Receptor Genotyping Research Articles

Maxiprep genomic DNA extractions for molecular epidemiology studies and biorepositories

Molecular epidemiology and genomic characterisation studies require the screening of large numbers of individuals to achieve statistical significance. Although many of the novel DNA extraction methods offer convenient, high-throughput capabilities, their use for the processing of larger sample volumes becomes very expensive. We are currently compiling the Mexican Genomic DNA Collection in order to address specific health priorities through molecular techniques. Our approach employs a low-cost laundry detergent based DNA extraction technique that maximizes DNA yield and quality. We have optimised four different modalities (maxiprep, midiprep, miniprep and microprep) for two different sources (leukocyte concentrates and whole blood). Our optimised protocol produces 4.5 mg of DNA from 15 ml of blood-bank discarded leukocyte concentrates with spectrophotometric quality, genomic integrity and PCR suitability that rivals that of phenol-chloroform extracted samples. We present evidence of many PCR applications that we have carried out on samples extracted with this technique including Killer-cell Immunoglobulin-like Receptor genotyping, Short Tandem Repeat profiling as well as nucleic acid screening for hepatitis B and human immunodeficiency type-1 viruses. This paper highlights many of the advantages that this DNA extraction technique provides over existing methodologies, whether it is used to establish large genomic DNA collections (as was our main intention) or as a routine DNA extraction method for PCR applications.

Ala67Thr mutation in the poliovirus receptor CD155 is a potential risk factor for vaccine and wild‐type paralytic poliomyelitis

Poliovirus infections can be asymptomatic or cause severe paralysis. Why some individuals develop paralytic poliomyelitis is unknown, but a role for host genetic factors has been suggested. To investigate if a polymorphism, Ala67Thr, in the poliovirus receptor, which has been found to facilitate increased resistance against poliovirus-induced cell lysis and apoptosis, is associated with increased risk of paralytic poliomyelitis, poliovirus receptor genotyping was undertaken among Italian subjects with vaccine-associated (n = 9), or with wild-type paralytic poliomyelitis (n = 6), and control subjects (n = 71), using RFLP-PCR and pyrosequencing. Heterozygous poliovirus receptor Ala67Thr genotype was found in 13.3% of the patients with paresis and in 8.5% of the controls (Odds Ratio = 1.667). The frequency of Ala67Thr among the controls is in agreement with earlier published data. It is concluded that the Ala67Thr mutation in the poliovirus receptor is a possible risk factor for the development of vaccine-associated or paralytic poliomyelitis associated with wild-type virus.

T Cell Receptor Genotyping and HOXA/TLX1 Expression Define Three T Lymphoblastic Lymphoma Subsets which Might Affect Clinical Outcome

T lymphoblastic lymphomas (T-LBL) are rare disorders of immature T cells which predominantly involve the mediastinum. Their oncogenic pathways and prognostic variables are not clear. We undertook a retrospective study of 41 cytoplasmic CD3+ T-LBL (nine cases aged <16 years) by assessing stage of maturation arrest based on T cell receptor (TCR) immunogenotyping, immunohistochemistry, and quantification of the oncogenes thought to be important in immature T cell malignancies. Application of a TCR-based immunogenetic classification allowed the identification of three subcategories: 11 immature IM0/D-LBL showed no TCR or only incomplete TCRD DJ rearrangement and corresponded to cytoplasmic CD3+ precursors of uncertain lineage. Sixteen mature TCRD(del)-LBL showed biallelic TCRD deletion and both TCRG and TCRB rearrangement, consistent with TCRalphabeta lineage restriction. Fourteen intermediate LBL (Int-LBL) showed complete TCRD VDJ and TCRG VJ rearrangement, with TCRB VDJ rearrangement in the majority. All Int-LBL expressed HOX11/TLX1 or HOXA9 transcripts and a proportion of the latter were associated with CALM-AF10 or NUP214-ABL fusion transcripts. IM0/D-LBL were restricted to adults with extrathymic disease and bone marrow involvement, whereas Int-LBL and TCRD(del)-LBL were found in children and adults with predominantly thymic disease. In adults, the Int-LBL subgroup was associated with a significantly superior clinical outcome. This subgroup can be identified either by TCR immunogenotyping or HOXA9/TLX1 transcript quantification. Application of this molecular classification will allow the prospective evaluation of prognostic effects within pediatric and adult protocols.

