DETECTION OF SUSCEPTIBILITY TO DIABETES

Abstract: The invention relates to the detection of genetic susceptibility and to the therapy and prophylaxis of diabetes. It has now been found out that certain specific HLA haplotypes exhibit a common genetic determinant for both IDDM, NIDDM and glucose intolerance. In accordance with the invention, the genetic susceptibility to diabetes can now be done by determining whether the person has said diabetogenic HLA haplotypes.

Description: DETECTION OF SUSCEPTIBILITY TO DIABETES FIELD OF THE INVENTION This invention relates to the detection of genetic sus- ceptibility to diabetes mellitus and to methods for the treatment and prevention of diabetes. BACKGROUND OF THE INVENTION Diabetes mellitus (DM) is a common disease worldwide which leads to hyperglycaemia. Its clinical manifestation ranges from insulin dependent diabetes mellitus (IDDM, type 1 dia¬ betes, childhood onset diabetes) to non-insulin dependent diabetes mellitus (NIDDM, type 2 diabetes, adult onset dia¬ betes) including the following subgroups: MODY diabetes (ma- turity-onset diabetes in the young) , malnutrition diabetes, gestational diabetes mellitus (GDM) (asymptomatic diabetes) , potential diabetes in pregnancy, secondary failures (diabetic patients whose blood sugar could be controlled by diet alone but who eventually need insulin) , impaired glucose tolerance (IGT) . Evidence indicates that both non-insulin dependent dia¬ betes mellitus (NIDDM) and insulin dependent diabetes melli¬ tus (IDDM) are multifactorial diseases with a strong genetic basis. Furthermore, it is believed that the genetic component in NIDDM appears to be stronger than that in IDDM. The progress in identifying genes involved in NIDDM has been disappointingly slow, in contrast to the rapid advances with regard to IDDM. In the mid-1970's the major genetic susceptibility to IDDM was found to be conferred by a gene (or genes) in the HLA (Human leukocyte system A) region located on chromosome 6 (1-3) . Linkage between the postulated susceptibility gene (or genes) and the HLA genes was estab¬ lished as the HLA haplotype distribution differed in families with two or more affected children from that in non-diabetic families (3-5) . The HLA region is thought to account for up to 70 % of the genetic susceptibility to IDDM (6-7) . Popu¬ lation studies of NIDDM in European and North American Cauca- soids have thus far failed to find a consistent association with HLA. Indeed this lack of an HLA association in NIDDM has been used as argument that NIDDM and IDDM are genetically two separate disease entities (8-10) . Some investigators have speculated that there could be a common genetic basis between IDDM and NIDDM. For instance, it has been suggested that the difference between these two types of diabetes could be ascribed to a double dosage of the gene in IDDM and a single dose in NIDDM (11) . In some populations weak and inconsistent associations have been found between HLA and NIDDM (12-18) . Additional HLA associations have also been reported (19- 21) . It is of interest that in all these studies those HLA antigens which are the most frequent ones in the background population were found to be increased in NIDDM patients. Furthermore, in certain isolated ethnic groups the background population shows a restricted HLA polymorphism and a high degree of homozygosity (22) . It is generally assumed that IDDM is an autoimmune dis¬ ease. One of the fundamental supporting arguments is that class II antigens are expressed on the pancreatic beta cells of IDDM patients. Diabetes leads to severe complications, especially due to vascular diseases causing kidney disease, blindness, heart disease, peripheral vascular disease of the lower ex¬ tremities. The excess of these complications in diabetic subjects cannot be explained by other factors than diabetes per se. It is obvious that to be effective the prevention of complications of diabetes must be related to the genetic mechanisms that control the onset and the course of diabetes. Other ways of therapy are only symptomatic and often do not have any influence on the prognosis of diabetic patients as shown by earlier studies. It would be most helpful if the genetic susceptibility of a person to diabetes could be detected as early as possible, before the possible onset of the symptons and complications. It would also be most helpful if the therapeutic and pro¬ phylactic treatment of diabetes could be targeted to correct the primary cause of the disease. The object of the present invention is to provide means for the above aims. DESCRIPTION OF THE INVENTION Summary of the invention We have now found out that certain specific HLA haplotypes exhibit a common genetic determinant for both IDDM, NIDDM and glucose intolerance. In accordance with the invention, the genetic suscep¬ tibility to diabetes can now be done by determining whether the person has said diabetogenic HLA haplotypes. The test is sufficiently sensitive and specific to detect the genetic susceptibility to diabetes. It can be used to detect the probable diabetes before its clinical manifestations in persons who have family history for diabetes and in the general population as well. Specific tests for different ethnic groups can also developed according to the findings from the groups that will be tested. The test can even be done for fetuses using amniocentesis. The test results can be used for genetic counselling of the families at risk of diabetes, and for individual counselling of people who carry known genetic susceptibility to diabetes to control the environmental determinants that may unmask diabetes. The invention also provides methods and preparations for the treatment or prophylaxis of diabetes in a person. In accordance with the invention, the expression of at least one gene in the diabetogenic haplotypes is prevented, or the action of at least one corresponding specifity is prevented, or at least one of the genes is replaced by another gene so that the haplotype is no more diabetogenic. In accordance with one embodiment, the interaction between the expression between of at least one of the genes and an enviromental agent or other genes is prevented. Thus the process leading to diabetes or worsening it is