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Background Complementary single-nucleotide polymorphisms (SNPs) may not be distributed equally between two DNA strands if the strands are functionally distinct, such as in transcribed genes. In introns, an excess of A↔G over the complementary C↔T substitutions had previously been found and attributed to transcription-coupled repair (TCR), demonstrating the valuable functional clues that can be obtained by studying such asymmetry. Here we studied asymmetry of human synonymous SNPs (sSNPs) in the fourfold degenerate (FFD) sites as compared to intronic SNPs (iSNPs). Results The identities of the ancestral bases and the direction of mutations were inferred from human-chimpanzee genomic alignment. After correction for background nucleotide composition, excess of A→G over the complementary T→C polymorphisms, which was observed previously and can be explained by TCR, was confirmed in FFD SNPs and iSNPs. However, when SNPs were separately examined according to whether they mapped to a CpG dinucleotide or not, an excess of C→T over G→A polymorphisms was found in non-CpG site FFD SNPs but was absent from iSNPs and CpG site FFD SNPs. Conclusion The genome-wide discrepancy of human FFD SNPs provides novel evidence for widespread selective pressure due to functional effects of sSNPs. The similar asymmetry pattern of FFD SNPs and iSNPs that map to a CpG can be explained by transcription-coupled mechanisms, including TCR and transcription-coupled mutation. Because of the hypermutability of CpG sites, more CpG site FFD SNPs are relatively younger and have confronted less selection effect than non-CpG FFD SNPs, which can explain the asymmetric discrepancy of CpG site FFD SNPs vs. non-CpG site FFD SNPs.

Aims/hypothesis In a recent linkage analysis of genome-wide gene-expression patterns in lymphoblastoid lines, the gene encoding the leukocyte-derived arginine aminopeptidase ( LRAP ) was identified as having its expression levels modulated by one of the most pronounced effects in cis , mapping to a single-nucleotide polymorphism (rs2762). As this enzyme has an important role in processing antigenic peptides for the HLA I molecules, a variant that drastically modulates its expression levels might affect risk of autoimmunity. This study was designed to confirm that LRAP expression in B-cell derived lines is controlled by a haplotype marked by rs2762 and to see whether this would be the basis of an association with type 1 diabetes (T1D). Methods Single-nucleotide primer extension (SNuPE) was adapted to determine the haplotype-specific expression. Genetic association was tested in 892 nuclear families with one T1D-affected offspring and two parents (2676 individuals). Results All nine heterozygous RNA samples showed an eight-fold higher level of one haplotype over the other (7.97 ± 0.99, p = 1.33 × 10 −9 ). However, no association of rs2762 with T1D was found by the transmission disequilibrium test (transmission ratio A/G = 377/388, p = 0.69). Conclusions/interpretation The genetically determined LRAP expression does not play significant roles in T1D. However, this dramatic genetic effect on LRAP expression justifies further investigation of association with other phenotypes, especially autoimmune and related to host defense to specific pathogens.

Recently, we have identified proinsulin (P-Ins) 73-90 as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with β-islet cell autoimmunity and of HLA-DR4/CD4 double-transgenic mice immunized with human P-Ins. We have compared the fine specificities of one human CD4 T cell clone and two mouse T cell hybridoma clones recognizing this epitope, and, although these three clones all recognized the same core region (LALEGSLQK), there were major differences in how they interacted with the peptide (p)/HLA complex, reflecting the fact that human P-Ins is a foreign antigen in the mouse and an autoantigen in the type 1 diabetes patient. The human T cell clone was forkhead transcription factor 3 (Foxp3)-positive, a marker for regulatory T cell lineages, and secreted predominantly IL-5, IL-10, and low levels of IFNγ in response to P-Ins 73-90 . This finding is compatible with the previously detected regulatory cytokine pattern in subjects with β-cell autoimmunity. However, added N- or C-terminal amino acids drastically changed HLA and tetramer binding capacity as well as T cell reactivity and the cytokine phenotype of the P-Ins 73-90 -specific human CD4 T cell clone, suggesting a potential for this P-Ins epitope as a target for therapeutic intervention in HLA-DR4-positive humans with β-islet cell autoimmunity or recent-onset type 1 diabetes.

Pediatric endocrine diseases and their adult consequences depend on interactions of environmental exposure factors with the genetic makeup of the individual. Much has been learned over the past two decades about the genetic component in cases of monogenic (Mendelian) disorders due to drastic disruption of a single gene. The majority of children consulting a pediatric endocrinologist, however, suffer from conditions not attributable to a single gene. The nature and mechanisms of more subtle alterations at multiple different genes that are responsible for the genetic component of most human morbidity and mortality is now only beginning to be elucidated, largely thanks to the vast amount of information that is becoming available through the human genome effort.The purpose of this review is to describe 1. the nature of the information on health-related human variation that is coming our of the genome effort; 2. how this variation can affect biology; 3. which research approaches show promise in linking DNA variation to disease; 4. how this variation is organized in genomic blocks of linkage disequilibrium; and 5. how this knowledge may, in the future, allow clinicians to individualize management of a specific patient according to his/her genetic makeup.

