Genetics
Abstract
The role of the epigenome in age-related neurodegenerative disorders remains understudied. Here, we analyzed circulating cell-free DNA (cfDNA) from blood to detect methylation changes as a liquid biopsy for Amyotrophic Lateral Sclerosis (ALS). Our study included 20 patients with sporadic ALS, 10 patients with C9orf72-associated ALS, 10 asymptomatic carriers of the C9orf72 repeat expansion mutation, and 21 nondisease control individuals. Following targeted enzymatic methyl-sequencing (EM-seq) of approximately 4 million CpG sites, we detected numerous differentially methylated genes, including several implicated in ALS disease risk and pathogenesis. By integrating multiple epigenetic features, we delineated a distinct epigenetic signature, which achieved an average area under the curve (AUC) of 0.91 ± 0.10 upon receiver operator characteristic (ROC) analysis, which enabled detection of approximately 70% of patients with ALS with close to 100% specificity. Furthermore, we also identified a set of genes whose methylation status significantly correlated with clinical disease progression and cerebrospinal fluid (CSF) neurofilament levels. Our results reveal the potential of cfDNA-based biomarkers to accurately diagnose ALS and potentially predict disease progression.
Authors
Sebastian Michels, Chaorong Chen, Wolfgang P. Ruf, M. Madhy Garcia Garcia, Frederick J. Arnold, Zhuoxing Wu, Craig L. Bennett, Daniel Shams, Leslie M. Thompson, Alyssa C. Walker, Dennis W. Dickson, Leonard Petrucelli, Johannes Dorst, Mercedes Prudencio, Wei Li, Albert R. La Spada
Abstract
Dilated cardiomyopathy (DCM) is a genetically heterogeneous disorder, characterized by ventricular dilatation and impaired systolic function, leading to heart failure and sudden cardiac death. Despite advances in genomic technologies, the genetic cause of DCM remains unidentified in more than half of the cases. Here, we performed an integrative analysis of genomic and transcriptomic data from patient-derived cardiac tissue to identify causative variants in genetically undiagnosed DCM. This approach enabled us to identify a homozygous splice-site variant (c.243+6T>A) in the sarcoglycan gene SGCB, which results in exon 2 skipping. This variant was significantly enriched in patients with DCM compared with the general population, with consistent genotype–phenotype correlations observed across multiple families. Protein-level analysis of cardiac tissue from homozygous individuals revealed loss of β-sarcoglycan, the protein product of SGCB, and destabilization of the sarcoglycan complex. Although SGCB has been previously associated with limb-girdle muscular dystrophy, these homozygous individuals showed no biochemical or clinical signs of skeletal muscle involvement, indicating an absence of myopathy. Compared with variant-negative patients with DCM, homozygous individuals also had a higher risk of early-onset adverse cardiac events. Together, these findings identify c.243+6T>A in SGCB as a cause of isolated DCM associated with unfavorable clinical outcomes.
Authors
Fangfang Li, Haruki Shinomiya, Yuki Kuramoto, Koshiro Kanaoka, Yuji Sakahashi, Yasuki Ishihara, Hidetaka Kioka, Seiko Ide, Yumi Yamaguchi-Kabata, Shu Tadaka, Ikuko N. Motoike, Kengo Kinoshita, Kinuko Ohneda, Hidetoshi Sakurai, Takahiro Okumura, Yohei Miyashita, Kota Jojima, Hisakazu Kato, Ken Matsuoka, Kazuya Tanabe, Shunsuke Nishimura, Seiji Takashima, Yoshihiro Asano, Yasushi Sakata
Abstract
BACKGROUND. Minimally invasive biomarkers predicting immunotherapy response in head and neck squamous cell carcinoma (HNSCC) remain an unmet clinical need. METHODS. Using patients from a prospective, multi-institutional phase II trial, we performed whole-genome sequencing of 185 longitudinal plasma cell-free DNA (cfDNA) samples from 68 patients with locally advanced, surgically resectable HNSCC who received neoadjuvant and adjuvant pembrolizumab. We developed the regional motif diversity score (rMDS), a fragmentomic metric that quantifies the entropy of cfDNA 5′-end motifs across genomic regions. RESULTS. Unsupervised analysis showed rMDS robustly distinguished responders from non-responders, outperforming established fragmentomic metrics and copy number alterations while remaining independent of technical confounders. Longitudinal rMDS changes localized to regions enriched for immune-, lectin-, and keratinization-related genes — hallmarks of squamous cell carcinoma — reflecting tumor–peripheral immunity interplay during treatment. The most dynamic regions clustered at telomere-proximal loci, suggesting a link between telomere biology and cfDNA fragmentation. An rMDS-based machine learning classifier achieved AUC 0.89–0.99 across validation settings, with the highest accuracy post-treatment, outperforming PD-L1 expression and tumor fraction in matched samples. Predicted responders showed improved disease-free survival (log-rank P = 0.035; HR 2.67, 95% CI 1.03–6.92). CONCLUSION. rMDS represents a biologically meaningful, clinically actionable biomarker for immunotherapy response in HNSCC, supporting integration into future risk assessment frameworks. TRIAL REGISTRATION. ClinicalTrials.gov NCT02641093. FUNDING. NHGRI R56HG012360 and startup funds from Cincinnati Children’s Hospital Medical Center, Northwestern University, and Robert H. Lurie Comprehensive Cancer Center (Y.L.); Science Olympiad Alumni Research Grant, Science Olympiad USA Foundation (R.B.); Merck Sharp & Dohme Corp. (T.W.D.).
