Alport syndrome (AS) is a progressive renal disease that is characterised by hematuria and progressive renal failure, and often accompanied by progressive high-tone sensorineural hearing loss and ocular changes in form of macular flecks and lenticonus. AS is a genetic heterogenous disease, and X-linked dominant in about 85% of the families. The autosomal recessive and dominant forms constitute about 15% of the cases. In the first part of the study is a multipoint linkage analysis of 12 families suspected of X-linked AS. The aim of that part of the study was to map a number of X-chromosomal polymorphic markers in relation to the locus for AS, in order to be able to perform carrier detection and prenatal diagnosis in the families. In addition, a more precise map of the region could form the basis for positional cloning of the gene for X-linked AS. In 1990 it was found that the X-linked form of AS is caused by mutation in the COL4A5 gene located at Xq22, and encoding the alpha 5-chain of type IV-collagen. The COL4A5 gene is a very large gene spanning 257 kb with a transcript of 6.5 kb distributed on 51 exons. In addition, two alternatively transcribed exons have been identified. In the second part of the study methods were set up for detection and characterisation of mutations in the COL4A5 gene in 135 patients suspected of AS. The aims of that part of the study were to develop an efficient and reliable approach for mutation detection, and to implement the results of the mutation analysis in clinical practice for carrier detection and prenatal diagnosis, in order to be able to offer a better genetic counselling to the families. Knowledge of a possible correlation between genotype and phenotype can be of help in predicting the prognosis. Samples from 135 probands suspected of AS and 359 of their relatives were collected, together with available clinical information. Southern blotting analysis and multiplex ligation-dependent probe amplification (MLPA) were used to screen for larger structural rearrangements (deletions and duplications). cDNA probes covering the entire coding region of the COL4A5 gene were hybridised on restriction enzyme digested genomic DNA on Southern blots. Three different rearrangements were found by Southern blotting, two of which were caused by single base substitutions, and also detected by the PCR-SSCP analysis. One larger rearrangement was found, an inversion of 21 Mb with a proximal breakpoint in COL4A5 intron 8 at Xq22.3, and a distal breakpoint in the RAB33A gene at Xq26.1. This rearrangement was exclusively ascertained by the Southern blotting analysis. Three deletions of >or= 2 exons were detected by MLPA. One of these was detected in a female proband. A deletion in heterozygous form will not be detected by PCR-SSCP or direct sequencing. A method based on the PCR-SSCP technique was set up for screening of the COL4A5 gene exon-by-exon for mutation. All 51 COL4A5 exons with flanking intronic sequences were screened by this technique. The two alternatively transcribed exons 41A and 41B were directly sequenced. The PCR-SSCP method was compared to direct sequencing in 15 of the cases. No difference in mutation detection rates were found. Finally, a method based on RT-PCR analysis of mRNA extracted from cultured skin fibroblasts was established. A mutation in a patient previously screened by PCR-SSCP analysis with normal result, was detected. Another advantage of analysing a skin biopsy is that it is also possible to perform immunostaining for the alpha 5(IV)-chain in the epidermal basement membrane on sections from the biopsy. Absence of the alpha 5(IV)-chain support a diagnosis of X-linked AS. A total of 64 different and putative disease causing mutations were found in 72 of the families. Half of the mutations identified were missense mutations. The most frequent mutations in AS were glycine substitutions in the Gly-Xaa-Yaa repeat sequence in the collagenous domain of the alpha 5(IV)-chain, accounting for 47% of all mutations and 89% if the missense mutations. Frame-shift mutations accounted for 17% of the mutations, splice site mutations for 13%, nonsense mutations for 11%, in-frame deletions for 4%, and larger structural rearrangements for 6%. In addition, 5 different non-pathogenic sequence variations, polymorphisms and mutations of unknown effect on the phenotype, were found. Nineteen of the mutations are new and have not previously been published, and 55 of the mutations have exclusively been detected in this material. Two of the mutations (3%) are de novo mutations, and it has been possible to trace the mutation back in six of the families, and to determine the parental origin of the mutation in these six families. The origin of the mutation was found to be paternal in 4 of the families (67%), and maternal in 2 of the families (33%). We have demonstrated a highly efficient and sensitive molecular diagnostic approach for analysing the COL4A5 gene in putative AS cases. Based on the present results and the litterature, an algorithm for molecular genetic analysis of the COL4A5 gene is suggested. The overall mutation detection rate was found to be 53%. The mutation detection rate was 72% in patients fulfilling >or= 3 of the clinical criteria for AS, and 82% in families clearly demonstrating X-linked inheritance. No COL4A5 mutation could be detected in 63 (47%) of the families. X-linked inheritance could be excluded in seven of these families solely based on a pedigree analysis, and a diagnosis of Epstein syndrome was established in one of the patients by MYH9 mutation analysis. We found that the underlying COL4A5 mutation, truncating or non-truncating, can significantly predict the age at ESRD in male patients. Truncating mutations, comprising nonsense mutations, frame-shifts, and larger structural rearrangements, were found to cause a juvenile form of the disease with a mean age at ESRD of 21.6 years, compared to 33.1 years in patients with a non-truncating mutation. The effect of non-truncating mutations is, however, less clear-cut. Glycine substitutions in the collagenous domain of the alpha 5(IV)-chain will result in an adult form of AS with a mean age at ESRD of 35.8 years. Missense mutations in the NC1-domain and in-frame deletions result in a juvenile form of the disease with a mean age at ESRD of 23.3 and 20.3 years, respectively, but the number of patients in each group is limited. We found no significant differences in the presence of hearing defects or ocular manifestations between patients with the different types of mutations. The lack of distinct genotype-phenotype correlations implies that the usefulness of the result of the COL4A5 mutation analysis to predict the prognosis is limited. Future technological improvements e.g. automated sequencing strategies and implementation of microarray technology , may increase the mutation detection rates, and lower the time and costs of the analyses. Functional studies are hampered by the restricted expression of the type IV collagen chains. The many different animal models for AS are obvious and promising targets for functional studies, and an important resource for gene therapy studies. This makes AS a reliable candidate for future gene therapy in humans.