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Letters to the Editor |
-Thalassemia Genotyping Resulting from a (G
C) Polymorphism at Nucleotide 71 3' to the 2-Globin Gene Termination Codon
1 Division of Hematology, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, ROC
2 Departments of Medicine, and Pathology, Boston University, School of Medicine, Boston, MA
3 Departments of Pediatrics, and Children’s Medical Institute, National University of Singapore, and Hospital, Singapore
4 Department of Pathology, Hong Kong Sanatorium, and Hospital, Hong Kong, ROC
aAddress correspondence to this author at: Division of Hematology, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, People’s Republic of China. Fax 852-2817-7565; e-mail amychan{at}pathology.hku.hk
To the Editor:
-Thalassemia is commonly diagnosed by PCR-based molecular methods, such as gap-PCR for deletional -thalassemia (1) and minisequencing (SNaPshotTM system) for nondeletional -globin gene mutations (2). However, "allele dropout" in the PCR amplification process can lead to erroneous results. We describe 3 individuals from 2 unrelated families in whom a hitherto unknown G
C single-nucleotide polymorphism (SNP) in the 2-globin gene led to apparent but erroneous diagnosis of homozygosity for -thalassemia mutations.
In family 1, a 3-year-old Chinese-American boy had a hemoglobin (Hb) of 118 g/L and a mean corpuscular volume of 62 fL. Initial study with the 7-deletion multiplex PCR system (1) showed only 1 single fragment diagnostic for the (– –SEA) -thalassemia deletion. This result indicated that the child was homozygous for the (– –SEA) deletion, which would be incompatible with life. Accordingly, his 2-globin gene was amplified with a different primer pair (3). Direct nucleotide sequencing showed a GC SNP at nucleotide 71 after the TAA termination codon. This SNP is located at the 5th position from the 3' end of the reverse primer (2-R) that was used in the 7-deletion multiplex PCR system. The child therefore was heterozygous for the Southeast Asian -thalassemia deletion (– –SEA/ ).
In family 2, a 9-year-old girl of Thai/Chinese ancestry with Hb H disease and her family were referred for molecular diagnosis. Their laboratory results are shown in Fig. 1
. The 7-deletion multiplex PCR system (1) showed that the proband and her father were heterozygous for the (– –SEA) deletion, and her sister was homozygous for the same deletion, which would be incompatible with life. The SNaPshot minisequencing system (2) revealed that both the proband and her mother appeared to be homozygous for Hb Constant Spring (HbCS). The latter diagnosis was not possible because this woman’s other daughter did not have HbCS (Fig. 1
). Accordingly, the 2-globin genes from the proband’s mother and sister were amplified with a different primer pair and sequenced (3). The same GC SNP was found in them as in family 1 and would account for the initial diagnostic results.
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The unexpected and erroneous genotyping results from all 3 individuals described in this report stem from the presence of a hitherto unknown GC SNP in their 2-globin gene, which happens to be within the annealing site of the reverse primer used in the 7-deletion multiplex PCR assay and the SNaPshot multiplex minisequencing assay, thereby causing allele dropout of the expected 2-globin gene allele during PCR. Only the 2-globin allele carrying the (– –SEA) deletion was amplified in the child in family 1 and the proband’s sister in family 2, leading to apparent homozygosity for the (– –SEA) deletion. Similarly, only the 2-globin allele containing the HbCS mutation was amplified in the proband’s mother in family 2, leading to apparent HbCS homozygosity.
We have since screened an additional 92 unrelated Chinese chromosomes (from 46 apparently healthy individuals) for this nucleotide change, and none of them harbored this SNP. Furthermore, in more than 50 nondeletional Hb H disease patients of Thai origin, we have not encountered any allele dropout of the 2-globin gene when performing the 7-deletion multiplex PCR assay. It is likely that this is a rare SNP among these 2 ethnic populations.
The present report underscores the importance of always correlating clinical and hematologic data, and family study if available, with DNA-based diagnostic results to arrive at the correct globin genotypes. Although the discrepancy is obvious when a molecular diagnostic result is incompatible with life, such as (– –SEA/– –SEA), other possible erroneous diagnoses, such as (CS /CS ) or (– 3.7/– 3.7), might not be as easily identified. Awareness of possible allele dropout as one diagnostic pitfall in PCR-based genotyping is essential. When necessary, alternative diagnostic protocols, such as repeating the PCR with other primer pairs and direct nucleotide sequencing, ought to be carried out for unexplained or discordant findings. This is particularly important in the setting of genetic counseling and prenatal diagnosis of -thalassemia so as to avoid misdiagnosis.
References
-thalassemia. Blood 2001;98:150-151.-thalassemia mutations. Clin Chem 2003;49:800-803. globin mutations by selective amplification and direct sequencing. Br J Haematol 1990;76:275-281.[Medline]
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, wild-type 
, – –SEA double-gene deletion allele; 
, 
, 
, ß-globin Hb E allele.