Jonathon Wasson

Genetic epidemiology of diabetes

Conventional genetic analysis focuses on the genes that account for specific phenotypes, while traditional epidemiology is more concerned with the environmental causes and risk factors related to traits. Genetic epidemiology is an alliance of the 2 fields that focuses on both genetics, including allelic variants in different populations, and environment, in order to explain exactly how genes convey effects in different environmental contexts and to arrive at a more complete comprehension of the etiology of complex traits. In this review, we discuss the epidemiology of diabetes and the current understanding of the genetic bases of obesity and diabetes and provide suggestions for accelerated accumulation of clinically useful genetic information.

Neuroblastoma: Effect of genetic factors on prognosis and treatment

Background and Methods. Genetic analysis of tumor tissue has provided considerable insight into mechanisms of malignant transformation and progression. Neuroblastomas have been studied by cytogenetics, flow cytometry, and molecular genetic techniques, and these studies have identified several specific abnormalities that allow subclassification of these tumors into genetic/ clinical subtypes.

An E23K single nucleotide polymorphism in the islet ATP-sensitive potassium channel gene (Kir6.2) contributes as much to the risk of Type II diabetes in Caucasians as the PPARγ Pro12Ala variant

2. Wren A, Seal L, Cohen M et al. (2001) Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 86: 5992–5995 3. Tschop M, Weyer C, Tataranni P, Devanarayan V, Ravussin E, Heiman M (2001) Circulating ghrelin levels are decreased in human obesity. Diabetes 50: 707–709 4. Ariyasu H, Takaya K, Tagami T et al. (2001) Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 86: 4753–4758 5. Hosoda H, Kojima M, Matsuo H, Kangawa K (2000) Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 279: 909–913 6. Shiiya T, Nakazato M, Mizuta M et al. (2002) Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 87: 240–244 7. Saad M, Bernaba B, Hwu C et al. (2002) Insulin regulates plasma ghrelin concentration. J Clin Endocrinol Metab 87: 3997–4000 8. Caixas A, Bashore C, Nash W, Pi-Sunyer F, Laferrere B (2002) Insulin, unlike food intake, does not suppress ghrelin in human subjects. J Clin Endocrinol Metab 87: 1902–1906

Cloning of human pancreatic islet large conductance Ca2+-activated K+ channel (hSlo) cDNAs: evidence for high levels of expression in pancreatic islets and identification of a flanking genetic marker

SummaryInsulin secretion from pancreatic beta cells is dependent on membrane potential changes that result from the concerted regulation of multiple ion channels. Among the distinct K+ channels known to be expressed in beta cells, large conductance Ca2+-activated K+ channels have been suggested to play an important role in stimulus-secretion coupling. In the course of a strategy to identify transcripts that are enriched in human pancreatic islet cells, we isolated a partial cDNA encoding a human large conductance Ca2+-activated K+ channel mRNA (hSlo). Northern analysis of mRNA showed that among a panel of human tissues hSlo is expressed at its highest levels in pancreatic islets. Screening of human insulinoma and islet cDNA libraries with the partial cDNA resulted in the isolation of 19 hSlo cDNAs. These comprised three splice variants: one shared the common underlying structure of previously reported Slo cDNAs, another variant encoded a novel 60-amino acid insertion in the putative Ca2+-sensing domain of hSlo, while the third group of clones had an alternate exon encoding eight amino acids in the predicted COOH-terminal end. Analysis of somatic-cell hybrids containing different portions of chromosome 10 indicated that hSlo maps to chromosome 10q22.2–q23.1. Furthermore, high resolution localization was obtained by analysis of genome-wide radiation hybrids and the CEPH “B” mega-YAC library, both of which identified for the first time a highly polymorphic genetic marker (D10S195) linked to hSlo. These studies provide tools with which to explore the physiological role of Ca2+-activated K+ channel proteins in pancreatic islets, and also to investigate the contribution of this locus to the inherited susceptibility to non-insulin-dependent diabetes mellitus.

