High-Resolution Melting Analysis Is a More Effective Approach for Screening TSC Genes Mutations

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with a prevalence of 1 in 95,136 in Taiwan. TSC is characterized by hamartomatous lesions in multiple organ systems. Genetic defects in TSC1 and TSC2 genes are the main causes of TSC. A molecular screening protocol using denaturing high-performance liquid chromatography (dHPLC) followed by DNA sequencing is currently performed to locate the genetic lesions in many clinical laboratories. The current screening approach is time consuming and inefficient. In this study, we analyzed all coding exons of TSC1 and TSC2 genes of 30 TSC patients and 47 unaffected family members using the traditional dHPLC protocol and our recently developed diagnostic platform based on high-resolution melting analysis (HRM) followed by bidirectional DNA sequencing. Data indicated that 20 mutations, including 5 mutations in TSC1 (2 sporadic, 1 familial mutation, and 2 of uncertain origin) and 15 mutations in TSC2 (14 sporadic and 1 familial mutation), 8 single-nucleotide polymorphisms (SNPs, including 3 SNPs found in irrelevant individuals without TSC phenotypes studied in the control group), and 3 variants with undetermined significance were identified, including 4 novel mutations. The sensitivities of HRM and dHPLC for TSC mutation screening were estimated as 95% and 75%, respectively. The specificities of HRM and dHPLC for TSC mutation screening were evaluated as 91% and 98%. In addition, results suggested our novel HRM screening protocol to be more economical. In conclusion, we successfully developed a superior approach for TSC genes mutation screening for clinical application.

Introduction

Tuberous sclerosis complex (TSC, OMIM 191100 and OMIM 613254) is an autosomal dominant multisystem disorder first described by Bourneville in 1880. TSC is characterized by benign tumors, namely hamartomas, in patients' brain, skin, heart, or kidney. Most children and adolescents suffering from TSC show seizures, mental retardation, autism, and/or behavioral abnormalities. Renal complications are most frequently responsible for TSC-related death (Curatolo et al., 2008). The prevalence of TSC is estimated to be 1 in 95,136 in Taiwan (Hong et al., 2009; Morrison, 2009).

Mutations in either the TSC1 or TSC2 gene are the main causes of TSC. Linkage analysis and positional cloning reveal that these two genes locate on chromosome 9q34 (Fryer et al., 1987; van Slegtenhorst et al., 1997) and 16p13.3 (Kandt et al., 1992; European Tuberous Sclerosis Consortium, 1993), respectively. TSC1 consists of 21 coding exons encoding a 130-kDa protein termed as hamartin (van Slegtenhorst et al., 1997). TSC2 is composed of 41 coding exons encoding a 200-kDa protein, namely tuberin, which contains a domain with GTPase-activating activity (European Tuberous Sclerosis Consortium, 1993; van Bakel et al., 1997). Hamartin and tuberin form a heterodimeric complex regulating mammalian target of rapamycin complex (mTORC) to control protein synthesis, cell growth, and proliferation. The hamartin-tuberin complex is regulated by the phosphoinositide 3-kinase/AKT pathway and extracellular signal-regulated kinase (ERK) (Jozwiak et al., 2008; Huang and Manning, 2009).

According to previous reports, approximately 80% of mutations in both genes are sporadic. Mutations in both genes are distributed over all coding exons, and no mutation hotspot has been reported (Jones et al., 1999b; Niida et al., 1999; Zhang et al., 1999; Yamashita et al., 2000; Dabora et al., 2001; Langkau et al., 2002; Emmerson et al., 2003; Hung et al., 2006; Au et al., 2007; Le Caignec et al., 2009). Although it has been documented that mutations occur most frequently in exon 15 of TSC1 and exon 16 of TSC2, the percentages of mutations in both exons are only 7% (Au et al., 2007). In addition, it is not clear whether this percentage is applicable in the Taiwanese TSC population.

