# X-Linked Myotubular Myopathy in a Female Patient with a Pathogenic Variant in the _Mtm1_ Gene
## 2 General Characteristics of the Family and Examination Methods  
We examined a nonconsanguineous family from Moscow Oblast, Russian Federation (family tree is presented in Figure 2A). The proband was examined in the counselling unit of the Research Centre for Medical Genetics at the age of 32 years. The proband's medical documents and biological materials (blood, fibroblast, and buccal epithelium samples), as well as blood samples of the proband's daughter, parents, sister, and brother, were provided (it was not possible to obtain a biopsy of the muscle tissue of the proband due to the complexity of the material sampling procedure).  
Molecular genetic analysis was carried out on samples of genomic DNA extracted from peripheral blood leukocytes, buccal epithelium cells, and fibroblasts, as well as on samples of RNA extracted from fresh peripheral blood leukocytes. Molecular genetic analysis was carried out using mass parallel sequencing (MPS) on a next-generation Ion S5(tm) sequencer (Thermo Scientific, Waltham, MA, USA). The probes were prepared using ultramultiplex PCR, followed by sequencing (AmpliSeq(tm)). We used the \"congenital muscular dystrophy\" target panel (developed based on the Ion Ampliseq technology) including coding sequences of the following genes: _SELENON_, _LMNA_, _TPM3_, _ACTA1_, _PLOD1_, _MYPN_, _ITGA7_, _STAC3_, _CNTN1_, _MYF6_, _CFL2_, _KBTBD13_, _TRIP4_, _CHST14_, _CCDC78_, _MYH2_, _KKRP_, _TNNTT1_, _RYR1_, _DNM2_, _COL6A3_, _KLHL41_, _BIN1_, _ZAK_, _SPEG_, _COL5A2_, _COL6A1_, _COL6A2_, _CHKB_, _ITGA9_, _KLHL40_, _LMOD3_, _MTMR14_, _MEGF10_, _LAMA2_, _COL12A1_, _DSE_, _FKBP14_, _AEBP1_, _TPM2_, _FKTN_, _COL5A1_, _MTM1_, and _VMA21_. The read depth of the detected variant in samples of DNA extracted from blood leukocytes exceeded \(\times 164\). The detected variant was verified by direct automated Sanger sequencing. The variants were analyzed using the hg19 genomic assembly, data from HGMD Professional v2022.1 [5], and the data interpretation guidelines [10]. The DNA samples were genotyped with PCR using the \(\kappa\)AmpFISTR Identifier Direct PCR Amplification Kit\(\ast\) (Applied Biosystems) for direct amplification of 16 loci of  
Figure 1: A schematic representation of _MTM1_ exons encoding myotubularin domains.  
human DNA according to the manufacturer's protocol, followed by amplification product separation on a 3130 xl Genetic Analyzer (Hitachi, Ibaraki, Japan). The ratio of inactivated X chromosomes was established by analyzing the methylation of the _AR_ promotor and _AR_ (gene located on the X chromosome) trinucleotide repeat quantity detection using specific primers GCTGTGAAAGGTTGTCTGTTCCTAT/AGGCAGCACCTCCGGGC. The X chromosome haplotypes were determined using the Investigator(r) Argus X-12 QS (Qiagen, Hilden, Germany) kit with subsequent amplification product separation on a 3130 xl Genetic Analyzer (Hitachi). The canonical transcript modification was demonstrated using mRNA analysis. The RNA was extracted from the proband's peripheral blood leucocytes using the QIAamp(r) RNA Blood Mini Kit (Qiagen). The quality of total extracted RNA was evaluated using agarose gel electrophoresis and spectrophotometric detection of the 260/280 ratio during the measurement of total RNA concentration. The reverse transcription of total RNA was carried out using the Quantifect Reverse Transcription Kit (Qiagen). Standard PCR for the proband's cDNA samples, as well as control samples of healthy people's cDNA, was carried out using specific primers GAACATATCAAGCTCTTTTGAC/GTCAAACAGTTCCCTACAGC.  
The detection of the pathogenic variant in the _XIST_ gene was carried out using direct automated Sanger sequencing on the 3130 xl Genetic Analyzer (Hitachi). Standard PCR for genomic DNA samples of the proband her sister was carried out using specific primers GTTCTTCTGATGCTTTGCTTTC/GTATCGGATACCTGATTCCC.