A splice site and copy number variant responsible for TTC25-related primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. With few exceptions, PCD is an autosomal recessive condition, and there are over 40 genes associated with the condition. We present a case of a newborn female with clinical features of PCD, specifically the Kartagener syndrome phenotype, due to variants in TTC25. This gene has been previously associated with PCD in three families. Two multi-gene panels performed as a neonate and at two years of age were uninformative. Exome sequencing was performed by the Care4Rare Canada Consortium on a research basis, and an apparent homozygous intronic variant (TTC25:c.1145+1G > A) was identified that was predicted to abolish the canonical splice donor activity of exon 8. The child's mother was a heterozygous carrier of the variant. The paternal sample did not show the splice variant, and homozygosity was observed across the paternal locus. Microarray analysis showed a 50 kb heterozygous deletion spanning the genes TTC25 and CNP. This is the first example of a pathogenic gross deletion in trans with a splice variant, resulting in TTC25-related PCD.

Mining the transcriptome for rare disease therapies: a comparison of the efficiencies of two data mining approaches and a targeted cell-based drug screen.

Most monogenic diseases can be viewed as conditions caused by dysregulated protein activity; therefore, drugs can be used to modulate gene expression, and thus protein level, possibly conferring clinical benefit. When considering repurposing drugs for loss of function diseases, there are three classes of genetic disease amenable to an increase of function; haploinsufficient dominant diseases, those secondary to hypomorphic recessive alleles, and conditions with rescuing paralogs. This therapeutic model then brings the questions: how frequently do such clinically useful drug-gene interactions occur and what is the most rapid and efficient route by which to identify them. Here we compare three approaches: (1) mining of pre-existing system-wide transcriptomal datasets such as Connectivity Map; (2) utilization of a proprietary causal reasoning engine knowledge base; and, (3) a targeted drug screen using clinically accepted agents tested against normal human fibroblasts. We have determined the validation rate of these approaches for 76 diseases (i.e., in vitro fibroblast mRNA increase); for the Connectivity Map, approximately 5% of tested putative drug-gene interactions validated, for causal reasoning engine knowledge base the rate was 10%, and for the targeted drug screen 9%. The degree of overlap between these methodologies was low suggesting they are complementary not redundant approaches to identify putative drug-gene interactions. Although the validation rate was low, a number of drug-gene interactions were successfully identified and are now being investigated for protein induction and in vivo effect. This analysis establishes potentially valuable therapeutic leads as well as useful benchmarks for the thousands of currently untreatable rare genetic conditions.

Stroke discharges from a rehabilitation unit: 1-year and 5-year domicile outcomes. Function is important.

BACKGROUND: Stroke units save lives, reduce disability and increase the chances of the person returning to their own home. Following the introduction of a stroke rehabilitation unit, we assessed the durability of stroke discharges over a 1-year period and predictors of early 'failed' home discharges. Stability of discharge domicile and survival over 5 years was also reviewed. METHODS: A 6-month cohort of all discharges was followed for 5 years. Changes in domicile, including entry into institutional care, were recorded out to 5 years or until death. Predictors of early (3 months) and later (1 year) discharge stability were assessed. RESULTS: There were 142 discharges. Fifty-eight (76%) of those who returned home were still at home 12 months later. In contrast, there was a high mortality of dependent patients who were discharged to high dependency care (9 (29%) and 13 (42%) at 3 and 12 months, respectively). The chance of an early failed discharge was associated with lower functional ability on discharge (P= 0.012). Lower function on discharge was also independently associated with death in the next 12 months (P < 0.0001). At 5 years the mortality for the whole sample was 55% (78 of 141) and 38 (61%) of the survivors still lived in the community whereas 24 (39%) resided in institutional care. CONCLUSION: Functional ability on discharge is a key predictor of ability to remain at home as well as survival and therefore every effort should be made to maximize function.

Developing unplanned care teams in Scotland: evaluating outcomes.

