LAMB3 Research Articles

Structural Defects of Laminin β3 N-terminus Underlie Junctional Epidermolysis Bullosa with Altered Granulation Tissue Response

Mutations in the laminin-332 (α3Aβ3γ2) genes cause junctional epidermolysis bullosa (JEB), a recessively inherited disease characterized by blistering and altered wound repair. In addition, specific mutations that affect the N-terminus of the α3A chain cause a JEB-related non-blistering condition characterized by chronic production of granulation tissue, suggesting a critical role of this region in epithelial-mesenchymal communication. We report here a 9-year-old patient with JEB with a few long-standing skin ulcers with prominent granulation tissue in the absence of active blistering. He bears a homozygous missense mutation, p.Gly254Asp, within the first laminin epidermal growth factor-like (LE) repeat of the β3 short arm. We show that p.Gly254Asp causes mis-folding of the LE motif, leading to reduced secretion of laminin-332 and structural alterations of the cutaneous basement membrane zone. These findings demonstrate, in a patient in vivo, that the β3 short arm is also involved in the outcome of the granulation tissue response.

A dystonia-like movement disorder with brain and spinal neuronal defects is caused by mutation of the mouse laminin β1 subunit, Lamb1.

A new mutant mouse (lamb1t) exhibits intermittent dystonic hindlimb movements and postures when awake, and hyperextension when asleep. Experiments showed co-contraction of opposing muscle groups, and indicated that symptoms depended on the interaction of brain and spinal cord. SNP mapping and exome sequencing identified the dominant causative mutation in the Lamb1 gene. Laminins are extracellular matrix proteins, widely expressed but also known to be important in synapse structure and plasticity. In accordance, awake recording in the cerebellum detected abnormal output from a circuit of two Lamb1-expressing neurons, Purkinje cells and their deep cerebellar nucleus targets, during abnormal postures. We propose that dystonia-like symptoms result from lapses in descending inhibition, exposing excess activity in intrinsic spinal circuits that coordinate muscles. The mouse is a new model for testing how dysfunction in the CNS causes specific abnormal movements and postures.

Mutational Spectrum of Stromal Genes By Whole Exome Sequencing and Stromal-Cellular Interaction in Diffuse Large B-Cell Lymphoma

