Linda C. Walker

Tissue specificity of a new splice form of the human lysyl hydroxylase 2 gene

In this study we present the first report of alternative RNA splicing in a gene for lysyl hydroxylase (LH) in a normal population. This splicing event, which we have observed in the LH2 gene, appears to be tissue specific. The LH2 isoform was recently cloned and sequenced from a human kidney cDNA library and predicted to encode a 737 amino acid protein. In the present study, we have isolated a cDNA for LH2 from human skin fibroblasts that codes for a protein of 758 amino acids, of which 21 amino acids are encoded by a new exon. This 63-bp exon, designated exon 13A, is located between exons 13 and 14 of the originally-described LH2 gene. Amplification of cDNAs by PCR, using primers from exons 13 and 14, showed the presence of two distinct LH2 mRNA populations. A 209-bp transcript was expressed in mRNAs isolated from all tissues examined and was the only transcript expressed in skin, lung, aorta and dura, whereas in mRNAs from spleen, cartilage, liver, kidney, frontal lobe and placenta, an additional shorter 146-bp transcript was amplified. DNA sequence analysis showed that these two mRNAs resulted from the alternative splicing of exon 13A. The transcript containing exon 13A is expressed as the major LH2 form in all tissues except kidney and spleen. Analysis of genomic DNA from skin, placenta and spleen showed that both transcripts were generated from the same LH2 gene. Both upstream (intron 13) and downstream (intron 13A) sequences bordering exon 13A had normal consensus sequences for the acceptor (ag) and donor (gt) splice sites. Preliminary studies indicated that only single transcripts which included exon 13A were amplified from normal fetal skin at different stages of gestation. This suggests that although exon 13A is variably expressed in different tissues, this alternative splicing event is not developmentally regulated.

Heterogeneous basis of the type VIB form of Ehlers–Danlos syndrome (EDS VIB) that is unrelated to decreased collagen lysyl hydroxylation

Skin fibroblasts from the majority of patients with the clinical diagnosis of Ehlers–Danlos syndrome type VI (EDS VI; kyphoscoliosis type), have significantly decreased lysyl hydroxylase (LH) activity due to mutations in the LH1 gene (classified as EDS VIA: OMIM no. 225400). A rare condition exists in which patients are clinically similar but have normal levels of LH activity (designated EDS VIB: OMIM no. 229200). To define the biochemical defect, we have examined cultured fibroblasts from four EDS VIB patients for changes in the levels of the mRNAs for LH1, LH2, and LH3, collagen cross‐linking patterns, and the extent of lysine hydroxylation of type I collagen α chains. Although normal levels of LH1 mRNA were observed in all four patients, in two patients the levels of LH2 mRNA were decreased by >50%, and a similar decrease was observed in LH3 mRNA in the other two patients. A distinct pattern of collagen cross‐links, indicative of decreased lysyl hydroxylation, could be identified in EDS VIA patients, but there was no clear correlation between collagen cross‐link pattern and changes in the individual LH mRNAs in EDS VIB patients. Linkage to tenascin‐X was excluded in these patients. This study suggests that the basis for this form of EDS VI is genetically heterogeneous, and that alternative pathways in addition to lysine hydroxylation of collagen may be affected.

Mutational analysis of the lysyl hydroxylase 1 gene (PLOD) in six unrelated patients with Ehlers‐Danlos syndrome type VI: Prenatal exclusion of this disorder in one family

Screening of full length cDNAs for lysyl hydroxylase 1 (LH1; also PLOD) amplified from dermal fibroblasts from six unrelated patients with the autosomal recessive disorder Ehlers‐Danlos syndrome type VI (EDS VI) has shown them to be both homozygous and compound heterozygous for mutations in the gene. These mutations, which were verified in genomic DNA, result in a deficiency of LH activity (<25% of normal) in the probands, who are clinically characterized by kyphoscoliosis and extensibility of skin and joints. Four novel mutations identified in these patients include a mutation of an inserted C in one homozygous patient (1702insC) and three point mutations resulting in premature termination codons (PTCs): Y142X, Q327X (in two patients), and R670X. In the family with the R670X mutation we have prenatally excluded EDS VI by the characterization of mutations and their allelic inheritance. We have identified two previously reported mutations in the new patients: a seven exon duplication (in two patients) and a point mutation that codes for a PTC, Y511X, (in two patients). Genotype analysis indicated that the Y511X mutation may originate from a common ancestral gene. Several alternative splicing pathways have been identified which bypass the PTCs and can also restore the open reading frame. Hum Mutat 16:90, 2000.

