Citations

BACKGROUND Diagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG. METHODS Using multistep mass spectrometry–based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag–based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion. RESULTS Of the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man 5 GlcNAc 2 , which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2 . CONCLUSION Complement C4–derived Man 5 GlcNAc 2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs. FUNDING U54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance)

Abstract Background Cholangiocarcinoma (CCA) is a heterogeneous malignancy. Platinum-based chemotherapy (Gemcitabine and Cisplatin) has been the standard treatment for over a decade and is the base for the current immunotherapy combinations. Response rates are typically between 20-30%. Herein, we present outcomes of preclinical efficacy trials on CCA PDX models in correlation with baseline multiomics profiling of CCA patient-derived xenograft (PDX) models to identify molecular features associated with response. Methods Tumor samples from twenty-eight CCA PDX were used to isolate RNA and protein to be included in multiomics analysis (Phosphoproteomics, Proteomics, and Transcriptomics). A subset of PDX models were treated with various doses of Gemcitabine and Cisplatin. The Median Efficacy Index (MEI) was calculated as the ratio of the difference in median of delta in tumor volume at the last day of the study between the treatment and the control arms divided by the median of delta in tumor volume of the control at the last day of the treatment (positive= growth, negative=inhibition, and 0=no efficacy). A Hierarchical All-Against-All algorithm was utilized to compare multiomics features to the MEI and pathway analysis completed for identified molecular features. Results After conducting preclinical efficacy trials, the five tested CCA PDX models were classified into sensitive, moderately sensitive, and resistant models based on the MEI on each treatment arm. The top up-regulated genes were identified for Gemcitabine response (SRPK2, GLK2, and RIOK2) and Cisplatin response (NTRK1, PRPF4B, and SRPK2). The top up-regulated genes related to Gemcitabine resistance were NTRK1, PIP5K1C, and CSNK2A2, while those related to Cisplatin resistance were SRPK1, PRPF4B, and CSNK2A1. Conclusion Cholangiocarcinoma multiomics signatures can predict therapeutic response to standard-of-care chemotherapy in preclinical models. Novel mechanisms of Gemcitabine and Cisplatin resistance were identified related to NTRK1, SRPK1 and SRPK2. Resistant signatures will be challenged with the appropriate targeted therapies on the resistant CCA PDX models. Citation Format: Amro M. Abdelrahman, Danielle M. Carlson, Erik Jessen, Dong-Gi Mun, Isaac Lynch, Alessandro Fogliati, Stella Konadu Adjei Antwi, Aushinie Abeynayake, Akhilesh Pandey, Mark J. Truty, Gregory J. Gores, Rory L. Smoot. Pretreatment multiomics in conjunction with preclinical trials on patient-derived xenograft models of cholangiocarcinoma can identify chemotherapy response signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6912.

AbstractAsparagine‐linked glycosylation 1 protein is a β‐1,4‐mannosyltransferase, is encoded by the ALG1 gene, which catalyzes the first step of mannosylation in N‐glycosylation. Pathogenic variants in ALG1 cause a rare autosomal recessive disorder termed as ALG1‐CDG. We performed a quantitative proteomics and N‐glycoproteomics study in fibroblasts derived from patients with one homozygous and two compound heterozygous pathogenic variants in ALG1. Several proteins that exhibited significant upregulation included insulin‐like growth factor II and pleckstrin, whereas hyaluronan and proteoglycan link protein 1 was downregulated. These proteins are crucial for cell growth, survival and differentiation. Additionally, we observed a decrease in the expression of mitochondrial proteins and an increase in autophagy‐related proteins, suggesting mitochondrial and cellular stress. N‐glycoproteomics revealed the reduction in high‐mannose and complex/hybrid glycopeptides derived from numerous proteins in patients explaining that defect in ALG1 has broad effects on glycosylation. Further, we detected an increase in several short oligosaccharides, including chitobiose (HexNAc2) trisaccharides (Hex‐HexNAc2) and novel tetrasaccharides (NeuAc‐Hex‐HexNAc2) derived from essential proteins including LAMP1, CD44 and integrin. These changes in glycosylation were observed in all patients irrespective of their gene variants. Overall, our findings not only provide novel molecular insights into understanding ALG1‐CDG but also offer short oligosaccharide‐bearing peptides as potential biomarkers.

Gastric cancer (GC) remains a leading cause of cancer-related mortality globally. This is due to the fact that majority of the cases of GC are diagnosed at an advanced stage when the treatment options are limited and prognosis is poor. The diffuse subtype of gastric cancer (DGC) under Lauren's classification is more aggressive and usually occurs in younger patients than the intestinal subtype. The concept of personalized medicine is leading to the identification of multiple biomarkers in a large variety of cancers using different combinations of omics technologies. Proteomic changes including post-translational modifications are crucial in oncogenesis. We analyzed the phosphoproteome of DGC by using paired fresh frozen tumor and adjacent normal tissue from five patients diagnosed with DGC. We found proteins involved in the epithelial-to-mesenchymal transition (EMT), c-MYC pathway, and semaphorin pathways to be differentially phosphorylated in DGC tissues. We identified three kinases, namely, bromodomain adjacent to the zinc finger domain 1B ( BAZ1B ), WNK lysine-deficient protein kinase 1 ( WNK1 ), and myosin light-chain kinase ( MLCK ) to be hyperphosphorylated, and one kinase, AP2-associated protein kinase 1 ( AAK1 ), to be hypophosphorylated. LMNA hyperphosphorylation at serine 392 (S392) was demonstrated in DGC using immunohistochemistry. Importantly, we have detected heparin-binding growth factor ( HDGF ), heat shock protein 90 ( HSP90 ), and FTH1 as potential therapeutic targets in DGC, as drugs targeting these proteins are currently under investigation in clinical trials. Although these new findings need to be replicated in larger study samples, they advance our understanding of signaling alterations in DGC, which could lead to potentially novel actionable targets in GC.

