test sites in screening for DSP. This will maximize
the probability of obtaining accurate and comparable
results at different assessment times.
1. Young MJ, Boulton AJM, Macleod AF, Williams DRR,
Sonksen PH. A multicentre study of the prevalence of diabetic
peripheral neuropathy in the United Kingdom hospital clinic
population. Diabetologia 1993;36:150–154.
2. Diabetes Control and Complications Trial Research Group.
The effect of intensive treatment of diabetes on the develop-
ment and progression of long-term complications in insulin-
dependent diabetes mellitus. N Engl J Med 1993;329:977–986.
3. UK Prospective Diabetes Study (UKPDS) Group. Intensive
blood-glucose control with sulfonylureas or insulin compared
with conventional treatment and risk of complications in
patients with type 2 diabetes (UKPDS 33). Lancet 1998;352:
4. Boulton A. Lowering the risk of neuropathy, foot ulcers and
amputations. Diabet Med 1998;15(suppl 4):S57–S59.
5. Rith–Najarian S, Stolusky T, Gohdes D. Identifying diabetic
patients at high risk for lower-extremity amputation in a pri-
mary health care setting. A prospective evaluation of simple
screening criteria. Diabetes Care 1992;15:1386–1389.
6. Perkins BA, Olaleye D, Zinman B, Bril V. Simple screening
tests for peripheral neuropathy in the diabetes clinic. Diabetes
7. Olaleye D, Perkins BA, Bril V. Evaluation of three screening
tests and a risk assessment model for diagnosing peripheral
neuropathy in the diabetes clinic. Diabetes Res Clin Prac
8. Bril V, Kojic J, Ngo M, Clark K. Comparison of a Neurothesi-
ometer and vibration in measuring vibration perception
thresholds and relationship to nerve conduction studies. Dia-
betes Care 1997;20:1360–1362.
9. Young M, Breddy J, Veves A, Boulton A. The prediction of
diabetic neuropathic foot ulceration using vibration perception
thresholds. A prospective study. Diabetes Care 1994;17:557–
10. Bril V, Perkins BA. Correlation of vibration perception thresh-
old values obtained with the Neurothesiometer and CASE IV
devices. Diabet Med 2002 (in press).
Mutations of the slow
gene, TPM3, are
a rare cause of
Abstract—The ?-tropomyosin-3 (TPM3) gene was screened in 40 unrelated
patients with nemaline myopathy (NM). A single compound heterozygous pa-
tient was identified carrying one mutation that converts the stop codon to a
serine and a second splicing mutation that is predicted to prevent inclusion of
skeletal muscle exon IX. TPM3 mutations are a rare cause of NM, probably
accounting for less than 3% of cases. The severity of cases with TPM3 mutations
may vary from severe infantile to late childhood onset, slowly progressive forms.
D. Wattanasirichaigoon, MD; K.J. Swoboda, MD; F. Takada, MD, PhD; H.-Q. Tong, MD; V. Lip, BS;
S.T. Iannaccone, MD; C. Wallgren-Pettersson, MD; N.G. Laing, PhD; and A.H. Beggs, PhD
Nemaline myopathy (NM) is a clinically and geneti-
cally heterogeneous disorder characterized by nema-
line rods and skeletal muscle weakness that ranges
in severity from a neonatally life-threatening disor-
der to mild muscle weakness of adulthood.1,2Nema-
line rods appear as abnormalities of the Z lines
associated with disruption of thin filament organiza-
tion suggesting that they may reflect primary abnor-
malities of thin filament proteins. The TPM3 gene
encodes the slow (type 1) fiber-specific isoform of
skeletal muscle ?-tropomyosin. A TPM3 missense
mutation, Met9Arg, was identified in an Australian
family with autosomal dominant transmission of a
late childhood onset, slowly progressive, form of
NM.3Follow-up studies of the TPM3 gene in 76 NM
cases have identified only 1 additional mutation, a
homozygous nonsense mutation in a severe infantile
case of NM.4In contrast, many cases result from
mutations in the nebulin or actin genes, whereas
troponin T (TNNT1) and ?-tropomyosin (TPM2) gene
mutations are rare causes of NM.1,5We report the
identification of the 3rd and the 4th distinct TPM3
mutations in an intermediate severity case of NM,
demonstrating further allelic and clinical heteroge-
neity among patients with TPM3 abnormalities.
