Introduction
Maturity-onset diabetes of the young (MODY) describes the dominantly inherited disorder of non-insulin-dependent diabetes typically diagnosed before 25 years that was first recognised by Tattersall [
1,
2]. MODY is the most common form of monogenic diabetes, accounting for an estimated 1–2% of diabetes in Europe [
3,
4], but is often misdiagnosed as type 1 or type 2 diabetes.
The term MODY is used to describe a group of clinically heterogeneous, often non-insulin-dependent forms of diabetes that are defined at the molecular genetics level by mutations in different genes. All show dominant inheritance and are disorders of beta cell dysfunction, but variable features include the age at onset, severity of the hyperglycaemia (and hence risk of complications) and associated clinical features. The most recent classification of diabetes by the American Diabetes Association and the World Health Organization recognises these discrete subtypes of MODY [
5].
Mutations in the
GCK and
HNF1A genes are the most frequent cause of MODY in all populations studied. They account for approximately 70% of cases (see Table
1). The ratio of
GCK to
HNF1A mutations varies between countries because of different recruitment strategies for genetic testing; blood glucose screening in young, asymptomatic individuals will identify a higher proportion of
GCK mutations.
Table 1
Genes in which mutations cause MODY
Protein | Glucokinase | Hepatocyte nuclear factor-1 alpha | Hepatocyte nuclear factor-4 alpha | Insulin promoter factor-1 | Neurogenic differentiation 1 | Hepatocyte nuclear factor-1 beta |
Chromosome locus | 7p13 | 12q24.31 | 20q13.12 | 13q12.2 | 2q31.3 | 17q12 |
Gene Accession no. | NM_000162.2 | NM_000545.4 | NM_000457.3a
| NM_000209.2 | NM_002500.2 | NM_000458.1 |
OMIM * (Gene) | 138079 | 142410 | 600281 | 600733 | 601724 | 189907 |
OMIM # (Phenotype) | 125851 | 600496 | 125850 | 606392 | 606394 | 137920 |
Mutation frequency (%) (not known in ~20% of cases) | 20–50 | 20–50 | ~5 | <1 | <1 | ~5 |
Heterozygous loss-of-function
GCK mutations result in mild, stable hyperglycaemia from birth. Microvascular complications are rare, reflecting the fact that HbA
1c is normally just above the upper limit of the normal range. Treatment with oral hypoglycaemic agents or insulin is not needed because it rarely changes HbA
1c [
6]. A genetic diagnosis is important for the small number of children misdiagnosed with type 1 diabetes and treated with insulin [
7]. The identification of
GCK mutations in women with gestational diabetes can be useful for obstetric management, since their babies who do not inherit the mutation are at risk of macrosomia [
8], and it can guide follow-up in the mothers.
Transcription factor mutations in the
HNF1A or
HNF4A genes cause a similar progressive diabetic phenotype although the penetrance of
HNF4A mutations is lower (S. Ellard and A. T. Hattersley, unpublished data). Sensitivity to sulfonylureas means that some patients can transfer from insulin to oral agents [
9,
10]. A low renal threshold for glucose is a feature of
HNF1A mutations [
11] and may provide a useful method of screening at-risk family members during childhood [
12].
Mutations identified in the
GCK,
HNF1A and
HNF4A genes include missense, nonsense, splicing, small deletions/insertions/duplications, and splice site and promoter region mutations [
13,
14]. Partial and whole deletions have recently been reported in
HNF1A and
GCK [
15]. The location of mutations within the
HNF1A gene influences the age at diagnosis; the average age at diagnosis for patients with exon 1–6 mutations that affect all three
HNF1A isoforms is younger than for those with mutations in exons 8–10 that affect only isoform
HNF1A(A) [
16,
17].
Rarer forms of MODY include heterozygous mutations in
PDX1 (also known as
IPF1; [
18,
19]) and
NEUROD1 [
20,
21], but analysis of these genes is not usually included in routine molecular genetic testing for MODY. Dominantly inherited syndromic forms of diabetes may also be described as MODY subtypes. The renal cysts and diabetes syndrome results from
HNF1B mutations, and other features include renal abnormalities, female genital malformations, hyperuricaemia, pancreatic atrophy and abnormal liver function tests [
22‐
24]. Mutations in the
CEL variable number tandem repeat cause a syndrome of diabetes and pancreatic exocrine dysfunction [
25]. These syndromes and maternally inherited diabetes and deafness caused by the mitochondrial m.3243A→G mutation are not included in these guidelines since testing is guided by the non-endocrine pancreatic or extra-pancreatic clinical features.
A molecular genetic diagnosis of a GCK, HNF1A or HNF4A mutation is important because it confirms a diagnosis of MODY, classifies the subtype, predicts the likely clinical course and may change the patient’s treatment. First-degree relatives will be at 50% risk of inheriting the mutation and asymptomatic individuals may be offered predictive genetic testing (after appropriate genetic counselling) in order to provide reassurance (for those shown not to carry the mutation) or regular blood glucose monitoring with early diagnosis and appropriate treatment (for mutation carriers).
Methods
A group of European clinicians and scientists met on 22 May 2007 at a workshop to formulate best practice guidelines for molecular genetic testing in MODY. Discussions focused on clinical criteria for selection of patients for testing, methodologies, interpretation of results and reporting those results to the referring clinicians.
A draft document was posted on 24 August 2007 and an online editing tool was used by participants to produce consensus guidelines.
