SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Rabbits (Oryctolagus cuniculus) increase caecal calcium absorption at increasing dietary calcium levels.

Hindgut fermenting herbivores from different vertebrate taxa, including tortoises, and among mammals some afrotheria, perissodactyla incl. equids, several rodents as well as lagomorphs absorb more calcium (Ca) from the digesta than they require, and excrete the surplus via urine. Both proximate and ultimate causes are elusive. It was suggested that this mechanism might ensure phosphorus availability for the hindgut microbiome by removing potentially complex-building Ca from the digesta. Here we use Ussing chamber experiments to show that rabbits (Oryctolagus cuniculus) maintained on four different diets (six animals/diet) increase active Ca absorption at increasing Ca levels. This contradicts the common assumption that at higher dietary levels, where passive uptake should be more prevalent, active transport can relax and hence supports the deliberate removal hypothesis. In the rabbits, this absorption was distinctively higher in the caecum than in the duodenum, which is unexpected in mammals. Additional quantification of the presence of two proteins involved in active Ca absorption (calbindin-D9K CB; vitamin D receptor, VDR) showed higher presence with higher dietary Ca. However, their detailed distribution across the intestinal tract and the diet groups suggests that other factors not investigated in this study must play major roles in Ca absorption in rabbits. Investigating strategies of herbivores to mitigate potential negative effects of Ca in the digesta on microbial activity and growth might represent a promising area of future research.

Relationships between neurotransmitter receptor densities and expression levels of their corresponding genes in the human hippocampus.

Neurotransmitter receptors are key molecules in signal transmission, their alterations are associated with brain dysfunction. Relationships between receptors and their corresponding genes are poorly understood, especially in humans. We combined in vitro receptor autoradiography and RNA sequencing to quantify, in the same tissue samples (7 subjects), the densities of 14 receptors and expression levels of their corresponding 43 genes in the Cornu Ammonis (CA) and dentate gyrus (DG) of human hippocampus. Significant differences in receptor densities between both structures were found only for metabotropic receptors, whereas significant differences in RNA expression levels mostly pertained ionotropic receptors. Receptor fingerprints of CA and DG differ in shapes but have similar sizes; the opposite holds true for their "RNA fingerprints", which represent the expression levels of multiple genes in a single area. In addition, the correlation coefficients between receptor densities and corresponding gene expression levels vary widely and the mean correlation strength was weak-to-moderate. Our results suggest that receptor densities are not only controlled by corresponding RNA expression levels, but also by multiple regionally specific post-translational factors.

Arginine- but not alanine-rich carboxy-termini trigger nuclear translocation of mutant keratin 10 in ichthyosis with confetti.

Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant-negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)-terminus, from poly-glycine to either a poly-arginine or -alanine tail. Previous studies on the type of C-terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C-termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C-terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine-rich C-terminus. However, this was not observed with K10 featuring an alanine-rich C-terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild-type and truncated forms of K10. This study demonstrates that, of the various 3' frameshift variants of KRT10, exclusively arginine-rich C-termini lead to nuclear localization of K10.

Risk of Cutaneous Squamous Cell Carcinoma Development in Renal Transplant Recipients Is Independent of TMC/EVER Alterations.

BACKGROUND: Renal transplant recipients (RTRs) have an increased risk of developing nonmelanoma skin cancer, mainly cutaneous squamous cell carcinoma (cSCC). Two genes (TMC6/EVER1 and TMC8/EVER2), mutated in epidermodysplasia verruciformis (EV) patients with an increased risk of cSCC development, contain numerous single-nucleotide polymorphisms (SNPs). AIM: To evaluate the effect of SNPs in both TMC/EVER genes on the different susceptibilities of RTRs to cSCC. METHOD: We determined the occurrence of cSCC in 105 RTRs who were transplanted at least 7 years previously and investigated the frequency of 26 SNPs within both TMC/EVER genes in severely affected (n = 16) as well as in nonaffected RTRs (n = 25). RESULTS: Our data did not indicate a significant association between any SNP genotype and risk of cSCC development in RTRs. CONCLUSION: To clarify the correlation between SNPs in both TMC genes and cSCC development in RTRs, integrated investigations of large cohorts including both RTRs and immunocompetent individuals with consideration of cSCC status, SNP genotype and human papillomavirus status might be necessary.

A mutation in a skin-specific isoform of SMARCAD1 causes autosomal-dominant adermatoglyphia.

