Diagnosis of the Accurate Genotype of HKαα Carriers in Patients with Thalassemia Using Multiplex Ligation-Dependent Probe Amplification Combined with Nested Polymerase Chain Reaction

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Diagnosis of the Accurate Genotype of HKαα Carriers in Patients with Thalassemia Using Multiplex Ligation-Dependent Probe Amplification Combined with Nested Polymerase Chain Reaction

Thalassemia, a globally prevalent inherited blood disorder, arises from mutations or deletions in the α- or β-globin genes, leading to imbalanced globin chain synthesis. In China, common α-thalassemia deletions include –SEA, -α³.⁷, and -α⁴.². However, rare genotypes such as the HongKongαα (HKαα) allele and -α³.⁷/αααanti-4.2 pose diagnostic challenges. Conventional methods like gap-polymerase chain reaction (Gap-PCR) and reverse dot blot (RDB) often misdiagnose these variants as -α³.⁷/αα, leading to inaccurate genetic counseling and clinical management. This study addresses these limitations by integrating nested PCR with multiplex ligation-dependent probe amplification (MLPA) to enhance diagnostic accuracy for HKαα and αααanti-4.2 carriers.

Methodology Overview

A cohort of 5,488 peripheral blood samples collected from July 2017 to October 2019 underwent systematic screening. Initial screening employed Gap-PCR to detect four α-globin deletions (-α³.⁷, -α⁴.², –SEA, –THAI) and RDB for three non-deletional α-thalassemia variants (Hb Constant Spring, Hb Quong Sze, Hb Westmead) and 17 β-thalassemia mutations. Samples identified with -α³.⁷ deletions were further analyzed using anti-4.2 multiplex-PCR to detect αααanti-4.2 triplications.

For ambiguous cases, a two-round nested PCR strategy was implemented. The first round amplified large genomic segments (4.0–4.5 kb) spanning recombination junctions, while the second round targeted specific fragments (1.5 kb) to confirm HKαα or αααanti-4.2. MLPA validated copy number variations across the α-globin cluster, distinguishing between deletions, duplications, and complex rearrangements. Statistical analysis using Fisher’s exact test compared the diagnostic accuracy of Gap-PCR alone versus the combined approach.

Key Findings

Among 5,488 samples, 2,544 (46.4%) were diagnosed with thalassemia: 1,190 (46.78%) α-thalassemia, 1,286 (50.55%) β-thalassemia, and 68 (2.67%) compound αβ-thalassemia. Gap-PCR initially identified 227 cases as -α³.⁷/αα, with two suspected HKαα carriers showing atypical banding patterns. Subsequent nested PCR and MLPA reclassified 21 cases:

  • 20 HKαα carriers: 15 HKαα/αα, three HKαα/αα with β-thalassemia coinheritance, one HKαα/–SEA, and one HKαα/-α⁴.2 with β-thalassemia.
  • 1 case of -α³.⁷/αααanti-4.2 with β-thalassemia coinheritance.

Statistical analysis revealed significant differences between Gap-PCR and the combined method in detecting HKαα (P < 0.05). The error rate for Gap-PCR alone was 9.17% (21/229), underscoring the necessity of supplemental techniques.

Technical Insights

Anti-4.2 Multiplex-PCR and Nested PCR

The anti-4.2 multiplex-PCR amplifies a 1.7 kb fragment spanning the X1/X2 hybrid junction, indicative of αααanti-4.2. For HKαα confirmation, nested PCR was designed to detect crossover junctions unique to the allele. The first-round primers (L-anti-4.2-F and L-α³.⁷-R) generated 4.0–4.5 kb products, while the second-round primers (AT4.2-F/R) yielded a 1.5 kb fragment specific to the HKαα recombination. Internal controls (LIS1-2.5 and LIS1-2.0 primers) ensured amplification fidelity.

MLPA for Copy Number Analysis

MLPA resolved ambiguities by quantifying exon-specific copy numbers across the α-globin cluster. Probes targeting regions upstream of HBA2 (HBA2-up) and HBA1 (HBA1-up) differentiated between deletions and triplications. For example:

  • HKαα/αα: HBA2-up (1.5 copies), HBA1-up (0.5 copies).
  • -α³.⁷/αααanti-4.2: HBA2-up (1.0 copy), HBA1-up (0.5 copies).

MLPA also identified coexisting mutations, such as the -α⁴.² deletion in one HKαα carrier, which Gap-PCR alone could not discern.

Clinical Implications

Misdiagnosing HKαα or αααanti-4.2 as -α³.⁷/αα has profound clinical consequences:

  1. HKαα Misdiagnosis: If a carrier’s spouse has –SEA/αα, offspring risk HKαα/–SEA, mimicking α⁰-thalassemia (Hb Bart’s hydrops fetalis). Unnecessary invasive prenatal testing increases miscarriage risk and patient anxiety.
  2. αααanti-4.2 and β-Thalassemia: Coinheritance exacerbates α/β chain imbalance, worsening β-thalassemia severity. The study identified one such case (-α³.⁷/αααanti-4.2 with β-IVS2-654), highlighting the need for accurate counseling.

Hematological Profiles

Most HKαα carriers exhibited near-normal hematological parameters (Table 2). Exceptions included:

  • Patient 7: Severe microcytic anemia (Hb 70 g/L, MCV 64.8 fL) due to concurrent iron deficiency.
  • Patient 16 (-α³.⁷/αααanti-4.2): Moderate anemia (Hb 126 g/L, MCV 77.5 fL) compounded by β-IVS2-654.

These findings align with prior reports that HKαα alone does not significantly alter red cell indices but may synergize with comorbidities or secondary mutations.

Methodological Advantages and Limitations

The nested PCR-MLPA combination offers several advantages:

  • High Specificity: Nested PCR discriminates HKαα from αααanti-4.2, while MLPA validates copy numbers and detects additional rearrangements.
  • Cost-Effectiveness: Compared to sequencing, this approach is economical for large-scale screening.

Limitations include:

  • Balanced Translocations: MLPA may misclassify translocations as deletions/duplications.
  • Rare Genotypes: HKαα/αααanti-4.2 and HKαα/αααanti-3.7 were not encountered, necessitating further validation.

Conclusion

This study demonstrates that integrating nested PCR with MLPA significantly improves diagnostic accuracy for HKαα and αααanti-4.2 in α-thalassemia carriers. The 9.17% misdiagnosis rate observed with Gap-PCR alone underscores the clinical necessity of supplemental testing. By enabling precise genotype-phenotype correlations, this approach enhances genetic counseling, reduces unnecessary interventions, and mitigates the risk of severe thalassemia in offspring. Future studies should expand sample diversity to refine detection protocols and explore the global prevalence of these elusive variants.

doi.org/10.1097/CM9.0000000000000768

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