The IDDM2 type 1 diabetes susceptibility locus was mapped to and identified as allelic variation at the insulin gene (INS) VNTR regulatory polymorphism. In Caucasians, INS VNTR alleles divide into two discrete size classes. Class I alleles (26 to 63 repeats) predispose in a recessive way to type 1 diabetes, while class III alleles (140 to more than 200 repeats) are dominantly protective. The protective effect may be explained by higher levels of class III VNTR-associated INS mRNA in thymus such that elevated levels of preproinsulin protein enhance immune tolerance to preproinsulin, a key autoantigen in type 1 diabetes pathogenesis. The mode of action of IDDM2 is complicated, however, by parent-of-origin effects and possible allelic heterogeneity within the two defined allele classes. We have now analysed transmission of specific VNTR alleles in 1,316 families and demonstrate that a particular class I allele does not predispose to disease when paternally inherited, suggestive of polymorphic imprinting. But this paternal effect is observed only when the father's untransmitted allele is a class III. This allelic interaction is reminiscent of epigenetic phenomena observed in plants (for example, paramutation; ref. 17) and in yeast (for example, trans-inactivation; ref. 18). If untransmitted chromosomes can have functional effects on the biological properties of transmitted chromosomes, the implications for human genetics and disease are potentially considerable.

The association between IL4R and type 1 diabetes has been tested in many studies in recent years, with contradictory results. The aim of this study was to re-evaluate the genetic association in type 1 diabetic nuclear families of mixed European background.We genotyped six non-synonymous single-nucleotide polymorphisms (SNPs) of the IL4R gene in 830 nuclear families as specified above, including a French Canadian subset.No association between type 1 diabetes and any SNP or haplotype was found by the transmission disequilibrium test.Previous positive reports may be due to population stratification as, according to HapMap data, allele frequencies in the IL4R region vary considerably by ethnicity.

The silencing of either the paternal or the maternal copy of the few human genes that are subject to parental imprinting may contribute to genetic disease or modify its inheritance pattern. Disruption of the normal process of imprinting may result in double gene dosage if both copies are expressed or in a null phenotype if neither is. When normally imposed, imprinting can play a role in disease by leaving only one of the two copies to be inactivated by mutation. This introductory review explains the various mechanisms and gives examples of the best-understood diseases where imprinting plays an important role. Keywords: imprinting; inheritance; Prader–Willi; Angelman; Beckwith–Wiedemann; transient neonatal diabetes; pseudohypoparathyroidism

Genetic susceptibility to the autoimmune B cell destruction that leads to Type 1 diabetes mellitus (T1D) is a complex trait.1 In recent years, many T1D associations have been reported, but only three (major histocompatibility complex, insulin, and cytotoxic T lymphocyte associated protein 4) have been confirmed in several independent studies.2,3 Independent confirmation is essential to eliminate artefacts of publication bias, multiple hypothesis testing, and, in findings of case–control studies, population stratification.4 Bottini et al recently found, by a case–control design in two independent populations, a novel association of T1D with a single nucleotide polymorphism (SNP) that caused a R620W aminoacid substitution (dbSNP rs2476601) in the lymphoid protein tyrosine phosphatase, non-receptor type 22 ( PTPN22 ) gene.5 PTPN22 encodes LYP, a non-receptor tyrosine phosphatase involved in lymphocyte function. This paper leaves two potential questions unanswered. Firstly, results of case–control studies are potentially artefacts of population stratification, no matter how well matched are the two groups. Secondly, although Bottini et al show that the T allele encodes a protein unable to bind to its important Csk partner, and postulate this as a very attractive candidate mechanism for the genetic effect, they did not address the question of the haplotype structure of the locus and the possibility that the association is due to linkage disequilibrium (LD) with another variant. Here we present the results of a study that confirms this association in a design impervious to population stratification, and in a preliminary step towards addressing the second question, we define the LD block that encompasses the PTPN22-R620W SNP and present a computationally generated list of potentially functional SNPs within the block. ### Subjects Genomic DNA was obtained after informed consent from 588 nuclear families with at least one T1D affected child and two parents. The research ethics board of the Montreal Children’s …

Electrochemical impedance measurements were used for the detection of single-strand DNA sequences using a peptide nucleic acid (PNA) probe layer immobilized onto Si/SiO2 chips. An epoxysilane layer is first immobilized onto the Si/SiO2 surface. The immobilization procedure consists of an epoxide/amine coupling reaction between the amino group of the PNA linker and the epoxide group of the silane. A 20-nucleotide sequence of PNA was used. Impedance measurements allow for the detection of the changes in charge distribution at the oxide/solution interface following modifications to the oxide surface. Due to these modifications, there are significant shifts in the semiconductor’s flat-band potential after immobilization and hybridization. The results obtained using this direct and rapid approach are supported by fluorescence measurements according to classical methods for the detection of nucleic acid sequences.

An individual’s predisposition to Type I diabetes (T1D) is largely determined by complex interactions between several genetic loci and other, nonheritable factors. In T1D, the HLA locus has been known for decades to contribute 50% of the inherited risk. Outside the HLA are many proposed candidate loci with smaller effects, but only two confirmed candidate genes, the INS-VNTR and the CTLA-4 genes, which together do not contribute more than 15% of the risk. Because of the high frequency of the disease-associated DNA variants of these genes, understanding the biological mechanisms of such DNA variation in the context of T1D can have tremendous impact on the development of preventive therapeutics. However, establishing a causal relationship between common DNA variations and disease-predisposing functional effects is not trivial and remains difficult, as the effects are expected to be subtle. The variable-number tandem-repeat (VNTR) region upstream of the insulin gene is known to mediate expression in the thymus and pancreas, whereas various polymorphisms in the 5 ′ and 3 ′ regulatory regions of CTLA-4 are thought to alter gene expression and a coding A49G polymorphism exerts effects on post-translational processing. This review details the latest efforts in elucidating the functional mechanisms that explain the genetic association of the INS-VNTR and CTLA-4 genes with T1D.

