Global DNA methylation changes in treated and untreated MS patients measured over time.

Multiple sclerosis (MS) is an autoimmune, neurological disease. We investigated genome-wide DNA methylation profiles of CD4+ and CD8+ T cells from MS patients and healthy controls at baseline and a follow-up visit. Patients were all treatment-naïve at baseline, and either on treatment or remained untreated at the follow-up visit. MS patients show more changes in their T cell DNA methylation profiles as compared to healthy controls over time, with the most pronounced differences observed in the untreated MS patients. These findings underline the potential of DNA methylation as biomarkers in MS.

Intermediate-term survival of robot-assisted versus open radical cystectomy for muscle-invasive and high-risk non-muscle invasive bladder cancer in The Netherlands.

BACKGROUND: Radical cystectomy with pelvic lymph node dissection is the recommended treatment in non-metastatic muscle-invasive bladder cancer (MIBC). In randomised trials, robot-assisted radical cystectomy (RARC) showed non-inferior short-term oncological outcomes compared with open radical cystectomy (ORC). Data on intermediate and long-term oncological outcomes of RARC are limited. OBJECTIVE: To assess the intermediate-term overall survival (OS) and recurrence-free survival (RFS) of patients with MIBC and high-risk non-MIBC (NMIBC) who underwent ORC versus RARC in clinical practice. METHODS AND MATERIALS: A nationwide retrospective study in 19 Dutch hospitals including patients with MIBC and high-risk NMIBC treated by ORC (n = 1086) or RARC (n = 386) between January 1, 2012 and December 31, 2015. Primary and secondary outcome measures were median OS and RFS, respectively. Survival outcomes were estimated using Kaplan-Meier curves. A multivariable Cox regression model was developed to adjust for possible confounders and to assess prognostic factors for survival including clinical variables, clinical and pathological disease stage, neoadjuvant therapy and surgical margin status. RESULTS: The median follow-up was 5.1 years (95% confidence interval ([95%CI] 5.0-5.2). The median OS after ORC was 5.0 years (95%CI 4.3-5.6) versus 5.8 years after RARC (95%CI 5.1-6.5). The median RFS was 3.8 years (95%CI 3.1-4.5) after ORC versus 5.0 years after RARC (95%CI 3.9-6.0). After multivariable adjustment, the hazard ratio for OS was 1.00 (95%CI 0.84-1.20) and for RFS 1.08 (95%CI 0.91-1.27) of ORC versus RARC. Patients who underwent ORC were older, had higher preoperative serum creatinine levels and more advanced clinical and pathological disease stage. CONCLUSION: ORC and RARC resulted in similar intermediate-term OS and RFS in a cohort of almost 1500 MIBC and high-risk NMIBC.

CD8+ T cell gene expression analysis identifies differentially expressed genes between multiple sclerosis patients and healthy controls.

BACKGROUND: Genetic and clinical observations have indicated T cells are involved in MS pathology. There is little insight in how T cells are involved and whether or not these can be used as markers for MS. OBJECTIVES: Analysis of the gene expression profiles of circulating CD8+ T cells of MS patients compared to healthy controls. METHODS: RNA from purified CD8+ T cells was sequenced and analyzed for differential gene expression. Pathway analyses of genes at several p-value cutoffs were performed to identify putative pathways involved. RESULTS: We identified 36 genes with significant differential gene expression in MS patients. Four genes reached at least 2-fold differences in expression. The majority of differentially expressed genes was higher expressed in MS patients. Genes associated to MS in GWAS showed enrichment amongst the differentially expressed genes. We did not identify enrichment of specific pathways amongst the differentially expressed genes in MS patients. CONCLUSIONS: CD8+ T cells of MS patients show differential gene expression, with predominantly higher activity of genes in MS patients. We do not identify specific biological pathways in our study. More detailed analysis of CD8+ T cells and subtypes of these may increase understanding of how T cells are involved in MS.

From genetic associations to functional studies in multiple sclerosis.

