Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Functional evaluation of the role of C-type lectin domain family 16A at the chromosome 16p13 locus.

The type 1 diabetes-associated 16p13 locus contains the CLEC16A gene. Its preferential immune cell expression suggests involvement in autoimmunity. Given its elevated expression in dendritic and B cells - known professional antigen-presenting cells (APCs) - we hypothesize that C-type lectin domain family 16 member A (CLEC16A) may be involved in T cell co-stimulation and consequent activation and proliferation. We also sought to identify CLEC16A's subcellular localization. The effect of the CLEC16A knock-down (KD) on B cell co-stimulation and activation of T cells was tested in human lymphoblastoid cell lines (LCLs) by co-culture with CD4(+) T cells. T cell activation and proliferation were determined by flow-cytometric analysis of CD69 and CD25 expression and carboxyfluorescein succinimidyl ester (CFSE) dilution, respectively. CLEC16A subcellular localization in K562 cells was examined by immunofluorescence. We show that the CLEC16A KD did not affect the tested indices of lymphoblastoid cell line (LCL) APC capacity. Additionally, the percentage of activated T cells following LCL co-culture was not affected significantly by the CLEC16A KD. T cells co-cultured with KD or control LCLs also exhibited similar cell division profiles. CLEC16A co-localized with an endoplasmic reticulum (ER) marker, suggesting that it may be an ER protein. In conclusion, CLEC16A may not be involved in T cell co-stimulation. Additional studies on CLEC16A, accounting for its ER localization, are needed to uncover its biological role.

A new paradigm emerges from the study of de novo mutations in the context of neurodevelopmental disease.

The study of de novo point mutations (new germline mutations arising from the gametes of the parents) remained largely static until the arrival of next-generation sequencing technologies, which made both whole-exome sequencing (WES) and whole-genome sequencing (WGS) feasible in practical terms. Single nucleotide polymorphism genotyping arrays have been used to identify de novo copy-number variants in a number of common neurodevelopmental conditions such as schizophrenia and autism. By contrast, as point mutations and microlesions occurring de novo are refractory to analysis by these microarray-based methods, little was known about either their frequency or impact upon neurodevelopmental disease, until the advent of WES. De novo point mutations have recently been implicated in schizophrenia, autism and mental retardation through the WES of case-parent trios. Taken together, these findings strengthen the hypothesis that the occurrence of de novo mutations could account for the high prevalence of such diseases that are associated with a marked reduction in fecundity. De novo point mutations are also known to be responsible for many sporadic cases of rare dominant mendelian disorders such as Kabuki syndrome, Schinzel-Giedion syndrome and Bohring-Opitz syndrome. These disorders share a common feature in that they are all characterized by intellectual disability. In summary, recent WES studies of neurodevelopmental and neuropsychiatric disease have provided new insights into the role of de novo mutations in these disorders. Our knowledge of de novo mutations is likely to be further accelerated by WGS. However, the collection of case-parent trios will be a prerequisite for such studies. This review aims to discuss recent developments in the study of de novo mutations made possible by technological advances in DNA sequencing.

Parental genomic imprinting of the human IGF2 gene.

The mouse igf2 gene, coding for the insulin-like growth factor II (IGF-II) is parentally imprinted, only the gene copy derived from the father is expressed. To know whether IGF2, the human homologue, is also imprinted, we used an ApaI polymorphism at the 3' untranslated region in order to distinguish between mRNA derived from each copy of the gene in placentae from heterozygote human fetuses, studied after careful removal of the decidua. Six term and two pre-term placentae of heterozygotes were studied, and in each case the cDNA contained only one of the two alleles present in the genomic DNA. In three cases the mother was homozygous for the non-expressed allele, allowing assignment of paternal origin to the transcribed gene copy. We conclude that, as in the mouse, human IGF2 is parentally imprinted.

Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus.

Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is due to autoimmune destruction of pancreatic beta-cells. Genetic susceptibility to IDDM is encoded by several loci, one of which (IDDM2) maps to a variable number of tandem repeats (VNTR) minisatellite, upstream of the insulin gene (INS). The short class I VNTR alleles (26-63 repeats) predispose to IDDM, while class III alleles (140-210 repeats) have a dominant protective effect. We have reported that, in human adult and fetal pancreas in vivo, class III alleles are associated with marginally lower INS mRNA levels than class I, suggesting transcriptional effects of the VNTR. These may be related to type 1 diabetes pathogenesis, as insulin is the only known beta-cell specific IDDM autoantigen. In search of a more plausible mechanism for the dominant effect of class III alleles, we analysed expression of insulin in human fetal thymus, a critical site for tolerance induction to self proteins. Insulin was detected in all thymus tissues examined and class III VNTR alleles were associated with 2- to 3-fold higher INS mRNA levels than class I. We therefore propose higher levels of thymic INS expression, facilitating immune tolerance induction, as a mechanism for the dominant protective effect of class III alleles.

Insulin VNTR allele-specific effect in type 1 diabetes depends on identity of untransmitted paternal allele. The IMDIAB Group.

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 effect of the MHC locus on autoantibodies in type 1 diabetes.

OBJECTIVE: To investigate whether the presence of autoantibodies specific for type 1 diabetes (T1D) is determined by the major genetic susceptibility locus for the disease at the HLA genes, using the T1D Genetics Consortium data. METHODS: We analysed anti-IA-2 and anti-GAD 65 autoantibody data from 2282 T1D patients from 1117 multiplex families. HLA genotyping was available for all cases and their parents and association with autoantibody positivity was tested by the transmission disequilibrium test. RESULTS: Association of anti-IA-2 with the HLA genes was detected at high statistical significance. HLA-DRB1*0401 confers both the strongest type 1 diabetes risk, and positive association of anti-IA-2, whereas the DRB1*03- DQA1*0501-DQB1*0201 haplotype, associated less strongly with T1D, showed a significant negative association with anti-IA-2 positivity. Interestingly, HLA-A*24 is also negatively associated with anti-IA-2, independently of the DRB1*03- DQA1*0501-DQB1*0201 haplotype. No statistically significant association was identified between anti-GAD65 and HLA. CONCLUSIONS: This study highlights that IA-2 as an autoantigen depends on HLA genotype and suggests new insights into the mechanism of loss of immune tolerance.

Association of RASGRP1 with type 1 diabetes is revealed by combined follow-up of two genome-wide studies.

BACKGROUND: The two genome-wide association studies published by us and by the Wellcome Trust Case-Control Consortium (WTCCC) revealed a number of novel loci, but neither had the statistical power to elucidate all of the genetic components of type 1 diabetes risk, a task for which larger effective sample sizes are needed. METHODS: We analysed data from two sources: (1) The previously published second stage of our study, with a total sample size of the two stages consisting of 1046 Canadian case-parent trios and 538 multiplex families with 929 affected offspring from the Type 1 Diabetes Genetics Consortium (T1DGC); (2) the Rapid Response 2 (RR2) project of the T1DGC, which genotyped 4417 individuals from 1062 non-overlapping families, including 2059 affected individuals (mostly sibling pairs) for the 1536 markers with the highest statistical significance for type 1 diabetes in the WTCCC results. RESULTS: One locus, mapping to a linkage disequilibrium (LD) block at chr15q14, reached statistical significance by combining results from two markers (rs17574546 and rs7171171) in perfect LD with each other (r2 = 1). We obtained a joint p value of 1.3 x 10(-6), which exceeds by an order of magnitude the conservative threshold of 3.26 x 10(-5) obtained by correcting for the 1536 single nucleotide polymorphisms (SNPs) tested in our study. Meta-analysis with the original WTCCC genome-wide data produced a p value of 5.83 x 10(-9). CONCLUSIONS: A novel type 1 diabetes locus was discovered. It involves RASGRP1, a gene known to play a crucial role in thymocyte differentiation and T cell receptor (TCR) signalling by activating the Ras signalling pathway.

Reassessment of the type I diabetes association of the OAS1 locus.

