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SAMD9L-related familial MDS
achieved a spontaneous remission as indicated by normal- ization of bone marrow morphology/cellularity and cyto- genetics (Figure 2, Table 1). A similar clinical picture was seen in P7 who was diagnosed with RCC with a -7 clone at the age of 2.1 years, and experienced rapid cytogenetic remission with normal marrow and blood counts until the last follow-up, 16 years after diagnosis (Table 1). Hematologic findings also normalized in P4, shortly after the initial manifestation; however, fluorescence in situ hybridization revealed chromosome 7 loss in bone mar- row, which gradually worsened and culminated, after 3.5 months, in the emergence of two independent clones with -7 and del(7q) (Table 1).
Due to high-risk cytogenetics and disease progression, five of the seven patients (P1, P3-6) underwent hematopoietic stem cell transplantation after myeloabla- tive conditioning. At last follow-up, six of the seven patients were alive, four after transplantation, and two without therapy.
Constitutional and acquired SAMD9L mutations Exploratory whole exome sequencing performed in family I identified two shared candidate variants in P1 and P2 evaluated as highly conserved and deleterious by in sil- ico prediction: SAMD9L (p.V1512M) and PTEN (p.Y188C) (Table 2, Online Supplementary Figure S1). Sequencing of DNA from hair follicles confirmed the constitutional nature of both novel mutations. The SAMD9L p.V1512M variant was inherited from the mother (Figure 1A) where- as PTEN p.Y188C was of paternal origin; both parents were asymptomatic and had normal complete blood counts at the time of testing. Finally, truncating acquired SAMD9L mutation p.R1188X (VAF 5.9%) was identified
in P1 in hematopoiesis (Table 2).
In pedigree II, targeted next-generation sequencing in P4
revealed SAMD9L p.R986H as the most plausible candi- date constitutional mutation predicted to be highly con- served and deleterious (Table 2, Figure 1A,B). This muta- tion was found in four individuals in ExAC (out of 120976
alleles). Additional missense variants in JAK3 p.A877V (ExAC: 11 individuals, 121372 alleles), and FANCM p.L57F (ExAC: 195 individuals, 121190 alleles) had lower and moderate pathogenicity scores, respectively (Table 2). Chromosomal breakage studies on P4 were negative thus arguing against a pathogenic role of the heterozygous FANCM variant. Germline analysis revealed SAMD9L and JAK3 variants in P3, P4, and their father, while the FANCM variant was transmitted from the mother only to P4. Both parents were asymptomatic.
In pedigree III, the SAMD9L p.R986C mutation was identified in P5 and the affected father, P6 (Figure 1A). This mutation has been reported in a family with ataxia- pancytopenia phenotype, with one of three carriers devel- oping MDS/-7 at the age of 18 months.20 The HLA-identi- cal brother of P5 was thoroughly evaluated as a potential bone marrow donor. He was clinically healthy and had a normal complete blood count, but he did not qualify as a donor because of hypocellular bone marrow with mild dysplastic features. He was also a carrier of the p.R986C mutation.
In P7 of pedigree IV, targeted next-generation sequenc- ing identified two SAMD9L mutations (Table 2): missense p.H880Q with a variant allele frequency of 27% out of 8139 reads (likely constitutional; this mutation was report- ed in multiple individuals within a family with ataxia pan- cytopenia but no MDS phenotype) and nonsense p.S1317RfsX21 likely acquired in a subclone as inferred from the much lower variant allele frequency of 10% (5934 reads). In summary, inherited SAMD9L mutations p.V1512M, p.R986H, and p.R986C were identified in three families (each with 2 individuals diagnosed with MDS/-7 and 1 healthy carrier Figure 1A,B), and p.H880Q in P7 who presented with transient monosomy 7.
Acquired mutations in known oncogenes
All patients with exception of P3 and P6 were evaluated for the presence of somatic mutations in leukemia-associ- ated genes using whole exome sequencing or targeted
Figure 2. Bone marrow findings in P1 and P2 at dif- ferent timepoints during the course of the disease. Hematoxylin and eosin staining of bone marrow (BM) at diagnosis of RCC in P1 showing dysplastic granulopoiesis with hypergranulation and a pseudo- Pelger cell (top left), myeloblast and dysplastic eosinophil (top right). BM at diagnosis in P2 (syn- chronous with monosomy 7) showing hypergranula- tion and vacuolization in myelocytes, and dysplastic erythropoiesis with double nuclei (bottom left). Normal BM morphology in P2, 15 years after initial BM confirming spontaneous phenotype reversion (bottom right).
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