Mapping of the human insulin receptor substrate-2 gene, identification of a linked polymorphic marker and linkage analysis in families with Type II diabetes: no evidence for a major susceptibility role

Summary Insulin receptor substrate 2 (IRS-2) is a substrate of the insulin receptor and mediates the action of the insulin. Disruption of the IRS-2 gene in mice results in peripheral insulin resistance and relative insulin deficiency. It is therefore possible that defects in the IRS-2 gene contribute to Type II (non-insulin-dependent) diabetes mellitus. We have examined the gene for evidence of linkage to Type II diabetes in Ashkenazi Jewish families. Radiation hybrid panel mapping was used to refine the map position of the IRS-2 gene and, in the absence of polymorphic markers within the gene, to identify nearby markers. The IRS-2 gene was placed 23cR from the marker D13S1265 on chromosome 13q34. 200 affected sibpairs were genotyped for three markers across the region. Nonparametric linkage analysis (GENEHUNTER) used with this data found no evidence of excess allele sharing in the IRS-2 gene region. We therefore concluded that variation in the IRS-2 gene is unlikely to contribute to Type II diabetes in this discrete Caucasian population. [Diabetologia (1998) 41: 1389–1391]

Candidate gene studies reveal that the WFS1 gene joins the expanding list of novel type 2 diabetes genes

Wolfram syndrome, originally described in 1938, is a rare, autosomal recessive disease that is characterised by young onset insulin-dependent diabetes, progressive sensorineural deafness, diabetes insipidus, autonomic nervous system dysfunction and, ultimately, brainstem atrophy and premature death [1]. The Wolfram gene (WFS1), which encodes wolframin, was mapped to chromosome 4p in families with multiple affected individuals [2], and cloned in 1998 [3]. Wolframin is a protein of 890 amino acids that is produced in a wide variety of tissues, most prominently in pancreatic beta cells and brain. Over 100 missense and non-sense mutations have been described patients. As these mutations are associated with a non-immune loss of beta cells and diabetes, the gene was subsequently evaluated in more common forms of diabetes.

Mapping Novel Pancreatic Islet Genes to Human Chromosomes

A strategy was developed to generate expressed sequence tags (ESTs) from human pancreatic islet gene products using differential display of mRNA. Screening of over 2,000 cDNA amplification products identified 42 cDNAs that were preferentially expressed in pancreatic islets relative to exocrine tissue. Public database analysis showed that 29 (69%) corresponded to novel genes, in contrast with only 66 of 250 (26.4%) cDNA clones randomly selected from a human islet library. Reverse transcription–polymerase chain reaction (RTPCR) and/or Northern analysis of RNA from multiple tissues confirmed that expression was enhanced in human islet cell RNA for 11 of 15 tested cDNAs. Sequencetagged sites developed from 19 islet cDNAs were used to map these genes to human chromosomes using a combination of monochromosomal somatic-cell hybrids, genome-wide radiation hybrids, and mega–yeast artificial chromosome analysis. These results indicate that this PCR-based cDNA selection strategy yields information on a distinct subset of pancreatic islet transcribed sequences, which complements ongoing human EST identification efforts based on random cDNA selection. These mapped ESTs may be used to assist in the positional cloning of diabetes susceptibility genes.

Isolation and characterization of the human AKT1 gene, identification of 13 single nucleotide polymorphisms (SNPs), and their lack of association with Type II diabetes

