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Neuromuscular Disorders 17 (2007) 587–596

Ullrich myopathy phenotype with secondary ColVI defectidentified by confocal imaging and electron microscopy analysis

Stefania Petrini a,*,1, Adele D’Amico a,1, Patrizio Sale b,c, Laura Lucarini d,Patrizia Sabatelli e, Alessandra Tessa a, Betti Giusti d, Margherita Verardo a,Rosalba Carrozzo a, Elisabetta Mattioli e, Marina Scarpelli f, Mon-Li Chu g,

Guglielmina Pepe d, Matteo Antonio Russo b,c, Enrico Bertini a

a Unit of Molecular Medicine, Department of Laboratory Medicine, Bambino Gesu Paediatric Hospital IRCCS, Rome, Italyb Department of Experimental Medicine and Pathology, University of Rome La Sapienza, Italy

c IRCCS San Raffaele, La Pisana, Roma, Italyd Department of Medical and Surgical Critical Care, University of Florence, Florence, Italy

e ITOI-CNR, Unit of Bologna c/o IOR, Bologna, Italyf Section of Pathological Anatomy and Histopathology, School of Medicine, Polytechnic University of the Marche Region, Ancona, Italy

g Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA

Received 3 January 2007; received in revised form 1 April 2007; accepted 22 April 2007

Abstract

Ullrich congenital muscular dystrophy (UCMD) is clinically characterized by muscle weakness, proximal contractures and distalhyperlaxity and morphologically branded by absence or reduction of collagen VI (ColVI), in muscle and in cultured fibroblasts. TheColVI defect is generally related to COL6 genes mutations, however UCDM patients without COL6 mutations have been recentlyreported, suggesting genetic heterogeneity. We report comparative morphological findings between a UCMD patient harboring ahomozygous COL6A2 mutation and a patient with a typical UCMD phenotype in which mutations in COL6 genes were excluded.The patient with no mutations in COL6 genes exhibited a partial ColVI defect, which was only detected close to the basal membraneof myofibers.

We describe how confocal microscopy and rotary-shadowing electron microscopy may be useful to identify a secondary ColVIdefect.� 2007 Elsevier B.V. All rights reserved.

Keywords: Confocal imaging; Rotary-shadowing electron microscopy; Ullrich congenital muscular dystrophy; Collagen VI defects

1. Introduction

Ullrich congenital muscular dystrophy (UCMD,OMIM 254090) is a congenital muscular dystrophy

0960-8966/$ - see front matter � 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.nmd.2007.04.010

* Corresponding author. Address: Molecular Medicine Unit,Department of Neurology, Bambino Gesu Paediatric Hospital IRCCS,P.zza S.Onofrio 4, 00165 Rome, Italy. Tel.: +39 6 68592105; fax: +39 668592024.

E-mail address: petrini.s@tiscalinet.it (S. Petrini).1 These authors contributed equally to this work.

(CMD) characterized by neonatal muscular weakness,joint contractures, distal hyperlaxity, protrudingcalcanei and normal intelligence. In many patients dis-tinctive cutaneous features are follicular hyperkeratosis,hyperhydrosis, and cheloid formation [1]. Histology ofthe muscle biopsy generally shows variation in fiber sizewith abundant endomysial connective tissue but rarephenomena of necrosis, and immunohistochemistrytypically shows reduction or absence of collagen VI(ColVI). Cultured fibroblasts show absent or reducedamount of ColVI and rotary-shadowing electron

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microscopy (REM) is able to show abnormal network ofsecreted ColVI microfibrils [2–6]. ColVI is an ubiquitousextracellular matrix protein made up of three distinctsubunits, a1(VI), a2(VI), and a3(VI) encoded by threedifferent genes COL6A1 (21q22.3), COL6A2 (22q23.3),and COL6A3 (2q37) [7]. Each subunit contains arelatively short triple-helical domain and N- andC-terminal globular regions, and ColVI monomersassemble into dimers, tetramers, and a final beadedmicrofibril structure [8].

The inheritance of UCMDs is generally autosomalrecessive and recessive homozygous or compoundheterozygous mutations are more frequently found inCOL6A2 and COL6A3 genes [9,10]. However,dominant heterozygous in-frame deletions or splice-sitemutations have also been reported as possible cause ofthe disease [9–11]. Moreover, in the last few yearspatients with a UCMD phenotype but withoutmutations in COL6 genes have also been described[12], and recently a new form of autosomal recessivecongenital muscular dystrophy with hyperlaxity,without ColVI defect, has been associated with a newlocus on chromosome 3p23-21.3 [13].

