Colleen A. Campbell

Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling.

Non‐typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet‐activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca2+ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell‐permeant intracellular Ca2+ chelator BAPTA‐AM (P = 0.022) or thapsigargin (P = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 (P < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019‐stimulated IP production in a dose‐dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol‐3‐kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX‐sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.

Linkage of otosclerosis to a third locus (OTSC3) on human chromosome 6p21.3-22.3

Clinical otosclerosis (OMIM 166800/605727) has a prevalence of 0.2-1% among white adults, making it the single most common cause of hearing impairment in this group. It is caused by abnormal bone homeostasis of the otic capsule with the consequent development of sclerotic foci that invade the stapedio-vestibular joint (oval window) interfering with free motion of the stapes. Impaired ossicular chain mobility results in a conductive hearing loss. We identified the first locus for otosclerosis (OTSC1) on chromosome 15 in 1998 and reported a second locus (OTSC2) on chromosome 7 last year. Here we present results of a genome wide linkage study on a large Cypriot family segregating otosclerosis. Results of this study exclude linkage to OTSC1 and OTSC2 and identify a third locus, OTSC3, on chromosome 6p. The defined OTSC3 interval covers the HLA region, consistent with reported associations between HLA-A/HLA-B antigens and otosclerosis.

Polymorphisms in KCNE1 or KCNE3 are not associated with Ménière disease in the Caucasian population.

Ménière disease (MD) is a complex disorder of unknown etiology characterized by the symptom triad of vertigo, sensorineural hearing loss, and tinnitus. Its reported incidence is 1–2 per 1,000 in Caucasians and 0.03–0.37 per 1,000 in Japanese. Doi et al. [Doi et al. ( 2005 ); ORL J Otorhinolaryngol Relat Spec 67:289–293] recently reported that two single nucleotide polymorphisms (SNPs) in KCNE1 and KCNE3 are associated with MD in Japanese subjects. Consistent with this possibility, these two genes encode potassium channels that are expressed in the stria vascularis and endolymphatic sac, respectively, and their role in ion transport suggests that they may be important in inner ear homeostasis. To establish whether a similar association exists in the Caucasian MD population, we sequenced the coding regions and exon–intron boundaries of both genes in 180 Caucasian persons with MD and 180 matched Caucasian controls. Neither of the two reported SNPs was significantly associated with MD when compared to the Caucasian controls (KCNE1, P = 0.55; KCNE3, P = 0.870). Comparison of allele frequencies between the Japanese MD population and our study population revealed no significant difference between groups (KCNE1, P = 0.90; KCNE3, P = 0.862), suggesting that the significant differences reported in the Japanese study arose from their control population. Six additional SNPs in both KCNE1 and KCNE3 were genotyped and none was associated with MD. Population stratification within our MD and Caucasian control population was excluded. Our data show that SNPs in KCNE1 and KCNE3 are not associated with MD in Caucasians.

Mutations in apoptosis-inducing factor cause X-linked recessive auditory neuropathy spectrum disorder

Background Auditory neuropathy spectrum disorder (ANSD) is a form of hearing loss in which auditory signal transmission from the inner ear to the auditory nerve and brain stem is distorted, giving rise to speech perception difficulties beyond that expected for the observed degree of hearing loss. For many cases of ANSD, the underlying molecular pathology and the site of lesion remain unclear. The X-linked form of the condition, AUNX1, has been mapped to Xq23-q27.3, although the causative gene has yet to be identified. Methods We performed whole-exome sequencing on DNA samples from the AUNX1 family and another small phenotypically similar but unrelated ANSD family. Results We identified two missense mutations in AIFM1 in these families: c.1352G>A (p.R451Q) in the AUNX1 family and c.1030C>T (p.L344F) in the second ANSD family. Mutation screening in a large cohort of 3 additional unrelated families and 93 sporadic cases with ANSD identified 9 more missense mutations in AIFM1. Bioinformatics analysis and expression studies support this gene as being causative of ANSD. Conclusions Variants in AIFM1 gene are a common cause of familial and sporadic ANSD and provide insight into the expanded spectrum of AIFM1-associated diseases. The finding of cochlear nerve hypoplasia in some patients was AIFM1-related ANSD implies that MRI may be of value in localising the site of lesion and suggests that cochlea implantation in these patients may have limited success.

Gene expression profiling analysis of the inner ear.

Recent developments in molecular genetics, including progress in the human genome project, have allowed identification of genes at an unprecedented rate. To date gene expression profiling studies have focused on identifying transcripts that are specifically or preferentially enriched within the inner ear on the assumption that they are more likely to be important for auditory and vestibular function. It is now apparent that some genes preferentially expressed in the cochleo-vestibular system are not crucial for hearing or balance or their functions are compensated for by other genes. In addition, transcripts expressed at low abundance in the inner ear are generally under-represented in gene profiling studies. In this review, we highlight the limitations of current gene expression profiling strategies as a discovery tool for genes involved in cochleo-vestibular development and function. We argue that expression profiling based on hierarchical clustering of transcripts by gene ontology, combined with tissue enrichment data, is more effective for inner ear gene discovery. This approach also provides a framework to assist and direct the functional characterization of gene products.

