Identification and characterization of disease-related copy number variations (CNVs) by high-dense SNP oligonucleotide microarrays

Genomic microarray analysis is rapidly replacing conventional chromosome analysis by molecular karyotyping due to the significant increase in the power to detect causative CNVs. Here, we extensively validated the HumanHap550 and Human610-Quadv1_B Illumina platforms for potential diagnostic application by using patients with undiagnosed intellectual disability (ID). The first and foremost goal of our application study was to use these arrays for reliable genome wide detection of rare CNVs in patients of three different cohorts: 1) patients with unexplained intellectual disability 2) patients with unknown diffuse congenital hyperinsulinism (CHI) and 3) a family with a distinctive diagnosis of Holt-Oram syndrome (HOS). We showed that SNP-based arrays allow the detection of intragenic deletions and duplications. The identification of a disease-CNV affecting only a single gene allowed us to consider that particular gene as a candidate for intellectual disability. This was the case for three unrelated patients with moderate intellectual disability, global developmental delay, and severe speech and language disorders in which a de novo deletion encompassing solely the FOXP1 gene was detected. To prove further the causality of the FOXP1 deletion following-up investigations were based on a screening of the entire coding region of FOXP1 for nucleotide changes in a panel of 883 probands with intellectual disability. Eight non-synonymous coding changes, three synonymous and nine non-coding variants were identified. In addition to the de novo cases of ID, also patients suffering from an autosomal recessive form of ID were found in our cohort. We detected three partial heterozygous deletions of the COH1 gene at locus 8q22 which is mutated in Cohen syndrome. After sequencing the entire coding region and the exon/intron boundaries of COH1 we identified a stop mutation, a frameshift and two missense mutations in the remaining allele, respectively. Therefore, three compound heterozygous mutations were identified in the COH1 gene, thus providing a distinctive Cohen Syndrome diagnose to three unrelated patients of our ID cohort. We studied the genetic basis of a rare human autosomal disorder such as diffuse Congenital Hyperinsulinsm (CHI) in a cohort of 40 patients with inconspicuous mutation screening of ABCC8 and KCNJ11 genes. Chromosomal abnormalities detected by SNP oligonucleotide arrays accounted for 20% of the studied cases. The most interesting rearrangement was a 970kb deletion at the chromosomal band 1p31.1 which was found to encompass the PTGER3 and ZRANB2 genes and the last exon of the NEGR1 gene. We hypothesized that the haploinsufficiency of PTGER3 gene induces a 50% reduction of the stimulation by PGE2, thus diminishing the inhibition of glucose-stimulated insulin secretion (GSIS) and resulting in elevated insulin secretion. The screening for point mutations in the candidate gene PTGER3 did not reveal any pathogenic variant neither in the second allele of the patient in which a de novo deletion was detected nor in a cohort of 39 unrelated patients with unexplained CHI. Instead we identified a novel polymorphic variant which was also detected in 18 individuals of our control cohort. CNV analysis in a family with both atypical Holt-Oram syndrome and additional mammary glands was performed allowing the detection of a contiguous heterozygous duplication at the chromosomal band 12q24.21. The maximal duplication size could be estimated as aproximately 345,6kb including the whole coding region of the TBX5 and TBX3 genes. Gene dosage assessment at specific genetic loci demonstrated the cosegregation of the duplication and the Holt-Oram syndrome/supernumerary mammary glands phenotype in this pedigree, this being a strong indicator of its pathogenecity. Up to date, this is the first report of a heterozygous duplication encompassing both TBX5 and TBX3 genes, and consequently the first report of a combined phenotype of Holt-Oram syndrome and supernumerary mammary glands