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HLA Genotyping Supports a Nonautoimmune Etiology in Patients Diagnosed With Diabetes Under the Age of 6 Months

¹ Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter, U.K
² Department of Clinical Science at North Bristol, University of Bristol, Bristol, U.K

Address correspondence and reprint requests to Dr. K.M. Gillespie, Medical School Unit, Southmead Hospital, Bristol BS10 5NB, U.K. E-mail: k.m.gillespie{at}bristol.ac.uk

Abbreviations: PNDM, permanent neonatal diabetes

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Children with permanent diabetes are usually assumed to have type 1 diabetes. It has recently been shown that there are genetic subgroups of diabetes that are often diagnosed during the neonatal period but may present later. A recent Italian study proposed that type 1 diabetes is rare before 6 months of age. We aimed to examine genetic susceptibility to type 1 diabetes in patients diagnosed with diabetes before the age of 2 years. We analyzed HLA class II genotypes, markers of autoimmune diabetes, in 187 children with permanent diabetes diagnosed at <2 years of age. Of the 79 subjects diagnosed at <6 months of age, 41% (95% CI 0.30–0.51) had type 1 diabetes–associated high-risk genotypes, a proportion similar to that in healthy population control subjects (44%, P = 0.56). This group included 32 patients with mutations in the KCNJ11 gene, which encodes Kir6.2 (44% high-risk HLA class II genotypes), and 47 in whom the etiology of diabetes was unknown (38% high-risk HLA class II genotypes). Of 108 patients diagnosed between 6 and 24 months of age, 93% (0.86–0.99) had high-risk HLA class II genotypes compared with 44% of the population control subjects (P < 0.0001). We conclude that infants diagnosed with diabetes before 6 months of age are unlikely to have autoimmune type 1 diabetes and are most likely to have a monogenic etiology.

Appropriate treatment regimens for diabetes require accurate characterization of disease (1). For patients diagnosed in infancy, this may be complicated; several different etiologies may cause diabetes, including type 1 diabetes and permanent neonatal diabetes (PNDM). Type 1 diabetes results from autoimmune destruction of insulin-producing ß-cells and is characterized by the presence of multiple islet autoantibodies and high-risk HLA haplotypes for type 1 diabetes. HLA DRB1*04-DQB1*0302 and/or HLA DRB1*03-DQB1*0201 are observed in >90% of affected children and in only 40% of the general population (2). The study of HLA genotypes may therefore help define which patients diagnosed in infancy are likely to have type 1 diabetes.

PNDM is typically defined as diabetes requiring permanent insulin treatment that is diagnosed in the first 3 months of life (3). Recent advances in determining the etiology of PNDM have demonstrated that heterozygous-activating mutations in the KCNJ11 gene encoding the ATP-sensitive K⁺ channel subunit Kir6.2 account for 40–64% of PNDM cases (4). To date, all activating KCNJ11 mutations have been identified in subjects diagnosed under the age of 6 months (5–11). Some of these patients are diagnosed outside the defined neonatal period of 3 months and therefore highlight a need to investigate the etiologies of diabetes in infancy. The identification of patients with KCNJ11 mutations is important, as most respond well to sulfonylureas and achieve better glycemic control following transfer from insulin to sulfonylurea tablets (6,8,12–14).

A recent Italian study suggested that children diagnosed with diabetes before 6 months of age do not have the genetic characteristics of autoimmune diabetes (15). Many patients from this cohort have since been diagnosed with KCNJ11 mutations (10), but the etiology of diabetes in the remainder is uncertain. To determine the likelihood of type 1 diabetes in children diagnosed in infancy, we investigated the frequency of high-risk HLA class II genotypes in a cohort of 187 subjects diagnosed with permanent diabetes under the age of 2 years, 32 with KCNJ11 mutations and 155 in whom mutations have been excluded.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
A total of 187 subjects were recruited worldwide. The inclusion criteria for this study were a diagnosis of diabetes before 24 months of age requiring continual insulin treatment from diagnosis. The physician’s classification was type 1 diabetes in the majority of cases. This cohort included 104 U.K. samples, of whom 58 subjects were recruited from the 1972–1981 British Diabetic Association Under 2s cohort (16) and 19 from the Bart’s Oxford study of childhood diabetes (17). Of the 187 samples, 79 were diagnosed at 0–6 months of age (22 of U.K. origin), 45 were diagnosed at 7–12 months (21 of U.K. origin), 32 were diagnosed at 13–18 months (30 of U.K. origin), and 31 were diagnosed at 19–24 months (31 of U.K. origin). Consent was obtained from all patients or their parents.

