Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines.

The interaction manner and biological function of Rab7 and its effector, Rab-interacting lysosomal protein (RILP), remain unclear in invertebrates. We provide a protocol for detecting the effects of Rab7 and RILP terminals on lysosome and autophagy in Spodoptera frugiperda Sf9 cells with overexpression and RNA interference. We describe steps for overexpressing plasmids, generating long double-stranded RNA, and transfecting them into Sf9 cells. We then detail procedures for cell immunofluorescence imaging with harmine treatment and fluorescence analysis. For complete details on the use and execution of this protocol, please refer to Cui et al. (2023).1.

KLHL3 deficiency in mice ameliorates obesity, insulin resistance, and nonalcoholic fatty liver disease by regulating energy expenditure.

Obesity is a growing global epidemic that can cause serious adverse health consequences, including insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD). Obesity development can be attributed to energy imbalance and metabolic inflexibility. Here, we demonstrated that lack of Kelch-like protein 3 (KLHL3) mitigated the development of obesity, IR, and NAFLD by increasing energy expenditure. KLHL3 mutations in humans cause Gordon's hypertension syndrome; however, the role of KLHL3 in obesity was previously unknown. We examined differences in obesity-related parameters between control and Klhl3-/- mice. A significant decrease in body weight concomitant with fat mass loss and improved IR and NAFLD were observed in Klhl3-/- mice fed a high-fat (HF) diet and aged. KLHL3 deficiency inhibited obesity, IR, and NAFLD by increasing energy expenditure with augmentation of O2 consumption and CO2 production. Delivering dominant-negative (DN) Klhl3 using adeno-associated virus into mice, thereby dominantly expressing DN-KLHL3 in the liver, ameliorated diet-induced obesity, IR, and NAFLD. Finally, adenoviral overexpression of DN-KLHL3, but not wild-type KLHL3, in hepatocytes revealed an energetic phenotype with an increase in the oxygen consumption rate. The present findings demonstrate a novel function of KLHL3 mutation in extrarenal tissues, such as the liver, and may provide a therapeutic target against obesity and obesity-related diseases.

The Clinical and Molecular Assessment of Iranian Families with Severe Congenital Neutropenia, Identification of HYOU1 and SHOC2 as Potential Novel Gene Defects.

Neutropenia congenita grave (SCN) is a rare disease with a genetically and clinically heterogeneous nature, usually diagnosed in childhood, with an elevated risk of infections such as otitis, skin infections, pneumonia, deep abscesses, and septicemia. Patients with SCN also have an increased risk of leukemia, and mutations in the ELANE and the HAX1 genes have been observed in those patients. This study was conducted to genetically screen six Iranian families with SCN who have at least one affected person. In the first step, all exons and intron boundaries of ELANE and HAX1 genes were sequenced in probands. Cases with no pathogenic mutations were tested through whole-exome sequencing (WES). Analysis showed five different variants in ELANE (c.377 C>T), HAX1 (c.130_131 insA), HYOU1 (c.69 G>C and c.2744 G>A) and SHOC2 (c.4 A>G) genes in four families. We found that two out of six families had mutations in ELANE and HAX1 genes. Moreover, we found two novel mutations at the HYOU1 gene that had not previously been reported, as well as a pathogenic mutation at SHOC2 with multiple phenotypes, that will contribute to determining the genetic basis for SCN. Our study revealed that WES could help diagnose SCN, improve the classification of neutropenia, and rule out other immunodeficiencies such as autoimmune neutropenia, primary immunodeficiency diseases, and inherited bone marrow failure syndromes.

Standardized evidence-based approach for assessment of oncogenic and clinical significance of NTRK fusions.

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.

Microsatellite Instability Assessment by Immunohistochemistry in Acute Myeloid Leukemia: A Reappraisal and Review of the Literature.

