A genome-wide association study identifies variants in KCNIP4 associated with ACE inhibitor-induced cough.

The most common side effect of angiotensin-converting enzyme inhibitor (ACEi) drugs is cough. We conducted a genome-wide association study (GWAS) of ACEi-induced cough among 7080 subjects of diverse ancestries in the Electronic Medical Records and Genomics (eMERGE) network. Cases were subjects diagnosed with ACEi-induced cough. Controls were subjects with at least 6 months of ACEi use and no cough. A GWAS (1595 cases and 5485 controls) identified associations on chromosome 4 in an intron of KCNIP4. The strongest association was at rs145489027 (minor allele frequency=0.33, odds ratio (OR)=1.3 (95% confidence interval (CI): 1.2-1.4), P=1.0 × 10(-8)). Replication for six single-nucleotide polymorphisms (SNPs) in KCNIP4 was tested in a second eMERGE population (n=926) and in the Genetics of Diabetes Audit and Research in Tayside, Scotland (GoDARTS) cohort (n=4309). Replication was observed at rs7675300 (OR=1.32 (1.01-1.70), P=0.04) in eMERGE and at rs16870989 and rs1495509 (OR=1.15 (1.01-1.30), P=0.03 for both) in GoDARTS. The combined association at rs1495509 was significant (OR=1.23 (1.15-1.32), P=1.9 × 10(-9)). These results indicate that SNPs in KCNIP4 may modulate ACEi-induced cough risk.

Genetic variation in the HLA region is associated with susceptibility to herpes zoster.

Herpes zoster, commonly referred to as shingles, is caused by the varicella zoster virus (VZV). VZV initially manifests as chicken pox, most commonly in childhood, can remain asymptomatically latent in nerve tissues for many years and often re-emerges as shingles. Although reactivation may be related to immune suppression, aging and female sex, most inter-individual variability in re-emergence risk has not been explained to date. We performed a genome-wide association analyses in 22,981 participants (2280 shingles cases) from the electronic Medical Records and Genomics Network. Using Cox survival and logistic regression, we identified a genomic region in the combined and European ancestry groups that has an age of onset effect reaching genome-wide significance (P>1.0 × 10(-8)). This region tags the non-coding gene HCP5 (HLA Complex P5) in the major histocompatibility complex. This gene is an endogenous retrovirus and likely influences viral activity through regulatory functions. Variants in this genetic region are known to be associated with delay in development of AIDS in people infected by HIV. Our study provides further suggestion that this region may have a critical role in viral suppression and could potentially harbor a clinically actionable variant for the shingles vaccine.

Presence of Borrelia burgdorferi sensu lato antibodies in the serum of patients with abdominal aortic aneurysms.

Infectious agents are likely to play a role in the pathogenesis of chronic inflammatory diseases, including abdominal aortic aneurysms (AAAs). The goal of this study was to determine if Borrelia burgdorferi sensu lato (sl), a microorganism responsible for Lyme disease, is involved in the etiology of AAAs. The presence of serum antibodies against B. burgdorferi sl was measured with enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blotting in 96 AAA and 108 peripheral artery disease (PAD) patients. Polymerase chain reaction (PCR) was used for the detection of Borrelia-specific DNA in the aneurysm wall. Among AAA patients 34% and among PAD patients 16% were seropositive for B. burgdorferi sl antibodies (Fisher's exact test, p = 0.003; odds ratio [OR] 2.79; 95% confidence interval [CI] 1.37-5.85). In the German general population, 3-17% are seropositive for Borrelia antibodies. No Borrelia DNA was detected in the aneurysm wall. Our findings suggest a relationship between AAAs and B. burgdorferi sl. We hypothesize that the underlying mechanism for B. burgdorferi sl in AAA formation is similar to that by the spirochete Treponema pallidum; alternatively, AAAs could develop due to induced autoimmunity via molecular mimicry due to similarities between some of the B. burgdorferi sl proteins and aortic proteins.

Developments in genomics to improve understanding, diagnosis and management of aneurysms and peripheral artery disease.