Estrogen Receptor Genotypes, Menopausal Status, and the Lipid Effects of Tamoxifen

Tamoxifen induces important changes in serum lipid profiles in some women; however, little information is available to predict which women will experience improved lipid profiles during tamoxifen therapy. As part of a multicenter prospective observational trial in 176 breast cancer patients, we tested the hypothesis that tamoxifen-induced lipid changes were associated with genetic variants in candidate target genes (CYP2D6, ESR1, and ESR2). Tamoxifen lowered low-density lipoprotein cholesterol (P<0.0001) by 23.5 mg/dl (13.5-33.5 mg/dl) and increased triglycerides (P=0.006). In postmenopausal women, the ESR1-XbaI and ESR2-02 genotypes were associated with tamoxifen-induced changes in total cholesterol (P=0.03; GG vs GA/AA) and triglycerides (P=0.01; gene-dose effect), respectively. In premenopausal women, the ESR1-XbaI genotypes were associated with tamoxifen-induced changes in triglycerides (P=0.002; gene-dose effect) and high-density lipoprotein (P=0.004; gene-dose effect). Our results suggest that estrogen receptor genotyping may be useful in predicting which women would benefit more from tamoxifen.

High-throughput killer cell immunoglobulin-like receptor genotyping by MALDI-TOF mass spectrometry with discovery of novel alleles

The killer cell immunoglobulin-like receptors (KIR) interact with major histocompatibility complex (MHC) class I ligands to regulate the functions of natural killer cells and T cells. Like human leukocyte antigens class I, human KIR are highly variable and correlated with infection, autoimmunity, pregnancy syndromes, and transplantation outcome. Limiting the scope of KIR analysis is the low resolution, sensitivity, and speed of the established methods of KIR typing. In this study, we describe a first-generation single nucleotide polymorphism (SNP)-based method for typing the 17 human KIR genes and pseudogenes that uses analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It is a high-throughput method that requires minute amounts of genomic DNA for discrimination of KIR genes with some allelic resolution. A study of 233 individuals shows that the results obtained by the SNP-based KIR/MALDI-TOF method are consistent with those obtained with the established sequence-specific oligonucleotide probe or sequence-specific polymerase chain reaction methods. The added sensitivity of the KIR/MALDI-TOF method allowed putative novel alleles of the KIR2DL1, KIR3DL1, KIR2DS5, and KIR2DL5 genes to be identified. Sequencing the KIR2DL5 variant proved it was a newly discovered allele, one that appears associated with Hispanic and Native American populations. This KIR/MALDI-TOF method of KIR typing should facilitate population and disease-association studies that improve knowledge of the immunological functions of KIR-MHC class I interactions.

Natural Killer Cell Immunoglobulin-like Receptors (KIR) Genotypes in two Arab Populations: Will KIR become a genetic landmark between nations?

Genotypic profiles of the Natural killer cell Immunoglobulin-like Receptors (KIR) have been reported to vary among different ethnic groups and this study represents a comparative report on its distribution between two Arabic populations in the Middle East: Lebanese and Palestinians. Our compared population samples included 120 unrelated healthy Lebanese (as per Mahfouz et al.) and a Palestinian population of 105 individuals (as per Norman et al.). All had their DNA typed using Sequence Specific Primer (SSP) technique for the presence of the different KIR loci. Similar to most published data, we observed that the two framework genes 2DL4 and 3DL2 are present in 100% of individuals from both communities, while 2DL2, 2DL3, 2DS1, 2DS2, 2DS3, 2DS5, 3DL1, and 3DS1 were very similar in frequency. However, significant differences were noted in the frequencies of 2DL1 and 2DS4. This report is comparing KIR genotyping distribution in two Arab populations that sheds additional light on the importance of this gene in delineating a possible geographic genetic demarcation among different ethnicities or even different communities among the same or close ethnic groups.