BACKGROUND: Microsatellite instability (MSI) is caused by defects in DNA mismatch repair (MMR) components. Inactivation of any MMR gene(s), including hMLH1, hMSH2, hMSH6, and hPMS2, can result in MSI. Immunohistochemistry (IHC) is a sensitive and specific screening tool for MSI that can detect loss of expression of one or more MMR components. Of the four MMR markers, hMLH1 and hMSH2 are considered most informative of MSI status. There has been renewed interest in MSI status in view of its favorable association with response to immune checkpoint inhibitors in some cancers. MMR expression patterns in acute myeloid leukemia (AML) have not been evaluated systematically. METHODS: We used clinically-validated IHC assays to assess the expression of hMLH1, hMSH2, hMSH6, and/or hPMS2 in formalin-fixed paraffin-embedded tissue sections of bone marrow core biopsies from patients diagnosed with AML. Mutation profiling was performed using next-generation sequencing to assess for mutations in MMR genes. RESULTS: The study group included 236 patients with AML, including a cohort treated on a clinical trial of azacitidine and nivolumab (NCT02397720). In addition, hMSH6, and/or hPMS2 expression was assessed in 99 AML patients with diploid karyotype. All patients, except two, had retained expression of all MMR markers assessed: One patient from the azacytidine+nivolumab group had zonal patchy loss of staining of hMLH1 and, to a lesser extent, a similar staining pattern of hMSH2; and one patient from the AML with diploid karyotype group had loss of hMSH2 but retained expression of hMLH1, hMSH6 and hPMS2. In addition, a retrospective analysis on a separate cohort of 139 patients with primary AML, on which next generation sequencing profiling was performed, identified 14 cases with alterations in MMR genes. CONCLUSION AND REMARKS: MMR loss is a rare event in AML, thus does not appear to underlie response patterns to anti-PD1 therapy.

Comprehensive Assessment of Skin Disorders in Patients with Common Variable Immunodeficiency (CVID).

BACKGROUND: Common variable immunodeficiency (CVID) is an inborn error of immunity (IEI) characterized by various clinical manifestations such as hypogammaglobulinemia, recurrent infections, and autoimmune diseases. Among different clinical manifestations, skin manifestations have been less reported in these patients. METHODS: In this study, we investigated the prevalence of dermatologic features in 387 CVID patients. Demographic information, clinical manifestations, laboratory data, and genetic findings were collected from medical records. All data were analyzed based on the presence or absence of skin disorders in CVID patients. RESULTS: We observed at least one skin manifestation in about 40% of these patients. Among these complications, skin infection (n = 64, 42.1%) was the most frequent presentation, followed by non-infectious skin lesions (n = 54, 35.6%). Among skin infections, abscesses (n = 34, 22.4%) were the most common complication. Skin infections such as cellulitis, impetigo, measles, and warts were also documented. Eczema (n = 34, 22.4%) was the most common complication in atopic lesions, and vitiligo (n = 13, 8.5%) was prevalent in autoimmune/pigmentation disorders. Among all the patients with genetic mutations, one-quarter had a deleterious mutation in the LRBA gene, relating to the autoimmune and atopic skin lesions. CONCLUSION: This rate of skin disorders in our cohort demonstrating these manifestations could be significant in CVID patients, and they are not rare. Low data of skin complications in CVID patients could be attributed to insufficient attention of physicians and also might alert dermatologists to perform immunological investigations in children with certain skin manifestations.

Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis.

We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation.

Quantitative assessment of glioblastoma phenotypes in vitro establishes cell migration as a robust readout of Crk and CrkL activity.

The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors. Here, we knocked down Crk, CrkL, or both using siRNAs in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes. The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells. These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.

National external quality assessment for next-generation sequencing-based diagnostics of primary immunodeficiencies.

Dutch genome diagnostic centers (GDC) use next-generation sequencing (NGS)-based diagnostic applications for the diagnosis of primary immunodeficiencies (PIDs). The interpretation of genetic variants in many PIDs is complicated because of the phenotypic and genetic heterogeneity. To analyze uniformity of variant filtering, interpretation, and reporting in NGS-based diagnostics for PID, an external quality assessment was performed. Four main Dutch GDCs participated in the quality assessment. Unannotated variant call format (VCF) files of two PID patient analyses per laboratory were distributed among the four GDCs, analyzed, and interpreted (eight analyses in total). Variants that would be reported to the clinician and/or advised for further investigation were compared between the centers. A survey measuring the experiences of clinical laboratory geneticists was part of the study. Analysis of samples with confirmed diagnoses showed that all centers reported at least the variants classified as likely pathogenic (LP) or pathogenic (P) variants in all samples, except for variants in two genes (PSTPIP1 and BTK). The absence of clinical information complicated correct classification of variants. In this external quality assessment, the final interpretation and conclusions of the genetic analyses were uniform among the four participating genetic centers. Clinical and immunological data provided by a medical specialist are required to be able to draw proper conclusions from genetic data.

MiR-216a-3p suppresses the proliferation and invasion of cervical cancer through downregulation of ACTL6A-mediated YAP signaling.

