Dgcr8 functions in the secondary heart field for outflow tract and right ventricle development in mammals.

The DGCR8 gene, encoding a critical miRNA processing protein, maps within the hemizygous region in patients with 22q11.2 deletion syndrome. Most patients have malformations of the cardiac outflow tract that is derived in part from the anterior second heart field (aSHF) mesoderm. To understand the function of Dgcr8 in the aSHF, we inactivated it in mice using Mef2c-AHF-Cre. Inactivation resulted in a fully penetrant persistent truncus arteriosus and a hypoplastic right ventricle leading to lethality by E14.5. To understand the molecular mechanism for this phenotype, we performed gene expression profiling of the aSHF and the cardiac outflow tract with right ventricle in conditional null versus normal mouse littermates at stage E9.5 prior to morphology changes. We identified dysregulation of mRNA gene expression, of which some are relevant to cardiogenesis. Many pri-miRNA genes were strongly increased in expression in mutant embryos along with reduced expression of mature miRNA genes. We further examined the individual, mature miRNAs that were decreased in expression along with pri-miRNAs that were accumulated that could be direct effects due to loss of Dgcr8. Among these genes, were miR-1a, miR-133a, miR-134, miR143 and miR145a, which have known functions in heart development. These early mRNA and miRNA changes may in part, explain the first steps that lead to the resulting phenotype in Dgcr8 aSHF conditional mutant embryos.

Crk and Crkl Are Required in the Endocardial Lineage for Heart Valve Development.

Background Endocardial cells are a major progenitor population that gives rise to heart valves through endocardial cushion formation by endocardial to mesenchymal transformation and the subsequent endocardial cushion remodeling. Genetic variants that affect these developmental processes can lead to congenital heart valve defects. Crk and Crkl are ubiquitously expressed genes encoding cytoplasmic adaptors essential for cell signaling. This study aims to explore the specific role of Crk and Crkl in the endocardial lineage during heart valve development. Methods and Results We deleted Crk and Crkl specifically in the endocardial lineage. The resultant heart valve morphology was evaluated by histological analysis, and the underlying cellular and molecular mechanisms were investigated by immunostaining and quantitative reverse transcription polymerase chain reaction. We found that the targeted deletion of Crk and Crkl impeded the remodeling of endocardial cushions at the atrioventricular canal into the atrioventricular valves. We showed that apoptosis was temporally increased in the remodeling atrioventricular endocardial cushions, and this developmentally upregulated apoptosis was repressed by deletion of Crk and Crkl. Loss of Crk and Crkl also resulted in altered extracellular matrix production and organization in the remodeling atrioventricular endocardial cushions. These morphogenic defects were associated with altered expression of genes in BMP (bone morphogenetic protein), connective tissue growth factor, and WNT signaling pathways, and reduced extracellular signal-regulated kinase signaling activities. Conclusions Our findings support that Crk and Crkl have shared functions in the endocardial lineage that critically regulate atrioventricular valve development; together, they likely coordinate the morphogenic signals involved in the remodeling of the atrioventricular endocardial cushions.

Crk/Crkl regulates early angiogenesis in mouse embryos by accelerating endothelial cell maturation.

Rationale: Ubiquitously expressed cytoplasmic adaptors CRK and CRKL mediate multiple signaling pathways in mammalian embryogenesis. They are also associated with cardiovascular defects occurring in Miller-Dieker syndrome and 22q11.2 deletion syndrome, respectively. The embryonic mesoderm contributes to the formation of the cardiovascular system, yet the roles that Crk and Crkl play there are not understood on a single cell level. Objectives: To determine functions of Crk and Crkl in the embryonic mesoderm during early mouse vascular development. Secondly, we will examine the molecular mechanisms responsible for early embryonic endothelial cell (EC) defects by performing single cell RNA-sequencing (scRNA-seq) and in vivo validation experiments. Methods and Results: Inactivation of both Crk and Crkl together using Mesp1 Cre resulted embryonic lethality with severe vascular defects. Although vasculogenesis appeared normal, angiogenesis was disrupted both in the yolk sac and embryo proper, leading to disorganized vascular networks. We performed scRNA-seq of the Mesp1 Cre mesodermal lineage and found that there was upregulation of a great number of angiogenesis and cell migration related genes in ECs in the mutants, including NOTCH signaling genes such as Dll4 and Hey1 . Further bioinformatic analysis of EC subpopulations identified a relative increase in the number of more differentiated angiogenic ECs and decrease in EC progenitors. Consistent with this, we identified an expansion of Dll4 expressing cells within abnormal arteries, in vivo . Also, our bioinformatic data indicates that there is dysregulated expression of lineage genes that promote EC differentiation causing accelerated cell fate progression during EC differentiation. Conclusions: Our results show that Crk and Crkl are crucial for regulating early embryonic angiogenesis. Combined inactivation of Crk/Crkl caused precocious EC maturation with an increase of atypical differentiated angiogenic ECs and failed vascular remodeling. This is in part due to increased NOTCH signaling and altered expression of cell migration genes.

