Lipids and amyotrophic lateral sclerosis: A two-sample Mendelian randomization study.

OBJECTIVE: Previous observational studies revealed a potential but partially controversial relation between lipid metabolism and the risk of amyotrophic lateral sclerosis (ALS), potentially prone to bias. Therefore, we aimed to study whether lipid metabolism involves genetically determined risk factors for ALS through Mendelian randomization (MR) analysis. METHODS: Using genome-wide association study summary-level data for total cholesterol (TC) (n = 188,578), high-density lipoprotein cholesterol (HDL-C) (n = 403,943), low-density lipoprotein cholesterol (LDL-C) (n = 440,546), apolipoprotein A1 (ApoA1) (n = 391,193), apolipoprotein B (ApoB) (n = 439,214), and ALS (12,577 cases and 23,475 controls), we implemented a bidirectional MR study to evaluate a genetic relation between lipids and ALS risk. We performed a mediation analysis to assess whether LDL-C is a potential mediator on the pathway from traits of LDL-C-related polyunsaturated fatty acids (PUFAs) to ALS risk. RESULTS: We identified genetically predicted increased lipid levels to be associated with the risk of ALS, whereby elevated LDL-C had the most potent effect (OR 1.028, 95% CI 1.008-1.049, p = 0.006). The effect of increased levels of apolipoproteins on ALS was similar to their corresponding lipoproteins. ALS did not cause any changes in lipid levels. We found no relation between LDL-C-modifying lifestyles and ALS. The mediation analysis revealed that LDL-C could act as an active mediator for linoleic acid, with the mediation effect estimated to be 0.009. CONCLUSIONS: We provided high-level genetic evidence verifying the positive link between preclinically elevated lipid and ALS risk that had been described in previous genetic and observational studies. We also demonstrated the mediating role of LDL-C in the pathway from PUFAs to ALS.

Immunohistochemical analysis of sex hormone receptors in squamous changes of the urothelium.

OBJECTIVE: Squamous cell transformation of the urinary bladder urothelium has various causes, symptoms, and few treatment options. The aim of this study was to analyze and compare the expression of sex hormone receptors in non-keratinized and keratinized squamous metaplasia (NKSM, KSM), squamous cell carcinoma (SCC), and healthy urothelium with regard to possible therapeutic approaches. METHODS: Biopsies from 26 patients with urothelial NKSM, KSM, and SCC were analyzed retrospectively. Tissue microarrays (TMA) of formalin-fixed paraffin-embedded (FFPE) bladder biopsies were stained with hematoxylin and eosin followed by immunohistochemical analysis with specific antibodies against estrogen, progesterone, and androgen receptors (ER, PR, AR) and assessment using the immunoreactive score. Statistical evaluations included the Wilcoxon signed-rank test and the Wilcoxon rank-sum test in the form of permutation tests. RESULTS: Of the 15 women and 11 men included in this explorative study, 17 had metaplasia: 15 (six men, nine women) had NKSM and two KSM (both men). A total of nine patients (three men, six women) had keratinized SCC or urothelial carcinoma with squamous differentiation. The comparison between normal urothelial cells and metaplasia showed a significantly stronger expression in the metaplastic tissue (P=0.0374). The invasive carcinoma showed significantly less PR than the extracellular matrix of the healthy urothelium (P=0.0026). Expression of AR was nearly absent in healthy and metaplastic urothelium. CONCLUSION: There appears to be an association between squamous metaplasia of the bladder mucosa and sex steroid hormone receptor expression, especially estrogen receptors. Topical hormone therapy should be considered.

T-cell prolymphocytic leukemia is associated with deregulation of oncogenic microRNAs on transcriptional and epigenetic level.

