Combination of Multiple Ligation-Dependent Probe Amplification and Illumina MiSeq Amplicon Sequencing for TSC1/TSC2 Gene Analyses in Patients with Tuberous Sclerosis Complex
2017; Elsevier BV; Volume: 19; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2016.10.009
ISSN1943-7811
AutoresNur Farrah Dila Ismail, Abdul Qawee Rani, Nik Mohd Ariff Nik Abdul Malik, Chia Boon Hock, Siti Nabilahuda Mohd Azlan, Salmi Abdul Razak, Wee Teik Keng, Lock Hock Ngu, Abdul Rashid Silawati, Nor Azni Yahya, Narazah Mohd Yusoff, Teguh Haryo Sasongko, Z.A.M.H. Zabidi-Hussin,
Tópico(s)Histiocytic Disorders and Treatments
ResumoTuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous disorder characterized by tumor growth in multiple organs and caused by mutations in either TSC1 or TSC2 genes. Because of their relatively large genomic sizes, absence of hotspots, and common type of mutations, mutation detection in TSC1 and TSC2 genes has been challenging. We devised a combination of multiple ligation-dependent probe amplification (MLPA) and amplicon sequencing (AS) to simplify the detection strategy, yet we come up with reasonably high detection rate. Thirty-four Malaysian patients diagnosed with TSC were referred to Human Genome Center, Universiti Sains Malaysia. We used a combination of MLPA to detect large copy number changes and AS to detect smaller mutations. TSC1 pathogenic or likely pathogenic mutations were found in 6 patients (18%) and TSC2 in 21 patients (62%), whereas 6 patients (18%) show no mutations and 1 patient (2%) showed only TSC2 missense variant with uncertain significance. Six of the mutations are novel. Our detection strategy costs 81% less and require 1 working week less than the conventional strategy. Confirmatory sequencing using Sanger method on a few representative mutations showed agreement with results of the AS. Combination of MLPA and Illumina MiSeq AS provides a simplified strategy and reasonably high detection rate for TSC1/TSC2 mutation, which suggested application of the strategies into clinical molecular diagnostics. Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous disorder characterized by tumor growth in multiple organs and caused by mutations in either TSC1 or TSC2 genes. Because of their relatively large genomic sizes, absence of hotspots, and common type of mutations, mutation detection in TSC1 and TSC2 genes has been challenging. We devised a combination of multiple ligation-dependent probe amplification (MLPA) and amplicon sequencing (AS) to simplify the detection strategy, yet we come up with reasonably high detection rate. Thirty-four Malaysian patients diagnosed with TSC were referred to Human Genome Center, Universiti Sains Malaysia. We used a combination of MLPA to detect large copy number changes and AS to detect smaller mutations. TSC1 pathogenic or likely pathogenic mutations were found in 6 patients (18%) and TSC2 in 21 patients (62%), whereas 6 patients (18%) show no mutations and 1 patient (2%) showed only TSC2 missense variant with uncertain significance. Six of the mutations are novel. Our detection strategy costs 81% less and require 1 working week less than the conventional strategy. Confirmatory sequencing using Sanger method on a few representative mutations showed agreement with results of the AS. Combination of MLPA and Illumina MiSeq AS provides a simplified strategy and reasonably high detection rate for TSC1/TSC2 mutation, which suggested application of the strategies into clinical molecular diagnostics. CME Accreditation Statement: This activity (“JMD 2017 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2017 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“JMD 