Introduction:Morphological sub-classification of DLBCL into germinal center B-cell-like (GCB) and activated B-cell like (ABC) lacks sufficient reproducibility for risk stratification and predicting survival based on International Prognostic Index (IPI). The importance of stromal cells and their cross-talk with lymphoma cells is important in lymphomagenesis and progression. We postulate that mutations in stromal genes may regulate the rate of tumor progression in DLBCL and understanding the stromal-cellular interaction in DLBCL may help evolve better markers of prognostication and identify novel therapeutic targets.Methods:The study was approved by the Institute ethics committee and patients were enrolled after an informed consent. Frozen samples from 6 cases which included 4 cases of de novo nodal DLBCL (2 cases each of GCB and ABC subtypes as per Han's algorithm) and two control cases of non-neoplastic reactive lymphoid hyperplasia were subjected to AmpliSeqExome - Whole Exome Sequencing(WES) using Ion TorrentTM and compared against human reference genome (hg19). Torrent suiteTM 4.4 version (Life Technologies) and Ion ReporterTM software was used to perform tumor vs normal analysis by choosing variants for missense, frameshift insertions/ deletions, stoploss and single nucleotide variations (SNP). Filtering for common SNPs were done by UCSC common SNPs followed by inclusion in dbSNP and COSMIC and further filtered by predictive functional scores of SIFT < 0.05 and Polyphen (0.15 - 1.0). Variants were visualized using Integrated Genome Viewer (IGV) software. Bioinformatics analysis was done using PANTHER, STRING and PCViz and data mined for mutations in extracellular matrix genes. Selected genes were validated in DLBCL cases using Sanger sequencing BigDye terminator v3.1 cycle sequencing kit (Life Technologies) and ABI PRISM 3130 analyzer (Life Technologies). Further, in-vitro experiments were carried out to evaluate the effect of the conditioned media of CD19- stromal cells on the CD19+ lymphoma cells. Fresh tissue from 5 cases of nodal DLBCL were collected in RPMI 1640, 20% FBS and 1% penicillin and streptomycin and subjected to primary culture at 37°C and 5% CO2 humidified atmosphere. After 24 hours, the cultured cells were sorted into CD19+ and CD19- cells (BD FACSAriaTM) which were re-cultured for 24 hours separately. After this, the conditioned media of CD19- cells (comprising stromal cells predominantly) was transferred onto CD19+ cells and incubated for further 48 hours. Thereafter, cytokine (IL-6, IL-10, TNF-α, IFN-γ, IL-2, IL-4, IL-17) analysis of conditioned media in comparison to mock controls was performed using BD THI/THII kit (560484).Results:Analysis of filtered mutated genes using Panther revealed thirty four genes coding for extracellular matrix mutated specifically in GCB subtype and 26 mutated genes in ABC DLBCL. These genes were then analyzed for association in STRING. STRING identified LAMC3, AGRN, LAMA2, LAMB2, COL5A2, COL13A1,FN1,TGFB3, LTBP1 andADAMTS16 in GCB phenotype whereas LAMB1,LAMA3,COL4A2, COL28A1,COL5A3, IBSP, FGA, MUC6, MUC2, MUC5B,SPARCL1, VWF, GAS6 and USH2A were mutated in ABC DLBCL (Figure 1A and B). Mutations of COL5A2, COL13A1, LAMB2, MUC2, MUC6, and MUC5B were further validated using Sanger sequencing in individual DLBCL cases. This advocated the role of collagen scaffolding and laminin cross-linking in DLBCL progression. In vitro experiments for effect of stromal cells on the secreted cytokine profile revealed increased IL-6, IL-10, TNF-α, IFN-γ, IL-4, IL-17 levels respectively in the media of CD19+ sorted lymphoma cells upon addition of conditioned media of CD19-stromal cells in comparison to controls.Conclusion:Mutational profile of COL5A2, COL13A1, LAMB2, MUC2, MUC6, and MUC5B stromal genes in DLBCL may help in refinement of risk stratification based on morphology. The cross-talk between neoplastic cells and interacting tumor microenvironment cells has implications for therapy in DLBCL.GCB Mutation ProfileABC Mutation Profile [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genome-wide association study identifies candidate loci associated with postoperative fentanyl requirements after laparoscopic-assisted colectomy.

Opioids are widely used as effective analgesics, but opioid sensitivity is well known to vary widely among individuals and the underlying genetic factors are not fully understood, thus hampering efficient pain treatment. We explored the genetic factors that contribute to individual differences in opioid sensitivity by performing a genome-wide association study. We conducted a multistage genome-wide association study in subjects who underwent laparoscopic-assisted colectomy (LAC). A nonsynonymous SNP in the LAMB3 gene region, rs2076222, was strongly associated with postoperative opioid requirements. The C allele of this best-candidate SNP was associated with lower opioid sensitivity and/or higher pain sensitivity in the patient subjects. Our findings provide valuable information for personalized pain treatment after LAC, in which the C allele of the rs2076222 SNP is associated with lower opioid sensitivity and requires more opioid analgesic after LAC.

Effects of acupuncture at Baihui (GV 20) and Zusanli (ST 36) on peripheral serum expression of MicroRNA 124, laminin and integrin β1 in rats with cerebral ischemia reperfusion injury.

