An interesting report of POPDC3 limb girdle muscular dystrophy R26 from India

Abstract

Introduction:

Popeye domain containing 3 (POPDC3) gene encodes a protein involved in membrane trafficking and is highly expressed in skeletal muscles. POPDC3 pathogenic variants are associated with LGMDR26. Only a few reports of POPDC3 LGMD exist worldwide and none from India. Herein, we describe the first case of POPDC3 LGMD26.

Methods:

This is a case report from a neurology referral center in India. All the clinical, laboratory and electrophysiological data were collected from the medical records.

Results:

A 34-year-old man born to non-consanguineous parents presented with progressive proximal weakness of lower limbs from 22 years of age. He developed calf muscle pain and recurrent falls on walking for 7 years. He had atrophy of calves (medial gastrocnemius more than lateral) along with weakness of hip extensor, adductors and knee flexors and normal upper limb power, resembling Miyoshi myopathy. Serum creatine kinase ranged from 3524 to 6531 U/L. Muscle MRI showed selective atrophy of gluteus maximus, quadriceps femoris, semimembranosus and gastrocnemius with sparing of rectus femoris, gracilis and sartorius. Muscle biopsy done elsewhere and reported to show dystrophic features and immunohistochemistry showed positive staining for dystrophins and sarcoglycans. Clinically the possibility of LGMDR2/Dysferlinopathy, was considered and whole exome sequencing was done which revealed a novel homozygous pathogenic nonsense premature termination codon (PTC) variant (NM_022361.5) c.316C > T (NP_079130.2:) p.Arg106Ter) in exon 2 of POPDC3 gene.

Conclusion:

This is the first report of POPDC3- LGMDR26 from India detected among a large cohort (461 genetically confirmed cases). POPDC3 gene variations should be considered in distal onset LGMDs with markedly elevated serum creatine kinase levels.

Introduction

Limb girdle muscular dystrophies (LGMDs) are a heterogenous group of genetically mediated muscle disorders, characterized by progressive symmetrical weakness of limb girdle muscles associated with elevated serum creatine kinase (CK) level, degenerative changes in muscle magnetic resonance imaging (MRI) and dystrophic features in muscle biopsy. ¹ Currently, there are more than 30 LGMDs described, and majority of them being autosomal recessive in inheritance. ² The various proteins involved in key muscle functions and structure such as sarcolemmal scaffolding and glycosylation, membrane repair and vesicle trafficking are implicated in causation of LGMD. Popeye domain containing (POPDC) family of genes consists of three members: blood vessel epicardial substance (BVES, or POPDC1), POPDC2, POPDC3.^3,4 The POPDC proteins consist of a short amino-terminus portion extracellularly which undergoes n-glycosylation and three transmembrane domains. The cytoplasmic portion consists of Popeye domain and a carboxy- terminus of variable length and isoform specificity. ⁵ POPDC proteins are a novel class of 3′-5′ cyclic adenosine monophosphate (cAMP) effector proteins and also interact with other molecules such as phosphodiesterase 4 (PDE4) and adenylyl cyclase 9 (AC9) involved in cAMP signalling. ⁶ Variations in POPDC1 and POPDC2 predominantly cause cardiac rhythm disturbances.^7,8 LGMDR25 previously described, is associated with homozygous pathogenic variations in BVES/POPDC1 gene.^9,10

LGMD-R26 is a recently described type of LGMD caused by pathogenic variations in POPDC3. To date only 6 families have been described in the English literature on POPDC3 related LGMD. Here we describe the first interesting case of POPDC3 LGMD-R26 from India.

Methods

This is a retrospective study of a patient evaluated at the Neurology department of a quaternary referral center for neurological disorders in South India. Till date we have 461 genetically confirmed LGMD cases and this is the only case with POPDC3 variation identified from this large cohort. Detailed clinical and laboratory reports were collected from the patient medical records. Informed consent was obtained from patient and his parents for publishing the clinical data and photographs with face recognition. Institutional ethics committee approval was obtained for the study [IEC no: NIMH/DO/(BS&NS) 2022].

