Penetrance Of Phenotype Research Articles

P0233 Prevalence and characteristics of short bowel syndrome in Crohn's disease: a retrospective observational single centre study

Abstract Background Despite a wide range of treatment options for Crohn’s Disease (CD), over half of patients require bowel resection within five years of diagnosis, with one-third needing additional surgery. Even with conservative surgical techniques, these patients remain at risk of Short Bowel Syndrome (SBS), defined as a residual small bowel length of less than 200 cm, potentially leading to intestinal failure (IF), characterized by reduced absorption of macronutrients, water or electrolytes. The prevalence and risk factors for SBS in CD patients remain unclear. Available retrospective studies report an SBS prevalence in operated CD patients ranging from 3.3% to 5.9 %. Our study aimed at describing the prevalence and characteristics of SBS, IF and Intestinal Functional Deficiency (IFD), defined as the need of any chronic enteral supplementation of macro and/or micronutrients, in a cohort of CD patients. Methods We conducted a retrospective observational cohort study, including all adult patients with CD referred to the Gastroenterology Department at IRCCS San Gerardo dei Tintori (Monza - Italy) between September 2014 and April 2022. Data were collected through the Electronic Medical Records. Results 456 CD patients were included with a mean age of 52 years (±18 SD). 221 (48.5%) patients underwent bowel surgery with a median resection length of 63 cm (IQR 60.75); the ileocecal valve was preserved in 49 (22.1%) patients. Four patients (1.8% of operated CD patients) developed SBS, after two to four surgeries over a median follow-up of 18 (IQR 15) years. Of these, three presented with type 2 SBS (jejuno-colonic anastomosis) and one with type 1 SBS (jejunostomy). Three patients had ileocolonic disease and one ileal disease, upper disease was present in three. Two SBS patients had stricturing disease, and the remaining two a penetrating phenotype. Perianal involvement was present in two patients. Only one patient (0.5%) developed permanent IF. On the other hand, 110 (49.7%) of operated patients presented post-surgical IFD: 109 (99.1%) received vitamins, 7 (6.4%) proteo-caloric, and 3 (2.7%) electrolytes supplements. These patients had a higher median age (62 vs 51.5 years, p=0.001), longer small bowel resections (60 vs 20 cm, p<0.0001) and more frequent ileocecal valve resections (91.8% vs 63.9%, p<0.0001) than patients without deficiency. Conclusion SBS and IF are rare conditions in CD patients following surgical bowel resection, prevalence is lower than previously reported. Whereas IFD affects about half of resected CD patients, highlighting the importance of prevention, early diagnosis and treatment of nutritional deficit.

Maternal, Paternal, and Dual-Parental Alcohol Exposures Result in Both Overlapping and Distinct Impacts on Behavior in Adolescent Offspring.

Emerging research reveals that alcohol use by fathers before conception can affect the growth and development of their offspring. Here, we used a C57BL/6J mouse model to study the effects of alcohol exposure on the behavior of the first-generation (F1) offspring, comparing the impacts of alcohol exposure by mothers, fathers, and both parents. Our goal was to determine how alcohol exposure by each parent or both parents influences the behavior of the offspring. We found that adolescent male offspring of alcohol-exposed fathers showed reduced anxiety-like behaviors as they spent more time in the center of the testing arena during the open field test. Both maternal and paternal alcohol exposure caused sex-specific increases in the nestlet shredding test while decreasing the number of buried marbles in the marble burying test. Interestingly, dual-parental alcohol exposure did not produce any significant changes in these same tests. However, during novel object recognition testing, we found that dual-parental male and female offspring exhibit an increased preference for novel objects, suggesting an increased risk preference. Finally, at sixteen weeks, male offspring of dual-exposed parents exhibited decreased voluntary physical activity on running wheels during the active phase, suggesting alterations in their circadian rhythms. Although differences in parental exposure histories between treatment groups make interpretation challenging, our findings suggest that exposure to alcohol by both parents may have unique effects on behavior and that studying both maternal and paternal alcohol use is essential for understanding the full range of factors influencing the penetrance and severity of alcohol-related phenotypes.

