Analysis of Genetic Inheritance in a Family Quartet by Whole-Genome Sequencing

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Science  30 Apr 2010:
Vol. 328, Issue 5978, pp. 636-639
DOI: 10.1126/science.1186802


We analyzed the whole-genome sequences of a family of four, consisting of two siblings and their parents. Family-based sequencing allowed us to delineate recombination sites precisely, identify 70% of the sequencing errors (resulting in > 99.999% accuracy), and identify very rare single-nucleotide polymorphisms. We also directly estimated a human intergeneration mutation rate of ~1.1 × 10−8 per position per haploid genome. Both offspring in this family have two recessive disorders: Miller syndrome, for which the gene was concurrently identified, and primary ciliary dyskinesia, for which causative genes have been previously identified. Family-based genome analysis enabled us to narrow the candidate genes for both of these Mendelian disorders to only four. Our results demonstrate the value of complete genome sequencing in families.

Whole-genome sequences from four members of a family represent a qualitatively different type of genetic data than whole-genome sequences from individual or sets of unrelated genomes. They enable inheritance analyses that detect errors and permit the identification of precise locations of recombination events. This leads in turn to near-complete knowledge of inheritance states through the precise determination of the parental chromosomal origins of sequence blocks in offspring. Confident predictions of inheritance states and haplotypes power analyses that include the identification of genomic features with nonclassical inheritance patterns, such as hemizygous deletions or copy number variants (CNVs). Identification of inheritance patterns in the pedigree permits the detection of ~70% of sequencing errors and sharply reduces the search space for disease-causing variants. These analyses would be far less powerful in studies that had fewer markers (such as standard genotype or exome data sets) or that had sequences from fewer family members.

DNA from each family member was extracted from peripheral blood cells and sequenced at CGI (Mountain View, California) with a nanoarray-based short-read sequencing-by-ligation technology (1), including an adaptation of the pairwise end-sequencing strategy (2). Reads were mapped to the National Center for Biotechnology Information (NCBI) reference genome (fig. S1 and tables S1 and S2). Polymorphic markers used for this analysis were single-nucleotide polymorphisms (SNPs) with at least two variants among the four genotypes of the family, averaging 802 base pairs (bp) between markers. We observed 4,471,510 positions at which at least one family member had an allele that varied from the reference genome. This corresponds to a Watterson’s theta (θW) of 9.5 × 10−4 per site for the two parents and the reference sequence (3), given the fraction of the genome successfully genotyped in each parent (fig. S1). This is a close match to the estimate of θW = 9.3 × 10−4 that we obtained by combining two previously published European genomes and the reference sequence (4). Of the 4.5 million variant positions, 3,665,772 were variable within the family; the rest were homozygous and identical in all four members. Comparisons to known SNPs show that 323,255 of these 3.7 million SNPs are novel.

For each meiosis in a pedigree, each base position in a resulting gamete will have inherited one of two parental alleles. The number of inheritance patterns of the segregation of alleles in gametes is therefore 2n, where n is the number of meioses in a pedigree. In a nuclear family of four, the Mendelian inheritance patterns can be grouped into four inheritance states for each variant position, with children receiving (i) the same allele from both the mother and the father (identical), (ii) the same allele from the mother but opposites from the father (haploidentical maternal), (iii) the same allele from the father, but opposites from the mother (haploidentical paternal), or (iv) opposites from both parents (nonidentical) (fig. S2). Adjacent variant base pairs in alignments of the family genomes have the same inheritance state unless a recombination has occurred between these bases in one of the meioses. This delineates inheritance blocks.

