Aniridia due to a novel microdeletion affecting PAX6 regulatory enhancers: case report and review of the literature.

Aniridia is a rare congenital ocular malformation that follows an autosomal dominant mode of inheritance. Most patients carry pathogenic point mutations in the paired box 6 gene (PAX6), but some carry deletions involving the 11p13 region, encompassing partly or completely PAX6 or the region downstream. We identified a novel deletion, ~564 kb in size located about 46.5 kb downstream of PAX6 in a family with bilateral aniridia and foveal hypoplasia using array-CGH and multiplex ligation-dependent probe amplification. We also reviewall of the reported deletions downstream of PAX6 in patients with aniridia and/or other congenital malformations and define the overlapping region that leads to aniridia when deleted.

Molecular analysis of Cypriot families with aniridia reveals a novel PAX6 mutation.

The present study investigated the clinical and mutational spectrum of aniridia in a cohort of 17 affected individuals from six families from Cyprus. Each proband was initially evaluated for copy number variants at the PAX6 locus and subsequently underwent PAX6 mutation screening. Sequence analysis of FOXC1 and PITX2 was performed in patients who did not carry a PAX6 mutation. The most common clinical features in the group of aniridia patients associated with aniridia were nystagmus, cataracts and glaucoma. PAX6 pathogenic mutations were identified in five out of six families (a diagnostic yield of 84%). Previously reported pathogenic mutations in PAX6 were identified in four families, which comprise p.R203*, p.R240* and p.R317*. In addition, a novel pathogenic variant (p.E220Gfs*23) was identified in a single family. No pathogenic mutations were detected in PAX6, FOXC1 or PITX2 in the only patient with a sporadic form of aniridia­like phenotype, confirming the genetic heterogeneity associated with this disease. To the best of our knowledge this is the first report on the mutational spectrum of PAX6 in aniridia patients of Cypriot ancestry. Mutational screening of PAX6 serves a crucial role in distinguishing isolated from syndromic forms of aniridia, and it may therefore eliminate the need for renal ultrasound scan surveillance, delineate the phenotype and improve genetic counseling.

Novel TBX3 mutation in a family of Cypriot ancestry with ulnar-mammary syndrome.

Ulnar-mammary syndrome (UMS) is an autosomal dominant disorder resulting from TBX3 haploinsufficiency. It typically affects limb, apocrine gland, hair, tooth and genital development and shows marked intrafamilial and interfamilial variability in phenotypic expression. We report a family (twin brothers and their father) affected with UMS because of a novel TBX3 mutation. The twin brothers showed classical features of UMS, whereas their father was mildly affected. The c.1423C>T (p.Q475*) nonsense mutation in exon 6 of the TBX3 gene identified in the patients by targeted Sanger sequencing is predicted to lead to premature termination of translation. This is the first report of a Cypriot family with UMS resulting from a novel TBX3 mutation. This report provides additional evidence in support of the rich variability in phenotypic expression, the mutational heterogeneity and ethnic diversity associated with this rare condition.

ABCB4 missense mutations D243A, K435T, G535D, I490T, R545C, and S978P significantly impair the lipid floppase and likely predispose to secondary pathologies in the human population.

Bile salts are natural detergents required to solubilise dietary fat and lipid soluble vitamins. They are synthesised in hepatocytes and secreted into the luminal space of the biliary tree by the bile salt export pump (BSEP), an ATP-binding cassette (ABC) transporter in the canalicular membrane. BSEP deficiency causes cytotoxic accumulation of bile salts in the hepatocyte that results in mild-to-severe forms of cholestasis. The resulting inflammation can also progress to hepatocellular cancer via a novel mechanism involving upregulation of proliferative signalling pathways. A second ABC transporter of the canalicular membrane is also critical for bile formation. ABCB4 flops phosphatidylcholine into the outer leaflet of the membrane to be extracted by bile salts in the canalicular space. These mixed micelles reduce the detergent action of the bile salts and protect the biliary tree from their cytotoxic activity. ABCB4 deficiency also causes cholestasis, and might be expected to cause cholangitis and predispose to liver cancer. Non-synonymous SNPs in ABCB4 have now been described in patients with liver cancer or with inflammatory liver diseases that are known to predispose to cancer, but data showing that the SNPs are sufficiently deleterious to be an etiological factor are lacking. Here, we report the first characterisation at the protein level of six ABCB4 variants (D243A, K435T, G535D, I490T, R545C, and S978P) previously found in patients with inflammatory liver diseases or liver cancer. All significantly impair the transporter with a range of phenotypes exhibited, including low abundance, intracellular retention, and reduced floppase activity, suggesting that ABCB4 deficiency is the root cause of the pathology in these cases.

