Severe viral infections of the skin can occur in patients with inborn errors of immunity (IEI). We report an all-in-one whole-transcriptome sequencing-based method by RNA-Seq on a single skin biopsy for concomitant identification of the cutaneous virome and underlying IEI. Skin biopsies were obtained from normal and lesional skin from patients with cutaneous infections suspected to be of viral origin. RNA-Seq was utilized as the first-tier strategy for unbiased human genome-wide rare variant detection. Reads unaligned to the human genome were utilized for the exploration of 926 different viruses in a viral genome catalog. In nine families studied, the patients carried pathogenic variants in six human IEI genes, including IL2RG, WAS, CIB1, STK4, GATA2, and DOCK8. Gene expression profiling also confirmed pathogenicity of the human variants and permitted genome-wide homozygosity mapping which assisted in identification of candidate genes in consanguineous families. This automated, all-in-one computational pipeline, called VirPy, enables simultaneous detection of the viral triggers and the human genetic variants underlying skin lesions in patients with suspicion of IEI and viral dermatosis.
Amir Hossein Saeidian, Leila Youssefian, Charles Y. Huang, Fahimeh Palizban, Mahtab Naji, Zahra Saffarian, Hamidreza Mahmoudi, Azadeh Goodarzi, Soheila Sotoudeh, Fatemeh Vahidnezhad, Maliheh Amani, Narjes Tavakoli, Ali Ajami, Samaneh Mozafarpoor, Mehrdad Teimoorian, Saeed Dorgaleleh, Sima Shokri, Mohammad Shenagari, Nima Abedi, Sirous Zeinali, Paolo Fortina, Vivien Béziat, Emmanuelle Jouanguy, Jean-Laurent Casanova, Jouni Uitto, Hassan Vahidnezhad
Spinocerebellar ataxia type1 (SCA1) is an adult-onset neurodegenerative disorder. As disease progresses motor neurons are affected, and their dysfunction contributes towards the inability to maintain proper respiratory function, a major driving force for premature death in SCA1. To investigate the isolated role of motor neurons in SCA1 we created a novel conditional SCA1 (cSCA1) mouse model. This model suppresses expression of the pathogenic SCA1 allele with a floxed stop cassette. cSCA1 mice crossed to a ubiquitous Cre line recapitulate all the major features of the original SCA1 mouse model, except they took twice as long to develop. We found that the cSCA1 mice produce less than half of the pathogenic protein compared to the unmodified SCA1 mice at 3 weeks of age. In contrast, restricted expression of the pathogenic SCA1 allele in motor neurons only leads to a decreased distance traveled of mice in the open field assay and did not affect body weight or survival. We conclude that a fifty percent or greater reduction of the mutant protein has a dramatic effect on disease onset and progression, and that expression of polyglutamine expanded ATXN1 at this level specifically in motor neurons is not sufficient to cause premature lethality.
James P. Orengo, Larissa Nitschke, Meike E. van der Heijden, Nicholas A. Ciaburri, Harry T. Orr, Huda Y. Zoghbi
Mosaic loss of chromosome Y (mLOY) in blood cells is one of the most frequent chromosome alterations in adult males. It is strongly associated with clonal hematopoiesis, hematopoietic malignancies, and other hematopoietic and nonhematopoietic diseases. However, whether there is a causal relationship between mLOY and human diseases is unknown. Here, we generated mLOY in murine hematopoietic stem and progenitor cells (HSPCs) with CRISPR/Cas9 genome editing. We found that mLOY led to dramatically increased DNA damage in HSPCs. Interestingly, HSPCs with mLOY displayed significantly enhanced reconstitution capacity and gave rise to clonal hematopoiesis in vivo. mLOY, which is associated with AML1-ETO translocation and p53 defects in patients with acute myeloid leukemia (AML), promoted AML in mice. Mechanistically, loss of KDM5D, a chromosome Y–specific histone 3 lysine 4 demethylase in both humans and mice, partially recapitulated mLOY in DNA damage and leukemogenesis. Thus, our study validates mLOY as a functional driver for clonal hematopoiesis and leukemogenesis.