HLA genotypes on 621 adult U.K. Caucasian control subjects from the general population with no history of autoimmune disease have been described previously (18). All control subjects gave written informed consent.

Sequencing of KCNJ11.
All subjects were sequenced for KCNJ11 as previously described (5–7,11 and personal communication with A.L. Gloyn, Diabetes Research Laboratories, University of Oxford, Oxford, U.K.).

HLA class II genotyping.
HLA-DRB1, -DQA1, and -DQB1 genotyping was performed using a Dynal reverse SSOP method (Dynal Biotech, Wirral, U.K.). Details of HLA class II genotypes in the 77 individuals from the Bart’s Oxford study/British Diabetic Association Under 2s study and 621 U.K. control subjects were already available (7,18). In the absence of parental samples, haplotypes were identified on the basis of well-established patterns of linkage disequilibrium between HLA DRB1 and DQB1.

For the purposes of this study, the genotypes HLA DRB1*04-DQB1*0302/X or HLA DRB1*03-DQB1*0201/X (where X was not DRB1*02-DQB1*0602) were defined as high risk. This includes the highest risk genotype HLA DRB1*04-DQB1*0302/HLA DRB1*03-DQB1*0201. All other genotypes including those containing the protective DRB1*02-DQB1*0602 were defined as low risk.

Statistical analysis.
To examine differences in the frequencies of HLA genotypes, ² analysis was used. All data were stored according to existing data protection regulations.

	RESULTS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
As previously reported, 32 of the 187 (17%) patients had heterozygous mutations in the KCNJ11 gene. All subjects with a KCNJ11 mutation were diagnosed with diabetes before 6 months of age (median 1.15 months [range 0.23–6]).

The overall results obtained are shown in Table 1. Of 32 individuals with a Kir6.2 mutation, 1 (3%) had the highest-risk HLA DRB1*04-DQB1*0302/HLA DRB1*03-DQB1*0201 genotype, whereas 16% had protective genotypes. These data are remarkably similar to those of the healthy control population, with frequencies of 3 and 17%, respectively. Of 47 children diagnosed under the age of 6 months with no mutation in the KCNJ11 gene, 4 (9%) carried the highest-risk genotype for type 1 diabetes and 17% were positive for protective genotypes, also very similar to the healthy control population. This is in marked contrast to 108 children diagnosed at >6 months of age, where 50% had the highest-risk HLA genotype and only 4% had protective genotypes. These data, including mutation status, country of origin, HLA data, and age at diagnosis, are available in an online appendix (available at http://diabetes.diabetesjournals.org.

View larger version (59K): [in this window] [in a new window]   FIG. 1. The prevalence of high-risk HLA class II genotypes (DR4-DQ8/DR3-DQ2, DR4-DQ8/X, and DR3-DQ2/X, where X is not DR2-DQ6) in all subjects from the international cohort (n = 187) diagnosed with diabetes under 24 months of age and in the normal control population. The group diagnosed under 6 months was compared with the three age-groups over 6 months. ****P < 0.00001.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
We have shown that HLA class II high-risk genotypes for susceptibility to type 1 diabetes are much more common in subjects diagnosed with diabetes over the age of 6 months than in those diagnosed before 6 months. The frequency of high-risk HLA class II genotypes in patients diagnosed before 6 months, regardless of KCNJ11 mutation status, was similar to that found in control subjects. Individuals diagnosed before 6 months of age are therefore unlikely to have type 1 diabetes, and even those who do not have mutations in the KCNJ11 gene are likely to have other forms of monogenic diabetes.

Our data support a previous elegant study (15) of HLA class II genotypes in 111 Italian subjects diagnosed under 12 months of age, which suggested that individuals diagnosed with diabetes under the age of 6 months frequently have protective HLA genotypes for type 1 diabetes. They also showed that patients diagnosed before 6 months of age were less likely to have ß-cell autoantibodies (n = 46) and more likely to have low birth weight (small for gestational age) than patients diagnosed after 6 months. Low birth weight is an indicator of reduced insulin secretion in utero and is consistent with a genetic cause of reduced insulin secretion in utero. This has been described with known monogenic causes of neonatal diabetes (19) such as heterozygous-activating KCNJ11 mutations (6) and homozygous glucokinase mutations (20). In contrast, there is evidence that HLA genotypes associated with type 1 diabetes are associated with high birth weight (21).