Mental health nursing, along with other professional groups, has had to adapt to new ways of delivering health services, often in response to government policy. Consequently, traditional professional boundaries and roles are being rapidly and consistently expanded, often requiring coordinated responses across strategic, educational and clinical domains to ensure service users experience high-quality mental health interventions. This paper explores and evaluates such a coordinated response in developing unplanned care services in Scotland. The evaluation, placed within a framework of realistic evaluation, highlights not only the efficacy of the provided training and education for new roles within unplanned care, but also that emotionally intelligent capabilities are required to successfully implement the level of change currently being experienced within the UK mental health services.

Persistent genital arousal disorder: a review of the literature and recommendations for management.

Persistent genital arousal disorder is a newly recognized condition that is poorly understood. There is a paucity of research in this area and there are concerns as to the validity of the results of what little research there has been. This article aims to draw together current literature on this topic and provide readers with guidance on the management of this condition. This includes a working definition, an exploration of possible aetiologies within the confines of current knowledge, practical advice regarding assessment, management and auditable outcomes of practice.

A literature review and case reports series on the use of phosphodiesterase inhibitors in the treatment of female sexual dysfunction.

The term 'female sexual dysfunction' (FSD) encompasses a number of different disorders, and while their aetiologies are not fully understood, the sub-classifications of this broad umbrella term are increasingly becoming more established and accepted. However, there is less consensus regarding the optimal treatment of these conditions. While it is known that phosphodiesterase (PDE5) is involved in the female sexual response, the clinical and research evidence supporting the unlicensed use of PDE5 inhibitors (PDE5i) in women is inconclusive and at times contradictory. In this article we explore this further by means of a comprehensive literature review on the use of PDE5i in the treatment of FSD and we also present our clinical experience of using these drugs in this context.

Healthcare-associated infection in acute hospitals: which interventions are effective?

This study investigated the potential factors linked to healthcare-associated infection (HCAI) rates in acute National Health Service hospitals, analysing mandatory surveillance data with existing data available to the Healthcare Commission, and supplemented by a bespoke questionnaire. A questionnaire was developed to cover important elements related to the management and control of HCAI. Additional data were collated from other sources. Infection outcomes comprised the mandatory surveillance data, for both meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia and Clostridium difficile-associated diarrhoea (CDAD). The response rate was 90%. A lower MRSA rate was linked to hand hygiene and isolation and a lower rate of CDAD to cleanliness, good antimicrobial prescribing practices and surveillance of infections. Lower rates of both organisms were related to strategic planned interventions, such as the inclusion of infection control in the staff development programme. However, certain interventions, for example increased levels of training, were related to a higher infection rate. These findings for MRSA and CDAD are supported by evidence from the infection control literature. We have found relationships between interventions and higher infection rates that are counterintuitive and that may represent examples of what we call 'reactive practice' to higher rates of infection. Whilst it is interesting to hypothesise that these interventions may be swift and simple to introduce and may not be sustained compared to more strategic and planned interventions linked to lower infection rates, they most probably simply represent the beginning of a culture change and embedding of infection control practice.

Post-exposure HIV prophylaxis following sexual exposure: a retrospective audit against recent draft BASHH guidance.

OBJECTIVES: To retrospectively audit the management of post-exposure HIV prophylaxis following sexual exposure (PEPSE) against the British Association for Sexual Health and HIV 2004 draft guidance. METHODS: A retrospective review of case notes from January 2000 to November 2004. The draft guidelines were not adopted into clinical practice during the study period. RESULTS: 76 patients received PEPSE. 79% (95% CI 68.08 to 87.46) of PEPSE prescriptions were given for exposures that were in accordance with the guidelines' recommended indications (target 90%). 87% (95% CI 77.13 to 93.51) of PEPSE was prescribed within 72 hours of risk exposure (target 90%). 91% (95% CI 81.94 to 96.22) of recipients received a recommended antiretroviral combination. 53% (95% CI 40.84 to 64.21) of recipients completed the PEPSE course (target 75%). 45% of patients attended for the 3 month follow up HIV test but only 12% (95% CI 5.56 to 21.29) attended for both the 3 month and 6 month HIV test (target 75%). CONCLUSION: PEPSE is predominantly being prescribed for recommended indications and is dispensed within 72 hours of risk exposure. PEPSE completion rates and attendance for 3 months and 6 months post-exposure HIV testing need improving, perhaps by introducing a PEPSE clinic.

