Page 74 - Haematologica3

V.B. Pastor et al.
Figure 4. Loss of mutated SAMD9L allele due to genomic deletion or mitotic recombination. Variant allelic frequency (VAF) scores for chromosome 7 in P1 and P2. single nucleotide polymorphisms and Indels detected using whole exome sequencing (~4000 variants with a VAF score >5% and <95%), show a complete loss of chromosome 7 in P1, as the VAF scores are either low or high. P2, unlike P1 demonstrates a partial loss of the chromosome 7 after position 7q11.22 towards the q terminal site. The read depth of the single nucleotide polymorphisms for P2 was maintained throughout for chromosome 7 with no loss thus confirming that loss of heterozygosity is due to UPD and not -7q. Whole exome sequencing VAF values are marked by a star within the graph. VAF: vari- ant allelic frequency; UPD: uniparental isodisomy. Blue line: centromere; red line: SAMD9L gene position; yellow dotted line: start of UPD. For P7, targeted next- generation sequencing identified 14 informative (heterozygous) polymorphisms located on chromosome 7q with an average depth of 1036 reads (Online Supplementary Table S1). Single nucleotide polymorphisms are represented in a VAF graph depicting the skewing of heterozytosity towards one allelle occurring after position g.66098482 (rs3764903).
germline mutations can be discerned (p.H880Q, p.I981T, p.R986H, p.R986C, p.C1196S, and p.V1512M). Of note, all these mutations cluster exclusively to the C-terminal half of the protein. Further, upon comparing reported mutations in the paralogue gene SAMD9 (p.R982H/C)22 with that of the present study in SAMD9L (p.R986H/C), we identified a potential mutational hotspot affecting highly conserved regions in both SAMD9L (p.984-989: GVRIIH) and SAMD9 (p980-985: GVRIIH) proteins.
The reported constitutional variants in both SAMD9/SAMD9L were classified as gain-of-function based on the observation of decreased cell proliferation in a 293T cell line.20-22 Similarly, we observed growth defi- ciency in 293T cells harboring SAMD9L p.V1512M and p.R986C. Based on these findings, one cautious specula- tion hints at a gain-of-function effect that is toxic to cells. This is supported by the discovery of an acquired stopgain SAMD9L mutation in P1 and P7 that likely “eliminates”
Figure 5. Clonal evolution and spontaneous reversion due to UPD7q. Clonal evolution model in P2 (D154) depicting disease history during an observation period of 20 years. At diagnosis, initial bone marrow harbored monosomy 7 (77% by fluorescence in situ hybridization and 51% by metaphase karyotyping). Blood counts normalized 3.7 years later and since then P2 maintained normal com- plete blood counts until last follow-up at the age of 22 years. From the age of 12 years, repeated yearly bone marrow examinations revealed normocellular hematopoiesis with no dysplasia and normal cytogenetics. Bone marrow collect- ed at the age of 17 years (*) was subjected to whole exome and targeted deep sequencing. Germline heterozygous SAMD9L mutation p.V1512M was detected at a variant allelic frequency (VAF) of ~20%, corresponding to a clonal size of ~40% in a diploid chromosome 7 background. Concurrently, a spontaneous genetic correction of the SAMD9L locus occurred resulting from uniparental isodisomy (UPD)7q of paternal origin. This self-corrected clone occurred either initially (dotted line) or after termination of monosomy 7 and contributed to nor- mal hematopoiesis. Abbreviations: Dx, diagnosis; pat, paternal origin; mat, maternal origin; UPD; uniparental isodisomy, LFU; last follow-up.
germline missense mutations. In the cases studied here, complete or partial deletion of chromosome 7 and also UPD7q was non-random and each time resulted in loss of the germline-mutated SAMD9L gene copy. Additional studies, which are essential to further define the effect of SAMD9L variants, might be challenging due to the growth inhibitory effect of the alterations. It also remains to be determined whether SAMD9L missense mutations lead to increased protein stability, alter protein structure, enhance an unknown functional domain, or exert a completely neomorphic effect.
We describe three silent mutation carriers from separate families demonstrating no previous relevant medical his- tory. Despite normal complete blood counts and mean corpuscular volume, the brother of P5 had a hypocellular marrow with mild dysplasia, evidently attributed to the identical pathogenic SAMD9L mutation. This finding emphasizes the need for thorough hematologic workup, including marrow studies, in potential sibling donors espe- cially when they lack a genetic marker for familial disease. The intrafamiliar heterogeneity regarding the hematologic presentation remains elusive; one can speculate that other yet unknown genetic or epigenetic mechanisms might act as modifiers.
Thus far there is only limited knowledge about the reg- ulation and cellular functions of SAMD9L. It has been pos- tulated that both SAMD9L and the adjacent paralogous SAMD9 gene share functional redundancy, shaped by a long-term, possibly virus-induced selective pressure.30
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haematologica | 2018; 103(3)