Faculty Bibliography

BACKGROUND:in HSCR and how they contribute to the pathogenesis of HSCR. METHODS:-acting elements in MAPK10 on miR-4516-mediated modulation and cell migration process. RESULTS:rescued the effect of miR-4516 on the migration of human neural cells. CONCLUSION/CONCLUSIONS:- and posttranscriptional modulation for HSCR pathogenesis.

Hundreds of loci have been associated with blood pressure traits from many genome-wide association studies. We identified an enrichment of these loci in aorta and tibial artery expression quantitative trait loci in our previous work in ~ 100 000 Genetic Epidemiology Research on Aging (GERA) study participants. In the present study, we sought to fine-map known loci and identify novel genes by determining putative regulatory regions for these and other tissues relevant to blood pressure. We constructed maps of putative cis-regulatory elements using publicly available open chromatin data for the heart, aorta and tibial arteries, and multiple kidney cell types. Variants within these regions may be evaluated quantitatively for their tissue- or cell-type-specific regulatory impact using deltaSVM functional scores, as described in our previous work. We aggregate variants within these putative cis-regulatory elements within 50Kb of the start or end of 'expressed' genes in these tissues or cell types using public expression data, and use deltaSVM scores as weights in the group-wise sequence kernel association test (SKAT) to identify candidates. We test for association with both blood pressure traits and expression within these tissues or cell types of interest, and identify the candidates MTHFR, C10orf32, CSK, NOV, ULK4, SDCCAG8, SCAMP5, RPP25, HDGFRP3, VPS37B, and PPCDC. Additionally, we examined two known QT interval genes, SCN5A and NOS1AP, in the Atherosclerosis Risk in Communities Study (ARIC), as a positive control, and observed the expected heart-specific effect. Thus, our method identifies variants and genes for further functional testing using tissue- or cell-type-specific putative regulatory information.

BACKGROUND:Over half of children with rare genetic diseases remain undiagnosed despite maximal clinical evaluation and DNA-based genetic testing. As part of an Undiagnosed Diseases Program applying transcriptome (RNA) sequencing to identify the causes of these unsolved cases, we studied a child with severe infantile osteopetrosis leading to cranial nerve palsies, bone deformities, and bone marrow failure, for whom whole-genome sequencing was nondiagnostic. METHODS:We performed transcriptome (RNA) sequencing of whole blood followed by analysis of aberrant transcript isoforms and osteoclast functional studies. RESULTS:We identified a pathogenic deep intronic variant in CLCN7 creating an unexpected, frameshifting pseudoexon causing complete loss of function. Functional studies, including osteoclastogenesis and bone resorption assays, confirmed normal osteoclast differentiation but loss of osteoclast function. CONCLUSION/CONCLUSIONS:This is the first report of a pathogenic deep intronic variant in CLCN7, and our approach provides a model for systematic identification of noncoding variants causing osteopetrosis-a disease for which molecular-genetic diagnosis can be pivotal for potentially curative hematopoietic stem cell transplantation. Our work illustrates that cryptic splice variants may elude DNA-only sequencing and supports broad first-line use of transcriptome sequencing for children with undiagnosed diseases.

DPH1variants have been associated with an ultra-rare and severe neurodevelopmental disorder, mainly characterized by variable developmental delay, short stature, dysmorphic features, and sparse hair. We have identified four new patients (from two different families) carrying novel variants in DPH1, enriching the clinical delineation of the DPH1 syndrome. Using a diphtheria toxin ADP-ribosylation assay, we have analyzed the activity of seven identified variants and demonstrated compromised function for five of them [p.(Leu234Pro); p.(Ala411Argfs*91); p.(Leu164Pro); p.(Leu125Pro); and p.(Tyr112Cys)]. We have built a homology model of the human DPH1-DPH2 heterodimer and have performed molecular dynamics simulations to study the effect of these variants on the catalytic sites as well as on the interactions between subunits of the heterodimer. The results show correlation between loss of activity, reduced size of the opening to the catalytic site, and changes in the size of the catalytic site with clinical severity. This is the first report of functional tests of DPH1 variants associated with the DPH1 syndrome. We demonstrate that the in vitro assay for DPH1 protein activity, together with structural modeling, are useful tools for assessing the effect of the variants on DPH1 function and may be used for predicting patient outcomes and prognoses.

Following the publication of the article, it was noted that the last column in Table 1, the total % should have read 5/8 (62.5) for the 'Epilepsy' row, and not 5.7 (71.4). This has now been amended in the HTML and PDF of the original article.