Genetic screens steadily reveal more loci that show robust associations to complex human diseases, including multiple sclerosis (MS). Although some of the identified genetic variants are easily interpreted into a biological function, most of the genetic associations are frequently challenging to interpret. Underlying these difficulties is the fact that chip-based assays typically detect single nucleotide polymorphisms (SNPs) representative of a stretch of DNA containing many genomic variants in linkage disequilibrium. Furthermore, a large proportion of the SNPs with strongest association to MS are located in regions of the DNA that do not directly code for proteins. Here we discuss challenges faced by MS researchers to follow up the large-scale genetic screens that have been published over the past years in search of functional consequences of the identified MS-associated SNPs. We discuss experimental design, tools and methods that may provide the much-needed biological insights in both disease etiology and disease manifestations.

The multiple sclerosis susceptibility genes TAGAP and IL2RA are regulated by vitamin D in CD4+ T cells.

Multiple sclerosis (MS) is an inflammatory, demyelinating disorder of the central nervous system that develops in genetically susceptible individuals. The majority of the MS-associated gene variants are located in genetic regions with importance for T-cell differentiation. Vitamin D is a potent immunomodulator, and vitamin D deficiency has been suggested to be associated with increased MS disease susceptibility and activity. In CD4+ T cells, we have analyzed in vitro vitamin D responsiveness of genes that contain an MS-associated single-nucleotide polymorphism (SNP) and with one or more vitamin D response elements in their regulatory regions. We identify IL2RA and TAGAP as novel vitamin D target genes. The vitamin D response is observed in samples from both MS patients and controls, and is not dependent on the genotype of MS-associated SNPs in the respective genes.

Knee and hip articular cartilage have distinct epigenomic landscapes: implications for future cartilage regeneration approaches.

OBJECTIVES: To elucidate the functional epigenomic landscape of articular cartilage in osteoarthritis (OA) affected knee and hip joints in relation to gene expression. METHODS: Using Illumina Infinium HumanMethylation450 BeadChip arrays, genome-wide DNA methylation was measured in 31 preserved and lesioned cartilage sample pairs (14 knees and 17 hips) from patients who underwent a total joint replacement due to primary OA. Using previously published genome-wide expression data of 33 pairs of cartilage samples, of which 13 pairs were overlapping with the current methylation dataset, we assessed gene expression differences in differentially methylated regions (DMRs). RESULTS: Principal component analysis of the methylation data revealed distinct clustering of knee and hip samples, irrespective of OA pathophysiology. A total of 6272 CpG dinucleotides were differentially methylated between the two joints, comprising a total of 357 DMRs containing 1817 CpGs and 245 unique genes. Enrichment analysis of genes proximal of the DMRs revealed significant enrichment for developmental pathways and homeobox (HOX) genes. Subsequent transcriptomic analysis of DMR genes exposed distinct knee and hip expression patterns. CONCLUSIONS: Our findings reveal consistent DMRs between knee and hip articular cartilage that marked transcriptomic differences among HOX genes, which were not reflecting the temporal sequential HOX expression pattern during development. This implies distinct mechanisms for maintaining cartilage integrity in adulthood, thereby contributing to our understanding of cartilage homeostasis and future tissue regeneration approaches.

Large-scale meta-analysis of interleukin-1 beta and interleukin-1 receptor antagonist polymorphisms on risk of radiographic hip and knee osteoarthritis and severity of knee osteoarthritis.