Genome-wide approaches, including microarray-based expression profiling, DNA linkage studies and genetic association studies, offer an unbiased way to identify genetic risk factors and biological processes leading to discoveries, which might help in the development of new diagnostic and therapeutic approaches for a wide range of diseases. Currently, the number of published genome-wide analyses for aneurysms and peripheral artery diseases is still limited, and it is difficult to generalise about the disease pathogenesis or genetic risk factors contributing to these diseases. Large multicentre studies are needed to provide sufficient statistical power, and replication studies are essential before these findings are used for defining clinical policies of diagnosis and treatment. The biggest future challenge will be to translate the genomic information to the clinical settings so that it will improve our understanding of the disease processes, help us to develop better diagnostic tools and lead to the design of new ways to manage aneurysms and peripheral artery disease in the era of personalised medicine. Characterisation of diseases at the molecular level is likely to lead to more accurate diagnoses and the use of 'genomic nosology' of diseases.

A mutation in the gene for type III procollagen (COL3A1) in a family with aortic aneurysms.

Experiments were carried out to test the hypothesis that familial aortic aneurysms, either thoracic or abdominal, are caused by mutations in the gene for type III procollagen (COL3A1) similar to mutations in the same gene that have been shown to cause rupture of aorta and other disastrous consequences in the rare genetic disorder known as Ehlers-Danlos syndrome type IV. A family was identified through a 37-yr-old female captain in the United States Air Force who was scrutinized only because many of her direct blood relatives had died of ruptured aortic aneurysms. The woman was heterozygous for a single-base mutation that converted the codon for glycine 619 of the alpha 1(III) chain of type III procollagen to a codon for arginine. Studies on cultured skin fibroblasts demonstrated the mutation caused synthesis of type III procollagen that had a decreased temperature for thermal unfolding of the protein. The same mutation was identified in DNA extracted from pathologic specimens from her mother who had died at the age of 34 and a maternal aunt who died at the age of 55 of aortic aneurysms. Examination of DNA from samples of saliva revealed that the woman's daughter, her son, a brother, and an aunt also had the mutation. The results demonstrated that mutations in the type III procollagen gene can cause familial aortic aneurysms and that DNA tests for such mutations can identify individuals at risk for aneurysms.

Abnormal copper metabolism and deficient lysyl oxidase activity in a heritable connective tissue disorder.

Biochemical abnormalities were studied in two brothers with bladder divericulas, inguinal hernias, slight skin laxity, and hyperelasticity and skeletal abnormalities including occipital exostoses. Lysyl oxidase activity was low in the medium of cultured skin fibroblasts, this abnormality being accompanied by reduced conversion of the newly synthesized collagen into the soluble form. Copper concentrations were markedly elevated in the cultured skin fibroblasts, but decreased in the serum and hair. Serum cerulophasmin levels were also low. The reduced lysyl oxidase activity is suggested to be responsible for ther clinical manifestations, but the deficiency in this copper-dependent enzyme may be secondary to the abnormalities in the metabolism of the cation. Nevertheless, a mutation directly affecting both lysyl oxidase and an intracellular copper transport protein cannot be excluded. The disease is tentatively classified as one subtype of the Ehlers-Danlos syndrome.

Secretion of lysyl oxidase by cultured human skin fibroblasts and effects of monensin, nigericin, tunicamycin and colchicine.

Lysyl oxidase is an extracellular enzyme that initiates crosslink formation in the major connective tissue proteins, the collagens and elastin. This enzyme activity accumulated in a fresh medium of cultured human skin fibroblasts for at least 24 h, but the accumulation was distinctly non-linear after the first 12 h. Most of the total enzyme activity was present in the medium, the activity found in the cell layer representing about 30% of the total activity at 4 h, and about 10-15% at 24 h. The bulk of the cell-layer-associated activity appeared to be extracellular, as more than half was lost upon trypsinization. Culturing of the cells for 8 h in the presence of either monensin or nigericin, ionophores known to inhibit the secretion of many proteins at the level of the Golgi complex, markedly reduced the accumulation of lysyl oxidase activity in the medium. Monensin was particularly effective, as it produced a distinct inhibition even at a 10 nM concentration, reaching 50% at 30 nM. Both ionophores also reduced enzyme activity in the cell layer, whereas no definite decrease was seen in the activity of the trypsinized cells. The effect of monensin was evidently not due to any general toxicity on the part of the drug, since even a 500 nM concentration gave no inhibition of the incorporation of [3H]leucine into total protein. Tunicamycin also reduced lysyl oxidase activity in the medium and to a lesser extent in the cell layer, but the effective dose, 1-10 micrograms/ml, also inhibited the incorporation of [3H]leucine into total protein. The reduced enzyme activity may therefore not be due to a direct effect of tunicamycin on the glycosylation of the lysyl oxidase protein itself but may be mediated through other actions of the drug. Colchicine caused no inhibition in lysyl oxidase activity secretion even at a 10 microM concentration, although it has been reported to inhibit collagen secretion at doses more than one order of magnitude lower.

