Whole-exome sequencing of the BDR cohort: evidence to support the role of the PILRA gene in Alzheimer's disease.

AIM: Late-onset Alzheimer's disease (LOAD) accounts for 95% of all Alzheimer's cases and is genetically complex in nature. Overlapping clinical and neuropathological features between AD, FTD and Parkinson's disease highlight the potential role of genetic pleiotropy across diseases. Recent genome-wide association studies (GWASs) have uncovered 20 new loci for AD risk; however, these exhibit small effect sizes. Using NGS, here we perform association analyses using exome-wide and candidate-gene-driven approaches. METHODS: Whole-exome sequencing was performed on 132 AD cases and 53 control samples. Exome-wide single-variant association and gene burden tests were performed for 76 640 nonsingleton variants. Samples were also screened for known causative mutations in familial genes in AD and other dementias. Single-variant association and burden analysis was also carried out on variants in known AD and other neurological dementia genes. RESULTS: Tentative single-variant and burden associations were seen in several genes with kinase and protease activity. Exome-wide burden analysis also revealed significant burden of variants in PILRA (P = 3.4 × 10-5 ), which has previously been linked to AD via GWAS, hit ZCWPW1. Screening for causative mutations in familial AD and other dementia genes revealed no pathogenic variants. Variants identified in ABCA7, SLC24A4, CD33 and LRRK2 were nominally associated with disease (P < 0.05) but did not withstand correction for multiple testing. APOE (P = 0.02) and CLU (P = 0.04) variants showed significant burden on AD. CONCLUSIONS: In addition, polygenic risk scores (PRS) were able to distinguish between cases and controls with 83.8% accuracy using 3268 variants, sex, age at death and APOE ε4 and ε2 status as predictors.

Capsular type K54, clonal group 29 and virulence plasmids: an analysis of K54 and non-K54 closely related isolates of Klebsiella pneumoniae.

Capsular type K54 of Klebsiella pneumoniae is associated with hypervirulence and we sought to discover the basis for this among isolates submitted to the UK reference laboratory between 2012 and 2017. Isolates were typed by variable number tandem repeat analysis, and capsular type and virulence elements sought by PCR. The most prevalent type found (15/31 isolates) corresponded to clonal group (CG) 29 and included five representatives carrying rmpA, rmpA2 (regulators of mucoid phenotype), iutA and iroD (from the aerobactin and salmochelin siderophore clusters) associated with virulence plasmids. These included isolate KpvK54, recovered from pus. The remaining isolates did not carry a virulence plasmid. We also noted 11 further related isolates, including NCTC 9159, not of capsular type K54, but nevertheless sometimes associated with sepsis and abscesses. Whole-genome sequencing showed that KpvK54 carried a large virulence plasmid and an ICEKp3-like structure carrying the yersiniabactin cluster, absent in NCTC 9159. Comparative chromosomal analysis with an additional four genomes showed that KpvK54 shared further genes with K1-ST23 hypervirulent isolates, and with LS358, a K54-ST29 isolate from liver abscess puncture fluid. While CG29 isolates displayed varying degrees of virulence, some, especially those with the virulence plasmid (all K54), were clearly associated with hypervirulence.

Ralstonia infection in cystic fibrosis.

This study aimed to determine prevalence of Ralstonia spp. in cystic fibrosis patients, look for any evidence of cross infection and to describe clinical outcomes for patients infected by Ralstonia spp. Prevalence of Ralstonia spp. was calculated annually from 2008 to 2016. Pulsed-field gel electrophoresis was performed on ⩾1 sample from patients with an isolation of Ralstonia spp. between 2008 and 2016. A prospective, longitudinal observational study of adult patients was performed with 12 months follow-up from recruitment. Prevalence of Ralstonia spp. rose from 0·6% in 2008 to 2·4% in 2016. In total 12 out of 14 (86%) patients with ⩾1 isolation of Ralstonia spp. developed chronic infection. A pair and a group of three unrelated patients with epidemiological connections shared strains of Ralstonia mannitolilytica. Lung function of Ralstonia spp. infected patients was moderately to severely impaired. Prevalence of Ralstonia spp. is low but increasing. The risk of a patient developing chronic Ralstonia spp. infection following first acquisition is high and cross-infection may be possible. Whether Ralstonia spp. infection causes increased pulmonary exacerbation frequency and lung function decline needs to be evaluated in larger prospective studies.

