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Abstract Background Although B-cell depleting therapy in rheumatoid arthritis (RA) is clearly effective, response is variable and does not correlate with B cell depletion itself. Methods The B-cell receptor (BCR) repertoire was prospectively analyzed in peripheral blood samples of twenty-eight RA patients undergoing rituximab therapy. Timepoints of achieved BCR-depletion and -repopulation were defined based on the percentage of unmutated BCRs in the repertoire. The predictive value of early BCR-depletion (within one-month post-treatment) and early BCR-repopulation (within 6 months post-treatment) on clinical response was assessed. Results We observed changes in the peripheral blood BCR repertoire after rituximab treatment, i.e., increased clonal expansion, decreased clonal diversification and increased mutation load which persisted up to 12 months after treatment, but started to revert at month 6. Early BCR depletion was not associated with early clinical response but late depleters did show early response. Patients with early repopulation with unmutated BCRs showed a significant decrease in disease activity in the interval 6 to 12 months. Development of anti-drug antibodies non-significantly correlated with more BCR repopulation. Conclusion Our findings indicate that rather than BCR-depletion it is repopulation with unmutated BCRs, possibly from naïve B cells, which induces remission. This suggests that (pre-existing) differences in B-cell turnover between patients explain the interindividual differences in early clinical effect.

Abstract Emm is a high incidence red cell antigen with eight previously reported Emm− probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm− brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm− individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm− brothers and by gnomAD frequency of

Abstract Beta-hemoglobinopathies become prominent after birth due to a switch from γ-globin to the mutated β-globin. Haploinsufficiency for the erythroid specific indispensable transcription factor Krueppel-like factor 1 (KLF1) is associated with high persistence of fetal hemoglobin (HPFH). The In(Lu) phenotype, characterized by low to undetectable Lutheran blood group expression is caused by mutations within KLF1 gene. Here we screened a blood donor cohort of 55 Lutheran weak or negative donors for KLF1 variants and evaluated their effect on KLF1 target gene expression. To discriminate between weak and negative Lutheran expression, a flow cytometry (FCM) assay was developed to detect Lu antigen expression. The Lu(a−b−) (negative) donor group, showing a significant decreased CD44 (Indian blood group) expression, also showed increased HbF and HbA2 levels, with one individual expressing HbF as high as 5%. KLF1 exons and promoter sequencing revealed variants in 80% of the Lutheran negative donors. Thirteen different variants plus one high frequency SNP (c.304 T > C) were identified of which 6 were novel. In primary erythroblasts, knockdown of endogenous KLF1 resulted in decreased CD44, Lu and increased HbF expression, while KLF1 over-expressing cells were comparable to wild type (WT). In line with the pleiotropic effects of KLF1 during erythropoiesis, distinct KLF1 mutants expressed in erythroblasts display different abilities to rescue CD44 and Lu expression and/or to affect fetal (HbF) or adult (HbA) hemoglobin expression. With this study we identified novel KLF1 variants to be include into blood group typing analysis. In addition, we provide further insights into the regulation of genes by KLF1.

Abstract Background Visual hallucinations are common in patients with Parkinson's disease and represent probably the major independent predictor for cognitive deterioration and nursing home placement. Objective To investigate if treatment of minor visual hallucinations in Parkinson's disease with rivastigmine delays the progression to psychosis. Methods A multicenter, randomized, double‐blind, placebo‐controlled trial was conducted which aimed to recruit 168 patients with Parkinson's disease reporting minor visual hallucinations 4 weeks before it. Important exclusion criteria were Parkinson's disease dementia, current delirium, and treatment with antipsychotics or drugs that have significant anti‐cholinergic side effects. Subjects were randomized to rivastigmine capsules, 3–6 mg twice a day, or placebo for 24 months. The primary outcome was the time to Parkinson's disease psychosis, which was defined as the need to start with antipsychotics. Results The trial was stopped prematurely because of slow recruitment. Ninety‐one patients were randomized: 46 patients were assigned to rivastigmine and 45 patients to placebo. No effect of rivastigmine could be demonstrated on the transition time to psychosis or dementia during the 24‐month follow‐up period. After 6 months of study treatment, cognition, mood, motor performance, and non‐motor performance did not differ significantly between the rivastigmine‐group and the placebo‐group. Conclusions Because the study was terminated early, it was insufficiently powered to properly evaluate the primary outcome. The limited data of the study favor a wait and see approach instead of early treatment with rivastigmine in PD patients with minor VH.