Ehlers-Danlos syndrome type IV: a single base substitution of the last nucleotide of exon 34 in COL3A1 leads to exon skipping.

The Ehlers-Danlos syndrome has been classified into nine phenotypic presentations. Type IV is a variant of particular importance because people affected with this genodermatosis are at great risk of spontaneous hemorrhage from vascular rupture or bowel perforation. Recent molecular advances have identified mutations in the gene for type III procollagen as responsible for Ehlers-Danlos syndrome type IV. We report a case of a 14-year-old male with a typical presentation of the type IV variant who was found to have markedly dilated fibroblast cisternae and varying collagen fibril diameter on ultrastructural study. A novel genetic defect was noted by polymerase chain reaction and DNA sequencing of genetic material isolated from skin fibroblast cultures. Analysis of the gene for type III procollagen revealed a single base mutation in the last nucleotide of exon 34. The mutation led to abnormal RNA splicing and skipping of exon 34 on the mRNA level.

A recombinant homotrimer of type I procollagen that lacks the central two D-periods. The thermal stability of the triple helix is decreased by 2 to 4 degrees C.

A D-period cassette system was developed that can be used to synthesize a variety of recombinant homotrimers of type I procollagen. A construct lacking the central two D-periods of pro alpha 1(I) chains was assembled and expressed as a recombinant protein in the mammalian cell line. The recombinant protein was purified to homogeneity and the thermal stability of the triple helix assayed by rapid protease digestion. The results indicated that deletion of the central 468 amino acids from the major triple helix lowered the thermal stability of the protein by 2 to 4 degrees C. The results therefore begin to define regions of the molecule that vary in their contributions to helical stability.

Genomic organization of the human COL3A1 and COL5A2 genes: COL5A2 has evolved differently than the other minor fibrillar collagen genes.

We report here on the complete structure of the human COL3A1 and COL5A2 genes. Collagens III and V, together with collagens I, II and XI make up the group of fibrillar collagens, all of which share a similar structure and function; however, despite the similar size of the major triple-helical domain, the number of exons coding for the domain differs between the genes for the major fibrillar collagens characterized so far (I, II, and III) and the minor ones (V and XI). The main triple-helical domain being encoded by 49-50 exons, including the junction exons, in the COL5A1, COL11A1 and COL11A2 genes, but by 43-44 exons in the genes for the major fibrillar collagens. Characterization of the genomic structure of the COL3A1 gene confirmed its association with the major fibrillar collagen genes, but surprisingly, the genomic organization of the COL5A2 gene was found to be similar to that of the COL3A1 gene. We also confirmed that the two genes are located in tail-to-tail orientation with an intergenic distance of approximately 22 kb. Phylogenetic analysis suggested that they have evolved from a common ancestor gene. Analysis of the genomic sequences identified a novel single nucleotide polymorphism and a novel dinucleotide repeat. These polymorphisms should be useful for linkage analysis of the Ehlers-Danlos syndrome and related disorders.

Analysis of coding sequences for tissue inhibitor of metalloproteinases 1 (TIMP1) and 2 (TIMP2) in patients with aneurysms.

Aneurysms are characterized by dilation, i.e. expansion and thinning of all the arterial wall layers, which is accompanied by remodeling of the connective tissue. Genes involved in the regulation of tissue remodeling are therefore candidate genes. We analyzed TIMP1 and TIMP2 coding sequences in 12 individuals with abdominal aortic aneurysms (AAA), one individual with AAA and intracranial aneurysms (IA), four individuals with IA and two clinically unaffected individuals. We identified two nucleotide variants in both the TIMP1 and the TIMP2 coding sequences. All differences occurred in the third base positions of codons and were neutral polymorphisms. A significant difference was observed in the frequency of TIMP2 nt 573 polymorphism between 168 alleles from AAA patients and 102 control alleles.

Synthesis and conformational properties of a recombinant C-propeptide of human type III procollagen.

