Enrichment Of Variants Research Articles

Analysis of 1386 epileptogenic brain lesions reveals association with DYRK1A and EGFR

Lesional focal epilepsy (LFE) is a common and severe seizure disorder caused by epileptogenic lesions, including malformations of cortical development (MCD) and low-grade epilepsy-associated tumors (LEAT). Understanding the genetic etiology of these lesions can inform medical and surgical treatment. We conducted a somatic variant enrichment mega-analysis in brain tissue from 1386 individuals who underwent epilepsy surgery, including 599 previously unpublished individuals with ultra-deep ( > 1600x) targeted panel sequencing. Here we confirm four known associations (BRAF, SLC35A2, MTOR, PTPN11), support eight associations without prior statistical support (FGFR1, PIK3CA, AKT3, NF1, PTEN, RHEB, KRAS, NRAS), and identify novel associations for two genes, DYRK1A and EGFR. Both novel genes show specific histopathological phenotypes, interact with LFE genes and pathways, and may represent promising candidates as biomarkers and potentially druggable targets.

Genetic Analysis of Heterotaxy in a Consanguineous Cohort.

Heterotaxy (HTX) is a group of clinical conditions with a shared pathology of dislocation of one or more organs along the left-right axis. The etiology of HTX is tremendously heterogeneous spanning environmental factors, chromosomal aberrations, mono/oligogenic variants, and complex inheritance. However, in the vast majority of cases, the etiology of HTX remains elusive. Here, we sought to describe the yield of genetic analysis and spectrum of variants in HTX in our highly consanguineous population. Twenty-four affected individuals, from 19 unrelated families, were consecutively recruited. Genetic analysis, with exome sequencing, genome sequencing, or multigene panel, detected 9 unique variants, 7 of which were novel, in 8 genes known to be implicated in autosomal recessive form of HTX (C1orf127, CCDC39, CIROP, DNAAF3, DNAH5, DNAH9, MMP21, and MNS1) providing a yield of 42.1%. Of note, 7 of the 9 variants were homozygous, while 2 were inherited in compound heterozygosity, including a heterozygous CNV deletion. A search for candidate genes in negative cases did not reveal a plausible variant. Our work demonstrates a relatively high yield of genetic testing in HTX in a consanguineous population with an enrichment of homozygous variants. The significant genetic heterogeneity observed herewith underscores the complex developmental mechanisms implicated in the pathogenesis of HTX and supports adopting a genome-wide analysis in the diagnostic evaluation of HTX.

The clinical and genetic spectrum of paediatric speech and language disorders.

Speech and language disorders are known to have a substantial genetic contribution. Although frequently examined as components of other conditions, research on the genetic basis of linguistic differences as separate phenotypic subgroups has been limited so far. Here, we performed an in-depth characterization of speech and language disorders in 52 143 individuals, reconstructing clinical histories using a large-scale data-mining approach of the electronic medical records from an entire large paediatric healthcare network. The reported frequency of these disorders was the highest between 2 and 5 years old and spanned a spectrum of 26 broad speech and language diagnoses. We used natural language processing to assess the degree to which clinical diagnoses in full-text notes were reflected in ICD-10 diagnosis codes. We found that aphasia and speech apraxia could be retrieved easily through ICD-10 diagnosis codes, whereas stuttering as a speech phenotype was coded in only 12% of individuals through appropriate ICD-10 codes. We found significant comorbidity of speech and language disorders in neurodevelopmental conditions (30.31%) and, to a lesser degree, with epilepsies (6.07%) and movement disorders (2.05%). The most common genetic disorders retrievable in our analysis of electronic medical records were STXBP1 (n = 21), PTEN (n = 20) and CACNA1A (n = 18). When assessing associations of genetic diagnoses with specific linguistic phenotypes, we observed associations of STXBP1 and aphasia (P = 8.57 × 10-7, 95% confidence interval = 18.62-130.39) and MYO7A with speech and language development delay attributable to hearing loss (P = 1.24 × 10-5, 95% confidence interval = 17.46-infinity). Finally, in a sub-cohort of 726 individuals with whole-exome sequencing data, we identified an enrichment of rare variants in neuronal receptor pathways, in addition to associations of UQCRC1 and KIF17 with expressive aphasia, MROH8 and BCHE with poor speech, and USP37, SLC22A9 and UMODL1 with aphasia. In summary, our study outlines the landscape of paediatric speech and language disorders, confirming the phenotypic complexity of linguistic traits and novel genotype-phenotype associations. Subgroups of paediatric speech and language disorders differ significantly with respect to the composition of monogenic aetiologies.

