Dp260 isoform of dystrophin is able to bind actin and produces a
Becker-like phenotype in transgenic mdx mice. L.E. Warner1,
C. Dello Russo1, R.W. Crawford1, J.M. Ervasti2,
J.S. Chamberlain1. 1) Dept Neurology, Univ Washington, Seattle,
WA; 2) Dept Physiology, Univ Wisconsin, Madison, WI.
Duchenne muscular dystrophy (DMD) is caused by defects in the
dystrophin gene. DMD is clinically characterized by severe, progressive muscle
degeneration and weakness. In muscle, dystrophin is thought to play a mechanical
role linking the actin cytoskeleton to the extracellular matrix through its
assembly in a larger complex called the dystrophin glycoprotein complex (DGC).
This link helps maintain muscle membrane integrity in part by dissipating the
forces of muscle contraction into the extracellular matrix. Loss of the DGC
results in contraction-induced injury and muscle degeneration in mdx mice
and DMD patients. Dystrophin has an N-terminal actin-binding domain (ABD) as
well as an internal ABD within the central rod region (repeats 11-17). Deletion
of the internal ABD does not significantly affect the ability of dystrophin to
bind actin or prevent dystrophy. However, it is not known whether the internal
ABD alone could bind actin in vivo and reverse the dystrophic phenotype
in mdx mice. To test this concept, we constructed transgenic mdx
mice expressing Dp260 in skeletal muscle. Dp260 is the retinal-specific isoform
of dystrophin and lacks the N-terminal ABD and the first 9 repeats of the rod
region, but retains the internal ABD. Our results indicated that the internal
ABD is able to bind actin in the absence of the N-terminal ABD and with similar
affinity. While Dp260 did not fully prevent dystrophy in mdx mice, it
slowed progression of the disease and prevented development of inflammation and
fibrosis. The difference in functionality between constructs deleted for the two
ABD may be related to differences in specificity of actin binding and the
particular repeats deleted. Importantly, Dp260 was highly effective in
protecting muscles from contraction-induced injury, demonstrating a mechanically
functional link with the cytoskeleton. In contrast, force development in
transgenic muscle was not different from mdx muscles. These results
suggest that the dystrophin rod domain may facilitate the formation of a muscle
architecture optimized for maximal force development.
Polyglutamine-expanded ataxin-7 induces a cone-rod dystrophy in transgenic
mice by antagonizing the function of the nuclear transcription factor CRX. A.R.
La Spada1, Y.H. Fu2, B.L. Sopher1, R.T. Libby1,
X. Wang3, L.Y. Li3, D.D. Einum4, J. Huang5,
D.E. Possin5, J.B. Hurley6,7, L.J. Ptacek4,8,
S. Chen3. 1) Dept Lab Medicine, Univ Washington, Seattle, WA; 2)
Dept Neurobiol & Anat, Univ Utah, Salt Lake City, UT; 3) Dept Ophthal &
Vis Sciences, Washington Univ, St Louis, MO; 4) Dept Human Genet, Univ Utah,
Salt Lake City, UT; 5) Dept Ophthal, Univ Washington, Seattle, WA; 6) Dept
Biochem, Univ Washington, Seattle, WA; 7) HHMI, Univ Washington, Seattle, WA; 8)
HHMI, Univ Utah, Salt Lake City, UT.
Spinocerebellar ataxia type 7 (SCA7) is caused by the
expansion of a CAG repeat. Although all polyglutamine diseases share a common
mutational motif, the basis of cell-type specificity in each disorder remains
elusive. To determine the mechanism of ataxin-7 neurotoxicity, we produced
transgenic mice that express ataxin-7 with 24 or 92 glutamines. Histological
analysis of the 92Q mice revealed periodic thinning of the photoreceptor cell
layer of the retina. Whole mounts and immunostaining with pigment-specific
antibodies indicated that this periodic thinning is due to preferential loss of
cones. ERGs performed on the 92Q mice revealed that they were blind. These
results indicate that the process of retinal degeneration in our SCA7 transgenic
mice is a cone-rod dystrophy phenotype remarkably akin to what occurs in SCA7
patients. When a yeast two-hybrid assay indicated that the cone-rod homeodomain
protein (CRX) interacts with ataxin-7, we performed further studies to assess
the significance of this interaction. We found that ataxin-7 and CRX co-localize
in nuclear aggregates and can be co-immunoprecipitated. We observed that
polyglutamine-expanded ataxin-7 can suppress CRX transactivation.
Electrophoretic mobility shift assays performed on retinal nuclear extracts
revealed a marked reduction in the ability of CRX to bind its consensus sequence.
We carried out real-time RT-PCR analysis on presymptomatic mice and found
significant decreases in the expression of genes regulated by CRX. Our results
suggest that CRX transcription interference accounts for SCA7 retinal
degeneration, and thus may provide an explanation for how cell-type specificity
is achieved in this disease.
Myotonia is associated with loss of transmembrane chloride conductance and
aberrant splicing of Clcn1, the skeletal muscle chloride channel, in a
transgenic model of myotonic dystrophy (DM1). A.K. Mankodi1,
M. Takahashi2, C. Beck3, S. Cannon2, C.A.
Thornton1. 1) Univ Rochester, Rochester, NY; 2) MGH Hospital,
Boston, MA; 3) Jefferson Univ, Philadelphia, PA.
DM1 is caused by expansion of an untranslated CTG repeat in
the DMPK gene. Pathogenic effects of this mutation are likely mediated, at least
in part, by the expanded CUG repeat in mutant mRNA. DM1 is characterized by
myotonia, a state of hyperexcitability in muscle fibers. In other disorders,
myotonia results from defects in the muscle chloride or sodium channels. The
pathophysiology of myotonia in DM1, however, is unclear. Lines of transgenic
mice (HSA-LR mice) that express expanded CUG repeats develop myopathy and
myotonia (Mankodi et al, 2000). We investigated the pathogenesis of myotonia in
HSA-LR mice. Intracellular recordings show that hyperexcitability is intrinsic
to the muscle fibers, independent of their innervation. Transmembrane chloride
conductance is reduced 7.5-fold in HSA-LR mice. Immunofluorescence studies show
segmental loss of full-length Clcn1 protein from muscle fibers. DM1 is
associated with aberrant splicing of cardiac troponin T (Phillips et al, 1998).
We examined the splicing of Clcn1 mRNA in HSA-LR skeletal muscle. RT-PCR
experiments show inclusion of a novel Clcn1 exon in HSA-LR mice, similar to the
pattern of splicing in muscle from neonatal wild-type mice. Inclusion of this
exon results in frame-shift and premature termination at codon 340 of 994.
Mis-splicing of this exon correlates with the presence of myotonia in different
HSA-LR founder lines. Aberrant splicing of Clcn1, however, is not confined to
this single exon. 29 Clcn1 cDNA clones generated from HSA-LR muscle show 11
different splice variants, none of which are observed in wild-type muscle. By
contrast, 19 of 20 alpha-sarcoglycan cDNA clones from HSA-LR mice are spliced
correctly, an indication that the fidelity of splicing is not universally
impaired. We conclude that the nuclear accumulation of expanded CUG repeats
triggers aberrant splicing of Clcn1 mRNA. The resulting loss of Clcn1 protein
and transmembrane chloride conductance is sufficient to account for the myotonia
in HSA-LR mice.
Myotonic Dystrophy Type 2 is Caused by a CCTG Expansion in Intron 1 of ZNF9.
L.P.W. Ranum1, C.L. Liquori1, M.L. Moseley1,
J.F. Jacobsen1, A.V. Phillips2, R. Savkur2, W.
Kress3, S.L. Naylor4, T.A. Cooper2, K. Ricker3,
J.W. Day1. 1) Institute of Human Genetics, Univ Minnesota,
Minneapolis, MN; 2) Department of Pathology, Baylor College of Medicine, Houston,
TX; 3) Department of Neurology, University of Würzburg, Germany; 4) Department
of Cellular and Structural Biology, University of Texas Health Science Center,
San Antonio, TX.
Myotonic dystrophy (DM) can be caused by a mutation on
chromosome 19 (DM1) or 3 (DM2/PROMM). DM1 is caused by a CTG expansion in the 3'
UTR of the dystrophia myotonica-protein kinase gene (DMPK).
Several mechanisms have been suggested to explain how this mutation causes the
multisystemic effects of DM including: DMPK haploinsufficiency; reduced
expression of regional genes (eg SIX5); and pathogenic effects of the CUG
expansion in RNA. Mouse models have suggested that each of these mechanisms
contributes to DM1 pathogenesis and that DM1 is a regional gene disorder. To
clarify the pathogenic mechanism of DM, we have identified a second human
mutation that causes the same multisystemic effects. Linkage disequilibrium
analysis (see Liquori, et al.) refined the DM2 region. One of our markers,
CL3N58, showed an aberrant segregation pattern by PCR. Southern analysis
demonstrated that all affected individuals in six DM2 families (LOD=31.6 at Q=0.00)
had an expanded allele that was not found in controls (n=1360). Sequence
analysis showed that the CCTG portion of the compound repeat (TG)n(TCTG)n(CCTG)n
expands in affected alleles. The range of expanded alleles is broad (~75-11,000
CCTGs, mean~5,000). The expansion is located in intron 1 of the zinc finger
protein 9 (ZNF9) gene, whose normal function as an RNA binding
protein appears unrelated to any of the genes found in the DM1 region. Similar
to the CUG RNA foci in DM1, intense CCUG-containing nuclear foci were found in
DM2 but not control muscle. RT-PCR of insulin receptor (IR) mRNA in skeletal
muscle showed a marked reduction in the IR-B isoform in DM2 (18%, n=3) and DM1
(21%, n=9) compared to controls (73%, n=5). Parallels between DM1 and DM2
indicate that the CUG and CCUG expansions in RNA can themselves be pathogenic
and cause the multisystemic features common to both diseases.
Muscular dystrophy and neuronal migration disorder caused by mutations in
a novel glycosyltransferase. K. Kobayashi1, A. Yoshida2,
H. Manya3, M. Mamoru4, T. Inazu4, H. Mitsuhashi2,
H. Topaloglu5, M. Takeuchi2, T. Endo3, T. Toda1.
1) Division of Functional Genomics, Department of Post-Genomics and Diseases,
Course of Advanced Medicine, Osaka University Graduate School of Medicine, Osaka,
Japan; 2) Central Laboratories for Key Technology, Kirin Brewery Co., Ltd.,
Yokohama, Japan; 3) Department of Glycobiology, Tokyo Metropolitan Institute of
Gerontology, Tokyo, Japan; 4) Research Department, The Noguchi Institute, Tokyo,
Japan; 5) Department of Paediatric Neurology, Hacettepe Children's Hospital,
Ankara, Turkey.
Muscle-eye-brain disease (MEB) is an autosomal recessive
disorder characterized by congenital muscular dystrophy, ocular abnormalities,
and lissencephaly. Mammalian O-Mannosyl glycosylation is a novel protein
modification observed in a limited number of glycoproteins of brain, nerve, and
skeletal muscle. Here we isolated a human cDNA for O-linked mannose b1,2-N-acetylglucosaminyltransferase
(OMGnT), which is a novel enzyme for mammalian type O-mannosyl glycan
synthesis. The cDNA encoded a putative type II membrane protein of 660 amino
acids having 23.2% sequence identity to human a-3-D-mannoside
b-1,2-N-acetylglucosaminyltransferase
I (GnT-I). By transfecting this cDNA into HEK293T cells, OMGnT activity
increased approximately 100-fold. However, OMGnT did not show any detectable
GnT-I activity. Northern blot analysis revealed that OMGnT is constitutively
expressed in all human tissues tested. These results clearly demonstrate that
OMGnT is a new b1,2-N-acetylglucosaminyltransferase
functionally different from GnT-I. Moreover, we identified two independent
mutations of this gene in two patients with MEB, indicating that the OMGnT gene
is responsible for MEB. These findings would suggest a novel pathomechanism,
glycosylation, on muscular dystrophy as well as neuronal migration disorder.