An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.

BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation.

Rare coding variants as risk modifiers of the 22q11.2 deletion implicate postnatal cortical development in syndromic schizophrenia.

22q11.2 deletion is one of the strongest known genetic risk factors for schizophrenia. Recent whole-genome sequencing of schizophrenia cases and controls with this deletion provided an unprecedented opportunity to identify risk modifying genetic variants and investigate their contribution to the pathogenesis of schizophrenia in 22q11.2 deletion syndrome. Here, we apply a novel analytic framework that integrates gene network and phenotype data to investigate the aggregate effects of rare coding variants and identified modifier genes in this etiologically homogenous cohort (223 schizophrenia cases and 233 controls of European descent). Our analyses revealed significant additive genetic components of rare nonsynonymous variants in 110 modifier genes (adjusted P = 9.4E-04) that overall accounted for 4.6% of the variance in schizophrenia status in this cohort, of which 4.0% was independent of the common polygenic risk for schizophrenia. The modifier genes affected by rare coding variants were enriched with genes involved in synaptic function and developmental disorders. Spatiotemporal transcriptomic analyses identified an enrichment of coexpression between modifier and 22q11.2 genes in cortical brain regions from late infancy to young adulthood. Corresponding gene coexpression modules are enriched with brain-specific protein-protein interactions of SLC25A1, COMT, and PI4KA in the 22q11.2 deletion region. Overall, our study highlights the contribution of rare coding variants to the SCZ risk. They not only complement common variants in disease genetics but also pinpoint brain regions and developmental stages critical to the etiology of syndromic schizophrenia.

Single-cell transcriptomics uncovers a non-autonomous Tbx1-dependent genetic program controlling cardiac neural crest cell development.

Disruption of cardiac neural crest cells (CNCCs) results in congenital heart disease, yet we do not understand the cell fate dynamics as these cells differentiate to vascular smooth muscle cells. Here we performed single-cell RNA-sequencing of NCCs from the pharyngeal apparatus with the heart in control mouse embryos and when Tbx1, the gene for 22q11.2 deletion syndrome, is inactivated. We uncover three dynamic transitions of pharyngeal NCCs expressing Tbx2 and Tbx3 through differentiated CNCCs expressing cardiac transcription factors with smooth muscle genes. These transitions are altered non-autonomously by loss of Tbx1. Further, inactivation of Tbx2 and Tbx3 in early CNCCs results in aortic arch branching defects due to failed smooth muscle differentiation. Loss of Tbx1 interrupts mesoderm to CNCC cell-cell communication with upregulation and premature activation of BMP signaling and reduced MAPK signaling, as well as alteration of other signaling, and failed dynamic transitions of CNCCs leading to disruption of aortic arch artery formation and cardiac outflow tract septation.

Tbx2 and Tbx3 regulate cell fate progression of the otic vesicle for inner ear development.

The morphogenesis of the otic vesicle (OV) to form inner ear organs serves as an excellent model system to understand cell fate acquisition on a single cell level. Tbx2 and Tbx3 (Tbx2/3) encode closely related T-box transcription factors that are expressed widely in the mammalian OV. Inactivation of both genes in the OV (Tbx2/3cKO) results in failed morphogenesis into inner ear organs. To understand the basis of these defects, single cell RNA-sequencing (scRNA-seq) was performed on the OV lineage, in controls versus Tbx2/3cKO embryos. We identified a multipotent population termed otic progenitors in controls that are marked by expression of the known otic placode markers Eya1, Sox2, and Sox3 as well as new markers Fgf18, Cxcl12, and Pou3f3. The otic progenitor population was increased three-fold in Tbx2/3cKO embryos, concomitant with dysregulation of genes in these cells as well as reduced progression to more differentiated states of prosensory and nonsensory cells. An ectopic neural population of cells was detected in the posterior OV of Tbx2/3cKO embryos but had reduced maturation to delaminated neural cells. As all three cell fates were affected in Tbx2/3cKO embryos, we suggest that Tbx2/3 promotes progression of multipotent otic progenitors to more differentiated cell types in the OV.

