| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
7ß1 in Muscular Dystrophy/Myopathy of Unknown Etiology
From the Neuromuscular Center,*Department of Neurological and Psychiatric Sciences, University of Padova, Padova, Italy; Department of Biomedical Sciences,University of Padova, Padova, Italy; the Department of Genetics, Biology, and Biochemistry,University of Torino, Torino, Italy; The Burnham Institute,La Jolla, California; and the Center for Genetic Medicine,¶Children’s Research Hospital, Washington, District of Columbia
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
7 in muscle pathology, we used a "candidate gene" approach in a large cohort of muscular dystrophy/myopathy patients. Antibodies against the intracellular domain of the integrin 7A and 7B were used to stain muscle biopsies from 210 patients with muscular dystrophy/myopathy of unknown etiology. Levels of 7A and 7B integrin were found to be decreased in 35 of 210 patients (
17%). In six of these patients no integrin 7B was detected. Screening for 7B mutation in 30 of 35 patients detected only one integrin 7 missense mutation (the mutation on the second allele was not found) in a patient presenting with a congenital muscular dystrophy-like phenotype. No integrin 7 gene mutations were identified in all of the other patients showing integrin 7 deficiency. In the process of mutation analysis, we identified a novel integrin 7 isoform presenting 72-bp deletion. This isoform results from a partial deletion of exon 21 due to the use of a cryptic splice site generated by a G to A missense mutation at nucleotide position 2644 in integrin 7 cDNA. This spliced isoform is present in about 12% of the chromosomes studied. We conclude that secondary integrin 7 deficiency is rather common in muscular dystrophy/myopathy of unknown etiology, emphasizing the multiple mechanisms that may modulate integrin function and stability.
and ß, associated by non-covalent interactions. In humans, at least 18 different and 8 different ß subunits are known, resulting in 22 distinct heterodimers.1,2
The 7 subunit is mainly expressed in skeletal and cardiac muscle, while the ß1 chain is expressed throughout the body and associated with other different subunits.3-6
In adults, the alternative ß1D isoform is restricted to skeletal and cardiac muscle.7-9
Integrin 7ß1 in skeletal and cardiac muscle binds via its extracellular domain to laminin 2 and 410-12
and via its cytoplasmic domain possibly to -actinin and talin ensuring continuity of structure and signaling between the cytoskeleton and the basal lamina.13,14
Integrins have a prominent role in myogenesis,15,16
differentiation, cell migration, and cell-cell interactions.2,17-19
Integrin 7 in skeletal muscle localizes at the sarcolemma, at the neuromuscular junctions, and, most prominently, at the myotendinous junctions where it provides an anchorage for laminin 2, conferring mechanical stability and traction resistance to the skeletal muscle fibers.5,9,20,21
The expression of several integrin 7 isoforms modulates the integrin 7ß1 ligand affinity and signaling specificity during myogenesis.20
All integrin isoforms are encoded by a single 7 gene (ITGA7) located on chromosome 12q1322
and result from alternatively spliced variants.20,23
In humans, different extracellular and cytoplasmic domain isoforms have been described.23,24
The 7X1 and 7X2 extracellular isoforms result from the alternative splicing of exons 5 or 6, in a variable region nearby the ligand binding site.5,24,25
The X2 isoform is the main variant expressed in adult muscle fibers.24,26
The cytoplasmic isoform 7A,20,24,27
corresponding to the normal exon 26 splicing, is temporarily up-regulated during myogenesis, while the 7B variant, the longest isoform resulting from the splicing out of exon 26, is the main isoform expressed in skeletal and cardiac muscle, smooth muscle cells, spleen, liver, and brain.20,28
To study the involvement of 7 integrin during myogenesis and its role in muscle integrity and function, a null allele of the gene for the 7 integrin subunit has been generated.29
Mice homozygous for the mutation showed features of a progressive muscular dystrophy in their muscle, suggesting that integrin 7 represents an indispensable linkage between the muscle fiber and the extracellular matrix,29
particularly at the myotendinous junction. To further support the role of integrin in connecting muscle cells with the surrounding extracellular matrix, it has been shown that 7ß1 is up-regulated in Duchenne muscular dystrophy (DMD) patients and in its animal model, mdx mice, to compensate for the reduced dystrophin-mediated linkage of fibers with the basal lamina.11,30