A randomized phase II trial comparing every 3-weeks carboplatin/paclitaxel with every 3-weeks carboplatin and weekly paclitaxel in advanced non-small cell lung cancer.

BACKGROUND: The optimal schedule of taxane administration has been an area of active interest in several recent clinical trials. METHODS: To address a pure schedule question, we randomized 161 patients with advanced stage IIIB or IV non-small-cell lung cancer (NSCLC) to either paclitaxel 225 mg/m2 every 3 weeks x 4 cycles or 75 mg/m2/week x 12 (cumulative dose on each arm = 900 mg/m2). Both arms received concurrent carboplatin AUC 6 every 3 weeks x 4 cycles. RESULTS: The two arms were well-balanced in terms of known prognostic factors. The overall response rate and survival outcomes were similar on the two arms. There was significantly more grade 3/4 thrombocytopenia and grade 2-4 anemia on the weekly arm but less severe myalgias/arthralgias and alopecia. No difference in the rates of peripheral neuropathy was observed; however, patients on the every 3 weeks arm reported significantly more taxane therapy-related side-effects on the functional assessment of cancer therapy taxane subscale. CONCLUSIONS: This randomized trial exploring schedule-related issues with carboplatin/paclitaxel confirms the versatility of this regimen.

GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila.

The tauopathies are a group of disorders characterised by aggregation of the microtubule-associated protein tau and include Alzheimer's disease (AD) and the fronto-temporal dementias (FTD). We have used Drosophila to analyse how tau abnormalities cause neurodegeneration. By selectively co-expressing wild-type human tau (0N3R isoform) and a GFP vesicle marker in motorneurons, we examined the consequences of tau overexpression on axonal transport in vivo. The results show that overexpression of tau disrupts axonal transport causing vesicle aggregation and this is associated with loss of locomotor function. All these effects occur without neuron death. Co-expression of constitutively active glycogen-synthase kinase-3beta (GSK-3beta) enhances and two GSK-3beta inhibitors, lithium and AR-A014418, reverse both the axon transport and locomotor phenotypes, suggesting that the pathological effects of tau are phosphorylation dependent. These data show that tau abnormalities significantly disrupt neuronal function, in a phosphorylation-dependent manner, before the classical pathological hallmarks are evident and also suggest that the inhibition of GSK-3beta might have potential therapeutic benefits in tauopathies.

From disease genes to cellular pathways: a progress report.

Mutations in a large number of retinal and retinal pigment epithelium (RPE) expressed genes can lead to the degeneration of photoreceptors and consequently the loss of vision. The genetic and phenotypic heterogeneity of retinal dystrophies poses a complex problem with respect to rational development of therapeutic strategies. Delineation of physiological functions of disease genes and identification of pathways that lead to disease pathogenesis represent essential goals towards developing a systematic and global approach to gene-based treatments. We are interested in identifying cellular pathways that are involved in photoreceptor differentiation, function and degeneration. We are, therefore, generating comprehensive gene expression profiles of retina and RPE of humans and mice using both cDNA- and oligonucleotide-based (Affymetrix) microarrays. Because of the under-representation of retinal/RPE genes in the public databases, we have constructed several unamplified cDNA libraries and produced almost twenty thousand expressed sequence tags (ESTs) that are being printed onto glass slides ('I-Gene' microarrays). In this presentation, we will report the microarray analysis of the rodless (and cone-enhanced) retina from the Nrl-knockout mouse as a paradigm to initiate the identification of cellular pathways involved in photoreceptor differentiation and function.

Nrl is required for rod photoreceptor development.

The protein neural retina leucine zipper (Nrl) is a basic motif-leucine zipper transcription factor that is preferentially expressed in rod photoreceptors. It acts synergistically with Crx to regulate rhodopsin transcription. Missense mutations in human NRL have been associated with autosomal dominant retinitis pigmentosa. Here we report that deletion of Nrl in mice results in the complete loss of rod function and super-normal cone function, mediated by S cones. The photoreceptors in the Nrl-/- retina have cone-like nuclear morphology and short, sparse outer segments with abnormal disks. Analysis of retinal gene expression confirms the apparent functional transformation of rods into S cones in the Nrl-/- retina. On the basis of these findings, we postulate that Nrl acts as a 'molecular switch' during rod-cell development by directly modulating rod-specific genes while simultaneously inhibiting the S-cone pathway through the activation of Nr2e3.

