A novel SERPINA12 variant and first European patients with diffuse palmoplantar keratoderma.

BACKGROUND: Hereditary palmoplantar keratodermas (hPPKs) comprise a heterogeneous group of skin disorders characterized by persistent palmoplantar hyperkeratosis. Loss-of-function variants in a serine peptidase inhibitor, SERPINA12, have recently been implicated in autosomal recessive diffuse hPPK. The disorder appears to share similarities with another hPPK associated with protease overactivity, namely Nagashima-type PPK (NPPK) caused by biallelic variants in SERPINB7. OBJECTIVES: The aim of this study was to enhance the understanding of the clinical and genetic characteristics of serine protease-related hPPKs caused by variants in SERPINA12 and SERPINB7. METHODS: Whole-exome sequencing (WES) was performed for hPPK patients. Haplotype analysis was completed for the patients with identified recessive SERPINA12 variants and their available family members. In addition, the current literature of SERPINA12- and SERPINB7-related hPPKs was summarized. RESULTS: The phenotype of SERPINA12-related hPPK was confirmed by reporting three new SERPINA12 patients, the first of European origin. A novel SERPINA12 c.1100G>A p.(Gly367Glu) missense variant was identified confirming that the variant spectrum of SERPINA12 include both truncating and missense variants. The previously reported SERPINA12 c.631C>T p.(Arg211*) was indicated enriched in the Finnish population due to a plausible founder effect. In addition, SERPINA12 hPPK patients were shown to share a similar phenotype to patients with recessive variants in SERPINB7. The shared phenotype included diffuse transgradient PPK since birth or early childhood and frequent palmoplantar hyperhidrosis, aquagenic whitening and additional hyperkeratotic lesions in non-palmoplantar areas. SERPINA12 and SERPINB7 hPPK patients cannot be distinguished without genetic analysis. CONCLUSIONS: Recessive variants in SERPINA12 and SERPINB7 leading to protease overactivity and hPPK produce a similar phenotype, indistinguishable without genetic analysis. SERPINA12 variants should be assessed also in non-Asian patients with diffuse transgradient PPK. Understanding the role of serine protease inhibitors will provide insights into the complex proteolytic network in epidermal homeostasis.

A novel desmoplakin mutation causes dilated cardiomyopathy with palmoplantar keratoderma as an early clinical sign.

BACKGROUND: PPKs represent a heterogeneous group of disorders with hyperkeratosis of palmar and/or plantar skin. PPK, hair shaft abnormalities, cardiomyopathy and arrhythmias can be caused by mutations in desmosomal genes, e.g. desmoplakin (DSP). PPK should trigger genetic testing to reveal mutations with possible related cardiac disease. OBJECTIVES: To report a large multigenerational family with a novel DSP mutation associated with early-onset PPK and adult-onset cardiomyopathy and arrhythmias. METHODS: A custom-designed in-house panel of 35 PPK related genes was used to screen mutations in the index patient with focal PPK. The identified DSP mutation was verified by Sanger sequencing. DNA samples from 20 members of the large multigenerational family were sequenced for the DSP mutation. Medical records were reviewed. Clinical dermatological evaluation was performed, including light microscopy of hair samples. Cardiac evaluation included clinical examination, echocardiography, cardiac magnetic resonance imaging (CMR), electrocardiogram (ECG), Holter monitoring and laboratory tests. RESULTS: We identified a novel autosomal dominant truncating DSP c.2493delA p.(Glu831Aspfs*33) mutation associated with dilated cardiomyopathy (DCM) with arrhythmia susceptibility and focal PPK as an early cutaneous sign. The mutation was found in nine affected family members, but not in any unaffected members. Onset of dermatological findings preceded cardiac symptoms which were variable and occurred at adult age. CONCLUSIONS: We report a novel truncating DSP mutation causing focal PPK with varying severity and left ventricular dilatation and ventricular extrasystoles. This finding emphasizes the importance of genetic diagnosis in patients with PPK for clinical counselling and management of cardiomyopathies and arrhythmias.

Hereditary palmoplantar keratoderma - phenotypes and mutations in 64 patients.