Abstract: In childhood immune thrombocytopenia (ITP), anti-platelet autoantibodies mediate platelet clearance through Fc-γ receptor (FcγR)–bearing phagocytes. In 75% to 90% of patients, the disease has a transient, self-limiting character. Here we characterized how polymorphisms of FcγR genes affect disease susceptibility, response to intravenous immunoglobulin (IVIg) treatment, and long-term recovery from childhood ITP. Genotyping of the FCGR2/3 locus was performed in 180 children with newly diagnosed ITP, 22 children with chronic ITP, and 180 healthy control children by multiplex ligation-dependent probe amplification. Children with newly diagnosed ITP were randomly assigned to a single administration of IVIg or observation, and followed for 1 year (Treatment With or Without IVIg for Kids With ITP [TIKI] trial). We defined transient ITP as a complete recovery (≥100 × 109/L) 3 months after diagnosis, including both self-limiting disease/IVIg responders and chronic ITP as absence of a complete recovery at 12 months. ITP susceptibility, as well as spontaneous recovery and response to IVIg, was associated with the genetic variants FCGR2C*ORF and FCGR2A*27W and the FCGR2B promoter variant 2B.4. These variants were overrepresented in patients with transient (N = 131), but not chronic (N = 43), disease. The presence of FCGR2C*ORF predisposed to transient ITP with an odds ratio of 4.7 (95% confidence interval, 1.9-14.3). Chronic ITP was associated with a deletion of FCGR2C/FCGR3B (copy number region 1) with an odds ratio of 6.2 (95% confidence interval, 1.8-24.7). Taken together, susceptibility to transient and chronic ITP is distinctly affected by polymorphic variants of FCGR2/3 genes. Our data suggest that genotyping of the FCGR2/3 locus may be useful for prognosis and guidance of treatment decisions in newly diagnosed childhood ITP.

Visuele problemen komen regelmatig voor bij de oudere patiënt enworden vaak veroorzaakt door oogheelkundige problemen. In deze klinische les laten we zien dat visuele problemen ook veroorzaakt kunnen worden door posterieure corticale atrofie (PCA). PCA is een klinisch-radiologisch syndroom dat vaak veroorzaakt wordt door een neurodegeneratieve aandoening zoals de ziekte van Alzheimer, dementie met Lewy-lichaampjes of corticobasale degeneratie. Het wordt gekenmerkt door progressieve achteruitgang in visuele informatieverwerking. Daarbij is er relatief behoud van geheugen en taal in vroege stadia. Bij beeldvorming van de hersenen wordt atrofie van de posterieure hersengebieden gezien.

Alloimmunization is currently the most frequent adverse blood transfusion event. Whilst completely matched donor blood would nullify the alloimmunization risk, this is practically infeasible. Current matching strategies therefore aim at matching a limited number of blood groups only, and have evolved over time by systematically including matching strategies for those blood groups for which (serious) alloimmunization complications most frequently occurred. An optimal matching strategy for controlling the risk of alloimmunization however, would balance alloimmunization complications and costs within the entire blood supply chain, whilst fulfilling all practical requirements and limitations. In this article the outline of an integrated blood management model is described and various potential challenges and prospects foreseen with the development of such a model are discussed.

