Genetic study of pocket a and b domain of rbl gene among malaysian children with retinoblastoma
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Date
2009
Authors
Ahmad Yusof@ Hanafi, Hanani
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Abstract
Retinoblastoma (RB) is the most common intraocular tumor mainly affecting children
under four years of age, with a prevalence of 1 in 15 000 to 20 000 live births.
Retinoblastoma is divided into hereditary and non-hereditary types. The development of
this malignancy requires both alleles of the tumor suppressor gene RB I to be knockedout.
Recent studies found mutation occurred more frequently in the pocket A and B
domain of RB 1 gene. The present study was undertaken to identify the mutation in the
pocket A and B domain of RB I gene among retinoblastoma patients in Malaysia, and
the association of the mutation to the laterality and stage of the tumor. DNA was
extracted from 50 RB patients and 50 healthy volunteers (ethnically matched to the
patient group) blood using commercial blood mini kit prior to PCR amplification which
was performed for exon 12 to 22 of RB 1 gene and its intronic flanking region. The PCR
products were screened for mutation through DHPLC analysis and sample with mutation
was SUbjected to DNA sequencing analysis. The ratio of boy to girl patient were 2 : I,
where 78% were Malays, 12% Indian and 10% Chinese. Most of the patients presented
with leukocoria and unilateral (only one eye affected) retinoblastoma. The mean age at
diagnosis of patients is 24.3 (16.9) [mean (SD)] months with an earlier diagnosis among
the bilateral cases [19.47(19.99)]. Thirty-one patient and control subjects presented with
mutation and single nucleotide polymorphism (SNP), which then were identified as
single nucleotide substitution which are located at seven distinct intronic regions, where
two of the mutations affected splice site region. Two novel mutations were identified
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(lYS13+184C>T and IYSI7-133T>C) i1l1 both patient and control group. No significant
association was found either between mutations and laterality or stage of the tumor.
Significant association was found between IVS 15-1 G>A and patient of Chinese origin,
while IVSI3+184C>T, IVSI7-133T>C and IYSI9-77A>G were found to be associated
with different ethnic groups in the control group. We postulated that the splice site
mutations, IVSI2+1G>A and rVSI5-1G>A might playa role in the predisposition to
retinoblastoma, as shown by previous studies. The other five mutations and SNPs are
probably important as genetic variant markers for population studies. However, a larger
sample size is needed for confirmation. To the best of our knowledge, this is the first
report of the mutations found within intronic region of pocket A and B domain of RB 1
gene among Malaysian children with retinoblastoma. In order to develop a more
complete spectrum of RB 1 mutations in Malaysian retinoblastoma patients, we suggest a
larger scale of target screening region should be conducted in future research.