inhibited. Such a treatment will prevent the insulin secretory defect of the pancreatic beta-cells or maintain normal insulin action in the tissues. In accordance with a second embodiment, the action of at least one of the genes on glucose metabolism and insulin action in the pancratic beta-sells or in other tissues is prevented. In accordance with a third embodiment, the expression of at least one of the genes is prevented in a fetus. Thus the normal developement of pancreatic beta-cells in the fetus is preserved, developement of a sufficient beta-cell mass assured and normal beta-cell function achieved. DETAILED DESCRIPTION OF THE INVENTION HLA System The HLA system (Human Leukocyte system A - it is now mostly thought to stand for Human Leukocyte Antigen system) is located on the short arm of chromosome 6 in the 6p21.3 band. It is a highly polymorphic system of closely linked loci situated in a 3.5 centimorgan long (3,500 kb) phylo- genetically conserved region. The HLA system is by now the most polymorphic system known in man. The extreme HLA polymorphism is thought to be advan¬ tageous as it increases the capacity of the immune system to respond to a large variety of possible antigens. HLA antigens are cell surface glycoproteins which are essential in the initiation of the immune response. In the HLA region the genes, the gene products (i.e. anti¬ gens or specificities), and the regions are known as class I, class II and class III. Class I genes (HLA-A, HLA-B, HLA-C, HLA-F, HLA-G) are located at the telomeric end of the HLA system. The order of the major class I genes is HLA-A, HLA-C, HLA-B and they code for the class I alpha chains of the various HLA-A, HLA-C, HLA-B antigens. The distance between the A and C locus gives a recombination fraction of nearly 1 %. There is a large gap in the genetic map between A and C and new genes can be still discovered in this area. The recombination fraction between A and C is nearly 1 %. According to our hypothesis there is a recombination hot spot between A and C. Three potentially functional genes - HLA-E (previously called 6.2), 6 and 5.4 lie between A and C. Only one of these genes is apparently expressed on the cell surface. The tissue distribution is different from the classical tissue distribution of mouse TL determinants. The human equivalents of the TL and the QA loci of the mouse have not yet been found. Each class I antigen consists of a polymorphic polypeptide chain coded by a gene in the HLA region and an invariate polypeptide chain which is beta-2 microglobulin coded on chromosome 15. The class II genes (prior called HLA-D region genes) are located at the centromeric end of the HLA system. Three major loci exist - HLA-DR, HLA-DQ and HLA-DP - including the various class II genes (DRA, DRB1, DRB2, DRB3, DRB4, DRB5 / DQA1, DQA2, DQB1, DQB2 / DPA1, DPA2, DPB1, DPB2) . In addition, the class II genes HLA-DOB and HLA-DNA exist. Most class II antigen consist of two different polypeptide chains, alpha and beta, and both of them are controlled by genes in the HLA region. The DR beta chain loci are the exception as three or four are found on most haplotypes. Of the four DQ genes DQA1 codes for the DQ alpha chain and DQB1 for the DQ beta 1 chain. The products of the DQA2 and the DQB2 genes are not expressed. The class III genes are situated between the class I and II genes. The class III gene products are the complement proteins C2 and C4 (C4A and C4B) of the classical complement pathway and properdin factor (Bf) of the alternative comp¬ lement pathway. C4A gene products are of the rapidly migrating acidic type (previously Rodgers blood group) and the C4B gene products of the slowly migrating basic type (previously Chido blood group) . During the last few years methods used in molecular bio¬ logy have allowed a detailed mapping of the HLA system. Apart from class I, II and III genes other genes exist in the HLA region. The two genes for 21 hydroxylase (210HA, 210HB) and the genes for tumour necrosis factor (TNF alpha, TNF beta) are situated between the class I and II genes. Also the genes for the major heat shock protein HSP70 and HLA-B associated transcripts (BAT 1-5) are between the class I and III region. In addition, pseudogenes exist in the HLA system and genes whose expression has not yet been detected. Class I antigens are expressed on all cells in the human body with the exception of the mature erythrocyte. The amount of class I antigens detectable on the cell surface is de¬ pendent on the turnover rate. It can be enhanced by inter- feron-gamma (INF-gamma) which is a cytokine produced by activated T lymphocytes. Also macrophage-derived tumour necrosis factor (TNF) enhances the expression of class I antigens. Class II antigens show tissue restriction and are mainly found on B lymphocytes, monocytes, macrophages, endothelial cells, activated T lymphocytes and other activated cells. Pancreatic beta cells from newly diagnosed IDDM patients have been shown to express augmented levels of class I anti¬ gens. In addition, they express class II molecules which are undetectable or in extremely low levels on non-diabetic pan¬ creatic beta cells. Expression of class II antigens can be induced on islet cells by the synergistic action of INF-gamma and TNF. Class II antigens are expressed on the pancreatic beta cells of IDDM patients. One has to bear in mind that also other cells, like for instance cancer cells, show detectable amounts of class II antigens once they are activated. So far it has not been shown whether the class II antigens expressed on the pancreatic beta cell correspond to the class II tissue type of that particular person or whether really new and aberrant class II antigens are expressed. Inheritance of HLA Antigens HLA genes are expressed in a co-dominant way and are gen¬ erally inherited according to Mendelian rules. HLA alleles are inherited en bloc in form of haplotypes. An allele is the contribution of one parent to a single locus and a haplotype is the contribution of one parent to a set of closely linked loci. Each person has two haplotypes one inherited from the father and one from the mother. By tissue typing all members of a family one can establish HLA genotypes and can get accurate information on the four different