Molecular mechanistic explanation for the spectrum of cholestatic disease caused by the S320F variant of ABCB4.

UNLABELLED: ABCB4 flops phosphatidylcholine into the bile canaliculus to protect the biliary tree from the detergent activity of bile salts. Homozygous-null ABCB4 mutations cause the childhood liver disease, progressive familial intrahepatic cholestasis, but cause and effect is less clear, with many missense mutations linked to less severe cholestatic diseases. ABCB4(S320F), in particular, is described in 13 patients, including in heterozygosity with ABCB4(A286V), ABCB4(A953D), and null mutants, whose symptoms cover the spectrum of cholestatic disease. We sought to define the impact of these mutations on the floppase, explain the link with multiple conditions at the molecular level, and investigate the potential for reversal. ABCB4(S320F), ABCB4(A286V), and ABCB4(A953D) expression was engineered in naïve cultured cells. Floppase expression, localization, and activity were measured by western blot, confocal microscopy, and lipid transport assays, respectively. ABCB4(S320F) was fully active for floppase activity but expression at the plasma membrane was reduced to 50%. ABCB4(A286V) expressed and trafficked efficiently but could not flop lipid, and ABCB4(A953D) expressed poorly and was impaired in floppase activity. Proteasome inhibition stabilized nascent ABCB4(S320F) and ABCB4(A953D) but did not improve plasma membrane localization. Cyclosporin-A improved plasma membrane localization of both ABCB4(S320F) and ABCB4(A953D), but inhibited floppase activity. CONCLUSION: The level of ABCB4 functionality correlates with, and is the primary determinant of, cholestatic disease severity in these patients. ABCB4(S320F) homozygosity, with half the normal level of ABCB4, is the tipping point between more benign and potentially fatal cholestasis and makes these patients more acutely sensitive to environmental effects. Cyclosporin-A increased expression of ABCB4(S320F) and ABCB4(A953D), suggesting that chemical chaperones could be exploited for therapeutic benefit to usher in a new era of personalized medicine for patients with ABCB4-dependent cholestatic disease.

Musculoskeletal health professional use of internet resources for personal and patient education: results from an online national survey.

OBJECTIVES: To study the current practice of computer use in musculoskeletal health professionals for their education and that of their patients. METHODS: A survey questionnaire, designed by a working group including representatives from Arthritis Research UK and the British Society for Rheumatology, was made available on surveymonkey.com and the link distributed by email. RESULTS: 190 health professionals responded. Rheumatology professionals made up two thirds of the participants. The modal age group of responders was under 40 years (37%). 97% had spent some educational time on a computer. Females were younger and spent more time using the computer for education purposes. The preferred learning modality was interactive online content (71%). The most common methods of educating patients were the Consultant and Specialist nurse while the web is used by 40% of the health professionals. The most common barrier to education was 'Insufficient resources for education groups'. Rheumatologists were more likely to log Continuous Professional Development (CPD) online, complete online modules and have mandatory training online. UpToDate and Arthritis Research UK were the highest rated websites for health professional and patient education respectively. CONCLUSIONS: This is the first national survey of E-learning in the musculoskeletal health profession, with a large proportion of Rheumatologists. Almost all use computer based learning. Use of the internet for patient education is low. Highly rated educational websites are available for both professionals and patients.

