Volume 8 Supplement 1
De novo mutations discovered in 8 Mexican American families through whole genome sequencing
© Wang and Zhu; licensee BioMed Central Ltd. 2014
Published: 17 June 2014
De novo mutations enrich the sequence diversity and carry the clue of evolutional selection. Recent studies suggest the de novo mutations could be one of the risk factors for complex diseases. We conducted a survey of de novo mutations using the whole genome sequence data but only available on the odd autosomes of Mexican American families provided by Genetic Analysis Workshop 18. We extracted 8 three-generation families who have sequencing data available from 20 large pedigrees. By comparing the known single nucleotide variants (SNVs) in dbSNP129 and the de novo variants transmitted in the Mexican American families, we were able to estimate a de novo mutation rate of 1.64(±0.42) × 10−8 per position per haploid genome. This result is consistent with the estimates in literature that required many extensive validation efforts, such as genotyping and further resequencing. Our analysis suggests the importance of using family samples for studying rare variants.
De novo mutations enrich the sequence diversity and carry the clue of evolutional selection . Because of the technological advances in whole genome sequencing, genome-wide de novo mutation survey becomes possible. Recent studies show that de novo mutations, including de novo copy number variations, are strongly associated with multiple diseases, such as autism and schizophrenia . Currently de novo mutations are often studied in family trios by comparing the parents' and child's whole genome sequence data, as well as the publicly available dbSNP database . Variants observed in offspring, but not in their parents, are often considered as potential de novo mutations. However, even highly accurate sequencing data will have inevitable errors that lead to false variant callings and possible mendelian errors. Therefore, the de novo mutation candidates observed by comparing offspring's and their parents' sequencing data can be false positive . Thus, researchers often resequence or genotype the candidates to confirm the true de novo mutations [1–4]. This procedure could be time and money consuming. Here we propose an approach using 3-generation families to detect de novo mutations (a) using the parents and grandparents to search for de novo mutation candidates, and (b) using offspring sequence data to confirm true de novo mutations. We applied this approach to the Genetic Analysis Workshop 18 (GAW18) data and found our results consistent with previous genotyping and further resequencing validation efforts. This result suggested our approach is reliable. With the continuously decreasing cost of whole genome sequencing, this approach should be efficient to detect de novo mutations.
Summary of de novo mutation numbers in each family.
Observed de novomutations N o
De novomutation rate µ
2.00 × 10−8
1.85 × 10−8
1.33 × 10−8
2.44 × 10−8
1.48 × 10−8
1.41 × 10−8
1.33 × 10−8
1.26 × 10−8
1.64(±0.42) × 10−8
We used the UCSC genome browser (http://genome.ucsc.edu/) [7, 8] and SIFT (http://sift.jcvi.org/)  to map and predict the protein functions of the 186 de novo mutations. Seven of them are in exon regions and 2 are nonsynonymous SNVs. One of the nonsynonymous SNVs is in the gene PDZ domain containing 2 (PDZD2) on chromosome 5; the other is in gene spastic ataxia of Charlevoix-Saguenay (sacsin) (SACS) on chromosome 13. PDZ domains are protein-protein recognition modules that play a central role in organizing diverse cell signaling assemblies, most often in the cytoplasmic tails of transmembrane receptors and channels. PDZD2 and its secreted form (sPDZD2) are possibly involved in functional maturation of human fetal PPC-derived ICCs and the early stages of prostate tumorigenesis [10, 11]. SACS encodes the sacsin protein, which is highly expressed in the central nervous system. Mutations in this gene will cause autosomal recessive spastic ataxia of Charlevoix-Saguenay, but the detail of its function is still unknown [12, 13].
CpG sites are known as the mutation hotspots in mammals . In the great apes, the de novo mutation rate on the CpG sites is estimated to be 11 times higher than that on the non-CpG sites [4, 15]. We extracted the CpG islands from UCSC genome browser and examined the locations of the identified de novo mutations. Of our confirmed 186 de novo mutations, only 1 is located on the CpG islands. Considering the coverage of CpG islands on the odd autosomes, we expect we underestimated the CpG mutations. In the remaining 185 non-CpG mutations, we observed 127 transition mutations and 58 transversion mutations. The transition-to-transversion ratio is 2.2, similar to previous estimates [4, 6].
Furthermore, we examined the relationships between the age of parents and the de novo mutation rate in the child using the first 2 generations in the 8 families by constructing linear models. In general, the de novo mutation rate in the child increases with the child's parents' ages, especially with the father's age. This is consistent with the previous report that the de novo mutation rate in offspring is positively correlated with the paternal age . Nevertheless, no significant association effect was observed because of the small sample size in this study.
We conducted an analysis of the whole genome sequences on odd autosomes of 8 three-generation families to identify de novo mutations. We found this 3-generation approach is efficient, although no further resequencing of the candidate variants was performed. In the 8 selected Mexican American families, we estimated a mutation rate of 1.64(±0.42) × 10−8 per position per haploid human genome, which is consistent with the previous estimates [4–6].
Among the 13,584 de novo mutation candidates observed in 8 three-generation families, only 186 are observed in grandchildren. This is remarkably less than the expected number of transmissions, suggesting that most de novo mutation candidates can be attributed to SNV calling errors. Because the goals in a whole genome sequencing project are to detect rare and possible de novo variants and test for association of these to a complex disease, how to account for the false-positive calls of SNVs is extremely important in an association study. Our analysis suggests sequencing family members is an efficient way to detect these SNV calling errors. For example, our analysis suggests that a variant observed in offspring but not in their parents in a simple trio can usually be treated as an SNV calling error, and should be excluded in downstream analyses. Previous studies suggest family data has many statistical advantages in detecting rare disease variants [16, 17]. Thus, our results suggest whole-genome sequencing family members is worthwhile when most current whole genome sequencing projects only focus on unrelated subjects. It should be pointed out that the recruitment of multigeneration pedigrees is more difficult than family trios. However, many multigeneration pedigrees have already been collected in traditional linkage studies, such as the pedigrees used here. We expect the proposed method can be useful in detecting de novo mutations.
The work was supported by the National Institutes of Health, grants HL086718 and HL053353 from National Heart, Lung, Blood Institute, and HG003054 and HG005854 from the National Human Genome Research Institute. The Genetic Analysis Workshop is supported by the National Institutes of Health, grant R01 GM031575.
The GAW18 whole genome sequence data were provided by the T2D-GENES Consortium, which is supported by NIH grants U01 DK085524, U01 DK085584, U01 DK085501, U01 DK085526, and U01 DK085545. The other genetic and phenotypic data for GAW18 were provided by the San Antonio Family Heart Study and San Antonio Family Diabetes/Gallbladder Study, which are supported by NIH grants P01 HL045222, R01 DK047482, and R01 DK053889. The Genetic Analysis Workshop is supported by NIH grant R01 GM031575.
This article has been published as part of BMC Proceedings Volume 8 Supplement 1, 2014: Genetic Analysis Workshop 18. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcproc/supplements/8/S1. Publication charges for this supplement were funded by the Texas Biomedical Research Institute.
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