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Investigation of the cooperation between Kmt2d and Tbx1 reveals shared developmental pathways in 22q11.2DS 

Clinical Genetics and Therapeutics
  • Primary Categories:
    • Basic Research
  • Secondary Categories:
    • Basic Research
Introduction:
In 22q11.2 deletion syndrome (22q11.2DS) there is a broad range of phenotypic presentation and severity despite ~90% patients sharing the same de novo 3 Mb deletion size on the 22nd chromosome. Some of its most common features include congenital heart disease (CHD), immunodeficiency, intellectual disability, and craniofacial dysmorphism, and yet these occur at varying rates between patients. TBX1 is an important causative gene of the 60-80% of CHD seen in 22q11.2DS, and yet its haploinsufficiency is not sufficient to explain the phenotypic variability. A possible explanation for this is that gene variants in the remainder of the genome impact pathways that have developmental overlap with those deleted in 22q11.2DS. In this way, 22q11.2DS represents a unique population in which to uncover genetic modifiers of congenital anomalies, as it provides a distinct genetic background with higher rates of pathology than the general population.

Methods:
To identify potential modifiers, our lab previously performed genome sequence analysis on 1,182 participants with 22q11.2DS and discovered variants in chromatin regulatory genes occurring in 8.5% of the subjects with CHD. This study aims to investigate the interaction of these genes with Tbx1, starting with the histone methyltransferase Kmt2d, as it can cause CHD and/or Kabuki syndrome when mutated in humans. To test this interaction, mouse crosses were generated with the conditional deletion of Kmt2d in the Tbx1 cell lineage and then assessed for phenotypic changes. Analysis was performed at multiple stages in developmental time to identify late-stage phenotypes (embryonic days (E) 15.5-17.5) and their earlier-stage developmental origins (E9.5-E11.5).

Results:
Conditional deletion of Kmt2d in the Tbx1 lineage leads to perinatal lethality in all mutant mice, with death occurring over the course of the first day post-birth (P0). A significant subset (~40%) present with aortic arch anomalies, including aberrant right subclavian artery and interrupted aortic arch type B. Assessment of the pharyngeal arch arteries (PAAs) earlier in developmental time at E10.5 using PECAM1 whole mount immunofluorescence revealed either absence or hypoplasia of the 4th PAAs in 100% of mutant embryos. Further characterization of the mutant phenotype has revealed thymic ectopia and/or hypoplasia in 100% of mutant embryos (n=20), as well as nonlethal but distinctive craniofacial dysmorphism that includes a narrow head and mandibular hypoplasia (p<0.05). Inspection of the pharyngeal apparatus in early development using a combination of frontal H&E sectioning and RNAscope in situ hybridization was suggestive of 3rd and 4th pharyngeal arch abnormalities. Finally, single cell RNA-sequencing was performed on control versus mutant embryos at E9.5, revealing changes in developmental pathways relevant to the phenotypes that were observed.

Conclusion:
The characterization of the Kmt2d conditional deletion in the Tbx1 lineage in mice overlaps with the presentation seen in patients of both 22q11.2DS and Kabuki syndrome. This is suggestive that Tbx1 and Kmt2d cooperate in shared developmental processes, including those involved in CHD. Additional functional genomics studies will delineate the shared and unique developmental roles of Kmt2d and Tbx1 which will further the understanding of the genetic architecture of both syndromic and non-syndromic developmental anomalies.

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