The patient underwent Magnetic Resonance Imaging (MRI) muscle using a 3 T Aera MRI system from Siemens Healthcare. The MRI sequences employed were axial T1-weighted, T2W and T2W fat-saturated images. The axial sections were obtained at three stations, covering (a) the hip to upper thigh, (b) the upper thigh to knee, and (c) the upper leg to ankle. The slice thickness was set at 5 mm, and the field of view (FOV) measured 330 × 184 mm. Muscles were assessed at pelvis, mid-thigh and leg levels. Muscle fatty infiltration was graded as per Mercuri grading ¹¹ and edema with Stramare grading. ¹² Cardiac function assessment was done with 12-lead Electrocardiogram, 2D Echocardiography and 24 h Holter monitoring. Muscle biopsy report was collected. DNA was extracted from blood and genetic testing was done by whole-exome sequencing (WES) a custom capture kit. The libraries were sequenced to mean depth of >80–100X on Illumina sequencing platform. The FASTQ reads were aligned to human reference genome (GRCh38) using BWA aligner, and variants were called as per the GATK best practices framework for identification of germline variants. Variant annotation was performed using VEP program ¹³ against the Ensembl release 104 human gene model. ¹⁴ In addition to SNVs and small Indels, copy number variants (CNVs) are detected from targeted sequence data using the Exome Depth method. Clinically relevant variants in both coding and non-coding regions were annotated using published variants in literature and a set of diseases databases: ClinVar, OMIM, HGMD, LOVD, DECIPHER (population CNV) and SwissVar. Common variants were filtered based on allele frequency in 1000Genome Phase 3, gnomAD v4.1.0, dbSNP v156, 1000 Japanese Genome, TOPMed (Freeze 8), GenomeAsia 100 K, and our internal Indian population database. Functional effects of the variants were predicted using multiple algorithms such as CADD, PolyPhen-2, SIFT, MutationTaster2 and LRT. Clinically significant variants were used for interpretation and reporting.

Case report

A 34-year-old man from southern India presented with progressive proximal lower limb weakness in May 2024. He noticed difficulty in rising from the floor and climbing stairs at the age of 22 years. He had exertion induced calf muscle pain and had buckling of knees for the past 7 years. There was no history of distal lower limb weakness or upper limb weakness. There was no history of myoglobinuria, cardiac or respiratory symptoms. He was born out of non-consanguineous parentage and there was no family history of similar illness.

On examination he had significant wasting of calves (medial more than lateral) resembling Miyoshi like muscular dystrophy (MMD) with slight wasting of quadriceps (Figure 1) There was mild scapular winging and no contractures. Cranial nerve examination was normal. The muscle power assessment according to modified Medical Research Council (MRC) grading showed normal power of iliopsoas (5), gluteus maximus (4), hip abductors (5), hip adductors (4), quadriceps (4+), hamstrings (4) with normal power of ankle plantar and dorsiflexors. He had normal proximal and distal upper limb power but with significant fatigability on arm abduction. Tendon reflexes were normal. Based on the clinical findings, possibilities of LGMDR2- dysferlinopathy /LGMDR12—anoctaminopathy were considered. Investigations showed elevated serum CK levels of 20–30 fold higher (3524, 5339, 6531 U/L on multiple occasions). ECG and 2D-Echocardiography were normal. 24 h Holter monitoring showed a few episodes of ectopic atrial rhythm. Pulmonary function test was normal. Muscle biopsy of right vastus lateralis reported at another center showed many atrophic fibers, myophagocytosis and membrane attack complex highlighting a few necrotic fibers. Enzyme histochemistry showed type 2 fiber grouping. Immunohistochemistry revealed positive staining for dystrophins and sarcoglycans. Muscle MRI showed selective atrophy of gluteus maximus, quadriceps femoris, semimembranosus and gastrocnemius with sparing of rectus femoris, gracilis and sartorius (Figure 2). WES identified a homozygous pathogenic nonsense variant (NM_022361.5) c.316C > T in exon 2 (chr6:g.105161594G > A), (NP_071756.2: p. Arg106Ter) of POPDC3 gene. This premature termination codon (PTC) variant is not reported in 1000 genomes, as well as our in-house database. The variant is observed in 41/1461876 (AF: 0.00002805) alleles from individuals gnomAD v4.1.0 exomes and 5/152034 (0.00003289) alleles from individuals of gnomAD v4.1.0 genomes in heterozygous state only. South Asian population had the highest allele frequency (6/91076, AF: 0.00006588). The PTC variant is located 2844 bp upstream of the last exon-intron junction of POPDC3, which is highly likely to trigger mRNA degradation through nonsense-mediated decay (Figure 3). The variant is situated in the critical Popeye domain (PFAM ID PF04831) of the protein, which is highly conserved in vertebrates. In addition, the clinical phenotype of the proband matches with that of the disorder caused by pathogenic variants in POPDC3 gene. For these reasons, this variant has been classified as Pathogenic (PM2 PVS1 PP4). Thus, the diagnosis of LGMD-R26 was confirmed (OMIM ID: 618848).

