Expression patterns of mismatch repair proteins in cervical cancer uncover independent prognostic value of MSH-2.

OBJECTIVE: Although early-detected cervical cancer is associated with good survival, the prognosis for late-stage disease is poor and treatment options are sparse. Mismatch repair deficiency (MMR-D) has surfaced as a predictor of prognosis and response to immune checkpoint inhibitor(s) in several cancer types, but its value in cervical cancer remains unclear. This study aimed to define the prevalence of MMR-D in cervical cancer and assess the prognostic value of MMR protein expression. METHODS: Expression of the MMR proteins MLH-1, PMS-2, MSH-2, and MSH-6 was investigated by immunohistochemical staining in a prospectively collected cervical cancer cohort (n=508) with corresponding clinicopathological and follow-up data. Sections were scored as either loss or intact expression to define MMR-D, and by a staining index, based on staining intensity and area, evaluating the prognostic potential. RNA and whole exome sequencing data were available for 72 and 75 of the patients and were used for gene set enrichment and mutational analyses, respectively. RESULTS: Five (1%) tumors were MMR-deficient, three of which were of neuroendocrine histology. MMR status did not predict survival (HR 1.93, p=0.17). MSH-2 low (n=48) was associated with poor survival (HR 1.94, p=0.02), also when adjusting for tumor stage, tumor type, and patient age (HR 2.06, p=0.013). MSH-2 low tumors had higher tumor mutational burden (p=0.003) and higher frequency of (frameshift) mutations in the double-strand break repair gene RAD50 (p<0.01). CONCLUSION: MMR-D is rare in cervical cancer, yet low MSH-2 expression is an independent predictor of poor survival.

Phosphatidylserine receptors enhance SARS-CoV-2 infection.

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

The Discovery of a LEMD2-Associated Nuclear Envelopathy with Early Progeroid Appearance Suggests Advanced Applications for AI-Driven Facial Phenotyping.

Over a relatively short period of time, the clinical geneticist's "toolbox" has been expanded by machine-learning algorithms for image analysis, which can be applied to the task of syndrome identification on the basis of facial photographs, but these technologies harbor potential beyond the recognition of established phenotypes. Here, we comprehensively characterized two individuals with a hitherto unknown genetic disorder caused by the same de novo mutation in LEMD2 (c.1436C>T;p.Ser479Phe), the gene which encodes the nuclear envelope protein LEM domain-containing protein 2 (LEMD2). Despite different ages and ethnic backgrounds, both individuals share a progeria-like facial phenotype and a distinct combination of physical and neurologic anomalies, such as growth retardation; hypoplastic jaws crowded with multiple supernumerary, yet unerupted, teeth; and cerebellar intention tremor. Immunofluorescence analyses of patient fibroblasts revealed mutation-induced disturbance of nuclear architecture, recapitulating previously published data in LEMD2-deficient cell lines, and additional experiments suggested mislocalization of mutant LEMD2 protein within the nuclear lamina. Computational analysis of facial features with two different deep neural networks showed phenotypic proximity to other nuclear envelopathies. One of the algorithms, when trained to recognize syndromic similarity (rather than specific syndromes) in an unsupervised approach, clustered both individuals closely together, providing hypothesis-free hints for a common genetic etiology. We show that a recurrent de novo mutation in LEMD2 causes a nuclear envelopathy whose prognosis in adolescence is relatively good in comparison to that of classical Hutchinson-Gilford progeria syndrome, and we suggest that the application of artificial intelligence to the analysis of patient images can facilitate the discovery of new genetic disorders.

Duplicated Enhancer Region Increases Expression of CTSB and Segregates with Keratolytic Winter Erythema in South African and Norwegian Families.

Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals.

Characterization of CEL-DUP2: Complete duplication of the carboxyl ester lipase gene is unlikely to influence risk of chronic pancreatitis.

