Characterization Of Copy Number Variations At The Single-base-pair Level Through WGS And The Mutational Mechanisms Revealed

Copy number variations（CNVs）,≥1 kb DNA segments that differ in terms of their copy number with respect to reference genome sequences. It is reported that CNV can lead to or predispose to human disease. Due to the limitation of detection technology, a large fraction of these CNVs have not been accurately characterized at the single-base-pair level. With the development of the next generation sequencing, whole genome sequencing has been proved to be an effective measure to detect CNV rapidly and precisely. CNVs play a critical role in the pathogenesis of MO, so we use the discovery of the CNV in MO as a template to explore the structure features and research strategy.Multiple osteochondromas （MO, OMIM:213700）, are characterised by the development of two or more cartilage capped bony outgrowths （osteochondromas） of the long bones. EXTl （OMIM# 608177） and EXT2 （OMIM# 608210） are the responsible genes in most MO patients.It is reported that MO can be caused by various kinds of mutations in EXT1 and EXT2, of which mainly focused on the point mutations. Research on CNVs only account for a small percentage and only the deletion CNVs have been discovered previously.In our MO study we not only identified point mutations, but also accurately described the breakpoints and associated mutational complexity of CNVs. Then we expand the scope of the study to 26 deletion CNVs, revealing the frequent occurrence of micro-mutations within the breakpoint-flanking regions and the related mechanisms. Besides, we also discovered and characterized a novel duplication for MO, as well as disclosed its hidden structure and underlying mutational mechanism.To address these questions, we conducted two researches in our article:Chapter 1:Accurate characterization of 26 deletion CNVs reveals the mutational complexity and the mutational mechanismsOriginally, We collected 12 MO families and plan to explore the genetic causes of the Chinese families, Sanger sequencing was employed to screen the coding regions and exon/intron boundaries of two known HMO genes, EXT1 （GeneBank NM₀00127.2） and EXT2 （GeneBank NM₂07122.1）, We detected 5 point mutations（3 nonsense mutations and 2 frameshift mutations）. For all Chinese MO and Dent disease families, which the genetic basis of the pathology remained unexplained after Sanger sequencing, linkage analyses, fluorescence in situ hybridization （FISH）, multiplex ligation-dependent probe amplification （MLPA） and/or chromosome microarray analysis （CMA） were performed. As a result, we discovered 3 novel pathogenic deletion CNVs. To accurately described the 3 CNVs, we performed whole genome sequencing and successfully characteriazed them at the single-base-pair level. Besides, we revealed additional mutational complexity of the 3 deletion CNVs, including small insertions in the breakpoint junctions, micro-mutations and the microhomologies in the flanking regions. This prompted us to expand the scope to additional 23 deletion CNVs ramdomly selected from previously reported Canadian autism spectrum disorder families. We found that breakpoint-flanking micro-mutations （within 22 bp of the breakpoint） are present in a significant fraction （5/26; 19%） of the deletion CNVs,which is the first time to specifically and collectively address this issue in the context of deletion CNVs in human. This analysis also provided evidence that a recently described error-prone form of DNA repair （i.e., repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome） not only causes human genetic disease but also impacts on human genome evolution.Chapter 2:Accurate characterization of the duplication CNV responsible for MO and its hidden structure and underlying mutational mechanismMultiple osteochondromas （MO）, the most frequent bone tumor disease, is an autosomal dominantly inherited skeletal disorder. Recently, copy number variations （CNVs） were found to be closely related with the pathogenesis of MO.The additional mutational complexity associated with CNVs can provide important clues as to the underlying mechanisms of CNV formation. Correct annotation of the structure and the additional mutational complexity of CNV is, however, a prerequisite for establishing the mutational mechanism. For a Chinese three-generation MO family, which the genetic basis of the pathology remained unexplained after regular Sanger sequencing, we employed the MLPA analysis and found a novel pathogenic EXT1 duplication. Though the WGS of the proband in this family, the～230kb duplication CNV has been accurately characterized. Moreover, WGS identified the CNV’s mutational complexity as having a 22-bp insertion at the breakpoint junction and, unprecedentedly, multiple breakpoint-flanking micromutations on both sides of the duplication. Further investigation revealed that this genomic rearrangement had a duplication-inverted triplication-duplication structure, the inverted triplication being a 41-bp sequence synthesized from a nearby template. This permitted the identification of the sequence determinants of both the initiation （an inverted Alu repeat） and termination （a triplex-forming sequence） of breakinduced replication and suggested a possible model for the repair of replication-associated double-strand breaks. It is the first report that duplication CNV can lead to MO and it had the mutational complexity.