Many algorithms can identify the boundaries of blocks, and theory-driven implementations are in wide use (57). For our complete genome sequence data, we developed an algorithm to identify all states, including non-Mendelian states. One non-Mendelian state will occur in regions where highly similar sequences are inadvertently compressed computationally (for example, during sequence assembly of CNVs). In such a “compression block,” many positions will appear to be heterozygous in all individuals, regardless of the inheritance patterns of the positions contributing to the compression. Other non-Mendelian patterns are seen in regions prone to errors in sequence calling or assembly or that have inherited hemizygous deletions. For both of these patterns, many positions will be observed as Mendelian inheritance errors (MIEs). Our algorithm identified six states: one for each of the four Mendelian inheritance states, one for a compression state, and one for a MIE-prone state (4). We identified 1.5% of the genome in this pedigree as 409 compression blocks and 1.7% as 126 error-prone blocks. Because these blocks are a source of false positives for recombination predictions, SNPs, and disease candidate alleles, their identification is important (Fig. 1). The power to precisely determine inheritance-state boundaries is striking in families of at least four and would be reduced had we sequenced fewer individuals (Fig. 2). Meiotic gene conversions could in principle be recognized in the same way as inheritance blocks; they would be indistinguishable from a short region flanked by meiotic recombinations in the same meiosis. We found that the great majority of candidate gene-conversion regions were caused by reads mismapped to repetitive DNA, such as CNVs or satellites, and did not conclusively identify gene-conversion regions.

Fig. 1

The landscape of recombination. Each chromosome in this schematic karyotype is used to represent information abstracted from the four corresponding chromosomes of the two children in the pedigree. It is vertically split to indicate the inheritance state from the father (left half) and mother (right half), as shown in the key. The three compound heterozygous (DHODH, DNAH5, and KIAA0556) and one recessive (CES1) candidate gene, depicted by red bands, lie in “identical” blocks. (Inset) Scatterplot of HapMap recombination rates (in centimorgans per megabase) within the predicted crossover regions. The maximum value of centimorgans per megabase found in each window is shown in red. The left histogram shows the size distribution of recombination windows (log10 value of –0.58 ± 0.92). The top graph shows the centimorgans per megabase distribution for the observed maximal values (red), for similarly sized windows shifted by 6 kb (orange), and for similarly sized windows randomly chosen from the entire genome (blue). A shift of 6 kb from the observed locations eliminates the correlation with hotspots. Of 155 recombination windows, 92 contained a HapMap site with >10 cM/Mb. Only five randomly picked windows are expected to contain such high recombination rates.

Fig. 2

Power of four. Shown are inheritance states for a single chromosome in six scenarios representing restrictions of the data set to the exome (for two siblings only or for the full family) or to subsets of the family (parents and one child, two siblings, or siblings and one parent), as compared with analysis with full data from all four family members. The most supported state for each bin is shown as a color; the height of each histogram bar is proportional to the number of informative markers supporting that state. The father has two regions of homozygosity (bottom, thin red lines) on the short arm of the chromosome, where it is not possible to distinguish the haploidentical maternal from identical states (fig. S2A, panel b). These regions are undetected when the mother’s genotypes are missing because all marker positions in the region are uninformative (second from bottom). A pedigree of two parents and one child has only one inheritance state and so provides no information on recombination. Red, identical; blue, nonidentical; green, haploidentical maternal; yellow, haploidentical paternal. Chromosome structure is annotated as in Fig. 1.

Recombination in maternal meioses is thought to occur 1.7 times more frequently than in paternal meioses (8). We inferred 98 crossovers in maternal and 57 in paternal meioses (count includes both offspring), which is consistent with this estimate. The median resolution of the 155 crossover sites was 2.6 kb, with a few sites localized within a 30-bp window (Fig. 1). Crossover sites were significantly correlated with hotspots of recombination as inferred from HapMap data, in which a hotspot is defined as a region with ≥10 centimorgan (cM)/Mb; 92 of the 155 recombinations took place in a hotspot.

By identifying inconsistencies across the 22% of the genomes of the two children in “identical” blocks, for which they are effectively twins, we computed an error rate of 1.0 × 10−5. We also determined error rate through other methods, including resequencing, which gave similar estimates, ranging from 8.1 × 10−6 to 1.1 × 10−5 (4). Furthermore, ~70% of the errors in a four-person pedigree can be detected as apparent MIEs and inconsistencies in inheritance state blocks, so the effective base-pair error rate in the context of a pedigree is ~3 × 10−6.

