Download Genotype–phenotype correlations in nemaline myopathy caused by

Neuromuscular Disorders 14 (2004) 461–470
www.elsevier.com/locate/nmd
Genotype –phenotype correlations in nemaline myopathy caused by
mutations in the genes for nebulin and skeletal muscle a-actin
Carina Wallgren-Petterssona,*, Katarina Pelina,1, Kristen J. Nowakb,c, Francesco Muntonid,
Norma B. Romeroe, Hans H. Goebelf, Kathryn N. Northg, Alan H. Beggsh,
Nigel G. Laingb,c, the ENMC International Consortium on Nemaline Myopathy
a
Department of Medical Genetics, University of Helsinki, and The Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland
b
Centre for Neuromuscular and Neurological Disorders, University of Western Australia, QEII Medical Centre, Nedlands, WA, Australia
c
Centre for Medical Research, West Australian Institute for Medical Research, Nedlands, WA, Australia
d
Department of Pediatrics, Imperial College London, Hammersmith Hospital, London, UK
e
Institut de Myologie, INSERM U.523, Hopital de la Salpetriere, 47 Boulevard de l’Hopital, 75013 Paris, France
f
Department of Neuropathology, Johannes Gutenberg University Medical Centre, Mainz, Germany
g
Neurogenetics Research Unit, Children’s Hospital at Westmead, Faculty of Medicine, University of Sydney, Sydney, NSW, Australia
h
Genetics Division, Children’s Hospital Boston and Harvard Medical School, Boston, MA, USA
Received 22 September 2003; received in revised form 24 February 2004; accepted 23 March 2004
Abstract
We present comparisons of the clinical pictures in a series of 60 patients with nemaline myopathy in whom mutations had been identified
in the genes for nebulin or skeletal muscle alpha-actin. In the patients with nebulin mutations, the typical form of nemaline myopathy
predominated, while severe, mild or intermediate forms were less frequent. Autosomal recessive inheritance had been verified or appeared
likely in all nebulin cases. In the patients with actin mutations, the severe form of nemaline myopathy was the most common, but some had
the mild or typical form, and a few showed other associated features such as intranuclear rods or actin accumulation. Most cases were
sporadic, but in addition there were instances of both autosomal dominant and autosomal recessive inheritance, while two families showed
mosaicism for dominant mutations. Although no specific phenotype was found to be associated with mutations in either gene, clinical and
histological features together with pedigree data may be used in guiding mutation detection. Finding the causative mutation(s) determines the
mode of inheritance and permits prenatal diagnosis if requested, but will not as such permit prognostication.
q 2004 Elsevier B.V. All rights reserved.
Keywords: Nemaline (rod) myopathy; Mutation; Mosaicism
1. Introduction
Nemaline myopathy is a clinically and genetically
heterogeneous congenital myopathy identified in 1958 by
Dr R.D.K. Reye [1] and then described five years later in
two separate, original reports [2,3]. This myopathy is
defined on the basis of muscle weakness and the presence in
the muscle fibres of nemaline bodies. For a review, see
North and co-workers [4].
* Corresponding author. Tel.: þ 358-9-315-5521; fax: þ358-9-315-5106.
E-mail address: [email protected] (C. Wallgren-Pettersson).
1
Present address: Department of Biological and Environmental Sciences,
University of Helsinki, Helsinki, Finland.
0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.nmd.2004.03.006
The International Database on Nemaline Myopathy was
established to answer clinical questions about nemaline
myopathy, to facilitate gene discovery and to serve as a
basis for genotype – phenotype correlations. Clinical and
histological data have been contributed by members of the
European Neuromuscular Centre (ENMC) International
Consortium on Nemaline Myopathy and other clinicians
and pathologists from various countries around the world.
To date, disease-associated mutations have been detected
in the genes for nebulin (NEB) or skeletal muscle alphaactin (ACTA1) in 60 of the more than 270 patients whose
clinical data have been entered into the database. Mutations
in these two genes appear to be the most common causes of
462
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
nemaline myopathy [5]; in one series of 97 patients, 23 had
nebulin mutations and 13 had actin mutations. The result is
based on mutation analysis of 25 –50% of the giant nebulin
gene, which has a transcript of 21 kb and a total of 183 exons
[6,7,8], and on sequencing of the six coding exons of
ACTA1 [9,10].
Mutations in genes encoding the other thin filament
proteins: tropomyosin 2 and 3 and troponin T1 appear to be
rare causes of nemaline myopathy. Mutations in the gene for
tropomyosin 3 (TPM3) have been identified in four kindreds
[11 –15], and mutations in the gene for tropomyosin 2
(TPM2) in three patients in two unrelated families [16]. A
severe form of nemaline myopathy with unusual associated
features due to a recessive mutation in the gene for fast
troponin T (TNNT1) has been described in children of a
Pennsylvanian Amish community [17]. In addition to the
genes currently known to be associated with nemaline
myopathy [18,19], on the basis of genetic linkage results, at
least one further gene is believed to exist. It also remains to
be elucidated whether some cases of the severe form
associated with the fetal akinesia sequence are caused by
mutations in a separate, hitherto unidentified gene [20].
These genes and the associated, rarer forms of nemaline
myopathy are beyond the scope of the present study.
Here we report on an extensive clinical comparison
between patients with nebulin mutations and those with
actin mutations, defining the range of clinical presentations
and the degree of overlap between phenotypes associated
with mutations in either gene.