To reassess the type I diabetes (T1D) association of the OAS1 locus, the Type I Diabetes Genetics Consortium (T1DGC) genotyped 11 tag single-nucleotide polymorphisms spanning approximately 41 kb from the 5' to 3' flanking region. For each sample obtained from over 2000 affected sib-pair families from nine cohorts, the genotyping was performed on both the Illumina Golden Gate and Sequenom iPlex platforms. The data suggest that there may be a weak association with T1D for two OAS1 polymorphisms, rs3741981 and rs10774671, in populations of European descent. The OAS1 locus is close to a recently identified T1D-associated linkage disequilibrium (LD) block in human chromosome 12q24. Extended LD in populations earlier examined may account for the prior observation of an association of T1D with OAS1 variants. This possibility needs to be addressed further by fine mapping of the T1D association represented in 12q24.

Remapping the type I diabetes association of the CTLA4 locus.

The Type I Diabetes Genetics Consortium genotyped 24 single-nucleotide polymorphisms (SNPs) in the CTLA4 locus in 2298 type I diabetes (T1D) nuclear families (11 159 individuals, 5003 affected) to evaluate the recognized T1D association. The 24 CTLA4 SNPs span approximately 43 kb from the 5' flanking to 3' flanking region of the gene in the middle of an extended region of linkage disequilibrium of more than 100 kb. The genotyping was performed using two technologies (Illumina GoldenGate and Sequenom iPlex) on the same samples. The genotype calls by both the methods were highly consistent (the majority >99%). Previously reported T1D association from both the +49G>A and the CT60 SNPs was replicated. The reported association of the -319C>T SNP was not replicated. Although associated with T1D risk, it is likely that neither SNP is causative, as the peak of T1D association was from the SNP rs231727 at 3' flanking of the CTLA4 gene. Comprehensive resequencing and fine mapping of the CTLA4 region are still needed to clarify the causal variants.

The type I diabetes association of the IL2RA locus.

To confirm and fine map previous reports of association, the Type I Diabetes (T1D) Genetics Consortium (T1DGC) assembled a large collection of DNA samples from affected sib-pair (ASP) families with T1D (5003 affected individuals) and genotyped polymorphic markers. One of these loci, involving the IL2RA gene, had been reported to be due to three independent effects. The T1DGC genotyped 69 single-nucleotide polymorphisms (SNPs) that span approximately 88 kb from the 5' flanking to 3' flanking region of the IL2RA locus. The most highly associated SNP reported earlier (ss52580101) was not included in the genotyping list; however, a 5-SNP (rs3134883, rs3118470, rs7072793, rs4749955 and rs12251307) haplotype (H5) was identified that strongly tagged its minor allele with r(2)=0.869 (95% CI, 0.850-0.885). This haplotype was significantly protective (P=3.2 x 10(-5)) in the T1D ASP families, with an odds ratio virtually identical to that reported for ss52580101. The SNP marking the second independent locus, (rs11594656) showed no association in the T1DGC set and the third (rs2104286) could not be distinguished, by conditional regression, from H5. Instead, the most significant independent effect was detected from the 5' flanking IL2RA SNP rs4749955, which remained significant after regression for H5. Thus, we confirm independent effects at the IL2RA locus.

rs2476601 T allele (R620W) defines high-risk PTPN22 type I diabetes-associated haplotypes with preliminary evidence for an additional protective haplotype.

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is the third major locus affecting risk of type I diabetes (T1D), after HLA-DR/DQ and INS. The most associated single-nucleotide polymorphism (SNP), rs2476601, has a C->T variant and results in an arginine (R) to tryptophan (W) amino acid change at position 620. To assess whether this, or other specific variants, are responsible for T1D risk, the Type I Diabetes Genetics Consortium analyzed 28 PTPN22 SNPs in 2295 affected sib-pair (ASP) families. Transmission Disequilibrium Test analyses of haplotypes revealed that all three haplotypes with a T allele at rs2476601 were overtransmitted to affected children, and two of these three haplotypes showed statistically significant overtransmission (P=0.003 to P=5.9E-12). Another haplotype had decreased transmission to affected children (P=3.5E-05). All haplotypes containing the rs2476601 T allele were identical for all SNPs across PTPN22 and only varied at centromeric SNPs. When considering rs2476601 'C' founder chromosomes, a second haplotype (AGGGGC) centromeric of PTPN22 in the C1orf178 region was associated with protection from T1D (odds ratio=0.81, P=0.0005). This novel finding requires replication in independent populations. We conclude the major association of PTPN22 with T1D is likely due to the recognized non-synonymous SNP rs2476601 (R620W).

