Staphylococcus aureus genotyping using novel real-time PCR formats

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of light upon extension (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the South West Pacific and Queensland community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 Aus-1/Aus-2 hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Non-major-histocompatibility-complex genetics of ankylosing spondylitis

There is strong evidence from twin and family studies indicating that a substantial proportion of the heritability of susceptibility to ankylosing spondylitis (AS) and its clinical manifestations is encoded by non-major-histocompatibility-complex genes. Efforts to identify these genes have included genomewide linkage studies and candidate gene association studies. One region, the interleukin (IL)-I gene complex on chromosome 2, has been repeatedly associated with AS in both Caucasians and Asians. It is likely that more than one gene in this complex is involved in AS, with the strongest evidence to date implicating IL-IA. Identifying the genes underlying other linkage regions has been difficult due to the lack of obvious candidates and the low power of most studies to date to identify genes of the small to moderate magnitude that are likely to be involved. The field is moving towards genomewide association analysis, involving much larger datasets of unrelated cases and controls. Early successes using this approach in other diseases indicates that it is likely to identify genes in common diseases like AS, but there remains the risk that the common-variant, common-disease hypothesis will not hold true in AS. Nonetheless, it is appropriate for the field to be cautiously optimistic that the next few years will bring great advances in our understanding of the genetics of this condition.

Identification of the semaphorin receptor PLXNA2 as a candidate for susceptibility to schizophrenia

The discovery of genetic factors that contribute to schizophrenia susceptibility is a key challenge in understanding the etiology of this disease. Here, we report the identification of a novel schizophrenia candidate gene on chromosome 1q32, plexin A2 (PLXNA2), in a genome-wide association study using 320 patients with schizophrenia of European descent and 325 matched controls. Over 25 000 single-nucleotide polymorphisms (SNPs) located within approximately 14 000 genes were tested. Out of 62 markers found to be associated with disease status, the most consistent finding was observed for a candidate locus on chromosome 1q32. The marker SNP rs752016 showed suggestive association with schizophrenia (odds ratio (OR) = 1.49, P = 0.006). This result was confirmed in an independent case control sample of European Americans (combined OR = 1.38, P = 0.035) and similar genetic effects were observed in smaller subsets of Latin Americans (OR = 1.26) and Asian Americans (OR = 1.37). Supporting evidence was also obtained from two family-based collections, one of which reached statistical significance (OR = 2.2, P = 0.02). High-density SNP mapping showed that the region of association spans approximately 60 kb of the PLXNA2 gene. Eight out of 14 SNPs genotyped showed statistically significant differences between cases and controls. These results are in accordance with previous genetic findings that identified chromosome 1q32 as a candidate region for schizophrenia. PLXNA2 is a member of the transmembrane semaphorin receptor family that is involved in axonal guidance during development and may modulate neuronal plasticity and regeneration. The PLXNA2 ligand semaphorin 3A has been shown to be upregulated in the cerebellum of individuals with schizophrenia. These observations, together with the genetic results, make PLXNA2 a likely candidate for the 1q32 schizophrenia susceptibility locus.

Further evidence of linkage at 7p22 with familial hyperaldosteronism type II

Primary aldosteronism (PAL) is caused by the autonomous over-production of aldosterone. Once thought rare, it is now reported to be responsible for 5–10% of hypertension. Familial hyperaldosteronism type II (FH-II), unlike familial hyperaldosteronism type I, is not glucocorticoid-remediable and not associated with the hybrid CYP11B1/CYP11B2 gene mutation. At least five times more common than FH-I, FH-II is clinically, biochemically and morphologically indistinguishable from apparently sporadic PAL, suggesting that its incidence maybe even higher. Studies performed in collaboration with C Stratakis (NIH, Bethesda) on our largest Australian FH-II family (eight affected members) demonstrated linkage at chromosome 7p22. Similar linkage at this region was also found in a South American FH-II family (DNA provided by MI New, Presbyterian Hospital, New York). Mutations in the exons and intron/exon boundaries of the PRKARIB gene (which resides at 7p22 and is closely related to PRKARIA gene mutated in Carney complex) have been excluded in our largest Australian FH-II family. Using more finely spaced markers, we have confirmed linkage at 7p22 in these 2 families, and identified a second Australian family with evidence of linkage at this locus. The combined multipoint LOD score for these 3 families is 4.87 (θ=0) with markers D7S462 and D7S2424, which exceeds the critical threshold for genome-wide significance suggested by Lander and Kruglyak (1995), providing strong support for this locus harbouring mutations responsible for FH-II. A newly identified recombination event in our largest Australian family has narrowed the region of linkage by 1.8 Mb, permitting exclusion of approximately half the genes residing in the original reported 5Mb linked locus. In addition, we have strongly excluded linkage to these key markers in two Australian families (maximum multipoint LOD scores −3.51 and −2.77), supporting the notion that FH-II may be genetically heterogeneous. In order to identify candidate genes at 7p22, more closely spaced markers will be used to refine the locus, as well as single nucleotide polymorphism analysis.