Deregulation of micro(mi)-RNAs is a common mechanism in tumorigenesis. We investigated the expression of 2083 miRNAs in T-cell prolymphocytic leukemia (T-PLL). Compared to physiologic CD4+ and CD8+ T-cell subsets, 111 miRNAs were differentially expressed in T-PLL. Of these, 33 belonged to miRNA gene clusters linked to cancer. Genomic variants affecting miRNAs were infrequent with the notable exception of copy number aberrations. Remarkably, we found strong upregulation of the miR-200c/-141 cluster in T-PLL to be associated with DNA hypomethylation and active promoter marks. Our findings suggest that copy number aberrations and epigenetic changes could contribute to miRNA deregulation in T-PLL.

Evaluation of Meal Carbohydrate Counting Errors in Patients with Type 1 Diabetes.

AIM: Correct estimation of meal carbohydrate content is a prerequisite for successful intensified insulin therapy in patients with diabetes. In this survey, the counting error in adult patients with type 1 diabetes was investigated. METHODS: Seventy-four patients with type 1 diabetes estimated the carbohydrate content of 24 standardized test meals. The test meals were categorized into 1 of 3 groups with different carbohydrate content: low, medium, and high. Estimation results were compared with the meals' actual carbohydrate content as determined by calculation based on weighing. A subgroup of the participants estimated the test meals for a second (n=35) and a third time (n=22) with a mean period of 11 months between the estimations. RESULTS: During the first estimation, the carbohydrate content was underestimated by -28% (-50, 0) of the actual carbohydrate content. Particularly meals with high mean carbohydrate content were underestimated by -34% (-56, -13). Median counting error improved significantly when estimations were performed for a second time (p<0.001). CONCLUSIONS: Participants generally underestimated the carbohydrate content of the test meals, especially in meals with higher carbohydrate content. Repetition of estimation resulted in significant improvements in estimation accuracy and is important for the maintenance of correct carbohydrate estimations. The ability to estimate the carbohydrate content of a meal should be checked and trained regularly in patients with diabetes.

Extreme clustering of type-1 NF1 deletion breakpoints co-locating with G-quadruplex forming sequences.

The breakpoints of type-1 NF1 deletions encompassing 1.4-Mb are located within NF1-REPa and NF1-REPc, which exhibit a complex structure comprising different segmental duplications in direct and inverted orientation. Here, we systematically assessed the proportion of type-1 NF1 deletions caused by nonallelic homologous recombination (NAHR) and those mediated by other mutational mechanisms. To this end, we analyzed 236 unselected type-1 deletions and observed that 179 of them (75.8%) had breakpoints located within the NAHR hotspot PRS2, whereas 39 deletions (16.5%) had breakpoints located within PRS1. Sixteen deletions exhibited breakpoints located outside of these NAHR hotspots but were also mediated by NAHR. Taken together, the breakpoints of 234 (99.2%) of the 236 type-1 NF1 deletions were mediated by NAHR. Thus, NF1-REPa and NF1-REPc are strongly predisposed to recurrent NAHR, the main mechanism underlying type-1 NF1 deletions. We also observed a non-random overlap between type-1 NF1-deletion breakpoints and G-quadruplex forming sequences (GQs) as well as regions flanking PRDM9A binding-sites. These findings imply that GQs and PRDM9A binding-sites contribute to the clustering of type-1 deletion breakpoints. The co-location of both types of sequence was at its highest within PRS2, indicative of their synergistic contribution to the greatly increased NAHR activity within this hotspot.

Effect of aggregation form on bioavailability of zeaxanthin in humans: a randomised cross-over study.