In merosin-negative congenital muscular dystrophy (CMD) and in its animal model, dy/dy mice, 7 integrin appears to be significantly reduced suggesting that lack of laminin 2 (2ß1
1) may down-regulate the expression or change the location of integrin 7 gene.11,12,30
The cloning of the full-length human ITGA7 cDNA5,26,31
allowed the identification of the first three patients with primary 7 integrin deficiency.31
The three patients reported were affected with congenital myopathy with variable clinical phenotype, and all showed a complete absence of integrin 7 in their muscle biopsies due to primary integrin 7 nonsense/splicing mutations or to a down-regulation of integrin 7 mRNA.31
Here we report a study of a large series of muscle biopsies from patients affected with unclassified muscular dystrophy/myopathy tested for integrin 7 deficiency by combined integrin 7 protein and gene studies.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Our muscle biopsy tissue bank at the Neuromuscular Center of the University of Padova was screened to search for patients meeting the following criteria: muscle weakness and/or hypotonia; muscle histopathology consistent with a myopathic or dystrophic process; and normal dystrophin, -sarcoglycan, calpain, dysferlin, and laminin 2 in their muscle biopsies. Inflammatory myopathies, neurogenic atrophies, and metabolic or mitochondrial myopathies were a priori excluded. Two hundred ten patients met the clinical and histopathological selection criteria and were chosen for integrin 7 screening. One hundred thirteen were muscular dystrophies, 24 CMD, and 73 carried a histopathological diagnosis of undetermined myopathy.
Case Report
Patient E.S. was delivered by Caesarian section at the 39th gestational week due to the threat of miscarriage. The child was oxygen-dependent and mechanically ventilated since birth. He presented with hypotonia and with hip, wrist, and ankle contractures. Karyotype study was normal. A brain computed tomography (CT) scan showed cortical atrophy and white matter signal abnormalities. Creatine kinase (CK) was 507 U/L (normal <250) and electromyography (EMG) myopathic. At one month of age a muscle biopsy was consistent with a congenital muscular dystrophy. Dystrophin, -sarcoglycan, and laminin 2 studies in his muscle biopsy were normal. The patient died at 13 months of age from respiratory failure. Integrin 7 was markedly reduced in the patient’s muscle biopsy.
Integrin 7 Immunohistochemistry
Serial cryosections, 4-µm thick, were fixed for 5 minutes in 100% ice-cold-acetone, air dried, and preincubated with phosphate-buffered saline containing 2% bovine serum albumin and 5% goat serum as blocking agent.31
Polyclonal anti-integrin 7B antibody (1:500) directed against the intracellular domain of the protein,12
polyclonal anti-integrin 7A antibody (1:500) directed against the COOH terminal peptide of the protein, polyclonal anti-ß1D (1:500),12
and monoclonal antibody directed against the carboxyl terminus region of the laminin 2 (1:1000)32
(Chemicon, Temecula, CA) were used to incubate the sections for 2 hours at room temperature. Appropriate Cy-3-conjugated (anti-rabbit or anti-mouse) secondary antibody (1:100) (Caltag Lab, Burlingame, CA) were used. The visualization of mounted sections was done on Zeiss Axioskop photomicroscope. Hematoxylin and eosin staining was done on parallel cryosections to check tissue integrity and histopathology.
Muscle biopsies were scored as integrin 7B negative, integrin 7B markedly reduced, or integrin 7B slightly reduced (based on the amount of integrin 7 immunostaining detected in each muscle biopsy). The same criteria were used to score 7A immunostaining.
Integrin 7ß Mutation Studies
RNA extraction
Approximately 50 mg of frozen muscle biopsy tissue was homogenized using a Kinematica Polytron PT 2100 homogenizer. RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) accordingly to the manufacturer’s instructions and stored in RNase-free water at -80°C.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
For cDNA synthesis, 1–2 micrograms of RNA was reverse transcribed using oligo-dT primers as previously described.33
cDNA was boiled to denature and inactivate reverse transcriptase. Eighteen sets of overlapping primers were designed to cover the entire 3414 bp of the integrin 7 coding sequence and the 5[prime]-UTR region (Table 1)
. PCR reactions contained about 50 ng of cDNA, 50 ng of each primer, 100 nmol/L of each dNTP, and 0.2 µCi -[32P] dATP in 12.5 µl total volume. Reaction conditions were 10 minutes at 94°C to denature; 30 seconds at 94°C, 30 seconds at 65°C, 30 seconds at 72°C for 35 cycles, with an extension of 10 minutes at 72°C for all primer sets except primer sets 1212F-1451R and 2068F-2245R where the annealing temperature was 50°C.