Natural killer cell crossmatch: Functional analysis of inhibitory killer immunoglobulin-like receptors and their HLA ligands

Attempts to predict outcome of bone marrow transplantation based on killer immunoglobulin-like receptor (KIR) and HLA genotyping have yielded discordant results. To better understand the factors involved, we investigated natural killer (NK) cell function and correlated it with genetics and expression of inhibitory KIR and HLA ligands in 20 normal allogeneic pairs. KIR expression was analyzed by flow cytometry to estimate the percentage of NK cells that could be inhibited by the HLA ligands in each pair combination. NK cytotoxicity against ConA blasts demonstrated a positive correlation between the number of KIR/HLA matches and the predicted number of NK cells that could be inhibited. When 50% or more of the NK cells could be inhibited, cytotoxicity was lower (8%) than when 25% or less of the NK cells expressed KIR with matched HLA (49%) (p < 0.0001). Our data suggest that the interaction between inhibitory KIR and HLA ligands can be correlated to some extent with NK cell function, but measurement of NK activity may provide the best information for analysis of clinical situations.

The progesterone receptor gene polymorphism, PROGINS, may be a factor related to the development of uterine fibroids

To assess the possible association between the polymorphic allele of the progesterone receptor gene, named PROGINS, and uterine leiomyomas. Case-control study. Department of Gynecology. Teaching hospital. One hundred twenty-two premenopausal women with fibroids and 125 postmenopausal controls not presenting the disease. The subjects were classified as White or non-White (Black and Mulatto) and the progesterone receptor genotyping was performed, with DNA extracted from uterus in cases and from peripheric blood in controls and submitted to polymerase chain reaction (PCR) and agarose gel electrophoresis. The presence of the PROGINS allele was recorded, and its frequency as well as the genotypic distribution among cases and controls were compared according to race. PROGINS-positive genotypes (heterozygous or mutant homozygous) were found in 19% of White and 11% of non-White women, and allelic frequency of PROGINS in the groups was 10.4% and 6.2%, respectively. Comparing patients and controls, we observed a significant difference among non-White women, both regarding presence of PROGINS-positive genotypes (4.9% vs. 25%, respectively), and PROGINS allele frequency (3.3% vs. 12.5%, respectively). There was no significant difference in PROGINS-positive genotypes among White cases and controls (16.4% vs. 20.6%, respectively), and in their allelic frequency (8.2% vs. 11.9%, respectively). The odds ratio showed reduced risk of fibroids related to PROGINS-positive genotypes in non-White women (odds ratio = 0.16, 95% confidence interval: 0.04-0.66), but not among White subjects (odds ratio = 0.76, 95% confidence interval: 0.33-1.74). The PROGINS polymorphism revealed to be protective in terms of uterine fibroids in Brazilian non-White women.

Androgen Receptor Genotyping in a Large Australasian Cohort with Androgen Insensitivity Syndrom? Identification of Four Novel Mutations

We genotyped the androgen receptor (AR) gene in 31 Australasian patients with androgen insensitivity syndrome (AIS). The entire coding region of AR was examined including analysis of polymorphic CAG and GGN repeats in all patients. AR defects were found in 66.7% (6/9) of patients with complete AIS (CAIS) and 13.6% (3/22) of patients with partial AIS (PAIS). A novel deletion (N858delG) leading to a premature stop codon was found in CAIS patient P1. CAIS patient P2 has a novel deletion (N2676delGAGT) resulting in a stop at codon 787. These mutations would result in inactivation of AR protein. A novel insertion of a cysteine residue in the first zinc finger of the AR DNA-binding domain (N2045_2047dupCTG) was found in CAIS patient P3. PAIS patient P4 has a novel amino acid substitution (Arg760Ser) in the AR ligand binding domain, which may impair ligand binding. Five patients were found to have previously reported AR mutations and no mutations were identified in the remaining patients.

