Year : 2001 | Volume
: 49 | Issue : 1 | Page : 37--42
Genetic profile of 81 retinoblastoma patients from a referral hospital in Southern India
Rajasekhar Harini1, Md Ata-ur-Rasheed2, Mahesh P Shanmugam3, Jacob Amali4, Debashish Das2, Govindasamy Kumaramanickavel5,
1 MS (MLT). Department of Genetics and Molecular Biology, Vision Reasearch Foundation, Chennai, India
2 MS (MLT). Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, India
3 MBBS, DO. Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, India
4 BSc, PGDMLT. Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, India
5 MD. Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, India
MS (MLT). Department of Genetics and Molecular Biology, Vision Reasearch Foundation, Chennai, India
Purpose: To determine chromosomal abnormalities and inheritance pattern in patients with retinoblastoma from a referral hospital in southern India.
Materials and Methods: Eighty-one retinoblastoma patients from 78 families were included in this study. Peripheral venous blood was taken for chromosomal analysis and pedigree was ascertained for segregation analysis.
Results: Male to female ratio was 1.7:1, 55.56% were bilateral retinoblastoma, the mean age of onset was 12.37 months in bilateral and 33.07 months in unilateral cases (p=0.048). Majority (90.12%) had sporadic inheritance and 6.17% had autosomal dominant inheritance. In chromosomal abnormalities, 8.33% had 13q14 deletion, three cases had de novo balanced translocations.
Conclusion: The age of onset of the disease was much earlier in the bilateral cases compared to unilateral cases. Sporadic inheritance was predominant while only a small percentage of patients had autosomal dominant inheritance. The percentage of patients with 13q14 deletion was higher than reported in the literature and three novel chromosomal translocations were observed. This is one of the largest series of cases reported from India.
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Harini R, Ata-ur-Rasheed M, Shanmugam MP, Amali J, Das D, Kumaramanickavel G. Genetic profile of 81 retinoblastoma patients from a referral hospital in Southern India.Indian J Ophthalmol 2001;49:37-42
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Harini R, Ata-ur-Rasheed M, Shanmugam MP, Amali J, Das D, Kumaramanickavel G. Genetic profile of 81 retinoblastoma patients from a referral hospital in Southern India. Indian J Ophthalmol [serial online] 2001 [cited 2020 Apr 7 ];49:37-42
Available from: http://www.ijo.in/text.asp?2001/49/1/37/22662
Retinoblastoma is the most common malignant intraocular tumour in the paediatric age group with an incidence of 1:12000 to 1:23000 live births amongst different races.[2-7] The tumour in retinoblastoma arises from the retinal precursor cells and therefore manifests as unilateral or bilateral tumour. If the pineal body is involved it is called trilateral retinoblastoma.[9,10] Retinoblastoma can develop as a result of a somatic or germinal mutation, but the susceptibility to the disease is inherited in an autosomal dominant manner. Knudson's "Two Hit Hypothesis" provides an explanation for the genetic basis of two forms of retinoblastoma:[11-13] (i) the heritable, which is bilateral and multifocal, and (ii) the non-heritable, which is unilateral and unifocal. Stallard gave the first clue to the location of retinoblastoma gene in 1962. High-resolution banding localised the retinoblastoma gene to sub-band 13q14.11.[15,16] The RBI gene expressed in a majority of cells has been well characterised as a tumour suppressor gene and both alleles must be inactivated or lost for tumourigenesis.[1,17-21] Only 5-6% of patients with retinoblastoma have cytogenetic deletions;[1,2] the remaining 95% of the mutations occurring in the retinoblastoma gene are submicroscopic.[1,2] In therapeutic experiments transgenic mice and gene therapy methods have been attempted to find a cure for retinoblastoma.
In the present study, conducted at a referral hospital in southern India, we propose to (i) determine the mode of inheritance and (ii) detect the cytogenetic aberrations in these 81 retinoblastoma patients.