BACKGROUND: Hereditary palmoplantar keratodermas (PPK) represent a heterogeneous group of rare skin disorders with epidermal hyperkeratosis of the palms and soles, with occasional additional manifestations in other tissues. Mutations in at least 69 genes have been implicated in PPK, but further novel candidate genes and mutations are still to be found. OBJECTIVES: To identify mutations underlying PPK in a cohort of 64 patients. METHODS: DNA of 48 patients was analysed on a custom-designed in-house panel for 35 PPK genes, and 16 patients were investigated by a diagnostic genetic laboratory either by whole-exome sequencing, gene panels or targeted single-gene sequencing. RESULTS: Of the 64 PPK patients, 32 had diffuse (50%), 19 focal (30%) and 13 punctate (20%) PPK. None had striate PPK. Pathogenic mutations in altogether five genes were identified in 31 of 64 (48%) patients, the majority (22/31) with diffuse PPK. Of them, 11 had a mutation in AQP5, five in SERPINB7, four in KRT9 and two in SLURP1. AAGAB mutations were found in nine punctate PPK patients. New mutations were identified in KRT9 and AAGAB. No pathogenic mutations were detected in focal PPK. Variants of uncertain significance (VUS) in PPK-associated and other genes were observed in 21 patients that might explain their PPK. No suggestive pathogenic variants were found for 12 patients. CONCLUSIONS: Diffuse PPK was the most common (50%) and striate PPK was not observed. We identified pathogenic mutations in 48% of our PPK patients, mainly in five genes: AQP5, AAGAB, KRT9, SERPINB7 and SLURP1.

Ectodysplasin is released by proteolytic shedding and binds to the EDAR protein.

Anhidrotic ectodermal dysplasia (EDA) is an X-linked disorder characterized by abnormal development of ectoderm and its appendices. The EDA gene encodes different isoforms of ectodysplasin, a transmembrane protein. The two longest isoforms, ectodysplasin-A1 and -A2, which differ by an insertion of two amino acids, are trimeric type II membrane proteins with an extracellular portion containing a short collagenous domain and a TNF ligand motif in the C-terminal region. We show that ectodysplasin is released from cells to the culture medium. Deletion constructs were used to localize the cleavage site and show that the putative recognition sequence of a furin-like enzyme is needed for the cleavage. Some EDA patients have missense mutations affecting this recognition sequence, suggesting that cleavage has biological significance in vivo. EDAR, a recently cloned member of the TNFR family and the product of the downless gene, is able to co-precipitate ectodysplasin, confirming that they form a ligand-receptor pair. In situ hybridization and immunostaining studies show that ectodysplasin and EDAR are expressed in adjacent or partially overlapping layers in the developing human skin. We conclude that as a soluble ligand, ectodysplasin is able to interact with EDAR and mediate signals needed for the development of ectodermal appendages.

Stromelysin-2 is upregulated during normal wound repair and is induced by cytokines.

Stromelysin-2 is a matrix metalloproteinase that degrades in vitro several protein components relevant to wound repair such as collagens III and IV, gelatin, nidogen, laminin-1, proteoglycans, and elastin. Furthermore, it can activate other matrix metalloproteinases, such as collagenase-1 (matrix metalloproteinase-1) and collagenase-2 (matrix metalloproteinase-8), as well as 92 kDa gelatinase. The aim of this study was to determine in a large variety of wounds (normally healing dermal and mucosal wounds, suction blisters, ex vivo cultures, diabetic, decubitus, rheumatic, and venous ulcers) and keratinocyte cultures, which factors contribute to stromelysin-2 expression and how it is induced in relation to other matrix metalloproteinases. Our results show that stromelysin-2 mRNA and protein are upregulated later (at 3 d) than matrix metalloproteinase-1 in normally healing wounds and ex vivo explants, in which stromelysin-2 is invariably expressed by keratinocytes migrating over dermal matrix. The number of keratinocytes expressing stromelysin-2 was greatest in chronic inflamed diabetic and venous ulcers compared with rheumatoid and decubitus ulcers, six of which had no signal. In keratinocyte cultures, tumor necrosis factor-alpha, epidermal growth factor, and transforming growth factor-beta1 induced stromelysin-2 expression as measured by quantitative reverse transcriptase-polymerase chain reaction, whereas different matrices did not upregulate the mRNA. Immunostaining demonstrated stromal transforming growth factor-beta1 in contact with the stromelysin-2-positive keratinocytes. Our results suggest that stromelysin-2 expression is important for the normal repair process and is upregulated by cytokines rather than cell-matrix interactions. Stromelysin-2 is most likely to participate in the remodeling of the newly formed basement membrane, and is not overexpressed in retarded wound healing.

The macrophage receptor MARCO.