Carotenoid bioavailability from plant and animal food is highly variable depending on numerous factors such as the physical deposition form of carotenoids. As the carotenoid zeaxanthin is believed to play an important role in eye and brain health, we sought to compare the human bioavailability of an H-aggregated with that of a J-aggregated deposition form of zeaxanthin encapsulated into identical formulation matrices. A randomised two-way cross-over study with sixteen participants was designed to compare the post-prandial bioavailability of an H-aggregated zeaxanthin and a J-aggregated zeaxanthin dipalmitate formulation, both delivering 10 mg of free zeaxanthin. Carotenoid levels in TAG-rich lipoprotein fractions were analysed over 9·5 h after test meal consumption. Bioavailability from the J-aggregated formulation (AUC=55·9 nmol h/l) was 23 % higher than from the H-aggregated one (AUC=45·5 nmol h/l), although being only marginally significant (P=0·064). Furthermore, the same formulations were subjected to an internationally recognised in vitro digestion protocol to reveal potential strengths and weaknesses of simulated digestions. In agreement with our human study, liberation of zeaxanthin from the J-aggregated formulation into the simulated duodenal fluids was superior to that from the H-aggregated form. However, micellization rate (bioaccessibility) of the J-aggregated zeaxanthin dipalmitate was lower than that of the H-aggregated zeaxanthin, being contradictory to our in vivo results. An insufficient ester cleavage during simulated digestion was suggested to be the root cause for these observations. In brief, combining our in vitro and in vivo observations, the effect of the different aggregation forms on human bioavailability was lower than expected.

Consideration of the haplotype diversity at nonallelic homologous recombination hotspots improves the precision of rearrangement breakpoint identification.

Precise characterization of nonallelic homologous recombination (NAHR) breakpoints is key to identifying those features that influence NAHR frequency. Until now, analysis of NAHR-mediated rearrangements has generally been performed by comparison of the breakpoint-spanning sequences with the human genome reference sequence. We show here that the haplotype diversity of NAHR hotspots may interfere with breakpoint-mapping. We studied the transmitting parents of individuals with germline type-1 NF1 deletions mediated by NAHR within the paralogous recombination site 1 (PRS1) or paralogous recombination site 2 (PRS2) hotspots. Several parental wild-type PRS1 and PRS2 haplotypes were identified that exhibited considerable sequence differences with respect to the reference sequence, which also affected the number of predicted PRDM9-binding sites. Sequence comparisons between the parental wild-type PRS1 or PRS2 haplotypes and the deletion breakpoint-spanning sequences from the patients (method #2) turned out to be an accurate means to assign NF1 deletion breakpoints and proved superior to crude reference sequence comparisons that neglect to consider haplotype diversity (method #1). The mean length of the deletion breakpoint regions assigned by method #2 was 269-bp in contrast to 502-bp by method #1. Our findings imply that paralog-specific haplotype diversity of NAHR hotspots (such as PRS2) and population-specific haplotype diversity must be taken into account in order to accurately ascertain NAHR-mediated rearrangement breakpoints.

Dysfunction of the MDM2/p53 axis is linked to premature aging.

The tumor suppressor p53, a master regulator of the cellular response to stress, is tightly regulated by the E3 ubiquitin ligase MDM2 via an autoregulatory feedback loop. In addition to its well-established role in tumorigenesis, p53 has also been associated with aging in mice. Several mouse models with aberrantly increased p53 activity display signs of premature aging. However, the relationship between dysfunction of the MDM2/p53 axis and human aging remains elusive. Here, we have identified an antiterminating homozygous germline mutation in MDM2 in a patient affected by a segmental progeroid syndrome. We show that this mutation abrogates MDM2 activity, thereby resulting in enhanced levels and stability of p53. Analysis of the patient's primary cells, genome-edited cells, and in vitro and in vivo analyses confirmed the MDM2 mutation's aberrant regulation of p53 activity. Functional data from a zebrafish model further demonstrated that mutant Mdm2 was unable to rescue a p53-induced apoptotic phenotype. Altogether, our findings indicate that mutant MDM2 is a likely driver of the observed segmental form of progeria.

Immune phenotypes predict survival in patients with glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM), a common primary malignant brain tumor, rarely disseminates beyond the central nervous system and has a very bad prognosis. The current study aimed at the analysis of immunological control in individual patients with GBM. METHODS: Immune phenotypes and plasma biomarkers of GBM patients were determined at the time of diagnosis using flow cytometry and ELISA, respectively. RESULTS: Using descriptive statistics, we found that immune anomalies were distinct in individual patients. Defined marker profiles proved highly relevant for survival. A remarkable relation between activated NK cells and improved survival in GBM patients was in contrast to increased CD39 and IL-10 in patients with a detrimental course and very short survival. Recursive partitioning analysis (RPA) and Cox proportional hazards models substantiated the relevance of absolute numbers of CD8 cells and low numbers of CD39 cells for better survival. CONCLUSIONS: Defined alterations of the immune system may guide the course of disease in patients with GBM and may be prognostically valuable for longitudinal studies or can be applied for immune intervention.

