The clinical features and TCAP mutation spectrum in a Chinese cohort of patients with limb-girdle muscular dystrophy R7

Abstract

Limb-girdle muscular dystrophy R7 (LGMDR7) is an autosomal recessive hereditary muscular dystrophy caused by mutations in titin-cap (TCAP). Here, we summarized the clinical characteristics and TCAP mutations in a Chinese cohort of 30 patients with LGMDR7. The onset age of Chinese patients was 19.89 ± 6.70 years old, which is later than European and South Asian patients (P < 0.05). Clinically speaking, 20.0% of patients presented with predominant distal weakness, and 73.3% of patients presented with predominant pelvic girdle weakness. Radiological study revealed semitendinosus and magnus adductor were severely involved in Chinese LGMDR7 patients. Rectus femoris, vastus lateralis, vastus intermedius, soleus and tibialis anterior were moderately to severely involved. The most prevalent mutation in this cohort is c.26_33dupAGGTGTCG, while c.165dupG and c.110 + 5G > A are unique in Chinese population as two of the common mutations. Besides, variant c.26_33dupAGGGTGTCG might be a founder mutation in Asian patients. Internal nuclei, lobulated fibers, and scattered rimmed vacuoles were typical morphological changes in Chinese LGMDR7 patients. This is the largest LGMDR7 cohort in the Chinese population and in the world. This article also expands the clinical, pathological, mutational and radiological spectrum of patients with LGMDR7 in China and in the world.

Introduction

Limb-girdle muscular dystrophy R7 (LGMDR7, OMIM 601954), also known as limb-girdle muscular dystrophy 2G, is a subtype of progressive muscular dystrophy (1). LGMDR7 is a rare hereditary myopathy that was first described by Eloisa S. Moreira in 1997 (2). In 2000, Eloisa S. Moreira found that the titin-cap (TCAP) gene is responsible for LGMDR7 (3). LGMDR7 patients typically present with proximal muscle weakness of the lower limbs, accompanied or followed by distal weakness of the lower limbs (4). Most patients presented predominantly pelvic-girdle involvement. A few patients were reported to present with distal myopathy (5,6). Rare symptoms such as facial muscle weakness (7), abdominal weakness (8) and ventricular extrasystoles (4) were observed in patients.

LGMDR7 is caused by mutations in TCAP, including microduplications, nonsense, missense and other frameshift mutations. TCAP gene encodes telethonin. To date, 67 patients have been reported (4,5,7–14). c.157C > T is a common mutation in Brazil, but c.75G > A is a common mutation in Bulgarian Muslims (13,14). However, it has been reported that c.165dupG or c.26_33dupAGGTGTCG is very common in China, according to two neuromuscular centers (6,11). However, the spectrum of TCAP mutations in the Chinese population has not yet been defined.

In this study, we screened 30 Chinese LGMDR7 patients to determine the clinical, pathological, mutational and radiological spectrum of LGMDR7 patients in China.