American neuromuscular clinics were enrolled after insti-
tutional review board–approved informed consent. Forty
unrelated probands included 7 with the severe congenital
Patients with NM from North
Additional material related to this article can be found on the Neurology
Web site. Go to www.neurology.org and scroll down the Table of Con-
tents for the August 27 issue to find the link for this article.
From the Genetics Division (Drs. Wattanasirichaigoon, Swoboda, Takada,
Tong, and Beggs, and V. Lip), Children’s Hospital, Harvard Medical School,
Boston, MA; Department of Neurology (Dr. Iannaccone), Texas Scottish
Rite Hospital, Dallas; Department of Medical Genetics (Dr. Wallgren-
Pettersson), University of Helsinki, and the Folkhälsan Department of
Medical Genetics, Helsinki, Finland; and the Centre for Neuromuscular
and Neurological Disorders (Dr. Laing), University of Western Australia,
Australian Neuromuscular Research Institute, QEII Medical Centre, Ned-
Supported by grants from the Muscular Dystrophy Association, the Joshua
Frase Foundation, and NIH (AR44345) (to A.H.B.) and the Australian Na-
tional Health and Medical Research Council Project Grant #970104 (to
Received June 1, 1999. Accepted in final form April 22, 2002.
Address correspondence and reprint requests to Dr. Alan H. Beggs, Genet-
ics Division, Children’s Hospital, 300 Longwood Ave., Boston, MA 02115;
Copyright © 2002 by AAN Enterprises, Inc.
form of nemaline myopathy, 15 with intermediate presen-
tations (did not achieve ambulation by 2 years or became
nonambulant by age 11 years), 15 with congenital onset of
myopathy and slowly or nonprogressive courses, and 3
cases of childhood onset NM.1,2All cases were either spo-
radic or consistent with autosomal recessive inheritance
with the exception of four families with dominant inheri-
tance. Patient 13-2 was a sporadic case who was hypotonic
at birth and walked at 17 months, but became wheelchair
bound at age 6 years. Muscle biopsy at age 5 years revealed
subsarcolemmal and intracytoplasmic nemaline rods in type
1 fibers, type 1 fiber predominance, central nuclei, and in-
creased endomysial connective tissue (figure 1; additional
clinical details are available in the Methods section of the
supplementary data; go to www.neurology.org).
Detailed methodology is available
as part of the supplementary data (additional material
related to this article can be found on the Neurology Web
site; go to www.neurology.org).
probands; however, single-strand conformation polymor-
phism analysis of exon IXsk revealed an aberrant con-
former in one patient, 13-2, and his unaffected father.
Sequence analysis revealed that this conformer contained
an A to C mutation at nucleotide 915 of the skeletal muscle
mRNA eliminating the normal skeletal muscle-specific
translational stop signal, replacing it with a serine residue
(TCA) at codon 285 (figure 2). Consequently, the mutated
protein includes an additional 57 amino acids (figure 3B).
Direct sequencing of the patient’s genomic PCR products
confirmed that he was heterozygous for this mutation and
identified a second heterozygous mutation, an AG to AA at
the acceptor splice site of the same exon (see figure 2A).
This change was evident in genomic DNA of the unaffected
mother, but not in the father. Neither mutation was found
in 109 unaffected control individuals.
To determine the effects of the presumed maternal
splice site mutation, rtPCR was performed using a reverse
primer in exon IXsk, sk986L. As expected if the maternal
exon IXsk is not properly spliced, rtPCR products from the
proband’s muscle contained only the mutant paternal tran-
script although the father’s muscle contained both mutant
and wild type transcripts (see figure 2B, C). In contrast,
rtPCR analysis of the proband’s mRNA using an exon Isk
forward primer and a nonmuscle exon VIIInm reverse
primer did reveal an abnormal splice product in which exon
VIIIsk skipped exon IXsk and was instead spliced to exon
VIIInm (see figure 2B, D). Remarkably, because of a frame
shift, this aberrant splice product recreates a normal stop
codon in the same location as encoded by exon IXsk. How-
ever, the clinical phenotype of Patient 13-2 suggests that this
aberrant splicing does not occur at sufficient frequency to
prevent rod formation and clinical weakness.
