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JNNP Online First, published on December 22, 2014 as 10.1136/jnnp-2013-307245 Neuromuscular
RESEARCH PAPER
Bethlem myopathy: long-term follow-up identifies COL6 mutations predicting severe clinical evolution N Deconinck,1,2 P Richard,3,4 V Allamand,5,6,7 A Behin,2,8 P Lafôret,2,8 A Ferreiro,2,5,9 A de Becdelievre,3 C Ledeuil,3 C Gartioux,5,6,7 I Nelson,5,6,7 R Y Carlier,10 P Carlier,8 K Wahbi,2,5,6,7 N Romero,2,8 M T Zabot,11 F Bouhour,12 V Tiffreau,13 A Lacour,14 B Eymard,2,8 T Stojkovic2,5,6,7,8 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jnnp-2013-307245). For numbered affiliations see end of article. Correspondence to Dr N Deconinck, Department of Neurology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Av. J.J. Crocq 15, Brussels 1020, Belgium; [email protected] Received 15 November 2013 Revised 29 September 2014 Accepted 3 December 2014
ABSTRACT Objective Mutations in one of the 3 genes encoding collagen VI (COLVI) are responsible for a group of heterogeneous phenotypes of which Bethlem myopathy (BM) represents the milder end of the spectrum. Genotype-phenotype correlations and long-term followup description in BM remain scarce. Methods We retrospectively evaluated the long-term clinical evolution, and genotype-phenotype correlations in 35 genetically identified BM patients (23 index cases). Results Nineteen patients showed a typical clinical picture with contractures, proximal weakness and slow disease progression while 11 presented a more severe evolution. Five patients showed an atypical presentation, namely a limb girdle muscle weakness in 2 and a congenital myopathy pattern with either no contractures, or only limited to ankles, in 3 of them. Pathogenic COL6A1-3 mutations were mostly missense or in frame exon-skipping resulting in substitutions or deletions. Twenty one different mutations were identified including 12novel ones. The mode of inheritance was, autosomal dominant in 83% of the index patients (including 17% (N=4) with a de novo mutation), recessive in 13%, and undetermined in one patient. Skipping of exon 14 of COL6A1 was found in 35% of index cases and was mostly associated with a severe clinical evolution. Missense mutations were detected in 39% of index cases and associated with milder forms of the disease. Conclusions Long-term follow-up identified important phenotypic variability in this cohort of 35 BM patients. However, worsening of the functional disability appeared typically after the age of 40 in 47% of our patients, and was frequently associated with COL6A1 exon 14 skipping.
INTRODUCTION
To cite: Deconinck N, Richard P, Allamand V, et al. J Neurol Neurosurg Psychiatry Published Online First: [ please include Day Month Year] doi:10.1136/ jnnp-2013-307245
Mutations in the COL6A1, COL6A2 and COL6A3 genes encoding three of the collagen VI (COLVI) chains are responsible for COLVI myopathies.1–3 The concept of COLVI myopathies as a specific condition has evolved over the years with the blurring of boundaries between two disorders initially described separately, but now recognised as the extreme ends of a continuous clinical spectrum: Bethlem myopathy (BM, OMIM#158810) and Ullrich congenital muscular dystrophy (UCMD, OMIM#254090).4–7 BM is a mild proximal myopathy mostly inherited dominantly, although recessive inheritance was previously reported.8 9 It was initially reported to begin within the first or second decade of life and
characterised by joint contractures that constitute a hallmark of this condition.10–13 The course is usually slow, with most of the patients (Pt) remaining ambulant although progression of muscle weakness may occur in the fifth decade.11 Respiratory failure and distal hyperlaxity are usually absent.12 13 In UCMD, lifespan is significantly reduced compared with BM, secondary to severe respiratory impairment.14 15 Intermediate phenotypes have been described and named ‘mild UCMD’ or ‘severe BM’, thereby reinforcing the idea of a clinical overlap between Ullrich and Bethlem phenotypes.6 7 16 Skin features such as follicular hyperkeratosis and hypertrophic scars or keloid formation are frequently observed in COLVI myopathies.7 15 17 Other common findings include normal cognitive abilities, normal or only slightly raised serum creatine kinase (almost systematically below 1000 UI/L) levels and absence of cardiac involvement.5 In the context of a retrospective study, we report the clinical presentation, course, immunocytochemical and genetic results of a cohort of 35 patients with BM.
MATERIALS AND METHODS Study group We retrospectively identified in the patients’ cohort of the Myology Institute (Paris, France) a series of 35 patients with BM; these composed of 23 index cases (the first medically identified patient in a family (Fam) with a particular condition) issued from 13 families (online supplementary figure S1) and 10 sporadic (Spo) cases (online supplementary table S1). All the reported patients were ultimately diagnosed with mutations in one of the COL6A1-3 genes. Diagnosis of BM was based on the following criteria: (1) occurrence of muscle weakness predominantly in the proximal muscles associated in most of the patients with early contractures, beginning in childhood or earlier with mildly delayed motor milestones in some of them, (2) the ability to walk independently, (3) normal intelligence, (4) the absence of ventilatory support and cardiac involvement and (5) a supportive COLVI muscle imaging pattern.12 18 19 Hip dislocation, torticollis and milestone delay were not considered as an exclusion criteria for BM diagnosis, as previously reported.12 13 The mean follow-up period for the 35 patients was 17.9 years±7.3 (median 18 years±8.1; range: 4–28 years).
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Neuromuscular From a methodological point of view, patients had been referred to the Myology Institute from several neurological centres for diagnosis purposes, but also for a regular standardised multidisciplinary follow-up: data regarding age and mode of onset, muscle performance, contractures, cardiac and respiratory functions and muscle biopsy findings were systematically collected (online supplementary table S1). Functional activity was systematically recorded in the patient’s clinical entries and was assessed using the Gardner, Medwin and Walton score (where 0=normal and 10=bed bound), which is easy to administer with an excellent inter-rater reliability.20 The number of follow-up visits varied from 2 to 9 per patient (every 2 or 3 years). Respiratory function systematically included forced vital capacity (FVC) measurement according to international standards.21 All procedures were carried out according to the Declaration of Helsinki. The research protocol was approved by the local ethical committee.
Muscle imaging study Thirty-three patients underwent a lower limb CT scan (N=11) or MRI (N=23) and among them, seven patients were also subjected to a whole-body muscle MRI using a previously described protocol.22 Images were analysed according to the Laminen-Mercuri classification for 105 muscles or muscle territories across the entire body.23
Statistical analysis Values are expressed as means±SD, or counts and percentages as appropriate. The existence of any correlation between age, Walton score and FVC was investigated using Spearman correlation. Patients’ values were compared using χ2 or Fisher’s exact test for difference in frequencies. All statistical analyses were performed using the R statistical software V.2.10.1 and a p value of less than 0.05 was considered statistically significant.
COLVI immunolabelling COLVI immunolabelling of skin fibroblasts from 20 index patients and controls was carried out, as previously reported, using either the protocols described by Demir et al3 or by Hicks et al.24 Slides were observed with an Axioplan 2 microscope (Zeiss) equipped with a HBO 100 mercury lamp (Zeiss).
table S1). The most frequent initial symptoms were ‘running and sports difficulties’ followed by the early report of ‘frequent falls’. Among them, five patients (14%) had motor milestones delay and did not achieve ambulation before the age of 2 years. Two patients presented with congenital hip dislocation without milestone delay and congenital torticollis for one of them. Four patients reported ankle contractures during the first decade. In the remaining 14% (Fam 6, Pt 13, Pt 14 and Pt 15; Fam 11, Pt 23 and Spo 3, Pt 28), the disease was recognised at a mean age of 39 years although some mild symptoms, such as long finger flexor (LFF) tightness and Achilles tendon tightness, may have appeared before.