|
Three different single strand conformational polymorphism (SSCP) conditions33
were used for screening for integrin 7 mutation. Conformers were re-amplified using the original amplification primer and PCR conditions as previously described33
and directly sequenced on automatic sequencer (CRIBI Biotechnology Center, Department of Biology, University of Padova).
The integrin 7 mutations identified were confirmed with the appropriate restriction endonuclease digestion if a gain or loss of restriction site was detected or with appropriate PCR primers designed to cover the mutated region and PCR product size-fractioned with denaturing polyacrylamide gel electrophoresis. Primers used to amplify exon 15 and exon 21 were as following: ex 15F 5'-TGCCCCTTATCTATGTCTCC-3'; ex 15R 5'-GAGAGGGCTTTCTCTCAATCC-3'; ex 21F 5'-TCCCCTCATACTCTCTTTTTCC-3', and ex 21R 5'-AACTCCCCATAACATCTGTAACC-3'.
Integrin 7 Gene Expression Studies
RNA extraction and cDNA synthesis were done as described above. Biopsies studied were from the 30 patients screened for integrin 7 mutation and from 10 integrin 7-positive myopathic control muscle biopsies.
Primer sets for co-amplification of integrin 7 RNA and -sarcoglycan RNA were designed, and for each set the forward primer was synthesized with fluorescein-linked 5' ends (Invitrogen). Primer sequences were as follows: -sarcoglycan ADH3F: 5'fluorescein-CTGCTCAACGTCACCTCTG-3'; ADH3R: 5'-CCGGCACTGACTTATCCAC-3'; and integrin 7 ITGA7.9F: 5'fluorescein-TTGATGGTGATGGGAAAGTCTTCA-3'; and ITGA7.12R: 5'-CAGACCCTTAGGTCCACACAGACC-3'.
Multiplex RT-PCR and quantitation of fluorescent PCR products were done as previously described.34
Briefly, cDNA corresponding to approximately 20 ng of total RNA was amplified with primers mixed for 22 cycles using standard conditions (denaturation at 95°C for 10 minutes, at 94°C for 1 minute, at 62°C for 1 minute, at 72°C for 1 minute, for 22 cycles; and extension at 72°C for 10 minutes). Fluorescent RT-PCR products were denatured and loaded on an ABI automated sequencer (Applied Biosystems, Foster City, CA) using a ROX/fluorescein matrix standard. ROX labeled markers were included as internal size standards. Peak areas corresponding to expected RT-PCR product sizes were determined, and peak area ratios of integrin 7 relative to internal control (-sarcoglycan) calculated. Two separate RT-PCR measurements were done for each patient’s biopsy. Statistical significance was done by Student’s t test using all calculated ratios.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
7-Deficient Patients
The entire database, about 5000 muscle biopsies, was searched for patients meeting the histopathological diagnosis of muscular dystrophy of unknown etiology (normal dystrophin, -sarcoglycan, calpain, dysferlin, and laminin 2). About 560 muscle biopsies, collected over 2 consecutive years, were searched for undetermined myopathy. Two hundred ten patients were selected for integrin 7 screening from the database search of muscle biopsies. One hundred thirty seven muscular dystrophy and 73 myopathies were tested with integrin 7 immunofluorescence. Muscle biopsies from patients diagnosed with muscular dystrophy showed marked fiber size variation, increased central nuclei, degeneration and regeneration, and a marked increase in the endomysial and perimysial connective tissue with fatty infiltration. Common features in the undetermined myopathy group of muscle biopsies included mild fiber size variation and scattered central nuclei, with little or absent muscle fiber degeneration or regeneration. Fibrofatty infiltration was absent or minimal.