parental haplo¬ types and their inheritance. In a healthy family the prob¬ ability of any pair of siblings being HLA identica, i.e. they inherited the same two parental haplotypes, or being HLA non- identical, i.e. they inherited two different parental haplo- types, is 25 %. The probability is 50 % that any pair of sibling is HLA haploidentical, i.e. they inherited one parental haplotype in common. Nomenclature of the HLA System The nomenclature for the HLA specificities identidied by serology or cellular typing is such that each antigen is defined by a letter characterizing the locus and a number identifying the specificity. Sometimes a "w" follows the letter for the locus indicating that it is a provisional "workshop" identification. This holds not true for the C locus where the "w" is kept to avoid confusion with the complement loci. The specificities of the A and B locus are numbered jointly for historical reasons. All other loci are numbered independently. The nomenclature for the genes in the HLA regions is as follows: 1) first the prefix HLA-, 2) then a letter speci¬ fying the locus, 3) followed by a letter for the subregion, 4) followed by either the letter A for the alpha chains or the letter B for the beta chains, 5) a number when there is more than one alpha or beta chain gene in the subregion. The nomenclature of the alleles in the HLA region is that after the gene name an asterisk follows and then a unique four digit number. The first two digits describe the most closely associated serological specificity in order to retain the relationship between alleles and HLA specificities de¬ fined by serology. In the present application, HLA specificities are used for designating haplotypes instead of complete allele names; i.e. B27 instead of HLA-B*2701. Also the loci will be re¬ ferred to as A, C, B instead of HLA-A, HLA-C, HLA-B. Special features of HLA Antigens Due to its extensive polymorphism the HLA system provides the best markers for anthropological studies. The frequencies of the many different HLA antigens vary between populations. Certain HLA specificities are exclusively found in particular ethnic groups. Therefore, also the antigen combinations found together as haplotypes differ between populations. Some haplotypes are exclusively found in one of the three major racial groups (Caucasoids, Blacks, Mongoloids) . Another striking feature of the HLA system is the strong linkage disequilibrium or gametic association between the genes of the various HLA loci. To understand the phenomenon of linkage disequilibrium is of prime importance for an understanding of the genetics of the HLA region and of the interaction between IDDM and HLA. Certain antigens of the various HLA loci occur more or less often together on a haplotype than is expected from the product of their gene frequencies. The difference between the product of the constituent allele frequencies and the observed haplotype frequency is measured by the δ value. It gives the extend of linkage disequilibrium and is 0 when two alleles are independent. The closer the loci are, the stronger is the linkage disequilibrium and the higher the δ value. Between certain alleles linkage disequilibrium seems to be nearly total. This is the reason why restriction fragment length polymorphism (RFLP) can be used to detect class II genes despite the fact that most restriction sites are located in the non-coding part of the DNA. Closely linked genes tend to remain together rather than undergo genetic randomization. Particular haplotypes are either favoured by natural selection or not enough time had elapsed for the alleles involved to become randomly dis¬ tributed in the population. Recombinations (cross-overs) reduce linkage disequilibrium by a factor of 1-θ per gen¬ eration, θ is the recombination frequency between the two loci in linkage disequilibrium. As the recombination fraction between closely linked loci is low, linkage disequilibrium between closely linked l ci will be conserved longer than that for more distant loci. Linkage disequilibrium between the same alleles varies between populations. It also varies between antigen combi- nations seen on haplotypes frequently found in IDDM patients ("diabetic haplotypes") and on haplotypes found in non- diabetic individuals ("non-diabetic" haplotypes) . One hypothesis is that the origin of a linked set of genes evolved through a series of duplication events. In the HLA region many genes with different but related functions are found most of which are in linkage disequilibrium. This results in specific gene combinations which we can crudely detect by determining "whole" HLA haplotypes including anti- gens of the A, C, B, DR and DQ loci as markers. This way genes whose gene products can not yet be detected but which are in linkage disequiblirum like for instance the putative susceptibility genes for IDDM can be assessed indirectly. Cross-reactivity between HLA specificities is a common phenomenon. Highly cross-reacting antigens might have evolved one from the other by recombination between alleles. The specificity of the various HLA antigens is determined only by a small fraction of the total HLA molecule. Even monoclonal HLA antibodies show the usual cross-reactions seen by sero- logy. Most originally defined HLA specificities coded both by class I and class II genes have been found later on to be heterogeneous. "Broad" antigens or "supertypic" specificities are usually "split" into several components either by using better defined antisera, or by testing them against different ethnic groups. One other common method to "split" antigens is to use a closely linked locus. Many B locus specificities can be "split" with the help of C locus specificities. Nowadays, the DQ locus is successfully used to subdivided DR specificities, especially DR4 both with conventional sero¬ logy and molecular methods. Using DQ-beta chain intron- specific probes and the restriction enzyme BamHI the DR4, DQwδ positive haplotypes are characterized by a 12 kb fragment and the DR4, DQw7 positive haplotypes by a 3.7 kb fragment. Detection of HLA antigens Conventional serology Class I antigens are defined by serology on isolated viable peripheral blood lymphocytes in a complement-dependent micro-cytotoxicity