Here, we report detailed comparative morphologicalfindings between a case of UCMD with no mutationsin the COL6 genes, and a mutated UCMD patientcross-matched by age. In both patients muscle biopsiesshowed a partial reduction of ColVI immunostainingwith reduced ColVI–collagen IV co-localization, andelectron microscopy demonstrated loss of associationof microfibrils with the basal lamina. However, confocalanalysis of the muscle biopsy and REM of culturedfibroblasts were very helpful to differentiate between aprimary and a secondary deficiency of collagen VI.

2. Patients and methods

2.1. Patients

The index case is patient A. Disease control musclesamples were selected from the following: a previousreported UCMD patient with homozygous missensemutation in the COL6A2 gene (patient D) [14], and 12muscle samples of patients with different muscledystrophies (2 DMD, 2 BMD, 2 MDC1A, 2c-sarcogly-canopathy, 1a-dystroglycan deficiency, 1 LGMDIA, 1caveolin-3 deficiency, and 1 calpain defect). All caseswere genetically confirmed.

2.2. Muscle biopsy

Muscle biopsy of patient A was performed onquadriceps muscle using standard techniques. Frozenmuscle sections were stained with standard histochemistryand immunohistochemistry. Immunohistochemicalstudies were performed with monoclonal antibodies

against ColVI (MAB1944, Chemicon), a-dystroglycan(Upstate Biotechnology, Waltham, MA), vonWillebrand factor (Novocastra, Newcastle, UK),dystrophin, b-dystroglycan and sarcoglycans (YLEM,Italy), tenascin (Sigma–Aldrich, St. Louis, Missouri),merosin, perlecan, fibronectin, and fibrillin-1(Chemicon), rabbit polyclonal antibodies against NG2and collagen IV (AB5320 and AB748, respectively, fromChemicon), nidogen (Calbiochem-Novabiochem Corp.,La Jolla, CA), decorin and biglycan (kindly providedby Dr. Larry W. Fisher), b1D (a generous gift of Dr.Eva Engvall), a7A and a7B integrins (kindly providedby Dr. Stephen J. Kaufman).

2.3. Skin biopsy and fibroblast cultures

Cultured fibroblasts were obtained from skin biopsyof patients D, A, and normal healthy subjects. Frozenskin sections were stained with standard immunohisto-chemistry using monoclonal antibodies against ColVI(MAB1944, Chemicon). Cells were grown in Dulbecco’smodified minimum essential medium (D-MEM)supplemented with 20% fetal calf serum (Life Technologies,Gaithersburgt, MD), penicillin and streptomycin.Confluent cultures were treated 5 days with 0.25 mMascorbic acid, fixed in cold methanol (�20 �C) andlabeled with anti-ColVI antibody (MAB1944, ChemiconInc., Temecula, CA,) diluted 1/50 followed by fluoresceinisothiocyanate (FITC) conjugated anti-mouse antibobydiluted 1/100 (DAKO, Glostrup, DK). After severalwashing with phosphate-buffer saline (PBS), thesamples were mounted with Pro-long anti-fade reagent(Molecular Probes, OR, USA) and observed with aNikon epifluorescence microscope.

2.4. TPE confocal microscopy analysis

Five micrometers muscle cryosections was co-labeledwith anti ColVI/collagen IV antibodies, respectively,and revealed by goat anti-mouse FITC and goatanti-rabbit TRITC antibodies (Chemicon). Imageacquisition was performed using a two-photon excitation(TPE) microscope built around a conventional confocallaser-scanning microscope Nikon-C1-Plus (NikonInstruments, Florence). TPE fluorescence of the dyeswas detected using a 750 nm Ti:sapphire ultrafast lasersource (Mai Tai Laser 750–850, Spectra Physics, CA)at approximately 6 mW of average power in the focalplane. The fluorescence signal, collected by the sameobjective and selected by a filter (HQ535-50, ChromaInc., Brattleboro, VT), is fed to a multimode fiber thatbrings the light to a photomultiplier (R928, Hamamatsu,Milan, Italy) in the C1 plus controller. The images ofsamples were acquired at (1024 * 1024 pixels) withresidence times in the range of 9.6 ms per pixel takesffi0.24s. and were analyzed with EZC1 software (Nikon

S. Petrini et al. / Neuromuscular Disorders 17 (2007) 587–596 589

Instruments Florence). To calculate the co-localizationwe used the software with the algorithm and thenumerical analysis with image J ver. 1.33u N.I.H. USA.

