De Novo Mutations Research Articles

Three intellectual disability-associated de novo mutations in MECP2 identified by trio-WES analysis

BackgroundTo date, at least 746 genes have been identified to cause intellectual disability (ID). Among them, mutations in the Methyl CpG binding protein 2 (MECP2) gene are the leading cause of Rett syndrome and associated ID.MethodsConsidering the large number of ID-associated genes, we applied trio-based whole-exome sequencing (trio-WES) and in silico analysis for genetic diagnosis of 294 children with ID.ResultsThree de novo heterozygous mutations [NM_004992.3: c.502C > T, p.(Arg168*), c.916C > T, p.(Arg306Cys), and c.879C > G, p.(Ile293Met)] in MECP2 were identified in three unrelated girls. The first two mutations were detected in two patients who were diagnosed as typical Rett syndrome, X-linked ID and psychomotor retardation. The third mutation (c.879C > G), a previously unreported, was found in a 6-year-old girl with ID, microcephaly, severe underweight and psychomotor retardation. Particularly, this extremely rare de novo mutation (DNM) is located in the transcriptional repression domain (TRD) of MECP2, where at least 62 different causal mutations are identified.ConclusionsWe identified three DNMs in MECP2 in a cohort of 294 individuals with ID. The novel c.879C > G mutation, as a likely pathogenic allele, may become a risk factor associated with X-linked ID, microcephaly and psychomotor retardation.

OR15-07 Novel Genes Involved in Sex Differentiation Identified by Whole-Exome Sequencing in a Cohort of Children with Disorders of Sex Development

Background: Disorders of sex development (DSD) are classified as a congenital discrepancy between external genitalia, gonadal and chromosomal sex. Despite extensive laboratory and imaging investigations, the etiology of DSD is unknown in more than 50% of patients and the diagnosis is often delayed to the second decade of life. Our objective was to evaluate the etiology of DSD by whole-exome sequencing (WES) in children in whom hormonal and candidate gene approaches had not identified the etiology. Methods: Nine patients diagnosed with DSD (eight 46,XY and one 46,XX) were enrolled. Patients underwent hormonal evaluation, including ACTH, GnRH and hCG tests. Candidate genes were sequenced in accordance with the hormonal results. WES was performed for the probands and their parents. Results: The eight 46,XY patients presented with micropenis, cryptorchidism and hypospadias at birth and the 46,XX patient with fusion of the labia majora. In six of the nine patients (66%), a pathogenic mutation was identified by WES that explained the phenotype: four known DSD-causing genes—POR, CHD7, HSD17B3 and WT1—and two novel genes—BMP4 and RFXP2. In three patients, variants of unknown significance were found. An 11-y-old boy had a novel de-novo mutation in BMP4. In humans, mutations in this gene, encoding bone morphogenetic protein 4, are associated with autosomal dominant microphthalmia. BMP4 is expressed in the urethral epithelium and has a role in the development of external genitalia and the pituitary. This is the first report of a BMP4 mutation in a child with DSD. A 12-y-old boy had a mutation in RFXP2, encoding insulin-like 3 hormone receptor, which has been previously reported in adult males with cryptorchidism. This is the first case of an RFXP2 mutation in a child with DSD. Conclusions: WES has a crucial role in early diagnosis of the etiology of DSD, making extensive endocrine testing unnecessary, and has important implications for sex of rearing decisions.

SAT-LB68 A Rare De Novo Mutation in LRP5 Gene in an Adolescent Female With Juvenile Onset Primary Osteoporosis

