Introduction

X-linked myotubular myopathy (XLMTM; MIM 310400) is a rare congenital muscular disorder that makes up the largest proportion of centronuclear myopathies (CNM) for the centrally located nuclei found in the muscle fibers [1]. The occurrence of XLMTM occurs 1 in 50,000 males annually, who tend to have the most severe phenotype [1]. Clinical manifestations are often in a homogenous pattern, arising early at birth and progresses rapidly with age. XLMTM is caused by the mutation in the MTM1 gene located on chromosome Xq28 [1].

The MTM1 gene is responsible for encoding the myotubularin protein, an endosomal phosphoinositide phosphatase that acts to dephosphorylate phosphoinositides, specifically key second messenger lipids PI3P and PI3, 5P2 [1]. Mutation in the MTM1 gene causes a deleterious effect in the myotubularin which fails to cause an electrical stimulus translation in the neuromuscular junction [2,3] therefore leading to calcium disregulation and muscle contractions and eventual abnormal trafficking of the effector proteins [2].

Currently, only a total of 8 genetically diagnosed MTM1 cases have been reported in the Chinese population abroad [47]. Classic phenotype of XLMTM include hypotonia, ventilator dependency, ophthalmoplegia, ptosis, undescended testes and arachnodactyly [8-13] whereas ventilator dependency and delayed gross motor milestones are prominent in severe cases [9]. To date at least 347 different mutations have been identified in the MTM1 gene, whereas missense/non-sense mutations made up of nearly half of the MTM1 mutations (http://www.hgmd.cf.ac.uk/ac/index. php2023.1). The variant c.1180dupG (p.Asp394GlyfsTer2) was searched for in published works and mutation databases such as ExAC(https://gnomad.broadinstitute.org/), Human Gene Mutation Database(http://www.hgmd.cf.ac.uk/ac/index.php) or 1000 Genomes（https://www.internationalgenome.org/data), accessed on Janurary, 2023 and to the best of our knowledge has not been mentioned so far. Herein, we report on a male neonate presented with neonatal hypotonia and persistent ventilator dependency whose whole exome gene test V6 (WES015) revealed a novel maternally inherited c.1180dupG (p.Asp394GlyfsTer2) MTM1 mutation.

Case Description

A male infant was delivered by cesarean section at 37 weeks to a non-consanguineous couple. Pregnancy complications included gestational hypertension, polyhydramnios and placental abruption. Family history for genetic disease was unremarkable. At birth, the infant was noted to have significant generalised hypotonia and weak respiratory response. He was incubated immediately and placed on mechanical ventilation. The Apgar scores were 7 and 8 at 1 and 5 minutes, respectively.

 Upon admission to the neonatal intensive care unit, his temperature, heart rate and blood pressure were normal. Respiration was 45 breaths/min (ventilation dependent). Birth weight was 2500g (below the 10th percentile), head circumference was 35cm

(above 90th percentile) and length was 49cm (50th percentile to 75th percentile). Physical features include cephalohematoma, sagged mouth due to the weak facial muscles, arthrogryposis and a single palmar crease found across the palm of the hand (Figure 1A). Auscultation of the lungs revealed bilateral moist rales. The heart sound was normal without any murmurs. The abdomen was soft and flat, the scrotum was small and the testes undescended. No spontaneous movements of limbs were observed.

On biochemical analysis, a complete blood count, electrolytes, alanine transaminase, asparate transaminase, blood urea nitrogen, creatinine, ammonia and lactate were all within the normal range. Chest X-ray showed thin ribs and clavicles with a collapsed lung encompassing 70% of the lung (Figure 1B). A chest tube was inserted and lung capacity returned to normal. Magnetic resonance imaging showed bilateral subdural hematoma. Ultrasonography of the heart was normal.

Figure 1: Clinical features of the patient. (A) Elongated face with decreased facial mass, involuntary opening of the mouth and small scrotum. (B) Chest radiography shows thin ribs.

