Guidelines & Recommendations for Laboratory Analysis in the Diagnosis & Management of Diabetes Mellitus

Genetic Markers

Noel K Maclaren MD and Zhong Sheng Sun Ph.D.
Department of Pediatrics
Weill College of Medicine of Cornell University
New York, NY 10021.

Use

Diagnosis/screening

Recommendation: In general, genetic markers are of limited clinical value to the evaluation and management of clinical diabetes, but this will change with future findings and the growing realization of diabetes prevention.

Genetic markers are however critical to the diagnosis of some particular forms of diabetes and they hold promise for the future in disease prediction, prevention, diagnosis and prognosis. Further, genetic testing for syndromic forms of diabetes may be more important in respect to the syndrome itself rather than to the associated diabetes as such, which may be secondary to the obesity of Prader-Willi syndrome mapping to 15 q, or the absence of adipose tissue inherent to recessive Seip-Berardinelli syndrome of generalized lipodystrophy mapping to 9q34. Indeed, there are over 60 distinct genetic disorders associated with glucose intolerance or frank diabetes. Diabetes is treated according to its’ severity and a physician’s notions of the relative contributions of insulin deficiency and insulin resistance to the degree of diabetes seen in individual patients.

Insulin Deficiency Diabetes:

For immune mediated (type-1) diabetes (IMD), HLA typing can be useful to indicate risk of diabetes and can assist in assigning a probability of the diagnosis of IMD. As indicated below, HLA-DR/DQ typing can be useful to indicate modified risk of IMD in persons with islet cell auto-antibodies. It is currently possible to screen new-born children to select out those who are destined to have a high risk of developing IMD. HLA-D genes are the most important in this regard, contributing as much as 50% of the genetic susceptibility. HLA-DQ genes appear to be central to the HLA associated risk of IMD. These antigens that are expressed on antigen presenting cells, B lymphocytes and platelets, and activated T cells, but not other somatic cells, are comprised of cis and trans complementated A and B chain heterodimers. Thus in any individual, four possible DQ are encoded. Positive risks for IMD are associated with A chains that have an amino acid arginine at residue 52, and B chains that lack an aspartic acid at residue 57. Persons at the highest genetic risk for IMD, are those where all 4 DQ combinations meet this criteria. 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 albeit those with DRB1*11 or 04 who also have DQB1*0301 (asp 57 +) are protected. HLA-DR are also involved in susceptibility to IMD in that in the B1*0401, and 0405 subtypes of DRB1*04 are susceptible while the 0403 and 0406 subtypes are strongly protective, even when found in HLA haplotypes of the susceptible DQA1*0301/DQB1*0302. DR molecules are heterodimers also, however the DRA chain is invariant in all persons. Additional DRB chains (B3, B4 and B5) have not been shown to be important. Class 2 MHC are involved in antigen presentation by antigen presenting cells, and the above associations will probably be explained in terms of defective affinities to islet cell antigenic 12 mer peptides, leading to persistence of T helper cells which escape thymic ablation. Class 1 HLA also are implicated in IMD. Persons with HLA-B9 amongst others seem to have an increased risk of IMD. If so, this would not be surprising, since cytotoxic CD8+ T cells that are thought responsible for the destruction of pancreatic b cells in the disease, see islet cell 9 mer peptide antigens on their surfaces in context of class 1 MHC. The next most important gene to those of the HLA region on chr. 6p, is the INS gene on chr.11q, or rather the variable nucleotide tandem repeat (VNTR) upstream from the gene. Long polymorphic alleles are protective, as related to expression of insulin on thymic dendrocytes, which could improve the recognition by potentially reactive T cells with receptors that have affinity for insulin peptides and their ablation in the thymus before they escape into the periphery. Typing new-born infants for both HLA/DR/DQ and the INS gene results in prediction of IMD to better than 1:10 in the general population. In HLA-identical siblings of a proband with IMD, their risk of IMD is 1:4, those with HLA-haplotype identity 1:15 and those with no shared haplotype 1:100.

Mutations in the insulin gene may result in early onset hyperglycemia with high levels of immunoreactive insulin. Diabetes due to mitochondrial DNA mutants has a matrilineal inheritance. Wolfram’s syndrome (optic atrophy, diabetes insipidus, diabetes mellitus and deafness) has been mapped to a gene on chr. 4p. For maturity onset diabetes of youth or MODY, the genes are generally understood and are identifiable, albeit the numbers of mutants are large. Persons at risk within MODY pedigrees can be identified through genetic means (see below), however for most patients the diabetes is mild and most often not associated with complications of diabetes. The research interest in MODY genes has been stimulated by hopes that they may provide insights into type-2 diabetes.