Objective. Our study was designed to establish in children with asthma the safety profile of repeated short courses of oral glucocorticoids on bone mineralization and metabolism and adrenal function.Methods. This cross-sectional study compared the bone density, bone metabolism, and adrenal function of children who were and were not exposed to bursts of oral glucocorticoids. Children were considered exposed when, in the preceding year, they received ≥2 courses of oral glucocorticoids and were prescribed the same therapy for the index exacerbation. Children were considered unexposed when they had no exposure to oral glucocorticoids and were not prescribed any for the index exacerbation. Indices of bone metabolism were measured during the subsequent month. Cortisol responses to adrenocorticotrophic hormone stimulation and bone density were assessed 30 days after the index exacerbation.Results. Eighty-three children (48 exposed, 35 unexposed) aged 2 to 17 years were enrolled. The median exposure level was 4 courses (range: 3–11) in the preceding year. Among exposed children, a transient decrease in serum osteocalcin was observed at the end of the 5-day course with a return to baseline by 30 days; no change was observed in urine pyridinoline cross-links. Mean bone density z score was similar in the exposed (−0.61 ± 1.0 [standard deviation]) and unexposed (−0.67 ± 0.9) groups. No cases of abnormal response to adrenocorticotrophic hormone suggestive of adrenal insufficiency were documented in the exposed (95% confidence interval: 0%–7%) or unexposed (0%–10%) groups.Conclusions. Repeated short courses of oral glucocorticoids in the treatment of asthma seem to be reasonably safe; this practice was not associated with any lasting perturbation in bone metabolism, bone mineralization, or adrenal function.

After more than six years of funding exclusive projects in forestry and agriculture, Genome Canada has now announced a $67.5 million funding competition for large-scale genomics projects in human health, with focus on personalised medicine.i The human genomics community of this country understandably rejoiced at this long overdue announcement that gives them, for the first time in years, the means to compete internationally (http://www.genomecanada.ca/en/portfolio/research/2012-competition.aspx). Canada has been fairly generous in funding human-health related research but mostly through the Canadian Institutes of Health Research (CIHR) or the Canada Foundation for Innovation, neither of which has within its mandate to fund the type of specific multimillion-dollar project that is usually thought of as genomics. The typical CIHR grant, for example, rarely exceeds $1 million (200 000 over 5 years) in direct costs. The elation over the announcement of this funding opportunity in late 2011 soon gave way to sober reflection when prospective applicants started looking at the fine print. The terms of reference made it clear that this is a call for proposals with a very strong utilitarian angle, ‘capable of concrete deliverables by the end of the funding period that will have clinical utility and/or practical applicability’ and ‘social and/or economic benefits … realised within a short time-frame after the end of the project’, to quote from the official announcement. The funding period is four years (only coincidentally, I am sure, the time to the next federal election). How short the additional ‘short time-frame’ might be, is left to interpretation but the context leaves little doubt that it cannot be more than a couple of years. How else can applicants ‘… demonstrate end-user engagement in the development and execution of the research plan’? Examples of end-users whose interest must be attracted sufficiently for them to participate in the ‘execution of the research’ and be …

A common T17A polymorphism in the signal peptide of the cytotoxic T-lymphocyte antigen 4 (CTLA-4), a T-cell receptor that negatively regulates immune responses, is associated with risk for autoimmune disease. Because the polymorphism is absent from the mature protein, we hypothesized that its biological effect must involve early stages of protein processing, prior to signal peptide cleavage. Constructs representing the two alleles were compared by in vitro translation, in the presence of endoplasmic reticulum membranes. We studied glycosylation by endoglycosidase H digestion and glycosylation mutant constructs, cleavage of peptide with inhibitors, and membrane integration by ultracentrifugation and proteinase K sensitivity. A major cleaved and glycosylated product was seen for both alleles of the protein but a band representing incomplete glycosylation was markedly more abundant in the predisposing Ala allele (32.7 +/- 1.0 versus 10.6% +/- 1.2 for Thr, p10(-9)). In addition, differential intracellular/surface partitioning was studied with co-transfection of the alleles fused to distinct fluorescent proteins in COS-1 cells. By quantitative confocal microscopy we found a higher ratio of cell surface/total CTLAThr(17) versus CTLAAla(17) (p = 0.01). Our findings corroborate observations, in other proteins, that the signal peptide can determine the efficiency of post-translational modifications other than cleavage and suggest inefficient processing of the autoimmunity predisposing Ala allele as the explanation for the genetic effect.

Type 1 diabetes results from autoimmune destruction of the insulin-producing pancreatic beta-cells. Evidence from our laboratory and others has suggested that the IDDM2 locus determines diabetes susceptibility by modulating levels of insulin expression in the thymus: the diabetes-protective class III alleles at a repeat polymorphism upstream of the insulin gene are associated with higher levels than the predisposing class I. To directly demonstrate the effect of thymic insulin expression levels on insulin-specific autoreactive T-cell selection, we have established a mouse model in which there is graded thymic insulin deficiency in linear correlation with insulin gene copy numbers, while pancreatic insulin remains unaltered. We showed that mice expressing low thymic insulin levels present detectable peripheral reactivity to insulin, whereas mice with normal levels show no significant response. We conclude that thymic insulin levels play a pivotal role in insulin-specific T-cell self-tolerance, a relation that provides an explanation for the mechanism by which the IDDM2 locus predisposes to or protects from diabetes.

