Detection of MEFV- Gene Mutations in Egyptian Children with Inflammatory Bowel Disease

Transcription

1 Detection of MEFV- Gene Mutations in Egyptian Children with Inflammatory Bowel Disease Thesis Submitted in the partial fulfillment of M.D.pediatrics By Yomna Mohamed Farag SUPERVISORS Prof.Dr Samia Salah ElDeen Mahmoud Professor of Pediatrics Faculty of Medicine-Cairo University Prof.Dr Mortada Hassan El Shabrawi Professor of Pediatrics Faculty of Medicine-Cairo University Prof.Dr Hala Fathi Shibah Professor of Clinical Pathology Faculty of Medicine-Cairo University Dr. Hala Mohamed Lotfy Ass. Professor of Pediatrics Faculty of Medicine-Cairo University Faculty of Medicine Cairo University 2012

2 Acknowledgement First of all, thanks to God for his grace and mercy, and for giving me the effort to complete this work. I was fortunate enough to carry out this work under the supervision of Prof. Dr Samia Salah, Professor of Pediatrics, Cairo University. I am grateful for her generous help and support not only in this work, but also throughout my whole working career. I would like to express my thanks to Prof. Dr Mortada Alshabrawy, Professor of Pediatrics, Cairo University for his continuous guidance and advice through this work. I would also like to thank Prof. Dr Hala Sheba, Professor of Clinical and Chemical Pathology, Cairo University for her help, especially in the laboratory part of the study. Words will never be able to express my deepest gratitude and appreciation to Ass. Prof. Dr Hala Lotfy, Assistant Professor of Pediatrics, Cairo University for her kindness, great patience, continuous support and unlimited help throughout the work. Special thanks to Dr Maha Abo Zekry, consultant of Pediatric Gastroentrology for her very generous help and advice especially during collection of patients. Last, but not least I would like to express my deepest thanks to my family, namely my dear parents, husband, brothers, and sisters-in-law for supporting me in this work and throughout my whole life. i

3 Abstract IBD and FMF share common clinical and biological features. This study aimed at determining the prevalence of MEFV gene mutation in IBD patients and to characterize FMF-IBD patients. It enrolled 17 patients with UC, 15 with CD, 10 with IC and 33 controls. All cases were screened for 12 MEFV gene mutations. 88.1% of the IBD patients carried the mutation versus 42.4% control cases. Data were comparable between MEFV positive and negative IBD patients. IBD patients living in countries where FMF is frequent should be screened for FMF. Keywords IBD-FMF-indeterminate colitis- MEFV gene ii

4 Contents List of Tables...v List of Figures.....vi List of Abbreviations......vii Introduction.1 Aim of the Work....3 Review of literature Chapter One: Familial Mediterranean Fever Definition History....4 Racial distribution...5 Etiology and pathogenesis Clinical manifestations 8 Complications 11 Laboratory investigations.. 12 Diagnosis...13 Differential Diagnosis Treatment.17 Genetics 20 Chapter Two: Inflammatory Bowel Disease Definition..25 Etiology. 25 Genotype-Phenotype Correlations in Pediatric IBD 28 Gut immunity in IBD 29 Epidemiology of IBD 30 Hygiene hypothesis.31 Natural History of Pediatric Indeterminate Colitis..32 Extra-intestinal manifestations. 33 Clinical features Differential diagnosis 37 Laboratory assessment Radiological assessment 41 Endoscopy and histology..42 Subclassification of UC and Crohn disease.45 iii

5 Treatment of IBD.46 Chapter Three: FMF and other inflammatory disorders 52 Patients and methods. 56 Results..59 Discussion 73 Conclusion and Recommendations..80 Summary...81 References 83 iv