The neonate struggled to wean of invasive mechanical ventilation due to the lack of spontaneous breathing even with low ventilation parameters. Limb movements and sucking reflex were unsatisfactory. Differential diagnosis included congenital muscular dystrophy, spinal muscular atrophy and Prader-Willi syndrome that were considered and excluded. In consideration of all the aforementioned manifestations, congenital muscular disease was highly suspected. Under the procedure authorized by the Institutional Review Board (registry number: LX20230305) of Shenzhen Luohu Maternal and Child Health Care Hospital, and informed consent obtained from the parents, DNA was extracted from the peripheral blood samples of both parents and infant. Whole exome gene test V6 (WES015) was performed. Whole genome sequencing in the proband revealed an inherited MTM1 mutation (NM_000252): c.1180dupG (p.Asp394GlyfsTer2) in both the mother and infant (Figure 2A).

Whole exome sequencing and data analysis for the patient and the patients were carried out in MyGenostics Inc, (Beijing, China). Genomic DNA was extracted using the DNA extraction kit (TIANGEN, Beijing, China) according to manufacturer’s instructions and was fragmented by Nanodrop 2000(Thermal Fisher Scientific, DE). A minimum of 3ug DNA was used for indexed Illumina libraries according to the manufacturers protocol (MyGenostics, Inc., Beijing, China). The sequence of MTM1 gene (NM_000252) was obtained from NCbigene database (https:// www.ncbi.nlm.nih.gov/). The DNA was fragmented and enriched for exon sequences using the Illumina NavoSeq6000 (Illumina, San Deigo, CA, USA) for paired end reads of 150bp. The average sequencing depth of the target region of exam sequencing was 200x. The variants with minor allele frequency (MAF) >1% in the Asian population and synonymous variants and were filtered out. Whole genome sequencing in the proband revealed a c.1180dupG (p.Asp394GlyfsTer2) mutation. Sanger sequencing confirmed the mutation (Figure 2A) in both the mother and infant.

Moreover, protein analysis revealed a duplication of base pair G at nucleotide 1180 of exon 11. The three-dimensional structure of wild-type and mutant (p.Asp394GlyfsTer2) of MTM1 was generated by the automated protein modeling server, Swiss-model (https:// swissmodel.expasy.org/interactive), and analyzed with PyMOL software. The change of aspartic acid by glycine at position 394 led to a stop codon at position 395(p.Asp394GlyfsTer2) resulting in a truncated protein (Figure 2B). In silico analysis by Mutation Taster confirmed the protein change. The mutation was searched for in published works and mutation databases such as ExAC, Human Gene Mutation Database or 1000 Genomes and to the best of our knowledge was not mentioned so far. A variant causing similar protein damage was found in an article published in 1997 [3], mentioning a similar frameshift mutation at the position 394 of the protein however was instead caused by an insertion mutation at the nucleotide 1233, instead of the nucleotide 1180 of exon 11 as such in our case. Soon after, the patient died from respiratory failure. Muscle biopsy and autopsy of the infant was denied by the parents.

Figure 2: Genetic findings in the XLMTM1 patient and parents (A) A hemizygous c.1180dupG mutation in the MTM1 gene was detected in the patient and his mother, as confirmed by Sanger sequencing. (B) The predicted myotubularin protein structure based on the crystal structure of the normal MTM1 protein and the truncated protein due to c.1180dupG mutation.

Discussion and Conclusions

Majority of MTM variants have a familial cause [2]. Mode of inheritance include autosomal dominant, autosomal recessive and X-linked recessive forms, the last being the most severe case [5] and as such affected males are more likely to exhibit a more severe phenotype. Of the 8 genetic-diagnosed MTM1 cases reported in the Chinese population, 2 were female and had a milder phenotype [4-6]. 5 male patients all had respiratory distress at birth, whereas absent in female carriers. On the other hand, the female carriers had prominent motor development delay and asymmetric facial and limb muscle weakness [4-7]. A study of 31 XLMTM male patients indicated that 94% had respiratory distress at birth, and 45% had a long and narrow face that were found consistent with our patient [14]. In addition, a study conducted with 116 patients included 67 patients with truncating mutation, 89% had a severe phenotype requiring respiratory support or found deceased [15].