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Association of insulin gene VNTR polymorphism with polycystic ovary syndrome
THE LANCET
Recurrent otitis media (which he also had) was a common childhood disorder and is mentioned because the insertion of grommets (and preceding full blood count) led to the diagnosis of thrombocytopenia. Although our patient’s kidney biopsy sample showed crescentic glomerulonephritis, Epstein’s syndrome is rare, with few reported cases worldwide. But variation between cases is a possibility. The elctronmicroscopic characteristics of the platelets in this patient agree with previously reported abnormalities. Our patient did not have ichthyosis, atopic dermatitis, leiomyomatosis, or ocular abnormalities. The comments of Pintavorn in no way detract from the important message of our papers: do not assume a diagnosis of refractory idiopathic thrombocytopenic purpura until other causes of thrombocytopenia have been excluded. *Mar y Cahill, Adrian Newland Depar tment of Haematology, Royal London Hospital, London E1 1BB, UK
Association of insulin gene VNTR polymorphism with polycystic ovary syndrome SIR—We have several concerns about the evidence for linkage and association of the insulin gene VNTR polymorphism with polycystic ovary syndrome (PCOS) that Waterworth and colleagues report (April 5, p 986).1 In a case-control study, they report an odds ratio of 8·2 for the association of the III/III genotype with anovulatory PCOS. For this association the correct two-sided p-value by Fisher’s exact test is 0·011, not 0·005 as they report. Even this probability should be interpreted with caution, since it is based not on testing a hypothesis specified in advance, but on post hoc restriction of the dataset to anovulatory cases and combining of the other two genotypes into a single category. The family-based association study would be an appropriate design in which to test the specific hypothesis raised by the case-control study: that the III/III genotype is associated with anovulatory PCOS. Assuming random mating, the genotypes constructed from pairs of untransmitted parental alleles should be in Hardy-Weinberg equilibrium, and representative of the population in which the cases arose. Analysis of table 3 shows, however, that the control genotype frequencies (15, 24, and 0 for genotypes I/I, I/III, and III/III, respectively) are not in HardyWeinberg equilibrium (2-test, p=0·006). This departure from
Vol 349 • June 14, 1997
expected frequencies could result from chance, systematic error in genotyping, or non-random mating. Whatever the reason for this strange result, the assumptions on which the family-based association study design depends2 are clearly violated, and any evidence of association in this dataset should be disregarded. With such doubtful evidence for an association, we may look more critically at the evidence for linkage of the INS gene region with PCOS. Of the five markers used in the linkage analysis, two—INS VNTR and HUMTH01—are only 0·01 cM apart and are in strong linkage disequilibrium.3 The Genehunter program4 used to test for linkage assumes that all markers used are in linkage equilibrium with each other. If this assumption is violated, probability distributions calculated by the program would be incorrect. The effect on detection of linkage is hard to predict, but disequilibrium between adjacent markers would mean that haplotypes identical by state would be shared within pedigrees more often than expected. This in turn could lead to overestimation of identity-by-descent sharing and false-positive evidence for linkage. To examine this possibility, Waterworth and colleagues could repeat the Genehunter test for linkage after excluding any markers that are in linkage disequilibrium with each other. Until these uncertainties are resolved, their proposal to designate the INS locus as PCOS1 is premature. *Paul McKeigue, Sarah Wild Depar tment of Epidemiology and Population Sciences, Epidemiology Unit, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
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2
3
4
Waterworth DM, Bennett ST, Gharani N, et al. Linkage and association of insulin gene VNTR regulatory polymorphism with polycystic ovary syndrome. Lancet 1997; 349: 986–90. Falk CT, Rubinstein P. Haplotype relative risks: an easy reliable way to construct a proper control sample for risk calculations. Ann Hum Genet 1987; 51: 227–33. Bennett ST, Lucassen AM, Gough SC, et al. Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 1995; 9: 284–92. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996; 58: 1347–63.