2.5. Electron microscopy analysis

Some fragments of muscle tissue from patient A andD and from controls were treated as reported [15]. Allsamples were dehydrated with ethanol and embeddedin Epon E812. Ultrathin sections were obtained fromseveral blocks, stained with lead citrate and uranylacetate. REM of fibroblast cultures was performed asdescribed previously [4], and the immunocytochemicalreaction was performed using an anti-mouse IgGsecondary antibody conjugated with 5 nm colloidal gold(Amersham Biosciences, UK). Epon embedded ultrathinsections and replicas obtained from rotary-shadowedfibroblast cultures were observed with a Philips EM400 transmission electron microscope operated at100 kV.

2.6. Genomic DNA analysis

Genomic DNA was extracted from whole blood ofpatient A and her parents in accordance to the localEthic Committees and informed consent was obtained.

Single exons were analyzed to characterize the nucle-otide variations found by sequencing the three COL6cDNAs. All the nucleotide variations were confirmedin the patient and were found among 100 controls bydHPLC, indicating that they are polymorphisms.

2.7. RT-PCR mutational analysis of the three COL6

cDNA and direct sequencing

Mutation screening analysis was performed by directsequencing of the entire coding regions of the threeCOL6 mRNAs (GenBank: COL6A1 – NM_001848;COL6A2 – NM_001849; COL6A3 – NM_004369).Total RNAs extracted from confluent fibroblasts(RNeasy-Qiagen) were treated with reverse transcrip-tase, and PCR amplified with specific oligonucleotidesas described [16], yielding 25 overlapping cDNAfragments. The PCR products were sequenced withforward and reverse primers used for the amplification.PCR analysis was carried out with denaturation at96 �C, annealing temperature between 58 �C and 66 �Cand extension at 72 �C for 60 s; all PCR amplifiedfragments were then subjected to direct sequencing. Thisanalysis allowed the detection of polymorphisms in thecoding sequences of the three genes.

2.8. DHPLC heteroduplex analysis

Denaturing high performance liquid chromatography(DHPLC) analysis was carried out on DNA PCR

amplified fragments from relatives and controls byan automated DHPLC instrument (WAVE DNAFragment Analysis System, Transgenomic, UK),equipped with a separation column (DNASep column,Transgenomic). Resulting chromatograms werecompared for variation in shape or retention time.

2.9. Northern blot analysis

Northern blot hybridization was performed using5 lg of total RNA prepared from confluent fibroblasts,separated on a 1% agarose gel containing formaldehydeand hybridized with [32P]dCTP-labeled cDNAs probesencoding the three COL6 chains, as reported [11].

2.10. Immunoprecipitation analysis

For immunoprecipitation of ColVI, confluentfibroblasts were cultured in the presence of 50 mg/mlL-ascorbic acid phosphate for 4 days. Fibroblasts werelabeled overnight with [35S]cysteine, and the resultingcells and culture medium were precipitated with apolyclonal antibody specific for the a3(VI) chain(antibody 1014) [2,17].

3. Results

3.1. Clinical history

Patient A. A 6-year-old girl, was the only child ofhealthy non-consanguineous parents with no familyhistory of neuromuscular diseases. Early motormilestones were within the normal range, and sheacquired autonomous walking at age 16 months,although she always had frequent falls, and difficultyin rising from the floor and going up stairs. When firstexamined at the age of 19 months she had axial and limbgirdle weakness and Gowers sign. She had marked jointlaxity of hands and feet and no contractures weredetected (Fig. 1B). Moreover, transitory dislocation ofher left elbow was reported. Skin examination showedmarked hyperlaxity with follicular hyperkeratosis.Serum CK levels were normal. At the age of 22months muscle biopsy was performed. The patient wasre-evaluated at the age of 5 years. She was able to walkindependently but had clear limb-girdle muscle weakness,and she was not able to stand up independently fromsitting. Skin hyperlaxity was persistent and she hadcheloid formation at the site of surgical biopsy(Fig. 1A). No proximal contractures were detected.Muscle MRI of their legs performed at the age of 6years, using T1 sequences through calves and thighs,showed a typical pattern of Ullrich disease [18] with adiffuse involvement of the thigh muscles and relativesparing of sartorius, gracilis, rectus femoris and of thecentral part of the vastus lateralis (Fig. 1C and D).