The tumor-suppressive role of microRNA-216a-3p (miR-216a-3p) has been evidenced in multiple tumors. Yet, the relevance of miR-216a-3p in cervical cancer remains undermined. The current study was designed to determine the expression and potential function of miR-216a-3p in cervical cancer. Expression of miR-216a-3p was markedly decreased in cervical cancer and functional assays revealed an inhibitory effect of miR-216a-3p on the proliferation, colony formation, and invasion of cervical cancer. Actin-like 6A (ACTL6A) was identified as a target gene of miR-216a-3p. Elevated ACTL6A expression was detected in cervical cancer, and ACTL6A inhibition exhibited a tumor-suppressive effect. ACTL6A inhibition increased yes-associated protein (YAP) phosphorylation and downregulated YAP-mediated transcriptional activity. ACTL6A restoration or YAP reactivation partially abrogated the miR-216a-3p-mediated antitumor effect in cervical cancer cells. Taken together, these data demonstrate that miR-216a-3p acts as a potential tumor-suppressive miRNA in cervical cancer, which exerts its function through inhibition of YAP signaling via targeting ACTL6A.

Simplified Molecular Subtyping of Medulloblastoma for Reduced Cost and Improved Turnaround Time.

Molecular subtyping of medulloblastoma (MB) has become increasingly important for prognosis and management. Typically this involves detailed molecular genetic testing which may not be available in all centers. The purpose of the present study was to find a simplified approach to assign molecular subtypes of MB for routine use in centers with more limited resources. The molecular subtypes of MBs from 32 Thai patients, aged 0.5 to 35 years, were first determined by NanoString. These results were then compared with those obtained using a combination of limited immunohistochemistry (IHC) (ß-catenin, GAB-1, YAP-1, p75-NGFR, OTX2) and CTNNTB exon 3 mutation analysis. By NanoString assay, there were 6 MBs (19%) in the wingless (WNT) group, 8 (25%) in the sonic hedgehog (SHH) group, 7 (22%) in group 3, and 11 (34%) in group 4. Although ß-catenin immunostaining missed 4/6 WNT MBs, CTNNTB mutation analysis confirmed all WNT MB cases with amplifiable DNA. The IHC panel correctly assigned all the other molecular subtypes, except for 1 MB in group 4. Thus, our protocol was able to correctly categorized 31/32 cases or 97% of cases. Our study is the first to report molecular subtypes of MB in Southeast Asia. We found that molecular subgroups of MBs can be reliably assigned using a limited IHC panel of ß-catenin, GAB-1, YAP-1, p75-NGFR, OTX2, together with CTNNTB exon 3 mutation analysis. This simplified approach incurs lower cost and faster turnaround time compared with more elaborate molecular methodologies and should be beneficial to centers with reduced laboratory resources.

Can social support during pregnancy affect maternal DNA methylation? Findings from a cohort of African-Americans.

BACKGROUND: While stress and the absence of social support during pregnancy have been linked to poor health outcomes, the underlying biological mechanisms are unclear. METHODS: We examined whether adverse experiences during pregnancy alter DNA methylation (DNAm) in maternal epigenomes. Analyses included 250 African-American mothers from the Boston Birth Cohort. Genome-wide DNAm profiling was performed in maternal blood collected after delivery, using the Infinium HumanMethylation450 Beadchip. Linear regression models, with adjustment of pertinent covariates, were applied. RESULTS: While self-reported maternal psychosocial lifetime stress and stress during pregnancy was not associated with DNAm alterations, we found that absence of support from the baby's father was significantly associated with maternal DNAm changes in TOR3A, IQCB1, C7orf36, and MYH7B and that lack of support from family and friends was associated with maternal DNA hypermethylation on multiple genes, including PRDM16 and BANKL. CONCLUSIONS: This study provides intriguing results suggesting biological embedding of social support during pregnancy on maternal DNAm, warranting additional investigation, and replication.

Systematic meta-analyses, field synopsis and global assessment of the evidence of genetic association studies in colorectal cancer.

OBJECTIVE: To provide an understanding of the role of common genetic variations in colorectal cancer (CRC) risk, we report an updated field synopsis and comprehensive assessment of evidence to catalogue all genetic markers for CRC (CRCgene2). DESIGN: We included 869 publications after parallel literature review and extracted data for 1063 polymorphisms in 303 different genes. Meta-analyses were performed for 308 single nucleotide polymorphisms (SNPs) in 158 different genes with at least three independent studies available for analysis. Scottish, Canadian and Spanish data from genome-wide association studies (GWASs) were incorporated for the meta-analyses of 132 SNPs. To assess and classify the credibility of the associations, we applied the Venice criteria and Bayesian False-Discovery Probability (BFDP). Genetic associations classified as 'positive' and 'less-credible positive' were further validated in three large GWAS consortia conducted in populations of European origin. RESULTS: We initially identified 18 independent variants at 16 loci that were classified as 'positive' polymorphisms for their highly credible associations with CRC risk and 59 variants at 49 loci that were classified as 'less-credible positive' SNPs; 72.2% of the 'positive' SNPs were successfully replicated in three large GWASs and the ones that were not replicated were downgraded to 'less-credible' positive (reducing the 'positive' variants to 14 at 11 loci). For the remaining 231 variants, which were previously reported, our meta-analyses found no evidence to support their associations with CRC risk. CONCLUSION: The CRCgene2 database provides an updated list of genetic variants related to CRC risk by using harmonised methods to assess their credibility.