P0721 Anti-TNFs have similar safety profile to vedolizumab and ustekinumab as first line biologic treatment in elderly patients with Crohn’s disease

Abstract Background The prevalence of Crohn’s disease (CD) in elderly patients is rising and is associated with aging-related challenges. Data on effectiveness and safety of first line biologic treatment (FLBT) in the elderly, are scarce. Our aim was to compare the safety, effectiveness, and drug persistence to FLBT in elderly patients with CD. Methods A retrospective, single tertiary center study, including all patients with CD who initiated their FLBT at age≥60 years, between 1/1/2010 to 31/12/2023. Clinical and demographic data were extracted and adverse events (AEs), treatment response and drug persistence were collected. Results A total of 214 patients with CD ≥60 years of age started FLBT, data were available for 168 (78.5%) patients. Anti-TNFs were initiated in 85 (50.6%) patients, and non anti-TNFs in 83 (49.4%) patients (71-vedolizumab, 12-ustekinumab). Patients starting anti-TNF were younger (67.3±6.1 vs 72.0±7.0 years, p<0.001), with lower Charlson comorbidity index (CCI: 3.6±1.7 vs 4.9±2.2, p<0.001), had higher rates of penetrating (31.8% vs 9.6%, p=0.002) and perianal (40.0% vs 18.1%, p=0.002) disease phenotype, and previous GI resections (37.6% vs 22.9%, p=0.038). Endoscopic disease severity was comparable [Table 1]. Drug persistence was calculated from 2014 (time of vedolizumabs’ approval for clinical use) and was similar between the groups (60 patients on anti-TNF: median 82.0 weeks [IQR 27.5-175.3] vs. 83 patients on non-anti-TNF median 89.7 weeks [IQR 41.0-183.9], p=0.765). Use of corticosteroids (CS) and immunomodulators (IMM) during 14-weeks of induction and 52-weeks of maintenance was comparable (p=NS). At 52-weeks, no difference was observed in clinical response and remission rates, nor in dose escalation and drug discontinuation rates (p=NS). Eighty-three AEs were recorded at 52-weeks: 50 (58.8%) in anti TNF vs. 33 (39.8%) in non-anti-TNF group. Serious infections: 7 (8.2%) patients in the anti-TNF group (3 hospitalized for sepsis: 1 urinary and 2 respiratory, 1 cellulitis, 1 pharyngitis, 1 systemic CMV and 1 clostridium difficile infection (CDI) vs 7 (8.2%) in the non-anti-TNF group (2 intra-abdominal abscess, 1 herpes zoster infection, 1 CDI, 1 cellulitis and 2 bronchitis, p=1). Hospitalization and surgery rates were 25.8% and 12.9% in anti-TNF vs 22.9% and 8.4% in non-anti-TNF groups respectively, (p=NS) [Table 2]. In a multivariate logistic regression analysis adjusted to type of FLBT, age, gender, CCI, dose escalation, and treatment with CS and IMM, no variable was significantly associated with the development of AEs (p=NS). Conclusion Anti-TNFs and vedolizumab/ustekinumab have similar safety and drug persistence as FLBT among elderly patients with CD. Larger prospective studies are needed to fortify our findings.

Polymyositis in Kooiker dogs is associated with a 39 kb deletion upstream of the canine IL21/IL2 locus.

Recently we characterized polymyositis in the Dutch Kooiker dog. The familial occurrence of the disease were suggestive of an inherited cause. Here we report the results of our molecular genetic investigation. A genome-wide association study of 33 cases and 106 controls indicated the involvement of a region on chromosome CFA19 (p = 4.7*10-10). Haplotype analysis indicated that the cases shared a 2.9 Mb region in the homozygous or the heterozygous state. Next Generation Sequencing of genomic DNA implicated a deletion of a 39 kb DNA fragment, located 10 kb upstream of the neighbouring interleukin genes IL21 and IL2. The frequency of the deletion allele was 0.81 in the available cases and 0.25 in a random sample of the Kooiker dog breed. Leukocytes of affected, untreated dogs that were homozygous for the deletion overexpress IL21 and IL2 upon stimulation with mitogens. We suggest that elements located 10-49 kb upstream of the IL21/IL2 locus play an important role in the regulation of the canine genes and that deletion of these elements is a risk factor for polymyositis in Kooiker dogs. Postulating causality, the penetrance of the disease phenotype was estimated at 10-20% for homozygous dogs and 0.5-2% for dogs that were heterozygous for the deletion. Our results suggest that distant variants upstream of IL21 could also be important for human autoimmune diseases that have been found to be associated with the IL21/IL2 chromosome region.