6846 Rare Variants in SEMA3A in Individuals with Constitutional Delay of Puberty (CDP)

Abstract Disclosure: W. He: None. E.C. Schafer: None. R. Brauner: None. A. Delaney: None. L. Dunkel: None. R. Grinspon: None. J.E. Hall: None. J.N. Hirschhorn: None. S. Howard: None. A. Latronico: None. A.A. Jorge: None. K. McElreavey: None. V. Mericq: None. P.M. Merino: None. M.R. Palmert: None. R.A. Rey: None. R.C. Rezende: None. S.B. Seminara: None. N. Shaw: None. T. Delayed Puberty Genetics Consortium: None. Y. Chan: None. Background: Constitutional delay of puberty (CDP), also known as self-limited delayed puberty, refers to late onset of puberty with relatively normal progression afterward. In contrast, idiopathic hypogonadotropic hypogonadism (IHH) results in a long-lasting defect in reproductive endocrine activity. Both CDP and IHH are highly heritable and are sometimes observed in different family members in the same pedigree. Previous work by our group and others has suggested genetic overlap between the two conditions. In this study, we aimed to identify specific IHH-related genes that are also genetic causes of CDP. Methods: The Delayed Puberty Consortium assembled exome data for 233 individuals of diverse ancestries with CDP, with onset of puberty between 12 and 18 y in girls and between 13.5 and 18 y in boys. For control data, we used exome sequencing data from gnomAD v2.1.1 (n=125,748). After data harmonization and quality control, we used a modification of the “TRAPD” method to perform gene-based burden testing for 63 genes associated with IHH. We analyzed rare variants (minor allele frequency < 0.01) with high impact (premature stop, frameshift, start-lost, stop-lost) in all individuals across ancestries (n=233), as allele frequencies for high-impact variants are unlikely to vary widely across populations. We also analyzed rare variants with moderate impact (missense, inframe insertions/deletions) predicted to be deleterious by the Primate AI algorithm (score >0.6) in non-Finnish European (NFE) individuals with CDP (n=99), then meta-analyzed the results for high- and moderate-impact variants. We considered p-values < 7.9 x 10-4 (0.05 with Bonferroni adjustment for 63 genes) significant. Results: Of the 63 genes analyzed, SEMA3A (which encodes semaphorin-3A) exhibited significant enrichment for moderate-impact variants in the NFE CDP cohort compared to controls (variant frequency in CDP vs control: 0.064 vs 0.0076, p = 1.3 x 10-4) and in the meta-analysis of both high- and moderate-impact variants (p = 3.5 x 10-6). Novel SEMA3A variants identified exclusively in CDP individuals but not in controls were a frameshift variant, p.F546fsX2, and a missense variant, I252R. Also, three missense variants in SEMA3A were observed at a higher frequency in NFE CDP individuals compared with controls: p.R66T, p.V461A, and p.T717I. No other IHH genes showed significant enrichment of rare variants in the CDP cohort. Conclusion: Rare variants in SEMA3A contribute to both CDP and IHH. While functional studies are required to assess the effects of these specific variants on protein function, previous in vitro studies of similar SEMA3A variants demonstrated defects in secretion or signaling activity. Subsequent studies employing individual-level ancestry-matched controls and larger CDP cohorts are planned to identify additional genes that contribute to CDP. Presentation: 6/1/2024

