Array-based sequence capture and next-generation sequencing for the identification of primary immunodeficiencies.

Primary immunodeficiencies are genetic disorders in which components of immunological pathways are either missing or dysregulated. With the advent of next-generation sequencing, testing for genes in conditions with a heterogeneous genetic background seems more promising. We designed a custom microarray with 385K probe capacity to capture exons of 395 human genes, known or predicted to be associated with primary immunodeficiency and immune regulation. Enriched target DNA was sequenced using a GS FLX Titanium 454 platform. The patients selected were likely to have an underlying immunodeficiency. In one patient with hepatosplenomegaly, recurrent infections and an elevated IgM level, sequence analysis of the patient and his two unaffected parents identified ATM (ataxia telangiectasia mutated) as the underlying defect. In a second child with a clinical SCID phenotype, we detected a mutation in the ARTEMIS gene after focusing on SCID-associated genes. 454 sequencing yielded 152,000-397,000 high-quality reads per patient. 78-99% of the targeted nucleotides were covered at least one time, 76-82% at least five times. Array-based sequence capture expands our capacities to sequence large targeted DNA regions in a less laborious and time-consuming approach. Our array was capable to find the underlying genetic defect in two patients with suspected primary immunodeficiency. Upcoming whole-exome sequencing definitely will add more valuable data, but bioinformatical analysis and validation of variants already pose major challenges.

TNFSF15 polymorphisms are associated with susceptibility to inflammatory bowel disease in a new European cohort.

OBJECTIVES: Inflammatory bowel disease (IBD), e.g., Crohn's disease (CD) and ulcerative colitis (UC), is a complex genetic disorder. Tumor necrosis factor (ligand) superfamily, member 15 (TNFSF15) has been previously identified as a susceptibility gene for CD in Japanese and UK cohorts. This replication study was designed in order to confirm and further validate the role of TNFSF15 in IBD. METHODS: A total of 666 IBD families (corresponding to 2,982 relatives) with European ancestry were genotyped for the rs6478108 and rs7869487 polymorphisms, which define the main TNFSF15 haplotypes previously associated with CD. An association between the main haplotypes and CD, UC and IBD was tested using the Genehunter TDT and Unphased statistics. Caspase recruitment domain 15 (CARD15)/TNFSF15 interaction and genotype/phenotype correlations were also studied. RESULTS: The previously reported "high-risk" haplotype (A) was associated with IBD (P=0.001) (OR=1.25 (1.05-1.50)) and CD (P=0.02) (OR=1.31 (1.03-1.67)) whereas the "protective" (B) haplotype was significantly less transmitted to IBD and CD patients. No interaction between CARD15 and TNFSF15 was detected. We also failed to define a clinical subgroup of CD patients specifically associated with TNFSF15 haplotype A. CONCLUSIONS: This study confirms that TNFSF15 or a closely linked gene is involved in the genetic predisposition to CD.

Treatment of Crohn's disease with fusidic acid: an antibiotic with immunosuppressive properties similar to cyclosporin.

Fusidic acid is an antibiotic with T-cell specific immunosuppressive effects similar to those of cyclosporin. Because of the need for the development of new treatments for Crohn's disease, a pilot study was undertaken to estimate the pharmacodynamics and tolerability of fusidic acid treatment in chronic active, therapy-resistant patients. Eight Crohn's disease patients were included. Fusidic acid was administered orally in a dose of 500 mg t.d.s. and the treatment was planned to last 8 weeks. The disease activity was primarily measured by a modified individual grading score. Five of 8 patients (63%) improved during fusidic acid treatment: 3 at two weeks and 2 after four weeks. There were no serious clinical side effects, but dose reduction was required in two patients because of nausea. Biochemically, an increase in alkaline phosphatases was noted in 5 of 8 cases (63%), and the greatest increases were seen in those who had elevated levels prior to treatment. All reversed to pre-treatment levels after cessation of treatment. The results of this pilot study suggest that fusidic acid may be of benefit in selected chronic active Crohn's disease patients in whom conventional treatment is ineffective. Because there seems to exist a scientific rationale for the use of fusidic acid at the cytokine level in inflammatory bowel disease, we suggest that the role of this treatment should be further investigated.

