Phenotype-Genotype Correlation of a Cohort of Patients with Congenital Myopathy: A Single Centre Experience from India

Abstract

Background:

Congenital myopathies (CMs) are a diverse group of inherited muscle disorders with broad genotypic and phenotypic heterogeneity. While the literature on CM is available from European countries, comprehensive data from the Indian subcontinent is lacking.

Objectives:

This study aims to describe the clinical and histopathological characteristics of a cohort of genetically confirmed CMs from India and attempts to do phenotype-genotype correlation.

Methods:

A retrospective chart review of genetically confirmed CMs was evaluated between January 2016 and December 2020 at the neuromuscular clinic. The clinical, genetic, and follow-up data were recorded in a pre-structured proforma as per the medical records, and the data was analyzed.

Results:

A total of 31(M: F = 14 : 17) unrelated patients were included. The median age at onset and duration of illness are 2.0(IQR:1–8) years and 6.0(IQR:3–10) years respectively. Clinical features observed were proximodistal weakness (54.8%), facial weakness (64.5%), and myopathic facies (54.8%), followed by ptosis (33.3%), and ophthalmoplegia (19.4%). Muscle histopathology was available in 38.7% of patients, and centronuclear myopathy was the most common histopathology finding. The pathogenic genetic variants were identified in RYR1 (29.0%), DNM2 (19.4%), SELENON (12.9%), KBTBD13 (9.7%), NEB (6.5%), and MYPN (6.5%) genes. Novel mutations were observed in 30.3% of the cohort. Follow-up details were available in 77.4% of children, and the median duration of follow-up and age at last follow-up was 4.5 (Range 0.5–11) years and 13 (Range 3–35) years, respectively. The majority were ambulant with minimal assistance at the last follow-up. Mortality was noted in 8.3% due to respiratory failure in Centronuclear myopathy 1 and congenital myopathy 3 with rigid spines (SELENON).

Conclusion:

This study highlights the various phenotypes and patterns of genetic mutations in a cohort of pediatric patients with congenital myopathy from India. Centronuclear myopathy was the most common histological classification and the mutations in RYR1 followed by DNM2 gene were the common pathogenic variants identified. The majority were independent in their activities of daily living during the last follow-up, highlighting the fact that the disease has slow progression irrespective of the genotype.

Keywords

Congenital myopathy phenotype-genotype histopathology creatine phosphokinase

INTRODUCTION

Congenital myopathies (CM) are clinically and genetically heterogeneous groups of inherited muscle disorders characterized by distinct histopathological features and, in general, having a relatively stable or slowly progressive clinical course [1, 2]. Though the exact prevalence of CMs is not known, the recent systematic review reports the pooled prevalence of CM in children to be 2.73 (95% CI, 1.34 – 4.12) per 1,00,000 [3, 4]. Since the initial description of CM in 1956 by Shy and Magee, the diagnosis was largely based on the muscle biopsy findings through which they were classified into core myopathies, nemaline, centronuclear, myosin storage, and congenital fiber type disproportion myopathy [5 –7].

With recent advances in gene panel testing and next-generation sequencing platforms, the diagnostic modality of choice is drifting away from muscle biopsy, which was standard clinical practice till a decade back [8]. Though a large number of genes are being described, a unique challenge in CMs is the wide heterogeneity between the clinical, histopathological, and genetic variants, even among children with features of myopathy belonging to the same family. This demands analysis of CMs from a comprehensively maintained database, which is available from European registries, however was limited from the Indian subcontinent [9 –11]. This is all the more pertinent as the Indian subcontinent has a more ethnically and genetically diverse gene pool, which, in general, can have wide heterogeneity [3 –6].

With the availability of Next Generation Sequencing (NGS) in our country since the last decade, increasing accessibility across the income range, availability of neuromuscular specialists, dedicated research centers, continuation of symptomatic care for floppy neonates, more and more children with CM are able to get a genetic diagnosis. The correlation of this genotypic information with the clinical and histological data provides crucial information for patient care, prognostication, and genetic counseling, and it would be the first step in exploring newer arenas for the treatment of CM. In India, with the advent of genetic testing, the knowledge about CMs’ clinical and genetic spectrum has been expanding. In this background, we undertook the present study to assess the clinical profile, histopathology, and mutational analysis in a cohort of pediatric patients with genetically confirmed CM and to determine their genotype-phenotype correlation.

MATERIALS AND METHODS

Study design

This is a retrospective study done from a quaternary care center for neurological disorders in South India (National Institute of Mental Health and Neurosciences, Bengaluru, India). A detailed chart review was done to identify and include the patients who (i) attended the neuromuscular clinic in the Neurology department between January 2016 and December 2020 (ii) were genetically confirmed to have congenital myopathy as per the “Gene table of monogenic neuromuscular disorders” guidelines [8] and (iii) were under the primary care of the authors. Patients were excluded from the analysis if (i) the diagnosis of CM was based on muscle biopsy, and genetic analysis was not available, and (ii) if the variants detected were benign or likely benign as per American College of Medical Genetics and Genomics (ACMG) guidelines. This study was approved by the Institute Ethics Committee (NIMHANS/IEC/2020-21). A prior informed consent was obtained from the participants at the time they underwent genetic testing. Written informed consent was obtained from parents/guardians for unmasking of the faces.

Patient cohort

Clinical data collection

The following data was recorded in a pre-structured proforma in an electronic database. Clinical details from their first visit to the hospital till their last clinical follow-up, either physical or tele-consultation, were noted. Clinical parameters included the demographic details, age at onset of symptoms, age at presentation, pattern of weakness-proximal/ distal/ axial/ combined, ocular-facial-bulbar, respiratory involvement, presence of ptosis, ophthalmoplegia, contractures, cardiac abnormalities, ambulatory status, and family history were noted. Creatine kinase (CK) levels and details of muscle biopsy were recorded wherever available. Follow-up details, including duration of illness, age at last follow-up, and clinical status, were recorded.

Genetic analysis methodology

All the study subjects who underwent genetic testing on a clinical basis were enrolled in the study at our center. Genomic DNA was extracted using standard procedures from peripheral blood samples. The libraries were sequenced as paired-end reads to mean > 80-100X coverage on Illumina sequencing platform (Illumina, CA). The sequences obtained were aligned to the human reference genome (GRCh37/hg19) using the BWA program, and gene annotation of the variants was performed using the VEP program [12 –15]. The variant annotation was done using published literature and the following databases: ClinVar, Online Mendelian Inheritance in Man (OMIM), Genome-Wide Association Study (GWAS), Human Gene Mutation Database (HGMD), and SwissVar. Variants were classified according to the principles outlined in the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology standards for interpretation of sequence variants. The clinically relevant variants were considered known if reported either in literature, ClinVar, or HGMD. Benign and likely benign variants were excluded from the study.

Statistical analysis

Data were analyzed using Statistical Package for Social Sciences (SPSS) software version 22.0 (Chicago, Illinois) using descriptive statistics for continuous variables like mean, median, interquartile range (IQR), and standard deviation for continuous variables. Frequency and percentages were used for categorical variables. For analysis of continuous variables among various subgroups, non-parametric tests were employed.

