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2.  Promising New Assays and Technologies for the Diagnosis and Management of Infectious Diseases 
Recent advancements in technology have led to the development of new techniques that hold promise for improved diagnosis and management of infectious diseases. Here, we review new assays that help better identify pathogens and tailor antibiotic therapy to patients' needs.
In the first decade of the 21st century, we have seen the completion of the human genome project and marked progress in the human microbiome project. The vast amount of data generated from these efforts combined with advances in molecular and biomedical technologies have led to the development of a multitude of assays and technologies that may be useful in the diagnosis and management of infectious diseases. Here, we identify several new assays and technologies that have recently come into clinical use or have potential for clinical use in the near future. The scope of this review is broad and includes topics such as the serum marker procalcitonin, gene expression profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and nucleic acid aptamers. Principles that underlie each assay or technology, their clinical applications, and potential strengths and limitations are addressed.
PMCID: PMC3657496  PMID: 23223587
procalcitonin; gene expression profiling; mass spectrometry; MALDI-TOF MS; sepsis
3.  Analysis of long-range chromatin interactions using Chromosome Conformation Capture 
Methods (San Diego, Calif.)  2012;58(3):10.1016/j.ymeth.2012.07.022.
Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.
PMCID: PMC3874837  PMID: 22903059
4.  Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes 
Albrechtsen, A. | Grarup, N. | Li, Y. | Sparsø, T. | Tian, G. | Cao, H. | Jiang, T. | Kim, S. Y. | Korneliussen, T. | Li, Q. | Nie, C. | Wu, R. | Skotte, L. | Morris, A. P. | Ladenvall, C. | Cauchi, S. | Stančáková, A. | Andersen, G. | Astrup, A. | Banasik, K. | Bennett, A. J. | Bolund, L. | Charpentier, G. | Chen, Y. | Dekker, J. M. | Doney, A. S. F. | Dorkhan, M. | Forsen, T. | Frayling, T. M. | Groves, C. J. | Gui, Y. | Hallmans, G. | Hattersley, A. T. | He, K. | Hitman, G. A. | Holmkvist, J. | Huang, S. | Jiang, H. | Jin, X. | Justesen, J. M. | Kristiansen, K. | Kuusisto, J. | Lajer, M. | Lantieri, O. | Li, W. | Liang, H. | Liao, Q. | Liu, X. | Ma, T. | Ma, X. | Manijak, M. P. | Marre, M. | Mokrosiński, J. | Morris, A. D. | Mu, B. | Nielsen, A. A. | Nijpels, G. | Nilsson, P. | Palmer, C. N. A. | Rayner, N. W. | Renström, F. | Ribel-Madsen, R. | Robertson, N. | Rolandsson, O. | Rossing, P. | Schwartz, T. W. | Slagboom, P. E. | Sterner, M. | Tang, M. | Tarnow, L. | Tuomi, T. | van’t Riet, E. | van Leeuwen, N. | Varga, T. V. | Vestmar, M. A. | Walker, M. | Wang, B. | Wang, Y. | Wu, H. | Xi, F. | Yengo, L. | Yu, C. | Zhang, X. | Zhang, J. | Zhang, Q. | Zhang, W. | Zheng, H. | Zhou, Y. | Altshuler, D. | ‘t Hart, L. M. | Franks, P. W. | Balkau, B. | Froguel, P. | McCarthy, M. I. | Laakso, M. | Groop, L. | Christensen, C. | Brandslund, I. | Lauritzen, T. | Witte, D. R. | Linneberg, A. | Jørgensen, T. | Hansen, T. | Wang, J. | Nielsen, R. | Pedersen, O.
Diabetologia  2012;56(2):298-310.
Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes.
The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m2 and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case–control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans.
Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10−14), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10−11) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10−10).
We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-012-2756-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC3536959  PMID: 23160641
Exome sequencing; Genetic epidemiology; Genetics; Lipids; Next-generation sequencing; Obesity; Type 2 diabetes
5.  A gene variant near ATM is significantly associated with metformin treatment response in type 2 diabetes: a replication and meta-analysis of five cohorts 
Diabetologia  2012;55(7):1971-1977.
In this study we aimed to replicate the previously reported association between the glycaemic response to metformin and the SNP rs11212617 at a locus that includes the ataxia telangiectasia mutated (ATM) gene in multiple additional populations.