2. Patients and methods
The 60 patients with NEB or ACTA1 mutations came
from 16 different countries. Clinical and histological data
were entered into the international database and each
patient’s nemaline myopathy was categorised according to
the classification approved by the ENMC International
Consortium on Nemaline Myopathy [21] (Table 1). The
present series comprised 26 patients with nebulin mutations
from 22 families and 34 patients with actin mutations from
Table 1
Summary of clinical classification of nemaline myopathy into six different
categories [18,21]
1. Severe congenital nemaline myopathy. Patients lacking spontaneous
movements or respiration at birth, or with severe contractures or fractures
at birth
2. Intermediate congenital nemaline myopathy. Patients breathing and
moving at birth but later unable to achieve respiratory independence or
ambulation
3. Typical congenital nemaline myopathy. Patients with onset in early
childhood, typical distribution of muscle weakness, milestones delayed but
reached, course slowly progressive or non-progressive
4. Mild childhood-onset nemaline myopathy
5. Adult-onset nemaline myopathy
6. Other forms of nemaline myopathy, with unusual associated features
30 families (for family history, clinical and histological
details, see Table 2). Fifteen of the nebulin mutations have
been reported previously [7,8], as have all of the actin
mutations [9,10,22– 24]. Based on detailed comparisons of
up to 166 clinical items per patient recorded in the
international database, we summarise in the present paper
the clinical correlates of these mutations.
3. Results
Among singleton patients with a very severe clinical
picture at birth, actin mutations dominated (20/30
probands) over nebulin mutations (6/22 probands) ðP ¼
0:0005Þ: Among probands with the typical or mild
clinical form, mutations in the nebulin gene (17/22)
were more common than mutations in the actin gene
(11/30) ðP ¼ 0:004Þ (Table 2).
There were altogether nine multiplex families with
nebulin mutations and five with actin mutations. The nonfamilial cases with identical mutations included two sharing
the same actin mutation, and one patient found to have the
same nebulin mutation as an affected sib pair in another
family. Among the patients sharing the same mutation in
either gene, clinical severity did show some variability, but
no fundamental differences were noted (Tables 2 and 3).
In both groups, muscle weakness was generalised,
usually symmetrical, and most pronounced in the neck
flexors (Figs. 1 and 2). In the nebulin group, the ankle
dorsiflexors were very weak also, while the extensors of the
knees were well preserved in comparison with the knee
flexors. In the actin group, on the other hand, the knee
extensors were weaker than the flexors, and there was better
preservation of the ankle dorsiflexors (Table 2).
Histologically, in both patient groups, predominance of
type 1 fibres and variability of fibre size was common. There
were also no apparent differences in the quantity and
distribution of nemaline bodies. Accumulations of actin
filaments, in the presence or absence of nemaline bodies,
were seen in a few patients with actin mutations, but none
were reported in the nebulin group. Three patients in the
actin group had intranuclear nemaline bodies while none
were observed in the nebulin group. In the nebulin group,
immunohistochemical studies of nebulin yielded inconsistent patterns [25].
Structural abnormalities of the heart were encountered in
one patient in each group. There were no instances of
cardiomyopathy.
4. Patients with nebulin mutations
Table 3A lists nine different hitherto unpublished
mutations in the nebulin gene, and 16 previously published
ones [7,8]. Most of the mutations found in the nebulin gene
are predicted to lead to premature truncation of the nebulin
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
463
Table 2
Clinical and histological details, and family history of altogether 60 patients with nemaline myopathy
Group
Nebulin
Actin
No. of families
No. of patients
Category of nemaline myopathy
Family history: no. of families
22
26 (M:F ¼ 12:14)
Severe 6, intermediate 3, typical 12, mild 5
Singleton pt 13, AR 9
Consanguinity
Patients deceased,
ages at death
Patients alive, (no.),
age range (median age)
4/22 Couples
5/26, 1 day–19 mo
30
34 (M:F ¼ 21:13)
Severe 18, typical 8, mild 3, other 5
Singleton pt 25, AR 2, AD 1, mosaic
parent 2
2/30 Couples
16/34, 1 day–16 yrs (median 4 mo)
Achieved walking
Course of the disease
Polyhydramnios
Fetal movements
Respiration neonatally
Antigravity movements neonatally
Hypotonia neonatally
Feeding difficulties neonatally
Major contractures neonatally
Apgar scores
Cardiac status
EMG findings
Muscle weakness, distribution
Muscle weakness, if not formally
tested
Asymmetry of muscle power
Muscle biopsy findings
Fibre type predominance
Nemaline bodies
Quantity
Location
Severe, (1), 0.6 yr; Intermed., (3), 9–30 yr;
Typical, (12), 1.3– 37 yr (median 7 yrs); Mild,
(5), 23–42 yr
15/16 (Latest 3.7 yr) (one not walking at 9 yr)
Improving 3/21, stable 11/21, deteriorating 4/21
4/18
Normal 13/21, weak and/or infrequent 8/21,
absent 0/21
Normal 16/22, insufficient 5/22, absent 1/22
Present 15/19, none 4/19
Severe 3/18, moderate 11/18, none 4/18
13/22
7/20
1 min: Mean 6.9, range 1– 10; 5 min: Mean 6.9,
range 1–10
Normal 16/18, structural abn. 1/18, right overload
1/18, cardiomyopathy none
Neurogenic 1/14, myogenic 1/14, mixed 6/14,
normal 4/14
Neck flexors and ankle dorsiflexors especially weak
Knee extensors , knee flexors
Proximal 8/9, distal 1/9
Severe, (7), 4 mo– 10 yr (median 1.5 yr);
Typical, (6), 1–42 yr (median 8 yr); mild,
(3), 18–45 yr; other, (1), 7.5 yr
9/9 (Latest 5 yrs)
Improving 5/18, stable 2/18, deteriorating 6/18
8/31
Normal 14/30, weak and/or infrequent 14/30,
absent 2/30
Normal 13/30, insufficient 11/30, absent 6/30
Present 15/30, none 15/30
Severe 20/29, moderate 7/29, none 2/29
26/31
8/25
1 min: Mean 5.6, range 1–9; 5 min: Mean 7.5,
range 5–10
Normal 17/19, structural abn. 1/19, right
overload 1/19, cardiomyopathy none
Neurogenic 7/10, myogenic 3/10
No 18/19, yes 1/19
No 19/20, yes 1/20
Type 1 14/16, type 2 2/16
Type 1 12/12, type 2 0
(þ þþ ) 8/16, (þ þ) 6/16, (þ ) 2/16
Internal 5/11, subsarc. 2/11, both 4/11
(þþ þ) 9/14, (þþ ) 3/14, (þ) 2/14
Internal 4/11, subsarc. 4/11, both 3/11,
intranuclear 2/10
Neck flexors especially weak
Knee extensors . knee flexors
Proximal 10/11, distal 1/11
M:F, males:females; category, form of nemaline myopathy as defined on the basis of the criteria outlined by the ENMC International Consortium on
Nemaline Myopathy [18,21], singleton pt, singleton patient, no other family members affected; AR, autosomal recessive; AD, autosomal dominant; structural
abn., structural abnormality. Nemaline bodies, quantity: (þ) small quantity of nemaline bodies in a small proportion of muscle fibres; (þþ ) nemaline bodies
present in more than one fourth of the fibres; (þ þ þ) numerous nemaline bodies in most fibres. Nemaline bodies, location: Subsarc., subsarcolemmal. Full
clinical details were not available on all patients. Thus, for the separate items, the number of patients in the relevant age group for whom data were known is
indicated. For example, children who died before they had reached the age at which they would normally have walked are not included in the numbers of the
section ‘Achieved walking’.
