Diagnosis of the Accurate Genotype of HKaa Carriers in Thalassemia Patients

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

Thalassemia, one of the world’s most common single-gene inherited disorders, disrupts hemoglobin production and affects millions—particularly in Southeast Asia and Southern China. For patients with alpha-thalassemia (a-thal), a major challenge is misdiagnosis: two rare genotypes—HongKongaa (HKaa) and -a3.7/aaaanti-4.2—often appear identical to the common -a3.7/aa deletion on standard tests. This mix-up can lead to unnecessary procedures, wrong genetic counseling, and worse health outcomes for families. A 2020 study from Sichuan Provincial People’s Hospital in China set out to solve this by combining advanced genetic tests to correctly identify these tricky genotypes.

Who Led the Study?

The research was led by Dong-Mei Chen (Clinical Medical School, Southwest Medical University) and Zheng-Lin Yang (Key Laboratory for Human Disease Gene Study of Sichuan Province, Sichuan Provincial People’s Hospital). The team included Shi Ma, Xiang-Lan Tang, and Ji-Yun Yang from the same institutions. The study followed ethical guidelines, with approval from the hospital’s Institutional Review Committee and informed consent from all participants.

How the Study Worked

From July 2017 to October 2019, the team analyzed 5,488 peripheral blood samples from patients tested for thalassemia. They used three tiers of testing:

  1. Standard Screen:

    • Gap-PCR: Detected common a-thal deletions (-a3.7, -a4.2, –SEA).
    • Reverse Dot Blot (RDB): Identified point mutations in a-globin (e.g., Hb Constant Spring) and 17 Chinese b-thal mutations (e.g., IVS-2-654).

  2. Targeted Follow-Up:
    For 229 patients initially labeled -a3.7/aa (one a-globin cluster with a 3.7-kb deletion, one normal), they added:

    • Nested PCR: A sensitive test to spot the HKaa allele (which has both -a3.7 and aaaanti-4.2 junctions on the same chromosome).
    • MLPA: A copy-number test to check for duplications (e.g., aaaanti-4.2) or additional deletions (e.g., –SEA).

Key Findings

Of 5,488 samples, 2,544 tested positive for thalassemia:

  • 1,190 (46.8%) had a-thal, 1,286 (50.6%) had b-thal, and 68 (2.7%) had combined ab-thal.

The critical discovery came from the 229 patients initially diagnosed as -a3.7/aa. Using nested PCR and MLPA, the team found:

  • 20 HKaa carriers: 15 had HKaa/aa (no other mutations), 3 had HKaa/aa + b-thal, 1 had HKaa/–SEA (severe a-thal), 1 had HKaa/-a4.2 + b-thal.
  • 1 patient with -a3.7/aaaanti-4.2: A deletion plus a duplication, combined with b-thal.

Statistically, the standard Gap-PCR test missed these genotypes—9.17% of -a3.7 patients were misdiagnosed. The difference between Gap-PCR alone and the combined tests (Gap-PCR + nested PCR + MLPA) was significant (P < 0.05).

Why Misdiagnosis Harms Patients

Mislabeling HKaa or -a3.7/aaaanti-4.2 as -a3.7/aa creates two major risks:

  1. Unnecessary Prenatal Risk:
    HKaa carriers have normal blood counts, but if misdiagnosed as -a3.7/aa, a spouse with –SEA/aa might trigger amniocentesis (a miscarriage risk) for a fetus that’s actually healthy (HKaa/–SEA, mild a-thal).

  2. Worse Outcomes for Offspring:
    For -a3.7/aaaanti-4.2 patients, a spouse with b-thal means a 1-in-4 chance of a child inheriting both aaaanti-4.2 (extra a-globin) and b-thal (low b-globin). This worsens anemia by unbalancing the a/b chain ratio—something misdiagnosis would miss.

Why Nested PCR + MLPA Is Better

Standard tests fail because HKaa and -a3.7/aaaanti-4.2 mimic -a3.7/aa. Nested PCR spots the unique HKaa sequence, while MLPA checks for duplications or extra deletions. Together, they:

  • Confirm if deletions/duplications are on the same chromosome (HKaa) or separate (-a3.7/aaaanti-4.2).
  • Uncover coinherited mutations (e.g., b-thal, –SEA) that change treatment.

Limitations and Next Steps

The study had gaps: no “gold standard” for HKaa testing, and some rare genotypes (e.g., HKaa/aaaanti-4.2) were missing. But the takeaway is clear: testing for HKaa and aaaanti-4.2 in -a3.7 carriers is non-negotiable for accurate care.

Conclusion

For patients with -a3.7 deletions—one of the most common a-thal findings—standard tests aren’t enough. Adding nested PCR and MLPA cuts misdiagnosis, provides reliable genetic counseling, and protects families from avoidable harm. As this study shows, HKaa is far more common than thought (8.81% of -a3.7 carriers here). Ignoring these genotypes puts patients at risk.

If you or a loved one has thalassemia, ask about testing for HKaa and aaaanti-4.2—especially if you have a -a3.7 deletion. Accurate diagnosis saves lives.

Dong-Mei Chen, Shi Ma, Xiang-Lan Tang, Ji-Yun Yang, Zheng-Lin Yang. Diagnosis of the Accurate Genotype of HKaa Carriers in Patients with Thalassemia Using Multiplex Ligation-Dependent Probe Amplification Combined with Nested Polymerase Chain Reaction. Chinese Medical Journal 2020;133(10):1175–1181. doi.org/10.1097/CM9.0000000000000768

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