title: Spectrum of germ-line RB1 gene mutations in Malaysian patients with retinoblastoma.
abstract: OBJECTIVE
  The availability of molecular genetic testing for retinoblastoma (RB) in Malaysia has enabled patients with a heritable predisposition to the disease to be identified, which thus improves the clinical management of these patients and their families. In this paper, we presented our strategy for performing molecular genetic testing of the RB1 gene and the findings from our first 2 years of starting this service.
  
  
  METHODS
  The peripheral blood of 19 RB probands, including seven bilateral and 12 unilateral cases, was obtained, and genomic DNA was extracted. Analysis of the RB1 exons and the promoter region was conducted first using PCR and direct sequencing. Next, multiplex ligation-dependent probe amplification (MLPA) analysis was performed for patients whom the first results were negative. For patients whom either the first or second method results were positive, parental samples were analyzed to determine the origin of the mutation.
  
  
  RESULTS
  Ten RB1 mutations were identified in ten (52.6%) of the 19 probands (seven bilateral and three unilateral cases), of which 30.0% (3/10) was identified with MLPA. The detection rates in the bilateral and unilateral cases were 100.0% (7/7) and 25.0% (3/12), respectively. Three new RB1 mutations were discovered, two in patients with bilateral RB and one in patient with unilateral RB. Interestingly, all mutations detected with the PCR-sequencing method were predicted to create a premature stop codon. Eight mutations were proven to be de novo while one mutation was inherited from the mother in a family with a positive history of RB.
  
  
  CONCLUSIONS
  Our results confirmed the heterogeneous nature of RB1 mutations and the predominantly de novo origin. The high prevalence of pathogenic truncating mutations was evident among local patients with RB. The combination of PCR sequencing and MLPA is recommended for sensitive identification of heritable RB cases.
authors.affiliation: Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Mohd Khairul Nizam
authors.initials: MK
authors.lastname: Mohd Khalid
authors.affiliation: Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Yusnita
authors.initials: Y
authors.lastname: Yakob
authors.affiliation: Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Rohani
authors.initials: R
authors.lastname: Md Yasin
authors.affiliation: Department of Genetics, Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Keng
authors.initials: K
authors.lastname: Wee Teik
authors.affiliation: Department of Genetics, Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Ch'ng Gaik
authors.initials: CG
authors.lastname: Siew
authors.affiliation: Department of Ophthalmology, Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Jamalia
authors.initials: J
authors.lastname: Rahmat
authors.affiliation: Department of Ophthalmology, Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Sunder
authors.initials: S
authors.lastname: Ramasamy
authors.affiliation: Department of Ophthalmology, Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur, Malaysia.
authors.forename: Joseph
authors.initials: J
authors.lastname: Alagaratnam
mesh_terms: D044466:Asian People; D002675:Child, Preschool; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D015870:Gene Expression; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D018095:Germ-Line Mutation; D006801:Humans; D007223:Infant; D007438:Introns; D008296:Malaysia; D008297:Male; D011401:Promoter Regions, Genetic; D012160:Retina / Q000378:metabolism / Q000473:pathology; D019572:Retinal Neoplasms / Q000208:ethnology / Q000235:genetics* / Q000473:pathology; D012175:Retinoblastoma / Q000208:ethnology / Q000235:genetics* / Q000473:pathology; D016160:Retinoblastoma Protein / Q000235:genetics*
chemical_list: D018389:Codon, Nonsense; D016160:Retinoblastoma Protein