BACKGROUND/OBJECTIVES: Carboxyl ester lipase is a pancreatic enzyme encoded by CEL, an extremely polymorphic human gene. Pathogenic variants of CEL either increases the risk for chronic pancreatitis (CP) or cause MODY8, a syndrome of pancreatic exocrine and endocrine dysfunction. Here, we aimed to characterize a novel duplication allele of CEL (CEL-DUP2) and to investigate whether it associates with CP or pancreatic cancer. METHODS: The structure of CEL-DUP2 was determined by a combination of Sanger sequencing, DNA fragment analysis, multiplex ligation-dependent probe amplification and whole-genome sequencing. We developed assays for screening of CEL-DUP2 and analyzed cohorts of idiopathic CP, alcoholic CP and pancreatic cancer. CEL protein expression was analyzed by immunohistochemistry. RESULTS: CEL-DUP2 consists of an extra copy of the complete CEL gene. The allele has probably arisen from non-allelic, homologous recombination involving the adjacent pseudogene of CEL. We found no association between CEL-DUP2 carrier frequency and CP in cohorts from France (cases/controls: 2.5%/2.4%; P = 1.0), China (10.3%/8.1%; P = 0.08) or Germany (1.6%/2.3%; P = 0.62). Similarly, no association with disease was observed in alcohol-induced pancreatitis (Germany: 3.2%/2.3%; P = 0.51) or pancreatic cancer (Norway; 2.5%/3.2%; P = 0.77). Notably, the carrier frequency of CEL-DUP2 was more than three-fold higher in Chinese compared with Europeans. CEL protein expression was similar in tissues from CEL-DUP2 carriers and controls. CONCLUSIONS: Our results support the contention that the number of CEL alleles does not influence the risk of pancreatic exocrine disease. Rather, the pathogenic CEL variants identified so far involve exon 11 sequence changes that substantially alter the protein's tail region.

Familial Infertility (Azoospermia and Cryptozoospermia) in Two Brothers-Carriers of t(1;7) Complex Chromosomal Rearrangement (CCR): Molecular Cytogenetic Analysis.

Structural aberrations involving more than two breakpoints on two or more chromosomes are known as complex chromosomal rearrangements (CCRs). They can reduce fertility through gametogenesis arrest developed due to disrupted chromosomal pairing in the pachytene stage. We present a familial case of two infertile brothers (with azoospermia and cryptozoospermia) and their mother, carriers of an exceptional type of CCR involving chromosomes 1 and 7 and three breakpoints. The aim was to identify whether meiotic disruption was caused by CCR and/or genomic mutations. Additionally, we performed a literature survey for male CCR carriers with reproductive failures. The characterization of the CCR chromosomes and potential genomic aberrations was performed using: G-banding using trypsin and Giemsa staining (GTG banding), fluorescent in situ hybridization (FISH) (including multicolor FISH (mFISH) and bacterial artificial chromosome (BAC)-FISH), and genome-wide array comparative genomic hybridization (aCGH). The CCR description was established as: der(1)(1qter->1q42.3::1p21->1q42.3::7p14.3->7pter), der(7)(1pter->1p2 1::7p14.3->7qter). aCGH revealed three rare genes variants: ASMT, GARNL3, and SESTD1, which were ruled out due to unlikely biological functions. The aCGH analysis of three breakpoint CCR regions did not reveal copy number variations (CNVs) with biologically plausible genes. Synaptonemal complex evaluation (brother-1; spermatocytes II/oligobiopsy; the silver staining technique) showed incomplete conjugation of the chromosomes. Associations between CCR and the sex chromosomes (by FISH) were not found. A meiotic segregation pattern (brother-2; ejaculated spermatozoa; FISH) revealed 29.21% genetically normal/balanced spermatozoa. The aCGH analysis could not detect smaller intergenic CNVs of few kb or smaller (indels of single exons or few nucleotides). Since chromosomal aberrations frequently do not affect the phenotype of the carrier, in contrast to the negative influence on spermatogenesis, there is an obvious need for genomic sequencing to investigate the point mutations that may be responsible for the differences between the azoospermic and cryptozoospermic phenotypes observed in a family. Progeny from the same parents provide a unique opportunity to discover a novel genomic background of male infertility.