test. Well characterized alloantibodies identified in sera from parous women are used to define the various HLA specificities and subgroups. A and B antigens show extensive serological cross-reactions. Two mutually exclusive supertypic specificities Bw4 and Bw6 exist which represent epitopes on the B molecules separate from the B antigen specificity. Some class II antigens are like the class I antigens defined by serology (DR, DQ) and others by cellular methods (D, DP) . A complement-dependent micro-cytotoxicity test is used for DR and DQ typing taking separated viable B-lympho- cytes as target cells. Sera from parous women (previously absorbed with thrombocytes which carry class I but not class II antigens) are mainly used as source of HLA antibodies. Also monoclonal antibodies against DR and DQ specificities exist. Serological cross-reactions between the various DR and between DQ specificities are common. Many different techniques exist to isolate T and B lympho- cytes from blood. In recent years a major breakthrough was made with the introduction of immunomagnetic beads. This not only made the test more sensitive but reduced the time needed for HLA-A, C, B, DR and DQ typing. In the future we will be able to develop specific test kits to be applied for diabetes associated haplotypes only. Sera from multiparous women are a good source for HLA antibodies. They are produced during pregnancy against the paternal HLA antigens (foreign antigenes) inherited by the fetus from the father. Sera which contain the specific alleles which make up the diabetogenic haplotypes will be defined and utilized. Isolated lymphocytes of the person to be tested are incubated with these HLA antibody containing sera in the presence of rabbit complement. Cell death will be assessed and the haplotypes deduced. A special kit of HLA antisera (around 60 to 120 different sera) or series of several kits which cover the necessary HLA specificities to define the diabetogenic haplotypes can be assembled. Such a kit may be based on the use of immunomagnetic beads combining several of the alleles into a single kit. Molecular methods in HLA A major breakthrough in molecular biology has made it possible to look at HLA at the genomic level. DNA can be isolated from blood samples or can be prepared from EB virus transformed cell lines. The polymerase chain reaction (PCR) is an in vivo method to replicate a specific nucleic acid target when it is present at a low copy number. The amplified product can be detected by gel electrophoresis and hybridiz- ation. PCR makes it possible to obtain large amounts of DNA. HLA Class I (A, C, B) and Class II (DR, DQ) genes, which make up the diabetogenic haplotype, can be defined by using sequence-specific primers (SSP) after the amplification of the DNA from the person to be tested. A kit or a series of kits which contains the necessary SSP's for defining the diabetogenic haplotypes can be developed. 1. Restriction fragment length polymorphism (RFLP) : Many different restriction enzymes are available which detect a large number of RFLP markers. Each restriction enzymes produces a number of fragments. These fragments are inherited according to Mendelian rules. The size of these fragments are often estimated differently by the various investigators which is very confusing. RFLP analysis reflects intron polymorphism. Using DQ-beta chain intron-specific probes and the re¬ striction enzyme BamHI the DR4, DQw8 positive haplotypes can be differentiated from the DR4, DQw7 positive haplotypes by a 12 kb and a 3.7 kb fragment respectively. All DR3, DQw2 positive haplotypes give a 4 kb fragment. 2. Oligonucleotide probes: The use of synthetic oligonucleotides for molecular HLA genotyping of the class II region has been very successful. The patterns obtained with locus-specific oligonucleotide probes are easier to interpret than patterns detected with cDNA probes. Exon specific oligonucleotide probes have been used to subdivide DQw3 on the genomic level into DQw7 (3.1) and into DQwδ (3.2). DQw7 has tyrosine and DQwδ leucine in position 26 of the first domain of the beta chain. 3. DNA sequencing DNA amplified by PCR can be cloned and sequenced. Nucleo- tide sequence data reveal a wide variety of alleles. The serologically defined HLA specificities often encompass the products of more than one allele. For instance, the serologi¬ cally defined specificity HLA-A2 is now known to include at least nine distinct proteins (A*0201 to A*0210) . Conventional serology versus molecular methods Conventional serology made HLA research possible and still remains the solid foundation of all molecular studies. The serological tests can be carried out fast and are relatively inexpensive whereas the methods used in molecular genetics are time consuming and expensive. The disadvantage of conventional serology is that fairly fresh blood samples are needed whereas DNA can be separated any time from frozen samples. Storage of DNA is simple whereas lymphocytes for tissue typing must be stored in liquid nitrogen after controlled freezing. Using methods of molecular biology one can detect genetic polymorphisms in the HLA region which are not apparent or not easily defined using conventional serolo y. Whatever is de- tected by the higher resolution of the molecular methods is still part of the "whole" HLA haplotype due to linkage dis¬ equilibrium in the HLA system. So far all siblings who were defined by conventional serology as being HLA identical have also been identical on the DNA level. The crucial question to be answered is whether HLA identical siblings or monozygotic twins always form the same DQ trans-associated heterodimers. Ideally, molecular methods should be applied outside the HLA system to look for possible additional genetic markers for IDDM. Examination showing the susceptibility Subjects and methods A cohort of 1711 men born in two Finnish communities between 1900 and 1919 were included in a multinational prospective cardiovascular disease (CVD) study, the Seven Countries Study, which started in 1959. The survivors of this cohort (n = 716) were first tested for glucose intolerance in 1984. In 1989, when the oral glucose tolerance test (OGTT) was repeated the men were 70 to 89 years old, i.e. old