A novel glycosyltransferase is mutated in a form of congenital muscular
dystrophy with secondary laminin a2
deficiency and abnormal glycosylation of a-dystroglycan.
M. Brockington1, D.J. Blake2, P. Prandini1,
S.C. Brown1, S. Torelli1,3, M.A. Benson2, C.P.
Ponting2, B. Estournet4, N. Romero5, T. Voit6,
C.A. Sewry7, P. Guicheney5, F. Muntoni1. 1)
The Dubowitz Neuromuscular Centre, ICSM,Hammersmith Hospital, London, UK; 2)
Department of Human Anatomy and Genetics, University of Oxford, UK; 3)
Department of Cytomorphology, University of Cagliari, Italy; 4) Hpital Raymond
Poincar, Garches, France; 5) Inserm U 523, Pitie-Salpetriere, Paris, France; 6)
Department of Paediatrics, University of Essen, Germany; 7) 7Department of
Histopathology, Robert Jones & Agnes Hunt Orthopaedic Hospital, Oswestry, UK.
The congenital muscular dystrophies (CMD) are a heterogeneous
group of autosomal recessive disorders, presenting in infancy with muscle
weakness, contractures and dystrophic changes on skeletal muscle biopsy. Brain
involvement is also present in several CMD syndromes. Approximately 40% of
patients have a primary deficiency of laminin a2
due to mutations in the LAMA2 gene. A secondary deficiency of laminin a2
is apparent in some CMD syndromes.
Here we identify a gene encoding a novel putative
glycosyltransferase. We report the genomic organization of this locus and its
pattern of tissue expression. Mutations in this gene have been identified in 7
families with a severe form of CMD that does not involve the brain. Patients
have markedly elevated serum creatine kinase and a secondary deficiency of
laminin a2. We observed a marked
decrease of muscle a-dystroglycan
immunostaining and a significant drop in its molecular weight on Western blot.
These observations are interpreted to be the result of altered glycosylation of
a-dystroglycan. This is likely to be integral to the pathology seen in these
patients.
Gene expression analysis of muscle from Duchenne muscular dystrophy
patients. J.N. Haslett, D. Sanoudou, S.A. Greenberg, H.G.W. Lidov, I.S.
Kohane, A.H. Beggs, L.M. Kunkel. Genetics Division, Children's Hospital,
Harvard Medical School, Boston, MA.
Muscular dystrophy (MD) refers to a clinically and genetically
heterogenous group of myopathyies defined by progressive degeneration of
skeletal muscle fibers, leading to loss of muscle function. Mutations in the
dystrophin gene were identified as causative of the most common muscular
dystrophy, Duchenne MD. Dystrophin is linked to a group of integral membrane
proteins, forming the dystrophin-associated protein complex (DAPC). To examine
the DMD pathogenic pathways and identify new or modifying factors involved in
muscular dystrophy we used Affymetrix oligonucleotide arrays to analyze the
expression patterns of 12,600 genes in 12 DMD patients. The DMD data were
compared to data from 10 normal skeletal muscle samples and to data obtained
from other myopathic samples. Conventional statistical and fold-ratio
comparisons and automated classification and clustering techniques were used to
analyze the data, allowing identification of both disease-specific changes and
changes due to the non-specific response of diseased muscle. This will enable
additional analysis of DMD pathophysioloical mechanisms and those of skeletal
muscle more generally. When compared to other datasets, the DMD samples show a
distinctive clustering pattern, presumed to reflect differences in the
pathogenic pathways. Fold-ratio analysis suggests approximately 80 of the genes
examined differ significantly in expression level between normal and DMD muscle.
More genes were found to be overexpressed than underexpressed in DMD,
potentially reflecting an increase in protein turnover, probably due to the
degenerative and regenerative nature of the disease. As expected dystrophin was
found to be substantially underexpressed in DMD muscle. In contrast, a number of
other muscle genes (myosin, troponinT, alpha- and beta-tubulin, alpha-actinin)
were found to be overexpressed in DMD muscle. The observed changes are being
subjected to further statistical analysis and classification, in addition to
experimental verification, to provide insight into the molecular mechanisms
behind the DMD pathogenic pathways.
The alternative splicing of DMPK is affected in cis by the CTG
expansion of Myotonic Dystrophy Type-1.. A.J. Gibb, M.G. Hamshere.
School of Life & Env. Sciences, University of Nottingham, Nottingham,
Nottinghamshire, UK.
Myotonic Dystrophy Type-1 (DM1) is the most common muscular dystrophy affecting
1/8000 live births in Western European and North American populations. The
condition is characteristically multisystemic with symptoms including myotonia,
progressive muscle weakness, cardiac conduction defects, cataracts, frontal
balding in males, testicular atrophy, diabetes, hypersomnia and mental
retardation. The condition segregates in an autosomal dominant manner and is
caused by a CTG triple-repeat expansion found in the 3UTR of the gene for a
serine-threonine protein kinase, DMPK.
DMPK has several alternative splice forms. The frequency of their use and
the nature of any effect mediated by the repeat, either in cis or in
trans, has not yet been fully investigated. Using affected and control
fibroblasts heterozygous for a SNP in Exon 10 of DMPK, we have been able
to develop a fluorescence based quantitative RT-PCR method in order to
discriminate between transcripts derived from the affected and unaffected
chromosomes. By a combination of fluorescence and size discrimination, we have
established that the profile of alternative splicing is affected in cis
by the myotonic dystrophy triplet repeat expansion.
Epidemiological impact of genetic counselling and predictive testing for
myotonic dystrophy. J. Mathieu, C. Tremblay, C. Prevost. Dept
Neurology, Complexe Hosp de la Sagamie, Chicoutimi, PQ., Quebec, Canada.
The prevalence of myotonic dystrophy type 1 (DM) is very high
in the Saguenay-Lac-Saint-Jean (SLSJ) region, a geographically isolated region
in the northeastern part of the province of Quebec (Canada). In SLSJ, a program
of genetic counselling for DM patients and their families was introduced in 1981
and a program of predictive testing is offered to the population since 1988. In
order to determine the effect of genetic counselling and predictive testing on
the prevalence of the DM phenotypes, we compared the demographic characteristics
and the phenotypes of the DM patients over the last 15-year interval
(1985-2000). During this period, the median age of the DM patients increased
from 34.5 years to 44.0 years (p<0.00001). The median age of the congenital,
childhood and classic DM patients increased respectively from 9 to 24 years
(p<0.001), 24 to 33 years (p=0.01) and 35 to 44 years (p<0.001). In this
time interval, the proportion of patients with a congenital or a childhood
phenotype remained stable but the proportion of patients with a classic
phenotype decreased from 75% to 61% and, the proportion of patients with a mild
phenotype increased from 6% to 20% (p<0.001). In the last 5 years, among the
63 newly diagnosed patients, 43% had a mild DM phenotype detected only by DNA
analysis. The reasons for the significant ageing of the DM population in SLSJ
region are complex and include a low natality rate throughout the Quebec
population, a reduction in births at risk due to genetic counselling and an
increase in the number of mildly affected patients often recognized at an older
age only by predictive testing. The shift in the distribution of the DM
phenotypes toward mildy affected individuals is expected to accelerate in the
future.
Founder effect in North-Eastern Quebec and the extent of the genetic
signature associated with the myotonic dystrophy mutation. V. Yotova1,
E. Zietkiewicz1, E. Lemieux-Blanchard1, M. Labuda1,
S. Bourgeois1, D. Labuda1, J. Fortin2, P.
Lepage2, T.J. Hudson2, A. Lescault3, C. Laberge3.
1) Research Center, Hopital Sainte-Justine, Montreal, Quebec, Canada; 2) MGH
Research Center, McGill University, Montreal Genome Center, Montreal, Quebec,
Canada; 3) Centre hospitalier, Universit Laval, Ste-Foy, Quebec, Canada.
Myotonic dystrophy is frequent in French-Canadian population
of North-Eastern Quebec (1:650). This dominant disorder is due to a CTG-triplet
expansion in the DMPK gene on chromosome 19q13 (OMIM160900). Rather than
directly analyzing the expansion, we wanted to define a unique haplotype
composed of single-nucleotide polymorphisms, a SNP signature, eventually
amenable to automatic screening. By the same token, we investigated the extent
of the ancestral carrier-haplotype conservation in a young population issued
from a founder effect and addressed the hypothesis of a single introduction of
the DM-mutation in North-Eastern Quebec. SNPs were found in silico as
well as experimentally in a population panel by DHPLC and subsequent sequencing.
Twenty of these SNPs were typed in 51 DM-families by allele-specific
oligonucleotide hybridization. The resulting haplotype extends over 2.3 Mb
according to the recent UCSC map. There are two groups of DM haplotypes. The
first, presumably young, is represented by a haplotype seen in 34 copies and a
minor recombinant (2 copies). The second is represented by four variants (5, 5,
3 and 2 copies) that can be mutually related through single recombinations.
These two groups could therefore represent two separate entries of the
DM-chromosomes. On the other hand, they share a shorter core haplotype,
suggesting a relatively recent common origin for both groups; genealogical data
will help to trace it back in time. In conclusion, the DM-mutation appears to be
associated with a limited number of unique SNP-signatures that are not shared
with non-affected chromosomes; shorter segments of these haplotypes are
relatively common in the population; our data demonstrate an important increase
in the extent of linkage disequilibrium that can be related to the young age of
the population of Quebec. (Supported by RMGA FRSQ).
Psychosocial impact of predictive testing for myotonic dystrophy. C.
Prevost1, J. Villeneuve1, M. Tremblay2, S.
Veillette3, M. Perron3, J. Mathieu1. 1)
Complexe Hosp de la Sagamie, Chicoutimi, Qc, Canada; 2) Quebec University at
Chicoutimi, Chicoutimi, Qc, Canada; 3) Cegep of Jonquiere, Qc, Canada.
In the Saguenay-Lac-Saint-Jean region (Quebec, Canada), a
predictive DNA-testing program for myotonic dystrophy type 1 (DM) has been
available as a clinical service since 1988. From 1 to 12 years (median, 5 years)
after receiving predictive testing, a total of 308 participants (44 carriers and
264 non carriers) answered a questionnaire to determine reasons for testing and
recall of test result, to assess their perception of the psychosocial impact of
predictive testing and to measure their actual general well-being, self-esteem
and psychological distress. The reasons for wanting to be tested were to learn
if children are at risk for DM or for reproductive decision making (75%) and to
relieve the uncertainty for themselves (17%). The majority of participants
(96.1%) remembered correctly their test result. Among carriers, 19% consider
themselves in a worse psychological state, 49% are more worried about their
future health, 25% feel less satisfaction about life, 25% present a lower
self-esteem and 53% are more concerned about their childrens risk. Among non
carriers, 36% find themselves in a better psychological situation, 49% are less
worried about their future health, 48% feel more satisfaction about life, 20%
present a better self-esteem and 85% are reassured about their childrens risk.
The actual general well-being, the self-esteem (Rosenberg Self-Esteem Scale) and
the psychological status (Psychiatric Symptom Index) are similar in carriers, in
non carriers and in the reference (Quebec) population; these results are not
influenced by the number of years elapsed since predictive testing was done. All
respondents believe that predictive testing should be available for the at-risk
population and the vast majority of carrier (95%) and of noncarriers (93%) would
recommend the use of predictive testing to their family members.
