Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation.

BACKGROUND: Alport syndrome (AS) is a clinically and genetically heterogeneous renal disorder, predominantly affecting the type IV collagen alpha 3/alpha 4/alpha 5 network of the glomerular basement membrane (GBM). AS can be caused by mutations in any of the three genes encoding these type IV collagen chains. The majority of AS families (85%) are X-linked (XL-AS) involving mutations in the COL4A5 gene. Mutations in the COL4A3 and COL4A4 genes cause autosomal recessive AS (AR-AS), accounting for approximately 14% of the cases. Recently, autosomal dominant AS (AD-AS) was linked to the COL4A3/COL4A4 locus in a large family. METHODS: COL4A3 and COL4A4 cDNAs were generated by nested reverse transcription-polymerase chain reaction and were analyzed by DNA sequence analysis. Denaturating high-performance liquid chromatography (DHPLC) was used for mutation and segregation analysis at the genomic DNA level. RESULTS: In the AD-AS family, a splice site mutation resulting in skipping of exon 21 of the COL4A3 gene was detected. The mutation does not alter the reading frame and is predicted to result in a COL4A3 chain with an internal deletion. CONCLUSION: As the NC domain is intact, this chain may be incorporated and distort the collagen triple helix, thereby causing the dominant effect of the mutation. The finding of a specific COL4A3 mutation in AD-AS completes the spectrum of type IV collagen mutations in all genetic forms of AS.

Identification of COL4A5 defects in Alport's syndrome by immunohistochemistry of skin.

BACKGROUND: The COL4A3-COL4A4-COL4A5 network in the glomerular basement membrane is affected in the inherited renal disorder Alport's syndrome (AS). Approximately 85% of the AS patients are expected to carry a mutation in the X-chromosomal COL4A5 gene and 15% in the autosomal COL4A3 and COL4A4 genes. The COL4A5 chain is also present in the epidermal basement membrane (EBM). It is predicted that approximately 70% of the COL4A5 mutations prevent incorporation of this chain in basement membranes. METHODS: We investigated whether or not COL4A5 defects could be detected by immunohistochemical analysis of the EBM. Punch skin biopsies were obtained from 22 patients out of 17 families and two biopsy specimens from healthy males were used as controls. RESULTS: In four cases with the COL4A5 frameshift or missense mutations, the COL4A5 chain was either lacking from the EBM (male) or showed a focally negative pattern (female). In three other patients with a COL4A5 missense mutation, a COL4A3 and a COL4A4 mutation, respectively, the COL4A5 staining was normal. A (focally) negative EBM-COL4A5 staining was found in three patients of six families with a diagnosis of AS and in one family of a group of four families with possible AS. CONCLUSIONS: The (focal) absence of COL4A5 in the EBM of skin biopsy specimens can be used for fast identification of COL4A5 defects. Combined with polymorphic COL4A5 markers, both postnatal and prenatal DNA diagnosis are possible in the family of the patient.

Differentiation of smooth muscle cells in human blood vessels as defined by smoothelin, a novel marker for the contractile phenotype.

Smoothelin is a constituent of the cytoskeleton specific for smooth muscle cells (SMCs) in a broad range of species. It has been postulated that smoothelin represents a marker of highly differentiated, contractile SMCs. Here, we present data on the presence of smoothelin in the human vascular system that support this hypothesis. For this purpose, smoothelin distribution was studied (1) during vasculogenesis of the placenta, (2) in normal adult blood vessels, and (3) in atherosclerotic lesions. Smoothelin was first observed in placental tissue at approximately week 10 to 11 of gestation. In full-term placenta, it was found in the SMCs of vessels in the large stem villi and in the chorionic plate. Furthermore, it was present in the fetal arteries of smaller stem villi, but it was not found in the veins. In adult blood vessels, a small population of aortic (approximately 10%) and large muscular artery (approximately 30% to 50%) SMCs was positive for smoothelin. In general, smoothelin and desmin were coexpressed in the same SMCs, but expression of desmin appeared to be less abundant. However, the majority of SMCs in these blood vessels were smoothelin- and desmin negative but expressed vimentin, whereas alpha-smooth muscle actin (alpha-SMA) was present in all SMCs. The SMCs in the media of small muscular arteries were positive for smoothelin and desmin (> 95%), whereas the vimentin-positive SMC type was scarce. Smoothelin was absent in capillaries, pericytic venules, and small veins but was occasionally observed in the SMCs of large veins. Thus, the distribution of smoothelin in the SMCs of the vascular system appears to be limited to blood vessels that are capable of pulsatile contraction. In atherosclerotic femoral arteries, smoothelin-positive cells were detected in the media, the atheromatous plaque, and the intimal thickening. Smoothelin-positive cells were present primarily at the luminal portion of advanced lesions. The presence of a considerable number of such smoothelin-positive cells at that location may indicate that these plaques are no longer expanding.

