Berthold Struk

Mutations of the gene encoding the transmembrane transporter protein ABC-C6 cause pseudoxanthoma elasticum

Abstract. We recently published the precise chromosomal localization on chromosome 16p13.1 of the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Here we report the identification of mutations in the gene encoding the transmembrane transporter protein, ABC-C6 (also known as MRP-6), one of the four genes located in the region of linkage, as cause of the disease. Sequence analysis in four independent consanguineous families from Switzerland, Mexico, and South Africa and in one non-consanguineous family from the United States demonstrated several different mis-sense mutations to cosegregate with the disease phenotype. These findings are consistent with the conclusion that PXE is a recessive disorder that displays allelic heterogeneity, which may explain the considerable phenotypic variance characteristic of the disorder.

The Y Chromosome: Epistatic and Ecogenetic Interactions in Genetic Hypertension

Previous studies have revealed conflicting evidence concerning a Y-chromosome effect on blood pressure (BP) in genetic crosses involving different strains of spontaneously hypertensive rats (SHR or SHRSP). We had previously found an approximately 16 mm Hg difference in systolic BP (P < 10(-7)) at baseline but not after dietary salt loading (P = .82) between F2 males derived from an SHRSPHD grandfather and a Wistar-Kyoto (WKYHD-0) grandmother and F2 males from a reciprocal cross (WKYHD-0 grandfather). When we examined F2 animals from reciprocal crosses between SHRSPHD and a congenic strain, WKYHD-1, which carries a 6-centimorgan-long SHRSPHD-homologous genomic fragment on chromosome 10 that contains a quantitative trait locus linked to BP (BP/SP-la), we found no significant differences either at baseline (P = .39) or after salt loading (P = .51) in the two reciprocal F2 cohorts. To test the hypothesis that Y-chromosome-autosomal epistasis accounts for the discrepant Y-chromosome effects on BP, we analyzed the interaction between BP/SP-1a and reciprocal cross status on BP in the two crosses. In the F2 (WKYHD-0xSHRSPHD) cross, no significant interaction was found for basal systolic BP (P = .89), arguing against a major influence of BP/SP-1a on the Y-chromosome effects on basal BP. However, a significant interaction between zygosity at the BP/SP-1a locus and reciprocal cross status for systolic BP after salt loading (P = .022) indicated that the BP/SP-1a-SHRSPHD allele exhibited a significant effect on BP after dietary excess salt only in males that inherited the SHRSP Y chromosome. These results support the relevance of a Y-chromosome effect on BP and suggest that a complex interplay of epistatic and ecogenetic interactions governs its effect on phenotype.

Role of the α-, β-, and γ-Subunits of Epithelial Sodium Channel in a Model of Polygenic Hypertension

The pathophysiological basis of Liddles syndrome, a rare autosomal dominant form of arterial hypertension, has been found to rest on missense mutations or truncations of the beta- and gamma-subunits of the epithelial sodium channel. The hypothesis has been advanced that molecular variants of these genes might also contribute to the common polygenic forms of hypertension. We tested this hypothesis by performing a cosegregation study in a reciprocal cross between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and a Wistar-Kyoto rat (WKY-1HD) reference strain. We carried out genetic mapping and chromosomal assignment of the alpha-, beta-, and gamma-subunits of the epithelial sodium channel using both linkage analysis and fluorescent in situ hybridization techniques. We demonstrate that in the rat, the beta- and gamma-subunits, as in humans, are in close linkage; they map to rat chromosome 1 and cosegregate with systolic pressure after dietary NaCl (logarithm of the odds [LOD] score, 3.7), although the peak LOD score of 5.0 for this quantitative trait locus was detected 4.4 cM away from the beta-/gamma-subunit locus. The alpha-subunit was mapped to chromosome 4 and exhibited no linkage to blood pressure phenotype. Comparative analysis of the complete coding sequences of all three subunits in the SHRSPHD and WKY-1HD strains revealed no biologically relevant mutations. Furthermore, Northern blot comparison of mRNA levels for all three subunits in the kidney showed no differences between SHRSPHD and WKY-1HD. Our results fail to support a material contribution of the epithelial sodium channel genes to blood pressure regulation in this model of polygenic hypertension.