Western blotting for tropomyosins in the muscle of Pa-
tient 13–2 revealed the presence of a novel reactive protein
approximately 6 kD larger than normal (~34 kD)
?-tropomyosin (see figure 3A). Interestingly, the ~36 kD
band for ?-tropomyosin, which migrates above that for
?-tropomyosin and is also detected by the CH1 antibody,6,7
appeared significantly reduced relative to ?-tropomyosin
levels (reflecting both TPM1 and TPM3 gene products).
TPM3 mutation screening was normal in 39
Figure 1. Pathologic findings in quadriceps muscle from
Patient 13-2. (A) Modified Gomori trichrome staining il-
lustrating intracytoplasmic rod aggregation (arrow), occa-
sional central nuclei, and increased endomysial fibrosis.
Original magnification 200X. (B) Electron micrograph re-
vealing central aggregation of rod bodies. Scale bar ? 5
?m. (C) Electron micrograph showing typical nemaline
rods emanating from Z lines and disrupting the normal
sarcomeric architecture. Scale bar ? 1 ?m.
August (2 of 2) 2002
Figure 2. TPM3 mutation analysis. Intron–exon and exon–exon boundaries are indicated by a vertical line and anno-
tated below each chromatogram. (A) DNA sequence analysis of genomic PCR products containing TPM3-exon IXsk and
adjacent intronic sequences illustrating wild type (top row), maternal splice acceptor site AG to AA mutation (second
row), paternal stop codon A915C mutation (third row), and double heterozygosity for these in the proband (bottom row).
(B) rtPCR analysis of normally spliced skeletal muscle transcripts (using primers sk22U and sk986L) (lanes 1–3) and ab-
normally spliced transcripts (using primers sk22U and nm829L) (lanes 4–6) in Patient 13-2 (lanes 1,4), his father (lanes
2,5), and a control subject (lane 3,6). (C) Sequence analysis of the exon VIIIsk-IXsk junction in rtPCR products from B,
lanes 1–3, from control (top), proband’s father’s (middle), and the proband’s (bottom) skeletal muscle. Although the pro-
band’s father expresses both mutant and wild type alleles at similar levels, only the mutant paternal allele is represented
in the proband when this combination of primers is used. (D) Sequence analysis of the proband’s skeletal muscle mRNA
using sk22U and nm829L primers (B, lane 4) illustrating the aberrant splice product containing exon VIIIsk spliced to
exon VIIInm. Notably, because of a frameshift this introduces an in-frame stop codon (TAA) at precisely the same location
as that of exon IXsk (indicated). (E) Schematic diagram of the TPM3 gene illustrating tissue-specific exons [red ? muscle
(“sk”), green ? nonmuscle (“nm”)] with the normal skeletal muscle and nonmuscle splicing patterns indicated above and
below. The aberrant exon VIIIsk-VIIInm splice product is indicated by dashed line. Approximate locations of PCR prim-
ers used in the rtPCR analysis are shown below.
August (2 of 2) 2002
mutations in rare cases of both autosomal dominant
and recessive forms of NM and demonstrates clinical
heterogeneity associated with TPM3 abnormalities.
Although it is impossible to draw firm conclusions
based on only three known instances of TPM3 muta-
tion, it may be that the relative degrees of severity
relate to the nature of the underlying mutations. The
mildest cases are all from the single family with the
missense mutation, Met9Arg.3In contrast, the most
profoundly weak patient was homozygous for a non-
sense mutation at codon 31 that prevented production
of stable and functional protein.4The intermediate
course of Patient 13-2 may be caused by his compound
heterozygosity for the exon IXsk acceptor splice site
mutation and the termination codon mutation leading
to production of a partially functional, enlarged protein
product. Because tropomyosins form heterodimers be-
tween different isoforms (including ?/? forms),8one
might predict that the *285Ser mutation would act in a
trans-dominant fashion. However, the lack of patho-
logic changes in the muscle or clinical disease in the
patient’s father, Subject 13-1, demonstrates that this is
not the case, perhaps suggesting that the extensive
disruption of ?-helical coiled–coil structure at the car-
boxy terminus of the *285Ser mutant protein prevents
Expression of TPM3 is limited to type 1 fibers,8as
was rod formation in Patient 13-2. Nevertheless, his
biopsy exhibited secondary type 1 fiber predomi-
nance, likely reflecting a dynamic pathologic process
associated with the patient’s loss of ambulation at
age 5 years. The apparent reduction in levels of
?-tropomyosin appears paradoxic in light of the be-
lief that type 1 fibers contain relatively higher ratios
This study confirms the role of TPM3
of ?- to ?-tropomyosin than type 2 fibers.8However,
Salviati et al.,7in protein analyses of single human
skeletal muscle fibers, have identified two popula-
tions of type 1 fibers, one of which expressed both
?-tropomyosin (e.g., TPM1 and TPM3) isoforms and
very little ?-tropomyosin.7The relative lack of
?-tropomyosin in the context of fiber type 1 predom-
inance suggests that muscle in Patient 13-2 prefer-
entially contains this subset of type 1 fibers.
