Introduction: Recessive mutations in the CAPN3 gene can lead to limb-girdle muscular dystrophy recessive 1 (LGMD R1). Targeted next-generation sequencing facilitates the discovery of new mutations linked with disease, owing to its ability to selectively enrich specific genomic regions. Methods: We performed targeted next-generation sequencing of all exons of the CAPN3 gene in 4 patients with sporadic limb-girdle muscular dystrophy (LGMD) and further analyzed the effects of the novel identified variant using various software tools. Results: We found 5 variants in CAPN3 gene in 4 patients, c.82_83insC (insertion mutation) and c.1115+2T>C (splicing mutation) are reported for the first time in CAPN3 (NM_000070.2). The bioinformatics analysis indicated that these two novel variants affected CAPN3 transcription as well as translation. Discussion: Our findings reveal previously unreported splicing mutation and insertion mutation in CAPN3 gene, further expanding the pathogenic gene profile of LGMD.

Limb-girdle muscular dystrophy (LGMD) is a group of hereditary muscular diseases characterized by weakness and muscle atrophy in the pelvic, shoulder girdle muscles, and proximal limb muscles, which were proposed by Walton and Nattrass in 1954 [1]. According to the 229th ENMC international workshop, LGMD is classified into autosomal dominant forms (LGMD D) and autosomal recessive forms (LGMD R), and the latter forms predominate in terms of both number of subtypes and individual prevalence [2]. LGMD R1 (previously known as LGMD2A) was first identified in a population on the Réunion Island by Richard et al. [3], which usually occurs in the second decade of life but can vary from 2.5 to 50 years old [4, 5]. The phenotypic manifestations of LGMD R1 are similar to other LGMDs with symmetrical and progressive weakness of girdle and proximal limb muscles [6]. According to the distribution of affected muscle and the age at onset, LGMD R1 could be divided into three subtypes: pelvi-femoral phenotype, scapula-humeral phenotype, and hyperCKemia [7, 8].

LGMD R1 is caused by mutations in CAPN3 gene, which is located in the chromosomal region 15q15.1-q21.1 and contains 24 exons [9]. It encodes the calpains 3 protein, a skeletal-muscle-specific calpain that processes substrates through limited and specific proteolysis to facilitate Ca2+ signal transduction and regulate various protein functions in cells [10, 11]. The development of targeted next-generation sequencing greatly advanced the identification of CAPN3 variants in recent years. To date, more than 500 unique pathogenic or likely pathogenic variants of CAPN3 have been reported [12]. Any types of mutation of CAPN3 can lead to the occurrence of LGMDR1, such as missense mutation, frameshift mutation, nonsense mutation, deletions/insertion, splice site mutation. Among these variants, missense mutations were the most common [13, 14]. Moreover, different types of CAPN3 gene mutations in LGMD R1 patients have been extensively studied across various countries, highlighting substantial regional and national differences. For instance, while the variant c.2120A>G is predominantly seen in China, c.550delA is more common among European populations [15].

In the present study, we applied target next-generation sequencing in four non-consanguineous Chinese families with preliminary clinical diagnoses of calpainopathy. This study expands the genotypic and phenotypic spectrum of LGMD R1.

Patients

This retrospective study consisted of 4 non-consanguineous Chinese LGMD patients who were referred to the neurology department of the First Affiliated Hospital of Soochow University. The main symptoms of these patients were proximal limb weakness and atrophy, the initial diagnosis depends on sufficient clinical assessment evidence (family history, age of onset, typical signs, muscle MRI, blood creatine kinase), and the final diagnosis depends on genetic test results. It is noteworthy that their family members did not present any similar signs or symptoms.

Genetic Analysis

Genomic DNA samples were isolated from peripheral blood using DNA Isolation Kit (CW2553, CWBio, Taizhou, China) and their concentrations were determined by Qubit dsDNA HS Assay Kit (Q32851, Invitrogen, Carlsbad, CA, USA). The quality control of samples was performed by agarose gel (1%) electrophoresis. Hybridization was performed using the IDT xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc., USA). Targeted DNA samples were sequenced by the Illumina NovaSeq 6000 System (Illumina, USA), and quality control was applied to the raw data (stored in FASTQ format) by Illumina Sequence Control Software (SCS). High-quality data were aligned to the human reference genome sequence hg19 using Burrows-Wheeler Aligner (v0.7.15) [16]. Consensus single nucleotide variants and insertion/deletions were filtered via Genome Analysis Toolkit v3.6 [17]. All single nucleotide variants and insertion/deletions information was annotated by the software ANNOVAR4. The pathogenicity of candidate mutations was analyzed based on the American College Medical Genetics and Genomics (ACMG) guidelines [18]. The CAPN3 mutations were finally selected according to their clinical relevance and pathogenicity.

