Page 88 - Haematologica Vol. 107 - September 2022

ARTICLE - Inherited cytopenias in children
O. Gilad et al.
switching. Thrombocytopenia was noted at birth. When referred to a pediatric hematology center (at age 6 years) he had macrocytosis (MCV 98fL), thrombocytopenia (48X109/L), substantially high fetal hemoglobin (30%) and a hypoplastic BM with dysplastic changes.
Patients presenting with thrombocytopenia
Thirty-three patients presented with thrombocytopenia and an inherited cause was found in 17 (51.5%). Of them, nine (52.9%) had IT that was known to affect platelet pro- duction and function and eight (47.1%) had variants in genes that were known to cause thrombocytopenia with a predisposition to leukemia (Figure 1; Table 5; Online Sup- plementary Table S3). Of the nine patients with IT due to platelet production or function defects, five had variants in the MYH9 gene, two in ACTN1 and one each in NBEAL2 and CYCS. Their thrombocytopenia ranged from mild to severe (7-125x109/L), with a normal to high MPV, 8-17.7 fL (normal values 5.6-12.1 fL33). Of the eight patients with thrombocytopenia and an inherited predisposition to MDS/AML, four had variants in the 5'UTR of ANKRD26, 2 in ETV6, 1 in RUNX1 and one in the GALE gene. Seven patients had mild to moderate thrombocytopenia (45-117x109/L), with a normal MPV, 8.5-12 fL (normal values 5.6-12.1 fL33). The patient diagnosed with a variant in GALE had giant and pale platelets (MPV 17 fL), as we previously de- scribed.34 He was of Bedouin origin and not related to the original family we described.
Patients presenting with neutropenia
Of the 51 patients presenting with neutropenia 6 (11.8%) were molecularly diagnosed (Figure 1; Table 6; Online Sup- plementary Table S3). Two patients had compound het- erozygous variants in the SBDS gene. One had a homozygous variant in a known neutropenia causing gene, JAGN135. One additional patient, presenting with familial neutropenia and recurrent infections, was subsequently diagnosed by WES with a novel SRP54 variant causing SCN and SBDS.25,36 This gene was not known to cause neu- tropenia at the time the NGS study was performed. We later incorporated this gene into the subsequent versions of our NGS panel and detected a variant in this gene in one more patient that had a re-do of the NGS panel. An additional patient who presented with isolated neutrope- nia was later diagnosed by WES with a homozygous vari- ant in UNC13D.
Twelve patients in our neutropenia group (8 patients of Muslim Arab origin and 4 of Yemenite Jewish origin), were homozygous to the known polymorphism in the DARK promoter (rs2814778, -30 T>C)37. They all had absolute neutrophil counts (ANC) >0.5x109/L (0.6-0.76x109/L) and no recurrent infections, compatible with the diagnosis of be- nign ethnic neutropenia. This same polymorphism was also found in one additional patient of Arabic origin, who
presented with severe neutropenia (ANC levels <0.2x109/L) and recurrent infections, and therefore underwent HSCT. His phenotype suggests the presence of a yet undis- covered gene causing congenital neutropenia; the patient underwent successful HSCT.
Patients referred with suspected severe acquired aplastic anemia
Thirty-one patients with suspected SAA were referred to rule out IBMFS or other MDS predisposing syndromes. A pathogenic variant in the GATA2 gene was found in one patient (3.2%) and his diagnosis was amended to IBMFS (Figure 1; Table 7; Online Supplementary Table S3). This pa- tient had mild to moderate neutropenia (0.6-1.2x109/L) 2 years prior to the diagnosis of SAA. He had normal base- line monocyte counts, which were reduced to 0.02-0.2 K/micL, together with the development of neutropenia.
Discussion
In this paper, we presented the results of the genetic di- agnosis of 189 children with prolonged cytopenias. P/LP germline variants were identified in 59 children (31.2%). Most of the diagnosed children (47/59, 79.7%) with per- sistent cytopenias had leukemia predisposition, while 12 children (20.3%) had either congenital thrombocytopenia with impairment of platelet production (9 children) or congenital neutropenia not currently known to predispose to malignant transformation (3 children).
In most children referred for genetic evaluation of cytope- nias, NGS diagnosis was performed upfront. In the minor- ity, when the clinical picture suggested a known disorder commonly caused by variants in a single gene, we initiated the genetic workup with Sanger sequencing. Of the vari- ous NGS methods, we used NGS panels as they offer a uniformly high depth of sequencing of the genes of inter- est. An advantage of our panel is that it was designed also to include non-protein coding regions such as the 5'UTR of ANKRD26 and DKC1; the RNA component of the telome- rase, encoded by the TERC gene; and intronic regions in GATA2. We report only P/LP sequence changes. The draw- back of the NGS panel method is that the time lag be- tween the identification of a new gene and its insertion into the panel requires frequent panel updating. We mod- ified our panel seven times during the 4-year study period. All children referred for a molecular workup had a pro- visional diagnosis determined by their treating hematol- ogist. For most patients, the genetic diagnosis supported the referral diagnosis. One patient with suspected SAA, who was evaluated to rule out an inherited disorder, was indeed diagnosed with a germline GATA2 variant; this changed the diagnosis to IBMFS. Three patients had pa- thogenic variants in the ERCC6L gene; two of them were
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