Monogenic disorders offer unique opportunities for researchers to shed light upon fundamental physiological processes in humans. We investigated a large family affected with autosomal-dominant adermatoglyphia (absence of fingerprints) also known as the "immigration delay disease." Using linkage and haplotype analyses, we mapped the disease phenotype to 4q22. One of the genes located in this interval is SMARCAD1, a member of the SNF subfamily of the helicase protein superfamily. We demonstrated the existence of a short isoform of SMARCAD1 exclusively expressed in the skin. Sequencing of all SMARCAD1 coding and noncoding exons revealed a heterozygous transversion predicted to disrupt a conserved donor splice site adjacent to the 3' end of a noncoding exon uniquely present in the skin-specific short isoform of the gene. This mutation segregated with the disease phenotype throughout the entire family. Using a minigene system, we found that this mutation causes aberrant splicing, resulting in decreased stability of the short RNA isoform as predicted by computational analysis and shown by RT-PCR. Taken together, the present findings implicate a skin-specific isoform of SMARCAD1 in the regulation of dermatoglyph development.

MicroRNA expression differs in cutaneous squamous cell carcinomas and healthy skin of immunocompetent individuals.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers, but the influence of microRNA (miRNA) expression has only been sporadically analysed. We hypothesized that miRNAs are differentially expressed in cSCC and hence influence its development. We therefore isolated total miRNA from well-differentiated cSCCs and from controls without SCC. Expression analyses of 12 miRNAs showed three significantly differentially expressed miRNAs. We identified a significant upregulation of the miR-21 and the miR-31, a proto-oncogene like miR-21. While the upregulated expression of miR-21 has been known for some time, the increased expression of miR-31 was never shown so clearly. Furthermore, we showed the upregulation of miRNA-205, which has never been described before. The miR-205 induces specific keratinocyte migration and could be a characteristic marker for cSCC. It has to be determined in following studies whether these upregulated expressions are specific for cSCC and if so, for which cSCC stages.

Recessive SERPING1 Variant Leads to Kinin-Kallikrein System Control Failure in a Consanguineous Brazilian Family with Hereditary Angioedema.

Background: Hereditary angioedema (HAE) is a severe and potentially life-threatening disease. The most common forms are caused by variants in SERPING1, resulting in C1-inhibitor (C1-INH) deficiency (HAE-C1-INH). C1-INH is a serine protease inhibitor (SERPIN) that regulates multiple proteases pathways, including the kallikrein-kinin system (KKS) and its complement. In HAE-C1-INH patients, C1-INH deficiencies affect KKS control, resulting in the development of kallikrein activity in plasma and the subsequent release of bradykinin (BK). While the overwhelming majority of disease-causing SERPING1 variants are dominant, very few recessive variants have been described. We present a large Brazilian HAE-C1-INH family with a recessive form of HAE-C1-INH. Methods: Blood samples of family members were investigated for protein levels of C1-INH, C4, C1q, and C1-INH function. The SERPING1 gene was sequenced. Results: In two severely affected sisters, we identified a homozygous missense variant in SERPING1 (NM_000062.3:c.964G>A;p.Val322Met). Fourteen family members were asymptomatic heterozygous carriers of the variant. Data regarding C1-INH function in the plasma showed that homozygous p.Val322Met strongly impacts C1-INH function to inhibit C1s and kallikrein (PKa). When heterozygously expressed, it affects the C1-INH control of C1s more than that of PKa. Conclusions: These studies of the variant's effects on the structure-function relationship reinforce prior observations suggesting that C1-INH deficiency is a conformational disease.

Prevalence of skin lesions in familial adenomatous polyposis: a marker for presymptomatic diagnosis?