12330 Rare Variants in SEMA3A in Individuals with Constitutional Delay of Puberty (CDP)

Abstract Disclosure: W. He: None. E.C. Schafer: None. R. Brauner: None. A. Delaney: None. L. Dunkel: None. R. Grinspon: None. J.E. Hall: None. J.N. Hirschhorn: None. S. Howard: None. A. Latronico: None. A.A. Jorge: None. K. McElreavey: None. V. Mericq: None. P.M. Merino: None. M.R. Palmert: None. R.A. Rey: None. R.C. Rezende: None. S.B. Seminara: None. N. Shaw: None. T. Delayed Puberty Genetics Consortium: None. Y. Chan: None. Background: Constitutional delay of puberty (CDP), also known as self-limited delayed puberty, refers to late onset of puberty with relatively normal progression afterward. In contrast, idiopathic hypogonadotropic hypogonadism (IHH) results in a long-lasting defect in reproductive endocrine activity. Both CDP and IHH are highly heritable and are sometimes observed in different family members in the same pedigree. Previous work by our group and others has suggested genetic overlap between the two conditions. In this study, we aimed to identify specific IHH-related genes that are also genetic causes of CDP. Methods: The Delayed Puberty Consortium assembled exome data for 233 individuals of diverse ancestries with CDP, with onset of puberty between 12 and 18 y in girls and between 13.5 and 18 y in boys. For control data, we used exome sequencing data from gnomAD v2.1.1 (n=125,748). After data harmonization and quality control, we used a modification of the “TRAPD” method to perform gene-based burden testing for 63 genes associated with IHH. We analyzed rare variants (minor allele frequency < 0.01) with high impact (premature stop, frameshift, start-lost, stop-lost) in all individuals across ancestries (n=233), as allele frequencies for high-impact variants are unlikely to vary widely across populations. We also analyzed rare variants with moderate impact (missense, inframe insertions/deletions) predicted to be deleterious by the Primate AI algorithm (score >0.6) in non-Finnish European (NFE) individuals with CDP (n=99), then meta-analyzed the results for high- and moderate-impact variants. We considered p-values < 7.9 x 10-4 (0.05 with Bonferroni adjustment for 63 genes) significant. Results: Of the 63 genes analyzed, SEMA3A (which encodes semaphorin-3A) exhibited significant enrichment for moderate-impact variants in the NFE CDP cohort compared to controls (variant frequency in CDP vs control: 0.064 vs 0.0076, p = 1.3 x 10-4) and in the meta-analysis of both high- and moderate-impact variants (p = 3.5 x 10-6). Novel SEMA3A variants identified exclusively in CDP individuals but not in controls were a frameshift variant, p.F546fsX2, and a missense variant, I252R. Also, three missense variants in SEMA3A were observed at a higher frequency in NFE CDP individuals compared with controls: p.R66T, p.V461A, and p.T717I. No other IHH genes showed significant enrichment of rare variants in the CDP cohort. Conclusion: Rare variants in SEMA3A contribute to both CDP and IHH. While functional studies are required to assess the effects of these specific variants on protein function, previous in vitro studies of similar SEMA3A variants demonstrated defects in secretion or signaling activity. Subsequent studies employing individual-level ancestry-matched controls and larger CDP cohorts are planned to identify additional genes that contribute to CDP. Presentation: 6/1/2024

Population-specific putative causal variants shape quantitative traits.

Human genetic variants are associated with many traits through largely unknown mechanisms. Here, combining approximately 260,000 Japanese study participants, a Japanese-specific genotype reference panel and statistical fine-mapping, we identified 4,423 significant loci across 63 quantitative traits, among which 601 were new, and 9,406 putatively causal variants. New associations included Japanese-specific coding, splicing and noncoding variants, exemplified by a damaging missense variant rs730881101 in TNNT2 associated with lower heart function and increased risk for heart failure (P = 1.4 × 10-15 and odds ratio = 4.5, 95% confidence interval = 3.1-6.5). Putative causal noncoding variants were supported by state-of-art in silico functional assays and had comparable effect sizes to coding variants. A plausible example of new mechanisms of causal variants is an enrichment of causal variants in 3' untranslated regions (UTRs), including the Japanese-specific rs13306436 in IL6 associated with pro-inflammatory traits and protection against tuberculosis. We experimentally showed that transcripts with rs13306436 are resistant to mRNA degradation by regnase-1, an RNA-binding protein. Our study provides a list of fine-mapped causal variants to be tested for functionality and underscores the importance of sequencing, genotyping and association efforts in diverse populations.