Two novel mutations in the POU1F1 gene generate null alleles through different mechanisms leading to combined pituitary hormone deficiency.

BACKGROUND: Mutations in the POU1F1 gene severely affect the development and function of the anterior pituitary gland and lead to combined pituitary hormone deficiency (CPHD). OBJECTIVE: The clinical and genetic analysis of a patient presenting with CPHD and functional characterization of identified mutations. PATIENT: We describe a male patient with extreme short stature, learning difficulties, anterior pituitary hypoplasia, secondary hypothyroidism and undetectable prolactin, growth hormone (GH) and insulin-like growth factor 1 (IGF1), with normal random cortisol. DESIGN: The POU1F1 coding region was amplified by PCR and sequenced; the functional consequence of the mutations was analysed by cell transfection and in vitro assays. RESULTS: Genetic analysis revealed compound heterozygosity for two novel putative loss of function mutations in POU1F1: a transition at position +3 of intron 1 [IVS1+3nt(A>G)] and a point mutation in exon 6 resulting in a substitution of arginine by tryptophan (R265W). Functional analysis revealed that IVS1+3nt(A>G) results in a reduction in the correctly spliced POU1F1 mRNA, which could be corrected by mutations of the +4, +5 and +6 nucleotides. Analysis of POU1F1(R265W) revealed complete loss of function resulting from severely reduced protein stability. CONCLUSIONS: Combined pituitary hormone deficiency in this patient is caused by loss of POU1F1 function by two novel mechanisms, namely aberrant splicing (IVS1+3nt (A>G) and protein instability (R265W). Identification of the genetic basis of CPHD enabled the cessation of hydrocortisone therapy without the need for further assessment for evolving endocrinopathy.

'Sink or swim': an evaluation of the clinical characteristics of individuals with high bone mass.

SUMMARY: High bone mineral density on routine dual energy X-ray absorptiometry (DXA) may indicate an underlying skeletal dysplasia. Two hundred fifty-eight individuals with unexplained high bone mass (HBM), 236 relatives (41% with HBM) and 58 spouses were studied. Cases could not float, had mandible enlargement, extra bone, broad frames, larger shoe sizes and increased body mass index (BMI). HBM cases may harbour an underlying genetic disorder. INTRODUCTION: High bone mineral density is a sporadic incidental finding on routine DXA scanning of apparently asymptomatic individuals. Such individuals may have an underlying skeletal dysplasia, as seen in LRP5 mutations. We aimed to characterize unexplained HBM and determine the potential for an underlying skeletal dysplasia. METHODS: Two hundred fifty-eight individuals with unexplained HBM (defined as L1 Z-score ≥ +3.2 plus total hip Z-score ≥ +1.2, or total hip Z-score ≥ +3.2) were recruited from 15 UK centres, by screening 335,115 DXA scans. Unexplained HBM affected 0.181% of DXA scans. Next 236 relatives were recruited of whom 94 (41%) had HBM (defined as L1 Z-score + total hip Z-score ≥ +3.2). Fifty-eight spouses were also recruited together with the unaffected relatives as controls. Phenotypes of cases and controls, obtained from clinical assessment, were compared using random-effects linear and logistic regression models, clustered by family, adjusted for confounders, including age and sex. RESULTS: Individuals with unexplained HBM had an excess of sinking when swimming (7.11 [3.65, 13.84], p < 0.001; adjusted odds ratio with 95% confidence interval shown), mandible enlargement (4.16 [2.34, 7.39], p < 0.001), extra bone at tendon/ligament insertions (2.07 [1.13, 3.78], p = 0.018) and broad frame (3.55 [2.12, 5.95], p < 0.001). HBM cases also had a larger shoe size (mean difference 0.4 [0.1, 0.7] UK sizes, p = 0.009) and increased BMI (mean difference 2.2 [1.3, 3.1] kg/m(2), p < 0.001). CONCLUSION: Individuals with unexplained HBM have an excess of clinical characteristics associated with skeletal dysplasia and their relatives are commonly affected, suggesting many may harbour an underlying genetic disorder affecting bone mass.

