Poor prognosis in familial acute myeloid leukaemia with combined biallelic CEBPA mutations and downstream events affecting the ATM, FLT3 and CDX2 genesписьмо
Аннотация: CEBPA is implicated in the delicate balance between myeloid cell proliferation and differentiation. Loss of CEBPA gene function, through biallelic point mutation, has been identified in up to 14% of patients with sporadic acute myeloid leukaemia (AML), and germline mutations have been reported in several families with autosomal dominant inheritance of AML and eosinophilia (Pabst et al, 2001; Gombart et al, 2002; Smith et al, 2004; Sellick et al, 2005). In both sporadic and familial AML, CEBPA point mutations are commonly biallelic (one germline and one acquired in the familial cases), consisting of a dominant negative N-terminal and a loss of DNA-binding C-terminal leucine zipper mutation (Preudhomme et al, 2002; Barjesteh van Waalwijk van Doorn-Khosrovani et al, 2003). Interestingly, in families carrying CEBPA mutations, the germline mutation is always N-terminal, whilst the acquired mutation is C-terminal, suggesting a role in leukaemia predisposition for the N-terminal mutation only. In 1978 a large familial aggregation of AML, with 14 diagnosed individuals over four generations, was reported (Gunz et al, 1978). The family is of Irish and English extraction, with the majority of documented members living in and around Sydney, Australia at the time of diagnosis. Contact with the family was lost in 1980, however at that time an additional three leukaemia cases had been diagnosed. Figure 1A shows the affected arm of the pedigree with numbering as in the original publication. Anticipation is notable: while diagnosis occurred at 66 years for individual II-13, the mean age at diagnosis decreased to 20 and 11 years in generations III and IV, respectively. Of the 17 diagnosed leukaemia cases, seven occurred in children under the age of 10 years, including one aged just three months (V-63a) whose mother (IV-89) had AML through the pregnancy. In addition, only five of the 17 individuals with leukaemia were known to be alive in 1980, three of whom were diagnosed shortly before contact was lost. The majority of the patients documented in the original article died within weeks to months of diagnosis, despite various treatment regimens (1968 onwards) including prednisone, cyclophosphamide and vincristine (Gunz et al, 1978). This suggests that the leukaemia predisposition in this family is both incredibly precocious and highly aggressive. Pedigree, germline and somatic CEBPA analysis. (A) Analysed branch of published pedigree, numbers as published (Gunz et al, 1978). Arrow depicts the proband; full symbols depict individuals diagnosed with leukaemia; diagonal bars depict deceased individuals. (B) Germline mutation in CEBPA in III-45. (C) Somatic mutation on the wild-type allele of CEBPA in III-45. DNA sequence variants are given relative to NM_004364, and protein variants relative to Swiss-Prot P49715. Due to the high morbidity of disease, and the length of time that has lapsed since contact was lost, only one somatic DNA sample was available for analysis; individual III-45. This individual presented at age 50 years with a 2-month history of worsening health with unexplained fevers and colds. Peripheral blood showed 406 × 109/l leucocytes with 99% blasts and a platelet count of 74 × 109/l, and cytogenetic analysis del(11q23) in all analysable metaphases. He was given a diagnosis of AML and treated with leucapheresis followed by chemotherapy including daunorubicin and cytosine arabinoside. One year after diagnosis he died from a relapse. Additional sample information is provided in Appendix S1. DNA sequencing of CEBPA revealed a single heterozygous base pair deletion (c.68delC) (Fig 1B). The affected poly-C string is a mutational hot spot and this identical mutation has been reported in both familial and sporadic AML cases (Barjesteh van Waalwijk van Doorn-Khosrovani et al, 2003; Smith et al, 2004). It leads to a p.P22fsX158 frameshift, causing truncation of the wild type 42 kDa protein and increased production of a 30 kDa dominant negative isoform (Pabst et al, 2001; Gombart et al, 2002). We also identified a probable acquired three base pair duplication in the C-terminal end of CEBPA (c.937_939dupAAG) (Fig 1C) in this same individual. The mutation was found in 25% of cloned alleles, indicative of it being an acquired mutation and only present in a proportion of peripheral blood cells. Long-range polymerase chain reaction demonstrated that the wild type CEBPA allele was affected. This mutation has previously been described in patients with sporadic AML and leads to an in-frame p.K313dup that is predicted to disrupt the leucine zipper domain (bZIP) (Gombart et al, 2002). (Method and primer sequence information is provided in Appendix S1). As discussed in the original publication (Gunz et al, 1978), the penetrance of the AML predisposition in this family differs across the pedigree, with the branch studied here having the highest penetrance. In addition, the high morbidity and aggressive nature