Recent evidence shows that Silver–Russell syndrome (SRS), the major functional deficit of which is limited growth, both intrauterine and postnatal, is due to a double dose of a gene within 7p11.2-p13 that is normally expressed exclusively from the maternal copy. Of the several growth-related genes in this chromosomal region, only GRB10 has been demonstrated to be imprinted; however, imprinting was limited to brain and muscle and was incomplete. Using reverse-transcript PCR, we now confirm GRB10 imprinting in these two tissues is isoform-specific and, more importantly, demonstrate absence of imprinting in growth plate cartilage, the tissue most directly involved in linear growth. Thus, it is unlikely that GRB10 is the gene responsible for SRS.

Type 1 diabetes results from autoimmune destruction of the insulin-producing pancreatic β cells. The insulin gene (INS) is also expressed in human thymus, an ectopic expression site likely involved in immune tolerance. The IDDM2 diabetes susceptibility locus maps to a minisatellite composed of a variable number of tandem repeats situated 0.5 kb upstream of INS. Chromosomes carrying the protective long INS variable number of tandem repeats alleles (class III) produce higher levels of thymic INS mRNA than those with the predisposing, short class I alleles. However, complete silencing of thymic INS transcripts from the class III chromosome was found in a small proportion of heterozygous human thymus samples. We hypothesized that the specific class III alleles found on these chromosomes silence rather than enhance thymic insulin expression. To test the prediction that these alleles are predisposing, we developed a DNA fingerprinting method for detecting two putative “silencing” alleles found in two thymus samples (S1, S2). In a set of 287 diabetic children and their parents we found 13 alleles matching the fingerprint of the S1 or S2 alleles. Of 18 possible transmissions, 12 of the S1–S2 alleles were transmitted to the diabetic offspring, a frequency of 0.67, significantly higher than the 0.38 seen in the remaining 142 class III alleles; P = 0.025. This confirms our prediction and represents an additional level of correlation between thymic insulin and diabetes susceptibility, which supports a thymic enhancer effect of the INS variable number of tandem repeats as the mechanism of IDDM2 and refines the contribution of IDDM2 genotyping to diabetes risk assessment.

Si/SiO(2) chips were used to detect the hybridization of immobilized Oligo d(T)(20) through impedance measurement. The immobilization procedure involved an aminopropyl silane grafted silicon oxide surface activated by glutaraldehyde and subsequently modified by an aminolinker supporting oligonucleotides. The immobilization procedure was optimized and, in the best conditions, the hybridization of the immobilized oligonucleotide was able to generate a 50 Omega impedance change at an applied dc potential of -300 mV. The optimized DNA sensor was then used to directly determine the immobilized oligonucleotide T(m) via impedance measurement in a continuous temperature control flow system. A reproducible and specific 65 Omega impedance change was observed at 32 degrees C with a step duration as low as 15 min. This value compared well with the 31.4 degrees C theoretical value calculated from the sequence base pair composition and length.

Type 1 diabetes results from the autoimmune destruction of pancreatic insulin-producing s-cells. Insulin is one of several s-cell molecules targeted as an autoantigen, but is the only autoantigen for which expression is specifically restricted to s-cells and the only autoantigen whose gene maps to a locus of genetic predisposition. In this chapter, we will review what is known about this locus and discuss the implications on the role of insulin as an autoantigen in autoimmune diabetes.

Publisher Summary This chapter discusses the programmed cell death (PCD) in the pathogenesis of autoimmune diabetes. Diabetes is one of the most common chronic diseases and a major cause of morbidity and mortality in the industrialized world. Diabetes manifests as a set of metabolic abnormalities, all of which can be attributed to insulin deficiency. This deficiency may be total and absolute (type 1 diabetes) or partial and relative to increased requirements resulting from insulin resistance. The insulin-secreting β-cells make up the bulk of the islets of Langerhans, small clusters of endocrine cells embedded within the acinae of the exocrine pancreas. Type 1 diabetes results from specific destruction of all or most of the β-cell mass over a relatively brief period of time. It is evident that this destruction has an autoimmune basis. The predisposition to autoimmune diabetes in both animal models is inherited as a polygenic trait and can be modified by a wide variety of environmental conditions. Patients with long-standing type 1 diabetes have very few or no detectable β-cells at autopsy, and that antibodies against several components of the β-cell can be detected in serum for some time before the onset of the metabolic manifestations of insulin deficiency.

Recently, we reported the localization of a gene for transient neonatal diabetes mellitus (TNDM), a rare form of childhood diabetes, to an approximately 5.4 Mb region of chromosome 6q24. We have also shown that TNDM is associated with both paternal uniparental disomy (UPD) of chromosome 6 and paternal duplications of the critical region. The sequencing of P1-derived artificial chromosome clones from within the region of interest has allowed us to further localize the gene and to investigate the methylation status of the region. The gene is now known to reside in a 300-400 kb region of 6q24 which contains several CpG islands. At one island we have demonstrated differential DNA methylation between patients with paternal UPD of chromosome 6 and normal controls. In addition, two patients with TNDM, in whom neither paternal UPD of chromosome 6 nor duplication of 6q24 have been found, show a DNA methylation pattern identical to that of patients with paternal UPD of chromosome 6. Control individuals show a hemizygous methylation pattern. These results show that TNDM can be associated with a methylation change and identify a novel methylation imprint on chromosome 6 associated with TNDM.