BACKGROUND AND AIMS: Benign skin tumors such as lipomas, fibromas, and epidermal cysts are among the extracolonic manifestations of familial adenomatous polyposis (FAP). Readily detectable by inspection, they could serve as presymptomatic diagnostic markers to identify FAP patients. We therefore prospectively determined the prevalence of cutaneous lesions in genetically confirmed adenomatous polyposis coli (APC) mutation carriers and assessed their potential usefulness in the identification of FAP patients. METHODS: Whole-skin examination was performed in 56 adult APC mutation carriers, compared with a control group (n = 116). In addition, FAP patients were investigated for the presence of congenital hypertrophy of the retinal pigment epithelium (CHRPE), an established clinical marker for FAP, and a detailed review of medical records was performed. RESULTS: Nearly half of all FAP patients (48.2%) had at least one FAP-associated skin lesion, compared with one third (34.5%) of controls. Only multiple lipomas and combined skin lesions were significantly more prevalent in APC mutation carriers. CHRPE was observed in 22 (43.1%) of 51 FAP patients, including 14 (37.8%) of 37 individuals with APC mutations outside the CHRPE-associated region between codons 311 and 1465. CONCLUSIONS: Despite a significantly higher prevalence of multiple lipomas, occurring at younger age, and combined skin lesions in APC mutation carriers, the low diagnostic sensitivity of FAP-associated skin lesions precludes their use as markers for FAP in clinical practice. Based on our findings, the common CHRPE-associated region should be extended to APC codons 148-2043.

The immigration delay disease: adermatoglyphia-inherited absence of epidermal ridges.

In the digital age, personal identification by fingerprints (epidermal ridges) has become more frequent and is often required for biometric passports. The more fingerprints are analyzed, the more variants in their formation are documented. Individuals completely missing fingerprints as an isolated finding are extremely rare. Only 4 kindreds have been described to date, with additional clinical features in most cases. We describe a female patient with missing epidermal ridges on the fingers, palms, toes, and soles as an isolated feature. Absent fingerprints, or adermatoglyphia, were inherited over 4 generations of her family in an autosomal dominant fashion. We present the clinical features of the index patient, and compare the case with previous reports in the literature. Because of problems in personal identification, this embryologic malformation caused the patient significant difficulties when traveling to other countries, which is why we name it the immigration delay disease.

Homozygosity for the c.917A→T (p.N306l) polymorphism in the EVER2/TMC8 gene of two sisters with epidermodysplasia verruciformis Lewandowsky-Lutz originally described by Wilhelm Lutz.

BACKGROUND: Epidermodysplasia verruciformis Lewandowsky-Lutz (EV) is a rare genodermatosis, characterised by development of numerous verrucous skin lesions caused by specific genotypes of human papillomaviruses belonging to the ß-papillomavirus genus. The EV loci were mapped to chromosome 2p21-p24 (EV2) and 17q25 (EV1). On chromosome 17, 2 adjacent related genes--EVER1/TMC6 and EVER2/TMC8--were identified. We reinvestigated 2 patients originally described by Wilhelm Lutz in 1946 with the aim to document the natural course of the disease and confirm his diagnosis. METHODS: PCR fragments specific for exons with short flanking intron sequences of EVER1/TMC6 and EVER2/TMC8 genes from patients' DNA were amplified using sequence information. The single-nucleotide polymorphism (SNP) rs7208422 was studied, using restriction fragment length polymorphism analysis. RESULTS: In the index patient, we identified a homozygous TT genotype in exon 8 of the EVER2/TMC8 gene (c.917A→T, p.N306I). The same mutation could thereafter be detected in her sister from paraffin-embedded skin. CONCLUSION: We have followed one of the first patients described with EV in Basel, Switzerland, in 1930 until today and demonstrated the TT genotype (SNP rs7208422) in the EVER2/TMC8 gene in this index patient and her sister. The results underline the possible relevance of SNP rs7208422 by influencing the susceptibility to ß-papillomaviruses and their oncogenic potential.

Buschke-Ollendorff syndrome in a three-generation family: influence of a novel LEMD3 mutation to tropoelastin expression.

Buschke-Ollendorff syndrome refers to the concomitant occurrence of connective tissue nevi, composed of elastic fibers in most cases, with osteopoikilosis. This autosomal dominant inherited disorder is caused by mutations in the gene LEMD3 on chromosome 12q14, which induces a rather heterogeneous clinical phenotype. Here, we report on the most proximal germline mutation found to date in the LEMD3 gene, p.Val94fs, in a three-generation Swiss family. Quantitative RNA analyses in affected and non-affected skin tissue from the proband demonstrate a comparable nonsense-mediated decay of mutant LEMD3 mRNA in both tissues; however, different levels of tropoelastin expression suggest that additional factors are involved in the development of the cutaneous lesions.

[Genodermatoses for practitioners--principles and concepts]. / Genodermatosen in der täglichen Praxis - die Rolle des Dermatologen.