Table 1

Clinical features of patients with TCAP variants in China

Family/Number of patients	Sex	Age at onset	Distribution of weakness	Prominent weakness of tibialis anterior	Joint retractions	Calf hypertrophy	CK (IU/L)	Cardiac involvement	TCAP mutation
P1 (15)	F	20	UL:prox>distal LL:prox = distal	Yes	No	Yes	666.5	NA	c.26_33dupAGGTGTCG/c.165dupG
P2	M	6	UL:prox>distal LL:prox>distal	Yes	Ankles	Yes	2062.3	NA	c.2 T > C
P3	M	21	LL: prox>distal	No	Ankles	Yes	1454	NA	c.110 + 5G > A
P4	F	26	UL:prox>distal LL:prox>distal	No	Ankles	No	1684	NA	c.26_33dupAGGTGTCG
P5	M	25	LL:prox = distal	Yes	NA	NA	1046	NA	c.110 + 5G > A
P6	F	NA	NA						c.110 + 5G > A
P7	F	28	LL:prox<distal	Yes	No	Yes	1058	NA	c.165dupG
P8	F	24	LL: prox<distal	Yes	No	Yes			c.165dupG
P9 (6)	F	22	UL:prox>distal LL:prox<=distal	Yes	No	No	592	Mild pulmonary valve regurgitation	c.165dupG
P10 (6)	F	22	UL:prox>distal LL:prox<=distal	Yes	No	No	940	No	c.165dupG
P11 (6)	M	6	LL:prox = distal	Yes	No	No	467	No	c.165dupG
P12	M	24	LL: prox<distal	Yes	No	No	1121	No	c.100del C/c. 110 + 5 G > A
P13	F	16	LL:prox = distal	Yes	No	No	3230	No	c.165dupG
P14	M	NA	NA	NA	NA	NA	NA	NA	c.165dupG
P15	M	15	UL:prox>distal LL:prox>distal	NA	Ankles	NA	2490	No	c. 110 + 5 G > A
P16 (19)	F	12	prox	NA	NA	No	474	No	c.26_33dupAGGTGTCG
P17 (19)	M	15	prox	NA	NA	NA	NA	Coronary artery disease	c.26_33dupAGGTGTCG
P18 (19)	M	21	prox	NA	NA	Yes	1441	No	c.26_33dupAGGTGTCG
P19 (19)	F	30	prox+distal	NA	NA	Yes	1403	No	c.26_33dupAGGTGTCG
P20	F	22	LL:prox<distal	Yes	Yes	No	678	No	c.26_33dupAGGTGTCG
P21	M	16	LL:prox>distal	No	No	No	2361	No	c.26_33dupAGGTGTCG
P22	F	19	LL: prox>distal	No	Yes	No	872	No	c.26_33dupAGGTGTCG
P23 (11)	M	17	UL: prox>distal; LL:prox>distal	Yes	No	No	408	No	c.110 + 5G > A
P24	M	22	UL:prox>distal LL:prox>distal	Yes	No	Yes	974	No	c.26_33dupAGGTGTCG
P25	F	31	UL:prox>distal; LL:prox>distal	No	No	Yes	NA	No	c.26_33dupAGGTGTCG
P26	F	35	LL:prox>distal	No	No	No	NA	No	c.26_33dupAGGTGTCG
P27 (11)	F	17	LL:prox>distal	Yes	No	No	375	No	c.26_33dupAGGTGTCG/c.110 + 5G > A
P28 (11)	M	14	prox>distal	No	No	No	700	No	c.26_33dupAGGTGTCG/c.110 + 5G > A
P29 (11)	M	17	LL:prox>distal	No	No	No	1088	No	c.26_33dupAGGTGTCG/c.110 + 5G > A
P30 (20)	F	14	LL:prox = distal	Yes	NA	NA	408	No	c.165dupG/c.100delC

F, female; M, male; P, patient; UL, upper limbs; LL, lower limbs; NA, not available; prox, proximal involvement; distal, distal involvement; CK, creatine kinase value

Results

Clinical features of LGMDR7 patients in the Chinese population

The detailed clinical findings of the LGMDR7 patients are summarized in Table 1. Patient characteristics are summarized in Table 2. The average age of onset of disease in the Chinese population was 19.89 ± 6.70 years old, with a range of 6–35 years. For 11 (36.7%) patients, onset occurred in the second decade of life (10–20 years old), and for 12 (40.0%) patients, onset occurred in the third decade of life (20–30 years old). The sex ratio was approximately 1.1:1 (16 females vs. 14 males). Six patients (20.0%) presented with predominant distal weakness, and 22 (73.3%) patients presented with predominant pelvic girdle weakness. Joint retractions were observed in six (20.0%) patients. Calf hypertrophy was observed in nine (30.0%) patients (Fig. 1A). Patient 9 had mild pulmonary valve regurgitation revealed through echocardiography. Patient 17 had been diagnosed as coronary artery disease and been treated with stent implantation.

Table 2

Summary of patient characteristics in the Chinese population

Clinical features	Number of patients (%)
Sex
Male	14/30(46.7)
Female	16/30(53.3)
Onset age
1–10y	2/30(6.7)
10–20y	11/30(36.7)
20–30y	12/30(40.0)
>30	3/30(10.0)
Unknown	2/30(6.7)
Phenotype
Predominant distal weakness	6/30(20.0)
Pelvic girdle weakness	22/30(73.3)
Undetermined	2/30(6.7)
Other symptoms
Joint retractions	6/30(20.0)
Calf hypertrophy	9/30(30.0)
Cardiac involvement	1/30(3.3)
CK value (IU/L)
0–1000	12/30(40.0)
1000–2000	8/30(26.7)
>2000	4/30(13.3)
Undetermined	6/30(20.0)

Figure 1

(A) Photograph of patient 3 showing calf hypertrophy muscle. (B) Muscle pathology of patient7 and patient 8. Bar = 20 mm. (a, c) H&E staining showing internal nuclei in patients 7 and 8. (b) NADH staining showing lobulated fibers in patient 7. (d) MGT staining showing rimmed vacuoles in patient 8. (C) Immunostaining of telethonin of patient 10 and control. Bar = 20 mm. (D) Western blot analysis showing absence of telethonin protein in patients 7, 8, 9, 12 and 14.