Background: Juvenile onset of primary osteoporosis is a rare skeletal disorder with a highly heterogenous clinical presentation and complex poorly understood genetic etiology. Low-density lipoprotein receptor-related protein 5 (LRP5), a Wnt-β-catenin pathway receptor involved in bone mineral density (BMD) regulation, has been reported in children with primary osteoporosis mainly due to missense and frame-shift mutations. To our knowledge, there is only one report of in-frame deletions in exon 21 possibly being implicated in pregnancy related osteoporosis (1). We report the first case of a rare heterozygous de novo in-frame deletion in exon 21 of LRP5 gene in a girl with juvenile onset primary osteoporosis. Clinical Case: A 7.5 yo previously healthy European-African-American female born to non-consanguineous family, was noted to have left ankle and bilateral wrists fractures during sport activities occurring over the span of 1 y. On exam, she was of normal stature, had normal sclerae and vision, but was found to have hypermobile joints. Initial blood work revealed an elevated iPTH 72 pg/mL (9-59), normal Ca 9.6 mg/dL (8.9-10.4), and low 25OH-Vitamin D 17 ng/mL (30-100), which normalized with supplementation to 30.4 ng/mL. By 10.5 yo, she had sustained multiple sports related and pathologic fractures of long bones with progressive bone pain and functional ambulatory impairment. Radiographic evaluation showed osteopenia, multiple healed long bone fractures with presumed non-ossifying fibroma in the distal right femoral metaphysis, and subtle height loss of T4-5 vertebrae. CT bone densitometry at 12 yo showed low mean cancellous lumbar vertebral BMD of 190 mg/cm3 (ref. 285 ± 45) and normal cortical BMD of the femoral midshaft of 1977 mg/cm3 (ref. 2000 ± 60). Genetic testing for Osteogenesis Imperfecta returned negative for COL1A1 and COL1A2 mutations. Whole exome sequencing (WES) revealed a rare, de novo heterozygous mutation in exon 21 that involved deletion of nucleotides 4454 to 4465 resulting in an in-frame deletion of amino acid 1485 to 1488. This mutation is predicted to be deleterious by in silico analysis. Conclusion: We present the first case of a young female with progressive pathologic fractures, functional ambulatory impairment, and low BMD in childhood, consistent with juvenile onset primary osteoporosis. WES revealed a rare de novo heterozygous in-frame deletion mutation in LRP5, providing a plausible biological mechanism for her clinical presentation, and further contributing to the increasing genetic heterogeneity of juvenile onset primary osteoporosis and LRP5-related bone disorders. Reference: (1) Cook, F., Mumm, S., Whyte, M., Wenkert, D. Pregnancy-associated osteoporosis with a heterozygous deactivating LDL receptor-related protein 5 (LRP5) mutation and a homozygous methylenetetrahydrofolate reductase (MTHRF) polymorphism. JBMR. 2014;29 (4): 922-8.

Co-localization between Sequence Constraint and Epigenomic Information Improves Interpretation of Whole-Genome Sequencing Data

The identification of functional regions in the noncoding human genome is difficult but critical in order to gain understanding of the role noncoding variation plays in gene regulation in human health and disease. We describe here a co-localization approach that aims to identify constrained sequences that co-localize with tissue- or cell-type-specific regulatory regions, and we show that the resulting score is particularly well suited for the identification of rare regulatory variants. For 127 tissues and cell types in the ENCODE/Roadmap Epigenomics Project, we provide catalogs of putative tissue- or cell-type-specific regulatory regions under sequence constraint. We use the newly developed co-localization score for brain tissues to score de novo mutations in whole genomes from 1,902 individuals affected with autism spectrum disorder (ASD) and their unaffected siblings in the Simons Simplex Collection. We show that noncoding de novo mutations near genes co-expressed in midfetal brain with high confidence ASD risk genes, and near FMRP gene targets are more likely to be in co-localized regions if they occur in ASD probands versus in their unaffected siblings. We also observed a similar enrichment for mutations near lincRNAs, previously shown to co-express with ASD risk genes. Additionally, we provide strong evidence that prioritized denovo mutations in autism probands point to a small set of well-known ASD genes, the disruption of which produces relevant mouse phenotypes such as abnormal social investigation and abnormal discrimination/associative learning, unlike the de novo mutations in unaffected siblings. The genome-wide co-localization results are available online.

Next generation sequencing of BRCA genes in glioblastoma multiform Egyptian patients: a pilot study

Background Germ line mutations of BRCA1 and BRCA2 were correlated with a variety of cancer Authors aimed to use next-generation sequencing (NGS) to detect BRCA1 and BRCA2 germ line mutations in glioblastoma multiform (GBM) Egyptian patients. Materials and methods Genomic DNA was extracted from six GBM cases, amplified using Ion AmpliSeq BRCA1 and BRCA2 panel. DNA libraries were pooled, barcoded and finally sequenced using Ion Torrent Personal Genome Machine sequencer. Results BRCA1 the previously reported rs1799966, rs1799950, rs16941 were found in five cases and they are in a linkage disequilibrium forming two distinct haplotypes, which might support their role in cancer predisposition. Out of the 18 reported variants in BRCA2, three denovo mutations were detected which leads to frame shift. Conclusion Further studies on large number of GBM patients and control cases to determine BRCA1 and BRCA2 germline mutations and haplotypes; diagnostic and prognostic role are encouraged.