Urinary excretion of Citrus flavanones and their major catabolites after consumption of fresh oranges and pasteurized orange juice: A randomized cross-over study.

SCOPE: Orange juice contains flavanones including hesperidin and narirutin, albeit at lower concentrations as compared to orange fruit. Therefore, we compared bioavailability and colonic catabolism of flavanones from orange juice to a 2.4-fold higher dose from fresh oranges. METHODS AND RESULTS: Following a randomized two-way cross-over design, 12 healthy subjects consumed a test meal comprising either fresh oranges or pasteurized orange juice, delivering 1774 and 751 µmol of total Citrus flavanones, respectively. Deglucuronidated and desulfated hesperetin, naringenin, and the flavanone catabolites 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid, 3-(3'-hydroxyphenyl)hydracrylic acid, 4-hydroxyhippuric acid, and hippuric acid were quantitated in 24-h urine by UHPLC-MS/MS. Differences in urinary hesperetin excretion were found to be nonsignificant (p = 0.5209) both after consumption of orange fruit (21.6 ± 8.0 µmol) and juice (18.3 ± 7.2 µmol). By analogy, postprandial flavanone catabolite excretions were highly similar between treatments. Excretion of 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid was inversely related to that of hesperetin, illustrating the catabolite/precursor relationship. CONCLUSION: Despite 2.4-fold higher doses, excretion of flavanones from ingested fresh orange fruit did not differ from that following orange juice consumption, possibly due to a saturation of absorption or their entrapment in the fiber-rich matrix of the fruit.

Analysis of a cohort of 101 CDAII patients: description of 24 new molecular variants and genotype-phenotype correlations.

Congenital dyserythropoietic anaemia type II (CDAII) is a rare autosomal recessive disease characterized by ineffective erythropoiesis, haemolysis, erythroblast morphological abnormalities, hypoglycosylation of some red blood cell membrane proteins, particularly band 3, and mutations in the SEC23B gene. We report the analysis of 101 patients from 91 families with a median follow-up of 23 years (range 0-65); 68 patients are newly reported. Clinical and haematological parameters were separately analysed in early infancy and thereafter, when feasible. Molecular analysis of the SEC23B gene confirmed the high heterogeneity of the defect, leading to the identification of 54 different mutations, 24 of which are newly described. To evaluate the genotype-phenotype correlation, patients were grouped according to their genotype (two missense mutations vs. one missense/one drastic mutation) and assigned to two different severity gradings based on laboratory data and on therapeutic needs; by this approach only a weak genotype-phenotype correlation was observed in the analysed groups.

De novo FUS mutations are the most frequent genetic cause in early-onset German ALS patients.

In amyotrophic lateral sclerosis (ALS) patients with known genetic cause, mutations in chromosome 9 open reading frame 72 (C9orf72) and superoxide dismutase 1 (SOD1) account for most familial and late-onset sporadic cases, whereas mutations in fused in sarcoma (FUS) can be identified in just around 5% of familial and 1% of overall sporadic cases. There are only few reports on de novo FUS mutations in juvenile ALS patients. To date, no systematic evaluation on the frequency of de novo FUS mutations in early-onset ALS patients has been conducted. Here, we screened a cohort of 14 early-onset sporadic ALS patients (onset age <35 years) to determine the frequency of mutations in C9orf72, SOD1, and FUS in this defined patient cohort. All patients were recruited prospectively by a single center in a period of 38 months. No mutations were detected in SOD1 or C9orf72; however, we identified 6 individuals (43%) carrying a heterozygous FUS mutation including 1 mutation that has not been described earlier (c.1504delG [p.Asp502Thrfs*27]). Genetic testing of parents was possible in 5 families and revealed that the mutations in these patients arose de novo. Three of the 6 identified patients presented with initial bulbar symptoms. Our study identifies FUS mutations as the most frequent genetic cause in early-onset ALS. Genetic testing of FUS thus seems indicated in sporadic early-onset ALS patients especially if showing predominant bulbar symptoms and an aggressive disease course.