protein [7,8]. Thus, it appeared likely that a majority of the
patients, homozygous or compound heterozygous for such
mutations, would lack the carboxy terminus of the
nebulin protein, preventing its anchoring into the Z discs.
The patients’ muscle biopsies should also then show no
immunohistochemical labelling with antibodies to the
C-terminal end of nebulin. Clinically, the patients might
have been expected to have an early-lethal form of nemaline
myopathy. Nevertheless, the typical form was the most
common (Fig. 1), and histologically, in most of the patients
studied using antibodies against nebulin, the C-terminal end
of the protein was detected [25].
Although the predominant clinical presentation of these
26 patients with nebulin mutations was the typical,
congenital-onset form of nemaline myopathy, there were
six severe cases [26] (Fig. 2) as well as three intermediate
and five mild cases (Table 2). Autosomal recessive
inheritance was either verified by the identification of
mutations on both alleles of the nebulin gene, or appeared
likely because one mutation detected in the patient(s) was
also found in one of the unaffected parents. In the latter
families, the search for the other mutation is ongoing in the
giant nebulin gene. It is to be noted that while the patients in
three consanguineous families showed homozygosity for
464
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
Table 3A
Patients with mutations in the nebulin (NEB) gene
Patient no.
Category
Type of mutation
Exona
Amino acid substitution
Homozygous/heterozygous
1.1
1.2
2
3.1
3.2
4
5
6
7
8.1
8.2
9.1
9.2
10
11
12
13
14
15
16
17
18
19
20
21
22
Typical
Typical
Typical
Severe
Severe
?Typical
Typical
Severe
?Typical
Mild
Mild
Typical
Intermediate
Typical
?Typical
Severe
Severe
Typical
Severe
?Typical
Intermediate
?Typical
?Mild
Mild
Mild
Intermediate
Frameshift
Frameshift
Frameshift Missense
Frameshift Frameshift
Frameshift Frameshift
Splicing
Nonsense
Frameshift
Frameshift
Frameshift
Frameshift
Missense Frameshift
Missense Frameshift
Missense Frameshift
Nonsense
Nonsense
Frameshift
Frameshift
Splicing Nonsense
Splicing
Nonsense Missense
Missense
Splice site
Missense
Nonsense Frameshift
Missense
156 (165)
156 (165)
163 (172) 122
163 (172) 177 (181)b
163 (172) 177 (181)b
154 (163)
181 (185)
180 (184)
171 (177D)b
171 (177D)b
171 (177D)b
151 (160) 122
151 (160) 122
151 (160) 61
152 (161)
180 (184)
180 (184)
171 (177D)b
Intron 147 (156) 148 (157)
Intron 156 (165)
163 (172) 122
142 (152)
Intron 148 (157)
147 (156)
170 (177C)b 7
167 (176)b
Stop at 58507
Stop at 58507
Stop at 61237 Ser4665Ile
Stop at 61237 Stop at 63917
Stop at 61237 Stop at 63917
Heterozygous
Heterozygous
Compound heterozygote
Compound heterozygote
Compound heterozygote
Homozygous
Homozygous
Homozygous
Homozygous
Homozygous
Homozygous
Compound heterozygote
Compound heterozygote
Compound heterozygote
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Compound heterozygote
Heterozygous
Compound heterozygote
Heterozygous
Heterozygous
Heterozygous
Compound heterozygote
Heterozygous
7
Glu6536Stop7
Stop at 65088
Stop in exon 1718
Stop in exon 1718
Stop in exon 1718
Thr5681Pro8 Stop at 4655
Thr5681Pro8 Stop at 4655
Thr5681Pro8 Stop at 2801
Tyr5689Stop8
Leu6523Stop8
Stop at 65128
Stop in exon 1718
Arg5564Stop8
8
Gln6105Stop Ser4665Ile
Pro5389Ala
Asp5528His
Stop in exon 170
Tyr6216Ser
Mutations found in the nebulin gene of 26 patients with nemaline myopathy. Category: form of nemaline myopathy as defined on the basis of the criteria
outlined by the ENMC International Consortium on Nemaline Myopathy [18,21].
a
Previous exon nomenclature, based on protein repeat domain number [43], in parentheses.
b
Alternatively spliced exon.
a mutation in the nebulin gene, compound heterozygosity
for nebulin mutations was found in a fourth consanguineous
family.
The majority of the nebulin mutations found to date
reside in the 30 end of the gene (Table 3A), encoding the
C-terminus of the protein, which is where the search for
mutations has initially concentrated. The C-terminal end of
the protein extends into the Z disc. Due to the extensive
alternative splicing of nebulin exons in this region, several
different nebulin isoforms are expressed.
In five patients with mild or typical phenotypes,
mutations were identified in exons 170 and 171 (previously
named 177C and 177D), which are known to be differentially expressed [7,27,28]. Patient 21, with a mild phenotype, is a compound heterozygote for a nonsense mutation in
exon 170 (former 177C) and a frameshift mutation in exon
7. The mutation in exon 7 is expected to cause truncation of
all nebulin isoforms at amino acid 227 in the N-terminal end
of the protein. The nonsense mutation in exon 170 (177C) is
expected to affect only isoforms expressing exon 170
(177C). Such isoforms seem to be rare [6].