OBJECTIVE: To clarify the role of common genetic variation in the Interleukin-1ß (IL1B) and Interleukin-1R antagonist (IL1RN) genes on risk of knee and hip osteoarthritis (OA) and severity of knee OA by means of large-scale meta-analyses. METHODS: We searched PubMed for articles assessing the role of IL1B and IL1RN polymorphisms/haplotypes on the risk of hip and/or knee OA. Novel data were included from eight unpublished studies. Meta-analyses were performed using fixed- and random-effects models with a total of 3595 hip OA and 5013 knee OA cases, and 6559 and 9132 controls respectively. The role of ILRN haplotypes on radiographic severity of knee OA was tested in 1918 cases with Kellgren-Lawrence (K/L) 1 or 2 compared to 199 cases with K/L 3 or 4. RESULTS: The meta-analysis of six published studies retrieved from the literature search and eight unpublished studies showed no evidence of association between common genetic variation in the IL1B or IL1RN genes and risk of hip OA or knee OA (P>0.05 for rs16944, rs1143634, rs419598 and haplotype C-G-C (rs1143634, rs16944 and rs419598) previously implicated in risk of hip OA). The C-T-A haplotype formed by rs419598, rs315952 and rs9005, previously implicated in radiographic severity of knee OA, was associated with reduced severity of knee OA (odds ratio (OR)=0.71 95%CI 0.56-0.91; P=0.006, I(2)=74%), and achieved borderline statistical significance in a random-effects model (OR=0.61 95%CI 0.35-1.06 P=0.08). CONCLUSION: Common genetic variation in the Interleukin-1 region is not associated with prevalence of hip or knee OA but our data suggest that IL1RN might have a role in severity of knee OA.

The role of plasma cytokine levels, CRP and Selenoprotein S gene variation in OA.

OBJECTIVE: Investigating the association between plasma levels of cytokines and chemokines, Selenoprotein S (SELS) gene variation and osteoarthritis (OA) subtypes. METHODS: The genetics of osteoarthritis and progression (GARP) study consists of 191 sibling pairs with symptomatic OA at multiple joint sites. We have measured plasma levels of 17 cytokines and chemokines and genetic variation at the SELS gene. RESULTS: Nine out of 17 serum markers could be assessed quantitatively, whereas eight markers were assessed qualitatively. Principal component analysis (PCA) on the quantitatively assessed markers and serum high sensitive C-reactive protein (S-HsCRP) revealed that three components underlie 61% of the total plasma variation. Three single nucleotide polymorphisms (SNPs) in the SELS gene revealed four common haplotypes, one of which, GAG (frequency 3.5%) showed significant association to an anti-inflammatory (P=0.019) and acute phase related (P=0.036) component. OA subtype analysis showed that one component (mainly representing chemokine variation) was significantly associated to hand OA and disc degeneration (P=0.029 and P=0.010 respectively) as well as a physical component score (PCS) (P=0.042). The CRP related component also showed a strong association to the PCS (P=0.007). SELS haplotypes showed no association to OA subtypes in the GARP study. CONCLUSION: Genetic variation in the SELS gene associates to components representing inflammatory signaling. Another component, representing chemokine variation, showed association to hand OA and disc degeneration in the GARP study indicating chemokines may contribute to OA pathogenesis.

Allelic variation at the C-reactive protein gene associates to both hand osteoarthritis severity and serum high sensitive C-reactive protein levels in the GARP study.

OBJECTIVE: To gain more insight into the role of genetic variation of the C-reactive protein (CRP) gene in serum CRP levels and osteoarthritis (OA). METHODS: Serum high sensitive CRP (S-HsCRP) levels were measured in the Genetics of osteoARthritis and Progression (GARP) study. Furthermore, to assess genetic variation of the CRP gene, genotypes of five tagging single nucleotide polymorphisms were assessed in the GARP study and a random control sample. RESULTS: A significant and consistent relation between S-HsCRP levels and observed haplotypes was identified. Additionally, a CRP haplotype, which also associated to a significantly higher expected phenotypic mean S-HsCRP level, was associated to severe hand OA. This haplotype was tagged by a single nucleotide polymorphism (rs3091244). Carriers of this allele have an increased risk for the presence of severe hand OA with an OR of 2.3 (95% confidence interval 1.2 to 4.3, p = 0.009). CONCLUSIONS: A haplotype of the CRP gene, associated to high basal S-HsCRP level, is also associated to severity of hand OA, indicating that innate high basal S-HsCRP levels may influence OA onset.

[Urethral meatal stenosis in boys easily overlooked]. / Urethrale-meatusstenose bij jongens gemakkelijk gemist.