The serum creatinine kinase (CK) levels in the patients ranged from slightly elevated to more than 18 times the upper limit of normal. The average of CK level was 1166.37 ± 727.80 IU/L, with a range of 375–3230 IU/L. The CK values were less than 2000 IU/L in most patients (20/30, 66.7%).

Fatty infiltration pattern in the lower limb muscles of Chinese patients with LGMDR7

Seven patients (patients 4, 9, 20, 22, 23, 28, 29) underwent proximal lower limbs MRI scans, and six patients (patients 4, 9, 20, 23, 28 and 29) underwent distal lower limbs MRI scans. The MRI scans of three patients (patients 4, 9 and 20) are depicted in Figure 2A, and the average FI score of some muscles of these patients were recorded in Table 3. In the proximal lower limb muscles of LGMDR7 patients, gracilis and sartorius showed mild to moderate involvement (average modified Mercuri scale 1.57 and 1.86, respectively). Rectus femoris, vastus lateralis and vastus intermedius were moderately to severely involved (average modified Mercuri scale 3.29, 3.50 and 3.71, respectively). Semitendinosus and magnus adductor were severely involved (average modified Mercuri scale of 4.14 and 4.14, respectively). In the distal lower limb muscles of LGMDR7 patients, tibialis posterior showed mild involvement (average modified Mercuri scale 0.08). Extensor digitorum longus, peroneus brevis, gastrocnemius lateralis and gastrocnemius medialis were moderately involved (average modified Mercuri scale of 2.00, 2.42, 2.00 and 2.67, respectively). Soleus and tibialis anterior were moderately to severely involved (average modified Mercuri scale of 3.75 and 3.67, respectively). Kruskal–Wallis test revealed significant differences in the FI score among the assessed lower limb muscles (n = 182, H = 78.046, P < 0.001). Further Kruskal–Wallis tests with Bonferroni correction for multiple comparisons were performed for the FI scores of muscles (Fig. 2B). Tibialis posterior was spared in five out of six (83.3%) patients (Table 3). Patients 9 and 20 showed prominent distal involvement; however, proximal muscles such as quadricep femoris, semitendinosus and magnus adductor were involved in patient 9. Quadricep femoris was spared in patient 20, but semitendinosus and magnus adductor were involved.

Figure 2

(A) MRI of patient 9, patient 20, and patient 4 showed fatty degenerative changes (red arrows). Sartorius, gracilis, and tibialis posterior (green arrows) were spared. (B) Percentages of FI per muscle in the lower limbs of patients with LGMDR7 in Chinese population. Pairwise comparisons between two muscles were analyzed by Kruskal–Wallis H test. Significance values was adjusted by the Bonferroni correction for multiple tests. ^*, P < 0.05. AM, adductor magnus; ED, extensor digitorum longus; Gl, gastrocnemius lateralis; Gm, gastrocnemius medialis; Gr, gracilis; RF, rectus femoris; S, soleus; Sa, sartorius; ST, semitendinosus; TA, tibialis anterior; TP, tibialis posterior; PB, peroneus brevis; VI, vastus intermedius; VL, vastus lateralis.

Table 3

Muscle imaging findings

Patient	RF	VL	VI	Sa	Gr	ST	AM	TA	ED	PB	TP	S	Gl	Gm
P4	1/2a	3	4	1	1	1/3	2	5	5	1	0	1	1	2
P9	1/2	2/3	3/5	1	1	5	5	3/4	1	2/5	0	2/5	2	1
P20	1	1	1	1	1	3	3	4/3	1	4	0	3/5	3	3
P22	5	4	4	1	1	5	4	NA	NA	NA	NA	NA	NA	NA
P23	5	5	5	5	5	5	5	4	3	3	0	4	2	4
P28	5	5	4	1	3	5	5	5	1	1	0	5	2	2
P29	4	4	4	1	1	4	5	1	1	2	1	5	2	4
Average	3.29	3.50	3.71	1.57	1.86	4.14	4.14	3.67	2.00	2.42	0.08	3.75	2.00	2.67

AM, adductor magnus; ED, extensor digitorum longus; Gl, gastrocnemius lateralis; Gm, gastrocnemius medialis; Gr, gracilis; NA, not assessed; RF, rectus femoris; S, soleus; Sa, sartorius; ST, semitendinosus; TA, tibialis anterior; TP, tibialis posterior; PB, peroneus brevis; VI, vastus intermedius; VL, vastus lateralis.