High-resolution melting analysis (HRM) and denaturing high-performance liquid chromatography (dHPLC) are powerful tools for mutation screening (Yu et al., 2006; Taylor, 2009). Both of them rely on heteroduplex formation of polymerase chain reaction (PCR) amplicons. HRM exploits saturated fluorescent dye to recognize the differential melting curves of hetero- and homoduplex amplicons. dHPLC utilizes the differential retention of partially denatured hetero- and homoduplex amplicons to distinguish sequence variants. dHPLC has been used for TSC mutation screening for 10 years (Jones et al., 1999a). Compared with former techniques, single-strand conformation polymorphism analysis and conventional heteroduplex analysis, which were used to identify TSC1 and TSC2 mutations, dHPLC was the one with highest sensitivity in the past (Jones et al., 2000). However, dHPLC protocol takes a long period of time for screening of all TSC1 and TSC2 coding exons even though it is a highly automated technique.

Vigabatrin is an antiseizure drug often prescribed for TSC-related epilepsy, especially for infantile spasms. However, it is useful only for patients with a mutant TSC1 gene but not for those with lesions in the TSC2 gene. In this study, blood samples from 30 TSC patients and 47 unaffected family members were collected and screened with the well-established dHPLC protocol (Jones et al., 2000) and our recently developed rapid molecular diagnostic platform based on HRM for TSC1 and TSC2 genes followed by DNA sequencing to demonstrate that our novel protocol provided a superior approach for clinical screening of TSC genes mutation.

Materials and Methods

Patient recruitment

With IRB approval, peripheral blood samples from 30 TSC patients and 47 unaffected family members were recruited from the Division of Medical Genetics and Neonatology, Department of Pediatrics, Chang Gung Memorial Hospital, Lin-Kou (J.-L. L.); Department of Pediatric Neurology, Chang Gung Memorial Hospital, Taipei (C.-Y. W. and P.-C. H.); and Department of Pediatrics, Mackay Memorial Hospital, Taipei (S.-P. L.). TSC diagnosis was made by clinicians according to the diagnostic criteria (Curatolo et al., 2008). All blood samples were subjected to genomic DNA extraction and were stored in −80°C until assay.

Genomic DNA extraction

Genomic DNA was extracted from each peripheral blood sample using Puregene Blood Core Kits (Qiagen, Hilden, Germany) according to the manufacturer's guide (Liou et al., 2004). The extracted DNA concentration and quality were measured using a GeneQuant 1300 Spectrophotometer (Piscataway, NJ).

Polymerase chain reaction before dHPLC and HRM analysis

For dHPLC screening, PCR was performed in a 50 μL reaction mixture containing 50 ng of genomic DNA, 1×PCR buffer (Protech Technology, Taipei, Taiwan), 0.2 mM of dNTP (Fermentas, Glen Burnie, MD), 0.3 M of each primer (Protech Technology), and 0.05 U of HotStart Taq DNA polymerase (Protech Technology). Amplification was carried out in T1 or TPersonal thermocyclers (Biometra, Goettingen, Germany). The reaction mixture was preheated at 95°C for 15 min and then cycled 38 times at 95°C for 40 s, 50 s for primer annealing, and elongation at 72°C for 30 s. An additional 7 min at 72°C was added to the final cycle to complete the extension. Primer sequences and annealing temperatures were adopted per Jones et al. (2000). There are 21 and 41 coding exons in TSC1 and TSC2 genes, respectively. Exon 15 and exon 23 of TSC1 were divided into three amplicons, exon 21 of TSC1 was divided into two amplicons, and exon 33 of TSC2 was divided into three amplicons. Totally, 69 amplicons need to be analyzed for each TSC case.

For HRM screening, PCR was carried out for 40 cycles of amplification in a 10 μL reaction mixture containing 10 ng of genomic DNA, 1×LCGreen Plus dye (Idaho Technology, Salt Lake City, UT), and other components as same as mentioned above.

