Genetic factors undoubtedly affect the development of congenital heart disease (CHD), but still remain ill-defined. We sought to identify genetic risk factors associated with CHD and to accomplish functional analysis of single nucleotide polymorphisms (SNP)-carrying genes. We performed a genome-wide association study of 4,034 Caucasian CHD patients and 8,486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified four highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence expression of WNT3, and variant rs870142 related to septal defects is proposed to influence expression of MSX1. The expression of all four genes was analyzed during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNAseq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3 and MSX1 play an essential functional role in heart development at the embryonic and newborn stage.
Harald Lahm, Meiwen Jia, Martina Dreßen, Felix F. M. Wirth, Nazan Puluca, Ralf Gilsbach, Bernard Keavney, Julie Cleuziou, Nicole Beck, Olga Bondareva, Elda Dzilic, Melchior Burri, Karl C. König, Johannes A. Ziegelmüller, Claudia Abou-Ajram, Irina Neb, Zhong Zhang, Stefanie A. Doppler, Elisa Mastantuono, Peter Lichtner, Gertrud Eckstein, Jürgen Hörer, Peter Ewert, James R. Priest, Lutz Hein, Rüdiger Lange, Thomas Meitinger, Heather J. Cordell, Bertram Müller-Myhsok, Markus Krane
Neonatal diabetes is caused by single gene mutations reducing pancreatic β cell number or impairing β cell function. Understanding the genetic basis of rare diabetes subtypes highlights fundamental biological processes in β cells. We identified 6 patients from 5 families with homozygous mutations in the YIPF5 gene, which is involved in trafficking between the endoplasmic reticulum (ER) and the Golgi. All patients had neonatal/early-onset diabetes, severe microcephaly, and epilepsy. YIPF5 is expressed during human brain development, in adult brain and pancreatic islets. We used 3 human β cell models (YIPF5 silencing in EndoC-βH1 cells, YIPF5 knockout and mutation knockin in embryonic stem cells, and patient-derived induced pluripotent stem cells) to investigate the mechanism through which YIPF5 loss of function affects β cells. Loss of YIPF5 function in stem cell–derived islet cells resulted in proinsulin retention in the ER, marked ER stress, and β cell failure. Partial YIPF5 silencing in EndoC-βH1 cells and a patient mutation in stem cells increased the β cell sensitivity to ER stress–induced apoptosis. We report recessive YIPF5 mutations as the genetic cause of a congenital syndrome of microcephaly, epilepsy, and neonatal/early-onset diabetes, highlighting a critical role of YIPF5 in β cells and neurons. We believe this is the first report of mutations disrupting the ER-to-Golgi trafficking, resulting in diabetes.
Elisa De Franco, Maria Lytrivi, Hazem Ibrahim, Hossam Montaser, Matthew N. Wakeling, Federica Fantuzzi, Kashyap Patel, Céline Demarez, Ying Cai, Mariana Igoillo-Esteve, Cristina Cosentino, Väinö Lithovius, Helena Vihinen, Eija Jokitalo, Thomas W. Laver, Matthew B. Johnson, Toshiaki Sawatani, Hadis Shakeri, Nathalie Pachera, Belma Haliloglu, Mehmet Nuri Ozbek, Edip Unal, Ruken Yıldırım, Tushar Godbole, Melek Yildiz, Banu Aydin, Angeline Bilheu, Ikuo Suzuki, Sarah E. Flanagan, Pierre Vanderhaeghen, Valérie Senée, Cécile Julier, Piero Marchetti, Decio L. Eizirik, Sian Ellard, Jonna Saarimäki-Vire, Timo Otonkoski, Miriam Cnop, Andrew T. Hattersley
Angiosarcomas are rare, clinically aggressive tumors with limited treatment options and a dismal prognosis. We analyzed angiosarcomas from 68 patients, integrating information from multiomic sequencing, NanoString immuno-oncology profiling, and multiplex immunohistochemistry and immunofluorescence for tumor-infiltrating immune cells. Through whole-genome sequencing (n = 18), 50% of the cutaneous head and neck angiosarcomas exhibited higher tumor mutation burden (TMB) and UV mutational signatures; others were mutationally quiet and non–UV driven. NanoString profiling revealed 3 distinct patient clusters represented by lack (clusters 1 and 2) or enrichment (cluster 3) of immune-related signaling and immune cells. Neutrophils (CD15+), macrophages (CD68+), cytotoxic T cells (CD8+), Tregs (FOXP3+), and PD-L1+ cells were enriched in cluster 3 relative to clusters 2 and 1. Likewise, tumor inflammation signature (TIS) scores were highest in cluster 3 (7.54 vs. 6.71 vs. 5.75, respectively; P < 0.0001). Head and neck angiosarcomas were predominant in clusters 1 and 3, providing the rationale for checkpoint immunotherapy, especially in the latter subgroup with both high TMB and TIS scores. Cluster 2 was enriched for secondary angiosarcomas and exhibited higher expression of DNMT1, BRD3/4, MYC, HRAS, and PDGFRB, in keeping with the upregulation of epigenetic and oncogenic signaling pathways amenable to targeted therapies. Molecular and immunological dissection of angiosarcomas may provide insights into opportunities for precision medicine.