6 List of Tables No. Table Page Table (1) Cytokines involved in chronic inflammation in FMF 7 Table (2) Clinical manifestations of familial Mediterranean fever 8 Table (3) Criteria for Diagnosis of Familial Mediterranean fever 14 Table (4) Tel-Hashimor criteria for the diagnosis of FMF 15 Table (5) Most frequent mutations observed in patients with FMF 22 Table (6) Non-classical findings at presentation in UC patients, which 33 do not exclude the diagnosis of UC Table (7) Factors contributing to growth impairment in children with 35 Crohn disease Table (8) Differences in clinical picture between UC and Crohn 37 disease Table (9) Tests currently utilized in the diagnosis of IBD 40 Table (10) Endoscopic and histological differences between UC and 43 Crohn s disease Table (11) Potential indications for capsule endoscopy in IBD 44 Table (12) Subclassification of Crohn disease, the Montreal 45 Classification Table (13) Demographic and Clinical Characteristics of IBD Patients 59 Table (14) Value of acute phase reactants (ESR&CRP) at time of 64 presentation Table (15) Different medications used for IBD patients 65 Table (16) Percentage of patients carrying MEFV gene mutations 65 among IBD patients Table (17) Distribution of IBD patients with positive mutations 66 Table (18) Types of gene mutations in IBD patients and controls 67 Table (19) Different MEFV gene mutations in IBD patients with extraintestinal 68 gene mutations, considering homozygous and compound heterozygous mutation Table (20) Comparison between genetic mutation test results between 69 IBD patients and control cases Table (21) Demographic and laboratory features ofpatients with MEFV 70 gene mutation andpatients without MEFV gene mutation Table (22) Studies discussing the prevalence and modifying effect of MEFV gene mutations on different types of IBD patient 75 v

7 List of Figures No. Figure Page Figure (1) Pyrin competitively binds with ASC via PYD, preventing 6 its binding to caspase-1 and the formation of the inflammasome, and also binds caspase-1 via B30.2 domain Figure (2) Pyrin and MEFV gene 21 Figure (3) Changing distribution of IBD 31 Figure (4) Barium meal follow-through demonstrates a long segment 42 of narrowed, ulcerated and nodular appearing ileum giving the characteristic cobblestone appearance Figure (5) Aphthous ulcers in CD 43 Figure (6) Ileo-cectectomy specimen characterized by a stricture in the area of the ileocecal valve 44 Figure (7) Potential implications of colchicine therapy in 54 atherogensis in FMF Figure (8) Colonoscopic ileal biopsy showing exudation of 61 neutrophils and mononuclear cells and wide expansion of the lymphoid components suggestive of active erosive ileitis of Crohn s disease Figure (9) Colonoscopic ileal biopsy showing wide surface erosions, 61 focally disorganized villi, wide exudation of neutrophils and MNCs and prominent hyperplasia of lymphoid follicle Figure (10) Colonoscopic biopsy from right colonic polyps showing 62 wide crypt distortion with cystic and branching forms, associated with cryptitis and crypt abscesses Figure (11) Different clinical manifestations of IBD patients at 63 presentation and during the course of illness (%) Figure (12) Percentage of occurrence of different extra-intestinal 63 manifestations Figure (13) Percentage of patients carrying MEFV gene mutations 66 among IBD patients Figure (14) Percentage of patients carrying different MEFV gene 67 mutations Figure (15) Percentage of MEFV gene mutations in the control cases 70 vi

8 Abbreviations 6-MP 6-Mercaptopurine 6-TG 6-thioguanin ABCB1 ATP binding cassette subfamily B member 1 AIH auto-immune hepatitis Anti-CBir Antibodies to CBir1 flagellin ATI antibodies to infliximab ASCA Anti-Saccharomices cerevisiae antibody ASC apoptosis-associated speclike-like protein AIDs auto-inflammatory diseases (AIDs) CARD caspase-recruitment domain CD Crohn disease CINCA Chronic infantile neurological, cutaneous, articular syndrome CRC Colo-rectal carcinoma CRP C-reactive protein DLG5 Drosophila Discs Large Homolog 5 gene EEN exclusive enteral feeding EIM extra-intestinal manifestations ELE erysipelas-like erythema EN enteral nutrition ENA78 Epithelial cell-derived neutrophil activator-78 ESR erythrocyte sedimentation rate ESPGHAN European Society of Pediatric Gastroenterology Hepatology and Nutrition FMF Familial Mediterranean Fever FCAS Familial cold autoimmune syndrome FUO Fever of unknown origin GM-CSF granulocyte monocyte colony stimulating factor HSP Henoch Schoenlin purpura IBD inflammatory bowel disease IC indeterminate colitis IDDM Insulin-dependent diabetes mellitus IFN-α interferon alpha vii