Authors
Ravi Bandaru, Hailu Fu, Haizi Zheng, Jocelyn Liang, Li Wang, Shuchi Gulati, Benjamin H. Hinrichs, Mingxiang Teng, Bin Zhang, Masha Kocherginsky, De-Chen Lin, David A. Hildeman, Francis P. Worden, Matthew O. Old, Neal E. Dunlap, John M. Kaczmar, Maura L. Gillison, Dalia El-Gamal, Trisha Wise Draper, Yaping Liu
Abstract
Authors
Fumihiko Urano, Bess A. Marshall, Stacy Hurst, Amy Robichaux-Viehoever, Saumel Ahmadi, Tamara Hershey, Gregory Van Stavern, Paulina Cruz Bravo, Jennifer Powers Carson, John Pesko, Kelly Fox, Nathalie Erpelding, Camille L. Bedrosian
Abstract
While radiation is an effective oncologic therapy, killing cancer by inducing DNA double-strand breaks (DSBs), it lacks specificity for neoplastic cells. We have previously adapted the CRISPR/Cas9 gene-editing technology as a cancer-specific treatment modality targeting somatic mutations in pancreatic cancer (PC). However, its tumoricidal potential remains unclear, especially in comparison with therapeutic doses of radiation. Here, we demonstrate that CRISPR/Cas9-induced DSBs are more cytotoxic in PCs than a comparable number of radiation-induced DSBs. We observed more than 90% tumor growth inhibition by targeting 9 sites with cancer-specific sgRNAs. Through both bioinformatics and cytogenetics analyses, we found that CRISPR/Cas9-induced DSBs triggered ongoing chromosomal rearrangements, with 87% of structural variants not directly produced from the initial CRISPR/Cas9-induced DSBs, and chromosomal instability peaking before cell death. By comparing the cytotoxicity of CRISPR/Cas9- and radiation-induced DSBs, we demonstrated that the number of DSBs required to achieve equitoxic effects was approximately 3 times higher for radiation than CRISPR/Cas9. Finally, we showed that PC cells that had survived CRISPR/Cas9 targeting retained susceptibility to subsequent CRISPR/Cas9-induced DSBs at different genomic sites with more than 87% growth inhibition. Together, our data support the therapeutic potential of CRISPR/Cas9 as an anticancer strategy.
Authors
Selina Shiqing K. Teh, Akhil Kotwal, Alexis Bennett, Eitan Halper-Stromberg, Laura Morsberger, Saum Zamani, Yanan Shi, Alyza Skaist, Qingfeng Zhu, Kirsten Bowland, Hong Liang, Ralph H. Hruban, Chien-Fu Hung, Robert A. Anders, Nicholas J. Roberts, Robert B. Scharpf, Michael Goldstein, Ying S. Zou, James R. Eshleman
Abstract
Based on the observation that loss-of-function mutations of KMT2C and KMT2D (KMT2C/D) are enriched and co-occur in gastric adenocarcinoma, we developed genetically engineered mouse model (GEMM) to conditionally knock out Kmt2c and Kmt2d in gastric epithelial cells. We observed that Kmt2c/d loss led to nuclear dysplasia, cellular crowding, and expansion of cells with mixed gastric lineage markers. When combined with Pten deletion, Kmt2c/d loss drove rapid development of muscle-invasive gastric adenocarcinoma as early as 3 weeks post Cre-mediated gene deletion. The adenocarcinoma exhibited decreased expression of gastric lineage markers and increased expression of intestinal differentiation markers, phenocopying human intestinal type gastric adenocarcinoma. Bioinformatic integration of single cell RNA-seq of our GEMMs and human gastric cancer datasets shows co-clustering of normal and of cancerous gastric epithelial cells. Kmt2c/d knockout in gastric epithelium reduced protein synthesis but upregulated transcription of ribosomal proteins, rendering the cells to be hypersensitive to mTORC1 inhibitors. Additionally, Kmt2c/d knockout increased MHC-I molecule expression and enhanced antigen presentation. Combination of mTORC1 inhibition and anti-PD1 immunotherapy markedly suppressed tumor growth in immune-competent mice. Together, these findings reveal the role of Kmt2c/d loss in gastric cancer initiation and suggest the potential therapeutic strategies for KMT2C/D-deficient gastric cancer.