Molecular analysis of Duchenne and Becker muscular dystrophy patients in
Saudi Arabia. R. Majumdar1, M. Al Jumah2, S. Al
Rajeh3, E. Chaves-Carballo4, M.M. Salih5, A.
Awada2, S. Shahwan6, S. Al Uthaim1. 1)
Neurogenetics Laboratory, Department of Medicine, King Fahad Natl Guard Hosp,
Riyadh, Saudi Arabia; 2) Division of Neurology, Department of of Medicine, King
Fahad Natl Guard Hosp, Riyadh, Saudi Arabia; 3) Division of Neurology, King Saud
University, Riyadh, Saudi Arabia; 4) Department of Neurosciences, King Faisal
Specialist Hospital & Research Center, Riyadh, Saudi Arabia; 5) Department
of Pediatrics, King Saud University, Riyadh, Saudi Arabia; 6) Department of
Pediatrics, Military Hospital, Riyadh, Saudi Arabia.
Objective:The deletion in the dystrophin gene has been
reported for many ethnic groups, but until now the mutations in this gene have
not been thoroughly investigated in Saudi Duchenne and Becker muscular dystrophy
(DMD/BMD) patients. Methods: We examined the deletion pattern in the
dystrophin gene of the Saudi patients applying multiplex-polymerase chain
reaction (PCR). Genomic DNA was isolated from twenty two patients with DMD/BMD
confirmed by dystrophin staining on muscle biopsy, eleven patients with clinical
suspicion of DMD without muscle biopsy, three patients with limb girdle muscular
dystrophy, twelve relatives of the patients, and five healthy Saudi volunteers.
Specific exons around the deletion prone regions (hot spots) of the dystrophin
gene were amplified. Results: The deletion of one or more exons was found
in sixteen of twenty two DMD/BMD patients. The deletion in the gene was detected
in seven of eleven patients with suspected DMD diagnosis, but not confirmed by
dystrophin staining of muscle biopsy. No deletion in the dystrophin gene was
detected in control Saudi volunteers, the limb girdle dystrophy patients, and
the relatives of patients, as expected. Conclusion: The present study
suggests that intragenic dystrophin gene deletions (70%) occur with the same
frequency in Saudi patients compared with other ethnic groups.
(This study was supported by King Fahad National Guard
Hospital and a grant from King Abdulaziz City for Science and Technology,
Project AT-18-03).
Choosing a novel strategy for point mutation detection in the dystrophin
gene : DHPLC or BESS ? A preliminary comparison. S. Chambert, C. Saquet,
M. Claustres, S. Tuffery-Giraud. Lab Genetique Moleculaire, CHU montpellier,
France.
In Duchenne and Becker Muscular Dystrophy (D/BMD), one third
of the mutations are not detectable as intragenic deletions or duplications of
the dystrophin gene. Because full gene sequencing (79 exons) would be laborious
and time-consuming in order to identify point mutations occurring in this gene,
we used an approach based on the analysis of dystrophin transcripts (RT-PCR)
coupled with the protein truncation test (PTT) during these last years. As a
result of these studies, the families of these patients are currently benefiting
from accurate carrier-status assessment. Also, some patients carrying a point
mutation have been selected for inclusion in clinical trials in view of therapy.
However, this technique was limited to a detection rate of 86% in our series. In
the aim at identifying point mutation in more patients, we set out to assess the
sensitivity of Denaturing High-Performance Liquid Chromatography (DHPLC) and
Base Excision Sequence Scanning (BESS) as an approach to mutation screening in
DMD. We first assessed a cohort of 15 patients with previously identified
mutations (frameshift or stop mutations) spread all over the gene. The
comparison of the two methods was based on the analysis of the same amplified
fragment of cDNA (~ 500bp) known to contain the mutation. A sequenced cDNA was
used as control, and for heteroduplex formation in DHPLC. The sensitivities of
DHPLC and BESS were very similar when respectively more than one column
temperature was used for the detection of mismatches, and when BESS T and BESS G
cleavage reactions were performed. Both allowed the rapid detection of single
base substitutions as well as small deletions/insertions. From these findings,
we conclude that these two methods provide a reliable alternative to the PTT. Up
to now, we have not yet established which of these two technology (BESS or DHPLC)
would be the most appropriate for the molecular diagnosis of DMD. We plan to
analyze DMD patients (clinical diagnosis confirmed by immunohistochemical
analysis of dystrophin) in whom extensive RT-PCR/PTT analysis failed to detect
the mutation. Supported in part by a grant of Association Franaise contre les
Myopathies (AFM).
A comprehensive molecular analysis of myotonic dystrophy in Iran. M.S.M.E.
Mousavi, K. Kahrizi, S.S Hosseini Amini, H. Najmabadi. Genetics Research
center, Universitiy of Social Welfare and Rehabilitation Sciences , Tehran, Iran.
Myotonic dystrophy (DM) is the most common form of adult
muscle dystrophies with varrying frequecies in different populations. So far
there has been no comprehensive population screening on this disease in Iran. DM
has multisystemic manifestatioin including muscle weakness and myotonia. It has
pattern of Trinucleotide (CTG) repeats, in 3' untranslated region of the serine
Thereonine protein kinase gene located on chromosome 19q13.3. Due to similarity
in clinical picture with the other dystrophic patients and the lack of reliable
diagnostics we decided to set up a molecular analysis technique for DM for the
first time in Iran. PCR and southern bloting were perfomed using nonradioactive
material. 100 patients were investigated. DM gene mutations were detected in 35%
of the clincally diagnosed DM patients.
Six novel point mutations in the Dystrophin gene identified in Brazilian
Duchenne patients. D.N.F. Vagenas1,2, A. Cerqueira1,
A. Torres1, R. Pavanello1, M.R. Passos-Bueno1,
M. Zatz1. 1) Centro de Estudo Genoma Humano, Universidade de So
Paulo, So Paulo, S.P, Brazil; 2) Centro em Interunidades em Biotecnologia,
Universidade de So Paulo, S.P. Brazil.
Duchenne (DMD) and Becker (BMD) type muscular dystrophies are
allelic X-linked conditions caused by mutations in the gene encoding dystrophin.
This gene contains 79 exons, which are distributed in approximately 2,400 Kb.
The phenotype is caused in about 60% of the cases by deletions, 5 - 6% by
duplications, while the remaining cases are due to point mutations or small
deletions or rearrangements. We have ascertained so far 1010 families with DMD
patients in our center. Among them 632 had deletions in the dystrophin gene. We
are currently trying to identify the mutations in the non-deleted cases through
SSCP, PTT (Protein Truncation Test) and sequencing of abnormal fragments. For
PTT analysis, the dystrophin gene was divided in 10 fragments, each one with 8
exons, encompassing the 79 exons. In 99 patients, about 4 fragments were
randomly analyzed leading to the identification of 10 mutations. Among these,
six are novel mutations: one deletion of exon 38, three frameshifting involving
the exons: 5 (494-497 delAGTA); 16 (2067 - 2068 del TG and 2080 - 2083 del ACAA);
and two nonsense in the exons 17 (2345 C®T;
Q 713 X) and 45 (6785 T®C; Q 2193 X).
We are also assessing the intellectual capacity in our patients in order to
verify what mutations/deletions are more commonly associated with mental
impairment. CEPID-FAPESP, PRONEX, CNPq, IAEA.
Resolution of equivocal molecular genetic test results in the diagnosis of
Facioscapulohumeral Muscular Dystrophy (FSHD). W.K. Seltzer1,
K. Felice2, J. Durocher1, A.R. Yesley1, M.A.
Boss1, S.E. Hallam1. 1) Athena Diagnostics, Inc,
Worcester, MA; 2) University of Connecticut Health Center, Farmington, CT.
Ninety to 95% of Facioscapulohumeral muscular dystrophy (FSHD)
cases are associated with a deletion within a repeat array. Athena Diagnostics,
Inc. performs FSHD analysis by EcoRI and EcoRI/Bln1 restriction endonuclease
digestions followed by Pulsed-Field Gel Electrophoresis to separate and resolve
the 8 to > 48 kb sized DNA fragments. Southern analysis is then performed
using probe, p13E-11, which hybridizes to repeat arrays at both 4q35 and 10q26.
Bln1 specifically digests the chromosome 10 repeat array to small fragments
allowing discrimination between chromosome 10 and chromosome 4 arrays. Twenty
percent of the general population carry a benign translocation resulting in
either three chromosome 4q35 repeat arrays and a single 10q26 array, or
alternatively, three 10q26 arrays and a single 4q35 array. This complicates
result interpretation since it is the location of the repeat array deletion, and
not its chromosomal origin, which determines the pathogenicity of the deletion.
The presence of these translocations can potentially lead to false positive and
false negative test results. We reviewed 275 cases of which 49 (18%) yielded
findings that could not be unequivocally interpreted. In order to achieve a
better understanding of the remaining equivocal results, we carried out XapI
analyses which specifically digest the chromosome 4 repeat array. This provided
significant utility in interpretation of these previously equivocal results.
New tools to study proteins involved in disease: FSHD as paradigm. S.
van Koningsbruggen1, H. de Haard2, R.W. Dirks1,
J.T. den Dunnen1, G.W. Padberg3, G.J. van Ommen1,
C.T. Verrips2, R.R. Frants1, S.M. van der Maarel2.
1) Center for Human & Clinical Genetics and Dept. of Molecular Cell Biology,
LUMC Leiden, Leiden, Netherlands; 2) Unilever Research, Vlaardigen, Netherlands;
3) Dept. of Neurology, UMCN Nijmegen, Nijmegen, The Netherlands.
Facioscapulohumeral muscular dystrophy (FSHD) is caused by a
complex genetic rearrangement. Likely, partial deletion of the subtelomeric D4Z4
repeat on 4qter causes the transcriptional deregulation of nearby genes by a
position effect variegation-like mechanism. Consequently, there are no obvious
structural mutations within the FSHD candidate genes, complicating their
positive identification.
Next to expression profiling of candidate genes, we have
embarked on protein profiling employing llama-derived phage-display single (heavy)
chain antibody fragments. Camelidae carry apart from a conventional
antibody repertoire, a unique repertoire of heavy-chain antibodies that only
consist of two heavy chains and are devoid of light chains. Using this antibody
repertoire as source for phage-display libraries circumvents the in vitro
combination of heavy and light chains, one of the major drawbacks of
conventional phage-display libraries. Moreover, these antibodies have unique
properties regarding affinity and stability. Current strategies are directed in
optimizing high-throughput selection and screening protocols and design of
phage-display vectors allowing uniform arraying, visualization, and
intracellular expression. Due to their small size (14kDa) and high affinities,
autofluorescent heavy-chain antibody fragments will be eminently suited for a
variety of applications.
As proof of principle, we successfully generated
immune-libraries raised against cocktails of proteins and whole human skeletal
muscle homogenate. We selected high-affinity antibody fragments against FRG1P,
the gene product of one of the candidate genes for FSHD. These antibody
fragments perform well in a series of immunological techniques: for example,
co-localization was observed for EGFP-tagged FRG1P and selected heavy-chain
antibody fragments.
Evidence of further genetic heterogeneity for both autosomal dominant and
autosomal recessive limb-girdle muscular dystrophy. A.L. Starling, M.
Vainzof, R. Pavanello, M. Canovas, A. Cerqueira, M.R. Passos-Bueno, M. Zatz.
Center for the study of the human genome, IBUSP, University of Sao Paulo, Sao
Paulo, Brazil.