Intestinal and extra-intestinal cancer in Crohn's disease: follow-up of a population-based cohort in Copenhagen County, Denmark.

AIM: To determine the long-term risk of intestinal and extra-intestinal malignancies in Crohn's disease patients in Copenhagen County, Denmark. METHODS: In Copenhagen County, a strictly population-based cohort of 374 patients with Crohn's disease diagnosed between 1962 and 1987 was followed until 1997 in order to determine the long-term risk of intestinal and extra-intestinal malignancies. Information on cancer occurrence was provided by the Danish National Cancer Registry and confirmed by the examination of hospital files. The observed number of cases was compared with the expected number, calculated from individually computed person-years at risk and 1995 cancer incidence rates for the background population. RESULTS: The risk of small bowel adenocarcinoma was significantly increased, independent of age and gender (standardized morbidity ratio, 66.7; 95% confidence interval, 18.1-170.7). The risk of colorectal cancer was not increased, either in the total group of patients or in patients with colonic Crohn's disease exclusively (standardized morbidity ratio, 1.64; 95% confidence interval, 0.20-5.92). Extra-intestinal cancer did not occur more frequently than expected. CONCLUSIONS: This population-based study of patients with Crohn's disease revealed no increase in colorectal cancer risk, possibly due to maintenance treatment with 5-aminosalicylic acid preparations and surgery in treatment failure. In contrast, the risk of small bowel cancer was increased more than 60-fold, but the numbers were small. The risk of extra-intestinal cancer was not increased and no lymphomas were observed.

Low-dose cyclosporin for Crohn's disease: implications for clinical trials.

Cyclosporin is a potent immunosuppressant, which has gained recent interest as a possible treatment for Crohn's disease. Chronic nephrotoxicity, however, has recently been demonstrated as a result of early treatment with high initial cyclosporin doses. We report the effect of a 3-month treatment with low-dose cyclosporin (5-7.5 mg kg-1 day-1) in 11 chronically active, therapy-resistant Crohn's disease patients. Eight of the 11 patients (72%) improved according to a clinical grading score and the Dutch Activity Index whereas 9/11 (82%) improved according to the Crohn's Disease Activity Index (P less than 0.05) after 1 month. Three patients were withdrawn despite clinical improvement. One developed arterial hypertension, one dropped out and one required surgical treatment due to a small bowel stricture. Five patients (45%) completed the treatment period with improved clinical scores. After tapering-off, two patients (18%) were better at follow-up. No serious side-effects were encountered and it is concluded that low-dose cyclosporin treatment should be further investigated in Crohn's disease.

Lifelong control with inflammatory bowel disease patients.

The benefits and the burden of a lifelong control scheme for patients with chronic inflammatory bowel disease are discussed. The benefits cover physical, psychologic, and social conditions. Valuable scientific gains can be yielded secondarily from the data collection.

Arthritis and the gut.

Mild arthritis/arthralgia is the most frequent extraintestinal manifestation in inflammatory bowel disease (IBD) and has been reported to occur in 10-35% of patients in different studies. A classification of peripheral arthropathy in relation to inflammatory bowel disease has recently been proposed. Type 1: pauciarticular, asymmetrical, preferably large joints, and related to IBD activity. Type 2: polyarticular, symmetrical, preferably small joints, and occurring independently of IBD activity. While this classification requires the presence of synovitis, arthralgia without swelling or other objective signs are of equal frequency but are not covered by this system. In this issue of the journal, Thomas et al. report a prospective study, incorporating strict endoscopic and histological criteria for pouchitis, which elucidates the correlation to arthropathy. Both pouchitis and symptoms of the joints occurred more frequently in ulcerative colitis patients than in patients with familial polyposis. Surprisingly, arthropathy was not more frequent among patients with pouchitis than among patients without pouchitis in this study. Extraintestinal manifestations of the joints are thus not likely to be a reactive arthritis secondary to pouchitis, which would have been an obvious explanation. Preoperative occurrence of extraintestinal manifestations from the joints does not seem to be predictive for the outcome of an ileo-anal pouch anastomosis and especially development of pouchitis. The arthritic symptoms were generally mild and not disabling.

