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An approach to localizing breakpoints with sequence-level precision employing massively parallel sequencing performed on libraries generated from haplotype-resolved chromosomes, genomic DNA, or molecular inversion probe–captured informative regions harboring paralog-distinguishing variants. The most common recurrent copy-number variants associated with autism, developmental delay and epilepsy are flanked by segmental duplications. Complete genetic characterization of these events is challenging because their breakpoints often occur within high-identity, copy-number polymorphic paralogous sequences that cannot be specifically assayed using hybridization-based methods. Here we provide a protocol for breakpoint resolution with sequence-level precision. Massively parallel sequencing is performed on libraries generated from haplotype-resolved chromosomes, genomic DNA or molecular inversion probe (MIP)-captured breakpoint-informative regions harboring paralog-distinguishing variants. Quantification of sequencing depth over informative sites enables breakpoint localization, typically within several kilobases to tens of kilobases. Depending on the approach used, the sequencing platform, and the accuracy and completeness of the reference genome sequence, this protocol takes from a few days to several months to complete. Once established for a specific genomic disorder, it is possible to process thousands of DNA samples within as little as 3–4 weeks.

The authors have stated that the coordinates of two deletions of the CFHR gene cluster on chromosome 1 represent the outer boundaries of the deletion interval based on patterns of sequence identity and included both copies of the duplicated sequence where breakpoint definition is difficult. For CNP147, this interval of 86.302kbp (chr1: 194988828–195075129) includes 1617 bp of 100% identical sequence located at the 5’ and 3’ breakpoints. Since the deletion allele retains a single copy of this sequence by virtue of non-allelic homologous recombination, the CNP147 deletion event in fact removes 84.685 kbp, a size consistent with the findings of Hughes et al. (2011) A 32 kb Critical Region Excluding Y402H in CFH Mediates Risk for Age-Related Macular Degeneration.

Genetic variants in the size range ∼1 kb-10 Mb altering the quantitative composition of a genome including insertions, duplications, and deletions, are known as copy-number variants (CNVs). This dissertation develops techniques addressing challenges in CNV detection and examines the population genetics of CNVs. To address challenges in integer assignment of CNV copy-number, four independent predictors of probe response are identified with potential utility in future array designs or data analyses. Additionally, an approach is developed to identify CNV breakpoints within segmental duplications (SDs), extended regions of highly identical sequence, using 17q21.31 deletions as a model locus. To examine the population genetics of CNVs, I characterized the prevalence copy-number variation and estimate its mutation rate in the general population. Among 2493 apparently normal individuals, large variants were collectively common with CNVs >500 kb observed in 5%–10% of individuals, and variants >1 Mb in 1%–2%. Conversely, correlations between the gene content, size, and frequency of CNVs suggested that such variation is generally deleterious. Underscoring the potential clinical impact of large CNVs, a meta-analysis of individuals with neuropsychiatric disease identified additional CNV loci (3q29, 16p12 and 15q25.2) for further investigation. Examining 386 trios unaffected by neuropsychiatric disease, I observe a genome-wide CNV mutation rate of μ = 1.2 × 10−2 CNVs per genome per transmission (μ = 6.5 × 10−3 for CNVs >500 kb), and infer that CNVs >500 kb are, on average, under significant purifying selection (s = 0.16). These observations suggest that large CNVs are fairly common in human populations due to a relatively high mutation rate but are constantly being removed by natural selection. Demonstrating how deleterious CNVs may manifest, identification of de novo CNVs in 3286 transmissions among 717 multiplex autism pedigrees revealed a fourfold enrichment for de novo CNVs in autism cases versus their unaffected siblings suggesting that many de novo CNVS contribute a subtle, but significant risk for autism. This work extends our ability to study CNVs in regions of the genome previously refractory to analysis, and highlights the importance of rare genetic variation in human disease.

We report the identification of a recurrent 520-kbp 16p12.1 microdeletion significantly associated with childhood developmental delay. The microdeletion was detected in 20/11,873 cases vs. 2/8,540 controls (p=0.0009, OR=7.2) and replicated in a second series of 22/9,254 cases vs. 6/6,299 controls (p=0.028, OR=2.5). Most deletions were inherited with carrier parents likely to manifest neuropsychiatric phenotypes (p=0.037, OR=6). Probands were more likely to carry an additional large CNV when compared to matched controls (10/42 cases, p=5.7×10-5, OR=6.65). Clinical features of cases with two mutations were distinct from and/or more severe than clinical features of patients carrying only the co-occurring mutation. Our data suggest a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity suggests that this two-hit model may be more generally applicable to neuropsychiatric disease.