Identificazione e caratterizzazione dei modificatori genetici della DMD

Background: Despite promising advances made in the past 30 years owing to the identification of the molecular bases of Duchenne muscular dystrophy (DMD), several obstacles remain for a full translation of novel therapies into clinical practice, and DMD patients and their families must still cope with severe disability and grim perspectives for the future. One of the most challenging hurdles to the success of clinical trials is the considerable inter-patient variability in terms of age at presentation, weakness progression, and degree of involvement of the central nervous system (CNS) observed in DMD, that is, the relevant phenotypic variability of DMD. The genetic background, that is, variations in genes different from disease genes, is increasingly believed to modulate the phenotype of Mendelian diseases. These trans-acting variants are called genetic modifiers. In this thesis, we aimed to deepen current understanding of these mechanisms. Therefore, the following aims were formulated. Aims: • In this project, we aimed to perform a genome-wide association study (GWAS) to search for DMD modifier loci, leveraging on clinical data and DNA samples from a large cohort of DMD patients followed by the Consortium of Italian Centers, which has collaborated to study DMD natural history over the past decade. In this thesis we present preliminary data on 265 DMD patients, but the final GWAS (still in progress, delayed by the Covid-19 pandemic) will include 700+ patients. • We also aimed to identify candidate variants involved in the modulation of the CNS phenotype in dystrophinopathies, by performing whole genome sequencing (WGS) in a pair of DMD siblings with discordant cognitive phenotypes and by filtering variants using a dedicated bioinformatic algorithm. • Lastly, we aimed to verify if known genetic modifiers of loss of ambulation (LoA) in DMD, both cis and trans acting, also affect the Performance of the Upper Limbs measured with the PUL test. Moreover, all associations were tested for validation in an independent cohort (i.e. Cooperative International Neuromuscular Research Group Duchenne Natural history Study, CINRG-DNHS) in which patients had been tested using the Brooke scale for upper limb function. Results: • We identified a list of SNPs with a suggestive p value, revealing the presence of a candidate locus in chromosome 1, at a distance of ~3,800 bp upstream of the C1orf21 locus, whose functional meaning needs to be further elucidated. • Regarding WGS in a pair of DMD siblings with discordant cognitive phenotypes, we focused our attention on two SNPs, and a deletion resulting from the breakdancer analysis. All of these variants reside within in a risk-genes for autism, and are co-expressed with dystrophin. The first one is a single SNP upstream of ANK3, and the second an intronic variant in NRXN3, encoding the dystrophin-associated glycoprotein neurexin 3. Both of these variants were previously identified in a WGS study of ASD twins. In our family, the younger brother (suffering from ASD) was heterozygous for both of these variants, while the elder was homozygous for the wild-type allele at both loci. • Finally, significant associations were observed between additive CD40 rs1883832 genotype and shoulder/distal PUL subscores, with a trend of association in the total score. Additive ACTN3 rs1815739 genotype was also significantly correlated with elbow and distal subscores Moreover, it was possible to assess a significant association of CD40 rs1883832 and SPP1 rs28357094 with the Brooke score in the CINRG-DNHS cohort.