RB1 gene mutations in Argentine retinoblastoma patients. Implications for genetic counseling

Abstract

Retinoblastoma (RB) is an inherited childhood ocular cancer caused by mutations in the tumor suppressor RB1 gene. Identification of RB1 mutations is essential to assess the risk of developing retinoblastoma in the patients´ relatives. Retinoblastoma is a potentially curable cancer and an early diagnosis is critical for survival and eye preservation. Unilateral retinoblastoma is mostly non-heritable and results from two somatic mutations whereas bilateral retinoblastoma is heritable and results from one germline and one somatic mutation, both have high penetrance, 90%. The purpose of this study was to identify causative RB1 mutations in RB patients with different clinical presentations. A comprehensive approach was used to study a cohort of 34 patients with unilateral, bilateral and trilateral retinoblastoma. Blood and tumor DNA was analyzed by sequencing and multiplex ligation-dependent probe amplification (MLPA) assay. Validation of an insertion mutation was performed by cloning the PCR product. Most of the patients in our cohort had unilateral RB, eight patients had bilateral RB and one patient had a trilateral tumor with ocular and suprasellar/sellar locations. Other tumors in addition to retinoblastoma were also found in the affected families. One patient had two syndromes, retinoblastoma and schwannomatosis, and another RB patient had a father with a retinoma. Five out of the 25 unilateral RB patients carried germinal mutations (20%), which were mostly missense mutations. The bilateral and trilateral patients carried splice-site, nonsense and frameshift mutations as well as a whole RB1 gene deletion. Missense mutations were associated with mild phenotype: unilateral retinoblastoma, retinoma or no tumor. In this study we identified causative RB1 mutations in most bilateral RB patients and in some unilateral RB patients, including five novel mutations. These data are crucial for genetic counseling and confirm the need to perform complete genetic screening for RB1 mutations in both constitutional and tumor tissues.

Fig 1. Sequence analysis of exon 14 in a unilateral RB patient (#668).

The heterozygous C to T transition generated a stop codon TGA, in which the mutant T peak was lower in height than the wild type C peak in DNA from blood, whereas in DNA from tumor the mutant T and wild type C peaks were similar suggesting a mosaic mutation.

Fig 2. Pedigree of a family with low penetrance retinoblastoma.

A unilateral RB patient (#661) carried a germline missense mutation in exon 7 which changed the amino acid leucine by proline (p.Leu223>Pro). The patient inherited this mutation from his father in whom this mutation led to a retinoma. Three out of nine of the father´s siblings also carried the mutation, but they were asymptomatic and none of them had children yet. OC: obligate carrier; half-blackened symbols: unilateral RB; dotted symbols: unaffected carriers; upper-left blackened symbol: retinoma; dashed symbol: deceased.

Fig 3. Sequence analysis of a 21-bp heterozygous insertion in exon 20 of RB1from a bilateral RB patient (#660).

Both, the mutant and wild type copies of exon 20 were retrieved by cloning. The site of insertion in the mutant copy is indicated between the arrows.