TSC1 and TSC2 genes mutation screening using dHPLC

dHPLC was carried out with a WAVE System (Transgenomic, Omaha, NE). In brief, PCR products were automatically analyzed in an ion-pair reversed-phase condition with 0.1 M triethylammonium acetate (Transgenomic), DNAsep column (Transgenomic) for stationary phase, and linear acetonitrile (Mallinckrodt Baker, Phillipsburg, NJ) gradient for mobile phase (Jones et al., 2000). Analytical temperatures were predicted using Navigator Software (Transgenomic).

TSC1 and TSC2 genes mutation screening using HRM

HRM was performed using a LightCycler 480 Instrument (Roche Applied Science, Mannheim, Germany) with Multiwell Plate 384 (Roche Applied Science). Fluorescence signals from LCGreen Plus dye (Idaho Technology) were acquired 25 times per degree continuously from 60°C to 95°C, with a rate of 0.02°C/s. Differential melting curves of hetero- and homoduplex amplicons were analyzed and displayed with Gene Scanning Software (Roche Applied Science).

DNA sequencing

Bidirectional DNA sequencing was performed in the DNA Sequencing Core Laboratory of Chang Gung Memorial Hospital, Lin-Kou.

Variant nomenclature and classification

DNA sequence variant nomenclature follows the recommendations of Human Genome Variation Society (www.hgvs.org/mutnomen/). The variant locus numbering is based on TSC1 and TSC2 coding sequences published by HUGO Gene Nomenclature Committee (www.genenames.org/index.html).

Mutation/polymorphism classification depends upon the mutation database of HUGO Gene Nomenclature Committee.

Cost and analytical time evaluation

For future clinical application, we evaluated the cost and analytical time for mutation screening of TSC1 and TSC2 genes. Cost evaluation includes the reagents in PCR, dHPLC, HRM, and DNA sequencing. Analytical time assessment contains the duration of dHPLC and HRM.

Results

Identification and characterization of TSC1 and TSC2 variants

Twenty-six TSC1 and TSC2 variants, including seven novel variants not previously reported, were detected in 30 TSC patients and 47 unaffected family members by dHPLC and/or HRM screening followed by bidirectional DNA sequencing validation. Twenty variants including 17 mutations, 1 single-nucleotide polymorphism (SNP), and 2 variants with undetermined significance (VUS) were detected in TSC patients. Two SNPs and 1 VUS were found in unaffected family members. Three SNPs were identified in the disease-free control group (Table 1). Four mutations were discovered in TSC1 gene including two sporadic and one familial mutation. The inheritance of the mutation in Case 38 could not be determined without family members' samples. Thirteen sporadic mutations were identified in TSC2 gene (Table 1). The mutation types are listed in Table 1.

Table 1.

TSC1 and TSC2 Variants Identified in Taiwanese Tuberous Sclerosis Complex Population by Denaturing High-Performance Liquid Chromatography and/or High-Resolution Melting Analysis Protocols

