Complete lung agenesis caused by complex genomic rearrangements with neo-TAD formation at the SHH locus

During human organogenesis, lung development is a timely and tightly regulated developmental process under the control of a large number of signaling molecules. Understanding how genetic variants can disturb normal lung development causing different lung malformations is a major goal for dissecting molecular mechanisms during embryogenesis. Here, through exome sequencing (ES), array CGH, genome sequencing (GS) and Hi-C, we aimed at elucidating the molecular basis of bilateral isolated lung agenesis in three fetuses born to a non-consanguineous family. We detected a complex genomic rearrangement containing duplicated, triplicated and deleted fragments involving the SHH locus in fetuses presenting complete agenesis of both lungs and near-complete agenesis of the trachea, diagnosed by ultrasound screening and confirmed at autopsy following termination. The rearrangement did not include SHH itself, but several regulatory elements for lung development, such as MACS1, a major SHH lung enhancer, and the neighboring genes MNX1 and NOM1. The rearrangement incorporated parts of two topologically associating domains (TADs) including their boundaries. Hi-C of cells from one of the affected fetuses showed the formation of two novel TADs each containing SHH enhancers and the MNX1 and NOM1 genes. Hi-C together with GS indicate that the new 3D conformation is likely causative for this condition by an inappropriate activation of MNX1 included in the neo-TADs by MACS1 enhancer, further highlighting the importance of the 3D chromatin conformation in human disease.

Identification and characterisation of a novel aberrant pattern of intron 1 inversion with concomitant large insertion and deletion within the F8 gene

SummaryIntron 1 inversion (Inv1) is a recurrent causative mutation of haemophilia A (HA) and is responsible for 1–5% of severe HA. Inv1 occurs as a result of intra-chromosomal homologous recombination between int1h-1 within intron 1 and int1h-2 located in approximately 125 kb telomeric to the F8 gene. In this report, we presented a previously undescribed aberrant type of Inv1 with complex genomic rearrangement in a pedigree with severe HA. The breakpoints of the rearrangement were identified by the genome walking technique; copy number variations (CNVs) of the F8 gene and X chromosome were detected by AccuCopy technique, Affymetrix CytoScan HD CNV assay and quantitative PCR (qPCR); the F8 transcripts related to the aberrant Inv1 were analysed by reverse transcription PCR (RT-PCR). We have characterised the exact breakpoints of the complex rearrangement, and determined the location and size of the insertion and deletion. The rearrangements can be summarised as an aberrant pattern of Inv1 with a deletion of 2.56 kb and a duplication of 227.3 kb inserted in the rejoining junction within the F8 gene. Our results suggested that this complex genomic rearrangement was generated by two distinct repair mechanisms of fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) and nonallelic homologous recombination (NAHR).