PhD PROGRAM IN TRASLATIONAL AND MOLECULAR MEDICINE DIMET UNIVERSITY OF MILANO-BICOCCA SCHOOL OF MEDICINE AND FACULTY OF SCIENCE Improving the Understanding of Shwachman-Diamond Syndrome Coordinator: Prof. Andrea Biondi Tutor: Prof. Andrea Biondi Co-Tutor: Dr. Giovanna D’Amico Dr. Valentina Isabella André Matr. No. 725278 XXIV CYCLE ACADEMIC YEAR 2010-2011 2 "It is the time you have wasted for your rose that makes your rose so important" (Antoine De Saint-Exup´ery) 3 4 Table of Contents 1 Introduction 9 1.1 Shwachman-Diamond syndrome: general intro- duction . .9 1.2 Clinical presentation . 10 1.2.1 Hematological abnormalities . 10 1.2.2 Gastrointestinal features . 16 1.2.3 Skeletal abnormalities . 16 1.2.4 Additional features . 17 1.3 Molecular pathogenesis . 18 1.3.1 SBDS gene: structure, functions and mu- tations . 18 1.4 Clinical management . 25 1.4.1 Hematology . 25 1.4.2 Hematopoietic stem cell transplantation 26 1.4.3 Gastroenterology . 28 1.4.4 Skeletal . 28 1.5 Mesenchymal stem cells . 28 1.5.1 Role of MSCs in supporting Neutrophil survival . 30 5 1.5.2 Role of MSCs in hematological disorders 31 References of Chapter 1 . 43 2 Aims of the study 45 3 Characterization of mesenchymal stem cells in Shwachman-Diamond syndrome reveals an altered gene expression profile 47 3.1 Rationale . 48 3.2 Methods . 50 3.2.1 Patients . 50 3.2.2 Isolation and culture of mesenchymal stem cells . 51 3.2.3 Proliferation assay . 54 3.2.4 Neutrophils and MSCs co-culture . 54 3.2.5 Co-culture of hematopoietic stem cells with mesenchymal cells layer . 56 3.2.6 Western blot analysis . 56 3.2.7 Karyotype and FISH analysis of MSCs . 58 3.2.8 RNA preparation and gene expression pro- filing . 59 3.2.9 Statistical analysis . 60 3.3 Results . 61 3.3.1 Evaluation of MSC-typical features in cells expanded from BM of SDS patients . 62 3.3.2 Functional characterization of SDS-MSCs 67 3.3.3 Analysis of SBDS protein in SDS-MSCs 73 3.3.4 Molecular genetics of SDS-MSCs . 73 6 3.4 Discussion . 78 References of Chapter 3 . 86 4 SNP array analysis in Shwachman-Diamond syn- drome for unraveling disease evolution 87 4.1 Rationale . 88 4.2 Methods . 89 4.2.1 Patients . 89 4.2.2 DNA preparation, copy number and SNP analysis . 90 4.3 Results . 92 4.3.1 Cytogenetics and SBDS mutations . 92 4.3.2 Copy number and SNP analyses . 92 4.4 Discussion . 96 References of Chapter 4 . 101 5 Summary, conclusion and future directions 103 References of Chapter 5 . 107 7 8 Chapter 1 Introduction 1.1 Shwachman-Diamond syndrome: general introduction In 1964, Shwachman-Diamond Syndrome (SDS, OMIM 260400) was described as a disease in a cohort of patients with cystic fibrosis (CF). The syndrome was first characterized in Eng- land by Bodian in May of 1964[1] and then later the same year the disease was reported by Shwachman, Diamond and Oski[2]. Shwachman-Diamond syndrome is a rare autosomal recessive inherited disorder characterized by bone marrow dysfunction, exocrine pancreatic insufficiency and an increased risk of devel- oping myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). In almost all affected patients, the disorder manifests a broad range of additional clinical features including skeletal abnormalities, neutropenia (recurrent infections, some- times fatal, are common) and cognitive impairment. It can be 9 diagnosed in children of all ages or in adults and is extremely heterogeneous. Data show that SDS is the third leading in- herited bone marrow failure syndrome after Diamond-Blackfan anemia and Fanconi anemia[3] and the most common cause of pancreatic insufficiency in children next to cystic fibrosis. The syndrome has an estimated incidence of 1 in 76000 (based on the observation that it is approximately 1/20th as frequent as CF in North America[4]) with the heterozygote frequency of 1/138[5] and has no gender or ethnic predilection. In 2003, Boocock et al. showed that approximately 90% of patients have hypomor- phic mutations in the Shwachman Bodian Diamond Syndrome gene (SBDS)[6] located on chromosome 7. The exact function of the SBDS protein remains unclear; however, recent studies in yeast and patient bone marrow cells show that SBDS gene is involved in RNA metabolism and ribosome biogenesis[7][8][9]. SBDS is an essential gene in embryogenesis and it is also impli- cated in cell division and cellular stress response[10][11]. 1.2 Clinical presentation In addition to case reports, several large cohort studies have summarized the clinical features associated with Shwachman syndrome. 