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GENETIC MARKERS
1. Use
A. Diagnosis/Screening
a. Type 1 diabetes
Recommendation: Routine measurement of genetic markers is not of value at this time for the diagnosis or management of patients with type 1 diabetes. For selected diabetic syndromes, valuable information can be obtained with definition of diabetes-associated mutations.
Level of evidence: E
Genetic markers are currently of limited clinical value in the evaluation and management of patients with diabetes. However, they hold promise for the future. For immune-mediated (type 1A) diabetes (IMD), HLA typing can be useful to indicate absolute risk of diabetes (see Table 6), as extended by insulin (INS) gene typing (and in some populations by CTLA-4 gene typing), and can assist in assigning a probability of the diagnosis of IMD to diabetes of uncertain etiology ADDIN EN.CITE Todd199713220Todd, J. A.1997Genetics of type 1 diabetesPathol Biol (Paris)453219-227Pathol Biol (Paris)AnimalChromosome MappingDiabetes Mellitus, Insulin-Dependent/*geneticsGenomeGenome, HumanHumanMiceSupport, Non-U.S. Gov't(196). As indicated below, HLA-DR/DQ typing can be useful to indicate modified risk of IMD in persons with positive islet cell autoantibodies, since protective alleles do not prevent the appearance of islet cell autoantibodies (most often as single autoantibodies), but do protect against clinical diabetes. Typing of class II major histocompatibility antigens or HLA DRB1, DQA1 and DQB1 is not diagnostic for IMD. However, some haplotypes form susceptibility, while others provide significant protection. Thus, HLA-DR/DQ typing can be used only to increase or decrease the probability of IMD presentation, and cannot be recommended for routine clinical diagnosis or classification ADDIN EN.CITE She199613100She, J. X.1996Susceptibility to type I diabetes: HLA-DQ and DR revisited [see comments]Immunol Today177323-9Immunol TodayDiabetes Mellitus, Insulin-Dependent/*genetics/immunologyDisease SusceptibilityHumanHLA-DQ Antigens/*genetics/immunologyHLA-DR Antigens/*genetics/immunologyLinkage Disequilibrium/geneticsRacial Stocks/geneticsSupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.(197).
It is possible to screen newborn children to identify those at increased risk of developing IMD ADDIN EN.CITE Ziegler199313170Ziegler, A. G.Bachmann, W.Rabl, W.1993Prophylactic insulin treatment in relatives at high risk for type 1 diabetesDiabetes Metabolic Review94289-293Diabetes Metab RevAnimalAutoimmunityDiabetes Mellitus, Experimental/prevention & controlDiabetes Mellitus, Insulin-Dependent/genetics/immunology/*prevention & controlFemaleHumanInsulin/administration & dosage/adverse effects/*pharmacologyMaleSupport, Non-U.S. Gov'tRewers199613210Rewers, M.Bugawan, T. L.Norris, J. M.Blair, A.Beaty, B.Hoffman, M.McDuffie, R. S., Jr.Hamman, R. F.Klingensmith, G.Eisenbarth, G. S.Erlich, H. A.1996Newborn screening for HLA markers associated with IDDM: diabetes autoimmunity study in the young (DAISY)Diabetologia397807-812DiabetologiaAutoimmune Diseases/epidemiology/*prevention & controlBiological Markers/bloodCaucasoid RaceCohort StudiesColorado/epidemiologyDiabetes Mellitus, Insulin-Dependent/epidemiology/*prevention & controlDNA Primers/chemistryFetal Blood/immunologyFollow-Up StudiesHumanHLA Antigens/*bloodIncidenceInfant, Newborn*Mass Screening/standardsMongoloid RaceNegroid RacePatient CompliancePolymerase Chain ReactionProspective StudiesQuality ControlRisk FactorsSupport, U.S. Gov't, P.H.S.(198, 199). This strategy cannot be recommended until there is a proven intervention available to delay or prevent the disease ADDIN EN.CITE Kukreja199914840Kukreja, A.Maclaren, N. K.1999Autoimmunity and diabetesJ Clin Endocrinol Metab84124371-8.J Clin Endocrinol MetabAntigen-Presenting Cells/immunology*Autoimmune Diseases/geneticsCytokinesDiabetes Mellitus, Insulin-Dependent/genetics/*immunologyHumanKiller Cells, Natural/immunologyMolecular Mimicry(200). The rationale for the approach is thus placed below under emerging considerations.