Although this is the largest study of HLA genotypes in patients aged between 0 and 2 years, it is difficult to be certain of an absolute cutoff at 6 months. The fact that the prevalence of high-risk HLA is slightly lower in thosediagnosed between 6 and 12 months of age compared with those between 12 and 24 months of age indicates that there may be a few patients with nonautoimmune diabetes in the 6- to 12-month age range.

Our data emphasize that, as expected, there is not a role for HLA class II genotyping in the classification of type 1 diabetes on an individual basis. While the absence of a high-risk HLA genotype makes type 1 diabetes unlikely, the presence of high-risk HLA does not exclude nonautoimmune diabetes. Approximately 40% of patients with KCNJ11 mutations have high-risk HLA genotypes, similar to their prevalence in the general population.

The risk associated with type 1 diabetes HLA haplotypes differs between continents (22). In this study, we assumed that HLA-DRB1*04-DQB1*0302 and HLA-DRB1*03-DQB1*0201 are high risk in all populations. We addressed this potential source of error by analyzing subjects of European Caucasian origin separately and obtained results similar to those of the international cohort.

We have shown that the frequency of HLA class II high-risk genotypes is significantly different in subjects with insulin-treated permanent diabetes diagnosed over and under 6 months of age. Those diagnosed under 6 months of age have a distribution of high-risk genotypes similar to that of the normal population. Our study confirms that children diagnosed with diabetes under 6 months of age are unlikely to have type 1 diabetes and should be screened for KCNJ11 gene mutations.

	ACKNOWLEDGMENTS

 
This study was funded by Diabetes UK and the Wellcome Trust at the University of Bristol and at Peninsula Medical School. A.T.H. is a Wellcome Trust Research Fellow.

We thank Prof. Steve Gough for providing HLA data on healthy control subjects.

	FOOTNOTES

 
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.

DOI: 10.2337/db06-0094

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication January 20, 2006 and accepted in revised form March 13, 2006