Antibodies directed against the 7A, 7B, and the ß1D subunit of integrin were used to characterize integrin expression in patients’ muscle biopsies. In parallel, laminin 2 immunostaining was done in each muscle biopsy to check the integrity of the muscle fibers. Three percent (6 of 210) of the muscle biopsies studied were completely deficient for integrin 7B (Table 2
; Figure 1G
) and about 14% (29 of 210) showed a (variable) reduction of integrin 7B immunostaining (Table 2
; Figure 1, M and Q
).
|
|
7B deficiency were all undetermined muscular dystrophy (Figure 1G)
. Three were diagnosed as CMD and 3 as limb-girdle muscular dystrophy. Marked integrin 7B reduction (ie, variable and barely detectable integrin 7 immunostaining in the majority of the muscle fibers with or without some scattered myofibers showing patchy but positive immunostaining) (Figure 1M)
was present in about 6% (8 of 137) of the muscular dystrophies and in 15% (11 of 73) of the myopathies studied (Table 2)
. A slight reduction in integrin 7B (ie, variable and faint integrin 7 immunostaining and/or a mosaic-like pattern) (Figure 1Q)
was detected in 5% (7 of 137) of muscular dystrophies and in about 4% (3 of 73) of unclassified myopathy (Table 2)
. Interestingly, the integrin 7-positive muscle fibers, in the subset of 7B partial deficiency, often showed a integrin 7-positive cytoplasmic immunostaining. Integrin 7A was reduced (Figure 1, H, N, R)
, and integrin ß1D mildly reduced (Figure 1, F, L, P)
in all muscle biopsies showing integrin 7B deficiency. In the six muscle biopsies showing complete integrin 7B deficiency, integrin 7A was more severely decreased than in the partial deficiency. Laminin 2 was normal in all biopsies studied (Figure 1, E, I, O)
.
Integrin 7B deficiency was present in about 39% (11 of 28) (Table 2)
of muscle biopsies from patients younger than 2 years, and only in 13% (24 of 182) (Table 2)
from patients older than 2 years. To determine whether integrin 7B deficiency in younger patients was a manifestation of integrin 7B being expressed at a lower level at a younger age, we selected a patient with a marked reduction of integrin 7B in muscle at 1 year of age who had two subsequent muscle biopsies obtained at 2 and 6.3 years of age. Integrin 7B immunostaining was performed in parallel with laminin 2 and integrin ß1D in the three-muscle biopsy. As shown in Figure 2
, the patient’s muscle biopsy performed at 2 years of age (Figure 2C)
contained scattered myofibers with a faint and variable integrin 7B immunostaining. Rare fibers showed a patchy and barely detectable integrin 7B immunostaining in the muscle biopsy performed at 6.3 years of age (Figure 2F)
. Integrin ß1D was mildly reduced (Figure 2, B and E)
, and laminin 2 was normal in these biopsies (Figure 2, A and D)
.
|
7 Mutation Studies
Adequate muscle tissue for RNA extraction was available for 30 of 35 muscle biopsies showing integrin 7B deficiency. Twenty were muscular dystrophies (including the six samples showing complete 7B deficiency) and 10 were myopathies. These 30 muscle biopsies were selected for integrin 7 mutation screening. RNA was extracted and RT-PCR/SSCP was performed using 18 sets of overlapping primers covering the entire integrin 7 coding sequence.
Integrin 7 Polymorphisms
Several conformers were identified and directly sequenced. Seven integrin 7 polymorphisms were identified. All of the identified polymorphisms were in the extracellular domain of the protein. Of the seven polymorphisms identified, six were silent nucleotide changes: G285T (Pro95Pro), G351A (Glu117Glu), A366G (Gln122Gln), G810A (Gly270Gly), T2307C (Ser769Ser), and C3018G (Ser1006Ser). An A1952G nucleotide change resulted in a histidine to an arginine amino acid change (His651Arg). The A1952G nucleotide change was present in 32 of 70 (46%) of the chromosomes studied.