Results

During the study period, a total of 31 (M: F- 14 : 17) patients with genetically confirmed congenital myopathy (CM) fulfilling the eligibility criteria were recruited from 31 unrelated families. The majority of the patients in our cohort were from South India (70.9%), followed by East India (22.6%) and North India (3.1%). Referral diagnoses were myopathy (25.8%), congenital myopathy (22.5%), limb-girdle muscular dystrophy (16.1%), Duchenne/Becker’s muscular dystrophy (9.6%), spinal muscular atrophy (3.2%), cerebral palsy, developmental delay, hereditary motor neuropathy, myotonic dystrophy, neuropathy, congenital myasthenic syndrome and brachial plexopathy (3% each). The clinical profile, histopathology, mutational analysis, and follow-up details of patients with genetically confirmed CM are summarized in Table 1.

Table 1

Table summarizing the clinical profile, histopathology, mutational analysis and follow up of patients with congenital myopathy of our cohort

P. number	Gender	Age at onset (Years)	Age at presentation (Years)	Pattern of weakness	Axial weakness	Facial	Ptosis	Myopathic facies	Extraocular movements	Ankle contractures	Consanguinity	CPK (U/L)	Histopathology	Loci of genetic mutation	Pattern of Inheritance	Age, at last, follow up (years)	Current clinical status
P- 1	Male	1	0.7	Proximal and distal	No	No	No	Yes	Restricted	No	No	103	NA	RYR1	AR	6	Assisted walking
P- 2	Male	2	9	Proximal and distal	No	Yes	Yes	Yes	Restricted	No	No	52	Centronuclear myopathy	RYR1	AR	10	Assisted walking
P- 3	Female	1	16	Proximal	No	No	Yes	Yes	Restricted	No	No	125	Centronuclear myopathy	RYR1	AR	22	Assistance to get up from floor
P- 4	Female	10	13	Proximal	No	No	No	No	Normal	No	Yes	925	NA	RYR1	AR	16	Assistance to get up from floor
P- 5	Male	1	5	Proximal	No	No	Yes	No	Normal	No	No	126	NA	RYR1	AR	8	Assisted walking
P- 6	Female	1	7	Proximal and distal	Yes	No	No	Yes	Normal	Yes	Yes	132	NA	RYR1	AD	11	Assisted walking
P- 7	Female	2	16	Proximal	No	Yes	No	No	Restricted	No	Yes	45	NA	RYR1	AR	19	Assistance to get up from floor
P- 8	Male	1	6	Proximal and distal	No	Yes	No	Yes	Normal	No	Yes	132	Centronuclear myopathy	RYR1	AR	12	Assisted walking
P- 9	Female	2	17	Proximal	No	Yes	No	Yes	Normal	No	No	67	NA	RYR1	AD	20	Independent walking
P- 10	Female	2	12	Proximal and distal	No	Yes	No	Yes	Normal	Yes	No	36	Centronuclear>Core> Nemaline rods	NEB	AR	18	Assisted walking
P- 11	Female	1	4	Proximal and distal	No	Yes	No	Yes	Normal	Yes	No	128	NA	NEB	AR	7	Assisted walking
P- 12	Male	14	16	Proximal	No	No	No	No	Normal	No	Yes	2077	NA	DNM2	AD	NA	NA
P- 13	Female	2	18	Proximal and distal	No	Yes	Yes	No	Restricted	Yes	No	19	Centronuclear myopathy	DNM2	AD	25	Assisted walking
P- 14	Female	8	9	Proximal and distal	No	Yes	Yes	Yes	Normal	Yes	No	103	Centronuclear myopathy	DNM2	AD	16	Walks independently
P- 15	Male	34	34	Proximal and distal	No	Yes	Yes	No	Normal	No	No	49	Centronuclear myopathy	DNM2	AR	35	Walks independently
P- 16	Female	11	13	Proximal	No	No	No	No	Normal	No	No	372	NA	DNM2	AD	NA	NA
P- 17	Male	11	13	Proximal	Yes	No	Yes	No	Normal	No	No	9082	Centronuclear myopathy	DNM2	AD	20	Died at 20 years of age
P- 18	Female	2	8	Proximal	No	Yes	No	Yes	Normal	No	No	90	NA	SELENON	AR	13	Died at 12 years due to respiratory failure
P- 19	Male	5	22	Proximal and distal	No	Yes	Yes	Yes	Normal	Yes	No	1516	NA	SELENON	AR	NA	NA
P- 20	Female	8	15	Proximal and distal	No	No	No	No	Normal	No	Yes	50	NA	SELENON	AR	NA	NA
P- 21	Female	1	8	Proximal	NA	NA	Yes	Yes	Normal	Yes	No	191	NA	SELENON	AR	12	Need support for climbing stairs
P- 22	Male	15	28	Proximal and distal	No	Yes	No	Yes	Normal	Yes	No	110	NA	MYPN	AR	NA	NA
P- 23	Male	13	23	Proximal and distal	No	No	No	No	Normal	No	Yes	33	NA	MYPN	AR	32	Assisted walking
P- 24	Female	1.5	8	Proximal	No	Yes	No	Yes	Normal	No	Yes	226	Nemaline Rods> Centronuclear myopathy> Central Cores	TPM3	AR	9	Assisted walking
P- 25	Female	1	5	Distal	No	Yes	No	Yes	Normal	No	No	154	Nemaline rod myopathy	TPM3	AR	13	Needs assistance to get up from floor
P- 26	Male	1	6	Proximal and distal	Yes	Yes	No	No	Normal	Yes	No	49	NA	TPM2	AD	9	Assisted walking
P- 27	Female	2	20	Proximal and distal	No	Yes	Yes	Yes	Normal	Yes	No	140	Central Core> centronuclear myopathy	MTM1	AR	25	Walks independently
P- 28	Male	1	15	Proximal and distal	No	Yes	Yes	No	Restricted	No	No	41	Central Core> centronuclear myopathy	KBTBD13	AD	7	Walks independently
P- 29	Female	7	14	Distal	No	Yes	No	No	Normal	No	No	57	NA	KBTBD13	AD	NA	NA
P- 30	Male	1.5	10	Distal	No	Yes	No	No	Normal	Yes	Yes	193	NA	KBTBD13	AD	NA	NA
P- 31	Male	0.5	5	Proximal and distal	No	Yes	No	Yes	Normal	No	No	118	NA	ACTA1	AD	3	Assisted walking

Abbreviations: AD- Autosomal Dominant; AR- Autosomal recessive; CPK – Creatine phosphokinase; DNM2 – Dynamin 2; KBTBD13 – Kelch repeat and BTB domain containing13; MTM1 – Myotubularin; MYPN – Myopalladin; NA – Not Available; NEB – Nebulin; RYR1 – Ryanodine receptor type 1; SELENON – Selenoprotein N; TPM2 – b-Tropomyosin; TPM3 – Tropomyosin 3;U/L – Units per liter.

