Diabetes mellitus John A. Todd O x f o r d University, Oxford, UK Developments on four fronts have contributed to an exciting year for the study of diabetes. These include advances in molecular genetic mapping, analysis of animal models of disease, understanding of disease phenotype, and the extension of statistical methods to the study of complex, non-Mendelian traits. Current Opinion in Genetics and Development 1992, 2:474-478
Introduction Clinical diabetes mellitus is defined as defective control of glucose homeostasis as a result of complete or partial insulin deficiency and can be broadly divided into three main types: type 1 or insulin-dependent diabetes mellitus (IDDM); type 2 or non-insulin-dependent diabetes mellims (NIDDM); and maturity-onset diabetes of the young (MODY). Type 1 diabetes is a T lymphocyte-dependent autoimmune disease of the insulin-producing [B-cells of the pancreas and is characterized by absolute insulin-dependency, childhood-onset (mostly less than 18 years) and the association of disease with the presence of certain alleles of the human leukocyte antigen (HLA) class II genes, HLA-DQ and HLA-DRWIthin the major histocompatibility complex (MHC). Type 2 diabetes affects up to 5% of global populations and is ten times more common than type 1 diabetes. The disease is not associated with autoimmunity or HLA markers, has a late 'age of onset' (mostly over 50 years of age) and is caused both by defects in the secretion of insulin by 13-cells and by defects in the ability of tissues to respond to insulin (insulin resistance). MODY is a form of NIDDM that usually occurs before the age of 25, accounts for only 5% of all diabetes, and has an autosomal dominant mode of inheritance that is in contrast to the other two types, the inheritance of which is not clear. In 1991, the first MODY gene was mapped [ 1 - ] using PCR technology and a variable number of tandemly repeated sequence or microsatellite marker locus on human chromosome 20. A large number of microsatellites have been developed as polymorphic markers in the mouse genome and used to map several type 1 diabetes loci outside the MHC [2.°]. The accumulation of these highly polymorphic markers in humans promises to accelerate the localization of further diabetes genes. In addition to requiring a highly informative map with markers at every 5 cM, gene mapping of complex dis-
ease also requires large numbers of nuclear pedigrees [3 °°] or, in the case of MODY, very large multigeneration pedigrees. The key papers [ 1 - - - 3 o,] highlight the exciting discoveries that await us over the next few years. In tl~is review, recent events are described in the context of advances in molecular genetic mapping, analysis of animal models of disease, understanding of disease phenotype, and the extension of statistical methods to the study of complex, non-Mendelian traits.
Advances in gene mapping The ability to map genes for complex traits depends mainly on the resolution of the map of the genome, that is the spacing and informativeness of the marker loci, the number and types of pedigree available and the amount of locus heterogeneity, that is the number of unlinked loci that can cause the disease and the degree to which they can be classified as essential. The development of microsatellites, which occur at least every 50kb in the human genome [4] and display size polymorphism that is the result of variation in the number of simple sequence repeats, first identified in human [5] and then in mouse [6], provides us with the ability to construct 5cM genome maps. Risch [7] demonstrated that cost-effective linkage studies using affected relative pairs and identity by descent (IBD) required that marker loci possess polymorphism information content (PIC) values greater than 0.7, equivalent to a marker locus with four alleles with equal population frequencies. At least 30% of microsatellites have PIC > 0.7 [8]. Bell et al. [1 o-] tested the linkage of 79 distinct DNA markers, which included at least one marker on every autosome except chromosome 18, to MODY in one very large pedigree. One of these marker loci, a tetranucleotide microsatellite repeat located in an Alu element in the adenosine deaminase gene on chromosome 20,
Abbreviations HI.A--human leukocyte antigen; IBD--identity by descent; IDDM--insulin-dependent diabetes mellitus; MHC--major histocompatibility complex; MODY--maturity-onset diabetes of the young; NIDDM--non-insulin-dependent diabetes mellitus; NOD~non-obese diabetic; PIC--polymorphism information content.