Abstract Background Lynch syndrome (LS) is an autosomal dominant inherited cancer syndrome characterized by early onset cancers of the colorectum, endometrium and other tumours. A significant proportion of DNA variants in LS patients are unclassified. Reports on the pathogenicity of the c.1852_1853AA>GC (p.Lys618Ala) variant of the MLH1 gene are conflicting. In this study, we provide new evidence indicating that this variant has no significant implications for LS. Methods The following approach was used to assess the clinical significance of the p.Lys618Ala variant: frequency in a control population, case-control comparison, co-occurrence of the p.Lys618Ala variant with a pathogenic mutation, co-segregation with the disease and microsatellite instability in tumours from carriers of the variant. We genotyped p.Lys618Ala in 1034 individuals (373 sporadic colorectal cancer [CRC] patients, 250 index subjects from families suspected of having LS [revised Bethesda guidelines] and 411 controls). Three well-characterized LS families that fulfilled the Amsterdam II Criteria and consisted of members with the p.Lys618Ala variant were included to assess co-occurrence and co-segregation. A subset of colorectal tumour DNA samples from 17 patients carrying the p.Lys618Ala variant was screened for microsatellite instability using five mononucleotide markers. Results Twenty-seven individuals were heterozygous for the p.Lys618Ala variant; nine had sporadic CRC (2.41%), seven were suspected of having hereditary CRC (2.8%) and 11 were controls (2.68%). There were no significant associations in the case-control and case-case studies. The p.Lys618Ala variant was co-existent with pathogenic mutations in two unrelated LS families. In one family, the allele distribution of the pathogenic and unclassified variant was in trans, in the other family the pathogenic variant was detected in the MSH6 gene and only the deleterious variant co-segregated with the disease in both families. Only two positive cases of microsatellite instability (2/17, 11.8%) were detected in tumours from p.Lys618Ala carriers, indicating that this variant does not play a role in functional inactivation of MLH1 in CRC patients. Conclusions The p.Lys618Ala variant should be considered a neutral variant for LS. These findings have implications for the clinical management of CRC probands and their relatives.

Abstract Background The Int7G24A variant of transforming growth factor-beta receptor type I (TGFBR1) has been shown to increase the risk for kidney, ovarian, bladder, lung and breast cancers. Its role in colorectal cancer (CRC) has not been established. The aims of this study were to assess the association of TGFBR1*Int7G24A variant with CRC occurrence, patient age, gender, tumour location and stage. Methods We performed a case-control study with 504 cases of sporadic CRC; and 504 non-cancerous age, gender and ethnically matched controls. Genotyping analysis was performed using allelic discrimination assay by real time PCR. Results The Int7G24A variant was associated with increased CRC incidence in an additive model of inheritance (P for trend = 0.005). No significant differences were found between Int7G24A genotypes and tumour location or stage. Interestingly, the association of the Int7G24A variant with CRC risk was significant in men (odds ratio 4.10 with 95% confidence intervals 1.41-11.85 for homozygous individuals; P for trend = 0.00023), but not in women. We also observed an increase in susceptibility to CRC for individuals aged less than 70 years. Conclusion Our data suggest that the Int7G24A variant represents a risk factor for CRC in the male Spanish population.

Abstract Background TGF-β receptor type I is a mediator of growth inhibitory signals. TGFBR1*6A (rs11466445) is a common polymorphic variant of the TGF-β receptor I gene and has been associated with tumour susceptibility. Nevertheless, the role of this polymorphism as a risk factor for colorectal cancer is controversial. The aim of this study was to assess the association between TGFBR1*6A and colorectal cancer, age, sex, tumour location and tumour stage in a Spanish population. Methods The case-control study involved 800 Spanish subjects: 400 sporadic colorectal cancer patients and 400 age-, sex-, and ethnic-matched controls. The odds ratio (OR) and 95% confidence interval (95% CI) for the TGFBR1*6A polymorphism were calculated using unconditional logistic regression adjusted for age and sex. Analysis of somatic mutations at the GCG repeat of TGFBR1 exon 1 and germline allele-specific expression were also conducted to obtain further information on the contribution of the TGFBR1*6A allele to CRC susceptibility. Results There was no statistically significant association between the TGFBR1*6A allele and CRC (p > 0.05). The OR was 1.147 (95% CI: 0.799–1.647) for carriers of the TGFBR1*6A allele and 0.878 (95% CI: 0.306–2.520) for homozygous TGFBR1*6A individuals compared with the reference. The frequency of the polymorphism was not affected by age, sex or tumour stage. The TGFBR1*6A allele was more prevalent among colon tumour patients than among rectal tumour patients. Tumour somatic mutations were found in only two of 69 cases (2.9%). Both cases involved a GCG deletion that changed genotype 9A/9A in normal DNA to genotype 9A/8A. Interestingly, these two tumours were positive for microsatellite instability, suggesting that these mutations originated because of a deficient DNA mismatch repair system. Allele-specific expression of the 9A allele was detected in seven of the 14 heterozygous 9A/6A tumour cases. This could have been caused by linkage disequilibrium of the TGFBR1*6A allele with mutations that cause allele-specific expression, as was recently suggested. Conclusion Our results suggest that the TGFBR1*6A allele does not confer an increased risk of colorectal cancer in the Spanish population.