To explore the effects of acupuncture at Baihui (GV 20) and Zusanli (ST 36) on the peripheral serum expression of microRNA 124 (miRNA 124), laminin and integrin β1 in rats with cerebral ischemia reperfusion injury (CIRI). Seventy-two healthy male Sprague-Dawley rats were randomized into a model group, an acupuncture group, and a sham-operated group using a random digits table, with 24 rats per group. Each group was further randomly divided into 1-, 3-, 5-, and 7-day subgroups based on the reperfusion time according to a random digits table, with 6 rats in each subgroup. In the model and acupuncture groups, CIRI was induced using the thread occlusion method. Electroacupuncture stimulation was applied daily to GV 20 and left ST 36 for 20 min at the indicated time points after successful operations. Serum was sampled for detecting laminin and integrin β1 protein via enzyme-linked immunosorbent assay, and serum miRNA 124 was examined using quantitative polymerase chain reaction. The serum level of miRNA 124 in the cerebral ischemia rats increased significantly, and the peak expression of miRNA 124 in both the model and acupuncture groups occurred at 3 days. The expression of miRNA 124 in the acupuncture group was higher than in the model group at the same time point (5.96±0.01 vs. 3.11±0.04, P <0.05). Laminin expression in serum from the cerebral ischemia group was higher than that in the sham-operated group. Compared with the model group, the level of laminin in the serum of the acupuncture group was significantly lower at each time point, especially at the 3-day, and 7-day time points (589.12±3.57 vs. 793.05±5.28, and 600.53±3.05 vs. 899.06±5.74, P <0.05). The level of integrin β1 in the serum from the acupuncture group was lower than that in the model group particularly at the 3-day and 7-day time points (208.66±0.95 vs. 280.83±1.77, and 212.36±0.95 vs. 316.77±2.42, P <0.05). Additionally, the model group and the acupuncture group showed dual peaks of integrin β1 and laminin expression at 3-day and 7-day. Acupuncture at GV 20 and ST 36 in rats alleviated CIRI and was associated with upregulated expression of miRNA 124 and with downregulated expression of integrin β1 and laminin in peripheral serum. These changes may represent one of the mechanisms underlying acupuncture's attenuation of CIRI.

Posterior vitreous detachment: more a case of the fibronectin interface than the inner limiting membrane?

PurposeUse the periodic acid shift and immunohistochemical staining to visualize the distribution of the inner limiting membrane components on eyes presenting Posterior Vitreal Detachment (PVD) as detected by Optical Coherence Tomography (OCT)MethodsPorcine eyes with PVD confirmed by OCT were processed into paraffin tissue blocks. Transverse, serial 15 mm thick sections were cut. After deparaffinization and antigen retrieval stages, slides were incubated with selected primary antibodies. Amplification stages were performed before fluorescence visualization of the specified marker. Among the markers stained were collagen II and IV, laminin beta 2 and fibronectin. Periodic acid shift staining was also performed on adjacent cross sections to assess potential structural change (PVD). Images were acquired on a Zeiss microscope and image analysis performed using Metamorph software (Leica).ResultsDifferent distribution patterns in the retina were observed for the selected markers. The inner retina blood vessels were labelled by laminin beta 2, collagen II and IV. Laminin beta 2 and collagen II was revealed on the Bruch membrane. Collagen II, IV, fibronectin and laminin beta 2 were detected at the inner limiting membrane. PVD area could be detected not only by periodic acid shift but also by fibronectin staining. Upon PVD, fibronectin staining completely migrated with the PVD front, cleanly detaching from the retinal cell layers.ConclusionsFibronectin staining can be used to detect and therefore confirm PVD presence in histological sections, even if the PVD has evolved beyond the point where it can still be detected by OCT.

Comparative proteome analysis of saccular intracranial aneurysms with iTRAQ quantitative proteomics

Objective. To screen differentially expressed proteins of saccular intracranial aneurysms and superficial temporal artery by the proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ) combined with reverse phase high-performance liquid chromatography. Methods. Collecting 17 samples from intracranial aneurysm patients undergoing aneurysmectomy as experiment group and 17 matched STA as control group. After quantification and enzymolysis of the protein, the iTRAQ were used to label the peptides of the 2 groups respectively. Then, the mixture of the peptides was fractioned by RP-HPLC and analyzed by LC-MS/MS to identify the differential expression proteins. Results. A total of 1699 proteins were identified from the ProteinPilot 4.5 software (AB SCIEX) using the Paragon database search algorithm. Comparing with STA, 54 proteins were significantly up-regulated (115:114<0.5-fold) and 37 were significantly down-regulated in sIAs (115:114>2.0-fold). Furthermore, Integrin β3, Secreted frizzled-related protein 2 were significantly up-regulated (2.3 fold and 2.1 fold, respectively), whereas MyosinIIb, Alpha-actinin-1, Laminin β2, and Carboxypeptidase A3 were down-regulated (3.01 fold, 2.1 fold, 2.07 fold, and 2.01 fold, respectively) in sIAs. GO Ontology analysis showed that most differential proteins expressed in cytoskeletal; up-regulated proteins in sIAs play an important role in inflammatory reaction, enzymatic hydrolysis, cell adhesion and invasion, and cellular immune reaction; down-regulated proteins in sIAs involved in cytoskeletal protein, enzyme, and structural protein. ITGB3, ACTN1 and MYL2 play a role in aneurysm formation via focal adhesion pathway. The results of Western-blot assay were consistent with the proteomic changes of those 6 proteins. Conclusion. The differentially expressed proteins in sIAs that showed aneurysm formation are related to cytoskeleton abnormal and extracellular matrix changes. The iTRAQ technology provides scientific foundation for the further study to explore the pathogenic mechanism of sIAs.

Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration.

The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade.

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression.

Androgen-Induced TMPRSS2 Activates Matriptase and Promotes Extracellular Matrix Degradation, Prostate Cancer Cell Invasion, Tumor Growth, and Metastasis

Dysregulation of androgen signaling and pericellular proteolysis is necessary for prostate cancer progression, but the links between them are still obscure. In this study, we show how the membrane-anchored serine protease TMPRSS2 stimulates a proteolytic cascade that mediates androgen-induced prostate cancer cell invasion, tumor growth, and metastasis. We found that matriptase serves as a substrate for TMPRSS2 in mediating this proinvasive action of androgens in prostate cancer. Further, we determined that higher levels of TMPRSS2 expression correlate with higher levels of matriptase activation in prostate cancer tissues. Lastly, we found that the ability of TMPRSS2 to promote prostate cancer tumor growth and metastasis was associated with increased matriptase activation and enhanced degradation of extracellular matrix nidogen-1 and laminin β1 in tumor xenografts. In summary, our results establish that TMPRSS2 promotes the growth, invasion, and metastasis of prostate cancer cells via matriptase activation and extracellular matrix disruption, with implications to target these two proteases as a strategy to treat prostate cancer.

Whole-exome sequencing reveals genetic variants associated with chronic kidney disease characterized by tubulointerstitial damages in North Central Region, Sri Lanka

The familial clustering observed in chronic kidney disease of uncertain etiology (CKDu) characterized by tubulointerstitial damages in the North Central Region of Sri Lanka strongly suggests the involvement of genetic factors in its pathogenesis. The objective of the present study is to use whole-exome sequencing to identify the genetic variants associated with CKDu. Whole-exome sequencing of eight CKDu cases and eight controls was performed, followed by direct sequencing of candidate loci in 301 CKDu cases and 276 controls. Association study revealed rs34970857 (c.658G>A/p.V220M) located in the KCNA10 gene encoding a voltage-gated K channel as the most promising SNP with the highest odds ratio of 1.74. Four rare variants were identified in gene encoding Laminin beta2 (LAMB2) which is known to cause congenital nephrotic syndrome. Three out of four variants in LAMB2 were novel variants found exclusively in cases. Genetic investigations provide strong evidence on the presence of genetic susceptibility for CKDu. Possibility of presence of several rare variants associated with CKDu in this population is also suggested.

125. Engineered Nucleases-Mediated In Situ Correction of a Genetic Defect By Homologous Recombination Into the Native Locus

Engineered nucleases specific for genomic targets are extensively used to generate DSBs that increase the rate and efficiency of homologous recombination (HR). We seek to determine the efficacy of nucleases in a clinical relevant genetic defect.The genetic defect we are addressing as model to test the nucleases-mediated genome editing technology is the junctional epidermolysis bullosa (JEB), a family of severe skin adhesion disorders due to autosomal recessive mutations in the LAMB3 gene coding for the laminin-332 heterotrimer, a key component of the dermal-epidermaljunction. Recently, we provided proof of principle that ZFN-mediated, AAVS1-targeted GFP addition can be achieved in human keratinocytes and in long-term repopulating epithelial stem cells in a validated preclinical model of xenotransplantation of human skin equivalents on immunodeficient mice.This project aims at the demonstration of a successful in situ correction of the LAMB3 gene in primary keratinocytes from Herlitz JEB patients. Recently TALEN-based gene correction for dystrophic EB has been reported. Similarly, we have developed a genome editing approach for JEB. In particular we have designed TALENs specific for the second intron of LAMB3 gene and a HR cassette including a splicible LAMB3 cDNA (from exon 3 to the end of the gene). In particular immortalized JEB keratinocytes were transfected with TALEN mRNAs and infected with an IDLV vector carrying the HR cassette. The in situ gene correction has been evaluated by site-specific PCR and knock-in expression of the corrected LAMB3 gene on bulk population. We then assessed targeting efficiency and specificity by extensive molecular analyses of single-cell clones isolated by limiting dilution from the TALENs/IDLV-treated immortalized JEB population. We isolated 256 clones and expanded 69 of them. Sixteen out of 69 clones showed an in vitro adhesion advantage, hosted the HR cassette correctly integrated into the predetermined locus, expressed the corrected LAMB3 gene and produced the laminin-332 protein. In parallel, CRISPR-Cas9 nuclease has been designed on the same locus to compare the transduction efficiency and cleavage activity and to translate the knock-in targeting platform to primary JEB keratinocytes.

