New nucleotide sequence variants of the DYSF gene, identified by the next-generation sequencing

Background. According to the frequency of occurrences dysferlinopathy occupies the second place in world among limb–girdle muscular dystrophy (LGMD) after calpainopathy. The disease has a relatively late manifestation and the similar clinical picture with other LGMD. In some cases, that creates significant difficulties in differential diagnostics. New generation sequencing (NGS) is method that quickly and efficiently allows to determine the variant of the DYSF gene that leads to violation of protein synthesis.

Aim: to determine the effectiveness of identifying variants of nucleotide sequences in the DYSF gene by the NGS in patients with aclinical diagnosis of diferlinopathy and also to evaluate the possibilities of this method in differential diagnostics of LGMD.

Methods. The search for genetic variants of the DYSF gene was performed in patients with detected phenotype of LGMD, as well as 10 to 100 times higher levels of creatine kinase (CK) and manifestation at the age of 2-73 years. In total, 157 patients with a clinical signs of LGMD were included in the study. Changes in the sequence of the DYSF gene were detected in 27 of them. 9 of 27 people – 34(15-58)% were examined with Sanger sequencing. 18 of them – 67(42-85)% were examined by the NGS method. In two patients only one changed variant in the DYSF gene was detected, in this regard multiplex ligation-dependent probe amplification (MLPA) was performed to search for large deletions and (or) duplications. The identified genetic variants were verified by the reference method – PCR. Histological and immunohistological examination was performed in four patients with ambiguous results. As a biopsy material for examination were used fragments (5 mm3) of the lateral head of the quadriceps femoral muscle.

Results and conclusions. Using the NGS method, in 27 patients 21 genetic variant of the DYSF gene was identified. 16 variants out of 21 belong to the category of previously described; thus, in almost a quarter of cases (5) – 25(0-52)% variants weren’t described before (novel). Among the 21 identified variants, based on ACMG criteria, 12 – 57(31-81)% variants were classified as pathogenic, 5 – 25(0-51)% as probably pathogenic and 4 – 20(0-46)% as variants with uncertain significance, however, all detected variants were accompanied by a detailed clinical signs of LGMD.

1. Chernova O.N., Mavlikeev M.O., Zeynalova A.K., et al. Reparativnyy rabdomiogistogenez u myshey, mutantnykh po genu DYSF [Reparative rhabdomyogenesis in mice with DYSF mutation]. Geny i kletki [Genes and cells]. 2019;14(2):32-39. (In Russ.)

2. Tsargush V.A., Bardakov S.N., Bagnenko S.S. et. al. Magnitnorezonansnyy pattern izmeneniy myshts tazovogo poyasa i nizhnikh konechnostey u patsiyentov s disferlinopatiyami [MRI pattern changes in pelvic muscle and lower limb in patients with dysferlinopathy]. Luchevaya diagnostika i terapiya [Diagnostic radiology and radiotherapy]. 2020;11(1):93-105. (In Russ.)

3. Guglieri M., Magri F., D’Angelo M.G. et al. Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients. Hum Mutat. 2008;29(2):258–266.

4. Bardakov S.N., Emelin A.М., Nikitin S.S., et al. Prichiny lozhnoy diagnostiki polimiozita u patsiyentov s disferlinopatiyey: klinicheskiy sluchay [Reasons for misdiagnosis of polymyositis in patients with dysferlinopathy: a clinical case]. Nervno-myshechnyye bolezni [Neuromuscular Diseases]. 2022;12(4):73-87. (In Russ.)

5. Bushby K.M. Making sense of the limb-girdle muscular dystrophies. Brain. 1999;122(8):1403–1420.

6. Diers A., Carl M., Stoltenburg-Didinger G. et al. Painful enlargement of the calf muscles in limb girdle muscular dystrophy type 2B (LGMD2B) with a novel compound heterozygous mutation in DYSF. Neuromuscular Disorders. 2007;17(2):157–162.