POLD1 Germline Mutations in Patients Initially Diagnosed with Werner Syndrome.

Segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ. A prototypic example is the Werner syndrome (WS), caused by biallelic germline mutations in the Werner helicase gene (WRN). While heterozygous lamin A/C (LMNA) mutations are found in a few nonclassical cases of WS, another 10%-15% of patients initially diagnosed with WS do not have mutations in WRN or LMNA. Germline POLD1 mutations were recently reported in five patients with another segmental progeroid disorder: mandibular hypoplasia, deafness, progeroid features syndrome. Here, we describe eight additional patients with heterozygous POLD1 mutations, thereby substantially expanding the characterization of this new example of segmental progeroid disorders. First, we identified POLD1 mutations in patients initially diagnosed with WS. Second, we describe POLD1 mutation carriers without clinically relevant hearing impairment or mandibular underdevelopment, both previously thought to represent obligate diagnostic features. These patients also exhibit a lower incidence of metabolic abnormalities and joint contractures. Third, we document postnatal short stature and premature greying/loss of hair in POLD1 mutation carriers. We conclude that POLD1 germline mutations can result in a variably expressed and probably underdiagnosed segmental progeroid syndrome.

Bioavailability of ß-cryptoxanthin is greater from pasteurized orange juice than from fresh oranges - a randomized cross-over study.

SCOPE: Orange fruits and products thereof represent important dietary sources of carotenoids, particularly ß-cryptoxanthin. Since previous studies reported a positive effect of vegetable processing on carotenoid absorption, our objective was to compare the bioavailability of ß-cryptoxanthin from either fresh navel oranges (Citrus sinensis L. Osbeck) or pasteurized orange juice. METHODS AND RESULTS: The study was designed as a randomized 2-way cross-over study. Twelve volunteers consumed two meals delivering 744 µg of ß-cryptoxanthin from either fresh navel oranges or pasteurized orange juice. Eight blood samples were collected over 9.5 h after test meal consumption and analyzed using HPLC-DAD. Additionally, carotenoid bioaccessibility was assessed after in vitro digestion of the same test foods. ß-cryptoxanthin bioavailability from pasteurized orange juice was 1.8-fold higher than from fresh oranges (P = 0.011). Similarly, mean absorption of the non-dose adjusted carotenoids lutein (P = 0.301), zeaxanthin (P = 0.216), and zeinoxanthin (P = 0.090) were slightly higher from orange juice, although not reaching statistical significance. The in vitro digestion revealed a 5.3-fold higher bioaccessibility of ß-cryptoxanthin from orange juice. Dietary fiber contents in the test foods were inversely associated with carotenoid bioavailability. CONCLUSION: Orange juice represents a more bioavailable source of ß-cryptoxanthin than fresh oranges.

Accuracy Evaluation of Four Blood Glucose Monitoring Systems in Unaltered Blood Samples in the Low Glycemic Range and Blood Samples in the Concentration Range Defined by ISO 15197.