474
(~ Current Biology Ltd ISSN 0959-437X
Diabetes mellitusTodd was tightly linked to MODY in this family. The study demonstrates the successful use of exclusion mapping in scanning the genome for a gene causing a monogenic trait. It also highlighted the care that must be taken in clinical diagnosis and inclusion criteria of affected individuals because this MODY family contained both IDDM and NIDDM cases, and inclusion of these would have lowered the lod scores obtained. (The lod score is determined by calculating the probability of obtaining the phenotypic data observed in a pedigree as a function of the parameter(s) of interest.) The definition of MODY is not clear cut, and perhaps in the presence of modifying genes, NIDDM may look like MODY and vice versa. The demonstration of this linkage is an important step in the genetics of diabetes. The next questions include how many other MODY pedigrees are affected as a result of mutation in this chromosome 20 gene, and what is the function of the gene product in [3-cell function and carbohydrate metabolism? Even if this gene contributes little to determining the overall frequenw of MODY, its characterization and the mapping of other loci in other MODY pedigrees will provide considerable insights into the aetiology of diabetes.
Development of linkage methods for complex traits The most important recent publications on this topic have been reviewed by Risch [9], but the paper by Hyer el aL [3"] is specifically relevant to the genetics of type 1 diabetes. These authors [3 °°] tested for linkage to human chromosome l l q based on the observations that chromosome 9 is weakly linked to IDDM in the non-obese diabetic (NOD) mouse, and that mouse chromosome 9 is homologous to human chromosome 11q. A set of 17 different polymorphic marker loci located at an average distance of 20 cM along the region of homology of chromosome 11q with mouse chromosome 9 were used in multipoint linkage analysis using the affected sib-pair method (where the affected pairs of sibs are exanlined for IBD at the marker locus). Importantly, they showed that the power to detect linkage (lod score > 3) depends on the IBD probabilities for allele sharing at the marker locus. The maximum lod score was < 0.5 for all marker loci tested. The question then asked was to what extent can the possibility that an IDDM susceptibility gene resides in the region be excluded? For all genetic models in which the probability of sibs sharing two alleles IBD at the presumed locus is P2 > 0.5, more than 90% of the region between the two most extreme markers can be excluded at a lod score < - 2 . Finally, theoretical affected sib-pair sample sizes were calculated to detect or exclude linkage. For example, the detection of a susceptibility gene with an effect on IBD probabilities equivalent to that of HLA loci would require a mean of 25-50 families with fully informative markers at 20 cM spacing. A sample of 200 affected sib-pairs may be adequate to exclude linkage for genetic models under IBD probability for P2 > 0.3. These analyses show that with a highly in-
formative map and 200 affected sib-pairs, it should be possible to detect IDDM susceptibility genes that exert 2-3-fold weaker effects than HI.A. These conclusions are consistent with previous theoretical estimations of the number of families required to detect gene effects that are weaker than or equivalent to HLA effects [10]. Unfortunately, very few marker systems, with the exception of a few variable number of repeat sequences and the HLA gene complex itself, are full), informative. To date, there are over 150 microsatellites published with PIC >0.7 [8], but most of these are PIC l~ R, BARNF'Iq"A, BMN S, BOITARD C, DESCHAMPS I, ET AI.: High Resolution Mapping for Susceptibility Genes in H u m a n Polygenic Disease; Insulin-dependent Diabetes Mellitus and C h r o m o s o m e l l q . Am J H u m Genet 1991, 48:243-259. An elegant and graphical analysis of the number of families that are required to enable the detection or exclusion of claromosom',d regions inw)lved in susceptibility to a complex, multifactorial disease. The authors provide a practical example by excluding linkage of 17 markers on human chromosome l l q to type 1 diabetes, and also use the established HLA linkage as a internal standard for this multipoint linkage approach. The power to detect linkage by affected sib-pair analysis is shown to be a function of IBD probabilities, and gives some indication of the range of gene effects, relative to the HLA, that might be detectable. 4.
STALIANGSRL, FORD AF, NELSON D, TORNEY DC, HILDEBR.PuND CE, MOYZlS RK: Evolution and Distribution of (GT)n Repetitive Sequences in Mammalian Genomes. Genomics 1991, 10:807-815.
5.
WEBERJL, POLYMEROPOUI.OSMH, MAY PE, KWITEKAE, XlAO H, MCPHERSON JD, WASMU'm JJ: Mapping of H u m a n Chromos o m e 5 Microsatellite DNA Polymorphisms. Genomics 1991, 11:695-700.