Clinical examination All patients except 5 (86%) presented a typical muscle weakness associated with contractures in adulthood. The latter affected most frequently LFF (77% of the patients) and ankles (74%), followed by elbows (46%) and hips (20%). Muscle weakness was systematically found in the proximal upper and lower limbs, in the neck in 80% of the patients, and almost always in the distal muscles of hands (26%), feet (11%) or both (51%), except in four patients (11%). Finger hyperlaxity was observed in 26% (9 patients), and follicular hyperkeratosis and/or keloids in only 14% (5 patients). Scoliosis was diagnosed in 29% of the patients, but was not particularly associated with an early onset of the disease and required only physiotherapy or bracing. Interestingly, five patients (14%) had an atypical clinical presentation. Three patients from family 5 presented a congenital myopathy-like clinical course with either no contractures or only limited to the ankles (Pt 12). This last patient even received an initial diagnosis of core myopathy based on the results of muscle biopsy. Two other patients (Pt 15 and Pt 27) had a limb girdle muscular dystrophy (LGMD) pattern with absent or very moderate distal and axial weakness, and adult onset of the disease. Therefore, in Pt 27, mutations in the SEPN1 and RYR1 genes had been previously excluded. Phenotypic variability was observed within family 6, with the coexistence of LGMD and classical BM phenotype with contractures or the coexistence of variable contractures and disability severity.
Genetic analysis After extraction from confluent fibroblast cultures using the BioRobot EZ1 Workstation (Qiagen), total RNA was retrotranscribed and the coding regions of the COL6A1 (NM_001848.2), COL6A2 (NM_001849.3) and COL6A3 (NM_004369.3) genes were amplified and sequenced on the ABI3730 system (Applied Biosystems, Foster City, California). One patient (Pt 30) was analysed by targeted gene sequencing with the next generation sequencing (Agilent Capture, HiSeq2000) in the context of the European FP7 NMD Chips project. The pathogenicity of novel mutations was assessed depending on the following criteria: the functional importance of the affected domain of the protein; the nature of the mutation (nonsense or insertion/deletion vs missense); its absence in variants’ databases (EVS, 1000G), in silico analyses using the Alamut (Interactive Biosoftwares), Polyphen (http://coot.embl.de/ PolyPhen), Mutation Taster and ALIGN GVCD softwares and its cosegregation with the disease in the available relatives.15
RESULTS Clinical characteristics and disease evolution Disease onset Onset of the disease was clearly noticed before the age of 11 years in 86% of the patients (30/35; online supplementary 2
Disease evolution The progression of the disease was monitored from the successive Walton recordings over long time periods for each patient (with the exception of 5 patients aged between 14 and 18 years; figure 1B). A significant positive correlation between age and Walton score at the last visit was detected, suggesting a progressive course of the disease over time (r=0.453; pA, c.1007A>G, c.1003−1G>C; c.1003−1 G>T) segregated in 8/12 index cases, three additional splicing mutations c.428+1G>T, c.739 −2A>G and c.1002+2T>C inducing the skipping of exons 3, 7 and 13, respectively; and lastly, 1 missense substitution (c.1022G>T/p.Gly341Val in the triple helical domain (THD)). Notably, conventional mRNA analysis did not allow detection of the causal mutation for Pt 31, but targeted gene capture and sequencing revealed a novel c.1002+2T>C splice mutation that induces skipping of exon 13. In COL6A2, 10 different mutations were identified; 3 mutations detected in four index patients exert a dominant negative effect: a splice mutation (c.736−2A>G) leading to exon 5 skipping and 2 missense substitutions p.Gly268Ser (c.802G>A) and p.Asp621Asn (c.1861G>A). Seven mutations acting as recessive were detected in four sporadic index patients: p.Thr590Met / p.Ala706Thr found in Pt 33, p.Gln819* (stop codon) homozygous in Pt 35 (despite no reported consanguinity, the patient’s parents originating from a small village in Italy were heterozygous carriers of the mutation). This same mutation was previously reported by Merlini et al28 in a homozygous patient presenting a myosclerosis myopathy. The two p.Asp135Asn/p. Trp1017Ar gmutations were found in Pt 32,in whom the transmission mode could not be ascertained since his parents were deceased. Either these two substitutions ( presenting all 4
criteria of disease-causing mutations) act in a recessive fashion or p.Trp1017Arg is the only disease-causing mutation and acts in a dominant de novo mode. Finally, the c.1751dupC duplication ( p.Gly585Trpfs*9), associated with intronic c.1970 −9G>A, responsible for an intronic 7-nucleotide long insertion within the mRNA, ( p.Gly657Alafs*18) were detected in Pt 34. We previously reported the c.1751dupC mutation as a potential ‘de novo’ mutation but we are now able to assess that it acts recessively.18 In COL6A3, inheritance was dominant in all three index cases harbouring missense substitutions located before the THD of the protein, p.Gly1679Trp, p.Gly1842Glu and p.Arg1970Cys. Overall, 12 novel mutations were described; 4 in COL6A1: the c.1002+2T>C causing exon 13 skipping, and 3 mutations (c.1003−1G>C, c.1003−1G>T, c.1007A>G) leading to exon 14 skipping. Five novel mutations affected COL6A2: c.736 −2A>G (skipping of exon 5), c.1751DupC (frame shift at the end of THD), and c.403G>A ( p.Asp135Asn), c.3049T>C ( p. Trp1017Arg) and c.2116G>A ( p.Ala706Thr) in the NH2– and COOH– globular domains Finally three novel mutations affecting the COOH– globular domain encoded by COL6A3: c.5035G>T ( p.Gly1679Trp), c.5525G>A ( p.Gly1842Glu) and c.5908C>T ( p.Arg1970Cys) were identified. The bioinformatics pathogenicity scores of missense mutations are indicated in table 1.
Genotype–phenotype correlations Since we observed a group of patients that showed a more severe disease evolution after the age of 40 (figure 1A), we divided the cohort of patients older than 40 years (N=19) into two groups, according to their Walton score (>3 for the severe group and ≤3 for the milder group) and looked at the distribution of mutations within both groups that consisted of 9 and 10
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Neuromuscular
Figure 3 Whole-body MRI of patient Pt 1 with a severe form of Bethlem myopathy (Walton 4 at 44 years of age). (A) The four T1-weighted axial views centred on masticatory muscles and tongue showing neither fatty replacement nor atrophy (arrows). (B) On the contrary, the axial T1-weighted views of the trunk and upper limbs revealing extensive involvement. However, the diagnosis could be suggested by the visualisation of concentric atrophy with a thin strip of preserved muscle in the centre of some muscles (arrows) like trapezius (top left), deltoid and great pectoral (top right) and triceps brachialis (middle left). (C) On the left on axial T1-weighted views of lower limbs there was either muscle fatty replacement or atrophy.