Distribution of killer cell immunoglobulin‐like receptors genotypes in the Lebanese population

This study represents the first report on the distribution of 16 Killer cell Immunoglobulin-like Receptors (KIR) genes in 120 unrelated healthy Lebanese individuals. We observed that 2DL2 frequency (61%) comes second highest after South Asians (64%) and 2DL5 frequency (58.3%) is the second highest reported so far after the South Asians (74%). Interestingly, a large number of AA1 genotype individuals with no loci for activating KIR and three completely new BB profiles not previously reported were found in our population with a group A : group B haplotypes ratio of 1.3:1. The frequency of the KIR loci suggests that the Lebanese population shares common general features with the Caucasoid populations studied before, but still has its own unique decreased or increased frequencies of several loci.

Association of Follicle Stimulating Hormone (FSH) Receptor Gene Polymorphisms With Outcome of Controlled Ovarian Stimulation in Patients Undergoing IVF

Controlled ovarian hyperstimulation (COH) using FSH is a widely used treatment in most assisted reproduction techniques. FSH triggers the maturation of follicles and plays a pivotal role in the recruitment of the dominant follicle. These important actions of FSH are mediated by the FSH receptor. In this study, we evaluated the association of FSH receptor gene polymorphisms with outcome of COH in patients undergoing IVF. Analysis of genetic screening data and clinical outcome Genomic DNA was extracted from peripheral blood of 202 women, including 141 ones were undergoing COH. We investigated the frequency of FSH receptor variants, Thr307Ala (T/A) and Asn680Ser (N/S) polymorphisms by using polymerase chain reactions and restriction fragment length polymorphism analysis. Their clinical outcome such as basal FSH level, estradiol level at administration of hCG, dosage of FSH treated, number of retrieved oocytes and pregnancy rates were compared related to their genotypes. In a population of 202 Korean women, the frequency of TT/NN, TA/NS and AA/SS for the variant Thr307Al a and Asn680Ser was 50.0%, 36.6% and 9.9%, respectively. The frequency of the unlinked genotypes, TA/NN, TT/NS, AA/NN and AA/NS were 2.0%, 0.5%, 0.5% and 0.5%, respectively. There was no significant difference in the basal FSH level, dosage of FSH treated and pregnancy rate among the COH-IVF patients with different genotypes. However, the proportion of good responder, which is higher estradiol level (> 2,000 pmol/L) at administration of hCG and number of retrieved oocytes (> 10 oocytes), in AA/SS genotype (81.8% and 63.6%) was higher than those of TT/NN (37.3% and 46.7%) and TA/NS (38.1% and 41.8%). We found that Korean AA/SS genotype of FSH receptor was good responder of FSH treatment in COH for IVF. This finding is contrast to the reports with other ethnic populations. Our data should be substantiated by the further studies of FSH receptor genotyping in the Korean population.

Follicle‐stimulating hormone receptor polymorphisms in a population of infertile women

There are two known polymorphisms of clinical relevance in the follicle-stimulating hormone (FSH) receptor exon 10, alanine or threonine at position 307, and asparagine or serine at position 680, giving rise to two discrete allelic variants: Thr307/Asn680 and Ala307/Ser680. At position 680, three FSH receptor variants are possible: Asn/Asn, Asn/Ser, and Ser/Ser. We hypothesized an association between FSH receptor polymorphisms and ovarian reserve. FSH receptor polymorphisms at position 680 were studied in a population of 68 infertile women. We used serum FSH level at cycle day 3 as a screening for ovarian reserve. DNA was extracted from peripheral leukocytes in whole blood by using PCR and DNA sequencing in order to determine the type of FSH receptor. The distribution of FSH receptor variants was Asn/Asn (AA) 35%, Asn/Ser (AS) 24%, and Ser/Ser (SS) 41%. In women with normal ovarian reserve, FSH levels at cycle day 3 were 5.6 +/- 1.9 (AA group), 6.7 +/- 1.3 (AS group), and 5.7 +/- 1.7 (SS group) (non-significant). Corresponding FSH levels at cycle day 10 were 6.9 +/- 1.9, 6.3 +/- 1.7, and 8.3 +/- 2.8 (P < 0.01, AA and AS vs. SS group). In the SS group, FSH levels at cycle day 10 were significantly higher than they were at cycle day 3 (P < 0.05). The results show that Ser/Ser-680 predominates in the studied infertile population. Furthermore, women with normal ovarian reserve and the Ser/Ser FSH receptor variant had significantly higher FSH levels, compared to women with Asn/Asn and Asn/Ser variants. FSH receptor genotyping may, thus, be interesting as an adjunct indicator of ovarian reserve for infertile women undergoing assisted reproduction, and may be helpful in the determination of the starting dosage of FSH in in vitro fertilization.

