Cellular strain avoidance is mediated by a functional actin cap - observations in an Lmna-deficient cell model.

In adherent cells, the relevance of a physical mechanotransduction pathway provided by the perinuclear actin cap stress fibers has recently emerged. Here, we investigate the impact of a functional actin cap on the cellular adaptive response to topographical cues and uniaxial cyclic strain. Lmna-deficient fibroblasts are used as a model system because they do not develop an intact actin cap, but predominantly form a basal layer of actin stress fibers underneath the nucleus. We observe that topographical cues induce alignment in both normal and Lmna-deficient fibroblasts, suggesting that the topographical signal transmission occurs independently of the integrity of the actin cap. By contrast, in response to cyclic uniaxial strain, Lmna-deficient cells show a compromised strain avoidance response, which is completely abolished when topographical cues and uniaxial strain are applied along the same direction. These findings point to the importance of an intact and functional actin cap in mediating cellular strain avoidance.

A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death.

Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.

Birt-Hogg-Dube syndrome is a novel ciliopathy.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Assessment of fibroblast nuclear morphology aids interpretation of LMNA variants.

The phenotypic heterogeneity of Lamin A/C (LMNA) variants renders it difficult to classify them. As a consequence, many LMNA variants are classified as variant of unknown significance (VUS). A number of studies reported different types of visible nuclear abnormalities in LMNA-variant carriers, such as herniations, honeycomb-like structures and irregular Lamin staining. In this study, we used lamin A/C immunostaining and nuclear DAPI staining to assess the number and type of nuclear abnormalities in primary dermal fibroblast cultures of laminopathy patients and healthy controls. The total number of abnormal nuclei, which includes herniations, honeycomb-structures, and donut-like nuclei, was found to be the most discriminating parameter between laminopathy and control cell cultures. The percentage abnormal nuclei was subsequently scored in fibroblasts of 28 LMNA variant carriers, ranging from (likely) benign to (likely) pathogenic variant. Using this method, 27 out of 28 fibroblast cell cultures could be classified as either normal (n = 14) or laminopathy (n = 13) and no false positive results were obtained. The obtained specificity was 100% (CI 40-100%) and sensitivity 77% (46-95%). We conclude that assessing the percentage of abnormal nuclei is a quick and reliable method, which aids classification or confirms pathogenicity of identified LMNA variants causing formation of aberrant lamin A/C protein.

No HLA-A gene detectable on one of the haplotypes in a Caucasian family.

An unusual haplotype was detected in a family of a caucasian transplant patient. Human leukocyte antigen (HLA) analysis of the family demonstrated the absence of HLA-A on one of the haplotypes present in two family members. One was serologically typed A24, the other A2. Because they had one haplotype in common, the HLA-A allele of the shared haplotype was supposed to be a null allele. Different molecular typing methods identified only one allele in both individuals. The results suggest a deletion of the complete HLA-A gene or a major part of it. For confirmation, microsatellite analysis of the HLA-A region was performed with six microsatellite markers. Both family members were heterozygous for all markers, and a deletion of HLA-A could not be proven. Fluorescent in situ hybridization (FISH) was performed with cosmid and PAC probes encompassing the HLA-A gene. Both probes demonstrated an identical normal distribution pattern for diploid results. The absence of any serologic and molecular reaction with the results of the microsatellite and FISH analysis make a deletion of a narrow region, encompassing the HLA-A gene, the most plausible explanation.

Soft substrates normalize nuclear morphology and prevent nuclear rupture in fibroblasts from a laminopathy patient with compound heterozygous LMNA mutations.

Laminopathies, mainly caused by mutations in the LMNA gene, are a group of inherited diseases with a highly variable penetrance; i.e., the disease spectrum in persons with identical LMNA mutations range from symptom-free conditions to severe cardiomyopathy and progeria, leading to early death. LMNA mutations cause nuclear abnormalities and cellular fragility in response to cellular mechanical stress, but the genotype/phenotype correlations in these diseases remain unclear. Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome. We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures. No malformations were seen in healthy control cells at any substrate stiffness. In addition, analysis of the actin cytoskeleton organization in this laminopathy cells demonstrates that the onset of nuclear abnormalities correlates to an increase in cytoskeletal tension. Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility. This assay may be useful in predicting patient-specific phenotypic development and in investigations on the underlying mechanisms of nuclear and cellular fragility in laminopathies.

