Genome-wide homozygosity mapping in Congenital Hyperinsulinism of Infancy (CHI) :a family based study

Congenital hyperinsulinism of infancy (CHI) is a pathology characterized by a profound hypoglicaemia related to a disregulated insulin secretion; the frequency of the disease is 1:30000-50000 live birth world –wide, but in same isolated populations with high consanguinity like the Arabic Peninsula, frequency of 1:2500 is reported. At present it is well known that dysfunctions of pancreatic - cells K-ATP channel are a common cause of CHI. The two subunits of the channel are coded by two genes, KCNJ11 and ABCC8. Mutations in ABCC8 gene are responsible of the 50-60% of focal and diffuse CHI patients; otherwise mutations in KCNJ11 are responsible for 10-15% of cases. Other less frequent molecular mechanism responsible for CHI are glutamate dehydrogenase (GDH), glucokinase and short- chain 3 hydroxyacyl Coenzyme A dehydrogenase (SCHAD) deficiency. CHI derives from a heterogeneous genetic background and many other genes involved in the pancreatic - cells metabolism are good candidate genes for this disease. In order to identify new disease susceptibility loci in a cohort of consanguineous and not consanguineous 34 CHI Italian families, we performed genome-wide homozygosity mapping using 250K NspI Gene Chip Affymetrix SNP microarrays and to reveal the number and the nature of homozygosity trait in common between probands to underline new regions containing candidate genes involved in the ethiopathogenesis of CHI. Further screening for mutations in candidate genes was performed and biochemical pathways involved in the disease has been analyzed. We used CNAG (v3.0) software to describe homozygosity traits present in 34 families with CHI affected probands. Consanguineous families bear a number of homozygous stretches much higher that not consanguineous ones and following investigations of the parental origin of homozygous stretches of more than 1 Mb in length has confirmed that they were not inherited and did not reveal any copy number loss, concluding that they were not deletions. Mendelian errors check (all below 0.1%, threshold due to genotyping errors and not transmission error) and parental haplotypes analysis has excluded uniparental disomy (UPD) and they must be considered as autozygosity traits. To further characterize homozygosity traits in CHI probands, we used dCHIP software to underline homozygosity regions in common between at least two patients. On Chromosome 11 we found five patients sharing a common homozygous trait on cytoband 11p15, where the two major CHI candidate genes are present. In two of these probands, where mutational screening of ABCC8 and KCNJ11 genes was completed, the causative mutation was revealed. Other relevant homozigosity region in common between 4 patients are present on chromosome 6 (p21.33-p21.31) and on chromosome 10 (q26.13- q26.2), while 18 common traits between 3 patients were revealed One of the numerous traits in common between two patient on chromosome 1 showed a homozygous region in 1p31, one patient bearing a long contiguous trait of 25 Mb. This common region contains medium-chain acyl-CoA dehydrogenase (MCAD) gene, involved in organic acid metabolism deficiency, and in that proband an homozygous synonymous mutation P195P in exon 8 of MCAD gene was identified (587 T >G); her parents and brother evidenced the same heterozygous nucleotidic variation. On chromosome 4 one probands showed the longest homozygosity stretch (from 4q13.2 to 4q31.21), which ranged 79,01 Mb and included 312 known genes in the region; this stretch contains the causative short- chain 3 hydroxyacyl Coenzyme A dehydrogenase gene (HADH). Direct sequencing of the coding region of HADH revealed a homozygous C to T transition in exon 6 causing a premature stop of the synthesized protein at codon 236 (706C>T), resulting in the nonsense mutation R236X. Mutation analysis of exon 6 of the HADH gene in the parents of the proband showed they were heterozygous carriers of the mutation. This is the fourth described mutation in HADHSC gene in a CHI patient, indicating the importance of -oxidation disregulation in this disease. A preliminary bioinformatics pathways analysis in CHI probands underlined that more than one pathway is involved in the disease, in particular type I diabetes mellitus, glycolysis and gluconeogenesis, fatty acid metabolism and amino acids metabolism. Homozygosity mapping in 34 CHI families has confirmed a relevant presence of autozygosity, due to presence of high inbreeding in at least 5 analysed families and to the restricted geographical provenience. Autozygosity excludes that deletions or UPD are at the molecular basis of the disease. Even if each patient has an homozygosity profile strictly peculiar, there are common region shared between at least two probands. These regions are good candidates for the discovery of new disease associated genes/loci. The discovery that CHI probands showing homozygosity traits on chromosome 11, in particular on 11p15.1, bear a mutation in known causatives genes and the recent finding of a not jet described stop mutation in HADH gene on chromosome 4 confirm the power of SNPs genome-wide genotyping technologies to underline loci with high mutational probability and to elucidate the molecular basis of autosomic recessive diseases. To our knowledge this is the first study in which molecular mechanism of CHI disease was investigated with genome-wide homozygosity mapping method in consanguineous and not consanguineous CHI families. Genome-wide homozygosity mapping in the present study has clearly evidenced 1p31.2-31.1, 4q27-28.1, 6p22.3-22.31, 11p15.4-15.2, and 18q12.2 chromosomal loci to be the most related to CHI disease and to better redefine that regions and to single out potential associated genes, we are planning to use SNPs genotypes calls generated from these 34 CHI family microarrays data to perform family based transmission disequilibrium analysis. On the other hand, we will increase the number of CHI families enrolled in the study in order to evidence by homozygosity mapping homozygosity traits which are strictly peculiar of the disease and not common in the normal population