LAMB1 polymorphism is associated with autism symptom severity in Korean autism spectrum disorder patients

Background: LAMB1 encodes laminin beta-1, which is expressed during early development of the human nervous system, and could be involved in the pathogenesis of neurodevelopmental disorders. Aims: In our study, we aimed to investigate whether single nucleotide polymorphisms (SNPs) in LAMB1 were associated with autism spectrum disorder (ASD) and with related clinical severities of ASD. Methods: Two coding SNPs (rs20556 and rs25659) and two intronic SNPs (rs2158836 and rs2237659) were compared between 180 patients with ASD and 147 healthy control subjects using direct sequencing. The Korean version of the Childhood Autism Rating Scale (K-CARS) was used to assess clinical severities. Multiple logistic regression models were employed to analyze genetic data, and associations with symptom severity were tested with the Kruskal–Wallis and the Mann–Whitney U tests. Results: None of the four examined SNPs was associated with ASD risk. However, the GG genotype of rs2158836 was associated with more severe symptoms for the “object use” and “non-verbal communication” measures. Conclusions: The results of our study suggest the association between rs2158836 polymorphisms and symptom severity in ASD.

Laminin β3 expression as a prognostic factor and a predictive marker of chemoresistance in colorectal cancer.

Laminin-332, a marker of epithelial-mesenchymal transition, is composed of a heterotrimer of α3, β3 and γ2 chains that regulates cell adhesion and migration. This study aimed to disclose the respective clinical significance of laminin β3 immunoexpression in colorectal cancer as a prognostic factor and a predictive marker of chemoresistance. Tissue specimens from 323 Stage II and 232 Stage III colorectal cancer patients who underwent curative resection were assessed using laminin β3 immunostaining. Among Stage III colorectal cancer patients, comparisons of 5-year disease-free survival rates revealed a poorer prognosis for the laminin β3-high group than for the laminin β3-low group (52.3 vs. 70.7%, P = 0.038), while there was no significant difference among Stage II patients. Among laminin β3-low Stage III patients, those who received adjuvant chemotherapy showed marginally better disease-free survival than those who did not receive it (75.8 vs. 62.8%; P = 0.096). Furthermore, multivariate analysis corroborated a distinct benefit of adjuvant chemotherapy in laminin β3-low patients (P = 0.035; hazard risk ratio = 1.66). Analyses of the laminin β3-high group, however, failed to show significance. Laminin β3 chain immunoreactivity was a poor prognostic factor for Stage III colorectal cancer patients, and laminin β3-high patients of Stage III colorectal cancer derived no survival benefit from adjuvant chemotherapy.

Increased proliferation and adhesion properties of human dental pulp stem cells in PLGA scaffolds via simulated microgravity.

To explore the possibility of utilizing a rotary cell culture system (RCCS) to model simulated microgravity and investigate its effects on the proliferation, adhesion, migration and cytoskeletal organization of human dental pulp stem cells (hDPSCs) on poly (lactic-co-glycolic acid) (PLGA) scaffolds. Isolated and identified hDPSCs grown on PLGA scaffolds were exposed to simulated microgravity (SMG) or normal gravity (NG) conditions for 3 days. MTT cell proliferation assays, BrdU incorporation assays, flow cytometry analysis and Western blotting were undertaken to identify the proliferation ability of hDPSCs under SMG conditions. Additionally, immunofluorescence detection, SEM observations and cell migration and adhesion assays were performed to compare adhesion, migration and cytoskeletal changes in hDPCSs subjected to SMG conditions. To further investigate the mechanisms, human pathway-focused matrix and adhesion PCR array analyses were performed. The Student's t-test was used for statistical analyses. SMG promoted proliferation and adhesion, decreased migration and reorganized the cytoskeletal organization of hDPSCs compared with the NG group. PCR array analyses revealed that following SMG treatment, ITGA6 (integrin alpha-6), ITGAV (integrin alpha-V), ITGB1 (integrin beta-1), LAMB1 (laminin beta-1) and TNC (tenascin-C) were significantly upregulated (P < 0.05). SMG may regulate the behaviour of hDPSCs grown in PLGA scaffolds in an integrin-mediated manner, which may contribute to tooth tissue engineering by increasing their expandability and scaffold adhesion.