Demographic and clinical profile:

The median age at onset and duration of illness were 2.0 (IQR:1–8) years and 6.0 (IQR:3–10) years, respectively. The median age at presentation was 12.5 (IQR: 7–16) years. At the time of presentation to us, 54.8% had features of both proximal and distal muscle weakness, 35.4% had a limb-girdle pattern of proximal muscle weakness, and 10% had isolated distal muscle weakness. Around 10% of children had predominant weakness of axial musculature. Clinical features of motor predominant delay were present in 64.5% of children, 19.4% had a skeletal deformity in the form of scoliosis, 35.5% had ankle contractures, and only 3.2% of children had features of bulbar weakness. Facial weakness was observed in 64.5% and ptosis in 33.3% of children. Myopathic facies and features of external ophthalmoplegia were noted in 54.8% and 19.4% of children, respectively. Consanguinity was observed in 29% and siblings of the patients were affected in 10%. The mean CK level was 547.4±1650 U/L. Representative clinical images are shown in Fig. 1.

Fig. 1

Representative photographs of children with congenital myopathy showing features of – A. dental mal-occlusion and bilateral ptosis (P-14), B: myopathic facies as elongated face and sunken cheeks (P-18), C. rigid spine (P-19), D. skeletal deformity- scoliosis (P-8), E bilateral ptosis (P-28), F. bifacial weakness(P-18), G. wasting of extensor digitorum brevis in both legs (P-19), H. features suggesting proximal lower limb weakness on trying to get up from floor (P-20). (Consent obtained)

Characterization of histopathological profile:

Muscle biopsy was available in 12/31 (38.7%) patients and their median age at the time of biopsy was 12.5 (IQR 8.6–16.5) years. Biopsy was obtained either from the biceps or quadriceps on the non-dominant side. About eighty percent of the patients had central nuclei (muscle fibre with central nuclei constituting > 30% of muscle fibre population) [Fig. 2 A, B, C, H, I] and more than half of these patients exhibiting multiple internalised nuclei. Rod bodies were detected on Modified Gomori Trichrome (MGT) stain [Fig. 2 D, E] and features of core myopathy on oxidative enzyme staining (NADH, SDH) [Fig. 2 F, G] in three patients (25%) each, of which two of them had both rods and cores (NEB, TPM3) in their muscle tissue. The biopsy in all the twelve patients showed variation in size comprising of variable density and distribution of both hypertrophic and atrophic fibers, and two-thirds of the biopsies exhibited well-preserved fascicular architecture in paraffin and cryosections. The rest showed focal/ partial effacement in the form of adipocytic infiltration and focal/ early fibrosis. Two patients (DNM2, MTM1) also exhibited non specific neurogenic changes in muscle biopsy, who had clinical phenotype of myopathy. In one of the patient’s biopsies (NEB), a few fibers displayed a rimmed vacuole on the MGT stain. All RYR1 and Centronuclear myopathy 1 myopathy patients had centronuclear myopathy in muscle biopsy. However, rods were predominantly noticed in patients with TPM3 mutation and cores in MTM1 and KBTBD13 myopathies. The details of histopathology are mentioned in Table 2, and representative images are shown in Fig. 2.

Fig. 2

Microphotograph showing transverse section of P-2, centronuclear myopathy with many muscle fibres (>30%) possessing central nuclei (H & E x100) [A, B] and radial staining pattern (NADH × 200) [C]. Microphotograph showing transverse section in P-25, of Nemaline myopathy with many muscle fibres displaying Nemaline rods of variable density, configuration and distribution (MGT × 200) [D]displaying Nemaline rods (black arrow) of variable density, configuration and distribution (MGT × 400) [E]. Microphotograph showing transverse section of P-27, central core myopathy with many muscle fibres displaying central areas (Cores) of absence of oxidative activity (SDH × 200) [F] and (NADH × 200) [G]. Note the type I fibre predominance. Microphotograph showing transverse section in P-15 of centronuclear myopathy with many muscle fibres (>30%) possessing central nuclei. Also observed is the adipocytic infiltration and fibrosis (H & E ×100) [H]. Microphotograph showing transverse section in P-17 of centronuclear myopathy with many muscle fibres (>30%) possessing central nuclei (H & E × 200) [I].

Table 2

Table describing the characteristic histopathological features in our cohort of patients with genetically confirmed congenital myopathy

Patient number	Histology							Enzyme Histochemistry			Rods	Cores	Others	Final interpretation	Gene mutation
	Fascicular archicture	Hypertrophic fibres	Atrophic fibres	Location of nuclei	Single nuclei	Multiple scattered nuclei	Inflammation	MGT	NADH/SDH	ATPase
P-2	Relatively PreservedEarly fibrosis+	Present	Present	Central	Absent	Present	Absent	No new changes	Subsarcolemmal staining	Mosaic pattern	Absent	Absent		CNM	RYR1
P-3	Preserved	Present	Present	Central	Present	Absent	Absent	No new changes	Absent	NA	Absent	Absent		CNM	RYR1
P-8	Preserved	Present	Present	Central	Present	Absent	Absent	No new changes	No cores	Type 1 fibre hypoplasiaType 2 fibre hypertrophy	Absent	Absent	Coexisting CFTD	CNM	RYR1
P-10	Preserved	Present	Present	Central	Present (40%)	Absent	Absent	Rods present- subsarcolemmal region	Type 1 fibre predominateFew unstained areas suggestive of cores	Nil	Present	Present	One myofiber with vacuole rimmed by basophilic granular material	CNM>Cores> Rods	NEB
P-13	Preserved	Present	Present	Central	Present	Present	Absent	Perinuclear staining	Type 1 fibre predominateType 2 fibre hypertrophy	Type 1 fibre predominateType 2 fibre hypertrophy	Absent	Absent	Coexisting CFTD	CNM	DNM2
P-14	Preserved	Present	Present	Central	Present	Absent	Absent	No inclusions	Type I fibre predominance	NA	Absent	Absent		CNM	DNM2
P-15	Partly effaced with adipocytic infiltration and focal fibrosis	Present	Present	Central	Present	Present	Absent	No new changes	No ragged blue fibres	Type 1 predominanceMosaic pattern maintained	Absent	Absent	Coexisting neurogenic changes	CNM	DNM2
P-17	Preserved	Present	Present	Central	Absent	Present	Absent	NA	NA	NA	Absent	Absent		CNM	DNM2
P-24	Partly effaced with adipocytic infiltration	Present	Present	Central	Present	Present	Absent	Numerous rod bodies in majority of fibres	Multiple cores notedType 1 fibrepredominace	Nil	Present	Present		Rods> CNM> Cores	TPM3
P-25	Effaced	Present	Present	Central	Present	PresentOccasional fibre with peripheral ring of nuclear arrangement seen, (necklace fibres)	Absent	Rods present	NA	NA	Present	Absent	Occasional Necklace fibre	Rods>CNM	TPM3
P-27	Effaced	Present	Present	Central	Present (20%)	Present (minimal)	Absent	No inclusions	No cores	Nil	Absent	Present	Coexisting neurogenic changes	Cores>CNM	MTM1
P-28	Effaced	Present	Present	Central	Present (40%)	Present (minimal)	Absent	No inclusions	Single, multicores	Type 1 fibre predominance	Absent	Present	Cores>CNM	KBTBD13

Abbreviations: CNM – centronuclear myopathy; CFTD- Congenital myopathy with fibre type disproportion; MGT – Modified Gomori Trichome; NA – Not Available; NADH/SDH – Reduced nicotinamide adenine dinucleotide/Succinate dehydrogenase.

