LXR directly regulates glycosphingolipid synthesis and affects human CD4+ T cell function.

The liver X receptor (LXR) is a key transcriptional regulator of cholesterol, fatty acid, and phospholipid metabolism. Dynamic remodeling of immunometabolic pathways, including lipid metabolism, is a crucial step in T cell activation. Here, we explored the role of LXR-regulated metabolic processes in primary human CD4+ T cells and their role in controlling plasma membrane lipids (glycosphingolipids and cholesterol), which strongly influence T cell immune signaling and function. Crucially, we identified the glycosphingolipid biosynthesis enzyme glucosylceramide synthase as a direct transcriptional LXR target. LXR activation by agonist GW3965 or endogenous oxysterol ligands significantly altered the glycosphingolipid:cholesterol balance in the plasma membrane by increasing glycosphingolipid levels and reducing cholesterol. Consequently, LXR activation lowered plasma membrane lipid order (stability), and an LXR antagonist could block this effect. LXR stimulation also reduced lipid order at the immune synapse and accelerated activation of proximal T cell signaling molecules. Ultimately, LXR activation dampened proinflammatory T cell function. Finally, compared with responder T cells, regulatory T cells had a distinct pattern of LXR target gene expression corresponding to reduced lipid order. This suggests LXR-driven lipid metabolism could contribute to functional specialization of these T cell subsets. Overall, we report a mode of action for LXR in T cells involving the regulation of glycosphingolipid and cholesterol metabolism and demonstrate its relevance in modulating T cell function.

Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

Multicenter study on the genetics of glomerular diseases among southeast and south Asians: Deciphering Diversities - Renal Asian Genetics Network (DRAGoN).

Multinational studies have reported monogenic etiologies in 25%-30% of children with steroid-resistant nephrotic syndrome. Such large studies are lacking in Asia. We established Deciphering Diversities: Renal Asian Genetics Network (DRAGoN) and aimed to describe the genetic and clinical spectrums in Asians. We prospectively studied a cohort of 183 probands with suspected genetic glomerulopathies from South and Southeast Asia, of whom 17% had positive family history. Using multi-gene panel sequencing, we detected pathogenic variants in 26 (14%) probands, of whom one-third had COL4A4 or COL4A5 variants (n = 9, 5%). Of those with COL4A5 defects, only 25% had features suggestive of Alport syndrome. Besides traditional predictors for genetic disease (positive family history and extrarenal malformations), we identified novel predictors, namely older age (6.2 vs. 2.4 years; p = 0.001), hematuria (OR 5.6; 95% CI 2.1-14.8; p < 0.001), and proteinuria in the absence of nephrotic syndrome (OR 4.6; 95% CI 1.8-11.8; p = 0.001) at first manifestation. Among patients who first presented with proteinuria without nephrotic syndrome, the genetic diagnostic rates were >60% when a second risk factor (positive family history or extrarenal manifestation) co-existed. The genetic spectrum of glomerulopathies appears different in Asia. Collagen IV genes may be included in sequencing panels even when suggestive clinical features are absent.

Performance evaluation of Cepheid Xpert Norovirus kit with a user-modified protocol.

The Cepheid Xpert® Norovirus kit automates sample processing, nucleic acid extraction, and real-time reverse transcription polymerase chain reactions (RT-PCRs) to detect norovirus genogroups I (GI) and II (GII). Eighty-five stool samples collected between February 2015 and May 2017 were used to compare the performance of a user-modified Xpert assay against a clinically validated laboratory-developed test (LDT). Of the 85 samples, 54 were previously archived in -80°C freezer. The remaining 31 were fresh samples tested concurrently with the LDT. The results of all samples tested using the Xpert kit and LDT were found to be concordant, including 12 GI- and 42 GII-positive samples, 1 GI and GII coinfection, and 30 negative samples. Comparison of the assays showed perfect concordance with a kappa coefficient score of 1.00 (95%CI from 1.00 to 1.00). Of the 30 negative stool samples tested, three samples were positive for rotavirus detected using an immunochromatographic assay, with no cross-reactivity shown in both LDT and Xpert assays. In-run sample processing control of the Xpert assay for all negative samples tested showed no/minor inhibition. Compared to the LDT, the Xpert assay produced similar or better Ct values for detection. It also showed better mitigation of PCR inhibition in stool sample testing.

One-step molecular detection of the MYD88 L265P mutation by unlabeled probe genotyping analysis.

The aim of our study was to establish an unlabeled probe genotyping approach for rapid detection of the MYD88 L265P mutation in the differential diagnosis of WaldenstrÓ§m macroglobulinemia patients. Analytical and clinical validation of the assay was performed using serially diluted amplicon-cloned standards, 14 clinical bone marrow aspirate samples, and 30 peripheral blood samples from healthy donors, respectively. The analytical validation results showed that the assay is able to reproducibly identify as low as 10% of the L265P mutant. Clinical validation results showed improved detection sensitivity for the L265P mutation compared to Sanger sequencing. With the simplicity, cost-effectiveness, specificity and rapidity, we foresee that the unlabeled probe HRM assay is a good alternative to substitute current established methods for routine diagnostic testing of the MYD88 L265P mutation.