patients, respectively, without difference in sex ratio (F/M=6/3 in the severe group and F/M=7/3 in the milder group). With regards to mutations localisation, the heterozygous skipping of exon 14 in the THD of α1(VI) was clearly more frequent in the severe group (4 patients, 44%, figure 4). Conversely, this mutation was only detected in one 43-year-old patient from the milder group (Pt 29). Interestingly, it was also detected in a much younger severely affected proband (Pt 26; Walton score: 5 at age 26 years). Seven patients from the milder group (70%) harboured missense mutations in globular domains of all three α(VI) chains (figure 4). Interestingly, four of these mutations affect domain C1 of α2(VI), which was altered in only one 65-year-old patient from the severe group (Pt 21; Walton score: 4). Finally, two patients harbouring dominant mutations, causing skipping of exons 3 and 7 in COL6A1 encoding part of the N-terminal domain very close to the THD, presented a mild phenotype (figure 4). In at least four cases, we observed a worsening of the phenotype between the parent who transmitted the mutation and the affected child (Fam 1, 4, 6 and 7, see pedigree online supplementary figure S1). As all the three COL6 genes are concerned and no instability in the sequence of these genes is known, we think that the increased severity of symptoms is
merely a reflection of the important intrafamilial phenotype variability for the same mutation, rather than a possible indication of anticipation.29
DISCUSSION We report a cohort of 35 genetically confirmed patients with BM, followed over a mean 18-year follow-up, allowing close monitoring of clinical presentation and evolution. Onset of the disease was clearly noticed in childhood, before the age of 11 years in 86% of the patients. Of them, seven patients had a clear onset of the disease before the age of 2 years, demonstrating either motor milestones delay or suffering from early congenital hip dislocation.13 30 In line with previous reports, the vast majority of patients had a slow disease evolution, implying that a majority of them were still able to walk as recorded, sometimes until the age of 59 years.13 However, within the group of 19 patients for which we had recordings after the age of 40 years, 9 (47%) had worsening of their symptoms since they had to use a cane and 1 patient (Pt 35) used a wheelchair constantly. This last patient, who originated from Italia, harboured the same homozygous mutation p.Gln819* in the COL6A2(VI) chain like the myosclerosis patient described by Merlini et al.28 Compared with this patient, our patient also had early occurrence of contractures in elbows, fingers and
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
5
6 Fam/ Spo
Summary of genetic data
Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt
4 4 7 10 2 2 2 5 3 Childhood 10 8 25 35 42 8 3 Childhood 7 6
44 15 14 14 52 24 53 31 58 23 47 35 61 40 49 15 18 58 59 31
COLVI secretion
Genetic data Gene
(4) (2) (4) (3) (6) (2) (4) (3) (6) (1) (2) (2) (3) (2) (3) (2) (2) (1) (3) (3)
AD
Dotted pattern
NA
COL6A1 c.1056 +1G>A
p.Gly335_Asp352del
Skipping exon 14
AD
NA Nl Nl
COL6A1 c.1007A>G
p.Gly335_Asp352del
AD
Very reduced +intracellular Reduced+intracellular
p.Gly335_Asp352del
AD
Dotted pattern
NA
AD
NA
NA
COL6A1 c.1003 −1G>C COL6A2 c.736 −2A>G COL6A2 c.802G>A
Skipping exon THD 14 Skipping exon THD 14 Skipping exon 5 THD
AD
Reduced
NA
COL6A2 c.1861G>A
p.Asp621Asn
AD
NA
NA
COL6A2 c.1861G>A
p.Asp621Asn
AD
Very reduced +intracellular
Nl
COL6A3 c.5035G>T
p.Gly1679Trp
7
65 (4)
AD
Reduced and dotted pattern
NA
COL6A3 c.5525G>A
p.Gly1842Glu
Fam 10 Pt22 Fam 11 Pt23
Childhood 45
43 (3) 61 (3)
AD AD
Nl Nl
COL6A1 c.428+1G>T p.Tyr77_Gly143del COL6A1 c.739−2A>G p.Cys247_Gln253del
Fam 12 Pt 24
2
18 (3)
AD
NA
Fam 13 Pt 25
Birth
60 (6)
AD
NA Very reduced and dotted pattern +intracellular Very reduced +intracellular Reduced+intracellular
Nl
Spo 1
Pt 26
4
26 (5)
Reduced+intracellular
NA
Spo 2
Pt 27
47
62 (3)
AD/parents not tested AD/parents not tested
Reduced+intracellular
NA
COL6A1 c.1056 +1G>A COL6A1 c.1056 +1G>A COL6A1 c.1056 +1G>A COL6A3 c.5908C>T
Spo 3
Pt 28
11
28 (3)
AD de novo
NA
Spo 4
Pt 29
3.5
43 (3)
AD de novo
Very reduced +intracellular Reduced+intracellular
Spo 5
Pt 30
Birth
29 (2)
AD de novo
Fam 2 Fam 3 Fam 4 Fam 5
Fam 6
Fam 7
Fam 8
Fam 9
Pt 21
Very reduced +intracellular
Nl NA
COL6A1 c.1003 −1G>T COL6A1 c.1056 +1G>A COL6A1 c.1022G>T
Protein/AA
p.Cys246_Lys267del p.Gly268Ser
p.Gly335_Asp352del
Polyphen score: 1 000Mutation Taster Score: 56 ALIGN GVGD: Class C0 Polyphen score: 0.999 Mutation Taster Score: 23 ALIGN GVGD: Class C15 Polyphen score: 0.999 Mutation Taster Score: 23 ALIGN GVGD: Class C15 Polyphen score: 0.999 Mutation Taster Score: 184 ALIGN GVGD: Class C65 Polyphen score: 0.999 Mutation Taster Score: 98 ALIGN GVGD: Class C65
Consequence
Protein domain
Muscle biopsy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Mutation
Pathogenicity scores (missense mutations)
Cultured fibroblasts
THD
Missense substitution
THD
Missense substitution
C1
Missense substitution
C1
Missense substitution
N2
Missense substitution
N1
Skipping exon 3 N1 Skipping exon 7 N1/THD
Skipping exon 14 p.Gly335_Asp352del Skipping exon 14 p.Gly335_Asp352del Skipping exon 14 p.Arg1970Cys Polyphen score: 0.791 Mutation Missense Taster Score: 180 ALIGN GVGD: substitution Class C35 p.Gly335_Asp352del Skipping exon 14 p.Gly335_Asp352del Skipping exon 14 p.Gly341Val Polyphen score: 1000 Mutation Missense Taster Score: 109 ALIGN GVGD: substitution Class C65
THD THD THD N1
THD THD THD
Continued
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Age at most recent examination (years); Walton score ()
Inheritance
Fam 1
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
Age at onset Patient (years)
Neuromuscular
Table 1
Continued COLVI secretion
Genetic data
Fam/ Spo
Age at onset Patient (years)
Age at most recent examination (years); Walton score ()
Inheritance
Cultured fibroblasts
Muscle biopsy
Gene
Spo 6
Pt 31
3
22 (3)
AD de novo
Reduced+intracellular
NA
Spo 7
Pt 32
Childhood
41 (6)
Unknown, parents not tested
No secretion +intracellular
Nl
COL6A1 c.1002 +2T>C COL6A2 c.403G>A
p.Asp135Asn
c.3049T>C
p.Trp1017Arg
Spo 8
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Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
Table 1
Pt 33
Childhood
75 (6)
AR
Reduced
Nl
Mutation
COL6A2 c.1769C>T c.2116G>A
Spo 9
Pt 34
Birth
32 (3)
AR
Reduced+intracellular
discontinuous staining
Spo 10
Pt 35
1.5
60 (7)
AR
Reduced+intracellular
discontinuous staining
COL6A2 c.1751DupC c.1970 −9G>A COL6A2 c.2455C>T
Protein/AA p.Gly320_Lys334Del
p.Thr590Met
p.Ala706Thr
p.Gly585Trpfs*9 p.Glu656Alafs*17
Pathogenicity scores (missense mutations)
Consequence
Skipping exon 13 Polyphen score: 0.988 Mutation Missense Taster Score: 23 ALIGN GVGD: substitution Class C0 Polyphen score: 0.997 Mutation Missense Taster Score: 101 ALIGN GVGD: substitution Class C0 Polyphen score: 0.998 Mutation Missense Taster Score: 81 ALIGN GVGD: substitution Class C0 Polyphen score: 0.999 Mutation Missense Taster Score: 58 ALIGN GVGD: substitution Class C55 Frameshift Frameshift
p.Gln819* Homozygote
Nonsense
Protein domain THD N1
C2
THD
C1
THD C1 C1/C2
Novel mutations are written in bold. Prediction sites: benign>malign; Polyphen: score of 0>1; Mutation Taster: score: 0>215; ALIGN GVGD: Class C0>C65. AD, autosomal dominant; AR, autosomal recessive; C, C-terminal domain; COLVI, collagen VI; Fam, family; N, N-terminal domain; NA, not available; Spo, sporadic; THD, triple helical domain.
