BACK TO INTRODUCTION
& GENETIC TESTING IN DIABETES
ROLE OF GLUCOSE MANAGEMENT IN DIABETES
& MINIMALLY INVASIVE GLUCOSE ANALYSIS
of Patients with Diabetes
& PRECURSORS, LEPTIN & AMYLIUN: IS THERE A ROLE?
FOR MICRO ALBUMINURIA
& Recommendations for Laboratory Analysis in the Diagnosis & Management
of Diabetes Mellitus
K Maclaren MD and Zhong Sheng Sun Ph.D.
Department of Pediatrics
Weill College of Medicine of Cornell University
New York, NY 10021.
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.
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.
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
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.
resistance is associated with acanthosis nigricans and hyperandrogenism,
especially when severe.
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.
Testing for MODY:
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.
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.
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.
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.
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.
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.