Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in theSLC22A5 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.

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

“…Although HRM curve analysis has been tested and shown to be successful in a number of research laboratories for clinical use [Chou et al, 2005;Dobrowolski et al, 2005Dobrowolski et al, , 2007aDobrowolski et al, , 2007bGrievink and Stowell, 2008;Kennerson et al, 2007;Krypuy et al, 2006;Lonie et al, 2006;Margraf et al, 2006;Poláková et al, 2008;Seipp et al, 2008;Smith et al, 2008;Willmore-Payne et al, 2006], we wanted to assess this method for detecting multiple sequence variants, as well as single and multiple variants (including both constitutional and somatic), within GC-rich fragments, as it would be applied in a diagnostic setting, with minimal expertise and time to manipulate assay design. This was achieved by screening five fragments for 13 variants in a total of 35 different combinations using the LightScanner s System and LC Green s PLUS DNA binding dye (Idaho Technology) as well as screening five GC-rich (Z65%) fragments for 12 variants in 22 combinations using the LightCycler s 480 and HRM Master dye (Roche).…”

“…Table S1, using either the 96-well formatted LightScanner s System (Idaho Technology) or the 384-well formatted LightCycler s 480 (Roche), to generate melt profiles from a change in fluorescence intensity that occurs when the product is heated. Melting data was normalized, temperature shifted, and displayed as derivative curves compared to the known wild-type samples [Dobrowolski et al, 2003[Dobrowolski et al, , 2005McKinney et al, 2004]. The ''auto-group'' function and the default sensitivity setting of the LightScanner s System Call-IT s software was applied to generate automatic genotype groups for five amplicons within the SERPINA1, CXCR7, MBL, and VDR genes.…”

Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling

“…As 420 of the mtDNA variants from healthy donors were homoplasmic, these studies help to address an outstanding issue in melt profiling, the efficiency to identify variation in the absence of heteroduplex molecules (e.g., homozygous, homoplasmic, hemizygous). We originally published that melt profiling identified 30% of homozygous variants in the SLC22A5 gene [Dobrowolski et al, 2005] but a more recent study of the phenylalanine hydroxylase gene indicated 80% of homozygous variants were identified [Dobrowolski et al, 2007a]. Improvements to instrument platforms, dyes, and software have facilitated an increased ability to recognize changes in the melt profile that do not involve heteroduplex molecules.…”

“…High-resolution melting (HRM or HRMA) is an effective method to screen for sequence variation and has been effectively used to assess genes involving inborn errors of metabolism, cancer susceptibility, and other genes whose dysfunction is associated with disease [Bastien et al, 2008;De Leeneer et al, 2008;Dobrowolski et al, 2007aDobrowolski et al, , 2007bDobrowolski et al, , 2005Erali et al, 2008;Laurie and George, 2009]. HRM profiling identifies the presence of sequence variants by deviation in the shape of a post-PCR melting profile using probe-based and amplicon-based strategies [Dobrowolski et al, 2007a[Dobrowolski et al, , 2007bMontgomery et al, 2007;Zhou et al, 2004].…”

Identification of Mutations and Evaluation of Cardiomyopathy in Turkish Patients with Primary Carnitine Deficiency

“…OCTN2 gene is located on chromosome 5q31 with ten exons encoding a 557-amino acid transmembrane protein consisting of 12 transmembrane domains and one ATP-binding domain and predicted molecular mass of 63 kDa (Saito et al 2002;Wu et al 1999). More than 90 mutations have been identified up to date (Amat di San Filippo C et al 2006aFilippo C et al , b, 2008Burwinkel et al 1999;Cederbaum et al 2002;Dobrowolski et al 2005;Koizumi et al 1999;Lamhonwah et al 2002;Li et al 2010;Makhseed et al 2004;Mayatepek et al 2000;Nezu et al 1999;Rahbeeni et al 2002;Spiekerkoetter et al 2003;Tang et al 1999;Vaz et al 1999;Wang et al 1999Wang et al , 2000aWang et al , b, 2001.…”