enough for the vast majority of potential diabetic subjects to have developed the disease. The participation rates in these surveys have been over 90 %. The survey procedures have been described elsewhere in detail (23-25) . Methods for HLA typing In 1989, we collected blood specimens for HLA typing from the 98 living men who had been classified as diabetic in the 1984 survey and from 74 control subjects who were non-dia- betic (normal glucose tolerance or IGT) according to the 1984 OGTT results. HLA-A, C, B, Bw, DR, DQ phenotypes were determined serologically in 172 unrelated survivors of the Finnish cohort of the Seven Countries Study using 180 HLA antisera (120 class I and 60 class II) and the same test as a previous research project "Childhood diabetes in Finland" (DiMe Study) (26, 27). In 3 elderly men with diabetes the DR and DQ results were not clear. HLA typing was done with a microlymphocytotoxic test using immunomagnetic beads ('Dyna- beads', Dynal AS, Oslo, Norway) coated with monoclonal antibodies against class I or class II for the isolation of T and B lymphocytes. All observed HLA phenotypes were transformed into the two most probable HLA haplotypes. The assignments of the most probably haplotypes were based on 3528 Finnish haplotypes derived from 3314 HLA genotyped individuals belonging to 882 different Finnish families (26, 27) . Between 1986-89 757 families consecutively ascertained through a newly diagnosed IDDM child (DiMe Study) and 125 non-diabetic control families had been HLA genotyped. Four- locus (A, C, B and DR) haplotype frequencies were compiled in the IDDM probands and the control children. Three-point delta values between the A, B and DR loci were calculated as de¬ scribed by Baur et al (28) . A computer program for HLA haplo¬ type analysis in families was especially developed by us for these studies. Definition of IDDM associated haplotypes In the recent DiMe Study which was the first population- based prospective family study in IDDM HLA genotypes and haplotypes were determined in 757 of 801 newly diagnosed children with IDDM aged 14 years or under and their families nationwide (26, 27) . Whole HLA haplotypes determined sero- logically by alleles of the class I loci A, C, B and the class II loci DR and DQ were used as markers for suscepti- bility to IDDM. HLA-DQ was not only defined by serology but for the 19 most frequently found Finnish haplotypes sequence- specific oligonucleotide typing for the 7 most important Finnish haplotypes (29) . In the DiMe Study several attempts were made to find the best way of classifying the 1470 unequivocally defined haplotypes found in the newly diagnosed IDDM probands (aged 14 years or under) according to their importance for conferring susceptibility to IDDM. "High-risk" IDDM as¬ sociated HLA haplotypes were defined as those haplotypes which had been seen in families either as the haplotype transmitted from an IDDM parent (n = 66) to an IDDM child or as the haplotype shared between haplo-identical affected IDDM siblings (n = 19) (Table I) . These thus defined IDDM associ¬ ated high risk haplotypes carry the susceptibility gene (or genes) for IDDM except in the rare event of recombination between the haplotype and the disease susceptibility locus (or loci) . 37 different high risk IDDM associated haplotypes were identified in Finland through the population-based DiMe Study (Table I) . These 37 high risk haplotypes explained the genetic susceptibility to IDDM in 83.8 % of the DiMe probands under the hypothesis that one high risk haplotype is suf¬ ficient to confer susceptibility to IDDM. Many of the haplotypes found in the 119 probands of the DiMe study who did not carry the above mentioned high risk IDDM associated haplotypes were different from these high risk haplotypes only by one allele at either the A, the C or the DR locus (Table II) . 13 of these such derived haplotypes which still conferred a risk for IDDM were also found in elderly Finnish men with NIDDM and IGT. These haplotypes had been modified by a recombination between either the A and C, the C and B, or the B and DR loci. Five additional haplotypes were considered diabetes associated once the classification was refined as they were only seen in elderly men with NIDDM and not in non-diabetic elderly men. These haplotypes were again derived from the original 37 IDDM associated high risk haplotypes allowing for one possible and for the particular haplotype likely sub- stitution at the A, the C or the DR locus. This original assignment of 50 diabetes associated hap¬ lotypes was then extended to 57 as 2 haplotypes (A3, Cw7, B7, Bw6, DRw6, DQw6 and A24, Cw7, B39, Bw6, DRw8, DQw4) trans¬ mitted from an NIDDM parent to an IDDM child (Table I) and 5 additional haplotypes (Table II) were considered diabetes associated. TABLE I. IDDM ASSOCIATED "HIGH RISK" HLA HAPLOTYPES (TABLE I CONT.) I (TABLE I CONT.) CO c CD ω H H c H m ω I m 51 n =» number of haplotype TABLE II. ADDITIONAL DIABETES ASSOCIATED "RISK" HLA HAPLOTYPES ) (TABLE II CONT.) D o H We predicted the glucose tolerance status using HLA haplotype data in 147 men who were classified into 2 groups: (i) Men with an IDDM associated high risk haplotype or a diabetes associated haplotype as defined in Tables I and II (n = 114) , (ii) Men without any diabetes associated haplo¬ types (n = 33) . In two non-diabetic elderly men two alterna¬ tive haplotypes (one diabetes associated and one non-diabetes associated) were equally probable and therefore prediction estimates were computed in two alternative way. The glucose tolerance status was defined based on the WHO 1985 criteria (10) and men were classified in the following way: (i) Diabetes (n = 90) , if the criteria for diabetes were met either in 1984 or in 1989. In 1984, 15 of the dia¬ betic men were treated with oral antidiabetic agents and one with insulin, and in 1989, 19 with oral agents, two with insulin and two had both oral agents and insulin. All these treated with drugs were classified as diabetic regardless of their blood blucose values, and many of these men did not take a glucose tolerance test; (ii) IGT (n = 34) if the WHO criteria for IGT (but not diabetic) were met either in 1984 or in 1989 and they had no antidiabetic drug treatment; (iii) Normal