Qi Zhang, Lei Zhao, Yi Yang, Shujun Li, Yu Liu, Chong Chen
The fibrous annulus of the mitral valve plays an important role in valvular function and cardiac physiology, while normal variation in the size of cardiovascular anatomy may share a genetic link with common and rare disease. We derived automated estimates of mitral valve annular diameter in the 4-chamber view from 32,220 MRI images from the UK Biobank at ventricular systole and diastole as the basis for GWAS. Mitral annular dimensions corresponded to previously described anatomical norms, and GWAS inclusive of 4 population strata identified 10 loci, including possibly novel loci (GOSR2, ERBB4, MCTP2, MCPH1) and genes related to cardiac contractility (BAG3, TTN, RBFOX1). ATAC-Seq of primary mitral valve tissue localized multiple variants to regions of open chromatin in biologically relevant cell types and rs17608766 to an algorithmically predicted enhancer element in GOSR2. We observed strong genetic correlation with measures of contractility and mitral valve disease and clinical correlations with heart failure, cerebrovascular disease, and ventricular arrhythmias. Polygenic scoring of mitral valve annular diameter in systole was predictive of risk mitral valve prolapse across 4 cohorts. In summary, genetic and clinical studies of mitral valve annular diameter revealed genetic determinants of mitral valve biology, while highlighting clinical associations. Polygenic determinants of mitral valve annular diameter may represent an independent risk factor for mitral prolapse. Overall, computationally estimated phenotypes derived at scale from medical imaging represent an important substrate for genetic discovery and clinical risk prediction.
Mengyao Yu, Catherine Tcheandjieu, Adrien Georges, Ke Xiao, Helio Tejeda, Christian Dina, Thierry Le Tourneau, Madalina Fiterau, Renae Judy, Noah L. Tsao, Dulguun Amgalan, Chad J. Munger, Jesse M. Engreitz, Scott M. Damrauer, Nabila Bouatia-Naji, James R. Priest
Primary ovarian insufficiency (POI) affects 1% of women and carries significant medical and psychosocial sequelae. Approximately 10% of POI has a defined genetic cause, with most implicated genes relating to biological processes involved in early fetal ovary development and function. Recently, Ythdc2, an RNA helicase and N6-methyladenosine (m6a) reader, has emerged as a novel regulator of meiosis in mice. Here, we describe homozygous pathogenic variants in YTHDC2 in three women with early-onset POI from two families: c. 2567C>G, p.P856R in the helicase-associated (HA2) domain; and c.1129G>T, p.E377*. We demonstrate that YTHDC2 is expressed in the developing human fetal ovary and is upregulated in meiotic germ cells, together with related meiosis-associated factors. The p.P856R variant results in a less flexible protein that likely disrupts downstream conformational kinetics of the HA2 domain, whereas the p.E377* variant truncates the helicase core. Taken together, our results reveal that YTHDC2 is a key new regulator of meiosis in humans and pathogenic variants within this gene are associated with POI.
Sinead M. McGlacken-Byrne, Ignacio del Valle, Polona Le Quesne Stabej, Laura Bellutti, Luz Garcia-Alonso, Louise A. Ocaka, Miho Ishida, Jenifer P. Suntharalingham, Andrey Gagunashvili, Olumide K. Ogunbiyi, Talisa Mistry, Federica Buonocore, GOSgene, Berta Crespo, Nadjeda Moreno, Paola Niola, Tony Brooks, Caroline E. Brain, Mehul T. Dattani, Daniel Kelberman, Roser Vento-Tormo, Carlos F. Lagos, Gabriel Livera, Gerard S. Conway, John C. Achermann
Hundreds of genetic variants in KCNQ2 encoding the voltage-gated potassium channel KV7.2 are associated with early onset epilepsy and/or developmental disability, but the functional consequences of most variants are unknown. Absent functional annotation for KCNQ2 variants hinders identification of individuals who may benefit from emerging precision therapies. We employed automated patch clamp recording to assess at an unprecedented scale the functional and pharmacological properties of 79 missense and 2 inframe deletion KCNQ2 variants. Among the variants we studied were 18 known pathogenic variants, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. We analyzed electrophysiological data recorded from 9,480 cells. The functional properties of 18 known pathogenic variants largely matched previously published results and validated automated patch clamp for this purpose. Unlike rare population variants, most disease-associated KCNQ2 variants exhibited prominent loss-of-function with dominant-negative effects, providing strong evidence in support of pathogenicity. All variants responded to retigabine, although there were substantial differences in maximal responses. Our study demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine, a proposed precision therapy for KCNQ2 developmental and epileptic encephalopathy.