Mutational analysis:

Among 31 patients, 41.9% of them had homozygous mutations, 19.4% had compound heterozygous, and the rest, 38.7%, had heterozygous mutations. A total of 33 different variants were observed in the CM-related genes. The majority of them among these 33 variants were missense variants (48.5%, 16/33), followed by frameshift (18.2%, 6/33), splice site (15.2%, 5/33), nonsense (9.1%, 3/33) and in-frame deletion (9.1%, 3/33). Among these 33 variants, 23 (69.7%) were previously reported and 10 (30.3%) were novel variants. Overall, 90.1% of variants (30/33) were classified as either pathogenic/ likely pathogenic, and the rest, 9.9% (3/23), were variants of uncertain significance (VUS). Among children with VUS, (i) P-17 with DNM2 mutation had muscle biopsy features consistent with CM supporting the diagnosis, (ii) P-31 had two variants in ACTA1 (Likely pathogenic) and MYH2 (VUS). Though the clinical phenotype was matching, due to lack of functional studies, pathogenicity among these two could not be ascertained, and (iii) P-12 had variant in DNM2 gene with matching clinical phenotype, as this was a novel variant, considered to be causative. A summary of the genetic variations observed in this study is shown in Table 3. The highest number of mutation variants were detected in RYR1 (36.4%), followed by DNM2 (15.2%), SELENON (12.1%), NEB (9.1%), KBTBD13 (6.1%), and MYPN (6.1%). Others were MYH2 , TPM3, ACTA1, MTM1, and TPM2, one (3%) each. The distribution of mutations identified in this study in the genes RYR1, DNM2, and SELENON are shown in Fig. 3. One variant in RYR1 (c.14126 C>T; p.Thr4709Met) was observed in three patients, homozygous state in two (P-7 and P-8) and heterozygous in one (P-2). In the subject P-31 heterozygous variants were detected in both MYH2 (c.5579 C>T; p.Thr1860Met; pathogenic) and ACTA1 (c.275_277del; p.Phe92del; likely pathogenic).