Every disease is a mirror of interactions between genes and the environment. In monogenic disorders only one mutation can lead to a specific phenotype. However the spectrum and the degree of manifestations depend on numerous factors from the environment. Ichthyosis vulgaris is caused by a mutation in the filaggrin gene. However the phenotype is much more pronounced in the winter months. In polygenic disorders such as atopic dermatitis numerous modifying genes influence the phenotype including a mutation in filaggrin. The skin is the organ of the human body which is most commonly involved in monogenic diseases. More than one third of all genetic diseases affect the integument. At the very moment more than 350 genodermatoses are identified with functional insights. The Human Genome Project was finished in 2001 with the aim that all genes can be identified for diagnostics, pharmacogenomics potential gene therapy and to understand the principle basis of diseases. The next project called ENCODE for Enzyclopedia of DNA Elements targets to identify all functional elements in the human genome sequence. MicroRNAs seem to have great importance for the regulation of genefunctions in the skin. At the moment epigenetics is at the epicentre of modern medicine. Epigenetics is the study of non-DNA sequence-related heredity. Epigenetics is an important tool to study the relationship between the genome and the environment. In the second part cases will be presented and the way of diagnosis making will be shown. It will be shown that it is very important to find clinical key features which may allow an allocation to a genetic pathway.

Gene expression is stable in a complete CIB1 knockout keratinocyte model.

Epidermodysplasia verruciformis (EV) is a genodermatosis characterized by the inability of keratinocytes to control cutaneous ß-HPV infection and a high risk for non-melanoma skin cancer (NMSC). Bi-allelic loss of function variants in TMC6, TMC8, and CIB1 predispose to EV. The correlation between these proteins and ß-HPV infection is unclear. Its elucidation will advance the understanding of HPV control in human keratinocytes and development of NMSC. We generated a cell culture model by CRISPR/Cas9-mediated deletion of CIB1 to study the function of CIB1 in keratinocytes. Nine CIB1 knockout and nine mock control clones were generated originating from a human keratinocyte line. We observed small changes in gene expression as a result of CIB1 knockout, which is consistent with the clearly defined phenotype of EV patients. This suggests that the function of human CIB1 in keratinocytes is limited and involves the restriction of ß-HPV. The presented model is useful to investigate CIB1 interaction with ß-HPV in future studies.

Mosaic manifestations of monogenic skin diseases.

A genetic mosaic is defined as an organism which is composed of genetically different cell lines which originate from a homogeneous zygote. Etiologically, cutaneous mosaics can be divided into two large categories, epigenetic mosaicism and genomic mosaicism. Genomic mosaics which have two or more genetically different cell populations are not inherited with the exception of para-dominant inheritance pattern. Epigenetic mosaics have a structurally homogeneous cell population but there are functional differences induced by modifying factors in the form of gene-steering retroviral elements that can be inherited. We distinguish five different manifestation patterns of mosaicism, including the Blaschko lines pattern, patchy pattern without midline separation, checkerboard pattern, phylloid pattern and lateralization pattern. All forms of epigenetic mosaicism, including the various patterns of X-inactivation, appear to be caused by the action of retrotransposons. A new concept is functional autosomal mosaicism transmittable through the action of retrotransposons

The use of a colon cancer associated nuclear antigen CCSA-2 for the blood based detection of colon cancer.

The early diagnosis of colorectal cancer (CRC) is central for effective treatment, as prognosis is directly related to the stage of the disease. Development of tumor markers found in the blood from patients, which can detect CRC at an early stage, should have a major impact in morbidity and mortality of this disease. The nuclear matrix is the structural scaffolding of the nucleus and specific nuclear matrix proteins (NMPs) have been identified as an "fingerprint" for various cancer types. Previous studies from our laboratory have identified four colon cancer associated NMPs termed colon cancer-specific antigen (CCSA)-2 to (CCSA)-5. The objective of the present study was to analyze the expression of one of these proteins, CCSA-2 in serum from various patient populations and to determine whether CCSA-2 antibodies could be used in a clinically applicable serum-based immunoassay specifically to detect colon cancer. Using an indirect ELISA, which detects CCSA-2, the protein was measured in the serum from 174 individuals, including healthy individuals, patients with colon cancer, patients with diverticulosis, colon polyps, inflammatory bowel disease (IBD) as well as other cancer types. With a predetermined cutoff absorbance of 0.6 OD we have successfully utilized this approach to develop an immunoassay that detected colon cancer. The immunoassay showed a sensitivity of 88.8% (24/27) and an overall specificity of 84.2% (106/127). This initial study showed the potential of CCSA-2 to serve as a highly specific blood based marker for colon cancer. Although potentially promising, the results of this study must be confirmed in larger independent validation studies.