Assessment of YAP gene polymorphisms and arsenic interaction in Mexican women with breast cancer.

The identification of gene-environment interactions related to breast cancer reveals the biological and molecular mechanisms underlying the disease and allows the distinction of women at high risk from women at lower risk, which could decrease the morbimortality of this neoplasm. The current study evaluated the association between polymorphisms rs1820453 and rs11225161 of the Yes-associated protein (YAP) gene in women with breast cancer exposed to arsenic (As) through drinking water. In total, 182 women were assessed for the frequency of YAP rs1820453 and rs11225161 polymorphisms and As urinary levels. The results demonstrated a positive and significant association between breast cancer and smoking, type of drinking water, and levels of AsIII , AsV and inorganic As (iAs) but not the YAP gene polymorphisms evaluated. In conclusion, our data showed that the source of drinking water and AsV and iAs urinary levels increased the risk for breast cancer, but no interactions between YAP gene polymorphisms and As urinary levels were found.

Peritumoral activation of the Hippo pathway effectors YAP and TAZ suppresses liver cancer in mice.

The Hippo signaling pathway and its two downstream effectors, the YAP and TAZ transcriptional coactivators, are drivers of tumor growth in experimental models. Studying mouse models, we show that YAP and TAZ can also exert a tumor-suppressive function. We found that normal hepatocytes surrounding liver tumors displayed activation of YAP and TAZ and that deletion of Yap and Taz in these peritumoral hepatocytes accelerated tumor growth. Conversely, experimental hyperactivation of YAP in peritumoral hepatocytes triggered regression of primary liver tumors and melanoma-derived liver metastases. Furthermore, whereas tumor cells growing in wild-type livers required YAP and TAZ for their survival, those surrounded by Yap- and Taz-deficient hepatocytes were not dependent on YAP and TAZ. Tumor cell survival thus depends on the relative activity of YAP and TAZ in tumor cells and their surrounding tissue, suggesting that YAP and TAZ act through a mechanism of cell competition to eliminate tumor cells.

Genome-Wide Assessment for Resting Heart Rate and Shared Genetics With Cardiometabolic Traits and Type 2 Diabetes.

BACKGROUND: High resting heart rate (RHR) occurs in parallel with type 2 diabetes (T2D) and metabolic disorders, implying shared etiology between them. However, it is unknown if they are causally related, and no study has been conducted to investigate the shared mechanisms underlying these associations. OBJECTIVES: The objective of this study was to understand the genetic basis of the association between resting heart rate and cardiometabolic disorders/T2D. METHODS: This study examined the genetic correlation, causality, and shared genetics between RHR and T2D using LD Score regression, generalized summary data-based Mendelian randomization, and transcriptome wide association scan (TWAS) in UK Biobank data (n = 428,250) and summary-level data for T2D (74,124 cases and 824,006 control subjects) and 8 cardiometabolic traits (sample size ranges from 51,750 to 236,231). RESULTS: Significant genetic correlation between RHR and T2D (rg = 0.22; 95% confidence interval: 0.18 to 0.26; p = 1.99 × 10-22), and 6 cardiometabolic traits (fasting insulin, fasting glucose, waist-hip ratio, triglycerides, high-density lipoprotein, and body mass index; rg range -0.12 to 0.24; all p < 0.05) were observed. RHR has significant estimated causal effect on T2D (odds ratio: 1.12 per 10-beats/min increment; p = 7.79 × 10-11) and weaker causal estimates from T2D to RHR (0.32 beats/min per doubling increment in T2D prevalence; p = 6.14 × 10-54). Sensitivity analysis by controlling for the included cardiometabolic traits did not modify the relationship between RHR and T2D. TWAS found locus chr2q23.3 (rs1260326) was highly pleiotropic among RHR, cardiometabolic traits, and T2D, and identified 7 genes (SMARCAD1, RP11-53O19.3, CTC-498M16.4, PDE8B, AKTIP, KDM4B, and TSHZ3) that were statistically independent and shared between RHR and T2D in tissues from the nervous and cardiovascular systems. These shared genes suggested the involvement of epigenetic regulation of energy and glucose metabolism, and AKT activation-related telomere dysfunction and vascular endothelial aging in the shared etiologies between RHR and T2D. Finally, FADS1 was found to be shared among RHR, fasting glucose, high-density lipoprotein, and triglycerides. CONCLUSIONS: These findings provide evidence of significant genetic correlations and causation between RHR and T2D/cardiometabolic traits, advance our understanding of RHR, and provide insight into shared etiology for high RHR and T2D.