1.2.1 Hematological abnormalities Hematological abnormalities are present in all SDS patients. In particular, neutropenia is the most common hematological 10 deficiency. This defect is cyclical, persist throughout life and is the main cause of morbidity and mortality. Neutropenia, absolute neutrophil count (ANC)<1500 cells/µL, occurs in up to 95% of patients[12] and it is most frequently intermittent rather than persistent[13]. Hematological manifestations other than neutropenia include anemia, thrombocytopenia, raised fe- tal hemoglobin (HbF) levels and defects in the lymphoid lin- eage. Anemia is the second most common cytopenia in SDS, recorded in up to 80% of patients. Red blood cells are usu- ally normochromic and normocytic but can also be macrocytic. Fetal hemoglobin was increased in 80% of patients[14]. Many patients (24% to 60%) develop a mild thrombocytopenia, de- fined by a platelet count<150×109/L. Pancytopenia with pro- gression to aplastic anemia has also been reported[15]. The de- velopment of aplastic anemia in patients with SDS suggests that the hematopoietic defect may reside at the level of an early hematopoietic stem cell. It has been demonstrated that patients with SDS had significantly lower number of CD34+ cells on bone marrow aspirates. In addition, SDS CD34+ cells showed markedly impaired colony production potential when plated in methylcellulose for clonogenic assays. The ability of marrow stromal cells from SDS patients to support normal CD34+ was also diminished. In addition, patients also have a generalized marrow dysfunction with abnormal bone marrow stroma in terms of its ability to produce fat clusters and to support and maintain hematopoiesi[16]. The pivotal mechanism of bone marrow failure in SDS appears mediated by increased 11 apoptosis. This increased propensity for apoptosis is linked to high expression of the Fas antigen on marrow cells and to hy- peractivation of the Fas signaling pathway[17][18]. Infection and immune abnormalities Patients with SDS are particularly susceptible to recurrent vi- ral, bacterial and fungal infections. Upper and lower respiratory tract infections and otits media are also frequently reported. Overwhelming sepsis is a well-recognized fatal complication of this disorder, particularly early in life. Neutropenia of variable degrees probably contributes to the susceptibility to infections. An additional mechanism possibly involves an inherited defect of neutrophil mobility, migration, and chemotaxis. Defects in lymphocyte mediated immunity have also been described. Sev- eral defects in B and T lymphocytes and natural killer (NK) cells were demonstrated[19]. B cells defects are comprised of low IgG production, low percentage of circulating B lympho- cytes and decreased in vitro B cells proliferation. Also T and NK abnormality are comprised of low percentage of total cir- culating cells. Hematopoiesis deficiency is probably linked to defects in the bone marrow. Risk of myelodysplastic syndrome and acute myeloid leukemia evolution Similar to other marrow failure syndromes, patients with SDS have an increased risk for MDS and malignant transformation, in particular development of AML. The risk of leukemic and 12 dysplastic transformation increases with age varying from 14% to 30%[20][21]. Myelodysplastic syndromes are clonal hemato- logic disorders characterized clinically and morphologically by ineffective hematopoiesis. In general, myelodysplastic condi- tions are pre-leukemic disorders but not all cases of myelodys- plasia evolve to AML[22]. Only if the percentage of leukemic blasts in the bone marrow is over 20% does the patient have leukemia, and not MDS. There is an increased frequency of clonal cytogenetic abnormalities in SDS, but estimates of the incidence of cytogenetic abnormalities in SDS patients are dif- ficult to ascertain from a review of case reports. In a cohort study[23], six of 88 patients developed clonal cytogenetic abnor- malities. Five of 12 patients had clonal abnormalities in another study[13]. A third longitudinal prospective study of 14 SDS pa- tients with up to 5 years of follow-up found clonal marrow cy- togenetic abnormalities consisting of i(7)(q10) or del(20)(q11) in four of 14 patients[24]. Abnormalities of chromosome 7, such as monosomy 7 or isochromosome 7q, are common. Other chro- mosome 7 abnormalities included monosomy 7, combined i(7q) and monosomy 7, and deletion or translocations involving 7q and accounted for 33% of the reported cytogenetic abnormali- ties in these patients. Deletion of long arm of chromosome 20 was the next most common chromosomal abnormality reported in 16% of cases. Although clonal cytogenetic abnormalities are typical in MDS and AML associated with SDS, the clinical sig- nificance of many of these cytogenetic abnormalities arising in the absence of morphologic MDS is unclear. There is some evi- 13 dence that i(7)(q10), resulting in duplication of 7q, is associated with a lower risk of MDS/AML[25] when compared with alter- ation commonly associated with MDS and rapid transformation to leukemia like monosomy of 7, deletion of 7q, derivative of 7, deletion of 5q, trisomy of 8 or complex abnormalities of 5q and 7q[26]. Alter reviewed 510 reported SDS cases, and no patient carrying the i(7)(q10) had developed MDS/AML, whereas pa- tients with overt evolution into MDS/AML, other chromosome changes, including complex karyotypes, were observed[21].