Fig. 1. (A and B) The cheloid formation at the site of surgical biopsy and the marked hyperlaxity of the fingers. Muscle MRI shows thediffuse involvement of the thigh muscles with relative sparing of sartorius, gracils, rectus femoris and of the central part of the vastus lateralis(C and D).

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Patient D. The clinical history of this patient hasalready been reported [14]. This UCMD patient is now7 years old; she is still able to walk independently butis not able to stand up alone from sitting. She has ahomozygous missense mutation c. 2624 C > A, R876Sin the COL6A2 gene and parents are healthy secondcousins, and are both asymptomatic heterozygouscarriers for the mutation. The muscle biopsy wasperformed at the age of 2 years.

3.2. Muscle biopsy

Muscle biopsy of patient A showed hypervariationof fiber size, several internal nuclei, type I fiberpredominance, rare necrotic and regenerative processes,and increase of connective tissue. Immunohistochemis-try disclosed a normal staining for merosin, dystrophin,sarco-dystroglycan complex, perlecan, von Willebrandfactor, NG2 proteoglycan, and a7B integrin subunit(Fig. 2). ColVI staining appeared normally expressed;however, double labeling for ColVI and nidogenconfirmed the integrity of myofiber basal lamina butrevealed a discontinuous distribution of ColVIaround myofibers and blood vessels (Fig. 2f). Decorin,biglycan, tenascin, collagen III, fibronectin, andfibrillin-1 were overexpressed owing to fibrosis, whilethe expression of a7A and b1D integrin subunits wereboth severely and moderately reduced (Fig. 2j and l).

In patient D the labeling for ColVI was significantlyreduced around the basal membrane and the endomys-ium (Fig. 3J) and also the NG2 proteoglycan stainingwas clearly reduced in contrast to patient A (Fig. 2p).

3.3. Confocal analysis

In patient A, ColVI labeling was mildly reducedaround myofibers but normal in the endomysial areas(Fig. 3F). However, collagen IV–VI co-localizationwas significantly decreased in basal lamina as well asin several capillaries (Fig. 3H) respect to normal controlmuscle (Fig. 3D). In patient D, ColVI expression wasseverely reduced in the endomysium (Fig. 3J) andcollagen IV/VI co-localization was drastically alteredaround myofibers and blood vessels (Fig. 3L). Diseasecontrol muscle samples, which had various degrees offibrosis, showed a normal co-localization of collagensVI and IV in myofibers and vessels, as observed innormal muscles (Fig. 3D).

3.4. Ultrastructural analysis

In normal and disease control myofibers (Fig. 4A) thebasement membrane was visualized as continuous layerssurrounding the cell constituted by: (i) the lamina rara,an electron transparent layer, (ii) the lamina densa, afelt-like structure, and (iii) the reticular lamina, whichcontains collagens, including ColVI and other fibrils.The ultrastructural examination of muscle samples ofpatients D and A revealed several areas in which thedensity between the reticular lamina and the laminadensa was reduced (Fig. 4B and C). Focal loss of micro-fibrils was also detected in these areas. These alterationswere found in about 10% of muscle fibers of patient Aand, in most patient D myofibers, but were neverdetected in the other controls. The lamina densa and

Fig. 2. Immunofluorescence images acquired by using an epifluorescence microscope. The expression of ColVI (b and e) and several components ofbasal lamina (nidogen: a and d), sarcolemma (a7A: g and j; a7B: h and k; b1D: i, l; NG2: m and p) and extracellular matrix (decorin: n and q;biglycan: o and r) in UCMD patient A (d–f, j–l, and p–r) and control (a–c, g–i, and m–o) muscles. The merged image (c and f) of ColVI (green) andnidogen (red) showed the integrity of the basal lamina but a discontinuous ColVI labeling around several myofibers in patient A (f) was observed.a7A and b1D integrin immunoreaction was reduced (j and l), while a7B subunit and NG2 proteoglycan were unaltered (k and p). Decorin andbiglycan staining (q and r) showed an increased intensity owing to connective tissue increment. Bar: 50 lm.