Activating Killer Cell Immunoglobulin-Like Receptor Gene KIR2DS1 Is Associated With Psoriatic Arthritis

Killer cell immunoglobulin-like receptor genotyping was performed on a cohort of American Caucasian patients with psoriasis to investigate any possible relationship between these chromosome 19 genes and autoimmune-linked disease. This patient cohort also contained a subgroup of patients who had been additionally diagnosed as positive for psoriatic arthritis (PsA). Because of the known association of human leucocyte antigen (HLA)-Cw*06 with psoriasis, the study concentrated on the five KIR genes that have HLA-C as their recognized ligand ( i.e., KIR2DL1, -2DL2, -2DL3, -2DS1, and -2DS2). An increase in the frequency of the activating KIR2DS1 gene was detected in the PsA patients, compared with psoriasis patients negative for PsA and an unaffected American Caucasian control group.

Comparison of Killer Ig-Like Receptor Genotyping and Phenotyping for Selection of Allogeneic Blood Stem Cell Donors

The repertoire of killer Ig-like receptors (KIRs) can be determined at the level of DNA, RNA, or surface protein expression for selection of blood stem cell donors. We compared genotyping and phenotyping of the four inhibitory KIRs that are important in transplantation for leukemia in 73 unrelated persons. In 5 (7%) of the 68 individuals in whom the KIR2DL1 gene was present and in 10 (15%) of the 67 in whom KIR3DL1 was present, the corresponding receptor was not expressed by NK cells, as determined by flow cytometry analysis. In contrast, one or both allelic forms of KIR2DL2/KIR2DL3 were expressed by a high proportion of NK cells in all 73 individuals. However if both KIR2DL2 and KIR2DL3 genes were present, KIR2DL3 was preferentially expressed, as transcripts of KIR2DL2 was not detectable by RT-PCR in 42% of these individuals. In total, repertoire assessment for the four KIRs by genotyping vs phenotyping was not in complete agreement in 18 (25%) of the 73 individuals. Furthermore, among the samples that tested positive for the expression of a certain KIR gene, the levels of transcripts and surface expression varied considerably as measured by both real-time quantitative PCR and flow cytometry analysis. Extension of this comparative analysis to include all 12 KIR family members showed that KIR2DL3 and KIR3DL2 were the only genes whose transcripts were consistently detectable. These results caution the use of genotyping alone for donor selection or leukemia-relapse prognostication because some KIRs may be expressed at a very low level.

Menopausal status and estrogen receptor genotypes influenced the severity of hot flashes after tamoxifen treatment

Background/Aims Hot flashes are the most common side effect of tamoxifen treatment. We conducted a prospective observational trial to evaluate factors that influenced hot flash severity after tamoxifen treatment. Methods Hot flashes frequency and severity were recorded in 7-day hot flashes diaries before, and 1, 4, 8, 12 months after tamoxifen treatment in 122 subjects. Hot flashes composit scores were calculated. Demographic information was collected at baseline, and estrogen receptor (ESR1 & 2) genotyping was also performed. Results Pre-menopausal women showed the biggest increase in hot flashes severity, from 5.2±10.5 at baseline to 28.5±51.6 at 4 month (P<0.0001), whereas there was no significant change in peri- or postmenopausal women. Twenty-two of the 122 women did not develop hot flashes. G allele carriers of the ESR2-02 SNP were 4.2 times more likely to develop hot flashes than homozygotes with AA genotype (P=0.008). Conclusion Pre-menopausal women were most likely to develop hot flashes after tamoxifen treatment. ESR2-02 genotype may also influence the risk of developing hot flashes after tamoxifen treatment. Clinical Pharmacology & Therapeutics (2005) 77, P4–P4; doi: 10.1016/j.clpt.2004.11.017

Killer Immunoglobulin-Like Receptors (KIRs) Genotypes in a Belgian Population.

