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Background: Type 2A hereditary haemochromatosis (type 2A HH) is a rare iron-loading disorder caused by mutations in the HFE2 gene, which encodes the HJV protein. We present characteristics, treatment and follow-up of subjects diagnosed with type 2A HH in the Netherlands to increase awareness of the disease and its treatment, and to define knowledge gaps. Methods: We collected clinical, biochemical and genetic data from seven patients (two female; five probands) from six families genetically diagnosed with type 2A HH at the Expertise Center for Iron Disorders, Radboud University Medical Centre between 2006 and 2016. Results: The five probands presented with heterogeneous complaints between the ages of 19 and 39. One of two patients with delayed clinical diagnosis developed hypogonadism and Y. enterocolitica sepsis. Diagnostic workup and follow-up varied. When assessed, elevated transferrin saturation (79-98%), ferritin (1400-6200 µg/l) and severely elevated liver iron levels were found, and in all subjects, phlebotomies were initiated. One subject was switched to erythrocytapheresis. Target ferritin levels varied. Despite long-term iron depletion, two subjects developed clinical complications. Sanger sequencing revealed two pathogenic HFE2 variants (homozygous or compound heterozygous) for the five families of Dutch descent and one new pathogenic variant in the family of non-Dutch descent. Conclusion: Three genetic variants caused type 2A HH in six families. Clinical diagnosis was delayed in two subjects. We observed variance in presentation, workup, follow-up and treatment. We found new complications in long-term iron-depleted patients. We recommend research and guidelines for optimal workup, follow-up and treatment of type 2A HH.

Type 3 hereditary haemochromatosis (HH) is a rare iron overload disorder caused by variants in the transferrin 2 receptor (TFR2) gene. We aim to present characteristics of patients diagnosed with TFR2-HH in the Netherlands, in order to increase knowledge and awareness of this disease.We collected clinical, biochemical and genetic data from four patients from three families diagnosed with HH type 3 in the Netherlands between 2009 and 2016.Three women and one man diagnosed with HH type 3 presented with arthralgia and elevated ferritin levels and transferrin saturation (TSAT) at ages 25-41 years. The hepcidin/ferritin ratio as measured in three patients was low. Liver iron content in two patients as assessed by MRI or liver biopsy was highly increased (250 and 362.7 μmol iron/g dry weight, respectively, reference35 μmol/g). DNA analysis revealed four different TFR2 pathogenic variants: one nonsense, one splicing and two missense variants, of which three are novel. Phlebotomy decreased the serum iron parameters but did not relieve the arthralgia.In patients with a combination of elevated TSAT and ferritin in the absence of anaemia, and after exclusion of HFE-related HH, rare forms of HH should be considered. In these cases, presentation with arthralgia in young adulthood, low hepcidin/ferritin ratio and/or liver iron content100 μmol/g form an indication for analysis of the TFR2 gene. Although type 3 HH is extremely rare, awareness of the disease among physicians is important in order to achieve an early diagnosis and prevent complications, such as liver damage.

Contains fulltext : 98140.pdf (Publisher’s version ) (Closed access) We present a patient with iron overload, who was initially diagnosed with hereditary haemochromatosis. Family analysis, however, established that the iron overload was secondary to congenital sideroblastic anaemia. The patient died of a hepatocellular carcinoma, likely a complication of iron overload, despite phlebotomies. Increased awareness, as well as development of evidence-based clinical guidelines, is required for timely diagnosis and adequate treatment. 4 p.

