The Diagnosis of HbS Genotypes and Identification of β-Thalassemia Mutations in Patients with Hbsβ-Thalassemia Using Next Generation Sequencing

The conventional diagnosis of sickle cell disease (SCD) depends on RBC morphology, Hb electrophoresis and/or high-performance liquid chromatography (HPLC). However, these methods are fraught with ambiguities especially in areas where HbSβ-thalassemia is common. The distinguishing laboratory feature between patients with HbSS and HbSb-thalassemia is the level of HbA₂. Unfortunately, when HbA₂ is estimated with HPLC in patients with Sβthalassemia, it tends to be spuriously high because it co-migrates with glycated HbS.

HbSβ-thalassemia accounts for 30 - 40% of the SCD patients being followed in Kuwait. Patients with a presumptive diagnosis of HbSS and/or Sβ-thalassemia, by HPLC were referred to the Hemoglobin Research Laboratory of Department of Pediatrics, Kuwait University for confirmatory molecular diagnosis.

The DNA samples collected from 1994 to 2018 were recently subjected to NGS. DNA was isolated from peripheral leukocytes using the phenol-chloroform method. The Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. For theHBBlocus, all β-globin mutations and variants were confirmed by arrayed primer extension (APEX) or Sanger sequencing methods. In addition, genotyping of modifier SNPs inBCL11Aon chromosome 2 and theHBS1L-MYBintergenic region on chromosome 6 was carried out to identify their influence on HbF expression among our patients. In all, 126 SNPs were genotyped. We hereby, report the false positive and false negative rates for the diagnosis of Sb-thalassemia with HPLC compared to NGS. We also report the spectrum of β-thal mutations among our patients with Sβ-thalassemia.

The DNA samples were from 232 patients aged from 1 to 59 with a mean of 12.7 ± 11.2 years. Analysis of the initial HPLC diagnosis showed that 27.7% were reported to have Sβ-thalassemia, while after NGS assessment, 29.4% were found to truly fit the diagnosis. Three individuals who were diagnosed as HbSS turned out to be HbAS and 1 each turned out to be HbAA and β-thalassemia trait respectively. Twelve patients that were originally thought to be Sβ-thalassemia turned out to be HbSS, i.e. a false positive rate of 5%, while 30 who were thought to be SS, turned out to be Sβ-thalassemia, i.e. a false negative rate of 13%.

A total of 12 mutations were identified in 61 Sβ-thalassemia patients. Of these, the most common were the β⁰ IVS-1 del 25 and the IVS-II-1 (G/A) in 9 (14.8%) each. The most common β⁺ mutation was the IVS-I-110 (G/A) in 8 (13.1%), followed by the IVS-I-5 (G/C) in 7 (11.5%). While the Sβ⁰-thalassemia patients had no HbA on HPLC, the Sβ⁺ were associated with varying concentrations of HbA, ranging from 0 for those carrying the IVS-I-5 (G/C) mutation to a mean of 15.5 +/- 6.2% in the patients with IVS-I-110 (G/A) and 25.1% in the Sβ⁺⁺patient with IVS-I-6 (C/T).

Indeed, HPLC is far from deal in detecting HbSβ-thalassemia with significant rates of false positivity and negativity. NGS is very versatile; it can interrogate thousands of genes simultaneously, making it ideal for use in SCD. The HbS genotype, β-thalassemia mutation, haplotype and different modifier polymorphisms can be determined in one run. It is therefore very useful for personalized diagnosis that can document prognostic factors, making for purposeful counseling and follow-up from an early age.