Benefits of Targeted Next Generation Sequencing (NGS) for Identification of Newborn Hemoglobinopathies: The California Experience
Authors: Singer Sylvia Titi , Hou Yu , Sharma Rajesh , Vichinsky Elliott P. , Meyer Adriane , Martinez Jennifer , Wu Cindy , Bishop Tracey , Tavassoli Shabnam 
Published: December 2024

Introduction
Hemoglobinopathies are a heterogenous group of disorders comprised of variants in α-globin (HBA), β-globin (HBB) genes or both. Newborn screening (NBS) is aimed to identify clinically significant hemoglobin disorders and facilitate early delivery of specialized care and education before the onset of clinical symptoms. The rapid growth in Asian and Middle Eastern segments in the US population has resulted in higher frequency of non-sickling thalassemia disorders and has affected NBS practices in some states; In California, due to such demographic changes, NBS for α thalassemia (Hb H disease) is mandated. In many other states the reporting for α thalassemia is of a presumptive diagnosis, mostly due to the complexity and expense of such testing, causing delays in diagnosing clinically significant forms of α thalassemia.
Our ‘Hb Reference Lab’ (https://hemoglobinlab.ucsf.edu) performs confirmatory testing following primary screening by HPLC (California NBS Program). The diagnostic approach targets the most common mutations or deletions (e.g., allele specific PCR) or analyzes the entire HBB and/or the HBA genes to identify the gene/s responsible for a Hb disorder (e.g. Sanger direct sequencing). Thus, different molecular techniques, in a sequential workflow, which cannot simultaneously assess both α and β genes, are required. It is therefore time consuming, a costly process and results in an extended turn-around time.
Next-generation sequencing (NGS) has a wide application in detecting disease-causing variants in genetic disorders; while not widely adopted for Hb disorders due to cost and technical challenges, NGS has proven helpful in carrier screening and diagnosing complex cases. We sought to assess the effectiveness of targeted NGS for confirmatory diagnosis of hemoglobinopathies in newborns.
Methods
Genomic DNA was extracted from peripheral blood and based on initial protein chemistry analysis (HPLC, IEF) molecular essays for HBA, HBB or both were performed and hemoglobinopathy determined. 98 samples with a known genotype were then analyzed by amplicon based NGS (Devyser Thalassemia NGS) which detects variants in HBA1, HBA2 and HBB genes.
Results
NGS successfully identified both deletions and point mutations including compound heterozygous conditions: HBB gene; Hb SS, SC, S/ β +, SE, S/β variant, S trait, E / β variants, homozygous β 0, Hb EE, β traits, β variants; E, D carriers. HBA genes; deletional or non-deletional Hb H disease, a case of 4 alpha gene deletion and several benign α-variants.
Previously unknown genotypes detected by NGS: A large β deletion making the diagnosis S/β0 thalassemia (instead of presumed Hb SS)3 cases of HPFH or a delta/beta deletion in patients with Hb SS9 homozygous and 26 heterozygous Xmn1 polymorphisms13 of 28 case of SCD had one alpha gene deletion (α3.7)3 cases of an α mutation carrier: Constant Spring (CS) and Hb Pakse.
Genotypes not detected by NGS: We could not specify if α variants were located on HbA1 or HBA2. However, unique mutation to HBA2, such as CS and Pakse were correctly identified.A case a coinheritance of α -4.2 deletion and α2 triplication was not identified (as they balance out each other)
Discussion
The use of NGS Thalassemia assay accurately detected mutations and deletions and provided a comprehensive hemoglobin profile for all NB samples flagged during initial screening. The essay also revealed variants that were not identified through our standard approach. This capability is particularly advantageous in regions with a high prevalence of thalassemia, where traditional methods may not detect these important genetic variants, in particular of α thalassemia. The ability to simultaneously analyze the HBA and HBB genes, is a significant benefit in reducing laborious processes of multiple essays and extended turn-around time. Additionally, it can limit or eliminate the need to analyze parents' samples (currently done on an elective basis for abnormal NBS results).
Beyond diagnosis of important NB hemoglobinopathies, the findings add clinically relevant data, such as presence of HPFH and a β deletion in patients diagnosed with Hb SS, which are therefore expected to have a milder phenotype, or knowledge of XMN1 status for considerations of hydroxyurea treatment. Some limitations exist due to a high degree of homology between HBA1 and HBA2 but have a limited impact on the accurate diagnosis of the expected clinical phenotype.