Incident users of metformin selected from the Diabetes Care System West-Friesland (DCS, n = 929) and the Rotterdam Study (n = 182) from the Netherlands, and the CARDS Trial (n = 254) from the UK were genotyped for rs11212617 and tested for an association with both HbA1c reduction and treatment success, defined as the ability to reach the treatment target of an HbA1c ≤7 % (53 mmol/mol). Finally, a meta-analysis including data from literature was performed.
In the DCS cohort, we observed an association between rs11212617 genotype and treatment success on metformin (OR 1.27, 95% CI 1.03, 1.58, p = 0.028); in the smaller Rotterdam Study cohort, a numerically similar but non-significant trend was observed (OR 1.45, 95% CI 0.87, 2.39, p = 0.15); while in the CARDS cohort there was no significant association. In meta-analyses of these three cohorts separately or combined with the previously published cohorts, rs11212617 genotype is associated with metformin treatment success (OR 1.24, 95% CI 1.04, 1.49, p = 0.016 and OR 1.25, 95% CI 1.33, 1.38, p = 7.8 × 10−6, respectively).
A gene variant near ATM is significantly associated with metformin treatment response in type 2 diabetic patients from the Netherlands and the UK. This is the first robustly replicated common susceptibility locus found to be associated with metformin treatment response.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-012-2537-x) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC3369131  PMID: 22453232
Genetics of type 2 diabetes; Human; Meta-analysis; Metformin; Oral pharmacological agents
6.  First-line chemotherapy for malignant peripheral nerve sheath tumor (MPNST) versus other histological soft tissue sarcoma subtypes and as a prognostic factor for MPNST: an EORTC Soft Tissue and Bone Sarcoma Group study 
Annals of Oncology  2010;22(1):207-214.
Background: The role of chemotherapy in advanced malignant peripheral nerve sheath tumor (MPNST) is unclear.
Patients and methods: Chemotherapy-naive soft tissue sarcomas (STS) patients treated on 12 pooled nonrandomized and randomized European Organization for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group trials were retrospectively analyzed. Clinical outcomes, overall survival, progression-free survival (PFS) and response were determined for MPNST and other STS histotypes and compared. Additionally, prognostic factors within the MPNST population were defined. Studied cofactors were demographics, sarcoma history, disease extent and chemotherapy regimen.
Results: After a median follow-up of 4.1 years, 175 MPNST out of 2675 eligible STS patients were analyzed. Outcome was similar for MPNST versus other STS histotypes, with a response rate, median PFS and overall survival of 21% versus 22%, 17 versus 16 weeks and 48 versus 51 weeks, respectively. Performance status was an independent prognostic factor for overall survival. Chemotherapy regimen was an independent prognostic factor for response (P < 0.0001) and PFS (P = 0.009). Compared with standard first-line doxorubicin, the doxorubicin–ifosfamide regimen had the best response, whereas ifosfamide had the worst prognosis.
Conclusion: This series indicates the role of chemotherapy in treatment of advanced MPNST. This first comparison showed similar outcomes for MPNST and other STS histotypes. The apparent superiority of the doxorubicin–ifosfamide regimen justifies further investigations of this combination in randomized trials.
PMCID: PMC3003614  PMID: 20656792
adriamycin; chemotherapy; ifosfamide; malignant peripheral nerve sheath tumor; soft tissue sarcoma
7.  CHECK (Cohort Hip and Cohort Knee): similarities and differences with the Osteoarthritis Initiative 
Annals of the rheumatic diseases  2008;68(9):1413-1419.
To describe the osteoarthritis study population of CHECK (Cohort Hip and Cohort Knee) in comparison with relevant selections of the study population of the Osteoarthritis Initiative (OAI) based on clinical status and radiographic parameters.
In The Netherlands a prospective 10-year follow-up study was initiated by the Dutch Arthritis Association on participants with early osteoarthritis-related complaints of hip and/or knee: CHECK. In parallel in the USA an observational 4-year follow-up study, the OAI, was started by the National Institutes of Health, on patients with or at risk of symptomatic knee osteoarthritis. For comparison with CHECK, the entire cohort and a subgroup of individuals excluding those with exclusively hip pain were compared with relevant subpopulations of the OAI.