AbstractThalassemias are a group of inherited monogenic disorders characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Delta‐beta (δβ‐) thalassemia has large deletions in the β globin gene cluster involving δ‐ and β‐globin genes, leading to absent or reduced synthesis of both δ‐ and β‐globin chains. Here, we used direct globin‐chain analysis using tandem mass spectrometry for the diagnosis of δβ‐thalassemia. Two cases from unrelated families were recruited for the study based on clinical and hematological evaluation. Peptides obtained after trypsin digestion of proteins extracted from red blood cell pellets from two affected individuals and their parents were analyzed using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Mass spectrometric analysis revealed a severe reduction in δ, β, and Aγ globin proteins with increased Gγ globin protein in the affected individuals. The diagnosis of Gγ(Aγδβ)0 ‐thalassemia in the homozygous state in the affected individuals and in the heterozygous state in the parents was made from our results. The diagnosis was confirmed at the genetic level using multiplex ligation‐dependent probe amplification (MLPA). Our findings demonstrate the utility of direct globin protein quantitation using LC‐MS/MS to quantify individual globin proteins reflecting changes in globin production. This approach can be utilized for accurate and timely diagnosis of hemoglobinopathies, including rare variants, where existing diagnostic methods provide inconclusive results.

Background:: Breast cancer is one of the leading causes of cancer deaths in women. Early diagnosis offers the best hope for a cure. Ductal carcinoma in situ is considered a precursor of invasive ductal carcinoma of the breast. In this study, we carried out microRNA sequencing from 7 ductal carcinoma in situ (DCIS), 6 infiltrating ductal carcinomas (IDC Stage IIA) with paired normal, and 5 unpaired normal breast tissue samples. We identified 76 miRNAs that were differentially expressed in DCIS and IDC. Methods:: Additionally, we provide preliminary evidence of miR-365b-3p and miR-7-1-3p being overexpressed, and miR-6507-5p, miR-487b-3p, and miR-654-3p being downregulated in DCIS relative to normal breast tissue. We also identified a miRNA miR-766-3p that was overexpressed in early-stage IDCs. The overexpression of miR-301a-3p in DCIS and IDC was confirmed in 32 independent breast cancer tissue samples. Results:: Higher expression of miR-301a-3p is associated with poor overall survival in The Can-cer Genome Atlas Breast Cancer (TCGA-BRCA) dataset, indicating that it may be associated with DCIS at high risk of progressing to IDC and warrants deeper investigation. Conclusion:: We also analyzed competing endogenous networks associated with differentially expressed miRNAs and identified LRRC75A-AS1 and MAGI2-AS3 as lncRNAs that potentially play an important role in early-stage breast cancers.

Abstract Despite intensive characterization of immune responses after COVID-19 infection and vaccination, research examining protective correlates of vertical transmission in pregnancy are limited. Herein, we profiled humoral and cellular characteristics in pregnant women infected or vaccinated at different trimesters and in their corresponding newborns. We noted a significant correlation between spike S1–specific IgG antibody and its RBD-ACE2 blocking activity (receptor-binding domain–human angiotensin-converting enzyme 2) in maternal and cord plasma (P < .001, R > 0.90). Blocking activity of spike S1–specific IgG was significantly higher in pregnant women infected during the third trimester than the first and second trimesters. Elevated levels of 28 cytokines/chemokines, mainly proinflammatory, were noted in maternal plasma with infection at delivery, while cord plasma with maternal infection 2 weeks before delivery exhibited the emergence of anti-inflammatory cytokines. Our data support vertical transmission of protective SARS-CoV-2–specific antibodies. This vertical antibody transmission and the presence of anti-inflammatory cytokines in cord blood may offset adverse outcomes of inflammation in exposed newborns.

Abstract Background Barth syndrome (BTHS) is an inborn error of phospholipid metabolism caused by pathogenic variants in the TAFAZZIN gene locus on a chromosome X. TAFAZZIN encodes a phospholipid transacylase enzyme, tafazzin, that promotes cardiolipin (CL) remodeling. Deficiency of tafazzin activity in BTHS results in accumulation of monolysocardiolipin (MLCL) and decreased abundance of mature CL, making an increased ratio of MLCL to CL as a diagnostic marker for BTHS. 52:2-MLCL to 72:8-CL in dried-blood spot (DBS) has been used for provisional identification BTHS using LC-MS/MS. A development of multiplexed method that targets several species of MLCL and CL can enhance the diagnosis of BTHS. Methods A targeted LC-MS/MS with scheduled multiple reaction monitoring (MRM) mode in negative electrospray ionization was developed using a triple quadrupole MS. DBS from patients with BTHS were obtained at Johns Hopkins University and provided by Barth Syndrome Foundation while control DBS were provided by Biochemical Genetics Laboratory at Mayo Clinic. Internal standards and chloroform:methanol (1:1, v/v) were added to DBS (1/8″ diameter) and sonicated. Supernatants were collected and dried under nitrogen gas. Followed by reconstitution in methanol, samples were subjected to LC-MS/MS analysis. Results More than 20 species of MLCL and CL species having various fatty acyl chains were detected and quantified. Remodeled CL species were decreased while MLCL and nascent CL species were found to be increased in known patients of BTHS (n = 8) compared to controls (n = 8). Notably, several ratios of lipids including 52:2-MLCL/72:8-CL and 54:2-MLCL/70:6-CL showed >300-fold elevation in the patients. Conclusion Various species of MLCL and CL that can be used to diagnose BTHS can be measured from an easily accessible specimen such as DBS using this method.