Materials and Methods
Retinoblastoma patients were referred to the Department of Genetics & Molecular Biology, Vision Research Foundation, Sankara Nethralaya, Chennai, where pedigree was drawn and analysed before counselling. Information obtained in this study included parental consanguinity, age of onset of retinoblastoma, family history, gender distribution and laterality. Autosomal dominant inheritance is labelled when one parent has retinoblastoma and 50% of children of either gender are affected with the same disease. Sporadic inheritance is labelled when no other family member is affected with retinoblastoma. Blood was collected from the affected individuals for chromosomal analysis. Statistical and cytogenetic analyses were done.
To confirm whether all these families showed an autosomal dominant mode of inheritance, Neel and Schull segregation analysis was used as follows:
where, O = observed and E = expected.
Lymphocyte culture was set with 0.4 ml of peripheral blood added to 10ml of RPMI medium (0.0103 mg / ml, Hi-media, India) containing 20% foetal calf serum (Hi-media, India), 6 mg L-glutamine (Hi-media, India), 2 mg penicillin (Hi-media, India) and 0.1 ml of the mitogen phytohemagglutinin (GibcoBRL,USA). After incubation at 37°C for 72 hours, 50 μl of methotrexate (4.5 x 10-6 mg/μl, Sigma, USA) was added and incubated at 37°C for 17 hours. The mixture was centrifuged at 1500 rpm for 10 minutes. The cells were washed twice with fresh unsupplemented medium and centrifuged again. Subsequently 50 μl of thymidine solution (2.5 x 10-4 mg/μl, Sigma, USA) was added to 10ml of the medium and incubated at 37°C for 5-6 hours. Finally, 10μl of colchicine (2 x 10-4 mg / μl, Sigma, USA) was added 10 minutes before harvesting the cells. The cells were fixed in methanol: acetic acid (3:1) and then the slides were prepared and stained using GTG-banding. Parents of 9 patients, however, refused cytogenetic study.
In the three and a half years between September 1995 and March 1999, we examined 81 patients from 78 retinoblastoma families referred from all over India. These included 51 males (62.96%) and 30 females (37.04%) (male to female ratio = 1.7:1); the age of onset of the disease according to the history ranged from 1 month to 24 years. Forty-five patients had bilateral manifestations (55.56%), while 36 had unilateral (44.44%). The mean age of onset for bilateral cases was 12.37 months (range = 10 days to 48 months, median = 6.5 months) whereas in the unilateral cases, it was nearly three times higher - 33.07 months (range = 1 to 288 months, median = 22.5 months). Age of onset of the disease differed significantly between unilateral and bilateral groups [Figure:1] with a 'p' value of 0.048. In the groups put together, the mean age of onset was 21.56 months (range = 10 days to 288 months, median = 12 months).
Only 5 families (8 patients) had positive family history for the disease (6.41%) (see pedigrees shown in [Figure:2]). The inheritance pattern was sporadic in 73 (90.12%), autosomal dominant (ad) in 5 (6.17%) and suspected ad in 3 (3.7%) patients. Nearly half, 36 out of 78 (46.15%) families were one-child families. There were 7 (8.97%) consanguineous marriages amongst the 78 families. On segregation analysis the x2 was 16.536, the value of x2 is significant with one degree of freedom if it is greater than 3.841 and therefore there was no significant departure from the expected number of normal and affected offspring assuming autosomal dominant inheritance. Six of 72 patients (8.33%) had 13q14 deletion. Two patients (2.78%) had 1q41 chromatid break. Three (4.33%) de novo balanced translocations, t(6:11) (q13:q25) [Figure:3], t(12:13) (q23:q33) and t(13:4) (q14:p16.3) were observed in three unrelated in three unrelated patients; all were mosaic with a normal choromosomal constitution as well.
In India, retinoblastoma is the leading paediatric tumour after Wilm's tumour and lymphoma,[25,26] and stands third in the five-year survival rate next to thyroid carcinoma and Hodgkin's disease. In a northern Indian study unilateral disease was 61.8%, median age at presentation was 12 months for bilateral and 41 months for unilateral retinoblastoma; male / female ratio was 1.41:1; consanguinity was 17% and positive family history was 1.7%. In our study, unilateral disease was 44.44%, median age of onset was 12.37 months for bilateral and 33.07 months for unilateral retinoblastoma; male/female ratio was 1.7:1; consanguinity was 8.97% and positive family history was present in 6.41% of families. We believe that the reason for the preponderance of males in this ad disease could be due to cultural and social influences.