Platelet findings in 22q11.2 deletion syndrome correlate with disease manifestations but do not correlate with GPIb surface expression.

Prior studies have demonstrated that patients with chromosome 22q11.2 deletion syndrome (22q11.2DS) have lower platelet counts (PC) compared to non-deleted populations. They also have an increased mean platelet volume. The mechanism for this has been postulated to be haploinsufficiency of the GPIBB gene. We examined platelet parameters, deletion size and factors known to influence counts, including status of thyroid hormone and congenital heart disease (CHD), in a population of 825 patients with 22q11.2DS. We also measured surface expression of GPIB-IX complex by flow cytometry. The major determinant of PC was deletion status of GP1BB, regardless of surface expression or other factors. Patients with nested distal chromosome 22q11.2 deletions (those with GP1BB present) had higher PCs than those with proximal deletions where GP1BB is deleted. Patients with 22q11.2DS also demonstrated an accelerated PC decrease with age, occurring in childhood. These data demonstrate that genes within the proximal deletion segment drive PC differences in 22q11.2DS and suggest that PC reference ranges may need to be adjusted for age and deletion size in 22q11.2DS populations. Bleeding did not correlate with either platelet count or GPIb expression. Further studies into drivers of expression of GPIb and associations with severe thrombocytopenia and immune thrombocytopenia are needed to inform clinical care.

Influence of Parent-of-Origin on Intellectual Outcomes in the Chromosome 22q11.2 Deletion Syndrome.

Learning and intellectual disabilities are hallmark features of 22q11.2 deletion syndrome. Data are limited, however, regarding influences on full-scale IQ (FSIQ). Here, we investigated possible 22q11.2 deletion parent-of-origin effects. In 535 individuals, we compared FSIQ (≥50), 481 with de novo and 54 with inherited 22q11.2 deletions. In the subsets with data available, we examined parent-of-origin effects on FSIQ. We used linear regression models to account for covariates. Median FSIQ was significantly higher in de novo vs. inherited deletions (77; range 50−116 vs. 67; range 50−96, p < 0.0001). Results remained significant using a regression model accounting for age at IQ testing, sex and cohort site. No significant parent-of-origin differences in FSIQ were observed for de novo deletions (n = 81, 63.0% maternal; p = 0.6882). However, median FSIQ was significantly lower in maternally than in paternally inherited familial deletions (65, range 50−86 vs. 71.5, range 58−96, respectively, p = 0.0350), with the regression model indicating an ~8 point decrement in FSIQ for this variable (p = 0.0061). FSIQ is higher on average in de novo than in inherited 22q11.2 deletions, regardless of parental origin. However, parent-of-origin appears relevant in inherited deletions. The results have potential clinical implications with further research needed to delineate possible actionable mechanisms.

Crk and Crkl have shared functions in neural crest cells for cardiac outflow tract septation and vascular smooth muscle differentiation.

CRK and CRKL encode cytoplasmic adaptors that contribute to the etiology of congenital heart disease. Neural crest cells (NCCs) are required for cardiac outflow tract (OFT) septation and aortic arch formation. The roles of Crk/Crkl in NCCs during mouse cardiovascular development remain unknown. To test this, we inactivated Crk and/or Crkl in NCCs. We found that the loss of Crk, rather than Crkl, in NCCs resulted in double outlet right ventricle, while loss of both Crk/Crkl in NCCs resulted in severe defects with earlier lethality due to failed OFT septation and severe dilation of the pharyngeal arch arteries (PAAs). We found that these defects are due to altered cell morphology resulting in reduced localization of NCCs to the OFT and failed integrity of the PAAs, along with reduced expression of Integrin signaling genes. Further, molecular studies identified reduced differentiation of vascular smooth muscle cells that may in part be due to altered Notch signaling. Additionally, there is increased cellular stress that leads to modest increase in apoptosis. Overall, this explains the mechanism for the Crk/Crkl phenotype.