Deletion of cysteine 369 in lysyl hydroxylase 1 eliminates enzyme activity and causes Ehlers-Danlos syndrome type VI.

This study describes the relative contribution of the 10 cysteine residues in lysyl hydroxylase 1 (LH1) to enzyme activity. We have identified a novel mutation of a 15-bp deletion in exon 11 in one LH1 allele, that codes for amino acids 367-371 (DLCRQ), in two unrelated compound heterozygous patients with Ehlers-Danlos type VI. The mutations in their other alleles were a C1119T change (exon 10) and a predicted Q49X (exon 2). We confirmed that the loss of cysteine 369 in the deleted sequence contributed to the diminished enzyme activity by structure/function analysis of mutant LH1 constructs, in which C369 and the nine other cysteines were individually mutated to serine by site-directed mutagenesis of a normal pAcGP67/LH1cDNA construct. Following their expression in an Sf9 insect cell/baculovirus system, SDS-PAGE and Western analysis showed that equivalent levels of correctly-sized (85-kDa) products were secreted. The mutation of residues C369 and also C375, C552 and C687 virtually eliminated LH activity, whereas mutations of C267, C270, and C680 had an intermediate effect. In contrast, the C204S, C484S and C566S constructs had normal activity. Although disulfide bond formation may affect the relative contribution of each cysteine to LH activity, catalytic activity does not appear to be directly related to dimerization of the enzyme.

TIA Nuclear Proteins Regulate the Alternate Splicing of Lysyl Hydroxylase 2

Synthesis of collagen, a major component of the extracellular matrix, is increased dramatically in fibrotic conditions such as scleroderma. This overaccumulation of collagen is associated with increased pyridinoline cross-links. These cross-links are derived by the action of the alternatively spliced long form of lysyl hydroxylase 2 (LH2), a collagen telopeptide LH. As LH2 (long) is reported to be overexpressed in scleroderma fibroblasts, the regulation of LH2 splicing suggests an important step in controlling fibrosis. Using an LH2 minigene, we have compared the regulation of the alternative splicing pattern of LH2, both endogenously and in the minigene, by the RNA-binding splicing proteins TIA-1 and TIAL1 (T-cell-restricted intracellular antigens). A decrease in the ratio of LH2 (long) to LH2 (short) was observed in fibroblasts from TIAL1 knockout mice, and in HEK293 cells knocked down for TIA-1 and TIAL1. As a corollary, overexpression of TIA-1/TIAL1 in HEK293 cells resulted in an increase in LH2 (long) minigene transcripts, accompanied by a decrease in LH2 (short). In scleroderma fibroblasts, a double TIA-1/TIAL1 knockdown reduced the ratio of LH2 (long) to LH2 (short) by over fivefold compared to controls. Identification of these TIA regulatory factors therefore suggests a tool to manipulate cellular LH2 levels in scleroderma so that potential intervention therapies may be identified.

Mutational analysis of 12 patients with the phenotype of Ehlers-Danlos syndrome type VIB shows no linkage to the zinc transporter gene SLC39A13.