Porokeratotic eccrine nevus may be caused by somatic connexin26 mutations.

Porokeratotic eccrine ostial and dermal duct nevus, or porokeratotic eccrine nevus (PEN), is a hyperkeratotic epidermal nevus. Several cases of widespread involvement have been reported, including one in association with the keratitis-ichthyosis-deafness (KID) syndrome (OMIM #148210), a rare disorder caused by mutations in the GJB2 gene coding for the gap junction protein connexin26 (Cx26). The molecular cause is, as yet, unknown. We have noted that PEN histopathology is shared by KID. The clinical appearance of PEN can resemble that of KID syndrome. Furthermore, a recent report of cutaneous mosaicism for a GJB2 mutation associated with KID describes linear hyperkeratotic skin lesions that might be consistent with PEN. From this, we hypothesized that PEN might be caused by Cx26 mutations associated with KID or similar gap junction disorders. Thus, we analyzed the GJB2 gene in skin samples from two patients referred with generalized PEN. In both, we found GJB2 mutations in the PEN lesions but not in unaffected skin or peripheral blood. One mutation was already known to cause the KID syndrome, and the other had not been previously associated with skin symptoms. We provide extensive functional data to support its pathogenicity. We conclude that PEN may be caused by mosaic GJB2 mutations.

Identification of chromosome 9 alterations and p53 accumulation in isolated carcinoma in situ of the urinary bladder versus carcinoma in situ associated with carcinoma.

Carcinoma in situ (CIS) of the urinary bladder is a flat, aggressive lesion and may be the most common precursor of invasive bladder cancer. Although chromosome 9 alterations are among the earliest and most prevalent genetic alterations in bladder cancer, discrepancy exists about the frequency of chromosome 9 losses in CIS. We analyzed 22 patients with CIS of the bladder (15 patients with isolated CIS, 7 patients combined with synchronous pTa or pT1 carcinomas) for gains and losses of chromosome (peri)centromere loci 1q12, 7p11-q11, 9p11-q12, and 9p21 harboring the INK4A/ARF locus (p16(INK4A)/p14(ARF)) and INK4B (p15(INK4B)) by multiple-target fluorescence in situ hybridization, and for p53 protein accumulation by immunohistochemistry. In 15 of 20 (75%) CIS lesions analyzed p53 overexpression was detected, whereas aneusomy for chromosomes 1 and 7 was identified in 20 of 22 (91%) CIS. In 13 of 22 (60%) CIS cases analyzed, 12 of which were not associated with a synchronous pTa or pT1 carcinoma, no numerical losses for chromosome 9 (p11-q12 and 9p21) were detected as compared with chromosomes 1 and 7. Furthermore 6 of 12 (50%) patients showed a metachronous invasive carcinoma within 2 years. In the remaining nine biopsies CIS lesions (40%) were recognized that showed losses of chromosome 9p11-q12 and 9p21, six of these were associated with a synchronous pTa or pT1 carcinoma. Three of these carcinomas were pTa and exhibited loss of 9q12 as well as a homozygous deletion of 9p21. The others were invasive carcinomas in which CIS lesions were also recognized that showed no numerical loss of chromosome 9, but did show an accumulation of p53. In conclusion our data demonstrate that predominantly isolated CIS lesions contained cells with no specific loss of chromosome 9, as opposed to CIS lesions with synchronous carcinomas that showed evidence of chromosome 9 loss. Furthermore our data strengthen the proposition that p53 mutations (p53 overexpression) precede loss of chromosomes 9 and 9p21 in CIS as precursor for invasive bladder cancer, as opposed to noninvasive carcinomas where chromosome 9 (9p11-q12) losses are early and frequently combined with homozygous deletions of 9p21.