In five other patients with typical or intermediate
phenotypes, compound heterozygosity was determined,
with one of the mutations in each patient being a missense
mutation, and the other a truncating mutation in a
constitutively expressed exon (Table 3A, patients 2, 9.1,
9.2, 10 and 17). Patients 2 and 17 are both compound
heterozygotes for mutations in exon 163 (172) expected to
cause truncation of the C-terminal end of nebulin, and a
missense mutation, Ser46651Ile, which changes a conserved SDXXYK-actin-binding motif. Patient 10, with a
typical phenotype, was found to be a compound heterozygote for a frameshift mutation in exon 61 and a missense
mutation, Thr5681Pro, in exon 151 (160). The frameshift
mutation is expected to cause truncation of all nebulin
isoforms at amino acid 2801. Exon 151 is expected to be
expressed in all nebulin isoforms, and the missense
mutation is predicted to cause a conformational change in
the protein [8]. The same missense mutation was also found
in an affected sib-pair (patients 9.1 and 9.2), who carried a
frameshift mutation in exon 122 on their other allele.
The splice-site mutations in patients 4, 16 and 19 are
predicted to generate transcripts otherwise normal, but
lacking one exon. In half the typical cases, only one
heterozygous mutation is known, precluding predictions
regarding what nebulin isoforms might be present in
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
465
Table 3B
Patients with mutations in the actin (ACTA1) gene
Patient no.
Clinical category
Type of mutation
Exon/codon change
Amino acid substitution
Homozygous/heterozygous
1
2
3
4
5
6.1
6.2
6.3
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25.1
25.2
25.3
26
27
28
29
30
Other form
Other form
Other form
Other form
Severe
Typical
?Typical
?Mild
Mild
Mild
Typical
Severe
Severe
Severe
Severe
Severe
?Typical
Typical
Severe
Severe
Other form
Severe
Typical
Severe
Typical
Severe
Severe
Severe
Severe
Severe
Severe
Severe
Typical
Severe
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Splice site
Splice site
Splice site
Missense
Missense
Missense
Missense
Missense
2/GGC-CGC
4/GTG-CTG
4/GTG-TTG
2/CAC-TAC
2/CTT-CCT 5/GAG-GTG
3/AAC-AGC
3/AAC-AGC
3/AAC-AGC
3/ATG-GTG
3/ATC-ATG
4/GGC-GAC
4/CGC-TGC
4/CGC-GGC
5/ATG-ACG
5/CGC-CAC
5/CAG-CTG
5/GGT-TGT
6/ATG-AGG
6/AAC-AAG
6/GAC-GGC
7/ATC-CTC
7/GTC-TTC
6/ATG-AAG
7/CGC-AGC
6/GCG-GAG
5/GAG-CAG
Intron 5 ag/GTAT-at/GTAT
Intron 5 ag/GTAT-at/GTAT
Intron 5 ag/GTAT-at/GTAT
4/GCG-GGG
3/GTG-GCG
2/GTG-CTG
7/AAA-CAA
3/GTC-TTC
Gly15Arg8
Val163Leu8
Val163Leu8
His40Tyr8
Leu94Pro8 Glu259Val8
Asn115Ser8,9
Asn115Ser8,9
Asn115Ser8,9
Met132Val8,21
Ile136Met9
Gly182Asp8
Arg183Cys8
Arg183Gly9
Met227Thr22
Arg256His8
Gln263Leu8,23
Gly268Cys9
Met269Arg22
Asn280Lys8
Asp286Gly8
Ile357Leu9
Val370Phe8
Met283Lys22
Arg372Ser22
Ala272Glu22
Glu224Gln22
– 22
– 22
– 22
Ala170Gly22
Val134Ala22
Val35Leu22
Lys373Gln22
Val43Phe22
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Compound heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous, father mosaic
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous, mother mosaic
Heterozygous
Heterozygous
Homozygous
Homozygous
Homozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Heterozygous
Mutations identified in the actin gene of 34 patients with nemaline myopathy. Category: form of nemaline myopathy as defined on the basis of the criteria
outlined by the ENMC International Consortium on Nemaline Myopathy [18,21].
the patients’ muscles. In most of these cases, the known
mutation is expected to result in a truncated protein.
In three out of four patients with a severe clinical picture
in whom both mutations were known, the mutations were
either frameshift or nonsense mutations. One patient with a
severe phenotype showed compound heterozygosity for a
frameshift mutation in the differentially expressed exon 177
(181), and another frameshift mutation in exon 163 (172).
Both mutations are expected to cause truncation of the
protein molecules, but due to the alternative usage of exon
177 (181), this allele is expected to result in the expression
of normal nebulin isoforms as well. A somewhat similar
situation was observed in another patient, also with severe
nemaline myopathy (Patient 15, Table 3A), found to be a
compound heterozygote for a nonsense mutation in exon
148 (157) and a splice-site mutation in intron 147 (156). The
nonsense mutation is predicted to cause the synthesis of a
truncated protein lacking all the simple repeats and the
unique C-terminal domains of nebulin, whereas the splice-
site mutation should allow the formation of both normal and
abnormal transcripts.
The remaining severe cases showed homozygous or
heterozygous truncating mutations in exon 180 (184), which
are expected to cause absence of the serine-rich domain and
the SH3 domain in the C-terminus [26].
5. Patients with actin mutations
Thirty-four patients had ACTA1 mutations. Most of these
mutations were de novo dominant, missense mutations,
predicted to result in the formation of an altered actin
molecule (Table 3B). The severe form of nemaline
myopathy was the most common clinical presentation
(Fig. 2), but eight out of the 34 patients had the typical
form (Table 2, Fig. 1) and three were mild cases [22]. Five
had unusual associated features, including three with
intranuclear rods and three whose histological picture was
466
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
Fig. 2. Two patients with severe nemaline myopathy, in one caused by a
mutation in the nebulin gene, in the other by a mutation in the actin gene.