INTRODUCTION: Systems for self-monitoring of blood glucose (SMBG) are expected to be accurate enough to provide reliable measurement results. Especially in the low glycemic range, adequate therapeutic decisions based on reliable results can alleviate complications associated with hypoglycemia. MATERIALS AND METHODS: The accuracy of four SMBG systems (system 1 was the ACCU-CHEK(®) Aviva [Roche Diagnostics GmbH, Mannheim, Germany], system 2 was the Contour(®) XT [Bayer Consumer Care AG, Basel, Switzerland], system 3 was the GlucoCheck XL [aktivmed GmbH, Augsberg, Germany], and system 4 was the GlucoMen(®) LX PLUS [A. Menarini Diagnostics S.r.l., Florence, Italy]) with three test-strip lots each was evaluated by calculating mean absolute relative differences (MARDs). Two datasets were evaluated: (1) 100 samples with blood glucose concentrations <70 mg/dL and (2) 100 samples distributed following International Organization for Standardization (ISO) standard 15197. Each sample was measured twice with each test-strip lot of each SMBG system. Comparison measurement results were obtained with a glucose oxidase method and a hexokinase method, both traceable according to ISO 17511. Analysis of variance of the MARD between the SMBG system and the comparison method was performed. RESULTS: MARD values ranged from 4.4% to 13.4% (<70 mg/dL) and 4.8% to 8.9% (ISO 15197-distributed) and differed significantly, with systems 1 and 2 showing lower MARDs than systems 3 and 4. MARD values deviated by up to 2.5% (corresponding to a relative deviation of approximately 40%) between the two comparison methods. CONCLUSIONS: The investigated SMBG systems showed a significant variation of accuracy (measured by MARD), especially with higher MARD values in the low glycemic range. The selected comparison method had an impact on the MARD and therefore on the apparent accuracy of the SMBG systems. Sufficient measurement accuracy in the low glycemic range is required to enable users to react adequately to hypoglycemia.

Normobaric Intermittent Hypoxia over 8 Months Does Not Reduce Body Weight and Metabolic Risk Factors--a Randomized, Single Blind, Placebo-Controlled Study in Normobaric Hypoxia and Normobaric Sham Hypoxia.

OBJECTIVE: Both a 1- to 4-week continuous or intermittent stay and moderate exercise in hypoxia versus normoxia can lead to weight loss. We examined the reproducibility and durability of added hypoxic exposure in a feasible health program of several months. METHODS: 32 obese persons, randomly assigned to either a hypoxia (age 50.3 ± 10.3 years, BMI 37.9 ± 8.1 kg/m(²)) or a normoxia (age 52.4 ± 7.9 years, BMI 36.3 ± 4.0 kg/m(²)) group, completed 52 exercise sessions within 8 months. Participants exercised for 90 min (65-70% HR(peak)) either at a simulated altitude of 3,500 m or in normoxia, and rested for further 90 min at 4,500 m or normoxia. Before, after 5 weeks, after 3 months, and after the intervention, body composition and exercise capacity were determined. Risk markers (e.g., blood pressure, cholesterol) were measured before, after 3 months, and after the intervention period. RESULTS: Body weight, BMI, waist and hip circumference, P(peak) and BP(sys) improved over time (p < 0.05) but without group difference. Fat mass reductions correlated with HDL changes (r = -0.427; p < 0.05) in the entire group. CONCLUSION: Long-term, moderate intensity exercise and rest in hypoxia does not lead to higher reductions in body weight than normoxia alone. Therefore, for weight loss and metabolic markers hypoxic exposure does not add effects at least when stimuli (i.e., hypoxia dose, exercise intensity/duration) are unaltered throughout the intervention.

Do orofacial clefts represent different genetic entities?

OBJECTIVE: To contribute to the understanding of potential genetic differences between different cleft types. METHOD: Analysis of family history concerning cleft type and search for cleft-type-specific associations in candidate genes performed in 98 individuals from 98 families. RESULTS: In a given family, the cleft type of a second case was more often identical to the index case than expected by chance. Each type of cleft (cleft lip [CL], cleft lip and palate [CLP], cleft palate only [CP], and submucous cleft palate only [SMCP]) was associated with different genes. CONCLUSION: Family history indicates some specificity of cleft types. The observed phenotype-genotype associations were compatible with this interpretation in that significant associations occurred with disjoint sets of genes in each cleft type. These observations indicate that CL, CLP, CP, and SMCP might represent genetically different entities.

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.