Dystrophinopathies are a group of X-linked recessive neuromuscular disorders due to mutations in the DMD gene. Truncating mutations, causing dystrophin absence in skeletal and cardiac muscle, cause the more severe form of dystrophinopathy, Duchenne muscular dystrophy (DMD). Conversely, mutations which respect the open reading frame, and give rise to quantitatively or qualitatively altered dystrophin, cause the milder allelic variant known as Becker muscular dystrophy (BMD).
DMD is a devastating disorder. Progressive muscle wasting and weakness causes disability since childhood, and the natural history is characterized by loss of independent ambulation (LoA) around 10 - 15 years of age, and reduced life expectancy because of respiratory and cardiac complications in young adults. Glucocorticoid corticosteroids (GCs) might delay disease progression, and there are promising novel molecular treatments, but a definitive cure remains elusive. Promising molecular treatments include antisense oligonucleotides (AONs) inducing exon skipping in out-of-frame deletions and premature stop codon readthrough compounds.
There is relevant variability in the severity and rate of progression of muscle wasting and weakness in DMD, which is not explained, if not in a minor proportion, by the disease-causing mutation, as all DMD patients have a complete, or near to complete dystrophin defect. Recently, our group and other authors have described genetic modifiers of DMD, i.e. common single nucleotide polymorphisms (SNPs) associated to more or less severe DMD expressivity. These include rs28357094, a SNP in the promoter of the SPP1 gene, enconding the cytokine osteopontin, and a coding haplotype in the LTBP4 gene, enconding Latent Transforming growth factor β-Binding Protein 4. These variants modulate the expression (SPP1), or alter the aminoacid sequence (for LTBP4) of corresponding proteins, both of which are involved in inflammatory and pro-fibrotic pathways. They were both identified by candidate gene approaches (respectively, expression profiling studies and a murine genome scan).
The first aim of this thesis was to provide an independent validation of the genetic association of the SPP1 rs28357094 SNP and the LTBP4 haplotype with age at LoA in DMD. This was achieved using data from the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS) of 340 DMD patients from 20 worldwide Centers. In this population, the minor allele G at rs28357094 was associated to a 2-year delay of LoA in CINRG-DNHS participants who had been treated with glucocorticoids (p < 0.05), and no significant effect in untreated patients, suggesting that the SNP might be a pharmacodynamic biomarker of GC response. Furthermore, the homozygous LTBP4 haplotype “IAAM” was associated to a 2-year delay of LoA in participants of European descent (p < 0.05), but not in the whole multi-ethinc CINRG-DNHS cohort, highlighting the relevance of population stratification in genetic modifier studies.
The second aim was to test for associations between specific DMD mutations and age at LoA in the CINRG-DNHS. We confirmed previous reports that deletions bordering exon 44 (and thus amenable to AON treatment for skipping of this exon), as well as the deletion of exons 3-7, were associated to later LoA (p < 0.01 and < 0.05 respectively). These findings have repercussions on clinical trial design and prognosis.
A third aim was to study age at LoA as a long-term outcome of several different GC regimens currently adopted in DMD. In this observational study, we found that the use of daily deflazacort was associated to 2.7-year later LoA than daily prednisone (p < 0.001), an unexpected finding that may be confirmed by ongoing randomized trials.
As a fourth aim, we genotyped 175/340 CINRG-DNHS participants with an Exome Chip, including thousands of functional (regulatory or coding) variants, and performed a genome-wide association study (GWAS) of age at LoA in a subgroup of 109 unrelated participants of European ancestry. While no SNP surpassed the Bonferroni-corrected significance threshold, we performed a hypothesis-driven prioritization of findings, focused on inflammatory and pro-fibrotic pathways, and identified a hit in a gene involved in inflammation and cell-mediated immunity. The GWAS association of earlier LoA with the minor allele at the identified locus (p < 9.9*10-5) was validated in a collaborative cohort of 660 DMD patients from the University of Padova, the European Bio-NMD network, and the United Dystrophinopathy Project in the USA (p < 0.05).
Finally, the fifth aim focused on BMD, which features a milder, but even more variable clinical picture than DMD. In BMD, most patients have in-frame deletions leading to internally deleted dystrophin protein. We quantified dystrophin by Western Blot, performed a retrospective study of LoA and loss of the ability to run, and a 1-year longitudinal study of motor function (6 Minute Walk Test [6MWT], North Star Ambulatory Assessment [NSAA], timed function tests [TFTs]) in 69 BMD patients at the University of Padova. We found that deletions bordering exon 45 were associated with frequent loss of the ability to run, risk of LoA in adults, and overt muscle weakness; while some deletion groups, like those bordering exon 51, or limited to exon 48, were preserved from these signs of disease progression. This is relevant not only to BMD prognosis and genetic counseling, but also to outcomes of exon skipping AON treatments which aim to reproduce the same deletions at the transcript level in DMD. Furthermore, we observed that NSAA and 6MWT, which we mutuated from DMD studies and had not been applied to BMD, were feasible, clinically meaningful, and able to identify disease progression at 1 year, suggesting their adequacy as outcome measure for future BMD clinical trials.
Altogether, the work presented here provides novel insights into the mechanisms of phenotypic and clinical variability in dystrophinopathy, which will be useful in delivering improved care for these disabling diseases