At baseline, CHECK included 1002 participants with in general similar characteristics as described for the OAI. However, significantly fewer individuals in CHECK had radiographic knee osteoarthritis at baseline when compared with the OAI (p<0.001). In contrast, at baseline, the CHECK cohort reported higher scores on pain, stiffness and functional disability (Western Ontario and McMaster osteoarthritis index) when compared with the OAI (all p<0.001). These differences were supported by physical health status in contrast to mental health (Short Form 36/12) was at baseline significantly worse for the CHECK participants (p<0.001).
Although both cohorts focus on the early phase of osteoarthritis, they differ significantly with respect to structural (radiographic) and clinical (health status) characteristics, CHECK expectedly representing participants in an even earlier phase of disease.
PMCID: PMC3134276  PMID: 18772189
8.  Importance of the First Postoperative Year in the Prognosis of Elderly Colorectal Cancer Patients 
Annals of Surgical Oncology  2011;18(6):1533-1539.
Elderly colorectal cancer patients have worse prognosis than younger patients. Age-related survival differences may be cancer or treatment related, but also due to death from other causes. This study aims to compare population-based survival data for young (<65 years), aged (65–74 years), and elderly (≥75 years) colorectal cancer patients.
All patients operated for stage I–III colorectal cancer between 1991 and 2005 in the western region of The Netherlands were included. Crude survival, relative survival, and conditional relative survival curves, under the condition of surviving 1 year, were made for colon and rectal cancer patients separately. Furthermore, 30-day, 1-year, and 1-year excess mortality data were compared.
A total of 9,397 stage I–III colorectal cancer patients were included in this study. Crude survival curves showed clear survival differences between the age groups. These age-related differences were less prominent in relative survival and disappeared in conditional relative survival (CRS). Only in stage III disease did elderly patients have worse CRS than young patients. Furthermore, significant age-related differences in 30-day and 1-year excess mortality were found. Thirty-day mortality vastly underestimated 1-year mortality for all age groups.
Elderly colorectal cancer patients who survive the first year have the same cancer-related survival as younger patients. Therefore, decreased survival in the elderly is mainly due to differences in early mortality. Treatment of elderly colorectal cancer patients should focus on perioperative care and the first postoperative year.
PMCID: PMC3087879  PMID: 21445672
9.  The heritability of beta cell function parameters in a mixed meal test design 
Diabetologia  2011;54(5):1043-1051.
We estimated the heritability of individual differences in beta cell function after a mixed meal test designed to assess a wide range of classical and model-derived beta cell function parameters.
A total of 183 healthy participants (77 men), recruited from the Netherlands Twin Register, took part in a 4 h protocol, which included a mixed meal test. Participants were Dutch twin pairs and their siblings, aged 20 to 49 years. All members within a family were of the same sex. Insulin sensitivity, insulinogenic index, insulin response and postprandial glycaemia were assessed, as well as model-derived parameters of beta cell function, in particular beta cell glucose sensitivity and insulin secretion rates. Genetic modelling provided the heritability of all traits. Multivariate genetic analyses were performed to test for overlap in the genetic factors influencing beta cell function, waist circumference and insulin sensitivity.
Significant heritabilities were found for insulinogenic index (63%), beta cell glucose sensitivity (50%), insulin secretion during the first 2 h postprandial (42–47%) and postprandial glycaemia (43–52%). Genetic factors influencing beta cell glucose sensitivity and insulin secretion during the first 30 postprandial min showed only negligible overlap with the genetic factors that influence waist circumference and insulin sensitivity.
The highest heritability for postprandial beta cell function was found for the insulinogenic index, but the most specific indices of heritability of beta cell function appeared to be beta cell glucose sensitivity and the insulin secretion rate during the first 30 min after a mixed meal.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-011-2060-5) contains supplementary material, which is available to authorised users.
PMCID: PMC3071945  PMID: 21311857
Gastro-entero pancreatic function; Genetics of type 2 diabetes; Human; Insulin secretion; Insulin sensitivity; Metabolic physiology
10.  Genetic association analysis of LARS2 with type 2 diabetes 
Diabetologia  2009;53(1):103-110.
LARS2 has been previously identified as a potential type 2 diabetes susceptibility gene through the low-frequency H324Q (rs71645922) variant (minor allele frequency [MAF] 3.0%). However, this association did not achieve genome-wide levels of significance. The aim of this study was to establish the true contribution of this variant and common variants in LARS2 (MAF > 5%) to type 2 diabetes risk.