Table 3

Summary of the genetic variants observed in our congenital myopathy cohort

Cases	Gene	Pattern of	#OMIM	Variant in HGVS	Location	Variant	Zygosity	Variant classification	Frequency in	HGMD ID	ClinVar
		Inheritance		format		Consequence		as per ACMG / AMP Criteria (2015)	Population Databases
											Interpretation	Variation ID	Phenotype
P- 1	RYR1	AR	255320	NM_000540.3:c.936delCNP_000531.2:p.F313Sfs*77	Exon 10 of 106	Frameshift	Het	PathogenicPM2 PVS1 PP4	gnomAD exomes-NovelgnomAD genomes-NovelInhouse database- Nove	NR	NR	NR	NR
				NM_000540.3: c.11707 C>TNP_000531.2: p.R3903W	Exon 85 of 106	Missense	Het	Likely PathogenicPM2 PM1 PP2 PM5 PP4	gnomAD exomes-3individuals only in het stategnomAD genomes-NovelInhouse database- Novel	NR	NR	NR	NR
P- 2	RYR1	AR	255320	NM_000540.3: c.1326dupGNP_000531.2: p.S443Efs*62	Exon 13 of 106	Frameshift	Het	PathogenicPM2 PVS1 PP4	gnomAD exomes-3individuals only in het stategnomAD genomes-NovelInhouse database- Novel	NR	NR	NR	NR
				NM_000540.3: c.14126 C>TNP_000531.2: p.T4709M	Exon 96 of 106	Missense	Het	Likely Pathogenic PP2 PP3 PM5 PP4 BS1 BS2	gnomAD exomes-36individuals only in het state gnomAD genomes-7 individuals only in het stateInhouse database-1 individuals only in homo state	CM073322	Pathogenic/Likely Pathogenic	RCV000555087/RCV000763429	RYR1-RD/CCO/CFTD/MMD
P- 3	RYR1	AR	255320	NM_000540.3: c.2348 C>TNP_000531.2: p.S783L	Exon 19 of 106	Missense	Het	Likely PathogenicPM2 PP2 PP3 PP4_Moderate	gnomAD exomes-3individuals only in het stategnomAD genomes-NovelInhouse database-Novel	NR	VUS	RCV000553747	RYR1-RD
				NM_000540.3: c.8888T>CNP_000531.2: p.L2963P	Exon 58 of 106	Missense	Het	Likley PathogenicPM2 PP2 PP3 PS1 PP4	gnomAD exomes-4individuals only in het state gnomAD genomes-NovelInhouse database-1 individuals only in het state	CM136222	Pathogenic	RCV000796219	RYR1-RD
P- 4	RYR1	AR	255320	NM_000540.3: c.3494 G>ANP_000531.2: p.G1165D	Exon 26 of 106	Missense	Het	Likely PathogenicPM1 PP2 PP3 PS1 PP4 BS2	gnomAD exomes-10individuals only in het stategnomAD genomes-2 individuals only in het stateInhouse database-7 individuals only in het state	CM117526	VUS	RCV001237337	RYR1-RD
				NM_000540.3: c.7444 + 1 G>A	Intron 46 of 105	Splice Donor	Het	PathogenicPM2 PVS1 PP4	gnomAD exomes-NovelgnomAD genomes-NovelInhouse database-1 individuals only in het state	NR	NR	NR	NR
P- 5	RYR1	AR	255320	NM_000540.3: c.8758 C>TNP_000531.2: p.R2920*	Exon 57 of 106	Stop Gained	Het	PathogenicPM2 PVS1 PP4	gnomAD exomes-Novel gnomAD genomes-Novel Inhouse database- Novel	CM144265	NR	NR	NR
				NM_000540.3: c.13090 G>ANP_000531.2: p.G4364S	Exon 91 of 106	Missense	Het	Likely PathogenicPM2 PM1 PP2 PP3 PM5 PP4_Moderate	gnomAD exomes-NovelgnomAD genomes-NovelInhouse database- Novel	NR	VUS	RCV000731208	NP
P- 6	RYR1	AD	117000	NM_000540.3: c.13913 G>TNP_000531.2: p.G4638V	Exon 95 of 106	Missense	Het	PathogenicPM2 PM1 PP2 PP3 PM5 PP4_Strong	gnomAD exomes-Novel gnomAD genomes-Novel Inhouse database- Novel	NR	Likely pathogenic/Pathogenic	RCV001198930/ RCV000056188	CFTD/ CCO
P- 7 P- 8	RYR1	AR	255320	NM_000540.3: c.14126 C>TNP_000531.2: p.T4709M	Exon 96 of 106	Missense	Homo	Likely Pathogenic PP2 PP3 PM5 PP4_Strong BS1 BS2	gnomAD exomes-36individuals only in het stategnomAD genomes-7 individuals only in het stateInhouse database-Novel	CM073322	Pathogenic/Likely Pathogenic	RCV000555087/RCV000763429	RYR1-RD/CCO/CFTD/MMD
P- 9	RYR1	AD	117000	NM_000540.3: c.14678 G>ANP_000531.2: p.R4893Q	Exon 102 of 106	Missense	Het	Pathogenic PM2 PM1 PP2 PP3 PM5 PP4_Strong	gnomAD exomes-NovelgnomAD genomes-Novel Inhouse database-Novel	CM030713	Pathogenic/Pathogenic	RCV001218792/RCV000056235	RYR1-RD/ CCD
P- 10	NEB	AR	256030	NM_001271208.2: c.1569 + 1 G>A	Intron 17 of 182	Splice Donor	Homo	PathogenicPM2 PVS1 PP4	gnomAD exomes-14 individuals only in het stategnomAD genomes-Novel Inhouse database- Novel	CS1413956	Pathogenic	RCV000667222	NEM2
P- 11	NEB	AR	256030	NM_001271208.2: c.10612 C>TNP_001258137.2:p.R3538*	Exon 73 of 183	Stop Gained	Het	PathogenicPM2 PVS1 PP5 PP4	gnomAD exomes-7 individuals only in het state gnomAD genomes-Novel Inhouse database- Novel	NR	Likely Pathogenic	RCV000986840	NEM2
				NM_001271208.2:c.24407_24410dupTGTTNP_001258137.2:p.L8137Ffs*18	Exon 173 of 183	Frameshift	Het	PathogenicPM2 PVS1 PP5 PP4	gnomAD exomes-9 individuals only in het stategnomAD genomes-NovelInhouse database-Novel	NR	Pathogenic/Likely Pathogenic	RCV000790962/RCV000781649	NEM2/ NEM
P- 12	DNM2	AD	160150	NM_001005360.3:c.808 G>ANP_001005360.1:p.D270N	Exon 6 of 21	Missense	Het	VUSPM2 PP2 PP3 PP4	gnomAD exomes-28 individuals only in het stategnomAD genomes- 2 individuals only in het stateInhouse database-Novel	NR	VUS	RCV001772330/RCV001122233	NP/ ADCM
P- 13 P- 14	DNM2	AD	160150	NM_001005360.3:c.1102 G>ANP_001005360.1:p.E368K	Exon 8 of 21	Missense	Het	PathogenicPM2 PM1 PP2 PP3 PS1 PP4	gnomAD exomes-NovelgnomAD genomes-Novel Inhouse database- Novel	CM053834	Pathogenic/Pathogenic	RCV000554046/RCV000145898	CNM/ CMTDIB
P- 15	DNM2	AR	160150	NM_001005360.3:c.1565 G>ANP_001005360.1:p.R522H	Exon 15 of 21	Missense	Het	Pathogenic PM2 PM1 PP2 PP3 PS1 PM5 PP4	gnomAD exomes-NovelgnomAD genomes-Novel Inhouse database-Novel	CM102028	Pathogenic/Pathogenic	RCV000552861/RCV000679888	CMTDIB/ CNM1
P- 16	DNM2	AD	160150	NM_001005360.3:c.1622_1627delACTGGTNP_001005360.1:p.Y541_W542del	Exon 15 of 21	In-frame Deletion	Het	Likely pathogenic PM2 PM4 PP3 PP4	gnomAD exomes-Novel gnomAD genomes-Novel Inhouse database- novel	NR	NR	NR	NR
P- 17	DNM2	AD	160150	NM_001005360.3:c.2392 G>ANP_001005360.1:p.V798M	Exon 20 of 21	Missense	Het	VUS PP2 PP4_Strong	gnomAD exomes- 7 individuals only in het stategnomAD genomes-Novel Inhouse database- Novel	NR	NR	NR	NR
P- 18	SELENON	AR	602771	NM_020451.3:c.249_250dupGGNP_065184.2:p.D84Gfs*17	Exon 2 of 13	Frameshift	Homo	Pathogenic PM2 PVS1 PP5 PP4	gnomAD exomes- 13 individuals only in het stategnomAD genomes- 1 individual in het state Inhouse database- 1 individual in het state	NR	Pathogenic/Pathogenic	RCV000799500/RCV000627410	RSMD1/ NP
P- 19	SELENON	AR	602771	NM_020451.3: c.1505_1508delACCA NP_065184.2: p.N502Rfs*23	Exon 12 of 13	Frameshift	Homo	Likley Pathogenic PM2 PVS1 PP4	gnomAD exomes-Novel gnomAD genomes-Novel Inhouse database- Novel	NR	NR	NR	NR
P- 20	SELENON	AR	602771	NM_020451.3: c.1552_1554delAAG NP_065184.2: p.K518del	Exon 12 of 13	In-frame Deletion	Homo	Likely Pathogenic PM2 PM4 PP3 PP4	gnomAD exomes-Novel gnomAD genomes-Novel Inhouse database- Novel	NR	NR	NR	NR
P- 21	SELENON	AR	602771	NM_020451.3:c.1574T>GNP_065184.2:p.M525R	Exon 12 of 13	Missense	Homo	Likely PathogenicPP3 PM5 PP4_Strong	gnomAD exomes-14 individuals only in het state gnomAD genomes-1 individuals only in het state Inhouse database- Novel	NR	VUS	RCV001326195	RSMD1
P- 22	MYPN	AR	617336	NM_032578.4:c.1973 + 1 G>C	Intron 10 of 19	Splice donor	Homo	Pathogenic PM2 PVS1 PP4	gnomAD exomes-NovelgnomAD genomes-Novel Inhouse database- 2 individuals only in het state	NR	NR	NR	NR
P- 23	MYPN	AR	617336	NM_032578.4:c.1974-2A>C	Intron 10 of 19	Splice Acceptor	Homo	Pathogenic PM2 PVS1_Strong PP4_Strong	gnomAD exomes-NovelgnomAD genomes-Novel Inhouse database- 4 individuals only in het state	NR	NR	NR	NR
P- 24 P- 25	TPM3	AR	609284	NM_152263.4:c.856T>ANP_689476.2:p.286Kext57	Exon 10 of 10	Stop Loss	Homo	Likely Pathogenic PM2 PM4 PP4_Strong	gnomAD exomes-NovelgnomAD genomes-NovelInhouse database- Novel	NR	NR	NR	NR
P- 26	TPM2	AD	609285	NM_003289.4:c.415_417delGAGNP_003280.2:p.E139del	Exon 4 of 9	In-frame Deletion	Het	Likely PathogenicPM2 PM4 PP3 PP5 PM5 PP4_Moderate	gnomAD exomes-NovelgnomAD genomes-Novel Inhouse database- Novel	NR	Pathogenic	RCV000013281/RCV000500415	CAPM2/NEM4
P- 27	MTM1	AR	310400	NM_000252.3:c.688T>CNP_000243.1:p.W230R	Exon 9 of 15	Missense	Homo	PathogenicPM2 PM1 PP2 PP3 PS1 PP4	gnomAD exomes-NovelgnomAD genomes-Novel Inhouse database- Novel	CM050296	Pathogenic/Likely pathogenic	RCV000146479	CNMX
P- 28	KBTBD13	AD	609273	NM_001101362.3:c.477delCNP_001094832.1:p.V160*	Exon 1 of 1	Frameshift	Het	Likely PathogenicPM2 PP4_Strong	gnomAD exomes- 1 individuals only in het state gnomAD genomes- 2 individuals only in het state Inhouse database- Novel	NR	VUS	RCV000532534	NEM6
P- 29 P- 30	KBTBD13	AD	609273	NM_001101362.3:c.677A>GNP_001094832.1:p.E226G	Exon 1 of 1	Missense	Het	Likely PathogenicPM2 PP3 PP4_Strong	gnomAD exomes- 4 individuals only in het state gnomAD genomes- novel Inhouse database- 2 individuals only in het state	CM184977	VUS	RCV001891752	NEM6
P- 31	ACTA1	AD	161800	NM_001100.4:c.275_277delTCTNP_001091.1:p.F92del	Exon 3 of 7	In-frame Deletion	Het	Likely PathogenicPM2 PM1 PM4 PP3 PP5 PM5 PP4_Moderate	gnomAD exomes-NovelgnomAD genomes-NovelInhouse database- Novel	NR	Pathogenic	RCV000691025	NEM3
	MYH2		605637	NM_017534.6:c.5579 C>TNP_060004.3:p.T1860M	Exon 39 of 40	Missense	Het	VUSPP2 PP3 PP4	gnomAD exomes- 79 individuals only in het stategnomAD genomes-10 individuals only in het state Inhouse database- 1 individuals only in het state	NR	VUS	RCV000538870	MYPOP