2017 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2017 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Tuberous sclerosis complex (TSC; Online Mendelian Inheritance in Man no. 191100 and no. 613254, https://www.omim.org, last accessed December 1, 2016) is an autosomal dominant neurocutaneous disorder characterized by tumor growth in multiple organs, including the brain, heart, skin, eyes, kidneys, lungs, and liver. It is also characterized by a broad phenotypic spectrum, including seizures, mental retardation, renal dysfunction, dermatologic abnormalities, and tumors. The clinical manifestations of TSC vary individually. Some patients may experience long-term symptoms, whereas others may have mild symptoms and can even appear asymptomatic.1Schwartz R.A. Fernandez G. Kotulska K. Jozwiak S. Tuberous sclerosis complex: advances in diagnosis, genetics, and management.J Am Acad Dermatol. 2007; 57: 189-202Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar TSC is caused by mutation in either one of the two disease-causing genes, TSC1 (tuberous sclerosis complex 1; chromosome 9q34; Gene ID 7248; RefSeq NC_000009.11) or TSC2 (tuberous sclerosis complex 2; chromosome 16p13.3; Gene ID 7249; RefSeq NC_000016.9), encoding for hamartin and tuberin, respectively.2European Chromosome 16 Tuberous Sclerosis ConsortiumIdentification and characterization of the tuberous sclerosis gene on chromosome 16.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1508) Google Scholar, 3van Slegtenhorst M. de Hoogt R. Hermans C. Nellist M. Janssen B. Verhoef S. et al.Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1392) Google Scholar Up to 10% to 25% of TSC patients showed no TSC1/TSC2 mutation identified by conventional genetic testing.4Northrup H. Krueger D.A. International Tuberous Sclerosis Complex Consensus GroupTuberous sclerosis complex diagnostic criteria update: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference.Pediatr Neurol. 2013; 49: 243-254Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar There are no mutation hot spots for both genes. The mutations happened at random location. Up to 90% of the mutations are small mutations involving one to several nucleotides, whereas the other 10% are gross changes in the genes. Mutations that have been reported to be found in TSC are various, including deletion, insertion, frame-shift, missense, and splice-site. According to the Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk/ac/index.php, last accessed 11 September 11, 2016), >300 and >900 unique mutations have been reported in TSC1 and TSC2, respectively.5Stenson P.D. Ball E.V. Mort M. Phillips A.D. Shiel J.A. Thomas N.S. Abeysinghe S. Krawczak M. Cooper D.N. The Human Gene Mutation Database (HGMD®): 2003 update.Hum Mutat. 2003; 49: 577-581Crossref Scopus (1346) Google Scholar The most recent consensus criteria (2012) for diagnosis of TSC emphasized the significance of TSC1/TSC2 mutation analyses in establishing the diagnosis. Regardless of other clinical features, identification of either a TSC1 or TSC2 pathogenic mutation in DNA from normal tissue is sufficient to make a definite diagnosis of TSC.4Northrup H. Krueger D.A. International Tuberous Sclerosis Complex Consensus GroupTuberous sclerosis complex diagnostic criteria update: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference.Pediatr Neurol. 2013; 49: 243-254Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar This is a new diagnostic criterion that has not appeared in previous diagnostic criteria.6Roach E.S. Gomez M.R. Northrup H. Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria.J Child Neurol. 