1 Centre for Molecular Genetics, Peninsula Medical School, Exeter, U.K
2 Department of Medicine, School of Medicine, Newcastle upon-Tyne, U.K
3 Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Infirmary, Oxford, U.K
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ABSTRACT |
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The protein small heterodimer partner (SHP, also called NR0B2) is an atypical orphan receptor that lacks a conserved DNA binding domain. SHP is expressed in the liver where it is involved in a nuclear receptor cascade involved in the regulation of cholesterol catabolism (1,2). Recent work has shown that SHP is also expressed in the pancreas and inhibits the transcriptional activity of hepatocyte nuclear factor (HNF)-4 (3). HNF4
is a key member of a regulatory network of maturity-onset diabetes of the young (MODY)-related transcription factors that are required for normal ß-cell function (4).
The regulation of HNF4 led Nishigori et al. (3) to investigate the role of the SHP gene in Japanese subjects with young-onset type 2 diabetes. They found five heterozygous mutations in 6 of 173 subjects (3.5%) who had reduced activity in functional studies. Further analysis showed that all subjects were moderately obese (BMI >25 kg/m2 is the Japanese criterion for obesity [5]), and within families, the mutations cosegregated with obesity (logarithm of odds [LOD] score 2.31) and not diabetes (LOD score
). SHP mutations were also found in 5.9% (6 of 101) of nondiabetic young subjects with obesity (BMI >25 kg/m2) but in none (0 of 116) of the nonobese control subjects. Birth weight was also shown to be markedly higher in mutation carriers (3,899 vs. 2,707 g, P = 0.008). These data suggested that genetic variation in SHP was the most common monogenic determinant of obesity and birth weight in Japanese subjects.
The work of Nishigori et al. was also important as it suggested that increased pancreatic insulin secretion might be a pathway in the etiology of obesity. Mutations reducing activity of SHP would result in reduced inhibition of HNF4 and, hence, could potentially increase insulin secretion. The finding of birth weight >1-kg higher than non-mutation carriers supports increased insulin secretion in utero; the birth weights were similar to that found in activating mutations of the sulfonylurea receptor (6). Replication of this initial finding is important, but no further studies of SHP have been reported.
The aim of our study was to investigate the role of SHP in type 2 diabetes, obesity, and birth weight in large U.K. Caucasian cohorts. As mutations had been found initially in obese, young-onset type 2 diabetic patients in Japan, we sequenced the whole coding region and intron/exon boundaries for SHP in 122 U.K. subjects with young-onset (diagnosis before 45 years) type 2 diabetes with a BMI >30 kg/m2 (Table 1). The only sequence variation detected was an amino-acid substituting polymorphism (G171A) in exon 1 in codon 171, where GGG encoding G was replaced by GCG encoding A. This polymorphism was present in 16 individuals (13%) and was in Hardy-Weinberg equilibrium with two subjects being homozygous for the rare A allele. This polymorphism had not been described in Japanese subjects (3).
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Although no mutations were detected, there was a common coding polymorphism G171A in exon 1 in U.K. subjects that had not been found in the Japanese study. The A allele of this polymorphism had an allele frequency of 7.2% (from control data) and was potentially significant, as G is conserved at this position in mouse and rat. However, our results showed no association of diabetes, obesity, or birth weight with the G171A allele or the heterozygous G/A genotype.
Subjects homozygous for the A allele of the G171A polymorphism are consistently more obese, although this observation was not significant in any single dataset. However, this suggests the G171A polymorphism may have a similar role as the heterozygous severe mutations seen in the Japanese. Due to small numbers of homozygotes, this is only significant if all cohorts are combined using Z scores. Given the multiple phenotypes analyzed and the modest association observed, this result should not be emphasized. If this result was replicated in further large cohorts, it would suggest this polymorphism was only altering function of SHP when it was homozygous and would also support the work of Nishigori et al., which suggested a role for SHP in the regulation of obesity. It is of interest that the only child homozygous for the A allele had a birth weight >4 kg. Further large association studies involving cohorts of over 3,000 subjects and functional studies are required to further assess the relationship with obesity and birth weight in subjects homozygous for the A allele.