We have previously shown that, in contrast to its murine homologue, the human IGF2R gene is not imprinted. However, in a small number of individuals, partial or complete repression of the paternal allele has been observed and it has been speculated that in man, IGF2R imprinting is a polymorphic trait. We have confirmed monoallelic IGF2R expression in one fetus and investigated whether genomic imprinting was involved in the silencing of the paternal allele. Two CpG rich regions, known to be important for the imprinted expression of Igf2r in mice, were examined for sequence and methylation changes. A 17 bp deletion was identified within the intronic CpG island. This deletion was shown to be polymorphic and without consequence for the expression of the relevant IGF2R allele. Furthermore, in this fetus, methylation patterns of the intronic and promoter CpG islands were identical to that of normal controls, including hypomethylation of the paternal promoter region. In mice, this region is hypermethylated on the paternal allele which is silenced. The absence of paternal promoter methylation indicates that paternal silencing in this particular fetus is by a mechanism other than parental imprinting or, alternatively, that promoter methylation is not necessary for IGF2R imprinting.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a rare disorder due to defective negative feedback regulation of insulin secretion by low glucose levels, is often familial. Most cases are recessively inherited, and mutations of the sulfonylurea receptor gene (SUR) or the closely linked KIR6.2 gene have been found in several families. Both of these genes encode components of the potassium channels responsible for glucose-regulated insulin release. However, in some families recessive PHHI is not linked to the SUR-KIR6.2 locus, suggesting genetic heterogeneity. We report here a French Canadian kindred with hypoglycemia in five first cousins. All five patients had documented hypoglycemia, and all responded well to diazoxide. In two, inappropriately elevated insulin levels during hypoglycemia were documented. This familial clustering strongly suggests the existence of an autosomal dominant form of PHHI. By preliminary linkage analysis, we tested the possibility of a dominant negative SUR or KIR6.2 mutant. The insulin (INS) and glucokinase (GCK) genes were also tested as additional candidates. Microsatellite markers closely linked to each gene were used, and large negative Lod scores were obtained at the known recombination fractions between all three genes studied and the corresponding marker. We conclude that mutation of a gene other than SUR or KIR6.2 is responsible for the dominant PHHI in this family, and this gene cannot be INS or GCK. We propose that a genome-wide search for this gene is important for elucidating this rare disorder and, more importantly, for determining its potential impact on understanding noninsulin-dependent diabetes mellitus and on the effort to develop bioengineered β-cells for transplantation.

Wilms’ tumor (WT) is an embryonal renal malignancy,which overexpresses insulin-like growth factor II (IGF-II), a fetal mitogen. Relaxation of parental imprintingof IGF2, the gene encoding IGF-II, is found in Wilms’tumors, suggesting an important role for IGF2 dosagein tumorigenesis. The IGF2R gene encodes a non-mitogenic receptor which targets IGF-II to thelysosomes for degradation and, therefore, inhibits themitogenic function of IGF-II. The human IGF2R isimprinted in a proportion of normal individuals. To testthe hypothesis that IGF2R imprinting predisposes toWilms’ tumor through the e•ect of decreased IGF2Rdosage on IGF-II inactivation, we examined IGF2Rimprinting in Wilms’ tumors. Two transcribed CArepeat polymorphisms were used to distinguish the twoalleles in the RT–PCR product. We observed that in 7/16 of Wilms’ tumor patients, the paternal IGF2R wasmarkedly but not completely repressed in both tumorand normal kidney. In one additional case, IGF2R waslikewise imprinted in the tumor but not in the normalkidney. A similar imprinting was observed in fetaltissues and placenta prior to 20 weeks fetal age but notin term placenta or postnatal blood cells, indicatingabnormal persistence of a fetal pattern in the kidneys ofWilms’ patients. Genetic analysis showed association ofthe imprinting with a cis-acting locus. The highfrequency of aberrant persistence of IGF2R imprintingin the kidneys of Wilms’ tumor patients, which may bean embryonic feature, suggests that it is a predisposingfactor in tumorigenesis. This is in accordance withevidence that IGF2R is a tumour suppressor in othertypes of malignancies.Keywords: imprinting; Wilms’ tumor; insulin-likegrowth factor II; receptor; gene dosageIntroductionWilms’ tumor is a childhood kidney cancer derivedfrom metanephric blastemal cells. It is usuallydiagnosed in the first 5 years of life. At the time ofresection, these tumors express IGF2 at levelscharacteristic of fetal tissues, which are much higherthan those found in the surrounding normal kidney(Reeve et al., 1985; Scott et al., 1985). This over-expression may, at least in part, be related todisruption of the parental genomic imprinting of theIGF2 gene.The term parental imprinting refers to the differ-ential behavior of the two copies of a gene dependingon the sex of the parent from which each wastransmitted. IGF2, one of the few mammalian genesknown to be imprinted, is normally expressedexclusively from the paternal copy in the mouse(DeChiara et al., 1991) and human (Giannoukakis etal., 1993). Interestingly Igf2r, the murine gene encodingone of the two receptors to which IGF-II binds, is alsoimprinted, but with exclusive maternal expression(Barlow et al., 1991). Thus IGF2 is reversiblyinactivated upon passage from the maternal germline,while Igf2r is inactivated upon passage from thepaternal one.The presence, in opposite parental sexes, of thisunusual property in two genetically unlinked butfunctionally coupled genes may be related to opposinge•ects of Igf2 and Igf2r expression on fetal growth(Haig and Graham, 1991). It is known that themitogenic actions of IGF-II are transmitted by thetype I IGF receptor (Kiess et al., 1987; Ellis et al.,1996), which binds both IGF-I and IGF-II and acts asa ligand-activated tyrosine kinase. By contrast, IGF2Rencodes a 280 kDa membrane receptor with no knownintracellular transduction mechanism, involved in thetargeting of lysosomal enzymes to the lysosomes(reviewed by Polychronakos, 1989). In addition to itsmannose 6-phosphate binding site through which itrecognizes the unique glycosylation pattern of lysoso-mal enzymes, this receptor has a high-a†nity specificIGF-II binding site, involved in the targeting of IGF-IIfor rapid lysosomal degradation (Oka et al., 1985; Elliset al., 1996). This may explain the lower mitogenicpotency of IGF-II in most biologic systems, although itbinds to the type I receptor with a†nity equal to thatof IGF-I (Germain-Lee et al., 1992). It may also berelated to a tumor suppressor function of IGF2R (DeSouza et al., 1995a,b; Hankins et al., 1996).It is not known whether the postnatal persistence ofthe high fetal levels of IGF2 expression is of etiologicsignificance in Wilms’ tumor, or merely a marker of itsembryonal nature. In favor of a causative role is therecent discovery of three completely independentmolecular pathologies, all with the potential toincrease IGF2 expression, and all occurring with highfrequency in Wilms’ tumors: (1) Paternal disomy of11p15.5, that would result in expression of IGF2 fromtwo instead of one gene copy, is sometimes seen inWilms’ tumors (Slater and Mannens, 1992) and theBeckwith-Wiedemann syndrome (Henry et al., 1991), acondition that predisposes to Wilms’ tumors. (2) Two-thirds of Wilms’ tumors maintaining heterozygosity atthe IGF2 locus show relaxation of imprinting (Rainieret al., 1993; Ogawa et al., 1993a), allowing expression