Genetic determinants associated with early age of diagnosis of IBD.

Inflammatory bowel disease (IBD) is typically diagnosed at 20 to 40 years of age. However, very young versus elderly patients with IBD may have different mechanisms of disease that may affect prognosis and care. The purpose of this work was to identify single nucleotide polymorphisms associated with age of onset of Crohn's disease and ulcerative colitis. Patients were genotyped using a custom microarray chip containing 332 IBD-associated single nucleotide polymorphisms. Age at diagnosis as a continuous variable was assessed using linear regression. Patients were then subgrouped by age at diagnosis and compared by the Fisher exact test. Bonferroni correction was used in all of the analyses. This study was conducted at a tertiary academic hospital. Sixty patients with Crohn's disease and 26 with ulcerative colitis were ≤ 16 years old, 259 patients with Crohn's disease and 248 with ulcerative colitis were 17-60 years old, and 10 patients with Crohn's disease and 20 with ulcerative colitis were >60 years old at diagnosis and included in this study. Age at diagnosis and single nucleotide polymorphism correlations were measured in this study. The NOD2 single nucleotide polymorphism rs2076756 was associated with younger age at Crohn's disease diagnosis (p = 0.0002). Patients with the AA/wild-type genotype were diagnosed at 31.9 ± 1.23 years, AG heterozygotes at 25.6 ± 0.99 years, and GG/at-risk allele homozygotes at 22.6 ± 1.32 years. Depending on age categories compared, single nucleotide polymorphisms in POU5F1, TNFSF15, and HLA DRB1*501 were associated with age of Crohn's disease diagnosis. No genetic associations were seen between ulcerative colitis and linear age at diagnosis; however, the G allele of the LAMB1 single nucleotide polymorphism rs886774 was found to be associated with ulcerative colitis diagnosed at ≤ 16 versus >17 years old (p = 0.008). This study was limited to known IBD single nucleotide polymorphisms. This analysis reaffirms the association between NOD2, a molecule of innate immunity, and early Crohn's disease onset. This is the first report of a possible association between early Crohn's disease and the POU5F1, TNFSF15, and HLA DRB1*501 genes. The LAMB1 gene, associated with mucosal basement membrane integrity, was associated with early onset ulcerative colitis and, thus, suggests a fundamentally different mechanism of early disease pathogenesis in ulcerative colitis versus Crohn's disease.

A New Mouse Model of Junctional Epidermolysis Bullosa: The LAMB3 628G>A Knockin Mouse

Junctional epidermolysis bullosa (JEB) is a group of recessively inherited genodermatoses, characterized by tissue separation in the epidermal basement membrane due to defective anchoring proteins. The lethal Herlitz type of this disorder is caused by absence of laminin332. Affected individuals suffer from widespread erosions of skin and mucous membranes and very often die within the first year of life. Mouse models lacking the a3or g2-chain of laminin-332 have been developed and a spontaneous s3-knockout mouse exists, but all die shortly after birth. We generated a new mouse model of JEB by knockin of the point mutation 628G>A (p.E210K) in the gene encoding the laminin-332 s3chain, LAMB3. In compound heterozygous humans, this mutation has always been associated with lifelong skin blistering without reduced life expectancy. Fourteen homozygous LAMB3 628G>A knockin mice were analyzed. Most of them showed skin blistering with tissue separation in the basement membrane soon after birth. Laminin-332 was almost completely absent. None of the homozygous LAMB3 628G>A knockin mice survived longer than 72 hours. LAMB3 gene expression levels in heterozygous and homozygous LAMB3 628G>A knockin mice, however, were similar to wild-type mice. Analysis of the LAMB3 transcript revealed alternative splicing in homozygous LAMB3 628G>A knockin mice: a 64 base-pair deletion of exon 7 led to a frame-shift and a premature termination codon. Due to alternative splicing, the phenotype of this new mouse model resembles that of knockout mice. The LAMB3 628G>A knockin mouse may contribute to a better understanding of the molecular basis of JEB. Authors’ details Department of Pediatrics and Adolescent Medicine, University Hospital Erlangen, Erlangen, Germany. Department of Biology, Nikolaus-FiebigerCenter for Molecular Medicine, Friedrich-Alexander-University ErlangenNurnberg, Erlangen, Germany.

