Renee Crooks

Serial measures of plasma homocyst(e)ine after acute myocardial infarction.

To determine whether plasma levels of homocyst(e)ine are affected by the acute phase response, we studied 10 subjects serially after acute myocardial infarction. Our data indicate that measurement of homocyst(e)ine in patients with myocardial infarction should ideally be deferred for 7 days if spuriously low levels are to be averted.

Mutation analysis of the STK11/LKB1 gene and clinical characteristics of an Australian series of Peutz–Jeghers syndrome patients

Peutz–Jeghers syndrome (PJS) is a rare cancer predisposition, which is characterized by the presence of hamartomatous polyposis and mucocutaneous pigmentation. A significant proportion of both familial and sporadic forms of this disorder are associated with mutations in the STK11 (serine/threonine kinase 11)/LKB1 gene. In this report we present a series of Australian PJS cases, which suggest that mutations in the STK11 gene do not account for many families or patients without a family history. The most likely explanation is either the presence of another susceptibility gene or genetic mosaicism in the non‐familial patients.

Hereditary non-polyposis colorectal cancer and the role of hPMS2 and hEXO1 mutations

Hereditary non‐polyposis colorectal cancer (HNPCC) is an autosomal dominant, inherited condition that is characterized primarily by the development of early‐onset colorectal cancer and a number of other epithelial malignancies. The underlying genetic basis of the disease is associated with a breakdown of DNA‐mismatch repair. There are many genes involved in DNA‐mismatch repair, and five of them have been implicated in HNPCC. Two of the genes (hMSH2 and hMLH1) account for the majority of HNPCC families (approximately 60%), and it is not known what the exact contributions of the remaining three genes (hPMS1, hPMS2, and hMSH6) are in relation to this condition. In addition, a sixth gene (hEXO1) has been associated with a disease phenotype that is consistent with HNPCC. Current estimates suggest that all four of these genes, combined, may account for up to 5% of families. In this report, we examine the contribution of hPMS2 and hEXO1 to a well‐defined set of families that fulfill the diagnostic criteria for HNPCC. The genes, hPMS2 and hEXO1, were studied by denaturing high performance liquid chromatography (DHPLC) analysis in 21 families that have previously been determined not to have mutations in hMSH2 or hMLH1. hPMS2 accounts for a small proportion of HNPCC families, and none were deemed to be associated with hEXO1. Mutations in hPMS2 appear to account for a small proportion of families adhering to the Amsterdam II criteria, whereas hEXO1 does not appear to be associated with HNPCC.

Mutation analysis of the MYH gene in an Australian series of colorectal polyposis patients with or without germline APC mutations

The MYH gene has recently been shown to be associated with a recessive form of colorectal adenomatous polyposis. Two common mutations in the MYH gene have been identified that lend themselves to rapid screening. We have examined a series of 302 individuals comprising 120 control subjects, 120 patients diagnosed with adenomatous polyposis but without germline mutations in the APC gene and 62 patients diagnosed with familial adenomatous polyposis all harbouring confirmed causative APC germline mutations. The results reveal that MYH accounts for 16 percent of polyposis patients without germline mutations in the APC gene and that it does not appear to be a modifier gene in FAP patients diagnosed with APC germline mutations.

Familial adenomatous polyposis: more evidence for disease diversity and genetic heterogeneity

Familial adenomatous polyposis (FAP) is characterised by the presence of profuse colonic carpeting of adenomas throughout the entire colon and rectum. The genetic basis of FAP has been shown to be primarily associated with germline mutations in theAPC gene. Notwithstanding, several reports have been published indicating that there is genetic heterogeneity in FAP and that the most likely explanation is the existence of another gene. In this report we further delineate the genotype/phenotype correlation in families that harbour germline mutations in theAPC gene and identify some previously unreported changes in the APC gene which predispose to an attenuated disease phenotype. From 53 index patients diagnosed with either FAP or attenuated FAP, 27 harboured changes in the APC gene. The remaining 26 patients were further subgrouped according to their colonic phenotype. There were nine patients with a mixed hyperplastic/adenomatous colonic phenotype and there were 17 patients with an adenomatous colonic phenotype. Evaluation of the disease characteristics of these patients and their families is presented which may aid in the identification of new genes associated with colonic polyposis.

An updated mutation spectrum in an Australian series of PJS patients provides further evidence for only one gene locus

The genetic predisposition Peutz–Jeghers Syndrome (PJS) has been shown to be associated with mutations in the serine threonine kinase 11 (STK11) gene but only a proportion of probands have been shown to harbour changes in the gene. The remaining patients were proposed to be either associated with a second PJS gene or they harboured more cryptic mutations within the STK11 gene itself. With the introduction of the multiplex ligation probe amplification (MLPA) assay, large sequence losses or gains can be more readily identified. In this report we have screened 33 PJS patients from unrelated families, employing a combination of denaturing high‐performance liquid chromatography, direct DNA sequencing and the MLPA assay to identify deleterious changes in the STK11 gene. The results revealed that 24 (73%) of patients diagnosed with PJS‐harboured pathogenic mutations in the STK11 gene, including 10 (36%) with exonic or whole‐gene deletions. No phenotypic differences were identified in patients harbouring large deletions in the STK11 gene compared to patients harbouring missense or nonsense mutations. Mutation analysis in PJS should include techniques such as MLPA to identify large exonic or whole‐gene deletions and rearrangements. The high proportion of families with identifiable mutations in the STK11 gene using this range of techniques suggests that most, if not all PJS, is attributable to mutations in the STK11 gene, perhaps including as yet undiscovered changes in promoter or enhancer sequences or other cryptic changes.

Germline Missense Changes in the APC Gene and Their Relationship to Disease

Familial adenomatous polyposis (FAP) is characterized by the presence of hundreds to thousands of adenomas that carpet the entire colon and rectum. Nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene account for the majority of mutations identified to date and predispose primarily to the typical disease phenotype. Some APC mutations are associated with a milder form of the disease known as attenuated FAP. Virtually all mutations that have been described in the APC gene result in the formation of a premature stop codon and very little is known about missense mutations apart from a common Ashkenazi Jewish mutation (1307 K) and a British E1317Q missense change. The incidence of missense mutations in the APC gene has been underreported since the APC gene lends itself to analysis using an artificial transcription and translation assay known as the Protein Truncation Test (PTT) or the In Vitro Synthetic Protein assay (IVSP).In this report we have used denaturing high performance liquid chromatography to analyse the entire coding sequence of the APC gene to determine if a cohort of patients adhering to the diagnostic criteria of FAP to assess the frequency of missense mutations in the APC gene. Altogether 112 patients were studied and 22 missense mutations were identified. From the total of 22 missense changes, 13 were silent changes and the remaining 9 resulted in amino acid substitutions. One or more of these changes were identified multiple times in 62.5% of the population under study.The results reveal that missense mutations in the APC gene appear not to radically alter protein function but may be associated with more subtle processing of RNA transcripts which in turn could result in the expression of differentially spliced forms of the APC gene which may interfere with the functional activity of the APC protein.

A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the Identification of 9 New Mutations Previously Unidentified by DGGE

Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC) has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE) with denaturing high performance liquid chromatography (DHPLC) in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE.From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.