Abstract P4-05-20: Case report - 16 year life history and genomic evolution of an ER+ HER2- breast cancer

Abstract Introduction Metastasis is the primary cause of cancer-related death, with genomic evolution between primary and metastatic breast cancer only recently being studied in smaller numbers. We reconstructed the tumor evolution of a female patient originally diagnosed with localized ER+ HER2- disease to investigate the molecular changes that triggered relapse after long term aromatase inhibitor (AI) therapy and tracked them during subsequent treatments. The patient was diagnosed in 2002, relapsed in 2012 (regional lymph node), progressed in 2015 (liver metastasis), and passed away in 2018. We performed DNA- and RNA-sequencing on FFPE tumor samples and multiple liquid biopsies. The patient underwent eight lines of therapy including genome guided targeted therapy. Materials and Methods Whole exome and transcriptome sequencing was performed on the patient's primary tumor site at diagnosis (T1) and a metastatic lymph node (M1). Targeted DNA sequencing was performed on a metastatic liver sample (M2, 315 genes) and on liquid biopsies (n=11, 70 genes) every 2-3 months from 2015-2018. Results The mutational landscape between timepoints T1, M1 and M2 revealed low mutational burden at diagnosis which ultimately doubled at progression with active mutational processes (APOBEC, POLE) showing concordance between timepoints. DNA-based analysis revealed multiple mutations in the PI3K-Akt signaling pathway. Two mutations were shared between the samples: a clonal PIK3CA Q546R and a TP53 E180K mutation detected as clonal in M1/M2 and subclonal in T1. Individual samples carried private driver mutations, ranging from n=4 in T1, n=7 in M1 to n=16 in M2. While T1 harbored a single clonal driver mutation, with additional driver mutations being subclonal, M1 and M2 harbored multiple clonal driver mutations and a smaller percentage of subclonal driver mutations. A phylogenetic tree showed branching off between T1 and M1/M2. Liquid biopsies, while initially negative, started to detect mutations found among T1, M1 and M2, with increasing allele frequency (AF) throughout time. By the end of 2016, an ESR1 E380Q mutation not previously detected was found at a low AF that had rapidly increased by the end of 2017. RNA-Seq analysis revealed 908 (209 up, 699 down) genes to be differentially expressed between T1 and M1. Most significantly downregulated genes in M1 were TFF1 and PGR indicating loss-of-sensitivity / resistance to AI therapy. Most upregulated genes in M1 included PTHLH, S100P, and SOX2 which have been implicated in promoting tumor growth and metastasis. Pathway analysis between T1 and M1 showed enrichment for the hallmark gene sets ‘Estrogen Response early and late’ and ‘Epithelial mesenchymal transition’. Conclusions This phylogenetic reconstruction of the life history of a single patient's cancer revealed an increased mutational rate over time between the patient's primary tumor at diagnosis and samples taken at relapse; and a high level of heterogeneity with metastatic sites sharing only one driver mutation with the primary tumor at diagnosis and having acquired multiple de-novo driver mutations. Transcriptome profiling aided in uncovering the mechanisms that lead to the patient's initial relapse, indicating expression profile changes that promote tumor progression and metastasis as well as reduced sensitivity / resistance to the patients AI based therapy. Monitoring tumor progression through frequent liquid biopsies allowed for the detection of mutations from multiple metastatic sites, in addition to the detection and growth of an AI resistant clone harboring an ESR1 E380Q mutation that was not present in any of the solid tissue samples. Citation Format: Bing Xu, Anu Amallraja, Padmapriya Swaminathan, Rachel Elsey, Christel Davis, Stephanie Theel, Sarah Viet, Jason Petersen, Amy Krie, Gareth E Davies, Casey B Williams, Erik Ehli, Tobias Meissner. Case report - 16 year life history and genomic evolution of an ER+ HER2- breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-20.

Polymorphisms and rare variants identified by next-generation sequencing confer risk for lung cancer in han Chinese population