Figure 1.

Clinical images of patient: A—Mild wasting of quadriceps (white arrow). B—wasting of medial > lateral calf muscles (white arrow).

Figure 2.

Muscle MRI images of patient: a – T1 weighted axial MRI images b – STIR axial MRI images At hip level: a1 - symmetrical moderate to severe atrophy of bilateral gluteus muscles, tensor fascia lata with b1 - mild edema noted within the muscles (white arrow- gluteus maximus). At thigh level: a2 - symmetrical severe atrophy of bilateral quadriceps femoris (white arrow in lateralis and medialis vastus muscles) except a3 – only mild atrophy of rectus femoris (white arrow), adductor group of muscles, biceps femoris muscles (arrow head) and moderate atrophy of semimembranosus muscle (white star). b2 – showed peripheral edema of quadriceps femoris (white arrow) with b3 showing edema in semimembranosus (white star). At calf level: a4 - symmetrical severe atrophy with near complete fatty replacement of bilateral gastrocnemius muscles (white arrow). Rest of the muscles were normal. At lower leg level: a5 and b5 - no significant muscle atrophy or signal changes.

Figure 3.

Gene diagram showing the genomic location of the c.316C > T p.(Arg106Ter) mutation in the POPDC3 variant. Coding exons are shown in black blocks. Previously reported ClinVar pathogenic variants are also indicated.

Discussion

We report the first case of POPDC3 related LGMDR26 from India with a homozygous nonsense pathogenic variation. In contrast to variations in POPDC1 and POPDC2 which primarily result in a cardiac phenotype, POPDC3 is expressed more selectively in skeletal muscles resulting in isolated MMD. ⁴ Comparison with previous studies is shown in Table 1. The patient reported in the current study presented with predominant pelvic girdle weakness and exertion induced myalgia of the calves with involvement of medial more than the lateral gastrocnemius. Till date only 6 families with LGMDR26 have been described. The first report of POPDC3 related LGMDR26 was described by Vissing et al. ¹⁵ in 5 patients from 3 families with varied ethnicities and age of onset ranging from 2^nd to early 5^th decade. All these patients had pelvic girdle weakness. Two other reports described patients in 2^nd and 3^rd decades respectively, with limb-girdle weakness, myalgia and markedly elevated serum CK.^16,17 In another report authors have described 4 patients from a consanguineous family, with age of onset ranging from 2^nd to late 4^th decade, with myalgia and only mild pelvic girdle weakness. ¹⁸ Exertion induced myalgia has been an important symptom in most of the cases, similar to the current patient. This is similar to the myalgia reported in LGMDR2/R9 and Facio-scapulo-humeral dystrophy and is hypothesized due to early inflammation or metabolic causes. ¹⁹ Significant cardiac involvement is not reported in most of the studies except in 2 patients described by De Ridder et al., ¹⁸ with asymptomatic nocturnal AV block. Our patient also showed asymptomatic few atrial ectopics. In addition to medial gastrocnemius atrophy described,^15,18 our patient also had other notable features of involvement of gluteus maximus, quadriceps femoris, semimembranosus and lateral gastrocnemius. There was also sparing of gracilis, sartorius and rectus femoris as reported by Vissing et al.,. ¹⁵ The muscle biopsy findings in LGMDR26 ranges from typical dystrophic features ¹⁵ as also noted in the current study to myopathic changes.^17,18 Till date, the described variants are most commonly missense located in the carboxy-terminal domain, specifically in Popeye domain^15,16 and two splice site variants.^17,18 This is the first report of a pathogenic PTC variant in POPDC3. The major patho-mechanism of the missense variants has been proposed and this is due to affection of cAMP affinity or the ability of mutant protein to cause ligand-induced conformational changes. POPDC3 is necessary for muscle structural integrity as suggested by the tail curling and muscle fiber detachment, seen in POPDC3 deficient zebrafish models. ¹⁵ All functions of POPDC3 protein are lost in case of loss of function (LOF) variants. However, which downstream abnormality exactly leads to vulnerability and loss of muscle fibers is yet to be elucidated.