Fig. 1. Two patients with the typical form of nemaline myopathy, one with a
mutation in the nebulin gene, the other with a mutation in the actin gene.
Reprinted from Journal of the Neurological Sciences, Volume 89, C.
Wallgren-Pettersson, Congenital nemaline myopathy: a clinical follow-up
study of twelve patients, 1–14, 1989, with permission from Elsevier.
dominated by an excess of thin filaments, i.e. actin
accumulation [9,29].
The majority of the cases with actin mutations were
sporadic, but it is to be noted that there were five multiplex
families. One of these families showed autosomal dominant
and two autosomal recessive inheritance, while in each of
the two other families, one of the parents was found to have
somatic mosaicism for the mutation. These families are the
only two showing mosaicism out of the total of nearly 80
families in which ACTA1 mutations have been identified
[23]. The mosaic parents had not sought medical attention
for any muscle complaint but neither did well in sports.
Two families with actin mutations were consanguineous.
In one of these, the nemaline myopathy was recessively
inherited, while in the other, the disorder was caused by a
new dominant mutation that had occurred in the patient.
6. Discussion
Although initial studies suggested that nebulin mutations
were associated with the typical form of nemaline myopathy
while actin mutations were found preferentially in severe
and mild cases [21], the present data clearly demonstrate a
significant overlap as mutations in either gene may be
associated with a wide range of severity (Figs. 1 and 2).
Nevertheless, in general, the severity and the course of the
disease appear milder in the group of patients with nebulin
mutations than in the group with actin mutations (Tables 2
and 3).
Familial cases showing an autosomal recessive inheritance pattern point in the direction of the nebulin gene,
although actin mutations are not a priori ruled out as
the cause of the disease. An autosomal dominant pattern is
likely to be due to a mutation in the actin gene rather than in
the nebulin gene, as to our knowledge no dominant
mutations have hitherto been identified in the nebulin gene.
Among sporadic cases, a singleton patient with a very
severe clinical picture at birth is more likely to have a de
novo dominant actin mutation (20/30 probands) than two
recessive nebulin mutations (6/22 probands) ðP ¼ 0:0005Þ:
Conversely, probands with the typical or mild clinical form
are more likely to have mutations in the nebulin gene
(17/22) than in the actin gene (11/30) ðP ¼ 0:004Þ:
As most new cases of nemaline myopathy are sporadic,
the family history often does not provide a clear indication
of the mode of inheritance. Thus, determining whether the
cause is a new dominant mutation, or two inherited
recessive mutations, requires identification of the causative
mutation(s) [18]. Although highly desirable for confirming
diagnosis and mode of inheritance in individual families,
mutation detection in nemaline myopathy is currently not
available as a service, with the exception of analysis of the
actin gene. For the giant nebulin gene, substantial efforts are
needed to establish practically feasible methods for
routine mutation detection. Possibly, this can be achieved
using denaturing high performance liquid chromatography
(Lehtokari et al. in preparation).
Both patient groups included neonatally severely affected
patients. Mortality was greater in the group with actin
mutations. In a number of these cases, active support was
withdrawn as it failed to lead to improvement over a period
of days or weeks, and prognosis was deemed hopeless. In
each group, those who improved and lived into late
childhood usually achieved walking. Detailed clinical
comparisons, however, did not identify any definite prognostic indicators. Thus, from these data, it does not appear
justified in individual cases to base predictions of prognosis
on which gene the mutation resides in. In other words, the
finding of a mutation in either of the two genes will not as
such facilitate decision-making in questions of active versus
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
passive treatment of severely affected infants. These difficult
situations thus need to be managed along the same lines as
for any neonate suffering from a severe disorder.
In both the nebulin group and the actin group, muscle
weakness was generalised and usually symmetrical, with a
predilection for the neck flexors. In the nebulin group, the
dorsiflexors of the ankles were especially weak also, and the
knee extensors were less severely affected than the knee
flexors, confirming previous findings [30,31]. In the actin
group, in contrast, the extensors of the knee were weaker
than the flexors. The reason for these subtle differences in
patterns of muscle involvement between the two groups is
not clear at present.
Detailed histological comparisons of the two groups with
mutations detected are in preparation. Those compiled in the
present paper are based on biopsy findings reported by the
respective pathologists involved in the diagnostic procedure, and are thus heterogeneous. Details such as the
presence or absence of intranuclear nemaline bodies had not
always been assessed. However, in summary, if the
histological picture is dominated by actin accumulations,
or if intranuclear nemaline bodies are present, it is worth
searching for a mutation in the actin gene [9], and where
labelling with different nebulin antibodies yields inconsistent patterns, the nebulin gene would be first priority [25].
Thus, the clinical and histological features of individual
patients may give a rough indication of where to start
molecular genetic analysis.
Disruption of the myofilamentous structure as seen on
muscle biopsy, and so-called neurogenic findings seen at
electromyography were more common in the actin group
than in the nebulin group, in which mixed patterns of both
‘myogenic’ and ‘neurogenic’ patterns are known from a
previous study to be common in older patients [32].
However, purely ‘neurogenic’ findings were mainly seen
in patients with the severe form, and as no patients with the
severe form due to nebulin mutations had undergone
electromyography, it is unclear whether these differences
are simply related to severity of the disease or whether they
are related to genetic etiology.
A better understanding of the disease pathogenesis from
mutations in the nebulin gene resulting in the various
phenotypes awaits a detailed characterisation of the
numerous human nebulin isoforms and their expression
patterns. Z discs vary in structure and width in different
muscle and fibre types, and during development. A positive
correlation has been shown between Z-disc width and
the number of nebulin C-terminal repeats [27]. It has
been suggested that differential expression of the nebulin
C-terminal repeats, together with the differentially
expressed titin N-terminal repeats, account for the full
range of Z-disc widths and structures [27]. Thus, a wide
nebulin isoform diversity appears to be required for normal
muscle development [6 –8].
The typical form of nemaline myopathy, clinically
milder than the severe form, appears to be associated with
467
the maintenance of a broad, albeit not full, isoform diversity.
