Page 72 - Haematologica3

V.B. Pastor et al.
next-generation sequencing. In P1 previously reported leukemia driver mutations SETBP1 p.D868N, EZH2 p.V582M, and KRAS p.Q61P were identified as somatic (Table 2, Online Supplementary Figure S2). Similarly, in P5 a somatic RUNX1 mutation c.413_427+5dup20bp was detected (Table 2). This is a novel splice-donor site muta- tion not present in databases. No additional mutations in leukemia driver genes were observed in other affected cases.
Clonal escape mechanisms from SAMD9L mutations are not random
In comparison with other constitutional variants, SAMD9L missense mutations showed significantly lower median allelic frequencies across all patients (53% versus 20%, P<0.05) (Table 2). This finding was corroborated by the consistent partial or complete loss of chromosome 7 (Figure 3, Table 1). In P1, -7 progressively expanded from 60% at the time that RCC was diagnosed to 95% at the time of progression to CMML. In addition, P1 and P7 har- bored subclones with acquired stop-gain SAMD9L muta- tions located upstream of the constitutional missense sub- stitutions (Figure 3). TA cloning confirmed the cis orienta-
tion of both mutations on the same chromosome in P1 (Figure 1C). Western blot of 293T cells transiently trans- fected with double-mutated (p.V1512M/p.R1188X) SAMD9L plasmid revealed stable expression of the trun- cated protein (data not shown). In P2 and P7, the initial -7 disappeared and was replaced by a large somatic clone with uniparental disomy (UPD) 7q that duplicated the paternal wild-type SAMD9L allele (Figures 3 and 4).
The natural history of SAMD9L-related myelodysplastic syndrome reveals divergent clinical outcomes
We next studied in detail the disease course in patients who were left untreated for longer periods of time. In P4, the evolution from normal karyotype to independent clones with -7 and del(7q) was rapid and occurred within a few months (Table 1). Cytogenetic progress was associ- ated with partial recovery of complete blood counts and normalization of bone marrow cellularity, pointing to the possibility of hematopoietic stem cell niche repopulation by -7/del(7q) retaining only the wild-type SAMD9L allele. P1 developed CMML 3.6 years after the initial diagnosis of MDS/-7. This patient carried somatic driver mutations representing major subclones co-existing on the -7 back-
Table 2. Overview of germline and somatic mutations.
Mutation type
Family I
Family II
Family III
Family IV
Patient # (UPN)
P1 (D084)
P2 (D154)
Father Mother
P3 (US1) P4 (US2)
Father
Mother
P5 (D637) P6 (father) Brother
P7 (SC054) P1 (D084)
P5 (D637) P7 (SC054)
Time -7% (FISH / point, age Metaphases)
Genotype
c.4534G>A p.V1512M
c.563A>G p.Y188C c.4534G>A p.V1512M
Myeloid sample
BM-GR
BM-GR BM-GR
BM-GR PB PB
PB PB PB PB PB PB PB PB
BM-GR PB PB
BM-GR
BM-GR
BM-GR BM-GR BM-GR BM-GR
BM-GR
VAF% (depth)
WES8.3% (277), DS13.3%(2755) WES57.5% (80) WES21.5% (424), DS19.3% (1261) WES59.7% (62) Sanger DS49.0% (13116)
Sanger
Sanger DS43.0% (252) DS48.1% (n.a.) DS49.0% (401) Sanger Sanger Sanger
DS7.5% (3422) Sanger Sanger
DS27% (8139)
WES5.9% (202) DS8.0% (884) WES37.7% (106) WES47.8% (355) WES69.2% (130) Sanger
DS10% (5934)
Germline source
-
-
HR (Sanger)
HR (Sanger) HR (Sanger) HR (Sanger)
HR (Sanger) HR (Sanger) HR (Sanger) HR (Sanger) HR (Sanger) HR (Sanger) HR (Sanger) HR (Sanger)
FB (Sanger) -
-
-
-
-
-
-
FB (Sanger)
-
Conservation/ Effect (SIFT / CADD- ExAC
7 yrs (CMML)
17 yrs
20 mo 15 mo
7.7 yrs 42 yrs
2.3 yrs
7 yrs (CMML)
7.7yrs
2.3yrs
60% / [6/16] 0% / -
-
-
16% / [3/21]
5.5% / [0/20]
-
-
- / [20/20] - / [11/16]
SAMD9Lm
PTENp SAMD9Lm
PTENp
PTEN
SAMD9L SAMD9Lp
JAK3p SAMD9Lp JAK3p FANCMm SAMD9L JAK3 FANCM
c.563A>G c.563A>G
p.Y188C
D/P/D/D(65%) 25.2 None
D/-/D/D(61%) 25.8 None D/P/D/D(65%) 25.2 None
D/-/D/D(61%) 25.8 None D/-/D/D(61%) 25.8 None D/P/D/D(65%) 25.2 None
PhysChem diff.