We combined genome-wide association data (n = 10,128) from the DIAGRAM consortium with independent data derived from a tagging single nucleotide polymorphism (SNP) approach in Dutch individuals (n = 999) and took forward two SNPs of interest to replication in up to 11,163 Dutch participants (rs17637703 and rs952621). In addition, because inspection of genome-wide association study data identified a cluster of low-frequency variants with evidence of type 2 diabetes association, we attempted replication of rs9825041 (a proxy for this group) and the previously identified H324Q variant in up to 35,715 participants of European descent.
No association between the common SNPs in LARS2 and type 2 diabetes was found. Our replication studies for the two low-frequency variants, rs9825041 and H324Q, failed to confirm an association with type 2 diabetes in Dutch, Scandinavian and UK samples (OR 1.03 [95% CI 0.95–1.12], p = 0.45, n = 31,962 and OR 0.99 [0.90–1.08], p = 0.78, n = 35,715 respectively).
In this study, the largest study examining the role of sequence variants in LARS2 in type 2 diabetes susceptibility, we found no evidence to support previous data indicating a role in type 2 diabetes susceptibility.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-009-1557-7) contains supplementary material, which is available to authorised users.
PMCID: PMC2789927  PMID: 19847392
Genetics; LARS2; Mitochondria; SNP; Type 2 diabetes
11.  Combined effects of single-nucleotide polymorphisms in GCK, GCKR, G6PC2 and MTNR1B on fasting plasma glucose and type 2 diabetes risk 
Diabetologia  2009;52(9):1866-1870.
Variation in fasting plasma glucose (FPG) within the normal range is a known risk factor for the development of type 2 diabetes. Several reports have shown that genetic variation in the genes for glucokinase (GCK), glucokinase regulatory protein (GCKR), islet-specific glucose 6 phosphatase catalytic subunit-related protein (G6PC2) and melatonin receptor type 1B (MTNR1B) is associated with FPG. In this study we examined whether these loci also contribute to type 2 diabetes susceptibility.
A random selection from the Dutch New Hoorn Study was used for replication of the association with FGP (2,361 non-diabetic participants). For the genetic association study we extended the study sample with 2,628 participants with type 2 diabetes. Risk allele counting was used to calculate a four-gene risk allele score for each individual.
Variants of the GCK, G6PC2 and MTNR1B genes but not GCKR were associated with FPG (all, p ≤ 0.001; GCKR, p = 0.23). Combining these four genes in a risk allele score resulted in an increase of 0.05 mmol/l (0.04–0.07) per additional risk allele (p = 2 × 10−13). Furthermore, participants with less than three or more than five risk alleles showed significantly different type 2 diabetes susceptibility compared with the most common group with four risk alleles (OR 0.77 [0.65–0.93], p = 0.005 and OR 2.05 [1.50–2.80], p = 4 × 10−6 respectively). The age at diagnosis was also significantly associated with the number of risk alleles (p = 0.009).
A combined risk allele score for single-nucleotide polymorphisms in four known FPG loci is significantly associated with FPG and HbA1c in a Dutch population-based sample of non-diabetic participants. Carriers of low or high numbers of risk alleles show significantly different risks for type 2 diabetes compared with the reference group.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-009-1413-9) contains supplementary material, which is available to authorised users.
PMCID: PMC2723681  PMID: 19533084
Fasting plasma glucose; Genetics; Single-nucleotide polymorphism; Type 2 diabetes
12.  The incidences of and consultation rate for lower extremity complaints in general practice 
Annals of the Rheumatic Diseases  2004;65(6):809-815.
To estimate the incidence and consultation rate of lower extremity complaints in general practice.
Data were obtained from the Second Dutch National Survey of General Practice, in which 195 general practitioners (GPs) in 104 practices recorded all contacts with patients during 12 consecutive months in computerised patient records. GPs classified the symptoms and diagnosis for each patient at each consultation according to the International Classification of Primary Care (ICPC). Incidence densities and consultation rates for different complaints were calculated.
During the registration period 63.2 GP consultations per 1000 person‐years were attributable to a new complaint of the lower extremities. Highest incidence densities were seen for knee complaints: 21.4 per 1000 person‐years for women and 22.8 per 1000 person‐years for men. The incidence of most lower extremity complaints was higher for women than for men and higher in older age.
Both incidences of and consultation rates for lower extremity complaints are substantial in general practice. This implies a considerable impact on the workload of the GP.