b. Type 2 diabetes and MODY
Recommendation: There is no role for routine genetic testing in patients with type 2 diabetes. These studies should be confined to the research setting and evaluation of specific syndromes.
Level of evidence: E
Type 2 diabetes: Fewer than 5% of patients with type 2 diabetes have been resolved on a molecular genetic basis and, not surprisingly, most of these have an autosomal dominant form of the disease or very high degrees of insulin resistance. Type 2 diabetes is a heterogenous polygenic disease with both resistance to the action of insulin and defective insulin secretion ADDIN EN.CITE Sacks19967660Sacks, D.B.McDonald, J. M.1996The pathogenesis of type II diabetes mellitus: a polygenic diseaseAm. J. Clin. Pathol.105149-156Am. J. Clin. Pathol.Reaven19887740Reaven, G. M.1988Role of insulin resistance in human diseaseDiabetesDiabetes371595-1607(3, 4). Multiple genetic factors interact with exogenous influences (e.g., environmental factors such as obesity) to produce the phenotype. Identification of the affected genes is therefore highly complex.
MODY: Mutation detection for maturity onset diabetes of youth (MODY) patients and their relatives is technically feasible. However, due to the high cost of establishing a facility to detect mutations and the high level of technical skill required for analysis, few laboratories perform these assays. As direct automated sequencing of genes becomes standard, it is likely that detection of specific diabetes mutations will become more common.
B. Monitoring/Prognosis
Although genetic screening may provide information about prognosis and could be useful for genetic counseling, genotype may not correlate with the phenotype. In addition to environmental factors, interactions among multiple quantitative trait loci expressions may be involved. Genetic identification of a defined MODY will have value for anticipating the prognosis.
Rationale
The HLA system, which has a fundamental role in the adaptive immune response, exhibits considerable genetic complexity. The HLA complex on chromosome 6 contains class I and II genes that code for several polypeptide chains ADDIN EN.CITE Klein200015470The HLA system. First of two partsKlein, J.Sato, A.Antigen Presentation/*physiologyHLA Antigens/chemistry/immunology/*physiologyHumanMajor Histocompatibility ComplexMolecular StructurePeptides/immunologyReceptors, Antigen, T-Cell/*immunologyThymus Gland/*immunologyN Engl J Med200034310702-9.(201). The major (classic) class I genes are HLA-A, B and C. The loci of class II genes are designated by three letters: the first (D) indicates the class, the second (M, O, P, Q or R) the family and the third (A or B) the chain. Both classes of molecules are heterodimers; class I consists of an ( chain and (2-microglobulin, while class II has ( and ( chains. The function of the HLA molecules is to present short peptides, derived from pathogens, to T cells to initiate the adaptive immune response ADDIN EN.CITE Klein200015470The HLA system. First of two partsKlein, J.Sato, A.Antigen Presentation/*physiologyHLA Antigens/chemistry/immunology/*physiologyHumanMajor Histocompatibility ComplexMolecular StructurePeptides/immunologyReceptors, Antigen, T-Cell/*immunologyThymus Gland/*immunologyN Engl J Med200034310702-9.(201). Genetic studies have revealed an association between certain HLA alleles and autoimmune diseases. These diseases include, but are not confined to, ankylosing spondylitis, celiac disease, Addisons disease and type 1 diabetes ADDIN EN.CITE Klein200015470The HLA system. First of two partsKlein, J.Sato, A.Antigen Presentation/*physiologyHLA Antigens/chemistry/immunology/*physiologyHumanMajor Histocompatibility ComplexMolecular StructurePeptides/immunologyReceptors, Antigen, T-Cell/*immunologyThymus Gland/*immunologyN Engl J Med200034310702-9.(201).