In silico splicing analysis of the PMS2 gene: exploring alternative molecular mechanisms in PMS2-associated Lynch syndrome

Lynch syndrome (LS) is one of the most common hereditary cancer syndrome in human populations, associated with germline variants in MLH1, MSH2/EPCAM, MSH6 and PMS2 genes. The advent of next generation sequencing has proven a significant impact in germline variant detection in the causative genes; however, a large proportion of patients with clinical criteria still receive uncertain or negative results. PMS2 is the least frequent reported gene, associated with up to 15% of LS cases with late-onset disease and low penetrance phenotype; however, the proportion of PMS2-LS cases is considered to be highly underestimated. In this context, our analysis aimed to improve the current diagnostic yield by focusing on missense and intronic PMS2 variants available in public clinical databases (ClinVar, LOVD). We performed an in silico assessment of the wild-type DNA sequence and the reported genetic variants, employing splicing bioinformatics tools known for their effectiveness in other genes. Splicing variants were predicted in silico and using GTEx short-read RNA expression data. Out of the 2384 missense variants discovered, 90% were classified with uncertain significance (VUS). 4.9% of missense variants were shown to have a potential splicing consequence (DS > 0.2) using SpliceAI. As described in the original publication, SpliceAI-visual was proven effective in annotation of short intronic variants (< 50 bp). Four short intronic variants were identified using SpliceAI-visual as potentially splicing disturbing, in spite of using a lower threshold (DS > 0.1). Exons 2, 3, 4, 5, 6, 7, 8, 11, 12 and 14 were consistently predicted in at least three out of eight software with weak canonical splice sites. Additionally, we noted that both Exonic Splicing Enhancers (ESEs) and Exonic Splicing Silencers (ESSs) contribute significantly to alternative splicing and exonic selection in PMS2 gene. Specifically, ESE motifs were consistently more abundant in highly expressed exons 5, 11 and 14, while ESS motifs played a fundamental role in exons 6, 7 and 10. Computational analysis performed in our study serves as a valuable filtering step for guiding further RNA experiments. Additional functional data is necessary to validate our findings.

Abstract 4127919: Disease-associated variants in DSP -encoded desmoplakin are common, yet low penetrant alleles associated with development of myocarditis and cardiomyopathy at a population level

Intro: DSP- encoded desmoplakin is implicated in a distinct form of arrhythmic cardiomyopathy (ACM), often involving the LV and characterized by myocardial inflammation. Conventional ACM diagnostic criteria has poor sensitivity for DSP -associated ACM, thus highlighting the need to identify the full spectrum of phenotypic risk associated with disease-associated DSP variants. Objective: To analyze the genotypic and phenotypic spectrum of DSP -mediated disease at a large population level. Methods: UK Biobank participants with exome sequencing (ES) were included. Variants were filtered by gene evidence category, yielding 3 groups: predicted deleterious (pDel); subset with predicted loss of function (LOF); and ultra-filtered subset by ClinVar 2* pathogenic/likely pathogenic (P/LP). Phenotypic penetrance was analyzed with DSP -neg individuals as control. Variant location analysis assessed LOFs by susceptibility to nonsense mediated decay region and missenses by amino acid sequence to identify mutational hotspots where P/LPs localize. Results: Out of 200,580 with ES, 1407 DSP carriers had pDel, 168 with LOF, and 44 with ClinVar 2* P/LP. DSP carriers had higher burden of myocarditis, cardiomyopathy (CM), and heart failure. A progressive enrichment in myocarditis and CM was observed by more stringent variant filtering in DSP carriers compared to control. A higher proportion of DSP carriers had myocarditis – 0.28% (4) of the pDels, 1.8% (3) of LOFs, and 4.5% (2) compared to control 0.07% (p&lt;0.05). A parallel trend noted the proportion of DSP carriers with CM growing from 1.4% (19) to 5.4% (9) and 6.8% (3) in the pDel, LOF, and ClinVar 2* P/LPs respectively compared to 0.6% in the control group (p&lt;0.01). Location analysis of missense variants revealed that P/LPs localized in higher proportions (74%) to key domains compared to non-P/LPs (50%; p&lt;0.001), highlighting prognostic value of predicting pathogenicity of missense DSP variants by mutation hotspots. Conclusions: While DSP variants are relatively common in the population, variant presence is associated with low penetrance. Enhancing clinical penetrance with increasingly stringent filtering of DSP variants emphasizes the importance of variant evaluation in clinical risk assessment. This first-in-kind population study of DSP provides insights into novel risk prediction factors, allowing us to predict pathogenicity by type and location of mutation and using variant filtering strategy to better assess phenotypic penetrance.

