Identification of a Linkage Disequilibrium Block in Chromosome 1q Associated With BMD in Premenopausal White Women

Osteoporosis is a complex disease with both genetic and environmental risk factors. A major determinant of osteoporotic fractures is peak BMD obtained during young adulthood. We previously reported linkage of chromosome 1q (LOD = 4.3) with variation in spinal areal BMD in healthy premenopausal white women. In this study, we used a two‐stage genotyping approach to identify genes in the linked region that contributed to the variation of femoral neck and lumbar spine areal BMD. In the first stage, 654 SNPs across the linked region were genotyped in a sample of 1309 premenopausal white women. The most significant evidence of association for lumbar spine (p = 1.3 × 10^?6) was found with rs1127091 in the GATAD2B gene. In the second stage, 52 SNPs around this candidate gene were genotyped in an expanded sample of 1692 white women. Significant evidence of association with spinal BMD (p < 10^?5), and to a lesser extent with femoral neck BMD, was observed with eight SNPs within a single 230‐kb linkage disequilibrium (LD) block. The most significant SNP (p = 3.4 × 10^?7) accounted for >2.5% of the variation in spinal BMD in these women. The 230‐kb LD block contains 11 genes, but because of the extensive LD, the specific gene(s) contributing to the variation in BMD could not be determined. In conclusion, the significant association between spinal BMD and SNPs in the 230‐kb LD block in chromosome 1q indicates that genetic factor(s) in this block plays an important role in peak spinal BMD in healthy premenopausal white women.     

Mapping of Quantitative Ultrasound of the Calcaneus Bone to Chromosome 1 by Genome-Wide Linkage Analysis

Quantitative ultrasound (QUS) may predict the risk of fracture independent of bone density. The aim of this study was to identify, using quantitative trait linkage analysis, chromosomal regions that might contain genes influencing variation in calcaneal ultrasound measures in a set of families from the general population. A genome-wide autosomal scan was conducted in 324 Caucasian families (1270 measured individuals) from the Framingham Osteoporosis Study, using a set of 401 Marshfield microsatellite markers with a 10 cM average density map. QUS measurements included broadband ultrasound attenuation (BUA), speed of sound (SOS), and quantitative ultrasound index (QUI). These phenotypes were regressed on age, age², body mass index, height, alcohol and caffeine consumption, smoking status, physical activity, and estrogen use in females, in each sex and generation separately. Adjusted QUS phenotypes demonstrated a strong heritability ranging from 0.45 (SOS) to 0.52 (BUA). By two-point variance components genome screening, phenotype-specific regions of possible linkage were identified on chromosomal regions 1p36.3 and 5p15.2. The maximum LOD score attained was 2.74 for BUA with D1S468 (4 cM) and 2.69 for SOS with D5S817 (23 cM). QUI, a linear combination of the SOS and BUA, showed linkage with both markers (LOD = 2.1 with D1S468 and LOD = 2.2 with D5S817). Results of two-point analysis were confirmed by multipoint linkage analysis only for BUA, with LOD = 2.4 at D1S468, but not for SOS or QUI. The results for QUS, adjusted for femoral and lumbar spinal bone mineral density, in addition to the above covariates, were virtually the same. In conclusion, our results suggest that there may be genetic determinants for BUA on 1p36.3. These results should encourage further investigations of the genetic source of QUS variability and candidate polymorphisms in this region. Received: 7 March 2002 / Accepted: 14 May 2002     

Novel Linkage Peaks Discovered for Diabetic Nephropathy in Individuals With Type 1 Diabetes

Genome-wide association studies (GWAS) and linkage studies have had limited success in identifying genome-wide significantly linked regions or risk loci for diabetic nephropathy (DN) in individuals with type 1 diabetes (T1D). As GWAS cohorts have grown, they have also included more documented and undocumented familial relationships. Here we computationally inferred and manually curated pedigrees in a study cohort of >6,000 individuals with T1D and their relatives without diabetes. We performed a linkage study for 177 pedigrees consisting of 452 individuals with T1D and their relatives using a genome-wide genotyping array with >300,000 single nucleotide polymorphisms and PSEUDOMARKER software. Analysis resulted in genome-wide significant linkage peaks on eight chromosomal regions from five chromosomes (logarithm of odds score >3.3). The highest peak was localized at the HLA region on chromosome 6p, but whether the peak originated from T1D or DN remained ambiguous. Of other significant peaks, the chromosome 4p22 region was localized on top of ARHGAP24, a gene associated with focal segmental glomerulosclerosis, suggesting this gene may play a role in DN as well. Furthermore, rare variants have been associated with DN and chronic kidney disease near the 4q25 peak, localized on top of CCSER1.     

