Evidence of Two Novel LAMA2 Variants in a Patient With Muscular Dystrophy: Facing the Challenges of a Certain Diagnosis

BackgroundBenefits and challenges resulting from advances in genetic diagnostics are two sides of the same coin. Facilitation of a correct and timely diagnosis is paralleled by challenges in interpretation of variants of unknown significance (VUS). Focusing on an individual VUS-re-classification pipeline, this study offers a diagnostic approach for clinically suspected hereditary muscular dystrophy by combining the expertise of an interdisciplinary team.MethodsIn a multi-step approach, a thorough phenotype assessment including clinical examination, laboratory work, muscle MRI and histopathological evaluation of muscle was performed in combination with advanced Next Generation Sequencing (NGS). Different in-silico tools and prediction programs like Alamut, SIFT, Polyphen, MutationTaster and M-Cap as well as 3D- modeling of protein structure and RNA-sequencing were employed to determine clinical significance of the LAMA2 variants.ResultsTwo previously unknown sequence alterations in LAMA2 were detected, a missense variant was classified initially according to ACMG guidelines as a VUS (class 3) whereas a second splice site variant was deemed as likely pathogenic (class 4). Pathogenicity of the splice site variant was confirmed by mRNA sequencing and nonsense mediated decay (NMD) was detected. Combination of the detected variants could be associated to the LGMDR23-phenotype based on the MRI matching and literature research.DiscussionTwo novel variants in LAMA2 associated with LGMDR23-phenotype are described. This study illustrates challenges of the genetic findings due to their VUS classification and elucidates how individualized diagnostic procedure has contributed to the accurate diagnosis in the spectrum of LGMD

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

Abstract The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.Peer reviewe

Funcional analyses of leupaxin in the initiation and progression of prostate carcinomas

Prostatakrebs ist die häufigste Krebsart und die zweithäufigste Ursache von Krebs-bezogenen Todesfällen in der westlichen männlichen Bevölkerung. Der Krebs entwickelt sich zunächst als androgen-abhängige Läsion in der Prostatadrüse, die erfolgreich durch operative Entfernung des Tumors und lokaler Bestrahlung therapiert werden kann. Patienten mit lokal fortgeschrittener und metastasierender Krankheit werden mit einer Androgen-unterdrückenden Therapie behandelt, die jedoch in 80% der Patienten zu einem androgen-unabhängigen, aggressiven und meist invasiven Rezidiv nach 18 bis 24 Monaten führt. Zur Zeit gibt es keine effektive Therapie für das hormon-refraktäre Prostatakarzinom, und die mittlere Überlebensdauer der Patienten beträgt durchschnittlich 12 Monate. Um geeignete Therapien entwickeln zu können, ist es obligatorisch, die Mechanismen zu verstehen, die zur Androgenunabhängigkeit und Invasivität des Prostatakarzinoms führen. Ein Kandidatengen stellt Leupaxin dar. Leupaxin ist ein zytoskelettales Adapterprotein, das hauptsächlich in hämtopoetischen Geweben und Zellen exprimiert wird. Leupaxin gehört zur Gruppe III der LIM-Domänen enthaltenden Proteine Paxillin und ARA55.In der vorliegenden Arbeit konnte gezeigt werden, dass Leupaxin in 22% der untersuchten Prostatakarzinome exprimiert wird. Dabei korrelierte die Leupaxin-Expression signifikant mit dem Gleason Score und Muster der Tumoren. Weiterhin konnten variierende Expressionslevel von Leupaxin in den Prostatakarzinomzelllinien LNCaP, PC-3 und DU 145 beobachtet werden. Leupaxin ist präferentiell an den Kontaktstellen dieser Zellen mit der extrazellulären Matrix (focal adhesion sites) lokalisiert. Über Mutationsanalysen mit GFP-Leupaxin-Fusionsproteinen konnte ein funktionelles Kernexportsignal nachgewiesen werden, welches für das Pendeln von Leupaxin zwischen Zytoplasma und Kern verantwortlich ist. Weiterhin konnte gezeigt werden, dass Leupaxin über die LIM-Domänen mit dem Androgen-, Glukokortikoid- und dem Progesteronrezeptor interagiert. Während die Interaktion mit dem Androgenrezeptor liganden-abhängig stattfindet, ist für die Interaktion von Leupaxin mit dem Glukokortikoid- und Progesteronrezeptor die Anwesenheit eines Liganden nicht notwendig. Zudem kann Leupaxin die transkriptionelle Aktivität dieser Steroidhormonrezeptoren erhöhen. Funktionelle Analysen mittels der RNA-Interferenz-Technologie ergaben weiterhin, dass androgen-abhängige LNCaP-Zellen mit reduzierter Leupaxin-Expression ihre Morphologie verändern, sich ablösen und apoptisch werden. Dabei scheint Leupaxin in die ERK-Signalkaskaden einzugreifen. Hingegen zeigen androgen-unabhängige PC-3- und DU 145-Zellen eine um 80% reduzierte Invasivität und eine stark beeinträchtigte Migrationsfähigkeit nach Herunterregulierung der Leupaxin-Expression.Die in vitro erhobenen Daten sollten zudem anhand eines transgenen Mausmodells verifiziert werden. Dazu wurden zwei transgene Mauslinien etabliert, die Leupaxin unter dem prostata-spezifischen Minimalpromotor des Ratten-Probasin-Gens exprimieren. Die Expression beschränkte sich auf die Epithelzellen der dorsalen und ventralen Lappen der Mausprostatae. In einer histologischen Analyse der Prostatae aus verschiedenen Altersstadien der transgenen Leupaxin-Mäuse konnte keine Veränderung im Prostataepithel beobachtet werden.Zusammenfassend zeigen die Ergebnisse der Arbeit, dass Leupaxin einen potentiellen Marker für die Progression von bestimmten Prostatakarzinomen darstellt. Als neuer Koaktivator des Androgenrezeptors könnte Leupaxin als putatives Ziel therapeutischer Interventionen des fortgeschrittenen Prostatakarzinoms dienen