High / Small
High / Large High / Small
High / Large High / Large High / Small
High / Small Weak / Small High / Small Weak / Small Weak / Small High / Small Weak / Small Weak / Small
High / Large High / Large High / Large
Weak / Small
MutTaster score browser /Polyphen2/ n/%in
PredictSNP)
population
p.Y188C c.4534G>A p.V1512M
c.2957G>A p.R986H c.2630C>T p.A877V c.2957G>A p.R986H c.2630C>T p.A877V c.171G>C p.L57F c.2957G>A p.R986H c.2630C>T p.A877V c.171G>C p.L57F
c.2956C>T p.R986C c.2956C>T p.R986C c.2956C>T p.R986C
c.2640C>A p.H880Q
c.3562C>T p.R1188X
c.182A>C p.Q61P c.2602G>A p.D868N c.1744G>A p.V582M
c.413_427 +5dup20bp
c.3951_ p.S1317RfsX21 3955delTAAAG*
D/D/D/D(87%) 26.5 T/N/B/N(74%) 23.3 D/D/D/D(87%) 26.5 T/N/B/N(74%) 23.3 D/D/B/D(52%) 17.9 D/D/D/D(87%) 26.5 T/N/B/N(74%) 23.3 D/D/B/D(52%) 17.9
4/0.003% 11/0.01% 4/0.003% 11/0.01% 195/0.32% 4/0.003% 11/0.01% 195/0.32%
SAMD9Lp SAMD9L - SAMD9Lp
D/N/B/D(87%) 21.7 None D/N/B/D(87%) 21.7 None D/N/B/D(87%) 21.7 None
T/-/D/-(-) 23.7 None
Novel, stop-gain 35 -
Known driver mut. 28.2 - Known driver mut. 26.7 - Known driver mut. 34 - Novel, splice donor n.a. -
Novel, stop-gain 28.8 -
- / [0/25] 60% / [6/16]
- / [20/20] - / [0/25]
SAMD9L SAMD9L
KRAS SETBP1 EZH2 RUNX1
SAMD9L
Mut: mutation; UPN: unique patient number; FISH: fluorescence in situ hybridization; BM: bone marrow; PB: peripheral blood; GR: granulocytes; HR: hair follicles; FB: skin fibroblast; m.: maternal ori- gin; p.: paternal origin; VAF: variant allelic frequency; WES: whole exome sequencing; DS: targeted deep sequencing; Sanger: identified by Sanger sequencing; n.a.: not available; +yrs/mo, years/months after diagnosis. Evolutionary conservation scores, Phylop and PhastCons; PhysChem diff., physicochemical difference between amino acids. In-silico prediction: SIFT: T-tolerated, D- deleterious; Mutation Taster: D-disease causing, N-polymorphism, P-polymorphism automatic; PolyPhen2: D-probably damaging, B- benign; PredictSNP consensus classifier: D-deleterious, N-neutral (% accuracy). Combined annotation-dependent depletion (CADD-score) of 20 means that a variant is among the top 1% of deleterious variants in the human genome; CADD-20=1%, CADD-30=0.1%, CADD-40=0.01%,CADD-50=0.001%.* mutation classified as acquired based on low allelic frequency.Gene annotations:SAMD9L (NM_001303500.1),EHZ2 (NM_152998),SETBP1 (NM_015559),KRAS (NM_004985.4), FANCM (NM_001308134), JAK3 (NM_000215), PTEN (NM_000314.4), RUNX1 (001001890).
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ACQUIRED
GERMLINE