6.
HEam~ECM, McAt.EER MA, LOVE JM, AITMAN TJ, CORNAU+ RJ, GHOSH S, KNIGHT AM, PRINS J-B, TODD JA: Additional Microsatellite Markers for Mouse G e n o m e Mapping. M a m m Genome 1991, 1:273-282.
19. •
MOSTHAFL, VOGT B, HARING HU, ULLRICHA: Altered Expression of Insulin Receptor Types A and B in the Skeletal Muscle of Non-insulin-dependent Diabetes Mellitus Patients. Proc Natl Ac ad Sci USA 1991, 88:4728--4730. Type 2 diabetes, NIDDM, is a genetically complex, heterogeneous disease. One route for studying the genetics of the disorder is to identify subphenoWpes that might have a more simple mode of inheritance and that are reproducibly typed in patients. Tests of defects associated with N1DDM, for example the oral glucose tolerance test, are notoriously Jrreproducible. Mosthaf et al. describe a PCR assay, of insulin-receptor RNA that is derived from the skeletal muscle of patients and control individuals and which detects alternatively spliced forms of the receptor. The two different receptor isoforms have different affinities for insulin and the authors find an increase in the level of the lower affinit3, form in skeletal muscle biopsies from patients with NIDDM. This observa. tion might be related to the insulin-resistant state observed in diabetic skeletal muscle. 20.
FORSGRENS, DAHL U, SODERSTROMA, HOLMBERGD, MATSUNAGA
•
T: The Phenotype of Lymphoid CeUs and Thymic Epithe-
lium Correlates with the D e v e l o p m e n t of A u t o i m m u n e Insulitis in NOD ,--* C57BL/6 Allophenic Chimeras. Ptx~c Nail Ac ad Sci USA 1991, 88:9335--9339. A good demonstration of the dissection of disease aetiology using a combinaUon of embryology and immunological studies in the NOD mouse.
477
478
Mammalian genetics 21.
PROCHAZKAM, SERREZEDV, FRANKELXX/N, LEITER EH: NOR/Lt Mice: MHC-matched Diabetes-resistant Control Strain for NOD Mice. Diabetes 1991, 41:98-106.
22.
CORNALLRJ, PP,INS J-B, TODD JA, PRE~EY A, DELARATO NH, WICKERIS, PETEILSONLB: Type 1 Diabetes in Mice is Linked to the lnterleukin-1 Receptor and Lsh/lry/Bcg Genes on Chromosome 1. Nature 1991, 353: 262-265. Expanding the work presented in [2"'], the authors describe an insulitis and diabetes gene located on mouse chromosome 1. Includes a demonstraUon of how linkage analysis can lead us to consider certain genes as candidate genes for disease susceptibility loci because they fail in the area of linkage to disease.
significant evidence that this gene also affects periinsulitis, the study is the first published use of an F2 outcross in the analysis of NOD autoimmunity, in which dominant suscepUbility genes can be detected. Backcrosses to NOD mice (see [2.% 22-]) cannot be used to detect fully dominant gene effects.
•
23. •
GARCHONH-J, BEDOSSA P, ELOY L, BACH J-F: Identification and Mapping to Chromosome 1 of a Susceptibility Locus for Periinsulitis in Non-obese Diabetic Mice. Nature 1991, 353:260-262. Using the microsateRites described in [2.,], Garchon and colleagues report evidence for a locus on chromosome 1 that is near Bcl.2 and which controls the development of sialiUs, the inffltraUon of the salivary #ands by cells of the immune system. Although they did not present
24.
TODDJA, BELLJl, McDEvlTT HO: HLA-D[3 Gene Contributes to Susceptibility and Resistance to Insulin-dependent Diabetes MelUtus. Nature 1987, 329:599--604.
25.
NADEAUJH: Maps of Linkage and Synteny Homologies Between Mouse and Man. Trends Genet 1989, 5:82--86.
26.
STRASSERA, WHITTINGHAM S, VAUX DL, BATH ML, ADAMS JM, CORY S, HARMS AW: Enforced BCL2 Expression in ~-Iymphoid Cells Prolongs Antibody Responses and Elicits Autoimmune Disease. Proc Natl Acad Sci USA 1991, 88:8661-8665.