Abbreviations: AR- Autosomal Recessive, AD- Autosomal Dominant,ACMG- American College of Medical Genetics; Ex-Exon; Hom- Homozygous; VUS- Variant of uncertain significance; RYR1-RD: RYR1-Related Disorders; NP- Not provided; NR-Not reported; Het- Heterozygous; CCO-Central core myopathy; CFTD-Congenital myopathy with fiber type disproportion; MMD – Multiminicore disease; In- Intron; SS – Splice site; NS- Nonsense; CCD – Central core myopathy; NEM – Nemaline myopathy; ADCM-Autosomal dominant centronuclear myopathy; CNM – Centronuclear myopathy; CMTDIB-Charcot-Marie-Tooth disease, dominant intermediate B;IFD-In-frame deletion; RSMD1 – Eichsfeld type congenital muscular dystrophy; CAPM2-Cap myopathy 2; CNMX- Severe X-linked myotubular myopathy; MYPOP – Myopathy, proximal, and ophthalmoplegia; OMIM – Online Mendelian Inheritance in Man.

Fig. 3

Schematic representation of the variations identified in our study in RYR1 gene (Panel A), DNM2 gene (Panel B) and SELENON gene (Panel C) with corresponding exons. The exons are represented as boxes with respective exonic numbers with non-coding regions shaded in black and grey at the ends. The novel variants are shaded in orange color. The filled dots represent homozygous variations, unfilled dots represent heterozygous variations, semi filled dot represent compound heterozygous variations and each dot represents number of variations.

Clinical follow-up details:

A total of 77.4% of children were available for follow-up. The median duration of follow-up was 4.5 (Range 0.5–11) years, and the median age at the last follow-up was 13 (Range 3–35) years. Among those on follow-up, 20.8% could walk independently without any support, 50% were walking with minimal assistance, and 20.8% required one-person support to get up from the floor and to climb stairs. Nonetheless, all were independent for activities of daily living. Two (8.3%) children expired (P-17, DNM2; P-18, SELENON) due to respiratory failure at the age of 20 years and 12 years respectively.

Phenotype – genotype correlation of selective CM

Overall, 61.3% of children had biallelic, and 38.7% had monoallelic patterns of gene mutation. The phenotypic details of some of the common genetic CMs observed in our cohort are described in Table 4.

Table 4

Table showing comparison of clinical features of common sub-types of congenital myopathy observed in our cohort

Variables	RYR1 (n = 9)	DNM2 (n = 6)	SELENON (n = 4)	KBTBD13 (n = 3)
Age at onset (Years) (Median;IQR)	1(IQR:1-2)	11.0(IQR:8.8-13.3)	3.5(IQR:1.8-5.8)	1.5(IQR:1.3-4.3)
Duration (Years) (Median;IQR)	4.0(IQR:3-14)	2.0(IQR:2-11)	7.5(IQR:7-9.8)	8.5(IQR:7.3-9.3)
Age at presentation (Years) (Median;IQR)	9.0(IQR:6-16)	14.5(IQR:13-17.5)	11.5(IQR:8-16.8)	14.0(IQR:12-14.5)
M: F	4 : 5	3 : 3	1 : 3	2 : 1
Pattern of weakness
Proximal and distal	44.4%	50%	50%	33.3%
Proximal	55.6%	50%	50%	0
Distal	0	0	0	66.4%
Axial weakness	11.1%	16.6%	0	0
Facial weakness	44.4%	50%	50%	100%
Ptosis	33.3%	66.6%	50%	33.3%
Myopathic facies	66.6%	16.6%	75%	0
External Ophthalmoplegia	44.4%	16.6%	0	33.3%
Contractures	11.1%	16.6%	50%	33.3%
Consanguinity	44.4%	16.6%	25%	33.3%
CK (U/L) (Mean±SD)	189.6±277.9	1950.3±3581.2	461.7±705.3	97±83.5
Histopathology	33.3% (All CNM)	66.6% (All CNM)	0	33.3% (Core myopathy)

Abbreviations: CNM- centronuclear myopathy; CK: Creatine phosphokinase; IQR- Inter quartile range; M- Male; n- number of patients; F- Female; U/L: SD- Standard Deviation; U/L-units per liter.

RYR1; Congenital myopathy 1A/congenital myopathy 1B [OMIM # 117000; 255320] (n = 9).

Among the children with RYR1 mutation, 89% had onset in the first two years of life, with a median age at presentation being 9 (IQR:6-16) years. About 44.4% of children had both proximal and distal weakness, and the remaining 55.6% had only proximal limb weakness in limb-girdle pattern. Nearly 66.6% of children had myopathic facies and external ophthalmoplegia was observed in 44.4%. Malignant hyperthermia and respiratory infections were not observed in our cohort. The mean CK was 189.6±277.9 U/L. Histopathology showed features of centronuclear myopathy in those who underwent muscle biopsy (33.3%). Nearly 77.7% of children had biallelic and 22.2% had monoallelic gene mutation. All the children were followed for a median duration of 4 (Range 1-7) years, with the median age at the last follow-up being 12 (Range 6-22) years. These children were ambulant, requiring minimal assistance to walk, and none of them were wheelchair-bound.

DNM2; Centronuclear myopathy 1 [OMIM# 160150] (n = 6).

Patients with Centronuclear myopathy 1 had a later age of presentation with a median age of onset of 14.5 (IQR:13–17.5) years. All except one (83.6%) had normal motor developmental milestones. Clinical phenotype was myopathy in all (100%) and the pattern of weakness was both proximal and distal in 50% of children and isolated limb girdle weakness in the remaining 50%. Ophthalmoparesis was seen in only 16.6% of children. Muscle biopsy was done in 66% and findings were suggestive of centronuclear myopathy. The mean CK was 1950.3±3581.2 U/L. All except one (83.6%) had autosomal dominant (AD) pattern of inheritance. One child died at the age of 20 years due to respiratory infection after seven years of follow-up, and the remaining were ambulant during the last follow-up. The median follow-up duration was 7 (Range 0.5–7) years, and the median age at the last follow-up was 23.0 (range 13–35) years.