BackgroundLung cancer is one of the leading causes of cancer death worldwide, and genetic risk factors account for a large part of its carcinogenesis. The low economic requirements and high efficiency of next-generation sequencing (NGS) make it widely used in detecting genetic alterations in pathogenesis. MethodsWe performed targeted panel sequencing in 780 Han Chinese lung cancer patients using a commercial probe, and the correlations between dozens of susceptible sites were verified in 1113 healthy controls. This study used Fisher's exact test and Benjamini-Hochberg FDR correction to analyze the mutual exclusion between mutated genes, and Pearson's p was used to verify the correlations between mutations and lung cancer susceptibility. ResultsOur results determined the mutation spectrum and showed that each lung cancer patient carried at least one DNA mutation. The most frequently mutated gene was BRCA2 (mutation rate,10.6 %.). The co-occurrence and mutual exclusion analysis of DNA damage related genes showed that gene ATM was mutually exclusive from MSH6. We conducted a further case-control study in different subtypes of lung cancer and the results described 14 mutations associated with adenocarcinoma, 9 with squamous cell carcinoma, and 4 with small cell lung cancer. These variants were novel de-novo germline mutations in lung cancer. Particularly, rs3864017 in FANCD2 showed a protective effect of lung adenocarcinoma for carriers (OR = 0.146, 95 % CI = 0.052∼0.405, Padjusted = 3.37 × 10−4). Conclusions18 candidate mutations might alter the risk of lung cancer in the Han Chinese population, including polymorphisms rs3864017(FANCD2), rs55740729(MSH6) and 16 rare variants. The underlying mechanisms of candidate genes in lung cancer remain unclear and we suggest more functional studies on exploring how these genes affect the risk of lung cancer.

Small ncRNA binding protein, PIWI: A potential molecular bridge between blood brain barrier and neuropathological conditions

The blood brain barrier (BBB) is a neuroprotective layer that maintains the homeostasis of central nervous system and provides an appropriate environment for neurons to execute their functions. The fundamental role of the dynamic semi-permeable BBB is selective and stringent transport of molecules from circulating blood and surrounding extracellular matrix across brain. Disruption of BBB has critical implications that can lead to various neuropathological disorders (NPDs) namely multiple sclerosis, Alzheimer’s disease, epilepsy, traumatic brain injuries and neuropsychiatric disorders, etc. Therapeutic management of NPDs is still a daunting challenge in the field of neuromedicine and there is a great need for identifying novel drug targets and biomarkers. Recently, noncoding RNAs (ncRNA) have emerged as promising prognostic markers in NPDs. Piwi interacting RNAs (piRNA), a family of short noncoding RNAs which in association with PIWI-like proteins have shown to regulate neuronal function and memory formation. In addition, piRNAs are differentially expressed in Alzheimer’s brain tissues and studies also revealed the association of denovo mutations in PIWI genes with autism. Moreover, the role of PIWI-like proteins in neuronal long-term potentiation and neurite outgrowth is now evident, confirming their importance in normal physiology of the brain. Notably, we have reported the significance of PIWI-like proteins in the maintenance of Blood Retinal Barrier (BRB) and its potential role in diseases like diabetic retinopathy through modulation of tight junction proteins. Further studies in hydra and cancer cells confirmed the important function of PIWI-like proteins in the organization of tight junctions. Interestingly, we also observed that loss of PIWI-like proteins affected the activity of Ephrin receptors which have an established functional link to tight junction assembly. Collectively, the evidences profoundly support the novel concept that piRNAs/PIWI-like proteins may have a potential role on the governance of BBB by altering the tight junctions through Ephrin effectors, commotion of which could be the preceding event to various NPDs. Here, we propose PIWI-like proteins and associated piRNAs as potential restorative drug targets for combating neuropathological conditions.

Analysis of De Novo Mutations in Sporadic Cardiomyopathies Emphasizes Their Clinical Relevance and Points to Novel Candidate Genes.

The vast majority of cardiomyopathies have an autosomal dominant inheritance; hence, genetic testing is typically offered to patients with a positive family history. A de novo mutation is a new germline mutation not inherited from either parent. The purpose of our study was to search for de novo mutations in patients with cardiomyopathy and no evidence of the disease in the family. Using next-generation sequencing, we analyzed cardiomyopathy genes in 12 probands. In 8 (66.7%), we found de novo variants in known cardiomyopathy genes (TTN, DSP, SCN5A, TNNC1, TPM1, CRYAB, MYH7). In the remaining probands, the analysis was extended to whole exome sequencing in a trio (proband and parents). We found de novo variants in genes that, so far, were not associated with any disease (TRIB3, SLC2A6), a possible disease-causing biallelic genotype (APOBEC gene family), and a de novo mosaic variant without strong evidence of pathogenicity (UNC45A). The high prevalence of de novo mutations emphasizes that genetic screening is also indicated in cases of sporadic cardiomyopathy. Moreover, we have identified novel cardiomyopathy candidate genes that are likely to affect immunological function and/or reaction to stress that could be especially relevant in patients with disease onset associated with infection/infestation.

