ABSTRACT

Introduction. MODY (Maturity Onset Diabetes of the Young) is a type of diabetes characterized by autosomal dominant inheritance and punctiform mutation or deletions of genes involved in b-cell differentiation and/or insulin secretion. It accounts for 1-2% of all diabetic patients and it is genetically confirmed in 30% of diabetic subjects with clinical characteristics suspected for MODY. Sardinia represents a geographical isolate and a unique model to study genetic diseases.

Materials and methods. Any positive patient with 1st degree family history of diabetes has been identified among 762 adult diabetic patients (out of 12563 patients) from the Diabetes Unit, A.O.B., Cagliari (Italy). Subjects highly suspected to have MODY underwent genetic test for MODY 2 (GCK) and MODY 3 (HNF-1α). Genetic analysis of GCK gene has been performed in one family only (Family 1: proband, mother, father and brother). DNA has been extracted with Amersham kit. Exons 1a, 5 and 6 of GKC (where more frequently the MODY mutations are found) have been amplified with PCR. Screening for mutations in the GCK gene have been performed with DHPLC method as described by Boutin.

Results. Among the 762 patients screened so far, 22% (166) had at least one first degree relative affected by diabetes and 7.3% (56) were highly suspected to have MODY according to our inclusion criteria (autosomal dominant inheritance, age at onset <50 yrs, negativity for islet related autoantibodies, insulin requirement <0.5 U/day). As the family 1, the clinical, immunological features and the HLA typing excluded the diagnosis of T1D. The DHPLC elution profile of the proband’s GCK exon 1a and 5 demonstrated the presence of omoduplexes, while the exon 6 produced an eteroduplex, suggesting the presence of different alleles on the two chromosomes.

Conclusions. The mutated allele could be responsible for the impaired glucose tolerance in the proband and the family relatives. It must be confirmed by the gene sequencing. The genetic screening confirmed the clinical suspicion of MODY in the only family analysed so far. The location of an heteroduplex on exon 6 of the GCK will restrict the sequencing only to this exon in the proband and in family members. Since, according to the literature, in only 30% of diabetic patients with the clinical characteristics of MODY corresponding mutations have been found, we are continuing typing our patients in order to assess the prevalence of MODY among our Sardinian diabetic population.

BACKGROUND

Sardinia is a large island in the middle of Mediterranean. It represents a geographical isolate and a unique model to study genetic diseases. Itrepresents an ideal basis for epidemiological studies because of it is homogeneus, stable, genetically well defined and relatively small population of 1,6 milions people.

MODY (Maturity Onset Diabetes of the Young) represents a genetically defined type of diabetes, characterized by autosomal dominant inheritance. It represents about 1% of the total diabetes cases. Today, several mutation in 6 different genes have been associated with such type of diabetes. The relative prevalence of distinct MODY suvbtypes differs substantially among thestudies in the different populations: mutations in GCK (MODY2) representing from 8 to 63% and HNF-1a mutations (MODY3) from 13% to 64% of all subject with MODY. Mutations in the HNF-4a, IPF-1, HF-1B and Neuro D1 have been recognised in single families only, while additional unknown MODY genes (MODY X) may be responsible for 16 to 45% of cases of MODY. These MODY mutations have not been identified yet.

The diagnosis of MODY is suspected when an autosomal dominant inheritance of impaired glucose metabolism is present, particularly when the impairment of glucose metabolism is mild, its onset occurrs during infancy or adolescence and no insulin is required. However the diagnosis is only confirmed by genetic analysis.

The confirmation of the diagnosis may lead to make some useful changes in the therapeutic approach.

Nothing is known about the prevalence of MODY in Sardinia nor on the relative prevalence of its mutations and whether there are mutations specific for the Sardinian population, which has been recognised to have peculiar characteristics compared to other populations due to the small number of founders and to the genetic drift.

MATERIALS AND METHODS

Family history of diabetes has been investigated in 762 out of 12563 patients from the Diabetes Unit, A.O.B., Cagliari (Italy). Patients with family history of impaired glucose metabolism (diabetes, IGT or IFG according the new ADA criteria and/or WHO criteria, or GDM) among 1st degree relatives in at least two consecutive generation (autosomal dominant inheritance) have been selected.

Further data were collected for each of these patients and they were classified as highly suspected for MODY if they had:

- Insulin requirement < 0.5 U/kg BW

- Undetectable ICA, GADA and IA2

Among the 762 patients screened so far, 22% (166) had at least one first degree relative affected by diabetes and 7.3% (56) were highly suspected to have MODY according to our inclusion criteria:

• age at diabetes onset < 50 years in at least one family member
• Insulin requirement < 0.5 U/kg BW
• Undetectable ICA, GADA and IA2

Thus far only one family (Family 1) has been genetically analysed.

So far, genetic analysis of MODY genes has been performed in only one family (Family 1) suspected to have MODY 2 from the clinical characteristics of the proband. Thus, exons 1a, 5 and 6 of GKC gene of the proband (where more frequently the MODY2 mutations are found) have been amplified with PCR and screened with DHPLC (Denaturing Hig-Performance Liquid Cromatography) for discriminating homoduplexes and heteroduplexes in PCR products by ion pair reverse-phase chromatografy under partially denaturing conditions (P. Boutin et al). Heteroduplex suggests the presence of an allele differing from the wild type.

Sequencing of the exon where the heteroduplex is found will be performed in the proband and in the family memebers.

AIMS

DISCUSSION

We selected 56 families highly suspected of MODY 2 or 3 (GCK or HNF-1a mutations). These represented 7.3% of the observed population.

According to the literature, only 30% of a population - selected by clinical criteria - is found to be carriers of a mutation in one of the MODY genes.

If this would be the case also in our Sardinian population, the estimated prevalence of MODY (2.2%) in our population should be comparable to the prevalence described among diabetic patients (1-3%) elsewhere.

However, the peculiarities of the Sardinian population and its high frequency of genetic diseases, do not allow us to apply this raw calculation without carrying out further investigations.

Furthermore, mutations of MODY genes in the Sardinian population could be different from other populations.

The genetic screening confirmed the clinical suspicion of MODY in the only family analysed so far. The location of an eteroduples on exon 6 of the GCK will restrict the sequencing only to this exon in the proband and in family members. Thus the screening approach with DHPLC allow us to save time and money, as described previously.

The mutated allele could be responsible for the impaired glucose tolerance in the proband and family relatives. However, it must be confirmed by the gene sequencing.

The correct identification of diabetes type, particularly the distinction between true Type 1 Diabetes and MODY, leads also to a correct therapeutic approach for the patients and his/her family members (MODY3 and liver aenomatosis). This suggests the need of a genetic screening in families with undefined type of diabetes or with a strong family history of impaired glucose metabolism.

This genetic screening is much more advisable In Sardinia, where many MODY patients could have been missdiagnosed as Type 1 patients because of the onset of both conditions in childhood or adolescence and because of the high incidence of type 1 diabetes in this island.

REFERENCES