Keywords
ORAI-1 - ophthalmoparesis - congenital myopathy - congenital fiber-type disproportion
Introduction
Calcium ion (Ca2+) is an important signaling molecule for regulating many cellular processes, especially
skeletal muscle contraction. A range of Ca2+ channels and transporters tightly regulate cytosolic Ca2+ concentration for the excitation-contraction coupling of skeletal muscles.[1] Store-operated Ca2+ entry (SOCE) is an important pathway involved in muscle contraction, which in turn
is mediated by two protein families: stromal interaction molecules (STIMs) 1 and 2
of endoplasmic reticulum and ORAI calcium release-activated calcium modulator proteins
forming Ca2+ release-activated calcium (CRAC) channel in the plasma membrane both of which regulate
the SOCE pathway.[2]
[3] ORAI-1 is the most important component of the CRAC channel, forming a hexameric
Ca2+-binding channel. A variety of null and loss-of-function (LoF) variations in ORAI-1 and STIM1 results in a distinct entity called “CRAC channelopathy,” a multisystem disorder
characterized by severe combined immunodeficiency (SCID) with recurrent infections,
autoimmune hemolytic anemia/thrombocytopenia, anhidrotic ectodermal dysplasia, and
nonprogressive muscle hypotonia. Gain-of-function (GoF) mutation causes Stormorken's
syndrome and York platelet syndrome, including bleeding diathesis, miosis, and tubular
aggregate myopathy.[4]
[5] Here, we report for the first time from India a unique homozygous ORAI-1 variation presenting clinically as congenital myopathy and histologically as congenital
fiber-type disproportion (CFTD).
Methods
This retrospective analysis was done at a quaternary neurology center in southern
India. Demographic and detailed clinical assessment data were recorded. Magnetic resonance
imaging (MRI) of the muscle with T1-weighted and T2/short tau inversion recovery sequences
was done to assess the fatty infiltration by modified Mercuri score and edema by Stramare
score.[6]
[7] Histopathological analysis of the muscle biopsy from quadriceps femoris was done
using hematoxylin and eosin, succinic dehydrogenase, and ATPase, and ultrastructural
analysis was performed. Genetic analysis was done by clinical exome sequencing with
coverage of 8332 clinical exome assay genes (critical genes analyzed are given in
[Supplementary Table S1]). DNA extracted from blood was used to perform targeted gene capture using a custom
capture kit. The libraries were sequenced to mean >80 to 100X coverage on Illumina
sequencing platform. The Genome Analysis Toolkit best practices framework for identifying
variants in the sample using Sentieon (v201808.01) were followed.[8] The sequences obtained were aligned to the human reference genome (GRCh37/hg19)
using Sentieon aligner and analyzed using Sentieon to remove duplicates, recalibration,
and realign indels. Sentieon haplotypecaller was used to identify variants that were
relevant to the clinical indication. Gene annotation of the variants was performed
using the VEP program[9] against the Ensemble release 91 human gene model.[10] Common variants are filtered based on allele frequency in 1000 Genome Phase 3, ExAC
(v1.0), gnomAD (v2.1), EVS, dbSNP (v151), 1000 Japanese Genome, and our internal Indian
population database. The nonsynonymous variants effect is calculated using multiple
algorithms such as PolyPhen-2, SIFT, MutationTaster2, and LRT. Only nonsynonymous
and splice site variants found in the clinical exome panel consisting of 8,332 genes
were used for clinical interpretation. The pathogenic variants are presented according
to standards of American College of Medical Genetics and Genomics (ACMG).[11] For the Sanger sequencing and segregation analysis of the second variant of unknown
significance, polymerase chain reaction (PCR) was performed using primers designed
to amplify the genomic region spanning the targeted variant in the exon. PCR products
were confirmed by gel electrophoresis followed by treatment with ExoSAP to digest
unutilized primers. The amplicons were subjected to cycle sequencing PCR using BigDye
Terminator v3.1 kit as per manufacturer instructions. Sanger sequencing was performed
on SeqStudio Genetic Analyzer. The variant at the targeted locus was ascertained by
visual inspection of the electropherogram, as well as comparing it with the reference
sequence. Informed consent for clinical details and patient figure publication was
obtained from the patient.
Results
An 18-year-old woman born to consanguineous parents presented with a 2-and-a-half-year
history of pain in the thighs and arms. Six months later, she developed progressive
lower limb proximal muscle weakness and in the next year noticed difficulty in walking.
There was no distal lower limb or upper limb weakness, ocular symptoms, or bulbar
weakness. She was noticed to be a slow runner since childhood. She also had a squint,
which was noticed by the parents. She did not have recurrent infections. There was
no significant family history.
On examination, she had a slender habitus, an elongated long face, and ophthalmoparesis.