Integrin 7 Alternatively Spliced Isoform
A unique conformer was detected with primer set 2607F-2868R in a single patient. Sequencing showed an in-frame deletion mutation starting at nucleotide position 2641 to nucleotide position 2712 of the integrin 7 coding sequence (Figure 3)
. The heterozygous in-frame deletion encompassed 72 bp in exon 21. This in-frame deletion detected at the cDNA level was absent at the genomic DNA level suggesting that the recognition of a cryptic splice site within exon 21 may be responsible for the skipping of about 40% of exon 21 directly to exon 22. To further characterize this in-frame deletion we performed SSCP on exon 21 in genomic DNA from the patient and from 60 controls. Identical conformers were detected in the patient and in 14 of 120 chromosomes studied (12%). Sequence analysis of the conformers showed a G to A substitution at position 2644 in cDNA (Figure 3)
. The G2644A base change resulted in a glutamic acid to lysine amino acid change (E822K) and it caused a gain of MseI restriction digestion site. Appropriate restriction digestion and RFLP analysis confirmed the mutation in the genomic DNA of the patient.
|
. When the score of the cryptic donor sequence was calculated using an algorithm based on the matrix compiled by Shapiro and Senapathy,35
the cryptic donor splice site AG/GTTA2644AG) scored 71.0 where the wild-type donor sequence (TG2712/GTGAGG) obtained 84.3 (accordingly to a Splice Site Score Calculator program supported by Genet. sickkids.on.ca).
|
7 Mutation
One of the 30 patients studied showed a heterozygous integrin 7 mutation. Patient E.S. showed a unique conformer with primer set 1850F-2083R (Figure 4)
. Direct sequencing of the aberrant conformers showed an heterozygous C to G nucleotide change at position 1969 in this patient (Figure 5)
. The C1969G nucleotide change resulted in a arginine to glycine amino acid change at position 657 of the integrin 7 protein. The Arg657Gly is located in the region containing the major ADP-ribosylation site. The C1969G nucleotide change resulted in a loss of a SmaI restriction-enzyme site. To verify the frequency of this mutation in the control population, integrin 7 exon 15 was PCR-amplified, and the PCR products were digested with SmaI. Digestion fragments were present in all of 200 control chromosomes but only in one allele of the patient’s DNA (heterozygous mutation) (Figure 5
, bottom).
|
7B Gene Expression Studies
The same 30 patients used for integrin 7 mutation studies were selected for RNA studies. Ten muscle biopsies with myopathic histopathology but with normal integrin 7B immunofluorescence were chosen as controls.
RNA was isolated from patients’ muscle biopsies and 100 ng of total RNA was reverse transcribed into cDNA using oligo dT10
primers. Approximately 20 ng of total cDNA was amplified using both integrin 7 and -sarcoglycan primers. Quantitative multiplex fluorescent RT-PCR (QMF-RT/PCR) products (22 cycles) were done in duplicate for each sample and electrophoresed on an ABI automatic sequencer (373A) to quantitate signals using peak area. Integrin 7 versus -sarcoglycan RNA ratios were determined. The patients’ muscle biopsies showed a normal amount of total integrin 7 RNA relative to controls in their muscles (Figure 6)
. Mean integrin 7 versus -sarcoglycan ratios were 24.2 ± 10 in the controls and 24.3 ± 11 in the patients’ group. Student’s t-test did not show any significant difference between patients’ and controls’ integrin 7 RNA levels (P = 0.8).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
7 candidate protein and gene analysis in our large cohort of muscular dystrophy/myopathy patients of unknown etiology. This approach was chosen since candidate protein and gene studies have shown impressive progress in dissecting the heterogeneous limb-girdle dystrophy group. To date, dystrophies caused by -, ß-, and 
-sarcoglycan have all been defined via candidate gene and protein approaches.36-40
The cloning of the integrin 7 gene,31
the preferential expression of integrin 7 in skeletal muscle,41-43
and the identification of causative integrin 7 mutations in a subset of Japanese patients presenting with congenital myopathy31
made integrin 7 an ideal candidate gene for screening a large bank of patient muscle biopsies. Candidate protein and gene analyses were conducted by using integrin 7 immunofluorescence in 210 muscle biopsies of patients affected with muscular dystrophy or myopathy of unknown etiology and followed by direct mutation screening of muscle biopsy RNA in 30 patients in which integrin 7 deficiency was identified. No integrin 7 mutations were identified in 29 of 30 patients showing various degrees of integrin 7 deficiency ranging from complete to slight deficiency. Only in one patient presenting at birth with CMD and showing, by immunofluorescence, a marked reduction of integrin 7 in his muscle biopsy, an heterozygous C1969G missense mutation resulting in an arginine to a glycine amino acid change (Arg657Gly) was identified. Despite our efforts, we were unable to identify in this patient a second mutation, consistent with the presumed recessive inheritance observed in CMD. We feel that the C1969G nucleotide change may be a causative mutation based on several lines of evidence. First, the C1969G results in an arginine to a glycine amino acid change (Arg657Gly). Arginine is a large, charged amino acid with a molecular weight of 174, whereas glycine is a small, (molecular weight 75) uncharged, polar amino acid. It is unlikely that such a dramatic amino acid change would have no functional consequences in an heterodimeric protein. Second, the C1969G mutation was not observed in 200 control chromosomes and it is located in a region of documented functional importance. The Arg657Gly mutation lies in the region containing the major ADP-ribosylation site44
that it is likely involved in the mechanisms modulating 7 integrin-mediated signaling pathways.