Insulin Resistance Diabetes:

Insulin resistance is associated with acanthosis nigricans and hyperandrogenism, especially when severe.

An autosomal dominant form of lipo-atrophic diabetes (Dunnigan’s syndrome) maps to 1q21-22, while the generalized recessive form to chr. 9p34. More than 70 mutations have been described in the insulin gene resulting in insulin resistance. Leprechaunism and the Robson-Mendenhall syndrome are recessive conditions with two inactivating mutations (severe and moderate respectively) in the insulin receptor gene. Other mutants can result in, decreased receptor biosynthesis, impaired transport of receptors to the cell surface, decreased insulin binding affinity,impaired receptor tyrosine kinase activity or accelerated degradation of the receptor. However all of these syndromes account only for a small fraction of patients loosely categorized as type-2 diabetes. It is increasingly probable that most forms of type-2 diabetes (which are usually strongly familial), will eventually be understood in defined genetic terms, but this is far from realized at present.

Genetic Testing for MODY:

Use:

A. Diagnosis/screening

Recommendation: Molecular diagnostic tests are generally derived from new discoveries made in the research laboratories. Application to routine clinical diagnosis requires implementation of methods of quality assurance more typical of clinical laboratories. This is a rapidly changing area, both from the technical and regulatory point of view.

The common form of type-2 diabetes is a strongly inherited form of the disease, resulting from the combination of problems of resistance to the action of insulin on the body, plus degrees of relative to absolute deficiencies in the capacity to secrete insulin. Genetic factors that cause type-2 diabetes still remain unclear, with a few exceptions. Therefore, genetic testing on the disease is not available yet. However, genes that cause several uncommon forms of type-2 diabetes, namely MODY, have been identified, and mutation detection for MODY patients and their relatives is technically feasible. Due to the high cost for setup the mutation detection facility and high standard of technical skill for performing such tests, so far only a few medical laboratories can perform them. Thus the genetic test of MODY is not recommended outside of a research setting.

B. Monitoring/prognosis

Genetic screening provides important information about possibility for relatives of MODY patients to develop MODY themselves. It has some value on monitoring the prognosis of MODYs patients, and it can direct the most optimal treatment to be used.

2. Rationale:

Five different MODYs have been identified. MODY-1, 3, 4, and 5 all result from mutations in the genes encoding transcription factors that regulated the expression of genes in the pancreatic b cell. They are hepatocyte nuclear factor-4a (HNF-4a) in MODY-1, HNF-1a in MODY-3, HNF-1b 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; heterozygous mutation in IPF-1 causes MODY-4. The modes of action of the HNF lesions in MODY is still not clear. It is likely that mutation in HNF-1a, 1b, and 4a cause diabetes because they impair insulin secretion. Unlike the MODYs mentioned above, MODY-2 is caused by mutations in the glucokinase gene. Glucokinase is an enzyme that plays an essential role in the glucose-sensing mechanism of the ² cells, and mutations in this gene lead to partial deficiencies of insulin secretion.

The mutation of MODY-1 has been characterized as C -- T substitution in code 268 of HNF-4a, which generates a nonsense mutation CAG (Gln)—TAG (stop codon) (Q268X). To date, 28 different mutations have been identified in the glucokinase gene in subjects with MODY-2. These mutations include 19 mis-sense mutations that change the sequence of glucokinase, 4 nonsense mutations that result in the synthesis of a truncated glucokinase molecule, and 5 deletions and mRNA splicing mutations which result in the synthesis of a mRNA molecule that cannot encode a normal functional protein. MODY-3 patients have genetically characterized to have one of 7 reported mutations in IFN-1a. Three of them are insertion or deletion mutations, leading to frame-shifts of the INF-1a gene, 2 are splicing mutants and 2 nonsense substitutions that also change protein sequences of INF-1a. Only one deletion mutation (Pro63fsdelC) has been identified for MODY-4. That mutation results in a frame-shift of coded protein of IPF-1 gene. MODY-5 has been attributed to the nonsense C—T mutation at code 177 of HNF-1b gene.

Analytical

Preanalytical:

Mutational detections are normally carried out by using peripheral blood genomic DNA that is extracted from the subject’s leukocytes. Blood samples should be drawn into test tubes containing EDTA and the DNA harvested within 3 days, albeit longer periods lower the yields. 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 ug DNA from 10 ml of whole blood.

Analysis:

Methods for mutational detection differ for different mutation types. In general, the mutants in MODYs are substitution, deletion and insertion of nucleotides in the coding region of the disease genes. The methods for the mutation detections on these genes are PCR based direct sequencing analysis. The detailed protocols for the detection of each specific mutation, are beyond the scope of this review. The interested readers can obtain information elsewhere.