Preliminary applicability tests of different methacrylic acid copolymers, type C NF, particularly relevant to spreading and film formation.

The intention of this study was to show under which conditions a film forming methacrylic acid copolymer coating excipient, corresponding to the requirements of pharmacopoeia, but obtained from different sources, can be substituted without severe problems. The mechanical properties of the film coats were investigated by dynamic-mechanical thermo-analysis (DMTA) experiments to determine with respect to the glass transition the storage modulus E', the loss modulus E'', and the loss factor tan delta. Further determinations concerned the surface tensions of the different coating dispersions. This attribute plays an important role in spreading, distribution and coalescence of the film forming preparations. Finally by a series of small experimental fluidized bed batches cores containing a model drug were coated with the different methacrylic acid copolymers. The resistance of these coated tablets in 0.1 N HC1 as well as their dissolution rates in artificial intestinal juice were tested. The coatings proved themselves so similar that in this case substitutions of products of different provenance are possible. The determinations of surface tension and the DMTA measurements seem to be useful and reliable preliminary applicability tests.

Lauroyldextran and crosslinked galactomannan as coating materials for site-specific drug delivery to the colon.

Lauroyldextran (LD) and crosslinked galactomannan (XGM) were investigated as microbiologically degradable film coating materials for site-specific drug delivery to the colon. LD was used with degrees of substitution between 0.12 and 0.40, and swelling in aqueous media between 195 and 50%, XGM-batches showed swelling between 309 and 520%. Theophylline tablets were coated in a Hüttlin Kugelcoater with coating quantities of 4-17 mg/cm2. Sprayable coating formulations were obtained with 4% aqueous dispersions of XGM or 4% dispersions of LD in a 1:1 mixture of 1-propanol and water with 10% glycerol (based on the polymer) as a plasticizer. Theophylline dissolution was monitored in a USP XXIII paddle dissolution apparatus with buffer pH 5.5. After 4 h, which is an average small intestine transit time, colon conditions were simulated by adding galactomannanase or dextranase, respectively. Results showed similar dissolution rates for all XGMs and high-swelling LDs during the first 4 h and a relatively quick disintegration after enzyme addition. Both parameters decreased with increasing coating quantities. Dissolution from low-swelling lauroyldextrans was very low but no disintegration was observed after enzyme addition. The disintegration rate was found to be proportional to the square root of the enzyme activity. All swollen materials exhibited low mechanical stability. XGM coatings, especially at higher coating quantities, showed small transient ruptures at the edges not caused by enzyme addition. This behaviour was explained by internal stress due to the high degree of swelling. In principle, materials of both types proved to be suitable as degradable coating materials. The ideal zero-dissolution before and quick disintegration after enzyme addition, however, was not realized with the present materials.

Familial occurrence and inheritance studies in inflammatory bowel disease.