^aRight/left when there are asymmetrical findings.

Muscle morphological characterization

About, 16 of the 30 (16/30, 53.3%) patients underwent muscle biopsy examination (Table 4). Muscle biopsy of these 16 patients showed an increased number of internal nuclei, lobulated fibers and scattered rimmed vacuoles (Fig. 1B). Among these sixteen patients, an increased number of internal nuclei was observed in 10 (10/16, 62.5%) patients. Lobulated fibers were observed in nine (9/16, 56.3%) patients, and eight (8/16, 53.3%) patients had rimmed vacuoles. These are the main morphological characteristics of LGMDR7 in this Chinese population. In this study, we conducted an analysis of the histological features, stratified by sex and mutation grouping. Our findings indicate that there was no statistically significant difference observed between the female and male groups. Furthermore, we did not observe any significant differences among patients with varying mutations, as presented in Supplementary Material, Table S2. Immunostaining and western blot analysis of telethonin revealed absent expressions in LGMDR7 patients (Fig. 1C and D).

Table 4

Muscle morphological characterization of Chinese LGMDR7 patients

	Nuclear internalization	Lobulated fibers	Rimmed vacuoles
Patient 1	−	−	−
Patient 2	−	−	+
Patient 3	+	+	−
Patient 4	+	+	−
Patient 7	−	+	−
Patient 8	+	−	+
Patient 9	+	+	+
Patient 11	+	+	+
Patient 12	+	−	+
Patient 13	+	+	+
Patient 15	+	+	+
Patient 20	−	−	−
Patient 21	−	−	+
Patient 22	+	−	−
Patient 23	−	+	−
Patient 28	+	+	−

Table 5

Mutation allelic frequencies of LGMDR7 patients in the Chinese population

Mutation	n of 60 alleles (total n = 30 × 2)	Patients of homozygote	Frequency (%)
c.2 T > C	2	1	3.3
c.110 + 5G > A	14	5	23.3
c.165dupG	16	7	26.7
c.100delC	2	0	3.3
c.26_33dupAGGTGTCG	26	11	43.3
Total	60	24	100.0

Molecular studies in China

Five variants were detected in 30 Chinese LGMDR7 patients. These variants included c.2 T > C, c.110 + 5G > A, c.165dupG, c.100delC, and c.26_33dupAGGTGTCG. According to the criteria of the American College of Medical Genetics (ACMG), these variants were classified as pathogenic variants: c.2 T > C (PVS1 + PS3 + PM2 + PM3), c.110 + 5G > A (PS3 + PM2 + PM3 + PP3 + PP4), c.165dupG (PVS1 + PS3 + PM2 + PM3), c.100delC (PVS1 + PS3 + PM2 + PM3), and c.26_33dupAGGTGTCG (PVS1 + PS3 + PM2 + PM3). Twenty-four patients (24/30, 80.0%) carried a homozygous mutation, and six (6/30, 20.0%) harbored compound heterozygous mutations. The c.26_33dupAGGTGTCG, c.165dupG, and c.110 + 5G > A variants were common in China, with allele frequencies of 43.3, 26.7 and 23.3%, respectively (Table 5). The c.26_33dupAGGTGTCG variant is hotspot variant (the most frequently occurring variant) in China. According to data from gnomAD (http://www.gnomad-sg.org/gene/ENSG00000173991?dataset=gnomad_r3), the allele frequencies of c.2 T > C, c.110 + 5G > A, c.165dupG, c.100delC and c.26_33dupAGGTGTCG in the East Asian population were 0, 0, 0, 0 and 0.0001926, respectively. According to the Hardy–Weinberg equation, 1/26957985, converting to approximately 37/1 000 000 000, individuals in the East Asian population are expected to suffer from LGMDR7. According to data from the Chinese inhouse database (a database of 6145 healthy Chinese Han people, Grandomics Biosciences), the allele frequencies of c.2 T > C, c.110 + 5G > A, c.165dupG, c.100delC and c.26_33dupAGGTGTCG are 0, 0.0000814, 0.0000814, 0 and 0.0003255, respectively. According to the Hardy–Weinberg equation, approximately 238/1 000 000 000 Chinese individuals are expected to suffer from LGMDR7. In addition, haplotype analysis was conducted in patients with LGMDR7. The analysis revealed that patients 3 and 15 did not exhibit a shared haplotype, despite carrying the same homozygous mutations c.110 + 5G > A. Additionally, homozygous variant c.165dupG was observed in patients 7, 9, 11, 13 and 14, all of whom belonged to consanguineous families. However, only patients 13 and 14 from the same family exhibited a shared haplotype (Supplementary Material, Fig. S1).