De novo mutations across 1,465 diverse genomes reveal mutational insights and reductions in the Amish founder population

De novo mutations (DNMs), or mutations that appear in an individual despite not being seen in their parents, are an important source of genetic variation whose impact is relevant to studies of human evolution, genetics, and disease. Utilizing high-coverage whole-genome sequencing data as part of the Trans-Omics for Precision Medicine (TOPMed) Program, we called 93,325 single-nucleotide DNMs across 1,465 trios from an array of diverse human populations, and used them to directly estimate and analyze DNM counts, rates, and spectra. We find a significant positive correlation between local recombination rate and local DNM rate, and that DNM rate explains a substantial portion (8.98 to 34.92%, depending on the model) of the genome-wide variation in population-level genetic variation from 41K unrelated TOPMed samples. Genome-wide heterozygosity does correlate with DNM rate, but only explains <1% of variation. While we are underpowered to see small differences, we do not find significant differences in DNM rate between individuals of European, African, and Latino ancestry, nor across ancestrally distinct segments within admixed individuals. However, we did find significantly fewer DNMs in Amish individuals, even when compared with other Europeans, and even after accounting for parental age and sequencing center. Specifically, we found significant reductions in the number of C→A and T→C mutations in the Amish, which seem to underpin their overall reduction in DNMs. Finally, we calculated near-zero estimates of narrow sense heritability (h2), which suggest that variation in DNM rate is significantly shaped by nonadditive genetic effects and the environment.

Novel genetic susceptibility loci identified by family based whole exome sequencing in Han Chinese schizophrenia patients

Schizophrenia (SCZ) is a highly heritable psychiatric disorder that affects approximately 1% of population around the world. However, early relevant studies did not reach clear conclusions of the genetic mechanisms of SCZ, suggesting that additional susceptibility loci that exert significant influence on SCZ are yet to be revealed. So, in order to identify novel susceptibility genes that account for the genetic risk of SCZ, we performed a systematic family-based study using whole exome sequencing (WES) in 65 Han Chinese families. The analysis of 51 SCZ trios with both unaffected parents identified 22 exonic and 1 splice-site de novo mutations (DNMs) on a total of 23 genes, and showed that 12 genes carried rare protein-altering compound heterozygous mutations in more than one trio. In addition, we identified 26 exonic or splice-site single nucleotide polymorphisms (SNPs) on 18 genes with nominal significance (P < 5 × 10−4) using a transmission disequilibrium test (TDT) in all the families. Moreover, TDT result confirmed a SCZ susceptibility locus on 3p21.1, encompassing the multigenetic region NEK4-ITIH1-ITIH3-ITIH4. Through several different strategies to predict the potential pathogenic genes in silico, we revealed 4 previous discovered susceptibility genes (TSNARE1, PBRM1, STAB1 and OLIG2) and 4 novel susceptibility loci (PSEN1, TLR5, MGAT5B and SSPO) in Han Chinese SCZ patients. In summary, we identified a list of putative candidate genes for SCZ using a family-based WES approach, thus improving our understanding of the pathology of SCZ and providing critical clues to future functional validation.

Genetic evidence of gender difference in autism spectrum disorder supports the female-protective effect

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a male-to-female prevalence of 4:1. However, the genetic mechanisms underlying this gender difference remain unclear. Mutation burden analysis, a TADA model, and co-expression and functional network analyses were performed on de novo mutations (DNMs) and corresponding candidate genes. We found that the prevalence of putative functional DNMs (loss-of-function and predicted deleterious missense mutations) in females was significantly higher than that in males, suggesting that a higher genetic load was required in females to reach the threshold for a diagnosis. We then prioritized 174 candidate genes, including 60 shared genes, 91 male-specific genes, and 23 female-specific genes. All of the three subclasses of candidate genes were significantly more frequently co-expressed in female brains than male brains, suggesting that compensation effects of the deficiency of ASD candidate genes may be more likely in females. Nevertheless, the three subclasses of candidate genes were co-expressed with each other, suggesting a convergent functional network of male and female-specific genes. Our analysis of different aspects of genetic components provides suggestive evidence supporting the female-protective effect in ASD. Moreover, further study is needed to integrate neuronal and hormonal data to elucidate the underlying gender difference in ASD.

Exome sequencing in schizophrenia-affected parent-offspring trios reveals risk conferred by protein-coding de novo mutations.