Neuromuscular
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Neuromuscular
Figure 4 Schematic localisation of the mutations identified in the α1(VI), α2(VI) and α3(VI) chains. Schematic representation of the three collagen VI chains. The legend for illustration of functional domains (N1-N10 and C1-C5 and THD for triple helical domain) is indicated in the square. The corresponding exons are indicated in each domain. The mutations are located according to their consequence at the protein level, taking into account the exonic and the intronic splicing mutations. ankles, and difficulties in sports activities in childhood. She was severely disabled, since she was the only patient from our cohort using a wheelchair at 50 years of age. However her FVC was almost normal at the age of 20 years (80% of predicted value) and declined at a very slow pace (FVC=60% of predicted value at the age of 60, online supplementary table S1), which was quite different from the case described by Merlini et al28 who had more severe reduction of FVC at the same age. Similarly, FVC was well preserved and no patient required mechanical ventilation. Interestingly, a very early onset of the disease and the isolated observation of congenital hip dislocation or torticollis were not correlated with a particularly severe clinical evolution.13 The majority of patients presented a typical phenotype with contractures involving most frequently LFFs, ankles but also typically elbows and hips. However, five patients showed an atypical phenotype: a LGMD muscle weakness pattern without contractures in two patients, as previously reported, and a congenital myopathy like pattern with no or very slight ankle contractures in three patients from the same family, one of whom had an initial diagnosis of core myopathy based on muscle biopsy.16 Minicores are not specific to one causative gene as they have been described in SEPN1-related, RYR1-related, MYH7-related disorders.31 32 Cores and nemaline bodies have recently been observed in a patient with BM harbouring a heterozygous mutation in the COL6A3 gene.33 Interestingly, we observed broad clinical variability within the same family ranging from LGMD or congenital myopathy phenotype with no contractures to a more classical phenotype with contractures and weakness.34 Modifier genes or haplotypes have already been described in some other neuromuscular 8
diseases and may also play a role in influencing phenotype presentation in COLVI myopathies.29 25 Clinical examination of other members of the family together with muscle imaging were instrumental in reorienting towards COL6 gene screening, particularly in the three patients with atypical phenotypes. Moreover, a convincing muscle imaging associated with typical phenotype prompted us to perform a multigene sequencing by targeted capture and next generation sequencing in patient Pt 31 in whom the COL6A1-3 mRNA chain Sanger sequencing showed no mutation.19 This allowed the identification of an intronic splice variant responsible for the deletion of COL6A1 exon 13. We hypothesise that this mutation failed to be detected on mRNA cultured fibroblasts due to the instability and very low level of the mutated transcript. Muscle imaging of lower limbs was compatible with the previously described characteristic patterns of COLVI myopathies.18 23 35 Interestingly, wholebody muscle MRI showed the typical pattern of fatty infiltration under the fascia in axial muscles and scapular muscles, which turned out to be particularly useful in the diagnostic workup of severely disabled patients with suspected COLVI myopathy and in whom lower or upper limb muscles may present with complete fatty infiltration.36 As compared with the UCMD cohort previously published by our group, mutations in COL6A1 (52%) were more represented than those in COL6A2 (35%) or COL6A3 (13%) in this cohort of 23 unrelated index patients.15 Confirming previous reports, deletion of COL6A1 exon 14 was identified in 30% of the index patients.27 37–40 Dominant de novo mutations were identified in 17% of all index patients (4/23) and 40% of sporadic patients (4/10), which is less frequent than in patients with
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Neuromuscular UCMD (60%). All are located in the THD of COL6A1, representing 33% of COL6A1 mutations. Recessive transmission was rarely observed and always associated with mutations in the COL6A2 gene, in line with previous reports.8 9 The spectrum and distribution of mutations also differed from those observed in UCMD, as in the latter were generally concentrated in the THD while in BM the distribution of mutations was more outstretched along the genes, except for COL6A1 in which exon 14 skipping predominates since only two patients had mutations out of the THD. Among the novel mutations that we described and particularly the mutations affecting COL6A1 mRNA splicing (especially those causing exon 14 deletion), several are novels at the nucleotide level, but the consequences at the protein level are similar to previously reported mutations. As expected, the phenotypes induced by these mutations are similar in our cohort and similar to the patients previously described.41 Importantly, we were able to infer genotype–phenotype correlations in patients over 40 years of age: the frequent exon 14 skipping (COL6A1) tended to be associated with a more severe clinical evolution most likely because it involves the THD, as is the case in patients with mutations acting recessively.8 9 Conversely, patients harbouring missense mutations often impacting the C1, N1 or N2 globular domain showed a much milder evolution course. Four patients from two families (Fam 5: Pt 10–12 and Spo 5: Pt 30), each carrying a dominant glycine substitution in the THD ( p.Gly268Ser in COL6A2 and p.Gly341Val in COL6A1), belonged to the milder group despite their early disease onset. Both mutations were outside the critical region from [Gly-X-Y] triplets 10–15 associated with more severe phenotypes recently described by others.26 In conclusion, our study highlights the importance of placing COLVI myopathies in the differential diagnosis of congenital myopathies and LGMD; indeed, within the same family, a limb girdle phenotype and the lack of contractures may all be considered for the molecular study of COL6 genes in the context of absence of cardiac involvement and a typical MRI muscle pattern. Although generally considered as a mild proximal myopathy, this long follow-up identified important phenotypic variability in the long term in this cohort. A quarter of the patients showed a worsening of the functional disability that appeared typically after the age of 40 years, frequently associated with COL6A1 exon 14 skipping.
Acknowledgements We thank Généthon and Patrick Nussbaum (Groupe hospitalier de Cochin, Laboratoire de Biochimie Génétique, Paris, France) for fibroblast cultures and Pr. Dan (Department of Neurology, Hôpital Universitaire des Enfants Reine Fabiola, ULB, Brussels, Belgium) for writing advice.
Author affiliations 1 Department of Neurology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Bruxelles, Belgium 2 AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Centre de référence des maladies neuromusculaires, Paris Est, France 3 AP-HP, Hôpitaux Universitaires La Pitié Salpêtrière—Charles Foix, U.F. Cardiogénétique et Myogénétique Moléculaire et Cellulaire, Paris, France 4 UMR_S 1166 Equipe “Génomique et Physiopathologie des Maladies Cardiovasculaires”, Sorbonne Universités, UPMC Univ Paris 06, Institute of Cardiometabolism and Nutrition (ICAN), Paris, France 5 Sorbonne Universités, UPMC Univ Paris 06, Institut de Myologie, Paris, France 6 CNRS, UMR7215, Paris, France 7 Inserm, U974, Paris, France 8 Groupe Hospitalier Pitié-Salpêtrière, Institut de Myologie, Paris, France 9 Inserm U787, Paris, France 10 AP-HP, Service de Radiologie, Hôpital Raymond Poincaré, Garches, France 11 Centre de biotechnologie cellulaire, CHU de Lyon-GH Est, Hospices Civils de Lyon, Bron, France 12 CHU de Lyon, G-H Est, Hôpital Pierre Wertheimer, Service d’explorations fonctionnelles neurologiques, Bron, France 13 CHRU de Lille, Hôpital Pierre Swynghedauw, Service de médecine physique et de réadaptation, Lille, France 14 CHRU de Lille, Hôpital Roger Salengro, Clinique neurologique, Lille, France
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Contributors ND contributed to data collection, analysis and drafting of the manuscript. PR and VA contributed to data collection, analysis and provided substantial help in the drafting of the manuscript. AB, PL, AF, MTZ, FB, AL and VT contributed to data collection. ADB, RYC and PC contributed to data collection, analysis and critical review of the manuscript. CL, CG and IN contributed to data collection and analysis. KW contributed to analysis and editing assistance. NR and BE contributed to data collection and critical review of the manuscript. TS contributed to data collection, analysis, supervision of the drafting and criticial review of the manuscript. Funding This work was supported by funds from Institut national de la santé et la recherche médicale (Inserm), Association Française contre les Myopathies (AFM), Assistance Publique–Hôpitaux de Paris and also partly by the NMD-CHIP Consortium, a FP7 HEALTH project of the European Commission (Development of Targeted DNAChips for High Throughput Diagnosis of Neuromuscular Disorders— Collaborative Project—FP7 Grant Agreement Number: HEALTH-F5-2008-223026; to IN and VA). Competing interests None. Patient consent Obtained. Provenance and peer review Not commissioned; externally peer reviewed.