To compare apolipoprotein B (apoB), non-high-density lipoprotein-cholesterol (non-HDL-c) and low-density lipoprotein-cholesterol (LDL-c) for identifying individuals with a deteriorated cardiovascular (CV) risk profile, including a panel of subclinical atherosclerosis measurements and prevalent cardiovascular disease (CVD) in a Dutch population-based cohort.Clinical and biochemical measurements and a panel of noninvasive parameters of subclinical atherosclerosis were determined in 1517 individuals, aged 50-70 years.Both men and women with increasing levels of apoB and non-HDL-c were more obese, had higher blood pressure and fasting glucose levels, and a more atherogenic lipid profile. Furthermore, compared to the reference group (composed of those with apoB, non-HDL-c and LDL-c levels in the bottom quartiles), participants with high apoB and high non-HDL-c levels had a lower ankle-brachial index at rest (-3.5% and -3.1%, respectively) and after exercise (-6.3% and -4.7%, respectively), a thicker near wall (+4.8% and +4.2%, respectively), far wall (both +6.2%), and mean intima-media thickness (+5.7% and +5.3%, respectively) and more plaques (+54.2% and +54.3%, respectively). In addition, they also showed increased stiffness parameters (e.g. pulse wave velocity both +3.6%). Less clear differences in CV risk profile and subclinical atherosclerosis parameters were observed when participants were stratified by LDL-c level. Furthermore, apoB but not LDL-c detected prevalent CVD, and non-HDL-c only detected prevalent CVD in men. The discriminatory power for prevalent CVD expressed as area under the receiver operating characteristic curve was 0.60 (P0.001) for apoB, 0.57 (P = 0.001) for non-HDL-c and 0.54 (P = 0.108) for LDL-c.Our data support the use of first apoB and secondly non-HDL-c above LDL-c for identifying individuals from the general population with a compromised CV phenotype.

Waist circumference is a clinical marker of obesity and an established risk factor for cardiovascular (CV) disease. Adiponectin, an adipocyte-derived hormone and new biomarker of obesity, was recently proposed as the missing link between obesity and increased cardiovascular risk. We evaluated waist and adiponectin in a middle-aged population-based cohort to compare the impact of both obesity-markers on subclinical atherosclerosis, in relation to other CV risk factors. DESIGN, SETTINGSUBJECTS: Seven noninvasive measurements of atherosclerosis (NIMA), as surrogate markers of (subclinical) atherosclerosis, were determined in 1517 participants of the Nijmegen Biomedical Study, aged 50-70 years, who were drawn from the Dutch community.Both men and women with a high waist (M104 cm; F95 cm) showed increased pulse wave velocity (PWV) (M: +9.4%; F: +8.3%) and thicker intima-media thickness (IMT) (M: +7.3%; F: +4.3%) and women also showed increased plaque thickness (+16.6%). After adjustment for other CV risk factors both men and women showed increased IMT (M: +4.8%; F: +2.8%) and men also showed increased PWV (+9.6%). Both men and women with a low adiponectin level (M2.2 mg L(-1); F3.5 mg L(-1)) showed a decreased ankle-brachial index after exercise (M: -9.5%; F: -3.9%) and increased IMT (M: +3.7%; F: +3.6%) and women also showed increased PWV (+6.8%), but after adjustment for other CV risk factors low adiponectin level was no longer associated with deteriorated outcomes of NIMA.Waist circumference showed independent associations with noninvasive measurements of subclinical atherosclerosis, whereas the association of adiponectin level with subclinical atherosclerosis was not independent of other CV risk factors. Prospective studies are needed to elucidate, if the atherogenic effect of a low adiponectin level is mediated by other CV risk factors and not by low adiponectin level intrinsically.

Contains fulltext : 80568.pdf (Publisher’s version ) (Closed access) Haemochromatosis should currently refer to hereditary iron overload disorders presenting with a definite and common phenotype characterised by normal erythropoiesis, increased transferrin saturation and ferritin and primarily parenchymal iron deposition related to innate low (but normally regulated) production of the hepatic peptide hormone hepcidin. Since the discovery of the haemochromatosis gene (HFE) in 1996, several novel gene defects have been detected, explaining the mechanism and diversity of iron overload diseases. Overall, at least four main types of hereditary haemochromatosis (HH) have been identified. This review describes the systematic diagnostic and therapeutic strategy and pitfalls for patients suspected for HH and their relatives.

