Disruption of OVOL2 Distal Regulatory Elements as a Possible Mechanism Implicated in Corneal Endothelial Dystrophy

The genetic architecture of corneal endothelial dystrophies remains unknown in a substantial number of affected individuals. The proband investigated in the current study was diagnosed in the neonatal period with bilateral corneal opacification due to primary endothelial cell dysfunction. Neither his parents nor his sister had signs of corneal disease. Conventional karyotyping revealed a de novo translocation involving chromosomes 3 and 20, t(3;20)(q25;p11-12). Following genome and targeted Sanger sequencing analysis, the breakpoints were mapped at the nucleotide level. Notably, the breakpoint on chromosome 20 was identified to lie within the same topologically associated domain (TAD) as corneal endothelial dystrophy-associated gene OVOL2, and it is predicted to disrupt distal enhancers. The breakpoint at chromosome 3 is located within intron 2 of PFN2, which is currently not associated with any human disease. Further interrogation of the proband’s genome failed to identify any additional potentially pathogenic variants in corneal endothelial dystrophy-associated genes. Disruption of a candidate cis-regulatory element and/or positional effects induced by translocation of OVOL2 to a novel genomic context may lead to an aberrant OVOL2 expression, a previously characterized disease mechanism of corneal endothelial dystrophy. Further research is necessary to explore how disruption of regulatory elements may elucidate genetically unsolved corneal endothelial dystrophies

The Potential Role of the Six1/Eya1 Pathway in the Establishment of Leukemia Stem Cells in MLL-ENL – Induced Leukemia

Abstract Abstract 1362 During the multistep pathogenesis of acute leukemia (AL), a pool of leukemia stem cells (LSCs) emerges that is capable of limitless self-renewal and ensuring disease maintenance. The molecular mechanism that controls the kinetics of cellular transformation and development of LSCs is largely unknown. Using our MLL-ENL-ERtm mouse model, we have previously shown (Takacova et al., Blood 2009, 114 (22): 947–947, ASH abstract) activation of the ATR/ATM-Chk1/Chk2-p53/p21 checkpoint leading to senescence at early stages of cellular transformation (myeloproliferation), thereby preventing AL development in vivo. Experimental ATM/ATR inhibition accelerated the transition to immature cell states, acquisition of LSC properties and AL development in these mice. The MLL-ENL-ERtm mouse model allows us to study the kinetics of MLL-ENL-ERtm LSC development. We raised the questions how the transformation process progresses from the pre-LSC to the LSC state, and how DNA damage response (DDR) - mediated senescence affects the transition in gene expression. Given that the threshold of DDR signaling events is rate-limiting, we determined the transcription profile of the pre- LSC–enriched cell states derived from bone marrow and spleen of the MLL-ENL-ERtm mice at the early disease stage, and we correlated this transcription profile with the level of DDR, proliferation rate and induction of senescence. Pair-wise comparisons revealed up-regulation of the Six1 transcription factor gene and its cofactor Eya1 in the MLL-ENL-ERtm pre-LSCs in association with aberrant proliferation in both tissues. The notable difference between the two tissues concerning the barrier induction was the higher threshold of DDR and senescence in the bone marrow due to cooperation with inflammatory cytokines that fine-tune the DDR level. Interestingly, the expression of Six1 and Eya1 genes was down-regulated in senescence exclusively in the bone marrow. Consistent with these in vivo data, we found Six1 expression decreased in response to inflammation/DDR-induced senescence in the MLL-ENL-ERtm bone marrow cells cultured in vitro and correlated with SA-beta-gal positivity and p16 up-regulation. Six1 mRNA level was decreased only transiently after ionizing radiation (4 Gy)-induced DDR in the same cell line. These data suggest that Six1 expression is down-regulated in response to high DDR and permanent cell-cycle arrest in the MLL-ENL-ERtm pre-LSCs. Furthermore, we identified the transcription profile of the LSC-enriched cell state after inhibition of DDR in caffeine-treated MLL-ENL-ERtm mice in vivo. Interestingly, the expression level of Six1 and Eya1 was significantly increased in the bone marrow and spleen of the MLL-ENL-ERtm AML mice compared to the early (preleukemia) stage. High expression of Six1 and Eya1 and higher cell number expressing these genes was further confirmed by immunohistochemical staining on tissue sections. The MLL-ENL-ERtm LSC-enriched spleen cells showed increased colony forming ability in vitro and leukemia-initiating potential in serial transplantation experiments compared to pre-LSCs. Moreover, we detected Six1 and Eya1 expression in the infiltrating leukemia cells in tissues of the caffeine-treated MLL-ENL-ERtm AML mice and in a subset of leukemia cells in transplanted mice. Based on these findings and correlations, we hypothesized that the Six1/Eya1 pathway might be involved in regulation of some of the aspects of LSC development as well as invasion and maintenance of leukemia in our MLL-ENL-ERtm mice. Notably, our data indicate that senescence represses a subset of the MLL-ENL-downstream transcription response and prevents full activation of self-renewal. Experiments leading to more detailed understanding of the role of the Six1/Eya1 pathway in the MLL-ENL-induced cellular transformation are ongoing. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Analysis of 31-year-old patient with SYNGAP1 gene defect points to importance of variants in broader splice regions and reveals developmental trajectory of SYNGAP1-associated phenotype: case report

