Rapid Detection of Deletion Mutations in Inherited Metabolic Diseases by Melting Curve Analysis with LightCycler

Abstract

Recently, non-gel electrophoresis-requiring, fluorophore probe-based rapid techniques have been introduced to detect known single-point mutations using the LightCyclerTM (Roche Molecular Biochemicals) (1)(2)(3)(4). This technique provides very rapid analytical time, real-time detection, and visualized images. Many inherited metabolic diseases are caused not only by single-point mutations but also by small deletion mutations. However, no studies have been reported on the detection of such deletion mutations using the LightCycler. Using melting curve analysis with the LightCycler, we have succeeded in rapidly detecting a 2-bp deletion mutation in genomic DNA of a patient with Fabry disease and a 9-bp deletion mutation in cDNA of a patient with carbamoyl-phosphate synthase I (CPS1; EC 6.3.4.16) deficiency. Fabry disease is an X-linked recessive disorder caused by the deficient activity of α-galactosidase (α-Gal; EC 3.2.1.22). A 15-year-old boy with classical Fabry disease who had suffered from angiokeratoma, acroparesthesias, and attacks of pain in his legs was referred to us. We extracted total RNA from his peripheral blood lymphocytes and analyzed the α-GAL gene ( GLA ; GenBank accession no. X14448) by reverse transcription-PCR (5). We sequenced a 1.3-kb PCR product covering the entire coding region and found a 2-bp deletion mutation at nucleotides 11 008–11 009. This change caused a frameshift mutation that had been described previously in another case of the disease (6). With written informed consent, we examined the patient’s relatives, including his mother, his unaffected brother, and his maternal grandmother, to determine whether they carry this mutation. Genomic DNAs were obtained from their peripheral blood lymphocytes, using QIAamp Blood Kit® (Qiagen) according to the manufacturer’s instructions. For fluorescence PCR analysis, we prepared two PCR primers (2Del-S and 2Del-AS) and two fluorescence probes (2Del-F and 2Del-LC; Table 1⇓ ).A 25mer oligonucleotide probe, 2Del-LC, synthesized by standard …