JA Todd, Nuffleld Department of Surgery, Oxford University, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.
Introduction Clinical diabetes mellitus is defined as defective control of glucose homeostasis as a result of complete or partial insulin deficiency and can be broadly divided into three main types: type 1 or insulin-dependent diabetes mellitus (IDDM); type 2 or non-insulin-dependent diabetes mellims (NIDDM); and maturity-onset diabetes of the young (MODY). Type 1 diabetes is a T lymphocyte-dependent autoimmune disease of the insulin-producing [B-cells of the pancreas and is characterized by absolute insulin-dependency, childhood-onset (mostly less than 18 years) and the association of disease with the presence of certain alleles of the human leukocyte antigen (HLA) class II genes, HLA-DQ and HLA-DRWIthin the major histocompatibility complex (MHC). Type 2 diabetes affects up to 5% of global populations and is ten times more common than type 1 diabetes. The disease is not associated with autoimmunity or HLA markers, has a late 'age of onset' (mostly over 50 years of age) and is caused both by defects in the secretion of insulin by 13-cells and by defects in the ability of tissues to respond to insulin (insulin resistance). MODY is a form of NIDDM that usually occurs before the age of 25, accounts for only 5% of all diabetes, and has an autosomal dominant mode of inheritance that is in contrast to the other two types, the inheritance of which is not clear. In 1991, the first MODY gene was mapped [ 1 - ] using PCR technology and a variable number of tandemly repeated sequence or microsatellite marker locus on human chromosome 20. A large number of microsatellites have been developed as polymorphic markers in the mouse genome and used to map several type 1 diabetes loci outside the MHC [2.°]. The accumulation of these highly polymorphic markers in humans promises to accelerate the localization of further diabetes genes. In addition to requiring a highly informative map with markers at every 5 cM, gene mapping of complex dis-
ease also requires large numbers of nuclear pedigrees [3 °°] or, in the case of MODY, very large multigeneration pedigrees. The key papers [ 1 - - - 3 o,] highlight the exciting discoveries that await us over the next few years. In tl~is review, recent events are described in the context of advances in molecular genetic mapping, analysis of animal models of disease, understanding of disease phenotype, and the extension of statistical methods to the study of complex, non-Mendelian traits.
Advances in gene mapping The ability to map genes for complex traits depends mainly on the resolution of the map of the genome, that is the spacing and informativeness of the marker loci, the number and types of pedigree available and the amount of locus heterogeneity, that is the number of unlinked loci that can cause the disease and the degree to which they can be classified as essential. The development of microsatellites, which occur at least every 50kb in the human genome [4] and display size polymorphism that is the result of variation in the number of simple sequence repeats, first identified in human [5] and then in mouse [6], provides us with the ability to construct 5cM genome maps. Risch [7] demonstrated that cost-effective linkage studies using affected relative pairs and identity by descent (IBD) required that marker loci possess polymorphism information content (PIC) values greater than 0.7, equivalent to a marker locus with four alleles with equal population frequencies. At least 30% of microsatellites have PIC > 0.7 [8]. Bell et al. [1 o-] tested the linkage of 79 distinct DNA markers, which included at least one marker on every autosome except chromosome 18, to MODY in one very large pedigree. One of these marker loci, a tetranucleotide microsatellite repeat located in an Alu element in the adenosine deaminase gene on chromosome 20,
Abbreviations HI.A--human leukocyte antigen; IBD--identity by descent; IDDM--insulin-dependent diabetes mellitus; MHC--major histocompatibility complex; MODY--maturity-onset diabetes of the young; NIDDM--non-insulin-dependent diabetes mellitus; NOD~non-obese diabetic; PIC--polymorphism information content.