Linkage and association with type 1 diabetes on chromosome 1q42

Type 1 diabetes is a complex disorder with multiple genetic loci and environmental factors contributing to disease etiology. In the current study, a human type 1 diabetes candidate region on chromosome 1q42 was mapped at high marker density in a panel of 616 multiplex type 1 diabetic families. To facilitate the identification and evaluation of candidate genes, a physical map of the 7-cM region surrounding the maximum logarithm of odds (LOD) score (2.46, P = 0.0004) was constructed. Genes were identified in the 500-kb region surrounding the marker yielding the peak LOD score and evaluated for polymorphism by resequencing. Single-nucleotide polymorphisms (SNPs) identified in these genes as well as other anonymous markers were tested for allelic association with type 1 diabetes by both family-based and case-control methods. A haplotype formed by common alleles at three adjacent markers (D1S225, D1S2383, and D1S251) was preferentially transmitted to affected offspring in type 1 diabetic families (nominal P = 0.006). These findings extend the evidence supporting the existence of a type 1 diabetes susceptibility locus on chromosome 1q42 and identify a candidate region amenable to positional cloning efforts.     

Effect of Endogenous GLP-1 on Insulin Secretion in Type 2 Diabetes

OBJECTIVE

The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) account for up to 60% of postprandial insulin release in healthy people. Previous studies showed a reduced incretin effect in patients with type 2 diabetes but a robust response to exogenous GLP-1. The primary goal of this study was to determine whether endogenous GLP-1 regulates insulin secretion in type 2 diabetes.

METHODS

Twelve patients with well-controlled type 2 diabetes and eight matched nondiabetic subjects consumed a breakfast meal containing d-xylose during fixed hyperglycemia at 5 mmol/l above fasting levels. Studies were repeated, once with infusion of the GLP-1 receptor antagonist, exendin-(9–39) (Ex-9), and once with saline.

RESULTS

The relative increase in insulin secretion after meal ingestion was comparable in diabetic and nondiabetic groups (44 ± 4% vs. 47 ± 7%). Blocking the action of GLP-1 suppressed postprandial insulin secretion similarly in the diabetic and nondiabetic subjects (25 ± 4% vs. 27 ± 8%). However, Ex-9 also reduced the insulin response to intravenous glucose (25 ± 5% vs. 26 ± 7%; diabetic vs. nondiabetic subjects), when plasma GLP-1 levels were undetectable. The appearance of postprandial ingested d-xylose in the blood was not affected by Ex-9.

CONCLUSIONS

These findings indicate that in patients with well-controlled diabetes, the relative effects of enteral stimuli and endogenous GLP-1 to enhance insulin release are retained and comparable with those in nondiabetic subjects. Surprisingly, GLP-1 receptor signaling promotes glucose-stimulated insulin secretion independent of the mode of glucose entry. Based on rates of d-xylose absorption, GLP-1 receptor blockade did not affect gastric emptying of a solid meal.Glucagon-like peptide 1 (GLP-1) is a gut-brain peptide that is a major component of the incretin effect and is essential for normal glucose tolerance (1). Based on studies in which synthetic GLP-1, or GLP-1 receptor (GLP-1r) agonists, is administered to humans, GLP-1 has a broad range of actions that promote glucose homeostasis, including stimulating insulin secretion (2), suppressing glucagon release (3–4), delaying gastric emptying (5), and increasing hepatic glucose balance (6–7). Importantly, and unlike other insulinotropic gut peptides, the effects of GLP-1 on glucose metabolism are retained in people with diabetes (8–10). This has led to the development of novel therapeutic compounds for use in diabetic patients that are based on GLP-1r signaling (11).The physiologic role of GLP-1 in individuals with diabetes has not been determined. However, there are several reasons to question whether the GLP-1 system is fully functional in this patient group. First, there is some evidence that GLP-1 secretion in response to meal ingestion in type 2 diabetes is impaired (12–15), although this finding has not been uniform (16–17). Second, the sensitivity of insulin secretion to exogenous GLP-1 is reduced in diabetic individuals (18). Finally, it has long been believed that the augmentation of glucose-stimulated insulin secretion during enteral glucose absorption, the incretin effect, is severely attenuated in type 2 diabetes, implying that incretins such as GLP-1 are not normally active in this group of subjects.In this study, we tested the hypothesis that the effect of endogenous GLP-1 to promote insulin secretion after meal ingestion is reduced in people with diabetes. Diabetic subjects and age- and weight-matched nondiabetic subjects were studied with and without infusion of the specific GLP-1r antagonist, exendin-(9–39) (Ex-9), during fixed hyperglycemia before and after a breakfast meal.     