Bioinformatic Analysis of Variant

Then, we use Splice AI (https://spliceailookup.broadinstitute.org/) [19] and Netgene2 (https://services.healthtech.dtu.dk/services/NetGene2-2.42/) [20] to predict the change of splice site of the variant. We utilized varSEAK Online (https://varseak.bio/) [21] to examine the sequence alterations following mutation and employed Adobe Illustrator to depict the structural diagram post-mutation. We used PyMOL software (http://www.pymol.org) to analyze the structure of calpain 3 in wild type and variants of c.82_83insC (p. Q28Pfs*6).

Clinical Presentation

All patients presented with proximal symmetric muscle weakness in upper or lower extremities in limb-girdle, atrophy, scapular winging, and hyperCKemia. The clinical presentation of all probands is summarized in Table 1. None of the individuals appeared to be dependent on a wheelchair or unable to walk. Additionally, none of them exhibited any cardiac or respiratory symptoms. Muscle MRI studied showed diffuse muscle atrophy at different extent, and fatty replacement of lower limbs (P1 with missing information, shown in Fig. 1). Laboratory test results showed elevated serum creatine kinase levels (shown in Table 1).

Table 1.

Clinical and biological observations for the patients of the four families harboring the CAPN3 variant

PatientsP1P2P3P4
Sex Male Male Female Male 
Family history Yes No No No 
Age at onset, years 10 25 35 29 
Age at exfrom 4 unrelated amination, years 22 65 48 33 
Weakness Low limbs Low limbs Proximal limbs Low limbs 
Atrophy Proximal limbs Low limbs Proximal limbs Low limbs 
Scapular winging Yes No Yes No 
Walking aids Yes Yes Yes Yes 
Serum CK, U/L 2,769 1,154 549.9 12,563 
PatientsP1P2P3P4
Sex Male Male Female Male 
Family history Yes No No No 
Age at onset, years 10 25 35 29 
Age at exfrom 4 unrelated amination, years 22 65 48 33 
Weakness Low limbs Low limbs Proximal limbs Low limbs 
Atrophy Proximal limbs Low limbs Proximal limbs Low limbs 
Scapular winging Yes No Yes No 
Walking aids Yes Yes Yes Yes 
Serum CK, U/L 2,769 1,154 549.9 12,563 
Fig. 1.

Muscle MRI of patients.

Fig. 1.

Muscle MRI of patients.

Close modal

Genotypes of Patients

Targeted next-generation sequencing was performed in all 4 probands. P1 carried a homozygous pathogenic variant, whereas P2, 3, and 4 had a compound heterozygous genotype as shown in Table 2. Totally five CAPN3 mutations have been identified (1 nonsense mutation, 1 missense mutation, 1 insertion, and 2 splice site mutations) (shown in Fig. 2). In these mutations, c.2120A>G, c.1194–9A>G, and c.734dup have been reported in previous studies [15, 22], while c.1115+2T>C and c.82_83insC were discovered and reported for the first time. Variant c.1115+2T>C result in a change in the splice site of intron 8, and c.82_83insC in exon 1 of the CAPN3 gene results in a translational frameshift of 6 amino acids and formation of a premature termination codon (shown in Table 2). In silico analysis and the ACMG Guidelines revealed that both c.1115+2T>C and c.82_83insC can be rated as pathogenic variants.

Table 2.