METTL23 Variants and Patients With Normal-Tension Glaucoma

This research confirms and further establishes that pathogenic variants in a fourth gene, METTL23, are associated with autosomal dominant normal-tension glaucoma (NTG). To determine the frequency of glaucoma-causing pathogenic variants in the METTL23 gene in a cohort of patients with NTG from Iowa. This case-control study took place at a single tertiary care center in Iowa from January 1997 to January 2024, with analysis occurring between January 2023 and January 2024. Two groups of participants were enrolled from the University of Iowa clinics: 331 patients with NTG and 362 control individuals without glaucoma. Patients with a history of trauma; steroid use; stigmata of pigment dispersion syndrome; exfoliation syndrome; or pathogenic variants in MYOC, TBK1, or OPTN were also excluded. Detection of an enrichment of METTL23 pathogenic variants in individuals with NTG compared with control individuals without glaucoma. The study included 331 patients with NTG (mean [SD] age, 68.0 [11.7] years; 228 [68.9%] female and 103 [31.1%] male) and 362 control individuals without glaucoma (mean [SD] age, 64.5 [12.6] years; 207 [57.2%] female and 155 [42.8%] male). There were 5 detected instances of 4 unique METTL23 pathogenic variants in patients with NTG. Three METTL23 variants-p.Ala7Val, p.Pro22Arg, and p.Arg63Trp-were judged to be likely pathogenic and were detected in 3 patients (0.91%) with NTG. However, when all detected variants were evaluated with either mutation burden analysis or logistic regression, their frequency was not statistically higher in individuals with NTG than in control individuals without glaucoma (1.5% vs 2.5%; P = .27). This investigation provides evidence that pathogenic variants in METTL23 are associated with NTG. Within an NTG cohort at a tertiary care center, pathogenic variants were associated with approximately 1% of NTG cases, a frequency similar to that of other known normal-tension glaucoma genes, including optineurin (OPTN), TANK-binding kinase 1 (TBK1), and myocilin (MYOC). The findings suggest that METTL23 pathogenic variants are likely involved in a biologic pathway that is associated with glaucoma that occurs at lower intraocular pressures.

Unveiling the role of IGF1R in autism spectrum disorder: a multi-omics approach to decipher common pathogenic mechanisms in the IGF signaling pathway.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition marked by impairments in social interaction, communication, and repetitive behaviors. Emerging evidence suggests that the insulin-like growth factor (IGF) signaling pathway plays a critical role in ASD pathogenesis; however, the precise pathogenic mechanisms remain elusive. This study utilizes multi-omics approaches to investigate the pathogenic mechanisms of ASD susceptibility genes within the IGF pathway. Whole-exome sequencing (WES) revealed a significant enrichment of rare variants in key IGF signaling components, particularly the IGF receptor 1 (IGF1R), in a cohort of Chinese Han individuals diagnosed with ASD, as well as in ASD patients from the SFARI SPARK WES database. Subsequent single-cell RNA sequencing (scRNA-seq) of cortical tissues from children with ASD demonstrated elevated expression of IGF receptors in parvalbumin (PV) interneurons, suggesting a substantial impact on their development. Notably, IGF1R appears to mediate the effects of IGF2R on these neurons. Additionally, transcriptomic analysis of brain organoids derived from ASD patients indicated a significant association between IGF1R and ASD. Protein-protein interaction (PPI) and gene regulatory network (GRN) analyses further identified ASD susceptibility genes that interact with and regulate IGF1R expression. In conclusion, IGF1R emerges as a central node within the IGF signaling pathway, representing a potential common pathogenic mechanism and therapeutic target for ASD. These findings highlight the need for further investigation into the modulation of this pathway as a strategy for ASD intervention.

Cancer associated variant enrichment CAVE, a gene agnostic approach to identify low burden variants in chronic lymphocytic leukemia

Intratumoral heterogeneity is an important clinical challenge because low burden clones expressing specific genetic alterations drive therapeutic resistance mechanisms. We have developed CAVE (cancer-associated variant enrichment), a gene-agnostic computational tool to identify specific enrichment of low-burden cancer driver variants in next-generation sequencing (NGS) data. For this study, CAVE was applied to TP53 in chronic lymphocytic leukemia (CLL) as a cancer model. Indeed, as TP53 mutations are part of treatment decision-making algorithms and low-burden variants are frequent, there is a need to distinguish true variants from background noise. Recommendations have been published for reliable calling of low-VAF variants of TP53 in CLL and the assessment of the background noise for each platform is essential for the quality of the testing. CAVE is able to detect specific enrichment of low-burden variants starting at variant allele frequencies (VAFs) as low as 0.3%. In silico TP53 dependent and independent analyses confirmed the true driver nature of all these variants. Orthogonal validation using either ddPCR or NGS analyses of follow-up samples confirmed variant identification. CAVE can be easily deployed in any cancer-related NGS workflow to detect the enrichment of low-burden variants of clinical interest.