Clinical application of targeted long read sequencing in prenatal beta-thalassemia testing and genetic counseling.

BACKGROUND: Beta thalassemia, related to HBB mutation and associated with elevated hemoglobin A2 (HbA2), is an important genetic hemoglobinopathy with high incidences of disease and carrier rates in Singapore. Carrier screening is essential to facilitate prenatal counseling and testing. However, when individuals with elevated HbA2 do not have an identifiable HBB disease-associated variant, there is ambiguity on risk to their offspring. METHODS: We describe a case report of a proband with elevated HbA2, no identifiable HBB disease-associated variant, whose partner was a beta thalassemia carrier. Through clinical HBB gene sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis, as well as targeted Nanopore long read sequencing of selected genes, we performed a complete analysis of HBB including the promoter region, 5'UTR and coding gene sequence, as well as evaluation for potential modifier variants and other rare structural variants. RESULTS: This process identified that the proband was heterozygous for KLF1:c.544T>C (p.Phe182Leu), a potential functional polymorphism previously known to be associated with benign elevated HbA2 levels. The presence of disease variants in the HBB locus was excluded. CONCLUSION: This finding provided clarity and enabled family planning for the proband and her family.

A De Novo Mosaic PHEX Variant Causing Sporadic X-Linked Hypophosphatemic Rickets in a 2-Year-Old Girl.

Pathogenic variants in the PHEX gene are causative of X-linked hypophosphatemic rickets (XLH). We present a case of a 2-year-old girl with hypophosphatemic rickets with genu varum and short stature without any family history of XLH. Next generation sequencing of the PHEX gene identified a splice donor variant, NM_000444.6:c.1173 + 5G > A in intron 10. This variant had a mosaic pattern with only 22% of the sequence reads showing the variant allele and was not present in the girl's parents, both of whom had a normal phenotype. This is a sporadic case of a de novo mosaic splice-site variant in the PHEX gene.

Targeting both rs12979860 and rs8099917 polymorphisms with a single-tube high-resolution melting assay for IL28b genotyping.

A rapid, duplex, high-resolution melting interleukin-28B gene (IL28B) genotyping assay, targeting both rs12979860 and rs8099917 polymorphisms, was developed and validated using 30 DNA samples from healthy volunteers. A linkage study on 300 healthy Singaporeans showed variable haplotypes. When the assay was applied to plasma DNA from 50 hepatitis C virus genotype-1 (HCV-1)-infected patients, five compound heterozygous types were detected.

A direct sequencing assay for pharmacogenetic testing of thiopurine-intolerant NUDT15 alleles in an Asian population.

OBJECTIVE: The nucleoside diphosphate linked moiety X (Nudix)-Type motif 15 (NUDT15) enzyme is involved in thiopurine metabolism. Genetic variants in the NUDT15 gene result in decreased NUDT15 activity, which in addition to decreased thiopurine S-methyltransferase (TPMT) activity, contributes to thiopurine toxicity. Current standard approaches of NUDT15 genetic analysis have mainly been targeting several common variants. We aimed to develop a clinical-grade DNA-based assay for genetic analysis of the NUDT15 gene using Sanger di-deoxy sequencing. RESULTS: Sanger sequencing results were fully concordant with the expected NUDT15 genotype in all 17 cell line samples with known NUDT15 variants (accuracy = 100%; 95% CI 80.49 to 100.00%). Precision studies showed 100% intra-run repeatability and 100% inter-run reproducibility, respectively. Genetic analysis of the NUDT15 gene was performed for 80 patients of Asian ethnicity with wildtype TPMT. 76% (N = 61) of the studied individuals had NUDT15 *1/*1 diplotype. 25% (N = 14) of Chinese and 36% (N = 5) of Malays were found to carry at least 1 non-functional NUDT15 allele. Our study confirmed a high frequency of NUDT15 c.415C>T and c.55_56insGAGTCG variants in the Chinese and Malay ethnic groups in Singapore, highlighting the importance of determining NUDT15 genotype prior to thiopurine dosing.

In silico analysis of BRCA1 and BRCA2 missense variants and the relevance in molecular genetic testing.