Authors’ reply SIR—We have repeated the Genehunter analysis for linkage of 11p15·5 to PCOS. Irrespective of whether HUMTH01, the INS VNTR, or both loci are excluded from the analysis, we obtain the same support for linkage at D11S922 (single point non-parametric linkage value [NPL]=2·77, p=0·005;
multipoint [excluding HUMTH01] NPL=3·2, p=0·002). Previous studies have reported associations of the INS VNTR class III/III genotype with type 2 diabetes and related disorders.1,2 Women with PCOS have increased risk of developing type 2 diabetes, and anovulatory PCOS is associated with hyperinsulinaemia. Therefore, our hypothesis was that the INS VNTR class III allele frequency would be higher in anovulatory PCOS patients than in controls. This prediction was confirmed in the casecontrol data set: the exact p-value obtained is 0·0059 (Statxact, Cytel Corporation, Cambridge, MA). The value cited by McKeigue and Wild (p=0·011) is conservative. We use the more conventional mid-point value. Analysis of the entire 3⫻3 table (our table 2) shows striking departure from independent assortment (p=0·008 for genotypes, Kruskal-Wallis test). The source of this departure lies predominantly in the comparison of normal versus anovulatory PCOS data sets (p=0·0027). We did a family-based association analysis with an independent family data set. The constructed AFBAC control genotypes are not in Hardy-Weinberg equilibrium, as pointed out by McKeigue and Wild. We believe this to be a chance event. Nevertheless, the trend is positive and consistent with the independent case-control analysis. Furthermore, we have analysed these families for evidence of linkage of PCOS to the insulin VNTR in the presence of association with the transmission disequilibrium test, in which allele transmission from heterozygous parents to affected offspring is measured and is not dependent on Hardy-Weinberg equilibrium. 3 Class III alleles were transmitted more often than class I VNTR alleles from class I/III heterozygous parents to offspring with anovulatory PCOS (27 transmissions, 17 non-transmissions; 61% transmission; 2-test, p=0·132). Because parent-of-origin effects have been observed at this locus in type 1 diabetes4 and there is evidence for gametic imprinting of this region of 11p15·5,5 we evaluated allele transmission from mothers and fathers, separately. Maternal transmissions did not depart from that expected for random transmission (12 transmissions, 12 nontransmissions; 50%). However, class III alleles were transmitted significantly more often than class I alleles from I/III fathers (15 transmissions, five nontransmissions; 75%; p=0·025). This analysis was extended to include transmissions of VNTR class III alleles in the 17 PCOS pedigrees used for the linkage study. In the combined data, class III alleles had a 47% transmission from mothers (14 transmissions, 16
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Recurrent otitis media (which he also had) was a common childhood disorder and is mentioned because the insertion of grommets (and preceding full blood count) led to the diagnosis of thrombocytopenia. Although our patient’s kidney biopsy sample showed crescentic glomerulonephritis, Epstein’s syndrome is rare, with few reported cases worldwide. But variation between cases is a possibility. The elctronmicroscopic characteristics of the platelets in this patient agree with previously reported abnormalities. Our patient did not have ichthyosis, atopic dermatitis, leiomyomatosis, or ocular abnormalities. The comments of Pintavorn in no way detract from the important message of our papers: do not assume a diagnosis of refractory idiopathic thrombocytopenic purpura until other causes of thrombocytopenia have been excluded. *Mar y Cahill, Adrian Newland Depar tment of Haematology, Royal London Hospital, London E1 1BB, UK
Association of insulin gene VNTR polymorphism with polycystic ovary syndrome SIR—We have several concerns about the evidence for linkage and association of the insulin gene VNTR polymorphism with polycystic ovary syndrome (PCOS) that Waterworth and colleagues report (April 5, p 986).1 In a case-control study, they report an odds ratio of 8·2 for the association of the III/III genotype with anovulatory PCOS. For this association the correct two-sided p-value by Fisher’s exact test is 0·011, not 0·005 as they report. Even this probability should be interpreted with caution, since it is based not on testing a hypothesis specified in advance, but on post hoc restriction of the dataset to anovulatory cases and combining of the other two genotypes into a single category. The family-based association study would be an appropriate design in which to test the specific hypothesis raised by the case-control study: that the III/III genotype is associated with anovulatory PCOS. Assuming random mating, the genotypes constructed from pairs of untransmitted parental alleles should be in Hardy-Weinberg equilibrium, and representative of the population in which the cases arose. Analysis of table 3 shows, however, that the control genotype frequencies (15, 24, and 0 for genotypes I/I, I/III, and III/III, respectively) are not in HardyWeinberg equilibrium (2-test, p=0·006). This departure from