Regulation of insulin gene expression by cytokines and cell-cell interactions in mouse medullary thymic epithelial cells.

AIMS/HYPOTHESIS: The expression of tissue-specific self-antigens in the thymus is essential for self-tolerance. Genetic susceptibility to type 1 diabetes correlates inversely with thymic insulin expression and, in mice, lowered levels of this expression result in T cell responses against insulin. This study was undertaken to examine whether thymic insulin expression is regulated by the same metabolic stimuli as in beta cells or by different inputs, possibly of an immune nature. METHODS: Ins2 mRNA changes in mouse thymus were evaluated in vivo, following intraperitoneal glucose injection. We also examined the effect of a high glucose concentration on Ins2 mRNA in clones of insulin-expressing medullary thymus epithelial cell lines (mTECs). The same in vitro system was used to evaluate the effect of IFN-gamma and cell-to-cell contact with thymocytes in co-culture. RESULTS: Ins2 mRNA was significantly increased in the pancreas following a glucose load, but remained unchanged in the thymus. Furthermore, stimulation of insulin-expressing mTECs in vitro with IFN-gamma, a cytokine involved in T cell negative selection, decreased levels of insulin expression even though expression of Aire was increased. Last, co-culture of mTECs with thymocytes resulted in an upregulation of both Aire and insulin expression. CONCLUSIONS/INTERPRETATION: We conclude that regulation of insulin transcription in the thymus is not dependent on metabolic stimuli but it may, instead, be under the control of cytokines and cell-to-cell interactions with lymphoid cells. That this regulation is not always coordinated with that of Aire, a non-specific master switch, suggests insulin-specific mechanisms.

The association between type 1 diabetes and the ITPR3 gene polymorphism due to linkage disequilibrium with HLA class II.

A fine mapping study of the MHC region in a Swedish case-control population sample reported a novel type 1 diabetes (T1D) association from the inositol 1-, 4-, 5-trisphosphate receptor type 3 gene (ITPR3) in a case-control study, reportedly independent of the HLA class II effect. We attempted to replicate this novel association in a family-based study of 1120 T1D families with at least one affected child, an approach immune to population stratification. We found association of the ITPR3 single nucleotide polymorphisms (SNPs) rs2296336 with T1D but in a direction opposite to that reported. Moreover, rs2296336 was in linkage disequilibrium (LD) with specific alleles of the HLA DQB1 gene. Conditional regression showed that all of the ITPR3 SNP T1D association could be accounted for by the DQB1 effect. Therefore, our findings do not support an obvious role of genetic variation of the ITPR3 gene in T1D risk.

Advances in type I diabetes associated tolerance mechanisms.

Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells by autoreactive T cells. The polygenic trait for T1D risk implicates many genes that have an impact on fundamental immunological processes such as central and peripheral tolerance. Several pieces of evidence have suggested that many of the genetic loci that are directly linked to type 1 diabetes susceptibility modulate the generation and/or the activation of autoreactive T-lymphocytes. We and others have proposed a critical role for medullary thymic epithelial cells (mTEC) forming the Hassall's corpuscles in T-cell tolerance. Indeed, mTEC have been found to express promiscuous self-antigens, used directly or through thymic dendritic cells to drive either negative selection of insulin-reacting precursors or their differentiation into naturally occurring regulatory Foxp3+ CD4+ CD25+ T cells. In the periphery, naturally occurring Foxp3+ CD4+ CD25+regulatory T (Treg) cells represent the master cells in dominant peripheral T-cell tolerance. The development and function of Treg cells are ultimately linked to IL-2 and Foxp3 expression. This review addresses recent literature and emerging concepts of central and peripheral T-cell tolerance with regards to T1D.

Minor contribution of SMAD7 and KLF10 variants to genetic susceptibility of type 2 diabetes.