7. Landrum M.J., Lee J.M., Benson M. et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 2018.

8. Vilchez J.J., Gallano P., Gallardo E. et al. Identification of a Novel Founder Mutation in the DYSF Gene Causing Clinical Variability in the Spanish Population. Archives of Neurology. 2005;62(8):1256.

9. Cagliani R., Fortunato F., Giorda R. et al. Molecular analysis of LGMD-2B and MM patients: identification of novel DYSF mutations and possible founder effect in the Italian population. Neuromuscular Disorders.2003;13(10):788–795.

10. Leshinsky-Silver E., Argov Z., Rozenboim L. et al. Dysferlinopathy in the Jews of the Caucasus: A frequent mutation in the dysferlin gene. Neuromuscular Disorders. 2007;17(11-12):950–954.

11. Illarioshkin S.N., Ivanova-Smolenskaya I.A., Greenberg C.R. et al. Identical dysferlin mutation in limb-girdle muscular dystrophy type 2b and distal myopathy. Neurology. 2000;55(12):1931–1933.

12. Konovalov F.A., Fedotov V.P., Umakhanova Z.R. et al. Molekulyarnaya diagnostika nasledstvennykh miopatiy metodom polnoekzomnogo sekvenirovaniya [Molecular diagnostics of hereditary myopathies by full-exome sequencing]. Meditsinskaya Genetika [Medical Genetics]. 2015;14(3):18-19. (In Russ.)

13. Ampleeva M., Tolmacheva E., Komarkov I. et al. NGS-based testing in diagnostics of hereditary neuro-muscular disorders: observations on a large cohort from a clinical bioinformatician’s perspective. Eur J Hum Genet. 2020. https://www.nature.com/articles/s41431-020-00739-z.

14. Sharkova I.V., Dadali E.L. Kliniko-geneticheskiye kharakteristiki i algoritm differentsial’noy diagnostiki progressiruyushchikh myshechnykh distrofiy, manifestiruyushchikh posle perioda normal’nogo motornogo razvitiya [Clinical and genetic characteristics and an algorithm for the differential diagnosis of progressive muscular dystrophies that manifest after a period of normal motor development]. Nervno-myshechnyye bolezni [Neuromuscular Diseases]. 2023;13(1):44-51. (In Russ.)

15. Shchagina O.A., Ryzhkova O.P., Chukhrova A.L., et al. Primeneniye ekzomnogo sekvenirovaniya dlya diagnostiki nasledstvennykh motornosensornykh neyropatiy Diagnostic utility of exome sequencing for inherited peripheral neuropathies. Nervno-myshechnyye bolezni Neuromuscular Diseases. 2020;10(4):12-26. (In Russ.)

16. Miller D.T., Lee K., Abul-Husn N.S., et al. ACMG SF v3.1 list for reporting of secondary findings in clinical exome and genome sequencing: A policy statement of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2022;24(7):1407-1414.

17. Harris E., Bladen C.L., Mayhew A. et al. The Clinical Outcome Study for dysferlinopathy: An international multicenter study. Neurol Genet. 2016;2(4):89.

18. Nguyen K., Bassez G., Bernard R. et al. Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies. Hum Mutat. 2005;26:165.

19. Nguyen K., Bassez G., Krahn M. et al. 2. Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of atypical phenotypes. Arch Neurol. 2007;64(8):1176–1182.

20. Aoki M., Liu J., Richard I. et al. Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy. Neurology. 2001;57(2):271-278.

21. Cagliani R., Magri F., Toscano A. et al. Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies. Hum Mutat. 2005;26(3):283.

22. Cacciottolo M., Numitone G., Aurino S. et al. Muscular dystrophy with marked Dysferlin deficiency is consistently caused by primary dysferlin gene mutations. European Journal of Human Genetics.2011;19(9):974–980.

23. Jin S.Q., Yu M., Zhang W. et al. Dysferlin Gene Mutation Spectrum in a Large Cohort of Chinese Patients with Dysferlinopathy. Chin Med J (Engl). 2016;129(19):2287-2293.