Genetic testing for syndromic forms of diabetes is the same as that for the underlying syndrome itself ADDIN EN.CITE American Diabetes Association19977560American Diabetes Association,1997Report of the expert committee on the diagnosis and classification of diabetes mellitusDiabetes Care201183-1201Diabetes Care(1). Such diabetes may be secondary to the obesity associated with Prader-Willi syndrome, which maps to chromosome 15 q, or to the absence of adipose tissue inherent to recessive Seip-Berardinelli syndrome of generalized lipodystrophy mapping to chromosome 9q34 ADDIN EN.CITE American Diabetes Association19977560American Diabetes Association,1997Report of the expert committee on the diagnosis and classification of diabetes mellitusDiabetes Care201183-1201Diabetes CareTaylor199913200Taylor, S. I.Arioglu, E.1999Genetically defined forms of diabetes in childrenJ Clin Endocrinol Metab84124390-6J Clin Endocrinol MetabAdultDiabetes Mellitus/*geneticsDiabetes Mellitus, Non-Insulin-Dependent/diagnosis/geneticsFemaleHumanInfantInsulin/deficiencyInsulin Resistance(1, 202). There are over 60 distinct genetic disorders associated with glucose intolerance or frank diabetes. Most forms of type 2 diabetes (which are usually strongly familial) will probably be understood in defined genetic terms, but this is far from realized at present. Some genes for MODY have been identified, but there are large numbers of individual mutants. Persons at risk within MODY pedigrees can be identified through genetic means. Depending on the specific MODY mutation, the disease can be mild (e.g., glucokinase mutation) and not usually associated with long term complications of diabetes or as severe as typical type 1 diabetes [e.g., hepatocyte nuclear factor (HNF) mutations] ADDIN EN.CITE Fajans199617120Fajans, S. S.Bell, G. I.Bowden, D. W.Halter, J. B.Polonsky, K. S.1996Maturity onset diabetes of the young (MODY)Diabet Med139 Suppl 6S90-5.Diabet MedBlood Glucose/metabolismChromosome Mapping*Chromosomes, Human, Pair 20*Chromosomes, Human, Pair 7Diabetes Mellitus, Non-Insulin-Dependent/drug therapy/*geneticsDiabetic Angiopathies/epidemiology/geneticsFemaleGenes, DominantGenetic MarkersGlucokinase/geneticsGlucose Intolerance/*geneticsHumanHyperglycemia/geneticsInsulin/blood/secretion/therapeutic useInsulin ResistanceIslets of Langerhans/secretionMalePedigreeSupport, U.S. Gov't, P.H.S.(203). The interest in the genetics of MODY is the hope that insight will be obtained into type 2 diabetes. (Note that MODY is not a form of type 2 diabetes.)
Five different MODYs have been identified. MODY-1, 3, 4, and 5 all result from mutations in the genes encoding transcription factors that regulate the expression of genes in pancreatic ( cells. These genes are hepatocyte nuclear factor-4( (HNF-4() in MODY-1, HNF-1( in MODY-3, HNF-1( in MODY-5, and insulin promoter factor-1 (IPF-1) in MODY-4. It has been shown that homozygous mutations of the IPF-1 gene leads to pancreatic agenesis and that heterozygous mutations of IPF-1 genes results in MODY-4 ADDIN EN.CITE Taylor199913200Taylor, S. I.Arioglu, E.1999Genetically defined forms of diabetes in childrenJ Clin Endocrinol Metab84124390-6J Clin Endocrinol MetabAdultDiabetes Mellitus/*geneticsDiabetes Mellitus, Non-Insulin-Dependent/diagnosis/geneticsFemaleHumanInfantInsulin/deficiencyInsulin Resistance(202). The modes of action of the HNF lesions in MODY is still not clear. It is likely that mutation in HNF-1(, 1(, and 4( cause diabetes because they impair insulin secretion. MODY-2 is caused by mutations in the glucokinase gene. The product of the gene is an essential enzyme in the glucose-sensing mechanism of the ( cells, and mutations in this gene lead to partial deficiencies of insulin secretion.