PMCID: PMC1798168  PMID: 16269430
leg pain; lower extremity; general practice
13.  Course and prognosis of elbow complaints: a cohort study in general practice 
Annals of the Rheumatic Diseases  2005;64(9):1331-1336.
Objective: To describe the course of new episodes of elbow complaints in general practice, and to identify predictors of short term and long term outcome in terms of pain intensity and functional disability.
Methods: 181 patients with elbow complaints filled in questionnaires at baseline and at 3, 6, and 12 months of follow up. Baseline scores of pain and disability, characteristics of the complaint, sociodemographic and psychosocial factors, physical activity, general health, and comorbidity were investigated as possible predictors of outcome. Outcome measures were analysed separately using multiple regression analyses.
Results: 13% of the patients reported recovery at the 3 month follow up and 34% at 12 months. Irrespective of outcome and length of follow up, a longer duration of the complaint before consulting the general practitioner, having musculoskeletal comorbidity, and using "retreating" as coping style increased the likelihood of an unfavourable outcome. Less social support was associated with an unfavourable outcome at 3 months, and having a history of elbow complaints and using "worrying" as coping style were associated with an unfavourable outcome at 12 months. The explained variance of the models ranged from 46% to 49%.
Conclusions: Recovery of patients with elbow complaints in general practice was poor. Besides characteristic of the complaint, passive coping and less social support were related to a worse prognosis. The results of this study may help general practitioners to provide patients with more accurate information about their prognosis.
PMCID: PMC1755654  PMID: 15708885
14.  Incidence and prevalence of complaints of the neck and upper extremity in general practice 
Annals of the Rheumatic Diseases  2005;64(1):118-123.
Objective: To study the incidence and prevalence of neck and upper extremity musculoskeletal complaints in Dutch general practice.
Methods: Data were obtained from the second Dutch national survey of general practice. In all, 195 general practitioners (GPs) from 104 practices across the Netherlands recorded all contacts with patients during 12 consecutive months. Incidence densities and consultation rates were calculated.
Results: The total number of contacts during the registration period of one year was 1 524 470. The most commonly reported complaint was neck symptoms (incidence 23.1 per 1000 person-years), followed by shoulder symptoms (incidence 19.0 per 1000 person-years). Sixty six GP consultations per 1000 person-years were attributable to a new complaint or new episode of complaint of the neck or upper extremity (incidence density). In all, the GPs were consulted 147 times per 1000 registered persons for complaints of the neck or upper extremity. For most complaints the incidence densities and consultation rates were higher for women than for men.
Conclusions: Neck and upper extremity symptoms are common in Dutch general practice. The GP is consulted approximately seven times each week for a complaint relating to the neck or upper extremity; of these, three are new complaints or new episodes. Attention should be paid to training GPs to deal with neck and upper limb complaints, and to research on the prognosis and treatment of these common complaints in primary care.
PMCID: PMC1755209  PMID: 15608309
15.  Internal consistency and validity of a new physical workload questionnaire 
Aims: To examine the dimensionality, internal consistency, and construct validity of a new physical workload questionnaire in employees with musculoskeletal complaints.
Methods: Factor analysis was applied to the responses in three study populations with musculoskeletal disorders (n = 406, 300, and 557) on 26 items related to physical workload. The internal consistency of the resulting subscales was examined. It was hypothesised that physical workload would vary among different occupational groups. The occupations of all subjects were classified into four groups on the basis of expected workload (heavy physical load; long lasting postures and repetitive movements; both; no physical load). Construct validity of the subscales created was tested by comparing the subscale scores among these occupational groups.
Results: The pattern of the factor loadings of items was almost identical for the three study populations. Two interpretable factors were found: items related to heavy physical workload loaded highly on the first factor, and items related to static postures or repetitive work loaded highly on the second factor. The first constructed subscale "heavy physical work" had a Cronbach's α of 0.92 to 0.93 and the second subscale "long lasting postures and repetitive movements", of 0.86 to 0.87. Six of eight hypotheses regarding the construct validity of the subscales were confirmed.
Conclusions: The results support the internal structure, internal consistency, and validity of the new physical workload questionnaire. Testing this questionnaire in non-symptomatic employees and comparing its performance with objective assessments of physical workload are important next steps in the validation process.
PMCID: PMC1740683  PMID: 15550603
16.  Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms 
Diabetologia  2007;50(12):2443-2450.
Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with type 2 diabetes and reduced insulin secretion. The transcription factor TCF7L2 is an essential factor for glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells. We studied whether a defect in the enteroinsular axis contributes to impaired insulin secretion in carriers of TCF7L2 polymorphisms.