1998; 13: 624-628Crossref PubMed Scopus (916) Google Scholar, 7Roach E.S. Smith M. Huttenlocher P. Bhat M. Alcorn D. Hawley L. Diagnostic criteria: tuberous sclerosis complex. Report of the Diagnostic Criteria Committee of the National Tuberous Sclerosis Association.J Child Neurol. 1992; 7: 221-224Crossref PubMed Scopus (203) Google Scholar This new development in diagnostic criteria entails challenges in molecular methods for the identification of mutations. TSC1 and TSC2 genes comprise a total 95 kb of genomic entity with 62 coding exons and transcribed into 14.1 kb of mRNA.2European Chromosome 16 Tuberous Sclerosis ConsortiumIdentification and characterization of the tuberous sclerosis gene on chromosome 16.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1508) Google Scholar, 3van Slegtenhorst M. de Hoogt R. Hermans C. Nellist M. Janssen B. Verhoef S. et al.Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1392) Google Scholar According to the HGMD5Stenson P.D. Ball E.V. Mort M. Phillips A.D. Shiel J.A. Thomas N.S. Abeysinghe S. Krawczak M. Cooper D.N. The Human Gene Mutation Database (HGMD®): 2003 update.Hum Mutat. 2003; 49: 577-581Crossref Scopus (1346) Google Scholar and the Leiden Open Variation Database (LOVD)8Fokkema I.F. Taschner P.E. Schaafsma G.C. Celli J. Laros J.F. den Dunnen J.T. LOVD v.2.0: the next generation in gene variant databases.Hum Mutat. 2011; 32: 557-563Crossref PubMed Scopus (688) Google Scholar (http://www.lovd.nl/3.0/home, last accessed September 11, 2016), all types of mutations has been found in both TSC loci involving small point mutations such as missense and nonsense, small deletions and insertions as well as large rearrangements, including whole gene deletion. No parts of both genes are particularly harboring most of the mutations. No mutations were found to be common enough across a significant number of patients. Taken together, the relatively large genomic region, absence of mutation hotspot, and absence of common mutations suggest that a single approach would not be sufficient to generate a significant mutation detection rate for TSC which is cost and time effective. We described here our mutation analysis strategy of TSC1 and TSC2 genes in patients with TSC involving a combination of multiple ligation-dependent probe amplification (MLPA) for detection of large copy number mutations and amplicon sequencing (AS) for detection of smaller nucleotide mutations. This is a cross-sectional study involving 34 Malaysian patients diagnosed with TSC and referred to our molecular genetics laboratory in Human Genome Center, Universiti Sains Malaysia. Patients were recruited from various hospitals in Malaysia between 2010 and 2014. Patients fulfilled clinical diagnosis based on the 2012 consensus criteria for TSC.4Northrup H. Krueger D.A. International Tuberous Sclerosis Complex Consensus GroupTuberous sclerosis complex diagnostic criteria update: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference.Pediatr Neurol. 2013; 49: 243-254Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar Informed consent was taken before blood taking, and the study was approved by Universiti Sains Malaysia Human Research Ethics Committee. Genomic DNA was extracted from whole blood. Clinical data were collected during recruitment. Data on TSC1/TSC2 gene mutations were obtained after the completion of mutation analyses. We initially screened the DNA of all patients for large copy number changes using MLPA. MLPA analysis was performed using commercially available kit, SALSA MLPA probemix P124-C1 for TSC1and SALSA MLPA probemix P046-C1 for TSC2 (MRC-Holland, Amsterdam, the Netherlands). The