Analysis of the CDKN2A, CDKN2B and CDK4 genes in 48 Australian melanoma kindreds

Germline mutations within the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and one of its targets, the cyclin dependent kinase 4 (CDK4) gene, have been identified in a proportion of melanoma kindreds. In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma. We have previously reported the identification of germline CDKN2A mutations in 7/18 Australian melanoma kindreds and the absence of the R24C CDK4 mutation in 21 families lacking evidence of a CDKN2A mutation. The current study represents an expansion of these efforts and includes a total of 48 melanoma families from Australia. All of these families have now been screened for mutations within CDKN2A and CDK4, as well as for mutations within the CDKN2A homolog and 9p21 neighbor, the CDKN2B gene, and the alternative exon 1 (E1beta) of CDKN2A. Families lacking CDKN2A mutations, but positive for a polymorphism(s) within this gene, were further evaluated to determine if their disease was associated with transcriptional silencing of one CDKN2A allele. Overall, CDKN2A mutations were detected in 3/30 (10%) of the new kindreds. Two of these mutations have been observed previously: a 24 bp duplication at the 5' end of the gene and a G to C transversion in exon 2 resulting in an M531 substitution. A novel G to A transition in exon 2, resulting in a D108N substitution was also detected. Combined with our previous findings, we have now detected germline CDKN2A mutations in 10/48 (21%) of our melanoma kindreds. In none of the 'CDKN2A-negative' families was melanoma found to segregate with either an untranscribed CDKN2A allele, an R24C CDK4 mutation, a CDKN2B mutation, or an E1beta mutation. The last three observations suggest that these other cell cycle control genes (or alternative gene products) are either not involved at all, or to any great extent, in melanoma predisposition.

Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or "indeterminate" 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition.

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 9p involved in the development of melanoma. Although LOH at 9p has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 9p. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations by single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele. Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748 the markers closest to CDKN2A. Of the remaining 11 tumors with LOH 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion. This report supports the conclusions of previous studies that a least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

Mutations in exon 3 of the beta-catenin gene are rare in melanoma cell lines

Mutations in exon 3 of the CTNNB1 gene encoding beta-catenin have been reported in colorectal cancer cell lines and tumours. Although one study reported mutations or deletions affecting beta-catenin in 20% of melanoma cell lines, subsequent reports detected a much lower frequency of aberrations in uncultured melanomas. To determine whether this difference in mutation frequency reflected an in vitro culturing artefact, exon 3 of CTNNB1 was screened in a panel of 62 melanoma cell lines. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect intragenic deletions affecting exon 3. One out of 62 (1.6%) cell lines was found to carry a mutation, indicating that aberration of the Wnt-1/wingless pathway through activation of beta-catenin is a rare event, even in melanoma cell lines.

Development and validation of novel methods for microbial source tracking based on Escherichia coli as an indicator of water quality

Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.

Evidence for time dependency of molecular rate estimates

Long-term changes in the genetic composition of a population occur by the fixation of new mutations, a process known as substitution. The rate at which mutations arise in a population and the rate at which they are fixed are expected to be equal under neutral conditions (Kimura, 1968). Between the appearance of a new mutation and its eventual fate of fixation or loss, there will be a period in which it exists as a transient polymorphism in the population (Kimura and Ohta, 1971). If the majority of mutations are deleterious (and nonlethal), the fixation probabilities of these transient polymorphisms are reduced and the mutation rate will exceed the substitution rate (Kimura, 1983). Consequently, different apparent rates may be observed on different time scales of the molecular evolutionary process (Penny, 2005; Penny and Holmes, 2001). The substitution rate of the mitochondrial protein-coding genes of birds and mammals has been traditionally recognized to be about 0.01 substitutions/site/million years (Myr) (Brown et al., 1979; Ho, 2007; Irwin et al., 1991; Shields and Wilson, 1987), with the noncoding D-loop evolving several times more quickly (e.g., Pesole et al., 1992; Quinn, 1992). Over the past decade, there has been mounting evidence that instantaneous mutation rates substantially exceed substitution rates, in a range of organisms (e.g., Denver et al., 2000; Howell et al., 2003; Lambert et al., 2002; Mao et al., 2006; Mumm et al., 1997; Parsons et al., 1997; Santos et al., 2005). The immediate reaction to the first of these findings was that the polymorphisms generated by the elevated mutation rate are short-lived, perhaps extending back only a few hundred years (Gibbons, 1998; Macaulay et al., 1997). That is, purifying selection was thought to remove these polymorphisms very rapidly.

UHT milk contains multiple forms of αS1-casein that undergo degradative changes during storage

Milk proteins are susceptible to chemical changes during processing and storage. We used proteomic tools to analyse bovine αS1-casein in UHT milk. 2-D gels of freshly processed milk αS1-casein was presented as five or more spots due to genetic polymorphism and variable phosphorylation. MS analysis after phosphopeptide enrichment allowed discrimination between phosphorylation states and genetic variants. We identified a new alternatively-spliced isoform with a deletion of exon 17, producing a new C-terminal sequence, K164SQVNSEGLHSYGL177, with a novel phosphorylation site at S174. Storage of UHT milk at elevated temperatures produced additional, more acidic αS1-casein spots on the gels and decreased the resolution of minor forms. MS analysis indicated that non-enzymatic deamidation and loss of the N-terminal dipeptide were the major contributors to the changing spot pattern. These results highlight the important role of storage temperature in the stability of milk proteins and the utility of proteomic techniques for analysis of proteins in food.

