E-selectin levels have been associated with the ABO blood group phenotype. METHODS AND RESULTS: We performed a high-resolution genome-wide association study of serum soluble E-selectin levels in 685 white individuals with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study to identify major loci influencing levels. Highly significant evidence for association (P=10(-29)) was observed for rs579459 near the ABO blood group gene, accounting for 19% of the variance in E-selectin levels. Levels of E-selectin were higher in O/O than O/A heterozygotes, which were likewise higher than A/A genotypes. Analysis of subgroups of A alleles reveals heterogeneity in the association, and even after this was accounted for, an intron 1 SNP remained significantly associated. We replicate the ABO association in nondiabetic individuals. CONCLUSIONS: ABO is a major locus for serum soluble E-selectin levels. We excluded population stratification, fine-mapped the association to sub-A alleles, and also document association with additional variation in the ABO region.

A genome-wide association study identifies a novel major locus for glycemic control in type 1 diabetes, as measured by both A1C and glucose.

OBJECTIVE: Glycemia is a major risk factor for the development of long-term complications in type 1 diabetes; however, no specific genetic loci have been identified for glycemic control in individuals with type 1 diabetes. To identify such loci in type 1 diabetes, we analyzed longitudinal repeated measures of A1C from the Diabetes Control and Complications Trial. RESEARCH DESIGN AND METHODS: We performed a genome-wide association study using the mean of quarterly A1C values measured over 6.5 years, separately in the conventional (n = 667) and intensive (n = 637) treatment groups of the DCCT. At loci of interest, linear mixed models were used to take advantage of all the repeated measures. We then assessed the association of these loci with capillary glucose and repeated measures of multiple complications of diabetes. RESULTS: We identified a major locus for A1C levels in the conventional treatment group near SORCS1 (10q25.1, P = 7 x 10(-10)), which was also associated with mean glucose (P = 2 x 10(-5)). This was confirmed using A1C in the intensive treatment group (P = 0.01). Other loci achieved evidence close to genome-wide significance: 14q32.13 (GSC) and 9p22 (BNC2) in the combined treatment groups and 15q21.3 (WDR72) in the intensive group. Further, these loci gave evidence for association with diabetic complications, specifically SORCS1 with hypoglycemia and BNC2 with renal and retinal complications. We replicated the SORCS1 association in Genetics of Diabetes in Kidneys (GoKinD) study control subjects (P = 0.01) and the BNC2 association with A1C in nondiabetic individuals. CONCLUSIONS: A major locus for A1C and glucose in individuals with diabetes is near SORCS1. This may influence the design and analysis of genetic studies attempting to identify risk factors for long-term diabetic complications.

Multiple superoxide dismutase 1/splicing factor serine alanine 15 variants are associated with the development and progression of diabetic nephropathy: the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Genetics study.

BACKGROUND: Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes. RESEARCH DESIGN AND METHODS: We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,362 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome. RESULTS: We observed association between rs17880135 in the 3' region of superoxide dismutase 1 (SOD1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64-4.18], P = 5.6 x 10(-5), q = 0.06) and persistent microalbuminuria (1.82 [1.29-2.57], P = 6.4 x 10(-4), q = 0.46). Sequencing and fine-mapping identified additional SOD1 variants, including rs202446, rs9974610, and rs204732, which were also associated (P < 10(-3)) with persistent microalbuminuria, whereas rs17880135 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum SOD activity, serum SOD1 mass, or SOD1 mRNA expression in lymphoblastoid cell lines. CONCLUSIONS: Multiple variations in SOD1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study.

Identification of single nucleotide polymorphisms in the human kallikrein 10 (KLK10) gene and their association with prostate, breast, testicular, and ovarian cancers.

BACKGROUND: The KLK10 gene (also known as the normal epithelial cell-specific 1 gene) is a member of the expanded human kallikrein gene family. Recently, it has been reported that KLK10 is a tumor suppressor gene and that its expression is downregulated in various forms of cancer and cancer cell lines. KLK10 is also upregulated in ovarian cancer. We thus hypothesized that the KLK10 gene may be a target for mutations in various cancers. METHODS: We sequenced the five coding exons of the KLK10 gene using genomic DNA from various tumors, normal tissues, and blood, by PCR amplification and automated sequencing. RESULTS: In none of the tumor-derived DNAs, we identified somatic mutations that could inactivate this gene. However, we identified a prevalent germline single nucleotide variation at codon 50 (exon 3) of this gene [GCC (alanine) to TCC (serine)]. The GCC genotype was less prevalent in prostatic cancer patients in comparison to control subjects (P = 0.027) but no differences were seen with testicular, ovarian, and breast cancer. We also identified four genetic variations in exon 4, at codons106 [GGC (glycine) to GGA (glycine)], codon 112 [ACG (threonine) to ACC (threonine)], codon 141 [CTA (leucine) to CTG (leucine)], and at codon 149 [CCG (proline) to CTG (leucine)]. None of these variations was significantly different between normal subjects and cancer groups. CONCLUSIONS: We found no evidence for somatic mutations of the KLK10 gene in cancers of the prostate, breast, ovary, and testis. The single nucleotide variation at codon 50 appears to be associated with prostate cancer risk.

Quantitative analysis of kallikrein 15 gene expression in prostate tissue.

PURPOSE: The newly discovered human kallikrein 15 gene KLK15 has been shown in preliminary analysis to be associated with more aggressive types of prostate cancer. We quantitatively measured and compared gene expression of KLK15 in malignant and benign prostate tissues. MATERIALS AND METHODS: Matched prostate tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 90 patients who underwent radical prostatectomy. Quantitative reverse transcriptase-polymerase chain reaction using SYBR Green I and the LightCycler system (Roche Applied Science, Mannheim, Germany) was performed. Associations of KLK15 expression with clinicopathological parameters were analyzed. RESULTS: KLK15 over expression in cancerous versus noncancerous tissue was found in 76 of the 90 patient samples (84.4%, p <0.001). The ratio of cancerous-to-noncancerous KLK15 expression tended to be higher in patients with stage pT3/4 versus pT2 tumors (p = 0.1). KLK15 expression tended to be higher in grade 3 than in grade 2 tumors and in Gleason score 7 or greater than in Gleason score less than 7 tumors (p = 0.18 and 0.23, respectively). A 1.7 cutoff at the 40th percentile provided a significant difference in stages pT2 and pT3/4 tumors (p = 0.029). CONCLUSIONS: On quantitative real-time polymerase chain reaction KLK15 expression was significantly higher in cancerous than in noncancerous tissue. Up-regulation of the KLK15 gene in advanced and more aggressive tumors may indicate a possible role for KLK15 protein as future serum marker for prostate cancer and for distinguishing tumor aggressiveness.