A normative chart for cognitive development in a genetically selected population.

Certain pathogenic genetic variants impact neurodevelopment and cause deviations from typical cognitive trajectories. Understanding variant-specific cognitive trajectories is clinically important for informed monitoring and identifying patients at risk for comorbid conditions. Here, we demonstrate a variant-specific normative chart for cognitive development for individuals with 22q11.2 deletion syndrome (22q11DS). We used IQ data from 1365 individuals with 22q11DS to construct variant-specific normative charts for cognitive development (Full Scale, Verbal, and Performance IQ). This allowed us to calculate Z-scores for each IQ datapoint. Then, we calculated the change between first and last available IQ assessments (delta Z-IQ-scores) for each individual with longitudinal IQ data (n = 708). We subsequently investigated whether using the variant-specific IQ-Z-scores would decrease required sample size to detect an effect with schizophrenia risk, as compared to standard IQ-scores. The mean Z-IQ-scores for FSIQ, VIQ, and PIQ were close to 0, indicating that participants had IQ-scores as predicted by the normative chart. The mean delta-Z-IQ-scores were equally close to 0, demonstrating a good fit of the normative chart and indicating that, as a group, individuals with 22q11DS show a decline in IQ-scores as they grow into adulthood. Using variant-specific IQ-Z-scores resulted in 30% decrease of required sample size, as compared to the standard IQ-based approach, to detect the association between IQ-decline and schizophrenia (p < 0.01). Our findings suggest that using variant-specific normative IQ data significantly reduces required sample size in a research context, and may facilitate a more clinically informative interpretation of IQ data. This approach allows identification of individuals that deviate from their expected, variant-specific, trajectory. This group may be at increased risk for comorbid conditions, such as schizophrenia in the case of 22q11DS.

Single cell multi-omic analysis identifies a Tbx1-dependent multilineage primed population in murine cardiopharyngeal mesoderm.

The poles of the heart and branchiomeric muscles of the face and neck are formed from the cardiopharyngeal mesoderm within the pharyngeal apparatus. They are disrupted in patients with 22q11.2 deletion syndrome, due to haploinsufficiency of TBX1, encoding a T-box transcription factor. Here, using single cell RNA-sequencing, we now identify a multilineage primed population within the cardiopharyngeal mesoderm, marked by Tbx1, which has bipotent properties to form cardiac and branchiomeric muscle cells. The multilineage primed cells are localized within the nascent mesoderm of the caudal lateral pharyngeal apparatus and provide a continuous source of cardiopharyngeal mesoderm progenitors. Tbx1 regulates the maturation of multilineage primed progenitor cells to cardiopharyngeal mesoderm derivatives while restricting ectopic non-mesodermal gene expression. We further show that TBX1 confers this balance of gene expression by direct and indirect regulation of enriched genes in multilineage primed progenitors and downstream pathways, partly through altering chromatin accessibility, the perturbation of which can lead to congenital defects in individuals with 22q11.2 deletion syndrome.

Genome-Wide Association Studies of Conotruncal Heart Defects with Normally Related Great Vessels in the United States.

Conotruncal defects with normally related great vessels (CTD-NRGVs) occur in both patients with and without 22q11.2 deletion syndrome (22q11.2DS), but it is unclear to what extent the genetically complex etiologies of these heart defects may overlap across these two groups, potentially involving variation within and/or outside of the 22q11.2 region. To explore this potential overlap, we conducted genome-wide SNP-level, gene-level, and gene set analyses using common variants, separately in each of five cohorts, including two with 22q11.2DS (N = 1472 total cases) and three without 22q11.2DS (N = 935 total cases). Results from the SNP-level analyses were combined in meta-analyses, and summary statistics from these analyses were also used in gene and gene set analyses. Across all these analyses, no association was significant after correction for multiple comparisons. However, several SNPs, genes, and gene sets with suggestive evidence of association were identified. For common inherited variants, we did not identify strong evidence for shared genomic mechanisms for CTD-NRGVs across individuals with and without 22q11.2 deletions. Nevertheless, several of our top gene-level and gene set results have been linked to cardiogenesis and may represent candidates for future work.

Common Variation in Cytoskeletal Genes is Associated with Conotruncal Heart Defects.