Identification of an apolipoprotein(e) variant associated with type III hyperlipoproteinaemia in an indigenous Australian

As a result of testing for lipid and apolipoprotein(e) (apo E) phenotype status of an indigenous Australian community, an apo E variant associated with type III hyperlipoproteinaemia has been identified. Apo E phenotype was determined by analysis of VLDL by isoelectric focusing, and genotype on DNA amplified by polymerase chain reaction, using two different restriction enzyme isotyping assays. Phenotypes and genotypes were discordant in samples from two subjects and an abnormal-sized restriction fragment was also observed in their genotyping gel patterns. DNA sequencing studies revealed this was due to a single nucleotide deletion. 3817delC, at amino acid 136 on apo E. This resulted in a new reading frame and the premature termination of the apo E protein due to a stop codon (TGA) at nucleotide 4105. The variant apo E null allele showed a recessive mode of inheritance and, in combination with the E2 allele, resulted in the type III hyperlipoproteinaemic phenotype but when inherited with the E4 allele had no marked effect on plasma lipids.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

Sequence variation in the proximity of IDE may impact age at onset of both Parkinson disease and Alzheimer disease

We recently reported that a linkage disequilibrium (LD) block on chromosome 10q encompassing the gene encoding insulin-degrading enzyme (IDE) harbors sequence variants that associate with Alzheimer disease (AD). Evidence also indicated effects upon a number of quantitative indices of AD severity, including age-at-onset (AAO). Since linkage of this immediate region to AAO has been shown in both AD and Parkinson disease (PD), we have explored the possibility that polymorphism within this LD block might also influence PD. Utilizing single nucleotide polymorphisms that delineate common haplotypes from this region, we observed significant evidence of association with AAO in an Australian PD case-control sample. Analyses were complemented with AAO data from two independent Swedish AD case samples, for which previously reported findings were replicated. Results were consistent between AD and PD, suggesting the presence of equivalent detrimental and protective alleles. These data highlight a genomic region in the proximity of IDE that may contribute to AD and PD in a similar manner.

Tau haplotypes regulate transcription and are associated with Parkinson's disease

A primary haplotype (H1) of the microtubule-associated protein Tau (MAPT) gene is associated with Parkinson's disease (PD). However, the mechanism for disease susceptibility remains unknown. We examined the promoter region of MAPT and identified single nucleotide polymorphisms and insertions of 1 to 11 nucleotides. These polymorphisms corresponded to the previously characterized haplotypes, H1 and H2, as well as a novel variant of the H1 haplotype, H1'. As observed in other studies, we demonstrated a significant association with the H1/H1 promoter genotype and PD in a cohort of 206 idiopathic late-onset cases. This is in contrast with a panel of 13 early-onset PD patients, for whom we did not detect any mutations in MAPT. By examining single nucleotide polymorphisms in adjacent genes, we showed that linkage disequilibrium does not extend beyond the MAPT haplotype to neighboring genes. To define the mechanism of disease susceptibility, we examined the transcriptional activity of the promoter haplotypes using a luciferase reporter assay. We demonstrated in two human cell lines, SK-N-MC and 293, that the H1 haplotype was more efficient at driving gene expression than the H2 haplotype. Our data suggest that an increase in expression of the MAPT gene is a susceptibility factor in idiopathic PD.