protocol was performed according to the manufacturer's description. Fragment analyses were subsequently done using capillary electrophoresis on ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Analysis for determination of copy number changes along TSC1 and TSC2 genes was done using Coffalyser.Net (MRC-Holland, https://coffalyser.wordpress.com, last accessed September 24, 2016). DNA of the patients without gross copy number changes detectable using MLPA was then subjected to AS using Illumina MiSeq (Illumina, San Diego, CA). This is for the detection of smaller nucleotide mutations. For the AS, long-range PCR amplifications were done to cover the whole exonic portion of TSC1 (four amplicons) and TSC2 (six amplicons) with sizes range from 2 to 9 kb. PCR amplicons of TSC1 and TSC2 are described in Table 1. The long-range PCR condition was 3 minutes at 94°C for initial denaturation, followed by 15 seconds at 94°C, 45 seconds at annealing temperature (Table 1), and 15 minutes at 68°C for 35 cycles, and a final extension done at 68°C for 7 minutes. Amplifications were performed in 60 μL of total volume containing 1 μg of genomic DNA, 1× Buffer S, 0.33 mol/L dNTPs, 0.33 μmol/L of each primer, and 1.5 U of MaxTaq polymerase (Vivantis Technology, Oceanside CA).Table 1Amplicon Fragments Generated for Sequencing in This StudyAmpliconsAnnealing temperature, °CExons∗Primer sequences were described elsewhere.9 First and last exon of each amplicon denoted location of forward and reverse primers, respectively, as described previously (eg, amplicon 1-TSC1 used forward primer for exon 3 and reverse primer for exon 89).Size, bpAmplicon 1-TSC1563–87687Amplicon 2-TSC1549–122089Amplicon 3-TSC15513–173829Amplicon 4-TSC15618–236442Amplicon 1-TSC2581–78264Amplicon 2-TSC2627–147934Amplicon 3-TSC2TD14–187816Amplicon 4-TSC26018–224361Amplicon 5-TSC2TD22–349284Amplicon 6-TSC2TD34–413792TouchDown (TD) PCR: annealing temperature started at 70°C and was gradually reduced by 0.03°C.∗ Primer sequences were described elsewhere.9Choi J.E. Chae J.H. Hwang Y.S. Kim K.J. Mutational analysis of TSC1 and TSC2 in Korean patients with tuberous sclerosis complex.Brain Dev. 2006; 28: 440-446Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar First and last exon of each amplicon denoted location of forward and reverse primers, respectively, as described previously (eg, amplicon 1-TSC1 used forward primer for exon 3 and reverse primer for exon 89Choi J.E. Chae J.H. Hwang Y.S. Kim K.J. Mutational analysis of TSC1 and TSC2 in Korean patients with tuberous sclerosis complex.Brain Dev. 2006; 28: 440-446Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Open table in a new tab TouchDown (TD) PCR: annealing temperature started at 70°C and was gradually reduced by 0.03°C. Each amplicon was normalized to 15 nmol/L and pooled at the equal volume. Pooled amplicons were subjected to library preparation using Nextera XT DNA Sample Preparation Kit (Illumina) and indexed according to the manufacturer's recommendation. Sequencing of paired-end 150 bp was done on Illumina MiSeq sequencer using the MiSeq Reagent Kit v2 (Illumina). PCR amplicon workflow was chosen for MiSeq Reporter to complete the tertiary analysis of the sequencing. We used GATK for the variant caller and BWA for the aligner. Filtering variables were set according to certain criteria. For insertion or deletion, variant was filtered out if it was found besides eight repeat motifa (1 or 2 bases). In general, variant was filtered out if its frequency was <0.20, if its genotype quality was <30, and if its quality was 90, read depths of >200, and allelic frequency of >0. Variant details were visualized