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.

The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42.

BACKGROUND: Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF. METHODS: Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation. RESULTS: Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16. CONCLUSION: These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.

Development of a novel compact sonicator for cell disruption.

Ultrasound microbial cell disrupters operating at around 20 kHz are often physically large and, due to significant heating, can be unsuitable for small sample volumes where biochemical integrity of the extracted product is required. Development of a compact device based on a 63.5-mm diameter, 6.5-mm thick tubular transducer for rapid cell disruption in small-volume samples in a high-intensity acoustic cavitation field with minimal temperature rises is described here. Suspensions of Saccharomyces cerevisiae were exposed to cavitation for various times in the compact device and a 20-kHz probe sonicator. Cell disruption was assessed by protein release and by staining. Yeast cell disruption was greater in the novel 267-kHz sonicator than in the 20-kHz probe sonicator for the same exposure time. A 1-dimensional (1-D) transfer matrix model analysis for piezoelectric resonators was applied to an axial cross-section of the tubular sonicator to predict frequencies of mechanical resonance in the sample volume associated with maximum acoustic pressure. Admittance measurements identified frequencies of electrical resonance. Ultrasonic cavitation noise peaks were detected by a hydrophone at both the mechanical and electrical resonances. Cell breakage efficiency was twice as great in terms of protein released per dissipated watt at the mechanical resonance predicted by the model, compared to those at the electrical resonance frequencies. The results form a basis for rational design of an ultrasound cell disruption technique for small-volume samples.

Cell-cell contact and membrane spreading in an ultrasound trap.

An ultrasonic standing wave trap [Langmuir 19 (2003) 3635] in which the morphologies of 2-D latex-microparticle aggregates, forming a pressure node plane, were characterised has been applied here to different cell suspensions with increasing order of specificity of cross-linking molecule, i.e. polylysine with chondrocytes; wheat germ agglutinin (WGA) with erythrocytes and surface receptors on neural cells. The outcome of initial cell-cell contact, i.e. whether the cells stuck at the point of contact (collision efficiency = 1) or rolled around each other (collision efficiency = 0), was monitored in situ by video-microscopy. The perimeter fractal dimensions (FD) of 2-D hexagonally symmetric, closely packed aggregates of control erythrocytes and chondrocytes were 1.16 and 1.18, respectively while those for the dendrititc aggregates formed initially by erythrocytes in 0.5microg/ml WGA and chondrocytes in 20 microg/ml polylysine were 1.49 and 1.66. The FDs for control and molecularly cross-linked cells were typical of reaction-limited aggregation (RLA) and transport diffusion-limited aggregation (DLA), respectively. The FDs of the aggregates of cross-linked cells decreased with time to give more closely packed aggregates without clear hexagonal symmetry. Suspensions of neural cells formed dendritic aggregates. Spreading of inter-cellular membrane contact area occurred over 15 min for both erythrocyte and neural cell dendritic aggregates. The potential of the technique to characterise and control the progression of cell adhesion in suspension away from solid substrata is discussed.

A phenocopy of CAII deficiency: a novel genetic explanation for inherited infantile osteopetrosis with distal renal tubular acidosis.

The rare bone thickening disease osteopetrosis occurs in various forms, one of which is accompanied by renal tubular acidosis (RTA), and is known as Guibaud-Vainsel syndrome or marble brain disease. Clinical manifestations of this autosomal recessive syndrome comprise increased bone density, growth failure, intracerebral calcification, facial dysmorphism, mental retardation, and conductive hearing impairment. The most common cause is carbonic anhydrase II (CAII) deficiency. Several different loss of function mutations in CA2, the gene encoding CAII, have been described. To date, there have been no exceptions to the finding of CAII deficiency in patients with coexistent osteopetrosis and RTA. Most often, the RTA is of mixed proximal and distal type, but kindreds are reported in which either distal or proximal RTA predominates. We report the molecular genetic investigation of two consanguineous kindreds where osteopetrosis and distal RTA (dRTA) were both manifest. One kindred harbours a novel homozygous frameshift alteration in CA2. In the other, CAII levels were normal despite a similar clinical picture, and we excluded defects in CA2. In this kindred, two separate recessive disorders are penetrant, each affecting a different, tissue specific subunit of the vacuolar proton pump (H(+)-ATPase), providing a highly unusual, novel genetic explanation for the coexistence of osteopetrosis and dRTA. The osteopetrosis is the result of a homozygous deletion in TCIRG1, which encodes an osteoclast specific isoform of subunit a of the H(+)-ATPase, while the dRTA is associated with a homozygous mutation in ATP6V1B1, encoding the kidney specific B1 subunit of H(+)-ATPase. This kindred is exceptional firstly because the coinheritance of two rare recessive disorders has created a phenocopy of CAII deficiency, and secondly because these disorders affect two different subunits of the H(+)-ATPase that have opposite effects on bone density, but which have only recently been determined to possess tissue specific isoforms.

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss.

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.