Authors
Naitao Wang, Dan Li, Tao Zhang, Mohini R. Pachai, Dana M. Schoeps, Yudi Bao, Woo Hyun Cho, Makhzuna N. Khudoynazarova, Kae Kristoff, Marion Liu, Laura Tang, Yelena Y. Janjigian, Ping Chi, Yu Chen
Abstract
Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease marked by progressive motor deficits and Purkinje cell (PC) degeneration, driven by polyglutamine expansion in ataxin-1. While oligodendroglial dysfunction precedes PC loss, its direct contribution toward SCA1 pathogenesis remains unclear. Here, using an oligodendroglia-specific SCA1 conditional knock-in mouse model, we demonstrate that mutant ataxin-1 in oligodendrocytes is sufficient to drive aspects of SCA1-related pathology, including dysregulated myelination, PC axonal shrinkage, and torpedo formation, ultimately impairing motor coordination. Transcriptomic analysis uncovers cerebellar oligodendrocyte subtypes with distinct gene expression signatures and aberrant abundance that contribute to demyelination. This, compounded by a progressive decline in the neuroprotective functions of a cerebellar-specific oligodendrocyte subtype, establishes a critical link between demyelination, axo-myelinic dysfunction, and axonal pathology in SCA1. Upstream transcriptional regulator analysis in oligodendroglia identifies TCF7L2 and HTT as key mediators of oligodendroglial dysfunction in SCA1, suggesting shared pathogenic mechanisms with other polyglutamine diseases. Collectively, these findings establish oligodendroglia as key mediators of SCA1 pathogenesis and underscore their critical role in preserving PC axonal integrity.
Authors
Changwoo Lee, Rosalie M. Grijalva, Leon Tejwani, Eunwoo Bae, Alison Chase, Hannah Ro, Hannah Kim, Victor Olmos, James P. Orengo, Janghoo Lim
Abstract
The human epidermal growth factor receptor 2 (HER2) is a major therapeutic target in cancer. While the oncogenic effects of HER2 hyperactivation are well-characterized, the biological consequences of its deficiency remain poorly defined. Here, through exome sequencing analyses of a cohort of 720 families affected by isolated or syndromic orofacial clefts, we unexpectedly identified five distinct rare germline HER2 variants in five unrelated families with growth deficits, orofacial clefts, and other craniofacial, skeletal, and auditory anomalies. In Xenopus embryos, these variants failed to recapitulate the developmental effects of wild-type HER2. In cultured cells, they disrupted HER2 protein stability, membrane localization, or site-specific phosphorylation, resulting in diminished ERK signaling. Strikingly, knock-in mice expressing a patient-derived HER2 variant and mice maternally exposed to Tucatinib, a recently approved anti-HER2 drug, both replicated patient phenotypes: retarded growth and diverse craniofacial abnormalities, including ocular dysgenesis, short jaws, and cleft palate. Collectively, our findings define a developmental disorder that we designate GRACE syndrome (Growth Retardation and Craniofacial Malformations Caused by HER2 Deficiency), establish HER2’s essential role in human growth and craniofacial morphogenesis, and reveal that HER2-targeted therapies during pregnancy can induce craniofacial defects and lifelong growth impairment in fetuses. 5
Authors
Huaxiang Zhao, Pan Wang, Yuhua Jiao, Huimei Huang, Min Yu, Qing He, Chengkai Pan, Shuang Guo, Wenbin Huang, Yunfei Jia, Qianying Kong, Huifang Peng, Yandong Han, Yuxia Hou, Zhanping Ren, Yongwei Tao, Fei Huang, Hongwei Jiang, Shan Sun, Yanying Dong, Jiuxiang Lin, Chunyan Yin, Xuechen Zhu, Feng Chen, Yi Ding
Abstract