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Fig. 3. TPE confocal microscopy of collagen IV and ColVI distribution and localization in unaffected control (A–D), patient A (E–H) and patient D(I–L) muscles. Muscle cryosections (5 lm) were co-labeled with anti-ColVI/collagen IV antibodies, and revealed by goat anti-mouse FITC and goatanti-rabbit TRITC conjugated antibodies. Confocal study showed a normal collagen IV expression (red) in the capillary and myofiber basal lamina incontrol (A), patient A (E), and D (I) muscles, whereas ColVI (green) staining was mildly reduced around most of myofibers in patient A (F), and itwas severely decrease in patient D muscle (J). The co-localization of two signals (red-green) and the merge signal (yellow) is showed in (C), (G), and(K), and the mask of co-localization is showed in (D), (H), and (L) (only co-localization points are reported in white). Collagen VI and IV co-localization was significantly reduced around myofibers and in some capillaries in UCMD patient A (H). A significantly defective co-localizationbetween ColVI and collagen IV was observed in UCMD patient D muscle (L). Bar: 30 lm.

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the plasma membrane of D and A patient’s myofiberswere well preserved.

3.5. Skin biopsy and cultured fibroblasts

Skin biopsy and cultured fibroblasts of patient Ashowed normal ColVI immunostaining (Fig. 4E), while inpatient D significant reduction of secreted ColVI in the

extracellular matrix and an intracellular accumulationof ColVI polypeptides was detected (Fig. 4F). Immunola-beled rotary-shadowed replicas of whole mountedcultured fibroblasts, did not reveal any considerablealteration of ColVI microfilaments or network abnor-mality in patient A compared to controls (Fig. 4H andG). In patient D the secreted ColVI microfilaments wasassembled in unusual large aggregates (Fig. 4I).

Fig. 4. Immunofluorescence and ultrastructural analyses of cultured fibroblasts and skeletal muscle from control (A, D, and G), and UCMD patientsA (B, E, and H) and D (C, F, and I). Control myofibers (A) showed that the basement membrane was well represented by continuous lamina layersand microfibrillar material of the reticular lamina normally leaned against the lamina densa. In patient’s A muscle (B), about 10% of myofibersshowed an electron-density reduction between reticular lamina and lamina densa with focal loss of microfibrils (arrows), as observed in most ofpatient’s D myofibers (C). (D–F) Immunofluorescence study of cultured fibroblasts treated 5 days with 0.25 mM ascorbic acid, fixed in cold methanoland labeled with anti-ColVI antibody. Normal amount of ColVI assembled in the extracellular matrix was observed in patient A cultured fibroblasts(E), and rotary-shadowed replicas analysis of whole mounted cultured fibroblasts (G–I) did not detected significant structural alterations of ColVImicrofilament network between control (G) and patient A (H) cells. Conversely, in patient D cultured fibroblasts (F and I), a decreased secretion andabnormal large aggregates of ColVI molecules were detected. Bars: 100 nm in (A–C), 200 nm in inset, 50 lm in (D–F) and 1.5 lm in (G–I).

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3.6. Genetic analyses and immunoprecipitation

No mutations were found in the three COL6 cDNAsof patient A (Fig. 5A). The analysis only yielded 11silent nucleotide polymorphisms and 4 single-nucleotidechanges that resulted in aminoacid substitutions (4 inCOL6A1, 5 in COL6A2, and 6 in COL6A3), some ofthem previously reported [11,19]. All of the nucleotidechanges were confirmed in the genomic DNA of thispatient and detected in controls of the Italianpopulation thus excluding pathogenetic significance. Inaddition we studied heterozygous single nucleotidepolymorphisms in the cDNA to exclude the presenceof large deletions or haploinsufficiency.

We also investigated the COL6 mRNA and proteinfrom patient’s A fibroblasts. Analysis of total RNA byNorthern blot hybridization showed that the COL6mRNA production for all three chains was comparableto control (Fig. 5B).

The protein synthesis was studied by immunoprecip-itation of metabolically labeled fibroblasts. The analysisshowed a normal amount of ColVI protein in patient Aculture medium compared to control (Fig. 5C, lanes 3

and 4) and to the reference protein (upper bands);further, the amount of ColVI protein in patient’s cellswas comparable to control (Fig. 5C, lanes 1 and 2). Asmaller amount of fibronectin was also detected inUCMD medium with respect to controls, as observedin fibroblasts from two other UCMD patients withknown collagen VI mutations (unpublished results,M.L. Chu).