A cDNA was prepared that coded for the signal peptide of type III procollagen linked to the complete C-propeptide of the protein. The cDNA was then used to express the protein in a baculovirus recombinant system. Recombinant protein was recovered as a trimer from the medium of transfected cells in a yield of 1 to 2.5 mg per liter. Mapping of peptide fragments with and without reduction indicated that the protein contained the expected interchain disulfide bonds. Analysis by circular dichroism suggested that the conformation of the protein corresponded to the native conformation. Therefore, the protein should be appropriate for further tests of its biological function and analysis of structure by X-ray diffraction.

Fibulin-2 exhibits high degree of variability, but no structural changes concordant with abdominal aortic aneurysms.

We used conformation sensitive gel electrophoresis and direct sequencing of PCR products to screen for mutations in the cDNA for fibulin-2, an extracellular matrix protein, from 11 patients with abdominal aortic aneurysms and two controls. When compared with the published reference sequence, a total of 14 single-base sequence variations were detected. Seven of the changes were neutral in that they did not result in an amino acid substitution. There were five missense changes at sites not conserved between human and mouse, and two missense changes at sites conserved between human and mouse. All but two of the sequence variants studied were also present in an additional set of 102 control alleles analyzed. One of these two changes was a missense mutation, but it did not segregate with abdominal aortic aneurysms in the family, whilst the other change was neutral. In conclusion, fibulin-2 has a large number of sequence variations in comparison with our previous analyses of type III collagen, and these variations will be useful in association studies. There was an excellent overall agreement between direct sequencing of PCR-products and conformation sensitive gel electrophoresis.

First-stage autosomal genome screen in extended pedigrees suggests genes predisposing to low bone mineral density on chromosomes 1p, 2p and 4q.

Osteoporosis is characterized by low bone density, and osteopenia is responsible for 1.5 million fractures in the United States annually. In order to identify regions of the genome which are likely to contain genes predisposing to osteopenia, we genotyped 149 members of seven large pedigrees having recurrence of low bone mineral density (BMD) with 330 DNA markers spread throughout the autosomal genome. Linkage analysis for this quantitative trait was carried out using spine and hip BMD values by the classical lod-score method using a genetic model with parameters estimated from the seven families. In addition, non-parametric analysis was performed using the traditional Haseman-Elston approach in 74 independent sib pairs from the same pedigrees. The maximum lod score obtained by parametric analysis in all families combined was +2.08 (theta = 0.05) for the marker CD3D on chromosome 11q. All other combined lod scores from the parametric analysis were less than +1.90, the threshold for suggestive linkage. Non-parametric analysis suggested linkage of low BMD to chromosomes 1p36 (Zmax = +3.51 for D1S450) and 2p23-24 (Zmax = +2.07 for D2S149). Maximum multi-point lod scores for these regions were +2.29 and +2.25, respectively. A third region with associated lod scores above the threshold of suggestive linkage in both single-point and multi-point non-parametric analysis was on chromosome 4qter (Zmax = +2.95 for D4S1539 and Zmax = +2.48 for D4S1554). Our data suggest the existence of multiple genes involved in controlling spine and hip BMD, and indicate several candidate regions for further screening in this and other independent samples.

Deficient production of lysyl oxidase in cultures of malignantly transformed human cells.

Lysyl oxidase activity in the culture medium of eight malignantly transformed human cell lines was very low compared with that in four control fibroblast lines, being 9-16% in five sarcoma cell lines and 7-11% in three other tumour cell lines. The low enzyme activity was probably due to deficient enzyme synthesis rather than impaired secretion into the cell medium, as low activity was also found in urea extracts of the cell pellets. Lysyl oxidase production thus appears to be closely regulated with deficient collagen gene expression in malignant transformation.

Founder mutations and the high prevalence of myotonia congenita in northern Finland.

OBJECTIVE AND BACKGROUND: To find an explanation at the molecular level for the high prevalence of myotonia congenita in northern Finland and the exceptional pattern of inheritance of the disease in many families, and to study genotype-phenotype correlation in the patients. METHODS: Forty-six patients with myotonia congenita and 16 unaffected relatives from 24 families were studied. All 23 exons and their flanking regions of the gene for the chloride channel protein (ClC-1) were sequenced from at least one patient from all families. RESULTS: There were three different mutations of ClC-1 in the patients: one in exon 11, a T-to-G transversion that resulted in the substitution of cysteine for phenylalanine at amino acid position 413 (F413C); one in exon 15, a C-to-T transition that resulted in the substitution of valine for alanine at amino acid position 531 (A531V); and one in exon 23, a C-to-T transition that resulted in the substitution of a stop codon for an arginine codon at amino acid position 894 (R894X). CONCLUSIONS: Molecular studies showed that even in families with apparent dominant inheritance, the actual mode of inheritance was autosomal recessive. This was explained not only by the observed consanguinity in some families but by an enrichment of three different mutations of the ClC-1 gene and a consequent high number of compound heterozygotes in the population. One of the mutations is unique to northern Finland. The conspicuous enrichment of the mutations is likely due to the founder effect and isolation by distance, as in other diseases in the Finnish heritage.