Genetic diversity in three redclaw crayfish (Cherax quadricarinatus, von Martens) lines developed in culture in China

The redclaw crayfish Cherax quadricarinatus (von Martens) accounts for the entire commercial production of freshwater crayfish in Australia. Two forms have been recognized, an 'Eastern' form in northern Queensland and a 'Western' form in the Northern Territory and far northern Western Australia. To date, only the Eastern form has been exported overseas for culture (including to China). The genetic structure of three Chinese redclaw crayfish culture lines from three different geographical locations in China (Xiamen in Fujian Province, Guangzhou in Guangdong Province and Chongming in Shanghai) were investigated for their levels and patterns of genetic diversity using microsatellite markers. Twenty-eight SSR markers were isolated and used to analyse genetic diversity levels in three redclaw crayfish culture lines in China. This study set out to improve the current understanding of the molecular genetic characteristics of imported strains of redclaw crayfish reared in China. Microsatellite analysis revealed moderate allelic and high gene diversity in all three culture lines. Polymorphism information content estimates for polymorphic loci varied between 0.1168 and 0.8040, while pairwise F ST values among culture lines were moderate (0.0020-0.1244). The highest estimate of divergence was evident between the Xiamen and Guangzhou populations.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson’s Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59–119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

The obesity-associated polymorphisms FTO rs9939609 and MC4R rs17782313 and endometrial cancer risk in non-Hispanic white women

Overweight and obesity are strongly associated with endometrial cancer. Several independent genome-wide association studies recently identified two common polymorphisms, FTO rs9939609 and MC4R rs17782313, that are linked to increased body weight and obesity. We examined the association of FTO rs9939609 and MC4R rs17782313 with endometrial cancer risk in a pooled analysis of nine case-control studies within the Epidemiology of Endometrial Cancer Consortium (E2C2). This analysis included 3601 non-Hispanic white women with histologically-confirmed endometrial carcinoma and 5275 frequency-matched controls. Unconditional logistic regression models were used to assess the relation of FTO rs9939609 and MC4R rs17782313 genotypes to the risk of endometrial cancer. Among control women, both the FTO rs9939609 A and MC4R rs17782313 C alleles were associated with a 16% increased risk of being overweight (p = 0.001 and p = 0.004, respectively). In case-control analyses, carriers of the FTO rs9939609 AA genotype were at increased risk of endometrial carcinoma compared to women with the TT genotype [odds ratio (OR) = 1.17; 95% confidence interval (CI): 1.03–1.32, p = 0.01]. However, this association was no longer apparent after adjusting for body mass index (BMI), suggesting mediation of the gene-disease effect through body weight. The MC4R rs17782313 polymorphism was not related to endometrial cancer risk (per allele OR = 0.98; 95% CI: 0.91–1.06; p = 0.68). FTO rs9939609 is a susceptibility marker for white non-Hispanic women at higher risk of endometrial cancer. Although FTO rs9939609 alone might have limited clinical or public health significance for identifying women at high risk for endometrial cancer beyond that of excess body weight, further investigation of obesity-related genetic markers might help to identify the pathways that influence endometrial carcinogenesis.

A DRD2 and ANKK1 haplotype is associated with nicotine dependence

To test the importance of the dopamine D2 receptor (DRD2) region in nicotine dependence, 150 smokers and 228 controls were genotyped for the DRD2 C957T, -141delC and ANKK1 TaqIA polymorphisms (rs6277, rs1799732 and rs1800497, respectively). The -141delC SNP did not show any association but both the C957T and TaqIA SNPs showed association at the allele, genotype, haplotype and combined genotype levels. The 957C/TaqI A1 haplotype was more than 3.5 times as likely to be associated with nicotine dependence compared with the 957T/TaqI A1 haplotype (P = 0.003). Analysis of the combined genotypes of both SNPs revealed that individuals who were homozygous for the 957C-allele (CC) and had either one or two copies of the TaqI A1-allele were 3.3 times as likely to have nicotine dependence compared to all other genotype combinations (P = 0.0003) and that these genotypes accounted for approximately 13% of the susceptibility to nicotine addiction in our population. Our findings suggest that the DRD2 C957T polymorphism and the ANKK1 TaqIA polymorphism are key contributors to the genetic susceptibility to nicotine dependence.

DRD2 C957T and TaqIA genotyping reveals gender effects and unique low-risk and high-risk genotypes in alcohol dependence

AIMS: As recent conflicting reports describe a genetic association between both the C- and the T-alleles of the dopamine D2 receptor (DRD2) C957T polymorphism (rs6277) in alcohol-dependent subjects, our aim was to examine this polymorphism and TaqIA (rs1800497) in Australian alcohol-dependent subjects. METHODS: The C957T polymorphism was genotyped in 228 patients with alcohol dependence (72 females and 156 males) and 228 healthy controls. RESULTS: The C-allele and C/C genotype of C957T was associated with alcohol dependence, whereas the TaqIA polymorphism was not. When analysed separately for C957T, males showed an even stronger association with the C-allele and females showed no association. The C957T and TaqIA haplotyping revealed a strong association with alcohol dependence and a double-genotype analysis (combining C957T and TaqIA genotypes) revealed that the relative risk of different genotypes varied by up to 27-fold with the TT/A1A2 having an 8.5-fold lower risk of alcohol dependence than other genotypes. CONCLUSION: Decreased DRD2 binding associated with the C-allele of the DRD2 C957T polymorphism is likely to be important in the underlying pathophysiology of at least some forms of alcohol dependence, and this effect appears to be limited to males only.