Mutations in the alternatively spliced exons 170 (177C) and
171 (177D) appear to correlate with a typical or even mild
phenotype, suggesting that exon 171 (177D) is expressed in a
few nebulin isoforms only. Our results from RT-PCR studies
of RNA isolated from different leg muscles indicate that this
is the case [6]. The majority of isoforms would thus be
normal in these patients. Other patients with the typical form
have homozygous or heterozygous splice-site mutations
leading to skipping of one exon, normally not alternatively
spliced out. Yet others show the presence of a missense
mutation in addition to a truncating one, permitting from that
allele the expression of full-length nebulin molecules.
One patient (patient 5, Table 3A) with typical nemaline
myopathy was found to be homozygous for a nonsense
mutation in exon 181 (185) of the nebulin gene, expected to
truncate the last 134 amino acids of the protein [7]. Unlike
such mutations in many other genes, the nebulin mutations
do not always lead to a severe form of the disorder. The
presence of the carboxy terminus of the protein despite
homozygous mutations predicted to cause truncation of the
protein is likely to be due to the expression only of isoforms
not containing the exon harbouring the mutation, or to
skipping of this exon to produce an internally deleted
protein. It is also possible that some of the mutations affect
exonic splicing enhancers, thus inducing exon skipping
[33]. In addition, immunohistochemical labelling with
antibodies specific to the C-terminal end of nebulin
indicate that truncated proteins are expressed in a subset
of fibres [7,25]. Apparently, this alternative nebulin
molecule is sufficient to maintain a muscle function
compatible with life, in many cases leading only to a
relatively mild muscle disorder.
Skipping of an exon harbouring a disease-causing
mutation to produce milder disease is a phenomenon
previously well documented. Among the muscle disorders
the evidence comes mainly from patients with out-of-frame
deletions of certain regions of the dystrophin gene causing
muscular dystrophy of the milder, Becker type instead of the
expected Duchenne-type severity [34]. In one family
segregating a mutation in the dystrophin gene the patients
exhibited a range of clinical pictures, the variation in
severity being explained by differences in levels of exon
skipping. The phenotypes varied from that of a healthy adult
man with mildly elevated serum creatine kinase activity
through a mildly affected young adult to an overtly affected
middle-aged man with cardiomyopathy and Becker-type
muscular dystrophy [35]. Alternative splicing of the AMP
deaminase gene apparently rescues the phenotype in some
persons with a common homozygous nonsense mutation
[36]. These examples might indicate hope for therapeutic
approaches such as inducing skipping of the exon harbouring the disease-causing mutation, e.g. through the use of
antisense oligonucleotides [37] or through enhanced
expression of alternative isoforms to maintain a broad
isoform diversity.
468
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
In half the typical cases and in one of the intermediate
cases with nebulin mutations, to date only one heterozygous
mutation has been identified. In most of these cases, the
mutation is predicted to lead to truncation of the nebulin
molecule. We therefore speculate that the other as yet
unidentified mutation is likely to be less severe, e.g. of
missense or splice-site nature or present in differentially
expressed exons.
Some patients with severe nemaline myopathy were
found to have mutations in exon 180 (184) of the nebulin
gene, or truncating mutations in exons expressed in the
majority of isoforms. The mutations in exon 180 (184) are
predicted to cause absence of the C-terminal serine-rich and
SH3 domains. These two domains are highly conserved
between species, and they are present in the Z discs of all
types of skeletal muscle, suggesting that they play important
roles in the structure of vertebrate Z discs [27,38]. These
domains do not however appear to be necessary for the
incorporation of nebulin C-terminal modules into the Z
discs [39]. This observation does not rule out the importance
of the nebulin serine-rich and SH3 domains in the regulation
and maintenance of the highly ordered structure of the Zdisc. SH3 domains closely related to the nebulin SH3
domain, such as those of the yeast actin-binding protein
ABP1 and cortactin, reside in proteins involved in the
regulation of the assembly of the actin cytoskeleton [38]. A
recently described protein, myopalladin, has been reported
to bind to the SH3-domain of nebulin and hence link nebulin
to a-actinin in the Z disc [40]. Furthermore, the nebulin
SH3-domain has been shown to have high binding affinity
for a stretch of titin known to colocalise with nebulin in the
Z disc [41].
The mutations in the actin gene are spread through all six
of the coding exons, with some being located within known
functional sites of the actin protein [10,42]. There appears to
be some clustering of mutations causing certain histological
phenotypes, e.g. that of actin myopathy with accumulation
of thin filaments [40]. The majority of mutations are
missense and cause dominant disease, suggesting that they
do so by producing a poison peptide, i.e. by a gain of
function. This is supported by two ACTA1 mutations that
effectively lead to a null allele resulting in recessive disease
[23]. The presence of these two mutations in unaffected
heterozygotes suggest that normal function can be achieved
in some cases by having one wild-type ACTA1 allele.
In each of two families with actin mutations, one parent
showed somatic mosaicism for the mutation, a phenomenon
not hitherto encountered in families with mutations in the
nebulin gene. This needs to be taken into account in the
genetic counselling of families in whom actin mutations
have been identified. Somatic mosaicism has been observed
in two out of nearly 80 families in total in which ACTA1
mutations have been identified [23]. Hitherto, there have
been no reports of proven gonadal mosaicism, but
the observed existence of somatic mosaicism makes
gonadal mosaicism likely.
A further aspect is notable in the genetic counselling of
families with nemaline myopathy. The occurrence in a
consanguineous family of a de novo dominant mutation in
the actin gene, and the compound heterozygosity in one of
the four consanguineous families with nebulin mutations
show that caution is warranted in drawing conclusions
regarding the mode of inheritance from pedigree data only.
In summary, it is possible that the nature of the mutation
and its effect on the protein determines the phenotype to a
greater extent than the gene in which the mutation has
occurred. Naturally, other factors also, both genetic and
non-genetic, contribute to the diversity of clinical pictures.
