Impact of RB1 gene screening from blood collected on a single day from 411 family members of 113 Retinoblastoma survivors in India

Abstract

Objectives: To analyse the profile and implication of genetic testing in a cohort of retinoblastoma (RB) patients and their families conducted on a single day during World Retinoblastoma Awareness Week 2017.

Methods: Retrospective analysis of blood samples were collected from 411 subjects, including 113 probands at a camp organised for RB awareness and were analysed for RB1 mutations by Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). If germline mutations were detected, the parents and siblings of the proband were tested for the same mutation.

Results: Germline RB1 mutations were identified in 61/113(54%) probands with a mutation detection rate of 96% (47/49) and 22% (14/64) for bilateral and unilateral RB, respectively. Ten novel pathogenic mutations were identified. Splice mutation was most common (31%) followed by nonsense mutation (26%). The mean age at RB diagnosis was significantly lower in patients having germline RB1 mutation (mean 10.7 months ±2.5) compared to those without (mean 27.2 months ±6.5) (p = <0.0001). Parental transmission of the mutant allele was detected in 15/61(25%) cases of which 11(18%) parents were unaffected indicating incomplete penetrance. The origin of the variant allele was both paternal (n = 7) and maternal (n = 4) wherein 5 were bilateral and 6 unilateral.

Conclusions: The detection of a germline mutation impacts the proband and family members due to its implications on change in prognosis, frequency of subsequent evaluations, screening for ocular and non-ocular cancers, and surveillance of family and future progeny.

Fig. 1. Distribution and schematic representation of mutations across RB1 gene.

A Types of mutations detected in our probands. B Mutational spectrum across RB1 gene: Mutation types with their frequencies are marked to relative exons. Novel mutations are highlighted in boxes. Gross RB1 deletion and duplications are represented in brown and yellow line bars, respectively.

Fig. 2. Representative pedigree of families with low penetrance RB1 mutation.

Family A: Father is a normal carrier of the heterozygous mutation: c.-1_1delCA; Frameshift mutation. The proband and two siblings carry the same heterozygous mutation. Family B: Father is an unaffected carrier of the heterozygous mutation: dup-exon 21-26; Exon Duplication. The proband and one among the two siblings carry the mutation.

Fig. 3. Post testing surveillance in familial RB.

A Right eye showing regressed retinoblastoma in a child with bilateral tumour with left eye enucleated. Unilateral retinoblastoma in left eye of the sibling of the above child, screened at 5 days of birth - Group B RB at presentation (B) and type 1 regression at final follow up (C).