Fifteen genes responsible for limb-girdle muscular dystrophy
have been identified to date including six autosomal dominant (LGMD1A-F) and
nine autosomal recessive (LGMD2A-I). We have ascertained three Brazilian
families that were excluded by linkage analysis (and protein studies when
possible) for these 15 LGMD causing genes. The first family with nine affected
members (7 males and 2 females) in 3 generations displays AD inheritance. Two of
the affected patients were submitted to muscle biopsy and electromyography which
showed a myopathic pattern in both exams. In all affected members the onset
occurred after age 30, with proximal weakness in the upper and lower limbs and a
variable presence of calf hypertrophy. Serum creatine kinase (CK) was increased
2 - 9 fold. The second family also with AD inheritance has four affected members
(3 males and 1 female) in 2 generations. All of them have a history of cardiac
problems, weakness of proximal limbs and a CK increase 3 - 6 fold. The age at
onset ranged from 25 to 30 years old. The last family, with four affected males
has an atypical inheritance. The affected patients have weakness of proximal
limbs (predominantly in the lower limbs in three patients but in the upper limbs
in the last one), calf hypertrophy and the age at onset ranged from 10 to 15
years old. The CK was grossly elevated (50-80 fold). Electromyography showed a
myopathic pattern and the biopsy showed a normal pattern for dystrophin, calpain,
dysferlin, the four sarcoglicans and telethonin. We are currently performing a
genome-wide scan to identify the disease loci in these three families. Supported
by FAPESP-CEPID, PRONEX and CNPq.
Mutations in the dystrophin gene are associated with sporadic dilated
cardiomyopathy. J. Yan1, J. Feng1, C.H. Buzin1,
J.A. Towbin*2, S.S. Sommer*1. 1) Department of
Molecular Genetics, City of Hope Medical Center, Duarte, CA; 2) Department of
Pediatrics (Cardiology), Baylor College of Medicine, Houston, TX. *These authors
contributed equally to the work.
Dilated cardiomyopathy is the major indication for heart
transplantation. Approximately 30% of all DCM is thought to be inherited, while
70% is sporadic. Mutations in the dystrophin gene have been associated with the
uncommon X-linked form of dilated cardiomyopathy. We hypothesized that missense
mutations and other less severe mutations might predispose to sporadic dilated
cardiomyopathy. To test this hypothesis, we examined 22 patients with sporadic
dilated cardiomyopathy by DOVAM-S (Detection of Virtually All Mutations-SSCP)(
Liu et al, 1999. BioTechniques 26:932-942; Buzin et al, 2000. BioTechniques
28:746-753), a form of SSCP in which there is sufficient redundancy to detect
virtually all mutations. Twenty-two kb of genomic dystrophin DNA was scanned in
the 22 patients with sporadic DCM, including all 79 coding sequences and splice
junctions, as well as six alternative exon 1 dystrophin isoforms. Three putative
new mutations (IVS5+1 G>T, N2299T and F3228L) and six polymorphisms were
identified. The splice site mutation IVS5+1 is predicted to cause skipping of
exon 5, a region containing an actin binding site. In the missense mutation
F3228L in exon 67, the amino acid phenylalanine is highly conserved in dog,
mouse, chicken, Torpedo, dogfish, starfish, scallop, and amphioxus dystrophins.
Screening of 141 control individuals failed to identify these 3 mutations,
hinting that milder mutations in the dystrophin gene are associated with
sporadic DCM, but clinical manifestation may require some environmental effector
or a modifying gene. These mutations may have reduced penetrance and generally
appear as sporadic DCM. Future studies are needed to confirm that
mutations in the dystrophin gene are a frequent cause of DCM and to analyze
families with these dystrophin mutations to search for environmental or genetic
modifiers that may help to generate the disease phenotype
Muscleblind localizes to nuclear foci of aberrant RNA in myotonic
dystrophy (DM) types 1 and 2. C.A. Thornton1, A. Mankodi1,
C. Urbinati2, R. Moxley1, V. Sansone1, M.
Swanson2. 1) Univ Rochester, Rochester, NY; 2) Univ Florida,
Gainesville, FL.
There is locus heterogeneity in DM. DM1 is caused by expansion
of a CTG repeat in the DMPK gene on chr. 19. DM2 is linked to chr. 3. Pathogenic
effects in DM1 are likely mediated, at least in part, by the expanded CUG repeat
in mutant mRNA. The mutant transcripts are retained in the nucleus in multiple
foci. Various proteins interact with CUG repeats in vitro, but none have been
shown to interact in vivo. We investigated the possibility that DM2 is caused by
expression of a CUG repeat or related sequence. RNase protection with a CAG-29
probe showed an expanded CUG repeat in DM1 but not in DM2 or normal controls
(n=4 in each group, myoblast RNA). However, FISH using CAG probes on sections of
muscle tissue showed nuclear foci in DM2 similar to those in DM1. Nuclear foci
were present in muscle tissue from all patients with symptomatic DM1 (n=9) or
DM2 (n=9) but not in disease or healthy controls (n=23). Foci were not seen with
CUG- or GUC-repeat probes. Foci in DM2 were distinguished from DM1 by lower
stability of the probe-target duplex, suggesting that a sequence related to CUG
repeats accumulates in the DM2 nucleus. Furthermore, muscleblind, homologue of a
protein required for muscle development in Drosophila, localized to the nuclear
foci in both DM1 and DM2. In contrast to previous studies showing hundreds of
RNA foci per nucleus in DM1 myoblasts, we found 1 to 3 foci per nucleus in
muscle tissue from DM1 and DM2 patients. The foci did not associate with a
specific nuclear structure, as determined by markers for nucleoli, coiled bodies,
PML bodies, or perinucleolar complex. To identify proteins that interact with
expanded CUG repeats in vivo, we examined muscle from transgenic mice that
express expanded CUG repeats. Among 9 proteins examined, including 7 dsRNA
binding proteins and CUGBP1, only muscleblind colocalized with foci of expanded
CUG repeats. These results support the idea that nuclear accumulation of mutant
mRNA is pathogenic in DM1, suggest that a similar disease process occurs in DM2,
and point to a role for muscleblind in the pathogenesis of both disorders.
Variable phenotype in monozygotic twins with Duchenne Muscular Dystrophy. K.
Radha Mani, G.R. Chandak. Centre for Cellular and Molecular Biology,
Hyderabad, Andhra Pradesh, India.
Duchenne and Becker Muscular Dystrophy are X-linked
recessive allelic neuromuscular disorders where affected individuals typically
have pseudohypertrophy of calf muscles with development of secondary atrophy and
contraction and cardiac or respiratory muscle involvement in the later stages.
The disease is caused by mutation in the DMD gene and approximately 65% of
patients have intragenic deletions, the rest have point mutations or
duplications. We report an interesting case of monozygotic twins with variable
phenotype on a similar genetic background. The patients presented to us at the
age of 12 years, with one of them almost immobilized with an age of onset at 4
years (Twin I) whereas the other fairly mobile with an age of onset only at the
age of 8 years (Twin II). Although both had similar pseudohypertrophy of the
calf muscles but the twin I had comparatively flabby muscles consistent with
degenerating muscles. The CPK levels were 28,000 and 13,000 respectively for
twin I and twin II. Cytogenetic study for both was consistent with a male
karyotype with a normal banding pattern. DNA fingerprinting using a 10-loci
microsatellite showed similar profile and matched with the parents thus
confirming paternity. Deletion analysis of the DMD gene showed exon 49-50
deletion out of 25 exons analysed. Analysis of entire DMD gene using cDNA probes
failed to show any other missing or abnormal restriction fragment. Both twins
showed deletion of 1.6 Kb and 3.7 Kb fragments on a HindIII blot hybridized with
cDNA 8 of the DMD gene. Neither the mother nor the relatives showed this
deletion suggesting the deletion to be a new mutation in this pair of
monozygotic twin. Two possible explanations can be advanced to account for this
variability on a seemingly similar genetic background. This may be an evidence
for somatic mosaicism or it may suggest the presence of other factors modulating
the severity of the DMD phenotype.
FSHD Myoblasts Possess Reduced Resistance to Oxidative Stress. K.A.
Barrett1,2, R. Tawil2, R.C. Griggs2, D.A.
Figlewicz1,2. 1) Dept Neurobiology & Anatomy, Univ Rochester,
Rochester, NY; 2) Dept Neurology, Univ Rochester, Rochester, NY.
Facioscapulohumeral muscular dystrophy (FSHD), the third most
common muscular dystrophy, is inherited in an autosomal dominant manner. A
variable deletion in a repeat region (D4Z4) of chromosome 4q35 has been
associated with the disorder, however, the pathogenesis of FSHD has yet to be
established. Myoblasts from FSHD patients possess a necrotic appearing
morphology, with a swollen cytoplasm and perinuclear vacuoles. Highly confluent
cells appear to lack the organization of normal myoblasts, and FSHD cells fuse
to form disorganized, swollen myotubes. Although necrotic appearing cells can be
found in cultures of normal myoblasts, a larger percentage of FSHD cells
possesses the phenotype (2.6 ±1.5% for normals, and 15.7± 6.1% for FSHD,
p=0.046, n=4.), controlling for cell density and replicative age. Preliminary
observations indicate that this phenotype can be elicited in normal cells
exposed to the superoxide anion generator, paraquat. Additionally, previous
studies have demonstrated an increased susceptibility of FSHD myoblasts to
paraquat relative to normal and disease control (other myopathies) cells. The
cyclin dependent kinase (cdk) inhibitor, p21, appears to be upregulated in FSHD
cells under normal growth conditions (22.2% of FSHD myoblast nuclei stain
strongly positive for p21 compared to 14.2% of normal myoblast nuclei, p=0.004,
n=4.); a similar phenomenon in fibroblasts was associated with oxidative stress.
Rescue from oxidative stress was attempted by exposing normal and FSHD myoblasts
to the membrane permeable anti-oxidant, glutathione ethyl ester (GSH-OEt). Low
concentrations of GSH-OEt (0.89mM) achieved full rescue of normal cells exposed
to 20mM paraquat. In contrast, GSH-OEt was unable to rescue FSHD cells exposed
to 10mM paraquat; the viability of FSHD myoblasts in test wells (containing
paraquat plus GSH-OEt) was equivalent to that in wells containing paraquat alone.
The current study demonstrates an enhanced irreversible vulnerability of FSHD
myoblasts to oxidative stress, suggesting a biochemical marker for FSHD early in
myocyte development
Development of a cDNA microarray to study musclar distrophy. T.
Tsukahara1, H. Nagasawa2, K. Arahata1, 3, 4.
1) Dept Neuromuscular Research, National Institute of Neuroscience, NCNP, Tokyo,
Japan; 2) Ebara Co., Kanagawa, Japan; 3) CREST, JST, Tokyo, Japan; 4) Dr. Kiichi
Arahata passed away on December 20, 2000.
Defects of plasma membrane or extracellular matrix associated
proteins, dystrophin or laminin alpha 2 and so on, due to fragile sarcolemma and
muscular dystrophy have been identified. However, there are some muscular
dystrophy genes whose products are not associated with the plasma membrane. In
particular, deficiencies in two nuclear membrane associated proteins, emerin and
lamin A/C, result from Emery-Dreifuss muscular dystrophy, EDMD. Moreover, genes
for some cytosolic enzymes, calpain 3 and myotonin protein kinase are also
responsible genes for muscular dystrophy. However, the relationship between
abnormalities in these genes and muscular dystrophy is unclear. Recently, the
characterization of pathological features by a comprehensive examination of gene
expression in the patients tissue has become possible. To clarify the gene
expression profile and to help therapeutic studies, we developed a human muscle
cDNA microarray.