Impact of a clonal outbreak of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in the development and evolution of bloodstream infections by K. pneumoniae and Escherichia coli: an 11 year experience in Oxfordshire, UK.

OBJECTIVES: The objectives of this study were: (i) to describe an outbreak of multidrug-resistant Klebsiella pneumoniae in our population; (ii) to identify the potential source of this outbreak by examining antibiotic resistance trends in urocultures; (iii) to evaluate the contribution of this outbreak to resistance patterns over time in the two commonest Gram-negative blood culture isolates, namely K. pneumoniae and Escherichia coli; and (iv) to assess risk factors for multidrug resistance and the impact of this resistance on mortality and length of stay. METHODS: We searched Microbiology and Patient Administration Service databases retrospectively and describe resistance trends in E. coli and K. pneumoniae bloodstream infections (BSIs) in Oxfordshire, UK, over an 11 year period. RESULTS: An outbreak of a multidrug-resistant, CTX-M-15 extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae clone was identified and shown by multilocus sequence typing to belong to a novel sequence type designated ST490. This was associated with a sporadic change in resistance rates in K. pneumoniae BSIs with rates of multidrug resistance (defined as resistance to three or more antibiotic classes) reaching 40%. A case-control study showed prior antibiotic exposure as a risk factor for infection with this organism. During the same time period, rates of ESBL-producing Klebsiella spp. isolated from urocultures increased from 0.5% to almost 6%. By contrast, the rate of multidrug resistance in E. coli rose more steadily from 0% in 2000 to 10% in 2010. CONCLUSIONS: Changes in resistance rates may be associated with outbreaks of resistant clones in K. pneumoniae. Changing resistance patterns may affect important health economic issues such as length of stay.

Molecular analysis of novel PROP1 mutations associated with combined pituitary hormone deficiency (CPHD).

OBJECTIVE: Homozygous mutations in the gene encoding the pituitary transcription factor PROP1 are associated with combined pituitary hormone deficiency (CPHD) in both mice and humans with a highly variable phenotype with respect to the severity and time of initiation of pituitary hormone deficiency. We have ascertained three pedigrees with PROP1 mutations from a large cohort of patients with variable degrees of CPHD who were screened for mutations in PROP1. RESULTS: Affected individuals from all three pedigrees were found to harbour novel PROP1 mutations. We have identified two siblings in one family who were homozygous for an intronic mutation (c.343-11C > G) that disrupts correct splicing resulting in the loss of exon 3 from the PROP1 transcript. Two siblings from a second, unrelated family are compound heterozygotes for two point mutations in the coding region, a missense mutation (p.R125W) that leads to impaired transcriptional activation, and a deletion of a single nucleotide (c.310delC) resulting in a frameshift and nonfunctional mutant protein. Additionally, we identified a homozygous deletion of the PROP1 locus in two patients born to consanguineous parents. CONCLUSION: Mutations in PROP1 are a frequent cause of familial CPHD. We have described four novel mutations in PROP1 in 3 pedigrees, all resulting in PROP1 deficiency by different mechanisms. The phenotypic variation observed in association with PROP1 mutations both within and between families, together with the evolving nature of hormone deficiencies and sometimes changing pituitary morphology indicates a need for continual monitoring of these patients.

Variable number tandem repeat loci providing discrimination within widespread genotypes of Acinetobacter baumannii.