Results
Clinical criteria for testing
Testing methodology
The mutation screening methodology should be described in the report [e.g. sequencing, denaturing high-performance liquid chromatography (dHPLC), conformation-sensitive capillary electrophoresis (CSCE)] together with the sensitivity. PCR primers should be checked for primer binding site single nucleotide polymorphisms (SNPs; a useful tool is available at
http://ngrl.man.ac.uk/SNPCheck/index.html). Gene dosage analysis may be useful if a diagnosis of MODY is strongly suspected and no mutation is found on mutation screening.
Interpretation of results
The textbox includes recommended interpretations for the most common reporting scenarios.
Reporting
Each laboratory has its own reporting format and general guidance on reporting is available from the European Molecular Genetics Quality Network (
http://www.emqn.org), the UK Clinical Molecular Genetics Society (
http://www.cmgs.org) and the Swiss Society of Medical Genetics (
http://www.ssgm.ch). A one page report is the preferred format.
The report should state the methodology and specify the gene, exons and/or mutations tested for. If promoter sequences are examined then the report should specify the nucleotides analysed. An estimation of the assay sensitivity is particularly useful for pre-screening techniques such as dHPLC, CSCE etc. The use of mutation nomenclature approved by the Human Genome Variation Society (
http://www.hgvs.org/mutnomen) is strongly recommended. The gene accession number (with version) is required in order to describe mutations unambiguously (see Table
1). The A nucleotide of the ATG start codon is numbered +1.
Reports describing novel variants should state that the variant is novel and include the evidence in support of pathogenicity. This might include the absence from a large series of ethnically matched controls or MODY patients (testing of 210 normal chromosomes is necessary to achieve at least 80% power to detect a polymorphism present in 1% of the population [
35]). Testing of other affected relatives is recommended in order to check for co-segregation and to calculate the LOD score in suitable sized pedigrees (LOD scores of ≥1 or ≥3 are suggestive or conclusive of linkage, respectively).
For missense variants the evidence for pathogenicity might include conservation across species and a significant amino acid substitution. Several programs are available that predict the pathogenicity of a missense variant based upon amino acid conservation (SIFT;
http://www.blocks.fhcrc.org/sift/SIFT.html) or the structure and function of the protein (PolyPHEN;
http://www.genetics.bwh.harvard.edu/pph) but they should be used to supplement other pieces of evidence rather than in isolation.
Both missense and silent variants can affect splicing if the mutation is within an exon splicing enhancer or exon splicing silencer. Splice predictor software programs (
http://www.fruitfly.org or
http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home) may aid interpretation. Base substitutions affecting the conserved splice donor (GT) site, splice acceptor (AG) site or the conserved A nucleotide within the branch site are highly likely to be pathogenic but splice predictor software may be useful in the interpretation of other intronic variants. Analysis of patient mRNA is often informative but lymphoblastoid cell lines are usually required because of the low levels of expression of the MODY genes in blood. Sequence analysis of RT-PCR products amplified from lymphoblastoid cell mRNA has demonstrated exon skipping, retention and the use of cryptic splice sites for a variety of intronic mutations in the
GCK,
HNF1A and
HNF1B genes [
36‐
38].
Novel promoter variants may be investigated by examination of known transcription binding sites or by in vitro transfection experiments [
39‐
42]. They may also alter mRNA expression levels, which may be measured by allele-specific real-time PCR [
43].
Polymorphisms
Some laboratories include details of polymorphisms detected in the report. The reasons for doing this include: (1) making all data available to the requesting clinician based on the rationale that a polymorphism may later be reclassified as a mutation; and (2) identifying heterozygous SNPs excludes a gene deletion involving the exon(s) in question.
However, this information can cause confusion or even misinterpretation of the result (A. T. Hattersley, unpublished data) and it certainly adds to the length of the report. While in some cases there may be reports in the literature of an association with type 2 diabetes or reduced insulin secretion, these polymorphisms do not cause MODY and we recommend that they should be excluded from the report.
Treatment
Individual treatment recommendations are outside the jurisdiction of a molecular genetics report since this is the referring clinician’s responsibility. It is useful to include an appropriate reference if there is evidence in the literature for a particular treatment (e.g. low-dose sulfonylureas in HNF1A/4A MODY) associated with the genetic diagnosis.
Other issues
Genetic counselling should be provided for all asymptomatic individuals requesting predictive testing. We recommend that unaffected relatives are offered a biochemical test first (fasting blood glucose for GCK mutations or OGTT for HNF1A/HNF4A mutations). If the biochemical test is consistent with a diagnosis of diabetes or hyperglycaemia then the genetic test will be diagnostic, not predictive.
For families requesting predictive testing for children too young to provide informed consent, referral to a specialist clinical genetics unit (or equivalent) is strongly recommended. Reasons for testing children include (1) to remove the uncertainty around the child’s status, and (2) to assist with management, as a negative test would mean that monitoring of blood glucose/glycosuria would not be necessary [
44].
Conclusions
Molecular genetic testing is useful in patients with MODY because it confirms a diagnosis of monogenic diabetes, predicts likely clinical course, defines risk for relatives and determines treatment.
At the present time, molecular genetic testing for MODY is relatively expensive and phenotypic selection prior to testing is normal practice. With the development of new technologies it is likely that these costs will decrease in time and that the analysis of genes associated with monogenic diabetes may become routine for all newly diagnosed patients. In the meantime we hope that these guidelines will be useful in determining which patients should be offered testing, and in the interpretation and reporting of the test results.
Acknowledgements
Funding for the best practice meeting was provided by the EuroGentest Network of Excellence Project 2005-EU Contract No FP6-512148 (
www.eurogentest.org) and the European Molecular Genetics Quality Network (
http://www.emqn.org). A. T. Hattersley is a Wellcome Trust Research Leave Clinical Fellow.