Case no.	Gene	Locus	Nucleotide change	Codon change	Variation type	Mutation type	Inheritance	Reference	dHPLC	HRM
40, 41	TSC1	Exon 8	c.682C→T	p.R228X	Mutation	Nonsense	S	Au et al. (2007)	P	P
25	TSC1	Exon 10	c.965T→C	p.M322T	SNP	—	—	Au et al. (2007)	P	P
C	TSC1	Exon 14	c.1335A→G	p.E445E	SNP	—	—	Au et al. (2007)	P	P
6, 6-2^a	TSC1	Exon 15	c.1884_1887delAAAG	—	Mutation	Frameshift	F	Hung et al. (2006)	P	P
38	TSC1	Exon 17	c.2103_2106dupGTTA^b	—	Mutation	Frameshift	N/A^c	—	N/A^d	N/A^d
7	TSC1	Exon 18	c.2283C→A	p.Y761X	Mutation	Nonsense	S	Hung et al. (2006)	P	P
29, 29-2^a	TSC1	Exon 19	c.2485A→T^b	p.S829C	VUS	Missense	—	—	P	P
40-1^a	TSC1	Exon 22	c.2829C→T	p.A943A	SNP	—	—	Au et al. (2007)	P	P
40-1^a	TSC1	Intron 3	c.106+15A→G	—	SNP	—	—	Hung et al. (2006)	P	P
19	TSC2	Exon 1	c.133_136delCTGA	—	Mutation	Frameshift	S	Hung et al. (2006)	P	P
2	TSC2	Exon 11	c.1226_1230delAACTG	—	Mutation	Frameshift	S	Hung et al. (2006)	P	P
5	TSC2	Exon 12	c.1336C→T	p.Q446X	Mutation	Nonsense	S	Hung et al. (2006)	P	P
11	TSC2	Exon 14	c.1565A→C^b	p.H522P	VUS	Missense	—	—	P	P
15	TSC2	Exon 18	c.2086T→C	p.C696R	Mutation	Missense	S	Hung et al. (2006)	N	P
22	TSC2	Exon 19	c.2103_2105dupTGA	p.D702dup	Mutation	In-frame	S	Hung et al. (2006)	P	P
11	TSC2	Exon 21	c.2461A→T	p.K821X	Mutation	Nonsense	S	Hung et al. (2006)	P	P
34	TSC2	Exon 23	c.2670delT^b	—	Mutation	Frameshift	S	—	N	P
28	TSC2	Exon 26	c.2974C→T	p.Q992X	Mutation	Nonsense	S	Hung et al. (2006)	P	P
27	TSC2	Exon 30	c.3693_3696delGTCT	—	Mutation	Frameshift	S	Au et al. (2007)	P	P
39-2^a	TSC2	Exon 33	c.4436C→T^b	p.A1479V	VUS	Missense	—	—	P	P
13	TSC2	Exon 33	c.4438_4439insA^b	—	Mutation	Frameshift	S	—	P	P
8	TSC2	Exon 40	c.5227C→T	p.R1743W	Mutation	Missense	S	Hung et al. (2006)	N/A^d	N/A^d
12, 14	TSC2	Exon 40	c.5238_5255delCATCAAGCGGCTCCGCCA	p.1746delHIKRLR	Mutation	In-frame	S	Au et al. (2007)	P	P
32	TSC2	Exon 41	c.5402_5403delAG^b	—	Mutation	Frameshift	S	—	P	P
C	TSC2	Intron 39	c.5161-9C→T	—	SNP	—	—	Hung et al. (2006)	P	P
C	TSC2	Intron 39	c.5161-10A→C	—	SNP	—	—	Au et al. (2007)	P	P

Unaffected family member.

Novel DNA sequence variant.

No samples of family members available.

Sample depleted.

C, control group; S, sporadic; F, familial; N/A, not applicable; P, positive; N: negative; SNP, single nucleotide polymorphism; VUS, Variant of undetermined significance; TSC, tuberous sclerosis complex.

After mutation screening, no mutation or VUS was identified from 10 of 30 TSC patients. These 10 patients were subjected to whole-gene DNA sequencing of the TSC1 and TSC2 genes. An additional three mutations and two SNPs were found (Table 2). Among them, one mutation was located in the TSC1 gene of Case 45. Because no other family members' samples were accessible, whether this mutation is sporadic or hereditary was undetermined. One familial and one sporadic mutation were identified in the TSC2 gene (Table 2). The mutation types are displayed in Table 2.

Table 2.