Osteosarcoma in a Patient With Pseudohypoparathyroidism Type 1b Due to Paternal Uniparental Disomy of Chromosome 20q

It is assumed that a persistent high level of parathyroid hormone (PTH) might have a relation with bone malignancy. However, there has been no report of osteosarcoma associated with pseudohypoparathyroidism type 1b (PHP1b), which is accompanied by high PTH. PHP1b is the result of resistance to PTH in certain end‐organ tissues, especially the kidney; the response in bone is unaffected because it normally expresses stimulatory G protein equally from both parental alleles. A 21‐year‐old male, presenting with gum swelling at the right mandible, was referred to a dental clinic. A curative surgical resection by segmental mandibulectomy was performed and the pathologic findings of the mass were consistent with osteoblastic osteosarcoma. His laboratory results showed a low calcium level despite high PTH, and he did not have any features of Albright hereditary osteodystrophy; therefore, PHP1b was suspected. Multiplex ligation‐dependent probe amplification and microsatellite marker analyses of chromosome 20 confirmed the diagnosis and identified paternal uniparental disomy of chromosome 20q (patUPD20). To the best of our knowledge, this is the first report of osteosarcoma in a patient with PHP1b due to patUPD20. © 2017 American Society for Bone and Mineral Research.     

Genome-Wide Association Study for Type 2 Diabetes in Indians Identifies a New Susceptibility Locus at 2q21

Indians undergoing socioeconomic and lifestyle transitions will be maximally affected by epidemic of type 2 diabetes (T2D). We conducted a two-stage genome-wide association study of T2D in 12,535 Indians, a less explored but high-risk group. We identified a new type 2 diabetes–associated locus at 2q21, with the lead signal being rs6723108 (odds ratio 1.31; P = 3.32 × 10⁻⁹). Imputation analysis refined the signal to rs998451 (odds ratio 1.56; P = 6.3 × 10⁻¹²) within TMEM163 that encodes a probable vesicular transporter in nerve terminals. TMEM163 variants also showed association with decreased fasting plasma insulin and homeostatic model assessment of insulin resistance, indicating a plausible effect through impaired insulin secretion. The 2q21 region also harbors RAB3GAP1 and ACMSD; those are involved in neurologic disorders. Forty-nine of 56 previously reported signals showed consistency in direction with similar effect sizes in Indians and previous studies, and 25 of them were also associated (P < 0.05). Known loci and the newly identified 2q21 locus altogether explained 7.65% variance in the risk of T2D in Indians. Our study suggests that common susceptibility variants for T2D are largely the same across populations, but also reveals a population-specific locus and provides further insights into genetic architecture and etiology of T2D.Type 2 diabetes (T2D) has developed into a major health problem, responsible for early morbidities and mortality that affects over a billion people worldwide (1). Developing countries such as India will be maximally affected by the T2D epidemic, both in terms of morbidity/mortality and socioeconomic loss in the coming decade (1,2). India, with one-sixth of the world’s population, typical risk phenotypes, and rapid socioeconomic transitions, provides an important resource for understanding the pathogenesis of T2D (1,3).Genome-wide association studies (GWAS) and subsequent meta-analyses have identified >56 susceptibility loci for T2D that collectively explain ∼10% of the disease risk (4–8). Although GWAS have greatly improved our understanding of the genetic basis of T2D, most of these studies have been performed in Europeans (9), and the studies involving South Asians are very limited (7,10). Interpopulation differences in allele frequencies and effect sizes have yielded the discovery of new loci in different populations (11). The relatively unexplored and high-risk Indian population provides an opportunity for genetic dissection of T2D and other metabolic disorders (3).In this study, we performed a two-staged GWAS of T2D involving a total of 12,535 Indians. The study involved a genome scan of 2,465 subjects and replication of top signals in two ethnic groups of India comprising 7,221 Indo-Europeans and 2,849 Dravidian subjects. These two ethnic groups of India are genetically diverse due to different ancestral backgrounds (12); hence, data for these two ethnic groups have been provided separately with the primary focus on Indo-Europeans. Our study identified a new T2D–associated locus on 2q21, with a lead signal at rs6723108 near TMEM163. Imputation analysis and genotyping revealed a stronger signal on 2q21 at rs998451 within TMEM163. Further analysis suggested a probable mechanism of influence of the TMEM163 variants on T2D susceptibility through insulin secretion.     