Three boys aged 8, 5, 3 and 9 years, respectively, appeared to have urethral meatal stenosis. In the first patient this appeared during a check-up following treatment for balanitis. Patient history revealed that his micturition duration was longer than before. In the second patient, who underwent surgical correction for hypospadia, it was discovered because he took longer to urinate than his brother. In the third patient stenosis was observed during an appointment for a retracted testicle; he had been circumcised earlier for cultural reasons. Meatomy was performed under anaesthesia in all 3 patients, after which the micturition duration and stream velocity were normal. The third patient continued to have an extremely large bladder capacity and residual volume. Meatal stenosis may lead to obstructive uropathy, urinary tract infection and eventually damage to renal parenchyma. Symptomatic presentation can be late. Diagnostic tests include urine analysis and culture, and uroflowmetry. Visual inspection by spreading the meatal dimple to visualise a pinhole urethra cannot be overemphasised.

Surgical findings in acquired undescended testis.

BACKGROUND/PURPOSE: The aim of this study was to retrospectively review the findings at orchidopexy in acquired undescended testis (UDT). METHODS: The authors reviewed a 14-year (1986 through 1999) surgical experience in 360 boys in whom 461 orchidopexies were performed for acquired-UDT. The operative notes were reviewed to determine at operation testis position and volume, persistence of patent processus vaginalis (PV), and attachment of the gubernaculum. Also, testis position after orchidopexy was evaluated. RESULTS: Age at operation ranged from 2 to 19 years (mean, 8.9 years), 205 of the 461 orchidopexies (44.5%) had been performed between 9 and 12 years of age. In 327 of the 461 cases (70.9%), testis position was documented as intraoperative; in 281 of these cases (86.0%), the testis was located in the superficial inguinal pouch (SIP). A note was made regarding the presence or absence of a hernial sac in 207 of the cases: 113 (54.6%) were associated with an open PV, which usually was slightly open. In 122 of the 461 cases (26.5%), the gubernacular attachment was assessed; in 121 of these (99.2%), a normal attachment of the gubernaculum was noted. At the end of orchidopexy, in 438 of the 461 cases (95.0%), testis position was recorded. Three hundred eighty-two of these testes (87.2%) were at the bottom of the scrotum. CONCLUSIONS: Acquired UDT usually is characterized by SIP position, closed or (small) open PV, and normal gubernaculum attachment. The results of surgery seem excellent.

Previous testicular position in boys referred for an undescended testis: further explanation of the late orchidopexy enigma?

OBJECTIVE: To investigate the significance of the acquired undescended testis (UDT), which is differentiated into congenital and acquired forms, by assessing the previous testicular position in affected boys. PATIENTS AND METHODS: The study comprised 261 boys who had been referred for a non-scrotal testis to the outpatient clinic during an 8-year period (1993-2000). There was a bimodal distribution of age, with peaks at 2.0 and 10.0 years. In each boy with UDT the previous testicular position was ascertained. RESULTS: On referral, 340 testes were not in the scrotum (182 uni- and 79 bilateral). Of the 340 testes, 82 (24%) in 61 boys were diagnosed as retractile, whereas the remaining 258 in 221 boys were undescended. The previous testicular position was known in 208 of 221 boys (94%), with 244 UDTs. In 65 of these 244 (26.6%) the testis had never been scrotal (congenital UDT); in 179 (73.4%) a previous intrascrotal position was recorded in early childhood (acquired UDT) at least once, in 149 (61%) at least twice and in 117 (48%) at least three times. The mean age at referral for congenital UDT was 2.1 years and for acquired UDT was 8.4 years. CONCLUSIONS: These results show that acquired UDT is frequent, and occurs at about three times the rate of congenital UDT. Because these boys are referred for treatment later in childhood, the acquired UDT probably accounts for the high rate of (late) orchidopexy.

Natural course of acquired undescended testis in boys.