MARCO (macrophage receptor with collagenous structure) belongs to the class A scavenger receptor molecules. The structure and function of the molecule is described. Although it is expressed on subsets of macrophages, it can be upregulated on other macrophages after bacterial infection. The strategic position of MARCO-expressing cells in lymphoid organs suggests an important role for this bacteria-binding molecule in removal of pathogens.

Ectodysplasin is a collagenous trimeric type II membrane protein with a tumor necrosis factor-like domain and co-localizes with cytoskeletal structures at lateral and apical surfaces of cells.

Anhidrotic ectodermal dysplasia (EDA) is a human genetic disorder of impaired ectodermal appendage development. The EDA gene encodes isoforms of a novel transmembrane protein, ectodysplasin. The sequence of the longest isoform includes an interrupted collagenous domain of 19 Gly-X-Y repeats and a motif conserved in the tumor necrosis factor (TNF)-related ligand family. In order to understand better the function of the ectodysplasin protein molecule and its domains, we have studied the processing and localization of wild-type and mutated isoforms in transfected human fetal kidney 293 and monkey kidney COS-1 cells. Similar to other members of collagenous membrane proteins and members of TNF-related ligands, ectodysplasin is a type II membrane protein and it forms trimers. The membrane localization of ectodysplasin is asymmetrical: it is found on the apical and lateral surfaces of the cells where it co-localizes with cytoskeletal structures. The TNF-like motif and cysteines found near the C-terminus are necessary for correct transport to the cell membrane, but the intracellular and collagenous domains are not required for the localization pattern. Our results suggest that ectodysplasin is a new member in the TNF-related ligand family involved in the early epithelial-mesenchymal interaction that regulates ectodermal appendage formation.

Structure and chromosomal localization of the human and murine genes for the macrophage MARCO receptor.

The structures of the human and mouse genes for the macrophage receptor with collagenous structure were determined. Both genes have 17 exons, of which exons 4-15 encode the collagenous domain. The transcription initiation sites in the mouse gene were identified using primer extension, SI nuclease mapping, and 5' capturing rapid amplification of cDNA ends assays. All three methods revealed two major initiation sites, one starting 27 bp downstream of a TATA box and another at positions -63 and -66 downstream of an AT-rich region. Several potential binding sites for transcription factors were identified in the promoter region, neither gene has a CAAT box or GC boxes. The human and mouse genes were localized to syntenic regions on chromosomes 2 and 1, respectively, using fluorescence in situ hybridization.

Roles of a macrophage receptor with collagenous structure (MARCO) in host defense and heterogeneity of splenic marginal zone macrophages.

Class A type I and type II macrophage scavenger receptors (MSR-A) and a macrophage receptor with collagenous structure (MARCO) are trimeric membrane glycoproteins mediating the uptake of chemically modified low density lipoproteins. MSR-A is expressed constitutively in several tissue macrophages and in liver sinusoidal endothelial cells, whereas MARCO is expressed constitutively in splenic marginal zone macrophages and in macrophages and endothelial cells in the lymphatic medullary sinuses of lymph nodes. The administration of LPS, zymosan, BCG, or L. monocytogenes to mice resulted in marked and transient MARCO expression and in the upregulation of MSR-A expression in the liver and spleen. In osteopetrotic (op) mutant mice defective in the production on M-CSF, ER-TR9-positive marginal zone macrophages and MOMA-1-positive marginal metallophilic macrophages were absent, whereas MARCO-expressing marginal zone macrophages were present, indicating the heterogeneity of marginal zone macrophages. Intravenous administration of BCG resulted in marked accumulation of BCG bacilli in the both marginal zone macrophages and marginal metallophilic macrophages in littermate control mice. In contrast, BCG bacilli were incorporated almost exclusively by MARCO-expressing marginal zone macrophages in op/op mice. These results indicate that MARCO is not only expressed constitutively in specific macrophage subpopulations but is also induced by various bacterial antigens and plays a role in host defense against bacteria.

Regulation and functional involvement of macrophage scavenger receptor MARCO in clearance of bacteria in vivo.

