First genome-wide association study investigating blood pressure and renal traits in domestic cats.

Hypertension (HTN) and chronic kidney disease (CKD) are common in ageing cats. In humans, blood pressure (BP) and renal function are complex heritable traits. We performed the first feline genome-wide association study (GWAS) of quantitative traits systolic BP and creatinine and binary outcomes HTN and CKD, testing 1022 domestic cats with a discovery, replication and meta-analysis design. No variants reached experimental significance level in the discovery stage for any phenotype. Follow up of the top 9 variants for creatinine and 5 for systolic BP, one SNP reached experimental-wide significance for association with creatinine in the combined meta-analysis (chrD1.10258177; P = 1.34 × 10-6). Exploratory genetic risk score (GRS) analyses were performed. Within the discovery sample, GRS of top SNPs from the BP and creatinine GWAS show strong association with HTN and CKD but did not validate in independent replication samples. A GRS including SNPs corresponding to human CKD genes was not significant in an independent subset of cats. Gene-set enrichment and pathway-based analysis (GSEA) was performed for both quantitative phenotypes, with 30 enriched pathways with creatinine. Our results support the utility of GWASs and GSEA for genetic discovery of complex traits in cats, with the caveat of our findings requiring validation.

Uromodulin gene variants and their association with renal function and blood pressure in cats: a pilot study.

OBJECTIVES: In humans, genome-wide association studies have identified variants in the uromodulin gene (UMOD) associated with blood pressure and renal function. This study aimed to evaluate the association of single nucleotide polymorphisms at the UMOD locus with renal function and blood pressure in cats. METHODS: We retrospectively identified cats aged 14 years that had participated in a geriatric monitoring program, and from which stored DNA samples were available, from a computerised database. We then measured the association of specific single nucleotide polymorphisms in the feline UMOD gene with renal function and systolic blood pressure as continuous variables and, also, the dichotomous outcome of azotaemic chronic kidney disease and systemic hypertension. RESULTS: Eight intronic single nucleotide polymorphisms, one 1372 base pairs upstream from UMOD and two exonic single nucleotide polymorphisms were evaluated in 227 cats with renal and blood pressure data. An analysis of 188 cats found four single nucleotide polymorphisms to be significantly associated (P<0·01) with systolic blood pressure although all were in linkage disequilibrium. No significant associations were identified between single nucleotide polymorphisms and renal function or chronic kidney disease. CLINICAL SIGNIFICANCE: Results of this pilot study suggest that genetic variation in UMOD might influence blood pressure in cats, similar to findings in humans. Validation of these results is required.

Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1ß and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

High quality genomic DNA extraction from postmortem fetal tissue.

OBJECTIVE: We examined the yield and quality of genomic deoxyribonucleic acid (DNA) extracted from various postmortem fetal tissues. METHODS: Fetal tissues were collected at the time of autopsy, and DNA was subsequently extracted. The yield and DNA quality was assessed using ultraviolet spectrometry and agarose gel electrophoresis. We used polymerase chain reaction (PCR) to assess the DNA extracted for genomic testing. RESULTS: The median (range) gestation of the fetuses was 22 (16-41) weeks and the postmortem interval was 5.5 (2-10) days. Non-degraded genomic DNA was successfully extracted from all fetal tissues. Liver tissue had the lowest quality and muscle the highest quality. DNA yield or purity was not influenced by the postmortem interval. CONCLUSION: High quality genomic DNA can be extracted from fetal muscle, despite postmortem intervals of several days.

Genes and hypertension.