Potential functional or clinical consequences of this
observation are presently unknown.
The last nine amino acid residues of skeletal mus-
cle ?-tropomyosin-3 determine the binding affinity of
unacetylated tropomyosin for actin.9Furthermore,
intact exon IXsk is required for the troponin complex
to promote the high affinity of tropomyosin for skele-
tal actin. Thus, alteration of carboxy-terminal se-
quences, such as the addition of a non–?-helical tail,
might be predicted to considerably alter the interac-
tions between tropomyosin and actin in thin fila-
ments, possibly accounting for the nemaline body
formation and muscle weakness in our patient. Al-
ternatively, by analogy to Tan et al.’s case,4rod for-
mation may simply result from perturbation of the
ratio of functional tropomyosin to other thin filament
components in type 1 fibers.
Both Patient 13-2 and the previously reported re-
cessive case4exhibited a more severe clinical pro-
gression than many of those with nebulin-associated
NM, whereas many patients with actin mutations
have had severe nemaline myopathy.1However, ad-
ditional patients with NM with TPM3, nebulin, and
actin mutations will need to be characterized before
any prognostic predictions can be made. Although the
proportion of NM cases caused by TPM3 mutations
Figure 3. (A) Western blot analysis of skeletal muscle tropomyosins from normal control (deltoid) muscle (lane 1) and
quadriceps muscle from Patient 13-2 (lane 2) using monoclonal anti-sarcomeric tropomyosin antibody clone CH1. Two
bands of apparent mw 34 kD (?-tropomyosin) and 36 kD (?-tropomyosin) are evident in the control muscle.7A novel band
of approximately 40 kD is evident in lane 2 (asterisk). (B) Secondary structure predictions for the mutant ?-tropomyosin 3
protein shown schematically at top. Filled portion includes residues 1–284 of the wild type skeletal muscle isoform.
Hatched area indicates the additional 57 amino acids predicted at the carboxy terminus of the abnormal protein. Shown
are predicted regions of alpha-helix (H), beta-pleated sheet (S), and turn (T), based on Chou-Fasman (CF), Robson-
Garnier (RG), and combined (CfRg) prediction algorithms. Note extensive ?-helical structure of residues 1–284 that con-
tribute to coiled–coil motifs in the native protein.
August (2 of 2) 2002
appears to be low (e.g., 3 of 116 studied families ? Download full-text
2.6%),3,4the relatively small size of the TPM3 gene and
transcript allows for rapid mutation testing, which is
now becoming an important part of the molecular ge-
netic workup for NM.
The authors thank all members of the ENMC International Con-
sortium for advice and comments; the many referring physicians
and patients who have contributed to this study; and the ENMC
and its main sponsors for organizational support.
1. Sanoudou D, Beggs AH. Clinical and genetic heterogeneity in
nemaline myopathy—a disease of skeletal muscle thin fila-
ments. Trends Mol Med 2001;7:362–368.
2. Ryan MM, Schnell C, Strickland CD, et al. Nemaline myop-
athy: a clinical study of 143 cases. Ann Neurol 2001;50:312–
3. Laing NG, Wilton SD, Akkari PA, et al. A mutation in the ?
tropomyosin gene TPM3 associated with autosomal dominant
nemaline myopathy. Nat Genet 1995;9:75–79.
4. Tan P, Briner J, Boltshauser E, et al. Homozygosity for a non-
sense mutation in the alpha-tropomyosin slow gene TPM3 in a
patient with severe infantile nemaline myopathy. Neuromuscul
5. Donner K, Ollikainen M, Ridanpaa M, et al. Mutations in the
beta-tropomyosin (TPM2) gene: a rare cause of nemaline myop-
athy. Neuromuscul Disord 2002;12:151–158.