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Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Bethlem myopathy: long-term follow-up identifies COL6 mutations predicting severe clinical evolution N Deconinck, P Richard, V Allamand, A Behin, P Lafôret, A Ferreiro, A de Becdelievre, C Ledeuil, C Gartioux, I Nelson, R Y Carlier, P Carlier, K Wahbi, N Romero, M T Zabot, F Bouhour, V Tiffreau, A Lacour, B Eymard and T Stojkovic J Neurol Neurosurg Psychiatry published online December 22, 2014
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JNNP Online First, published on December 22, 2014 as 10.1136/jnnp-2013-307245 Neuromuscular
RESEARCH PAPER
Bethlem myopathy: long-term follow-up identifies COL6 mutations predicting severe clinical evolution N Deconinck,1,2 P Richard,3,4 V Allamand,5,6,7 A Behin,2,8 P Lafôret,2,8 A Ferreiro,2,5,9 A de Becdelievre,3 C Ledeuil,3 C Gartioux,5,6,7 I Nelson,5,6,7 R Y Carlier,10 P Carlier,8 K Wahbi,2,5,6,7 N Romero,2,8 M T Zabot,11 F Bouhour,12 V Tiffreau,13 A Lacour,14 B Eymard,2,8 T Stojkovic2,5,6,7,8 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jnnp-2013-307245). For numbered affiliations see end of article. Correspondence to Dr N Deconinck, Department of Neurology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Av. J.J. Crocq 15, Brussels 1020, Belgium; [email protected] Received 15 November 2013 Revised 29 September 2014 Accepted 3 December 2014
ABSTRACT Objective Mutations in one of the 3 genes encoding collagen VI (COLVI) are responsible for a group of heterogeneous phenotypes of which Bethlem myopathy (BM) represents the milder end of the spectrum. Genotype-phenotype correlations and long-term followup description in BM remain scarce. Methods We retrospectively evaluated the long-term clinical evolution, and genotype-phenotype correlations in 35 genetically identified BM patients (23 index cases). Results Nineteen patients showed a typical clinical picture with contractures, proximal weakness and slow disease progression while 11 presented a more severe evolution. Five patients showed an atypical presentation, namely a limb girdle muscle weakness in 2 and a congenital myopathy pattern with either no contractures, or only limited to ankles, in 3 of them. Pathogenic COL6A1-3 mutations were mostly missense or in frame exon-skipping resulting in substitutions or deletions. Twenty one different mutations were identified including 12novel ones. The mode of inheritance was, autosomal dominant in 83% of the index patients (including 17% (N=4) with a de novo mutation), recessive in 13%, and undetermined in one patient. Skipping of exon 14 of COL6A1 was found in 35% of index cases and was mostly associated with a severe clinical evolution. Missense mutations were detected in 39% of index cases and associated with milder forms of the disease. Conclusions Long-term follow-up identified important phenotypic variability in this cohort of 35 BM patients. However, worsening of the functional disability appeared typically after the age of 40 in 47% of our patients, and was frequently associated with COL6A1 exon 14 skipping.
INTRODUCTION
To cite: Deconinck N, Richard P, Allamand V, et al. J Neurol Neurosurg Psychiatry Published Online First: [ please include Day Month Year] doi:10.1136/ jnnp-2013-307245
Mutations in the COL6A1, COL6A2 and COL6A3 genes encoding three of the collagen VI (COLVI) chains are responsible for COLVI myopathies.1–3 The concept of COLVI myopathies as a specific condition has evolved over the years with the blurring of boundaries between two disorders initially described separately, but now recognised as the extreme ends of a continuous clinical spectrum: Bethlem myopathy (BM, OMIM#158810) and Ullrich congenital muscular dystrophy (UCMD, OMIM#254090).4–7 BM is a mild proximal myopathy mostly inherited dominantly, although recessive inheritance was previously reported.8 9 It was initially reported to begin within the first or second decade of life and
characterised by joint contractures that constitute a hallmark of this condition.10–13 The course is usually slow, with most of the patients (Pt) remaining ambulant although progression of muscle weakness may occur in the fifth decade.11 Respiratory failure and distal hyperlaxity are usually absent.12 13 In UCMD, lifespan is significantly reduced compared with BM, secondary to severe respiratory impairment.14 15 Intermediate phenotypes have been described and named ‘mild UCMD’ or ‘severe BM’, thereby reinforcing the idea of a clinical overlap between Ullrich and Bethlem phenotypes.6 7 16 Skin features such as follicular hyperkeratosis and hypertrophic scars or keloid formation are frequently observed in COLVI myopathies.7 15 17 Other common findings include normal cognitive abilities, normal or only slightly raised serum creatine kinase (almost systematically below 1000 UI/L) levels and absence of cardiac involvement.5 In the context of a retrospective study, we report the clinical presentation, course, immunocytochemical and genetic results of a cohort of 35 patients with BM.
MATERIALS AND METHODS Study group We retrospectively identified in the patients’ cohort of the Myology Institute (Paris, France) a series of 35 patients with BM; these composed of 23 index cases (the first medically identified patient in a family (Fam) with a particular condition) issued from 13 families (online supplementary figure S1) and 10 sporadic (Spo) cases (online supplementary table S1). All the reported patients were ultimately diagnosed with mutations in one of the COL6A1-3 genes. Diagnosis of BM was based on the following criteria: (1) occurrence of muscle weakness predominantly in the proximal muscles associated in most of the patients with early contractures, beginning in childhood or earlier with mildly delayed motor milestones in some of them, (2) the ability to walk independently, (3) normal intelligence, (4) the absence of ventilatory support and cardiac involvement and (5) a supportive COLVI muscle imaging pattern.12 18 19 Hip dislocation, torticollis and milestone delay were not considered as an exclusion criteria for BM diagnosis, as previously reported.12 13 The mean follow-up period for the 35 patients was 17.9 years±7.3 (median 18 years±8.1; range: 4–28 years).
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Neuromuscular From a methodological point of view, patients had been referred to the Myology Institute from several neurological centres for diagnosis purposes, but also for a regular standardised multidisciplinary follow-up: data regarding age and mode of onset, muscle performance, contractures, cardiac and respiratory functions and muscle biopsy findings were systematically collected (online supplementary table S1). Functional activity was systematically recorded in the patient’s clinical entries and was assessed using the Gardner, Medwin and Walton score (where 0=normal and 10=bed bound), which is easy to administer with an excellent inter-rater reliability.20 The number of follow-up visits varied from 2 to 9 per patient (every 2 or 3 years). Respiratory function systematically included forced vital capacity (FVC) measurement according to international standards.21 All procedures were carried out according to the Declaration of Helsinki. The research protocol was approved by the local ethical committee.
Muscle imaging study Thirty-three patients underwent a lower limb CT scan (N=11) or MRI (N=23) and among them, seven patients were also subjected to a whole-body muscle MRI using a previously described protocol.22 Images were analysed according to the Laminen-Mercuri classification for 105 muscles or muscle territories across the entire body.23
Statistical analysis Values are expressed as means±SD, or counts and percentages as appropriate. The existence of any correlation between age, Walton score and FVC was investigated using Spearman correlation. Patients’ values were compared using χ2 or Fisher’s exact test for difference in frequencies. All statistical analyses were performed using the R statistical software V.2.10.1 and a p value of less than 0.05 was considered statistically significant.
COLVI immunolabelling COLVI immunolabelling of skin fibroblasts from 20 index patients and controls was carried out, as previously reported, using either the protocols described by Demir et al3 or by Hicks et al.24 Slides were observed with an Axioplan 2 microscope (Zeiss) equipped with a HBO 100 mercury lamp (Zeiss).
table S1). The most frequent initial symptoms were ‘running and sports difficulties’ followed by the early report of ‘frequent falls’. Among them, five patients (14%) had motor milestones delay and did not achieve ambulation before the age of 2 years. Two patients presented with congenital hip dislocation without milestone delay and congenital torticollis for one of them. Four patients reported ankle contractures during the first decade. In the remaining 14% (Fam 6, Pt 13, Pt 14 and Pt 15; Fam 11, Pt 23 and Spo 3, Pt 28), the disease was recognised at a mean age of 39 years although some mild symptoms, such as long finger flexor (LFF) tightness and Achilles tendon tightness, may have appeared before.
