Evaluated in germline dna cd3 t cells from peripheral

evaluated in germline DNA (CD3
+
T cells from peripheral
blood or hair follicles).
Eleven of 38 patients had moderate, 12 severe, and 15
very severe AA. Median age of AA patients at diagnosis
was 30 years (range 9-79 years). Three patients (8%) had
abnormal cytogenetics ( Online Supplementary Table S1 ). In
haematologica 2014; 99:e165
L
ETTERS TO THE
E
DITOR
Table 1. Mutations in aplastic anemia patients. Sequence numbering is according to the protein sequence of Ensembl protein ID
ENSP00000266058 (SLIT1), ENSP00000282030 (SETBP1) and ENSP00000364839 (ASXL1), ENSP00000217026 (MYBL2),
ENSP00000442788 (TET2), of genome build GRCh37, release 73.
Characteristic Somatic mutations Germline SNVs
Patient ID 1 2 3 4
Age at diagnosis (years) 61 14 42 45
Age at sampling (years) 61 28 58 45
Sex M M F F
Severity VSAA SAA MAA SAA
Treatment ATG/ ATG/CSA, alloHSCT CSA, ATG/CSA Syngeneic HSCT
Time from diagnosis to first treatment (months) 0.5 16 1 4
Best response (CR, PR) CR CR PR CR
Time from last treatment to last follow up (years) 1.9 8.3 4.3 9
PNH clone (granulocytes, %) 4 1.4 0 0
Telomere length (average T/S ratio) unknown unknown 1.25 unknown
Source of DNA BM PB BM PB
Karyotype 46,XY 46,XY 46,XX, 46,XX
developed
trisomy 8
Affected gene SLIT1 SETBP1, MYBL2 TET2
ASXL1
Variation, nucleotide level c.1252C>A c.2602G>A c.844T>C c.2981A>G
c.1934dupG
Variation, protein level p.Q418K p.D868N**, p.S282P p.H994R*
p.G645fs***
Germline affected No No Yes Yes
Variant allele frequency (%) 50
§
35
§
38.7 50
25
§
Survival from diagnosis 1.5, alive 21.6, alive 16.8, alive 7.5, alive
(years), status
M: male; F: female; SAA: severe aplastic anemia;VSAA: very severe aplastic anemia; MAA: moderate aplastic anemia; ATG: anti-thymocyte globulin; CSA: ciclosporin; alloHSCT:
allogeneic hematopoietic stem cell transplantation; PB: peripheral blood; BM: bone marrow; n.a.: not applicable. *previously identified as somatic SNV COSM1426210 **pre-
viously identified as somatic SNV in SETBP1, COSM1318401 ***previously identified as somatic insertion in ASXL1, COSM34210
§
estimated from Sanger sequencing trace.

17 patients (44.7%), a GPI-deficient clone suggesting
PNH/AA overlap syndrome was present. Twenty-two
patients underwent allogeneic or syngeneic transplanta-
tion. The median duration of follow up from diagnosis of
patients alive was seven years. Thirty-five of 38 patients
were alive at last follow up. In support of the diagnosis of
AA, telomeres in peripheral blood leukocytes were signif-
icantly shorter in AA patients (n=13) than in age-matched
healthy controls (n=20) (median age AA, 44 years; median
age healthy controls, 43 years, P =0.43; mean T/S ratio AA
patients 1.43, mean T/S ratio healthy controls, 2.48,
P <0.001) ( Online Supplementary Figure S1 ). Next generation
sequencing yielded an average coverage of 2015 reads per
amplicon. In total, 3 somatic mutations were identified in
2 patients in the examined MDS candidate genes (5.3%)
(Table 1 and Online Supplementary Figure S2 ). One patient
had a somatic missense mutation in SLIT1, and one
patient with severe AA had two somatic mutations, one
missense mutation in SETPB1 (D868N) and one frameshift
mutation in ASXL1 ( G645fs ). This patient was diagnosed
with severe AA at the age of 14 years and received four
courses of immunosuppressive therapy. After the first
cycle of ATG, the patient was in remission for four years.