Hypotonia of lower limbs was noted. According to the modified Medical Research Council
grading, muscle strength of the neck and upper limbs was grade 4. Bilateral hip flexion,
extension, adduction, abduction, knee flexion, and extension were grade 3; ankle dorsiflexion
and plantar flexion were grade 4. Tendon reflexes and sensory examination were normal.
She had a waddling gait.
Her investigations showed normal complete blood count, serum creatine kinase, and
renal and liver functions. Muscle MRI of lower limbs showed fatty infiltration with
Mercuri scale grade 3 (washed-out appearance) in glutei and grade 2b (late moth-eaten
appearance) in quadriceps femoris muscles ([Fig. 1]). Histopathological analysis of muscle biopsy from quadriceps femoris showed type
1 fiber predominance which were hypotrophic with the absence of rods or cores in electron
microscopy suggestive of CFTD ([Fig. 2A–D]). On electron microscopy, a few fibers with focal Z-band disorganization and aggregation
of Z-band material resembling minicore-like areas were observed. There was an absence
of rods, cores, and tubular aggregates ([Fig. 2E–H]).
Fig. 1 Muscle MRI of the patient: (A) and (B) show muscle MRI–T2 sequences that show fatty
infiltration with atrophy of the glutei (blue arrow) and quadriceps (yellow arrow).
MRI, magnetic resonance imaging.
Fig. 2 Histopathological images of muscle biopsy of the patient: (A) The hematoxylin and
eosin–stained section shows an admixture of larger myofibers with several smaller
fibers. (B), (C), and (D) reveal the more numerous hypotrophic, type 1 fibers that
are dark on succinic dehydrogenase stain (B), light on ATPase 9.4 (C), and dark on
ATPase ph4.6. Note somewhat fairly uniform size of the type 1 fibers. Ultrastructure
of muscle biopsy: (E) Low power view of two adjoining myofibers separated by endomysial
connective tissue. (F) A portion of a muscle fiber showing a subsarcolemmal myonucleus
and focal disorganization of a myofilament (red star). No abnormal subsarcolemmal
tubular aggregates or other deposits are seen. (G) Transverse and (H) longitudinal
sections displaying disorganization of myofilamentous architecture with Z-band streaming
and aggregation (red star). (Magnification included.)
A novel pathogenic homozygous premature termination codon in exon 1 of the ORAI-1 gene NC_000012.12 (NM_032790.3): c.205G > T; NP_116179.2: p.Glu69Ter (ACMG criteria:
PM2 PVS1 PP4 as per ACMG guidelines[11]) was detected at a depth of 228x ([Fig. 3]). This variant is novel (not reported in any individuals) in gnomAD and has also
not been reported in the 1000 Genomes, ExAC, TOPMed, and our internal databases. This
variant is predicted to cause loss of normal protein function through protein truncation.
This is a stop-gained variant that occurs in an exon of ORAI-1 upstream of where nonsense-mediated decay is predicted to occur. There are seven
downstream pathogenic LoF variants, with the furthest variant being 200 residues downstream
of this variant. This indicates that the region is critical to protein function. The
p.Glu69Ter variant is an LoF variant in the gene ORAI-1, which is intolerant of LoF variants, as indicated by the presence of existing pathogenic
LoF variants NP_116179.2:p.S49Pfs*52 and NP_116179.2:p.S71Hfs*17. Sanger validation
and segregation analysis revealed the variant to be segregating in a heterozygous
state in the asymptomatic parents. In addition, the proband's clinical phenotype matches
that of the disorder caused by pathogenic variants in the ORAI-1 gene. For these reasons, this variant has been classified as pathogenic.
Fig. 3 Electropherogram of patient and parents showing homozygous variant c.205G > T (p.Glu69Ter)
in ORAI-1 gene.
Discussion
The clinical features described in our patient were suggestive of a slowly progressive
congenital myopathy, which revealed features of CFTD on muscle biopsy with ophthalmoparesis.