A number of studies have demonstrated aggregation of cases of ulcerative colitis or Crohn's disease in families, and of cases of both diseases within the same families, suggesting that patients share a genetic background. Perhaps because of differences in the selection of patients, study design and diagnostic criteria, different patterns of occurrence of inflammatory bowel disease (IBD) have been found among relatives of patients with these disorders. In recent years, however, several studies have been carried out, aiming by epidemiological methods to reveal (1) the frequency of familial occurrence of IBD among patients with ulcerative colitis and Crohn's disease, and (2) the prevalence of IBD among 1(0) relatives to patients with these diseases. Results from these studies show a relatively uniform pattern of family occurrence in about 10% of patients with ulcerative colitis and Crohn's disease, and a prevalence among 1(0) relatives of about 10 times that of the background population. A twin study reported a significantly higher concordance rate for Crohn's disease than for ulcerative colitis in monozygotic twins. By use of complex segregation analyses in 3 different studies, a very similar model of inheritance was found to fit for ulcerative colitis, namely a major dominant or additive gene with a low penetrance. For Crohn's disease the best-fitting model was a major recessive gene, with a high penetrance. This difference strongly supports the concept of ulcerative colitis and Crohn's disease as two separate disease entities. The occurrence of both diseases within the same families in certain members of the affected families is difficult to explain. The search for distinct associations of HLA genes with inflammatory bowel disease has shown a positive correlation between DR2 and ulcerative colitis and a negative association with DR4 and DRw6, compared with ethnically matched controls. In contrast, in Crohn's disease a positive association with the combination of DR1 and DQw5 alleles was revealed, thus indicating genetically different disease susceptibility for the two disorders. In general, however, no consistent pattern has been revealed from studies of association of HLA-A or -B antigens or blood group and serum protein markers. In two French families with several members affected with Crohn's disease no evidence for an HLA haplotype association could be revealed. Possible inherited markers of ulcerative colitis or Crohn's disease have been sought but without convincing success. Increased intestinal permeability, presence of anticolon antibodies and presence of antineutrophil leukocyte antibodies have been proposed, but not proved. Thorough studies are now needed of multimember families with disease for linkage studies to identify loci which contribute to increased liability. Such studies are in progress in different centres.

Investigation of inheritance of chronic inflammatory bowel diseases by complex segregation analysis.

OBJECTIVE: To investigate the mode of inheritance of ulcerative colitis and Crohn's disease by complex segregation analysis. DESIGN: Cross sectional population based survey of familial occurrence of chronic inflammatory bowel disease. SETTING: Population of the Copenhagen county in 1987. SUBJECTS: 662 patients in whom inflammatory bowel disease had been diagnosed before 1979, of whom 637 (96%) provided adequate information. Of 504 patients with ulcerative colitis, 54 had 77 relatives with ulcerative colitis and of 133 patients with Crohn's disease, five had seven relatives with Crohn's disease. MAIN OUTCOME MEASURES: Patterns of segregation of either disease as assessed by complex segregation analysis performed with the computer program POINTER. RESULTS: The analysis suggested that a major dominant gene with a penetrance of 0.20-0.26 is present in 9-13% of adult patients with ulcerative colitis. The analysis did not allow for other components in the familial aggregation. For Crohn's disease the best fitting model included a major recessive gene with complete penetrance, for which 7% of the patients are homozygous. However, this model was not significantly different from a multifactorial model. CONCLUSIONS: The segregation pattern indicates that a major dominant gene has a role in ulcerative colitis, and suggests that a major recessive gene has a role in Crohn's disease.

[Therapeutic principles for chronic inflammatory bowel disease]. / Behandlingsprincipper for de kroniske inflammatoriske tarmsygdomme.

Whereas the incidence of ulcerative colitis has remained stable at around 8-9/10(5), the incidence of Crohn's disease has increased from below 1 to more than 5/10(5) per year during the last three decades. The new disease entities, collagenous colitis and lymphocytic colitis, are now covered by the term, chronic inflammatory bowel disease. The general principles of treatment of these diseases are to induce remission of outbreaks and to prevent outbreaks during remission. Available pharmaceutical products are 5-aminosalicylic acid preparations, with different delivery profiles in the gastrointestinal tract, glucocorticoids, and other immunosuppressants, especially azathioprine. New immunomodulating agents, with a specific effect on intracellular processes in the inflammatory cascade are now being developed, and infliximab, a TNF-alpha antibody, is now an accepted agent for use in severe, treatment-resistant cases of fistulising Crohn's disease. When medical treatment fails, surgical treatment is an option. In ulcerative colitis, colectomy is, in principle, curative, but it leaves the patient with either a permanent ileostomy or an ileal pouch, which serves as an artificial rectum after ileoanal anastomosis. This latter procedure has the obvious advantage of giving the patient a normal bowel continuity, but complications in the form of intractable "pouchitis" have been experienced in a small number of patients, thus necessitating removal of the pouch. Patients with Crohn's disease, who do not respond to medical treatment or present signs of stenosis in either the small or the large bowel, must be given surgical treatment, although an operation is less curative than in ulcerative colitis. Surgical resections for Crohn's disease must therefore be more conservative, so as to preserve the bowel and only remove macroscopically affected tissue.