Some genotypes of Acinetobacter baumannii, defined by pulsed-field gel electrophoresis (PFGE), have been found in many hospitals. Our aim was to find variable number tandem repeat (VNTR) loci capable of providing discrimination among isolates with highly similar or identical PFGE profiles, to gain insights into the epidemiology. Thirteen loci identified in A. baumannii ATCC 17978 were tested using a panel of isolates that included multiple representatives of genotypes belonging to the three European clonal lineages. Two loci, with repeat units of 9 and 6 bp respectively were selected. Repeat numbers varied between 3 and 29, and 9 and 26 respectively at the two loci. The repeat numbers of representatives of each genotype often differed between hospitals, providing a means of tracking patient transfers and possible transmissions between patients. The results suggest that this analysis accurately reflects the known epidemiological information, and provides a valuable tool for cross-infection studies.

Phagocyte-specific S100 proteins are released from affected mucosa and promote immune responses during inflammatory bowel disease.

Phagocyte-derived S100 proteins are endogenous activators of innate immune responses. S100A12 binds to the receptor for advanced glycation end-products, while complexes of S100A8/S100A9 (myeloid-related proteins, MRP8/14; calprotectin) are ligands of toll-like receptor 4. These S100 proteins can be detected in stool. In the present study we analyse the release of S100A12 and MRP8/14 from intestinal tissue. Specimens from patients with Crohn's disease (CD; n = 30), ulcerative colitis (UC; n = 30), irritable bowel syndrome (IBS; n = 30) or without inflammation (n = 30) were obtained during endoscopy. After 24 h culture, S100A12 and MRP8/14 were analysed in supernatants. Endoscopic, histological, laboratory and clinical disease activity measures were documented. We found an increased spontaneous release of S100A12 from tissue in inflammatory bowel disease (IBD). The release of S100A12 into the supernatants was 28-fold enhanced in inflamed tissue when compared to non-inflamed tissue (mean 46.9 vs. 1.7 ng/ml, p < 0.0001). In active CD, release of S100A12 and MRP8/14 was strongly dependent on localization, with little release from sites of active ileal inflammation compared to colonic inflammation. This difference was more pronounced for S100A12 than for MRP8/14. S100A12 and MRP8/14 provoked up-regulation of adhesion molecules and chemokines on human intestinal microvascular endothelial cells (HIMECs) isolated from normal colonic tissue. The direct release of phagocyte-derived S100 proteins from inflamed tissues may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (MRP8/14). Via activation of pattern recognition receptors, these proteins promote inflammation in intestinal tissue. The enhanced mucosal release can explain the correlation of fecal markers with disease activity in IBD.

Identification of superoxide dismutase as a potential urinary marker of carbon tetrachloride-induced hepatic toxicity.

The aim of this study was the identification of a novel protein marker of hepatotoxicity in rat urine. Rats were dosed by gavage with carbon tetrachloride (CCl(4)) to induce acute liver injury. Surface enhanced laser desorption/ionisation (SELDI) ProteinChip technology revealed the appearance of a 15.7 kDa protein in the CCl(4)-treated rat urine. One-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) identified an 18.4 kDa protein in the CCl(4)-treated rat urine. The appearance of either protein was coincident over a time course during which they first appeared at 12h post-dosing, peaked at 36h and had disappeared again within 3 days post-dosing. The protein was identified by in-gel digestion and nano-electrospray (nano-ES)-tandem mass spectrometry as Cu/Zn superoxide dismutase (SOD-1). SOD activity was found to be increased by 61.4-fold in CCl(4)-treated rat urine. Western blots of tissue homogenates from the rats revealed a time-dependent loss of SOD-1 from the livers of CCl(4)-treated rats matching the time course of SOD-1 appearance in urine. SOD-1 is not specifically located in liver; however, its appearance in urine in response to acute CCl(4)-induced hepatotoxicity is a novel finding; this coupled with loss from the liver following injury suggests urinary SOD-1 may be a potential marker of hepatotoxicity.

Does screening neonates in the neonatal intensive care unit for Pseudomonas aeruginosa colonization help prevent infection?

