Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using next‐generation sequencing

Abstract

Summary Introduction Congenital haemolytic anaemia (CHA) refers to a group of genetically heterogeneous disorders, mainly caused by changes in genes encoding globin chains, cytoskeletal proteins and red cell enzymes, in which accurate diagnosis can be challenging with conventional techniques. Methods To set‐up a comprehensive assay for detecting mutations that could improve aetiological diagnosis, we designed a custom panel for sequencing coding regions from 40 genes known to be involved in the pathogenesis of CHA, using the Ion Torrent™ (Thermo Fisher Scientific, S.L. Waltham, MA, USA) Personal Genome Machine (PGM) Sequencer. A control group of 16 samples with previously known mutations and a test group of 10 patients with unknown mutations were included for assay validation and application, respectively. Results In the test group, we identified pathogenic mutations in all cases: four patients had novel mutations in genes related to membrane defects (SPTB, ANK1, SLC4A1 and EPB41), four were homozygous or compound heterozygous for mutations in genes related to enzyme deficiencies (GPI, TPI1 and GSS), one had a mutation in the HBB gene and another presented a homozygous mutation in the ADAMTS13 gene. Conclusions Ion PGM sequencing with our custom panel is a highly efficient way to detect mutations causing haemolytic anaemia, including new variations. It is a high‐throughput detection method that is ready for application in clinical laboratories.

Deciphering molecular heterogeneity of Indian families with hereditary spherocytosis using targeted next‐generation sequencing: First South Asian study

“…Previous studies have used different targeted NGS panels for the diagnosis of haemolytic anaemia on different platforms (Del Orbe Barreto et al , ; Kim et al , ). NGS is efficient and rapid for providing a correct diagnosis in clinical settings.…”

Combined study of ADAMTS13 and complement genes in the diagnosis of thrombotic microangiopathies using next‐generation sequencing

“…Therefore, the expectation that the whole‐exome sequencing approaches could identify other genes involved in aHUS is high; however, these studies still remain under investigation . Conversely, NGS‐targeted gene panels are being introduced into clinical practice providing substantial benefits for definitive diagnoses in hematological diseases …”

“…The workflow procedure of in silico analysis, the genetic databases consulted for validation of rare variants, the criteria for assessment of the pathogenic mutations, and, finally, their validation by Sanger sequencing were in accordance with those previously described in our recent work and explained in detail in the Supplementary Materials.…”

Diagnostic approaches for inherited hemolytic anemia in the genetic era

“…Additionally, the usefulness of Sanger sequencing is limited for the diagnoses of complex, multi-gene disorders or those with locus heterogeneity. Recent advances in molecular technologies have helped to identify unexpected candidate genes in numerous inherited disorders including IHA [438990]. Various NGS-based methods have been developed, including whole genome sequencing, exome sequencing, and gene panels [91].…”