There is strong evidence for a genetic contribution to non-syndromic congenital heart defects (CHDs). However, exome- and genome-wide studies conducted at the variant and gene-level have identified few genome-wide significant CHD-related genes. Gene-set analyses are a useful complement to such studies and candidate gene-set analyses of rare variants have provided insight into the genetics of CHDs. However, similar analyses have not been conducted using data on common genetic variants. Consequently, we conducted common variant analyses of 15 CHD candidate gene-sets, using data from two common types of CHDs: conotruncal heart defects (1431 cases) and left ventricular outflow tract defects (509 cases). After Bonferroni correction for evaluation of multiple gene-sets, the cytoskeletal gene-set was significantly associated with conotruncal heart defects (ßS = 0.09; 95% confidence interval (CI) 0.03-0.15). This association was stronger when analyses were restricted to the sub-set of cytoskeletal genes that have been observed to harbor rare damaging genotypes in at least two CHD cases (ßS = 0.32, 95% CI 0.08-0.56). These findings add to the evidence linking cytoskeletal genes to CHDs and suggest that, for cytoskeletal genes, common variation may contribute to the risk of CHDs.

Genetic contributors to risk of schizophrenia in the presence of a 22q11.2 deletion.

Schizophrenia occurs in about one in four individuals with 22q11.2 deletion syndrome (22q11.2DS). The aim of this International Brain and Behavior 22q11.2DS Consortium (IBBC) study was to identify genetic factors that contribute to schizophrenia, in addition to the ~20-fold increased risk conveyed by the 22q11.2 deletion. Using whole-genome sequencing data from 519 unrelated individuals with 22q11.2DS, we conducted genome-wide comparisons of common and rare variants between those with schizophrenia and those with no psychotic disorder at age ≥25 years. Available microarray data enabled direct comparison of polygenic risk for schizophrenia between 22q11.2DS and independent population samples with no 22q11.2 deletion, with and without schizophrenia (total n = 35,182). Polygenic risk for schizophrenia within 22q11.2DS was significantly greater for those with schizophrenia (padj = 6.73 × 10-6). Novel reciprocal case-control comparisons between the 22q11.2DS and population-based cohorts showed that polygenic risk score was significantly greater in individuals with psychotic illness, regardless of the presence of the 22q11.2 deletion. Within the 22q11.2DS cohort, results of gene-set analyses showed some support for rare variants affecting synaptic genes. No common or rare variants within the 22q11.2 deletion region were significantly associated with schizophrenia. These findings suggest that in addition to the deletion conferring a greatly increased risk to schizophrenia, the risk is higher when the 22q11.2 deletion and common polygenic risk factors that contribute to schizophrenia in the general population are both present.

Using common genetic variation to examine phenotypic expression and risk prediction in 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome (22q11DS) is associated with a 20-25% risk of schizophrenia. In a cohort of 962 individuals with 22q11DS, we examined the shared genetic basis between schizophrenia and schizophrenia-related early trajectory phenotypes: sub-threshold symptoms of psychosis, low baseline intellectual functioning and cognitive decline. We studied the association of these phenotypes with two polygenic scores, derived for schizophrenia and intelligence, and evaluated their use for individual risk prediction in 22q11DS. Polygenic scores were not only associated with schizophrenia and baseline intelligence quotient (IQ), respectively, but schizophrenia polygenic score was also significantly associated with cognitive (verbal IQ) decline and nominally associated with sub-threshold psychosis. Furthermore, in comparing the tail-end deciles of the schizophrenia and IQ polygenic score distributions, 33% versus 9% of individuals with 22q11DS had schizophrenia, and 63% versus 24% of individuals had intellectual disability. Collectively, these data show a shared genetic basis for schizophrenia and schizophrenia-related phenotypes and also highlight the future potential of polygenic scores for risk stratification among individuals with highly, but incompletely, penetrant genetic variants.

Optical mapping of the 22q11.2DS region reveals complex repeat structures and preferred locations for non-allelic homologous recombination (NAHR).