Kevin M Sweet and Ron C Michaelis. Published by Springer Science, 2011, 211 pp., ISBN: 978-94-007-1146-4 As genetic testing increasingly enters into the daily clinic, a short reference text meticulously explaining jargon ought to be a valuable resource for the practitioner. This short volume achieves most of the objectives such a book should aim for, with particularly good coverage of cardiology and oncology. It approaches genetics in focused, well balanced detail for clinicians. Each chapter has an abstract at the beginning and small sections that make it easy to follow through, especially for the busy clinician. The book's first few chapters set a good foundation by going …

The search for expression quantitative trait loci has traditionally centred entirely on the process of transcription, whereas variants with effects on messenger RNA translation have not been systematically studied. Here we present a high-throughput approach for measuring translational cis-regulation in the human genome. Using ribosomal association as proxy for translational efficiency of polymorphic messenger RNAs, we test the ratio of polysomal/non-polysomal messenger RNA level as a quantitative trait for association with single nucleotide polymorphisms on the same messenger RNA transcript. We identify one important ribosomal distribution effect, from rs1131017 in the 5'-untranslated region of RPS26, that is in high linkage disequilibrium with the 12q13 locus for susceptibility to type 1 diabetes. The effect on translation is confirmed at the protein level by quantitative western blots, both ex vivo and after in vitro translation. Our results are a proof-of-principle that allelic effects on translation can be detected at a transcriptome-wide scale.

One of the loci encoding susceptibility to insulin-dependent diabetes mellitus (IDDM) is IDDM2, mapped to a variable number of tandem repeats (VNTR) polymorphism situated 596 bp upstream of the insulin gene (INS). The shorter alleles (class I) predispose to IDDM, while the longer class III alleles are protective. Besides INS, it is possible that transcription levels of IGF2, the nearby gene encoding the insulin-like growth factor II, may be modulated by allelic forms of the VNTR. In an effort to define the pathophysiologic mechanism of the IDDM2 effect, we examined the effect, in cis, of VNTR genotype on steady-state mRNA levels of INS in samples of human fetal pancreas, and of IGF2 in leucocytes of diabetic children. Relative levels of mRNA transcripts derived from each chromosome carrying a defined VNTR allele were measured by RT-PCR, taking advantage of transcribed polymorphisms at the 3' untranslated region of each gene. In 10 samples of human fetal pancreas, INS transcripts from chromosomes carrying a class III VNTR were slightly but significantly (P = 0.015) lower than those from class I (13% lower, 95% confidence limits 3-21%). In 10 leucocyte samples, mRNA from both IGF2 alleles was seen, indicating relaxation of the parental imprinting of IGF2 in these cells. However, this relaxation was incomplete as maternal allele mRNA was systematically at a lower level than paternal. The paternal/maternal ratio varied widely among individual subjects. Two of the most extreme cases, demonstrating almost complete repression of the maternal allele, were identical twins, suggesting that this variable relaxation of imprinting is genotype-dependent. However, this genotype-dependence cannot be accounted for by the maternal VNTR, as the mean ratios of paternal/maternal IGF2 mRNA levels were not statistically different in individuals with a maternal VNTR of class I vs. class III (3.2 +/- 1.5 vs. 3.89 +/- 0.94). Thus, we present evidence that: (a) class III VNTR alleles are associated with lower INS mRNA in fetal pancreas than class I alleles. The biologic importance of this difference remains to be determined; and (b) the variable relaxation of IGF2 imprinting seen in human leucocytes is not dependent on the presence of a class I vs. a class III VNTR.