[Long-term prognosis of patients with ulcerative colitis]. / Langtidsprognosen for patienter med colitis ulcerosa.

A regional inception cohort of 1161 ulcerative colitis patients was followed from diagnosis to the end of 1987. Follow-up rate for death and occurrence of cancer was 99.9%. Median observation time 11.7 years, range 0-26 years. One hundred and forty-one deaths were observed, 26 due to ulcerative colitis or complications thereof. No significant excess mortality was found after the first year, but in the year of diagnosis the relative risk of death was 2.4 (p < 0.001). The cumulative colectomy rate was 32.4% 25 years after diagnosis. The initial extent of disease significantly influenced the colectomy probability, being 35% in total colitis, 19% in substantial colitis and 9% in distal colitis within the first five years after diagnosis. Six patients developed colorectal cancer within the observation period. Compared to the expected number of 6.6 the relative risk for patients with ulcerative colitis was 0.9. The calculated cumulative cancer incidence was 3.1% after 25 years (95% confidence limits 0.0-6.8). The calculated lifetime risk (0 to 74 years) for development of colorectal cancer was 3.5% for the patients compared to 3.7% for the Danish population. We conclude that with an active approach to medical and surgical treatment as was practiced here, those patients who are left with their colon intact bear no significantly increased risk of colo-rectal malignancy.

[Increased incidence of Crohn disease in the county of Copenhagen]. / Stigende incidens af morbus Crohn i Københavns Amt.

The incidence of Crohn's disease was assessed in a population based study in Copenhagen County from 1962-1987. The incidence increased six fold during the study period from 0.62/10(5) in 1962 to a mean of 4.1/10(5) from 1979-1987, equally in both sexes. The highest incidence was found in the age group 15-24 years: 12.8/10(5) for women and 6.0/10(5) for men (as mean of the period 1979-1987). The prevalence at the end of the study was 54/10(5). The clinical symptoms and extent of disease at diagnosis did not change over the time, but a significantly higher disease activity was found in the 1980's, 74% (1962-1979) vs 84% (1980-1987), p = 0.04. The increase in incidence of Crohn's disease is well established in most parts of the industrialized world. The analysis shows that it is not due to a new disease entity, since the extent of disease, the age and sex distribution, and the symptoms remained unchanged during the 26 year study period.

[Inflammatory bowel diseases in children]. / Inflammatorisk tarmsygdom hos børn.

From an incidence cohort diagnosed during 1962-1987 we identified all patients with onset of IBD before the age of 15 in order to describe the course and to compare course and prognosis with adult onset IBD. The mean incidence of IBD among children below 15 years was 2.2/10(5), 2.0 for ulcerative colitis (UC), and 0.2 for Crohns disease (CD). At diagnosis, UC children had more extensive disease compared to adults (p < 0.05). Abdominal pains were also more frequent. The cumulative colectomy probability was 6% after one year and 29% after 20 years, not different from adults. Regarding disease activity, it was found that 60-70% of UC patients were in remission in the first 10 years of disease, for CD about 50% were in remission. One UC patient developed carcinoma of the sigmoid colon. Time between onset and development of carcinoma was 12 years. For CD no differences in clinical appearance at diagnosis and course between children and adults were found. No deaths occurred among CD patients. Three CD patients were found to have severe growth retardation already at diagnosis. In conclusion, the incidence of IBD is low in childhood. At diagnosis children with UC have more widespread disease than adults. Children with CD do not differ in clinical presentation, course or prognosis compared to adult onset CD. However, growth retardation is a problem among CD patients.

