# X-Linked Myotubular Myopathy in a Female Patient with a Pathogenic Variant in the _Mtm1_ Gene
## 3 Results
### Molecular Genetic Analysis  
We analyzed the genes included in the \"congenital muscular dystrophy\" target panel, which also includes congenital myopathy genes, using MPS. The analysis detected a heterozygous c.1261-10A\(>\)G nucleotide sequence variant, previously described as pathogenic [11; 12; 13], in intron 11 of the _MTM1_ gene (NM_000252) (Figure 3A). Figure 3: (**A**). The result of the MPS of genomic DNA extracted from peripheral blood leukocytes of the proband. A replacement in the 11 th intron of the _MTM1_ gene is presented. (**B**). The result obtained in the study of intron 11 of the _MTM1_ gene by [PERSON] sequencing in genomic DNA extracted from peripheral blood leukocytes for: (**a**) the proband, (**b**) the proband’s mother, (**c**) the proband’s father, and (**d**) the proband’s daughter. The c.1261-10A\(>\)G nucleotide sequence variant was detected in the proband and her daughter in a heterozygous state. According to the literature data and the Splice AI prediction program, the detected variant leads to a disruption in the acceptor splice site of exon 12 and causes the activation of the cryptic site, which leads to a nine-nucleotide elongation of exon 12 [11], thus adding three extra amino acids on the protein level. The read depth of this point was \(\times\)164. After that, we searched for the detected variant in genomic DNA samples (extracted from peripheral blood leukocytes) of the proband's parents, brother, sister, and daughter using Sanger sequencing. The patient's daughter has the pathogenic c.1261-10A\(\times\)G variant in intron 11 of the _MTM1_ gene (NM_000252) in a heterozygous state. This variant was not detected in the proband's parents and siblings. The relationship establishment showed both parents to be true biological parents; therefore, the nucleotide sequence variant detected in the proband is de novo (Figure 3B). After that, we determined the methylation pattern in the (CAG)n repeat region in exon 1 of the \(AR\) gene in samples of genomic DNA (extracted from peripheral blood leukocytes) of the proband, her mother, sister, and daughter, as well as in samples of genomic DNA extracted from the proband's buccal epithelium cells and fibroblasts. The analysis of genomic DNA extracted from peripheral blood leukocytes showed the skewed X chromosome inactivation in the proband and her sister (proband--96%: 4%, (CAG)n repeats 19/24; the proband's sister--80%: 20%; (CAG)n repeats 19/24) (Figure 4). Figure 4: (**A**). A schematic representation of the X chromosome with locations of markers included in the Investigator® Argus X-12 QS kit. (**B**). A schematic representation of the X chromosome haplotypes (genomic DNA isolated from peripheral blood lymphocytes was studied). The proband's father has 24 CAG repeats in exon 1 of the \(AR\) gene, while the mother has 19 (highlighted in red). The proband’s maternal X chromosome containing the allele with 19 repeats is inactivated by 96%, while the paternal chromosome containing the allele with 24 repeats is inactivated by 4% and is therefore active. The proband’s sister has the 19-repeat chromosome inactivated by 80%, and the 24-repeat by 20%. The proband’s daughter did not have skewed inactivation. Colors indicate the X chromosome haplotypes in the family. As shown by the presented data, the _MTM1_ gene is located below the DXS10134 marker, and as a result of crossover, the X chromosomes exchanged the loci containing the _MTM1_ gene. The proband's daughter did not have skewed X chromosome inactivation (40%: 60%, (CAG)n repeats 18/19). The analysis of the proband's genomic DNA extracted from buccal epithelium cells and fibroblasts also showed skewed X chromosome inactivation (90%: 10%, (CAG)n repeats 19/24 and 99%: 1%, (CAG)n repeats 19/24, respectively). It was impossible to evaluate the X chromosome inactivation ratio in the proband's mother because of the presence of CAG repeats in a homozygous state ((CAG)n 19/19).