To develop a low-background microarray, we constructed a
highly nonredundant human singleton database for virtual cDNAs expressed in
skeletal or cardiac muscle. The database was constructed from public sequence
data, and then homologous sequences with rRNA and with mtDNA, and with
repetitive sequences were excluded to increase the reliability of each probe.
PCR primers were designed as amplification lengths of 450550b in the vicinity of
3 ends of each singleton. Each fragment of genes in the database was amplified
with specific primers and muscle cDNA pools, cloned and confirmed by sequencing.
1536 clones were then amplified, purified and spotted on a CMT-GAPS coated
slideglass to make microarrays.
To confirmed sensitivity and reproducibility of our microarray,
RNAs were labeled and then analyzed by using tyramide signal amplification
system and the ScanArray 5000. One microgram of total RNA was enough to analyze,
and our microarray showed low background and good resolution. This microarray
was considered to be a suitable device for gene expression analysis of the
muscular disease with biopsied samples.
Lamin C mutation associated with variable expression of dilated
cardiomyopathy and conduction-system disease (D-CM) and limb girdle muscular
dystrophy type 1B (LGMD1B) in four generations of a large kindred. S.
Dyack1,2, K. Collins1, P. Welch2, C. Riddell3,4.
1) Division of Medical Genetics, IWK Health Centre, Halifax, NS, Canada; 2)
Department of Pediatrics, IWK Health Centre, Halifax, NS, Canada; 3) Department
of Laboratory Medicine, IWK Health Centre, Halifax, NS, Canada; 4) Department of
Pathology, Dalhousie University, Halifax, NS, Canada.
LMNA mutations have been described in four distinct medical
conditions including dilated cardiomyopathy with conduction-system defects (D-CM),
autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD), limb girdle
muscular dystrophy type 1B (LGMD1B), and familial partial lipodystrophy (FPLD).
Lamin A and C are nuclear proteins that are encoded by the LMNA gene and differ
from each other only through alternative splicing of exon 10. We report here a
unique family with a novel mutation, R541S, in exon 10 of the lamin C coding
region of LMNA. There is variable expression of D-CM and LGMD1B in affected
family members. Three affected individuals had severe, progressive D-CM
resulting in sudden cardiac death. A 13 year old male with severe D-CM is now
the youngest reported child with a LMNA mutation to develop D-CM and to require
a cardiac transplant. Two members presented with a myopathy, subsequently
discovered to be LGMD1B, and had less severe D-CM. One affected individual with
mild LGMD1B had delayed ambulation as a child. Several asymptomatic individuals
have cardiac conduction defects. Many affecteds have muscle pain and most have
had an elevated CPK. Also noted is a distinct physical phenotype in females with
a decrease in adipose tissue of the extremities, but not associated with the
biochemical features of insulin resistance as seen in FPLD. This family
illustrates that a single LMNA mutation may lead to the expression of several
distinct phenotypes and, in light of these findings, it is suggested that
individuals with LMNA mutations should be investigated for features of all of
the above named conditions. In particular, it should be recognized that
alterations in the lamin C sequence can lead to the development of severe D-CM
and children with these mutations may require a heart transplant at a young age.
Functional analysis of lamin A/C mutations leading to dilated
cardiomyopathy, Emery-Dreifuss muscular dystrophy and partial lipodystrophy.
G.L. Brodsky1,2, A.C. Moss2, L. Mestroni1,2,
M.R.G. Taylor1,2. 1) Dept Cardiology/Medicine, Univ Colorado
Health Sci Ctr, Denver, CO; 2) University of Colorado Cardiovascular Institute,
Aurora, CO.
Lamin A/C gene mutations have been identified in four
different human diseases including Emery Dreifuss muscular dystrophy (EDMD),
limb-girdle muscular dystrophy (LGMD), dilated cardiomyopathy (DCM) and partial
lipodystrophy (PLD). The lamin A/C gene encodes two proteins which are the
primary components of the nuclear lamina; a proteinascious meshwork lining the
nucleoplasmic face of the inner nuclear membrane. Lamins have also been shown to
form intranuclear filaments. The nuclear lamina has been implicated in nuclear
structural integrity, chromatin organization, cell cycle regulation and
transcriptional unit organization.
As a first step in elucidating the molecular pathway by which
lamin A/C mutations lead to human disease, we have examined the expression and
localization of disease causing mutant lamin A proteins in mammalian cell
culture. Single nucleotide substitutions responsible for EDMD, DCM and PLD were
introduced into a wild type lamin A cDNA and transfected into mouse F9 cells.
The expression and sub-cellular localization of the mutant lamin A proteins were
determined by indirect immunofluorescence deconvoluting confocal microscopy.
All of the mutant lamin A proteins examined were found to be
expressed and localized to the cell nucleus. The mutant proteins responsible for
EDMD and DCM produced a wild type pattern of distribution, localizing to the
nuclear lamina as well as intranuclear filaments. In contrast, the mutant lamin
A proteins responsible for PLD were found to be concentrated in disk-like or
punctate structures at the nuclear periphery. No diffuse lamina staining or
intranuclear filaments were observed.
This is the first demonstration of altered lamin A function
resulting from disease causing mutations. Furthermore, these results support a
model where expression of dominant negative lamin proteins is responsible for
the observed disease phenotypes.
Histone Hypoacetylation of Subtelomeric DNA Repeats (D4Z4) Whose Deletion
Causes Fascioscapulohumeral Muscular Dystrophy. G. Jiang, F. Tsien, M.
Ehrlich. Human Genetics, Tulane Cancer Center, Tulane Medical School, New
Orleans, LA.
Fascioscapulohumeral muscular dystrophy (FSHD) is an unusual
autosomal dominant syndrome caused by the loss of some copies of a complex
repeat (D4Z4) in the subtelomeric region of one chromosome 4 homologue. The
number of copies of this 3.3-kb repeat at 4q35 arm is polymorphic. Unaffected
individuals have 11 to about 95 copies on each 4q35 while > 90% of FSHD
patients have <10 copies at one 4q35. Many investigators have proposed that
normally this region is heterochromatic but that when the number of tandem
copies of D4Z4 is <10, the region loses its condensed chromatin structure.
This is hypothesized to induce inappropriate gene expression in the affected
muscle cells. However, there have been no reports of the chromatin structure in
this region. Because constitutive heterochromatin has hypoacetylated core
histones, which lead to chromatin compaction, we are testing the acetylation of
D4Z4 repeat chromatin using chromatin immunoprecipitation (ChIP) with an
antibody for tetra-acetylated histone H4 followed by PCR with various amounts of
immunoprecipitated sample for DNA amplification. We compared H4 acetylation of
chromatin containing the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
gene, D4Z4 repeats, or centromeric satellite a
repeats by ChIP assays on a normal lymphoblastoid cell line. GAPDH served
as a euchromatic standard and satellite a
repeats as the heterochromatic standard. H4 acetylation was quantitated from the
immunoprecipitated chromatin normalizing for the PCR efficiency. The relative
amounts of H4 acetylation were 1.0, 0.01, and <0.01, for GAPDH, D4Z4,
and satellite a, respectively, which is
consistent with heterochromatinization of D4Z4. We will analyze chromatin
containing D4Z4, satellite a, or various
euchromatic genes for mono- and tetra-acetylation of histone H4 to determine if
D4Z4 chromatin is normally hypoacetylated compared to bulk euchromatin, as is
satellite a heterochromatin. We will
also compare cells from FSHD patients and analogous cells from unaffected
individuals to determine if the D4Z4 repeats from the deletion-containing 4q35
are abnormally hyperacetylated in FSHD cells. Supported by FSH Society Grant
FSHS-MB-06.
Myotonic dystrophy in the North of Portugal: a significant but still
overlooked problem. T. Coelho1, A. Tuna2, M. Freijo2,
M.C. Palmares3, R. Santos4, M. Santos5, A.
Sousa6. 1) Neurophysiology, HGSA; 2) Neurology, HGSA; 3) Centro
Gentica Clinica; 4) Inst. Genetica Medica Jacinto Magalhaes; 5) Neuropediatria,
Hospital Maria Pia; 6) Inst. Ciencias Biomedicas Abel Salazar, Porto, Portugal.
Myotonic dystrophy, one of the most frequent hereditary
muscular disorders has never been systematically studied in Portugal. Complete
molecular diagnosis became available only recently. Since 1991, 74 cases of
myotonic dystrophy from --35 different families were diagnosed at one paediatric
(Hospital Maria Pia) and one adult (Hospital Santo Antnio) integrated
neuromuscular outpatient clinics in Porto. Recently 32 families (69 patients, 37
female and 32 male) had molecular confirmation of the CTG expansion in
chromosome 19. The size of this expansion was quantified in 36 patients.
Clinical type, age-of-onset, family history and molecular studies were
reappraised in these 69 patients. Age-at-onset was established in 57: congenital
form presented in 10 patients (8 probands) and varied from 1 to 63 years in the
non-congenital forms. Concerning parental transmission, 28 patients inherited
the disease from their fathers and 29 from their mothers; 12 cases had
unidentified transmitter. Mean age-of-onset was higher (23.3 years) in offspring
of affected fathers than in offspring of affected mothers (13.8 years). Myotonic
dystrophy is a relevant problem in the North of Portugal. Systematic molecular
diagnosis of all patients will help in ascertainement of families and genetic
counselling.
Mutation screening of caveolin-3 and myotilin in autosomal dominant Limb
Girdle Muscular Dystrophy and other dominant myopathies. R.D. Dancel1,
J.M. Stajich1, J.M. Vance1, M.A. Pricak-Vance1,
P.C. Gaskell1, J.M. Gilchrist2, R.W. Tim3, M.C.
Speer1, M.A. Hauser1. 1) Section of Medical Genetics,
Department of Medicine, Duke University Medical Center, Durham, NC; 2) Rhode
Island Hospital, Providence, RI; 3) Raleigh Neurology Associates, Raleigh, NC.
Limb-girdle muscular dystrophy (LGMD) is a genetically
heterogeneous condition with both autosomal dominant and autosomal recessive
forms and extensive heterogeneity demonstrated within each of the two forms. In
the autosomal dominant form of LGMD, loci have been identified on chromosomes 5
(myotilin), 3 (caveolin-3), 1, and 6. To date, only one family has been
identified as having a mutation in myotilin. Thus, we undertook a screen of
myotilin and caveolin-3 in 32 autosomal dominant LGMD families. We also
investigated patients affected with other autosomal dominant myopathies
including individuals affected with non-chromosome 4 linked facioscapulohumeral
muscular dystrophy, scapuloperoneal muscular dystrophy, and other non-specific
dominant myopathies. Each of the 9 coding exons in the myotilin gene and 2
coding exons in the caveolin-3 gene was PCR amplified from genomic DNA in pools
of 5 unrelated individuals. The PCR products were analyzed by denaturing high
performance liquid chromatography using the Transgenomic WAVE DHPLC system.
Several different temperatures were tested for each PCR amplicon to optimize
mutation detection sensitivity. Any pools displaying altered retention time were
separated and the constituent samples were reanalyzed individually. All observed
changes were confirmed by sequencing both strands using the Beckman CEQ2000
capillary electrophoresis sequencer. A single polymorphism, present in both
affected and control individuals, was detected in the caveolin-3 gene. To date,
no caveolin-3 or myotilin mutations have been detected, excluding mutations in
these genes as a common underlying cause of disease inthese families.
LAMA2 mutation thought to be molecular basis of congenital muscular
dystrophy in the merosin-deficient dy2j mouse is found to be splicing
variant present in normal mice. D.M. Pillers, J. Pang. Dept
Pediatrics and Molecular & Medical Genetics, Oregon Health Sci Univ,
Portland, OR.