We genotyped 1,110 non-diabetic German participants for five single nucleotide polymorphisms in TCF7L2. All participants underwent an OGTT; GLP-1 secretion was measured in 155 participants. In 210 participants, an IVGTT combined with a hyperinsulinaemic–euglycaemic clamp was performed. In another 160 participants from the Netherlands and 73 from Germany, a hyperglycaemic clamp (10 mmol/l) was performed. In 73 German participants this clamp was combined with a GLP-1 infusion and an arginine bolus.
The OGTT data confirmed that variants in TCF7L2 are associated with reduced insulin secretion. In contrast, insulin secretion induced by an i.v. glucose challenge in the IVGTT and hyperglycaemic clamp was not different between the genotypes. GLP-1 concentrations during the OGTT were not influenced by the TCF7L2 variants. However, GLP-1-infusion combined with a hyperglycaemic clamp showed a significant reduction in GLP-1-induced insulin secretion in carriers of the risk allele in two variants (rs7903146, rs12255372, p < 0.02).
Variants of TCF7L2 specifically impair GLP-1-induced insulin secretion. This seems to be rather the result of a functional defect in the GLP-1 signalling in beta cells than a reduction in GLP-1 secretion. This defect might explain the impaired insulin secretion in carriers of the risk alleles and confers the increased risk of type 2 diabetes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-007-0753-6) contains supplementary material, which is available to authorised users.
PMCID: PMC2063563  PMID: 17661009
GLP-1; Insulin secretion; Polymorphism; TCF7L2
17.  Clinimetric evaluation of shoulder disability questionnaires: a systematic review of the literature 
Annals of the Rheumatic Diseases  2004;63(4):335-341.
Methods: Systematic literature searches were performed to identify self administered shoulder disability questionnaires. A checklist was developed to evaluate and compare the clinimetric quality of the instruments.
Results: Two reviewers identified and evaluated 16 questionnaires by our checklist. Most studies were found for the Disability of the Arm, Shoulder, and Hand scale (DASH), the Shoulder Pain and Disability Index (SPADI), and the American Shoulder and Elbow Surgeons Standardised Shoulder Assessment Form (ASES). None of the questionnaires demonstrated satisfactory results for all properties. Most questionnaires claim to measure several domains (for example, pain, physical, emotional, and social functioning), yet dimensionality was studied in only three instruments. The internal consistency was calculated for seven questionnaires and only one received an adequate rating. Twelve questionnaires received positive ratings for construct validity, although depending on the population studied, four of these questionnaires received poor ratings too. Seven questionnaires were shown to have adequate test-retest reliability (ICC >0.70), but five questionnaires were tested inadequately. In most clinimetric studies only small sample sizes (n<43) were used. Nearly all publications lacked information on the interpretation of scores.
Conclusion: The DASH, SPADI, and ASES have been studied most extensively, and yet even published validation studies of these instruments have limitations in study design, sample sizes, or evidence for dimensionality. Overall, the DASH received the best ratings for its clinimetric properties.
PMCID: PMC1754942  PMID: 15020324
18.  Comparison of the responsiveness of the Harris Hip Score with generic measures for hip function in osteoarthritis of the hip 
Annals of the Rheumatic Diseases  2003;62(10):935-938.
Objective: To compare responsiveness of the Harris Hip Score with generic measures (that is, the Short Form-36 (SF-36), and a test of walking speed and pain during walking) in patients with osteoarthritis (OA) of the hip.
Method: The first 75 cases within the population of a randomised clinical trial on manual therapy and exercise therapy were selected for secondary analysis. Experienced (self reported) recovery by the patients after treatment (five weeks) was used as an external criterion for clinically relevant improvement. Responsiveness was evaluated by comparing responsiveness ratios and receiver operating characteristic curves.
Results: The responsiveness ratio for the Harris Hip Score was high (1.70) compared with walking speed (0.45), pain during walking (0.66), and the subscales of the SF-36—"bodily pain" (0.42) and "physical functioning" (0.36). The area under the curve also was highest for the Harris Hip Score (0.92) compared with walking speed (0.71), pain during walking (0.73), and the SF-36 subscales—bodily pain and physical functioning (both 0.66).
