Megaconial congenital muscular dystrophy due to novel CHKB variants: a case report and literature review

Abstract

Background: Choline kinase beta (CHKB) catalyzes the first step in the de novo biosynthesis of phosphatidyl choline and phosphatidylethanolamine via the Kennedy pathway. Derangement of this pathway might also influence the homeostasis of mitochondrial membranes. Autosomal recessive CHKB mutations cause a rare form of congenital muscular dystrophy known as megaconial congenital muscular dystrophy (MCMD).

Case presentation: We describe a novel proband presenting MCMD due to unpublished CHKB mutations. The patient is a 6-year-old boy who came to our attention for cognitive impairment and slowly progressive muscular weakness. He was the first son of non-consanguineous healthy parents from Sri Lanka. Neurological examination showed proximal weakness at four limbs, weak osteotendinous reflexes, Gowers' maneuver, and waddling gate. Creatine kinase levels were mildly increased. EMG and brain MRI were normal. Left quadriceps skeletal muscle biopsy showed a myopathic pattern with nuclear centralizations and connective tissue increase. Histological and histochemical staining suggested subsarcolemmal localization and dimensional increase of mitochondria. Ultrastructural analysis confirmed the presence of enlarged ("megaconial") mitochondria. Direct sequencing of CHKB identified two novel defects: the c.1060G > C (p.Gly354Arg) substitution and the c.448-56_29del intronic deletion, segregating from father and mother, respectively. Subcloning of RT-PCR amplicons from patient's muscle RNA showed that c.448-56_29del results in the partial retention (14 nucleotides) of intron 3, altering physiological splicing and transcript stability. Biochemical studies showed reduced levels of the mitochondrial fission factor DRP1 and the severe impairment of mitochondrial respiratory chain activity in patient's muscle compared to controls.

Conclusions: This report expands the molecular findings associated with MCMD and confirms the importance of considering CHKB variants in the differential diagnosis of patients presenting with muscular dystrophy and mental retardation. The clinical outcome of MCMD patients seems to be influenced by CHKB molecular defects. Histological and ultrastructural examination of muscle biopsy directed molecular studies and allowed the identification and characterization of an intronic mutation, usually escaping standard molecular testing.

Keywords: Choline kinase beta (CHKB); Enlarged mitochondria; Megaconial congenital muscular dystrophy; Mitochondrial dynamics.

Fig. 1

Histological, immunohistochemical, and ultrastructural studies in patient’s muscle. A Light microscopy observations of MTG showed moderately increased variation in fiber size with a consistent number of hypotrophic fibers and the presence of abnormal subsarcolemmal granules in several fibers. B Staining for COX activity shows the presence of large mitochondria at the periphery of fibers and central areas lacking any activity (* indicates lack of COX activity due to mitochondrial depletion in those areas/fibers). Immunofluorescence staining for p62 (C) and LC3A (D) was positive in some fibers. Caveolin-3 was used for membrane staining and nuclei were counterstained with DAPI. E–H Representative images of the main alterations in skeletal muscle and in mitochondria morphology. E Minor abnormalities of the of Z-line (indicated by an arrow), mitochondrial alterations including giant mitochondria (F), mitochondria with globular inclusions (G), and swollen mitochondria (H). Scale bars: A, 400 µm; B–D, 250 µm, E–F 2.27 µm; G, 0.83 µm; H, 1.43 µm

Fig. 2

Genetic and biochemical findings. A Pedigree of the family investigated. Electropherogram showing the CHKB nucleotide substitutions detected in our patient and segregation analysis in his unaffected parents. B Transcript analysis of CHKB in patient’s muscle. Electropherograms show the sequences of RT-PCR amplicons obtained before (left) and after (right) subcloning. Subcloning experiment confirmed the partial intronic retention in CHKB ORF in patient’s muscle. C Sequence electropherograms of the intronic region between CHKB exons 3 and 4 and subcloning experiments confirming the the presence of a small deletion. D Western blot analysis of Mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) protein levels in muscle samples from patient and controls. Actin levels were used as reference. E Graph showing respiratory chain enzymatic activities in Patient’s muscle compared to control levels assessed through spectrophotometric analysis (experiment in duplicate). Values are normalized to citrate synthetase (CS) activity and expressed as pmol/min/mg protein. F Western blot analysis of representative subunits of mitochondrial respiratory chain. No difference was observed between patients’ and control muscle samples. Actina and porin (VDAC) levels were used as reference

Fig. 3

Genotype–phenotype correlation in CHKB-mutated patients. A Scheme of CHKB gene (above) and the encoded choline kinase beta (bottom). Mutations identified in MCMD patients are indicated in red (group 1: non-sense/frameshift mutations), blue (group 2: missense/in-frame mutations) or black (mutations disclosed in our proband). B Distribution of age at onset (in years) in MCMD patients. C Relative comparison of best motor achievements in MCMD patients stratified according to genotype. D Prevalence of intellectual disability in MCMD patients stratified according to genotype. E Prevalence of heart disease (mainly dilated cardiomyopathy) in MCMD patients stratified according to genotype