Analysis of the mutation rate, including germline and early embryonic somatic mutations, requires highly accurate sequence data. Even with such data, however, most apparent aberrations in allele inheritance will be due to errors in the data and not to mutation. Our data had thousands of such false-positive candidates for each true de novo mutation. Our initial data encompassed 2.3 billion bases and contained 49,720 candidate MIEs that were consistent with the presence of a single-nucleotide mutation. After excluding sites in MIE-prone and compression states as well as sites that were unsuitable for probe design, 33,937 potential mutations among 1.83 billion bases remained. We resequenced each of these candidates and applied a stringent base-calling algorithm to confirm 28 candidates as de novo mutations. In a final confirmation step, we verified all 28 mutations with mass spectrometry (table S3) (4), corresponding to a mutation rate of 3.8 × 10−9 per position per generation per haploid genome.

Because the raw estimate of 3.8 × 10−9 does not account for the true mutations that were not conclusively identified through resequencing, we estimated a false-negative rate by applying the base-calling algorithm to 5 Mb of independent resequencing data, divided into 25 randomly selected regions of the genome. A comparison of the resequencing data with the complete genome sequence for the same regions provided a de novo mutation false negative rate of 0.662 [95% confidence interval (CI) 0.644 to 0.680]. Adjusting for the false-negative rate produced an unbiased mutation rate estimate of 1.1 × 10−8 per position per haploid genome, corresponding to approximately 70 new mutations in each diploid human genome (95% CI of 6.8 × 10−9 to 1.7 × 10−8) (4). In great apes, CpG sites are reported to mutate at a rate 11 times higher than other sites (9). We observed five CpG mutations, closely matching this estimate. Of the remaining 23 mutations, seven were transversions and 16 were transitions. This yields a transition-to-transversion ratio of 2.3 (table S3), which is once again similar to a previous estimate of 2.2 for non-CpG sites (10).

Although both the observed transition-to-transversion ratio and the proportion of CpG mutations in our data match predictions, our estimated human mutation rate is lower than previous estimates, the most widely cited of which is 2.5 × 10−8 per generation (10) based on three parameters: a human-chimpanzee nucleotide divergence per site (Kt) of 0.013, a species divergence time of 5 million years ago, and an ancestral effective population size of 10,000. More recent estimates indicate a nucleotide divergence of 0.012 (9), species divergence time between 6 and 7 million years ago (1115), and ancestral effective population size between 40,000 and 148,000 (1619). With these parameter ranges and a generation length of 15 to 25 years, the mutation rate estimate is between 7.6 × 10−9 and 2.2 × 10−8 per generation, which is consistent with our intergenerational estimate of 1.1 × 10−8. Our estimate is within 1 SD of an earlier estimate of 1.7 × 10−8 (SD of 9 × 10−9) based on 20 disease-causing loci (20). The rate we report is for autosomes and should be substantially lower than that of the Y chromosome because in the male germ line, more cell divisions occur per generation. Although our rate differs approximately as expected from the recently reported estimate of 3.0 × 10−8 (95% CI, 8.9 × 10−9 to 7.0 × 10−8) for the Y chromosome, this difference is not significant (21).

Genomic inheritance analysis facilitates the identification of alleles that cause genetic disorders. Because genome sequences from a family of four provide near-exact determination of inheritance-state boundaries, the number of false-positive disease-gene candidates is greatly reduced as compared with those of analyses lacking the context of a pedigree or complete genome sequence (Fig. 3 and tables S3 and S4). Two disorders in this family—Miller syndrome and primary ciliary dyskinesia, which affect both offspring but neither parent—provided an opportunity to test this application. A parsimonious explanation is that each phenotype arises from defects in a single gene or a site regulating a single gene. The inheritance mode is undetermined, but a recessive mode is more consistent with observed data. We therefore examined each candidate variant by testing each of three inheritance modes: dominant, simple recessive, or compound heterozygote (a subcategory of recessive).

Fig. 3

The power of family genome inheritance analysis. The number of false-positive candidates drops exponentially as the number of family members increases. (A) Number of candidate SNPs that are consistent with a simple recessive inheritance mode. (B) Number of candidate genes that are consistent with a compound heterozygous model. The different groupings of parents (large silhouettes) and children (small silhouettes) are depicted below. Dashed lines join the average values of each grouping. For this figure, “probably detrimental” includes missense, nonsense, splice defect, and non-initiation; “possibly detrimental” also includes UTR, noncoding, and splice region. A block of SNPs so that all SNPs in the block are within 5 kb of another SNP in the block is counted only once because together these are likely to encode at most one phenotype. “A,” all probably detrimental SNPs; “B,” all possibly detrimental SNPs; “C,” rare possibly detrimental SNPs; “D,” rare probably detrimental SNPs.