In conclusion, we have shown that mutations in SHP are less common in U.K. obese type 2 diabetic subjects than in Japanese obese type 2 subjects. We have identified a common G171A coding polymorphism that is present in 14.1% of U.K. subjects. The A allele or G/A genotype is not associated with obesity or increased birth weight. Our preliminary results suggest subjects who are homozygous for the rare A allele may be predisposed to moderate obesity and possibly increased birth weight, but further studies are required to confirm this.
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RESEARCH DESIGN AND METHODS |
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Genotyping.
We assessed variation in the SHP coding region by sequencing both exons in 122 young-onset type 2 diabetic subjects (BMI >30 kg/m2) (Table 1). The SHP exons were PCR amplified in three amplicons from genomic DNA using the following primer pairs: forward 5'-CAGAACACAGAGCCAGAGAG-3' and reverse 5'-CTCAAAGGTCACAGCATCTT-3'; forward 5'-CAAGACAGTGGCCTTCCT-3' and reverse 5'-GAGGACCCAATGAGATAACA-3'; and forward 5'-GCCAGTCTTGTCCTTTGG-3' and reverse 5'-CTCTGCCCACCTGATCTC-3'. PCR was performed in a 50-µl volume containing, in addition to the standard reagents, 1 mol/l betaine, 5% DMSO, deaza GTP, and 0.5 units AmpliTaq Gold (Applied Biosystems). PCR cycling conditions were denaturation at 95°C for 12 min followed by 40 cycles of denaturation at 95°C for 1 min and annealing at 55°C for 1 min and extension at 72°C for 2 min, with a final 10 min extension at 72°C. PCR products were purified using the QIAquick PCR purification kit (QIAgen) before both strands were sequenced using an ABI 377 DNA sequencer according to the manufacturers instructions (Applied Biosystems).
We used tetra-primer ARMS-PCR (10) to screen for the G171A polymorphism in our populations using the following primers: forward inner 5'-CCAAGGAATATGCCTGCCTGAATGC-3'; reverse inner 5'-ACCGGGGTTGAAGAGGATGGACC-3'; forward outer 5'-CTATGTGCACCTCATCGCACCTGC-3'; and reverse outer 5'-CTGGGTGACAGAGTGAGACTCTGTCTCAGA-3'.
PCR was performed in a 10-µl volume containing the standard reagents and 0.1 pmol/µl of each outer primer, 1 pmol/µl of forward inner primer, and 2 pmol/µl of reverse inner primer. PCR cycling conditions were denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 94°C for 45 s and annealing at 65°C for 45 s and extension at 72°C for 45 s, with a final 10 min extension at 72°C. We resolved the tetra-primer ARMS-PCR products on 2% agarose gel stained with ethidium bromide.
Statistical methods.
We assessed Hardy-Weinberg equilibrium of the G171A polymorphism in our cohorts by 2 comparisons of observed genotype frequencies with expected genotype frequencies inferred from observed allele frequencies. All cohorts were in Hardy-Weinberg equilibrium.
The significance of allele and genotype frequency differences were calculated using 2 analysis, with overall allele numbers used to calculate odds ratios and 95% confidence intervals using 2x2 contingency tables. TDT in parent offspring trios for individual variants was performed using TDT/S-TDT program (11).
To assess the effect of the polymorphism in our populations as a whole, we log-transformed BMI data to make it normally distributed and then generated cohort-, sex-, and generation-specific Z scores. We combined the Z scores from each cohort and stratified these scores by genotype. We assessed the significance of the effect of SHP171 genotype on BMI by using a Mann-Whitney U test.
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ACKNOWLEDGMENTS |
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T.M.F. is a career scientist of the South and West National Health Service Research Directorate. K.R.O. is supported by a Diabetes U.K. Clinical Training Fellowship. A.T.H. is a Wellcome Trust Career Leave Research fellow.
We thank all of the people who took part in these collections and the many nurses, general practitioners, and physicians who assisted with them. We also thank Dr. Sian Ellard for laboratory organization and Diane Jarvis for the DNA extraction.
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FOOTNOTES |
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Received for publication 9 August 2002 and accepted in revised form 20 December 2002.
HNF, hepatocyte nuclear factor; LOD, logarithm of odds; MODY, maturity-onset diabetes of the young; SHP, small heterodimer partner; TDT, transmission disequilibrium test.
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REFERENCES |
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