Clinical examination All patients except 5 (86%) presented a typical muscle weakness associated with contractures in adulthood. The latter affected most frequently LFF (77% of the patients) and ankles (74%), followed by elbows (46%) and hips (20%). Muscle weakness was systematically found in the proximal upper and lower limbs, in the neck in 80% of the patients, and almost always in the distal muscles of hands (26%), feet (11%) or both (51%), except in four patients (11%). Finger hyperlaxity was observed in 26% (9 patients), and follicular hyperkeratosis and/or keloids in only 14% (5 patients). Scoliosis was diagnosed in 29% of the patients, but was not particularly associated with an early onset of the disease and required only physiotherapy or bracing. Interestingly, five patients (14%) had an atypical clinical presentation. Three patients from family 5 presented a congenital myopathy-like clinical course with either no contractures or only limited to the ankles (Pt 12). This last patient even received an initial diagnosis of core myopathy based on the results of muscle biopsy. Two other patients (Pt 15 and Pt 27) had a limb girdle muscular dystrophy (LGMD) pattern with absent or very moderate distal and axial weakness, and adult onset of the disease. Therefore, in Pt 27, mutations in the SEPN1 and RYR1 genes had been previously excluded. Phenotypic variability was observed within family 6, with the coexistence of LGMD and classical BM phenotype with contractures or the coexistence of variable contractures and disability severity.
Genetic analysis After extraction from confluent fibroblast cultures using the BioRobot EZ1 Workstation (Qiagen), total RNA was retrotranscribed and the coding regions of the COL6A1 (NM_001848.2), COL6A2 (NM_001849.3) and COL6A3 (NM_004369.3) genes were amplified and sequenced on the ABI3730 system (Applied Biosystems, Foster City, California). One patient (Pt 30) was analysed by targeted gene sequencing with the next generation sequencing (Agilent Capture, HiSeq2000) in the context of the European FP7 NMD Chips project. The pathogenicity of novel mutations was assessed depending on the following criteria: the functional importance of the affected domain of the protein; the nature of the mutation (nonsense or insertion/deletion vs missense); its absence in variants’ databases (EVS, 1000G), in silico analyses using the Alamut (Interactive Biosoftwares), Polyphen (http://coot.embl.de/ PolyPhen), Mutation Taster and ALIGN GVCD softwares and its cosegregation with the disease in the available relatives.15
RESULTS Clinical characteristics and disease evolution Disease onset Onset of the disease was clearly noticed before the age of 11 years in 86% of the patients (30/35; online supplementary 2
Disease evolution The progression of the disease was monitored from the successive Walton recordings over long time periods for each patient (with the exception of 5 patients aged between 14 and 18 years; figure 1B). A significant positive correlation between age and Walton score at the last visit was detected, suggesting a progressive course of the disease over time (r=0.453; pA, c.1007A>G, c.1003−1G>C; c.1003−1 G>T) segregated in 8/12 index cases, three additional splicing mutations c.428+1G>T, c.739 −2A>G and c.1002+2T>C inducing the skipping of exons 3, 7 and 13, respectively; and lastly, 1 missense substitution (c.1022G>T/p.Gly341Val in the triple helical domain (THD)). Notably, conventional mRNA analysis did not allow detection of the causal mutation for Pt 31, but targeted gene capture and sequencing revealed a novel c.1002+2T>C splice mutation that induces skipping of exon 13. In COL6A2, 10 different mutations were identified; 3 mutations detected in four index patients exert a dominant negative effect: a splice mutation (c.736−2A>G) leading to exon 5 skipping and 2 missense substitutions p.Gly268Ser (c.802G>A) and p.Asp621Asn (c.1861G>A). Seven mutations acting as recessive were detected in four sporadic index patients: p.Thr590Met / p.Ala706Thr found in Pt 33, p.Gln819* (stop codon) homozygous in Pt 35 (despite no reported consanguinity, the patient’s parents originating from a small village in Italy were heterozygous carriers of the mutation). This same mutation was previously reported by Merlini et al28 in a homozygous patient presenting a myosclerosis myopathy. The two p.Asp135Asn/p. Trp1017Ar gmutations were found in Pt 32,in whom the transmission mode could not be ascertained since his parents were deceased. Either these two substitutions ( presenting all 4
criteria of disease-causing mutations) act in a recessive fashion or p.Trp1017Arg is the only disease-causing mutation and acts in a dominant de novo mode. Finally, the c.1751dupC duplication ( p.Gly585Trpfs*9), associated with intronic c.1970 −9G>A, responsible for an intronic 7-nucleotide long insertion within the mRNA, ( p.Gly657Alafs*18) were detected in Pt 34. We previously reported the c.1751dupC mutation as a potential ‘de novo’ mutation but we are now able to assess that it acts recessively.18 In COL6A3, inheritance was dominant in all three index cases harbouring missense substitutions located before the THD of the protein, p.Gly1679Trp, p.Gly1842Glu and p.Arg1970Cys. Overall, 12 novel mutations were described; 4 in COL6A1: the c.1002+2T>C causing exon 13 skipping, and 3 mutations (c.1003−1G>C, c.1003−1G>T, c.1007A>G) leading to exon 14 skipping. Five novel mutations affected COL6A2: c.736 −2A>G (skipping of exon 5), c.1751DupC (frame shift at the end of THD), and c.403G>A ( p.Asp135Asn), c.3049T>C ( p. Trp1017Arg) and c.2116G>A ( p.Ala706Thr) in the NH2– and COOH– globular domains Finally three novel mutations affecting the COOH– globular domain encoded by COL6A3: c.5035G>T ( p.Gly1679Trp), c.5525G>A ( p.Gly1842Glu) and c.5908C>T ( p.Arg1970Cys) were identified. The bioinformatics pathogenicity scores of missense mutations are indicated in table 1.
Genotype–phenotype correlations Since we observed a group of patients that showed a more severe disease evolution after the age of 40 (figure 1A), we divided the cohort of patients older than 40 years (N=19) into two groups, according to their Walton score (>3 for the severe group and ≤3 for the milder group) and looked at the distribution of mutations within both groups that consisted of 9 and 10
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Neuromuscular
Figure 3 Whole-body MRI of patient Pt 1 with a severe form of Bethlem myopathy (Walton 4 at 44 years of age). (A) The four T1-weighted axial views centred on masticatory muscles and tongue showing neither fatty replacement nor atrophy (arrows). (B) On the contrary, the axial T1-weighted views of the trunk and upper limbs revealing extensive involvement. However, the diagnosis could be suggested by the visualisation of concentric atrophy with a thin strip of preserved muscle in the centre of some muscles (arrows) like trapezius (top left), deltoid and great pectoral (top right) and triceps brachialis (middle left). (C) On the left on axial T1-weighted views of lower limbs there was either muscle fatty replacement or atrophy.