The two recessive modes require that both offspring have identical dysfunctional variants for which the parents are heterozygous and which may come either from the same position (simple recessive) or occur at distinct positions within the same gene (compound heterozygote). Genes that are consistent with these two recessive modes must lie in “identical” inheritance blocks because both offspring are affected, limiting the search space to the 22% of the genome in these blocks. Because the phenotypes are rare, they are likely to be encoded by rare variants, which further limits the possibilities. Only two missense SNPs in the CES1 gene matched the simple recessive mode (table S4), whereas three genes fit the compound heterozygote mode: DHODH, DNAH5, and KIAA0556 (Fig. 1). A small number of possibly detrimental variants outside exons also matched the simple recessive mode: two in highly conserved regions, one in an intronic sequence near a splice site, five in non–protein-coding transcripts, and one in an untranslated region (UTR). Concurrent with this study, the core exomes of the two affected offspring were sequenced along with those of two unrelated individuals with Miller syndrome (22). Compared with that study of only affected individuals, our analysis of just two affected offspring and their unaffected parents reduced the number of gene candidates in the core exome from nine to four; had we not sequenced the parents, we would have had 34 rather than four candidates (Fig. 3 and table S5). The exome study supported DHODH as the primary gene for Miller syndrome. DNAH5 had been previously identified as a cause of primary ciliary dyskinesia, and so is probably the cause in these offspring as well (23).

Family genome analysis can clearly be effective for finding candidate genes that encode Mendelian traits because sequence accuracy is enhanced. In addition, delineation of recombination sites identifies inherited chromosome segments precisely and reduces the chromosomal search space for candidate genes (in this case to 22% of the genome). The ability to identify large effects of very rare alleles in small pedigrees can complement the power of genome-wide association studies in identifying weak effects of common alleles in large populations. An unknown fraction of important phenotypes in humans are encoded by nonexonic variants identified only by means of whole-genome sequencing. When the cost of recruiting additional families is expensive relative to sequencing costs, sequencing genomes of families will be an economical strategy for the identification of many disease-causing genes. Constraining searches to very rare variants can provide considerable power, as recently demonstrated for Freeman-Sheldon syndrome and congenital chloride diarrhea (24, 25). De novo mutations can be assayed, either as we have reported here or through family sequencing of more than two generations. As our knowledge of gene function increases, we will be able to use the power of family genome analysis rapidly to identify disease-gene candidates. These data, along with relevant environmental and medical information, will characterize the integrated medical records of the future.

Supporting Online Material

Materials and Methods

Figs. S1 to S5

Tables S1 to S5


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. This study was supported by the University of Luxembourg–Institute for Systems Biology Program and by these NIH grants: Center for Systems Biology GM076547 (L.H. and L.R.), RO1GM081083 (A.F.S. and G.G.), R01HL094976 and RZ1HG004749 (J.S.), RC2HG005608 (M.D. and J.S.), and R01HD048895 (M.B.). H. Tabor assisted with ethical review. J. Xing performed the principal components analysis. H. Mefford performed CNV analysis. A. Bigham and K. Buckingham evaluated candidate genes in unrelated individuals. D. Ballinger, A. Sparks, A. Halpern, and G. Nilsen assisted with sequencing and analysis. R. Bressler, S. Dee, and D. Mauldin assisted with bioinformatics. S. Ng and R. Qiu performed the capture array. S. Bloom obtained the resequencing data on the Illumina Genome Analyzer. M. Janer and S. Li performed Sequenom analysis. D. Cox commented on an early version of the manuscript. R. Durbin and D. Altshuler granted permission for our use of 1000 genomes SNP data. CGI employees (R.D. and K.P.) have stock options in the company. J.S. has consulted for CGI. L.H. is a scientific advisor to CGI and holds stock in the company. The dbGAP accessions can be found at (accession phs000244.v1.p1).
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