Functional Variants in NOS1 and NOS2A Are Not Associated with Progressive Hearing Loss in Ménière's Disease in a European Caucasian Population

Hearing loss in Ménières disease (MD) is associated with loss of spiral ganglion neurons and hair cells. In a guinea pig model of endolymphatic hydrops, nitric oxide synthases (NOS) and oxidative stress mediate loss of spiral ganglion neurons. To test the hypothesis that functional variants of NOS1 and NOS2A are associated with MD, we genotyped three functional variants of NOS1 (rs41279104, rs2682826, and a cytosine-adenosine microsatellite repeat in exon 1f) and the CCTTT repeat in the promoter of NOS2A gene (rs3833912) in two independent MD sets (273 patients in total) and 550 controls. A third cohort of American patients was genotyped as replication cohort for the CCTTT repeat. Neither allele nor genotype frequencies of rs41279104 and rs2682826 were associated with MD, although longer alleles of the cytosine-adenosine microsatellite repeat were marginally significant (corrected p = 0.05) in the Mediterranean cohort but not in a second Galicia cohort. Shorter numbers of the CCTTT repeat in NOS2A were significantly more frequent in Galicia controls (OR = 0.37 [CI, 0.18-0.76], corrected p = 0.04), but this finding could not be replicated in Mediterranean or American case-control populations. Meta-analysis did not support an association between CCTTT repeats and risk for MD. Severe hearing loss (>75 dB) was also not associated with any functional variants studied. Functional variants of NOS1 and NOS2A do not confer susceptibility for MD.

Ketogenic diet – A novel treatment for early epileptic encephalopathy due to PIGA deficiency

We describe the presentation and workup of two brothers with early-onset epileptic encephalopathy who became seizure-free on a ketogenic diet. Extensive testing culminated in whole exome sequencing, which led to the diagnosis of phosphatidyl inositol glycan biosynthesis class A protein (PIGA) deficiency. This familial case highlights the importance of genetic testing for early-onset epileptic encephalopathies and underscores the potential value of a ketogenic diet in the treatment of this condition.

DNM1 encephalopathy A new disease of vesicle fission

Objective: To evaluate the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling. Methods: We reviewed phenotypic data of 21 patients (7 previously published) with DNM1 mutations. We compared mutation data to known functional data and undertook biomolecular modeling to assess the effect of the mutations on protein function. Results: We identified 19 patients with de novo mutations in DNM1 and a sibling pair who had an inherited mutation from a mosaic parent. Seven patients (33.3%) carried the recurrent p.Arg237Trp mutation. A common phenotype emerged that included severe to profound intellectual disability and muscular hypotonia in all patients and an epilepsy characterized by infantile spasms in 16 of 21 patients, frequently evolving into Lennox-Gastaut syndrome. Two patients had profound global developmental delay without seizures. In addition, we describe a single patient with normal development before the onset of a catastrophic epilepsy, consistent with febrile infection-related epilepsy syndrome at 4 years. All mutations cluster within the GTPase or middle domains, and structural modeling and existing functional data suggest a dominant-negative effect on DMN1 function. Conclusions: The phenotypic spectrum of DNM1-related encephalopathy is relatively homogeneous, in contrast to many other genetic epilepsies. Up to one-third of patients carry the recurrent p.Arg237Trp variant, which is now one of the most common recurrent variants in epileptic encephalopathies identified to date. Given the predicted dominant-negative mechanism of this mutation, this variant presents a prime target for therapeutic intervention.

Screening of living kidney donors for genetic diseases using a comprehensive genetic testing strategy

Related living kidney donors (LKDs) are at higher risk of end‐stage renal disease (ESRD) compared with unrelated LKDs. A genetic panel was developed to screen 115 genes associated with renal diseases. We used this panel to screen six negative controls, four transplant candidates with presumed genetic renal disease and six related LKDs. After removing common variants, pathogenicity was predicted using six algorithms to score genetic variants based on conservation and function. All variants were evaluated in the context of patient phenotype and clinical data. We identified causal variants in three of the four transplant candidates. Two patients with a family history of autosomal dominant polycystic kidney disease segregated variants in PKD1. These findings excluded genetic risk in three of four relatives accepted as potential LKDs. A third patient with an atypical history for Alport syndrome had a splice site mutation in COL4A5. This pathogenic variant was excluded in a sibling accepted as an LKD. In another patient with a strong family history of ESRD, a negative genetic screen combined with negative comparative genomic hybridization in the recipient facilitated counseling of the related donor. This genetic renal disease panel will allow rapid, efficient and cost‐effective evaluation of related LKDs.

Ultrasound visualization of ingested tablets: a pilot study

Study Objective. To evaluate the ability of ultrasonography as a radiographic technique to visualize ingested tablets in the human stomach.