Molecular studies of LGMDR7 patients worldwide

Thirteen TCAP variants were detected in LGMDR7 patients in the world (Fig. 3A). According to the Sequence Ontology, these variants were divided into four groups: start lost variant (c.2 T > C), frameshift variant (c.26_33dupAGGTGTCG, c.165dupG, c.100delC and c.90_91del), stop gained (c.32C > A, c.75G > A, c.157C > T, c.172C > T, c.244C > T and c.255C > A) and intronic variant (c.100_100 + 1 del and c.110 + 5G > A) (Fig. 3B). Almost all LGMDR7 patients carried frameshift variants (37.7%) or stop gained variants (48.1%) (Fig. 3B). The start lost variant was only reported in one patient (15).

Figure 3

(A) A total of thirteen TCAP variants have been detected globally in LGMDR7 patients, spanning various regions including Spain, India, China, Greece, Brazil, France and Singapore, among others. Notably, five of these variants have been identified in Chinese patients. TCAP mutation spectrum of all LGMDR7 patients in the worldwide (labeled red). (B) The correlation between variant types (missense variant, frameshift variant, stop gained variant, and intron variant) and variants. (C) The age at onset of the different ethnicities of patients. ^* Indicates P < 0.05; ^** indicates 0.001 < P < 0.05.

Ethnicity analysis of the clinical features of LGMDR7 patients

To date, 51 LGMDR7 patients have been diagnosed with LGMDR7 in countries other than China. The clinical findings are summarized in Supplementary Material, Table S1. In countries other than China, disease onset occurred during the first or second decade of life for 39 (76.5%) patients, and disease onset occurred during the third decade of life for five (9.8%) patients. The sex ratio was approximately 1:1.5 (20 females vs. 30 males) in countries other than China (the sex of one patient was unknown). The age of onset was 15.80 ± 6.64, 6.33 ± 3.09, 19.89 ± 6.70 and 22 ± 6.89 years in European, South Asian, Chinese and Southeast Asian populations, respectively. The onset age of Chinese patients was later than that of European and South Asian populations (P < 0.05) (Fig. 3C). The age of disease onset for patients in South America was 9–15 years, which is younger than that of Chinese patients. No significant difference in onset age was observed between Chinese and Southeast Asian patients.

Ethnicity analysis of TCAP mutations in global LGMDR7 patients

For all LGMDR7-related mutations, Chinese patients accounted for the majority of the dataset (37.0%), followed by European (30.2%) and South American patients (18.5%) (Fig. 4A). Mutations originating from South Asian, Southeast Asian and West Asian populations are rare, accounting for 14.2% together. The hotspot variant varies from ethnicity to ethnicity. In China, the hotspot variant is c.26_33dupAGGTGTCG. Conversely, the hotspot variant in Europe, South America and South Asia are c.75G > A, c.157C > T, and c.32C > A, respectively (Fig. 4A). Notably, c.26_33dupAGGTGTCG has solely been detected in Asian patients, whereas c.75G > A has exclusively been identified in European patients, and c.32C > A has solely been found in South Asian patients (Fig. 4B). Of interest, the c.157C > T variant is shared by European and South American patients, albeit to varying degrees: while only two European patients carried the c.157C > T variant, 15 South American patients harbored this genetic mutation. This geographical phenomenon may be attributed to a founder effect. In addition, Chinese patients shared the c.26_33dupAGGGTGTCG variant with South Asian and Southeast Asian patients and shared the c.110 + 5G > A variant with Southeast Asian patients (Fig. 4B). Moreover, European patients shared the c.90_91del variant with West Asian patients (Fig. 4B).