The development of the gut from endodermal tissue to an organ with multiple distinct structures and functions occurs over a prolonged time during embryonic days E10.5-E14.5 in the mouse. During this process, one major event is innervation of the gut by enteric neural crest cells (ENCCs) to establish the enteric nervous system (ENS). To understand the molecular processes underpinning gut and ENS development, we generated RNA-sequencing profiles from wild-type mouse guts at E10.5, E12.5, and E14.5 from both sexes. We also generated these profiles from homozygous Ret null embryos, a model for Hirschsprung disease (HSCR), in which the ENS is absent. These data reveal 4 major features: 1) between E10.5 and E14.5 the developmental genetic programs change from expression of major transcription factors and its modifiers to genes controlling tissue (epithelium, muscle, endothelium) specialization; 2) the major effect of Ret is not only on ENCC differentiation to enteric neurons but also on the enteric mesenchyme and epithelium; 3) a muscle genetic program exerts significant effects on ENS development; and 4) sex differences in gut development profiles are minor. The genetic programs identified, and their changes across development, suggest that both cell autonomous and nonautonomous factors, and interactions between the different developing gut tissues, are important for normal ENS development and its disorders.

BACKGROUND:Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by the absence of enteric ganglia in the distal part of the gut. RET is the major causative gene and contains > 80% of all known disease-causing mutations. RESULTS:To determine the incidence of RET pathogenic variants, be they Mendelian inherited, mosaic in parents or true de novo variants (DNVs) in 117 Chinese families, we used high-coverage NGS and droplet digital polymerase chain reaction (ddPCR) to identify 15 (12.8%) unique RET coding variants (7 are novel); one was inherited from a heterozygous unaffected mother, 11 were DNVs (73.3%), and 3 full heterozygotes were inherited from parental mosaicism (2 paternal, 1 maternal): two clinically unaffected parents were identified by NGS and confirmed by ddPCR, with mutant allele frequency (13-27%) that was the highest in hair, lowest in urine and similar in blood and saliva. An extremely low-level paternal mosaicism (0.03%) was detected by ddPCR in blood. Six positive-controls were examined to compare the mosaicism detection limit and sensitivity of NGS, amplicon-based deep sequencing and ddPCR. CONCLUSION/CONCLUSIONS:Our findings expand the clinical and molecular spectrum of RET variants in HSCR and reveal a high frequency of RET DNVs in the Chinese population.

Disruptions in gene regulatory networks (GRNs), driven by multiple deleterious variants, potentially underlie complex traits and diseases. Hirschsprung disease (HSCR), a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and 7 chromosomal loci, with RET and EDNRB as its major genes. We previously demonstrated that RET transcription in the ENS is controlled by an extensive GRN involving the transcription factors (TF) RARB, GATA2 and SOX10 and other HSCR genes. We now demonstrate, using human and mouse cellular and animal models, that EDNRB is transcriptionally regulated in the ENS by GATA2, SOX10 and NKX2.5 TFs. Significantly, RET and EDNRB expression is regulated by their shared use of GATA2 and SOX10 and, in turn, these TFs are controlled by EDNRB and RET in a dose-dependent manner. This study expands the ENS development GRN to include both RET and EDNRB, uncovers the mechanistic basis for RET-EDNRB epistasis and emphasizes how functionally different genes associated with a complex disorder can be united through a common GRN.

OBJECTIVES/OBJECTIVE:Families affected by Hirschsprung disease (HSCR) have opportunities to learn recurrence risks to their children from statistical genetic and empiric studies and, in some cases, prenatal genetic testing or preimplantation genetic diagnosis (PGD). This study aimed to assess interest in reproductive genetic information for HSCR and factors that predict this interest in two groups with elevated risk of having a child with HSCR. METHODS:Adult HSCR patients and parents of children with HSCR were surveyed about their interest in learning reproductive genetic information regarding HSCR through genetic counseling, prenatal testing, and PGD. Covariates assessed in this cross-sectional study included quality of life, illness perceptions, depressive symptoms, and adaptation to the condition. Bivariate analyses assessed differences between affected adults and parents. Logistic modeling was used to identify predictors of interest in reproductive genetic information. RESULTS:Study participants (n = 368) reported high interest in reproductive genetic information through genetic counseling (yes = 60%/unsure = 16%), prenatal testing (yes = 59%/unsure = 16%), and PGD (yes = 43%/unsure = 18%). Illness perceptions differed between affected adults and parents, but perceived severity of HSCR was high among all participants (μ = 3.42, SD = 0.67, 4 point scale). Interest in reproductive information was associated with being an affected adult, not having a family history of HSCR, negative emotional representations, and adaptation to the condition. CONCLUSIONS:Findings from this study support a desire among the surveyed HSCR patient groups to attend genetic counseling and be offered testing when available. Exploration of perceptions and experiences with the condition should be incorporated into the counseling to insure informed preference-based decision making.