Page 84 - Haematologica Vol. 107 - September 2022

ARTICLE - Inherited cytopenias in children
The study was approved by the Rabin Medical Center In-
stitutional Review Board.
Sanger sequencing
We initiated the genetic workup by Sanger sequencing in patients with a clear clinical presentation and when vari- ants in a single gene were a common cause of the specific suspected diagnosis. This approach included the genes FANCA for Fanconi anemia (FA), ELANE for severe congen- ital neutropenia (SCN), RPS19 for Diamond Blackfan ane- mia (DBA), SBDS for Shwachman Bodian Diamond syndrome (SBDS) and DKC1 for dyskeratosis congenita (DC). Sanger sequencing was also used to confirm NGS findings and family segregation.
Next-generation sequencing
Our custom-made NGS panel is continuously updated. The latest version included 226 known genes that cause inherited cytopenias (Online Supplementary Table S1). Im- portantly, the panel includes the TERC gene encoding for the RNA component of the telomerase, the non-coding 5'UTR regions of DKC1 and ANKRD26, and intron 5 of GATA2, in which disease-causing variants have been de- scribed.23 Panel design, library preparation and sequencing were performed as previously described.24 The DNA used for genetic analysis was extracted from peripheral blood. Germline variants were identified by the variant allele fre- quency. In patients for whom it was uncertain whether the variant was of a germline origin, we performed Sanger sequencing on DNA extracted from fibroblasts.
Whole exome sequencing
WES was performed as described in Goldberg et al.25 Table 1. Referrals and genetic diagnoses in our cohort.
O. Gilad et al.
Sequencing results and bioinformatics pipeline
Sequencing reads were aligned against a reference ge- nome (GRCh37/UCSC hg19) and variants were called and annotated using the SureCall software (v.3.5.1.46; Agilent Technologies). SNP filtering was established as pre- viously described,24 using both an in-house platform and the Emedgene AI-based genomic analysis platform (Emedgene Technologies, Tel-Aviv, Israel). Genetic vari- ants were reported according to the American College of Medical Genetics guidelines.26,27 Only P/PL results were reported.
Copy number variant detection
Copy number variant (CNV) analysis was performed using “Rainbow”-Genoox CNV Caller. The Rainbow caller employs a machine-learning based anomaly detection algorithm, in which variants are determined based on exon-level coverage, using a cohort of samples (n>30). The model considers several factors including GC con- tent, coverage variance over multiple samples and neighboring gene coverage (Genoox,Tel-Aviv, Israel). CNV findings were confirmed by multiplex ligation-depend- ent probe amplification (MLPA, MRC, Amsterdam, Hol- land) available for: FANCA, RPS19, RPL5, RPS26, RPL11, RPS17, RPL35A, TERT and DKC1 and by cytoscan-high density SNP-array.28
Statistical analysis
Fisher exact test was used to compare the clinical and demographic characteristics between patients who were and were not diagnosed with inherited disorders. A P-value of <0.05 was considered statistically signifi- cant.
Referral diagnosis
N of pts
N of genetically diagnosed pts (%)
Diagnosis according to molecular findings (N of pts)
IBMFS
48
29 (60.4)
IBMFS (29)
MDS
26
6 (23.1)
MDS predisposition (3)
IBMFS (2)
IT with MDS predisposition (1)
SAA
31
1 (3.2)
IBMFS (1)
Isolated thrombocytopenia
33
17 (51.5)
Classical IT (9)
IT with MDS predisposition (8)
Isolated neutropenia
51
6 (11.8)
Congenital neutropenia (6)
Total
189
59 (31.2)
IBMFS: inherited bone marrow failure syndromes; IT: inherited thrombocytopenia; MDS: myelodysplastic syndrome; SAA: severe aplastic ane- mia; No: number; pts: patients.
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