Identifying genetic variants associated with chromatin looping and genome function

Here we present a comprehensive HiChIP dataset on naïve CD4 T cells (nCD4) from 30 donors and identify QTLs that associate with genotype-dependent and/or allele-specific variation of HiChIP contacts defining loops between active regulatory regions (iQTLs). We observe a substantial overlap between iQTLs and previously defined eQTLs and histone QTLs, and an enrichment for fine-mapped QTLs and GWAS variants. Furthermore, we describe a distinct subset of nCD4 iQTLs, for which the significant variation of chromatin contacts in nCD4 are translated into significant eQTL trends in CD4 T cell memory subsets. Finally, we define connectivity-QTLs as iQTLs that are significantly associated with concordant genotype-dependent changes in chromatin contacts over a broad genomic region (e.g., GWAS SNP in the RNASET2 locus). Our results demonstrate the importance of chromatin contacts as a complementary modality for QTL mapping and their power in identifying previously uncharacterized QTLs linked to cell-specific gene expression and connectivity.

Complex trait associations in rare diseases and impacts on Mendelian variant interpretation

Emerging evidence implicates common genetic variation - aggregated into polygenic scores (PGS) - in the onset and phenotypic presentation of rare diseases. Here, we comprehensively map individual polygenic liability for 1102 open-source PGS in a cohort of 3059 probands enrolled in the Genomic Answers for Kids (GA4K) rare disease study, revealing widespread associations between rare disease phenotypes and PGSs for common complex diseases and traits, blood protein levels, and brain and other organ morphological measurements. Using this resource, we demonstrate increased polygenic liability in probands with an inherited candidate disease variant (VUS) compared to unaffected carrier parents. Further, we show an enrichment for large-effect rare variants in putative core PGS genes for associated complex traits. Overall, our study supports and expands on previous findings of complex trait associations in rare diseases, implicates polygenic liability as a potential mechanism underlying variable penetrance of candidate causal variants, and provides a framework for identifying novel candidate rare disease genes.

Predictive Prioritization of Enhancers Associated with Pancreas Disease Risk.

Genetic and epigenetic variations in regulatory enhancer elements increase susceptibility to a range of pathologies. Despite recent advances, linking enhancer elements to target genes and predicting transcriptional outcomes of enhancer dysfunction remain significant challenges. Using 3D chromatin conformation assays, we generated an extensive enhancer interaction dataset for the human pancreas, encompassing more than 20 donors and five major cell types, including both exocrine and endocrine compartments. We employed a network approach to parse chromatin interactions into enhancer-promoter tree models, facilitating a quantitative, genome-wide analysis of enhancer connectivity. With these tree models, we developed a machine learning algorithm to estimate the impact of enhancer perturbations on cell type-specific gene expression in the human pancreas. Orthogonal to our computational approach, we perturbed enhancer function in primary human pancreas cells using CRISPR interference and quantified the effects at the single-cell level through RNA FISH coupled with high-throughput imaging. Our enhancer tree models enabled the annotation of common germline risk variants associated with pancreas diseases, linking them to putative target genes in specific cell types. For pancreatic ductal adenocarcinoma, we found a stronger enrichment of disease susceptibility variants within acinar cell regulatory elements, despite ductal cells historically being assumed as the primary cell-of-origin. Our integrative approach-combining cell type-specific enhancer-promoter interaction mapping, computational models, and single-cell enhancer perturbation assays-produced a robust resource for studying the genetic basis of pancreas disorders.

Network Analysis of Enhancer-Promoter Interactions Highlights Cell-Type-Specific Mechanisms of Transcriptional Regulation Variation.

Gene expression is orchestrated by a complex array of gene regulatory elements that govern transcription in a cell-type-specific manner. Though previously studied, the ability to utilize regulatory elements to identify disrupting variants remains largely elusive. To identify important factors within these regions, we generated enhancer-promoter interaction (EPI) networks and investigated the presence of disease-associated variants that fall within these regions. Our study analyzed six neuronal cell types across neural differentiation, allowing us to examine closely related cell types and across differentiation stages. Our results expand upon previous findings of cell-type specificity of enhancer, promoter, and transcription factor binding sites. Notably, we find that regulatory regions within EPI networks can identify the enrichment of variants associated with neuropsychiatric disorders within specific cell types and network sub-structures. This enrichment within sub-structures can allow for a better understanding of potential mechanisms by which variants may disrupt transcription. Together, our findings suggest that EPIs can be leveraged to better understand cell-type-specific regulatory architecture and used as a selection method for disease-associated variants to be tested in future functional assays. Combined with these future functional characterization assays, EPIs can be used to better identify and characterize regulatory variants' effects on such networks and model their mechanisms of gene regulation disruption across different disorders. Such findings can be applied in practical settings, such as diagnostic tools and drug development.