Congenital myopathies are clinically, histologically, and genetically heterogeneous
structural disorders of skeletal muscles. The classification of congenital myopathies
is primarily based on muscle biopsy features and, recently, on genetic characterization.[12] CFTD is a type of congenital myopathy defined histologically by disproportionately
smaller and numerous type 1 fibers compared with type 2 fibers in the absence of other
abnormal features such as rods or cores. It has an onset of symptoms mostly in the
first decade of life with a very slowly progressive course. Clinically affected children
present with mild facial dysmorphism, delayed motor milestones, muscle hypotonia,
absent tendon reflexes, and limb-girdle type of muscle weakness.[13] These characteristic features were seen in our patient. Interestingly, our patient
also had ophthalmoparesis, which has been previously described and considered specific
for RYR-1-associated CFTD.[14] Several genotypic variations have been described in CFTD. The most common is an
autosomal dominant or recessive mutation in α tropomyosin-3 (TPM-3),[15] followed by ryanodine receptor (RYR-1) variation.[14] Other variations described include selenoprotein-N (SEPN-1), α-actin-1 (ACTA-1), myosin heavy chain-7 (MYH-7), and tropomyosin-2 (TPM-2).[12]
Most of the LoF ORAI-1 variations are due to frameshifts, splice site defects, or substitutions involving
the transmembrane domains and occur as null variations that interfere with protein
expression, folding of α-helical transmembrane domains, and protein stability.[4] SCID-like immunodeficiency is common and noted in most patients with ORAI-1 LoF
variation, characterized by recurrent and life-threatening viral, bacterial, and fungal
infections.[4]
[5] However, the presence of autoimmunity is less common with ORAI-1 than with STIM1 variations.[5]
[16] The absence of obvious manifestations of nonmuscular features of CRAC channelopathy,
such as ectodermal defects and immune abnormalities, can be noted in patients with
heterozygous LoF ORAI-1 variants owing to retained residual SOCE function.[4]
[17] However, it is interesting that patient in the current study presented with a pure
muscular phenotype despite a homozygous ORAI-1 variant which has not been reported previously.
Myopathies associated with ORAI-1 variations have been described with both LoF and GoF mutations. The nonprogressive
muscle hypotonia of LoF mutation of ORAI-1 is characterized by poor muscle strength and endurance since infancy, including respiratory
muscle weakness and hypernasal voice.[5]
[16] Frequently, muscle hypotonia is associated with iris hypoplasia and mydriasis.[5]
[18] Muscle biopsy shows nonspecific findings such as atrophy of type 2 fibers with a
predominance of type 1 fibers.[5] To date, about eight different LoF mutations have been described in ORAI-1.[19] Ophthalmoparesis reflecting the lack of concerted eye muscle contraction due to
a defective SOCE pathway has been described in the GoF variation of ORAI-1.[20]
[21] No previous reports of LoF ORAI-1 variants are associated with CFTD and ophthalmoparesis,
as noted in our patient ([Table 1]).
Table 1
Comparison of previous studies on ORAI-1 myopathy
|
S. no.
|
Authors (year of study)
|
No. of patients
|
Age at onset
|
Clinical presentation
|
Muscle MRI
|
Muscle biopsy
|
Genotypic features
|
|
Infections
|
Autoimmunity
|
Myopathy
|
Others
|
|
1
|
Lacruz and Feske (2015)[4]
|
1
|
<1
|
+
|
+
|
|
|
−
|
−
|
c.271C > T (p.Arg91Trp)
|
|
2
|
McCarl et al (2009)[5]
|
6
|
<1
|
+1/2
+
+
|
−
+
−
|
Congenital muscular hypotonia in all
|
Ectodermal anomalies (all three genotypes), mydriasis
|
–
|
Variation in muscle fiber size with a predominance of type 1 fibers and atrophic type
2 fibers (c.271C > T)
|
c.271C > T (p.Arg91Trp) (2)
p.Ala88SerfsX25 (1)
c.308C > A (p. Ala103Glu)/c.581T > C (p. Leu194Pro) (1)
Not done in two
|
|
3
|
Maul-Pavicic et al (2011)[22]
|
2
|
<1
|
+
|
+
|
Congenital muscular hypotonia
|
Ectodermal anomalies
|
–
|
–
|
c.271C > T (p.Arg91Trp)
|
|
4
|
Chou et al (2015)[23]
|
1
|
<1
|
+
|
+
|
Congenital muscular hypotonia
|
–
|
–
|
–
|
c.493_494insC (p.H165Pfs)
|
|
5
|
Lian et al (2018)[24]
|
4
|
<1
|
+
|
+
Absent (p.V181SfsX8)
|
Congenital muscular hypotonia
|
Ectodermal anomalies
|
–
|
Predominance of type 1 fibers and atrophic type 2 fibers (p.V181SfsX8)
|
p.V181SfsX8, c.581T > C (p. Leu194Pro), c.292G > C (p. Gly98Arg)
|
|
6
|
Our study (2022)
|
1
|
15–16
|
–
|
–
|
CFTD and ophthalmoparesis
|
–
|
Fatty infiltration of glutei and quadriceps femoris
|
CFTD—type 1 fiber predominance which was hypotrophic with absence of rods or cores
in electron microscopy
|
c.205G > T (p.Glu69Ter)
|
Abbreviations: CFTD, congenital fiber-type disproportion; MRI, magnetic resonance
imaging.
Conclusion
We present an extremely rare case of slowly progressive myopathy and ophthalmoparesis
with CFTD morphology on histology due to ORAI-1 variation. Though ORAI-1 is expressed in many tissues, our patient manifested a limited phenotype of only
congenital myopathy. Further functional studies are required to identify the modulators
of disease expression and reveal new therapeutic avenues. The current case expands
the clinical and histological spectrum of ORAI-1-associated disorders.