The Role of Laboratory Testing in Differentiating Type 1 Diabetes from Type 2 Diabetes in Patients Undergoing Bariatric Surgery

Background

It may be difficult to distinguish between adults with type 1 diabetes and type 2 diabetes by clinical assessment. In patients undergoing bariatric surgery, it is critical to correctly classify diabetes subtype to prevent adverse perioperative outcomes including diabetic ketoacidosis. This study aimed to determine whether testing for C-peptide and islet cell antibodies during preoperative evaluation for bariatric surgery could improve the classification of type 1 versus type 2 diabetes compared to clinical assessment alone.

Methods

This is a retrospective analysis of the Improving Diabetes through Lifestyle and Surgery trial, which randomized patients with clinically diagnosed type 2 diabetes and BMI 30–40 kg/m² to medical weight loss or bariatric surgery; one participant was discovered to have type 1 diabetes after experiencing postoperative diabetic ketoacidosis. Using blood samples collected prior to study interventions, we measured islet cell antibodies and fasting/meal-stimulated C-peptide in all participants.

Results

The participant with type 1 diabetes was similar to the 11 participants with type 2 diabetes in age at diagnosis, adiposity, and glycemic control but had the lowest C-peptide levels. Among insulin-treated participants, fasting and stimulated C-peptide correlated strongly with the C-peptide area-under-the-curve on mixed meal tolerance testing (R = 0.86 and 0.88, respectively). Three participants, including the one with type 1 diabetes, were islet cell antibody positive.

Conclusions

Clinical characteristics did not correctly identify type 1 diabetes in this study. Preoperative C-peptide testing may improve diabetes classification in patients undergoing bariatric surgery; further research is needed to define the optimal C-peptide thresholds.

     