Multitrait Analysis to Decipher the Intertwined Genetic Architecture of Neuroanatomical Phenotypes and Psychiatric Disorders

BackgroundThere is increasing evidence of shared genetic factors between psychiatric disorders and brain magnetic resonance imaging (MRI) phenotypes. However, deciphering the joint genetic architecture of these outcomes has proven to be challenging, and new approaches are needed to infer the genetic structures that may underlie those phenotypes. Multivariate analyses are a meaningful approach to reveal links between MRI phenotypes and psychiatric disorders missed by univariate approaches. MethodsFirst, we conducted univariate and multivariate genome-wide association studies for 9 MRI-derived brain volume phenotypes in 20,000 UK Biobank participants. Next, we performed various complementary enrichment analyses to assess whether and how univariate and multitrait approaches could distinguish disorder-associated and non–disorder-associated variants from 6 psychiatric disorders: bipolar disorder, attention-deficit/hyperactivity disorder, autism, schizophrenia (SCZ), obsessive-compulsive disorder, and major depressive disorder. Finally, we conducted a clustering analysis of top associated variants based on their MRI multitrait association using an optimized k-medoids approach. ResultsA univariate MRI genome-wide association study revealed only negligible genetic correlations with psychiatric disorders, while a multitrait genome-wide association study identified multiple new associations and showed significant enrichment for variants related to both attention-deficit/hyperactivity disorder and SCZ. Clustering analyses also detected 2 clusters that showed not only enrichment for association with attention-deficit/hyperactivity disorder and SCZ but also a consistent direction of effects. Functional annotation analyses of those clusters pointed to multiple potential mechanisms, suggesting a role of neurotrophin pathways in particular in both MRI and SCZ. ConclusionsOur results show that multitrait association signature can be used to infer genetically driven latent MRI variables associated with psychiatric disorders, thereby opening paths for future biomarker development.

Regulatory transposable elements in the encyclopedia of DNA elements

Transposable elements (TEs) comprise ~50% of our genome, but knowledge of how TEs affect genome evolution remains incomplete. Leveraging ENCODE4 data, we provide the most comprehensive study to date of TE contributions to the regulatory genome. We find 236,181 (~25%) human candidate cis-regulatory elements (cCREs) are TE-derived, with over 90% lineage-specific since the human-mouse split, accounting for 8–36% of lineage-specific cCREs. Except for SINEs, cCRE-associated transcription factor (TF) motifs in TEs are derived from ancestral TE sequence more than expected by chance. We show that TEs may adopt similar regulatory activities of elements near their integration site. Since human-mouse divergence, TEs have contributed 3–56% of TF binding site turnover events across 30 examined TFs. Finally, TE-derived cCREs are similar to non-TE cCREs in terms of MPRA activity and GWAS variant enrichment. Overall, our results substantiate the notion that TEs have played an important role in shaping the human regulatory genome.

Role of miRNA Gene Variants (miR-22 and miR-155) as the Factors Affecting Susceptibility to Panic Disorder.

Variants within genes encoding microRNAs (miRNAs) may alter the expression of both miRNAs and their target genes, thus contributing to the etiology of psychiatric disorders. The involvement of miRNAs in neuronal differentiation and synaptic plasticity supported this hypothesis. We aimed to investigate the links between miR-155 rs767649/miR-22 rs8076112 and the risk of panic disorder (PD) in a sample of Turkish population. In this experimental study, 134 PD patients and 140 healthy controls were recruited. Genotyping was carried out using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. To evaluate PD phenotypes, Panic Disorder Severity Scale (PDSS) was also administered to patients to clarify possible associations between the scale and risk variants analyzed. The genotype analysis of miR-155 rs767649 did not show an association with PD risk and it was not related to the disease severity. For miR-22 rs8076112 variant, a statistically significant association was determined; CC genotypes were lower in patients compared to controls. Logistic regression analysis proved the highly protective effect (80.4%) of CC genotype against PD (p = 0.041; OR = 0.196, 95% CI = 0.041-0.934). Though its significance in disease liability, miR-22 rs8076112 was not associated with the disease severity. Our findings firstly report the combined analysis of miR-155 rs767649 and miR-22 rs8076112 in PD in terms of both disease susceptibility and severity. These findings await replication in independent cohorts with enrichment of other miRNA gene variants. Thus, certain miRNAs and their target genes involved in the etiology and phenotypes of PD could be enlightened.