The Variants of TSC1 and TSC2 Found in Taiwanese Tuberous Sclerosis Complex Cases with Normal Mutation Screening with Direct DNA Sequencing

Case no.	Gene	Locus	Nucleotide change	Codon change	Variation type	Mutation type	Inheritance	Reference
18	TSC1	Exon 5	c.231C→T	p.N77N	SNP	—	—	—
45	TSC1	Exon 15	c.1525C→T	p.R509X	Mutation	Nonsense	N/A^b	Au et al. (2007)
46	TSC1	Exon 15	c.1726T→C	p.L576L	SNP	—	—	Au et al. (2007)
42, 42-1^a, 42-2^a	TSC2	Exon 13	c.1372C→T	p.R458X	Mutation	Nonsense	F	Au et al. (2007)
26	TSC2	Exon 26	c.3094C→T	p.R1032X	Mutation	Nonsense	S	Au et al. (2007)

Affected family member.

No samples of family members available.

Sensitivities and specificities of dHPLC and HRM screening

Because of limited DNA samples available, Cases 8 and 38 were analyzed with direct DNA sequencing. Excluding the 2 variants in exhausted cases, 24 TSC variants were detected by HRM protocol during screening of TSC1 and TSC2 coding exons, and 22 of them were revealed by the dHPLC protocol (Table 1). Repeated experiments demonstrated that TSC2 c.2086T→C and TSC2 c.2670delT (data not shown) could be identified using HRM screening rather than dHPLC screening (Fig. 1A). According to DNA sequencing, we calculated the number of true-positive, false-negative, true-negative, and false-positive PCR amplicons during dHPLC and HRM screening for the 30 TSC patients (Table 3). Sensitivity was calculated through a formula that the number of true-positive amplicons was divided by the number of true-positive plus false-negative amplicons. Specificity was calculated through a formula that the number of true-negative amplicons was divided by the number of true-negative and false-positive amplicons. The data are summarized in Table 3.

FIG. 1.

Detection results of TSC1/TSC2 gene variants. (A) Typical data of dHPLC, HRM, and DNA sequencing of mutation detection. In dHPLC and HRM data, blue line indicates control without known mutation or SNP, whereas red line indicates samples with denoted mutation or SNP. Green line in dHPLC data is negative control for PCR amplification. In DNA sequencing data, arrow indicates the sequence variant detected by forward DNA sequencing, whereas arrowhead points out the sequence variant detected by reverse DNA sequencing. Green, red, blue and black peaks represent A, T, C and G, respectively. (B) Schematic diagram of representative sequence variant locations within the amplicon and detection rates of HRM screening for them. TSC2 c.5161-10 is the 60th nucleotide of exon 40 amplicon (281 bp). TSC1 c.682 is the 64th nucleotide of exon 8 amplicon (197 bp). TSC2 c.2670 is the 94th nucleotide of exon 23 amplicon (193 bp). TSC2 c.1336 is the 109th nucleotide of exon 12 amplicon (180 bp). TSC1 c.106+15 is the 239th nucleotide of exon 3 amplicon (298 bp). TSC2 c.133_136 are the 207th to 210th nucleotides of exon 1 amplicon (253 bp). Detection rates of HRM screening for each variant were assessed through repeated experiments for 10 times. Color images available online at www.liebertonline.com/gtmb.

Table 3.

Sensitivities and Specificities of Denaturing High-Performance Liquid Chromatography and High-Resolution Melting Analysis

	True positive (amplicon)	False negative (amplicon)	Sensitivity (%)
dHPLC	18	6	75
HRM	20	1	95

	True negative (amplicon)	False positive (amplicon)	Specificity (%)
dHPLC	463	10	98
HRM	186	19	91

dHPLC, denaturing high-performance liquid chromatography; HRM, high-resolution melting analysis.

To evaluate whether genetic lesion location within the amplicon would affect HRM screening efficiency, we chose six variants residing near the 5′-terminus, near the 3′-terminus, or in the middle of an amplicon: (1) TSC2 c.5161-10A→C and TSC1 c.682C→T represent the variants near the 5′-end of an amplicon; (2) TSC2 c.2670delT and TSC2 c.1336C→T stand for the variants in the middle of an amplicon; (3) TSC1 c.106+15A→G and TSC2 c.133_136delCTGA symbolize the variants near the 3′-end of an amplicon. Reproducibility of HRM screening for each variant was assessed through repeated experiments for 10 times. Data revealed that except for the two variants near the ends of the amplicon, HRM screening protocol can detect all of them (Fig. 1B).