A novel Q378X mutation exists in the transmembrane transporter protein ABCC6 and its pseudogene : implications for mutation analysis in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is an inherited disorder of the elastic tissue with characteristic progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Recently mutations in the ABCC6 gene, encoding a transmembrane transporter protein, were identified as cause of the disease. Surprisingly, sequence and RFLP analysis for exon 9 with primers corresponding to flanking intronic sequence in diseased and haplotype negative members from all of our families and in a control population revealed either a homozygous or heterozygous state for the Q378X (1132C→T) nonsense mutation in all individuals. With the publication of the genomic structure of the PXE locus we had identified the starting point of a large genomic segmental duplication within the locus in the cytogenetic interval defined by the Cy19 and Cy185 somatic cell hybrid breakpoints on chromosome 16p13.1. By means of somatic cell hybrid mapping we located this starting point telomeric to exon 10 of ABCC6. The duplication, however, does not include exon 10, but exons 1–9. These findings suggest that one or several copies of an ABCC6 pseudogene (ψABCC6) lie within this large segmental duplication. At least one copy contains exons 1–9 and maps to the chromosomal interval defined by the Cy163 and Cy11 breakpoints. Either this copy and/or an additional copy of ψABCC6 within Cy19-Cy183 carries the Q378X mutation that masks the correct identification of this nonsense mutation as being causative in pseudoxanthoma elasticum. Long-range PCR of exon 9 starting from sequence outside the genomic replication circumvents interference from the ψABCC6 DNA sequences and demonstrates that the Q378X mutation in the ABCC6 gene is associated with PXE in some families. These findings lead us to propose that gene conversion mechanisms from ψABCC6 to ABCC6 play a functional role in mutations causing PXE.

Evidence for primary genetic determination of heart rate regulation: chromosomal mapping of a genetic locus in the rat.

BACKGROUND We investigated whether an accelerated heart rate (HR), observed in the stroke-prone spontaneously hypertensive rat (SHRSP(HD)), is a primary, genetically determined trait and whether it contributes to blood pressure (BP) regulation in this model of polygenic hypertension. METHODS AND RESULTS We measured BP and HR in SHRSP(HD) and normotensive Wistar-Kyoto rats (WKY), as well as in F2 hybrids bred from crossing the two strains, at baseline and after 12 days of dietary NaCl loading. Random marker genome screening and cosegregation analysis were performed on F2 hybrids derived from SHRSP(HD)/WKY-0(HD) (n=115) and SHRSP(HD)/WKY-1(HD) (n=139) crosses (WKY-0(HD) and WKY-1(HD) are two congenic WKY strains). HR in SHRSP(HD) was significantly higher than in WKY-0(HD) both at baseline (404+/-30 versus 375+/-46 bpm; P=.0034) and after NaCl (437+/-23 versus 364+/-40 bpm; P=10(-9)). BP in F2 hybrids showed no significant correlation with HR either at baseline or after NaCl loading. HR after NaCl loading but not at baseline was significantly linked in a recessive fashion to a locus on chromosome 3: in animals homozygous for the SHRSP(HD) allele, HR was 414+/-49 compared with 383+/-44 bpm in heterozygotes and WKY homozygotes (F(210,1)=19.7, P=1.4x10(-5), lod score=5.9). The putative BP-relevant gene at this locus, termed HR-SP1, showed no evidence of linkage to any of the BP parameters measured. CONCLUSIONS Our results demonstrate that a genetic locus on rat chromosome 3, HR-SP1, contributes directly to the regulation of HR in SHRSP(HD) but exhibits no effect on BP. Thus, in addition to its modulation by reflex-mediated neurohumoral mechanisms, HR is also under the direct influence of primary genetic factors.

Pseudoxanthoma elasticum: evaluation of diagnostic criteria based on molecular data

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Statistical Considerations for Genome-Wide Scans: Design and Application of a Novel Software Package POLYMORPHISM

Objective: Given the cost and complexity of genome-wide scans, optimization of study design is of critical importance. Available algorithms only partially satisfy this need. We designed a software package called ‘POLYMORPHISM’ to meet these needs. Methods: The program is designed to calculate linkage parameters for both ‘single-point’ and ‘two-point’ settings that are applicable also to incompletely informative microsatellite markers. In single-point analysis, the heterozygosity, polymorphism information content (PIC) and linkage information content (LIC) statistics based on marker allele frequencies are provided. In two-point analysis, joint PIC values for two markers, the conditional probability of detecting linkage phase, the frequency of double heterozygotes and the expected number of informative meioses are calculated. Results: Results were obtained using S.A.G.E./DESPAIR (Design of Linkage Studies Based on Pairs of Relatives) in addition to applying this program to a Centre d’Etude du Polymorphisme pedigree-derived genotyping data set, which estimated critical parameters used in a two-stage genome scan. A single nucleotide polymorphism (SNP)-based one-stage genomic screen strategy is also considered. Conclusions: LIC values are crucial for getting accurate estimates on those parameters that are important for a two-stage genome screening study. Optimization of the cost-effectiveness of an SNP-based genomic screen strategy is possible by modeling a balance between marker information content and marker density.