BACKGROUND: Pseudomonas aeruginosa (PA) is a Gram-negative environmental organism that can cause severe infection in immunosuppressed patients, including preterm neonates. In recent years, it has become common practice to screen neonates for PA colonization. AIM: To assess the value of screening neonates for PA in (1) predicting the risk of developing severe PA infection and (2) directing infection control practice. METHODS: Between August 2012 and September 2015, babies admitted to the neonatal intensive care unit (NICU) at North Bristol NHS Trust were screened routinely for PA colonization on admission and weekly thereafter. Data were also collected on babies who developed PA infection. Environmental samples from the NICU were tested for the presence of PA. Variable number tandem repeat (VNTR) typing was performed on all strains of PA from babies and the environment. FINDINGS: No babies with positive screens subsequently developed PA infection. There was no VNTR strain evidence supporting cross-infection from the environment or other babies. CONCLUSION: Screening neonates for PA did not identify babies who subsequently developed PA infection. Following cessation of screening in September 2015, there was no increase in the number of babies identified with PA infection.

Pseudomonas aeruginosa intensive care unit outbreak: winnowing of transmissions with molecular and genomic typing.

BACKGROUND: Pseudomonas aeruginosa healthcare outbreaks can be time consuming and difficult to investigate. Guidance does not specify which typing technique is most practical for decision-making. AIM: To explore the usefulness of whole-genome sequencing (WGS) in the investigation of a P. aeruginosa outbreak, describing how it compares with pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) analysis. METHODS: Six patient isolates and six environmental samples from an intensive care unit (ICU) positive for P. aeruginosa over two years underwent VNTR, PFGE and WGS. FINDINGS: VNTR and PFGE were required to fully determine the potential source of infection and rule out others. WGS results unambiguously distinguished linked isolates, giving greater assurance of the transmission route between wash-hand basin water and two patients, supporting the control measures employed. CONCLUSION: WGS provided detailed information without the need for further typing. When allied to epidemiological information, WGS can be used to understand outbreak situations rapidly and with certainty. Implementation of WGS in real-time would be a major advance in day-to-day practice. It could become a standard of care as it becomes more widespread due to its reproducibility and lower costs.

An outbreak of multidrug-resistant Pseudomonas aeruginosa in a burns service in the North of England: challenges of infection prevention and control in a complex setting.

BACKGROUND: Burns patients are at high risk of nosocomial infection, and Pseudomonas aeruginosa is one of the most common causes of wound and systemic infections resulting in significant morbidity and mortality in burns patients. AIM: To describe an outbreak of multidrug-resistant P. aeruginosa (MDR-Pa) at a specialist burns service and highlight the challenges in identifying the reservoir of infection despite extensive epidemiological, microbiological, and environmental investigations. METHODS: Multi-disciplinary outbreak control investigation. FINDINGS: Following an inter-hospital transfer of a burns patient from another country, an admission screen revealed that the patient was colonized with MDR-Pa. Subsequently nine more patients contracted MDR-Pa in the period from November 2015 to September 2017. Given the relatively long gap between confirmation of the index and subsequent cases, it was not possible to identify with certainty the reservoirs and mechanisms of spread of infection, although contamination of the burns service environment and equipment are likely to be contributory factors. CONCLUSION: Preventing infection transmission in specialist burns services is highly challenging, and it may not always be possible to identify and eradicate the reservoirs of infection for P. aeruginosa outbreaks. Our study supports the literature, providing additional evidence that inanimate, common contact surfaces play an important role in nosocomial transmission of P. aeruginosa. These surfaces should either be decontaminated efficiently between patient contacts or be single patient use. Enhanced vigilance is crucial, and, with strict adherence to infection prevention and control procedures, it is possible to reduce the risk of acquisition and spread of infection in patients.

Use of sequence-based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii.

Representatives (n = 31) of outbreak strains of Acinetobacter baumannii from five countries fell into three clear groups, designated Groups 1-3, based on their ompA (outer-membrane protein A), csuE (part of a pilus assembly system required for biofilm formation) and bla(OXA-51-like) (the intrinsic carbapenemase gene in A. baumannii) gene sequences. With the exception of the closely related alleles within the Group 1 clonal complex, alleles at each locus were highly distinct from each other, with a minimum of 14 nucleotide differences between any two alleles. Isolates within a group shared the same combination of alleles at the three loci, providing compelling evidence that the outbreak strains investigated belonged to three clonal lineages. These corresponded to the previously identified European clones I-III. Sequence differences among the alleles were used to design multiplex PCRs to rapidly assign isolates belonging to particular genotypes to sequence groups. In the UK, genotypes belonging to the Group 1 clonal complex have been particularly successful, accounting for the vast majority of isolates referred from hospitals experiencing problems with Acinetobacter.