Cost and analytical time evaluation

The costs of dHPLC and HRM protocol for analyzing one TSC amplicon were estimated to be around 23 and 21 American dollars (USDs), respectively, including 18 USDs for variant validation by bidirectional DNA sequencing. All together, there are 69 amplicons of TSC1 and TSC2 gene per TSC case for screening, which required 372 and 237 USDs for dHPLC and HRM protocol, respectively, including 19 USDs for DNA sequence variants validation (Table 4).

Table 4.

Cost and Analytical Time for Tuberous Sclerosis Complex Mutation Detection

Cost	dHPLC (USD )	HRM (USD)	Direct sequencing (USD)
For one amplicon	23^a	21^a	19^b
For one TSC case	372^a	237^a	1,288

Analytical time	dHPLC (min)	HRM (min)	Direct sequencing (min)
For one amplicon	105	36	120
For one TSC case	7,204	36	240

Including 18 USDs for bidirectional DNA sequencing.

Including 1 USD for PCR before bidirectional DNA sequencing.

USD, American dollar.

Regarding the analytical time for one TSC amplicon, it required 105 and 36 min for dHPLC and HRM screening, respectively, to analyze the disease-free control, TSC sample, and negative control of PCR. For one TSC case, it took 7,204 and 36 min for dHPLC and HRM, respectively, to analyze 69 TSC amplicons. Because the 69 TSC amplicons could be analyzed simultaneously by HRM within a 384-well plate, it massively shortened the analytical time needed (Table 4).

Discussion

TSC1 and TSC2 genes are large genes with complicated genomic structure. To analyze all coding exons of both genes efficiently, we developed an HRM protocol for screening all coding exons of both genes followed by DNA sequencing. In the meantime, we evaluated the sensitivity, cost, and analytical time of this novel method versus the existing dHPLC protocol, which is a widely used approach for TSC gene mutation screening.

In this study, we discovered 20 mutations, including 5 mutations in TSC1 and 15 mutations in TSC2. Our data suggested a wide mutation spectrum in both genes and no mutation hotspot. Only three mutations, TSC1 c.682C→T, TSC1 c.1884_1887delAAAG, and TSC2 c.5238_5255delCATCAAGCGGCTCCGCCA, appeared twice. Together with a previous study by Hung et al. (2006), mutations most frequently occur in TSC1 exon 15 (7%) and TSC2 exon 40 (11%) in the Taiwanese TSC population. This assessment is not completely concordant with that proposed by Au et al. (2007), who declare that mutations most frequently appear in exon 15 of TSC1 and exon 16 of TSC2. Of the 20 identified mutations in this study, 4 mutations were not reported before. They are likely disease-causing mutations because they are frameshift mutations. It was speculated that these mutations might be specific among the Taiwanese TSC patients. In addition, three unclassified variants were identified. According to bioinformatic information, the substituted amino acids resulting from TSC2 c.1565A→C (p.H522P) and TSC2 c.4436C→T (p.A1479V) are not involved in protein interaction, GTPase activation, and protein phosphorylation (NCBI database, Category Protein, NP_000539.2), whereas the amino acid affected by TSC1 c.2485A→T locates in a region similar to Snf7, which is involved in protein sorting and transport from endosome to lysosome (NCBI database, Category Protein, NP_000359.1). Functional analysis will be performed in future to elucidate whether they are disease-causing mutations.