Background: Pancreatic ductal adenocarcinoma (PDAC) is usually diagnosed in late adulthood; therefore, many patients suffer or have suffered from other diseases. Identifying disease patterns associated with PDAC risk may enable a better characterization of high-risk patients. Methods: Multimorbidity patterns (MPs) were assessed from 17 self-reported conditions using hierarchical clustering, principal component and factor analyses in 1705 PDAC cases and 1084 controls from a European population. Their association with PDAC was evaluated using adjusted logistic regression models. Time since diagnosis of morbidities to PDAC diagnosis/ recruitment was stratified into recent (<3 years) and long term (⩾3 years). The MPs and PDAC genetic networks were explored with DisGeNET bioinformatics-tool which focuses on gene-diseases associations available in curated databases. Results: Three MPs were observed: gastric (heartburn, acid regurgitation, Helicobacter pylori infection and ulcer), metabolic syndrome (obesity, type-2 diabetes, hypercholesterolemia and hypertension) and atopic (nasal allergies, skin allergies and asthma). Strong associations with PDAC were observed for ⩾2 recently diagnosed gastric conditions [odds ratio (OR), 6.13; 95% confidence interval CI 3.01-12.5)] and for ⩾3 recently diagnosed metabolic syndrome conditions (OR, 1.61; 95% CI 1.11-2.35). Atopic conditions were negatively associated with PDAC (high adherence score OR for tertile III, 0.45; 95% CI, 0.36-0.55). Combining type-2 diabetes with gastric MP resulted in higher PDAC risk for recent (OR, 7.89; 95% CI 3.9-16.1) and long-term diagnosed conditions (OR, 1.86; 95% CI 1.29-2.67). A common genetic basis between MPs and PDAC was observed in the bioinformatics analysis. Conclusions: Specific multimorbidities aggregate and associate with PDAC in a time-dependent manner. A better characterization of a high-risk population for PDAC may help in the early diagnosis of this cancer. The common genetic basis between MP and PDAC points to a mechanistic link between these conditions. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to validate a molecular classification of colorectal cancer (CRC) based on microsatellite instability (MSI), CpG island methylator phenotype (CIMP) status, BRAF, and KRAS and investigate each subtype's response to chemotherapy., Design: This retrospective observational study included a population-based cohort of 878 CRC patients. We classified tumours into five different subtypes based on BRAF and KRAS mutation, CIMP status, and MSI. Patients with advanced stage II (T4N0M0) and stage III tumours received 5-fluoruracil (5-FU)-based chemotherapy or no adjuvant treatment based on clinical criteria. The main outcome was disease-free survival (DFS)., Results: Patients with the combination of microsatellite stable (MSS) tumours, BRAF mutation and CIMP positive exhibited the worst prognosis in univariate (log rank P<0.0001) and multivariate analyses (hazard ratio 1.75, 95% CI 1.05-2.93, P = 0.03) after adjusting for age, sex, chemotherapy, and TNM stage. Treatment with 5-FU-based regimens improved prognosis in patients with the combination of MSS tumours, KRAS mutation and CIMP negative (log rank P = 0.003) as well as in patients with MSS tumours plus BRAF and KRAS wild-type and CIMP negative (log-rank P<0.001). After adjusting for age, sex, and TNM stage in the multivariate analysis, only patients with the latter molecular combination had independently improved prognosis after adjuvant chemotherapy (hazard ratio 2.06, 95% CI 1.24-3.44, P = 0.005)., Conclusion: We confirmed the prognostic value of stratifying CRC according to molecular subtypes using MSI, CIMP status, and somatic KRAS and BRAF mutation. Patients with traditional chromosomally unstable tumours obtained the best benefit from adjuvant 5-FU-based chemotherapy., Competing Interests: The authors have declared that no competing interests exist.