The most prevalent microdeletion in humans occurs at 22q11.2, a region rich in chromosome-specific low copy repeats (LCR22s). The structure of this region has defied elucidation due to its size, regional complexity, and haplotype diversity, and is not well represented in the human genome reference. Most individuals with 22q11.2 deletion syndrome (22q11.2DS) carry a de novo hemizygous deletion of ~ 3 Mbp occurring by non-allelic homologous recombination (NAHR) mediated by LCR22s. In this study, optical mapping has been used to elucidate LCR22 structure and variation in 88 individuals in thirty 22q11.2DS families to uncover potential risk factors for germline rearrangements leading to 22q11.2DS offspring. Families were optically mapped to characterize LCR22 structures, NAHR locations, and genomic signatures associated with the deletion. Bioinformatics analyses revealed clear delineations between LCR22 structures in normal and deletion-containing haplotypes. Despite no explicit whole-haplotype predisposing configurations being identified, all NAHR events contain a segmental duplication encompassing FAM230 gene members suggesting preferred recombination sequences. Analysis of deletion breakpoints indicates that preferred recombinations occur between FAM230 and specific segmental duplication orientations within LCR22A and LCR22D, ultimately leading to NAHR. This work represents the most comprehensive analysis of 22q11.2DS NAHR events demonstrating completely contiguous LCR22 structures surrounding and within deletion breakpoints.

Gene-based analyses of the maternal genome implicate maternal effect genes as risk factors for conotruncal heart defects.

Congenital heart defects (CHDs) affect approximately 1% of newborns. Epidemiological studies have identified several genetically-mediated maternal phenotypes (e.g., pregestational diabetes, chronic hypertension) that are associated with the risk of CHDs in offspring. However, the role of the maternal genome in determining CHD risk has not been defined. We present findings from gene-level, genome-wide studies that link CHDs to maternal effect genes as well as to maternal genes related to hypertension and proteostasis. Maternal effect genes, which provide the mRNAs and proteins in the oocyte that guide early embryonic development before zygotic gene activation, have not previously been implicated in CHD risk. Our findings support a role for and suggest new pathways by which the maternal genome may contribute to the development of CHDs in offspring.

Atypical chromosome 22q11.2 deletions are complex rearrangements and have different mechanistic origins.

The majority (99%) of individuals with 22q11.2 deletion syndrome (22q11.2DS) have a deletion that is caused by non-allelic homologous recombination between two of four low copy repeat clusters on chromosome 22q11.2 (LCR22s). However, in a small subset of patients, atypical deletions are observed with at least one deletion breakpoint within unique sequence between the LCR22s. The position of the chromosome breakpoints and the mechanisms driving those atypical deletions remain poorly studied. Our large-scale, whole genome sequencing study of >1500 subjects with 22q11.2DS identified six unrelated individuals with atypical deletions of different types. Using a combination of whole genome sequencing data and fiber-fluorescence in situ hybridization, we mapped the rearranged alleles in these subjects. In four of them, the distal breakpoints mapped within one of the LCR22s and we found that the deletions likely occurred by replication-based mechanisms. Interestingly, in two of them, an inversion probably preceded inter-chromosomal 'allelic' homologous recombination between differently oriented LCR22-D alleles. Inversion associated allelic homologous recombination (AHR) may well be a common mechanism driving (atypical) deletions on 22q11.2.

The 22q11 low copy repeats are characterized by unprecedented size and structural variability.

Low copy repeats (LCRs) are recognized as a significant source of genomic instability, driving genome variability and evolution. The Chromosome 22 LCRs (LCR22s) mediate nonallelic homologous recombination (NAHR) leading to the 22q11 deletion syndrome (22q11DS). However, LCR22s are among the most complex regions in the genome, and their structure remains unresolved. The difficulty in generating accurate maps of LCR22s has also hindered localization of the deletion end points in 22q11DS patients. Using fiber FISH and Bionano optical mapping, we assembled LCR22 alleles in 187 cell lines. Our analysis uncovered an unprecedented level of variation in LCR22s, including LCR22A alleles ranging in size from 250 to 2000 kb. Further, the incidence of various LCR22 alleles varied within different populations. Additionally, the analysis of LCR22s in 22q11DS patients and their parents enabled further refinement of the rearrangement site within LCR22A and -D, which flank the 22q11 deletion. The NAHR site was localized to a 160-kb paralog shared between the LCR22A and -D in seven 22q11DS patients. Thus, we present the most comprehensive map of LCR22 variation to date. This will greatly facilitate the investigation of the role of LCR variation as a driver of 22q11 rearrangements and the phenotypic variability among 22q11DS patients.