9 IGF-1 Insulin-like growth factor 1 IgM immunoglobulin M IL-1b JIA LRR LTB4 MAGUK MAPK MCTD MDR1 MEFV MIF MNCs NACHT NADPH NF-κB NK NSAID OCTN PAN PFAPA PN PYD RA ROS SAA1 sicam SLE interleukin 1b juvenile idiopathic arthritis leucine-rich repeat domain leukotriene B4 membrane-associated guanylate kinase mitogen activated protein kinase Mixed connective tissue disease The multi-drug resistance gene MEditerranean FeVer gene macrophage migration inhibiting factor mononuclear cells Neuronal apoptosis inhibitory protein CIITA, HET-E, and TP1 Nicotinamide adenine dinucleotide phosphate nuclear factor-kappa beta natural killer cells non-steroidal anti-inflammatory drugs organic cation transporter proteins Polyarteritis nodosa Periodic fever, aphthous stomatitis, pharyngitis and adenopathy syndrome parenteral nutrition pyrin domain rheumatoid arthritis Reactive oxygen species type 1 serum amyloid A protein soluble intracellular adhesion molecule systemic lupus erythematosus TNF Tumour necrosis factor TPMT Thiopurine methyltransferase enzyme TRAPS TNF receptor-associated periodic syndrome TRPM2 Transient receptor potential cation channel, subfamily M, member 2 UC Ulcerative colitis viii

10 Introduction Inflammatory bowel disease (IBD) presenting with diarrhea, abdominal pain, and rectal bleeding is the idiopathic inflammation of the gastrointestinal tract (Ferguson et al., 2008). Ulcerative colitis (UC) and Crohn s disease (CD) are the most common forms of IBD. Several susceptibility gene loci for IBD have been identified, termed IBD1 7, by conducting genome-wide scan analyses (Weersma et al., 2007). Familial Mediterranean fever (FMF) is the most common disease in the periodic inflammatory clinical syndromes. FMF is an autosomal recessive disease not only affecting ethnic groups living around the Mediterranean basin, but also reported throughout the world s populations. It is characterized by acute episodes of fever, polyserositis (peritonitis, arthritis and pleuritis), myalgia and erysipelas-like skin lesions (Livneh et al., 1997). Mediterranean fever gene (MEFV), responsible for FMF, is located at the short arm of chromosome 16 (16p13.3) and encodes pyrin (marenostrin), which plays a role in the regulation of neutrophil activity. Importantly, MEFV gene mutations were found responsible for subclinical inflammation in apparently healthy subjects and were associated with myocardial infarction in the general population, severe course of inflammation with arthritis in inflammatory bowel disease, vascular involvement in Behçet s disease (Giaglis et al.,2006). IBD and FMF share common clinical and biological features. Abdominal inflammation with infiltration by neutrophils at the site of injury, periodicity and relapse of clinical features, as well as the involvement of joints are some examples (Podolsky, 2002). Data suggests that abnormal regulation of apoptosis along with the involvement of IL-1β and NF-κB pathway could be implicated in both IBD and 1

11 FMF. Moreover, the epidemiologic data in non-askhenazi Jews indicates that IBD is more common and severe in patients with FMF. These data suggest that the genes responsible for FMF (MEFV) could have a potential modifying effect on the IBD (Karban et al.,2005). 2

12 Aim of the work The aim of this study is the detection of the prevalence of MEFV gene mutations in 42 unrelated patients having inflammatory bowel disease (Ulcerative colitis, Crohn disease and indeterminate colitis) and following up at the Tropical Pediatric Gastroenterology Clinic, Abo El-Reesh hospital, from 2004 to 2011, and in a control group of 33 healthy individuals with no family history or clinical manifestations suggestive of FMF or IBD, and the investigation of the effect of these mutations, if present, on the clinical picture of IBD. 3