Molecular evidence for Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn's disease correlates with enhanced TNF-alpha secretion.

BACKGROUND: Support for a role of Mycobacterium avium subspecies paratuberculosis in Crohn's disease is largely based on epidemiological evidence, as no data on mechanisms linking the presence of M. avium subspecies paratuberculosis with gut damage is available. AIMS: To determine whether the presence of M. avium subspecies paratuberculosis contributes to the pathogenesis of Crohn's disease by promoting cytokine secretion within gut mucosa. PATIENTS AND METHODS: A total of 235 subjects were recruited: 63 with Crohn's disease, 53 with ulcerative colitis, 45 with irritable bowel syndrome and 74 normal controls. M. avium subspecies paratuberculosis status was defined by nested PCR using IS900 sequence. Gut mucosal organ cultures were established to detect cytokine secretion patterns. RESULTS: Significantly higher tumour necrosis factor-alpha concentrations were found in culture supernatants for Crohn's disease compared to ulcerative colitis (p<0.05), irritable bowel syndrome (p<0.01) and controls (p<0.0001). When tumour necrosis factor-alpha levels were correlated with the presence of M. avium subspecies paratuberculosis, significantly greater concentrations were only found in M. avium subspecies paratuberculosis-positive Crohn's disease patients (p<0.05). Tumour necrosis factor-alpha levels in M. avium subspecies paratuberculosis-positive Crohn's disease were significantly higher than in M. avium subspecies paratuberculosis-positive ulcerative colitis (p<0.01), M. avium subspecies paratuberculosis-positive irritable bowel syndrome (p<0.05) and M. avium subspecies paratuberculosis-positive controls (p<0.01) and all M. avium subspecies paratuberculosis-negative specimens. CONCLUSIONS: The data link M. avium subspecies paratuberculosis with a pathogenic mechanism in Crohn's disease and is consistent with abnormal macrophage handling of M. avium subspecies paratuberculosis.

Epidemiology of infections caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp.: a nested case-control study from a tertiary hospital in London.

Information on risk factors for acquisition of extended-spectrum ss-lactamase (ESBL)-producing organisms and their outcomes in patients with invasive infections is scant. The objectives of this study were to evaluate risk factors and all-cause mortality associated with infection due to ESBL-producing organisms using a nested case-control design, and to document transmission within a hospital employing molecular and conventional epidemiological methods. From December 2003 to April 2005, 50 patients with bloodstream infections (BSIs) due to ESBL-producing E. coli and Klebsiella spp. were recruited. Controls (N=50) were chosen, within the same period, from patients with non-ESBL-producing BSIs by simple random sampling; account was taken of potential confounding factors. Cases and controls were followed-up until November 2005, and outcomes were recorded as discharged or deceased. Molecular methods, supported by conventional epidemiology, were used to study the transmission of organisms. Logistic regression analyses showed prior ss-lactam antibiotics [odds ratio (OR) 11.57; 95% confidence intervals (CI) 2.31-51.15; P=0.003], hospital stay >15 days (OR 2.63; 95% CI 1.01-6.89; P=0.04) and prior admission to the intensive care unit (OR 13.98; 95% CI 1.88-19.15; P=0.006) to be independent risk factors for the acquisition of ESBL-producing organisms. In the first 15 days of follow-up, a significant proportion of patients with ESBL-producing organisms died; however, there was no difference in mortality between cases and controls at the end of the follow-up period. Molecular epidemiology identified five clusters amongst the ESBL-producing isolates. Conventional epidemiological analyses supported the evidence of transmission in three of these clusters.

Novel Lethal Form of Congenital Hypopituitarism Associated With the First Recessive LHX4 Mutation.