Figure 4

(A) The correlation between ethnicities and genetic variants identified in this study, using the online Circos tool. The thickness of the connecting lines between ethnicities and variants indicates the proportion of variants present in patients of a particular ethnicity. The variants depicted include: A, c.157C > T; B, c.26_33dupAGGTGTCG; C, c.90_91del; D, c.75G > A; E, c.100_100 + 1del; F, c.172C > T; G, c.244C > T; H, c.255C > A; I, c.32C > A; J, c.2 T > C; K, c.110 + 5G > A; L, c.165dupG; M, c.100delC. (B) The common coexisting variants in patients from various ethnic groups (European, Chinese, South Asian, South American, and Southeast Asian populations) were identified using a Venn diagram tool. The different color blocks in the diagram represent the different ethnicities, with green indicating Europeans, blue representing West Asians, red representing South Americans, yellow representing South Asians, and brown representing Southeast Asians. The overlap of two or more-color blocks reflects the shared variants among these ethnicities, and the figure indicates the number of variants shared. Mutations labeled red are the hotspot variants in different ethnicities.

Discussion

We report 30 genetically confirmed LGMDR7 patients in China. To our knowledge, this is the largest LGMDR7 cohort in the Chinese population and in the world. LGMDR7 is a rare myopathy with an estimated disease prevalence of 238/1,000,000,000 in Chinese population. This study also expands the clinical, pathological, mutational, and radiological spectrum of Chinese patients with LGMDR7.

It used to be reported that LGMDR7 onset in the first or second decade of life (4). However, most patients in China present muscle weakness in the second or third decade of life. Age of onset was similar between Chinese patients and Southeast Asian patients. However, the age of onset of Chinese patients was later than that of European and South Asian patients. This phenomenon suggests that genetic background or some modifying genes might be involved in LGMDR7. Regarding the muscle involvement pattern, 73.5% of patients presented with pelvic girdle weakness, and 20.0% of patients presented with distal myopathy. LGMDR7 patients presenting with distal myopathy might be misdiagnosed with GNE myopathy. Atypical GNE myopathy is a differential diagnosis for LGMDR7 (6). Patients 9 and 20 exhibited notable distal involvement in their lower limbs, while magnetic resonance imaging (MRI) also revealed involvement of the proximal muscles. Radiological study revealed the most seriously affected muscles were the rectus femoris, vastus lateralis, vastus intermedius, semitendinosus, magnus adductor, soleus and tibialis anterior. Sartorius, gracilis, and tibialis posterior were relatively spared in Chinese LGMDR7 patients.

Cardiac involvement is a rare manifestation of LGMDR7. To date, only a few cases of right bundle conduction disturbance and ventricular extrasystoles have been reported in LGMDR7 patients (4,16). In addition, mild pulmonary valve regurgitation was observed in patient 9, and coronary artery disease was identified in patient 17. The relationship between these cardiac phenotypes and LGMDR7 remains uncertain. LGMDR7 is an autosomal recessive disorder, and its mode of inheritance and mutation type are distinct from hypertrophic cardiomyopathy 25 (OMIM 607487), an autosomal dominant disease caused by missense mutation of TCAP (17). In the aforementioned documented case, the proband received a diagnosis of hypertrophic cardiomyopathy during adulthood. A missense heterozygous mutation, namely c.171C > G (p.C57W), was identified in both the patient and her affected brother. This particular mutation was situated within the overlapping domains of titin-cap/telethonin, which play a significant role in the interaction between titin and muscle LIM protein. These two proteins, in turn, are crucial for the development of cardiomyopathies. Additionally, it was hypothesized that this mutation interfered with the binding of titin’s Z1 and Z2 domains, as well as muscle LIM protein (MLP) (17). Nevertheless, given that a deficiency in the TCAP can cause hypertrophic cardiomyopathy 25, regular cardiac monitoring is crucial for individuals with LGMDR7. Some patients presented joint retractions and calf hypertrophy.

LGMDR7 is caused by mutations in TCAP, including microduplications, nonsense, missense and other frameshift mutations. The hotspot variant in Europe, South America and South Asia are c.75G > A, c.157C > T, and c.32C > A, respectively. In this study, the most common mutation in Chinese LGMDR7 patients was c.26_33dupAGGTGTCG. This geographical phenomenon might be explained by a founder effect. Moreover, Chinese patients shared the c.26_33dupAGGGTGTCG variant with South Asian and Southeast Asian patients. Thus, the c.26_33dupAGGGTGTCG variant might be a founder mutation in Asian populations. Moreover, c.165dupG and c.110 + 5G > A are both common in China, with frequencies of 26.7 and 23.3%, respectively.

We summarized the morphological characteristics of LGMDR7 patients in the Chinese population. Nuclear internalization, lobulated fibers, and scattered rimmed vacuoles are the main morphological features in Chinese LGMDR7 patients, which is consistent with that of foreign patients (14). Lobulated fibers were recognized as abnormal mitochondria positioning. This illustrates that mitochondrial positioning is abnormal in LGMDR7. Immunostaining and western blotting analysis revealed absent or reduced telethonin expression. These findings may provide diagnostic clues for LGMDR7.