Limitations of IL-2 and Rapamycin in Immunotherapy of Type 1 Diabetes

Administration of low-dose interleukin-2 (IL-2) alone or combined with rapamycin (RAPA) prevents hyperglycemia in NOD mice. Also, low-dose IL-2 cures recent-onset type 1 diabetes (T1D) in NOD mice, partially by boosting pancreatic regulatory T cells (T_reg cells). These approaches are currently being evaluated in humans. Our objective was to study the effect of higher IL-2 doses (250,000–500,000 IU daily) as well as low-dose IL-2 (25,000 IU daily) and RAPA (1 mg/kg daily) (RAPA/IL-2) combination. We show that, despite further boosting of T_reg cells, high doses of IL-2 rapidly precipitated T1D in prediabetic female and male mice and increased myeloid cells in the pancreas. Also, we observed that RAPA counteracted IL-2 effects on T_reg cells, failed to control IL-2–boosted NK cells, and broke IL-2–induced tolerance in a reversible way. Notably, the RAPA/IL-2 combination failure to cure T1D was associated with an unexpected deleterious effect on glucose homeostasis at multiple levels, including β-cell division, glucose tolerance, and liver glucose metabolism. Our data help to understand the therapeutic limitations of IL-2 alone or RAPA/IL-2 combination and could lead to the design of improved therapies for T1D.In type 1 diabetes (T1D), the immune system destroys the pancreatic β-cells (1). At clinical onset, ∼30% of β-cells are still able to produce insulin (2), thus stopping autoimmune destruction, which at this stage is a promising approach (3). Along the same lines, there is a growing list of phase I/II clinical trials based on immunomodulation that are currently being conducted in T1D patients (4).NOD mice, which develop spontaneous T1D, represent an accepted model for testing new therapies (5), the gold standard being that treatments that cure overt hyperglycemia in these mice may be most appropriate for translation into the clinic, as was the case for anti-CD3 antibodies (Abs) (6), which have been tested in patients with promising results (7). In addition, results from our own group showing that low-dose interleukin-2 (IL-2) can prevent (8) and revert disease in NOD mice (9) have led to the translation of this strategy into clinical trials in T1D patients (clinical trial reg. no. {"type":"clinical-trial","attrs":{"text":"NCT01353833","term_id":"NCT01353833"}}NCT01353833, clinicaltrials.gov).We have shown that in NOD mice, administration of low-dose IL-2 for 5 days induced the remission of new-onset T1D by specifically boosting regulatory T cells (T_reg cells) in the pancreas without activating pathogenic effector T cells (T_eff cells). However, remission was obtained in only 60% of treated mice, and half of them became diabetic again during the following months (9). Consequently, improving IL-2 therapy by optimizing dosing or combining IL-2 with other immunomodulatory drugs, such as rapamycin (RAPA), could be of great importance for the goal of translating this therapy to humans.RAPA has been used in clinical transplantation for many years (10), and it has been safely administered to T1D patients during islet transplantation (11,12). In mice, RAPA monotherapy can prevent T1D development (13); however, it is unable to induce disease reversal (14). Moreover, RAPA and IL-2 were found to be synergistic for the prevention of diabetes in NOD mice (13). Consequently, we decided to test whether RAPA could synergize with short-term IL-2 therapy to reverse T1D and reinforce the development of long-term tolerance.In this work, we have further studied the mechanisms of action of IL-2 and RAPA alone or in combination in the NOD model of T1D.     

HLA Class I and Genetic Susceptibility to Type 1 Diabetes: Results From the Type 1 Diabetes Genetics Consortium

OBJECTIVE

We report here genotyping data and type 1 diabetes association analyses for HLA class I loci (A, B, and C) on 1,753 multiplex pedigrees from the Type 1 Diabetes Genetics Consortium (T1DGC), a large international collaborative study.

RESEARCH DESIGN AND METHODS

Complete eight-locus HLA genotyping data were generated. Expected patient class I (HLA-A, -B, and -C) allele frequencies were calculated, based on linkage disequilibrium (LD) patterns with observed HLA class II DRB1-DQA1-DQB1 haplotype frequencies. Expected frequencies were compared to observed allele frequencies in patients.

RESULTS

Significant type 1 diabetes associations were observed at all class I HLA loci. After accounting for LD with HLA class II, the most significantly type 1 diabetes–associated alleles were B*5701 (odds ratio 0.19; P = 4 × 10⁻¹¹) and B*3906 (10.31; P = 4 × 10⁻¹⁰). Other significantly type 1 diabetes–associated alleles included A*2402, A*0201, B*1801, and C*0501 (predisposing) and A*1101, A*3201, A*6601, B*0702, B*4403, B*3502, C*1601, and C*0401 (protective). Some alleles, notably B*3906, appear to modulate the risk of all DRB1-DQA1-DQB1 haplotypes on which they reside, suggesting a class I effect that is independent of class II. Other class I type 1 diabetes associations appear to be specific to individual class II haplotypes. Some apparent associations (e.g., C*1601) could be attributed to strong LD to another class I susceptibility locus (B*4403).

