1 Steno Diabetes Center, Gentofte, Denmark
2 Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts
3 Obstetric Clinic, National University Hospital, Rigshospitalet, Copenhagen, Denmark
4 Research Centre for Prevention and Health, Glostrup University Hospital, Glostrup, Denmark
5 Faculty of Health Science, Aarhus University, Aarhus, Denmark
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Type 2 diabetes is a common metabolic disease with a pathophysiology characterized by decreased insulin secretion and insulin resistance. There is substantial evidence for genetic components in the pathogenesis of the nonautoimmune forms of diabetes, specifically for the autosomal dominantly inherited maturity-onset diabetes of the young (MODY), where the impaired glucose regulation is due to a deficiency in pancreatic ß-cell insulin secretion. Several subtypes of MODY are recognized as being caused by single heterozygous mutations in genes encoding various transcription factors: hepatocyte nuclear factors (HNFs), along with insulin promoting factor-1, neurogenic differentiation factor D, and the glycolytic enzyme glucokinase (GCK) (17). Mutations in the known MODY genes explain only 5070% of all MODY cases (810). Thus, other genes, so-called MODY-X genes, are needed to explain the remaining MODY cases.
A targeted knock-out of the novel oxidoreductase Ncb5or in mice was recently reported in which it was shown that homozygous Ncb5or/ mice developed a diabetic phenotype (11). The enzyme is a widely expressed cytosolic cytochrome b-type NAD(P)H oxidoreductase containing cytochrome b5 and b5 reductase domains (12). Young Ncb5or/ mice (aged 4 weeks) had impaired glucose tolerance and low insulin secretion, and after 7 weeks a considerable rise in blood glucose levels was observed when compared with Ncb5or+/+ mice. Ncb5or+/ heterozygote mice had normal blood glucose levels and normal glucose tolerance (NGT). For older Ncb5or/ mice there was a modest reduction in the number of islets, which in addition were distinctly depleted of ß-cells. Furthermore, islets from 4-week-old Ncb5or/ mice showed a 40% decrease in insulin content compared with size-matched islets from Ncb5or+/+ mice. Ncb5or/ mice at age 7 weeks also had elevated serum triglycerides and cholesterol levels and were hyperphagic (11).
Since absence of Ncb5or expression resulted in a diabetic phenotype with a noninflammatory ß-cell deficiency, we hypothesized that variation in human NCB5OR might be related to MODY-X or type 2 diabetes. Furthermore, owing to the relatively low age at the development of the phenotype, we included various early-onset diabetes subtypes in this supposition. Indeed, in a genome-wide scan (13) of MODY-X families, a linkage peak was identified on chromosome 6 (flanked by markers D6S1017 and D6S460) close to the chromosomal localization of human NCB5OR (6q14.2). Moreover, a genome-wide scan of Finnish affected sibling pairs showed strong linkage of a region on chromosome 6 close to NCB5OR with late-onset type 2 diabetes and HDL/total cholesterol ratios (14). In this study we investigated the entire coding region of human NCB5OR, including intron-exon boundaries for variation using denaturing high-performance liquid chromatography (dHPLC) and subsequent nucleotide sequencing in 183 diabetic patients including probands from MODY-X families, early-onset type 2 diabetic patients, late-onset type 2 diabetic patients, and women diagnosed with gestational diabetes mellitus (GDM). A total of 22 variants were identified (Fig. 1). Three variants were further investigated by genotyping in a group of 717 type 2 diabetic patients and in a group of 529 control subjects with NGT. The Gln187Arg (identified in one patient) and His223Arg (in four patients) variants were chosen due to their potential impact on NCB5OR protein function and the IVS5+7del(CT) (one patient) variant because it is located near a predicted splice site and may therefore influence mRNA expression and/or stability. The most frequent variant was IVS1472T>C with a minor allele frequency (MAF) of 8% followed by IVS14166C>T and IVS15259C>A with approximate MAFs of 5% each. All other variants had MAFs of
3% or lower (estimated from the primary mutation analysis of 183 subjects). The degree of linkage disequilibrium between the variants was estimated. The IVS215T>C and IVS3+81G>A variants were in complete linkage disequilibrium. The IVS5245A>G and IVS6+26A>G variants were also in complete linkage disequilibrium and furthermore in tight linkage disequilibrium with the IVS8196A>G (R2 = 0.8). There was no evidence of linkage between any of the remaining variants. This may be due to the relatively low allele frequencies of the variants.
|
|
|
Three intronic variants (IVS489A>T, IVS5131T>C, and IVS659A>G) were identified in only one patient each. These patients were the probands of three MODY-X families, and although these variants are unlikely to have any functional impact on NCB5OR, they may be in linkage disequilibrium with a yet unidentified causative variant located outside the area included in the present mutation analysis. DNA was obtained from the remaining family members who were genotyped for the respective variants using nucleotide sequencing and examined in a cosegregation study with disease status. No evidence of cosegregation with the MODY diabetes subtype was found in the three families (data not shown). A fourth intronic variant (IVS795G>A) was identified only in a proband from a family with early-onset type 2 diabetes and was investigated in a similar manner; however, no evidence of cosegregation with disease status or age of disease onset was observed in this family (data not shown). Furthermore, one MODY-X family that was examined in the mentioned genome scan (13) and contributed to the chromosome 6 linkage peak was also included in the present mutation analysis. No variants were observed in this family.
