Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles

Dobrowolski, Steven F.; Ellingson, Clinton; Coyne, Thomas; Grey, Jesse L.; Martin, Ranae; Naylor, Edwin W.; Koch, Richard; Levy, Harvey L.

doi:10.1016/j.ymgme.2007.03.010

Cited by 43 publications

(42 citation statements)

References 29 publications

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2009

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Mutation detection has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to design. Nongel-based systems are therefore important to increase simplicity and improve turn around time without compromising assay sensitivity and accuracy, especially in the diagnostic/ clinical setting. In this study, we assessed the latest of the nongel-based methods, namely high-resolution melt (HRM) curve analysis. HRM is a closed-tube method that incorporates a saturating dye during DNA amplification followed by a monitoring of the change in fluorescence as the DNA duplex is denatured by an increasing temperature. We assessed 10 amplicons derived from eight genes, namely SERPINA1, CXCR7, MBL, VDR, NKX3A, NPY, TP53, and HRAS using two platforms, the LightScanner s System using LC Green s PLUS DNA binding dye (Idaho Technology, Salt Lake City, UT, USA) and the LightCycler s 480 using the HRM Master dye (Roche Diagnostics, Indianapolis, IN, USA). DNA variants (mutations or polymorphims) were previously identified using denaturing gradient gel electrophoresis (DGGE) a method, similarly to HRM, based upon the different melting properties of doublestranded DNA. Fragments were selected based on variant and fragment complexity. This included the presence of multiple sequence variants, variants in alternate orientations, and single or multiple variants (constitutional or somatic) in GC-rich fragments. We demonstrate current limitations of the HRM method for the analysis of complex DNA regions and call for caution when using HRM as the sole method to make a clinical diagnosis based on genetic analysis.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2009

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

“…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]. Recently, incorporation of ''melt calibration'' to ampliconbased genotyping enabled the resolution of alternative homozygotes with basepair-neutral changes (e.g., C4G variant that inverts the basepair C:G to G:C) despite nearest neighbor pairs prediction indicating that no melt temperature (Tm) difference exists between alternative homozygotes [Gundry et al, 2008;Liew et al, 2004].…”

Section: Introductionmentioning

confidence: 99%

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

Dobrowolski¹,

Hendrickx

Bosch

et al. 2009

Hum. Mutat.

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Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. Highresolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301-658 bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A4G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A4G, and m.1555A4G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1 hr.

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“…[9][10][11][12][13][14] PAH mutations are spread throughout the gene and the spectrum of mutations varies among PKU patients from different geographical regions. For example, in the heterogeneous American population the spectrum of mutations is exceedingly broad with p.R408W present at B18% and a small number of other mutations are observed at 6-8%.…”

Section: Introductionmentioning

confidence: 99%

A limited spectrum of phenylalanine hydroxylase mutations is observed in phenylketonuria patients in western Poland and implications for treatment with 6R tetrahydrobiopterin

et al. 2009

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Phenylketonuria (PKU) is an autosomal recessive defect in hepatic metabolism of phenylalanine, which is secondary to mutations in the phenylalanine hydroxylase (PAH) gene. Sixty-seven ethnically Polish PKU patients, followed at the Outpatient Department of Pediatrics and Developmental Medicine in Poznan, Poland, were assessed for mutations in the PAH gene. Two mutations were identified in 61 of 67 patients and a single mutation was identified in the remaining six patients. The four most prevalent mutations (p.R408W, 68%; c.1066-11G4A, 6%; c.1315+1G4A, 5.2%; c.822-832delGCCCATGTATA, 3.7%) accounted for 83% of the mutant alleles. Fifteen additional mutations were identified of which most (13/15) were observed in an individual patient. Before knowledge of PAH genotypes, 19 patients were challenged with a 20 mg kg À1 dose of 6R tetrahydrobiopterin (BH 4 ) and serum phenylalanine concentration was monitored in hospital over 24 h. Two patients responded to the BH 4 challenge with a reduction of serum phenylalanine concentration 430% from baseline. PAH genotypes of the two responsive patients uld have been predicted, as they contained mutations recognized as BH 4 responsive, whereas the 17 patients who were unresponsive would have been predicted as their mutations were either recognized as non-responsive or were highly deleterious frame-shift mutations. Overall, only 7.5% (5/ 67) of patients had PAH mutations recognized as responsive to co-factor therapy. Among the PKU patients from western Poland, PAH mutations responsive to BH 4 therapy are poorly represented; therefore, genotyping may be useful for identifying candidate patients likely to respond to BH 4 before physiological challenge.

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Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles

Cited by 43 publications

References 29 publications

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

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

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

A limited spectrum of phenylalanine hydroxylase mutations is observed in phenylketonuria patients in western Poland and implications for treatment with 6R tetrahydrobiopterin

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