Extended clinical series, further experimental studies and
wider knowledge of the normal isoform diversity and
structure – function relationships of the sarcomeric proteins
in question are needed to answer remaining questions
regarding genotype– phenotype correlations.
Acknowledgements
We thank the following colleagues for contributing data
and samples on one or two patients each: Dr Janice
Anderson, Prof. Corrado Angelini, Dr Emilia Bijlsma, Prof.
Peter Van den Bergh, Prof. Kate Bushby, Prof. Carsten
Bönnemann, Prof. Angus Clarke, Drs Basil Darras and
Martin Eswara, Prof. Michel Fardeau, Drs Maria Luisa
Giovanucci Uzielli, Nathalie Goemans, Claudio Graziano,
Carolyn Green and Margaret Grunnet, Prof. Folker
Hanefeld, Drs Arvid Heiberg, Jens Michael Hertz, Marc
D’Hooghe and Imelda Hughes, Prof. Christoph Hübner,
Prof. Susan Iannaccone, Prof. Constantin von Kaisenberg,
Drs Martin Lammens, Carol Leicher, Hanns Lochmüller,
Meriel McEntagart, Julie McGaughran, Thor Ruud-Hansen,
Rolf Schlösser and Gudrun Schreiber, Prof. Rebecca
Sutphen, Drs Kathryn Swoboda and Tal Thomas, Prof.
Haluk Topaloglu, Prof. Andoni Urtizberea, Drs Christophe
Vial, Jaqueline Vigneron and Sheila Wallace. We are
grateful to the other members of the ENMC International
Consortium (Drs Anthony Akkari, Olli Carpén and Avril
Castagna, Prof. Victor Dubowitz, Drs Baziel van Engelen,
Marc Fiszman, Claudio Graziano and Edna Hardeman, Prof.
Susan T. Iannaccone, Dr Heinz Jungbluth, Prof. Siegfried
Labeit, Drs Martin Lammens and Carmen Navarro,
Prof. Ikuya Nonaka, Drs Berardino Porfirio and Norma
B. Romero, Prof. Caroline Sewry, Prof. Lars-Eric Thornell,
Dr Mariz Vainzof) for continuing inspiring collaboration, to
the same persons and to clinicians around the world for
contributing data and samples not used in this study, and to
the European Neuromuscular Centre (ENMC) for organisational support. We thank Patricia Zilliacus, MSc, for
assistance with the international database on nemaline
myopathy, and Dr Pankaj B. Agrawal for statistical
calculations. KP was supported by grants to CWP from
the University of Helsinki, the Association Francaise contre
les Myopathies, the Sigrid Jusélius Foundation, the Finska
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
Läkaresällskapet and the Medicinska understödsföreningen
Liv och Hälsa, AHB by NIH AR44345, the Muscular
Dystrophy Association of the USA and the Joshua Frase
Foundation. NGL is supported by the Australian National
Health and Medical Research Council (Fellowship 139170,
Project grants 139039 and 110242) and the Association
Francaise contre les Myopathies. KJN is a CJ Martin Fellow
(ID No. 212086) with the Australian National Health and
Medical Research Council.
References
[1] Schnell C, Kan A, North KN. An artefact gone awry: identification of
the first case of nemaline myopathy by Dr R.D.K. Reye. Neuromuscul
Disord 2000;10:307 –12.
[2] Shy GM, Engel WK, Somers JE, Wanko T. Nemaline myopathy: a
new congenital myopathy. Brain 1963;86:793– 810.
[3] Conen PE, Murphy EG, Donohue WL. Light and electron microscopic
studies of myogranules in a child with hypotonia and muscle
weakness. Can Med Assoc J 1963;89:983–6.
[4] North KN, Laing NG, Wallgren-Pettersson C, the ENMC International Consortium on Nemaline Myopathy. Nemaline myopathy:
current concepts. J Med Genet 1997;34:705–13.
[5] Wallgren-Pettersson C, Laing NG. 83rd ENMC International Workshop: Fourth Workshop on Nemaline Myopathy, 22–24 September,
2000, Naarden, The Netherlands. Neuromuscul Disord 2001;11:
589–95.
[6] Donner K, Sandbacka M, Lehtokari V-L, Wallgren-Pettersson C,
Pelin K. Complete genomic structure of the human nebulin gene and
identification of alternatively spliced isoforms. Eur J Hum Genet,
in press.
[7] Pelin K, Hilpelä P, Sewry C, et al. Mutations in the nebulin gene
associated with autosomal recessive nemaline myopathy. Proc Natl
Acad Sci, USA 1999;96:2305–10.
[8] Pelin K, Donner K, Holmberg M, Jungbluth H, Muntoni F, WallgrenPettersson C. Nebulin mutations in autosomal recessive nemaline
myopathy: an update. Neuromuscul Disord 2002;12:680 –6.
[9] Nowak KJ, Wattanasirichaigoon D, Goebel HH, et al. Mutations in the
skeletal muscle alpha actin gene in patients with actin myopathy and
nemaline myopathy. Nat Genet 1999;23:208–12.
[10] Ilkovski B, Cooper ST, Nowak K, et al. Nemaline myopathy caused
by mutations in the muscle alpha-skeletal-actin gene. Am J Hum
Genet 2001;68:1333 –43.
[11] Laing NG, Majda BT, Akkari PA, et al. Assignment of a gene (NEM1)
for autosomal dominant nemaline myopathy to chromosome 1. Am J
Hum Genet 1992;50:576–83.
[12] Laing NG, Wilton SD, Akkari PA, et al. A mutation in the alphatropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy NEM1. Nat Genet 1995;9:75–9.
[13] Tan P, Briner J, Boltshauser E, et al. Homozygosity for a nonsense
mutation in the alpha-tropomyosin slow gene TPM3 in a patient with
severe infantile nemaline myopathy. Neuromuscul Disord 1999;9:
573–9.
[14] Wattanasirichaigoon D, Swoboda KJ, Takada F, et al. Mutations of
the slow muscle alpha-tropomyosin gene, TPM3, are a rare cause of
nemaline myopathy. Neurology 2002;59:613–7.