KIRs (Killer cell Immunoglobulin-like Receptors) are expressed on NK (Natural Killer) cells and a subpopulation of T lymphocytes namely memory CD8+ T cells. The distribution of KIR genes varies among individuals and populations. These genes are encoded on chromosome 19 (19q13.4). Till now 17 KIR genes and pseudogenes have been identified. KIRs recognise groups of HLA class I alleles. NK activity is partially controlled through the interaction between KIRs and their HLA ligands. Several studies report that KIRs may affect the outcome of Hematopoietic Stem-Cell Transplantations.We performed KIR typing of 17 genes and pseudogenes in 100 healthy Belgian unrelated individuals in “West-Vlaanderen” from Caucasoid origin using a PCR-SSP method. Three genes (KIR3DL3, 2DL4 and 3DL2), named frame-work genes, and the pseudogene 3DP1 were found in all individuals. KIR2DL1 and 2DP1 genes were present in a frequency of 99%. In addition, KIR3DL1 and 2DS4 genes represented a frequency of 97. The KIR2DL3 was found in 90% and the frequencies of other genes varied between 56% and 24%. The individual KIR gene content ranged from 8 to 17 genes. A total of 19 KIR locus profiles was observed. The most common KIR locus profile (32%) consisted of a combination of genes characterising A haplotypes (KIR2DL1 and 2DL3) without the presence of genes characteristic of B haplotypes (KIR2DL2 and 2DS2). The second most common KIR locus profile, accounting for 20% contained a combination of genes characteristics for both A and B haplotypes. The allele KIR2DS4*003 was found in 89% and KIR2DS4*00101/00102/002 only in 41%. KIR3DP1*00301/00302 was present in all individuals and KIR3DP1*001/002 only in 35%.Our results show that frequencies of most KIR loci in our Belgian population were comparable to literature data of other Caucasian populations. Only KIR2DS1 was lower (27% vs 47.7% with a p-value of 0.0002).In the future, KIR typing of donor and patient before a Hematopoietic Stem-cell Transplantation may be necessary, due to the diversity in KIR genotypes.

Development of a multiplex PCR-SSP method for Killer-cell immunoglobulin-like receptor genotyping.

Killer-cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells recognize groups of HLA class I alleles. Recent work suggests that KIR genotype may affect the outcome of hematopoietic stem-cell transplants and that prospective KIR typing maybe of benefit in future matching of donors and recipients. A simple and informative KIR genotyping method was developed using a multiplex polymerase chain reaction-sequence-specific primer strategy. This method contains four multiplex reactions for detecting all functional KIR genes, including some 2DS4 variants that harbor a common deletion. Primer pairs were designed to provide short amplicons (108-565 bp) that can be analyzed by agarose gel electrophoreses or by automated electrophoretic systems. This method was evaluated in a blinded survey with the NK/KIR Phase II QC Panel (a total of 16 cell lines) from the 14th International Histocompatibility Workshop (IHWS), and the results are 100% concordant with the consensus genotype. Results in further KIR genotyping of 20 reference cell lines from the 10th IHWS were consistent with previously published genotypes, matching those of one study in instances where different genotypes have been previously reported. The genotypes obtained in this study may be helpful to other labs developing KIR genotyping methods in resolving typing discrepancies and in detecting common deletion variants of 2DS4. This method can save labor and reagent costs. It provides good results from partially degraded template DNA due to short amplicons in this method. It is convenient to use in both clinical and research laboratories.