Smoothelin expression characteristics: development of a smooth muscle cell in vitro system and identification of a vascular variant.

Recently we described a protein, smoothelin, that has been exclusively found in smooth muscle cells (SMC). The human cDNA has been cloned from a colon cDNA library and the putative protein sequence was deduced. Smoothelin does not belong to a known protein family but shows a partial homology with members of the spectrin family. Transfection studies revealed that smoothelin has an affinity for actin and is either capable of forming filamentous structures or colocalizes with such structures. The protein is expressed in visceral as well as vascular tissues of all vertebrate classes. A study on the distribution of smoothelin in the vascular and placental system showed that smoothelin expression was largely restricted to the muscular pulsating blood vessels. Therefore, we hypothesized that smoothelin is expressed in contractile SMC only (36, 37). No expression of smoothelin was observed in established cell lines of SMC. In tissue explants smoothelin mRNA concentration decreases to undetectable levels within 12 hours after dissection as was in general the case in primary cell cultures. Here we report on continued smoothelin expression for several passages observed in a human prostate primary cell culture system. Smoothelin was demonstrated to colocalize with actin stress fibers but not with desmin filaments. This culture system offers opportunities to study the cytological localization of smoothelin, interactions with other proteins and should provide a system to test the promoter of the smoothelin gene. On immunoblots the molecular weight of smoothelin differed between visceral and vascular smooth muscle tissue with apparent molecular weights of respectively 59 kDa and 94 kDa. There is no evidence for the existence of another gene coding for the 94 kDa smoothelin. Thus, posttranslational modification, alternative splicing and dual promoter control are the alternatives for the expression of two isoforms of smoothelin.

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

The characterization of a novel 59-kD cytoskeletal protein is described. It is exclusively observed in smooth muscle cells by Northern blotting and immunohistochemical analysis and therefore designated "smoothelin." A human smooth muscle cDNA library was screened with the monoclonal antibody R4A, and a full-size cDNA of the protein was selected. The cDNA was sequenced and appeared to contain a 1,113-bp open reading frame. Based on the cDNA sequence, the calculated molecular weight of the polypeptide was 40 kD and it was demonstrated to contain two N-glycosylation sites. Computer assisted analysis at the protein level revealed a 56-amino acid domain with homologies of approximately 40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, beta-spectrin and alpha-actinin. In situ hybridization demonstrated that human smoothelin is encoded by a single copy gene which is located on chromosome 22. Immunohistochemistry and Western blotting revealed synthesis of smoothelin in smooth muscle of species evolutionarily as far apart as human and teleost. Northern blotting indicated that sequence as well as size of the mRNA (approximately 1,500 bases) are conserved among vertebrates. Cell fractionation studies and differential centrifugation showed that the protein cannot be extracted with Triton X-100, which indicates that it is a part of the cytoskeleton. Transfection of the human cDNA into smooth muscle cells and COS7 cells produced a protein of 59 kD, which assembled into a filamentous network. However, in rat heart-derived myoblasts association with stress fibers was most prominent. Smoothelin was not detected in primary or long term smooth muscle cell cultures. Also, transcription of smoothelin mRNA was almost instantly halted in smooth muscle tissue explants. We conclude that smoothelin is a new cytoskeletal protein that is only found in contractile smooth muscle cells and does not belong to one of the classes of structural proteins presently known.

Integration of titin into the sarcomeres of cultured differentiating human skeletal muscle cells.

Titin is amongst the first sarcomeric proteins to be detected in the process of myofibrillogenesis of striated muscle. During embryogenesis this high molecular weight protein is initially observed in a punctate staining pattern in immunohistochemical studies, while during maturation titin organizes into a cross-striated pattern. The dynamic process of titin assembly up to its integration into the sarcomeres of cultured human skeletal muscle cells has been studied in subsequent stages of differentiation with antibodies to four well-defined titin epitopes. Since in maturated muscle cells these epitopes are clearly distinguishable on the extended titin molecule we wondered how these epitopes reorganize during myofibrillogenesis, and whether such a reorganization would reveal important clues about its supramolecular organization during development. Immunofluorescence staining of postmitotic mononuclear myoblasts indicate that the investigated epitopes of the titin molecule are displayed in a punctate pattern with neighboring, but clearly separate spots in the cytoplasm of the cells. During elongation and fusion of the cells, these titin spots associate with stress fiber-like structures to finally reach their position at either the Z-line, the A-I junction or the A-band. We propose that during this transition the large titin molecule is unfolded, with the amino terminus of the molecule migrating in the direction of the Z-line and the carboxy terminus moving towards the M-line. In maturated, fused myotubes the final cross-striated patterns of all investigated titin epitopes are observed. While this process of unfolding of the titin molecule progresses, other compounds of the Z-line and the A-band migrate to their specific positions in the nascent sarcomere. A-band components such as sarcomeric myosin and C-protein, are also observed as dot-like aggregates during initial stages of muscle cell differentiation and organize into a cross-striated pattern in the sarcomere virtually simultaneously with titin. The Z-line associated component desmin organizes into a cross-striated pattern at a later stage.