Management of patients with Crohn's disease – from history to modern approaches

Since the isolation of Crohn's disease as a separate pathology, significant progress has been made both in the diagnosis of the disease and in approaches to the treatment of patients with this pathology. The active introduction of biologics and the top-down approach into clinical practice in patients with negative prognosis factors has led to a significant improvement in treatment results and prognosis in patients. The use of the most effective anti-inflammatory therapy during the «therapeutic window of opportunity» – up to 18 months – is the key to achieving remission, or minimal activity of Crohn's disease by suppressing inflammation and preventing irreversible structural damage to the intestinal wall and, as a result, the development of complications. The article presents an excursion into the history of the discovery of this disease, which is still far from its completion. The approaches to the treatment of patients with this pathology are highlighted with an explanation of the current position based on data from meta-analyses and systematic reviews. A demonstration of a patient with a penetrating phenotype of Crohn's disease in the form of a perianal lesion and a history of oncopathology is presented. Ustekinumab therapy can be considered as an optimal management strategy for patients with Crohn's disease with unfavorable prognosis factors.

Myh9b is a critical non-muscle myosin II encoding gene that interacts with myh9a and myh10 during zebrafish development in both compensatory and redundant pathways.

Non-muscle myosin (NMII) motor proteins have diverse developmental functions due to their roles in cell shape changes, cell migration, and cell adhesion. Zebrafish are an ideal vertebrate model system to study the NMII encoding myh genes and proteins due to high sequence homology, established gene editing tools, and rapid ex utero development. In humans, mutations in the NMII encoding MYH genes can lead to abnormal developmental processes and disease. This study utilized zebrafish myh9a, myh9b, and myh10 null mutants to examine potential genetic interactions and roles for each gene in development. It was determined that the myh9b gene is the most critical NMII encoding gene, as myh9b mutants develop pericardial edema and have a partially penetrant lethal phenotype, which was not observed in the other myh mutants. This study also established that genetic interactions occur between the zebrafish myh9a, myh9b, and myh10 genes where myh9b is required for the expression of both myh9a and myh10, and myh10 is required for the expression of myh9b. Additionally, protein analyses suggested that enhanced NMII protein stability in some mutant backgrounds may play a role in compensation. Finally, double mutant studies revealed different and more severe phenotypes at earlier timepoints than single mutants, suggesting roles for tissue specific genetic redundancy, and in some genotypes, haploinsufficiency. These mutants are the first in vivo models allowing for the study of complete loss of the NMIIA and NMIIB proteins, establishing them as valuable tools to elucidate the role of NMII encoding myh genes in development and disease.

8639 Clinical characterization and germline DNA methylation analysis of a large cohort of patients with paraganglioma syndrome type 4

Abstract Disclosure: R. Halperin: None. R. Horowitz: None. A. Jabarin: None. A. Tirosh: None. Background: Patients harboring germline pathogenic variant (PV) in the SDHB gene (Paraganglioma syndrome type 4, PPGL4) have a higher risk of developing paragangliomas and pheochromocytomas. PPGL4 is considered a higher-risk syndrome for aggressive and metastatic abdominal-thoracic paragangliomas compared with other paraganglioma syndromes. The penetrance in PPGL4 reaches 65% by the age of 80 years. Aims: To assess possible germline DNA epigenetic alterations in patients harboring the SDHB PV. Materials and Methods: A large cohort of patients with PPGL4 was characterized clinically in our clinic and genetically via germline DNA sequencing for the familial variant (SDHB c.640C&amp;gt;T p.Q214Ter, n=144) and whole genome methylation analysis compared 19 patients vs. 129 healthy controls. The control group consisted of four SDHB PV-negative family members and an additional 125 samples retrieved from a public database (GSE153712). Analysis was performed on Rstudio, using the ChAMP suite for normalization, imputation, differentially methylated probes (DMP), and regions (DMR) analyses. Pathway analysis and visualization were executed using the clusterprofiler and enrichplot packages. Promoter regions were defined by the transcription start site (TSS1500). Results: The cohort includes three kindreds, and each presented independently with an index patient: Index patient in kindred A, a 12-year-old boy, with thoracic paraspinal and testicular paragangliomas, non-secretory, requiring several surgeries due to spinal compression with subsequent improvement of neurological deficit. Index patient in kindred B, a 26-year-old woman with abdominal paraganglioma, metastatic to the lungs and spine. Index patient in kindred C, a 41-year-old man with a locally aggressive abdominal paraganglioma, metastatic to the skull and spine. A total of 114 (44.2% females) patients underwent genetic evaluation (45/50, 16/28, and 25/36 in kindreds A, B and C, respectively). Of those, 50 (58.1%) harbored the familial SDHB PV (68.9%, 56.2%, and 40.0%, respectively). Twenty-six patients underwent at least partial clinical evaluation: Eight had paraganglioma (4/8 had metastatic disease), three had pheochromocytoma, and two patients had neck masses that are currently being evaluated. Pathway analysis revealed enrichment of the germline DNA alterations to the regulation of cell differentiation and development, metabolic processes, and cellular component organization (p&amp;lt;0.03 for all pathways). Conclusions: We report one of the largest cohorts of patients with SDHB PV, showing typical low penetrance and aggressive phenotype. Genomic analysis shows distinct germline DNA methylation in SDHB PV-positive compared with the control group. Future studies may reveal an association between specific epigenetic alterations and penetrance/phenotypic patterns in this rare hereditary syndrome. Presentation: 6/2/2024