List of CAPN3variants in the proband

PatientsChr-startcDNAAmino acidExonZygosityFrequencyClassificationACMG evidenceClinVar
P1 chr15-42686541 c.1115+2T>C Splice-5 Intron 8 Hom Pathogenic PSV1, PM2, PP3 Yes 
P2 chr15-42681225 c.734dup p.S246* Exon 5 Het Pathogenic PSV1, PM2, PM5, PP3 No 
chr15-42702630 c.2120A>G p. D707G Exon 20 Het 0.00015 Pathogenic PSV1, PM2, PP3 Yes 
P3 chr15-42652082 c.82_83insC p. Q28Pfs*6 Exon 1 Het Pathogenic PSV1, PM2, PP3 No 
chr15-42702630 c.2120A>G p. D707G Exon 20 Het 0.00015 Pathogenic PSV1, PM2, PP3 Yes 
P4 chr15-42691681 c.1194–9A>G Splice Intron 9 Het 0.00001 Pathogenic PSV, PS1, PM2, PP3 Yes 
chr15-42702630 c.2120A>G p. D707G Exon 20 Het 0.00015 Pathogenic PSV1, PM2, PP3 Yes 
PatientsChr-startcDNAAmino acidExonZygosityFrequencyClassificationACMG evidenceClinVar
P1 chr15-42686541 c.1115+2T>C Splice-5 Intron 8 Hom Pathogenic PSV1, PM2, PP3 Yes 
P2 chr15-42681225 c.734dup p.S246* Exon 5 Het Pathogenic PSV1, PM2, PM5, PP3 No 
chr15-42702630 c.2120A>G p. D707G Exon 20 Het 0.00015 Pathogenic PSV1, PM2, PP3 Yes 
P3 chr15-42652082 c.82_83insC p. Q28Pfs*6 Exon 1 Het Pathogenic PSV1, PM2, PP3 No 
chr15-42702630 c.2120A>G p. D707G Exon 20 Het 0.00015 Pathogenic PSV1, PM2, PP3 Yes 
P4 chr15-42691681 c.1194–9A>G Splice Intron 9 Het 0.00001 Pathogenic PSV, PS1, PM2, PP3 Yes 
chr15-42702630 c.2120A>G p. D707G Exon 20 Het 0.00015 Pathogenic PSV1, PM2, PP3 Yes 

CAPN3 gene reference genome version: GRCh37/hg19, NM_000070.2. The Genome Aggregation Database (GnomAD) version: v2.1.1. Hom, homozygous; het, heterozygous. Frequency results are based on genome aggregation database. Variation Classification refers to the American College of Medical Genetics and Genomics guidelines. ACMG evidence: PVS1-null variant (nonsense, frameshift, canonical ± 1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where loss of function is a known mechanism of disease; PS2 de novo (both maternity and paternity confirmed) in a patient with the disease and no family history; PS3, well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (a variant performed <40% in a minimum of 2 functional assays in our study; moderate PS3 well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product (a variant performed <40% in only 1 of our assays); PM1, variants located in a mutational hotspot and/or critical and well-established functional domain; PM2, absent from controls (or at an extremely low frequency if recessive) as shown in the Exome Sequencing Project; PM5, a novel missense change at an amino acid residue where a different missense change was determined to be pathogenic and has been seen before; PP1, cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease; PP3, multiple lines of computational evidence that support a deleterious effect on the gene or gene product (evolutionary impact, splicing, etc.); BS2, observed in a healthy, adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age; BS3, well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing; BS4, the lack of segregation in affected family members.

Fig. 2.

Sequence chromatograms of the CAPN3 variants.

Fig. 2.

Sequence chromatograms of the CAPN3 variants.

Close modal

Prediction of Novel CAPN3 Variants c.1115+2T>C and c.82_83insC

For c.1115+2T>C, the results of Splice AI showed that the donor splice site of the intron 8 has moved 2bp backward compared with the reference sequence (shown in Fig. 3a). The results of Netgene2 showed that the neural network donor site score of the original splice site decreased after the mutation, while the neural network donor site score of “GT” sequence in intron 8 increased (shown in Table 3). This is consistent with the results of Splice AI and varSEAK Online. In addition, the observation that the neural network coding score at the splice donor site decreased from 0.945 to 0.1 after the mutation strongly suggests that the mutation has disrupted the ability of the splicing enzyme to accurately cut the intron at the correct location and facilitate the correct connection of the exons. This disruption in splicing can have significant consequences for the formation of mature mRNA molecules, potentially leading to aberrant protein products or even loss of gene function (shown in Fig. 3b). The structural analysis of calpain 3 in wild type and variants of c.82_83insC (p. Q28Pfs*6) showed that the mutation is a novel frameshift variant, which caused early termination of protein synthesis and loss of protein function after the catastrophe point (shown in Fig. 4).

Fig. 3.