glucose tolerance (n = 23) if the two OGTT's in 1984 and 1989 were both normal. Men whose glucose tolerance status had not been determined either in 1984 or in 1989 (n = 22) and the three men whose DR typing results were inconclusive were excluded from certain analyses. Statistical analyses The assignment of the HLA status (diabetes associated or no diabetes associated HLA haplotypes) was done in the 172 HLA phenotyped elderly men using HLA haplotypes without access to any clinical or biochemical data. The classi¬ fication "diabetes associated" was made when at least one of the IDDM associated haplotypes - either one of the 37 high risk or one of the 13 other IDDM associated haplotypes - was present. A person without any IDDM associated haplotype was classifies as "no diabetes associated". Sensitivity, speci¬ ficity and predictive power according to the HLA haplotype status among men with diabetes or normal glucose tolerance were calculated in two different ways; separately for diabetic men only and for the diabetes and IGT categories together. In order to assess the overall impact of the HLA haplotype status on blood glucose concentration in the study subjects we computed fasting and 2-hour post-challenge blood glucose distributions and mean values for men with diabetes as¬ sociated HLA haplotypes in comparison with those in men who had no diabetes associated HLA haplotypes. Analyses were done with SAS computer software using the general linear models procedure. Blood glucose values whenever available were included in this analysis which was done separately for blood glucose in 1984 and 1989 and for the average of the two measurement in each subjects. Differences in HLA antigen frequencies between diabetic men and men with normal glucose tolerance were tested for significance with the X 2 -test with Yates correction or Fisher's exact test when the number of cases per cell was less than 5. Results Prediction of abnormal and glucose tolerance by IDDM associated haplotypes In 131 of the 147 elderly men (89 %) who had complete biochemical data the diagnosis was correctly predicted by the HLA haplotype data (Table III) . Diabetes associated haplo¬ types were present in 94 % (85/90) of diabetic subjects, 79 % (27/34) of IGT subjects and only (17 %) (4/23) of non- diabetics (X 2 = 65.4; p < 0.0001; Cramers V = 0.67). Sensitivity, i.e. the probability of correctly identifying truly diabetic men with diabetes associated HLA haplotypes was high for diabetic men (94 %) . It was only slightly lower for the combination of men with IGT together with diabetic men (90 %) . Specificity, i.e. the probability of correctly identifying non-diabetic men by possessing no diabetes associated HLA haplotypes only was also high: 91 % or 83 % depending how the two men with unclear haplotypes were con- TABLE III. PREDICTION OF ABNORMAL GLUCOSE TOLERANCE STATUS BY THE HLA HAPLOTYPE STATUS sidered. Predictive power for diabetes associated HLA haplo¬ types varied between 95 % and 98 % for the two alternative ways of calculation. Distribution of the IDDM associated haplotypes Of the 37 different IDDM associated high risk haplotypes found in Finnish children with IDDM 26 (70 %) were also seen in elderly Finnish men with NIDDM, 12 (32 %) in elderly men with IGT and 3 (8 %) in the non-diabetic men (Table I) . 5 of the 11 IDDM associated high risk haplotypes not seen in the elderly men with NIDDM carried A24, and 9 had a low haplotype frequency (under 0.7 %) in the IDDM probands. Using the alleles of the most polymorphic HLA locus - the B locus - as determinants 13 basic haplotypes are the origin for all 37 IDDM associated high risk haplotypes (Table I) . At the A locus A2, A24, A28 and A3 were consistently found on the 13 basic haplotypes. A32 was seen on B39 and B44 containing haplotypes and infrequently Aw33 on B14, and A30 on B18 containing haplotypes. Al was only seen on one haplotype wich conferred the lowest risk of all B8, DR3 positive haplotypes. At the C locus Cwl, Cw3, Cw7 were the most frequent alleles. Most IDDM associated high risk haplotypes were Bw6 positive and carried either DR4 or DR3 (83 %) . HLA antigen frequencies Overall, the differences in HLA phenotype frequencies between the three strata of men (diabetic, IGT and normal) were generally small (Table IV) . With regard to the A-locus we found no statistically significant differences between the glucose tolerance strata. The frequency of B51 was signifi¬ cantly lower (p = 0.042) in diabetic men compared with non- diabetic men. The frequency of DR4 (p = 0.0005) and that DRw8 (p = 0.042) were significantly higher and that of DR1 lower (p = 0.035) in diabetic men. Blood glucose concentration and HLA haplotypes Average fasting blood glucose was significantly higher (p = 0.037) in men with diabetes associated HLA haplotypes than in men with no diabetes associated haplotypes (Table V) . The 2-hr glucose challenge resulted in only a moderate shift towards higher values in men with no diabetes associated HLA haplotypes (the mean value moved from fasting 5.6 mmol/1 to 2-hr 6.6 mmol/1) . In men with diabetes associated HLA haplotypes the shift of the blood glucose distribution was very drastic (the mean value moved from fasting 5.6 mmol/1 to 2-hr 10.7 mmol/1) . The differences in the 2-hour post- challenge blood glucose mean values between the two strata of men according to the HLA haplotype status was highly significant (p < 0.0001), and much greater than that for fasting blood glucose. The increment in blood glucose according to the HLA halotype status was essentially the same in 1984 and 1989. Some of the known diabetic subjects did not want to take a glucose load and, therefore, 2-hr blood TABLE IV. HLA PHENOTYPES IN FINNISH ELDERLY MEN (TABLE 17 CONT.) (TABLE IV CONT. ) I I * P < 0.05; *** P < 0.001 between diabetic and normal subjects. TABLE V - MEAN VALUES (GEOMETRIC MEAN) AND STANDARD DEVI¬ ATIONS OF BLOOD GLUCOSE CONCENTRATION BY YEAR AND HLA HAPLO¬ TYPE STATUS * = after logarithmic transformation of data glucose values were missing in 20 subjects in 1984 and in 22 subjects in 1989. Most of them were on antidiabetic drug therapy. Their mean fasting blood glucose was 8.5 mmol/1 in 1984 and 11.5 mmol/1 in 1989. The lack of 2-hr blood