Page 68 - Haematologica3

V.B. Pastor et al. Introduction
Germline predisposition has been recognized as an underlying cause for the development of myelodysplastic syndromes (MDS) in children. Recently, it has also been gaining importance in the etiology of adult myeloid neo- plasia, particularly in cases with a positive family history. Various genes are known to be associated with heritable forms of MDS and acute myeloid leukemia,1,2 e.g., GATA2,3 CEBPA,4 RUNX1,5 ANKRD26,6 ETV67 and DDX41,8 in addition to inherited bone marrow failure syn- dromes. Germline mutations in DDX41 can result in adult-onset myeloid neoplasia, while aberrations in RUNX1 and GATA2 are associated with myeloid neoplasia in younger individuals. We recently reported that GATA2 deficiency is the most common genetic cause of primary childhood MDS, accounting for 15% of all cases of advanced MDS, and 37% of MDS with monosomy 7 (MDS/-7).9 However, in the majority of cases of pediatric MDS, and also in a considerable number of cases of famil- ial myeloid neoplasia, the presumed germline cause has not yet been discovered.10,11
Monosomy 7 is the most frequent cytogenetic lesion in children with MDS and, unlike in adults, it often occurs as the sole cytogenetic abnormality.12 Due to the rapid and progressive course of the disease, it is considered an urgent indication for hematopoietic stem cell transplanta- tion.13 However, transient monosomy 7 has occasionally been documented in childhood MDS.14-16 Considering that complete (-7) and partial [del(7q)] deletion of chromosome 7 are common aberrations in MDS, extensive efforts have been undertaken to discern causative tumor suppressor genes located on chromosome 7. Asou and colleagues identified SAMD9 (Sterile Alpha Motif Domain-contain- ing 9), its paralogue SAMD9L (SAMD9-like), and Miki/HEPACAM2 as commonly deleted genes within a 7q21 cluster in patients with myeloid neoplasia.17 Notably Samd9l-haploinsufficient mice were shown to develop myeloid malignancies characterized by different cytope- nias and mimicking human disease with monosomy 7.18
In line with these findings, germline heterozygous gain- of-function SAMD9L mutations p.H880Q, p.I891T, p.R986C, and p.C1196S were recently discovered in four pedigrees with variable degrees of neurological symptoms (ataxia, balance impairment, nystagmus, hyperreflexia, dysmetria, dysarthria) and hematologic abnormalities (single to tri-lineage cytopenias, MDS/-7). For most carri- ers, the clinical presentation was compatible with the diagnosis of ataxia-pancytopenia syndrome.19,20 Similarly, in two recent studies, we and others reported de novo gain- of-function mutations in SAMD9 in a total of 18 patients with MIRAGE syndrome (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital pheno- types, and enteropathy), of whom four notably also devel- oped MDS/-7.21,22 However, not all patients develop the full MIRAGE disease spectrum, as documented in one family with SAMD9-related MDS.23 The SAMD9 and SAMD9L genes share 62% sequence identity and apart from their putative role as myeloid tumor suppressors, their general function and their specific effect pertaining to hematopoiesis are not well-understood.18
In this study we aimed to identify the genetic cause in pedigrees with non-syndromic familial MDS. We discov- ered constitutional SAMD9L mutations associated with non-random patterns of clonal escape leading to loss of
the mutant allele. We further demonstrate in two cases that SAMD9L–related disease can be associated with tran- sient -7, occurring as a one-time clonal event followed by somatic correction of hematopoiesis achieved by UPD7q with double wild-type SAMD9L.
Methods
Patients
The diagnosis of MDS was established according to World Health Organization criteria.24,25 Patients 1 (P1), 2 (P2), 5 (P5), and 7 (P7) were enrolled in prospective study 98 of the European Working Group of MDS in Childhood (EWOG-MDS) (www.clin- icaltrials.gov; #NCT00047268). Patient 6 (P6) was the father of P5 (family III). Family II (P3 and P4) was referred for evaluation of familial MDS from Phoenix Children´s Hospital, USA. The study had been approved by an institutional ethics committee (University of Freiburg, CPMP/ICH/135/95 and 430/16). Written informed consent to participation had been obtained from patients and parents.
Genomic studies and bioinformatics
Exploratory whole exome sequencing was performed in bone marrow granulocytes in P1 and P2, as outlined in the Online Supplementary Material. Targeted deep sequencing for SAMD9/SAMD9L and genes related to MDS/inherited bone mar- row failure syndromes was performed in other patients, except for P3 and P6 due to unavailability of material. All relevant variants were validated using Sanger sequencing. For germline confirma- tion, DNA was extracted from skin fibroblasts and/or hair follicles, and targets were amplified and sequenced as previously described.9 The degree of deleteriousness was calculated using the combined annotation-dependent depletion scoring system (CADD-score).26 The variants with CADD-scores higher than 20 were further evaluated for their role in hematologic disease or can- cer, thereby focusing on the top 1% most deleterious variants in the human genome. In addition, pathogenicity calculations were performed using standard prediction tools. The evolutionary con- servation across species and the physicochemical difference between amino acids were estimated by PhyloP, PhastCons and the Grantham score, respectively.27 Mutant clonal size was inferred from allelic frequencies and the total number of next-gen- eration sequencing reads normalized to the ploidy level. Further details are provided in the Online Supplementary Methods.
Evaluation of variant allelic configuration
Genomic DNA of P1 collected at the time of progress to chronic myelomonocytic leukemia (CMML) was amplified to obtain a 1333 bp region encompassing both SAMD9L mutations: p.V1512M (germline) and p.R1188X (acquired). Polymerase chain reaction products were TA-cloned and sequenced as previously reported.28 Sequences of 170 colonies were evaluated for the pres- ence of SAMD9L mutations.
Cellular and functional studies
Metaphase karyotyping and interphase fluorescence in situ hybridization were performed using bone marrow specimens according to standard procedures.12 Human colony-forming cell assays were performed in P1 (at CMML disease stage) and in P7 (at diagnosis) as previously described.29 Furthermore, to evaluate the effect of the patient-derived SAMD9L p.V1512M and p.R986C mutations on cellular proliferation, 293FT cells were dye-labeled and consequently transfected with wild-type or mutant teal fluo- rescent protein (TFP)-SAMD9L as previously described.20 The
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haematologica | 2018; 103(3)