4. Discussion

ColVI is a critical component of skeletal muscle,localized in the basement membrane zone likely throughits interaction with collagen IV [20,21]. Complete absenceor partial deficiency of ColVI is generally observed inUCMD patients [2,3,11,22,23] and mutations inCOL6A1, COL6A2, and COL6A3 have been reportedin most patients. Nevertheless COL6 gene mutationsare not found in a few subjects with a UCDMphenotype [12,24], and in one of these reported patients,it has been clearly demonstrated by biosynthetic andassembly in vitro studies, that the ColVI deficiency isnot the primary cause of the disease [12]. In a series of

Fig. 5. Genetic analyses and immunoprecipitation in UCMD patientA. (A) Polymorphisms in the three COL6 genes of UCMD patient A.The analysis only yielded 11 silent nucleotide polymorphisms and 4single-nucleotide changes that resulted in aminoacid substitutions (4 inCOL6A1, 5 in COL6A2, and 6 in COL6A3). All of the aminoacidsubstitutions were confirmed in the patient genomic DNA and detectedin controls thus demonstrating the non pathogenicity. (B) Northernblot analysis of total RNA (5 lg) from patient A and control confluentfibroblasts using [32P]dCTP-labeled cDNA probes. The probes hybrid-ized with the three COL6 mRNAs of 4.2, 3.5, and 9–10 kb,respectively, plus an alternatively spliced COL6A2 mRNA (COL6A2a)of 6.0 kb. (C) Immunoprecipitation of cell layer (left) and culturemedia (right) from control and patient A fibroblasts. Confluentfibroblasts were cultured in the presence of 50 lg/ml L-ascorbic acidphosphate for 4 days. Fibroblasts were labeled overnight with[35S]cysteine; then, resulting cells and culture medium were precipitatedwith a polyclonal antibody specific for the a3(VI) collagen chain(antibody 1014), so that only a1(VI) and a2(VI) chains that areassociated with the a3(VI) chain would be coimmunoprecipitated. Theanalysis showed a normal amount of ColVI protein in patient Aculture medium (lanes 3 and 4) and cells (lanes 1 and 2) compared tocontrol, and to the reference protein (upper bands).

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UCMD patients with no COL6 genes mutations, themorphological data are limited to the description ofreduced or absent immunostaining of ColVI insarcolemma with loss of association of microfibrils withthe basal lamina at the level of the reticular lamina [24].

Nevertheless, these abnormalities have also beenreported in UCMD mutated patients with partialdeficiency of ColVI [3,5]. Therefore, immunohisto-chemistry is never sufficient to discriminate betweenColVI-linked or unlinked UCMD, nor is it a geneticscreening test. Moreover, very recently, a new locus onchromosome 3p23-21 was mapped in some patients,from 11 French–Canadian families, affected by aUCMD-like phenotype but without immunohistochem-ical muscle ColVI defect [13]. Therefore, summarizing itappears that the UCMD phenotype is a geneticallyheterogeneous condition, in which muscle immunohisto-chemistry can show a ColVI deficiency both in patientsharbouring COL6 genes mutations, but also in otherpatients without detectable mutations in COL6 genesand with normal level of ColVI protein and mRNA.In addition some patients may show a UCMDphenotype and have no genetic and morphologicalevidence of a ColVI defect.

Our patient had a typical UCMD phenotype, with atypical pattern of muscle group degeneration by MRI.

Although immunohistochemistry already revealed amild ColVI deficiency at the level of the basal laminaof myofibers and blood vessels, confocal microscopyclearly confirmed the marked reduction in the ColVI/collagen IV co-localization only at the level of basallamina. Thus, confocal microscopy was more sensitivein detecting alterations than the simple epifluorescencemicroscopy. In patient D conversely, confocal analysisconfirmed the strongly defective co-localization of thetwo collagens in all skeletal muscle areas, includingbasal lamina, endomysium, and perimysium.

In patient A muscle ultrastructure was able to detectalterations in 10% of myofibers showing a focal loss ofmicrofibrils at the reticular lamina of the basal laminavery similar to the alterations detected in patient Dharbouring the homozygous missense mutation inCOL6A2, with a partial ColVI defect. These ultrastruc-tural aspects have been already described in myofibersof mutated and not mutated Ullrich patients [5,24],but were not detected in our 12 patients with other mus-cle dystrophies, and have never been detected in otherforms of CMD [24,25].