To date, there is no approved treatment for LGMDR7. However, our previous study revealed that idebenone improves the phenotypes of LGMDR7 in zebrafish models (18). Further studies should be performed to explore other treatments for LGMDR7.

In summary, this is the largest scale report on LGMDR7 in the Chinese population and in the world. We analysed the clinical and genetic findings of 30 Chinese LGMDR7 patients, the radiological findings in six patients and the morphopathological findings in 16 patients. The c.26_33dupAGGTGTCG, c.165dupG, and c.110 + 5G > A mutations are common mutations in the Chinese population. Compared to the other populations, the age of onset occurred later in the Chinese population. Furthermore, we summarized the typical morphological changes in Chinese LGMDR7 patients, such as internal nuclei, lobulated fibers and scattered rimmed vacuoles. These findings further expand the clinical, pathological and genetic characteristics of Chinese LGMDR7 patients.

Materials and Methods

Subjects

We screened 30 LGMDR7 patients from Peking University First Hospital, Huashan Hospital, Qilu Hospital, China-Japan Friendship Hospital, Fujian Medical University, and Kaohsiung Medical University Hospital. This cohort included two previously reported patients from Xiangya Hospital (15), six patients from Qilu Hospital of Shandong University (6), four patients from Kaohsiung Medical University Hospital (19), one patient from China-Japan Friendship Hospital (20) and four patients from Fujian Medical University (11). The patient inclusion criteria included the following: (1) progressive muscle weakness; (2) DNA sequencing revealed recessive mutations in TCAP; and (3) myopathic changes in muscle biopsy. All recruited patients met criteria 1 and 2, with or without 3. Patient performed CK value detection in the hospital. We collected the detailed clinical findings, muscle strength results, CK values, magnetic resonance imaging (MRI) examination results, pathological characteristics, and genetic analysis results for the 30 patients. Muscle strength was assessed by the Medical Research Council (MRC) grading scale.

We searched the PubMed database for previous reports about LGMDR7 patients in foreign countries. The keywords used for the database search were as follows: [LGMDR7/limb-girdle muscular dystrophy R7/limb-girdle muscular dystrophy]. We reviewed detailed information about onset age, CK level, ethnicity and mutations.

Muscle magnetic resonance imaging

Patients 4, 9, 20, 23, 28 and 29 underwent magnetic resonance imaging (MRI) examination MRI of human muscle was performed using a 3.0 T MRI spectrometer (Siemens, Magnetom Verio 3.0, Germany). T1-weighted imaging (T1WI) and fat-suppressed T2-weighted imaging (FS-T2WI) sequences were performed in these patients. The degree of FI was scored using a modified Mercuri’s scale (0–5 score; from normal appearance to complete FI) (21). The T1 images were evaluated by two neurologists and a radiologist, each with over 10 years of clinical experience. To avoid bias, the observers were blinded to the clinical data and scored the data independently. The degree of FI within a specific muscle group was assessed through the computation of the mean scale derived from the evaluation of all muscles among the patient population. The extent of FI was then divided into five categories: mild involvement (0 ≤ mean score ≤ 1), mild–moderate involvement (1 < mean score < 2), moderate involvement (2 ≤ mean score ≤ 3), moderate–severe involvement (3 < mean score < 4), and severe involvement (4 ≤ mean score ≤ 5). To determine whether there were any differences in FI scales (FI score) in muscles of the lower limbs, a Kruskal–Wallis test was conducted.

Muscle pathological examination

Some patients underwent pathological examination and routine histochemical stainings such as hematoxylin–eosin (H&E) staining, Modified Gomori Trichrome (MGT) staining, and reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) staining were performed. The H&E staining protocol was performed using a hematoxylin–eosin (HE) Stain Kit (G1120, Solarbio, China). Initially, tissue sections were immersed in Harris’ hematoxylin for 30 s and subsequently rinsed in tap water for three minutes. Then, the sections were stained with eosin for 30 s.

The following is the protocol for MGT staining. Firstly, immerse the sections in Harris’ hematoxylin for a duration of 10 minutes. Subsequently, rinse the sections in running water and subsequently immerse them in a Gomori trichrome mixture for 1 hour. The Gomori trichrome mixture comprises of chromotrope 2R (0.6 g, C7710, Solarbio, China), fast green (0.3 g, F8130, Solarbio, China), phosphotungstic acid (0.6 g, YS153691, Solarbio, China), glacial acetic acid (1.0 mL, 64-19-7, Supelco, Germany) and distilled water 100 mL. Finally, adjust the pH of the mixture to 3.4.

