A Common CHAT Gene Mutation of Congenital Myasthenic Syndrome Found in Kadazandusun Children

Abstract

Congenital myasthenic syndrome (CMS) is an uncommon inherited neuromuscular junction disease. The clinical presentation of this disorder is diverse. Typically patients with this disorder present with early-onset swallowing difficulty and apnea in infancy, fluctuating ocular palsies and fatigable proximal muscle weakness during childhood, and late-onset form involving progressive weakness in adulthood. Difficulty in performing neurophysiology studies in children and the absence of a pathognomonic investigation marker increase the challenges in diagnosis of this disorder. The emergence of next-generation sequencing technology has circumvented these challenges somewhat, and has contributed to the discovery of novel mutations. We present here diagnostic odyssey of three CMS patients from two unrelated Kadazandusun kinships and their follow-up treatment. A rare homozygous mutation c.916G > C (p.Val306Leu) in CHAT gene was found in two siblings born of a consanguineous marriage. Third patient had compound heterozygous mutations c.406G > A (p.Val136Met) and c.916G > C (p.Val306Leu) in CHAT gene. We postulate that p.Val306Leu may be a founder mutation in the Kadazandusuns, an indigenous ethnic minority of Borneo Island.

Introduction

Congenital myasthenic syndrome (CMS) is an inherited neuromuscular junction disorder, characterized by muscle weakness and fatigability. There is a bewildering array of phenotypes, in which the manifestation depends on the underlying genetic mutation affecting neuromuscular transmission. Patients may present with arthrogryposis at birth; hypotonia, feeding difficulty, and episodic apnea in the neonatal period; and ptosis, ophthalmoplegia, sudden respiratory distress, fatigability, or limb girdle weakness during childhood or adolescence. At present, CMS is still considered an orphan disease, with an estimated prevalence of 25:1,000,000 to 123:1,000,000 ¹ ; however, these figures could be underestimated.

Background

CMS is classified into presynaptic, synaptic, and postsynaptic types according to the location of the protein defects. Presynaptic CMS is the rarest type, comprising only 6 to 7% of CMS. ² To date, 8 protein defects have been identified to be associated with presynaptic CMS. ³ Of these, choline acetyltransferase (ChAT) deficiency is the most common one. CHAT gene encodes ChAT; a defective ChAT results in deficient resynthesis of acetylcholine following its reuptake at the nerve terminal. Forty-eight CHAT gene mutations have been identified for causing presynaptic CMS. ⁴

Clinical Manifestation

CHAT- CMS ( CHAT mutations-associated CMS) typically present with episodic apnea in infancy and early childhood. Common triggers could be due to trivial respiratory infection, emotion, or, at times, without any obvious reasons. As a result, patients are at risk of hypoxic brain injury if the acute life-threatening event is not recognized and responded promptly. Despite this clinical feature, apneic spell is not a pathognomonic for CHAT -CMS, as other subtypes, such as sodium-channel myasthenia and RAPSYN-related mutations, could present with a similar manifestation. ⁵

Diagnosis

Neurophysiologic tests, particularly repetitive nerve stimulation and single fiber electromyography are commonly performed to elucidate neuromuscular junction disorder. ^{5 6} However, performing these tests is challenging in young children due to discomfort and the length of the procedures. In addition, anti-acetylcholine receptor (anti-AChR) antibody is known to be negative in this context as CMS is not an autoimmune disease. As such, it makes difficult to diagnose CMS in children.

In the past few decades, the diagnostic odyssey of CMS has been circumvented with the availability of genomic testing. The knowledge of molecular medicine has helped in understanding the disease and this in turn provides a better genotype–phenotype correlation in this disease. ⁷

Case Reports

We describe two kinships with a background of Kadazandusun sharing a common CHAT gene mutation.

Family 1

The proband V-2, is the second child of a Kadazandusun family born to a consanguineous parents ( Fig. 1 ). He had a normal birth and development till 1 year. At the age of 1 year old, he was diagnosed with “bronchial asthma” after an episode of “pneumonia” requiring invasive mechanical ventilation. At the age of 18 months, he was noted to have muscle weakness and ptosis. These were intermittent but more pronounced in cold environment and at the end of the day. Despite compliance with prophylactic inhaled glucocorticoid, he had another admission at 3 years old for respiratory distress requiring assisted ventilation. His first neurogenetic examination was at 4 years old. Physical examination at that time revealed normal neurology, in particular tone, reflexes, and power were normal and no ptosis or ophthalmoplegia were observed at rest. However, proximal muscle power was diminished and ptosis appeared after some exertion (running around the clinic). Investigations such as metabolic blood and urine workup, forearm ischemic lactate-ammonia test, nerve conduction study, and repetitive nerve stimulation tests were normal. CMS was suspected and a trial of pyridostigmine and salbutamol led to improved muscle strength. At the age of 11 years old, the underlying molecular defect was uncovered by whole exome sequencing (WES). WES revealed a homozygous mutation in CHAT gene c.916G > C (p.Val306Leu). At his current age of 15 years old, his weakness and ptosis are appreciable in the afternoons and evenings or after school, despite taking pyridostigmine and salbutamol. He does not require prophylactic inhaled glucocorticoid and has not experienced difficulty in breathing.

Fig. 1.

Pedigree of family 1 showing two siblings (V2 and V6) with homozygous mutation, c.916G > C(p.Val306Leu).

His younger brother, V-6, was brought to medical attention when he was 2.5 years old because of similar symptoms. He started having intermittent bilateral ptosis at 9 months old. At the age of 13 months, he was admitted for a viral respiratory tract infection. In the ward, he struggled while receiving nebulization and had apnea. He required invasive ventilation. Subsequently, he had two further apneic attacks with intravenous line insertions and required bag-valve-mask ventilation.