6. Lin JJ-C, Chou C-S, Lin JL-C. Monoclonal antibodies against
chicken tropomyosin isoforms: production, characterization,
and application. Hybridoma 1985;4:223–242.
7. Salviati G, Betto R, Danieli Betto D, Zeviani M. Myofibrillar-
protein isoforms and sarcoplasmic-reticulum Ca2?-transport
activity of single human muscle fibres. Biochem J 1984;224:
8. Schiaffino S, Reggiani C. Molecular diversity of myofibrillar
proteins: gene regulation and functional significance. Physiol
9. Hammell RL, Hitchcock-DeGregori SE. Mapping the functional
domains within the carboxyl terminus of alpha-tropomyosin
encoded by the alternatively spliced ninth exon. J Biol Chem
inclusions in CADASIL
Abstract—Three siblings with genetically assessed cerebral autosomal dom-
inant arteriopathy with subcortical infarcts and leukoencephalopathy
(CADASIL) with core-like lesions and mitochondrial abnormalities in muscles
are described. Involvement of the Ryanodine receptor 1 gene was excluded. In
the current cases, the relation between molecular genetic lesion and muscle
fiber abnormalities remains to be determined, but the Notch3 gene may
influence mitochondrial metabolism.
A. Malandrini, MD; F. Albani, BSc; S. Palmeri, MD; F. Fattapposta, MD; S. Gambelli, MD; G. Berti, BSc;
A. Bracco, BSc; A. Tammaro, BSc; S. Calzavara, BSc; M. Villanova, MD; M. Ferrari, MD; A. Rossi, MD;
and P. Carrera, PhD
To date, deposition of granular osmiophilic material
in the smooth muscle cells of precapillary arterioles
of the skin, muscle, peripheral nerve, heart, and kid-
ney has been regarded as the only extracerebral in-
arteriopathy with subcortical infarcts and leukoen-
cephalopathy (CADASIL).1,2These lesions do not
have clinical manifestations. Clinical signs are con-
fined to the CNS even in advanced stages. Here, we
describe distinctive but asymptomatic skeletal mus-
cle involvement in three siblings with CADASIL.
Methods and results.
for this family have been reported previously.3
For all three patients, brain MRI showed many bilat-
eral and almost symmetric areas of hyperdensity of the
cerebral white matter on T2-weighted images. None of the
three patients had EMG changes or elevated serum creat-
ine kinase levels.
A 52-year-old man had right facial paresthe-
sia and headache from the age of 37 years. Similar epi-
sodes had since occurred about once a year. From the age
of 40 to 41 years, he had progressive mental deterioration.
Occasional episodes of transient weakness on the right
side and aphasia had occurred in recent years. Neurologic
examination showed mild impairment of cognitive function
(IQ, 80) and tendon reflexes accentuated on the right side.
A 58-year-old woman had frequent episodes
of headache with dizziness from approximately age 40
years. At 52 years old, she presented with severe headache
and disorientation. In recent years, episodes of amnesia
and slight behavior disturbances also have been noticed.
A 60-year-old woman had occasional epi-
sodes of headache, visual impairment, speech disturbance,
Clinical and laboratory findings
From Istituto Scienze Neurologiche (Drs. Malandrini, Palmeri, Gambelli,
Villanova, and Rossi, and G. Berti), Università di Siena; I.R.C.C.S. Osped-
ale S. Raffaele (Drs. Ferrari and Carrera, F. Albani, and S. Calzavara),
Laboratorio Biologia Molecolare Clinica, Milano; Istituto di Clinica delle
Malattie Nervose e Mentali (Dr. Fattapposta), Università di Roma; Centro
per lo Studio dell’Ipertermia Maligna (A. Bracco and A. Tammaro), AORN
“A. Cardarelli,” Napoli, Italy.
Received May 22, 2001. Accepted in final form April 26, 2002.
Address correspondence and reprint requests to Alessandro Malandrini,
MD, Institute of Neurological Sciences, University of Siena, Viale Bracci 2,
53100 Siena, Italy; e-mail: email@example.com
Copyright © 2002 by AAN Enterprises, Inc.