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Chinese Group Develops Multicolor Melt Curve Test for Non-Deletional Alpha-Thalassemia Mutations

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NEW YORK (GenomeWeb) – A group of researchers in China has developed a method to detect a set of mutations which lead to the blood disorder alpha-thalassemia.

The disorder is caused by deletional and non-deletional mutations in the alpha-globin gene cluster and it is considered one of the most common monogenic disorders in the world. There are two loci for the alpha-globin gene, and symptoms increase in severity with the number of affected alleles, with four mutations often being lethal at birth.

The disorder also has prevalence rates of up to 17 percent in Southern China, but non-deletional mutations have proven challenging to detect.

Now, a group led by Qingge Li, director of the engineering research center for molecular diagnostics at Xiamen University, has shown that multi-color melt-curve analysis (MMCA) could be the best way to tackle this problem.

In a study published last month in Clinical Chemistry and Laboratory Medicine, Li and his team showed an MMCA assay for non-deletional mutations was reproducible and 100 percent accurate when compared to Sanger sequencing.

Unlike systems using hybridization probes or adjacent probes, MMCA uses independent unpaired probes that can be easily synthesized and can be detected by individual channels in real-time PCR machines, Li told GenomeWeb in an email.

The group had previously developed a diagnostic assay for beta-thalassemia based on the MMCA concept. The assays use "dual-labeled, self-quenched probes, and can be used in any multi-color real-time PCR machine," Li said.

The beta-thalassemia test has since been made into a Chinese Food and Drug Administration-approved diagnostic kit called MeltPro HBB, which is marketed by Zeesan Biotech of Xiamen, China.

The alpha-thalassemia assay thus reflects sustained efforts to develop diagnostic assays for all variants of thalassemia, Li explained.

Zeesan also markets a Xiamen University-developed tuberculosis drug-resistance assay which uses MMCA technology and was recently clinically validated, as previously reported. The MMCA method has been granted patents in China, Europe, and the US, Li noted.

Alpha-globin genes have two highly homologous types, HBA2 and HBA1, which are located sequentially on chromosome 16, Li said.

The disease-causing mutations are on HBA2, so specific primer pairs must be designed to amplify HBA2 only.

"Such a design yields a PCR amplicon of 685 bp, which is much longer than that recommended for real-time PCR; moreover, the amplicon has a GC-content of 68.5 percent, which also exerts a challenge for efficient amplification," Li said.

These technical difficulties provided extra inspiration to develop and perfect the MMCA technique. The assay was ultimately able to detect six non-deletional alpha-thalassemia mutations within the 685-bp region in a single closed-tube test. 

In comparison with other current methods, the assay also has the advantages of being rapid and easy to use, with a lower chance for PCR product contamination, Li said, adding, "These advantages confer its immediate applicability in clinical settings."

The group is now working on integrating the non-deletional alpha thalassemia test with a recently developed deletional alpha-thalassemia assay. The combined assay will cover over 99 percent of mutations associated with the disorder, Li said.

The group is also developing assays for several other serious inherited diseases in China, including a hearing loss assay now undergoing a multicenter evaluation and a phenylketonuria assay expected to be finished this year.

"All these assays are under the name of MeltPro and are expected to be used for neonatal screening in China," he said.

The beta-thalassemia mutation, meanwhile, has the distinction of being the first gene in the human germline to be edited using the CRISPR/Cas9 genome editing system. 

Alpha-thalassemia is also potentially one of the targets of Boston-headquartered Veritas Genetics' R&D center in China, which is also developing genetic products targeting the Asian population.

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