Infantile hypertonic myofibrillar myopathy (MFM) causes the rapid development of rigid muscles, respiratory insufficiency soon after birth, loss of myelinated fibers and the presence of giant axons with thin myelin sheets [1]. It was first reported in several Canadian patients and was predicted to be transmitted by autosomal recessive inheritance [2].

Del Bigio et al. [1] analyzed the DNA of eight Canadian patients with infantile hypertonic MFM. They observed a homozygous deletion c.60delC in the alpha B-crystallin ($\alpha$BC) expressing gene, CRYAB, in all patients. The mutation led to the loss of $\alpha$BC expression in muscle fibers. It resulted in patients’ pathological features, including endomysial fibrosis, different sizes of myofiber, homogeneous eosinophilic inclusions and peculiar powdery, granular transformation Z-bands [2]. It has also been demonstrated that heterozygous mutations of CRYAB (different from CRYAB mutations identified in infantile hypertonic MFM) are the cause of MFM2, a specific form of MFM [3, 4, 5]. MFM2 occurs at middle age (an average age ranged from 35 years to 45 years) and is characterized by distinct clinical features compared to the infantile hypertonic MFM [5, 6, 7].

Patients with MFM2 typically develop proximal and distal limb muscle weakness associated with neck, trunk and palatopharyngeal muscle involvement, pulmonary insufficiency associated with paresis of the diaphragm, weakness of the cervical, cardiomyopathy and cataracts can be associated features sometimes [5]. So far, about 19 heterozygous CRYAB mutations have been described in association with MFM2 [3, 4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. However, the infantile hypertonic MFM caused by homozygous CRYAB mutations is extremely rare. Since the first description c.60delC in 2011, only other two CRYAB mutations have been reported, including a female Caucasian infant with the homozygous mutation c.343delT and a male Chinese infant with the homozygous mutation, c.3G$>$A [20, 21]. We need more cases to further identify and understand the disease as well as improve the diagnosis.

This case report described infantile hypertonic MFM characteristics in four Chinese infants who developed the disease shortly after birth. The same homozygous mutation, c.3G$>$A in the CRYAB gene, was identified in all patients. This may be a critical hotspot mutation that contributes to the disease’s pathogenesis in Chinese infants.

Infantile hypertonic MFM (OMIM 613869) is characterized by the early-onset of severe respiratory distress and hypertonic myopathy, mainly involving the trunk and neck muscles. This was first reported in several Canadian patients, and the pathology was named infantile hypertonic dystrophy at that time [2]. Muscular dystrophy usually first manifests as a weakness in the limbs; however, these individuals first experienced weakness in the trunk muscles. Thus, this disease was re-defined as a new type of early-onset MFM, and researchers suggested that it was a different disorder than a typical MFM or muscular dystrophy. So far, only limited cases have been reported, and the clinical diagnosis strategy is still unclear [1, 2]. Here, we analyzed four patients’ clinical characteristics and summarized the disease features to improve the understanding of this disease and assist in diagnosing other cases.

The disease onset occurred at a few months old for all the infancy patients, which differs from typical MFM diseases since the onset usually occurred in adults [23]. All patients displayed a sudden onset of rigidity in their abdomen, neck, or back. In the early stage of the disease, we identified muscle lumps in 50% of patients, suggesting the fibrosis of affected myofiber. Additionally, we always observed abnormal sweating, which may be due to the energy consumption caused by muscle rigidity and neurological dysfunction. We also noticed an acute progression of respiratory distress, which was probably caused by the rapid progression of endomysial fibrosis, especially in the rectus abdominis, which mainly supports infants’ abdominal breathing [24]. The intercostal chest muscles that participate in the chest breathing of adolescents and elder children were also affected, which may also contribute to respiratory distress progression. The abnormal respiratory function affected the coughing and sputum excretion of patient 3. The pulmonary infection repeatedly occurred, leading to pulmonary hypertension and the right ventricular enlargement, which was detected by the electrocardiogram examination. What should be mentioned is that patients 2 and 4 showed an increase in anti-Acetylcholine (Ach) receptor antibodies in the serum. The elevated anti-Ach receptor antibodies were suspected to indicate MG [25].

However, the patient did not present with typical features of MG. So, the patient has not been diagnosed with MG; no MG treatments have been given to the patients. The anti-Ach receptor antibodies have been reported to be detected in patients with muscular disorders, including myotonic dystrophy [26]. This could be coincidental. The presence of Ach receptor antibodies reflects the breaking of immune tolerance to Ach receptors due to autoinflammation secondary to muscle fiber degeneration [27]. However, the relationship between anti-Ach receptor antibody and these diseases have not been reported. We still could not explain the presence of Abs in a genetically diagnosed muscular myopathy.

Z-disk-related proteins such as desmin, B-crystallin, myotilin, ZASP, filamin C, and Bag3 are functionally affected by mutations in the MFM associated genes [26, 27]. CRYAB knockout mice develop kyphosis and myopathies involving mostly the posterior tongue and axial muscles but rarely the extremities [28]. Multiple MFM cases with CRYAB mutations have been reported, and it was identified as the genetic cause of MFM in both adults and infants [1, 27].