[15] Durling HJ, Reilich P, Müller-Höcker J, et al. De novo missense mutation in a constitutively expressed exon of the slow
alpha-tropomyosin gene TPM3 associated with atypical,
sporadic case of nemaline myopathy. Neuromuscul Disord 2002;
12:947–51.
469
[16] Donner K, Ollikainen M, Ridanpää M, et al. Mutations in the btropomyosin (TPM2) gene in two families diagnosed with nemaline
myopathy. Neuromuscul Disord 2002;12:151–8.
[17] Johnston JJ, Kelley RI, Crawford TO, et al. A novel nemaline
myopathy in the Amish caused by a mutation in troponin T1. Am J
Hum Genet 2000;67:814–21.
[18] Wallgren-Pettersson C, Hilpelä P, Donner K, et al. Clinical and
genetic heterogeneity in autosomal recessive nemaline myopathy.
Neuromuscul Disord 1999;9:564– 72.
[19] Wallgren-Pettersson C. Gene table: the congenital myopathies. Eur J
Paed Neurol 2001;5:87–8.
[20] Lammens M, Moerman PH, Fryns JP, et al. Fetal akinesia
sequence caused by nemaline myopathy. Neuropediatrics 1997;28:
116–9.
[21] Wallgren-Pettersson C, Laing NG. Report of the 70th ENMC
International Workshop: nemaline myopathy. Neuromuscul Disord
2000;10:299–306.
[22] Jungbluth H, Sewry CA, Nowak KJ, et al. Mild phenotype of
nemaline myopathy with sleep hypoventilation due to a mutation in
the skeletal muscle actin (ACTA1) gene. Neuromuscul Disord
2001;11:35 –40.
[23] Sparrow JC, Nowak KJ, Durling HJ, et al. Muscle disease caused by
mutations in the skeletal muscle alpha-actin gene (ACTA1).
Neuromuscul Disord 2003;13:519 –31.
[24] Buxmann H, Schlosser R, Schlote W, et al. Congenital nemaline
myopathy due to ACTA1-gene mutation and carnitine insufficiency: a
case report. Neuropediatrics 2001;32:267 –70.
[25] Sewry C, Brown SC, Pelin K, et al. Abnormalities in the expression of
nebulin in chromosome-2 linked nemaline myopathy. Neuromuscul
Disord 2001;11:146–53.
[26] Wallgren-Pettersson C, Donner K, Sewry C, et al. Mutations in the
nebulin gene can cause severe congenital nemaline myopathy.
Neuromuscul Disord 2002;12:674 –9.
[27] Millevoi S, Trombitas K, Kolmerer B, et al. Characterization of
nebulette and nebulin and emerging concepts of their roles for
vertebrate Z-discs. J Mol Biol 1998;282:111 –23.
[28] Pelin K, Labeit S, Wallgren-Pettersson C. Nebulin, Human.
Encyclopedia of Molecular Medicine, vol. 5. Chichester: Wiley;
2002. p. 2223–5.
[29] Goebel HH, Anderson JR, Hübner C, Oexle K, Warlo I. Congenital
myopathy with excess of thin myofilaments. Neuromuscul Disord
1997;7:160 –8.
[30] Wallgren-Pettersson C. Congenital nemaline myopathy: a clinical
follow-up study of twelve patients. J Neurol Sci 1989;89:1–14.
[31] Wallgren-Pettersson C, Kivisaari L, Jääskeläinen J, Lamminen A,
Holmberg C. Ultrasonography, CT and MRI of muscles in congenital
nemaline myopathy. Pediatr Neurol 1990;6:20–8.
[32] Wallgren-Pettersson C, Sainio K, Salmi T. Electromyography in
congenital nemaline myopathy. Muscle Nerve 1989;12:587–93.
[33] Cartegni L, Chew SL, Krainer AR. Listening to silence and
understanding nonsense: exonic mutations that affect splicing. Nat
Rev Genet 2002;3:285–98.
[34] Chelly J, Gilgenkrantz H, Lambert M, et al. Effect of dystrophin gene
deletions on mRNA levels and processing in Duchenne and Becker
muscular dystrophies. Cell 1990;63:1239 –48.
[35] Ginjaar IE, Kneppers ALJ, vd Meulen J-DM, et al. Dystrophin
nonsense mutation induces different levels of exon 29 skipping and
leads to variable phenotypes within one BMD family. Eur J Hum
Genet 2000;8:793 –6.
[36] Morisaki H, Morisaki T, Newby LK, Holmes EW. Alternative
splicing: a mechanism for phenotypic rescue of a common inherited
defect. J Clin Invest 1993;91:2275 –80.
[37] van Deutekom JCT, Bremmer-Bout M, Janson AAM, et al.
Anitsense-induced exon skipping restores dystrophin expression in
DMD patient derived muscle cells. Hum Mol Genet 2001;10:
1547– 54.
470
C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470
[38] Politou AS, Millevoi S, Gautel M, Kolmerer B, Pastore A. SH3 in
muscles: solution structure of the SH3 domain from nebulin. J Mol
Biol 1998;276:189–202.
[39] Ojima K, Lin ZX, Bang M-L, et al. Distinct families of Z-line
targeting modules in the COOH-terminal region of nebulin. J Cell
Biol 2000;150:553–66.
[40] Bang M-L, Mudry RE, McElhinny AS, et al. Myopalladin: a novel
145-kilodalton sarcomeric protein with multiple roles in Z-disc and Iband protein assemblies. J Cell Biol 2001;153:413–27.
[41] Politou AS, Spadaccini R, Joseph C, et al. The SH3 domain of
nebulin binds selectively to type II peptides: theoretical
prediction and experimental validation. J Mol Biol 2002;316:
305 –15.
[42] Sheterline P, Clayton J, Sparrow JC. Actin. Oxford: Oxford
University Press; 1999.
[43] Pfuhl M, Winder SJ, Castiglione Morelli MA, Labeit S, Pastore A.
Correlation between conformational and binding properties of nebulin
repeats. J Mol Biol 1996;257:367– 84.