Molecular genetics of familial hypercholesterolemia in Spain: Ten novel LDLR mutations and population analysis.

Mutations underlying FH in Spain are largely unknown because only a few and limited surveys have been carried out on Spanish FH patients up to now. To gain information on this issue, we have analysed a group of 113 unrelated Spanish FH patients from an eastern area of Spain (Valencian Community). We have screened the LDLR gene by Southern blot and PCR-SSCP analysis to detect large rearrangements and small mutations, respectively. In addition, we have screened the Apo B gene for mutations known to cause FDB by PCR-SSCP analysis. We have identified a total of 47 different mutations in the LDLR gene (5 large rearrangements, and 42 small mutations, which were characterized by DNA sequencing), 19 of which have not been described in other populations (Valencia-1 to -4, 112insA, P160R, 790DelATGA, 920insTCAG, G642E, and the ten novel mutations E246A, 884delT, I289T, S305F, Q328X, Y354C, I603del, 2312-3C>A, V779M, and N804K). Three of these mutations (15%) were present in more than 1 proband, being mutation 112insA the most prevalent (frequency approximately 8%) in our sample. The Apo B gene R3500Q mutation was found in only one patient and no underlying defect was found in about 27% of patients. Our data support the notion that Spaniards represent a heterogeneous population with its own spectrum of LDLR gene mutations and that, in our population, FDB has a lower frequency or a milder expression than in central Europe countries.

Seven DNA polymorphisms in the LDL receptor gene: application to the study of familial hypercholesterolemia in Spain.

We have performed restriction fragment length polymorphism (RFLP) analysis at the low density lipoprotein receptor (LDLR) locus in order to investigate the molecular genetics of familial hypercholesterolemia (FH) in Spain. Firstly, a sample of 50 unrelated patients with a clinical diagnosis of FH was screened for the presence of major rearrangements at this locus by Southern blot analysis of BglII digested genomic DNA. Four different mutations were detected, accounting for 8% of the mutant alleles in the Spanish FH sample. Then, we determined the relative allele frequency and estimated linkage disequilibrium between seven RFLPs of the LDLR gene in the remaining 46 FH patients and in 61 normolipidemic controls. HincII, AvaII, PvuII, MspI, and NcoI are the most polymorphic sites with individual PIC values higher than 0.28, whereas the TaqI and StuI sites display low levels of polymorphism. The usefulness of the seven RFLPs to confirm a clinical diagnosis of FH was investigated in 15 FH-families, consisting of 118 individuals, in whom the presence of Familial Defective Apolipoprotein B-100 (FDB) due to the apoB3500 mutation was excluded. Independent haplotypes were constructed for 71 chromosomes: 15 FH and 56 control haplotypes. A total of 14 different haplotypes was found. In 12 families, clinical diagnosis of FH was confirmed by cosegregation analysis, which makes these RFLPs useful for studying the inheritance of the LDLR gene in 80% of Spanish families with FH. Comparison of haplotypes found in the Spanish sample with those found in Swiss and Norwegians suggests heterogeneity of haplotypes among European populations.

A three-allelic polymorphic system in exon 12 of the LDL receptor gene is highly informative for segregation analysis of familial hypercholesterolemia in the Spanish population.

We have screened exon 12 of the low density lipoprotein (LDL) receptor gene from 46 familial hypercholesterolemia (FH) heterozygotes and 64 normolipidemic controls for two polymorphisms, HincII, which is caused by a T to C substitution at base 1773, and a C to T transition at base 1725, by using single strand conformation polymorphism (SSCP) analysis. Our results indicate that polymorphism at base 1725, previously reported as very rare from a Japanese sample, is quite frequent in the Spanish population and that it is closely linked to the presence of the HincII site (HincII+). Thus, both polymorphisms constitute a system of three alleles, typed HincII- C1725, HincII+ C1725, and HincII+ T1725, whose frequencies in the FH sample were 0.489, 0.347, and 0.164, respectively. No significant differences were found in the allele frequencies between the FH and control samples. This three-allelic polymorphic system provides a higher information content (PIC value) than the HincII RFLP alone (0.537 versus 0.373, respectively); therefore, it is an extremely useful marker for linkage analysis of FH in Caucasian populations.

Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.

Tandem transcription termination sites in the dnaN gene of Escherichia coli.

The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes. We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. However, the recF gene has its own promoter region located in the middle of the dnaN structural gene. In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon. These sites are located in the dnaN gene, downstream from the recF promoter region. Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters. As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon. The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity. In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes. We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli.

The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB. Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene. In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene. Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene. Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters. Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription. In this report, we present evidence that only one of them is dependent on the Rho protein. Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

Transcriptional organization of the dnaN and recF genes of Escherichia coli K-12.

The dnaN gene of Escherichia coli determines the beta subunit of DNA polymerase III, a multisubunit enzyme responsible for most of the replicative DNA synthesis. The dnaN gene maps between the dnaA and recF genes. We have characterized the regulatory region of the dnaN gene by screening DNA restriction fragments for promoter activity, S1 mapping of mRNAs, deletion analysis, and in vivo dnaN complementation tests. There are at least three dnaN promoters located in the second half of the dnaA coding region. The one closest to the dnaN structural gene is the weakest, but it provides sufficient dnaN expression for complementation when the gene is present on a multicopy plasmid. Deletion of sequences needed for initiation of dnaN translation or introduction of nonsense codons into dnaN causes reduction of recF expression. However, a deletion inactivating dnaN without changing the reading frame of the gene does not affect expression of the recF gene. These results indicate that the dnaN and recF genes are organized in an operon. We have previously shown the presence of termination signals within the dnaN coding region (Armengod, M.E., and Lambíes, E. (1986) Gene (Amst.) 43, 183-196). Therefore, we propose that the polarity produced by nonsense mutations in dnaN is primarily transcriptional. The uncoupling of transcription and translation of the dnaN gene (when translation is interrupted by premature nonsense codons or by other mechanisms) probably results in transcription termination at termination signals in dnaN.