474
(~ Current Biology Ltd ISSN 0959-437X
Diabetes mellitusTodd was tightly linked to MODY in this family. The study demonstrates the successful use of exclusion mapping in scanning the genome for a gene causing a monogenic trait. It also highlighted the care that must be taken in clinical diagnosis and inclusion criteria of affected individuals because this MODY family contained both IDDM and NIDDM cases, and inclusion of these would have lowered the lod scores obtained. (The lod score is determined by calculating the probability of obtaining the phenotypic data observed in a pedigree as a function of the parameter(s) of interest.) The definition of MODY is not clear cut, and perhaps in the presence of modifying genes, NIDDM may look like MODY and vice versa. The demonstration of this linkage is an important step in the genetics of diabetes. The next questions include how many other MODY pedigrees are affected as a result of mutation in this chromosome 20 gene, and what is the function of the gene product in [3-cell function and carbohydrate metabolism? Even if this gene contributes little to determining the overall frequenw of MODY, its characterization and the mapping of other loci in other MODY pedigrees will provide considerable insights into the aetiology of diabetes.
Development of linkage methods for complex traits The most important recent publications on this topic have been reviewed by Risch [9], but the paper by Hyer el aL [3"] is specifically relevant to the genetics of type 1 diabetes. These authors [3 °°] tested for linkage to human chromosome l l q based on the observations that chromosome 9 is weakly linked to IDDM in the non-obese diabetic (NOD) mouse, and that mouse chromosome 9 is homologous to human chromosome 11q. A set of 17 different polymorphic marker loci located at an average distance of 20 cM along the region of homology of chromosome 11q with mouse chromosome 9 were used in multipoint linkage analysis using the affected sib-pair method (where the affected pairs of sibs are exanlined for IBD at the marker locus). Importantly, they showed that the power to detect linkage (lod score > 3) depends on the IBD probabilities for allele sharing at the marker locus. The maximum lod score was < 0.5 for all marker loci tested. The question then asked was to what extent can the possibility that an IDDM susceptibility gene resides in the region be excluded? For all genetic models in which the probability of sibs sharing two alleles IBD at the presumed locus is P2 > 0.5, more than 90% of the region between the two most extreme markers can be excluded at a lod score < - 2 . Finally, theoretical affected sib-pair sample sizes were calculated to detect or exclude linkage. For example, the detection of a susceptibility gene with an effect on IBD probabilities equivalent to that of HLA loci would require a mean of 25-50 families with fully informative markers at 20 cM spacing. A sample of 200 affected sib-pairs may be adequate to exclude linkage for genetic models under IBD probability for P2 > 0.3. These analyses show that with a highly in-
formative map and 200 affected sib-pairs, it should be possible to detect IDDM susceptibility genes that exert 2-3-fold weaker effects than HI.A. These conclusions are consistent with previous theoretical estimations of the number of families required to detect gene effects that are weaker than or equivalent to HLA effects [10]. Unfortunately, very few marker systems, with the exception of a few variable number of repeat sequences and the HLA gene complex itself, are full), informative. To date, there are over 150 microsatellites published with PIC >0.7 [8], but most of these are PIC l~ R, BARNF'Iq"A, BMN S, BOITARD C, DESCHAMPS I, ET AI.: High Resolution Mapping for Susceptibility Genes in H u m a n Polygenic Disease; Insulin-dependent Diabetes Mellitus and C h r o m o s o m e l l q . Am J H u m Genet 1991, 48:243-259. An elegant and graphical analysis of the number of families that are required to enable the detection or exclusion of claromosom',d regions inw)lved in susceptibility to a complex, multifactorial disease. The authors provide a practical example by excluding linkage of 17 markers on human chromosome l l q to type 1 diabetes, and also use the established HLA linkage as a internal standard for this multipoint linkage approach. The power to detect linkage by affected sib-pair analysis is shown to be a function of IBD probabilities, and gives some indication of the range of gene effects, relative to the HLA, that might be detectable. 4.
STALIANGSRL, FORD AF, NELSON D, TORNEY DC, HILDEBR.PuND CE, MOYZlS RK: Evolution and Distribution of (GT)n Repetitive Sequences in Mammalian Genomes. Genomics 1991, 10:807-815.
5.
WEBERJL, POLYMEROPOUI.OSMH, MAY PE, KWITEKAE, XlAO H, MCPHERSON JD, WASMU'm JJ: Mapping of H u m a n Chromos o m e 5 Microsatellite DNA Polymorphisms. Genomics 1991, 11:695-700.
6.