Since the provisional diagnosis of his elder brother was CMS at this juncture, he was also investigated for the same disorder. His molecular analysis also revealed the same CHAT homozygous mutation as his brother. Pyridostigmine was prescribed and oral salbutamol was added as an adjunctive therapy later. He has never developed any apneic episode or respiratory insufficiency since. Compared with the index case, he has noticeable ptosis earlier in the day and effort intolerance while on treatment.

Family 2

The third patient in this report is a 6-year-old girl, the second child of mixed Chinese and Kadazandusun parentage ( Fig. 2 ). She had normal birth and development. She was admitted three times for cyanosis without apparent precipitating cause at the age of 3, 6, and 17 months, respectively. During her third admission, she required prolonged ventilatory support and was noticed to have ptosis. Retrospective history revealed that the ptosis has been present since her first birthday.

Fig. 2.

Pedigree of family 2 showing II2 with compound heterozygous mutation, c.916G > C(p.Val306Leu) and c.406G > A(p.Val136Met, from her mother and father, respectively.

Among the series of investigations, she had a positive Tensilon test and anti-AChR antibody (0.64 nmol/L; normal range < 0.5 nmol/L). A clinical diagnosis of myasthenia gravis was made initially and she received a course of immunoglobulin and prednisolone, as well as commencement of pyridostigmine. With this treatment, her respiratory status stabilized and she was successfully extubated.

She had a 4th admission at the age of 21 months for respiratory depression secondary to viral pneumonia. She escaped invasive mechanical ventilation with increment of her pyridostigmine dose from 15 mg three times daily to 30 mg five times a day. A repeat anti-AChR antibody was positive at > 8.4 nmol/L.

She was referred to us for further investigations and treatment of myasthenia gravis at the age of 2 years. In view of the early onset of symptoms, history of episodic apnea and ptosis, a differential diagnosis of CMS was considered. A CMS gene panel test of 22 genes uncovered compound heterozygous mutation in CHAT gene, c.406G > A (p.Val136Met) and c.916G > C(p.Val306Leu). Parental testing revealed that c.406G > A(p.Val136Met) was inherited from her Chinese father and c.916G > C (p.Val306Leu) was inherited from her Kadazandusun mother. At present, she has no apneic spell with pyridostigmine, but she gets tired quickly if she has to walk for a long distance.

Discussion

In this report, we present the diagnostic course of three CMS patients with recurrent apneic spells in their early lives, intermittent ptosis, and proximal weakness. The diagnosis of the first patient in family 1 was confirmed 10 years after his initial presentation. Although a neuromuscular diagnosis was suspected, the repetitive nerve stimulation was not supportive of CMS. This reiterates the current knowledge that patients with mild to moderate ChAT deficiency may have normal repetitive nerve stimulation findings, especially between episodes of exacerbation because of adequate amount of acetylcholine in the nerve terminal. ⁸ The test outcome may become positive after 5 minutes of stimulation at 10 Hz but this is often not tolerated by young children. Similarly, single fiber electromyography was not pursued further because of the technical difficulty in this age group.

In the same thread, anti-AChR antibody is a common test in investigating neuromuscular diseases and expected to be negative in CMS. Nevertheless, this was repeatedly positive in our patient in family 2. This has been reported in literature, ^{9 10} albeit uncommonly, and the exact underlying mechanism remains unknown. In addition to inconsistent investigative findings, the unique symptom of weakness exacerbated by cold is often not picked up or associated with CMS. This clinical feature has been reported by Maselli et al, who explained that cold temperature reduces the level of acetylcholine formed from the mutant ChAT protein which leads to poor synaptic neurotransmission. ¹¹ All these, added to the rarity of CMS, present as barriers and result in delayed diagnosis.

The high cost of molecular testing like gene panel testing and WES presents a challenge in our resource-limited setting. Nevertheless, it is important to do genetic testing to confirm clinically suspected CMS cases for accurate treatment and genetic counseling. The CHAT mutation c.406G > A (p.Val136Met) present in family 2 is a reported known pathogenic variant. ¹² On the other hand, c.916G > C (p.Val306Leu) mutation present in unrelated Kadazandusun individuals of families 1 and 2 is classified as a likely pathogenic variant ( Fig. 3 ). p.Val306Leu mutation replaces the highly conserved valine residue with leucine and is predicted to be pathogenic by the in silico prediction software, Sorting Intolerant From Tolerant and PolyPhen-2. This mutation has been reported previously in one other Kadazandusun family. ¹³ Therefore, c.916G > C(p.Val306Leu) mutation is likely to be causing CMS in these patients and we postulate that it may be a founder mutation in the Kadazandusuns.

Fig. 3.

Schematic diagram of the CHAT gene (upper) and protein (lower) with localization of mutations. The c.406G > A(p.Val136Met) and c.916G > C(p.Val306Leu) mutations are located in exon 4 and 8, respectively. Both variants locate within the CoA-dependent acyltransferase domain, which includes amino acids from position 128 to 508. Consequently, these variants might have impaired the function of acetyl coenzyme-dependent acyltransferase domain in our patients. Exons (orange box), introns (blue line), and protein domain were not drawn precisely to scale.

In conclusion, the diagnosis of CMS can be challenging. High index of suspicion, early diagnosis, and treatment are essential to minimize the mortality and morbidity. If available, gene panel testing reduces the diagnostic odyssey and has a clear advantage over other more invasive and nonspecific investigations. Molecular confirmation of CMS in these two families will help in genetic counseling with regards to recurrence risk in subsequent pregnancy and option for prenatal testing.

Funding Statement

Funding None.