Carlos G. Vanoye, Reshma R. Desai, Zhigang Ji, Sneha Adusumilli, Nirvani Jairam, Nora Ghabra, Nishtha Joshi, Eryn Fitch, Katherine L. Helbig, Dianalee McKnight, Amanda S. Lindy, Fanggeng Zou, Ingo Helbig, Edward C. Cooper, Alfred L. George Jr.
BACKGROUND. Prostate cancer is multifocal with distinct molecular subtypes. The utility of genomic subtyping has been challenged due to inter- and intra-focal heterogeneity. We sought to characterize the subtype-defining molecular alterations of primary prostate cancer across all tumor foci within radical prostatectomy (RP) specimens and determine the prevalence of collision tumors. METHODS. From the Early Detection Research Network cohort, we identified 333 prospectively collected RPs from 2010 to 2014 and assessed ERG, SPINK1, PTEN, and SPOP molecular status. We utilized dual ERG/SPINK1 immunohistochemistry, fluorescence in situ hybridization to confirm ERG rearrangements and characterize PTEN deletion, and high-resolution melting curve analysis and Sanger sequencing to determine SPOP mutation status. Analysis of biochemical recurrence-free of patients with collision tumors was conducted using Kaplan-Meier method. RESULTS. Based on index focus alone, ERG, SPINK1, PTEN, and SPOP alterations were identified in 47.5%, 10.8%, 14.3%, and 5.1% of RP specimens, respectively. In 233 multifocal RPs with ERG/SPINK1 status in all foci, 139 (59.7%) had discordant molecular alterations between foci. Collision tumors, as defined by discrepant ERG/SPINK1 status within a single focus, were identified in 29 (9.4%) RP specimens. CONCLUSION. Interfocal molecular heterogeneity was identified in ~60% of multifocal RP specimens and collision tumors were present in ~10%. We present this phenomenon as a model for the intra-focal heterogeneity observed in previous studies and propose future genomic studies screen for collision tumors to better characterize molecular heterogeneity.
Jacqueline Fontugne, Peter Y. Cai, Hussein Alnajar, Bhavneet Bhinder, Kyung Park, Huihui Ye, Shaham Beg, Verena Sailer, Javed Siddiqui, Mirjam Blattner-Johnson, Jaclyn A. Croyle, Zohal Noorzad, Carla Calagua, Theresa Y. MacDonald, Ulrika Axcrona, Mari Bogaard, Karol Axcrona, Douglas S. Scherr, Martin G. Sanda, Bjarne Johannessen, Arul M. Chinnaiyan, Olivier Elemento, Rolf I. Skotheim, Mark A. Rubin, Christopher E. Barbieri, Juan M. Mosquera
Defective primary cilia cause a range of diseases called ciliopathies, which include hearing loss (HL). Variants in human oxysterol binding protein like 2 (OSBPL2/ORP2) are responsible for autosomal dominant nonsyndromic HL (DFNA67). However, the pathogenesis of OSBPL2 deficiency has not been fully elucidated. In this study, we showed that the Osbpl2-knockout (KO) mice exhibited progressive HL and abnormal cochlea development with defective cilia. Further research revealed that OSBPL2 was located at the base of kinocilia in hair cells (HCs) and primary cilia in supporting cells (SCs), and functioned in the maintenance of ciliogenesis by regulating the homeostasis of PI(4,5)P2 on the cilia membrane. OSBPL2 deficiency led to a significant increase of PI(4,5)P2 on the cilia membrane, which could be partially rescued by the overexpression of INPP5E. In addition, the key molecules in Sonic Hedgehog (Shh) signaling pathway (SMO and GLI3) were detected to be down-regulated in Osbpl2-KO HEI-OC1 cells. Our findings revealed that OSBPL2 deficiency resulted in ciliary defects and abnormal Shh signaling transduction in auditory cells, which helped to elucidate the underlying mechanism of OSBPL2 deficiency in HL.