patients, respectively, without difference in sex ratio (F/M=6/3 in the severe group and F/M=7/3 in the milder group). With regards to mutations localisation, the heterozygous skipping of exon 14 in the THD of α1(VI) was clearly more frequent in the severe group (4 patients, 44%, figure 4). Conversely, this mutation was only detected in one 43-year-old patient from the milder group (Pt 29). Interestingly, it was also detected in a much younger severely affected proband (Pt 26; Walton score: 5 at age 26 years). Seven patients from the milder group (70%) harboured missense mutations in globular domains of all three α(VI) chains (figure 4). Interestingly, four of these mutations affect domain C1 of α2(VI), which was altered in only one 65-year-old patient from the severe group (Pt 21; Walton score: 4). Finally, two patients harbouring dominant mutations, causing skipping of exons 3 and 7 in COL6A1 encoding part of the N-terminal domain very close to the THD, presented a mild phenotype (figure 4). In at least four cases, we observed a worsening of the phenotype between the parent who transmitted the mutation and the affected child (Fam 1, 4, 6 and 7, see pedigree online supplementary figure S1). As all the three COL6 genes are concerned and no instability in the sequence of these genes is known, we think that the increased severity of symptoms is
merely a reflection of the important intrafamilial phenotype variability for the same mutation, rather than a possible indication of anticipation.29
DISCUSSION We report a cohort of 35 genetically confirmed patients with BM, followed over a mean 18-year follow-up, allowing close monitoring of clinical presentation and evolution. Onset of the disease was clearly noticed in childhood, before the age of 11 years in 86% of the patients. Of them, seven patients had a clear onset of the disease before the age of 2 years, demonstrating either motor milestones delay or suffering from early congenital hip dislocation.13 30 In line with previous reports, the vast majority of patients had a slow disease evolution, implying that a majority of them were still able to walk as recorded, sometimes until the age of 59 years.13 However, within the group of 19 patients for which we had recordings after the age of 40 years, 9 (47%) had worsening of their symptoms since they had to use a cane and 1 patient (Pt 35) used a wheelchair constantly. This last patient, who originated from Italia, harboured the same homozygous mutation p.Gln819* in the COL6A2(VI) chain like the myosclerosis patient described by Merlini et al.28 Compared with this patient, our patient also had early occurrence of contractures in elbows, fingers and
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
5
6 Fam/ Spo
Summary of genetic data
Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt Pt
4 4 7 10 2 2 2 5 3 Childhood 10 8 25 35 42 8 3 Childhood 7 6
44 15 14 14 52 24 53 31 58 23 47 35 61 40 49 15 18 58 59 31
COLVI secretion
Genetic data Gene
(4) (2) (4) (3) (6) (2) (4) (3) (6) (1) (2) (2) (3) (2) (3) (2) (2) (1) (3) (3)
AD
Dotted pattern
NA
COL6A1 c.1056 +1G>A
p.Gly335_Asp352del
Skipping exon 14
AD
NA Nl Nl
COL6A1 c.1007A>G
p.Gly335_Asp352del
AD
Very reduced +intracellular Reduced+intracellular
p.Gly335_Asp352del
AD
Dotted pattern
NA
AD
NA
NA
COL6A1 c.1003 −1G>C COL6A2 c.736 −2A>G COL6A2 c.802G>A
Skipping exon THD 14 Skipping exon THD 14 Skipping exon 5 THD
AD
Reduced
NA
COL6A2 c.1861G>A
p.Asp621Asn
AD
NA
NA
COL6A2 c.1861G>A
p.Asp621Asn
AD
Very reduced +intracellular
Nl
COL6A3 c.5035G>T
p.Gly1679Trp
7
65 (4)
AD
Reduced and dotted pattern
NA
COL6A3 c.5525G>A
p.Gly1842Glu
Fam 10 Pt22 Fam 11 Pt23
Childhood 45
43 (3) 61 (3)
AD AD
Nl Nl
COL6A1 c.428+1G>T p.Tyr77_Gly143del COL6A1 c.739−2A>G p.Cys247_Gln253del
Fam 12 Pt 24
2
18 (3)
AD
NA
Fam 13 Pt 25
Birth
60 (6)
AD
NA Very reduced and dotted pattern +intracellular Very reduced +intracellular Reduced+intracellular
Nl
Spo 1
Pt 26
4
26 (5)
Reduced+intracellular
NA
Spo 2
Pt 27
47
62 (3)
AD/parents not tested AD/parents not tested
Reduced+intracellular
NA
COL6A1 c.1056 +1G>A COL6A1 c.1056 +1G>A COL6A1 c.1056 +1G>A COL6A3 c.5908C>T
Spo 3
Pt 28
11
28 (3)
AD de novo
NA
Spo 4
Pt 29
3.5
43 (3)
AD de novo
Very reduced +intracellular Reduced+intracellular
Spo 5
Pt 30
Birth
29 (2)
AD de novo
Fam 2 Fam 3 Fam 4 Fam 5
Fam 6
Fam 7
Fam 8
Fam 9
Pt 21
Very reduced +intracellular
Nl NA
COL6A1 c.1003 −1G>T COL6A1 c.1056 +1G>A COL6A1 c.1022G>T
Protein/AA
p.Cys246_Lys267del p.Gly268Ser
p.Gly335_Asp352del
Polyphen score: 1 000Mutation Taster Score: 56 ALIGN GVGD: Class C0 Polyphen score: 0.999 Mutation Taster Score: 23 ALIGN GVGD: Class C15 Polyphen score: 0.999 Mutation Taster Score: 23 ALIGN GVGD: Class C15 Polyphen score: 0.999 Mutation Taster Score: 184 ALIGN GVGD: Class C65 Polyphen score: 0.999 Mutation Taster Score: 98 ALIGN GVGD: Class C65
Consequence
Protein domain
Muscle biopsy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Mutation
Pathogenicity scores (missense mutations)
Cultured fibroblasts
THD
Missense substitution
THD
Missense substitution
C1
Missense substitution
C1
Missense substitution
N2
Missense substitution
N1
Skipping exon 3 N1 Skipping exon 7 N1/THD
Skipping exon 14 p.Gly335_Asp352del Skipping exon 14 p.Gly335_Asp352del Skipping exon 14 p.Arg1970Cys Polyphen score: 0.791 Mutation Missense Taster Score: 180 ALIGN GVGD: substitution Class C35 p.Gly335_Asp352del Skipping exon 14 p.Gly335_Asp352del Skipping exon 14 p.Gly341Val Polyphen score: 1000 Mutation Missense Taster Score: 109 ALIGN GVGD: substitution Class C65
THD THD THD N1
THD THD THD
Continued
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Age at most recent examination (years); Walton score ()
Inheritance
Fam 1
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
Age at onset Patient (years)
Neuromuscular
Table 1
Continued COLVI secretion
Genetic data
Fam/ Spo
Age at onset Patient (years)
Age at most recent examination (years); Walton score ()
Inheritance
Cultured fibroblasts
Muscle biopsy
Gene
Spo 6
Pt 31
3
22 (3)
AD de novo
Reduced+intracellular
NA
Spo 7
Pt 32
Childhood
41 (6)
Unknown, parents not tested
No secretion +intracellular
Nl
COL6A1 c.1002 +2T>C COL6A2 c.403G>A
p.Asp135Asn
c.3049T>C
p.Trp1017Arg
Spo 8
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Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
Table 1
Pt 33
Childhood
75 (6)
AR
Reduced
Nl
Mutation
COL6A2 c.1769C>T c.2116G>A
Spo 9
Pt 34
Birth
32 (3)
AR
Reduced+intracellular
discontinuous staining
Spo 10
Pt 35
1.5
60 (7)
AR
Reduced+intracellular
discontinuous staining
COL6A2 c.1751DupC c.1970 −9G>A COL6A2 c.2455C>T
Protein/AA p.Gly320_Lys334Del
p.Thr590Met
p.Ala706Thr
p.Gly585Trpfs*9 p.Glu656Alafs*17
Pathogenicity scores (missense mutations)
Consequence
Skipping exon 13 Polyphen score: 0.988 Mutation Missense Taster Score: 23 ALIGN GVGD: substitution Class C0 Polyphen score: 0.997 Mutation Missense Taster Score: 101 ALIGN GVGD: substitution Class C0 Polyphen score: 0.998 Mutation Missense Taster Score: 81 ALIGN GVGD: substitution Class C0 Polyphen score: 0.999 Mutation Missense Taster Score: 58 ALIGN GVGD: substitution Class C55 Frameshift Frameshift
p.Gln819* Homozygote
Nonsense
Protein domain THD N1
C2
THD
C1
THD C1 C1/C2
Novel mutations are written in bold. Prediction sites: benign>malign; Polyphen: score of 0>1; Mutation Taster: score: 0>215; ALIGN GVGD: Class C0>C65. AD, autosomal dominant; AR, autosomal recessive; C, C-terminal domain; COLVI, collagen VI; Fam, family; N, N-terminal domain; NA, not available; Spo, sporadic; THD, triple helical domain.