Several integrin 7 polymorphisms were also identified. In particular, we report a novel alternatively spliced integrin 7 isoform, generated from the use of a cryptic splice site in exon 21 (AG/GTTA2644AG), that was derived from a G2644A nucleotide change. The use of this cryptic splice site resulted in the skipping of about 40% of exon 21 directly to exon 22. This splice variant is present in about 12% of the chromosomes studied. A Senpathy score of at least 70 is required for donor splice site recognition.45
The AG/GTTA2644AG cryptic donor site sequence scored only 71, thus the splicing theory would predict that the AG/GTTA2644AG sequence would be a weak donor site. Since both normal and cryptic mRNA are observed, both splice sites are used. However, the ratio between the normal and cryptic RNA favors the normal transcript (Figure 3)
, suggesting that the utilization of the cryptic AG/GTTA2644AG splice is incomplete. It is difficult to predict the functional importance of this alternatively spliced integrin 7 isoform that removes 27 amino acids at a region where a major ADP-ribosylation site and a potential integrin leucyl-aspartyl-valine (LDV) binding site have been located.26
While it is possible that the spliced variant may act as a modifying factor in a predisposing genetic background, it is also possible that it is without any functional and/or structural importance. Among the several integrin 7 isoforms previously reported,5,20,23-28
the 7D, resulting from a partial deletion of exon 15 and the entire exon 16, is particularly interesting encompassing the major ADP-ribosylation site.23,26
Unfortunately, the exact role of ADP-ribosylation on 7 integrins remains to be determined,26
thus the potential functions of these isoforms are still largely unknown.
It has been reported that the expression of the cytoplasmic splice variant 7B is developmentally regulated, and that it is detectable in skeletal muscle only after the age of 2 years.30
Since we used an antibody against the integrin 7B variant, we checked if the integrin 7 deficiency was related to the age of the patients at the time of the biopsy. Indeed, 39% (11 of 28) of the muscle biopsies of patients younger than 2 years compared to 13% (24 of 182) of patients older than 2 years showed integrin 7 deficiency. Even if this difference is significant, we feel that factors other than developmental stage may affect the sarcolemmal expression of the 7B integrin subunit in diseased skeletal muscle, as suggested by Cohn et al.30
In the only patient where consecutive muscle biopsies were available, the integrin 7 deficiency detected in the muscle biopsy performed at 1 year of age was not rescued in the subsequent muscle biopsies done at 2 and 6.3 years of age. Moreover, 60% (17 of 28) of patients younger than 2 years showed normal integrin 7 immunostaining. The positive integrin 7 immunostaining detected in patients younger than 2 years of age may also be interpreted as some yet undefined compensatory mechanisms acting in dystrophic/myopathic muscle as suggested by the expression of integrin 7B in DMD and in the hypertrophic muscle fibers in SMA (spinal muscular atrophy) patients younger than 2 years.30
To better investigate the discrepancy between the integrin 7 protein deficiency detected through immunofluorescence analysis of patients’ muscle biopsies and the lack of identification of integrin 7 gene mutations, we conducted a series of experiments.
Our inability to detect integrin 7 gene mutation in integrin 7-deficient muscle samples is not likely due to technical reasons. We cannot rule out that mutations in the promotor region or in some intronic sequences that we did not analyze, underlie the observed integrin 7 deficiency. However, we feel that our mutation detection system is quite sensitive (we calculated about 93% sensitivity in a previous study).46
Since laminin 2 deficiency, both partial or total, is a well-known cause of secondary integrin 7 deficiency,11,12,30
all of the 210 muscle biopsies included in this study were checked by laminin 2 immunofluorescence, and all were normal.