Protein-coding de novo mutations (DNMs) are significant risk factors in many neurodevelopmental disorders, whereas schizophrenia (SCZ) risk associated with DNMs has thus far has been modest. We analyzed DNMs from 1,695 SCZ affected trios and 1,077 published SCZ affected trios to better understand the contribution to SCZ risk. Among 2,772 SCZ probands, exome-wide DNM burden remains modest. Gene set analyses reveal that SCZ DNMs are significantly concentrated in genes either highly brain expressed, under strong evolutionary constraint, and/or overlap with genes identified in other neurodevelopmental disorders. No single gene surpasses exome-wide significance, however sixteen genes are recurrently hit by protein-truncating DNMs, a 3.15-fold higher rate than the mutation model expectation (permuted 95% CI=1–10 genes, permuted p=3e-5). Overall, DNMs explain a small fraction of SCZ risk, and larger samples are needed to identify individual risk genes, as coding variation across many genes confer risk for SCZ in the population.

Autism risk in offspring can be assessed through quantification of male sperm mosaicism.

De novo mutations (DNMs) arising on the paternal chromosome make the largest known contribution to autism risk, and correlate with paternal age at the time of conception. The recurrence risk for autism spectrum disorders (ASD) is substantial, leading many families to decline future pregnancies, but the potential impact of assessing parental gonadal mosaicism has not been considered. We measured sperm mosaicism using deep whole genome sequencing, both for variants present in an offspring and evident only in father’s sperm, and identified single nucleotide, structural, and short tandem repeat variants. We found that mosaicism quantification can stratify ASD recurrence risk due to DNMs into the vast majority with near 0% recurrence and a small fraction with a substantially higher and quantifiable risk, and we identify novel mosaic variants at risk for transmission to a future offspring. Thus, this suggests that genetic counseling would benefit from the addition of sperm mosaicism assessment.

Sex-Based Analysis of De Novo Variants in Neurodevelopmental Disorders

While genes with an excess of de novo mutations (DNMs) have been identified in children with neurodevelopmental disorders (NDDs), few studies focus on DNM patterns where the sex of affected children is examined separately. We considered ∼8,825 sequenced parent-child trios (n ∼26,475 individuals) and identify 54 genes with a DNM enrichment in males (n = 18), females (n = 17), or overlapping in both the male and female subsets (n = 19). A replication cohort of 18,778 sequenced parent-child trios (n = 56,334 individuals) confirms 25 genes (n= 3 in males, n = 7 in females, n = 15 in both male and female subsets). As expected, we observe significant enrichment on the X chromosome for females but also find autosomal genes with potential sex bias (females, CDK13, ITPR1; males, CHD8, MBD5, SYNGAP1); 6.5% of females harbor a DNM in a female-enriched gene, whereas 2.7% of males have a DNM in a male-enriched gene. Sex-biased genes are enriched in transcriptional processes and chromatin binding, primarily reside in the nucleus of cells, and have brain expression. By downsampling, we find that DNM gene discovery is greatest when studying affected females. Finally, directly comparing de novo allele counts in NDD-affected males and females identifies one replicated genome-wide significant gene (DDX3X) with locus-specific enrichment in females.Our sex-based DNM enrichment analysis identifies candidate NDD genes differentially affecting males and females and indicates that the study of females with NDDs leads to greater gene discovery consistent with the female-protective effect.