Jason Yongsheng Chan, Jing Quan Lim, Joe Yeong, Vinod Ravi, Peiyong Guan, Arnoud Boot, Timothy Kwang Yong Tay, Sathiyamoorthy Selvarajan, Nur Diyana Md Nasir, Jie Hua Loh, Choon Kiat Ong, Dachuan Huang, Jing Tan, Zhimei Li, Cedric Chuan-Young Ng, Thuan Tong Tan, Mikio Masuzawa, Ken Wing-Kin Sung, Mohamad Farid, Richard Hong Hui Quek, Ngian Chye Tan, Melissa Ching Ching Teo, Steven George Rozen, Patrick Tan, Andrew Futreal, Bin Tean Teh, Khee Chee Soo
BACKGROUND. Transcriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq guided method to diagnose individuals across a wide range of ages and clinical phenotypes. METHODS. One hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network (UDN) clinical site from 2014-2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis. RESULTS. The transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts. CONCLUSION. For our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA.
David R. Murdock, Hongzheng Dai, Lindsay C. Burrage, Jill A. Rosenfeld, Shamika Ketkar, Michaela F. Müller, Vicente A. Yépez, Julien Gagneur, Pengfei Liu, Shan Chen, Mahim Jain, Gladys Zapata, Carlos A. Bacino, Hsiao-Tuan Chao, Paolo Moretti, William J. Craigen, Neil A. Hanchard, Brendan Lee
Germ cell tumors (GCTs) are the most common cancer in men between the ages of 15-40. While most patients are cured, those with disease arising in the mediastinum have distinctly poor outcomes. One in every 17 patients with primary mediastinal non-seminomatous GCTs develop an incurable hematologic malignancy and prior data intriguingly suggests a clonal relationship exists between hematologic malignancies and GCTs in these cases. To date however, the precise clonal relationship between GCTs and the diverse additional somatic malignancies arising in such individuals has not been determined. Here, we traced the clonal evolution and characterized the genetic features of each neoplasm from a cohort of fifteen patients with GCTs and associated hematologic malignancies. We discovered that GCTs and hematologic malignancies developing in such individuals evolved from a common shared precursor, nearly all of which harbored allelically imbalanced TP53 and/or RAS pathway mutations. Hematologic malignancies arising in this setting genetically resembled mediastinal GCTs rather than de novo myeloid neoplasms. Our findings argue that this scenario represents a unique clinical syndrome, distinct from de novo GCTs or hematologic malignancies, initiated by an ancestral precursor which gives rise to the parallel evolution of GCTs and blood cancers in these patients.