3. Analytical Considerations
A detailed review of analytical issues will not be attempted here, since genetic testing for diabetes outside of a research setting is currently not recommended for clinical care. Serological HLA typing should be replaced by molecular methods, such as sequence specific priming, since antibodies with a mixture of specificities and cross reactivities have been estimated to give inaccurate results in approximately 15% of typings.
Preanalytical
Detection of mutations is performed using genomic DNA extracted from peripheral blood leukocytes. Blood samples should be drawn into test tubes containing EDTA and the DNA preparations should be harvested within 3 days; longer periods both lower the yield and degrade the quality of the DNA obtained. Genomic DNA can be isolated from fresh or frozen whole blood by lysis, digestion with proteinase K, extraction with phenol, and then dialysis. The average yield is 100 to 200 (g DNA from 10 ml of whole blood. DNA samples are best kept at 80 (C in Tris-EDTA solution, where the integrity of the sample lasts virtually indefinitely.
Analytical
Methods for the detection of mutations differ for different types of mutation. The MODYs have substitution, deletion or insertion of nucleotides in the coding region of the genes. These are detected by PCR. The detailed protocols for the detection of specific mutations are beyond the scope of this review.
5. Emerging considerations
To screen for the propensity for IMD in general populations, HLA-D genes are the most important, contributing as much as 50% of the genetic susceptibility ADDIN EN.CITE Todd199713220Todd, J. A.1997Genetics of type 1 diabetesPathol Biol (Paris)453219-227Pathol Biol (Paris)AnimalChromosome MappingDiabetes Mellitus, Insulin-Dependent/*geneticsGenomeGenome, HumanHumanMiceSupport, Non-U.S. Gov't(196). HLA-DQ genes appear to be central to the HLA associated risk of IMD, albeit DR genes may be independently involved (for review, see ADDIN EN.CITE Atkinson200117160Atkinson, M. A.Eisenbarth, G. S.2001Type 1 diabetes: new perspectives on disease pathogenesis and treatmentLancet3589277221-9.LancetAdultAge of OnsetAutoantibodies/immunologyAutoimmune Diseases/diagnosisBlood Glucose Self-MonitoringChild*Diabetes Mellitus, Insulin-Dependent/epidemiology/etiology/genetics/therapyGenetic Predisposition to DiseaseHumanIncidenceInsulin/therapeutic useIslets of Langerhans TransplantationHarrison200117110Harrison, L. C.2001Risk assessment, prediction and prevention of type 1 diabetes.Pediatric Diabetes271-82Pediatric Diabetes(204, 205). The heterodimeric proteins that are expressed on antigen presenting cells, B lymphocytes, platelets and activated T cells, but not other somatic cells, are composed of cis and trans complementated ( and ( chain heterodimers. Thus, in any individual four possible DQ dimers are encoded. Positive risks for IMD are associated with ( chains that have an arginine at residue 52, and ( chains that lack an aspartic acid at residue 57. Persons at the highest genetic risk for IMD are those in whom all four DQ combinations meet this criterion. Thus, persons heterozygous for HLA-DRB1*04 -DQA1*0301-DQB1*0302 and DRB1*03- DQA1*0501-DQB1*0201 are the most susceptible, with an absolute life-time risk of IMD in the general population of about 1:12. Persons who are protected from IMD are those with DRB1*15-DQA1*0201-DQB1*0602 (Asp 57+) haplotypes in particular ADDIN EN.CITE Redondo200015650Redondo, M. J.Kawasaki, E.Mulgrew, C. L.Noble, J. A.Erlich, H. A.Freed, B. M.Lie, B. A.Thorsby, E.Eisenbarth, G. S.Undlien, D. E.Ronningen, K. S.2000DR- and DQ-associated protection from type 1A diabetes: comparison of DRB1*1401 and DQA1*0102-DQB1*0602*J Clin Endocrinol Metab85103793-3797.J Clin Endocrinol MetabCell LineDNA/geneticsDiabetes Mellitus, Insulin-Dependent/*geneticsHLA-DQ Antigens/*geneticsHLA-DR Antigens/*geneticsHistocompatibility TestingHumanLinkage (Genetics)/geneticsSupport, Non-U.S. Gov'tSupport, U.S. Gov't, P.H.S.(206), albeit those with DRB1*11 or 04 who also have DQB1*0301 (Asp 57 +) are protected. HLA-DR is also involved in susceptibility to IMD in that the B1*0401 and 0405 subtypes of DRB1*04 are susceptible, while the 0403 and 0406 subtypes are protective, even when found in HLA haplotypes of the susceptible DQA1*0301-DQB1*0302. DR molecules are heterodimers also, however the DR( chain is invariant in all persons. Additional DR( chains (B3, B4 and B5) are not important.