In most non-neoplastic tissues studied to date, IGF2 is expressed only from the paternal allele and H19 is expressed only from the maternal allele. The choroid plexus, the only normal tissue to date where IGF2 is expressed from both parental alleles, does not express H19. We present an additional situation in which biallelic IGF2 expression is associated with the absence of H19 transcription in normal tissue: blood cells. In blood cells, functional IGF2 imprinting was found to be a polymorphic trait among individuals: expression was biallelic in 79 out of 85 individuals, but the remaining 6 expressed a single allele. Only the latter expressed H19. Finally, the familial clustering of functional IGF2 imprinting in blood cells suggests that the trait may be genotype-dependent.

If you are wondering why a Polychronakos would have any personal interest in the existence and universal adoption of a code that uniquely and unambiguously identifies authors of scientific papers, just do a PubMed search on my surname without my initial. There are four of us currently publishing: in addition to my C there is my brother V (an elementary-particle physicist), cousin A (ophthalmology) and another A, a prolific physicist whom I have never met. This does not include my late cousin D whose publications in obscure German or Greek journals can still be found at the chronological fringes of the PubMed search, or my father (yet another A) whose papers in French and Greek radiology …

One of the basic tenets of Mendelian genetics is that autosomal traits are equivalently transmitted from each of the two parents. In recent years the molecular corollary of this has also been demonstrated for the majority of the genes studied: Both copies of each autosomal gene are equivalently expressed at the mRNA level, regardless of the parent from which each was derived.

The power of massively parallel sequencing (MPS) combined with target enrichment technologies has led, in the space of barely 2 years, to a true revolution in our ability to explore the genome for sequence changes responsible for phenotypes of interest. While the resequencing efforts in search of low-frequency variants involved in complex traits have only generated hopes to date, targeted sequencing can claim one resounding success: the rate at which reports are appearing that identify the gene mutated in rare monogenic diseases is astounding and growing by the month. The sequencing of coding exons and adjacent splicing elements, sites of the vast majority of mutations responsible for Mendelian diseases, is now a routine and relatively inexpensive procedure, blurring the distinction between discovery and service to the individual. Of the several examples of exome sequencing results highlighted in this issue, the report by Majewski et al 1 is a case in point. An adult female with a solid clinical diagnosis of Leber's congenital amaurosis (LCA) …

It is difficult to believe how astonishing the idea appeared, only five short years ago, that megabase-scale structural changes in the genome, until then believed to be confined to rare contiguous-gene syndromes, are actually plentiful in phenotypically healthy individuals.1 Routine use of high-resolution array-CGH and, more recently, massively parallel sequencing is revealing a pattern of structural variation in the general population that, in numbers of nucleotides affected, rivals the ubiquitous SNPs. An ever increasing number of pathologies are now associated with copy-number variation on the basis of evidence whose rigour tends to vary among studies. As might be expected, JMG has been receiving increasing numbers of submissions linking copy-number variations to phenotypes and we have been happy to publish those that meet our standards for proof of causality. In …

Type 1 diabetes (T1D) is a strongly heritable autoimmune disease due to the destruction of pancreatic β-cells by self-reactive T-cells. The third strongest genetic locus for T1D involves a single-nucleotide polymorphism (SNP) causing an arginine(R) to triptophan(W) substitution at codon 620 (620R>W) in PTPN22, the gene encoding the tyrosine phosphatase LYP, an important negative regulator of T-cell receptor (TCR) activation. However, the cellular effects of the 620R>W risk allele remain controversial, as different studies report either a gain or a loss of function in its ability to inhibit TCR signaling. To study this variant in an animal model, we created a knock-in mouse by introducing the 620R>W substitution in, the murine LYP orthologue, PEP encoded by Ptpn22. The knock-in analyzed on the autoimmune-resistant C57Bl/6 background showed a mild but significant increase in CD4+ helper T-cell activation and proliferation upon weak TCR activation. Conversely, upon strong TCR stimulation, the tendency seemed to be reversed as the 620W allele showed a slightly decreased responsiveness compared to the 620R allele. Phenotypic analysis of other immune cellular compartments, including memory T cells, showed no significant difference between genotypes. There was no evidence of autoimmunity in this genetic background. This dependence of allelic effect on the strength of the TCR signal may reconcile previous observations and indicate a complex role of R620>W in autoimmunity.

The murine genes coding for insulin-like growth factor II (Igf2) and its specific receptor (Igf2r) are parentally imprinted, with exclusive expression from the paternal (Igf2) or maternal (Igf2r) gene copy. We have demonstrated that the human IGF2 gene is imprinted, like its murine homologue. To examine whether the human IGF2R is also imprinted, we used CA repeat polymorphisms of the cDNA sequence to distinguish expression from each copy. Unlike the mouse, most subjects equally expressed both gene copies. However, two out of 14 informative fetuses showed exclusive expression from the maternal gene copy. We conclude that the human IGF2R gene is parentally imprinted, like its murine homologue, but only in a minority of individuals. This is the first direct demonstration of imprinting as a polymorphic trait, a property that had been observed in transgene methylation and predicted from the behavior of certain human tumors.