Single-spin density-gradient ultracentrifugation (DUC) has proven to be a reproducible method for detection of low-density-lipoprotein (LDL) heterogeneity. Recently another method has been described for this: gradient gel electrophoresis (GGE) of serum, a method that might be more suitable for screening. To gain insight into the relationship of GGE to DUC and into their reproducibility, we determined LDL heterogeneity by DUC and GGE in 41 healthy individuals. In 90.2% (n = 37) of the subjects, the number of LDL subfractions found by both methods agreed. In addition, the density and the relative migration distance of the predominant LDL subfraction observed with the respective methods showed a strong correlation (Pearson correlation, r = 0.85, P less than 0.0001). Although it was not possible to compare for all aspects of LDL heterogeneity, these data suggest that GGE is a valid method of analysis for LDL heterogeneity. In screening programs, it may be necessary to store samples. Therefore, we studied in 24 sera the influence of storage at -80 degrees C for one, four, and 12 weeks on the LDL subfraction distribution detected by each method. LDL heterogeneity was maintained during storage under these conditions.

HFE-related hereditary haemochromatosis (HH) is an iron overload disease attributed to the highly prevalent homozygosity for the C282Y mutation in the HFE gene. The pathophysiology of this error in iron metabolism is not completely elucidated yet, although deficiency of the iron regulatory hormone hepcidin appears to play a role. Ways of diagnosing iron overload include measurement of the serum iron parameters, i.e. serum transferrin saturation and serum ferritin, by a liver biopsy or by calculating the amount of mobilisable body iron withdrawn by phlebotomies. Clinical signs attributed to HFE-related HH include liver failure, arthralgia, chronic fatigue, diabetes mellitus and congestive heart failure. organ failure can be prevented by phlebotomies starting before irreversible damage has occurred. Therefore, screening to facilitate early diagnosis is desirable in individuals at risk of developing HFE-related iron overload. over time it appeared that the clinical penetrance of the HFE mutations was much lower than had previously been thought. This changed the opinion about a suitable screening modality from case detection, via population screening, to family screening as the most appropriate method to prevent HFE-related disease. However, before the implementation of family screening it is vital to have thorough information on the relevance of the specific health problem involved, on the clinical penetrance of C282Y homozygosity and on the effectiveness of the screening approach.

Family screening has been suggested as a sophisticated model for the early detection of HFE-related hereditary haemochromatosis (HH). However, until now, controlled studies on the morbidity and mortality in families with HH are lacking.Data on iron parameters, morbidity and mortality were collected from 224 dutch C282Y-homozygous probands with clinically overt HH and 735 of their first-degree family members, all participating in the HEmochromatosis fAmily study (HEfAs). These data were compared with results obtained from an age- and gender-matched normal population. HEfAs and controls filled in similar questionnaires on demographics, lifestyle factors, health, morbidity and mortality.A significantly higher proportion of the HEfAs first-degree family members reported to be diagnosed with haemochromatosis-related diseases: 45.7 vs 19.4% of the matched normal population (McNemar p0.001). Mortality among siblings, children and parents in the HEFAS population was similar to that in the relatives of matched control.In this study we show that, morbidity among first-degree family members of C282Y-homozygous probands previously diagnosed with clinically proven HH is higher than that in an age- and gender-matched normal population. Further studies are needed to definitely connect these increase morbidity figures to increase prevalenc of the C282Y mutated HFE-gene and elevated serum iron indices.