Association between polymorphisms in the progesterone receptor gene and endometriosis

The progesterone receptor (PR) is a candidate gene for the development of endometriosis, a complex disease with strong hormonal features, common in women of reproductive age. We typed the 306 base pair Alu insertion (AluIns) polymorphism in intron G of PR in 101 individuals, estimated linkage disequilibrium (LD) between five single-nucleotide polymorphisms (SNPs) across the PR locus in 980 Australian triads (endometriosis case and two parents) and used transmission disequilibrium testing (TDT) for association with endometriosis. The five SNPs showed strong pairwise LD, and the AluIns was highly correlated with proximal SNPs rs1042839 ({Delta}2 = 0.877, D9 = 1.00, P < 0.0001) and rs500760 ({Delta}2 = 0.438, D9 = 0.942, P < 0.0001). TDT showed weak evidence of allelic association between endometriosis and rs500760 (P = 0.027) but not in the expected direction. We identified a common susceptibility haplotype GGGCA across the five SNPs (P = 0.0167) in the whole sample, but likelihood ratio testing of haplotype transmission and non-transmission of the AluIns and flanking SNPs showed no significant pattern. Further, analysis of our results pooled with those from two previous studies suggested that neither the T2 allele of the AluIns nor the T1/T2 genotype was associated with endometriosis.

Effects of Variation at the ALDH2 Locus on Alcohol Metabolism, Sensitivity, Consumption, and Dependence in Europeans

Background: The low-activity variant of the aldehyde dehydrogenase 2 (ALDH2) gene found in East Asian populations leads to the alcohol flush reaction and reduces alcohol consumption and risk of alcohol dependence (AD). We have tested whether other polymorphisms in the ALDH2 gene have similar effects in people of European ancestry. Methods: Serial measurements of blood and breath alcohol, subjective intoxication, body sway, skin temperature, blood pressure, and pulse were obtained in 412 twins who took part in an alcohol challenge study. Participants provided data on alcohol reactions, alcohol consumption, and symptoms related to AD at the time of the study and subsequently. Haplotypes based on 5 single-nucleotide polymorphisms (SNPs) were used in tests of the effects of variation in the ALDH2 gene on alcohol metabolism and alcohol's effects. Results: The typed SNPs were in strong linkage disequilibrium and 2 complementary haplotypes comprised 83% of those observed. Significant effects of ALDH2 haplotype were observed for breath alcohol concentration, with similar but smaller and nonsignificant effects on blood alcohol. Haplotype-related variation in responses to alcohol, and reported alcohol consumption, was small and not consistently in the direction predicted by the effects on alcohol concentrations. Conclusions: Genetic variation in ALDH2 affects alcohol metabolism in Europeans. However, the data do not support the hypothesis that this leads to effects on alcohol sensitivity, consumption, or risk of dependence.

Analysing diversity in sugarcane resistance gene analogues

As resistance genes have been shown to contain conserved motifs and cluster in many plant genomes, the identification of resistance gene analogues can be used as a strategy for both the discovery of DNA markers linked to disease resistance loci and the map-based cloning of disease resistance genes. Sugarcane suffers from many important diseases and an analysis of resistance gene analogues offers a means to identify DNA markers linked to resistance loci. However, sugarcane has the most complex genome of any crop plant and initially it is important to understand the extent of resistance gene analogue diversity in the sugarcane genome before genetic analysis. We review herein how more than 100 expressed sequence tags with homology to different resistance genes have been identified in sugarcane with many mapped as single-dose restriction fragment length polymorphism markers. Importantly, some of these resistance gene analogues have been shown to be linked to disease resistance genes or disease quantitative trait loci. In an attempt to more efficiently analyse additional resistance gene analogues in sugarcane, we report on experiments aimed at investigating the molecular diversity of several resistance gene analogue families using a modified form of a technique termed Ecotilling. Using Ecotilling, we were able to rapidly detect single nucleotide polymorphisms in fragments amplified by PCR from four different resistance gene analogue families, SoRP1D, SoPTO, SoXa21 and SoHs1pro-1. An analysis of a diverse set of sugarcane varieties, including modern sugarcane cultivars and several S. officinarum and S. spontaneum clones, indicated that all amplicons, apart from SoHs1pro-1, contained significant polymorphism within the gene region studied. However, a comparison among these sugarcane clones, including between the parents of two sugarcane mapping populations, indicated that most polymorphisms were multi-dose, not single-dose, preventing their genetic map location or association with disease susceptibility or resistance from being determined.

