A Targeted Next-Generation Sequencing-Based Diagnosis for Hereditary Anaemias - European Medical Journal


A Targeted Next-Generation Sequencing-Based Diagnosis for Hereditary Anaemias

2 Mins
*Roberta Russo,1,2 Immacolata Andolfo,1,2 Francesco Manna,2 Antonella Gambale,1,2 Roberta Marra,1,2 Barbara Eleni Rosato,1,2 Paola Caforio,1,2 Valeria Pinto,3 Piero Pignataro,1 Kottayam Radhakrishnan,4 Sule Unal,5 Giovanna Tomaiuolo,6 Gian Luca Forni,3 Achille Iolascon1,2

The authors have declared no conflicts of interest.


This work was supported by grants from the Italian Ministry of Education, Universities and Research, by PRIN to Dr Andolfo (20128PNX83), by SIR to Dr Russo (RBSI144KXC), and by grants from Regione Campania (DGRC2362/07).

EMJ Hematol. ;6[1]:50-52. Abstract Review No. AR5.

Each article is made available under the terms of the Creative Commons Attribution-Non Commercial 4.0 License.

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Anaemia affects 1.6 billion people worldwide, with roughly 10% of these individuals affected by rare anaemias, of which 80% are hereditary.1 Hereditary anaemias (HA) encompass a highly heterogeneous group of disorders characterised by anaemia of variable degrees and by complex genotype–phenotype correlations. Differential diagnosis, classification, and patient stratification among HA is often very difficult.

To date, the major current application of next-generation sequencing (NGS) in diagnostics is through disease-targeted tests, for which multiple causal genes are known. Some studies have already demonstrated the utility of a targeted NGS (t-NGS) approach in the study of specific subtypes of HA patients. In this study, we described the diagnostic workflow based on t-NGS that we developed for the diagnosis of patients affected by HA. Within this wide group of disorders, we included a) hyporegenerative anaemias, such as congenital dyserythropoietic anaemias (CDA); b) haemolytic anaemias due to red cell membrane defects, such as hereditary spherocytosis (HS) and stomatocytosis (HSt); and c) haemolytic anaemias due to enzymatic defects, such as pyruvate kinase (PK) deficiency.2-5

We generated two consecutive versions of the same custom gene panel: the first included 34 genes, the second 71 genes. The probe design was performed by SureDesign (Agilent Technologies, Santa Clara, California, USA). Sample preparation was obtained by HaloPlex Target Enrichment kit for Illumina Sequencing (Agilent Technologies), and high-throughput sequencing was performed by Illumina NextSeq 500 (Illumina Inc., San Diego, California, USA). For bioinformatic analyses, we used Agilent SureCall software (v, Agilent Technologies). The pathogenicity of each variant was evaluated according to the guidelines of the American College of Medical Genetics and Genomics (ACMG).6,7

We investigated 74 probands with clinical suspicion of HA. Our approach revealed a diagnostic yield of 64.9% of analysed patients. Genetic data by t-NGS analysis confirmed the clinical suspicion in 54.2% of patients. Of note, most of these patients were originally suspected to have red cell membrane disorders (HSt or HS).

Conversely, t-NGS analysis modified the original diagnosis in 45.8% of patients; 81.8% of these patients were clinically suspected to have CDA. Of note, among the 22 patients originally classified as CDA, we identified 45.5% of cases with a conclusive genetic diagnosis of congenital haemolytic anaemias due to enzymatic defects. Indeed, we diagnosed one case with biallelic mutations in GPI, the causative gene of haemolytic non-spherocytic anaemia due to glucose phosphate isomerase deficiency; another case due to mutations in AK1, the causative locus of haemolytic anaemia due to adenylate kinase deficiency; and eight cases due to mutations in PKLR, the causative gene of PK deficiency.7

Our observation regarding congenital haemolytic anaemia patients misdiagnosed as CDA is highly relevant; it underlines how t-NGS analysis is valuable not only for achieving a correct and conclusive diagnosis but also for guiding possible treatment of HA patients. This is mainly true for the treatment of PK deficient-patients, for whom there is an allosteric activator of PK enzyme available that can increase the enzymatic activity of patient erythrocytes treated ex vivo.8

Hertz L et al. Is increased intracellular calcium in red blood cells a common component in the molecular mechanism causing anemia? Front Physiol. 2017;8:673. Gambale A et al. Diagnosis and management of  congenital dyserythropoietic anemias. Expert Rev Hematol. 2016;9(3):283-96. Andolfo I et al. New insights on hereditary erythrocyte membrane defects. Haematologica. 2016;101(11):1284-94. Andolfo I et al. Hereditary stomatocytosis: An underdiagnosed condition. Am J Hematol. 2018;93(1): 107-21. Canu G et al. Red blood cell PK deficiency: An update of PK-LR gene mutation database. Blood Cells Mol Dis. 2016;57:100-9. Al-Riyami AZ et al. Targeted next generation sequencing identifies a novel β-spectrin gene mutation A2059P in two Omani children with hereditary pyropoikilocytosis. Am J Hematol. 2017;92(10):E607-9. Russo R et al. Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias. Am J Hematol. 2018;93(5):672-82. Kung C et al. AG-348 enhances pyruvate kinase activity in red blood cells from patients with pyruvate kinase deficiency. Blood. 2017;130(11):1347-56.