Sphingosine-1-phosphate lyase (SPL) insufficiency syndrome (SPLIS) or nephrotic syndrome type 14 (NPHS14), is an autosomal recessive multisystem disorder caused by loss-of-function mutations in SGPL1, encoding the enzyme responsible for the terminal degradation of sphingosine-1-phosphate (S1P). We investigated a patient carrying a previously undescribed c.1084T>A (p.Ser362Thr) SGPL1 variant and analyzed the metabolic and cellular consequences of SPL deficiency using patient fibroblasts, SGPL1-knockout HEK293T cells, and Sgpl1–/– and Sgpl1rosa+fl/fl mice. Metabolic stable isotope labelling revealed that SPL deficiency does not invariably result in S1P accumulation. Instead, SPL-deficient cells maintain near-normal S1P levels through (i) feedback regulation of de novo sphingolipid synthesis via the ORMDL–ceramide axis and (ii) increased diversion of excess ceramides into glycosphingolipids. However, perturbation of sphingolipid homeostasis — either by exogenous sphingolipid load or disruption of compensatory regulation — induces pathological intracellular S1P accumulation. In vivo, Sgpl1–/– mice exhibited pronounced urinary S1P excretion and renal S1P enrichment, accompanied by cytoskeletal disorganization and impaired epithelial morphogenesis. Mechanistically, we identify aberrant Rho–ROCK signaling as a key mediator of S1P-driven cytoskeletal dysregulation. Pharmacological ROCK inhibition with Fasudil mitigated renal cytoskeletal defects in Sgpl1–/– and Sgpl1rosa+fl/fl mice and partially restored epithelial architecture. These findings redefine the metabolic consequences of SPL deficiency and identify S1P-driven Rho–ROCK hyperactivation as a tractable therapeutic target in SPLIS.
Authors
Adam Majcher, Ranjha Khan, Kathrin Buder, Florence Bourquin, Julie D. Saba, Thorsten Hornemann
Abstract
BORCS5 encodes a subunit of the BLOC-One-Related Complex (BORC), which is known to promote anterograde movement and fusion of lysosomes. We identified 16 individuals from nine families with bi-allelic BORCS5 variants, revealing a spectrum of neurodevelopmental and neurodegenerative phenotypes. Carriers of homozygous protein-truncating variants (PTVs), resulting in complete loss of BORCS5, presented with prenatally lethal arthrogryposis multiplex congenita, brain malformations, and neuropathological evidence of neuroaxonal dystrophy. Individuals with missense or splice-site variants presented differently, with microcephaly, developmental epileptic encephalopathy, optic atrophy, spasticity, and progressive movement disorders. In this group, brain MRI showed diffuse hypomyelination, corpus callosum abnormalities, as well as progressive global cerebral atrophy, consistent with neurodegeneration. Borcs5 knockout in zebrafish exhibited microcephaly, motor deficits, and increased seizure susceptibility, mirroring the patients’ clinical presentation. At the cellular level, only BORCS5 PTVs, but not missense variants, led to perinuclear lysosomal clustering and impaired lysosomal axonal trafficking in induced pluripotential stem cell-derived forebrain neurons. However, both PTVs and missense variants were associated with reduced lysosomal proteolysis and activity of lysosomal hydrolases glucocerebrosidase and cathepsin B, indicating lysosomal dysfunction. Our study reveals a role for BORCS5 in modulation of lysosomal function, in addition to its known role in lysosome movement and fusion, possibly underlying the diverse clinical manifestations in individuals with BORCS5-related disorders.
Authors
Niccolò E. Mencacci, Georgia Minakaki, Reza Maroofian, Raffaella De Pace, Adeline Paimboeuf, Tiago Branco Fonseca, Tatiana Abramova, Patrick Shannon, David Chitayat, Francesca Magrinelli, Wesley J. Peng, Diptaman Chatterjee, Sara H. Eldessouky, Julia Baptista, Tamas Marton, Julie Vogt, Juan Dario Ortigoza-Escobar, Loreto Martorell, Marta Gómez-Chiari, Ingrid M. Wentzensen, Erik-Jan Kamsteeg, Maha S. Zaki, Annarita Scardamaglia, Giovanni Zifarelli, Zuhair Nasser Al-Hassnan, Elka Miller, Shiri Shinar, Lova S. Matsa, Sri Hari Chandan Appikonda, Ghada A. Otaify, Khalid Al-Thihli, Almundher Al-Maawali, Michael Schwake, Mariasavina Severino, Henry Houlden, Shunmoogum A. Patten, Juan S. Bonifacino, Kailash P. Bhatia, Dimitri Krainc
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)