Germline Variants in Patients Affected by Both Uveal and Cutaneous Melanoma.

Uveal melanoma (UM) and nonacral cutaneous melanoma (CM) are distinct entities with varied genetic landscapes despite both arising from melanocytes. There are, however, similarities in that they most frequently affect people of European ancestry, and high penetrance germline variants in BAP1, POT1 and CDKN2A have been shown to predispose to both UM and CM. This study aims to further explore germline variants in patients affected by both UM and CM, shedding light on the underlying genetic mechanism causing these diseases. Using exome sequencing we analysed germline DNA samples from a cohort of 83 Australian patients diagnosed with both UM and CM. Eight (10%) patients were identified that carried pathogenic mutations in known melanoma predisposition genes POT1, MITF, OCA2, SLC45A2 and TYR. Three (4%) patients carried pathogenic variants in genes previously linked with other cancer syndromes (ATR, BRIP1 and MSH6) and another three cases carried monoallelic pathogenic variants in recessive cancer genes (xeroderma pigmentosum and Fanconi anaemia), indicating that reduced penetrance of phenotype in these individuals may contribute to the development of both UM and CM. These findings highlight the need for further studies characterising the role of these genes in melanoma susceptibility.

Targeting the IKs Channel PKA Phosphorylation Axis to Restore Its Function in High-Risk LQT1 Variants.

The KCNQ1+KCNE1 (IKs) potassium channel plays a crucial role in cardiac adaptation to stress, in which β-adrenergic stimulation phosphorylates the IKs channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear. Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/IKs channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific-induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions. By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of IKs. We refer to this pattern as the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell-derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities. Our findings not only elucidate the molecular mechanism of PKA-dependent IKs channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.

Mutational cooperativity of RUNX1::RUNX1T1 isoform 9a and oncogenic NRAS in zebrafish myeloid leukaemia.

RUNX1::RUNX1T1 (R::RT1) acute myeloid leukaemia (AML) remains a clinical challenge, and further research is required to model and understand leukaemogenesis. Previous zebrafish R::RT1 models were hampered by embryonic lethality and low penetrance of the malignant phenotype. Here, we overcome this by developing an adult zebrafish model in which the human R::RT1 isoform 9a is co-expressed with the frequently co-occurring oncogenic NRASG12D mutation in haematopoietic stem and progenitor cells (HSPCs), using the Runx1+23 enhancer. Approximately 50% of F0 9a+NRASG12D transgenic zebrafish developed signs of haematological disease between 5 and 14 months, with 27% exhibiting AML-like pathology: myeloid precursor expansion, erythrocyte reduction, kidney marrow hypercellularity and the presence of blasts. Moreover, only 9a+NRASG12D transplant recipients developed leukaemia with high rates of mortality within 40 days, inferring the presence of leukaemia stem cells. These leukaemic features were rare or not observed in animals expressing either the NRAS or 9a oncogenes alone, suggesting 9a and NRAS cooperation drives leukaemogenesis. This novel adult AML zebrafish model provides a powerful new tool for investigating the basis of R::RT1 - NRAS cooperativity with the potential to uncover new therapeutic targets.