Class II MHC is involved in antigen presentation to CD4 helper cells, and the above associations are likely to be explained by defective affinities to islet cell antigenic peptides, leading to persistence of T helper cells which escape thymic ablation. Class I HLA is also implicated in IMD. Multiple non-HLA loci also contribute to susceptibility to type 1 diabetes ADDIN EN.CITE Atkinson200117160Atkinson, M. A.Eisenbarth, G. S.2001Type 1 diabetes: new perspectives on disease pathogenesis and treatmentLancet3589277221-9.LancetAdultAge of OnsetAutoantibodies/immunologyAutoimmune Diseases/diagnosisBlood Glucose Self-MonitoringChild*Diabetes Mellitus, Insulin-Dependent/epidemiology/etiology/genetics/therapyGenetic Predisposition to DiseaseHumanIncidenceInsulin/therapeutic useIslets of Langerhans Transplantation(204). For example, the variable nucleotide tandem repeat (VNTR) upstream from the insulin (INS) gene on chromosome 11q is also useful for predicting the development of IMD, with alleles with the longest VNTR having protective effects. Typing newborn infants for both HLA/DR/DQ, and to a lesser degree the INS gene, results in prediction of IMD to better than 1:10 in the general population. The risk of IMD in HLA-identical siblings of a proband with IMD is 1:4, while siblings who have HLA-haplotype identity have a 1:12 risk and those with no shared haplotype a 1:100 risk ADDIN EN.CITE Harrison200117110Harrison, L. C.2001Risk assessment, prediction and prevention of type 1 diabetes.Pediatric Diabetes271-82Pediatric Diabetes(205). The numerous other putative genomic intervals suggested to be linked to IMD remain to be confirmed in multiple data sets, and discussion of these is outside the scope of the article. The sequencing of the human genome and the formation of consortia should result in advances in the identification of the genetic bases for both type 1 and type 2 diabetes. This progress should ultimately result in family counseling, prognostic information and the selection of optimal treatment ADDIN EN.CITE Taylor199913200Taylor, S. I.Arioglu, E.1999Genetically defined forms of diabetes in childrenJ Clin Endocrinol Metab84124390-6J Clin Endocrinol MetabAdultDiabetes Mellitus/*geneticsDiabetes Mellitus, Non-Insulin-Dependent/diagnosis/geneticsFemaleHumanInfantInsulin/deficiencyInsulin ResistanceMaclaren200115487Maclaren, N.K.Kukreja, A.2001Type 1 diabetesSly, W.S. The metabolic and molecular bases of inherited disease.St. LouisMcGraw-Hill1471-14888th(202, 207) .
PAGE 1
NACB: Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus
David B. Sacks, David E. Bruns, David E. Goldstein, Noel K. Maclaren, Jay M. McDonald, Marian Parrott
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