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.

Keywords: congenital hemolytic anemia; differential diagnosis; pathogenic variants; red blood cells; targeted-NGS.

FIGURE 1

Diagnostic suspect after first and second level laboratory investigation and final diagnosis after t-NGS in Group 1 (A) and Group 2 (B) patients.

FIGURE 2

Families with erythrocyte membrane defects and complex phenoptypes and/or intrafamily variability.

FIGURE 3

Advandages and limitations of laboratory approach vs. t-NGS technologies in congenital hemolytic anemias. The laboratory tests/parameters specifically useful for diagnosis of different diseases are reported. Brilliant green, exhaustive diagnostic approach; Green, requiring other approaches to confirm the diagnosis; gray, insufficient laboratory markers to reach the diagnosis.