title: Combination of Multiple Ligation-Dependent Probe Amplification and Illumina MiSeq Amplicon Sequencing for TSC1/TSC2 Gene Analyses in Patients with Tuberous Sclerosis Complex.
abstract: 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.
authors.affiliation: Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; Center for Neuroscience Services and Research, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia.
authors.forename: Nur Farrah Dila
authors.initials: NF
authors.lastname: Ismail
authors.affiliation: Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; Department of Pediatrics, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia.
authors.forename: Abdul Qawee
authors.initials: AQ
authors.lastname: Rani
authors.affiliation: Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; Center for Neuroscience Services and Research, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia.
authors.forename: Nik Mohd Ariff
authors.initials: NM
authors.lastname: Nik Abdul Malik
authors.affiliation: Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia.
authors.forename: Chia
authors.initials: C
authors.lastname: Boon Hock
authors.affiliation: Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia.
authors.forename: Siti Nabilahuda
authors.initials: SN
authors.lastname: Mohd Azlan
authors.affiliation: Department of Pediatrics, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia.
authors.forename: Salmi
authors.initials: S
authors.lastname: Abdul Razak
authors.affiliation: Hospital Kuala Lumpur, Kuala Lumpur, Malaysia.
authors.forename: Wee Teik
authors.initials: WT
authors.lastname: Keng
authors.affiliation: Hospital Kuala Lumpur, Kuala Lumpur, Malaysia.
authors.forename: Lock Hock
authors.initials: LH
authors.lastname: Ngu
authors.affiliation: Hospital Sultanah Aminah, Johor Bahru, Malaysia.
authors.forename: Abdul Rashid
authors.initials: AR
authors.lastname: Silawati
authors.affiliation: Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia.
authors.forename: Nor Azni
authors.initials: NA
authors.lastname: Yahya
authors.affiliation: Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Malaysia.
authors.forename: Narazah
authors.initials: N
authors.lastname: Mohd Yusoff
authors.affiliation: Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; Center for Neuroscience Services and Research, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; Division of Human Biology, School of Medicine, International Medical University, Kuala Lumpur, Malaysia. Electronic address: teguhhs@usm.my.
authors.forename: Teguh Haryo
authors.initials: TH
authors.lastname: Sasongko
authors.affiliation: Center for Neuroscience Services and Research, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; Department of Pediatrics, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia.
authors.forename: Z A M H
authors.initials: ZA
authors.lastname: Zabidi-Hussin
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D003430:Cross-Sectional Studies; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods / Q000592:standards; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods / Q000592:standards; D010641:Phenotype; D014402:Tuberous Sclerosis / Q000175:diagnosis* / Q000235:genetics*; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D055815:Young Adult
chemical_list: C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins