Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience Monocentric Study

Abstract

Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHAs; 122 patients from 105 unrelated families were investigated and the results compared with conventional laboratory pathway. Patients were divided in two groups: 1) cases diagnosed with hematologic investigations to be confirmed at molecular level, and 2) patients with unexplained anemia after extensive hematologic investigation. The overall sensitivity of t-NGS was 74 and 35% for families of groups 1 and 2, respectively. Inside this cohort of patients we identified 26 new pathogenic variants confirmed by functional evidence. The implementation of laboratory work-up with t-NGS increased the number of diagnoses in cases with unexplained anemia; cytoskeleton defects are well detected by conventional tools, deserving t-NGS to atypical cases; the diagnosis of Gardos channelopathy, some enzyme deficiencies, familial siterosterolemia, X-linked defects in females and other rare and ultra-rare diseases definitely benefits of t-NGS approaches.

Identification of variants in 94 Chinese patients with hereditary spherocytosis by next‐generation sequencing

“…In recent years, new discoveries have been made using targeted NGS technology 19–21 . Compared with Sanger sequencing, NGS with a preselected gene panel has higher diagnostic efficiency and can quickly conduct comprehensive gene analysis for patients suspected as having an erythrocyte membrane disease 22–24 . Diagnosis of HS is based on clinical data, family history, and phenotypic testing.…”

“…[19][20][21] Compared with Sanger sequencing, NGS with a preselected gene panel has higher diagnostic efficiency and can quickly conduct comprehensive gene analysis for patients suspected as having an erythrocyte membrane disease. [22][23][24] Diagnosis of HS is based on clinical data, family history, and phenotypic testing. Taken together, NGS comes across as a reliable supplement to the previous experimental diagnostic methods.…”

Effect of primary lesions in cytoskeleton proteins on red cell membrane stability in patients with hereditary spherocytosis

“…The DNA samples were analysed on an NGS-targeted panel containing 43 genes associated with congenital hemolytic anemia (Supplemetary Table S1) (Fermo et al, 2021b). Libraries were obtained by HaloPlexHS Target Enrichment System Kit (Agilent Technologies, Santa Clara, United States) and sequenced on a MiSeq platform (Illumina, San Diego, United States).…”

“…The defective RBC membrane proteins identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), have been considered for many years a confirmatory test for membrane defects in HS; however, it is known that a in about 10% of the patients, the protein deficiency remains unclassified by this technique (Mariani et al, 2008;Bianchi et al, 2012). The development of next generation sequencing (NGS) techniques and targeted-NGS panels may be useful to unravel complex cases, since the genes encoding for RBC membrane proteins are now usually included in the targeted panels specifically designed for the diagnosis of hereditary hemolytic anemias; this resulted in a rapidly growing knowledge of the molecular basis of HS and of the genotype phenotype correlation (He et al, 2017;Russo et al, 2018;van Vuren et al, 2019;Xue et al, 2019;Bianchi et al, 2020;Qin et al, 2020;Fermo et al, 2021b;Vives-Corrons et al, 2021). However, few data are available regarding the direct consequences of the various genetic defects on the assembly and dynamic interactions of the RBC cytoskeleton.…”

Facilitating EMA binding test performance using fluorescent beads combined with next‐generation sequencing

“…To facilitate HS diagnosis, targeted next‐generation sequencing (tNGS) is used to detect germline mutations in genes encoding for RBC cytoskeleton proteins [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. tNGS is less time consuming than traditional sequencing techniques, but the technique is associated with high costs and long turnaround.…”