Software prediction results of splice site mutation. a Top: reference and variant of partial sequence of CAPN3gene exons 8, 9, and intron 8 (purple: splice site). Middle: schematic of CAPN3 gene exon 8, 9 and intron 8. Bottom: schematic of CAPN3 gene structure. b The figure “Coding” is the activity of an ensemble of coding predicting networks, values close to 0.0 indicate intron region, while values close to 1.0 indicate exon. Black: reference; red: variant (results from NetGene2-2.42).

Fig. 3.

Software prediction results of splice site mutation. a Top: reference and variant of partial sequence of CAPN3gene exons 8, 9, and intron 8 (purple: splice site). Middle: schematic of CAPN3 gene exon 8, 9 and intron 8. Bottom: schematic of CAPN3 gene structure. b The figure “Coding” is the activity of an ensemble of coding predicting networks, values close to 0.0 indicate intron region, while values close to 1.0 indicate exon. Black: reference; red: variant (results from NetGene2-2.42).

Close modal
Table 3.

Prediction results of NetGene2 of c.1115+2T>C

34842G34826G
normalmutationnormalmutation
Neural network donor site score 0.945 0.100 0.964 0.986 
Neural network acceptor site score 
Neural network coding score 0.059 0.126 0.010 0.015 
Neural network frame score 
90% sensitivity level cutoff value for donor site predictions 0.689 0.650 0.744 0.704 
90% sensitivity level cutoff value for acceptor site predictions 0.521 0.544 0.487 0.511 
Confidence of the donor site prediction 0.846 0.079 0.832 0.895 
Confidence of the acceptor site prediction 
34842G34826G
normalmutationnormalmutation
Neural network donor site score 0.945 0.100 0.964 0.986 
Neural network acceptor site score 
Neural network coding score 0.059 0.126 0.010 0.015 
Neural network frame score 
90% sensitivity level cutoff value for donor site predictions 0.689 0.650 0.744 0.704 
90% sensitivity level cutoff value for acceptor site predictions 0.521 0.544 0.487 0.511 
Confidence of the donor site prediction 0.846 0.079 0.832 0.895 
Confidence of the acceptor site prediction 
Fig. 4.

Predicted mutational impact of p.Q28Pfs*6 on the CAPN3 protein structure. Image representation of CAPN3 structures in its wild type (residues 1–821) and the p.Q28Pfs*6 (residues 1–821) mutants (AlphaFold code: AF-P20807-F1). Red, yellow, and green are marked helices, sheets, and loops, respectively. The translation of loop structure after mutation is colored blue. Residues Q28 and their mutation (P28) are shown as purple sticks. P26 and K30, which make contact with Q28 or P28, are shown as orange sticks. Lower panel, the mutation of Q28 to P28 mutations disrupts the hydrogen bond between amino acids 26 and 30, destabilizing the original hydrogen bonding framework and interfering with normal conformational transmission.

Fig. 4.

Predicted mutational impact of p.Q28Pfs*6 on the CAPN3 protein structure. Image representation of CAPN3 structures in its wild type (residues 1–821) and the p.Q28Pfs*6 (residues 1–821) mutants (AlphaFold code: AF-P20807-F1). Red, yellow, and green are marked helices, sheets, and loops, respectively. The translation of loop structure after mutation is colored blue. Residues Q28 and their mutation (P28) are shown as purple sticks. P26 and K30, which make contact with Q28 or P28, are shown as orange sticks. Lower panel, the mutation of Q28 to P28 mutations disrupts the hydrogen bond between amino acids 26 and 30, destabilizing the original hydrogen bonding framework and interfering with normal conformational transmission.

Close modal

In this study, we identified 4 patients with LGMD R1 and found a total of five variants in the CAPN3 gene. Among these variants, c.2120A>G is the most common variant in the Chinese population [15]. In our report, the c.734dup variant also occurs on exon 5 of the CAPN3 gene but turns Serine residue in position 246 of the protein to ochre termination codon, producing a truncated protein (p.S246*). This is similar to the previous report (c.734dupC, p.S246Ter) [22]. This suggests that c.734dup is equally likely to be pathogenic. The c.82_83insC is not recorded in either gnomAD or ClinVar. Although C.1115+2T>C has been recorded as likely pathogenic in ClinVar and pathogenic in LOVD. However, clinical cases of the C.1115+2T>C variant have not yet been published to our knowledge.