Justin Taylor, Mark T.A. Donoghue, Caleb Ho, Kseniya Petrova-Drus, Hikmat A. Al-Ahmadie, Samuel A. Funt, Yanming Zhang, Umut Aypar, Pavitra Nagesh Rao, Shweta S. Chavan, Michael Haddadin, Roni Tamari, Sergio Giralt, Martin S. Tallman, Raajit K. Rampal, Priscilla Baez, Rajya Kappagantula, Satyajit Kosuri, Ahmet Dogan, Satish K. Tickoo, Victor E. Reuter, George J. Bosl, Christine A. Iacobuzio-Donahue, David B. Solit, Barry S. Taylor, Darren R. Feldman, Omar Abdel-Wahab
BACKGROUND. Current methods for the detection and surveillance of bladder cancer (BCa) are often invasive and/or possess suboptimal sensitivity and specificity, especially in early stage, minimal, residual tumors. METHODS. We developed a novel method for the detection of urine tumor DNA Methylation at multiple genomic regions by Mass Array, termed utMeMA. We identified the BCa-specific methylation markers by combined analyses of Sun Yat-sen Memorial Hospital (SYSMH), TCGA and GEO cohorts. The BCa diagnostic model was built in a retrospective cohort (n=313) and validated in a multicenter, prospective cohort (n=175). The performance of this diagnostic assay was analyzed and compared with urine cytology and FISH. RESULTS. We first discovered 26 significant methylation markers of BCa in combined analyses. We build and validate a two-marker-based diagnostic model that discriminated patients with BCa with high accuracy (86.7%), sensitivity (90.0%) and specificity (83.1%). Furthermore, utMeMA based assay achieved a great improvement in sensitivity over urine cytology and FISH, especially in the detection of early stage (Ta and low grade tumor, 64.5% vs. 11.8%, 15.8%), minimal (81.0% vs. 14.8%, 37.9%), residual (93.3% vs. 27.3%, 64.3%) and recurrent (89.5% vs. 31.4%, 52.8%) tumors. The urine diagnostic score (UD-score) from this assay was better associated with tumor malignancy and burden. CONCLUSIONS. Urine tumor DNA methylation assessment for early diagnosis, minimal, residual tumor detection and surveillance in bladder cancer is a rapid, high-throughput, non-invasive and promising approach, which may reduce the burden of cystoscopy and blind second surgery.
Xu Chen, Jingtong Zhang, Weimei Ruan, Ming Huang, Chanjuan Wang, Hong Wang, Zeyu Jiang, Shaogang Wang, Zheng Liu, Chunxiao Liu, Wanlong Tan, Jin Yang, Jiaxin Chen, Zhiwei Chen, Xia Li, Xiaoyu Zhang, Peng Xu, Lin Chen, Ruihui Xie, Qianghua Zhou, Shizhong Xu, Darryl Irwin, JIAN-BING FAN, Jian Huang, Tianxin Lin
Gain-of-function mutations in the WNK1 and WNK4 genes are responsible for Familial Hyperkalemic Hypertension (FHHt), a rare inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the KLHL3-CUL3 E3 ubiquitin ligase complex have shed light on the importance of WNKs cellular degradation on renal ion transport. Using full exome sequencing in a four-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants at the WNK1 gene, clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early-onset and a marked hyperkalemic phenotype, but normal blood pressure values. Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex, rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR-Cas9 engineered mouse model recapitulated both the clinical and biological phenotype. Renal investigations showed increased activation of the SPAK-NCC phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Altogether, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
Helene Louis-Dit-Picard, Ilektra Kouranti, Chloe Rafael, Irmine Loisel-Ferreira, Maria Chavez-Canales, Waed Abdel Khalek, Eduardo Argaiz, Stephanie Baron, Sarah Vacle, Tiffany Migeon, Richard Coleman, Marcio Do Cruzeiro, Marguerite Hureaux, Nirubiah Thurairajasingam, Stéphane Decramer, Xavier Girerd, Kevin M. O'Shaughnessy, Paolo Mulatero, Gwenaelle Roussey, Ivan Tack, Robert J. Unwin, Rosa Vargas-Poussou, Olivier Staub, P. Richard Grimm, Paul A. Welling, Gerardo Gamba, Eric Clauser, Juliette Hadchouel, Xavier Jeunemaitre
Males and females differ in body composition and fat distribution. Using a mouse model that segregates gonadal sex (ovaries and testes) from chromosomal sex (XX and XY), we showed that XX chromosome complement in combination with a high-fat diet led to enhanced weight gain in the presence of male or female gonads. We identified the genomic dosage of Kdm5c, an X chromosome gene that escapes X-chromosome inactivation, as a determinant of the X chromosome effect on adiposity. Modulating Kdm5c gene dosage in XX female mice to levels that are normally present in males reduced body weight, fat content, and food intake to a similar degree as altering the entire X chromosome dosage. In cultured preadipocytes, the levels of KDM5C histone demethylase influenced chromatin accessibility (ATAC-seq), gene expression (RNA-seq), and adipocyte differentiation. Both in vitro and in vivo, Kdm5c dosage influenced gene expression involved in extracellular matrix remodeling, which is critical for adipocyte differentiation and adipose tissue expansion. In humans, adipose tissue KDM5C mRNA levels and KDM5C genetic variants were associated with body mass. These studies demonstrate that the sex-dependent dosage of Kdm5c contributes to male/female differences in adipocyte biology, and highlight X-escape genes as a critical component of female physiology.