Abstract Background Whole exome sequencing is a powerful tool for the analysis of genetically heterogeneous conditions. The prioritization of variants identified often focuses on nonsense, frameshift and canonical splice site mutations, and highly deleterious missense variants, although other defects can also play a role. The definition of the phenotype range and course of rare genetic conditions requires long-term clinical follow-up of patients. Case presentation We report an adult female patient with severe intellectual disability, severe speech delay, epilepsy, autistic features, aggressiveness, sleep problems, broad-based clumsy gait and constipation. Whole exome sequencing identified a de novo mutation in the SYNGAP1 gene. The variant was located in the broader splice donor region of intron 10 and replaced G by A at position +5 of the splice site. The variant was predicted in silico and shown experimentally to abolish the regular splice site and to activate a cryptic donor site within exon 10, causing frameshift and premature termination. The overall clinical picture of the patient corresponded well with the characteristic SYNGAP1-associated phenotype observed in previously reported patients. However, our patient was 31 years old which contrasted with most other published SYNGAP1 cases who were much younger. Our patient had a significant growth delay and microcephaly. Both features normalised later, although the head circumference stayed only slightly above the lower limit of the norm. The patient had a delayed puberty. Her cognitive and language performance remained at the level of a one-year-old child even in adulthood and showed a slow decline. Myopathic facial features and facial dysmorphism became more pronounced with age. Although the gait of the patient was unsteady in childhood, more severe gait problems developed in her teens. While the seizures remained well-controlled, her aggressive behaviour worsened with age and required extensive medication. Conclusions The finding in our patient underscores the notion that the interpretation of variants identified using whole exome sequencing should focus not only on variants in the canonical splice dinucleotides GT and AG, but also on broader splice regions. The long-term clinical follow-up of our patient contributes to the knowledge of the developmental trajectory in individuals with SYNGAP1 gene defects

Peritoneal dialysis induces alterations in the transcriptome of peritoneal cells before detectible peritoneal functional changes