Furthermore, patient A showed a normal secretion,assembly and structure of the ColVI microfibril networkin the fibroblast culture detected by immunofluorescenceand REM. These findings have not been so far describedin UCMD patients with secondary ColVI deficiency,and they also distinguish our patient from the UCMDpatient described by Baker and co-workers, who showedunusual large aggregates in the cultured medium. On thecontrary, patient D fibroblasts showed a severe defect inColVI secretion and accumulation. We can confirm byour personal experience, and following previousobservations [3,5,6], that REM is always abnormal inUCMD fibroblasts from patients with COL6 genes

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mutations. Our genetic analysis demonstrated absenceof pathogenetic mutations in the three COL6 cDNAs,and the finding of heterozygous single nucleotidepolymorphisms in the cDNA excluded the presence oflarge deletions or haploinsufficiency. Additionally, wedemonstrated that ColVI was normal both at thetranscriptional and the protein level.

We can reasonably believe that ColVI mRNA levelsobserved in the fibroblast culture can reflect skeletalmuscle ColVI mRNA levels, as it has been shown thatunlike muscle interstitial fibroblasts, myogenic cells(i.e. myoblasts and myotubes) do not contribute to thedeposition of ColVI in the skeletal muscle [26].

To disclose the primary abnormality in patient A weinvestigated some proteins that potentially bind toColVI, such as biglycan, perlecan, a-dystroglycan, a7b1integrin complex subunits, decorin, fibrillin-1, and vonWillebrand factor. The immunostaining of theseproteins showed a normal pattern. Staining for thea7A and b1D subunits was reduced in the sarcolemmaof myofibers, but the study of the muscle-specific a7b1integrin heterodimer in several UCMD muscles revealeda variable pattern of expression that seems not related tothe degree of ColVI reduction [1,14]. The primary a7deficiency is a rare cause of muscular dystrophy, andso far only few cases have been demonstrated to carrymutations in the integrin a7 gene, albeit the presenceof a large number of patients with immunoistochemical-ly markedly reduced integrin a7 [27,28]. Furthermorethese patients, although not described to have a Ullrichphenotype, have not been reported in detail, and inaddition no immunocytochemical/electron microscopicfindings have been reported to allow comparison.

NG2 proteoglycan revealed a normal immunoreac-tion in the sarcolemma of patient A, conversely to whatwe observed in UCMD mutated muscles with totalor partial ColVI deficiency [14], in whom NG2 isabnormally reduced. By Western blotting, we furtherconfirmed the normal expression of NG2 core proteinlevels (not shown) in patient A. Thus, the normalexpression of NG2 may also be considered as anadditional marker that helps to differentiate forms ofUCMD without mutations in COL6 genes from theother mutated UCMD patients. In addition, byimmunofluorescence we excluded a reduction oftenascin. Moreover, we can indirectly exclude aTenascin-X (TNX) mutation because in TNX-nullfibroblasts the mRNA expression level of all ColVIchains has been found to be remarkably decreased [29].

Our data confirm the genetic heterogeneity of theUCMD phenotype. The characteristic abnormality withselective reduction of ColVI microfilaments at the levelof the reticular lamina of the basal membrane reinforcesthe idea that other proteins closely interacting withColVI may be implicated in the pathogenesis of UCMD.Finally, our findings suggest that an extensive morpho-

logical analysis supported by confocal imaging andREM of cultured fibroblasts, supported by biochemicalfindings are useful methods to predict UCMD patientswith a secondary collagen VI defect.

Acknowledgments

We wish to thank Dr. Eva Engvall for providinganti-b1D integrin antibody, Prof. Stephen J. Kaufmanfor the generous gift of a7A and a7B integrin antibodies,and Prof. Larry W. Fisher for providing antibodiesagainst decorin and biglycan. We thank PompilioStroppiccioli, Pietro Cirigliano Stefano Di Meo, SilvanoDe Pascalis and Michele Signore for their continuoussupport and advice. This work was supported by fundsfrom the Telethon Italy (Grant n� GGP04113), theAssociation Francaise contre les Myopathies (AFM,Grant n� 9398), the Italian Ministry for Universityand Research (FIRB Project n� RBNE01JJ45_005). Agrant from the Italian Ministry of Health on Muscledystrophy is also acknowledged.

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