Angela van Daal

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

Christopher Howard — Mon, 21 Sep 2009 17:18:58 PDT

Extended-spectrum B-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 B-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the blaSHV-2a gene and seven strains carried the blaSHV-12 gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL blaSHV-11 gene and one strain carried the non-ESBL blaSHV-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-ESBLencoding gene in addition to the blaSHV-2a or blaSHV12 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 blaSHV-2a from strains carrying blaSHV-12. In addition, this method was used to demonstrate an association between the relative copy numbers of blaSHV genes in individual strains and the levels of antibiotic resistance.

Novel PCR-EIA method for the detection of Chlamydia pneumoniae in respiratory specimens

J Inman-Bamber — Wed, 18 Feb 2009 13:47:01 PST

We report the development of a microtitre plate-based PCR-EIA assay (ELAHA; Enzyme Linked Amplification and Hybridization Assay) for the sensitive and specific detection of Chlamydia pneumoniae in sputum samples from patients with chronic obstructive airways disease (COAD). Following PCR amplification of a segment of the chlamydial heat shock 60 protein gene, the 587 bp sized amplicon is captured onto the streptavidin coated surface of a microtitre plate using a C. pneumoniae specific biotinylated probe and the level of captured product is subsequently determined via a colorimetric reaction using an automated plate reader. The ELAHA is a simple, rapid and inexpensive method for detection of low levels of infectious agents and is readily adaptable to current clinical laboratory equipment. The assay was evaluated with a cohort of hospital respiratory patients: (i) COAD patients with acute exacerbation, (ii) COAD patients without exacerbation (stable) and (iii) a non-respiratory control group. The ELAHA produced 6/12 (50%) C. pneumoniae positives in the COAD with exacerbation group, 3/13 (23%) positives in the COAD without exacerbation group and only 1/6 (17%) positives in the control non-respiratory group. This sensitive and robust PCR-EIA method can provide clinically relevant diagnostic evidence of current C. pneumoniae infection contributing to serious respiratory tract diseases such as COAD.

Melanocortins and their receptors and antagonists

J Voisey — Wed, 18 Feb 2009 13:41:00 PST

The melanocortins are a group of small protein hormones derived by post-translational cleavage of the proopiomelanocortin (POMC) gene product. The known melanocortin hormones include α-melanocyte stimulating hormone (MSH), β-MSH, γ-MSH and adrenocorticotropic hormone (ACTH). Five melanocortin receptors (MC1R through to MC5R) have been identified and most of these show tissue-specific expression patterns, as well as different binding affinities for each of the melanocortin hormones. The central melanocortin system consists of α-MSH, agouti-related protein (AGRP), MC3R and MC4R. AGRP and α-MSH are believed to be the natural antagonist and agonist respectively of MC3R and MC4R. This central melanocortin system is thought to play a fundamental role in the control of feeding and body weight. Knock-out mice models and genetic studies have pointed to the importance of the melanocortins in complex human pathways such as pigmentation, lipolysis, food intake, thermogenesis, sexual behaviour, memory and inflammatory response. Recently the melanocortins and their receptors have been the target for drug-based treatment of human physiological processes. MC3R and MC4R are likely targets for controlling body weight; MC1R may be used in the treatment of inflammation and MC2R for the treatment of glucocortical deficiency. A role for MC5R still remains unclear, but the evidence suggests an exocrine gland function.

Interrogation of multimeric DNA amplification products by competitive primer extension using Bst DNA polymerase (large fragment)

J Voisey — Wed, 18 Feb 2009 13:36:51 PST

Linear dsDNA composed of tandem repeats may, be exponentially amplified by the strongly strand-displacing Bst DNA polymerase (large fragment) and two primers specific for opposite strands. When the repetitive DNA is derived from rolling circle replication of a circular template, the reaction is termed cascade rolling circle amplification (CRCA). We have developed a variant of CRCA in which one primer is attached to the surface of a microwell and the other is labeled, thus enabling detection of amplified material using an ELISA-like protocol. The circular template is derived by annealing and ligation of a padlock on target DNA. It was found that there was good correlation between the synthesis of amplified material and signal. The specificity of the reaction with respect to single-nucleotide polymorphisms was investigated, and it was found that Bst DNA polymerase is prone to extension from primers with mismatched 3' ends. Reliable single nucleotide specificity was only obtained when pre-synthesized amplified material was interrogated by competitive primer extension.

Association of functionally different RUNX2 P2 promoter alleles with BMD

James D. Doecke — Thu, 12 Feb 2009 12:31:55 PST

RUNX2 gene SNPs were genotyped in subjects from the upper and lower deciles of age- and weight-adjusted femoral neck BMD. Of 16 SNPs in RUNX2 and its two promoters (P1 and P2), only SNPs in the P2 promoter were significantly associated with BMD. These P2 promoter SNPs were functionally different in gel-shift and promoter activity assays.