SELENON; Congenital myopathy 3 with rigid spine [OMIM #602771] (n = 4).

The median age at onset of symptoms in children with SELENON mutation was 3.5 (IQR:1.8–5.8) years. The median age at presentation was 11.5 (IQR:8–16.8) years, with features of proximal and distal limb weakness in 50%, and the remaining 50% had only limb-girdle pattern of weakness, of which one (25%) had recurrent respiratory infections. The majority (75%) had myopathic facies. Scoliosis was present in two (50%) children, of which one had the feature of a rigid spine. The mean CK was 461.8±705.3 U/L. All children had AR pattern of inheritance. One child died at the age of 12 years due to recurrent chest infections and respiratory failure.

KBTBD13; Nemaline myopathy 6 [OMIM # 609273) (n = 3).

Three children with KBTBD13 mutation presented with motor predominant delay. AD inheritance pattern was observed in all. They had difficulty in getting up from the floor with the median age at onset of this symptom being 1.5 (IQR:1.3–4.3) years. The median age at presentation to us was 14 (IQR:12–14.25) years. On examination, features of proximal and distal limb weakness were present in all (100%) and with sluggish tendon reflexes. The mean CK was 97±83.5 U/L. At one year follow-up, the clinical status has remained the same.

Other Congenital Myopathies: (n = 9).

Two children with NEB(P-10,11) and TPM3(P-24,25) gene mutation each had presented with motor predominant delay in the first two years of life. Children with TPM3 mutation had myopathic faces with bifacial weakness and generalized hypotonia. Children with MYPN mutation (P-22,23) presented with features of limb-girdle weakness. A child with ACTA1 mutation (P-31) had manifested with bulbar weakness at onset associated with developmental delay. Antenatal history of decreased foetal movements was present in a child with TPM2 mutation (P-26). This child had recurrent respiratory infections and later on, developed limb-girdle weakness and had myopathic facial features (elongated facies, low set ears, high arch palate, and bifacial weakness) with exaggerated lumbar lordosis. An adolescent girl with MTM1 mutation (P-27), had presented with difficulty in running at 20 years of age, who at the time of birth had a history of hypophonia and bulbar weakness and had features of non-fatigable asymmetric ptosis and elbow contracture with proximal limb weakness. Most of these patients gradually gained motor milestones and were able to walk with or without assistance.

DISCUSSION

In the present study, we attempted to describe the clinical profile, histopathology, mutational analysis and follow up of a large pediatric cohort with genetically confirmed CM from a single neurology centre, at south India. As this is the first such study from Asian region, we have compared our results with European population. Largely, patients were from southern part followed by eastern part of the country. The predominant clinical manifestations observed were both proximal and distal muscle weakness, in the majority associated with motor-predominant delay followed by facial weakness, ophthalmoparesis, and skeletal abnormalities. In our cohort, centronuclear myopathy was the commonest histopathological subtype, and RYR1 gene-associated CM was the commonest genetic subtype, which was in coherence with the previously published literature [9, 16]. Most of the patients in our study had a slow progression with majority (around 90%) being ambulant with or without assistance at the last median follow-up of 4.5 years. It is interesting to note that only one-fifth of the children referred to us had an initial referral diagnosis of CM, highlighting the lack of awareness among clinicians regarding CM.

The median age at onset of symptoms was the second year of life in our cohort, unlike previous studies where onset in the neonatal/infantile period is common. Antenatal or neonatal onset ranged between 56% to 76% in prior studies, with nearly one-third requiring respiratory support [11, 16]. The later age at the onset of symptoms in our cohort could probably be due to institutional bias with the lack of neonatal care at our institute. The other plausible explanation could be the lesser proportion of patients with mutations in ACTA1 and MTM1 genes, which tend to have an earlier onset, and the majority die by the first year of life, as observed in a previous study [16]. Most of the children had delayed developmental milestones similar to the Denmark study, except in centronuclear myopathy 1, where the majority had normal milestones with later age of onset [10]. Proximal or proximo-distal weakness observed in most were similar to the observations reported in European studies [9, 11]. It is also worthwhile to note that two of three patients with KBTBD13 mutation had distal predominant weakness. Children with respiratory distress requiring ventilatory support were less commonly observed in our study, in contrast to the study from London, where nearly 25–30% of the children required assisted ventilation [9, 16]. This could be due to the difference in patient population, genetic spectrum, institutional bias, and non-availability of follow-up in some patients. Axial weakness was observed in children with mutations in RYR1, DNM2, and TPM2 genes. While facial weakness and contractures were non-specific and observed in all the genes, ptosis was pronounced in those with RYR1, DNM2 and SELENON gene mutations. In comparison to the other non-RYR1 subtypes of myopathies, consanguinity and external ophthalmoplegia were commonly observed in RYR1 myopathy. Similarly, features of bulbar palsy and siblings being affected are more often observed in non-RYR1 subtypes of CM. However, there were no significant differences in the clinical profile among RYR1 and non-RYR1 subtypes of congenital myopathies. The mean CK value was mildly elevated, up to threefold, with the highest in those with DNM2 mutations.

As we included only those with genetically confirmed CMs, histopathology was available in less than half of the cohort, with the commonest subtype observed being centronuclear myopathy. Nonetheless, core myopathies have been reported frequently in studies from UK and Spain [9, 11]. The genetic heterogeneity described in CMs was evident in our cohort, with mutations in both RYR1 and DNM2 resulting in the same histopathology, i.e., centronuclear myopathy. Nemaline myopathy was seen in those with TPM3 mutations. Core myopathy was noticed in patients with mutations in MTM1 and KBTBD13 genes. Interestingly, two of our patients also had dual pathology with the presence of both rods and cores, which were delineated clearly with genetic testing as nemaline myopathy 2 and congenital myopathy 4A/congenital myopathy 4B. Previously, such dual pathology has been reported in RYR1 myopathy [17]. At times, even genetically established myopathy can have non-specific histopathological observations, as evidenced by prior studies where these changes ranged from 3.8% to 22% [9, 11]. These variations can also be due to dynamic changes based on the site of the biopsy, and the findings can be absent if a biopsy is done very early in the course of the illness, highlighting the development of new histopathological changes with aging [18]. Owing to these variations, it is essential to have a more comprehensive approach for making the diagnosis of CM. The yield of genetic testing in histology-confirmed CMs ranged from 56% to 79% across studies from the UK, Spain, and Denmark [9 –11]. The yield was reportedly higher in those with specific histopathology findings (presence of cores, nemaline rods, central nuclei, or fibre type disproportion).