Neuromuscular
7
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Neuromuscular
Figure 4 Schematic localisation of the mutations identified in the α1(VI), α2(VI) and α3(VI) chains. Schematic representation of the three collagen VI chains. The legend for illustration of functional domains (N1-N10 and C1-C5 and THD for triple helical domain) is indicated in the square. The corresponding exons are indicated in each domain. The mutations are located according to their consequence at the protein level, taking into account the exonic and the intronic splicing mutations. ankles, and difficulties in sports activities in childhood. She was severely disabled, since she was the only patient from our cohort using a wheelchair at 50 years of age. However her FVC was almost normal at the age of 20 years (80% of predicted value) and declined at a very slow pace (FVC=60% of predicted value at the age of 60, online supplementary table S1), which was quite different from the case described by Merlini et al28 who had more severe reduction of FVC at the same age. Similarly, FVC was well preserved and no patient required mechanical ventilation. Interestingly, a very early onset of the disease and the isolated observation of congenital hip dislocation or torticollis were not correlated with a particularly severe clinical evolution.13 The majority of patients presented a typical phenotype with contractures involving most frequently LFFs, ankles but also typically elbows and hips. However, five patients showed an atypical phenotype: a LGMD muscle weakness pattern without contractures in two patients, as previously reported, and a congenital myopathy like pattern with no or very slight ankle contractures in three patients from the same family, one of whom had an initial diagnosis of core myopathy based on muscle biopsy.16 Minicores are not specific to one causative gene as they have been described in SEPN1-related, RYR1-related, MYH7-related disorders.31 32 Cores and nemaline bodies have recently been observed in a patient with BM harbouring a heterozygous mutation in the COL6A3 gene.33 Interestingly, we observed broad clinical variability within the same family ranging from LGMD or congenital myopathy phenotype with no contractures to a more classical phenotype with contractures and weakness.34 Modifier genes or haplotypes have already been described in some other neuromuscular 8
diseases and may also play a role in influencing phenotype presentation in COLVI myopathies.29 25 Clinical examination of other members of the family together with muscle imaging were instrumental in reorienting towards COL6 gene screening, particularly in the three patients with atypical phenotypes. Moreover, a convincing muscle imaging associated with typical phenotype prompted us to perform a multigene sequencing by targeted capture and next generation sequencing in patient Pt 31 in whom the COL6A1-3 mRNA chain Sanger sequencing showed no mutation.19 This allowed the identification of an intronic splice variant responsible for the deletion of COL6A1 exon 13. We hypothesise that this mutation failed to be detected on mRNA cultured fibroblasts due to the instability and very low level of the mutated transcript. Muscle imaging of lower limbs was compatible with the previously described characteristic patterns of COLVI myopathies.18 23 35 Interestingly, wholebody muscle MRI showed the typical pattern of fatty infiltration under the fascia in axial muscles and scapular muscles, which turned out to be particularly useful in the diagnostic workup of severely disabled patients with suspected COLVI myopathy and in whom lower or upper limb muscles may present with complete fatty infiltration.36 As compared with the UCMD cohort previously published by our group, mutations in COL6A1 (52%) were more represented than those in COL6A2 (35%) or COL6A3 (13%) in this cohort of 23 unrelated index patients.15 Confirming previous reports, deletion of COL6A1 exon 14 was identified in 30% of the index patients.27 37–40 Dominant de novo mutations were identified in 17% of all index patients (4/23) and 40% of sporadic patients (4/10), which is less frequent than in patients with
Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Neuromuscular UCMD (60%). All are located in the THD of COL6A1, representing 33% of COL6A1 mutations. Recessive transmission was rarely observed and always associated with mutations in the COL6A2 gene, in line with previous reports.8 9 The spectrum and distribution of mutations also differed from those observed in UCMD, as in the latter were generally concentrated in the THD while in BM the distribution of mutations was more outstretched along the genes, except for COL6A1 in which exon 14 skipping predominates since only two patients had mutations out of the THD. Among the novel mutations that we described and particularly the mutations affecting COL6A1 mRNA splicing (especially those causing exon 14 deletion), several are novels at the nucleotide level, but the consequences at the protein level are similar to previously reported mutations. As expected, the phenotypes induced by these mutations are similar in our cohort and similar to the patients previously described.41 Importantly, we were able to infer genotype–phenotype correlations in patients over 40 years of age: the frequent exon 14 skipping (COL6A1) tended to be associated with a more severe clinical evolution most likely because it involves the THD, as is the case in patients with mutations acting recessively.8 9 Conversely, patients harbouring missense mutations often impacting the C1, N1 or N2 globular domain showed a much milder evolution course. Four patients from two families (Fam 5: Pt 10–12 and Spo 5: Pt 30), each carrying a dominant glycine substitution in the THD ( p.Gly268Ser in COL6A2 and p.Gly341Val in COL6A1), belonged to the milder group despite their early disease onset. Both mutations were outside the critical region from [Gly-X-Y] triplets 10–15 associated with more severe phenotypes recently described by others.26 In conclusion, our study highlights the importance of placing COLVI myopathies in the differential diagnosis of congenital myopathies and LGMD; indeed, within the same family, a limb girdle phenotype and the lack of contractures may all be considered for the molecular study of COL6 genes in the context of absence of cardiac involvement and a typical MRI muscle pattern. Although generally considered as a mild proximal myopathy, this long follow-up identified important phenotypic variability in the long term in this cohort. A quarter of the patients showed a worsening of the functional disability that appeared typically after the age of 40 years, frequently associated with COL6A1 exon 14 skipping.
Acknowledgements We thank Généthon and Patrick Nussbaum (Groupe hospitalier de Cochin, Laboratoire de Biochimie Génétique, Paris, France) for fibroblast cultures and Pr. Dan (Department of Neurology, Hôpital Universitaire des Enfants Reine Fabiola, ULB, Brussels, Belgium) for writing advice.
Author affiliations 1 Department of Neurology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Bruxelles, Belgium 2 AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Centre de référence des maladies neuromusculaires, Paris Est, France 3 AP-HP, Hôpitaux Universitaires La Pitié Salpêtrière—Charles Foix, U.F. Cardiogénétique et Myogénétique Moléculaire et Cellulaire, Paris, France 4 UMR_S 1166 Equipe “Génomique et Physiopathologie des Maladies Cardiovasculaires”, Sorbonne Universités, UPMC Univ Paris 06, Institute of Cardiometabolism and Nutrition (ICAN), Paris, France 5 Sorbonne Universités, UPMC Univ Paris 06, Institut de Myologie, Paris, France 6 CNRS, UMR7215, Paris, France 7 Inserm, U974, Paris, France 8 Groupe Hospitalier Pitié-Salpêtrière, Institut de Myologie, Paris, France 9 Inserm U787, Paris, France 10 AP-HP, Service de Radiologie, Hôpital Raymond Poincaré, Garches, France 11 Centre de biotechnologie cellulaire, CHU de Lyon-GH Est, Hospices Civils de Lyon, Bron, France 12 CHU de Lyon, G-H Est, Hôpital Pierre Wertheimer, Service d’explorations fonctionnelles neurologiques, Bron, France 13 CHRU de Lille, Hôpital Pierre Swynghedauw, Service de médecine physique et de réadaptation, Lille, France 14 CHRU de Lille, Hôpital Roger Salengro, Clinique neurologique, Lille, France
12
Contributors ND contributed to data collection, analysis and drafting of the manuscript. PR and VA contributed to data collection, analysis and provided substantial help in the drafting of the manuscript. AB, PL, AF, MTZ, FB, AL and VT contributed to data collection. ADB, RYC and PC contributed to data collection, analysis and critical review of the manuscript. CL, CG and IN contributed to data collection and analysis. KW contributed to analysis and editing assistance. NR and BE contributed to data collection and critical review of the manuscript. TS contributed to data collection, analysis, supervision of the drafting and criticial review of the manuscript. Funding This work was supported by funds from Institut national de la santé et la recherche médicale (Inserm), Association Française contre les Myopathies (AFM), Assistance Publique–Hôpitaux de Paris and also partly by the NMD-CHIP Consortium, a FP7 HEALTH project of the European Commission (Development of Targeted DNAChips for High Throughput Diagnosis of Neuromuscular Disorders— Collaborative Project—FP7 Grant Agreement Number: HEALTH-F5-2008-223026; to IN and VA). Competing interests None. Patient consent Obtained. Provenance and peer review Not commissioned; externally peer reviewed.
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Deconinck N, et al. J Neurol Neurosurg Psychiatry 2014;0:1–10. doi:10.1136/jnnp-2013-307245
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Bethlem myopathy: long-term follow-up identifies COL6 mutations predicting severe clinical evolution N Deconinck, P Richard, V Allamand, A Behin, P Lafôret, A Ferreiro, A de Becdelievre, C Ledeuil, C Gartioux, I Nelson, R Y Carlier, P Carlier, K Wahbi, N Romero, M T Zabot, F Bouhour, V Tiffreau, A Lacour, B Eymard and T Stojkovic J Neurol Neurosurg Psychiatry published online December 22, 2014
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