glucose in these diabetic men, most of whom had a diabetes associated HLA haplotype, is likely to reduce the real difference in 2- hr blood glucose between the groups. The distributions of fasting blood glucose of men in the two strata of the HLA haplotype status were largely over¬ lapping (Figure 1) . The shape of these distributions was very similar and they were both positively skewed. The median values of fasting blood glucose were 5.2 mmol/1 and 5.6 mmol/1 for men with diabetes associated haplotypes and men with no diabetes associated haplotypes, respectively. However, the entire distribution of 2-hr blood glucose in men with diabetes associated HLA haplotypes was markedly shifted to the right. In contrast, in men with no diabetes associated haplotypes the 2-hr blood glucose distribution hardly differed from the distribution of fasting blood glucose. The median values for 2-hr post-challenge blood glucose were 5.7 mmol/1 and 10.0 mmol/1 for the two HLA haplotype strata of men, respectively. It is obvious from the Figure 1 that in men with no diabetes associated haplotypes the distributions for fasting and 2-hr post-challenge blood glucose are strik¬ ingly similar, suggesting that even in elderly subjects with no susceptibility genes for diabetes the glucose load does not affect the post-challenge blood glucose concentration much. Discussion Our study clearly demonstrates that the susceptibility genes for diabetes (for IDDM, NIDDM and IGT) are common and located in the HLA region on the short arm of chromosome 6 (6p21.3 band). Moreover, our data show that the general belief that the genetic background of IDDM and NIDDM is different has been largely incorrect. Most of the HLA haplotypes which carried a high risk for IDDM also carried risk for NIDDM. The question is open why some of the gen¬ etically susceptible individuals get the disease at an earlier age and in a more severe form than others who carry the same HLA haplotype. Recombinations are a way to change IDDM associated high risk haplotypes into haplotypes which carry less or no risk and vice versa. Through the population-based DiMe Study it became clear that recombinations between certain loci do not occur at random but that only a limited number of recombi¬ nations are possible either in order to preserve the as- sociation with IDDM or to escape it. According to our hypo¬ thesis high risk IDDM associated haplotypes can be modified by a recombination between either the A and C, C and B, or B and DR loci. For instance, A29, Cw2 were not seen on diabetes associated haplotypes. In contrast, the A locus specificity A24 increases the risk of a particular haplotype and might make it more potentially pathogenic for IDDM. We would like to propose that not only pathogen-driven selection is operating in creating the polymorphism of the HLA system but that also such long existing diseases like diabetes have an important impact on the diversity of the HLA system. Our cohort of elderly men in Finland has been renowned for its extremely high incidence and mortality of CVD. Selection through CVD mortality may indeed have removed some of the subjects with certain IDDM associated high risk HLA haplo¬ types. Four such haplotypes which all carried A24 and DR4, DQwδ were not seen in elderly men with NIDDM. These might be haplotypes which also carry a high risk for CVD mortality. Such a selection, if anything should work against our hypothesis. Our data suggest that the major histocompatibility complex (MHC) contains a gene (or genes) which regulates glucose homeostasis. The gene density in the class II region is as high as one gene every 25kb whereas in the class I region, sized 1500kb, comparatively few genes have been identified. We favour the hypothesis that susceptibility to β-cell damage is probably also determined by a gene(s) in the HLA class I region and that this is a common genetic factor in the aetiology of both NIDDM and IDDM. The difference between the two diseases is that in NIDDM the β-cell secretory capacity not sufficient to produce overt disease. Abnormal glucose control would then be unmasked by another factor (genetic or environmental) affecting insulin secretion or action. Gestational diabetes is a time of acquired insulin re- sistance and some studies have found an association between it and HLA (30) . These studies had been previously hard to understand as the majority of women with gestational diabetes eventually go on to develop NIDDM. The cause of worsening glucose tolerance with age is poorly understood. However, it is clear from the elderly men study that NIDDM is not an inevitable consequence of ageing and the predisposition is determined by genes within the major histocompatibility complex. It has been suggested that ageing leads to a reduction in beta-cell function and a consequent deterioration of glucose tolerance. Our data does not provide support for such an argument in all individuals. We think that ageing alone may not be a sufficient to cause for the deterioration of glucose tolerance which will only manifest in those individuals with diabetes associated HLA haplotypes. In our study, the dis¬ tribution of 2-hr post-challenge blood glucose was not only shifted towards higher values, but its shape was relatively normal, slightly positively skewed. This can be taken as strong evidence that these blood glucose values belong to a distinctly different subpopulation. The bimodality we dem¬ onstrated by the HLA haplotype status leaves little doubt that abnormal glucose tolerance is a genetically determined trait and that persons who do not have genetic predisposition to diabetes may show little abnormality in their blood glu¬ cose concentration even during an OGTT. We also attempted to obtain clinical information for those subjects for whom we had HLA haplotype data, but one of the two OGTT's was missing and we could not assign the final category of glucose intolerance. All available hospital records, laboratory data and death certificates of deceased cases were received for 19 of these men. No suggestion of diabetes or glucose intolerance was found for 15 of these men of whom 11 had no diabetes associated HLA haplotypes. All three diabetic men and the one with probable IGT had a dia¬ betes associated