Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries

Abstract

Objective: Genetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) and Becker muscular dystrophies (BMD), which are due to dystrophin (DMD) gene mutations, either for disease prevention or personalized therapies. To evaluate the ethnic-related genetic assortments of DMD mutations, which may impact on DMD genetic diagnosis pipelines, we studied 328 patients with DMD and BMD from non-European countries.

Methods: We performed a full DMD mutation detection in 328 patients from 10 Eastern European countries (Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia) and 2 non-European countries (Cyprus and Algeria). We used both conventional methods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specific sequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients where DMD mutations were already identified by standard techniques. WES output was also interrogated for DMD gene modifiers.

Results: We identified DMD gene mutations in 222 male patients. We identified a remarkable allele heterogeneity among different populations with a mutation landscape often country specific. We also showed that WES is effective for picking up all DMD deletions and small mutations and its adoption could allow a detection rate close to 90% of all occurring mutations. Gene modifiers haplotypes were identified with some ethnic-specific configurations.

Conclusions: Our data provide unreported mutation landscapes in different countries, suggesting that ethnicity may orient genetic diagnosis flowchart, which can be adjusted depending on the mutation type frequency, with impact in drug eligibility.

Figure 1. Distribution of DMD Mutations in Patients With DMD in Countries

Overview of mutation distribution in patients with DMD from Eastern Europe (A) and Algeria (B). Nonsense mutations were the most frequently occurring mutations in Eastern European patients, accounting for 31% of mutation types in the patients with DMD, followed by deletions (29%), frameshifting (18%), duplications (11%), and splicing canonical sites (9%); missense and consensus splicing are the least frequent (1% each). Deletions were the most frequent mutations in Algerian patients (77%), whereas nonsense (5%), frameshifting (5%), splicing canonical sites (5%), duplication (5%), and missense (3%) variations were the least frequent. No splicing consensus sequence mutations were identified in patients from Algeria. The reported numbers include known and novel, likely pathogenic, mutations/variations, but not VUS. Among the 28 females tested for carrier detection, we found 11 carriers, all heterozygous for large deletions (6) and small mutations (5). The remaining females were not carriers. The 2 patients from Cyprus were not included in the statistical analysis as 1 patient showed an exon 2 duplication, whereas the other resulted negative. DMD = Duchenne muscular dystrophy; VUS = variant of uncertain/unknown significance.

Figure 2. Country Distribution of DMD Mutations in Patients With DMD in Eastern European Countries

Geographical distribution of DMD mutations in Eastern European countries and relative number of patients (in brackets). The percentages for each type of DMD mutation are shown for each country. In countries with more than 30 patients with DMD carrying a DMD mutation, differences can be noted because Polish and Ukrainian patients have a similar mutation landscape with a very high frequency of small mutations (average of 55%), whereas in Romania, 31% of mutation types are small mutations with deletions, and 69% account for duplications. DMD = Duchenne muscular dystrophy.

Figure 3. Integrative Genome Viewer (IGV) Visualization

(A) IGV visualization of sample 2533/18. Visualization of sample 2533/18 with deletion of exon 51: (a) there is no coverage or readings of exon 51. (b and c) Visualization of coverage and reads of exons 50 and 52, which precede and follow the deletion of exon 51, respectively. (B) IGV visualization of sample 2526/18. Visualization of sample 2526/18 with deletion of exons 18–19. (a) This figure shows that there is no coverage or readings corresponding to exons 18 and 19. (b and c) Visualization of coverage and reads of the flanking exon 17 preceding the deletion and of exon 20 following the deletion.