Significance of coprophagy for the fatty acid profile in body tissues of rabbits fed different diets.

Four groups of eight New Zealand hybrid rabbits were fattened with ad libitum access to the following pelleted experimental diets: ryegrass meal or alfalfa meal fed either alone or with oats meal in a ratio of 1:1. After 25 weeks they were slaughtered and dissected. Fatty acid (FA) profiles of caecotrophs (re-ingested fermentation products of the caecum), perirenal adipose tissue and intramuscular fat in the Musculus quadriceps were determined. With high proportions of branched-chain FA (BFA) and trans FA, and increased proportions of saturated FA relative to the diets, the caecotroph FA profile showed a clear fingerprint of anaerobe microbial lipid metabolism including biohydrogenation. By contrast, the FA profiles of adipose and lean tissue comprised high proportions of polyunsaturated FA (PUFA), whilst BFA and trans FA occurred in much lower proportions compared to the caecotrophs. Thus, coprophagy did not substantially modify the FA composition of the tissues investigated. Use of forage-only diets, compared to the oats supplemented diets, led to extraordinary high proportions of n-3 PUFA (including 18:3 and long-chain n-3) in the fat of adipose (21.3 vs. 6.7%) and lean tissue (15.4 vs. 5.7%). The forage type diet (grass vs. alfalfa) had smaller effects on the FA profiles. Indications of diet effects on endogenous desaturation, chain elongation and differential distribution of functional FA between the two tissues investigated were found.

A leaky voltage sensor domain of cardiac sodium channels causes arrhythmias associated with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a structural heart disease that causes dilatation of cardiac chambers and impairs cardiac contractility. The SCN5A gene encodes Nav1.5, the predominant cardiac sodium channel alpha subunit. SCN5A mutations have been identified in patients with arrhythmic disorders associated with DCM. The characterization of Nav1.5 mutations located in the voltage sensor domain (VSD) and associated with DCM revealed divergent biophysical defects that do not fully explain the pathologies observed in these patients. The purpose of this study was to characterize the pathological consequences of a gating pore in the heart arising from the Nav1.5/R219H mutation in a patient with complex cardiac arrhythmias and DCM. We report its properties using cardiomyocytes derived from patient-specific human induced pluripotent stem cells. We showed that this mutation generates a proton leak (called gating pore current). We also described disrupted ionic homeostasis, altered cellular morphology, electrical properties, and contractile function, most probably linked to the proton leak. We thus propose a novel link between SCN5A mutation and the complex pathogenesis of cardiac arrhythmias and DCM. Furthermore, we suggest that leaky channels would constitute a common pathological mechanism underlying several neuronal, neuromuscular, and cardiac pathologies.

Epidermodysplasia Verruciformis: Inborn Errors of Immunity to Human Beta-Papillomaviruses.

Epidermodysplasia verruciformis (EV) is an autosomal recessive skin disorder with a phenotype conditional on human beta-papillomavirus (beta-HPV) infection. Such infections are common and asymptomatic in the general population, but in individuals with EV, they lead to the development of plane wart-like and red or brownish papules or pityriasis versicolor-like skin lesions, from childhood onwards. Most patients develop non-melanoma skin cancer (NMSC), mostly on areas of UV-exposed skin, from the twenties or thirties onwards. At least half of the cases of typical EV are caused by biallelic loss-of-function mutations of TMC6/EVER1 or TMC8/EVER2. The cellular and molecular basis of disease in TMC/EVER-deficient patients is unknown, but a defect of keratinocyte-intrinsic immunity to beta-HPV is suspected. Indeed, these patients are not susceptible to other infectious diseases and have apparently normal leukocyte development. In contrast, patients with an atypical form of EV due to inborn errors of T-cell immunity invariably develop clinical symptoms of EV in the context of other infectious diseases. The features of the typical and atypical forms of EV thus suggest that the control of beta-HPV infections requires both EVER1/EVER2-dependent keratinocyte-intrinsic immunity and T cell-dependent adaptive immunity.

The human CIB1-EVER1-EVER2 complex governs keratinocyte-intrinsic immunity to ß-papillomaviruses.

Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human ß-papillomaviruses (ß-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to ß-HPVs of EV patients.