As a group of rare muscle genetic diseases, LGMD has high genetic heterogeneity [23]. The phenotypes of LGMDR1 demonstrate variability in some degree related to gender and mutation type, the nonsense mutations lead to a more severe phenotype, while compound heterozygous patients are the least affected [24]. In our study, the 3 probands carrying compound heterozygous mutations all presented with mature adult disease onset and mild clinical manifestation. Compared with these 3 cases, both the age at onset and the appearance time of scapular winging are earlier in case 1 who carry the homozygous mutation. This could be attributed to the complex heterozygote scenario where two different mutated proteins are produced, and their interaction facilitates compensation for each other [25].

Donor and acceptor splice site variants typically disrupt protein function [26], and dysfunction variants in CAPN3are known to be pathogenic [27]. mRNA splicing analysis showed the c.1115+2T>C variant in the CAPN3gene disrupts the canonical splice donor site in intron 8, resulting in the exonization of part of intron sequences, which likely leads to an abnormal protein product. Besides, the retained sequences also can serve as abnormal information mediating mRNA decay. Although we did not perform transcriptional studies to confirm this prediction, the data we surmised suggested that this mutation is pathogenic.

When a number of nucleotides, not divisible by three, is inserted into the DNA, it results in the misreading of downstream codons. This kind of insertion mutation disrupts the reading frame, leading to potential loss of protein function [28]. Extra-base insertion leads to frameshift which causes amino acid translation to stop prematurely and produce nonfunctional calpains 3. The ClinVar database has recorded 65 insertional mutations in CAPN3, most of which are pathogenic mutations. Herein, we found a compound heterozygous mutation (c.82_83insC, p. Q28Pfs*6; c.2120A>G, p.D707G) of CAPN3 gene in Chinese adult with LGMDR1. CAPN3 is Ca2+-dependent cysteine protease composed of 821 amino acid residues, the 28th amino acid is located in the loop region of protein sequence [29]. Using the PyMOL software, we found the mutation of Gln-28 disrupts hydrogen bond formation between residues 26 and 30, leading to the instability of the original hydrogen bonding framework.

Although we reported two novel pathogenic mutations in LGMD patients for the first time, the shortcoming of this study is that the influence of mRNA level after mutation is not further verified through appropriate experiments. DNA sequence variants affect pre-mRNA processing through exon skipping, cryptic splicing, intron inclusion, leaky splicing, and other methods [30]. Therefore, it is difficult to accurately predict the specific cause of pathogenicity by artificial intelligence. Experimental mRNA and cDNA analysis are necessary for accurately assessing the effects of splicing mutation [31].

Totally 5 mutations have been found in our four LGMDR1 probands, three of the four probands carrying the hotspot c.2120A>G mutation, which is consistent with the previous reports. Another two mutations, c.734dup, p.S246* and c.1194-9A>G, were previously reported in Chinese LGMDR1 patients. More importantly, two novel mutations, c.1115+2T>C and c.82_83insC in the CAPN3 gene, have been identified in patients who were clinically diagnosed as LGMDR1. Although the mRNA and protein levels of calpain 3 were not further verified, bioinformatics analysis showed that these mutations resulted in abnormal CAPN3 protein synthesis, thereby providing valuable information on the diagnosis of LGMDR1 and expanding the spectrum of disease-causing CAPN3mutations.

Authors are grateful to all the families for their participation in this study.

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was reviewed and approved by the First Affiliated Hospital of Soochow University (NO. 2023082). All participants provided written informed consent to participate in this study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This study was supported by the National Natural Science Foundation of China (Nos. 82071300 to Q.F. and 82001219 to D.D.) and the Natural Science Foundation of Jiangsu Province (BK20190183).

Lulu Zhang, Yi Zhang, and Chunru Han participated in the material preparation and medical data collection. Xiuying Cai and Liqiang Yu provided genetic information of patient. Huan Qi performed bioinformatics analysis of mutations with the support of Jianhua Jiang and Juean Jiang. The first draft of the manuscript was written by Lulu Zhang. Qi Fang and Dongxue Ding contributed to the interpretation of the findings and critical revision of the manuscript. All authors contributed to the study conception and design and read and approved the final manuscript.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author (D.X.D.) upon reasonable request.

