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Variant discovery and breakpoint region prediction for studying the human 22q11.2 deletion using BAC clone and whole genome sequencing analysis.

ABSTRACT: Velo-cardio-facial syndrome/DiGeorge syndrome/22q11.2 deletion syndrome (22q11.2DS) is caused by meiotic non-allelic homologous recombination events between flanking low copy repeats termed LCR22A and LCR22D, resulting in a 3 million base pair (Mb) deletion. Due to their complex structure, large size and high sequence identity, genetic variation within LCR22s among different individuals has not been well characterized. In this study, we sequenced 13 BAC clones derived from LCR22A/D and aligned them with 15 previously available BAC sequences to create a new genetic variation map. The thousands of variants identified by this analysis were not uniformly distributed in the two LCR22s. Moreover, shared single nucleotide variants between LCR22A and LCR22D were enriched in the Breakpoint Cluster Region pseudogene (BCRP) block, suggesting the existence of a possible recombination hotspot there. Interestingly, breakpoints for atypical 22q11.2 rearrangements have previously been located to BCRPs To further explore this finding, we carried out in-depth analyses of whole genome sequence (WGS) data from two unrelated probands harbouring a de novo 3Mb 22q11.2 deletion and their normal parents. By focusing primarily on WGS reads uniquely mapped to LCR22A, using the variation map from our BAC analysis to help resolve allele ambiguity, and by performing PCR analysis, we infer that the deletion breakpoints were most likely located near or within the BCRP module. In summary, we found a high degree of sequence variation in LCR22A and LCR22D and a potential recombination breakpoint near or within the BCRP block, providing a starting point for future breakpoint mapping using additional trios.

SUBMITTER: Guo X

PROVIDER: S-EPMC5216616 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Variant discovery and breakpoint region prediction for studying the human 22q11.2 deletion using BAC clone and whole genome sequencing analysis.

Guo Xingyi X Delio Maria M Haque Nousin N Castellanos Raquel R Hestand Matthew S MS Vermeesch Joris R JR Morrow Bernice E BE Zheng Deyou D

Human molecular genetics 20160719 17

Velo-cardio-facial syndrome/DiGeorge syndrome/22q11.2 deletion syndrome (22q11.2DS) is caused by meiotic non-allelic homologous recombination events between flanking low copy repeats termed LCR22A and LCR22D, resulting in a 3 million base pair (Mb) deletion. Due to their complex structure, large size and high sequence identity, genetic variation within LCR22s among different individuals has not been well characterized. In this study, we sequenced 13 BAC clones derived from LCR22A/D and aligned t ...[more]

PMID: 27436579

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Project description:BACKGROUND: Individuals affected with DiGeorge and Velocardiofacial syndromes present with both phenotypic diversity and variable expressivity. The most frequent clinical features include conotruncal congenital heart defects, velopharyngeal insufficiency, hypocalcemia and a characteristic craniofacial dysmorphism. The etiology in most patients is a 3 Mb recurrent deletion in region 22q11.2. However, cases of infrequent deletions and duplications with different sizes and locations have also been reported, generally with a milder, slightly different phenotype for duplications but with no clear genotype-phenotype correlation to date. METHODS: We present a 7 month-old male patient with surgically corrected ASD and multiple VSDs, and dysmorphic facial features not clearly suggestive of 22q11.2 deletion syndrome, and a newborn male infant with cleft lip and palate and upslanting palpebral fissures. Karyotype, FISH, MLPA, microsatellite markers segregation studies and SNP genotyping by array-CGH were performed in both patients and parents. RESULTS: Karyotype and FISH with probe N25 were normal for both patients. MLPA analysis detected a partial de novo 1.1 Mb deletion in one patient and a novel partial familial 0.4 Mb duplication in the other. Both of these alterations were located at a distal position within the commonly deleted region in 22q11.2. These rearrangements were confirmed and accurately characterized by microsatellite marker segregation studies and SNP array genotyping. CONCLUSION: The phenotypic diversity found for deletions and duplications supports a lack of genotype-phenotype correlation in the vicinity of the LCRC-LCRD interval of the 22q11.2 chromosomal region, whereas the high presence of duplications in normal individuals supports their role as polymorphisms. We suggest that any hypothetical correlation between the clinical phenotype and the size and location of these alterations may be masked by other genetic and/or epigenetic modifying factors.

Project description:Background22q11.2 deletion syndrome (22q11.2DS) is the most common micro-deletion syndrome. The associated 22q11.2 deletion conveys the strongest known molecular risk for schizophrenia. Neurodevelopmental phenotypes, including intellectual disability, are also prominent though variable in severity. Other developmental features include congenital cardiac and craniofacial anomalies. Whereas existing mouse models have been helpful in determining the role of some genes overlapped by the hemizygous 22q11.2 deletion in phenotypic expression, much remains unknown. Simple model organisms remain largely unexploited in exploring these genotype-phenotype relationships.MethodsWe first developed a comprehensive map of the human 22q11.2 deletion region, delineating gene content, and brain expression. To identify putative orthologs, standard methods were used to interrogate the proteomes of the zebrafish (D. rerio), fruit fly (D. melanogaster), and worm (C. elegans), in addition to the mouse. Spatial locations of conserved homologues were mapped to examine syntenic relationships. We systematically cataloged available knockout and knockdown models of all conserved genes across these organisms, including a comprehensive review of associated phenotypes.ResultsThere are 90 genes overlapped by the typical 2.5 Mb deletion 22q11.2 region. Of the 46 protein-coding genes, 41 (89.1 %) have documented expression in the human brain. Identified homologues in the zebrafish (n = 37, 80.4 %) were comparable to those in the mouse (n = 40, 86.9 %) and included some conserved gene cluster structures. There were 22 (47.8 %) putative homologues in the fruit fly and 17 (37.0 %) in the worm involving multiple chromosomes. Individual gene knockdown mutants were available for the simple model organisms, but not for mouse. Although phenotypic data were relatively limited for knockout and knockdown models of the 17 genes conserved across all species, there was some evidence for roles in neurodevelopmental phenotypes, including four of the six mitochondrial genes in the 22q11.2 deletion region.ConclusionsSimple model organisms represent a powerful but underutilized means of investigating the molecular mechanisms underlying the elevated risk for neurodevelopmental disorders in 22q11.2DS. This comparative multi-species study provides novel resources and support for the potential utility of non-mouse models in expression studies and high-throughput drug screening. The approach has implications for other recurrent copy number variations associated with neurodevelopmental phenotypes.

Dataset Information

Variant discovery and breakpoint region prediction for studying the human 22q11.2 deletion using BAC clone and whole genome sequencing analysis.

Publications

Variant discovery and breakpoint region prediction for studying the human 22q11.2 deletion using BAC clone and whole genome sequencing analysis.

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