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• PCR-RFLP assay for W24X mutation detection in non-syndromic hearing loss subjects
  Madhu Khullar
  Published on: 6 September 2005

• Published on: 6 September 2005

  PCR-RFLP assay for W24X mutation detection in non-syndromic hearing loss subjects

  • Madhu Khullar, Additional Professor
  • Other Contributors:
    • Seema Bhalla, Rajni Sharma, B S Sehrawat, Naresh K Panda

  Dear Editor,

  We are studying the genetic basis of non-syndromic hearing loss in North Indian population and we performed the PCR-RFLP assay described by authors for the detection of W24X mutation in this article. The assay was carried out using the primers (1F and 1R) and the restriction enzyme Alu1, as described by the authors. However, we have observed a distinctly different RFLP pattern for this mutation as compared...

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  Dear Editor,

  We are studying the genetic basis of non-syndromic hearing loss in North Indian population and we performed the PCR-RFLP assay described by authors for the detection of W24X mutation in this article. The assay was carried out using the primers (1F and 1R) and the restriction enzyme Alu1, as described by the authors. However, we have observed a distinctly different RFLP pattern for this mutation as compared to that reported by Ramshankar et al, 2003.

  The authors have reported that the presence of W24X mutation introduces an Alu1 restriction site. According to them, Alu1 digestion of 286bp PCR amplified product from unaffected subjects produces a single fragment of 286bp, whereas the presence of W24X mutation in homozygous genotype would produce two fragments of 182bp and 104bp and in W24X heterozygotes, it would generate three fragments of 286bp, 182bp and 104bp.

  However, we observed that the presence of W24X mutation in homozygous state produced three fragments of 168bp 104bp and 14bp, whereas in W24X heterozygotes we found four fragments of 272bp, 168bp 104bp and 14bp, which suggested that there may be an additional Alu1 site in the 286bp PCR product. An in-silico PCR amplification followed by restriction digestion using REBASE (http://rebase.neb.com) showed that the 286bp fragment amplified by the given primers carries another Alu1 restriction site independent of the one created by the presence of W24X mutation (Fig.1).

  Hence, even in the absence of W24X mutation, the Alu1 restriction digestion of 286 amplicon gives two fragments of 272bp and 14bp (Fig.2). Indeed, we have observed these RFLP patterns in control and W24X positive samples. Thus, our results confirm the presence of an additional Alu1 site in the 286bp amplified PCR product giving RFLP pattern different from that reported by these authors.

  Figure 1. Alu1 restriction sites in 286bp amplicon (Part of NC_004004).

  

  Figure 2. RFLP patterns for W24X mutation.

  

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  Conflict of Interest:
  None declared.

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