BACKGROUND: LHX4 encodes a member of the LIM-homeodomain family of transcription factors that is required for normal development of the pituitary gland. To date, only incompletely penetrant heterozygous mutations in LHX4 have been described in patients with variable combined pituitary hormone deficiencies. OBJECTIVE/HYPOTHESIS: To report a unique family with a novel recessive variant in LHX4 associated with a lethal form of congenital hypopituitarism that was identified through screening a total of 97 patients. METHOD: We screened 97 unrelated patients with combined pituitary hormone deficiency, including 65% with an ectopic posterior pituitary, for variants in the LHX4 gene using Sanger sequencing. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) were consulted upon identification of variants. RESULTS: We identified the first novel homozygous missense variant (c.377C>T, p.T126M) in two deceased male patients of Pakistani origin with severe panhypopituitarism associated with anterior pituitary aplasia and posterior pituitary ectopia. Both were born small for gestational age with a small phallus, undescended testes, and mid-facial hypoplasia. The parents' first-born child was a female with mid-facial hypoplasia (DNA was unavailable). Despite rapid commencement of hydrocortisone and T4 in the brothers, all three children died within the first week of life. The LHX4(p.T126M) variant is located within the LIM2 domain, in a highly conserved location. The absence of homozygosity for the variant in over 65 000 controls suggests that it is likely to be responsible for the phenotype. CONCLUSION: We report, for the first time to our knowledge, a novel homozygous mutation in LHX4 associated with a lethal phenotype, implying that recessive mutations in LHX4 may be incompatible with life.

Optimum effectiveness of intestinal alpha-glucosidase inhibitors: importance of uniform distribution through a meal.

A major barrier to the widespread clinical use of an alpha-glucosidase inhibitor such as Acarbose, is the unpleasant gastrointestinal symptoms of carbohydrate malabsorption associated with its use. Acarbose is usually administered as a tablet and eaten with the first mouthful of the meal, making its uniform distribution through the meal unlikely. In the present study, Acarbose was crushed to a powder and mixed through a test meal before it was consumed. Six healthy young men consumed test meals containing 75 g carbohydrate either as whole brown rice or as ground brown rice. When Acarbose was uniformly mixed through a ground rice meal prior to digestion it produced dose-dependent reductions in the postprandial glucose, insulin and GIP responses which were evident at doses as low as 12.5 mg. The responses to whole brown rice were intermediate between those to 12.5 and 25 mg Acarbose in ground brown rice. In tablet form Acarbose was only one quarter as effective in flattening the post prandial glucose and insulin responses as it was in powder form. These results highlight the importance of uniform distribution of Acarbose through a carbohydrate meal in order to achieve maximum effectiveness in delaying digestion and absorption and yet not promoting carbohydrate malabsorption.

Detection of residual donor leucocytes in leucoreduced red blood cell components using a fluorescence microplate assay.

In November 1999, universal leucoreduction of blood components was introduced in the UK to minimise the risk of variant Creutzfeldt-Jakob Disease (vCJD) transmission by blood transfusion. The UK specifications for leucodepletion processes state that 99% of leucodepleted components should contain < 5 x 10(6) leucocytes/unit, within 95% confidence limits. However, this leucocyte concentration is below the detection limits of standard haematology analysers. The development of a fluorometric immunoassay to detect the residual donor leucocytes in leucoreduced blood components is described. Monoclonal antibodies to leucocyte-specific cell surface antigens, CD45 and CD15, were adsorbed to the well surface in 96-well microplates. Red blood cell samples containing low numbers of leucocytes were added to the wells and the cells of interest captured by the monoclonal antibodies. Since leucocytes are the only nucleated cells found in significant numbers in blood components they were quantified using PicoGreen, a fluorescent stain specific for dsDNA. In comparison to flow cytometry, the method currently used to detect low numbers of leucocytes, the microplate assay demonstrated excellent sensitivity (1.00) and acceptable specificity (0.81) when standard leucodepleted samples were tested. There was no significant difference between the two methods (p < or = 0.175). In conclusion, the fluorescence microplate assay represents a simple, high throughput alternative to flow cytometry for monitoring leucodepletion compliance in blood banks.