Approximately 15% of TSC patients have no mutation identified. These patients show differential phenotypic characteristics compared with TSC patients carrying TSC1 or TSC2 mutations (Camposano et al., 2009). In patients who have no mutation identified, TSC progression may be due to epigenetic silencing (e.g., promoter methylation), mutations in a putative TSC3 gene (OMIM 191100), or deregulation of upstream regulators of the TSC protein complex (e.g., protein kinase B [AKT] and extracellular signal-regulated kinase [ERK]) (Jozwiak et al., 2008). TSC patients with such alterations could not be screened molecularly with the novel HRM-based protocol or traditional dHPLC method.

Recently, HRM-based protocols have been widely applied to detect DNA sequence variation and suggested as a high-throughput technique with high sensitivity. Sensitivity comparison of HRM versus dHPLC has been performed in other studies. Sensitivity of HRM is consistently higher than that of dHPLC for cystic fibrosis transmembrane conductance regulator (CFTR) and neurofibromatosis type 2 (NF2) genes mutation screening (Chou et al., 2005; Sestini et al., 2008). In this study, sensitivities and specificities of HRM and dHPLC for TSC genes mutation screening were explored. Sensitivities of HRM and dHPLC were estimated to be 95% and 75%, respectively. Specificities of HRM and dHPLC were evaluated to be 91% and 98%. Although the sensitivity of HRM for TSC genes is high, however, we found that sequence variants near the ends of amplicon were slightly difficult to be identified using the HRM protocol.

For future clinical application, we compared the consumable cost and analytical time of dHPLC versus HRM for TSC gene mutation screening. Including PCR amplification, mutation screening, and bidirectional DNA sequencing, it required about 372 and 237 USDs for the dHPLC and the HRM protocol, respectively, to analyze one TSC case. Here, we just calculated the consumable cost regardless of labor cost, which depends on analytical time. The difference in total cost between dHPLC and HRM protocol was actually more dramatically significant than the difference in consumable cost between them. Regarding the analytical time for analyzing all coding exons of each TSC case, it took roughly 200-fold more time using the dHPLC protocol than the HRM protocol. In contrast to dHPLC, which analyzes samples one by one, HRM can simultaneously screen all coding exons of TSC1 and TSC2 genes using a 384-well plate and greatly shorten analytical time for each TSC case. This high-throughput capacity is one of the many advantages the HRM protocol possesses.

In our hospital, TSC-associated epilepsy is treated with an antiseizure drug, vigabatrin, especially in infantile spasms. However, only patients with TSC1 gene mutations respond well to vigabatrin. This is why a rapid molecular diagnostic platform to identify the mutations in TSC1 or TSC2 is crucial for clinicians. In this study, we established an HRM protocol with high-throughput capacity and demonstrated that HRM is a superior screening tool for mutations in TSC genes.

Footnotes

Acknowledgment

Grant support: Chang Gung Memorial Hospital (CMRPD170391, 170392, 170393).

Disclosure Statement

No competing financial interests exist.

References

, Williams

, Roach

et al. 2007. Genotype/phenotype correlation in 325 individuals referred for a diagnosis of tuberous sclerosis complex in the United States. Genet Med, 9:88-100.

Bourneville

. 1880. Sclérose tubéreuse des circonvolutions cérébrales. Arch Neurol, 1:81-91.

Camposano

, Greenberg

, Kwiatkowski

et al. 2009. Distinct clinical characteristics of tuberous sclerosis complex patients with no mutation identified. Ann Hum Genet, 73:141-146.

Chou

, Lyon

, Wittwer

. 2005. A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning: cystic fibrosis transmembrane conductance regulator gene as a model. Am J Clin Pathol, 124:330-338.

Curatolo

, Bombardieri

, Jozwiak

. 2008. Tuberous sclerosis. Lancet, 372:657-668.

Dabora

, Jozwiak

, Franz

et al. 2001. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am J Hum Genet, 68:64-80.

Emmerson

, Maynard

, Jones

et al. 2003. Characterizing mutations in samples with low-level mosaicism by collection and analysis of DHPLC fractionated heteroduplexes. Hum Mutat, 21:112-115.

