Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies

Abstract

Retinoblastoma, the most common childhood eye cancer, presents in two forms: heritable or sporadic. Heritable retinoblastoma is caused by a germline mutation in the RB1 gene. Early diagnosis of children at risk of inheriting an RB1 mutation is crucial to achieve optimal clinical outcome. Currently, the majority of genetic testing is performed on newborns, which has multiple disadvantages for both families and the healthcare system. We have developed a non-invasive prenatal diagnosis (NIPD) service for retinoblastoma, available from 8 weeks' gestation, which uses a combination of massively parallel sequencing (MPS) techniques, dependent on the inheritance model. Detection of paternal or suspected de novo RB1 variants is achieved through amplicon-based MPS. NIPD of a fetus at risk of maternal inheritance is performed using capture-based targeted sequencing and relative haplotype dosage analysis. In addition, we show proof of principle of how capture-based sequencing can be used for de novo variants unsuitable for amplicon-based testing. In total, we report the NIPD of 15 pregnancies, results of which show 100% concordance with all postnatal testing performed at the time of publication (n = 12) with remaining pregnancies ongoing. NIPD of retinoblastoma therefore offers a viable alternative to newborn genetic testing.

Keywords: cell-free DNA; non-invasive; prenatal diagnosis; retinoblastoma.

Figure 1

Graphical representation of the mutant allele % detected in both maternal cfDNA (green) and maternal gDNA (blue) for each family tested by amplicon-based MPS. The maternal cfDNA mutant allele % is an average of two duplicate samples. The maximum mutant allele % detected in the five control cfDNA samples is also shown (black dotted line). Tests reported as predicted to be affected are indicated with a star.

Figure 2

Graphic representation of NIPD by relative haplotype dosage analysis (RHDO) result for (a) Family L and (b) Family M at risk of maternal inheritance of retinoblastoma. Haplotype blocks are represented by contiguous arrows spanning a 5 Mb genomic region on chromosome 13 (black line) containing RB1 (orange bar). Red arrows indicate the fetus has inherited the mutated maternal (M-ma) allele. Blue arrows indicate the fetus has inherited wild-type (N-ma) alleles. The position of informative single nucleotide polymorphisms (SNPs) used to identify haplotype blocks is shown for both alpha (green triangles) and beta (green crosses) SNPs.

Figure 3

Graphic representation of the paternal allele frequency (PAF%) across the 5 Mb region of chromosome 13 containing RB1 (orange bar) within the previous affected child (proband) gDNA (blue line) and maternal cfDNA (red line) of Family N. The affected child is hemizygous for chr13:47,515,019–49,482,240. The maternal cfDNA had a calculated fetal fraction of 6% and an average of 2.83% paternal alleles across the region, indicating that the fetus is unaffected.