PIK3CA mutations are specifically localized to lymphatic endothelial cells of lymphatic malformations.

Lymphatic malformations (LM) are characterized by the overgrowth of lymphatic vessels during pre- and postnatal development. Macrocystic, microcystic and combined forms of LM are known. The cysts are lined by lymphatic endothelial cells (LECs). Resection and sclerotherapy are the most common treatment methods. Recent studies performed on LM specimens in the United States of America have identified activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene in LM. However, whole tissue but not isolated cell types were studied. Here, we studied LM tissues resected at the University Hospitals Freiburg and Regensburg, Germany. We isolated LECs and fibroblasts separately, and sequenced the commonly affected exons 8, 10, and 21 of the PIK3CA gene. We confirm typical monoallelic mutations in 4 out of 6 LM-derived LEC lines, and describe two new mutations i.) in exon 10 (c.1636C>A; p.Gln546Lys), and ii.) a 3bp in-frame deletion of GAA (Glu109del). LM-derived fibroblasts did not possess such mutations, showing cell-type specificity of the gene defect. High activity of the PIK3CA-AKT- mTOR pathway was demonstrated by hyperphosphorylation of AKT-Ser473 in all LM-derived LECs (including the ones with newly identified mutations), as compared to normal LECs. Additionally, hyperphosphorylation of ERK was seen in all LM-derived LECs, except for the one with Glu109del. In vitro, the small molecule kinase inhibitors Buparlisib/BKM-120, Wortmannin, and Ly294002, (all inhibitors of PIK3CA), CAL-101 (inhibitor of PIK3CD), MK-2206 (AKT inhibitor), Sorafenib (multiple kinases inhibitor), and rapamycin (mTOR inhibitor) significantly blocked proliferation of LM-derived LECs in a concentration-dependent manner, but also blocked proliferation of normal LECs. However, MK-2206 appeared to be more specific for mutated LECs, except in case of Glu109 deletion. In sum, children that are, or will be, treated with kinase inhibitors must be monitored closely

Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2

Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug

Amygdalin influences bladder cancer cell adhesion and invasion in vitro

The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type

Proliferation studies with multi-kinase inhibitor Sorafenib on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).

<p>0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.</p

Phospho-ERK Western blot analysis of lymphatic endothelial cells.

<p>Note that phospho-ERK (pERK) is highly expressed in lymphatic malformation-derived Ly-LECs (Ly-LEC1, 10, and 12), but not in normal HD-LECc3 and c4. Ly-LEC2, which contain a Glu109del in <i>PIK3CA</i>, are similar to normal LECs, which contain clear levels of total ERK (tERK). Antibodies against α-tubulin were used as loading control.</p

Proliferation studies with PIK3CD inhibitor CAL-101 (Idealisib) on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).

<p>0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.</p