Exclusion of mutations in the gene for type III collagen (COL3A1) as a common cause of intracranial aneurysms or cervical artery dissections: results from sequence analysis of the coding sequences of type III collagen from 55 unrelated patients.

We performed detailed DNA sequencing analysis on type III collagen cDNA from 58 patients with either intracranial artery aneurysms or cervical artery dissections. The 58 patients were of seven different nationalities; among the patients were three pairs of relatives, so that 55 were unrelated, and of these, 29 had at least one blood relative with either an intracranial artery aneurysm or a cervical artery dissection. The age of the patients at the time of diagnosis ranged from 15 to 68 years (mean +/- SD = 40.3 +/- 11.0). The study group consisted of 25 males and 33 females. The analysis covered 3,232 nucleotides of significant (nonredundant) sequences per allele; therefore, we analyzed as many as 355,520 nucleotides. Mutations in the coding sequences for the triple-helical domain of type III collagen were excluded in 40 individuals with intracranial aneurysms and 18 individuals with cervical artery dissections. Direct sequencing of polymerase chain reaction products allowed mutations to be excluded with a high degree of confidence. Mutations that markedly decreased expression from one allele were also excluded in 42 of the 58 individuals, since the presence of both bases at one or more polymorphic sites in the 42 patients showed that two alleles were transcribed. The results indicated that mutations in the gene for type III procollagen (COL3A1) are not a common cause of either intracranial artery aneurysms or cervical artery dissections.

The effect of proteoglycans, collagen and lysyl oxidase on the metabolism of low density lipoprotein by macrophages.

To study the interactions of lipoproteins, connective tissue components and cells, mouse peritoneal macrophages were incubated in the presence of human low density lipoproteins (LDL) that had been complexed with pig aortic proteoglycans (PG) or incubated in the presence of soluble collagen and/or lysyl oxidase, which catalyses the formation of cross-linkages in collagen and elastin by oxidising epsilon-amino groups of lysine residues to aldehydes. Soluble and insoluble PG-LDL complexes increased the incorporation of [3H]oleate into cellular cholesteryl esters (CE) 1.6- and 2.8-fold, respectively, while LDL incubated with collagen and lysyl oxidase had no effect compared to control LDL. As judged on the basis of incubations with fucoidin, spermine and 125I-labelled lipoproteins, the mechanism of internalisation of the PG-LDL complexes is different from that of acetylated LDL or dextran sulphate-LDL complexes. The formation of PG-LDL complexes in the arterial intima may lead to an increased uptake of lipoproteins by intimal macrophages during the early phase of atherogenesis.

Robotic automation of dideoxyribonucleotide sequencing reactions.

We developed a robot to carry out standard Sanger dideoxyribonucleotide sequencing reactions efficiently and with minimal human intervention. A commercial robot was adapted to our design and specifications, and we programmed it to perform up to 240 sequencing reactions in a single unattended run of 7 h. The robot configuration can be easily altered to allow 480 reactions to be performed in an unattended run of 14 h. The special features of our robot include cooled reagent reservoirs and cooled chambers for storage of DNA templates and completed reactions as well as reproducible aspiration of small volumes by using a sensing algorithm. The robot has successfully performed over 3500 DNA reactions in about 30 separate runs in our DNA core facility.

Substitution of arginine for glycine at position 154 of the alpha 1 chain of type I collagen in a variant of osteogenesis imperfecta: comparison to previous cases with the same mutation.

A substitution of arginine for glycine at amino acid position 154 of the alpha 1(I) collagen chain was found in a father and his three children. The phenotype of the patients includes manifestations of types I and III/IV osteogenesis imperfecta, but appears to be milder than that of the previously described two unrelated patients that had the identical mutation in the alpha 1(I) collagen chain. The variability in the phenotype raises the possibility of epistatic loci or environmental effects on expression of the disorder.