using Integrated Genome Viewer version 2.3 which was freely available from The Broad Institute (Cambridge, MA; http://www.broadinstitute.org/igv). The analysis was based on the latest GRCh38 Genome Reference Consortium Human Reference 38 (GCA_000001405.15; https://genome-euro.ucsc.edu/cgi-bin/hgGateway?db=hg38&redirect=manual&source=genome.ucsc.edu, last accessed June 1, 2016). To validate the results of AS, we directly sequenced the TSC2 exons of a few patients using the Sanger method, representing different types of mutations identified through the AS. Direct DNA Sequencing was done to confirm the mutation findings using ABI Prism 3100 Genetic Analyzer (Applied Biosystems). We manually studied each variant found in the patients that passed our filtering variables. We determined variant's pathogenicity based on American College of Medical Genetics and Genomics Standards and Guidelines.10Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. Voelkerding K. Rehm H.L. ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (14616) Google Scholar Pathogenicity of the missense variants was also predicted using several online tools for variant pathogenicity prediction as suggested by American College of Medical Genetics and Genomics Standards and Guidelines. Missense variants that were reported previously were compared with LOVD version 3.08Fokkema I.F. Taschner P.E. Schaafsma G.C. Celli J. Laros J.F. den Dunnen J.T. LOVD v.2.0: the next generation in gene variant databases.Hum Mutat. 2011; 32: 557-563Crossref PubMed Scopus (688) Google Scholar (Leiden University Medical Center, Leiden, the Netherlands; http://chromium.lovd.nl/LOVD2/TSC). We described here the clinical features and TSC1/TSC2 mutations in 34 Malaysian patients with TSC (Tables 2 and 3). Patients are of Malaysian Malay and Chinese ethnicities and were referred to the Human Genome Center Universiti Sains Malaysia from various hospitals across Malaysia.Table 2Clinical Features and TSC1/TSC2 Mutations and Missense Variants in Malaysian Patients with Tuberous Sclerosis ComplexPatient IDSexAge, yearsFamily historyEpilepsyMRSkin manifestationsBrain tumorsOther tumorsOther manifestationsTSC1/TSC2 Mutations/VariantsReference to mutation/variantFAHMSPFPUFCTSENSEGACRRA001-007-001∗Clinically diagnosed as definite TSC.M23NoYesNo√√√TSC1 Exon 3:c.65G>A;p.R22QTSC1†LOVD, TSC1: http://chromium.lovd.nl/LOVD2/TSC/home.php?select_db=TSC1.003-010-001∗Clinically diagnosed as definite TSC.F1YesYesNo√√TSC1 Exon 17:c.2074C>T;p.R692XTSC1003-011-001∗Clinically diagnosed as definite TSC.F5NoNoNo√√TSC1 Exon18 :c.2272C>T;p.Q758XTSC1003-011-002∗Clinically diagnosed as definite TSC.M6NoNoNo√√TSC1 Exon 18:c.2272C>T;p.Q758XTSC1003-011-003∗Clinically diagnosed as definite TSC.M3NoNoNo√√TSC1 Exon 18:c.2272C>T;p.Q758XTSC1002-004-001∗Clinically diagnosed as definite TSC.M2NoYesNo√√TSC1 Exon15:c.1525C>T;p.R509X3van Slegtenhorst M. de Hoogt R. Hermans C. Nellist M. Janssen B. Verhoef S. et al.Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1392) Google Scholar, 11Sancak O. Nellist M. Goedbloed M. Elfferich P. Wouters C. Maat-Kievit A. Zonnenberg B. Verhoef S. Halley D. van den Ouweland A. Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: genotype–phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex.Eur J Hum Genet. 2005; 13: 731-741Crossref PubMed Scopus (360) Google Scholar, 12Rendtorff N.D. Bjerregaard B. Frodin M. Kjaergaard S. Hove H. Skovby F. Brondum-Nielsen K. Schwartz M. Danish Tuberous Sclerosis GroupAnalysis of 65 tuberous sclerosis complex (TSC) patients by TSC2 DGGE, TSC1/TSC2 MLPA, and TSC1 long-range PCR sequencing, and report of 28 novel mutations.Hum Mutat. 