CONCLUSIONS

These data indicate that HLA class I alleles, in addition to and independently from HLA class II alleles, are associated with type 1 diabetes.Type 1 diabetes is an autoimmune disease characterized by progressive T-cell–mediated destruction of the pancreatic β-cells. Both genetic and environmental factors are involved in disease susceptibility; the major genetic susceptibility determinants are the highly polymorphic HLA loci on chromosome 6p21—more specifically the class II loci, HLA-DRB1, HLA-DQB1/DQA1 (see the study by Erlich et al. [1] and references therein), and, to a lesser extent, HLA-DPB1/DPA1 (2–6). These genes, however, cannot completely explain the association between type 1 diabetes and the HLA region. Several studies have shown that HLA class I genes (A, B, and C) are associated with type 1 diabetes (7–11). Products of the HLA class I genes bind and present peptide antigens. The HLA class I/peptide antigen complexes function both in shaping the T-cell repertoire in the thymus and in initiating antigen-specific T-cell–mediated cytotoxicity, providing a plausible immunological rationale to explain the genetic association. The extremely high linkage disequilibrium (LD) within the HLA region, combined with the strong susceptibility effects of the HLA DR- and DQ-encoding loci, can confound association studies of any loci in the region. Thus, apparent susceptibility effects of HLA class I alleles may, in some cases, be attributable to their presence on highly protective or predisposing HLA DRB1-DQA1-DQB1 haplotypes.Compared with the hundreds of studies of HLA class II association with type 1 diabetes, only a handful of reports focus on HLA class I and type 1 diabetes (7–12), and only a subset of these include molecular genotyping and consideration of LD with class II in association analyses. Some alleles have appeared consistently associated with type 1 diabetes both at the serologic and allele level, including A*24(02) and B*39(06), with and without conditioning on DR-DQ. HLA class I loci are extremely polymorphic, with a total of 2,893 alleles assigned for the three loci as of October 2009. Thus, large sample sizes are crucial to generate sufficient class I data for adequately powered disease association studies. The Type 1 Diabetes Genetics Consortium (T1DGC) is an international collaborative project that has ascertained the largest set of multiplex type 1 diabetes families in existence for the study of the genetic basis of type 1 diabetes susceptibility. All samples collected by the T1DGC are genotyped at all classical HLA loci (DRB1, DQA1, DQB1, DPA1, DPB1, A, B, and C) as well as for single nucleotide polymorphisms (SNPs) in the insulin and CTLA4 genes that have repeatedly been shown to be associated with type 1 diabetes. Subsets of the T1DGC collection have been genotyped for candidate gene SNPs reported to be associated with type 1 diabetes (the “Rapid Response” project), genome-wide microsatellites, and genome-wide SNPs (www.T1DGC.org).     

Absence of Linkage for Bone Mineral Density to Chromosome 12q12-14 in the Region of the Vitamin D Receptor Gene

Polymorphisms in the region of the gene for the vitamin D receptor (VDR) (chromosome 12q12-14) have been associated with differences in bone mineral density (BMD) in some studies but not in others. Because linkage analysis assesses allele sharing identical-by-descent among relatives instead of the association of a particular allele of an anonymous marker, we have performed a linkage study for bone BMD using microsatellite markers flanking the VDR locus. The present study explores whether or not relatives who share the chromosomal region containing the VDR gene have more similar bone density. Participants in the Framingham Osteoporosis Study (aged 37–89 years) who had undergone BMD testing were used to test for concordance of genotype with phenotype in the hip (femoral neck, Ward's area, trochanter) and lumbar spine (L2-L4) with adjustment for covariates. Multipoint quantitative trait linkage analysis using variance components methods was conducted with microsatellite markers flanking the VDR locus (GATA91H06, GATA5A09, GGAT2G06) in 332 extended families containing 1062 individuals with both bone density measures and marker data. In addition, quantitative trait sib-pair linkage analysis, with a marker (AFM345xf1) in close proximity to the VDR locus, was performed in a second sample of 169 sibships (n = 413), comprising 284 full-sib pairs. Neither analysis revealed evidence for linkage of this region to femoral neck, Ward's area, lumbar spine, and trochanter in age or sex BMI, and height-adjusted bone density measures. Additional adjustment for alcohol intake, caffeine consumption, smoking status, and estrogen supplement (female only) did not alter the results. The present study could not demonstrate linkage of BMD to chromosome 12q12-14. These findings suggest that neither the VDR gene nor other genes at this locus are likely to have a substantial impact upon bone density. Received: 23 February 2000 / Accepted: 3 August 2000 / Online publication: 22 December 2000