A 3-year-old boy from Tamil Nadu presents with severe hemolytic anemia, hepatosplenomegaly, and bone deformities. Peripheral blood smear shows hypochromic microcytic RBCs with target cells and nucleated RBCs. Which investigation is most appropriate to confirm the diagnosis of β-thalassemia major and determine the molecular defect?

Question

Accepted Answer

B. DNA sequencing of β-globin gene. ## Investigation Strategy for Thalassemia Diagnosis

### Diagnostic Hierarchy

**Key Point:** While HPLC and hemoglobin electrophoresis confirm the hemoglobin phenotype (elevated HbF and HbA2 in β-thalassemia major), DNA sequencing of the β-globin gene is the gold standard for identifying the specific molecular defect and enabling genetic counseling and prenatal diagnosis.

### Role of Each Investigation

| Investigation | Purpose | Limitation |
|---|---|---|
| **HPLC** | Quantifies HbA, HbF, HbA2 levels; confirms thalassemia phenotype | Does not identify the underlying mutation |
| **Hemoglobin electrophoresis** | Separates hemoglobins by charge; detects abnormal variants | Less sensitive than HPLC; does not identify molecular defect |
| **DNA sequencing (β-globin gene)** | Identifies point mutations, deletions, insertions; enables genetic counseling | Gold standard for molecular diagnosis and family planning |
| **Osmotic fragility test** | Assesses RBC membrane integrity | Not specific for thalassemia; used for hereditary spherocytosis |

### Clinical Context

**High-Yield:** In β-thalassemia major, HPLC will show:
- Elevated HbF (fetal hemoglobin, 50–90%)
- Elevated HbA2 (α2δ2, >3.5%)
- Absent or severely reduced HbA (normal adult hemoglobin)

However, the question asks for confirmation AND determination of the **molecular defect** — only DNA sequencing provides this.

**Clinical Pearl:** Knowing the specific β-globin mutation is essential for:
- Genetic counseling of parents
- Prenatal diagnosis in future pregnancies
- Identifying patients eligible for gene therapy trials
- Predicting severity (some mutations cause milder disease)

**Warning:** Do not confuse HPLC (phenotypic diagnosis) with DNA sequencing (genotypic diagnosis). NEET PG frequently tests this distinction.

Answer

A. Hemoglobin electrophoresis on cellulose acetate

Answer

C. High-performance liquid chromatography (HPLC)

Answer

D. Osmotic fragility test

Explanation

Investigation Strategy for Thalassemia Diagnosis

Diagnostic Hierarchy

Role of Each Investigation

Clinical Context

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Investigation	Purpose	Limitation
HPLC	Quantifies HbA, HbF, HbA2 levels; confirms thalassemia phenotype	Does not identify the underlying mutation
Hemoglobin electrophoresis	Separates hemoglobins by charge; detects abnormal variants	Less sensitive than HPLC; does not identify molecular defect
DNA sequencing (β-globin gene)	Identifies point mutations, deletions, insertions; enables genetic counseling	Gold standard for molecular diagnosis and family planning
Osmotic fragility test	Assesses RBC membrane integrity	Not specific for thalassemia; used for hereditary spherocytosis