title: Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene.
abstract: Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate dye-binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA binding dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate dye-binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration.
authors.affiliation: Idaho Technology, Salt Lake City, Utah, USA.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Dobrowolski
authors.forename: Jason T
authors.initials: JT
authors.lastname: McKinney
authors.forename: Cristina
authors.initials: C
authors.lastname: Amat di San Filippo
authors.forename: Keow
authors.initials: K
authors.lastname: Giak Sim
authors.forename: Bridget
authors.initials: B
authors.lastname: Wilcken
authors.forename: Nicola
authors.initials: N
authors.lastname: Longo
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002331:Carnitine / Q000172:deficiency* / Q000378:metabolism; D002648:Child; D018389:Codon, Nonsense; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis / Q000379:methods*; D004200:Diseases in Twins / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis*; D006358:Hot Temperature; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007438:Introns / Q000235:genetics; D008297:Male; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009691:Nucleic Acid Denaturation; D027701:Organic Cation Transport Proteins / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D000074058:Solute Carrier Family 22 Member 5; D014162:Transfection
chemical_list: D018389:Codon, Nonsense; D005456:Fluorescent Dyes; D027701:Organic Cation Transport Proteins; D011993:Recombinant Fusion Proteins; C492020:SLC22A5 protein, human; D000074058:Solute Carrier Family 22 Member 5; D002331:Carnitine