Influence of number of CAG repeats on local control in the RTOG 86–10 protocol

To determine the influence of CAG repeat length on response to hormones in the RTOG 86–10 patients. The length of the CAG repeats in the N- terminal of the androgen receptor gene correlates inversely with AR transcriptional activity, and shorter CAG repeats are associated with a higher probability of developing prostate cancer. The effect of number of CAG repeats on outcome after androgen deprivation is less clear and will be studied. The cohort described was from RTOG 86–10, a phase III study in which patients were randomized to receive either radiotherapy (RT) alone or RT and short term androgen deprivation (RT + HT). Androgen receptor (AR) genotyping was done on archival diagnostic specimens with sufficient tissue. AR was also quantified by flow cytometry in a smaller subset of men to determine antigen density and percent positive receptors. The resultant values were studied for association with local failure (LF), distant metastases (DM), prostate cancer survival (PSS), and overall survival (OS) in univariate and multivariate analyses. Of the 456 analyzable cases in RTOG 86–10 (parent cohort), 94 (21%) had CAG repeat values determined (test cohort). The distributions of patients by pretreatment characteristics or assigned treatment (RT vs. H +RT) were not statistically significantly different between the parent and the test groups. However, cases with determined CAG values had a significantly higher risk of death (p =0.049), suggesting that the cases with determined CAG may not be representative of the entire cohort of patients on the study. The median CAG repeat was 19. The distribution of disease stage and assigned treatment were not significantly different in either the high or low CAG subgroups. There was a trend (p = 0.09) toward more Gleason 7–10 scores in both assigned treatments among the patients with CAG 19 or greater, but this trend was not seen in patients with CAG <19. CAG was not show be significant within the entire test cohort either in univariate or multivariate analysis of each of the four outcomes. However, subset analysis revealed that patients with CAG < 19 who received H+RT had improved LF compared to those who got RT alone (p = 0.026, 5-year rates 4.6% vs. 36.4%) and improved LF compared to patients with CAG 19 or greater that received H+RT (p = 0.028). No similar effect on OS was seen in these groups, but in the group with CAG 19 or greater, patients that received H+RT had improved OS compared to patients getting RT (p = 0.033, 5-year rates 69.0% vs. 53.1%). Measurement of CAG repeats may aid in selection of patients who achieve local control benefit from androgen deprivation

Caution with ?1-adrenergic receptor genotyping*1

Caution with ?1-adrenergic receptor genotyping*1

Neutrophil FcgammaRIIIb allelic polymorphism in anti-neutrophil cytoplasmic antibody (ANCA)-positive systemic vasculitis.

Neutrophils constitutively express FcgammaRIIa and FcgammaRIIIb receptors. Both receptors exhibit allelic variants which have different quantitative functional capacities: the biallelic FcgammaRIIa-R131 and -H131 alleles, and the neutrophil antigen (NA) NA1/NA2 alleles. ANCA activation of neutrophils requires ligation of FcgammaRIIa receptor, but recent data have shown that ANCA can also bind FcgammaRIIIb receptor. The aim of this study was to determine whether the FcgammaRIIIb polymorphism was a risk factor for the development of ANCA-associated systemic vasculitis, or the associated nephritis. FcgammaRIIIb receptor genotyping was determined by allele-specific polymerase chain reaction. Genomic DNA was extracted from 101 Caucasian patients with ANCA+ vasculitis (of whom 84 had renal disease) and 100 ethnically matched controls. Of the patients with ANCA+ systemic vasculitis, 71 had ANCA with specificity for proteinase 3 and 30 with specificity for myeloperoxidase (MPO). Overall no significant difference in genotype distribution or allele frequencies was found between patients and controls, or between patients with renal disease and controls. However, there was a trend for an increase in homozygosity for the NA1 allele in patients with a vasculitis and this was significant in patients who had anti-MPO antibodies. The FcgammaRIIIb receptor polymorphism is not a major factor predisposing to the development of ANCA+ systemic vasculitis or the associated nephritis. The over-representation of the FcgammaRIIIb homozygous NA1 allele in patients with anti-MPO antibodies may have implications for disease susceptibility.