Machine Learning Unveils Proteotypic Mimicry in Genetically Defined SCN Variants

Background Novel computational algorithms for multi-omics analysis bear great potential to highlight pathomechanisms of monogenic diseases. We recently defined the in-depth proteome of primary human neutrophil granulocytes (PMID 30630937). Here, we ask the question whether proteotypic patterns differ between defined genetic subtypes associated with severe congenital neutropenia (SCN). We focus on two novel genetic variants in constituents of the signal recognition particle (SRPRA and SRP19) and previously reported SCN genotypes SRP54, HAX1, and ELANE. Methods We analyzed proteomes of highly purified neutrophil granulocytes from a total of 26 SCN patients, including 5 with homozygous splice site mutations in SRP19, one patient with a de-novo heterozygous missense mutation in SRPRA (using 5 biological replicates collected months apart) as well as 6 patients with SRP54, 8 with HAX1 and 6 with ELANE mutations. Samples of 70 healthy donors (HD) served as controls. Whole cell proteome analysis was based on data-independent acquisition using a Thermo Fisher QExactive HF mass spectrometer. Data analysis was performed in R and Cytoscape, machine learning approaches included lasso regression and random forest. Results Differential expression analysis in comparison to HD showed in all genotypes overexpression of ribosomes, the translational apparatus, mitochondria, cell-substrate junctions and response to unfolded proteins. Underexpressed proteins showed genotype specific enrichment for granule subsets. Whereas ELANE showed deficiency of primary and secretory granules, HAX1 showed deficiency of specific, tertiary and secretory granules. All SRP genotypes showed markedly reduced abundance of proteins in all granule subsets. Principal component analysis showed clear separation of healthy and diseased proteotypes on the first component, whereas the separation of patient genotypes became clear only using five dimensions. We derived genotype specific proteome signatures by lasso regression, consisting of 26 (minimal specific set) to 128 (comprehensive signature) proteins, and a signature of 48 proteins when joining the SRP genotypes as one group. This signatures allow for perfect separation of the genotypes, demonstrating a clear genotype specific effect on protein abundance levels. We asked the question if the genotypes SRP19 and SRPRA show more similar proteomic profile to SRP54 than the other genotypes (ELANE, HAX1) by training a random forest model on proteome data from SRP54, HAX1, ELANE and HD and subsequently testing if the other SRP samples get classified as SRP54. We observe only few misclassifications using either all proteins (7/10) or using the lasso derived genotype defining proteins (8/10). This strongly supports our hypothesis that mutations in different subunits of the same complex lead to similar proteotype changes, a phenomenon we propose to call "proteotypic mimicry". For a systems biology perspective we selected proteins that were exclusively regulated in the SRP genotypes and restricted a network of interactions to these proteins together with their direct interactors (based on APID level 1). The resulting network contained 464 proteins and 3587 interactions. MCODE analysis identified 16 clusters that were consequently annotated using BINGO enrichment analysis. The SRP specific network shows features of the translational apparatus, the proteasome, the septin complex, splicing and cell-metabolic processes. Further studies to dissect specific pathomechanisms are under way. Conclusion Here we provide for the first time evidence for the correlation between SCN causing genotypes and their corresponding neutrophil proteotypes. In particular, we demonstrate significant overlap of all SRP related proteotypes, indicating a phenomenon we propose to be called "proteotypic mimicry". Studies on similarities and disparities of neutrophil proteotypes will help to raise new hypothesis on distinct cellular dysfunction in defined genetic defects of neutrophil granulocytes. Disclosures No relevant conflicts of interest to declare.

Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring.

The emergence of the CRISPR-Cas9 system has triggered a technical revolution in mammalian genome editing. Compared to traditional gene-targeting strategies, CRISPR-Cas9 technology offers a more efficient and cost-effective approach for generating genetically modified animal models. However, off-target cleavage in CRISPR-mediated genome editing is a major concern in the analysis of phenotypes as well as the selection of therapeutic targets. Here, we analyzed whole-genome sequencing (WGS) data from two knock-out (KO) mouse strains generated by using the CRISPR-Cas9 system targeting the Mmd and Paqr8 loci. A total of nine individuals were sequenced including two parents, four F1 offspring and three uninjected control mice. Using GATK and bcftools software, we identified two off-target events in the founder mice. The two CRISPR-Cas9-induced off-target events were predictable using Cas-OFFinder and were not passed on to the offspring that we investigated. In addition, our results indicated that the number of CRISPR-Cas9-induced mutations was not statistically distinguishable from the background de novo mutations (DNMs).

De Novo Mutations in FOXJ1 Result in a Motile Ciliopathy with Hydrocephalus and Randomization of Left/Right Body Asymmetry

Hydrocephalus is one of the most prevalent form of developmental central nervous system (CNS) malformations. Cerebrospinal fluid (CSF) flow depends on both heartbeat and body movement. Furthermore, it has been shown that CSF flow within and across brain ventricles depends on cilia motility of the ependymal cells lining the brain ventricles, which play a crucial role to maintain patency of the narrow sites of CSF passage during brain formation in mice. Using whole-exome and whole-genome sequencing, we identified an autosomal-dominant cause of a distinct motile ciliopathy related to defective ciliogenesis of the ependymal cilia in six individuals. Heterozygous de novo mutations in FOXJ1, which encodes a well-known member of the forkhead transcription factors important for ciliogenesis of motile cilia, cause a motile ciliopathy that is characterized by hydrocephalus internus, chronic destructive airway disease, and randomization of left/right body asymmetry. Mutant respiratory epithelial cells are unable to generate a fluid flow and exhibit a reduced number of cilia per cell, as documented by high-speed video microscopy (HVMA), transmission electron microscopy (TEM), and immunofluorescence analysis (IF). TEM and IF demonstrate mislocalized basal bodies. In line with this finding, the focal adhesion protein PTK2 displays aberrant localization in the cytoplasm of the mutant respiratory epithelial cells.