Jenny C. Link, Carrie B. Wiese, Xuqi Chen, Rozeta Avetisyan, Emilio Ronquillo, Feiyang Ma, Xiuqing Guo, Jie Yao, Matthew Allison, Yii-Der I. Chen, Jerome I. Rotter, Julia S. El-Sayed Moustafa, Kerrin S. Small, Shigeki Iwase, Matteo Pellegrini, Laurent Vergnes, Arthur P. Arnold, Karen Reue
Nearly all breast cancer deaths result from metastatic disease. Despite this, the genomic events that drive metastatic recurrence are poorly understood. We performed whole-exome and shallow whole-genome sequencing to identify genes and pathways preferentially mutated or copy-number altered in metastases compared with the paired primary tumors from which they arose. Seven genes were preferentially mutated in metastases — MYLK, PEAK1, SLC2A4RG, EVC2, XIRP2, PALB2, and ESR1 — 5 of which are not significantly mutated in any type of human primary cancer. Four regions were preferentially copy-number altered: loss of STK11 and CDKN2A/B, as well as gain of PTK6 and the membrane-bound progesterone receptor, PAQR8. PAQR8 gain was mutually exclusive with mutations in the nuclear estrogen and progesterone receptors, suggesting a role in treatment resistance. Several pathways were preferentially mutated or altered in metastases, including mTOR, CDK/RB, cAMP/PKA, WNT, HKMT, and focal adhesion. Immunohistochemical analyses revealed that metastases preferentially inactivate pRB, upregulate the mTORC1 and WNT signaling pathways, and exhibit nuclear localization of activated PKA. Our findings identify multiple therapeutic targets in metastatic recurrence that are not significantly mutated in primary cancers, implicate membrane progesterone signaling and nuclear PKA in metastatic recurrence, and provide genomic bases for the efficacy of mTORC1, CDK4/6, and PARP inhibitors in metastatic breast cancer.
Matt R. Paul, Tien-chi Pan, Dhruv K. Pant, Natalie N.C. Shih, Yan Chen, Kyra L. Harvey, Aaron Solomon, David Lieberman, Jennifer J.D. Morrissette, Danielle Soucier-Ernst, Noah G. Goodman, S. William Stavropoulos, Kara N. Maxwell, Candace Clark, George K. Belka, Michael Feldman, Angela DeMichele, Lewis A. Chodosh
The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur cluster (Fe-S) biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial co-chaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent two-iron, two-sulfur ([2Fe-2S]) clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in red blood cell hemoglobinization, the development of siderocytes, and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.
Andrew Crispin, Chaoshe Guo, Caiyong Chen, Dean R. Campagna, Paul J. Schmidt, Daniel A. Lichtenstein, Chang Cao, Anoop K. Sendamarai, Gordon J. Hildick-Smith, Nicholas C. Huston, Jeanne Boudreaux, Sylvia S. Bottomley, Matthew M. Heeney, Barry H. Paw, Mark D. Fleming, Sarah Ducamp