The identification and characterisation of alleles of sucrose phosphate synthase gene family III in sugarcane

Little is known about the extent of allelic diversity of genes in the complex polyploid, sugarcane. Using sucrose phosphate synthase (SPS) Gene (SPS) Family III as an example, we have amplified and sequenced a 400 nt region from this gene from two sugarcane lines that are parents of a mapping population. Ten single nucleotide polymorphisms (SNPs) were identified within the 400 nt region of which seven were present in both lines. In the elite commercial cultivar Q165(A), 10 sequence haplotypes were identified, with four haplotypes recovered at 9% or greater frequency. Based on SNP presence, two clusters of haplotypes were observed. In IJ76-514, a Saccharum officinarum accession, 8 haplotypes were identified with 4 haplotypes recovered at 13% or greater frequency. Again, two clusters of haplotypes were observed. The results suggest that there may be two SPS Gene Family III genes per genome in sugarcane, each with different numbers of different alleles. This suggestion is supported by sequencing results in an elite parental sorghum line, 403463-2-1, in which 4 haplotypes, corresponding to two broad types, were also identified. Primers were designed to the sugarcane SNPs and screened over bulked DNA from high and low Sucrose-containing progeny from a cross between Q165(A) and IJ76-514. The SNP frequency did not vary in the two bulked DNA samples, suggesting that these SNPs from this SPS gene family are not associated with variation in sucrose content. Using an ecotilling approach, two of the SPS Gene Family III haplotypes were mapped to two different linkage groups in homology group 1 in Q165(A). Both haplotypes mapped near QTLs for increased sucrose content but were not themselves associated with any sugar-related trait.

KRAS variation and risk of endometriosis

Endometriosis is a common gynaecological disease with symptoms of pelvic pain and infertility which affects 7-10% of women in their reproductive years. Activation of an oncogenic allele of Kirsten rat sarcoma viral oncogene homologue (KRAS) in the reproductive tract of mice resulted in the development of endometriosis. We hypothesized that variation in KRAS may influence risk of endometriosis in humans. Thirty tagSNPs spanning a region of 60.7 kb across the KRAS locus were genotyped using iPLEX chemistry on a MALDI-TOF MassARRAY platform in 959 endometriosis cases and 959 unrelated controls, and data were analysed for association with endometriosis. Genotypes were obtained for most individuals with a mean completion rate of 99.1%. We identified six haplotype blocks across the KRAS locus in our sample. There were no significant differences between cases and controls in the frequencies of individual single-nucleotide polymorphisms (SNPs) or haplotypes. We also developed a rapid method to screen for 11 common KRAS and BRAF mutations on the Sequenom MassARRAY system. The assay detected all mutations previously identified by direct sequencing in a panel of positive controls. No germline variants for KRAS or BRAF were detected. Our results demonstrate that any risk of endometriosis in women because of common variation in KRAS must be very small.

Genetic basis of inosine triphosphate pyrophosphohydrolase (ITPA) deficiency

Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138GMA, 561GMA, 708GMA) and two associated with ITPase deficiency (94CMA, IVS2+21AMC). Homozygotes for the 94CMA missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94CMA heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21AMC homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94CMA (allele frequency: 0.06), 24 were heterozygotes for IVS2+21AMC (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21AMC heterozygotes and 94CMA/IVS2+21AMC compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed.

Ethanol and gene expression in brain

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Izuru Matusmoto and Peter A. Wilce. The presentations were (1) GABA receptor subunit expression in the human alcoholic brain, by Tracey Buckley and Peter Dodd; (2) NMDAR gene expression during ethanol addiction, by Jorg Puzke, Rainer Spanagel, Walther Zieglgansberger, and Gerald Wolf; (3) Differentially expressed gene in the nucleus accumbens from ethanol-administered rat, by Shuangying Leng; (4) Expression of a novel gene in the alcoholic brain, by Peter A. Wilce; and (5) Investigations of haplotypes of the dopamine Da-receptor gene in alcoholics, by Hans Rommelspacher, Ulrich Finckh, and Lutz G. Schmidt.