The combination of investigation of rare Mendelian forms of hypertension, candidate gene studies, comparative mapping and genome-wide screening in both animal models and man has led to significant progress in determining new mechanisms of blood pressure control. In this review, the newly discovered blood pressure/cardiovascular genes, WNK kinases and angiotensin converting enzyme 2 and the development of a new anti-hypertensive agent PST2238 are discussed. Major genes causing essential hypertension have yet to be discovered, however, there are now over 20 published genome-wide screens for blood pressure controlling genes. Several regions demonstrate suggestive linkage to the trait and there is some overlap of regions between the different studies. It is hoped that new blood pressure genes will ultimately be discovered using this method. Pharmacogenetic studies in hypertension have only been initiated recently, some are described in this paper. Small studies upon single candidate genes, suggest that the contribution of genetics to the inter-individual variation in blood pressure response to anti-hypertensive therapy, is small, approximately 3-5%. Recently micro-arrays with multiple polymorphisms in multiple genes have been used. After accounting for the additive affects of multiple blood pressure loci, an individual's genetic profile appeared to explain up to 50% of the variation in blood pressure response to therapy. Knowledge of the genetic variants that cause hypertension and influence response to anti-hypertensive therapy will ultimately provide a greater understanding of the molecular mechanisms underlying blood pressure control.

Human chromosome 17 in essential hypertension.

Hypertension affects up to 30% of the adult population in Western societies and is a major risk factor for kidney disease, stroke and coronary heart disease. It is a complex trait thought to be influenced by a number of genes and environmental factors, although the precise aetiology remains unknown at this time. A number of methods have been successfully used to identify mutations that cause Mendelian traits and these are now being applied to the investigation of complex diseases. This review summarises the data gathered, using such approaches, that suggest there is a gene or genes on chromosome 17 causing human essential hypertension. Studies in rodent models are discussed first, followed by studies of human hypertension that include the investigation of pseudohypoaldosteronism type II, a monogenic trait that manifests with hypertension alongside other phenotypic variables. In addition, candidate gene studies, genome screens and linkage studies based on comparative mapping are outlined. To date no gene has been identified on human chromosome 17 that influences blood pressure and causes human essential hypertension. However, results of ongoing fine mapping and candidate gene studies in both rodents and man are eagerly awaited.

Identification of a transactivation motif in the CLN3 protein.

A transactivation motif has been identified in the neurodegenerative disease protein, CLN3. The C-terminal domain (residues 394-438) of CLN3 can function as a transcriptional activator when fused to the DNA binding domain, LexA. A series of deletion and substitution constructs have been generated to identify the essential region for transactivation. A similar motif is also present in the POU domain transcription factor, nubbin. However, this domain alone does not activate transcription, allowing further localisation of the critical residues in CLN3 required for activity.

Analysis of CLN3-protein interactions using the yeast two-hybrid system.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood neurodegenerative disease that is caused by mutations in the CLN3 gene. The protein encoded by CLN3 has no homology with any proteins of known function and its cellular role remains elusive. In order to investigate the role played by the CLN3 protein we aimed to identify interacting proteins. Here, we describe the yeast two-hybrid system as the approach taken to investigate such protein-protein interactions. CLN3 was expressed as a fusion protein with a DNA-binding domain and used to screen a library of human fetal brain cDNAs fused to a transcriptional activation domain. Owing to low level expression of the full length CLN3 fusion protein, truncated regions corresponding to the predicted hydrophilic regions were also tested. No proteins that interact with CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions. Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the major component of the storage material that accumulates in Batten disease patients, was also tested. No interaction was detected suggesting that the accumulation of subunit c does not result from loss of a process that requires a direct interaction with CLN3. We conclude that either CLN3 does not interact with other proteins or such interactions cannot be detected using the two-hybrid system.

Pseudoexon activation as a novel mechanism for disease resulting in atypical growth-hormone insensitivity.

Inherited growth-hormone insensitivity (GHI) is a heterogeneous disorder that is often caused by mutations in the coding exons or flanking intronic sequences of the growth-hormone receptor gene (GHR). Here we describe a novel point mutation, in four children with GHI, that leads to activation of an intronic pseudoexon resulting in inclusion of an additional 108 nt between exons 6 and 7 in the majority of GHR transcripts. This mutation lies within the pseudoexon (A(-1)-->G(-1) at the 5' pseudoexon splice site) and, under in vitro splicing conditions, results in inclusion of the mutant pseudoexon, whereas the wild-type pseudoexon is skipped. The presence of the pseudoexon results in inclusion of an additional 36-amino acid sequence in a region of the receptor known to be involved in homo-dimerization, which is essential for signal transduction.