To determine whether the decreased accumulation of integrin 7 protein might be the result of protein instability or due to the lack of integrin 7 gene expression or RNA processing, we quantitated total RNA from muscle biopsies of integrin 7-deficient patients. QMF-RT/PCR has been shown to have a relatively low experimental error, and has the additional advantage of using internal control RNA for each test.47
Our results showed normal integrin 7 mRNA levels in the patients studied suggesting that the loss of integrin 7 in their muscle biopsies occurs at the protein level. It is possible that the observed secondary integrin 7 deficiency may be the result of an incorrect processing and/or location of a normally synthesized integrin 7 protein in the patients’ muscles. The cytoplasmic integrin 7-positive immunostaining observed in some of our patients’ muscle biopsies further support the hypothesis of a integrin 7 moiety that is properly produced but is mislocated or unstable at the membrane level in absence, for example, of some yet unknown ligand. Quantitation of integrin 7, such as by immunoblotting analysis, may be informative, but unfortunately the small size of our diagnostic biopsies is a limiting factor. The biopsies are not sufficient to obtain a membrane-enriched pellet, and this limitation did not allow us to perform such an analysis in this study.
Taken together, our results suggest that secondary integrin 7 deficiency is rather common in muscular dystrophy/myopathy of unknown etiology. This is not surprising considering the central role played by 7ß1D integrin in anchoring the intracellular cytoskeleton via actin, to the extracellular matrix, via laminin, and the potential for signal transduction possibly mediated by extracellular matrix, soluble growth factors, and/or associated transmembrane molecules. "Inside-out" and "inside-in" signals can mutually modulate the activation of cell proliferation and migration, and if this signaling is malfunctioning, the lack of appropriate cell matrix interaction can lead to cell death via apoptosis.48
Analyses of mutant phenotypes provide further evidence of the key role of integrins in adhesion-mediated events in vertebrates. Germ-line mutations in genes coding for other laminin binding integrin result in disease. For example, mutations in either the 6 or the ß4 subunit of the epithelial hemidesmosomes integrin result in a variant of the usually lethal skin blistering disorder, epidermolysis bullosa. 3 integrin in epithelia plays a direct role in the assembly of the basement membrane as shown by the 3-null mice that show basement membrane abnormalities in the kidney, lung, and skin and die soon after birth.48
Further work is necessary to obtain more insight into the role of integrin-mediated adhesion and signaling and their modulating factors.
|
Footnotes |
|---|
Supported by Italian Telethon grant 1114.
Accepted for publication March 13, 2002.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
7ß1: functional characterization and expression in normal and malignant melanocytes. Cell Regul 1991, 2:805-817[Medline]
6ß1 for binding to the E8 and T8 fragments of laminin. J Biol Chem 1991, 266:23593-236017 is a novel integrin chain that is developmentally regulated during skeletal myogenesis. J Cell Biol 1992, 117:643-6577ß1 integrin in muscle development and disease. Cell Tissue Res 1999, 296:183-190[Medline]
1, 7A, and 7B integrins with the neuromuscular junction. Dev Biol 1996, 174:125-139[Medline]
7 integrin mediates cell adhesion and migration on specific laminin isoforms. J Biol Chem 1996, 271:25598-256037ß1 integrin in human and murine muscular dystrophies. J Cell Sci 1997, 110:2873-2881[Abstract]
7ß1) in muscle function and survival: disrupted expression in merosin-deficient congenital muscular dystrophy. J Clin Invest 1997, 100:1870-1881[Medline]
-actinin and the ß1 integrin subunit in vitro. J Cell Biol 1990, 111:721-7297 integrin cytoplasmic domains during skeletal muscle development: alternate forms, conformational change, and homologies with serine/threonine kinases and tyrosine phosphatases. J Cell Sci 1993, 106:1139-1152[Abstract]