2005; 26: 374-383Crossref PubMed Scopus (41) Google Scholar, 13Au K.S. Williams A.T. Roach E.S. Batchelor L. 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Tuberous sclerosis type 1: three novel mutations detected in exon 15 by a combination of HDA and TGGE.Hum Mutat. 2000; 16: 88Crossref PubMed Google Scholar002-002-001∗Clinically diagnosed as definite TSC.F5NoYesNo√√√√Multiple renal cystTSC1 Exon 13:c.1273A>G;p.M425V‡Pathogenicity of the missense variants was described in Table 3.NovelTSC2 Exon 8:c.833A>C;p.H278P‡Pathogenicity of the missense variants was described in Table 3.TSC2§LOVD, TSC2: http://chromium.lovd.nl/LOVD2/TSC/home.php?select_db=TSC2.001-016-001¶Clinically diagnosed as possible TSC.M8NoNoNo√TSC2 Exon 3: c.266T>A;p.L89XNovel001-020-001FNANANANATSC2 Exon 7: c.705insC;p.S235SFsX337Novel002-005-001¶Clinically diagnosed as possible TSC.M1NoYesNo√TSC2 Exon 8: c.826_827delAT;p.M276VFsX33711Sancak O. Nellist M. Goedbloed M. Elfferich P. Wouters C. Maat-Kievit A. Zonnenberg B. Verhoef S. Halley D. van den Ouweland A. Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: genotype–phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex.Eur J Hum Genet. 2005; 13: 731-741Crossref PubMed Scopus (360) Google Scholar, 17Dabora S.L. Jozwiak S. Franz D.N. Roberts P.S. Nieto A. Chung J. Choy Y.S. Reeve M.P. Thiele E. Egelhoff J.C. Kasprzyk-Obara J. Domanska-Pakiela D. Kwiatkowski D.J. 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. 2001; 68: 64-80Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar, 18Jang M.A. Hong S.B. Lee J.H. Lee M.H. Chung M.P. Shin H.J. Kim J.W. Ki C.S. Identification of TSC1 and TSC2 mutations in Korean patients with tuberous sclerosis complex.Pediatr Neurol. 2012; 46: 222-224Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 19Niida Y. Lawrence-Smith N. Banwell A. Hammer E. Lewis J. Beauchamp R.L. Sims K. Ramesh V. Ozelius L. Analysis of both TSC1 and TSC2 for germline mutations in 126 unrelated patients with tuberous sclerosis.Hum Mutat. 1999; 14: 412-422Crossref PubMed Scopus (130) Google Scholar005-001-001∗Clinically diagnosed as definite TSC.F2YesNoNo√√√TSC2 Exon 23: c.2656_2657insGT;p.V886GFsX895NovelTSC2 Exon 9:c.856A>G;p.M286V‡Pathogenicity of the missense variants was described in Table 3.12Rendtorff N.D. Bjerregaard B. Frodin M. Kjaergaard S. Hove H. Skovby F. Brondum-Nielsen K. Schwartz M. Danish Tuberous Sclerosis GroupAnalysis of 65 tuberous sclerosis complex (TSC) patients by TSC2 DGGE, TSC1/TSC2 MLPA, and TSC1 long-range PCR sequencing, and report of 28 novel mutations.Hum Mutat. 2005; 26: 374-383Crossref PubMed Scopus (41) Google Scholar, 20Dabora S.L. Sigalas I. Hall F. Eng C. Vijg J. Kwiatkowski D.J. Comprehensive mutation analysis of TSC1 using two-dimensional DNA electrophoresis with DGGE.Ann Hum Genet. 1998; 62: 491-504Crossref PubMed Google Scholar, 21Jansen F.E. Vincken K.L. Algra A. Anbeek P. Braams O. Nellist M. Zonnenberg B.A. Jennekens-Schinkel A. van den Ouweland A. Halley D. van Huffelen A.C. van Nieuwenhuizen O. Cognitive impairment in tuberous sclerosis complex is a multifactorial condition.Neurology. 2008; 70: 916-923Crossref PubMed Scopus (162) Google Scholar, 22Hodges A.K. Li S. Maynard J. Parry L. Braverman R. Cheadle J.P. DeClue J.E. Sampson J.R. Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin.Hum Mol Genet. 