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Updated: Nov 3, 2025
Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience
Elisa Fermo1, Cristina Vercellati1, Anna Paola Marcello1
1UOS Fisiopatologia delle Anemie, UOC Ematologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
Insights
Targeted Next Generation Sequencing (t-NGS) significantly improves diagnosis for rare congenital hemolytic anemias (CHAs). This genetic testing approach aids in identifying new variants and managing unexplained anemia cases effectively.
Area of Science:
- Hematology
- Genetics
- Rare Diseases
Background:
- Congenital hemolytic anemias (CHAs) are rare, heterogeneous disorders with complex pathophysiology and limited diagnostic tools.
- Understanding the genetic basis of CHAs is crucial for accurate diagnosis, clinical management, and patient stratification.
- Current diagnostic pathways often struggle with rare and atypical CHA presentations.
Purpose of the Study:
- To evaluate the diagnostic yield and clinical utility of a 43-gene targeted Next Generation Sequencing (t-NGS) panel for congenital hemolytic anemias (CHAs).
- To compare the effectiveness of t-NGS with conventional laboratory diagnostic methods in a monocentric cohort.
- To identify novel genetic variants and improve the diagnosis of unexplained anemia cases.
Main Methods:
- A 3-year monocentric study involving 122 patients from 105 unrelated families with suspected CHAs.
- Implementation of a 43-gene targeted Next Generation Sequencing (t-NGS) panel.
- Comparison of t-NGS results with conventional hematologic investigations, dividing patients into confirmed and unexplained anemia groups.
Main Results:
- The overall sensitivity of t-NGS was 74% for patients with initially diagnosed CHAs and 35% for those with unexplained anemia.
- t-NGS identified 26 new pathogenic variants, confirmed by functional evidence.
- The integration of t-NGS enhanced diagnostic rates, particularly for rare conditions and atypical presentations not detected by conventional methods.
Conclusions:
- Targeted Next Generation Sequencing (t-NGS) is a valuable tool for diagnosing congenital hemolytic anemias (CHAs), especially for unexplained cases and rare genetic defects.
- t-NGS complements conventional diagnostic approaches, improving the identification of genetic variants and enabling better patient management.
- The study highlights the benefit of t-NGS for diagnosing specific conditions like Gardos channelopathy, enzyme deficiencies, and X-linked defects in females.
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.