13 Definition Chapter One Familial Mediterranean Fever The term auto-inflammatory disease or periodic fever syndrome has been proposed to describe a group of disorders characterized by attacks of unprovoked inflammation without significant levels of either autoantibodies or autoreactive T-cells. The study of autoinflammatory disease has progressed from clinical characterization to genetic analysis and to definition of the functional defects (Padeh et al.,2005). FMF is the most frequent periodic fever syndrome and autosomal recessive disease not only affecting ethnic groups living around the Mediterranean basin, but also reported throughout the world s populations. It is characterized by recurrent inflammatory febrile attacks, abdominal, chest or joint pain, myalgia and erysipelas-like skin lesions (Guz et al.,2009) History FMF was first described in 1908 by Janeway and Mosenthal in a 6- year-old Jewish girl presenting with abdominal pain and recurrent fever (Janeway & Mosenthal,1908). In 1945 Siegal, a Jewish allergologist, described it as benign paroxystic peritonitis, his own clinical picture was similar to features presented by other five Jewish patients: they all had cutaneous signs, recurrent peritonitis and periodic fever attacks (Siegal, 1964). In 1950, two French researchers, Mamou and Cattan, studied the familiar pattern of this disease in Sephardic Jews (Mamou &cattan,1952). In the same period, Reimann et al(1954) reports this disease has been described as benign paroxystic polyserositis, Armenian s disease, Siegal-Cattan-Mamou s syndrome and Reimann s periodic disease. In 1958,Heller defined the overall 4

14 FMF clinical picture and studied its autosomal recessive inheritance with the associated nephropathy deriving from amyloidosis: he also created the name FMF ( Heller et al.,1958).. This is the most common auto-inflammatory disease and the first one with the causing gene identified in 1997 (Babior & Matzner,1997). Racial distribution It has been estimated that about 170,000 people worldwide are affected with FMF, though a restricted ethnic distribution has been observed in people living around the Mediterranean basin and in the ancient Mesopotamia: Armenians, non-ashkenazi Jews (above all Sephardic Jews), Turks and Arabs (mostly North-Africans such as Moroccons) (Ozen, 2003). However because of many migrations during the past centuries the gene causing FMF has also been spread in Western Europe and patients have been reported in Brazil, China, Australia, New Zealand and Japan (Kostantopoulos et al., 2004). Carrier frequency is very different among the same populations. It is around 1:3 in Armenians and fluctuates in a range of 1:5-1:16 in Sephardic Jews who immigrated to the Iberian Peninsula. Males and females are equally affected, even though there is a slightly higher prevalence in males. A selective biological advantage for carriers of mutations in the FMF gene has been suggested, similarly to the advantage of sickle cell trait against malaria (Gershoni-Baruch et al., 2001). Unfortunately, the incidence of individuals heterozygous for MEFV mutations is to date unknown in Egypt. In Turkey, for example, the estimated prevalence of FMF is 1/1,000 and 20% of the individuals carry MEFV mutations (Yalcinkaya et al., 2007). The prevalence of FMF is 1 per 250 to 1 per 1000 in non-ashkenazi Jews. Non-Ashkenazi Jews originated in North Africa and emigrated to Israel and France after World War II (Cattan et al., 2000). 5

15 Etiology and Pathogenesis In 1997, two groups independently traced the genetic background of FMF to a gene on the short arm of chromosome 16, dubbed the MEditteranean FeVer (MEFV) gene (French FMF Consortium, 1997). The MEFV gene encodes for a protein known as pyrin or marenostrin. Pyrin is expressed as a cytoplasmic protein in mature monocytes in association with microtubules, but is predominantly found in the nucleus in granulocytes, dendritic cells, and synovial fibroblasts. The expression of pyrin is induced by inflammatory mediators such as interferon-α and tumour necrosis factor (TNF). The pyrin domain is shared by many proteins involved in apoptosis and inflammation and is a member of the death-domain superfamily. Pyrin binds specifically to other proteins that contain a pyrin domain, as in figure (1), which include the adaptor protein apoptosis-associated speclike-like protein with a CARD (ASC) (Centol et al., 2000). Figure (1) Pyrin competitively binds with ASC via PYD, preventing its binding to caspase-1 and the formation of the inflammasome, and also binds caspase-1 via B30.2 domain (IL-1b: interleukin 1b; PYD: pyrin domain; NACHT: domain 6