In a majority of retinoblastoma cases, the symptoms become evident before the age of three. It was similar in our patients (mean age of onset: 21.56 months). It has been reported that the mean age for bilateral cases is 12 months whereas in unilateral cases it is 23 months. In our study the average age of onset for bilateral cases was 12.37 months and for unilateral cases, 33.07 months. The higher age of onset in the unilateral group was due to the fact that two patients in our series reported unilateral retinoblastoma after 20 years of age. Cases of late onset of retinoblastoma however have been reported earlier.[31,32]
A majority of the patients (90.12%) in our study showed sporadic inheritance. Five individuals of two unrelated families showed autosomal dominant inheritance. However, in three unrelated individuals it could possibly be an autosomal dominant inheritance; particularly, in one family where a second cousin was affected. We presume this family could have low penetrance of the retinoblastoma gene as reported earlier.[33,34] Even though majority of cases were sporadic, segregation analysis showed autosomal dominant inheritance. This statistical correlation was possibly because nearly half of the families (46.15%) had only one child.
The consanguinity observed in these families could be due to the high incidence of consanguineous marriages in our population and therefore could be coincidental. The reported incidence of 13q14 deletion has been from 4 to 5%.[15,16,36-38] In our study it was 8.33% considerably higher than these reports. Potluri et al reported non-random chromosomal aberrations in 82 patients, where they observed monosomies and deletions of chromosome 13 in a few cases and additional copies of 1q, 6p or extra chromosome 6 in a few others. We observed three (4.33%) de novo autosomal balanced translocations, t(6:11) (q13:q25), t(12:13) (q23:q33) and t(13:4) (q14:p16.3). The first was a male but the other two were females. Interestingly, all three patients were mosaics with normal cells as well and such mosaicism has been reported earlier both in 13q14 deletions and in balanced translocations.[42,43] Autosomal de novo balanced translocations reported were t(1:13) (p22:q12) t(13:18) (q14.1:q12.2) and t(2:13) (p24.3:q14.2). Only further molecular genetic studies could prove whether the translocation case we report with non-involvement of chromosome 13[t(6:11)] could be coincidental or not. Rosenberg et al applied flourescent-in-situ-hybridisation (FISH) to study a constitutive rearrangement involving chromosomes 4 and 13. This technique helps to detect translocations and micro-deletions easily. We also had two patients with chromatid break in 1q41 region; deletion 1q41\?\ter has been reported earlier.
In our study, except for one 1q41 chromatid break, all other patients with a cytogenetic abnormality had bilateral retinoblastoma. Patients with bilateral retinoblastoma have an increased incidence of second primary tumours and this rate is further increased in those treated with external radiotherapy. Our study period was too short to assess for any second primary tumours. The 13q14 region has been shown to harbour the osteosarcoma gene as well, however, recently this region has generated considerable interest and shown to harbour genes for prostate, pituitary, breast and bladder cancers too.[50-53] In the west, molecular genetic techniques like FISH, DNA fragment analysis, and mutational screening are presently being used in the diagnosis and genetic counselling for patients and families with retinoblastoma. This could in time be made safer and cheaper than conventional ophthalmic screening.[54- Currently molecular genetic studies are in progress in these patients; such methods could help one-child families to have normal children.
In conclusion, in the 81 retinoblastoma patients we found: (i) earlier onset (mean age of onset: 12.37 months) of the disease in bilateral retinoblastoma cases than unilateral (mean age of onset: 33.07 months), (ii) higher incidence of 13q14 chromosomal deletion (8.33%) than in earlier reports, (iii) sporadic inheritance of the disease in a majority of the patients (90.12%) and (iv) three novel de novo balanced translocations.
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