Hereditary haemochromatosis (HH) is a disease related to mutations in the HFE gene and can lead to progressive iron accumulation, especially in the liver, eventually resulting in organ damage. We have developed guidelines for the diagnosis and treatment of this disease according to CBO methodology (dutch institute for Healthcare Quality). The prevalence of clinical symptoms such as fatigue, arthropathies, impotence and diabetes mellitus among homozygotes was similar to that in a control population. Nevertheless, we recommend the assessment of serum iron indices when these symptoms remain unexplained. When transferrin saturation is45% and ferritin exceeds local reference ranges, HFE mutations should be investigated. Homozygosity for the C282Y mutation or combined C282Y/H63d mutation confirms the diagnosis of HFE-related HH. Liver biopsy is recommended when ferritin exceeds 1000 microg/l to establish the presence or absence of cirrhosis, which will affect prognosis and management. iron accumulation confirmed by magnetic resonance imaging (MRI) in the absence of the homozygous C282Y mutation or the combined C282Y/H63d genotype may justify a search for rare hereditary forms of non-HFE HH in a specialised centre. The literature supports the benefits of adequate phlebotomy and the screening of first-degree relatives of index patients with clinically overt HH. overall, the guidelines presented here are to a great extent based on the expert opinion of the working party, as the quantity of evidence that met predefined criteria posed by the evidence-based approach was small. We therefore recommend world-wide efforts to collaboratively address these remaining issues.

In 1998 a clinical guideline for the targeted, accurate and early detection and treatment of HFE-related hereditary haemochromatosis (HH), which comprises a test for the causative HFE-gene mutations, was introduced in our outpatient department.The impact of this guideline was evaluated retrospectively. Data were acquired from medical records of patients with discharge diagnosis codes suggestive of HH (n=878 patients), obtained from a period before (n=422) and after guideline introduction (n=456).Combined measurements of serum transferrin saturation and serum ferritin rose from 12.2% (n=53) to 29.5% (n=138, p0.001), leaving 70% of the patients eligible for HH not tested for iron parameters. The HFE-gene mutation detection test was correctly used in II (40.7%) of 27 tested patients and improperly interpreted in six (22.2%) of these 27 patients. Five new HH patients were diagnosed before and 13 after introduction. Seven of these 13 patients appeared to be incorrectly diagnosed, due to misinterpretation of laboratory results. Diagnostic costs of case detection for each accurately diagnosed patient were euro 2380 before and euro 2600 after introduction of the guideline.Evaluation of the introduction of a practical guideline for targeted HH detection reveals a low compliance with the guideline, resulting in both a small percentage of patients tested for HH and overdiagnosis of HH. Therefore, the introduction of the guideline should be combined with a more appropriate implementation strategy which includes education on its most critical points, i.e. the indication and interpretation of the iron parameters and the HFE genotype.

Expression of the inhibitor of apoptosis protein survivin is up-regulated in many tumors of epithelial origin and frequently shows a relationship with disease prognosis.We investigated survivin mRNA expression in 32 urothelial cell carcinomas by use of real-time quantitative PCR. Expression values were normalized to transcript levels of the housekeeping gene cyclophilin.All bladder tumor tissues expressed survivin mRNA. The median normalized survivin mRNA expression values were 0.26 for superficial tumors (n = 17) and 0.78 for invasive tumors (n = 15). A significant relationship with increasing pathological stage (p0.001) and grade (p0.001) was observed. Although survivin mRNA expression did not relate to disease progression or the patient survival period, patients with superficial bladder tumors and normalized survivin values over 0.26 had an increased risk of recurrence (log-rank test: p = 0.018).Our results suggest that quantitative measurement of survivin mRNA 1) can identify invasive and high-grade urothelial cell carcinomas and 2) may be used as an indicator for early recurrence of superficial tumors.

To determine whether patients previously diagnosed as chronic fatigue syndrome (CFS) actually have primary haemochomatosis (PH).The setting was a Dutch referral centre. Transferrin saturation (TS) was retrospectively evaluated in banked blood samples of 88 patients diagnosed as CFS. Patients with elevated TS values were asked to provide a new overnight fasting blood sample for a second determination of TS and measurement of serum ferritin. The DNA was investigated for mutations in the HFE gene when one of these iron parameters was elevated.For 19 out of 88 patients with CFS an elevated TS was found. A new blood sample was obtained from 11 of these 19: six had increased TS and two had elevated serum ferritin values. These eight patients were neither C282Y homozygotes nor compound C282Y-H63D heterozygotes. In the eight cases where no new blood samples could be obtained, the TS was50% for two of the five men and45% for the three female patients.In a group of 88 CFS patients we could exclude PH in all but two of them (prevalence 2.3%; 95% confidence interval 0-5.5%). In our population of CFS patients PH is not more common than in a control population of northern European descent (prevalence 0.25-0.50%).