Hairong Shi, Hongshun Wang, Cheng Zhang, Yajie Lu, Jun Yao, Zhibin Chen, Guangqian Xing, Qinjun Wei, Xin Cao
Myosin heavy chain 7 (MYH7) is a major causative gene for hypertrophic cardiomyopathy, but the affected signaling pathways and therapeutics remain elusive. In this research, we identified ventricle myosin heavy chain like (vmhcl) as a zebrafish homolog of human MYH7, and we generated vmhcl frameshift mutants. We noted vmhcl-based embryonic cardiac dysfunction (VEC) in the vmhcl homozygous mutants and vmhcl-based adult cardiomyopathy (VAC) phenotypes in the vmhcl heterozygous mutants. Using the VEC model, we assessed 7 known cardiomyopathy signaling pathways pharmacologically and 11 candidate genes genetically via CRISPR/Cas9 genome editing technology based on microhomology-mediated end joining (MMEJ). Both studies converged on therapeutic benefits of mTOR or mitogen-activated protein kinase (MAPK) inhibition of VEC. While mTOR inhibition rescued the enlarged nuclear size of cardiomyocytes, MAPK inhibition restored the prolonged cell shape in the VEC model. The therapeutic effects of mTOR and MAPK inhibition were later validated in the VAC model. Together, vmhcl/myh7 loss of function is sufficient to induce cardiomyopathy in zebrafish. The VEC and VAC models in zebrafish are amenable to both efficient genetic and chemical genetic tools, offering a rapid in vivo platform for discovering candidate signaling pathways of MYH7 cardiomyopathy.
Haisong Bu, Yonghe Ding, Jiarong Li, Ping Zhu, Yu-Huan Shih, Mingmin Wang, Yuji Zhang, Xueying Lin, Xiaolei Xu
BACKGROUND. >1,500 variants in the ABCA4 locus underlie a heterogeneous spectrum of retinal disorders ranging from aggressive childhood-onset chorioretinopathy to milder, late-onset macular disease. Genotype-phenotype correlation studies have been limited in clinical applicability as patient cohorts are typically small and seldom capture the full natural history of individual genotypes. To overcome these limitations, we constructed a genotype-phenotype correlation matrix that provides quantifiable probabilities of long-term disease outcomes associated with specific ABCA4 genotypes from a large, age-restricted patient cohort. METHODS. The study included 112 unrelated patients ≥50 years of age in whom 2 pathogenic variants were identified after sequencing of the ABCA4 locus. Clinical characterization was performed using the results of best-corrected visual acuity, retinal imaging and full-field electroretinogram testing. RESULTS. Four distinct prognostic groups were defined according to the spatial severity of disease features across the fundus. Recurring genotypes were observed in milder prognoses including those associated with a newly defined class of rare hypomorphic alleles. PVS1 (predicted null) variants were enriched in the most severe prognoses; however, missense variants comprised a larger than expected fraction of these patients. Analysis of allele combinations and their respective prognostic severity, showed that certain variants such as p.(Gly1961Glu), and both rare and frequent hypomorphic alleles, are “clinically dominant” with respect to patient phenotypes irrespective of the allele in trans. CONCLUSION. These results provide much needed structure to the complex genetic and clinical landscape of ABCA4 disease and adds a tool to the clinical repertoire to quantitatively assess individual genotype-specific prognoses in patients.
Winston Lee, Jana Zernant, Pei-Yin Su, Takayuki Nagasaki, Stephen H. Tsang, Rando Allikmets
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