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. 2017 Nov 15;7(1):15677.
doi: 10.1038/s41598-017-15814-7.

Genomic mosaicism in paternal sperm and multiple parental tissues in a Dravet syndrome cohort

Xiaoxu Yang  1 , Aijie Liu  2 , Xiaojing Xu  2 , Xiaoling Yang  2 , Qi Zeng  2 , Adam Yongxin Ye  1   3   4 , Zhe Yu  3   5 , Sheng Wang  6   7 , August Yue Huang  1 , Xiru Wu  2 , Qixi Wu  8   9   10 , Liping Wei  11   12 , Yuehua Zhang  13   14
Affiliations

Genomic mosaicism in paternal sperm and multiple parental tissues in a Dravet syndrome cohort

Xiaoxu Yang et al. Sci Rep. .

Abstract

Genomic mosaicism in parental gametes and peripheral tissues is an important consideration for genetic counseling. We studied a Chinese cohort affected by a severe epileptic disorder, Dravet syndrome (DS). There were 56 fathers who donated semen and 15 parents who donated multiple peripheral tissue samples. We used an ultra-sensitive quantification method, micro-droplet digital PCR (mDDPCR), to detect parental mosaicism of the proband's pathogenic mutation in SCN1A, the causal gene of DS in 112 families. Ten of the 56 paternal sperm samples were found to exhibit mosaicism of the proband's mutations, with mutant allelic fractions (MAFs) ranging from 0.03% to 39.04%. MAFs in the mosaic fathers' sperm were significantly higher than those in their blood (p = 0.00098), even after conditional probability correction (p' = 0.033). In three mosaic fathers, ultra-low fractions of mosaicism (MAF < 1%) were detected in the sperm samples. In 44 of 45 cases, mosaicism was also observed in other parental peripheral tissues. Hierarchical clustering showed that MAFs measured in the paternal sperm, hair follicles and urine samples were clustered closest together. Milder epileptic phenotypes were more likely to be observed in mosaic parents (p = 3.006e-06). Our study provides new insights for genetic counseling.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
DS cohort analyzed in the study and mDDPCR results identifying parental mosaicism in blood samples. (a) Description of the cohort analyzed in this study. A total of 719 DS affected families identified since 2005 were included. Sanger sequencing, panel NGS sequencing and MLPA detected SCN1A mutations from probands in 591 families. Peripheral blood samples from both parents are available for 242 of the families, and there are 8 probands among these families that inherited mutations from their parents according to the Sanger screening results. A total of 132 of the families provided blood samples and agreed to be included in the mDDPCR screening. TaqMan genotyping assays were able to be conducted for 112 families. (b) Overview of mDDPCR results that identified “de novo” mutations in families. The y-axis shows the maximum likelihood estimates of MAFs, and the error bars show the 95% binomial CIs calculated from the mDDPCR results. The probands have corrected MAFs between 40% and 60%, whereas the allele frequencies detected in the parents and negative controls were all under the detection limit. (c) Overview of MAFs measured in candidate parental mosaic families. Blood samples from family members are plotted. For each parental mosaic family, only one mosaic parent had an MAF 95% binomial CI between 50% and 0%. Blood samples from the non-mosaic parent and the negative control show MAFs of approximately 0%. (d) A representative result for parental mosaicism identified in family DS314 is shown. The PCR Sanger sequencing chromatogram, mDDPCR flow cytometry scatter plots and PASM raw IGV views and CI calculations after Bayesian modeling for the blood samples from the DS314 family are provided. Detailed mDDPCR flow cytometry scatter plots for all parental mosaic families are provided in Supplementary Fig. S7. (e) Histogram of the MAF distribution for parental blood samples from parental mosaic families.
Figure 2
Figure 2
Mutant allelic fractions (MAFs) detected by mDDPCR in paternal sperm samples were significantly higher than those measured in blood. (a) MAF in paternal sperm versus blood samples from the same individuals. Each color represents a different father. The MAFs were higher in sperm than in blood, and the difference was statistically significant (p = 0.00098 by a paired single-tailed Wilcoxon rank-sum test). In three of the families, only MAFs detected in the sperm sample exceeded the cutoff value of 10−4. (b) In families DS203, DS296 and DS308, parental mosaicism was only identified in the fathers’ sperm samples and not in the fathers’ blood samples. (c) Square-root transformed MAFs measured in parental sperm samples are positively correlated with those in blood (R2 = 0.87, p = 8.873e-05 by an F test). The 95% CI of the regression line is shown in blue curves; 95% prediction intervals are shown in orange curves.
Figure 3
Figure 3
MAFs measured in multiple peripheral tissue samples of the mosaic parents. The color of each cell represents a different MAF. Each column represents a family affected by DS, sorted by the MAFs in the mosaic parents’ blood. Each row represents a tissue or sample type. Hierarchical clustering of square-root transformed MAFs from different sample types shows that samples from the same mosaic parent cluster together, and parental blood and saliva have more similar MAFs than oral epithelia. Hair follicle samples from mosaic parents cluster closer to the paternal sperm samples than to the urine samples. Blood samples from controls and non-mosaic parents all have MAFs of approximately 0% and are clustered together. The heterozygous probands have MAFs of approximately 50%. Multiple tissue and control MAFs analyzed by mDDPCR are shown in Supplementary Fig. S8.
Figure 4
Figure 4
Parental mosaicism contributes to the parents’ epileptic phenotypes. Epileptic phenotype data were collected from all family members by clinicians from Peking University First Hospital. (a) Of mosaic parents, 41% report having had an epileptic seizure. Parents with epileptic phenotypes were significantly more likely to be observed among parents with detectable mosaic mutations (odds ratio = 10.8, p = 3.0e-06 by a two-tailed Fisher’s exact test). (b) Among parents in the “de novo” families, 6% have previously had epileptic seizures. (c) No non-mosaic parents from mosaic families reported having had any epileptic seizures. (d) Among families with detected parental mosaicism, MAFs in the mosaic parents with epileptic phenotypes at any time in their lives were significantly higher than MAFs in epilepsy-free mosaic parents (p = 0.010 by a one-tailed Mann-Whitney U test with continuity correction).
Figure 5
Figure 5
Proband mosaicism is confirmed and influences phenotypic characters. (a) The proband from the DS315 family turned out to be a mosaic proband with an MAF of 32.98% after homology correction. Proband DS178 and DS315 share the same point mutation, NM_001165963.1: c.1837C > T. DS178 had an onset of seizures at 6 months of age; however, the mosaic proband DS315 has his first seizure at 10 months. (b) The proband from the DS330 family was found to have an MAF of 26.48% after homology correction. No mutation signals were found in his parents. (c) The distribution of the ages of onset for all other probands carrying de novo SCN1A mutations are shown; arrows on dashed red lines indicate that the ages of onset of DS330 (9.5 months, p = 0.04816 by a single-tailed Wilcoxon rank sum test with continuity correction) and DS315 (10 months, p = 0.04816 by a single-tailed Wilcoxon rank sum test with continuity correction) are significantly later than those of nearly all probands carrying other de novo SCN1A mutations. Dashed blue lines describe the 95% confidence intervals of the distribution of proband ages of onset.
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