BACKGROUND: Although orchidopexy is commonly performed for acquired undescended testis, therapy is still controversial. A prospective study on the natural course of acquired undescended testis in boys was initiated. METHODS: At present, the study includes 63 boys with 74 acquired undescended testes in whom treatment and follow-up has been completed. In 15 boys with 20 acquired undescended testes, orchidopexy was performed before the onset of puberty, mainly at the request of the parents. In the remaining 48 boys with 54 acquired undescended testes, the onset of puberty was awaited. Of these, four boys with four acquired undescended testes were lost to follow-up. RESULTS: In 42 of 50 boys the testis descended spontaneously at puberty with a testicular volume appropriate for age. In the remaining eight boys the testis failed to descend at puberty and orchidopexy was performed. CONCLUSION: The preliminary results of this study indicate that spontaneous descent at puberty commonly occurs in boys with acquired undescended testes, with testicular volume appropriate for age. It is suggested that surgical intervention before onset of puberty may not always be necessary in acquired undescended testis.

A new clinical classification for undescended testis.

A new classification for undescended testis (UDT), suitable for a clinical setting, is proposed. UDT is categorized into congenital and acquired forms. Congenital forms include intra-abdominal, intra-canalicular, supra-scrotal and ectopic testes. Acquired forms can be divided into primary and secondary types. Primary forms are described as either ascending testes, i.e. those which cannot be manipulated into the scrotum, or high scrotal testes, i.e. those which can still be brought through the scrotal entrance into a high scrotal (unstable) position. Secondary forms are the result of ipsilateral groin surgery and are termed "trapped testes". Congenital forms of UDT should be treated surgically at an early age, preferably at 1 year. Therapy for primary acquired forms remains controversial. Therapeutic modalities include orchidopexy, hormonal treatment (preferably with human chorionic gonadotrophin) or waiting for spontaneous descent during the peripubertal period ("laissez faire policy"). Secondary acquired forms are probably best treated surgically.

[Previous testicular position in boys who underwent orchidopexy due to undescended testis at the Alkmaar Medical Center, the Netherlands, during the period 1986-1999]. / De vroegere testisligging van jongens bij wie orchidopexie was verricht wegens niet-ingedaalde testis in het Medisch Centrum Alkmaar (1986-1999).

OBJECTIVE: To determine the previous testicular position in boys, in whom orchidopexy was performed for undescended testis. DESIGN: Retrospective, descriptive. METHOD: All boys, aged 0-18 years, who underwent orchidopexy in the Alkmaar Medical Centre, the Netherlands, during the period 1986-1999, were studied. The following information was obtained from the hospital medical records: indication for operation, date of the operation, laterality (unilateral or bilateral), the surgical findings and whether previous testicular position played a role in the decision to perform orchidopexy. For each boy who underwent orchidopexy for undescended testis, previous testicular positions up until the date of the operation were obtained from the appropriate youth health care institutions. RESULTS: Hospital records were available for 851 boys who had undergone orchidopexy. The operation for undescended testis was performed in 717 boys and previous testicular positions were obtained for 565 boys. On a per testicle basis, 707 operations were carried out (142 bilaterally, 205 left-sided, 218 right-sided). From these 707 testes, a previous intrascrotal position was found at least once in 572 (80.9%), at least twice in 493 (69.7%) and at least three times in 419 (59.3%); 135 (19.1%) testes had never been intrascrotal. The majority of previously undescended testes were operated on at 3 years of age; most operations on previously descended testes were performed at 10.5 years of age. For 344 (48.7%) out of 707 testes, previous testis localisation was known in the hospital's medical records, for 96 (13.6%) testes registration was unclear and in 267 (37.8%) testes it was not reported. In 8 (1.4%) boys, testis registration after the birth was used on referral to document previous testicular position. CONCLUSION: In total 80.9% of all orchidopexy operations were performed on testes that had previously been diagnosed as having descended normally. These probably included retractile testes as well as acquired forms. In 51.3% of the cases, previous testicular position was not known in the hospital's medical records at the time of operation.

Increase in incidental renal cell carcinoma in the northern part of the Netherlands.