Unlocking the Genetic Secrets of Acromegaly: Exploring the Role of Genetics in a Rare Disorder.

Acromegaly is a rare endocrine disorder characterized by the excessive production of growth hormone (GH) in adulthood. Currently, it is understood that certain pituitary neuroendocrine tumors (PitNETs) exhibit a hereditary predisposition. These tumors' genetic patterns fall into two categories: isolated and syndromic tumors. The isolated forms are characterized by molecular defects that predispose exclusively to PitNETs, including familial isolated pituitary adenomas (FIPAs) and sporadic genetic defects not characterized by hereditary predisposition. All the categories involve either germline or somatic mutations, or both, each associated with varying levels of penetrance and different phenotypes. This highlights the importance of genetic testing and the need for a more comprehensive view of the whole disease. Despite the availability of multiple treatment options, diagnosis often occurs after several years, and management is still difficult. Early detection and intervention are crucial for preventing complications and enhancing the quality of life for affected individuals. This review aims to elucidate the molecular, clinical, and histological characteristics of GH-secreting PitNETs, providing insights into their prevalence, treatment nuances, and the benefits of genetic testing for each type of genetic disorder associated with acromegaly.

Prevalence of and gene regulatory constraints on transcriptional adaptation in single cells

BackgroundCells and tissues have a remarkable ability to adapt to genetic perturbations via a variety of molecular mechanisms. Nonsense-induced transcriptional compensation, a form of transcriptional adaptation, has recently emerged as one such mechanism, in which nonsense mutations in a gene trigger upregulation of related genes, possibly conferring robustness at cellular and organismal levels. However, beyond a handful of developmental contexts and curated sets of genes, no comprehensive genome-wide investigation of this behavior has been undertaken for mammalian cell types and conditions. How the regulatory-level effects of inherently stochastic compensatory gene networks contribute to phenotypic penetrance in single cells remains unclear.ResultsWe analyze existing bulk and single-cell transcriptomic datasets to uncover the prevalence of transcriptional adaptation in mammalian systems across diverse contexts and cell types. We perform regulon gene expression analyses of transcription factor target sets in both bulk and pooled single-cell genetic perturbation datasets. Our results reveal greater robustness in expression of regulons of transcription factors exhibiting transcriptional adaptation compared to those of transcription factors that do not. Stochastic mathematical modeling of minimal compensatory gene networks qualitatively recapitulates several aspects of transcriptional adaptation, including paralog upregulation and robustness to mutation. Combined with machine learning analysis of network features of interest, our framework offers potential explanations for which regulatory steps are most important for transcriptional adaptation.ConclusionsOur integrative approach identifies several putative hits—genes demonstrating possible transcriptional adaptation—to follow-up on experimentally and provides a formal quantitative framework to test and refine models of transcriptional adaptation.

Advance of research on 22q11.2 deletion syndrome

22q11.2 deletion syndrome (22q11.2DS) is the most common chromosomal microdeletion disorder. Its phenotype is highly variable with incomplete penetrance. 22q11.2DS is a rare disease, and the research progress is relatively slow, which has restricted its treatment and intervention. In recent years, much progress has been made in the pathogenic mechanism and genome-wide association study of 22q11.2DS. In this review, the pathogenesis of 22q11.2DS was summarized. Thereafter, the molecular and pathological mechanisms of TBX1 and DGCR8 genes were clarified. Finally, factors affecting the penetrance of cardiac and immune system phenotypes were reviewed. This review may enhance the understanding of 22q11.2DS and has important clinical implications on the prenatal diagnosis, genetic counseling, treatment and intervention of this disease.

Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development.

Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes - Fgf3, Fgf4, and Fgf15 - that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes - for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

Abstract Mo080: Clinically Variable Penetrant MYH7 G256E Mutation Shows Gene-Dose-Dependent HCM Disease Phenotype on a Transcriptomic, Proteomic, and Functional Level

Introduction: Hypertrophic cardiomyopathy (HCM) is a monogenic disorder caused by heterozygous (HET) mutations in sarcomere genes such as MYH7 . Recently, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying HET HCM mutations have shed new insights into the molecular mechanisms of HCM. However, the cause of phenotype variability and the role of allelic imbalance in phenotype penetrance of HET missense mutations such as MYH7 G256E remain unclear. Hypothesis: We hypothesize that hiPSC CMs carrying clinically variable penetrant HCM missense mutations such as MYH7 G256E show a gene dose-dependent disease phenotype indicating their susceptibility for allelic imbalance as a cause for variable disease phenotype penetrance. Methods: We generated HET and homozygous (HOM) hiPSC-lines for variable penetrant HCM mutation MYH7 G256E. We performed multiplexed single-cell RNA sequencing and quantitative proteomics analyses on wildtype (WT), HET, and HOM iPSC-CMs at day 30 of cardiac differentiation. Additional Digital Droplet PCR studies were performed to investigate the MYH7 mutant allele expression. The functional contractile phenotype was assessed with Sony SI8000 Cell motion imaging system. Results: We detected a large clone-to-clone and batch-to-batch variability in HET iPSC-CMs on a transcriptomic and proteomic level that obscured the identification of a consistent mutant phenotype in HET MYH7 G256E vs WT CMs. These phenotypes correlated with a 1:1 bulk ratio of mutant vs WT mRNA and protein expression, while overall MYH7 gene and protein expression remained comparable. Transcriptomic and proteomic analyses of WT, HET, and HOM CMs show a gene dose-dependent regulation of known hypertrophy response markers such as NPPB (mRNA: HET vs WT: +1.76x p .adj=7.0*10 -7 , HOM vs WT: +5.42x p.adj=1.5*10 -48 ; protein: HET vs WT: +1.15x, 95% CI [0.86, 1.58]; HOM vs WT: +1.51x, 95% CI [1.12, 2.01]) which functionally correlated with enhanced contractility (contraction velocity in μm/s: WT: 5.84 +/-2.24, HET: 6.04 +/-2.89, HOM: 7.77 +/-3.352, p=0.3*10 -2 ANOVA) and the more frequent occurrence of arrhythmias (WT: 1/46, HET: 0/55, HOM: 5/41). Conclusions: Hypertrophic disease phenotype in MYH7 G256E mutation iPSC-CMs shows a gene dose-dependent penetrance on a transcriptomic, proteomic, and functional level. This indicates susceptibility to allelic imbalance as a potential cause for variable disease penetrance.

Prenatal detection of distal 18p deletion by chromosomal microarray analysis: Three case reports and literature review.

Chromosome 18p deletion syndrome is caused by total or partial deletion of the short arm of chromosome 18 and associated with cognitive impairment, growth retardation and mild facial dysmorphism. However, most studies on the genotype-phenotype correlations in the 18p region are diagnosed postnatally. Prenatal reports involving 18p deletions are limited. Three pregnant women opted for invasive prenatal testing due to noninvasive prenatal testing indicating high risk for chromosome 18 abnormalities. Karyotypic analysis and chromosomal microarray analysis (CMA) were performed simultaneously. The pregnancy outcomes for all cases were followed up. Meanwhile, we also made a literature review on prenatal phenotypes of 18p deletions. G-banding analysis showed that 2 fetuses presented abnormal karyotypes: 45,XN,der(18)t(18;21)(p11; q11),-21 (case 2) and 46,XN,18p- (case 3). The karyotype of case 1 was normal. Meanwhile, CMA detected 4.37 Mb (case 1), 7.26 Mb (case 2) and 14.97 Mb (case 3) deletions in chromosome 18p region. All 3 pregnancies were terminated finally according to genetic counseling based upon abnormal CMA results. Prenatal diagnosis of 18p deletion syndrome is full of challenges due to the phenotypic diversity, incomplete penetrance and lack of prenatal phenotypes. Increased nuchal translucency and holoprosencephaly are common prenatal phenotypes of distal 18p deletion. For fetuses carrying 18p deletions with atypical sonographic phenotypes, noninvasive prenatal testing could be adopted as an effective approach.