RYR1 mutation was the most common subtype of CM in our cohort. Similar observations were noted in all previously reported large global cohorts both in children [10, 19] and adults [20]. The higher prevalence of Congenital myopathy 1A/congenital myopathy 1B (RYR1) in most of the studies could be due to complete sequencing of this large gene, unlike nebulin, which is often difficult to identify in conventional studies [9]. Congenital myopathy 1A/congenital myopathy 1B (RYR1) has been described as having both AD and AR patterns of inheritance and sometimes with sporadic mutations [21, 22]. Centronuclear myopathy 1 (DNM2) was the second most common subtype, similar to the observations of the Denmark study [10], and unlike the study from the United Kingdom, where none had DNM2 mutation [9]. Also, it is important to note that all centronuclear myopathy 1 (DNM2) children were sporadic, which strengthens the hypothesis of de novo mutations in these subgroups [23]. SELENON myopathy was the third most common subtype of CM, unlike the Spanish population where TTN myopathy was seen in higher proportion, while SELENON mutations were absent. Variation in the proportion of various genetic subtypes in the study population of our groups, including those from the United Kingdom and Spain, suggests geographic variability among different subtypes of CM, though RYR1 mutation remains the most prevalent globally.

The majority of our children had an AR pattern of inheritance. In Congenital myopathy 1A/congenital myopathy 1B (RYR1), both recessive and dominant patterns were observed, and there was not much difference between these two subtypes. However, the sample was too small to make any inference. However, severely affected phenotype was observed in recessive forms in previous studies [10]. AD pattern was observed mainly in DNM2 and KBTBD13 gene-associated myopathy. An Italian study also showed de-novo dominant pattern of inheritance in DNM2 myopathy [23]. Similar observations have been made in Nemaline myopathy 6 (KBTBD13). The type of gene mutations was varied and mainly had missense and frameshift mutations, and no single mutation hot spot was observed, similar to the results observed in the study from Denmark, United Kingdom [9, 10]. However, studies with larger samples of each genetic subtype are needed for any inference. Table 5 summarizes the previously published literature and our findings on genetically confirmed cases of congenital myopathy.

Table 5

A comparison table describing the summary of previously published literature on genetically confirmed cases of congenital myopathy and our study

Parameters	Maggi et al., 2013 [9]	Colombo et al., 2015 [16]	Witting et al., 2017 [10]	Benito et al., 2021 [11]	Present study
Place	United Kingdom	London	Denmark	Spain	India
No of subjects	66	125	82	104	31
Male: Female	37 : 29	61 : 64	41 : 41	57 : 47	14 : 17
Age at onset (years)	0.8	Neonatal onset 76%	NA*	Neonatal onset-56%	2
Age at presentation (years)	4.7	Neonatal presentation-60% Infantile presentation 16%	27.8	12.5	13
Pattern of weakness
Proximal limb weakness	71.2%	NA	NA	55%	35.4%
Proximal and distal limb weakness	6.1%	40%	54.8%
Distal predominant weakness	–	–	10%
Axial predominant weakness	19.7%	–	10%
Ophthalmoplegia	10.6%	20.7%	12.5%	54.8%
Facial involvement	NA	64.6%	66%	64.5%
Bulbar palsy	36.4%	46.4%	NA	14.9%	3.2%
Skeletal abnormalities	13.6%	40%	25.6%	42%	19.3%
Histopathology
Number of subjects	54/66	104/125	46/82	95/104	12/31
Core myopathy	54%	37.5%	17%	42%	16.6%
Nemaline rod myopathy	17%	31.8%	15%	16%	16.6%
Myotubular/centronuclear myopathy	13%	17.3%	18%	14%	66.6%
Congenital fibre type disproportion	4%	4.8%	33%	3%	No
Isolated type 1 predominance	11%	4.8%	No	No	No
Mixed core–rod myopathy	2%	No	No	No	No
Nonspecific myopathic changes	11.1%	3.8%	17%	22%	No
Gene mutations
Number of subjects	44/66	99/125	46/82	65/104	31/31
RYR1	59%	44.4%	22.0%	23.1%	29%
ACTA1	16%	17.2%	4.8%	1.9%	3.2%
SELENON	16%	16.2%	3.6%	–	12.9%
MTM1	5%	8.1%	3.6%	6.7%	3.2%
NEB	2%	8.1%	7.3%	3.8%	6.5%
TPM3	2%	2.1%	4.8%	1.9%	6.5%
DNM2	–	1%	7.3%	2.9%	19.4%
TTN	–	–	1.2%	7.7%	–
TPM2	–	1%	Included in TPM3	1.9%	3.2%
KBTBD13	–	–	–	–	9.7%
SELENON	–	–	–	6.7%	–
MYH3	–	–	–	1%	–
MYPN	–	–	–	–	6.5%
Follow up
Number of subjects	66/66	125/125	80/82	NA	24/31
Median duration of follow up (years)	5	10	NA		4.5 (IQR:3-6.5)
Stable	86%	–	73.8%		94%
Deteriorated	6%	–	15%
Expired	8%	12%	–		5.9%

*NA- Not available.

Among children, who were available for follow-up, all continued to be independent for their daily activities, highlighting the slowly progressive nature of the CMs irrespective of subtype. Similar observations were noted in studies from Denmark and the United Kingdom [9, 10]. In contrast to this, a higher mortality rate was noted in a study from London, especially those with neonatal onset and specific subtypes like Congenital myopathy-2A/2B/2 C (ACTA1) and X-linked centronuclear myopathy (MTM1), probably due to severity of illness and associated respiratory dysfunction and infections. This suggests a hypothesis that those CMs with later onset tend to have slow progression.

The strengths of this study are (i) This is the first Indian study on phenotypic and genotypic characterization of genetically confirmed CM patients while also describing the follow-up and natural history, (ii) one of the large cohorts of various sub-types of genetically confirmed CM, reported from Asian region, and (iii) This study also emphasizes the utility of genetic testing in children with suspected congenital myopathy.

The study limitations include (i) inherent flaws of a retrospective study like lack of uniformity in clinical assessments, data collection and short follow up, (ii) detailed muscle charting and functional testing could not be done as majority were young, not co-operative, (iii) assessment of co-morbidities like cardiac involvement, respiratory function and cognitive levels in all the children were not available, (iv) current pediatric cohort cannot be considered as a reflection of the prevalence of CM in the country as majority of patients were from southern parts of the country which could be due to institutional bias,(v) long term follow up with inclusion of adults would have been ideal for better understanding of disease course, (vi) segregation analysis and functional validation of the variants were not done and (vii) lack of histopathological confirmation in few cases.

FUTURE DIRECTIONS

The current study provides in-depth insights into the genetic spectrum of CMs from the Indian subcontinent. The advances in genetic therapeutics seen particularly in the field of neuromuscular diseases provide ample signs that patients with CM may also be future candidates. Knowledge about the genetic spectrum and maintenance of CM registries from India will ensure trial readiness as and when novel gene therapy options become available. With a unique gene pool that is ethnically and genetically diverse, multicentric registries from across the country would be the way forward.

CONCLUSIONS

Our study gives an insight into the clinical presentations and genetic mutational patterns of congenital myopathy from Indian sub-continent. Majority had symptom onset by 2 years of age with centronuclear myopathy being the most common histological classification. Mutations in RYR1 followed by DNM2 genes were the leading pathogenic variants identified. Majority were independent for their activities of daily living during the last follow-up, highlighting the fact that the disease has slow progression irrespective of the genotype.

ACKNOWLEDGMENTS – FUNDING

Nil.

CONFLICT OF INTEREST

Nil.

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