The scavenger receptors expressed by macrophages are thought to play an important role in the immune response against bacteria by mediating binding and phagocytosis. A novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (MARCO), has recently been identified. In this study we have generated a panel of mAbs with specificities for different domains of this receptor. Two of those reacting with the C-terminal cysteine-rich domain block ligand binding of MARCO. The in vivo expression of this murine receptor is normally restricted to distinct populations of macrophages in the spleen and lymph nodes. During bacillus Calmette-Guérin (BCG) infection, during bacterial sepsis, or after the injection of purified LPS, however, the expression of MARCO is rapidly induced on macrophages in other tissues, including Kupffer cells in the liver. Using the mouse macrophage cell line J774.2, it was shown that LPS stimulation up-regulates surface expression of MARCO in a dose- and time-dependent fashion. The proinflammatory cytokines IL-1, IL-6, TNF-alpha, and IFN-gamma had little or no effect. Using inhibitory mAbs, the relevance of MARCO for the clearance of circulating bacteria in vivo was determined. Although the overall elimination of live Escherichia coli and Staphylococcus aureus from the blood did not appear to be affected by treatment with these Abs, the capturing of heat-killed bacteria by macrophages in the marginal zone areas of the spleen was clearly inhibited. This study suggests a role for MARCO in the host antibacterial defense.

Structure of the human macrophage MARCO receptor and characterization of its bacteria-binding region.

The primary structure of human macrophage receptor with collagenous structure (MARCO) was determined from cDNA clones and shown to be highly similar to that of mouse (Elomaa, O., Kangas, M., Sahlberg, C. , Tuukkanen, J., Sormunen, R., Liakka, A., Thesleff, I., Kraal, G., and Tryggvason, K. (1995) Cell 80, 603-609). Features such as potential carbohydrate attachment sites in the extracellular spacer domain III and the interruption of Gly-Xaa-Yaa repeats in the collagenous domain IV were conserved between the two species. However, the human MARCO polypeptide chain lacked the intracellular cysteine present in mouse, as well as two extracellular cysteines that form interchain disulfide bonds in the murine protein. In situ hybridization showed MARCO to be strongly expressed in macrophages of several tissues of human individuals with sepsis. No expression was observed in other cell types. The bacteria-binding region of MARCO was determined in binding studies with full-length and truncated variants of MARCO, and localized to a region proximal to the cysteine-rich part of the COOH-terminal domain V. The intrachain disulfide bond pattern of domain V was established showing that these bonds are between cysteine pairs C1-C5, C2-C6, and C3-C4.

Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages.

A novel murine plasma membrane protein has been identified in subpopulations of macrophages. It has an intracellular N-terminal domain, a transmembrane domain, and an extracellular region with a short spacer, an 89 Gly-Xaa-Yaa repeat-containing collagenous domain, and a C-terminal cysteine-rich domain. In situ hybridization and immunohistochemical staining have localized the protein to a subset of macrophages in the marginal zone of the spleen and the medullary cord of lymph nodes. No expression was observed in macrophages of liver or lung. Transfected COS cells synthesized a native trimeric plasma membrane protein that bound labeled bacteria and acetylated LDL, but not yeast or Ficoll. The results suggest that the novel protein is a macrophage-specific membrane receptor with a role in host defense, as it shows postnatal expression in macrophages, which are considered responsible for the binding of bacterial antigens and phagocytosis.

Human alpha 1 (XIII) collagen gene. Multiple forms of the gene transcripts are generated through complex alternative splicing of several short exons.

The structure of the gene for the human alpha 1(XIII) collagen chain (COL13A1) was determined from genomic clones spanning 140,000 base pairs (bp), including about 3,000 bp of the 5'-end-flanking region and 5,000 bp of the 3'-end-flanking region. The gene was shown to contain 39 exons. There were eight exons of 27 bp, five of 36 bp, four of 54 bp, three of 45 bp, and two of 42 bp. The rest of the exons coding for translated sequences had sizes varying between 24 and 153 bp. The genomic clones did not contain exons 3 and 4 whose sizes could, however, be estimated from cDNA clones. S1 nuclease mapping and primer extension analyzes indicated five closely spaced initiation sites of transcription. Sequencing of the 5'-end-flanking region did not reveal a typical TATA box but a four times repeated TATTTAT sequence that may serve as true TATA boxes. Two CCAAT boxes were found starting at positions-13 and -194, and furthermore, the promoter region contains two GC boxes. Previous studies on alpha 1 (XIII) collagen cDNA and genomic clones showed that the primary transcript undergoes complex alternative splicing generating at least four different forms of mRNAs. The present work demonstrated that sequences of seven exons are alternatively used. These exons contain sequences coding for pure collagenous regions, pure noncollagenous regions, and an exon coding for a junction of a collagenous and noncollagenous domain.