The observation that homozygous Ncb5or/ knock-out mice present with an early-onset diabetic phenotype with a ß-cell defect as a primary observation makes human NCB5OR a credible biological candidate gene for a mutation analysis in relation to type 2 diabetes and various diabetes subtypes related to ß-cell dysfunction. This is further strengthened by evidence from a MODY-X and an affected sibling pair genome scan showing linkage with a chromosomal area close to the NCB5OR locus (13,14).
Alignment of the Gln187Arg and His223Arg variants showed that both the Gln- and His-alleles were conserved between the human and murine NCB5OR proteins. All three amino acids are polar and hydrophilic, but whereas histidine and arginine are both positively charged and an exchange of these is not considered to confer a major change in the protein function as such, the glutamine to arginine substitution results in a change from an amide to an alkaline amino acid. The His223Arg variant is located in the b5 reductase domain of NCB5OR and the Gln187Arg variant in the hinge region connecting the cytochrome b5 and b5 reductase domains. The Gln187Arg substitution was genotyped in 1,246 participants; however, no additional carriers were identified. The patient for whom the variant was originally identified was a woman diagnosed with glucose intolerance during her second pregnancy. In pregnancies complicated by GDM the increased demand for insulin due to impaired tissue insulin sensitivity is not readily met because of reduced pancreatic ß-cell function (rev. in 15). Furthermore, a positive family history of diabetes may pose an increased risk of developing GDM in women >30 years of age (16). We genotyped 140 women with GDM in order to investigate the role of the Gln187Arg variant in GDM, but we failed to identify any additional carriers. At the time of her examination, the 49-year-old patient had a BMI of 30 kg/m2, her fasting serum lipids were normal, and she was hypertensive. There was no known history of diabetes in her family, and no family members were available for examination. Given her anthropometric variables, her diabetes may be precipitated by obesity-induced insulin resistance. Whether the Gln187Arg variant is involved in decreased ß-cell function is not clear from our data.
The His223Arg variant did not show any association with type 2 diabetes or diabetes-related intermediary phenotypes. On the contrary, the IVS5+7del(CT) variant had a lower frequency among type 2 diabetic patients than among glucose-tolerant subjects and correspondingly a slightly lower relative number of mutation carriers. However, none of the quantitative traits investigated in this study were significantly associated with the variant, although we did observe some suggestive tendencies in fasting serum lipid and insulin levels. For both of the variants this may be due to the low allele frequency and the resulting relatively low power to detect quantitative trait differences; larger study populations are clearly needed. Also, there is a need for replication of the initial finding of an association between the IVS5+7del(CT) variant and type 2 diabetes. The observation that the IVS5+7del(CT) variant confers a modestly reduced risk of developing type 2 diabetes is somewhat counterintuitive, and considering the total number of tests performed in the present study a correction for multiple testing is appropriate. Consequently, the differences in allele frequencies and genotype distribution are not statistically significant. Moreover, we were unable to ascertain a specific metabolic phenotype for this observation. Thus, we conclude that the IVS5+7del(CT) variant is not an important contributor in the pathogenesis of type 2 diabetes in the examined population.
A putative relationship between NCB5OR and MODY of as yet unknown genetic cause was investigated by analyzing 63 MODY-X patients for mutations and genotyping all promising variants in the patients family members. However, there was no evidence in the present study to suggest that variation in NCB5OR is involved in MODY or early-onset type 2 diabetes. The assessment of NCB5OR as a positional candidate gene for MODY was also done by including one MODY-X family in the mutation analysis, which contributed to the previously described chromosome 6 linkage peak. Likewise, this part of the study did not point to any role of NCB5OR in the etiology of MODY. A limitation to this conclusion is, however, that variation in regulatory sequences in the NCB5OR locus may exist and possibly have an impact on the expression of the protein, thereby influencing diabetes-related traits.
Even though the Ncb5or/ mice presented with a very convincing nonautoimmune diabetic phenotype, the lack of an association between the identified NCB5OR variants and type 2 diabetes and/or quantitative phenotypic traits could be explained by the fact that the heterozygote Ncb5or+/ mice had NGT. On the contrary, for the Hnf1 knock-out mouse it was also observed that the heterozygote Hnf1
+/ mice appeared to have NGT (17), while heterozygote mutations in human HNF1
are the cause of MODY3 (2), and a frequent Ala98Val polymorphism in this gene has been suggested to associate with intermediary prediabetic phenotypes such as reduced postoral glucose tolerance test (OGTT) serum insulin and C-peptide release (18,19).