1.
Walton
JN
,
Nattrass
FJ
.
On the classification, natural history and treatment of the myopathies
.
Brain
.
1954
;
77
(
2
):
169
231
.
2.
Straub
V
,
Murphy
A
,
Udd
B
;
LGMD workshop study group
.
229th ENMC international workshop: limb girdle muscular dystrophies - nomenclature and reformed classification Naarden, The Netherlands, 17-19 March 2017
.
Neuromuscul Disord
.
2018
;
28
(
8
):
702
10
.
3.
Richard
I
,
Roudaut
C
,
Saenz
A
,
Pogue
R
,
Grimbergen
JE
,
Anderson
LV
, et al
.
Calpainopathy-a survey of mutations and polymorphisms
.
Am J Hum Genet
.
1999
;
64
(
6
):
1524
40
.
4.
Dincer
P
,
Leturcq
F
,
Richard
I
,
Piccolo
F
,
Yalnizoglu
D
,
de Toma
C
, et al
.
A biochemical, genetic, and clinical survey of autosomal recessive limb girdle muscular dystrophies in Turkey
.
Ann Neurol
.
1997
;
42
(
2
):
222
9
.
5.
Fardeau
M
,
Hillaire
D
,
Mignard
C
,
Feingold
N
,
Feingold
J
,
Mignard
D
, et al
.
Juvenile limb-girdle muscular dystrophy. Clinical, histopathological and genetic data from a small community living in the Reunion Island
.
Brain
.
1996
;
119 ( Pt 1)
(
Pt 1
):
295
308
.
6.
Angelini
C
,
Giaretta
L
,
Marozzo
R
.
An update on diagnostic options and considerations in limb-girdle dystrophies
.
Expert Rev Neurother
.
2018
;
18
(
9
):
693
703
.
7.
Kyriakides
T
,
Angelini
C
,
Schaefer
J
,
Sacconi
S
,
Siciliano
G
,
Vilchez
JJ
, et al
.
European Federation of Neurological S: EFNS guidelines on the diagnostic approach to pauci- or asymptomatic hyperCKemia
.
Eur J Neurol
.
2010
;
17
(
6
):
767
73
.
8.
Fanin
M
,
Nascimbeni
AC
,
Aurino
S
,
Tasca
E
,
Pegoraro
E
,
Nigro
V
, et al
.
Frequency of LGMD gene mutations in Italian patients with distinct clinical phenotypes
.
Neurology
.
2009
;
72
(
16
):
1432
5
.
9.
Ono
Y
,
Ojima
K
,
Shinkai-Ouchi
F
,
Hata
S
,
Sorimachi
H
.
An eccentric calpain, CAPN3/p94/calpain-3
.
Biochimie
.
2016
;
122
:
169
87
.
10.
Chen
L
,
Tang
F
,
Gao
H
,
Zhang
X
,
Li
X
,
Xiao
D
.
CAPN3: a muscle-specific calpain with an important role in the pathogenesis of diseases (Review)
.
Int J Mol Med
.
2021
;
48
(
5
):
203
.
11.
Ojima
K
,
Ono
Y
,
Hata
S
,
Koyama
S
,
Doi
N
,
Sorimachi
H
.
Possible functions of p94 in connectin-mediated signaling pathways in skeletal muscle cells
.
J Muscle Res Cell Motil
.
2005
;
26
(
6–8
):
409
17
.
12.
Ye
Q
,
Campbell
RL
,
Davies
PL
.
Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation
.
J Biol Chem
.
2018
;
293
(
11
):
4056
70
.
13.
Park
HJ
,
Jang
H
,
Lee
JH
,
Shin
HY
,
Cho
SR
,
Park
KD
, et al
.
Clinical and pathological heterogeneity of Korean patients with CAPN3 mutations
.
Yonsei Med J
.
2016
;
57
(
1
):
173
9
.
14.
Fanin
M
,
Angelini
C
.
Progress and challenges in diagnosis of dysferlinopathy
.
Muscle Nerve
.
2016
;
54
(
5
):
821
35
.
15.
Zhong
H
,
Zheng
Y
,
Zhao
Z
,
Lin
P
,
Xi
J
,
Zhu
W
, et al
.
Molecular landscape of CAPN3 mutations in limb-girdle muscular dystrophy type R1: from a Chinese multicentre analysis to a worldwide perspective
.
J Med Genet
.
2021
;
58
(
11
):
729
36
.
16.
Li
H
,
Durbin
R
.
Fast and accurate short read alignment with Burrows-Wheeler transform
.