Aims/hypothesis. AKT1, a serine/threonine protein kinase, is an important downstream target of the insulin-signalling pathway, with both anti-apoptotic and peripheral metabolic effects. Because impaired insulin signalling is a major hallmark of Type II (non-insulin-dependent) diabetes mellitus, we considered whether the AKT1 gene could be a candidate gene involved in susceptibility of this condition. To test this possibility, we isolated and characterized the human AKT1 gene. We also looked for single nucleotide polymorphisms in the gene and examined their association with Type II diabetes mellitus in the Ashkenazi Jewish population. Methods. Human BAC/P1 genomic libraries were screened to isolate the AKT1 gene. To obtain structural information and the sequences of the exon-intron boundaries, BAC/P1 clones were directly sequenced. Identification of single nucleotide polymorphisms was done by polymerase chain reaction of each exon, followed by denaturing high performance liquid chromatography. Six single nucleotide polymorphisms were genotyped in Ashkenazi Jewish patients with Type II diabetes mellitus and in control subjects. Results. The human AKT1 gene was at least 24.6 kb in length and comprised 14 exons. Altogether 13 putative intragenic single nucleotide polymorphisms, with minor-allele frequencies ranging from 0.011 to 0.354, were identified. The allelic and the genotypic frequencies of 6 single nucleotide polymorphisms were the same in diabetic patients and in control subjects. Conclusion/interpretation. The results of our studies show that the AKT1 gene is not a major contributor to susceptibility to Type II diabetes mellitus in Ashkenazi Jews. [Diabetologia (2001) 44: 910–913]

Characterization of the LIM/Homeodomain Gene Islet-1 and Single Nucleotide Screening in NIDDM

Islet-1 (Isl-1) is a unique transcription factor that binds to the enhancer region of the insulin gene. To evaluate this gene in non-insulin-dependent diabetes mellitus (NIDDM), a full-length human Isl-1 cDNA was isolated and the genomic structure was characterized. The cDNA [2,395 bp plus additional poly(A) residues] contained an open reading frame from an initiator methionine at nucleotide 240 to an opal stop codon at nucleotide 1,286 (GenBank accession number UO7559), encoding a predicted protein of 349 amino acids (39 kDa). From their ends, 23 additional clones were sequenced, revealing 15 incomplete cDNAs and 8 intron-containing partially processed precursors. As determined by Northern blotting and reverse transcriptase–polymerase chain reaction analysis, Isl-1 was most abundantly expressed as a 2.4-kb mRNA in human islets, with a restricted pattern of expression in other adult human tissues. Analysis of genomic clones revealed that Isl-1 is encoded by six exons, varying in size from 168 bp (exon 5) to 1,230 bp (exon 6). Exons 2 and 3 each encode a LIM domain, while the homeodomain is completely contained within exon 4. The sequence of the proximal promoter region, including 426 bp upstream of the 5′ -end of the cDNA, revealed two potential regulatory elements, GCCAGCCGG (–414 to –406) and GCCACAGG (–357 to – 350), each differing by one base form a homologous sequence in the insulin gene (GCCACCGG) that has been shown to be a major positive regulatory element (Boam DSW, Clark AR, Docherty L: Positive and negative regulation of the human insulin gene by multiple trans-acting factors. J Biol Chem 265:8285–8296, 1990). A search by single-strand conformational polymorphism analysis for variants in the Isl-1 gene was undertaken in NIDDM patients. Three variants were identified in the cDNA, none of which alter the predicted amino acid sequence. No variants were found in the promoter. The results of these studies thus indicate that the Isl-1 gene is not a major contributor to NIDDM susceptibility in this Northern European Caucasian population.

Allele quantification and DNA pooling methods.

Studies utilizing differences in single-nucleotide polymorphism allele frequencies between cases and controls have been widely used in genetic analyses to locate putative genes or chromosomal regions that may be associated with a disease. In these studies the assessment of allele frequencies can be expedited and the genotyping costs reduced by the use of DNA pools. There have been multiple studies that have reported the accuracy of Pyrosequencing for the assessment of allele frequencies in DNA pools. In addition, there are an increasing number of other types of studies that make use of allele quantification to evaluate a disease status or to make a clinical diagnosis. In this chapter, the making of DNA pools is described, as well as the use of Pyrosequencing to quantify alleles. The ease of use, short run, and analysis times make Pyrosequencing the preferred method.