A polymorphism in the agouti signalling protein (ASIP) is associated with decreased levels of mRNA

Joanne Voisey — Thu, 12 Feb 2009 12:31:54 PST

To date, a role for agouti signalling protein (ASIP) in human pigmentation has not been well characterized. It is known that agouti plays a pivotal role in the pigment switch from the dark eumelanin to the light pheomelanin in the mouse. However, because humans do not have an agouti banded hair pattern, its role in human pigmentation has been questioned. We previously identified a single polymorphism in the 3'-untranslated region (UTR) of ASIP that was found at a higher frequency in African-Americans compared with other population groups. To compare allele frequencies between European-Australians and indigenous Australians, the g.8818A → G polymorphism was genotyped. Significant differences were seen in allele frequencies between these groups (P < 0.0001) with carriage of the G allele highest in Australian Aborigines. In the Caucasian sample set a strong association was observed between the G allele and dark hair colour (P = 0.004) (odds ratio 4.6; 95% CI 1.4-15.27). The functional consequences of this polymorphism are not known but it was postulated that it might result in message instability and premature degradation of the transcript. To test this hypothesis, ASIP mRNA levels were quantified in melanocytes carrying the variant and non-variant alleles. Using quantitative real-time polymerase chain reaction the mean ASIP mRNA ratio of the AA genotype to the AG genotype was 12 (P < 0.05). This study suggests that the 3'-UTR polymorphism results in decreased levels of ASIP and therefore less pheomelanin production. � 2006 Blackwell Synergy

Gene polymorphisms and their effects in the melanocortin system

Levi J. Carroll — Thu, 12 Feb 2009 12:31:54 PST

In addition to its role in human pigmentation, components of the melanocortin system regulate appetite, energy homeostasis and hormone production. Recent studies have suggested possible roles of this system in immunity, transmission of pain signals, and reproductive potential. A number of polymorphisms have been identified in genes of the melanocortin system and are associated with pigmentation in humans, as well as being causative of disorders of adrenal hormone production and obesity. This review gives an outline of these polymorphisms, their functional significance and possible application to or impact on diagnosis and pharmacotherapy based on melanocortin pathways.

Linkage disequilibrium analysis identifies an FGFR1 haplotype-tag SNP associated with normal variation in craniofacial shape

Angela Van Daal — Thu, 12 Feb 2009 12:31:54 PST

Mutations in FGFR1 and TWIST1 have been reported to affect the timing of calvarial suture fusion resulting in craniosynostosis and facial abnormalities. We screened nonpathologic populations for genetic polymorphisms that may associate with normal craniofacial variation. We identified 17 single-nucleotide polymorphisms (SNPs) in FGFR1, 6 of which were novel (g.8591855G→A, g.8593685G→A, g.8602303C→T, g.8602475A→G (p.Ile293Val), g.8605849C→T, g.8607868G→A). No SNPs were found in TWIST1. FGFR1 SNP haplotypes were reconstructed for Caucasian, Asian, Australian Aboriginal, and African American populations. All populations shared two linkage disequilibrium blocks, with one haplotype-tag SNP (htSNP) tagging each block. The htSNP g.8592931G→C was found to have a significant negative correlation with the cephalic index for all populations (R = −0.187, p = 0.036), with larger correlations in Asians and females. This finding is a starting point in the identification of a set of SNPs that can be genotyped to determine both normal and disease craniofacial phenotypes.

Single nucleotide polymorphisms in the MATP gene are associated with normal human pigmentation variation

Justin Graf — Thu, 12 Feb 2009 12:31:53 PST

Human physical pigmentation is determined by the type and amount of melanin and the process of pigmentation production probably involves more than 100 genes. A failure to synthesize melanin results in oculocutaneous albinism(OCA). A recently identified form of OCA results from mutations in the Membrane Associated Transporter Protein (MATP) gene. The role of MATP in human pigmentation is not clear. We investigated the role of two nonpathogenic nonsynonymous single nucleotide polymorphisms (SNPs) in the MATP gene to determine if they are associated with normal human skin, hair, and eye colour variation. A total of 608 individuals from four different population groups (456 Caucasians, 31 Asians, 70 African-Americans, and 51 Australian Aborigines) were genotyped for c.814G>A (p.Glu272Lys) and c.1122C>G (p.Phe374Leu). Results indicate that the allele frequencies of both polymorphisms are significantly different between population groups. The two alleles, 374Leu and 272Lys, are significantly associated with dark hair, skin, and eye colour in Caucasians. The odds ratios (ORs) of the LeuLeu genotype for black hair and olive skin are 25.63 and 28.65, respectively, and for the LysLys genotype are 43.23 and 8.27, respectively. The OR for eye colour is lower at 3.48 for the LeuLeu and 6.57 for LysLys genotypes. This is the first report of this highly significant association of MATP polymorphisms with normal human pigmentation variation.

Mouse models of obesity

Carroll Levi — Thu, 12 Feb 2009 12:31:52 PST

Insights into the etiology of human obesity have arisen from the study of animal models. Animal models of obesity are also important for the development of future treatments of obesity. An agouti mouse mutation resulting in obese, yellow mice was described over a century ago and in 1992 agouti was cloned, making it the first obesity gene characterized at the molecular level. The lethal yellow mouse mutation is one of five dominant agouti mutations and is an excellent model for human obesity. The molecular categorization of agouti was responsible for the elucidation of the melanocortin system's involvement in hypothalamic weight regulation. As genetic knowledge increases many transgenic mice have been created with genes either over-expressed or deleted, models which further enhance the understanding of obesity.