Canalicular ABC transporters and liver disease.

Bile is a complex mixture that includes bile salts, the membrane phospholipid phosphatidylcholine (PC), cholesterol and various endobiotic and xenobiotic toxins, each of which is secreted across the canalicular membrane of the hepatocyte by different ATP-binding cassette (ABC) transporters. The bile salts are essential for the emulsification of dietary fat and lipophilic vitamins. They are synthesized from cholesterol in the hepatocyte and their secretion by the bile salt export pump (BSEP or ABCB11) drives bile flow and is the starting point for the enterohepatic cycle. The detergent nature of bile salts that is key to their physiological role also means that they are inherently cytotoxic, and failure to secrete bile (intraheptic cholestasis) can precipitate severe liver disease and mortality. Such progressive familial intrahepatic cholestasis (PFIC) comes in three types of autosomal recessive disease. PFIC2 is caused by mutation to ABCB11. PFIC3 is caused by mutation of a closely related ABC transporter, ABCB4, which flops PC into the outerleaflet of the canalicular membrane. The flopped PC is extracted by the bile salts in the canaliculus to form a mixed micelle that reduces bile salt detergent activity. The third protein that is essential for bile flow from the hepatocyte is a member of a different class of transporter protein, a P-type ATPase, ATP8B1. Mutation of ATP8B1 causes PFIC1, but ATP8B1 does not transport a component of bile into the canaliculus. Data from different laboratories, published this year, suggests two different roles for ATP8B1 in the hepatocyte: a lipid flippase, that counterbalances the deleterious effects of ABCB4 on barrier function of the canalicular membrane; and an anchor of the actin cytoskeleton necessary to form the microvilli of the brush border. These latest discoveries are described, along with a spectrum of cholestatic disorders whose aetiologies lie in these and other transporters of the canalicular membrane.

Structure of ABC transporters.

ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.

Influence of ROBO1 and RORA on risk of age-related macular degeneration reveals genetically distinct phenotypes in disease pathophysiology.

ROBO1 is a strong candidate gene for age-related macular degeneration (AMD) based upon its location under a linkage peak on chromosome 3p12, its expression pattern, and its purported function in a pathway that includes RORA, a gene previously associated with risk for neovascular AMD. Previously, we observed that expression of ROBO1 and RORA is down-regulated among wet AMD cases, as compared to their unaffected siblings. Thus, we hypothesized that contribution of association signals in ROBO1, and interaction between these two genes may be important for both wet and dry AMD. We evaluated association of 19 single nucleotide polymorphisms (SNPs) in ROBO1 with wet and dry stages of AMD in a sibling cohort and a Greek case-control cohort containing 491 wet AMD cases, 174 dry AMD cases and 411 controls. Association signals and interaction results were replicated in an independent prospective cohort (1070 controls, 164 wet AMD cases, 293 dry AMD cases). The most significantly associated ROBO1 SNPs were rs1387665 under an additive model (meta P = 0.028) for wet AMD and rs9309833 under a recessive model (meta P = 6 × 10(-4)) for dry AMD. Further analyses revealed interaction between ROBO1 rs9309833 and RORA rs8034864 for both wet and dry AMD (interaction P<0.05). These studies were further supported by whole transcriptome expression profile studies from 66 human donor eyes and chromatin immunoprecipitation assays from mouse retinas. These findings suggest that distinct ROBO1 variants may influence the risk of wet and dry AMD, and the effects of ROBO1 on AMD risk may be modulated by RORA variants.

Symptoms of nicotine dependence and other predictors of student smoking at school: implications for school smoking policy.

