Mutations in the gene of lysyl hydroxylase of patients with Ehlers-Danlos syndrome type VI

Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and in the other collagen like proteins. Hydroxylysine participates in the formation of cross-links between collagen molecules and can bind to the carbohydrates, galactose and glucosylgalactose. Patients with the type VIA Ehlers-Danlos syndrome (EDS) have characteristically a deficiency in hydroxylysine of collagen in their skin that is caused by reduced activity of lysyl hydroxylase 1. In this work the mutations were studied in detail in four different Ehlers-Danlos VIA patients. The first patient characterized in this study had a duplication of seven exons in the lysyl hydroxylase gene 1. The mutation was caused by homologous recombination of two identical 44-nucleotide regions of Alu sequences in introns 9 and 16 in the gene. This study also suggests that uniparental isodisomy does not explain the homozygosity of the mutation. The second patient was found to have two mutations in the gene for lysyl hydroxylase 1 in a compound heterozygote state. The study resulted in the discovery of the first deletion mutation in the gene. The deletion was caused by an Alu-Alu recombination that removes about 3 kb from the gene including all the exon 17 sequences. The other mutation causes deletion of exon 16 from the mRNA. Deletion of the penultimate nucleotide of intron 15 destroys the consensus sequence of the intron/exon boundary and thus causes the deletion. The third patient was described to have a nonsense codon in exon 14 of one allele which causes a reduction in the amount of lysyl hydroxylase mRNA and leads to aberrant RNA splicing in the cell. The other allele was concluded to be operationally null. In the last work two novel null mutations were found in the gene for lysyl hydroxylase 1. The first was a one nucleotide deletion in the acceptor splice site of intron 4 and the other an insertion of a C nucleotide in exon 2. The abnormal alleles lead to markedly decreased lysyl hydroxylase mRNA levels. This work revealed many exon deleted splicing variants of lysyl hydroxylase mRNA which were first discovered in affected cells, but traces of similarly spliced mRNA species were also found in the cytoplasm of normal human skin fibroblasts. These data indicate that the splicing machinery of the cell is leaky. In this thesis, several types of stuctural mutations in the DNA were found to be responsible for lysyl hydroxylase deficiency in patients with type VIA variant of EDS. The different mechanisms causing these mutations were also studied in detail

A compound heterozygote patient with Ehlers-Danlos syndrome type VI has a deletion in one allele and a splicing defect in the other allele of the lysyl hydroxylase gene.

We report the first deletion mutation and the first splicing defect in the lysyl hydroxylase gene in a compound heterozygote patient with Ehlers-Danlos syndrome type VI with markedly reduced lysyl hydroxylase activity. Northern analysis of the RNA isolated from skin fibroblasts of the patient demonstrated the presence of a truncated lysyl hydroxylase mRNA. PCR and sequence analysis confirmed the truncation and indicated that the cells contain two types of shortened mRNAs, one lacking the sequences corresponding to exon 16 and the other lacking that corresponding to exon 17 of the lysyl hydroxylase gene. Analysis of genomic DNA revealed deletion of the penultimate adenosine from the 3\u27 end of intron 15 from one allele. This defect was probably responsible for the skipping of exon 16 sequences from the transcript. The other allele, inherited from the mother, contains an Alu-Alu recombination with a deletion of about 3,000 nucleotides from the gene; this abnormality explains the lack of exon 17 sequences. The identified mutations in exon 16 and exon 17 do not alter the reading frame of the transcripts