1990-2000: progress in determining high blood pressure genes.

INTRODUCTION: This article attempts to summarise the genetic research that has taken place during the past decade to determine the identity of genes causing high blood pressure. METHODS: Candidate gene studies and genome-wide scanning have been the methods primarily employed, and studies have been performed in both experimental models (rats and mice) and human volunteers (sibling-pairs and case-control). Key studies from the past 10 years are discussed, in addition to the congenic strains. RESULTS: Genome-wide scans and candidate gene studies in both rat and man have generated many chromosomal regions and loci involved in blood pressure regulation. However, much work is still required to fine map the large chromosomal regions found in the genome-wide scans and to isolate variants in candidate genes and prove that they are disease-causing. CONCLUSIONS: It is anticipated that within the next 5 to 10 years at least one blood pressure susceptibility gene will be identified in rat and possibly some in man. It is hoped that the identification of genes controlling blood pressure will enable investigators to determine physiological/biochemical pathways defective in hypertensive patients. This information may then be utilised to identify specific hypertensive phenotypes, to tailor therapy appropriately for patients and hopefully to develop novel therapeutic agents for hypertension.

Full-field ERG in patients with Batten/Spielmeyer-Vogt disease caused by mutations in the CLN3 gene.

PURPOSE: To investigate, using full-field ERG, the retinal function in patients with Batten/Spielmeyer-Vogt disease caused by mutations in the CLN(3) gene. METHODS: Batten disease status of five patients was confirmed by the presence of vacuolated lymphocytes in peripheral blood and the identification of mutations in the Batten disease gene (CLN(3)). Visual acuity, fundus appearance, and full-field ERG were examined in all patients (age 4-19 years). The examination was repeated in one patient after 16 months. RESULTS: Three unrelated patients were homozygous for the most common mutation in CLN(3), the 1.02 kb deletion; two patients (sisters) were heterozygous for the 1.02 kb deletion and an as yet unidentified mutation in the CLN(3) gene. Full-field ERG recordings in all five patients demonstrated no rod responses and only small remaining cone responses, which could be detected with 30 Hz-flicker stimulation. Re-examination of a six-year-old girl after 16 months revealed a fast progression of the retinal degeneration. CONCLUSION: Full-field ERG recordings in Batten disease patients, both homozygous and heterozygous for the 1.02 kb deletion in the CLN( 3) gene, confirm retinal degeneration to be severe, widespread, and with a rapid progression early in the disease course. The onset of visual failure may be delayed when compared to the classic disease course, particularly in patients who are not homozygous for the most common CLN(3) mutation, a 1.02 kb deletion. In that case, the disease progression in terms of other symptoms may also be further delayed.

Genetics of hypertension.

In the past year, substantial progress has been made in both mapping and fine mapping the genes involved in blood pressure regulation. Genome scans have been carried out in humans and mice and these reveal many new potential chromosomal locations for blood pressure susceptibility loci. The chromosomal regions containing blood pressure genes for many of the inbred hypertensive rat models have been refined using new congenic strains. Further genetic studies support a role for antiotensinogen, aldosterone synthase and a region close to the epithelial sodium channel in blood pressure regulation. Finally, comprehensive single-nucleotide polymorphism analysis of cardiovascular genes has been undertaken using chip technology.

Molecular basis of the neuronal ceroid lipofuscinoses: mutations in CLN1, CLN2, CLN3, and CLN5.