Conclusion: The Harris Hip Score is more responsive than the test of walking speed, pain, and subscales for function of the SF-36 in patients with OA of the hip. The Harris Hip Score seems to be a suitable instrument to evaluate change in hip function in patients with OA of the hip.
PMCID: PMC1754316  PMID: 12972470
19.  Effectiveness of exercise in patients with osteoarthritis of hip or knee: nine months' follow up 
Annals of the Rheumatic Diseases  2001;60(12):1123-1130.
OBJECTIVE—To determine whether the effects of an exercise programme in patients with osteoarthritis of hip or knee are sustained at six and nine months' follow up.
METHODS—A randomised, single blind, clinical trial was conducted in a primary care setting. Patients with osteoarthritis of hip or knee (ACR criteria) were selected. Two intervention groups were compared. Both groups received treatment from their general practitioner, including patient education and drug treatment if necessary. The experimental group also received exercise treatment from a physiotherapist in primary care. The treatment period was 12 weeks, with an ensuing 24 week follow up. The main outcome measures were pain, drug use (non-steroidal anti-inflammatory drugs), and observed disability.
RESULTS—201 patients were randomly allocated to the exercise or control group, and 183 patients completed the trial. At 24 weeks exercise treatment was associated with a small to moderate effect on pain during the past week (difference in change between the two groups −11.5 (95% CI −19.7 to −3.3). At 36 weeks no differences were found between the groups.
CONCLUSIONS—Beneficial effects of exercise decline over time and finally disappear.

PMCID: PMC1753453  PMID: 11709454
20.  Relation of H pylori to gastric mucins and gastric surface mucous gel layer 
Gut  2001;48(6):869-871.
PMCID: PMC1728332  PMID: 11394387
21.  H pylori colocalises with MUC5AC in the human stomach 
Gut  2000;46(5):601-607.
BACKGROUND—The bacterium Helicobacter pylori is able to adhere to and to colonise the human gastric epithelium, yet the primary gene product responsible as a receptor for its adherence has not been identified.
AIMS—To investigate the expression of the gastric mucins MUC5AC and MUC6 in the gastric epithelium in relation to H pylori colonisation in order to examine their possible roles in the binding of H pylori.
PATIENTS—Seventy two consecutive patients suspected of having H pylori infection.
METHODS—MUC5AC, MUC6, and H pylori were detected in single sections of antral biopsy specimens using immunohistochemical triple staining.
RESULTS—MUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric pits. The expression of both mucins in the epithelium was complementary. In each patient, there was a sharply delineated transition between MUC5AC and MUC6 producing cell populations. In all H pylori positive patients there was a striking colocalisation of H pylori and MUC5AC; more than 99% of the bacteria were associated with either extracellular MUC5AC or the apical domain of MUC5AC producing cells.
CONCLUSIONS—H pylori is very closely associated with extracellular MUC5AC and epithelial cells that produce MUC5AC. This indicates that MUC5AC, but not MUC6, plays a role in the adhesion of H pylori to the gastric mucosa.

Keywords: Helicobacter pylori; gastric mucin; MUC5AC; MUC6; stomach
PMCID: PMC1727935  PMID: 10764701
22.  Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis 
Gut  1999;44(3):387-393.
BACKGROUND—Decreased synthesis of the predominant secretory human colonic mucin (MUC2) occurs during active ulcerative colitis. 
AIMS—To study possible alterations in mucin sulphation and mucin secretion, which could be the cause of decreased mucosal protection in ulcerative colitis. 
METHODS—Colonic biopsy specimens from patients with active ulcerative colitis, ulcerative colitis in remission, and controls were metabolically labelled with [35S]-amino acids or [35S]-sulphate, chase incubated and analysed by SDS-PAGE, followed by quantitation of mature [35S]-labelled MUC2. For quantitation of total MUC2, which includes non-radiolabelled and radiolabelled MUC2, dot blotting was performed, using a MUC2 monoclonal antibody. 
RESULTS—Between patient groups, no significant differences were found in [35S]-sulphate content of secreted MUC2 or in the secreted percentage of either [35S]-amino acid labelled MUC2 or total MUC2. During active ulcerative colitis, secretion of [35S]-sulphate labelled MUC2 was significantly increased twofold, whereas [35S]-sulphate incorporation into MUC2 was significantly reduced to half. 
