Polymorphisms and mutational analysis of the NOTCH3 gene in a large cohort of patients affected by leukoencephalopathy

Objective: The aim of the present study was to analyse the coding region and intron-exon boundaries of the NOTCH3 gene in a large cohort of patients af- fected by leukoencephalopathy to investigate the presence of genetic vari- ants. Patients and methods: We analyzed the exons 2-23, exons 3-4, exons 3-4-6-8 respectively in 157, 684 and 542 patients affected by leukoen- cephalopathy. Genomic DNA was extracted from peripheral-blood leuco- cytes using the salting out method. Twenty-two exons (2-23) out of 33 of the NOTCH3 gene and their intronic flanking sequences were amplified by PCR with sets of oligonucleotide primers specific for NOTCH3. The amplicons were then analyzed by Denaturing High Performance Liquid Chromatogra- phy; patients' chromatograms for each exon were compared with corre- sponding normal controls and the exons showing an abnormal DHPLC eluition profile were directly sequenced in both forward and reverse direc- tions on an ABI Prism 3130XL genetic analyzer. Results: The molecular analysis revealed several nucleotide alterations in comparison to the wild type sequence. In particular we identified 20 differ- ent mutations in 45 subjects from 30 families, 23 polymorphisms, 11 of them were novel, and seven genetic variants of unknown pathological meaning never been reported previously. Discussion and conclusion: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a cere- brovascular disease caused by mutations in the NOTCH3 gene. Most CADASIL associated mutations result in a gain or loss of a cysteine residue in one of the 34 EGF-like repeats in the extracellular domain of the Notch3 protein, thus sparing the number of cysteine residues within a domain. To date, more than 130 different mutations in the NOTCH3 gene have been re- ported in CADASIL patients, the 95 % being missense point mutations. Many polymorphisms have also been identified in the NOTCH3 coding sequence some of them leading to amino acids substitutions. Here, we report a summary of 20 mutations, 23 polymorphisms and 7 new nucleotide variations in a cohort of 684 patients affected by leukoen- cephalopathy and we hope this NOTCH3 gene mutational analysis in a so significant number of unrelated and related patients may help in molecular screening for NOTCH3 gene and it may contribute to enlarge the NOTCH3 gene variations database.