The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a group of neurodegenerative disorders characterised by the accumulation of an autofluorescent lipopigment in many cell types. Different NCL types are distinguished according to age of onset, clinical phenotype, ultrastructural characterisation of the storage material, and chromosomal location of the disease gene. At least eight genes underlie the NCLs, of which four have been isolated and mutations characterised: CLN1, CLN2, CLN3, CLN5. Two of these genes encode lysosomal enzymes, and two encode transmembrane proteins, at least one of which is likely to be in the lysosomal membrane. The basic defect in the NCLs appears to be associated with lysosomal function.

The molecular basis of GROD-storing neuronal ceroid lipofuscinoses in Scotland.

Two distinct clinical subtypes of neuronal ceroid lipofuscinosis caused by mutations in the PPT gene, INCL and vJNCL/GROD, occur at a high frequency in the central region of Scotland. In this paper we summarize the clinical details and the molecular basis underlying the disease in the Scottish patients. Comparison of the combination of mutations in the different clinical types reveals a clear genotype-phenotype correlation.

Delayed classic and protracted phenotypes of compound heterozygous juvenile neuronal ceroid lipofuscinosis.

OBJECTIVE: To correlate the phenotypes with the genotypes of 10 Finnish juvenile neuronal ceroid lipofuscinosis (JNCL; late-onset Batten disease) patients who all are compound heterozygotes for the major 1.02-kb deletion in the CLN3 gene. METHODS: The mutations on the non-1.02-kb deletion chromosomes were screened in 6 patients; in the other 4 patients the mutations were known (one affecting a splice site, two missense mutations, and one deletion of exons 10 through 13). Clinical features were examined, and MRI, MRS, somatosensory evoked magnetic field (SEF), and overnight polysomnography (PSG) studies were performed. RESULTS: A novel deletion of exons 10 through 13 was found in 6 patients belonging to three families. In the patients carrying the deletions of exons 10 through 13 the clinical course of the disease was fairly similar. Variation was greatest in the time course to blindness. In these patients the mental and motor decline was slower than in classic JNCL, but more severe than in the two patients with missense mutations in exons 11 and 13. MRI showed brain atrophy in 4 patients. One patient had hyperintense periventricular white matter, otherwise brain signal intensities were normal. SEFs were enhanced in patients older than 14 years, whereas in PSG all but the youngest 6-year-old patient showed epileptiform activity in slow-wave sleep. CONCLUSIONS: JNCL can manifest as at least three different phenotypes: classic, delayed classic, and protracted JNCL with predominantly ocular symptoms. Finnish compound heterozygotes have the delayed classic or the protracted form of JNCL.

Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome.

Keutel syndrome (KS, MIM 245150) is an autosomal recessive disorder characterized by abnormal cartilage calcification, peripheral pulmonary stenosis and midfacial hypoplasia. A genome search using homozygosity mapping provided evidence of linkage to chromosome 12p12.3-13.1 (maximum multipoint lod score, 4.06). MGP was a candidate on the basis of its localization to this chromosomal region and the known function of its protein. MGP maps to chromosome 12p near D12S363. Human MGP is a 10-kD skeletal extracellular matrix (ECM) protein that consists of an 84-aa mature protein and a 19-aa transmembrane signal peptide. It is a member of the Gla protein family, which includes osteocalcin, another skeletal ECM protein, and a number of coagulation factors (factors II, VII, IX, X and proteins S and C). All members of this family have glutamic acid residues modified to gamma-carboxyglutamic acids (Gla) by a specific gamma-carboxylase using vitamin K as a cofactor. The modified glutamic acid residues of Gla proteins confer a high affinity for mineral ions such as calcium, phosphate and hydroxyapatite crystals, the mineral components of the skeletal ECM. The pattern and tissue distribution of Mgp expression in mice suggest a role for Mgp in regulating ECM calcification. Mglap-deficient mice (Mglap-/-) have been reported to have inappropriate calcification of cartilage. Mutational analysis of MGP in three unrelated probands identified three different mutations: c.69delG, IVS1-2A-->G and c.113T-->A. All three mutations predict a non-functional MGP. Our data indicate that mutations in MGP are responsible for KS and confirm its role in the regulation of extracellular matrix calcification.