CONCLUSIONS—During active ulcerative colitis, less MUC2 is secreted, because MUC2 synthesis is decreased while the secreted percentage of MUC2 is unaltered. Furthermore, sulphate content of secreted MUC2 is unaltered by a specific compensatory mechanism, because sulphated MUC2 is preferentially secreted while sulphate incorporation into MUC2 is reduced. 

Keywords: ulcerative colitis; mucin; MUC2; sulphation; secretion
PMCID: PMC1727426  PMID: 10026326
23.  Expression of a marker for colonic crypt base cells is correlated with poor prognosis in human colorectal cancer 
Gut  1998;42(1):63-70.
Background—There is a need for markers in colorectal cancer which will allow subclassification of stage groups into subgroups with high versus low risk of recurrent disease. 
Aims—To develop monoclonal antibodies that recognise antigens on immature crypt base cells, on the assumption that in a neoplasm undifferentiated but not the terminally differentiated cells will be responsible for tumour progression. 
Methods—Colon crypt cells which were isolated from human colonic mucosa by EDTA/EGTA incubation were studied. By stepwise harvesting, crypt base cell enriched fractions were obtained, and after incubation with antibodies against dominant antigens, used as immunogens. 
Results—Of one crypt base cell specific antibody (5E9), the reactive epitope appeared to be a non-terminal carbohydrate in the mucin O-glycans of the colon. The epitope did not seem to be colon specific, but was expressed in a variety of other tissues. In colorectal carcinomas, 5E9 immunoreactivity identified a subgroup of patients with a tendency for worse prognosis.
Conclusion—A mucin associated maturation epitope was identified in colonic crypt base cells, the expression of which in Dukes' stage B3 colorectal carcinoma may be associated with poor prognosis. 

Keywords: colorectal adenocarcinoma; maturation epitope; stem cell; prognostic marker; mucin
PMCID: PMC1726940  PMID: 9505887
24.  Complete nucleotide sequence of simian endogenous type D retrovirus with intact genome organization: evidence for ancestry to simian retrovirus and baboon endogenous virus. 
Journal of Virology  1997;71(5):3666-3676.
A complete endogenous type D viral genome has been isolated from a baboon genomic library. The provirus, simian endogenous retrovirus (SERV), is 8,393 nucleotides long and contains two long terminal repeats and complete genes for gag, pro, pol, and env. The primer binding site is complementary to tRNA(Lys)3, like in lentiviruses. The env GP70 protein is highly homologous to that of baboon endogenous virus (BaEV). PCR analysis of primate DNA showed that related proviral sequences are present in Old World monkeys of the subfamily Cercopithecinae but not in apes and humans. Analysis of virus and host sequences indicated that the proviral genomes were inherited from a common ancestor. Comparison of the evolution of BaEV, exogenous simian retrovirus types 1 to 3 (SRV1 to SRV3), and SERV suggests that SERV is ancestral to both BaEV and the SRVs.
PMCID: PMC191515  PMID: 9094640
25.  Two regions within the DNA binding domain of nuclear factor I interact with DNA and stimulate adenovirus DNA replication independently. 
Molecular and Cellular Biology  1996;16(8):4073-4080.
The cellular transcription factor nuclear factor I (NFI) stimulates adenovirus DNA replication by up to 50-fold. The NFI DNA binding domain (NFI-BD) is sufficient for stimulation and interacts with the viral DNA polymerase, thereby recruiting the precursor terminal protein-DNA polymerase complex (pTP-pol) to the origin of replication. The mechanism of DNA binding by NFI is unknown. To examine DNA binding and stimulation of adenovirus DNA replication by NFI-BD in more detail, we generated a series of deletion mutants and show that the DNA binding domain of NFI consists of two subdomains: a highly basic N-terminal domain that binds nonspecifically to DNA and a C-terminal domain that binds specifically but with very low affinity to the NFI recognition site. Both of these subdomains stimulate DNA replication, although not to the same extent as the intact DNA binding domain. The N-terminal domain has an alpha-helical structure, as shown by circular dichroism spectroscopy. The C-terminal domain interacts with the pTP-pol complex and is able to recruit the pTP-pol complex to DNA, which leads to pTP-pol-dependent stimulation of replication. The N-terminal domain also stimulates replication in a pTP-pol-dependent manner and enhances binding of pTP-pol to DNA. Since we could not detect a direct protein-protein interaction between pTP-pol and the N-terminal domain, we suggest that this domain stimulates replication by inducing structural changes in the DNA.
PMCID: PMC231403  PMID: 8754805

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