Laminin is a trimeric extracellular glycoprotein that is a
component of the extracellular matrix. It plays an important role in cell
adhesion and migration, in addition to signaling through transmembrane molecules
such as integrin and dystroglycan. Laminin-2, also known as merosin, is critical
in skeletal muscle development and function. Mutations in laminin-a2
are associated with human congenital muscular dystrophy (CMD) and with muscular
dystrophy in the mouse models dy and dy2j. A G to A splice donor
mutation at exon 2 in dy2j was defined by Xu et al. (1994) as the site of
the mutation resulting in the muscular dystrophy phenotype based upon the
creation of a group of aberrant laminin-a2
transcripts. In one, exon 2 is skipped but the reading frame is retained. In
others, a stop codon is predicted to result in a truncated protein. We performed
RT-PCR analysis of the alternatively spliced products for laminin-a2
and found that the spliced-in fragment described by Xu et al. is present in both
normal C57Bl/6J mice and the muscular dystrophy mouse model dy2j
suggesting that the mutation may actually be a polymorphism. Furthermore, we
found that the truncated products predicted by Xu et al. could be translated
into near full-length proteins, lacking only domain VI of the laminin-a2
subunit which contains the binding sites for heparin and cell surface receptors.
We also found that the skipping pattern of exon 2 resulted in skipping the
entire exon 2 while retaining the reading frame, as opposed to skipping part of
exon 2 and the entire exon 3 as suggested by Xu et al. Thus, we propose that the
splicing-in variant is expressed in normal mouse and likely has a specific
function. In addition, we propose that the alternative splice pattern occurs
independent of the G to A polymorphism and is the result of a novel regulatory
mechanism.
Evaluation of real-time quantitative PCR for carrier detection of
dystrophin gene rearrangements. S. Tuffery-Giraud, C. Saquet, S. Chambert,
M. Claustres. Lab Genetique Moleculaire, CHU Montpellier, France.
Large deletions and duplications in the dystrophin gene are
detected in about two third of patients with Duchenne (DMD) or Becker (BMD)
muscular dystrophy. This enables accurate carrier detection and prenatal
diagnosis in those families. Diagnosis of heterozygous deletions is mainly based
on haplotype analysis, involving detection of loss of heterozygosity for
microsatellite alleles or other polymorphic markers, whereas Southern blot is
the method currently used to assess gene dosage for duplications. To meet the
need for an easy-to-perform, rapid direct diagnosis of BMD/DMD carrier status,
we report the development of a method based on measurement of gene copy number.
Gene copy number analysis by real-time quantitative PCR can be targeted directly
to a selected sub region of the gene of interest, and does not depend on the
availability of informative flanking markers or parental DNA. Also, it involves
no radioisotopes and requires no post-PCR handling. Heterozygosity for
duplication increases the gene dose from two to three, whereas heterozygosity
for a deletion reduces the gene dose from two to one. The amplification was
performed in the Light-CyclerTM PCR system using the LightCycler FastStart DNA
master SYBR Green I kit. In order to demonstrate the accuracy and reliability of
the method for genetic testing, we analyzed 15 patients and relatives females
with previously identified deletions or duplications including either exon 11 or
44. Fifteen normal samples were also tested as controls. All samples were run in
triplicate. In order to correct for differences in the amount of input genomic
DNA, the method involves amplification of a reference locus with known copy
number and the use of a common standard DNA for amplification of the test and
reference loci. The presence or absence of the genomic deletion or duplication
was unambiguously diagnosed in all individuals. Hence, our initial data
demonstrated the effectiveness of this approach, which could be extended to the
analysis of multiple dystrophin exons in a cost- effective manner. Support: the
Association Franaise contre les Myopathies (AFM).
DNA rearrangements at the D4Z4 locus in South African facioscapulohumeral
muscular dystrophy families. A. van der Merwe1, C-M. Schutte1,
S.M. van der Maarel2, M. Alessandrini1, E. Honey1,
R.R. Frants2, A. Olckers1. 1) University of Pretoria,
South Africa; 2) Leiden University Medical Centre, The Netherlands.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal
dominant, late-onset degenerative neuromuscular disorder. Disease onset occurs
generally within the first or second decade of life. The phenotypical expression
varies in the age of onset and clinical severity. FSHD is characterised at onset
by the progressive weakness and atrophy of the facial and shoulder girdle
muscles. Linkage was established to the sub-telomeric region of chromosome 4q35.
This sub-telomeric region contains a polymorphic repeat array locus (D4Z4)
consisting of 3.3 kb repeats. An integral number of repeats are deleted in
affected individuals. Integrated haplotypes were constructed for 177 individuals
from five extended South African FSHD families. Southern Blot analysis was
performed to determine DNA rearrangements in 162 individuals from this selected
group of families. Deletion fragments were observed in all families investigated.
A BlnI resistant deletion fragment of 24 kb segregated with the postulated
FSHD-haplotype in families F10, F30 and F40. The FSHD-haplotype in all three
families was identical, with the entire nine-allele haplotype segregating in one
sub-family of F10 (F15) and in family F30. Six alleles of this haplotype
co-segregated with the FSHD phenotype in F13 (sub-family of F10) and three
alleles co-segregated in F40. It was previously reported that an identical six
allele FSHD-haplotype segregated in families F20 and F60. The deletion fragments
segregating in these families were also of identical size. To date, only two
FSHD haplotypes have been observed in the South African population - each
co-segregating with a deletion fragment of a specific size.
Temporal expression profiling in a progressive disease, Duchenne muscular
dystrophy. Y.-W. Chen1, M. Bakay1, R. Borup1,
S. Toppo2, G. Valle2, G. Lanfranchi2, E.P.
Hoffman1. 1) Center for Genetic Medicine, Children's Natioanl
Medical Center, Washington, DC; 2) CRIBI Biotechnology Center, University of
Padua, Padua, Italy.
Duchenne muscular dystrophy (DMD) is a progressive muscular
disorder caused by mutations in the dystrophin gene. Although the primary
biochemical defect is known, the downstream biochemical changes leading to later
patient weakness and death are not understood. To identify differentially
expressed genes involved in the progression of the disease, we report expression
profiling of pre-symptomatic, early-stage and mid- to end-stage DMD muscles by
using custom made Affymetrix MuscleChip containing approximately 1000
full-length genes and 2000 muscle ESTs. Among the total 4,654 probe sets on the
MuscleChip, expression of 3688 (80%) genes was detected by the MucleChip at
least once out of 46 chips in this study. We found a large number of significant
gene expression changes in fetal muscles (193 up- and 8 down-regulated genes)
years before the onset of symptoms. By applying both correlation and K-means
analysis, 48 genes were identified up-regulated during the progression of the
DMD while down-regulated in the age-matched control individuals. One hundred and
fifty seven genes were down-regulated during disease progression while
up-regulated in control individuals. Among the down-regulated genes, 55% of the
known genes were mitochondrial and metabolic genes. Genes involved in regulation
of oxidative stress, protein metabolism were also identified. The major
up-regulated genes are genes involved in fibrosis and extracellular matrix
remodeling, muscle fiber structure, protein synthesis and degradation.
Interestingly, expression levels of many genes that we previously reported
misregulated by comparing pooled 5-9-year-old DMD vs. control (ex. a-cardiac
actin and chondroitin sulfate proteoglycan versican) showed correlation to the
progression of the disease. By comparing the pre-symptomatic, early- and mid- to
late-stage DMD profiles, we set the stage for progression-specific therapeutic
targets for this common and lethal inherited disease.
Novel Dysferlin Mutations in Brazilian LGMD2B Patients. F. Paula1,
M. Vainzof1, E.S. Moreira1, M.R. Passos-Bueno1,
K. Bushby2, R. Bashir2, M. Zatz1. 1) Ctr
Study Human Genome, Univ de São Paulo, São Paulo, SP, Brazil; 2) Dept of
Biochemistry and Genetics, Newcastle Upon Tyne, Newcastle Univ, UK.
The limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi
myopathy (MM) are caused by allelic mutations in the dysferlin gene. Only few
pathogenic changes have been reported to date. In order to establish a
genotype-phenotype correlation we are analyzing the distribution of dysferlin
mutations in patients from 18 LGMD2B and MM Brazilian families , classified by
linkage analysis (10) or dysferlin deficiency (8).
Through SSCP, dHPLC and sequencing of abnormal fragments we
identified to date 5 different mutations in 7 unrelated families: 2 missense
changes in heterozigosity (the L189V in 2 families and D396H in one, exons 6 and
13 respectively), one stop codon in exon 28 (W999X) in 2 families (one
homozigous and another in heterozigous state) and 2 frameshift mutations in the
last 2 families: 3522-3523delTC (in one allele) and 3446-3453insCAGTGCTT (a
duplication of 8pb in homozigosity state), both in exon 29. Dysferlin protein
analyzed in muscle from all of these families showed absence of this protein. No
hot spot for mutations was identified.
All these patients related weakness first in the distal
muscles of the lower limbs and involvement in upper limbs on average 10 years
later , but with a variable course even in patients belonging to the same family.
All showed slow progression and on average, the phenotype in patients with
nonsense mutations was not more severe than among those with missense mutations.
In one of the families with the W999X mutation , the parents (deceased in their
sixties) who were first degree cousins, were both affected. They had 10 children,
also with a variable course. The intrafamilial variability , despite the high
degree of consanguinity in this unusual family gives further support to the
existence of modifying factors modulating the severity of the phenotype.
Supported by FAPESP/CEPID, CNPq, PRONEX and IAEA.
.
Expression profiling in facioscapulohumeral muscular dystrophy (FSHD)
indicates a defect in myogenic differentiation. S. Winokur1,
J. Martin1, Y-W. Chen2, J. Ehmsen1, K. Flanigan3.
1) Dept Biological Chemistry, Univ California, Irvine, CA; 2) Children's
National Medical Center, Washington, D.C; 3) Eccles Institute of Genetics,
University of Utah, Salt Lake City, UT.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal
dominant neuromuscular disorder resulting from integral deletions of a 3.3 kb
tandem repeat (D4Z4) in the subtelomeric region of chromosome 4q. Although the
specific genes affected by this deletion have not been identified, a regional
disruption of chromatin structure affecting local gene expression is thought
underlie the pathophysiology. The global gene expression profiling of mature
muscle tissue in FSHD presented here elucidates aberrant cellular processes
previously concealed by this unusual molecular mechanism. FSHD expression
profiles were compared to those generated from normal muscle and other types of
muscular dystrophies (DMD, aSGD, JDM) in order to determine FSHD specific
changes. In addition, matched biopsies (affected and unaffected muscle) from
patients with FSHD served to monitor expression changes during the progression
of the disease and to reduce non-specific changes resulting from individual
variability. Among 15 genes upregulated in an FSHD specific and highly
significant manner were several involved in cellular differentiation and
proliferation. These include muscle LIM protein (MLP), delta homolog (DLK1) and
the mitotic inhibitor huWEE1. As MLP and DLK were not increased further in the
affected to unaffected FSHD muscle, these may represent a primary change in FSHD
rather than an effect of progressing dystrophy. Increased MLP and DLK expression
was confirmed by both real time RT-PCR and a second GeneChip format. The
majority of FSHD-specific genes with reduced expression reflect a diminished
capacity to buffer oxidative stress. These FSHD specific downregulated genes
include SNAPC1 (HHCPA78 homolog) and nuclear factor of kappa light polypeptide
gene alpha (NFKB1A). Enhanced vulnerability to oxidative stress is a
characteristic previously demonstrated in cultured FSHD undifferentiated
myoblasts. We propose a model linking aberrant myogenic differentiation and
oxidative stress to subtelomeric shortening in FSHD.