Protective actions of PJ34, a poly(ADP-ribose)polymerase inhibitor, on the blood–brain barrier after traumatic brain injury in mice

Poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays an important role in traumatic brain injury (TBI). The objective of this study was to investigate whether PARP activation participated in the blood–brain barrier (BBB) disruption and edema formation in a mouse model of controlled cortical impact (CCI). N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) (10mg/kg), a selective PARP inhibitor, was administered intraperitoneally at 5min and 8h after experimental CCI. After 6h and 24h of CCI, the permeability of the cortical BBB was determined after Evans Blue administration. The water content of the brain was also measured. Treatment with PJ34 markedly attenuated the permeability of the BBB and decreased the brain edema at 6h and 24h after CCI. Our data showed the up-regulation of nuclear factor-κB in cytosolic fractions and nuclear fractions in the injured cortex, and these changes were reversed by PJ34. Moreover, PJ34 significantly lessened the activities of myeloperoxidase and the levels of matrix metalloproteinase-9, enhanced the levels of occludin, laminin, collagen IV and integrin β1, reduced neurological deficits, decreased the contusion volume, and attenuated the necrotic and apoptotic neuronal cell death. These data suggest the protective effects of PJ34 on BBB integrity and cell death during acute TBI.

Modifications in integrin expression and extracellular matrix composition in children with biliary atresia.

The aetiology of biliary atresia (BA) is still unresolved. The study's aim was to investigate the distribution of extracellular matrix proteins and cellular adhesion molecules in children with BA compared to other cholestatic liver disease (CLD) and normal liver architecture (NLA). Liver biopsies were obtained from children with BA (n=13), CLD (n=6) and NLA (n=8). We systematically analysed ultra thin frozen sections from the liver hilum stained with 25 monoclonal antibodies for cellular characterisation, extracellular matrix proteins and adhesion molecules. 2 changes were specifically found in BA: laminin beta1 was reduced in children with BA vs. NLA and CLD. Conversely, integrin alpha 3 was increased in BA vs. NLA and CLD (p<0.05). Furthermore, we detected changes in a similar pattern for both BA and CLD vs. NLA: in BA and CLD perlecan was increased. On the contrary, integrin beta1 and entactin were decreased vs. NLA (p<0.05). Extracellular matrix proteins and adhesion molecules mediate cellular polarity and integrity, development of tubular structures, and proliferation. Therefore, our findings can be important for the understanding of the genesis of BA. The composition of extracellular matrix proteins and adhesion molecules in children with BA differs from NLA and other CLD in distribution of laminin beta1 and integrin alpha 3, which may have implications for genetic, immunologic and environmental associations in BA.

Pierson syndrome: A case report with a neonatal cardiac association based on a novel mutation in the LAMB2 gene

Congenital nephrotic syndrome (CNS) combined with eye abnormalities including microcoria (small pupils that don't respond to light) and abnormal lens shape can suggest a clinical diagnosis of Pierson syndrome (which mainly affects the kidneys and eyes). Mutations in the genes NPHS1, NPHS2, and WT1 are known to account for the majority of CNS cases, whereas a definitive diagnosis of Pierson syndrome can be established by the detection of a causative mutation in both copies of a patient's LAMB2 gene (encoding laminin β2). CNS can manifest in utero (ultrasound may reveal hyperechogenic kidneys and oligohydramnios), or during the first 3 months of life. Pierson syndrome is an autosomal recessive disorder comprised of CNS and distinct ocular abnormalities. The prognosis of this extremely rare disorder is very poor, with the most babies developing end-stage renal disease. Those who do survive tend to show the neurodevelopmental delay and visual loss. We report a case of neonatal Pierson syndrome in conjunction with complex cyanotic cardiac disease in which a novel homozygous mutation in the LAMB2 gene was detected. The clinical association of Pierson syndrome with heart manifestation is a novel finding, reported here for the 1 st time.