Table 1.

Comparison with previous studies.

S.no	Authors/year	Ethnicity	Age at onset (years)/sex	Clinical presentation	Serum CK (U/L)	Muscle MRI	Muscle biopsy features	Type of mutational variants	POPDC3 pathogenic variants
1	Vissing et al./ 2019	I-1 Denmark	40/M	All had pelvic girdle muscle weakness. Calf hypertrophy in I-1, I-2 & III-2	3500 (mean) 1050–9227 (range)	Fatty atrophy of thigh muscles and medial gastrocnemius with prominent sparing of gracilis, sartorius and rectus femoris	Typical features of dystrophy with increased internal nuclei. EM—normal ultrastructure of myofibrils.	All missense	c.782G > A (p.Arg261Gln)
		I-2 Denmark	≈15/F						c.782G > A (p.Arg261Gln)
		II-1 Iran	25/M						c.464T > A (p.Leu155His)
		III-1 Spain	20/M						c.651A > T (p.Leu217Phe)
		III-2 Spain	35/M						c.651A > T (p.Leu217Phe)
2	Ullah et al/2021	Pakistani	13/M	Pelvic girdle muscle weakness with myalgia	4500–5500 (range)	Not done	Not done	Missense	c.460A > G; (p.Lys154Glu)
3	Zhang et al/2022	Chinese	25/F	Pelvic girdle muscle weakness	525	Mild atrophy of posterior thigh muscles	Myogenic damage with fiber size variability, partial atrophy, and central nuclei.	Splice site	c.486–1G > A (intron-2)
4	De Ridder et al/2023	Belgian	1–14/M 2–25/M 3–36/M 4–38/M 12,3—siblings 4—distant relative	All had Myalgia with exercise intolerance. Only 4—had mild limb weakness Patients 2& 3 had nocturnal episodes of first-degree AV block on Holter monitoring	1025–3938 (range)	Selective muscle atrophy of medial gastrocnemius and posterior thigh muscles were noted only in patient-2	Predominantly myopathic features with fiber-2 predominance and pyknotic nuclear clumps.	Splice site	c.486–6T > A (intron-2)
5	Current study/2024	Indian	34/M	Pelvic girdle muscle weakness with exertional myalgia of calf muscles	3524–6531 (range)	Selective atrophy of gluteus maximus, quadriceps femoris, semimembranosus and gastrocnemius—medialis and lateralis with sparing of rectus femoris, gracilis and sartorius.	Atrophic fibers, myophagocytosis and membrane attack complex highlighting a few necrotic fibers with type-2 fiber grouping.	Nonsense	c.316C > T (p. Arg106Ter)

Footnotes: AV—Atrio-Ventricular, CK—Creatine kinase, EM—Electron Microscopy, F-Female, M-Male, MRI—Magnetic Resonance Imaging.

Conclusion

This is the first case of POPDC3 related LGMDR26 in India. It is important to consider the diagnosis in patients with early calf atrophy and exertion induced myalgia mimicking MMD (dysferlinopathy/anoctaminopathy).

Supplemental Material

Supplemental Material - An interesting report of POPDC3 limb girdle muscular dystrophy R26 from India

Supplemental Material for An interesting report of POPDC3 limb girdle muscular dystrophy R26 from India by Dipti Baskar, Kiran Polavarapu, Ananthapadmanabha Kotambail, Gautham Arunachal, Seetam Kumar Tumulu, Madhulika Kotra, Darshan Gowda, Atchayaram Nalini and Seena Vengalil in Journal of Neuromuscular Diseases