Bioinformatics
.
2009
;
25
(
14
):
1754
60
.
17.
Van der Auwera
GA
,
Carneiro
MO
,
Hartl
C
,
Poplin
R
,
Del Angel
G
,
Levy-Moonshine
A
, et al
.
From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline
.
Curr Protoc Bioinformatics
.
2013
;
43
(
1110
):
11.10.1
33
.
18.
Richards
S
,
Aziz
N
,
Bale
S
,
Bick
D
,
Das
S
,
Gastier-Foster
J
, et al
.
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of medical genetics and genomics and the association for molecular pathology
.
Genet Med
.
2015
;
17
(
5
):
405
24
.
19.
Jaganathan
K
,
Kyriazopoulou Panagiotopoulou
S
,
McRae
JF
,
Darbandi
SF
,
Knowles
D
,
Li
YI
, et al
.
Predicting splicing from primary sequence with deep learning
.
Cell
.
2019
;
176
(
3
):
535
48.e24
.
20.
Hebsgaard
SM
,
Korning
PG
,
Tolstrup
N
,
Engelbrecht
J
,
Rouzé
P
,
Brunak
S
.
Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information
.
Nucleic Acids Res
.
1996
;
24
(
17
):
3439
52
.
21.
Gulilat
M
,
Lamb
T
,
Teft
WA
,
Wang
J
,
Dron
JS
,
Robinson
JF
, et al
.
Targeted next generation sequencing as a tool for precision medicine
.
BMC Med Genomics
.
2019
;
12
(
1
):
81
.
22.
Yu
M
,
Zheng
Y
,
Jin
S
,
Gang
Q
,
Wang
Q
,
Yu
P
, et al
.
Mutational spectrum of Chinese LGMD patients by targeted next-generation sequencing
.
PLoS One
.
2017
;
12
(
4
):
e0175343
.
23.
Nallamilli
BRR
,
Chakravorty
S
,
Kesari
A
,
Tanner
A
,
Ankala
A
,
Schneider
T
, et al
.
Genetic landscape and novel disease mechanisms from a large LGMD cohort of 4656 patients
.
Ann Clin Transl Neurol
.
2018
;
5
(
12
):
1574
87
.
24.
Richard
I
,
Hogrel
JY
,
Stockholm
D
,
Payan
CA
,
Fougerousse
F
,
Calpainopathy Study Group
,
Eymard
B
, et al
.
Natural history of LGMD2A for delineating outcome measures in clinical trials
.
Ann Clin Transl Neurol
.
2016
;
3
(
4
):
248
65
.
25.
Sáenz
A
,
Ono
Y
,
Sorimachi
H
,
Goicoechea
M
,
Leturcq
F
,
Blázquez
L
, et al
.
Does the severity of the LGMD2A phenotype in compound heterozygotes depend on the combination of mutations
.
Muscle Nerve
.
2011
;
44
(
5
):
710
4
.
26.
Baralle
D
,
Baralle
M
.
Splicing in action: assessing disease causing sequence changes
.
J Med Genet
.
2005
;
42
(
10
):
737
48
.
27.
Saenz
A
,
Leturcq
F
,
Cobo
AM
,
Poza
JJ
,
Ferrer
X
,
Otaegui
D
, et al
.
LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene
.
Brain
.
2005
;
128
(
Pt 4
):
732
42
.
28.
Mahillon
J
,
Chandler
M
.
Insertion sequences
.
Microbiol Mol Biol Rev
.
1998
;
62
(
3
):
725
74
.
29.
Sorimachi
H
,
Imajoh-Ohmi
S
,
Emori
Y
,
Kawasaki
H
,
Ohno
S
,
Minami
Y
, et al
.
Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and μ-types
.
J Biol Chem
.
1989
;
264
(
33
):
20106
11
.
30.
Siavriene
E
,
Petraityte
G
,
Burnyte
B
,
Morkuniene
A
,
Mikstiene
V
,
Rancelis
T
, et al
.
Compound heterozygous c.598_612del and c.1746-20C > G CAPN3 genotype cause autosomal recessive limb-girdle muscular dystrophy-1: a case report
.
BMC Musculoskelet Disord
.
2021
;
22
(
1
):
1020
.
31.
Krawczak
M
,
Thomas
NS
,
Hundrieser
B
,
Mort
M
,
Wittig
M
,
Hampe
J
, et al
.
Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing
.
Hum Mutat
.
2007
;
28
(
2
):
150
8
.