Samarth Bhatt

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis. Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.

Exposure to stress early in life permanently shapes activity of the hypothalamic-pituitary-adrenocortical (HPA) axis and the brain. Prenatally, glucocorticoids pass through the placenta to the fetus with postnatal impacts on brain development, birth weight (BW), and HPA axis functioning. Little is known about the biological mechanisms by which prenatal stress affects postnatal functioning. This study addresses this gap by examining the effect of chronic stress and traumatic war-related stress on epigenetic changes in four key genes regulating the HPA axis in neonatal cord blood, placenta, and maternal blood: CRH, CRHBP, NR3C1, and FKBP5. Participants were 24 mother-newborn dyads in the conflict-ridden region of the eastern Democratic Republic of Congo. BW data were collected at delivery and maternal interviews were conducted to assess culturally relevant chronic and war-related stressors. Chronic stress and war trauma had widespread effects on HPA axis gene methylation, with significant effects observed at transcription factor binding (TFB) sites in all target genes tested. Some changes in methylation were unique to chronic or war stress, whereas others were observed across both stressor types. Moreover, stress exposures impacted maternal and fetal tissues differently, supporting theoretical models that stress impacts vary according to life phase. Methylation in several NR3C1 and CRH CpG sites, all located at TFB sites, was associated with BW. These findings suggest that prenatal stress exposure impacts development via epigenetic changes in HPA axis genes.

Genomic copy number variation (CNV) plays a major role in various human diseases as well as in normal phenotypic variability. For some recurrent disease-causing CNVs that convey genomic disorders, the causative mechanism is meiotic, non-allelic, homologous recombination between breakpoint regions exhibiting extensive sequence homology (e.g. low-copy repeats). For the majority of recently identified rare pathogenic CNVs, however, the mechanism is unknown. Recently, a model for CNV formation implicated mitotic replication-based mechanisms, such as (alternative) non-homologous end joining and fork stalling and template switching, in the etiology of human pathogenic CNVs. The extent to which such mitotic mechanisms contribute to rare pathogenic CNVs remains to be determined. In addition, it is unexplored whether genomic architectural features such as repetitive elements or sequence motifs associated with DNA breakage stimulate the formation of rare pathogenic CNVs. To this end, we have sequenced breakpoint junctions of 30 rare pathogenic microdeletions and eight tandem duplications, representing the largest series of such CNVs examined to date in this much detail. Our results demonstrate the presence of (micro)homology ranging from 2 to over 75 bp, in 79% of the breakpoint junctions. This indicates that microhomology-mediated repair mechanisms, including the recently reported fork stalling and template switching and/or microhomology-mediated break-induced replication, prevail in rare pathogenic CNVs. In addition, we found that the vast majority of all breakpoints (81%) were associated with at least one of the genomic architectural features evaluated. Moreover, 75% of tandem duplication breakpoints were associated with the presence of one of two novel sequence motifs. These data suggest that rare pathogenic microdeletions and tandem duplications do not occur at random genome sequences, but are stimulated and potentially catalyzed by various genomic architectural features.