Functional consequences of seven novel mutations in the CYP11B1 Gene: four mutations associated with nonclassic and three mutations causing classic 11 -Hydroxylase Deficiency

Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling i

Plasma oxalate: comparison of methodologies

Measurement of oxalate in the blood is essential for monitoring primary hyperoxaluria patients with progressive renal impairment and on dialysis prior to transplantation. As no external quality assurance scheme is available for this analyte, we conducted a sample exchange scheme between six laboratories specifcally involved with the investigation of primary hyperoxaluria to compare results. The methodologies compared were gas chromatography/mass spectrometry (GCMS), ion chromatography with mass spectrometry (ICMS), and enzymatic methods using oxalate oxidase and spectrophotometry. Although individual laboratories performed well in terms of reproducibility and linearity, there was poor agreement (absolute values) between centres as illustrated by a longer-term comparison of patient results from two of the participating laboratories. This situation was only partly related to diferences in calibration and mainly refected the lower recoveries seen with the ultrafltration of samples. These fndings lead us to conclude that longitudinal monitoring of primary hyperoxaluria patients with deteriorating kidney function should be performed by a single consistent laboratory and the methodology used should always be defned. In addition, plasma oxalate concentrations reported in registry studies and those associated with the risk of systemic oxalosis in published studies need to be interpreted in light of the methodology used. A reference method and external quality assurance scheme for plasma oxalate analysis would be benefcial

Next generation sequencing reveals novel genetic variants (SRY, DMRT1, NR5A1, DHH, DHX37) in adults with 46,XY DSD

Context: The genetic basis of human sex development is slowly being elucidated and more than 40 different genetic causes of differences (or disorders) of sex development (DSD) have now been reported. However, reaching a specific diagnosis using traditional approaches can be difficult, especially in adults where limited biochemical data may be available. / Objective: We used a targeted next-generation sequencing approach to analyze known and candidate genes for DSD in individuals with no specific molecular diagnosis. / Partcipants and Design: We studied 52 adult 46,XY women attending a single-center adult service, who were part of a larger cohort of 400 individuals. Classic conditions such as17β-hydroxysteroid dehydrogenase deficiency type 3, 5α-reductase deficiency type 2 and androgen insensitivity syndrome were excluded. The study cohort had broad working diagnoses of complete gonadal dysgenesis (CGD) (n=27) and partially-virilised 46,XY DSD (pvDSD) (n=25), a group that included partial gonadal dysgenesis (PGD) and those with a broad ”partial androgen insensitivity syndrome” label. Targetted sequencing of 168 genes was undertaken. / Results: Overall a likely genetic cause was found in 16/52 (30.8%) individuals (22.2% CGD; 40.0% pvDSD). Pathogenic variants were found in SRY (n=3), DMRT1 (n=1), NR5A1/SF-1 (n=1) and DHH (n=1) in the CGD group, and in NR5A1 (n=5), DHH (n=1) and DHX37 (n=4) in the pvDSD group. / Conclusions: Reaching a specific diagnosis can have clinical implications and provides insight into the role of these proteins in sex development. Next-generation sequencing approaches are invaluable, especially in adult populations or where diagnostic biochemistry is not possible

Nephrolithiasis related to inborn metabolic diseases

Nephrolithiasis associated with inborn metabolic diseases is a very rare condition with some common characteristics: early onset of symptoms, family history, associated tubular impairment, bilateral, multiple and recurrent stones, and association with nephrocalcinosis. The prognosis of such diseases may lead to life threatening conditions, not only because of unabated kidney damage but also because of progressive extra-renal involvement, either in a systemic form (e.g. primary hyperoxaluria type 1, requiring combined liver and kidney transplantation), or in a neurological form (Lesch–Nyhan syndrome leading to auto-mutilation and disability, phosphoribosyl pyrophosphate synthetase superactivity, which is associated with mental retardation). Patients with other inborn metabolic diseases present only with recurrent stone formation, such as cystinuria, adenine phosphoribosyl-transferase deficiency, xanthine deficiency. Finally, nephrolithiasis may be secondarily part of some other metabolic diseases, such as glycogen storage disease type 1 or inborn errors of metabolism leading to Fanconi syndrome (nephropathic cystinosis, tyrosinaemia type 1, fructose intolerance, Wilson disease, respiratory chain disorders, etc.). The diagnosis is based on highly specific investigations, including crystal identification, biochemical analyses and DNA study. The treatment of nephrolithiasis requires hydration as well as specific measures. Compliance is a major issue regarding the progression of renal damage, but the overall outcome mainly depends on extra-renal involvement in relation to the metabolic defect

Rumours, sects and rallies : the ethnic politics of recent Hmong Millenarian movements in Vietnam’s highlands

Contrary to modernist assumptions, millenarianism has not died out but continues to influence the politics of many marginalised groups in upland Southeast Asia, including the Hmong. This article summarises and analyses post-World War II Hmong millenarian activity in Vietnam, focusing on three case studies from the 1980s onwards, within the political backdrop of ongoing government suspicions of ethnic separatism and foreign interference. Far from being isolated or peripheral, Hmong millenarian rumours and movements interact with overseas diasporas, human rights agencies and international religious networks to influence state responses, sometimes in unexpected ways

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Background The effectiveness of using point-of-care (POC) urine culture in primary care on appropriate antibiotic use is unknown. Aim To assess whether use of the Flexicult™ SSI-Urinary Kit, which quantifies bacterial growth and determines antibiotic susceptibility at the point of care, achieves antibiotic use that is more often concordant with laboratory culture results, when compared with standard care. Design and setting Individually randomised trial of females with uncomplicated urinary tract infection (UTI) in primary care research networks (PCRNs) in England, the Netherlands, Spain, and Wales. Method Multilevel regression compared outcomes between the two groups while controlling for clustering. Results In total, 329 participants were randomised to POC testing (POCT) and 325 to standard care, and 324 and 319 analysed. Fewer females randomised to the POCT arm than those who received standard care were prescribed antibiotics at the initial consultation (267/324 [82.4%] versus 282/319 [88.4%], odds ratio [OR] 0.56, 95% confidence interval [CI] = 0.35 to 0.88). Clinicians indicated the POCT result changed their management for 190/301 (63.1%). Despite this, there was no statistically significant difference between study arms in antibiotic use that was concordant with laboratory culture results (primary outcome) at day 3 (39.3% POCT versus 44.1% standard care, OR 0.84, 95% CI = 0.58 to 1.20), and there was no evidence of any differences in recovery, patient enablement, UTI recurrences, re-consultation, antibiotic resistance, and hospitalisations at follow-up. POCT culture was not cost-effective. Conclusion Point-of-care urine culture was not effective when used mainly to adjust immediate antibiotic prescriptions. Further research should evaluate use of the test to guide initiation of ‘delayed antibiotics’

Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation

BACKGROUND: Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear. METHODS: PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays. RESULTS: PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation. CONCLUSIONS: A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.British Journal of Cancer advance online publication, 14 November 2017; doi:10.1038/bjc.2017.391 www.bjcancer.com