Basal lamina genes affected in leiomyomatosis and congenital muscular dystrophy : structure and mutation analyses of the collagen COL4A6 and laminin LAMA2 genes

Basal laminae (basement membranes) are flexible thin sheets of specialized extracellular matrix separating organ cells from the underlying or surrounding connective tissues. They are found beneath epithelial and endothelial cells amd surrounding different cells, such as muscle fibers and nerves. Basal laminae are mainly composed of type IV collagen laminin, heparan sulfate proteoglycan, and nidogen that are specific for these structures. These proteins are not only important for forming the structural substrate for the cells positioned on the of basal laminae, but also have several other biological functions. For example, they promote cell differentiation and adhesion, serve as a specific highway for cell migration, determine cell polarity, and are involved in regeneration processes as well as in the filtration of body fluids. In addition, changes in the basal laminae can lead to numerous acquired and genetic diseases.

To facilitate studies on basal laminae and, particularly, their involvement in diseases such as Alport syndrome, leiomyomatosis, and also their possible role in congenital muscular dystrophy, the complete exon intron structure of the gene for the human type IV collagen a6 chain (COL4A6) and laminin a2 chain (LAMA2) were determined.

The COL4A6 was shown to span about 425 Kb and contain 46 exons, of which exon IB was found to be alternatively used (1). The COL4A6 characterized in this study is the largest collagen to be analyzed thus far. The large size of COL4A6 is mainly contributed to by a single 340 Kb second intron, while the sizes of other introns only range between 0.164 and 16 kb. Based on the exon-intron structure of COL4A6, the possible involvement of this gene in patients with Alport syndrome was investigated. Sequencing of four exons (exons 2, 12, 13, and 14) in 250 patients with Alport syndrome revealed no mutations, showing that mutations in the a6(1V) collagen chain gene are not likely to cause Alport syndrome (II). In contrast, 17 different mutations were found in the same study in ten exons in COL4A5, which is separated from COL4A6 by a 425 bp sequence.

These results and other studies that have described numerous mutations in all regions of COL4A5 gene supported our hypothesis that mutations in COL4A6 do not lead to Alport syndrome. Furthermore, the tissue distribution studies of the a5(1V) and a6(1V) chains using chain specific antibodies have demonstrated differences between the two chains (Ninomiya et al. 1995, Peissel et al. 1995, Hino et al. 1996). In fact, unlike the a3, a4 and a5, the a6(1V) chain has never been found in glomerular base membrane (GBM). Furthermore, in spite of the close location of the two genes, COL4A6 is transcribed from two alternative promoters dependent on in which tissues it is to be expressed (Sugimoto et al. 1994). These differences indicate that the fimction of COL4A6 differs from that of COL4A5. It has been shown that some deletions of COL4A6 could cause Alport syndrome associated leiomyomatosis in patients. This observation suggests that COL4A6 has a more important role in basal laminae of smooth muscle than in the GBM.

Congenital muscular dystrophies (CMDs) are a clinically and genetically heterogeneous group of autosomal recessive neuromuscular diseases. These diseases include a so-called classical congenital muscular dystrophy. Immunocytochemical analysis of the laminina2 chain (merosin chain) in skeletal muscle of some of these patients demonstrated absence of this chain (Tome et al. 1994). Homozygosity mapping and linkage analysis showed that the merosin-negative CMD locus was located on chromosome 6q2 (Hillaire et al. 1994), where the laminin a2 chain gene had been localized (Vuolteenaho et al. 1994). These data strongly made the laminin a2 chain gene a candidate for merosin-negative CMD. The complete exon-intron structure of LAMA2 described in this study (III) provided information essential for mutation screening in patients with CMD. The LAMA2 itself was shown to be over 260 Kb and to have 64 exons, two of which, exon 43 and 52, were extremely small or six and twelve base pairs, respectively.

The gene structure indicated that the LAMA2 was more closely related to the LAMA4 than to the LAMB and LAMC. The LAMB and LAMC are a family of genes coding for laminin B and y chains, respectively. The first two mutations identified in human CMD were identified in this thesis study (IV). One was an A to T transversion at the -2 position of consensus acceptor splice site of exon 31, resulting in the deletion of 192 bp and in a premature stop codon at the beginning of the domain II. The second one was a nonsense mutation changing a glutamine (CAA) to a TAA stop codon in domain IVa. Mutations in the LAMA2 gene have also been shown to cause muscular dystrophy in mice (Xu et al. 1994). To date, 16 different mutations have been reported in CMD patients based on the knowledge of the LAMA2 structure determined in the thesis work.

These mutations which all have been detected in later collaborative studies include small deletions, nonsense mutations, splice mutations, and single base mutations (IV, Nissinen et al. 1996, Allamand et al. 1997, Guicheney et al. 1997a, 1997b). In most cases, the mutations lead to premature stop codon and truncation of the a2 chain lacking domain 1/11, and G domain or part of the G domain. It has been shown that domains 1/11 of three laminin chains (a2,B1 and y 1 for laminin-2) are involved in a coiled coil formation of the longarm, while the G domain provides the binding sites to a dystroglycan. The attachment of muscle to extracellular matrix is important to protect muscle cell from stresses during muscle contraction. Dysfunction and/or lack of the laminin a2 chain certainly affects the integrity of the linkage between extracellular and cytoskeleton resulting in muscular dystrophy.

In conclusion, the studies of this thesis work have for the first time described the complete exon-intron structures of the type IV collagen a6 chain and the laminin a2 chain genes from any species. Secondly, they show that COL4A6 is not likely to be involved in AS, and finally, these studies demonstrate that congenital muscular dystrophy is caused by mutations in LAMA2. The results have provided new information on the role of basal laminae for muscle development and function, and in particular, new diagnostic methods for leiomyomatosis and congenital muscular dystrophy. The work may also lay the ground for gene therapy.