fructose corn syrup (80HFCS), 56g HFCS plus 24g inulin (inulin), or 56g HFCS (56HFCS) using a randomized, single-blind, cross-over design. A standard lunch was served 4 hours after the test drink. The treatments were designed to distinguish between the effects of adding inulin to HFCS (56HFCS vs. Inulin) or partially substituting inulin for HFCS (80HFCS vs. Inulin). The addition or substitution of inulin did not alter glucose and insulin responses. Serum acetate, propionate and butyrate were significantly higher after Inulin than both 56HFCS and 80HFCS beginning at 4 hours. FFAs fell at a similar rate following all test drinks but Inulin resulted in significantly lower serum FFA at 4 hours compared to 56HFCS. Plasma GLP-1 was higher 30 minutes after inulin than 56HFCS, while plasma ghrelin was significantly lower 4, 4.5 and 6 hours after Inulin than 56HFCS and 80HFCS. The results of the study support the hypothesis that SCFA generated from the colonic fermentation of dietary fibre can influence serum FFA and certain gut hormones involved in the regulation of body weight. Therefore, they may provide a link between dietary fibre intake and prevention of type 2 diabetes through a SCFA-mediated reduction in insulin resistance, a reduction in food intake and/or obesity. This work was supported by the Canadian Institutes of Health Research.

Four transplantable cell lines (PA-I, II, III, and IV) derived from four Lobund-Wistar (L-W) rats that manifested spontaneous prostate cancer have demonstrated metastatic capacity in visceral organs. Interestingly, PA-III cells, when deposited over the scapula or calvarium of the Lobund-Wistar rat, could produce lytic and blastic reactions on rat skeleton. Since growth factors and growth factor receptors have been implicated in bone remodeling, cancer biology, and metastatic growth of cancer cells, we have examined 1) the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on the proliferation of PA-III cells; and 2) the presence of specific receptors for these peptides. IGF-I (0.5 to 100 ng/ml), IGF-II (0.5 to 100 ng/ml), and insulin (0.5 to 10 micrograms/ml) stimulated tritiated thymidine uptake and increased the number of PA-III cells in culture. Receptor studies demonstrated the presence of specific bindings sites for IGF-I and II but not for insulin. The number and affinity of the receptor sites were: IGF-I (nb = 675 fmol/100 g protein, Kd = 0.56 nmol) and IGF-II (nb = 225 fmol/100 g protein, Kd = 0.71 nmol). Molecular characterization of IGF binding sites by polyacrylamide gel electrophoresis under denaturing conditions indicated only the presence for the type I IGF receptor. The presence of the IGF-I receptor and the absence of IGF-II and insulin receptors are discussed in relation to the capacity of PA-III cells to produce bone lesions on the L-W rat.

Insulinlike growth factor I (IGF-I) has been shown to be a potent mitogen for breast cancer cells in vitro, and IGF-I receptors have been demonstrated on human primary breast neoplasms. In a randomized, placebo-controlled study, we document that administration of the antiestrogen tamoxifen to patients with breast cancer was associated with a statistically significant (P = .002) reduction in the serum level of IGF-I. The mean IGF-I level was 1.4 U/mL in the placebo-treated group and 0.9 U/mL in the tamoxifen-treated group. Because serum IGF-I level is growth hormone (GH) dependent and because data suggest that the pubertal surge in GH and IGF-I levels is sex steroid dependent, we speculate that the mechanism underlying our observation may involve blockade by tamoxifen of estrogen action in the hypothalamic-pituitary axis. We conclude that tamoxifen treatment reduces IGF-I levels and that this reduction may contribute to the therapeutic effect of the drug.

The insulin-like growth factor-II (IGF-II) and lysosomal enzymes containing a mannose-6-phosphate (M6P) recognition site bind to different sites of the same receptor molecule. We have observed that M6P increases the receptor-mediated uptake of IGF-II into IM-9 cells. We now confirm this phenomenon in a different line, the H-35 rat hepatoma cells, and present additional characterization of the underlying mechanisms. When incubated in the presence of radiolabeled IGF-II, H-35 cells accumulated, in a time-dependent fashion, radioactivity that was resistant to removal by trypsin digestion at 15 C, indicating that it was endocytosed. In the presence of 3 mM M6P, endocytosed counts were approximately 2-fold higher after 5 min of incubation, an enhancement that peaked at 10 min, then declined, but was still evident after 40 min (1.5-fold). The rate of release of cell-associated IGF-II, degraded or intact, as measured in a chase experiment, was not affected by M6P. These observations indicate that M6P increased accumulation of IGF-II by accelerating its rate of endocytosis rather than by interfering with IGF-II degradation or with the recycling of intact hormone-receptor complexes to the cell surface. Electrophoresis after affinity cross-linking of labeled cells demonstrated that the enhancement in radioactivity could be located at a molecular size of approximately 250 kDa, corresponding to IGF-II-receptor complexes. Preincubation with M6P did not significantly alter the specific binding of IGF-II to the cell surface of H-35 cells, as measured by a binding assay at 4 C. Finally, pretreatment with cycloheximide for up to 8 h, to remove all newly synthesized lysosomal enzymes bound to the M6P/IGF-II receptor, did not affect IGF-II endocytosis beyond what could be accounted for by some loss of receptor, suggesting that the observed effect of M6P is due to the binding of M6P itself to the receptor and not to displacement of lysosomal enzymes. We conclude that simultaneous occupancy of the M6P/IGF-II receptor by ligands on both binding sites enhances its rate of endocytosis.

Rodriguez et al. (1) are right in pointing out that our findings (2) do not rule out with absolute certainty a role for rs689 in type 1 diabetes through the effects on splicing under conditions not reflected in the tissues we examined. In biological science, absolute certainty is difficult to attain. …