Quantifying variant contributions in cystic kidney disease using national-scale whole-genome sequencing.

BACKGROUNDCystic kidney disease (CyKD) is a predominantly familial disease in which gene discovery has been led by family-based and candidate gene studies, an approach that is susceptible to ascertainment and other biases.METHODSUsing whole-genome sequencing data from 1,209 cases and 26,096 ancestry-matched controls participating in the 100,000 Genomes Project, we adopted hypothesis-free approaches to generate quantitative estimates of disease risk for each genetic contributor to CyKD, across genes, variant types and allelic frequencies.RESULTSIn 82.3% of cases, a qualifying potentially disease-causing rare variant in an established gene was found. There was an enrichment of rare coding, splicing, and structural variants in known CyKD genes, with statistically significant gene-based signals in COL4A3 and (monoallelic) PKHD1. Quantification of disease risk for each gene (with replication in the separate UK Biobank study) revealed substantially lower risk associated with genes more recently associated with autosomal dominant polycystic kidney disease, with odds ratios for some below what might usually be regarded as necessary for classical Mendelian inheritance. Meta-analysis of common variants did not reveal significant associations, but suggested this category of variation contributes 3%-9% to the heritability of CyKD across European ancestries.CONCLUSIONBy providing unbiased quantification of risk effects per gene, this research suggests that not all rare variant genetic contributors to CyKD are equally likely to manifest as a Mendelian trait in families. This information may inform genetic testing and counseling in the clinic.

The Role of Cilia and the Complex Genetics of Congenital Heart Disease.

Congenital heart disease (CHD) can affect up to 1% of live births, and despite abundant evidence of a genetic etiology, the genetic landscape of CHD is still not well understood. A large-scale mouse chemical mutagenesis screen for mutations causing CHD yielded a preponderance of cilia-related genes, pointing to a central role for cilia in CHD pathogenesis. The genes uncovered by the screen included genes that regulate ciliogenesis and cilia-transduced cell signaling as well as many that mediate endocytic trafficking, a cell process critical for both ciliogenesis and cell signaling. The clinical relevance of these findings is supported by whole-exome sequencing analysis of CHD patients that showed enrichment for pathogenic variants in ciliome genes. Surprisingly, among the ciliome CHD genes recovered were many that encoded direct protein-protein interactors. Assembly of the CHD genes into a protein-protein interaction network yielded a tight interactome that suggested this protein-protein interaction may have functional importance and that its disruption could contribute to the pathogenesis of CHD. In light of these and other findings, we propose that an interactome enriched for ciliome genes may provide the genomic context for the complex genetics of CHD and its often-observed incomplete penetrance and variable expressivity.

Rapidly Inducible Yeast Surface Display for Antibody Evolution with OrthoRep.

We recently developed "autonomous hypermutation yeast surface display" (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence-activated cell sorting. AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 h of induction. Here we report an updated version of the AHEAD platform that utilizes a synthetic β-estradiol-induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system and traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.

Comprehensive network modeling approaches unravel dynamic enhancer-promoter interactions across neural differentiation

BackgroundIncreasing evidence suggests that a substantial proportion of disease-associated mutations occur in enhancers, regions of non-coding DNA essential to gene regulation. Understanding the structures and mechanisms of the regulatory programs this variation affects can shed light on the apparatuses of human diseases.ResultsWe collect epigenetic and gene expression datasets from seven early time points during neural differentiation. Focusing on this model system, we construct networks of enhancer-promoter interactions, each at an individual stage of neural induction. These networks serve as the base for a rich series of analyses, through which we demonstrate their temporal dynamics and enrichment for various disease-associated variants. We apply the Girvan-Newman clustering algorithm to these networks to reveal biologically relevant substructures of regulation. Additionally, we demonstrate methods to validate predicted enhancer-promoter interactions using transcription factor overexpression and massively parallel reporter assays.ConclusionsOur findings suggest a generalizable framework for exploring gene regulatory programs and their dynamics across developmental processes; this includes a comprehensive approach to studying the effects of disease-associated variation on transcriptional networks. The techniques applied to our networks have been published alongside our findings as a computational tool, E-P-INAnalyzer. Our procedure can be utilized across different cellular contexts and disorders.