The Ehlers–Danlos syndrome (EDS) encompasses a group of heterogeneous inherited connective tissue disorders in which patients are clinically characterized by fragile and hyperextensible skin and joint hypermobility. EDS has been divided into six distinct clinical subtypes [Beighton et al., 1998], one of which is the autosomal recessive kyphoscoliosis type (EDS VIA) (OMIM# 225400). EDS VIA is attributed to mutations in the lysyl hydroxylase 1 (LH1) gene (also known as PLOD1: procollagen-lysine, 2oxoglutarate 5-dioxygenase) [Yeowell and Walker, 2000], which codes for a post-translational modifying enzyme lysyl hydroxylase (LH) important in collagen biosynthesis [Yeowell and Pinnell, 1993; Steinmann et al., 2002]. Two other enzymes in the LH family include LH2 [Valtavaara et al., 1997], which exists in alternatively spliced forms, LH2 (short) and LH2 (long) [Yeowell and Walker, 1999], and LH3 [Passoja et al., 1998; Valtavaara et al., 1998]. A class of patients has been identified (EDS VIB) (OMIM# 229200) that has a similar clinical phenotype to EDS VIA but with normal LH activity. An earlier study on four EDS VIB patients showed no linkage to mutations in the genes for LH1, 2, and 3 [Walker et al., 2004a]. A second study on three LH2-deficient patients, with phenotypes ofmixed EDSVI and normal LHactivity, showed that their deficiency of LH2 was unrelated to mutations in either the coding or proximal promoter region of the LH2 gene [Walker et al., 2004b]. More recently, reports [Fukada et al., 2008; Giunta et al., 2008] have linked three families with EDS-like phenotypes to homozygous mutations in the zinc transporter gene (SLC39A13NM_152264.2). These included a homozygous 9 bp inframe deletion in exon 4 (c.483–491 del9) in two families [Giunta et al., 2008] and a homozygousG toA transition at nucleotide c.221 (c.221G>A) in exon 2 predicting the non-conservative amino acid substitution G74D in SLC39A13 in the third family [Fukada et al., 2008]. In addition to the typical EDS characteristics of hyperelastic, thin, bruisable skin, and hypermobile joints, the patients with the 9 bp deletion [Giunta et al., 2008] showed a generalized skeletal dysplasia and abnormalities of the hands, leading to the authors’ description of this disorder as the ‘‘spondylocheiro dysplastic form of EDS (SCD-EDS).’’ Although the lysyl and prolyl collagen residues were underhydroxylated in these patients, their cultured skin fibroblasts showed normal enzyme activities. In vivo, this underhydroxylation could be attributed to an increased internal Zn2þ content which competed with Fe2þ, a cofactor required for lysyl and prolyl hydroxylation. In contrast, in the in vitro enzyme assay [Murad et al., 1985], addition of supplementary Fe2þ together with the other required cofactors resulted in normal LH and prolyl hydroxylase (PH) activities. Based on these results, we screened 12 cell lines in our cell repository from EDS VI-like patients, with normal LH activity measured in their cultured skin fibroblasts, for mutations in this gene. These included the fourEDSVIB cell lines previously reported in this journal [Walker et al., 2004a], the three EDSVI-like cell lines reported in a separate publication [Walker et al., 2004b], and five additional cell lines. The patients were selected on the basis of their general EDS VI-like clinical characteristics as described in Table I. Although not all of the clinical characteristics of this genetically

Mutational analysis of the lysyl hydroxylase 1 gene (PLOD) in six unrelated patients with Ehlers-Danlos syndrome type VI: Prenatal exclusion of this disorder in one family Communicated by: Darwin J. Prockop Online Citation: Human Mutation, Mutation in Brief #340(2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/340.pdf

Screening of full length cDNAs for lysyl hydroxylase 1 (LH1; also PLOD) amplified from dermal fibroblasts from six unrelated patients with the autosomal recessive disorder Ehlers-Danlos syndrome type VI (EDS VI) has shown them to be both homozygous and compound heterozygous for mutations in the gene. These mutations, which were verified in genomic DNA, result in a deficiency of LH activity (<25% of normal) in the probands, who are clinically characterized by kyphoscoliosis and extensibility of skin and joints. Four novel mutations identified in these patients include a mutation of an inserted C in one homozygous patient (1702insC) and three point mutations resulting in premature termination codons (PTCs): Y142X, Q327X (in two patients), and R670X. In the family with the R670X mutation we have prenatally excluded EDS VI by the characterization of mutations and their allelic inheritance. We have identified two previously reported mutations in the new patients: a seven exon duplication (in two patients) and a point mutation that codes for a PTC, Y511X, (in two patients). Genotype analysis indicated that the Y511X mutation may originate from a common ancestral gene. Several alternative splicing pathways have been identified which bypass the PTCs and can also restore the open reading frame.