of the disease in this family is in contrast to the good prognosis normally associated with sporadic and familial CEBPA mutations (Preudhomme et al, 2002; Smith et al, 2004; Sellick et al, 2005). Environmental as well as germline and somatic genetic events in this current family might be contributing to the high penetrance, early onset and mortality observed. To further investigate a possible additional genetic contribution, we performed a genomic single nucleotide polymorphism chip copy number analysis. Excluding copy number aberrations that were present in normal unrelated DNA samples of European, Asian or American ancestry (Redon et al, 2006), and considered to be polymorphic, we identified two putatively pathogenic acquired deletions in individual III-45 (see Appendix S2). The first is a 357 Kb deletion on chromosome 13q12 affecting the FLT3 tyrosine kinase gene, as well as the ParaHox A cluster of homeobox genes (GSX1, PDX1 and CDX2) (Fig 2A). FLT3 is targeted by activating heterozygous mutations in about 30% of AML, leading to constitutive activation of the receptor and growth-factor independent cell growth and perturbed differentiation (Yamamoto et al, 2001). The ParaHox A gene cluster member, CDX2, is overexpressed in 90% of patients with AML (Scholl et al, 2007). The deletion of both CDX2 and FLT3 in III-45 is therefore in contrast to the overexpression (CDX2 and FLT3) and constitutive activation (FLT3) normally observed for these genes in AML. This may suggest an alternative role for these genes in the pathogenesis of this disease. (A) Copy number analysis identified a deletion on chromosome 13q12 that targets the homeobox genes GSX1, PDX1 and CDX2 and the FLT3 gene. (B) Copy number analysis confirmed a large deletion on chromosome 11q (breakpoints mapped to chr.11:82,252,843-114,704,989) that contains approximately 180 known genes, including the ATM. Horizontal axis represents distance along chromosome and vertical axis represents the log ratio value for each SNP. The second deletion identified is 32·4 Mb in size on chromosome 11q14.1-23.2, (Fig 2B), containing the Ataxia Telangiectasia Mutated gene (ATM) among others (Appendix S3). This gene is frequently targeted in lymphoid neoplasms, with biallelic loss of function often obtained through deletion of one allele and point mutation of the other. Sequencing of the remaining alleles of ATM and FLT3, as well as several genes with known roles in haematopoietic stem cell biology and/or in leukaemogenesis, including RUNX1, KIT, JAK2 and the homeobox genes MEIS1 and MLL, did not identify any additional mutations (for non-pathogenic sequence variants and exons analysed, see Appendix S1). Studies that utilize genome wide approaches, such as copy number analysis or high throughput sequencing analyses of candidate genes, have the potential to provide insight into the genomic instability occurring during myeloid leukaemogenesis. Our findings confirmed mutated CEBPA as a causative factor for leukaemia predisposition, however we demonstrated for the first time that mutation of this gene is not necessarily associated with a good prognosis in familial AML. We also identified deletion of ATM, FLT3 and CDX2 genes as possible disease modifiers in familial myeloid leukaemia. The authors would like to thank the late Dr Frederick W Gunz of the Kanematsu Institute in Sydney for his initial description of the family, and Cheryl Paul from his laboratory for providing the sample analysed in this paper. Affymetrix array analyses were performed by the Australian Genome Research Facility, which was established through the Commonwealth-funded Major National Research Facilities program. This work was supported by grants from the National Health and Medical Research Council of Australia (program grants 257501 (HSS) and 219176 (HSS), fellowships 171601 (HSS) and 461204 (HSS), and a Dora Lush Postgraduate Award (CLC) Leukaemia Foundation of Australia (Grant in Aid to HSS, Postdoctoral fellowship to CLC), the Cancer Council of South Australia (HSS), and MedVet Pty Ltd (HSS). CC, EJW, CNH, RE and HSS designed the research; CC, EJW, HB and CNH performed the experimentation, data analysis and interpretation; TPS, MSH, PCV and GY contributed to the research and the experimental design; CC, RE and HSS wrote the paper; and all authors checked the final version of the manuscript. The authors declare no competing financial interests. Appendix S1. Sample information and sequencing analysis. Appendix S2. Copy number analysis. Appendix S3. Gene involved in 11q deletion. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Год издания: 2010
Авторы: Catherine Carmichael, Ella Wilkins, Henrik Bengtsson, Marshall S. Horwitz, Terence P. Speed, Paul Vincent, Graham Young, Christopher N Hahn, Robert Escher, Hamish S. Scott
Издательство: Wiley
Источник: British Journal of Haematology
Ключевые слова: Acute Myeloid Leukemia Research, Cancer-related gene regulation, Genomics and Chromatin Dynamics
Другие ссылки: British Journal of Haematology (HTML)
PubMed (HTML)
PubMed (HTML)
Открытый доступ: bronze
Том: 150
Выпуск: 3
Страницы: 382–385