HEam~ECM, McAt.EER MA, LOVE JM, AITMAN TJ, CORNAU+ RJ, GHOSH S, KNIGHT AM, PRINS J-B, TODD JA: Additional Microsatellite Markers for Mouse G e n o m e Mapping. M a m m Genome 1991, 1:273-282.
19. •
MOSTHAFL, VOGT B, HARING HU, ULLRICHA: Altered Expression of Insulin Receptor Types A and B in the Skeletal Muscle of Non-insulin-dependent Diabetes Mellitus Patients. Proc Natl Ac ad Sci USA 1991, 88:4728--4730. Type 2 diabetes, NIDDM, is a genetically complex, heterogeneous disease. One route for studying the genetics of the disorder is to identify subphenoWpes that might have a more simple mode of inheritance and that are reproducibly typed in patients. Tests of defects associated with N1DDM, for example the oral glucose tolerance test, are notoriously Jrreproducible. Mosthaf et al. describe a PCR assay, of insulin-receptor RNA that is derived from the skeletal muscle of patients and control individuals and which detects alternatively spliced forms of the receptor. The two different receptor isoforms have different affinities for insulin and the authors find an increase in the level of the lower affinit3, form in skeletal muscle biopsies from patients with NIDDM. This observa. tion might be related to the insulin-resistant state observed in diabetic skeletal muscle. 20.
FORSGRENS, DAHL U, SODERSTROMA, HOLMBERGD, MATSUNAGA
•
T: The Phenotype of Lymphoid CeUs and Thymic Epithe-
lium Correlates with the D e v e l o p m e n t of A u t o i m m u n e Insulitis in NOD ,--* C57BL/6 Allophenic Chimeras. Ptx~c Nail Ac ad Sci USA 1991, 88:9335--9339. A good demonstration of the dissection of disease aetiology using a combinaUon of embryology and immunological studies in the NOD mouse.
477
478
Mammalian genetics 21.
PROCHAZKAM, SERREZEDV, FRANKELXX/N, LEITER EH: NOR/Lt Mice: MHC-matched Diabetes-resistant Control Strain for NOD Mice. Diabetes 1991, 41:98-106.
22.
CORNALLRJ, PP,INS J-B, TODD JA, PRE~EY A, DELARATO NH, WICKERIS, PETEILSONLB: Type 1 Diabetes in Mice is Linked to the lnterleukin-1 Receptor and Lsh/lry/Bcg Genes on Chromosome 1. Nature 1991, 353: 262-265. Expanding the work presented in [2"'], the authors describe an insulitis and diabetes gene located on mouse chromosome 1. Includes a demonstraUon of how linkage analysis can lead us to consider certain genes as candidate genes for disease susceptibility loci because they fail in the area of linkage to disease.
significant evidence that this gene also affects periinsulitis, the study is the first published use of an F2 outcross in the analysis of NOD autoimmunity, in which dominant suscepUbility genes can be detected. Backcrosses to NOD mice (see [2.% 22-]) cannot be used to detect fully dominant gene effects.
•
23. •
GARCHONH-J, BEDOSSA P, ELOY L, BACH J-F: Identification and Mapping to Chromosome 1 of a Susceptibility Locus for Periinsulitis in Non-obese Diabetic Mice. Nature 1991, 353:260-262. Using the microsateRites described in [2.,], Garchon and colleagues report evidence for a locus on chromosome 1 that is near Bcl.2 and which controls the development of sialiUs, the inffltraUon of the salivary #ands by cells of the immune system. Although they did not present
24.
TODDJA, BELLJl, McDEvlTT HO: HLA-D[3 Gene Contributes to Susceptibility and Resistance to Insulin-dependent Diabetes MelUtus. Nature 1987, 329:599--604.
25.
NADEAUJH: Maps of Linkage and Synteny Homologies Between Mouse and Man. Trends Genet 1989, 5:82--86.
26.
STRASSERA, WHITTINGHAM S, VAUX DL, BATH ML, ADAMS JM, CORY S, HARMS AW: Enforced BCL2 Expression in ~-Iymphoid Cells Prolongs Antibody Responses and Elicits Autoimmune Disease. Proc Natl Acad Sci USA 1991, 88:8661-8665.
JA Todd, Nuffleld Department of Surgery, Oxford University, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.