haplotype. We believe that in NIDDM the main mechanism leading to glucose intolerance is related to impaired beta-cell function. Due to various conditions the capacity of β-cells in genetically susceptible individuals may be exceeded, as demonstrated in women with gestational diabetes and elderly people. Our results are in accord with the previous findings that fasting blood glucose is a far less sensitive indicator of abnormal glucose tolerance and diabetes than the 2-hr post- challenge blood glucose is. Whilst the standardized OGTT is accepted as the main criterion of diabetes, its reproduci- bility especially for IGT is questioned. Recent data seem to indicate that half of the subjects with IGT defined by a single OGTT may be false positives. However, many of these elderly men with IGT in our study had a diabetes associated haplotype (27/34) and therefore we may speculate that IGT is also determined by genetic factors. The present data on bimodality of 2-hour post-challenge blood glucose strongly related to the HLA haplotype status underlines the diagnostic importance of the abnormality in glucose tolerance during OGTT. It is also known that a considerable proportion of subjects with IGT will develop NIDDM later on. Therefore, the category of asymptomatic IGT seems to be justified. People with IGT are considered to be a particular target group for measures to prevent NIDDM. In general, scientific data on primary prevention of diabetes are very scarce. Our present findings will make it possible to identify subjects who are genetically at risk of diabetes and, there¬ fore, it will open a new avenue for studies of prediction and real primary prevention of diabetes. These findings show, that the strength of association between NIDDM and HLA will be missed when phenotype data are used instead of haplotype data. This explains why earlier studies of HLA and NIDDM have been inconclusive. As at least 70 % of those with the genetic susceptibility to IDDM never develop disease and as Finland has the highest incidence of IDDM in the world, it follows that there must be a high frequency of IDDM susceptibility genes in the general population. Could this be the explanation of the observed HLA association with NIDDM and IGT in our elderly study popu¬ lation? We do believe this is likely to be the case. Most of the commonest IDDM associated high risk HLA haplotypes found in the Finnish study, except the A2, Cwl, B56, Bw6, DR4, DQ8 haplotype (26) , have also been reported to be present in IDDM patients in other populations. From the published HLA pheno¬ type data one can postulate assuming similar linkage dis- equilibrium between certain HLA antigens in these popu¬ lations, that the diabetes associated haplotypes seen in Finland are present also in NIDDM patients elsewhere. Popu¬ lation-based studies are needed to find out how many other diabetes associated HLA haplotypes exist worldwide, in addition to those that we have identified in Finland. Our educated guess is that the list will not be very long. The number of phenotyped elderly men in our study was not very large and the study needs to be extended, especially the group of non-diabetic men. In addition, studies of elderly women are needed. In conclusion, HLA in the Finnish population is a major common genetic determinant for IDDM, NIDDM and IGT. These observations carry important implications for the patho- genesis of this disease. Still, environmental factors and non-HLA genes influence the phenotypic expression of diabetes in genetically predisposed individuals. The central principle of the present discovery is that the glucose secretory function and glucose regulation of the pan- creatic beta-cells depend on and is controlled by the HLA haplotype. The prophylaxis of diabetes can therefore be carried out in fetus with diabetes an associated haplotype. This can be done in-utero in order to assure that beta-cells will develop normally and that a baby is born with intact beta-cell secretory funtion. After the birth the prophylaxis can be implemented in subjects with diabetes associated haplotypes in order to preserve the existing beta-cell function with a preventive therapy based on the control of the action of genes in the diabetogenic haplotype. Treatment of subjects who have diabetes associated haplotypes and elevated blood glucose concentration can be developed on the basis of glucose secretory function and glucose control of the pancreatic beta-cells. Such a novel therapy will be based on the original principles of the genetic regulation of glucose control in man and therapy it provides the most natural and the safest way to treat diabetes and glucose in¬ tolerance. REFERENCES INCORPORATED 1. Singal DP, Blajchman MA. 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Claims: CLAIMS 1. Method for detecting the susceptibility to diabetes of a person, characterized in that it is determined whether the person's HLA haplotype is diabetes associated; the diabetes associated HLA haplotypes being especially following: C B Bw DR DQ 2 1 56 6 4 8 3 1 56 6 4 8 24 51 4 8 24 1 39 6 4 8 2 X 51 4 4 8 2 24 3 2 2. Diagnostic kit for detecting the susceptibility to diabetes of a person, characterized in that the kit contains means for determining the HLA haplotypes according to claim 1 from a sample of the person. 3. Method for the treatment or prophylaxis of diabetes in a person, characterized in that the expression of at least one gene in the haplotypes according to claim 1 is prevented, or the action of at least one corresponding specifity is prevented, or at least one of the genes is replaced by another gene so that the haplotype is no more diabetogenic. 4. Preparation for the treatment or prophylaxis of diabetes in a person, characterized in that it contains an active amount of substance capable of preventing the ex- pression of at least one gene in the haplotypes according to claim 1, or an active amount of substance capable of pre¬ venting the action of at least one of the corresponding specifities.

Inventors: TUOMILEHTO JAAKKO, TUOMILEHTO-WOLF EVA, TUOMILEHTO, JAAKKO, TUOMILEHTO-WOLF, EVA
Applicants: TUOMILEHTO JAAKKO, TUOMILEHTO WOLF EVA, TUOMILEHTO, JAAKKO, TUOMILEHTO-WOLF, EVA
Application year: 1993
Main classification: C12Q1
IPC codes: C12Q1