Genome analysis and pleiotropy assessment using causal networks with loss of function mutation and metabolomics.

BACKGROUND: Many genome-wide association studies have detected genomic regions associated with traits, yet understanding the functional causes of association often remains elusive. Utilizing systems approaches and focusing on intermediate molecular phenotypes might facilitate biologic understanding. RESULTS: The availability of exome sequencing of two populations of African-Americans and European-Americans from the Atherosclerosis Risk in Communities study allowed us to investigate the effects of annotated loss-of-function (LoF) mutations on 122 serum metabolites. To assess the findings, we built metabolomic causal networks for each population separately and utilized structural equation modeling. We then validated our findings with a set of independent samples. By use of methods based on concepts of Mendelian randomization of genetic variants, we showed that some of the affected metabolites are risk predictors in the causal pathway of disease. For example, LoF mutations in the gene KIAA1755 were identified to elevate the levels of eicosapentaenoate (p-value = 5E-14), an essential fatty acid clinically identified to increase essential hypertension. We showed that this gene is in the pathway to triglycerides, where both triglycerides and essential hypertension are risk factors of metabolomic disorder and heart attack. We also identified that the gene CLDN17, harboring loss-of-function mutations, had pleiotropic actions on metabolites from amino acid and lipid pathways. CONCLUSION: Using systems biology approaches for the analysis of metabolomics and genetic data, we integrated several biological processes, which lead to findings that may functionally connect genetic variants with complex diseases.

Assessment of expression of RELN signaling pathway in multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Nearly 85% of MS patients are recognized with relapsing-remitting MS (RRMS), a typical clinical course of disease which is distinguished by several episodes of relapses, separated by remissions of neurological impairment. Failure of repair mechanisms is a main factor in progression of neurological dysfunction in MS. Several lines of evidence suggest that Reelin (RELN) signaling pathway can contribute in the regulation of repair mechanisms in MS patients. In the present study, we assessed expression levels of RELN and Disabled-1 (DAB1), two key genes in RELN signaling pathway, in peripheral blood of 50 RRMS patients and 50 matched healthy subjects. RELN was significantly down-regulated in total MS patients, and total female patients compared with the matched controls. However, no statistically significant difference was found in DAB1 mRNA expression between MS patients and controls. Furthermore, considerable correlations were detected between expression levels of RELN and DAB1 in the patients group. There were no significant correlations between expression levels of genes and EDSS, disease duration or age at onset. Our study provides evidences for the role of RELN signaling pathway in the pathogenesis of MS. Further studies are required to clarify the exact clinical significance of this pathway in MS patients.

A Prospective Assessment of Periprosthetic Bone Mineral Density and Osteoimmunological Biomarkers Variations After Total Knee Replacement Surgery.

Aseptic loosening is a major cause of premature failure of total knee replacement (TKR). Variations in periprosthetic bone mineral density (BMD) and osteoimmunological biomarkers levels could help to quantify prosthesis osteointegration and predict early aseptic loosening. The gene expression of 5 selected osteoimmunological biomarkers was evaluated in tibial plateau bone biopsies by real-time polymerase chain reaction and changes in their serum levels after TKR were prospectively evaluated with enzyme-linked immunosorbent assay for 1 yr after surgery. These variations were correlated to changes in periprosthetic BMD. Sixteen patients were evaluated. A statistically significant decrease in serum levels of Sclerostin (p = 0.0135) was observed immediately after surgery. A specular pattern was observed between dickkopf-related protein 1 and osteoprotegerin expression. No statistically significant changes were detectable in the other study biomarkers. Periprosthetic BMD did not change significantly across the duration of the follow-up. Prosthetic knee surgery has an impact on bone remodeling, in particular on sclerostin expression. Although not showing statistically significant changes, in the patterns of dickkopf-related protein 1, osteoprotegerin, and the ligand of the receptor activator of nuclear factor kappa-B symmetries and correspondences related to the biological activities of these proteins could be identified. Variation in osteoimmunological biomarkers after TKR surgery can help in quantifying prosthesis osteointegration.