Challenges for molecular diagnosis of familial early-onset diabetes in unexplored populations.

Dear Editor-in-Chief

Understanding the genetic causes of monogenic forms of diabetes is important for prognostic, follow up, and treatment of the patients (1). MODY (Maturity-onset diabetes of the young) is a form of monogenic diabetes clinically and genetically heterogeneous. Indeed, to-date there are at least 13 different forms of MODY, each one is due to mutations in a specific gene (MODY1-HNF4A, MODY2-GCK, MODY3-HNF1A, MODY4-IPF1, MODY5-HNF1B, MODY6-NEUROD1, MODY7-KLF11, MODY8-CEL, MODY9-PAX4, MODY10-INS, MODY11-BLK, MODY12-ABCC8 and MODY13-KCNJ11) (2). Besides, MODY is not due to a recurrent mutation (1). Molecular diagnosis of MODY is often based on mutation screening using Sanger sequencing and MLPA (Multiplex ligation-dependent probe amplification) (3). In the Tunisian population and in other populations, the genetic causes of familial early-onset diabetes remain unknown (3).

In this report, we described the difficulties encountered in determining MODY genetic etiologies, after a mutation screening of PAX4(Paired box gene 4) gene insix MODY-X Tunisian families, using classical methods and suggesting some solutions.

The studied families had been previously found negative for mutations and deletions in GCK, HNF1A, HNF4A, HNF1B, INS, IPF1 and NEUROD1 genes (3). The molecular analysis was carried out after taking informed written consent from all patients and after approval of the local ethics committee.

We chose to sequence PAX4 gene because of the resemblance between the clinical characteristics of the six families’ probands (Table 1) and the reported MODY9-PAX4 patients (4, 5). Moreover, PAX4 gene encodes a transcription factor which is part of the regulatory mechanism balancing the development of pancreatic beta cells (6).

Table 1

The clinical and molecular characteristics of Tunisian patients selected for PAX4 mutation screening and MODY9 patients of other studies

Study	Patient	Age at diagnostic	Diabetic generations	BMI Kg/m2	Glycaemia mmol/l	HbA1c %	Traitement	PAX4 SNPs	Consequence
Our study	F2	16	3	25,3	14,1	12.8	INS	rs327516rs2233579rs712701	c.-221G>Cc.412+35G>Ap.H321P
	F4	27	2	23,5	6,2	6	INS	rs327516	c.-221G>C
	F5	24	3	22,8	10,6	6.6	OHA->INS	rs327516rs2233579rs712701	c.-221G>Cc.412+35G>Ap.H321P
	F6	29	3	18	12,2	14.9	Diet->INS	rs327516rs712701	c.-221G>Cp.H321P
	F8	26	2	37,2	12,1	11.3	OHA->INS+OHA	rs327516rs2233578rs2233579	c.-221G>Cp.R133Wc.412+35G>A
	F15	22	3	23,5	9	8.2	INS	rs327516rs712701	c.-221G>Cp.H321P
Thai study(4)	Proband	20	2	--	--	--	OHA	--	p.R164W
Japanese study(5)	Proband father	1530	22	1832	35--	14.5--	INSDiet	--	c.374-412del39

The sequencing of all nine exons of PAX4 showed four common polymorphisms and nocausal mutations (Table 1). Thus, the six families were negative for an eighth gene out of thirteen (reported to be responsible for MODY forms). The familial early-onset diabetes observed in these patients could be explained by mutations in other reported genes (ABCC8, KCNJ11…) or in unknown genes. This clearly shows the difficulty and time consuming to determine the molecular causes behind MODY-X using Sanger sequencing based on clinical findings. Hence, the need for fast and less expensive techniques that screen all 13 known MODY genes in one step. PCR enrichment in microdroplets combined with next generation sequencing, or targeted next generation sequencing assay could be used as a first step in the research of mutations in known MODY genes (7, 8). In case of mutation absence in these genes, whole exome and genome sequencing combined with high-throughput genotyping and linkage analysis could be useful to identify new genes responsible for monogenic diabetes (2). Otherwise, setting up a research strategy based on metabolomics and biomarkers might be of great interest for molecular diagnostic of MODY.

The genetic etiologies of familial early-onset diabetes are still unidentified in many populations. Undeniably, introducing a next generation sequencing-based strategy will reduce costs, increase the throughput, allow gaining precious time and elucidate new pathways underlying the pathophysiology of the disease.