Vol 349 • June 14, 1997
expected frequencies could result from chance, systematic error in genotyping, or non-random mating. Whatever the reason for this strange result, the assumptions on which the family-based association study design depends2 are clearly violated, and any evidence of association in this dataset should be disregarded. With such doubtful evidence for an association, we may look more critically at the evidence for linkage of the INS gene region with PCOS. Of the five markers used in the linkage analysis, two—INS VNTR and HUMTH01—are only 0·01 cM apart and are in strong linkage disequilibrium.3 The Genehunter program4 used to test for linkage assumes that all markers used are in linkage equilibrium with each other. If this assumption is violated, probability distributions calculated by the program would be incorrect. The effect on detection of linkage is hard to predict, but disequilibrium between adjacent markers would mean that haplotypes identical by state would be shared within pedigrees more often than expected. This in turn could lead to overestimation of identity-by-descent sharing and false-positive evidence for linkage. To examine this possibility, Waterworth and colleagues could repeat the Genehunter test for linkage after excluding any markers that are in linkage disequilibrium with each other. Until these uncertainties are resolved, their proposal to designate the INS locus as PCOS1 is premature. *Paul McKeigue, Sarah Wild Depar tment of Epidemiology and Population Sciences, Epidemiology Unit, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
1
2
3
4
Waterworth DM, Bennett ST, Gharani N, et al. Linkage and association of insulin gene VNTR regulatory polymorphism with polycystic ovary syndrome. Lancet 1997; 349: 986–90. Falk CT, Rubinstein P. Haplotype relative risks: an easy reliable way to construct a proper control sample for risk calculations. Ann Hum Genet 1987; 51: 227–33. Bennett ST, Lucassen AM, Gough SC, et al. Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 1995; 9: 284–92. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996; 58: 1347–63.
Authors’ reply SIR—We have repeated the Genehunter analysis for linkage of 11p15·5 to PCOS. Irrespective of whether HUMTH01, the INS VNTR, or both loci are excluded from the analysis, we obtain the same support for linkage at D11S922 (single point non-parametric linkage value [NPL]=2·77, p=0·005;
multipoint [excluding HUMTH01] NPL=3·2, p=0·002). Previous studies have reported associations of the INS VNTR class III/III genotype with type 2 diabetes and related disorders.1,2 Women with PCOS have increased risk of developing type 2 diabetes, and anovulatory PCOS is associated with hyperinsulinaemia. Therefore, our hypothesis was that the INS VNTR class III allele frequency would be higher in anovulatory PCOS patients than in controls. This prediction was confirmed in the casecontrol data set: the exact p-value obtained is 0·0059 (Statxact, Cytel Corporation, Cambridge, MA). The value cited by McKeigue and Wild (p=0·011) is conservative. We use the more conventional mid-point value. Analysis of the entire 3⫻3 table (our table 2) shows striking departure from independent assortment (p=0·008 for genotypes, Kruskal-Wallis test). The source of this departure lies predominantly in the comparison of normal versus anovulatory PCOS data sets (p=0·0027). We did a family-based association analysis with an independent family data set. The constructed AFBAC control genotypes are not in Hardy-Weinberg equilibrium, as pointed out by McKeigue and Wild. We believe this to be a chance event. Nevertheless, the trend is positive and consistent with the independent case-control analysis. Furthermore, we have analysed these families for evidence of linkage of PCOS to the insulin VNTR in the presence of association with the transmission disequilibrium test, in which allele transmission from heterozygous parents to affected offspring is measured and is not dependent on Hardy-Weinberg equilibrium. 3 Class III alleles were transmitted more often than class I VNTR alleles from class I/III heterozygous parents to offspring with anovulatory PCOS (27 transmissions, 17 non-transmissions; 61% transmission; 2-test, p=0·132). Because parent-of-origin effects have been observed at this locus in type 1 diabetes4 and there is evidence for gametic imprinting of this region of 11p15·5,5 we evaluated allele transmission from mothers and fathers, separately. Maternal transmissions did not depart from that expected for random transmission (12 transmissions, 12 nontransmissions; 50%). However, class III alleles were transmitted significantly more often than class I alleles from I/III fathers (15 transmissions, five nontransmissions; 75%; p=0·025). This analysis was extended to include transmissions of VNTR class III alleles in the 17 PCOS pedigrees used for the linkage study. In the combined data, class III alleles had a 47% transmission from mothers (14 transmissions, 16
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