For NADH-TR staining, sections were incubated in incubating solution for 30 minutes and were rinsed in the running water. The NADH incubating solution utilized in this study was composed of a NADH stock solution (1 mL) and NADH (1 mg, N8120, Solarbio, China). The NADH stock solution was prepared by combining 6.25 mL of 1% nitroblue tetrazolium solution (N8140, Solarbio, China), 6.25 mL of 0.2 M Tris buffer (pH 7.4), 1.25 mL of 0.5 M cobalt chloride (H9060, Solarbio, China), and 8.75 mL of distilled water. All the sections were pictured using an Olympus microscope (Olympus BX41, Japan).

Immunohistochemistry and telethonin protein-expression studies

Telethonin (G-11:sc-25 327; Santa Cruz Biotechnology, Santa Cruz, USA) primary antibody was used for immunohistochemistry with standard procedures as previously reported (22). Patients ultimately diagnosed with cerebrovascular stenosis were the control of immunohistochemistry and western blot analysis. The protocol of telethonin protein-expression analysis was as previously reported (6). All the sections were pictured using an Olympus microscope (Olympus BX41, Japan).

Genetic analysis

The genomic DNA of patients diagnosed in Qilu Hospital was extracted as previously described. The DNA samples were screened for targeted next-generation sequencing (NGS), which revealed variants in the TCAP gene. The variants have also been classified according to the criteria of the American College of Medical Genetics (ACMG). The TCAP gene sequence refers to NM_003673.3. According to the Sequence Ontology, the variants discovered in LGMDR7 patients were divided into four groups: missense variant, frameshift variant, stop gained and intron variant. The correlation between the variant and the variant type was shown using the Circos online tool (http://circos.ca/).

Haplotype analysis

A haplotype sharing analysis was conducted to investigate the variants c.26_33dupAGGTGTCG, c.110 + 5G > A, and c.165dupG using three short tandem repeat (STR) markers, namely D17S800, D17S1299, and D17S1818, located within a 2 Mb region surrounding the TCAP gene. Within our cohort of patients, a total of four D17S800 haplotypes, three D17S1299 haplotypes and four D17S1818 haplotypes were identified. The sequence of primer used for haplotype analysis are as followed. The forward primer 5′-GGTCTCATCCATCAGGTTTT-3′ and reverse primer 5′-ATAGACTGTGTACTGGGCATTGA-3′ were for D17S800. The forward primer 5′-TAGCACTTGAGCACACATGG-3′ and reverse primer 5′-GTGCATTATGGGGACCATTA-3′ were for D17S1299. The forward primer 5′-CATAGGTATGTTCAGAAATGTGA-3′ and reverse primer 5′-TGCCTACTGGAAACCAGA-3′ were for D17S1818.

Ethnicity analysis

We divided all LGMDR7 patients into six groups: Chinese, Europeans, South Asians, West Asians, South Americans and Southeast Asians. The correlation between onset age and mutation type was plotted with the Ciros online tool (http://circos.ca/). A Venn diagram (https://bioinformatics.psb.ugent.be/webtools/Venn/) was used to screen for common variants between groups.

Statistical analysis

Data about the age at disease onset are expressed as the mean ± Standard Deviation (SD). The mean value was used to express the FI scale of each muscle. The statistical analysis was conducted using the Statistical Package for the Social Sciences (SPSS) software version R26.0.0.0. A Student’s t-test was employed to compare continuous variables among different groups, whereas Fisher’s exact test was utilized to make pairwise comparisons for categorical variables between the groups. In order to determine whether there were any differences in the FI scales (FI score) between the muscles in the lower limbs, the Kruskal–Wallis test was performed.

Acknowledgements

We want to thank all the participants who participated this study. Furthermore, we would like to express our appreciation to Professor Wen-Chen Liang for diligently following up on the cardiac phenotype of patient 17.

Conflict of Interest statement. The authors declare that they have no conflict of interest.

Funding

This study was funded by National Natural Science Foundation of China [grant no. 82271436], Shandong Provincial Natural Science Foundation [grant no. ZR2022MH190] and Qingdao Science and Technology Benefit People Demonstration Guide Special Project [grant/award number: 22-8-7-smjk-1-nsh].

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author notes

Xiaoqing Lv, Feng Lin contributed equally to the work.