Objective Analysis of the levels of cell-free fetal and total DNA in serum of women carrying a male fetus affected by trisomy 21, and comparison of these levels with those in women carrying a normal male fetus. Methods DNA was extracted from archived second-trimester maternal serum samples collected as part of a prenatal screening program. A total of 10 cases with trisomy 21 male fetuses were compared with 10 controls (male fetuses) with samples matched for duration of storage and gestational age. Real-time quantitative PCR of the SRY and albumin genes was used to quantify fetal and total DNA respectively. Results The median fetal DNA level in the group of 10 pregnancies with trisomy 21 was 31.98 cell-equivalents per mL compared to 34.06 in the control group. The difference was not significant. The median total DNA level in women with a trisomy 21 fetus was significantly higher (P = 0.029) than that in controls (36 152.6 vs 5832.81 cell-equivalents per mL). Conclusions Although we could not confirm previous studies of an increased amount of fetal DNA in pregnancies affected by trisomy 21, we did find increased levels of total DNA. The possible reasons for these observations are discussed with respect to previous findings. Larger studies are needed to elucidate the true value, if any, of measurement of fetal and total DNA in maternal serum in the context of prenatal screening for chromosomal abnormalities. Copyright © 2003 John Wiley & Sons, Ltd.

In 25% of patients diagnosed with colorectal cancer, hepatic metastases are not detected at presentation of the colorectal primary but develop during follow-up. Early detection of these metastases may improve the chance of cure by surgical resection. We hypothesised that in patients with occult hepatic metastases, tumour DNA might be detected in bile which could be collected during resection of the colorectal primary. To test this hypothesis, bile from the gall bladder was collected from 17 patients scheduled for resection of evident hepatic metastases (2 cm3) from a previously resected colorectal primary. Mutation analysis of the metastases identified five patients (34%) with a K-ras gene mutation in the tumour tissue. These cases were selected for bile analysis for mutant K-ras. Non-mutated DNA could be amplified from all the bile samples, but mutant K-ras could only be detected in bile from one patient. False negative results due to technical deficits could be ruled out by control experiments showing a high DNA isolation efficiency and high sensitivity of the mutation detection method. It is concluded that hepatic metastases, in contrast to pancreatic cancers, do not (regularly) shed mutated DNA into the bile. Hence, molecular screening of bile seems of only limited clinical value for the detection of occult liver metastases.

Telomerase reverse transcriptase (hTERT) messenger RNA has been detected in 95% of bladder tumors using RT-PCR. In this study, we quantified the expression of hTERT in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia using a real-time quantitative PCR assay. hTERT expression was detected in all 35 urothelial cell carcinomas of varying grade and stage, but not in normal tissue samples. An increase in both pathological grade and clinical stage as prognostic parameters correlated with increased hTERT expression. Using different cutoff values for grades and stages, normalized hTERT expression values could discriminate among low, medium, and high grade tumors and between superficial and muscle-invasive tumors. We conclude that standardized real-time measurement of hTERT expression can be used for early tumor detection and may be used for determination of prognosis in urothelial cell carcinomas of the bladder.