Detection of Mutations in the Dystrophin Gene Via Automated DHPLC
Screening and Direct Sequencing. R.R. Bennett1, J. denDunnen2,
K. O"Brien3, B. Darras1, L. Kunkel1,3,4.
1) Division of Genetics, Children's Hospital, Boston, MA; 2) Department of Human
and Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands; 3)
Department of Genetics, Harvard Medical School, Boston, MA; 4) Howard Hughes
Medical Institute, Childrens Hospital, Harvard Medical School, Boston,
Massachusetts,USA.
Duchenne Muscular Dystrophy (DMD) is a genetic disorder which
manifests itself in one of every 1600 to 2000 live births. One in 3200 to 4000
will be severly disabled boys and one in 3200 to 4000 will be carrier females
with reproductive complications and potential late-onset cardiac complications.
Approximately 65% of mutations causing this disease are large deletions or
duplications, most of which are found by the existing set of multiplex PCR
primers. The other 35% have remained undetected in most patients. The purpose of
this project was to develop an efficient and inexpensive process for detecting
mutations large and small. We have obtained sequence upstream and downstream of
each exon as well as the 5' and 3' UTRs from the NIH human genome database and
have designed primers to include 30 to 100 bases on either side of each exon.
These primers were designed to produce a single clean band on agarose gel, to
create DNA fragments with melting characteristics appropriate for DHPLC analysis
using the WAVE from Transgenomic Inc. for DNA variation screening, and finally
to sequence well on automated DNA sequencers. We have tested eight patients --selected
from medical records as clearly having DMD but no large mutation-- using these
new primers in conjuction with the WAVE and PE-ABI 373 sequencer. We have
detected six disease-causing mutations. The remaining two patients have been
completely sequenced. Several polymorphisms have been detected in each, most of
which are known to be non disease-causing. Further investigation is required to
determine the cause of DMD in these two patients.
MAPH detection of deletions/duplications in Duchenne/Becker Muscular
Dystrophy: an alternative to quantitative Southern blotting. M.H.
Breuning, S. White, M. Kalf, M. Villerius, E. Bakker, G.-J.B. van Ommen, J.T.
den Dunnen. Center for Human and Clinical Genetics, LUMC, Leiden,
Netherlands.
Currently most genetic diagnostic protocols are PCR-based and
do not readily yield quantitative data. As a consequence, potential deletions
and duplications of the regions analyzed go undetected unless specific methods
are applied. Southern blotting is most commonly used, but is time consuming and
laborious. Recently an alternative method was published, called Multiplex
Amplifiable Probe Hybridization (MAPH). In this technique a series of short DNA
fragments are cloned in such a way that all can be PCR-amplified using one pair
of primers. The probes are hybridized to genomic DNA immobilized on nylon
filters, and after stringent washing the probes are recovered off the filters
and PCR- amplified in a quantitative manner. We have applied this technique to
the diagnosis of Duchenne/Becker Muscular Dystrophy (DMD/BMD), diseases caused
by mutations in the dystrophin gene. This gene is the largest known, covering
2.4 Mb, containing 79 exons. In approximately 65 % of cases the mutation is a
deletion or duplication of one or more exons. Frame-shift mutations cause the
lethal DMD, whereas maintenance of the reading frame leads to the less severe
BMD. For this reason it is important to assess the boundaries of rearrangements,
a potentially arduous task. A technique allowing simultaneous analysis of all
exons would greatly simplify this procedure. We cloned all 79 exons into the
same vector and divided the PCR products into 2 pools. Following hybridization
the secondary PCR was performed using a fluorescently labeled primer, allowing
the products to be analyzed on a 96 capillary sequencer. This allows parallel
analysis of 96 samples in ~48 hours. The number of copies of each exon could be
determined by comparing the appropriate peaks between controls and patients.
Using this technique we were able to detect exon deletions and duplications
missed using current methods. Further probe sets are being developed to cover
other areas of interest such as breast cancer and deletion syndromes. The
possibility of analyzing many more regions simultaneously using microarrays is
being investigated.
Point mutations in the dystrophin gene: a super hot spot of mutation at a
CpG dinucleotide and a difference in the pattern of microdeletions. C.H.
Buzin1, J. Yan1, J. Feng1, J.R. Mendell2,
S.S. Sommer1. 1) Molecular Genetics, City of Hope Medical Center
, Duarte, CA; 2) Ohio State University Medical Center, Columbus, OH.
About 35% of patients with Duchenne muscular dystrophy have
point mutations in the dystrophin gene, one of the largest known genes that
spans nearly 2.4 Mb of genomic DNA, contains 79 exons, and encodes a 14 kb
transcript. Comprehensive mutation scanning was performed with DOVAM-S, a
partially automated method that detects virtually all mutations. In this study,
genomic DNA from 141 patients with DMD, ascertained both clinically and by
muscle biopsy, was scanned in all coding exons, associated intronic splice
regions, and promoter sequences. Although prior screening had excluded most
patients with large deletion and duplication mutations, eight of the patients
were found to have a large deletion. Of the remaining 133 patients, 98 (74%) had
truncating point mutations, including nonsense, small deletions or insertions,
and splice mutations. Of the 98 causative point mutations, 94 were independent
as determined by haplotype analysis. No causative missense mutations were found,
although many missense polymorphisms were discovered, all of which were seen
also as second site changes in patients in which a truncating mutation was found.
For frameshift and nonsense mutations (86 independent mutations), a mutation
target could be determined within the coding region. One super hot spot mutation,
a transition at CpG in exon 59 (arg2905ter), was detected in six patients, all
with different haplotypes. When the point mutation types are normalized for
their target, the relative rates of microdeletions/insertions, transitions and
transversions at and not at CpG were similar to the factor IX gene. However, the
size distribution of microdeletions was significantly different in DMD and
factor IX.
Laminin is required to produce membrane instability in muscular dystrophy
mediated by sarcoglycan loss. E.M. McNally1, K. Lapidos2,
M. Hadhazy1, A.A. Hack2. 1) Department of Medicine,
The University of Chicago, Chicago, IL; 2) Department of Molecular Genetics Cell
Biology, The University of Chicago, Chicago, IL.
The sarcoglycans, a group of transmembrane proteins in muscle
and heart, are part of the dystrophin glycoprotein complex (DGC). Mutations in
sarcoglycan genes cause cardiomyopathy and muscular dystrophy in humans and in
mice by producing instability of the remaining sarcoglycans while leaving
dystrophin intact. In contrast, mutations in dystrophin cause a reduction in not
only the sarcoglycans, but also in additional DGC proteins such as the
syntrophins and dystrobrevins. We used a genetic analysis to evaluate whether
sarcoglycan is sufficient to mediate membrane instability and by generating mice
lacking both dystrophin (mdx) and g-sarcoglycan.
Mice lacking both g- sarcoglycan and
dystrophin (gdx) had an identical phenotype to mice lacking only g-sarcoglycan
with a similar degree of central nucleation and serum creatine kinase elevation.
This demonstrates that sarcoglycan loss is sufficient to produce alterations in
membrane permeability and the dystrophic process. In parallel experiments, we
evaluated the role of the extracellular matrix protein laminin in the dystrophic
process by producing mice mutant for both g-sarcoglycan
and laminin-a2. Laminin-a2
is the major site of attachment to the extracellular matrix for the DGC. Mice
mutant for both g-sarcoglycan and laminin-a2,
(gdy), surprisingly displayed a severe dystrophic process yet had little
evidence for disruption of membrane integrity. Little to no Evans Blue Dye
uptake was seen and serum creatine kinase was only minimally elevated in (gdy)
mice. These genetic analyses demonstrate that sarcoglycan loss is sufficient to
produce the dystrophic process, and that laminin is required for disruption of
membrane integrity and increased membrane permeability that is seen when
sarcoglycan is absent.
PFGE analysis of 4qter-10qter interchromosomal exchanges in Italian
families with Facioscapulohumeral muscular dystrophy (FSHD). L. Felicetti1,2,
G. Galluzzi1,2, L. Colantoni1,2, M. Rossi1,2,
B. Merico2, F. Mangiola1, P. Tonali2, E. Ricci2.
1) Ctr Neuromuscular Diseases, UILDM, Rome, Italy; 2) Institute of Neurology,
Catholic University, Rome , Italy.
In 95% of FSHD patients, molecular diagnosis is based on the
detection by probe p13E-11 of EcoRI, BlnI-resistant fragments of 10 to 35 kb,
shorter than those found in normal individuals (35-300 kb). The size reduction
is due to a deletion of a variable number of KpnI repeats at 4q35 region. 4q-10q
interchromosomal exchanges occur both in normal and FSHD subjects, resulting in
the reshuffling of 4q-type BlnI-resistant and 10q-type BlnI-sensitive repeats
from one chromosome to the other. We analyzed the segregation of BlnI-resistant
and BlnI-sensitive alleles in 55 FSHD Italian families for a total of 230
individuals (116 affected, 70 unaffected and 44 spouses). DNA extraction and
subsequent restriction steps with EcoRI, BlnI and Tru9I were performed directly
in agarose blocks. After separation by Pulsed Field Gel Electrophoresis (PFGE),
the alleles were identified with p13E-11 and KpnI cloned sequences as probes. We
observed different types of exchanges: total 4q to 10q transfers resulting in
trisomy and tetrasomy; partial 10q to 4q transfers leading to monosomy and
nullisomy; complex rearrangements such as multiple translocations, even in
subjects with p13E-11 standard allele configuration. In addition, we detected
two patients carrying 10q-4q translocations involving the short fragments
associated with FSHD. Among 8 sporadic cases carrying a de novo p13E-11 small
fragment (BlnI-resistant), we found four patients, with no evidence of somatic
mosaicism, with one or both parents showing various types of rearrangements
(trisomy, monosomy, etc.). The remaining four were somatic mosaics, all carrying
4q-10q translocations. Our results show the high frequency of 4qter-10qter
interchromosomal exchanges in FSHD families and confirm that the instability of
subtelomeric regions can play a role in the molecular mechanism of the disease.
Telethon Italy grant n.1296.
Detection of expanded CAG repeats at the myotonic dystrophy (DM) locus in
single cells by triplet primed PCR (TP-PCR) for preimplantation genetic
diagnosis (PGD). P.F. Ray1, N. Frydman2, R. Frydman2,
M. Vekemans1, A. Munnich1. 1) Departement de genetique
medicale, Hopital Necker, PARIS; 2) Service de genicologie-obstetrique, Hopital
Antoine Beclere, Clamart, France.
The amplification of large number of triplet repeats is
challenging from microgram amounts of DNA but is impossible from single cells.
Large pathologic alleles responsible for myotonic dystrophy (DM) can expand up
to several thousands triplets but a maximum of 50-100 repeats can reliably be
amplified from single cells. Thus, genetic analysis in the course PGD for
couples at risk of transmitting myotonic dystrophy has so far mostly been based
on the detection of the normal allele of the transmitting parent. This
semi-indirect strategy, however, is only applicable when that allele is
different in size from the proposituss partner two alleles, from our experience,
only about one fourth of the couples. Warner et al. (1996)* described a
fluorescent assay in which a primer internal to the triplet repeat itself was
used and allowed the detection of expanded alleles. We utilized this strategy
and developed a single cell hemi nested TP-PCR assay. The outer reaction mix
contained a DM specific forward primer and the TP primer made of 7 CAG repeats
tailed with an overhanging 20 bp specific primer. Aliquots of this reaction were
reamplified with the same 5 fluorescently labeled primer and the 20 nucleotides
specific primer. Fluorescent TP-PCR products were analyzed in an ABI 310 DNA
analyzer. Expanded alleles were detected in 100% of the control lymphocytes
analyzed and one PGD cycle was carried out. Following this procedure two embryos
which had not shown amplification of the expanded allele were transferred.