BACKGROUND: Transgenic mice over-expressing SMAD7 in pancreatic beta-cells develop type 2 diabetes (T2D). The expression of SMAD7 is affected by KLF11, which contains gene variants that have previously been shown to be involved in genetic susceptibility to T2D, and by the highly homologous KLF10. This study aims to assess the genetic contribution of SMAD7 and KLF10 gene variants to T2D susceptibility in the French population. METHODS: We screened both genes to identify rare and frequent variants by direct sequencing and then genotyped these variants. Six frequent variants of SMAD7 and six of KLF10 were analyzed in 349 T2D patients and 349 normoglycaemic adult subjects. Variants with statistically significant differences in allele and/or genotype distribution were further analyzed in a population sample of 1.712 T2D patients and 1.072 normoglycaemic subjects. RESULTS: Two variants showed a significant association under a recessive model: The intronic SMAD7 IVS2 -21 had an odds ratio of 0.62 (P=0.007, 95% CI=0.44-0.88; P=0.034 when adjusting for age, sex and BMI by logistic regression), and the KLF10 3'UTR +1002 variant had an Odds Ratio of 0.81 (P=0.009, 95% CI=0.69-0.95; P=0.042 when adjusting for age, sex and BMI). CONCLUSION: Although the observed association of SMAD7 and KLF10 gene variants with T2D is modest, they may weakly contribute to a particular genetic background that increases the susceptibility to development of T2D.

Type 1 diabetes and the OAS gene cluster: association with splicing polymorphism or haplotype?

BACKGROUND: The 2',5'-oligoadenylate synthetase genes (OAS1, OAS2, and OAS3) map to human chromosome 12q24 and encode a family of enzymes pivotal to innate antiviral defence. Recently, the minor allele of an OAS1 single nucleotide polymorphism (SNP) that alters splicing (rs10774671) was found to be associated with increased enzymatic activity and, in a case-sibling control study, with type 1 diabetes (T1D). METHODS: We have confirmed this T1D association in 784 nuclear families (two parents and at least one affected offspring) by the transmission disequilibrium test (TDT; G:A = 386:329, p = 0.033). However, because of linkage disequilibrium within OAS1 and with the other two OAS genes, functional attribution of the association to this SNP cannot be assumed. To help answer this question, we also genotyped two non-synonymous SNPs in OAS1 exons 3 and 7. RESULTS: All three SNPs showed significant transmission distortion. Three of the eight possible haplotypes accounted for 98.4% of parental chromosomes and two of them carried the non-predisposing A allele at rs10774671. Parents heterozygous for these two haplotypes showed significant transmission distortion (p = 0.009) despite being homozygous at rs10774671. CONCLUSIONS: We confirm the T1D association with rs10774671, but we conclude that it cannot be attributed (solely) to the splicing variant rs10774671. A serine/glycine substitution in OAS1 exon 3 is more likely a functional variant.

Impedance-based detection of DNA sequences using a silicon transducer with PNA as the probe layer.

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.

Insulinlike growth factor I: a potent mitogen for human osteogenic sarcoma.

Insulinlike growth factor I (IGF-I) is among the peptide mitogens that play key roles in the regulation of normal skeletal growth. To investigate the possibility that certain skeletal neoplasms retain a sensitivity to mitogenic stimulation by IGF-I, we studied the effects of this growth factor on human osteosarcoma. Competitive-binding assays and affinity-labeling experiments on membranes prepared from MG-63 immortalized human osteosarcoma cells and primary human osteogenic sarcoma cells demonstrate the presence of specific IGF-I receptors. Furthermore, we show that IGF-I is a potent stimulator of proliferation of MG-63 cells in vitro and is active at concentrations as low as 10(-10) M. A blocking antibody against the IGF-I receptor (alpha-IR3) significantly reduces IGF-I-stimulated proliferation in a dose-dependent manner. These results are consistent with the hypothesis that at least a subset of human osteogenic sarcomas are responsive to IGF-I and indicate that it may be possible to exploit this responsiveness therapeutically.

Effect of tamoxifen on serum insulinlike growth factor I levels in stage I breast cancer patients.

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.