Expression of the hTERT gene, which codes for the catalytic subunit of telomerase, is associated with malignancy. We recently developed a real-time reverse transcription-PCR assay, based on TaqMan technology, for accurate and reproducible determination of hTERT mRNA expression (Lab Investig 1999;79:911-2). This method may be of interest for molecular tumor diagnostics in tissues and corresponding body fluids, washings, or brushes.In this study, we measured hTERT expression in a subset of healthy tissues and tumors to select those tumor types with the best potential for quantification of hTERT in corresponding body fluids. To demonstrate the use of the method in body fluids, we quantified hTERT expression in voided urine of patients with bladder cancer and controls.Real-time measurement of hTERT expression could discriminate between all healthy and malignant tissue samples from pancreas, lung, esophagus, and bladder, but not for colon tissues. Moreover, in five of nine (55%) urine samples, hTERT could be quantified.The present study demonstrates that accurate quantitative measurement of hTERT expression has high potential for discrimination between healthy and tumor cells in tissues and urine and supports future measurements in pancreatic fluid, bronchoalveolar lavage fluid, esophageal brushings, and urine or bladder washings.

The metabolism of three low density lipoprotein (LDL) subfractions by human monocyte-derived macrophages (HMDM) through the LDL receptor pathway was studied. The three LDL subfractions, very light LDL1, light LDL2 and heavy LDL3, were isolated from serum pools of normolipidemic subjects by density gradient ultracentrifugation. The LDL subfractions were shown to differ in molecular size and chemical composition but not in electrophoretic mobility on agarose gel. Cell specific association, cell specific degradation and stimulation of cholesteryl esterification were determined in parallel after incubation of HMDM with increasing amounts of LDL-protein of the three LDL subfractions. The experiments were repeated four times with freshly prepared LDL subfractions. Both the cell specific association and degradation increased more with increasing LDL-protein concentration for LDL1 than for LDL3 (p0.001). The results for LDL2 were intermediate between those for LDL1 and LDL3 and differed significantly from both (p0.05). For the stimulation of cholesteryl ester formation, the curves for LDL1 and LDL2 increased more with increasing LDL-protein concentration than that for LDL3 (p0.001); the results for LDL1 and LDL2 did not differ significantly from each other. These differences between LDL subfractions in cholesteryl esterification were independent of the cholesterol content of the LDL subfractions. The results show that LDL subfractions have different rates of LDL receptor-mediated catabolism by HMDM. As HMDM play an important role in the formation of atherosclerotic plaques, the differences between the LDL subfractions in catabolism by HMDM, may result in differences in atherogenicity between LDL subfractions isolated from normolipidemic subjects.

Single-spin density-gradient ultracentrifugation (DUC) has proven to be a reproducible method for detection of low-density-lipoprotein (LDL) heterogeneity. Recently another method has been described for this: gradient gel electrophoresis (GGE) of serum, a method that might be more suitable for screening. To gain insight into the relationship of GGE to DUC and into their reproducibility, we determined LDL heterogeneity by DUC and GGE in 41 healthy individuals. In 90.2% (n = 37) of the subjects, the number of LDL subfractions found by both methods agreed. In addition, the density and the relative migration distance of the predominant LDL subfraction observed with the respective methods showed a strong correlation (Pearson correlation, r = 0.85, P less than 0.0001). Although it was not possible to compare for all aspects of LDL heterogeneity, these data suggest that GGE is a valid method of analysis for LDL heterogeneity. In screening programs, it may be necessary to store samples. Therefore, we studied in 24 sera the influence of storage at -80 degrees C for one, four, and 12 weeks on the LDL subfraction distribution detected by each method. LDL heterogeneity was maintained during storage under these conditions.

A single spin density gradient ultracentrifugation method in a swinging bucket rotor has been applied for the detection and isolation of low density lipoprotein (LDL) subfractions. The visualization of the LDL heterogeneity was facilitated by prestaining the serum with Coomassie Brilliant Blue R prior to density gradient ultracentrifugation for 19.5 hr. A total of 13 human serum pools was analyzed. In each pool, two LDL subfractions, a lighter LDL1 subfraction, occasionally showing a subdivision into two bands, LDL1A and LDL1B, and a heavier LDL2 could be clearly distinguished by the banding pattern in the density gradient. Physicochemical characteristics of the isolated LDL subfractions were determined. The simple method for detection and isolation of these subfractions presented here may facilitate future studies on LDL heterogeneity.