Multi-omics study in monozygotic twins confirm the contribution of de novo mutation to psoriasis

BackgroundGenome-wide association studies have identified over 120 risk loci for psoriasis. However, most of the variations are located in non-coding region with high frequency and small effect size. Pathogenetic variants are rarely reported except HLA-C*0602 with the odds ratio being approximately 4.0 in Chinese population. Although rare variations still account for a small proportion of phenotypic variances in complex diseases, their effect on phenotypes is large. Recently, more and more studies focus on the low-frequency functional variants and have achieved a certain amount of success. MethodWhole genome sequencing and sanger sequencing was performed on 8 MZ twin pairs discordant for psoriasis to scan and verified the de novo mutations (DNMs). Additionally, 665 individuals with about 20 years' medical history versus 2054 healthy controls and two published large population studies which had about 8 years’ medical history (including 10,727 cases versus 10,582 controls) were applied to validate the enrichment of rare damaging mutations in two DNMs genes. Besides, to verify the pathogenicity of candidate DNM in C3, RNA-sequencing for CD4+, CD8+ T cells of twins and lesion, non-lesion skin of psoriasis patients were carried out. Meanwhile, the enzyme-linked immunosorbent assay kit was used to detect the level of C3, C3b in the supernatant of peripheral blood. ResultA total of 27 DNMs between co-twins were identified. We found six of eight twins carry HLA-C∗0602 allele which have large effects on psoriasis. And it is interesting that a missense mutation in SPRED1 and a splice region mutation in C3 are found in the psoriasis individuals in the other two MZ twin pairs without carrying HLA-C*0602 allele. In the replication stage, we found 2 loss-of-function (LOF) variants of C3 only in 665 cases with about 20 years' medical history and gene-wise analysis in 665 cases and 2054 controls showed that the rare missense mutations in C3 were enriched in cases (OR = 1.91, P = 0.0028). We further scanned the LOF mutations of C3 in two published studies (about 8 years’ medical history), and found one LOF mutation in the case without carrying HLA-C*0602. In the individual with DNM in C3, RNA sequencing showed the expression level of C3 in skin was significant higher than healthy samples in public database (TPM fold change = 1.40, P = 0.000181) and ELISA showed protein C3 in peripheral blood was higher (~2.2-fold difference) than the other samples of twins without DNM in C3. ConclusionTo the best of our knowledge, this is the first report that DNM in C3 is the likely pathological mutations, and it provided a better understanding of the genetic etiology of psoriasis and additional treatments for this disease.

De Novo Damaging DNA Coding Mutations Are Associated With Obsessive-Compulsive Disorder and Overlap With Tourette’s Disorder and Autism

BackgroundObsessive-compulsive disorder (OCD) is a debilitating neuropsychiatric disorder with a genetic risk component, yet identification of high-confidence risk genes has been challenging. In recent years, risk gene discovery in other complex psychiatric disorders has been achieved by studying rare de novo (DN) coding variants. MethodsWe performed whole-exome sequencing in 222 OCD parent-child trios (184 trios after quality control), comparing DN variant frequencies with 777 previously sequenced unaffected trios. We estimated the contribution of DN mutations to OCD risk and the number of genes involved. Finally, we looked for gene enrichment in other datasets and canonical pathways. ResultsDN likely gene disrupting and predicted damaging missense variants are enriched in OCD probands (rate ratio, 1.52; p = .0005) and contribute to risk. We identified 2 high-confidence risk genes, each containing 2 DN damaging variants in unrelated probands: CHD8 and SCUBE1. We estimate that 34% of DN damaging variants in OCD contribute to risk and that DN damaging variants in approximately 335 genes contribute to risk in 22% of OCD cases. Furthermore, genes harboring DN damaging variants in OCD are enriched for those reported in neurodevelopmental disorders, particularly Tourette’s disorder and autism spectrum disorder. An exploratory network analysis reveals significant functional connectivity and enrichment in canonical pathways, biological processes, and disease networks. ConclusionsOur findings show a pathway toward systematic gene discovery in OCD via identification of DN damaging variants. Sequencing larger cohorts of OCD parent-child trios will reveal more OCD risk genes and will provide needed insights into underlying disease biology.