Over the years since the genetic testing of BRCA1 and BRCA2 has been conducted for research and later introduced into clinical practice, a high number of missense variants have been reported in the literature and deposited in public databases. Polymorphism Phenotyping v2 (PolyPhen-2) and Sorting Intolerant from Tolerant (SIFT) are two widely applied bioinformatics tools used to assess the functional impacts of missense variants. A total of 2605 BRCA1 and 4763 BRCA2 variants from the ClinVar database were analysed with PolyPhen2 and SIFT. When SIFT was evaluated alongside PolyPhen-2 HumDiv and HumVar, it had shown top performance in terms of negative predictive value (NPV) (100%) and sensitivity (100%) for ClinVar classified benign and pathogenic BRCA1 variants. Both SIFT and PolyPhen-2 HumDiv achieved 100% NPV and 100% sensitivity in prediction of pathogenicity of the BRCA2 variants. Agreement was achieved in prediction outcomes from the three tested approaches in 55.04% and 68.97% of the variants of unknown significance (VUS) for BRCA1 and BRCA2, respectively. The performances of PolyPhen-2 and SIFT in predicting functional impacts varied across the two genes. Due to lack of high concordance in prediction outcomes among the two tested algorithms, their usefulness in classifying the pathogenicity of VUS identified through molecular testing of BRCA1 and BRCA2 is hence limited in the clinical setting.

A novel de novo androgen receptor nonsense mutation in a sex-reversed 46,XY infant.

An infant with 46,XY karyotype, and unambiguous female phenotype was found to have testes in the inguinal regions. Capillary sequencing of the androgen receptor (AR) gene identified a hemizygous de novo mutation (NM_000044.6:c.1621G > T) in exon 2 resulting in a termination codon p.(Glu541*) at the DNA binding domain (DBD). This novel nonsense mutation adds to the compendium of AR mutations which result in complete androgen insensitivity syndrome (AIS).

Laboratory Verification of a BRCA1 and BRCA2 Massively Parallel Sequencing Assay from Wet Bench to Bioinformatics for Germline DNA Analysis.

Introduction A robust genetic test for BRCA1 and BRCA2 genes is necessary for the diagnosis, prognosis, and treatment of patients with hereditary breast and ovarian cancer. We evaluated a commercial amplicon-based massively parallel sequencing (MPS) assay, BRCA MASTR Plus on the MiSeq platform, for germline BRCA genetic testing. Methods This study was performed on 31 DNA from cell lines and proficiency testing samples to establish the accuracy of the assay. A reference cell line DNA, NA12878 was used to determine the reproducibility of the assay. Discordant MPS result was resolved orthogonally by the current gold-standard Sanger sequencing method. Results The analytical accuracy, sensitivity, and specificity for variant detection were 93.55, 92.86, and 100.00%, respectively. Both sequencing depth and variant allele frequencies were highly reproducible by comparing the NA12878 DNA tested in three separate runs. The single discordant result, later confirmed by Sanger sequencing was due to the inability of the MASTR Reporter software to identify a 40-bp deletion in BRCA1 . Conclusion The BRCA MASTR Plus assay on the MiSeq platform is accurate and reproducible for germline BRCA genetic testing, making it suitable for use in a clinical diagnostic laboratory. However, Sanger sequencing may still serve as a confirmatory method to improve diagnostic capability of the MPS assay.

Validation of the Hemochromatosis (2SNP+) Direct EUROArray Assay for the Molecular Diagnosis of HFE-Related Hereditary Hemochromatosis.

Aim: Two missense variants in the HFE gene, c.845G>A (p.Cys282Tyr) and c.187C>G (p.His63Asp), are commonly screened as part of the diagnostic workup for HFE-related hereditary hemochromatosis (HH) and iron overload. Identification of the two variants can be achieved by polymerase chain reaction (PCR)-based laboratory tests and other methods. Evaluation of the analytical performance of the test is essential to ensure that the assay is precise and accurate. The aim of this study was to evaluate the analytical performance of the DNA microarray-based Hemochromatosis (2SNP+) Direct assay on the EUROArray test system (EUROIMMUN, Lübeck, Germany). Materials and Methods: Evaluation of the commercial assay was performed on 50 clinical blood samples and 26 retrospective College of American Pathologists (CAP)-provided external quality assurance (EQA) DNA samples and compared to a laboratory-developed PCR-restriction enzyme digestion (PCR-RE) test and DNA sequencing. Results and Discussion: HFE genotyping results obtained from both Hemochromatosis (2SNP+) Direct and PCR-RE assays were 100% concordant with nucleotide sequencing for all clinical samples evaluated. One hundred percent accuracy was also achieved on the retrospective CAP EQA samples. Precision studies performed on wild type and c.845G>A/c.187C>G compound heterozygous whole blood samples showed 100% intra-run repeatability (N = 3) and 100% inter-run reproducibility (N = 3), respectively. Conclusion: The Hemochromatosis (2SNP+) Direct EUROArray test provides a rapid and accurate method of detection for both the c.845G>A and c.187C>G variants for molecular diagnosis of HFE-related HH.

Genetic testing of GCK-MODY identifies a novel pathogenic variant in a Chinese boy with early onset hyperglycemia.

Glucokinase-maturity-onset diabetes of the young (GCK-MODY or MODY 2), caused by a heterozygous inactivating variant in the Glucokinase (GCK) gene, is a common form of MODY. Here, we present a case of GCK-MODY in a young Chinese boy, his sister and his father with a novel pathogenic variant in exon 8 of the GCK gene, NM_000162.5:c.1015del, p.(Glu339Argfs*14), which is predicted to cause a significant change in protein structure and function.