The c.3G$>$A mutation in CRYAB was detected in all four patients. This mutation affects the initiation of $\alpha$-B crystal protein translation and causes a functional protein’s loss of expression. A previous study indicated that the absence of $\alpha$-B crystal protein or the presence of a mutant protein would increase the aggregation of desmin [29]. Our study showed a significantly increased accumulation of desmin and large aggregates in the patient’s rectus abdominis myocytes. Additionally, we observed a severely disturbed sarcomere structure with muscle vesicles using an EM. The smearing of Z-disk and autophagic vacuoles, together with the accumulation of cytoplasmic filaments, indicated a fibrosis degeneration with phagocytosis and necrosis [30]. These histological images all presented a specific morphological change of MFM, which confirms the pathogenicity of the c.3G$>$A mutation in CRYAB.

Mutations in the CRYAB gene are also responsible for other autosomal dominant inheritance associated with multisystemic diseases such as cardiomyopathy, cataract genesis, and myopathic features [5, 7, 31, 32]. We observed an enlargement of the right ventricle in one of our patients, suggesting a possible cardiac function involvement. This was probably due to cardiac ischemia leading to sustained resting cardio muscle tension, as shown in murine models [33, 34]. However, we did not observe any cataract diseases among the affected patients.

It is interesting that all Canadian patients carry the same homozygous CRYAB mutation (c.60delC), while we identified the mutation c.3G$>$A in our 4 patients, as well as in another reported case of a Chinese infant with similar clinical manifestations [21]. This indicates that the hot spot mutation c.3G$>$A may be enriched in the Chinese population. The five Chinese patients are from the south of China, so we also speculated that the c.3G$>$A mutation could be a founder mutation in this region. All the reported Chinese patients are male, which suggested males experiencing a more severe disease presentation and being thus more easily detected or less likely to be misdiagnosed. Further analyses, including more cases and complete family information, are required to validate these hypotheses.

Our study shows the homozygous mutation c.3G$>$A of CRYAB induced a fatal infantile hypertonic MFM. The patients were always born healthy but showed a disease onset early in infancy. In the early stage, the infants may present dysphagia, dyspnea, and hypertonia of the neck and trunk muscles mostly. The disease progressed rapidly, leading to irreversible respiratory failure, mild weakness of proximal limb muscles, vacuolization, muscle fiber necrosis, and poor prognosis. Muscle biopsies and genetic analysis could help in an earlier diagnosis. Then a better treatment might prevent the development of breathing difficulties and respiratory tract infections. We could also replace the defective gene in patients. Performing genome editing using various CRISPR/Cas-based therapeutics or providing a functional mRNA explicitly delivered to muscle cells provides an excellent potential for treating infantile hypertonic MFM in the foreseeable future [35, 36].

In this study, we reported 4 pediatric cases with symptoms similar to those of the fatal infantile hypertonic MFM with severe respiratory distress and a mutation in CRYAB reported in another Chinese patient [21]. Here, we speculate that the c.3G$>$A mutation is a possible hot spot mutation specific to the Chinese population. We also analyzed the clinical characteristics of our cases to improve the diagnosis of the disease. Further functional studies are required to confirm the clinical relevance of the c.3G$>$A mutation and the pathogenicity of the disease.

$\alpha$BC, Alpha B-crystallin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; CKMB, creatine kinase myocardial bound; CRD, complex repetitive discharges; EM, electron microscopy; EMG, Electromyogram; IHC, immunohistochemistry; MFM, myofibrillar myopathy; MFM2, alpha-B crystallin-related myofibrillar myopathy (myofibrillar myopathy-2); MRI, magnetic resonance imaging.

CH designed and supervised the project and reviewed the manuscript. XL summarized the patient’s information, reviewed the literature, and drafted the discussion of the manuscript. UY enrolled patients, arranged the clinical evaluation, and wrote the clinical portion of the manuscript. JM performed clinical tests. JL performed the biopsy staining and pathological assessment. YH analyzed the sequencing data, reviewed and revised the manuscript.

The Ethics Committee approved the project of Shenzhen Children’s hospital. All participants have provided written informed consent with the agreement to participate in this study. Informed consent was obtained.

We would like to thank the patients and their families for their cooperation. We are appreciated Running Gene Inc. for professional genetic sequencing. We thank LH for proofreading and editing the manuscript. We also want to thank colleagues in our department for their assistance.

This work was supported by the Shenzhen Science and Technology Innovation Commission funding project (ID: JCYJ20140416141331487), the Sanming Project of Medicine in Shenzhen (ID: SZSM201812005), Shenzhen Key Medical Discipline Construction Fund (No. SZXK033) and the Shenzhen Fund for Guangdong Provincial Highlevel Clinical Key Specialties (No. SZGSP012).

The authors declare that they have no competing interests.