Multiple phosphorylated isoforms of NRL are expressed in rod photoreceptors.

NRL, a bZIP transcription factor of the Maf subfamily, interacts with the homeodomain protein CRX and synergistically regulates rhodopsin expression. Here we report that six isoforms of NRL (29-35 kDa) are generated by phosphorylation and expressed specifically in the mammalian retina. The anti-NRL antibody also cross-reacts with a cytosolic 45-kDa protein, which is detected in neuronal tissues but is not encoded by the NRL gene. In both human retinal cell cultures and sections of fetal and adult human retina, NRL is present in the nuclei of developing and mature rods but not cones. We propose that NRL regulates rod photoreceptor-specific gene expression and is involved in rod differentiation.

Five novel RPGR mutations in families with X-linked retinitis pigmentosa.

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe because of their early onset, often leading to significant visual impairment before the fourth decade. RP3, genetically localized at Xp21.1, accounts for 70% of XLRP in different populations. The RPGR (Retinitis Pigmentosa GTPase Regulator) gene that was isolated from the RP3 region is mutated in 20% of North American families with XLRP. From mutation analysis of 27 independent XLRP families, we have identified five novel RPGR mutations in 5 of the families (160delA, 789 A>T, IVS8+1 G>C, 1147insT and 1366 G>A). One of these mutations was detected in a family from Chile. Hum Mutat 17:151, 2001.

Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15.

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.

Axenfeld-Rieger syndrome resulting from mutation of the FKHL7 gene on chromosome 6p25.

Mutations in the forkhead-like 7 (FKHL7) gene have been recently shown to cause juvenile glaucoma and anterior segment anomalies. We report on a three-generation family with Axenfeld-Rieger syndrome (ARS), harboring an alteration in the FKHL7 gene. Genetic linkage analyses excluded the ARS phenotype from chromosomes 4q25 and 13q14, the locations of the PITX2 and RIEG2 loci, respectively. Evidence of linkage was observed with markers at 6p25, near the FKHL7 gene. Direct sequencing of FKHL7 detected a C67T mutation that segregated with the ARS phenotype in this family, but was not detected in over 80 control chromosomes. This mutation is predicted to cause a nonsense mutation of the FKHL7 protein (Gln23Stop) upstream of the forkhead DNA-binding domain, and thus to generate a truncated FKHL7 protein product. This discovery broadly implicates FKHL7 in ocular, craniofacial, dental, and umbilical development.

Mutations of the forkhead/winged-helix gene, FKHL7, in patients with Axenfeld-Rieger anomaly.

Genetic linkage, genome mismatch scanning, and analysis of patients with alterations of chromosome 6 have indicated that a major locus for development of the anterior segment of the eye, IRID1, is located at 6p25. Abnormalities of this locus lead to glaucoma. FKHL7 (also called "FREAC3"), a member of the forkhead/winged-helix transcription-factor family, has also been mapped to 6p25. DNA sequencing of FKHL7 in five IRID1 families and 16 sporadic patients with anterior-segment defects revealed three mutations: a 10-bp deletion predicted to cause a frameshift and premature protein truncation prior to the FKHL7 forkhead DNA-binding domain, as well as two missense mutations of conserved amino acids within the FKHL7 forkhead domain. Mf1, the murine homologue of FKHL7, is expressed in the developing brain, skeletal system, and eye, consistent with FKHL7 having a role in ocular development. However, mutational screening and genetic-linkage analyses excluded FKHL7 from underlying the anterior-segment disorders in two IRID1 families with linkage to 6p25. Our findings demonstrate that, although mutations of FKHL7 result in anterior-segment defects and glaucoma in some patients, it is probable that at least one more locus involved in the regulation of eye development is also located at 6p25.