In summary, we discovered a total of 22 novel nucleotide variants in NCB5OR, and in epidemiological studies we invstigated a selection of these variants along with two additional SNPs identified in silico. The IVS5+7del(CT) variant showed significant association with type 2 diabetes in a case-control study, but we failed to relate this finding with prediabetic and/or obesity-related quantitative traits. Further studies of this variant are needed and may involve functional in vitro studies and genotyping of the variant in even larger study samples in order to explore its role in NCB5OR function and expression.
![]() |
RESEARCH DESIGN AND METHODS |
---|
![]() ![]() ![]() ![]() |
---|
Biochemical assays.
Blood samples for measurement of serum insulin and plasma glucose levels were drawn after a 12-h overnight fast. The plasma glucose, serum specific insulin, and serum lipid levels (triglyceride and cholesterol) and HbA1c were measured using Steno Diabetes Center routine methods.
Mutation detection.
The 16 exons of NCB5OR including intron-exon boundaries (in total 9,007 bp) was divided into 15 segments (552719 nucleotides) and amplified using PCR in a total volume of 25 µl with primer sequences, MgCl2 concentration, and annealing temperatures as listed in Table 3. All primers were HPLC purified. Equal amounts of known wild-type PCR and sample PCR products were mixed in order to identify homozygous variants. The mixed PCR products were denatured at 95°C for 5 min, renatured at room temperature for 30 min, and analyzed by dHPLC using the Wave DNA Fragment Analysis System with DNASep Technology (Transgenomic, San Jose, CA). WaveMaker 4.1 was applied to analyze the segments for optimal melting profile. The temperatures are listed in Table 3. Aberrantly migrating samples were bidirectionally sequenced using fluorescent chemistry (MWG-Biotech, Ebersberg, Germany).
|
Statistical analysis.
Fishers exact test was applied to examine differences in allele frequencies and genotype distributions between diabetic and nondiabetic subjects. A general linear model was used to test variables (or transformed variables) for differences between genotype groups in the sample of unrelated NGT subjects. Genotype and sex were considered fixed factors and age and BMI as covariates. All phenotype analyses were performed using Statistical Package for Social Science (SPSS, Chicago, IL) version 11.5. A P value <0.05 was considered significant. The area under the curve was calculated using the trapezoidal method in 30-min intervals. The insulinogenic index was calculated as fasting serum insulin (in picomoles per liter) subtracted from 30-min post-OGTT serum insulin (in picomoles per liter) and divided by 30-min post-OGTT plasma glucose (in millimoles per liter). The homeostasis model of assessment for insulin resistance was calculated as fasting plasma glucose (in millimoles per liter) multiplied by fasting serum insulin (in picomoles per liter) divided by 22.5. Linkage disequilibrium was estimated as R2, where R2 = 1 in the case of complete linkage and R2 = 0 in the case of no linkage. R2 was calculated as described at www.ekstroem.com.
In silico data analysis.
The human NCB5OR nucleotide sequence was retrieved at the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov) with accession numbers BC025380, AL139232, and AL034347. Intron-exon boundary structure was defined at NCBI (Evidence Viewer) and confirmed at Ensembl (www.ensembl.org). A search for additional SNPs was performed using LocusLink at NCBI (www.ncbi.nlm.nih.gov/LocusLink). Protein translations were performed using Baylor College of Medicine Search Launcher (searchlauncher.bcm.tmc.edu/seq-util/seq-util.html) and confirmed with the protein as it was described previously (12). Protein and nucleotide sequence alignments of the human and murine (NM_024195 and BC025438) protein sequences were performed using ClustalW version 1.82 (www.ebi.ac.uk/clustalw/index.html). Primers for PCRs were designed using Primer3 (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Restriction sites for endonu-cleases were obtained from Webcutter version 2.0 (www.firstmarket.com/cutter/cut2.html). A genetic map relating the chromosomal localization of human NCB5OR with the chromosome 6 MODY-X linkage peak was obtained from the Center for Medical Genetics (www.marshfieldclinic.org/research/genetics/) using markers D6S1017 at 63.28 cM, D6S460 at 89.83 cM, and D6S1634 at 92.25 cM (Kosambi cM).
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank Professor P.D. Meyts for valuable supervision of G. Andersens PhD project.
Address correspondence and reprint requests to Gitte Andersen, MSc, Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 2, NSH2.16, DK-2820 Gentofte, Denmark. E-mail: gtta{at}steno.dk
Received for publication June 1, 2004 and accepted in revised form July 21, 2004
dHPLC, denaturing high-performance liquid chromatography; GCK, glucokinase; GDM, gestational diabetes mellitus; HNF, hepatocyte nuclear factor; MAF, minor allele frequency; MODY, maturity-onset diabetes of the young; NCBI, National Center for Biotechnology Information; NGT, normal glucose tolerance; OGTT, oral glucose tolerance test; OHA, oral hypoglycemic agent; RFLP, restriction fragmentlength polymorphism; RG, restriction site generating; SNP, single nucleotide polymorphism
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Diabetes | Diabetes Care | Clinical Diabetes | Diabetes Spectrum | DOC News |