Incidence of colonic cancer in inflammatory bowel disease.

In a cohort of 783 regional patients with ulcerative colitis treated in specialized clinics from the onset of the disease, the intestinal cancer risk was estimated. Seven patients developed colonic cancer from 4 to 44 years after onset of disease (median, 14 years). Eighteen years after onset of the disease the calculated risk was 1.4% (0.7-2.8%). After completing this follow-up study a new study was carried out with colonoscopy of all long-standing cases (greater than or equal to 15 years) of ulcerative colitis which had not been operated on. Of 100 patients examined, 2 showed epithelial dysplasia, and 98 did not. In a similar cohort of 185 patients with Crohn's disease, 1 showed small-bowel cancer. This gives a calculated risk of 0.6% (0.1-3.1%) at 10 years after the disease onset.

Epidemiology in gastroenterology.

A review of the literature on gastrointestinal epidemiology during the last 25 years shows a comprehensive contribution from Danish studies. This is partly explained by very favourable conditions in Denmark for epidemiological research, due to various valid registers and a long tradition for longitudinal population and patient cohorts. Data are presented on prevalence, incidence, demography, clinical risk factors, clinical course, and prognosis of various gastrointestinal disorders. Main emphasis is put on Danish studies, which are compared with international results. Present possibilities for prophylaxis against gastrointestinal diseases and delimitation of treatment of benign diseases are discussed. It is expected that research within gastrointestinal epidemiology in Denmark will increase in the coming years.

MiR-125 in normal and malignant hematopoiesis.

MiR-125 is a highly conserved microRNA throughout many different species from nematode to humans. In humans, there are three homologs (hsa-miR-125b-1, hsa-miR-125b-2 and hsa-miR-125a). Here we review a recent research on the role of miR-125 in normal and malignant hematopoietic cells. Its high expression in hematopoietic stem cells (HSCs) enhances self-renewal and survival. Its expression in specific subtypes of myeloid and lymphoid leukemias provides resistance to apoptosis and blocks further differentiation. A direct oncogenic role in the hematopoietic system has recently been demonstrated by several mouse models. Targets of miR-125b include key proteins regulating apoptosis, innate immunity, inflammation and hematopoietic differentiation.

Highly skewed T-cell receptor V-beta chain repertoire in the bone marrow is associated with response to immunosuppressive drug therapy in children with very severe aplastic anemia.

One of the major obstacles of immunosuppressive therapy (IST) in children with severe aplastic anemia (SAA) comes from the often months-long unpredictability of bone-marrow (BM) recovery. In this prospective study in children with newly diagnosed very severe AA (n=10), who were enrolled in the therapy study SAA-BFM 94, we found a dramatically reduced diversity of both CD4+ and CD8+ BM cells, as scored by comprehensive V-beta chain T-cell receptor (TCR) analysis. Strongly skewed TCR V-beta pattern was highly predictive for good or at least partial treatment response (n=6, CD8+ complexity scoring median 35.5, range 24-73). In contrast, IST in patients with rather moderate reduction of TCR V-beta diversity (n=4, CD8+ complexity scoring median 109.5, range 82-124) always failed (P=0.0095). If confirmed in a larger series of patients, TCR V-beta repertoire in BM may help to assign children with SAA up-front either to IST or to allogeneic stem-cell transplantation.

Hsa-mir-125b-2 is highly expressed in childhood ETV6/RUNX1 (TEL/AML1) leukemias and confers survival advantage to growth inhibitory signals independent of p53.

MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose-dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic function of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT-PCR in four types of childhood acute lymphoblastic leukemia (ALL) characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly, high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies showed that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together, these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53-independent manner.