European Tuberous Sclerosis Consortium. 1993. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell, 75:1305-1315.

Fryer

, Chalmers

, Connor

et al. 1987. Evidence that the gene for tuberous sclerosis is on chromosome 9. Lancet, 1:659-661.

10.

Hong

, Darling

, Lee

. 2009. Prevalence of tuberous sclerosis complex in Taiwan: a national population-based study. Neuroepidemiology, 33:335-341.

11.

Huang

, Manning

. 2009. A complex interplay between Akt, TSC2 and the two mTOR complexes. Biochem Soc Trans, 37:217-222.

12.

Hung

, Su

, Chien

et al. 2006. Molecular and clinical analyses of 84 patients with tuberous sclerosis complex. BMC Med Genet, 7:72.

13.

Jones

, Austin

, Hansen

et al. 1999a. Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clin Chem, 45:1133-1140.

14.

Jones

, Sampson

, Hoogendoorn

et al. 2000. Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis. Hum Genet, 106:663-668.

15.

Jones

, Shyamsundar

, Thomas

et al. 1999b. Comprehensive mutation analysis of TSC1 and TSC2 and phenotypic correlations in 150 families with tuberous sclerosis. Am J Hum Genet, 64:1305-1315.

16.

Jozwiak

, Jozwiak

, Wlodarski

. 2008. Possible mechanisms of disease development in tuberous sclerosis. Lancet Oncol, 9:73-79.

17.

Kandt

, Haines

, Smith

et al. 1992. Linkage of an important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic kidney disease. Nat Genet, 2:37-41.

18.

Langkau

, Martin

, Brandt

et al. 2002. TSC1 and TSC2 mutations in tuberous sclerosis, the associated phenotypes and a model to explain observed TSC1/TSC2 frequency ratios. Eur J Pediatr, 161:393-402.

19.

Le Caignec

, Kwiatkowski

, Kury

et al. 2009. Three independent mutations in the TSC2 gene in a family with tuberous sclerosis. Eur J Hum Genet, 17:1165-1170.

20.

Liou

, Chu

, Cheng

et al. 2004. Human chromosome 21-specific DNA markers are useful in prenatal detection of Down syndrome. Ann Clin Lab Sci, 34:319-323.

21.

Morrison

. 2009. Tuberous sclerosis: epidemiology, genetics and progress towards treatment. Neuroepidemiol, 33:342-343.

22.

Niida

, Lawrence-Smith

, Banwell

et al. 1999. Analysis of both TSC1 and TSC2 for germline mutations in 126 unrelated patients with tuberous sclerosis. Hum Mutat, 14:412-422.

23.

Sestini

, Provenzano

, Bacci

et al. 2008. NF2 mutation screening by denaturing high-performance liquid chromatography and high-resolution melting analysis. Genet Test, 12:311-318.

24.

Taylor

. 2009. Mutation scanning using high-resolution melting. Biochem Soc Trans, 37:433-437.

25.

van Bakel

, Sepp

, Ward

et al. 1997. Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT) Hum Mol Genet, 6:1409-1414.

26.

van Slegtenhorst

, de Hoogt

, Hermans

et al. 1997. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 277:805-808.

27.

Yamashita

, Ono

, Okada

et al. 2000. Analysis of All Exons of TSC1 and TSC2 genes for germline mutations in Japanese patients with tuberous sclerosis: report of 10 mutations. Am J Med Genet, 90:123-126.

28.

, Sawyer

, Chiu

et al. 2006. DNA mutation detection using denaturing high-performance liquid chromatography (DHPLC) Curr Protoc Hum Genet Chapter 7:Unit7, 10.

29.

Zhang

, Nanba

, Yamamoto

et al. 1999. Mutational analysis of TSC1 and TSC2 genes in Japanese patients with tuberous sclerosis complex. J Hum Genet, 44:391-396.