Titin expression as an early indication of heart and skeletal muscle differentiation in vitro. Developmental re-organisation in relation to cytoskeletal constituents.

Established myogenic cell lines of different species and tissue origin have been used to study expression and organisation of muscle-specific proteins during differentiation. Furthermore, primary cultures of rat myocard cells were used to examine these same processes during dedifferentiation. In particular, we were interested in the general mechanism that underlies the changes in the supramolecular organisation of titin during in vitro myogenesis. It became obvious that in the differentiating muscle cell cultures the redistribution of desmin, actin and myosin in a typical, differentiation state dependent fashion, always showed a certain delay when compared to titin. The sequence of changes in the assembly of cytoskeletal and sarcomeric structures observed during differentiation of the cell lines was reversed during the process of dedifferentiation in cultured rat myocard cells. These results all indicate that titin is an early marker of myogenic differentiation, both in vivo and in vitro, and the typical reorganisation of this giant molecule is independent of species or muscle cell type.

Rearrangement of intercellular junctions and cytoskeletal proteins during rabbit myocardium development.

A direct and close association between desmosomes and intermediate-sized filaments of the keratin type exists in embryonic and in adult epithelial tissues. Cardiomyocytes are interconnected by spot-desmosomes, which are found in the intercalated disks and can be immunocytochemically detected by antibodies to desmoplakins. In this study, at the light microscopical level, we describe an interaction of keratin filaments with desmoplakins during rabbit myocardiogenesis. In the early stages (0-1 somites), desmoplakins are more abundant in the heart anlagen than in the adjacent intra- and extraembryonic mesoderm. During development of the myocardium, desmoplakin expression gradually rearranges from an apicolateral into an intercalated disk localization in later states. Keratin expression in the developing myocardium of the rabbit heart decreases with the age of the embryo. Keratin filaments are gradually lost via dot-like aggregates which colocalize with desmoplakin-positive clusters. Our results suggest a role for keratins in the developmental rearrangement of desmoplakins into the intercalated disks. A direct relation of desmin and titin reorganization to desmoplakin rearrangement, which was examined because of the dominant role of these proteins in cardiogenesis, was not found.

Cultured pig rhabdomyosarcoma cells with a deletion of the Xq24-qter chromosome region: an immunochemical and cytogenetic characterization.

A pig rhabdomyosarcoma cell line (PRUM59) was established, and the immuno(histo)chemical and cytogenetic characterization of these cells was determined. At various swine farms in the Netherlands, pigs were observed that had solitary or multiple skin nodules, which were diagnosed as rhabdomyosarcomas. Cells of a tumor derived from a 3.5-week-old female pig were cultured for immunochemical and cytogenetic analyses. The cell line had characteristic features of undifferentiated muscle cells, similar to those observed in tumor tissue sections; they contained titin, a high-molecular weight protein specific for striated muscle, as dot-like aggregates and as filaments, desmin filaments and cross-striations, smooth muscle actin stress fibers, and vimentin filaments. The cells stained positively for striated muscle actin and tropomyosin as well. The immunohistochemical staining results were supported by results of immunoblotting experiments. Karyotyping of the cells revealed a deletion of a major part of Xq24-qter, a part of the long arm of 1 of the 2 X chromosomes. The other X chromosome and all autosomes appeared to be normal.

Expression and organization of muscle specific proteins during the early developmental stages of the rabbit heart.

The expression and intracellular distribution patterns of muscle-specific proteins were studied during rabbit embryo development (7-13 dpc) using monoclonal antibodies against titin, myosin, tropomyosin and actin, as well as the intermediate filament proteins desmin, keratin and vimentin. From our panel, titin appeared to be the first muscle-specific protein to be exclusively expressed in the embryonic rabbit heart. Upon differentiation (myocyte and myotube formation), titin reorganizes from dot-like aggregates into a cross-striated pattern (in 9- to 30-somite embryos) via a transiently filamentous distribution. When the expression and organization of the other muscle proteins was studied in relation to titin, it became apparent that tropomyosin followed upon titin with respect to its exclusive expression in the heart anlagen and its organization into a striated pattern. Myosin and desmin were organized into cross-striated patterns after titin and tropomyosin, but this arrangement had not reached its final form in 13-dpc embryos. Actin, keratin and vimentin were distributed in cytoplasmic filaments in the embryonic stages we investigated. Since the first pulsations are already detected in 3-somite embryos, we conclude that the organization of titin, tropomyosin, myosin and desmin into a striated pattern does not seem to be essential for the initiation of muscle cell contraction in the heart anlagen. Furthermore, this study shows that, in comparison with studies on mouse, chick and rat, the sequence of expression of muscle-specific and intermediate filament proteins during cardiomyogenesis is species-dependent, and that their expression and organization varies in time in different regions of the developing heart.