16 present in neuronal apoptosis inhibitory protein CIITA, HET-E, and TP1; LRR: leucine-rich repeat domain; ASC: apoptosis-associated speck-like protein; CARD:caspase-recruitment domain) (Guz et al.,2009). The pro-inflammatory cytokine interleukin (IL)-1beta is central in the pathogenesis of FMF. This cytokine is expressed as an active precursor, which is cleaved by caspase-1 to yield the active IL-1β. The sequestration hypothesis holds that pyrin has an inhibitory effect on caspase-1-mediated activation of IL-1beta. In summary, pyrin seems to modulate the activity of the apoptotic proteins and signal transduction pathway, playing a crucial role in the inflammatory pathways of the innate immune system (Chae et al., 2006). Other cytokines involved in chronic inflammation in FMF are included in table (1) (Ben-Zvi & Levni,2011). Table (1): Cytokines involved in chronic inflammation in FMF (Ben-Zvi & Levni,2011). Cytokine Function Relationship of cytokine level to FMF disease status IL-6 Promotes release of Serum levels increase in acute-phase proteins attacks, decrease after IL-8 Chemoattractant of colchicines therapy IL-12 IL-18 TNF neutrophis Activates NK cells & phagocytic activity Induces IFN-γ, enhances TH1 immune response Initiation & perpetuation of inflammation IFN-γ Activates NKcells,dendritic cells and neutrophils. MIF Upregulates macrophage function sicam-1 Mediates leucocyte migration LTB4 Chemoattractant of neutrophis 7 Serum levels increase in attacks and remission (levels similar in each phase) Serum levels increase in remission, decrease after colchicines therapy Serum levels increase in attacks and remission (highest in attack) Serum levels increase in remission Serum levels increase in attacks and remission (levels similar in each phase) Urinary levels increase in attacks and remission(similar

17 levels) IL-1β Initiation & perpetuation of inflammation No change in serum level IL-10 Inhibits TNF, IL-1 &6 Serum levels increase in remission (similar levels) (Abbreviations:LTB4: leukotriene B4; MIF: macrophage migration inhibiting factor; sicam: soluble intracellular adhesion molecule; TNF: tumour-necrosis factor). Clinical manifestations FMF is characterized by short recurrent self-resolving attacks of fever, abdominal, thoracic or joint pain and systemic inflammation with inter-critical period of apparent wellness. Clinical picture and disease severity may vary among different ethnic groups (Livneh et al.,1996). Onset of symptoms begins in about 50% of cases during the first decade and only in 5% after the third decade. In few cases symptoms might appear during the first months of life. Features of a typical attack, as seen in Table (2), comprise fever and possibility of serositis or arthritis, lasting from 1 to 3 days, resolving spontaneously. Attacks do not have a regular periodism in consequence of a frequency varying from once a week to once every three-four months or more; intervals between attacks are different both among patients and in the same patient too (Tamir et al.,1999) Table (2): Clinical manifestations of familial Mediterranean fever (Rigante et al.,2006) Fever is present in 96% of inflammatory episodes: body temperature can reach very high values, even over 40 C; it usually appears suddenly and lasts from 12 to 72 hours and is preceded by shivers in about 20-30% of 8

18 patients. Rarely fever is the only FMF manifestation without other signs of localized inflammation. Between attacks, patients are perfectly well (Rigante et al.,2006). Abdominal pain is present in about 90% patients and is the first manifestation in 50% of them. It is due to peritoneal inflammation with a clinical outline simulating an acute abdominal pathology, like positive Blumberg s sign. Sometimes there are reduced peristalsis, air-fluid levels on abdominal X-ray film and small amounts of ascites upon ultrasound investigation. Patients might undergo unnecessary surgery in 30-40% of cases. Some authors recommend a laparoscopic appendectomy because the exclusion of a true acute appendicitis is not easy at a merely clinical level. Constipation may be present in some patients, but diarrhea is more common in children. Recurrent peritonitis can cause adhesions compromising female fertility (Ben- Chetrit & Levi, 2003). Serositis: Mono or bilateral pleural effusion can be demonstrated in 50-60% patients and is characterized by quick resolution, whilst recurrent pericarditis can be observed in a small percentage of FMF patients (1-2.5%) during febrile attacks (Kees et al.,1997). Intense scrotal pain simulating a testicular torsion is frequent in children due to inflammation of tunica vaginalis. It presents as unilateral, red, tender swelling of the scrotum associated with pain and fever and lasting from few hours to four days (Eshel et al.,1994). Joints: The third most frequent FMF clinical manifestation is articular involvement which might present as transient arthralgia, monoarthritis or oligoarthritis: short lasting attacks begin without prodromes, involving large joints in lower limbs (hip, knee, and ankle) or upper limbs (wrist) and suddenly disappearing in about hours with no sequelae. The affected joint is hot, tender and red. Rarely it is possible to observe prolonged arthritis lasting for more than one week or even destructive chronic arthritis in 2-5% of cases (Dudkiewicz et al., 2001). 9