Positive hCG were detected two weeks later, indicating a successful uterine
implantation of an embryo but the pregnacy was not sustained. Reliable detection
of expanded CAG repeats was achieved from single lymphocytes and blastomeres by
fluorescent TP-PCR. This single cell analysis technique is applicable to all
couples at risk of transmitting myotonic dystrophy and similar protocols could
be developed for the diagnosis of other trinucleotide expansion diseases. * J
Med Genet, 1996, (33) 1022-6.
Functional correction of adult mdxmouse muscle using gutted
adenoviral vectors expressing full-length dystrophin. C. DelloRusso1,3,
J. Scott3, D. Hartigan-O'Connor3, C. Barjot2,
G. Salvatori2, A. Robinson2, S. Brooks1, J.
Chamberlain3. 1) Departments of Physiology and; 2) Human
Genetics, University of Michigan, Ann Arbor; 3) Department of Neurology,
University of Washington, Seattle.
Duchenne muscular dystrophy is a degenerative lethal muscle
disorder caused by mutations in the dystrophin gene. Adenoviral vectors are
promising tools that may be used to express dystrophin in affected muscle. We
have constructed 3 gutted adenoviral vectors devoid of all viral genes and
containing a full-length human (HDys) or mouse (MDys and GEbDys)
dystrophin cDNA driven by a muscle specific promoter. Virus was injected into 1
year old mdxmouse TA muscles and a novel lengthening contraction (LC)
protocol was used to test for restored muscle function. This protocol reveals
the high susceptibility of mdxTA muscles to contraction-induced injury;
after 1 LC, wild type muscle force is reduced by 10% while mdxmuscles
show a 72% force loss. After 5 days, HDys injected muscles were significantly
protected from injury and expressed high levels of dystrophin in contrast to
sham injected control muscles. However, 25 days after injection, significant
decreases in force generating capacity were detected. This loss of force was
similar to that observed after injection of a first generation virus containing
a LacZ transgene (CNb). MDys and GEbDys
injected muscles demonstrated high levels of dystrophin protein expression and
no functional defects after 25 days. In addition, MDys injected muscles were
able to produce 62% of wild type force levels after 1 LC that, in contrast,
reduced mdxand sham injected control muscles to 38 and 29% of wild type
force, respectively. FACS sorting revealed the least amount of CD4+ and CD8+
cells in MDys injected muscles, 1/3 more immune cells in HDys injected muscles,
and the highest number in muscles injected with CNb. Collectively, these data demonstrate 1) gutted adenoviral
vectors are successful in transducing dystrophin in TA muscles of 1 year old mdxmice,
2) evidence for an immune response against human dystrophin that causes atrophy
and loss of function, and 3) a partial functional correction of adult mdxmouse
muscle after delivery of full-length mouse dystrophin
Delivery of Functional Four-Repeat Micro-Dystrophin to Mdx Muscle
Via Different AAV Serotypes. S. Harper1, C. DelloRusso1,
R. Crawford1, H. Harper1, J. Engelhardt2, D.
Duan2, J. Chamberlain1. 1) Dept of Neurology,
University of Washington, Seattle, WA; 2) Dept of Anatomy and Cell Biology,
University of Iowa, Iowa City, IA.
The goal of this study was to evaluate the correction of
Duchenne muscular dystrophy (DMD) in the mdx mouse model using different
serotypes of AAV carrying extremely small dystrophin genes (~3.5 kb vs. the
full-length 14 kb). The rod domain of dystrophin is composed of 24 spectrin-like
repeats, and natural, in-frame deletions of this region lead to a milder form of
dystrophy called Becker MD. Based on our prior analysis in transgenic mice, we
created several rod domain and C terminal deletions that resulted in dystrophin
cDNAs small enough to fit into AAV. AAV is a highly efficient vector that does
not elicit an immune response in dystrophic muscle if it is carrying a gene
driven by a muscle-specific promoter. AAV-2 is the most prevalent and
well-characterized system, but recent studies show that serotypes 1, 5, and 6
boost gene expression 100- to 1000-fold in mouse muscle. We generated transgenic
mice expressing several different micro-dystrophin clones containing only four
repeats, and analyzed their ability to correct muscular dystrophy by both
morphological and physiological assays. Two of three transgenes significantly
reduced the amount of dystrophy observed in the mdx limb and diaphragm
muscles. The best transgene (DR4-R23)
produced muscle with wild-type levels of central nuclei, membrane integrity,
resistance to contraction-induced injury, and the ability to run on a treadmill.
No areas of fibrosis nor monocyte infiltration were observed. We subsequently
demonstrated that highly functional micro-dystrophins can be successfully
delivered via AAV-2 to muscles of young adult mdx mice. We are currently
testing gene expression of our best micro-dystrophin with alternate AAV
serotypes, a more efficient Kozak sequence, and a highly active, mutant form of
the muscle creatine kinase promoter (CK6). Functional correction of muscular
dystrophy using these vectors will be compared to results obtained with our
gutted adenovirus vector system that expresses full-length dystrophin.
Amelioration of dystrophic phenotypes in transgenic mdx mice
expressing truncated dystrophin cDNA. M. Sakamoto, K. Yuasa, T. Yokota,
S. Masuda, Y. Miyagoe-Suzuki, S. Takeda. Molecular Therapy, Natl Inst
Neurosci NCNP, Kodaira, Tokyo, Japan.
Duchenne Muscular Dystrophy (DMD) is an X-linked, lethal
disorder caused by a defect in the dystrophin gene. Progressive muscle
weakness, cardiomyopathy and early death characterize the disease. Dystrophin is
localized at the inner surface of the plasma membrane and forms the complex with
dystrophin-associated proteins (DAPs) to stabilize the sarcolemma by linking the
cytoskeleton to the extracellular matrix. Dystrophin has a central rod domain,
which consists of 24 triple-helical repeats and 4 hinge segments, and accounts
for 76% of the molecule. It has been shown that a large in-frame deletion in
this domain results in a mild allelic form of the disease, Becker muscular
dystrophy (BMD).
Adeno-associated virus (AAV) vector-mediated micro-dystrophin
cDNA transfer is one of attractive approaches for the treatment of DMD. AAV
vector allows long-term expression of the transferred gene without significant
immune responses, while it has a limited insertion size up to 4.7-4.9 kb.
Therefore, we cannot accommodate a full-length dystrophin cDNA (14 kb) into AAV
vector. To find a functional, but small-sized dystrophin, we generated a series
of rod-truncated micro-dystrophin cDNAs with one rod repeat and two hinges
(named M3 construct), three rod repeats and two hinges (AX11 construct), and
four rod repeats and three hinges (CS1 construct). We have generated transgenic mdx
mice expressing micro-dystrophins and evaluated their function to improve
dystrophic phenotype. Histological examination showed that CS1 construct
recovered DAPs at the sarcolemma and significantly reduced muscle degeneration
in the hind limb muscles and diaphragm. On the other hand, M3 construct could
not ameliorate mdx phenotype in spite of recovery of DAPs at the
sarcolemma. These data suggest that the rod structure, especially its length is
critical for function of micro-dystrophin. We are now investigating the
phenotypes of AX11-transgenic mdx mice. Our goal is to identify a
functional micro-dystrophin and transfer it into dystrophin-deficient dystrophic
muscles using AAV vector
Modulation of muscular dystrophy by inhibitors of apoptosis. S.
Abmayr, R.W. Crawford, J.S. Chamberlain. Dept Neurology, Univ Washington,
Seattle, WA.
Duchenne muscular dystrophy (DMD) is a human X-linked
recessive disorder caused by mutations in the dystrophin gene. DMD is
characterized by a progressive loss of muscle function, leading to death in the
early twenties. The relationship between the protein deficiency and the late
clinical onset of muscle pathology remains unclear. Several lines of evidence
support the hypothesis that cell death of dystrophin deficient muscle might be
initiated by apoptosis, followed by necrotic processes. We have been studying
the role of proteins that repress apoptosis or/and enhance muscle regeneration
for their ability to modulate the dystrophic phenotype in conjunction with gene
replacement therapy. We have generated transgenic mice that overexpress ARC, an
apoptosis inhibitor, almost exclusively expressed in skeletal muscle and heart.
ARC was shown previously to interact selectively with caspases and to prevent
hypoxia-induced release of cytochrome c from mitochondria. Morphology studies
were performed to determine the level of de-and regeneration of muscle fibers of
ARC transgenic/mdx mice in comparison with mdx controls. Tg/mdx
mice displayed a clear pattern of mdx pathology. We performed
immunohistochemistry analysis to compare the level of activated caspase-3 in mdx
and transgenic/mdx muscle and detected no significant difference in the
levels of activation. Caspase-3 positive fibers displayed membrane damage as
assessed by uptake of the vital dye Evans blue, suggesting apoptotic pathways
may be activated as a result of membrane leakage. Together the results suggest
that overexpression of ARC does not overtly protect mdx muscle from
dystrophic pathology. Currently we are investigating if ARC plays an inhibitory
role in hypoxia-induced apoptosis in muscle, which might contribute to improved
muscle function in less sedentary animals. In a complementary approach, we are
examining the role of IGF-I in muscle and its ability to modulate the mdx
muscle pathology. IGF-I has been shown to enhance muscle regeneration and to
prevent age-related declines in muscle mass and function. We have generated
adenoviral vectors expressing IGF-I and dystrophin and are studying their
effects in preventing and reversing dystrophic pathology in mdx mice.
Adenoviral Vector Systems for Delivery of Dystrophin cDNA Expression
Constructs to Mouse Skeletal Muscle. J.M. Scott, D.J. Hartigan-O'Connor,
C. Barjot, S.Q. Harper, A.S. Robinson, C. DelloRusso, R.W. Crawford, J.S.
Chamberlain. Neurology, University of Washington, Seattle, WA.
Gene therapy using viral vectors holds promise for several
human genetic diseases but much remains to be learned about which vector system
will meet all of the requirements of such a treatment. Our laboratory has
focused on modification of adenoviral (Ad) vectors in order to improve the
efficiency of delivering expression constructs to skeletal muscle with respect
to transgene expression levels, persistence, immune response, and
tissue-specific vs. constitutive promoters. Improvements in first generation Ad
vectors that carry mini-dystrophin cassettes was achieved by deleting the viral
polymerase gene which rendered the vector replication defective and eliminated
viral late gene expression in transduced cells. We have also made gutted Ad
vectors lacking all viral ORFs, and adeno-associated virus (AAV) containing a
micro-dystrophin expression cassette. These systems are compared using the mdx
mouse model of Duchenne muscular dystrophy whereby skeletal muscle is injected
and assays are performed to evaluate dystrophin protein expression levels,
persistence of the transgene, immune cell infiltration, and integrity of the
muscle fiber membranes. Our results indicate that gutted Ad vectors containing a
dystrophin expression cassette driven by a strong muscle-specific promoter
(modified MCK) lead to significantly increased transgene expression and
decreased immune response compared to the first generation viruses. We find that
the MCK promoter is highly effective in reducing the antigen-specific immune
response against Ad vectors despite the muscle cell death and antigen-presenting
cell infiltration in dystrophic muscle. A gutted Ad vector expressing
full-length utrophin is also being compared with the dystrophin vector for
function, persistence and immunogenicity. Additional improvements have been
noted using an AAV vector which exhibits robust expression of dystrophin and
even less immune response than that elicited by Ad vectors. These results show
that modified Ad and AAV vectors can lead to significant and prolonged gene
expression in dystrophic muscle.