OBJECTIVES: Evaluating in a retrospective survey the incidence of incidental and symptomatic renal cell carcinoma (RCC) between 1977 and 1994 in the northern part of the Netherlands and the mode of their detection. PATIENTS AND METHODS: Retrospectively, 173 patients surgically treated for RCC were divided into two groups according to the period of detection, 1977-1987 (n = 87) and 1987-1994 (n = 86). Because of the increase in abdominal ultrasound in 1987, this year was used as the cutoff date. In both periods the patients were grouped according to whether the tumor was found incidentally or whether the tumor was suspected. The mode of detection was recorded together with the tumor stage at presentation and survival. RESULTS: The incidental detection rate was 33% (29/87) in the 1977-1987 group and 49% (42/86) in the 1987-1994 group, showing a significant difference (p = 0.038). In the 1977-1987 group incidental tumors were detected with ultrasound in 83% and symptomatic tumors with ultrasound in 36%. Of the cases in the 1987-1994 group this percentage was 91 and 43%, respectively. Disease-free survival rates after a mean follow-up of 10 years were 63% in the incidental RCC group and 37% in the symptomatic RCC group (p = 0.0159). CONCLUSIONS: There is an increase in incidental tumors in this part of the Netherlands with ultrasound as the mode of detection. The disease-free survival is significantly better in the incidental tumor group.

Genetic analysis of 2 cases of clear cell renal cancer in 2 sisters.

Two sisters affected with renal cell carcinoma (RCC) is an extremely rare finding, and may indicate a hereditary pattern or the presence of other predisposing factors. We describe here 2 sisters presenting with clear cell renal cell cancer. Examination for von Hippel-Lindau (VHL)-related features and tuberous sclerosis (M. Bourneville) was negative and both had a normal constitutional karyotype. Cytogenetic analysis of the tumor tissue of both patients showed a translocation involving chromosomes 3 and 5, resulting in loss of 3p sequences and gain of part of 5q. The 5q breakpoints were similar, but the breakpoints at 3p appeared to differ. Allelic imbalance analysis supported our observations. Microsatellite analysis revealed that both sisters inherited different chromosome 3 parental alleles. For chromosome 5, 3 different haplotypes could be deduced, but the chromosome 5 alleles overrepresented in the different tumor tissues were from different parental origin. The development of the 2 RCCs in these 2 sisters thus cannot be explained by the inheritance of a mutated VHL gene located at 3p25, nor by the inheritance of other gene defects at chromosomes 3p or 5q. Although the chance that 2 sisters develop sporadic RCC is very low, in the presented case it is probably coincidental or related to another genetic predisposition.

Can duplex Doppler ultrasound replace computerized tomography in staging patients with renal cell carcinoma?

The purpose of this study was to evaluate the accuracy and reliability of duplex Doppler ultrasound (US) and computerized tomography (CT) in staging patients with renal cell carcinoma (RCC). Sixty-six patients were evaluated pre-operatively with duplex Doppler ultrasound and CT. The results were compared with the surgical and histopathological findings. T stage was determined correctly with duplex Doppler US and CT in 56 and 50 cases respectively. In 4 patients with nodal disease duplex Doppler US was correct in 2 patients, 1 was false positive. With CT, 3 patients were staged correctly and 3 were false positive. Of the 14 patients with vascular tumour thrombi, 13 were staged correctly with duplex Doppler US and 12 with CT scan. False positive vascular tumour invasion was seen only with CT in 4 cases. Based on these results we conclude that duplex Doppler US is at least as accurate as CT scanning in the staging of RCC. Also in patients with renal or caval thrombi, duplex Doppler US is highly accurate in establishing the diagnosis and in the determination of the extent of the thrombus.

Spontaneous caval tumor thrombus necrosis and regression of pulmonary lesions in renal cell cancer.

Idiopathic regression of metastases is one of the features of the unpredictable behaviour of renal cell carcinoma. We report a patient with pulmonary metastases and a tumor thrombus in the inferior vena cava with spontaneous regression of the lung lesions and necrosis of the thrombus before any therapy was instituted.