2001; 10: 2899-2905Crossref PubMed Scopus (99) Google ScholarTSC2 Exon 16: c.1825A>G;p.S609G‡Pathogenicity of the missense variants was described in Table 3.Novel004-001-001∗Clinically diagnosed as definite TSC.F9NoNoNo√√√TSC2 Intron 12: c.1361+1G>ATSC2001-015-001¶Clinically diagnosed as possible TSC.F6NoYesNo√Pits in dental enamelTSC2 Exon 15: c.1714C>T;p.Q572XTSC2003-009-001∗Clinically diagnosed as definite TSC.M45NoYesYes√√√√√√Confetti skin lesionTSC2 Exon 37:c.4925G>A;p.G1642D‡Pathogenicity of the missense variants was described in Table 3.23Hoogeveen-Westerveld M. Wentink M. van den Heuvel D. Mozaffari M. Ekong R. Povey S. den Dunnen J.T. Metcalfe K. Vallee S. Krueger S. Bergoffen J. Shashi V. Elmslie F. Kwiatkowski D. Sampson J. Vidales C. Dzarir J. Garcia-Planells J. Dies K. Maat-Kievit A. van den Ouweland A. Halley D. Nellist M. Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex.Hum Mutat. 2011; 32: 424-435Crossref PubMed Scopus (66) Google Scholar003-002-001∗Clinically diagnosed as definite TSC.M3NoNoNo√√√√Retinal acromic patchTSC2 Exon 40:c.5228G>A;p.R1743Q‡Pathogenicity of the missense variants was described in Table 3.11Sancak O. Nellist M. Goedbloed M. Elfferich P. Wouters C. Maat-Kievit A. Zonnenberg B. Verhoef S. Halley D. van den Ouweland A. Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: genotype–phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex.Eur J Hum Genet. 2005; 13: 731-741Crossref PubMed Scopus (360) Google Scholar, 23Hoogeveen-Westerveld M. Wentink M. van den Heuvel D. Mozaffari M. Ekong R. Povey S. den Dunnen J.T. Metcalfe K. Vallee S. Krueger S. Bergoffen J. Shashi V. Elmslie F. Kwiatkowski D. Sampson J. Vidales C. Dzarir J. Garcia-Planells J. Dies K. Maat-Kievit A. van den Ouweland A. Halley D. Nellist M. Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex.Hum Mutat. 2011; 32: 424-435Crossref PubMed Scopus (66) Google Scholar, 24Hoogeveen-Westerveld M. Ekong R. Povey S. Mayer K. Lannoy N. Elmslie F. Bebin M. Dies K. Thompson C. Sparagana S.P. Davies P. van Eeghen A.M. Thiele E.A. van den Ouweland A. Halley D. Nellist M. Functional assessment of TSC2 variants identified in individuals with tuberous sclerosis complex.Hum Mutat. 2013; 34: 167-175Crossref PubMed Scopus (46) Google Scholar001-011-001∗Clinically diagnosed as definite TSC.F9NoYesNo√√√√TSC2 Exon 29: c.3581G>A;p.W1194X25Lee J.S. Lim B.C. Chae J.H. Hwang Y.S. Seong M.W. Park S.S. Kim K.J. Mutational analysis of paediatric patients with tuberous sclerosis complex in Korea: genotype and epilepsy.Epileptic Disord. 2014; 16: 449-455PubMed Google Scholar001-002-003∗Clinically diagnosed as definite TSC.M21YesNoNo√√√TSC2 Exon 30: c.3755C>A;p.S1252XNovel001-012-001¶Clinically diagnosed as possible TSC.F16YesYesNo√TSC2 Exon 30: c.3755C>A;p.S1252XNovel003-003-002∗Clinically diagnosed as definite TSC.F31YesNoNo√√√TSC2 Exon 30: c.3755C>A;p.S1252XNovel003-004-001∗Clinically diagnosed as definite TSC.M19NoYesYes√√√√√√√Confetti skin lesionTSC2 Exon 32: c.3999C>A;p.Y1333XNovel008-001-001¶Clinically diagnosed as possible TSC.F1NoNoNo√TSC2 Exon 33: c.4344_4345insC;p.S1448FsX1523Novel003-005-001∗Clinically diagnosed as definite TSC.F4NoNoNo√√√√TSC2 Exon 39: c.5074_5075insG;p.E1692GFsX1706TSC2TSC2 Exon18:c.2032G>A;p.A678T‡Pathogenicity of the missense variants was described in Table 3.TSC2002-007-001∗Clinically diagnosed as definite TSC.M5NoYesYes√√√√√TSC2 Exon 38: c.4993C>T;p.Q1665X26Maheshwar M.M. 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Genotype/phenotype correlation in 325 individuals referred for a diagnosis of tuberous sclerosis complex in the United States.Genet Med. 2007; 9: 88-100Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 18Jang M.A. Hong S.B. Lee J.H. Lee M.H. Chung M.P. Shin H.J. Kim J.W. Ki C.S. Identification of TSC1 and TSC2 mutations in Korean patients with tuberous sclerosis complex.Pediatr Neurol. 2012; 46: 222-224Abstract Full Text Full Text
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