Absence of linkage of the epithelial sodium channel to hypertension in black Caribbeans.

Hypertensives of African origin have low-renin, sodium-sensitive blood pressure and respond poorly to treatment with angiotensin converting enzyme inhibitors. The epithelial sodium channel may be important in the pathogenesis of essential hypertension in this population. This is supported by the identification of mutations within this channel, which lead to excess sodium reabsorption and hypertension in Liddle's syndrome. In this study we tested whether there was linkage of the genes encoding the three subunits of the epithelial sodium channel to essential hypertension in 63 affected sibling pairs of West African origin from St. Vincent and the Grenadines. We found no support for linkage of the epithelial sodium channel to essential hypertension in this population. However, further studies will be needed in larger populations of African ancestry to exclude a contribution of the genes encoding the epithelial sodium channel to hypertension.

Mutations in the palmitoyl-protein thioesterase gene (PPT; CLN1) causing juvenile neuronal ceroid lipofuscinosis with granular osmiophilic deposits.

A subtype of neuronal ceroid lipofuscinosis (NCL) is well recognized which has a clinical course consistent with juvenile NCL (JNCL) but the ultrastructural characteristics of infantile NCL (INCL): granular osmiophilic deposits (GROD). Evidence supporting linkage of this phenotype, designated vJNCL/GROD, to the INCL region of chromosome 1p32 was demonstrated (pairwise lod score with D1S211 , Z max = 2.63, straight theta = 0.00). The INCL gene, palmitoyl-protein thioesterase (PPT ; CLN1), was therefore screened for mutations in 11 vJNCL/GROD families. Five mutations in the PPT gene were identified: three missense mutations, Thr75Pro, Asp79Gly, Leu219Gln, and two nonsense mutations, Leu10STOP and Arg151STOP. The missense mutation Thr75Pro accounted for nine of the 22 disease chromosomes analysed and the nonsense mutation Arg151STOP for seven. Nine out of 11 patients were shown to combine a missense mutation on one disease chromosome with a nonsense mutation on the other. Mutations previously identified in INCL were not observed in vJNCL/GROD families. Thioesterase activity in peripheral blood lymphoblast cells was found to be markedly reduced in vJNCL/GROD patients compared with controls. These results demonstrate that this subtype of JNCL is allelic to INCL and further emphasize the correlation which exists between genetic basis and ultrastructural changes in the NCLs.

Spectrum of mutations in the Batten disease gene, CLN3.

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis [JNCL]) is an autosomal recessive condition characterized by accumulation of lipopigments (lipofuscin and ceroid) in neurons and other cell types. The Batten disease gene, CLN3, was recently isolated, and four disease-causing mutations were identified, including a 1.02-kb deletion that is present in the majority of patients (The International Batten Disease Consortium 1995). One hundred eighty-eight unrelated patients with JNCL were screened in this study to determine how many disease chromosomes carried the 1.02-kb deletion and how many carried other mutations in CLN3. One hundred thirty-nine patients (74%) were found to have the 1.02-kb deletion on both chromosomes, whereas 49 patients (41 heterozygous for the 1.02-kb deletion) had mutations other than the 1.02-kb deletion. SSCP analysis and direct sequencing were used to screen for new mutations in these individuals. Nineteen novel mutations were found: six missense mutations, five nonsense mutations, three small deletions, three small insertions, one intronic mutation, and one splice-site mutation. This report brings the total number of disease-associated mutations in CLN3 to 23. All patients homozygous for mutations predicted to give rise to truncated proteins were found to have classical JNCL. However, a proportion of the patients (n = 4) who were compound heterozygotes for a missense mutation and the 1.02-kb deletion were found to display an atypical phenotype that was dominated by visual failure rather than by severe neurodegeneration. All missense mutations were found to affect residues conserved between the human protein and homologues in diverse species.