Students who violate school smoking policies present a difficult health and disciplinary issue for school officials. Professionals know little about the characteristics of students who smoke at school. In a prospective study of 679 students in two cities in central Massachusetts, researchers examined how nicotine dependence contributes to the problem of smoking at school. After three years of follow up, smoking at school was reported by 10.3% of students. Among subjects who admitted to smoking at school, 63% reported that symptoms of nicotine dependence preceded their smoking at school. After adjusting for other variables, student smokers with symptoms of nicotine dependence were nine times more likely to report smoking in school (OR 9.1, 95% CI 2.9-28.5) than were student smokers without symptoms. Smoking at school was more common among daily smokers and those who paid for their own cigarettes. Age, gender, race, and parental smoking status were not significantly associated with students' reports of smoking at school. These data suggest nicotine dependence as an important contributor to the problem of smoking at school, but not the only reason why students violate school smoking policies. Disciplinary action against students caught violating school smoking policies should be supplemented with an offer of treatment for nicotine dependence.

Neurokinin-1 receptor and substance P messenger RNA levels increase during intraabdominal adhesion formation.

BACKGROUND: Intraabdominal peritoneal adhesions are a significant cause of postoperative morbidity and remain one of the major long-term complications associated with abdominal surgery. Adhesion formation at the molecular level involves a complex interaction of cytokines, growth factors, cell adhesion molecules, and neuropeptides, as well as many other factors secreted by cells proximate to the traumatized area. Limited studies exist which investigate the molecular processes involved in adhesion formation. Therefore, the aim of the present study was to determine the pattern of gene expression for substance P, the neurokinin-1 receptor, and downstream mediators of substance P action during the early stages of adhesion formation in the rat. METHODS: Four ischemic buttons were created on one side of the peritoneum in male Wistar rats. Animals were sacrificed at 3, 6, and 12 h and 1, 3, 5, and 7 days following surgery. Peritoneal tissue from ischemic buttons and from the opposite sidewall was harvested for total RNA isolation. Semiquantitative RT-PCR was used to measure changes at the transcript level for the neurokinin-1 receptor, substance P, TGFbeta-1, and the cell adhesion molecules ICAM-1 and VCAM-1. RESULTS: Messenger RNA levels for substance P, neurokinin-1 receptor, TGFbeta-1, ICAM-1, and VCAM-1 were significantly increased in peritoneal tissue taken from ischemic button sites (P < 0.05) when compared with controls. In the peritoneal tissues taken from the opposite sidewall, there was a significant (P < 0.05) early increase in substance P mRNA levels. TGF-beta1, neurokinin-1 receptor, and ICAM-1 mRNA levels were also significantly (P < 0.05) increased when compared to controls, while the mRNA levels for VCAM-1 did not change. CONCLUSIONS: The increased levels of mRNA for substance P, the neurokinin-1 receptor, and the downstream mediators of substance P action, TGF-beta1, ICAM-1, and VCAM-1, in peritoneal tissue associated with intraabdominal adhesions support a role for substance P in adhesion formation.

Firefighters' blood pressure and employment status on hazardous materials teams in Massachusetts: a prospective study.

We evaluated the association between hypertension and changes in employment status in 334 hazardous materials firefighters. Firefighters were categorized by blood pressure (BP) at baseline (1996 or 1997) and subsequent follow-up examinations (1997, 1998, and 1999). They were followed up for a maximum of 4 years for possible adverse outcomes (death, placement on "injured-on-duty" status, termination of duty, resignation, retirement, or incident cardiovascular disease). In several analytic models, we found that firefighters with stage II hypertension (BP > or = 160/100 mm Hg) were consistently 2 to 3 times more likely to experience an adverse outcome compared with those with normal BP. Cox proportional-hazards regression was used to adjust for age, body mass index, smoking, cholesterol, and antihypertensive medication. In these models, the hazard ratio for stage II hypertension was 3.2 (95% confidence interval [CI], 1.50 to 7.04, P = 0.003) and for untreated stage II hypertension, it was 4.6 (95% CI, 2.08 to 10.11, P = 0.0002). Firefighters with a BP > or = 160/100 mm Hg should receive further evaluation and demonstrate improved BP control before being determined fit for duty.