We report a new seminested PCR method for detection of Legionella pneumophila based on the simultaneous amplification of a 387 bp fragment of the dotA gene and a 827 bp recombinant fragment that serves as an internal positive control. This new detection system was validated and its specificity and detection limit were determined. Parallel analysis of 90 environmental samples to compare this method, a real-time PCR method and the standard culture isolation method, demonstrated that this seminested method is useful for the study of L. pneumophila.

Background/aims: Genetic abnormalities of the p53 gene may play a major role in the carcinogenesis of gastric stump carcinomas (GSC) and intestinal-type primary gastric carcinomas (IPGC). Also, they may modulate P-gp expression producing chemoresistance. The aim of this article is to analyze p53 genetic abnormalities and the influence of p53 gene status on P-gp expression in both types of carcinomas., Methodology: Forty-two paraffin-embedded samples of gastric carcinomas corresponding to 17 GSC and 25 IPGC were studied. P53 genetic abnormalities in exon 5-9 were screened by direct sequencing of PCR products. P53 and P-glycoprotein (P-gp) were assessed by a standard streptavidin-biotin immunoperoxidase method. Anti-p53 DO7 and anti-P-gp C494 were used as primary antibodies., Results: Fourteen p53 mutations were found, 5 in GSC (29%) and 9 in IPGC (36%). Thirteen mutations were base-pair substitutions that produced a change in the amino acid sequence. Eight mutations were located at exon 7 (57%). P53 nuclear immunopositivity was observed in 12 GSC (71%) and 15 IPGC (60%). Only two carcinomas (1 IPGC and 1 GSC) harboring a p53 mutation did not show any p53 expression. All except one of the gastric carcinomas having a p53 mutation showed medium or high P-gp expression. However, there was no difference in P-gp expression between tumors with and without p53 mutation., Conclusions: The p53 genetic alterations found in GSC and IPGC could originate from a similar pathogenetic pathway. No association was demonstrated between p53 gene status and P-gp expression, although most of the carcinomas harboring a p53 mutation showed medium or high P-gp expression.

Small cell lung cancer (SCLC) expresses neuroectodermal markers including HuD, the best characterized member of the Hu gene family. The aim of this study is to optimize a simple and sensitive reverse transcriptase-polymerase chain reaction assay to detect circulating HuD-expressing cells for the early detection of SCLC recurrences. HuD-specific primers that selectively amplify the three HuD isoforms allowed the detection of one tumor cell/10(6) non-tumor cells. However, HuD transcripts were also detected in the mononuclear fraction of all samples from normal individuals (n=6) and patients with SCLC (n=5). By contrast, HuD protein was not detected in these fractions using Western blotting. More quantitative assays are necessary to examine the value of HuD transcript detection for the identification of tumor recurrences.

Unlabelled: The 18q21 region is frequently altered in gastrointestinal tumors. Three candidate tumor suppressor genes have been identified in it: DCC, Smad4/DPC4 and Smad2; the mechanisms involving their inactivation have not been completely elucidated. In this study, genetic losses at 18q21 and expression of DCC and DPC4 in colorectal (n=12) and pancreatic (n=16) cell lines and in colorectal tissues (n=10) were analyzed. The status of the 18q21 region was assessed using microsatellite analysis and duplex PCR of exonic sequences; expression was analyzed by RT-PCR; mutational analysis of DPC4 cDNA was performed in selected cases. Homozygous losses of microsatellite markers at 18q21 were not observed in colon or pancreas lines; however, a higher proportion of apparent homozygosity than expected was found. DCC and DPC4 transcripts were detected in 11/12 and 12/12 colorectal cancer lines, respectively. In tumors, homozygous losses at 18q21 were detected in three cases, without affecting DCC. All tumors retained DCC and DPC4 mRNA expression. In pancreatic lines, DPC4 was inactivated through homozygous deletion (n=5), intragenic mutation (n=3), and lack of protein (n=2)., In Conclusion: (1) microsatellite analysis does not provide adequate information regarding homozygous losses at 18q21; (2) approximately 65% of pancreas cancer lines show inactivation of DPC4; and (3) loss of DCC and DPC4 occur independently.