Personalized recurrence risk assessment following the birth of a child with a pathogenic de novo mutation

Abstract

Following the diagnosis of a paediatric disorder caused by an apparently de novo mutation, a recurrence risk of 1-2% is frequently quoted due to the possibility of parental germline mosaicism; but for any specific couple, this figure is usually incorrect. We present a systematic approach to providing individualized recurrence risk. By combining locus-specific sequencing of multiple tissues to detect occult mosaicism with long-read sequencing to determine the parent-of-origin of the mutation, we show that we can stratify the majority of couples into one of seven discrete categories associated with substantially different risks to future offspring. Among 58 families with a single affected offspring (representing 59 de novo mutations in 49 genes), the recurrence risk for 35 (59%) was decreased below 0.1%, but increased owing to parental mixed mosaicism for 5 (9%)-that could be quantified in semen for paternal cases (recurrence risks of 5.6-12.1%). Implementation of this strategy offers the prospect of driving a major transformation in the practice of genetic counselling.

Fig. 1. Stratification of DNMs into seven categories.

Establishing the origin (paternal [blue], maternal [pink] or post-zygotic [proband, green]), and timing of the mutational events (purple colour indicates mutant cells), yields widely different recurrence risks in different families. See main text, Supplementary Fig. S1 and Supplementary Note 1.

Fig. 2. Flow chart describing the three-tier sample analysis in the PREGCARE study.

Following collection of up to 14 different biological samples per family and verification of the familial relationships between the 3 individuals of the trio, the DNM site was deep-sequenced in all family samples (performed in triplicate reactions together with 3 unrelated controls) to detect low levels of parental mosaicism or instances of post-zygotic mosaicism in the proband. For those families without evidence of overt mosaicism, haplotyping using long-read sequencing (MinION platform from Oxford Nanopore Technologies (ONT)) was performed to resolve the parental origin of the DNM and further stratify the recurrence risk (RR). Refer to Fig. 1 for category (Cat) description.

Fig. 3. Mutation levels observed in the families presenting with mosaicism.

Variant allele frequencies (VAF) in different samples from the family member in whom mosaicism was detected by ultra-deep Illumina Sequencing (Deep-NGS). Family number, gene, cDNA coordinates of the DNM and the origin of the different samples are indicated on the x-axis in the same order for each family. The category classification is indicated at the top of the figure and the colours reflect the parental/post-zygotic origin of the DNM (blue [somatic paternal tissues], turquoise [paternal sperm], pink [somatic maternal tissues] or green [post-zygotic/proband tissues]). The ╳ represents a sample failure. Full data for the other family members and controls are presented in Supplementary Fig. S2 and Supplementary Data 2. FAM17 and FAM60 are the two families with multiple affected pregnancies and belong to Category F (maternal mixed mosaicism); note the low VAF in the maternal blood samples (M3) for both families. Each bar represents a single VAF calculated from the sum of 3 technical replicates, then corrected using measurements from 3 unrelated controls (corrected VAFs, see “Methods”). Error bars represent the 95% binomial confidence intervals. Abbreviations: F father; M mother; C child; 1 = buccal mucosa (left); 2 = buccal mucosa (right); 3 = blood; 4 = saliva; 5 = urine; 6 = sperm; 7 = genomic DNA from original testing.

Fig. 4. Overview of the results of the PREGCARE study showing refinement of individual recurrence risk for all families.

a Summary table of the PREGCARE results for the 59 DNMs analyzed in this study and overview of the refined recurrence risk (RR). b Personalized recurrence risk (RR%) estimates for each of the 60 families (61 DNMs) enrolled in the PREGCARE study represented on a logarithmic scale. The red dotted line represents the generic population RR given to couples who have had a child with a DNM (~1.5%). Individual family numbers are indicated on the x-axis. Each bar represents the new refined RR for a given family enrolled into the PREGCARE study. For ease of visualization, bars are coloured according to the origin of the DNM [blue (paternal), pink (maternal), green (post-zygotic/proband) or grey (the parental origin could not be resolved)]. The RR can be quantified for DNMs of paternal (Categories A–C, via semen analysis, blue) and post-zygotic (Category G, green) origin (represented by block colours); note that to be conservative in our estimates of RR, the plotted bars represent the upper 95% binomial CI from the corrected VAF measured in sperm samples by Deep-NGS for each of the paternally-derived DNMs (Supplementary Data 2). The RR can only be estimated for maternal (pink) or haplotype-unresolved cases (grey). These estimates are represented by stripes, with error bars representing the upper and lower 95% CI—see Supplementary Note 6 for details on estimate calculations. Note that the DNM of FAM54 was analyzed by allele-specific PCR (Supplementary Note 2) and that Category F includes the two additional families with multiple affected pregnancies (FAM17 and FAM60).