7ß1 integrin is a component of the myotendineous junction on skeletal muscle. J Cell Sci 1993, 106:579-590[Abstract]
7 integrin gene (ITGA7) on human chromosome 12q13: clustering of integrin and Hox genes implies parallel evolution of these gene families. Genomics 1995, 26:568-570[Medline]
7 gene (ITGA7). Biochem Biophys Res Commun 1999, 260:357-364[Medline]
7 subunit are differentially expressed during development. J Biol Chem 1993, 268:26773-267837 integrin receptor is defined by developmentally regulated splicing in the extracellular domain. Mol Biol Cell 1997, 8:1723-1734[Abstract]
7 subunit generated by alternative intron splicing. Biochem Biophys Res Commun 1998, 243:317-325[Medline]
7ß1 is developmentally regulated in skeletal muscle. J Biol Chem 1993, 268:19019-190247 integrin and desmin define the primary and secondary myogenic lineages. Dev Biol 1993, 156:202-229
7 causes a novel form of muscular dystrophy. Nat Genet 1997, 17:318-323[Medline]
7B integrin in laminin 2-deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle. J Neurol Sci 1999, 163:140-152[Medline]
7 gene cause congenital myopathy. Nat Genet 1998, 19:94-97[Medline]
2 muscular dystrophy: genotype/phenotype study in 22 patients. Neurology 1998, 51:101-110
-sarcoglycan in chromosome 13 muscular dystrophy. Science 1995, 270:819-822-sarcoglycan gene. Nat Genet 1996, 14:195-198[Medline]
7 integrin. J Biol Chem 1996, 271:2071-20757 integrin receptor in differentiated smooth muscle cells. J Cell Sci 1997, 110:1477-1487[Abstract]
7 in skeletal muscle myotubes. J Biol Chem 1995, 270:9227-9233This article has been cited by other articles:
 |
|
 |
|
  M. D. Boppart, S. E. Volker, N. Alexander, D. J. Burkin, and S. J. Kaufman Exercise promotes {alpha}7 integrin gene transcription and protection of skeletal muscle Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2008; 295(5): R1623 - R1630. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Peat, J. M. Smith, A. G. Compton, N. L. Baker, R. A. Pace, D. J. Burkin, S. J. Kaufman, S. R. Lamande, and K. N. North Diagnosis and etiology of congenital muscular dystrophy Neurology, July 29, 2008; 71(5): 312 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liu, D. J. Burkin, and S. J. Kaufman Increasing {alpha}7{beta}1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression Am J Physiol Cell Physiol, February 1, 2008; 294(2): C627 - C640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Gheyara, A. Vallejo-Illarramendi, K. Zang, L. Mei, R. St.-Arnaud, S. Dedhar, and L. F. Reichardt Deletion of Integrin-Linked Kinase from Skeletal Muscles of Mice Resembles Muscular Dystrophy Due to {alpha}7 1-Integrin Deficiency Am. J. Pathol., December 1, 2007; 171(6): 1966 - 1977. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Jethanandani and R. H. Kramer {alpha}7 Integrin Expression Is Negatively Regulated by {delta}EF1 during Skeletal Myogenesis J. Biol. Chem., October 28, 2005; 280(43): 36037 - 36046. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yonekawa and J. M. Harlan Targeting leukocyte integrins in human diseases J. Leukoc. Biol., February 1, 2005; 77(2): 129 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Burkin, G. Q. Wallace, D. J. Milner, E. J. Chaney, J. A. Mulligan, and S. J. Kaufman Transgenic Expression of {alpha}7{beta}1 Integrin Maintains Muscle Integrity, Increases Regenerative Capacity, Promotes Hypertrophy, and Reduces Cardiomyopathy in Dystrophic Mice Am. J. Pathol., January 1, 2005; 166(1): 253 - 263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Koch, J. Schulze, U. Hansen, T. Ashwodt, D. R. Keene, W. J. Brunken, R. E. Burgeson, P. Bruckner, and L. Bruckner-Tuderman A Novel Marker of Tissue Junctions, Collagen XXII J. Biol. Chem., May 21, 2004; 279(21): 22514 - 22521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Inlow and L. L. Restifo Molecular and Comparative Genetics of Mental Retardation Genetics, February 1, 2004; 166(2): 835 - 881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xiao, P. Jethanandani, B. L. Ziober, and R. H. Kramer Regulation of {alpha}7 Integrin Expression during Muscle Differentiation J. Biol. Chem., December 12, 2003; 278(50): 49780 - 49788. [Abstract] [Full Text] [PDF]
|
|
2641–2712) of the integrin 