	REFERENCES

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Pearson ER, Starkey BJ, Powell RJ, Gribble FM, Clark PM, Hattersley AT: Genetic aetiology of hyperglycaemia determines response to treatment in diabetes. Lancet 362:1275–1281, 2003[Medline]
2. Devendra D, Eisenbarth GS: 17. Immunologic endocrine disorders. J Allergy Clin Immunol 111 (Suppl. 2):S624–S636, 2003
3. Shield JP: Neonatal diabetes: new insights into aetiology and implications. Horm Res 53 (Suppl. 1):7–11, 2000
4. Hattersley AT, Ashcroft FM: Activating mutations in Kir6.2 and neonatal diabetes: new clinical syndromes, new scientific insights, and new therapy. Diabetes 54:2503–2513, 2005[Abstract/Free Full Text]
5. Gloyn AL, Cummings EA, Edghill EL, Harries LW, Scott R, Costa T, Temple IK, Hattersley AT, Ellard S: Permanent neonatal diabetes due to paternal germline mosaicism for an activating mutation of the KCNJ11 gene encoding the Kir6.2 subunit of the beta-cell potassium adenosine triphosphate channel. J Clin Endocrinol Metab 89:3932–3935, 2004[Abstract/Free Full Text]
6. Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, Slingerland AS, Howard N, Srinivasan S, Silva JM, Molnes J, Edghill EL, Frayling TM, Temple IK, Mackay D, Shield JP, Sumnik Z, van Rhijn A, Wales JK, Clark P, Gorman S, Aisenberg J, Ellard S, Njolstad PR, Ashcroft FM, Hattersley AT: Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med 350:1838–1849, 2004[Abstract/Free Full Text]
7. Edghill EL, Gloyn AL, Gillespie KM, Lambert AP, Raymond NT, Swift PG, Ellard S, Gale EA, Hattersley AT: Activating mutations in the KCNJ11 gene encoding the ATP-sensitive K⁺ channel subunit Kir6.2 are rare in clinically defined type 1 diabetes diagnosed before 2 years. Diabetes 53:2998–3001, 2004[Abstract/Free Full Text]
8. Sagen JV, Raeder H, Hathout E, Shehadeh N, Gudmundsson K, Baevre H, Abuelo D, Phornphutkul C, Molnes J, Bell GI, Gloyn AL, Hattersley AT, Molven A, Sovik O, Njolstad PR: Permanent neonatal diabetes due to mutations in KCNJ11 encoding Kir6.2: patient characteristics and initial response to sulfonylurea therapy. Diabetes 53:2713–2718, 2004[Abstract/Free Full Text]
9. Vaxillaire M, Populaire C, Busiah K, Cave H, Gloyn AL, Hattersley AT, Czernichow P, Froguel P, Polak M: Kir6.2 mutations are a common cause of permanent neonatal diabetes in a large cohort of French patients. Diabetes 53:2719–2722, 2004[Abstract/Free Full Text]
10. Massa O, Iafusco D, D’Amato E, Gloyn AL, Hattersley AT, Pasquino B, Tonini G, Dammacco F, Zanette G, Meschi F, Porzio O, Bottazzo G, Crino A, Lorini R, Cerutti F, Vanelli M, Barbetti F: KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes. Hum Mutat 25:22–27, 2005[Medline]
11. Flanagan SE, Edghill E, Gloyn AL, Stals K, Ellard S, Hattersley AT: Kir6.2 gene mutations are a common cause of diabetes before 6 months and the presence of neurological features varies with the site of the mutataion. Diabet Med 22: 10, 2005
12. Klupa T, Edghill EL, Nazim J, Sieradzki J, Ellard S, Hattersley AT, Malecki MT: The identification of a R201H mutation in KCNJ11, which encodes Kir6.2, and successful transfer to sustained-release sulphonylurea therapy in a subject with neonatal diabetes: evidence for heterogeneity of beta cell function among carriers of the R201H mutation. Diabetologia 48:1029–1031, 2005[Medline]
13. Zung A, Glaser B, Nimri R, Zadik Z: Glibenclamide treatment in permanent neonatal diabetes mellitus due to an activating mutation in Kir6.2. J Clin Endocrinol Metab 89:5504–5507, 2004[Abstract/Free Full Text]
14. Codner E, Flanagan S, Ellard S, Garcia H, Hattersley AT: High-dose glibenclamide can replace insulin therapy despite transitory diarrhea in early-onset diabetes caused by a novel R201L Kir6.2 mutation. Diabetes Care 28:758–759, 2005[Free Full Text]
15. Iafusco D, Stazi MA, Cotichini R, Cotellessa M, Martinucci ME, Mazzella M, Cherubini V, Barbetti F, Martinetti M, Cerutti F, Prisco F: Permanent diabetes mellitus in the first year of life. Diabetologia 45:798–804, 2002[Medline]
16. Botha JL, Parker H, Raymond NT, Swift PG: Diabetes diagnosed before the age of 2 years: mortality in a British cohort 8–17 years after onset. Int J Epidemiol 21:1132–1137, 1992[Abstract/Free Full Text]
17. Bingley PJ, Bonifacio E, Williams AJ, Genovese S, Bottazzo GF, Gale EA: Prediction of IDDM in the general population: strategies based on combinations of autoantibody markers. Diabetes 46:1701–1710, 1997[Abstract]
18. Simmonds MJ, Howson JM, Heward JM, Cordell HJ, Foxall H, Carr-Smith J, Gibson SM, Walker N, Tomer Y, Franklyn JA, Todd JA, Gough SC: Regression mapping of association between the human leukocyte antigen region and Graves disease. Am J Hum Genet 76:157–163, 2005[Medline]
19. Hattersley AT, Tooke JE: The fetal insulin hypothesis: an alternative explanation of the association of low birth weight with diabetes and vascular disease. Lancet 353:1789–1792 1999
20. Njolstad P, Sovik O, Cuesta-Munoz A, Bjorkhaug L, Massa O, Barbetti F, Undlien D, Shiota C, Magnuson M, Molven A, Matschinsky F, Bell G: Neonatal diabetes mellitus due to complete glucokinase deficiency. N Engl J Med 344:1588–1592, 2001[Free Full Text]
21. Larsson HE, Lynch K, Lernmark B, Nilsson A, Hansson G, Almgren P, Lernmark A, Ivarsson SA: Diabetes-associated HLA genotypes affect birthweight in the general population. Diabetologia 48:1484–1491, 2005[Medline]
22. Ronningen KS, Keiding N, Green A: Correlations between the incidence of childhood-onset type I diabetes in Europe and HLA genotypes. Diabetologia 44 (Suppl. 3):B51–B59, 2001

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