19 A pathognomonic skin manifestation for FMF diagnosis is erysipelaslike erythema (ELE) which is often localized over legs or feet, sometimes accompanied with arthritis. Tender, hot, swollen, sharply bordered red lesions appear on the skin of lower extremities. The dermatitis is often accompanied by abrupt elevation of body temperature and lasts hours. Histological examination of the lesions reveals edema of the superficial dermis and sparse perivascular infilterate without vasculitis. In all cases, direct immunofluorescence shows deposits of C3 in the wall of the small vessels of the superficial vascular plexus (Brazilai et al., 2000) Muscular manifestations can present with febrile myalgia having three different patterns: spontaneous, effort-induced and prolonged with an overall duration of some weeks. In 1994, Langevitz et al first described FMF patients with a syndrome of protracted febrile myalgia, which is characterized by severe debilitating myalgia, prolonged fever, and abdominal pain, a higher sedimentation rate than commonly found in FMF (around 100mm in the first hour), leukocytosis, and hyperglobulinemia. In patients treated with NSAIDs, the attack lasts 6 to 8 weeks, but subsided promptly after prednisone is added (Langevitz et al., 1994). Other non-specific FMF clinical signs are aseptic meningitis, cryptogenic liver cirrhosis, splenomegaly and oral aphthae. Splenomegaly is seen in around 30% of patients and is usually not associated with amyloidosis. Recurrent oral aphthae are often reported, unassociated with attacks. (Majeed et al., 2002) Moreover some non-granulomatous Th-1 polarized vasculitis have been associated with FMF such as Henoch-Schönlein purpura (in 2.6-5% cases), nodous polyarteritis (0.8-1% cases) and Behçet disease (0.5% cases) (Bakkaloglu et al.,2004) Inflammatory bowel disease is 8 to 14 times more frequent in FMF patients and its clinical course appears usually more aggressive (Lange-Sperandio et al.,2004) 10

20 FMF disease phenotypes can be divided into three groups; phenotype I: clinically overt disease, manifested with episodes of fever and arthritis, or peritonitis or pleuritis or erysipelas-like erythema, or a combination of these and with amyloid A (AA) amyloidosis. Genetic diagnosis is not required. Phenotype II: the only manifestation is AA amyloidosis. Diagnosis is based on family history of phenotype I disease, or presence of MEFV mutations. Phenotype III: genetic positive ( 2 mutations), but clinically silent disease (Ben-Zvi & Lvni, 2011). Complications Amyloidosis occurs frequently in untreated patients with FMF. It is the most severe manifestation, resulting from the deposition of amyloid, which is presumed to be a cleavage product of serum amyloid A, an acute-phase reactant produced by the liver. It usually presents in FMF patients with persistent heavy proteinuria leading to nephrotic syndrome. Amyloid nephropathy has been reported in a child as young as 5 years of age (Oner et al., 2003). Moreover, 2 genes, namely SAA1 (type 1 serum amyloid A protein) and MICA (Major Histocompatibility Complex (MHC) class-i-chain related gene A) have been investigated in FMF patients and were found to have an effect on amyloidosis and on the course of the disease respectively. The genotype alpha/alpha of the SAA1 gene was associated with a sevenfold increase in the incidence of renal amyloidosis, especially in patients homozygous for M694V, whereas the MICA alleles A4 and A9 were associated with a diminished attacks frequency and an earlier age of onset, respectively, notably in patients homozygous for M694V (Gershoni-Baruch et al.,2003). The prevalence of amyloidosis among FMF patients is considered to be independent of the frequency, duration and intensity of FMF flares. Other organs involved by amyloid deposition are gut, spleen, liver and heart. The pathogenic role of environmental factors in amyloidosis occurrence such as the 11