Long-term peritoneal dialysis (PD) is associated with functional and structural alterations of the peritoneal membrane. Inflammation may be the key moment, and, consequently, fibrosis may be the end result of chronic inflammatory reaction. The objective of the present study was to identify genes involved in peritoneal alterations during PD by comparing the transcriptome of peritoneal cells in patients with short- and long-term PD. Peritoneal effluent of the long dwell of patients with stable PD was centrifuged to obtain peritoneal cells. The gene expression profiles of peritoneal cells using microarray between patients with short- and long-term PD were compared. Based on microarray analysis, 31 genes for quantitative RT-PCR validation were chosen. A 4-h peritoneal equilibration test was performed on the day after the long dwell. Transport parameters and protein appearance rates were assessed. Genes involved in the immune system process, immune response, cell activation, and leukocyte and lymphocyte activation were found to be substantially upregulated in the long-term group. Quantitative RT-PCR validation showed higher expression of CD24, lymphocyte antigen 9 (LY9), TNF factor receptor superfamily member 4 (TNFRSF4), Ig associated-α (CD79A), chemokine (C-C motif) receptor 7 (CCR7), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), and IL-2 receptor-α (IL2RA) in patients with long-term PD, with CD24 having the best discrimination ability between short- and long-term treatment. A relationship between CD24 expression and genes for collagen and matrix formation was shown. Activation of CD24 provoked by pseudohypoxia due to extremely high glucose concentrations in dialysis solutions might play the key role in the development of peritoneal membrane alterations

Validation of rs2956540:G&gt;C and rs3735520:G&gt;A association with keratoconus in a population of European descent

Corneal ectasias, among which keratoconus (KC) is the single most common entity, are one of the most frequent reasons for corneal grafting in developed countries and a threatening complication of laser in situ keratomileusis. Genome-wide association studies have previously found lysyl oxidase (LOX) and hepatocyte growth factor (HGF) associated with susceptibility to KC development. The aim of our study was to validate the effects of seven single-nucleotide polymorphisms (SNPs) within LOX and HGF over KC. Unrelated Czech cases with KC of European descent (108 males and 57 females, 165 cases in total) and 193 population and gender-matched controls were genotyped using Kompetitive Allele Specific PCR assays. Fisher's exact tests were used to assess the strength of associations. Evidence for association was found for both of the tested loci. It was strongest for rs3735520:G&gt;A near HGF (allelic test odds ratio (OR)=1.45; 95% confidence interval (CI), 1.06-1.98; P=0.018) with A allele being a risk factor and rs2956540:G&gt;C (OR=0.69; 95% CI, 0.50-0.96; P=0.024) within LOX with C allele having a protective effect. This first independent association validation of rs2956540:G&gt;C and rs3735520:G&gt;A suggests that these SNPs may serve as genetic risk markers for KC in individuals of European descent.</p

Peritoneal dialysis induces alterations in the transcriptome of peritoneal cells before detectible peritoneal functional changes

Long-term peritoneal dialysis (PD) is associated with functional and structural alterations of the peritoneal membrane. Inflammation may be the key moment, and, consequently, fibrosis may be the end result of chronic inflammatory reaction. The objective of the present study was to identify genes involved in peritoneal alterations during PD by comparing the transcriptome of peritoneal cells in patients with short- and long-term PD. Peritoneal effluent of the long dwell of patients with stable PD was centrifuged to obtain peritoneal cells. The gene expression profiles of peritoneal cells using microarray between patients with short- and long-term PD were compared. Based on microarray analysis, 31 genes for quantitative RT-PCR validation were chosen. A 4-h peritoneal equilibration test was performed on the day after the long dwell. Transport parameters and protein appearance rates were assessed. Genes involved in the immune system process, immune response, cell activation, and leukocyte and lymphocyte activation were found to be substantially upregulated in the long-term group. Quantitative RT-PCR validation showed higher expression of CD24, lymphocyte antigen 9 ( LY9), TNF factor receptor superfamily member 4 ( TNFRSF4), Ig associated-α ( CD79A), chemokine (C-C motif) receptor 7 ( CCR7), carcinoembryonic antigen-related cell adhesion molecule 1 ( CEACAM1), and IL-2 receptor-α ( IL2RA) in patients with long-term PD, with CD24 having the best discrimination ability between short- and long-term treatment. A relationship between CD24 expression and genes for collagen and matrix formation was shown. Activation of CD24 provoked by pseudohypoxia due to extremely high glucose concentrations in dialysis solutions might play the key role in the development of peritoneal membrane alterations.</jats:p