Polymorphisms of α-Globin Genes Compromise Polymerase Chain Reaction-Based α-Thalassemia Genotyping in Three Chinese
Manna Sun1, Jiwu Lou1, Ying Zhag1
1a Department of Prenatal Diagnosis Center , Dongguan Maternal and Child Health Hospital , Dongguan , Guangdong Province , People's Republic of China.
Insights
Molecular diagnostic pitfalls in alpha-thalassemia (α-thal) genotyping were identified in three Chinese families due to DNA polymorphisms. These genetic variations caused allele drop-out and assay failures, highlighting the need for careful monitoring in α-thal diagnosis.
Area of Science:
- Molecular Genetics
- Hematology
- Diagnostic Medicine
Background:
- Gap-polymerase chain reaction (gap-PCR) and reversed dot-blot are standard molecular diagnostic methods for alpha-thalassemia (α-thal) genotyping.
- Accurate α-thal genotyping is crucial for genetic counseling and clinical management.
- Unexplained results during routine molecular diagnosis can indicate underlying genetic complexities.
Purpose of the Study:
- To investigate the causes of diagnostic pitfalls encountered in three unrelated Chinese families during α-thal genotyping.
- To identify specific genetic variations leading to assay failures in gap-PCR and reversed dot-blot methods.
- To emphasize the importance of vigilance in molecular diagnostics to prevent misdiagnosis.
Main Methods:
- Collection of blood or chorionic villus samples from affected individuals and family members.
- Performance of hematological investigations.
- Execution of genetic tests, including gap-polymerase chain reaction (gap-PCR) and reversed dot-blot assays, to analyze α-globin gene cluster variations.
Main Results:
- Family 1: A polymorphism (HBA2: c.301-24delinsCTCGGCC) at the primer annealing site caused allele drop-out in PCR-reverse dot-blot.
- Family 2: A synonymous mutation (HBA2: c.376C>T) interfered with the detection of the HBA2: c.377T>C (Hb Quong Sze) mutation.
- Family 3: The HBA2: c.-771_-428del mutation resulted in a PCR fragment size similar to the -α4.2 deletion and was linked to HBA1: c.-673A>G.
Conclusions:
- Genetic polymorphisms and mutations within the α-globin gene cluster can lead to significant diagnostic challenges in α-thalassemia genotyping.
- Technical pitfalls and inherent DNA sample properties can cause diagnostic errors, necessitating careful monitoring.
- Comprehensive genetic analysis and awareness of potential assay limitations are essential to ensure accurate α-thal diagnosis.
Abstract:
In practice, gap-polymerase chain reaction (gap-PCR) and reversed dot-blot are the two most frequently used molecular diagnostic methods for α-thalassemia (α-thal) genotyping. Here, we describe three Chinese individuals from three unrelated families in whom a polymorphism on the α-globin gene cluster led to diagnostic pitfalls. During general molecular diagnosis of thalassemia, three individuals with unexplained results were found. Blood or chorionic villus samples were collected from these three individuals and their family members. Hematological investigations and genetic tests were performed. In Family 1, a polymorphism of HBA2: c.301-24delinsCTCGGCC at the annealing site of the forward primer used in the PCR-reverse dot-blot assay was identified, leading to allele drop-out during the PCR amplification process. In Family 2, a synonymous mutation of C>T substitution at codon 125 of the α2 gene (HBA2: c.376C>T) was identified, leading to the failure of PCR-reversed dot-blot for the HBA2: c.377T>C (Hb Quong Sze or Hb QS) mutation. In Family 3, the size of the PCR fragment from the α2-globin allele carrying the HBA2: c.-771_-428del mutation was smaller and nearly equal to the size of the fragment corresponding to the -α4.2 (leftward) deletion; we also found that the HBA2: c.-771_-428del mutation was linked to a known HBA1: c.-673A>G mutation in this family. In conclusion, diagnostic errors may be caused by technical pitfalls or inherent properties of the DNA sample. All logical steps should be taken to monitor and thus preclude such events.
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