O-135 The consequences of paternal pre-conceptional health for offspring health and pregnancy outcomes: involvement of the germ-cell epigenome and non-germ cell factors

Abstract Study question Paternal health at conception is important for offspring health. The susceptibility windows within the male reproductive tract and the molecular mechanisms are not completely understood. Summary answer Using mouse genetics, (epi)genomics and in-depth phenotyping, we show that the germ-cell epigenome and components of the seminal fluid are critical for paternal intergenerational effects. What is known already Paternal intergenerational effects are highly conserved across mammals and lower organisms including flies and nematodes. In mammals, the sperm epigenome, as well as the composition of the seminal fluid are sensitive to environmental challenges and influence offspring development and adult phenotypic trajectories. The best known of these mechanisms are DNA methylation and small non-coding RNAs in spermatozoa and the balance between inflammatory and tolerogenic cytokines in the seminal fluid. Small non-coding RNAs are mostly acquired by maturing spermatozoa during the epididymal transit from extracellular vesicle of epididymal epithelial origin, and have the potential to influence embryonic development and offspring health. Study design, size, duration We use two paternal paradigms of pre-conceptional challenge in wild-type C57BL6 mice: high-fat diet feeding for 2 weeks and circadian disruption for 4 weeks before conception. The duration of the environmental challenges has been chosen to meet timing of sperm maturation in the mouse (high-fat diet) or to be the minimum sufficient to induce circadian disruption without overt side effects. Exposed males and their offspring have undergone deep metabolic and molecular phenotyping. Participants/materials, setting, methods We used both bulk and single cell (epi)genomics on exposed males as well as male and female offspring. The molecular phenotyping has been instructed by the results of the metabolic phenotyping performed in collaboration with the German Mouse Clinic, the biggest mouse clinic in Europe, using highly standardized and robust procedures. Importantly, we have used a family-based approach for the data analysis, according to which the experimental n is the exposed father. Main results and the role of chance Paternal circadian disruption achieved by four weeks of day-restricted feeding (food only during the light phase), leads to hyperphagia, hyperglycemia, hypercorticosteronemia and partial circadian disruption in unexposed male offspring. The phenotypes are gender-specific and the females feature partial protection from the paternal effects. Mechanistically, mouse seminal fluid contains corticosterone, whose appearance dynamic follows the organismal circadian rhythm and is blunted upon circadian disruption. Offspring phenotypes are sensitive to the levels of corticosterone in the seminal fluid and consequential to impaired placentation, reduced placental efficiency and fetal growth restriction. Paternal exposure to an acute high-fat diet challenge, instead, leads to a 30% penetrant glucose intolerance phenotype exclusively in male offspring. Mechanistically, this is linked to diet-induced mitochondrial dysfunction in somatic and germ cells and a compensatory burst of mitochondrial DNA transcription in spermatozoa. Mitochondrial DNA encoded small non-coding RNAs accumulate in spermatozoa, are inherited at fertilization, and reprogram early embryo transcription towards premature activation of oxidative metabolism, a predisposing factor to adult-onset glucose intolerance. Importantly, phenotypes and mechanisms are conserved across highly characterized human cohorts. These studies strengthen the importance of paternal health at conception for pregnancy and offspring health and identify germ-cell and non-germ cell factors as potential mechanistic mediators. Limitations, reasons for caution Although we have shown for the first time that stress hormones are present in the seminal fluid and relay paternal stress to the offspring and provided the first example of epigenetic inheritance of sperm small non-coding RNAs, mechanisms are still to be completely uncovered. Complementary mechanisms cannot be excluded. Wider implications of the findings Our findings strengthen the importance of paternal health at conception for pregnancy and offspring health. They also provide potential male tract biomarkers and novel strategies to monitor paternal health at conception and preconception lifestyle interventions aimed at improving pregnancy outcomes and preventing the spread of metabolic disorders through epigenetic inheritance. Trial registration number not applicable

Exploring penetrance of clinically relevant variants in over 800,000 humans from the Genome Aggregation Database.

Incomplete penetrance, or absence of disease phenotype in an individual with a disease-associated variant, is a major challenge in variant interpretation. Studying individuals with apparent incomplete penetrance can shed light on underlying drivers of altered phenotype penetrance. Here, we investigate clinically relevant variants from ClinVar in 807,162 individuals from the Genome Aggregation Database (gnomAD), demonstrating improved representation in gnomAD version 4. We then conduct a comprehensive case-by-case assessment of 734 predicted loss of function variants (pLoF) in 77 genes associated with severe, early-onset, highly penetrant haploinsufficient disease. We identified explanations for the presumed lack of disease manifestation in 701 of the variants (95%). Individuals with unexplained lack of disease manifestation in this set of disorders rarely occur, underscoring the need and power of deep case-by-case assessment presented here to minimize false assignments of disease risk, particularly in unaffected individuals with higher rates of secondary properties that result in rescue.