Blood steroids are associated with prognosis and fat distribution in endometrial cancer.

BACKGROUND: Despite being a hormone dependent cancer, there is limited knowledge regarding the relation between level of steroids in blood and prognosis for endometrial cancer (EC) patients. METHODS: In this study we investigated plasma levels of 19 steroids using liquid-chromatography tandem mass-spectrometry in 38 postmenopausal EC patients, 19 with long, and 19 with short survival. We explored if estradiol levels were associated with specific abdominal fat distribution patterns and if transcriptional alterations related to estradiol levels could be observed in tumor samples. RESULTS: The plasma steroid levels for DHEA, DHEAS, progesterone, 21 OH progesterone and E1S were significantly increased (all p < 0.05) in patients with long survival compared to short. Estradiol levels were significantly positively correlated with visceral fat percentage (p = 0.035), and an increased expression of genes involved in estrogen related signaling was observed in tumors from patients with high estradiol levels in plasma. CONCLUSION: Several of the identified plasma steroids represent promising biomarkers in EC patients. The association between increased estradiol levels and a high percentage of visceral fat indicates that visceral fat is a larger contributor to estradiol production compared to subcutaneous fat in this population.

Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and their Molecular Characteristics.

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3, TM4SF18, CFI, GPX8, and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process.

RRAD, IL4I1, CDKN1A, and SERPINE1 genes are potentially co-regulated by NF-κB and p53 transcription factors in cells exposed to high doses of ionizing radiation.

BACKGROUND: The cellular response to ionizing radiation involves activation of p53-dependent pathways and activation of the atypical NF-κB pathway. The crosstalk between these two transcriptional networks include (co)regulation of common gene targets. Here we looked for novel genes potentially (co)regulated by p53 and NF-κB using integrative genomics screening in human osteosarcoma U2-OS cells irradiated with a high dose (4 and 10 Gy). Radiation-induced expression in cells with silenced TP53 or RELA (coding the p65 NF-κB subunit) genes was analyzed by RNA-Seq while radiation-enhanced binding of p53 and RelA in putative regulatory regions was analyzed by ChIP-Seq, then selected candidates were validated by qPCR. RESULTS: We identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes, namely IL4I1, SERPINE1, and CDKN1A, an antagonistic effect of the TP53 and RELA silencing was consistent with radiation-enhanced binding of both p53 and RelA. This suggested the possibility of a direct antagonistic (co)regulation by both factors: activation by NF-κB and inhibition by p53 of IL4I1, and activation by p53 and inhibition by NF-κB of CDKN1A and SERPINE1. On the other hand, radiation-enhanced binding of both p53 and RelA was observed in a putative regulatory region of the RRAD gene whose expression was downregulated both by TP53 and RELA silencing, which suggested a possibility of direct (co)activation by both factors. CONCLUSIONS: Four new candidates for genes directly co-regulated by NF-κB and p53 were revealed.

Screening for viral nucleic acids in vestibular schwannoma.

To investigate if viruses are involved in the pathogenesis of vestibular schwannomas (VS), we have screened biopsies from VS patients using different molecular techniques. Screening for the presence of known viruses using a pan-viral microarray assay (ViroChip) indicated the presence of several viruses including human endogenous retrovirus K (HERV-K) and human herpes virus 2 (HHV2). But with the exception of HERV-K, none of the findings could be verified by other methods. Whole transcriptome sequencing showed only the presence of HERV-K transcripts and whole genome sequencing showed only the presence of Epstein-Barr virus, most likely originating from infiltration of lymphocytes. We therefore conclude that it is less likely that viruses are involved in the pathogenesis of vestibular schwannomas.