Novel and Recurrent PNPLA1 Mutations in Spanish Patients with Autosomal Recessive Congenital Ichthyosis; Evidence of a Founder Effect.

Autosomal recessive congenital ichthyosis (ARCI) is a group of rare non-syndrome diseases that affect cornification. PNPLA1 is one of the 12 related genes identified so far. Mutation screening of this gene has resulted in the identification of 13 individuals, from 10 families, who carried 7 different PNPLA1 mutations. These mutations included 2 missense, 2 frame-shift and 3 nonsense, 3 of them being novel. One of the identified variants, c.417_418delinsTC, was highly prevalent, as it was found in 6 out of 10 (60%) of our ARCI families with PNPLA1 mutations. Clinical manifestations varied significantly among patients, but altered sweating; erythema, palmar hyperlinearity and small whitish scales in flexor-extensor and facial areas were common symptoms. Haplotype analyses of c.417_418delinsTC carriers confirmed the existence of a common ancestor. This study expands the spectrum of the PNPLA1 disease, which causes variants and demonstrates that the c.417_418delinsTC mutation has founder effects in the Spanish population.

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

The role of lipoxygenases in epidermis.

Lipoxygenases (LOX) are key enzymes in the biosynthesis of a variety of highly active oxylipins which act as signaling molecules involved in the regulation of many biological processes. LOX are also able to oxidize complex lipids and modify membrane structures leading to structural changes that play a role in the maturation and terminal differentiation of various cell types. The mammalian skin represents a tissue with highly abundant and diverse LOX metabolism. Individual LOX isozymes are thought to play a role in the modulation of epithelial proliferation and/or differentiation as well as in inflammation, wound healing, inflammatory skin diseases and cancer. Emerging evidence indicates a structural function of a particular LOX pathway in the maintenance of skin permeability barrier. Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 have been found to represent the second most common cause of autosomal recessive congenital ichthyosis and targeted disruption of the corresponding LOX genes in mice resulted in neonatal death due to a severely impaired permeability barrier function. Recent data indicate that LOX action in barrier function can be traced back to the oxygenation of linoleate-containing ceramides which constitutes an important step in the formation of the corneocyte lipid envelope. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Novel transglutaminase 1 mutations in patients affected by lamellar ichthyosis.

Lamellar Ichthyosis (LI) is a form of congenital ichthyosis that is caused by mutations in the TGM1 gene that encodes for the transglutaminase 1 (TG1) enzyme. Functional inactivation of TG1 could be due to mutations, deletion or insertions. In this study, we have screened 16 patients affected by LI and found six new mutations: two transition/transversion (R37G, V112A), two nonsense mutations and two putative splice site both leading to a premature stop codon. The mutations are localized in exons 2 (N-terminal domain), 5, 11 (central catalytic domain), and none is located in the two beta-barrel C-terminal domains. In conclusion, this study expands the current knowledge on TGM1 mutation spectrum, increasing the characterization of mutations would provide more accurate prenatal genetic counselling for parents at-risk individuals.

Finding homes for orphan cytochrome P450s: CYP4V2 and CYP4F22 in disease states.

The cytochrome P450 (CYP) 4 family of enzymes contains several recently identified membersthat are referred to as "orphan P450s" because their endogenous substrates are unknown.Human CYP4V2 and CYP4F22 are two such orphan P450s that are strongly linked to ocular andskin disease, respectively. Genetic analyses have identified a wide spectrum of mutations in the CYP4V2gene from patients suffering from Bietti's crystalline corneoretinal dystrophy, and mutations in theCYP4F22 gene have been linked to lamellar ichthyosis. The strong gene­disease associations provideunique opportunities for elucidating the substrate specificity of these orphan P450s and unraveling thebiochemical pathways that may be impacted in patients with CYP4V2 and CYP4F22 functional deficits.

Involvement of corneodesmosome degradation and lamellar granule transportation in the desquamation process.

Desquamation in the mammalian skin is a well-balanced process of producing corneocytes and shedding them from the surface of the skin. The corneodesmosome, which is a modified desmosome, is the main adhesive structure in the cornified cell layer. The major extracellular constituents of corneodesmosomes are desmoglein 1, desmocollin 1, and corneodesmosin. Proteases involved in the degradation of corneodesmosomes and their inhibitors are secreted from lamellar granules in the granular cell layer. Genetic defects in corneodesmosin and protease inhibitors result in accelerated desquamation and severe barrier impairment. Abnormalities in transportation and secretion of lamellar granules underlie ichthyosis seen in certain human diseases.

Premature terminal differentiation and a reduction in specific proteases associated with loss of ABCA12 in Harlequin ichthyosis.

One of the primary functions of skin is to form a defensive barrier against external infections and water loss. Disrupted barrier function underlies the most severe and often lethal form of recessive congenital ichthyosis, harlequin ichthyosis (HI). HI is associated with mutations in the gene that encodes the ABC transporter protein, ABCA12. We have investigated the morphological and biochemical alterations associated with abnormal epidermal differentiation and barrier formation in HI epidermis. An in vitro model of HI skin using human keratinocytes retrovirally transduced with shRNA targeting ABCA12 in a three-dimensional, organotypic co-culture (OTCC) system has also been developed. A robust reduction in ABCA12 expression had a dramatic effect on keratinocyte differentiation and morphology comparable with that observed in HI skin, including a thicker epidermis and abnormal lipid content with a reduction in nonpolar lipids. As seen in HI epidermis, proteins that are normally expressed in late differentiation were highly dysregulated in the ABCA12-ablated OTCC system. These proteins were expressed in the stratum basale and also in the stratum spinosum, indicative of a premature terminal differentiation phenotype. Expression of the proteases kallikrein 5 and cathepsin D was dramatically reduced in both HI epidermis and the OTCC model. These data suggest that ABCA12 is a key molecule in regulating keratinocyte differentiation and transporting specific proteases associated with desquamation.

Chanarin-Dorfman syndrome: deficiency in CGI-58, a lipid droplet-bound coactivator of lipase.

Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive disease of lipid metabolism; it is associated with congenital ichthyosis typed as non-bullous congenital ichthyosiform erythroderma (NCIE). CDS is characterized by the presence of an abnormally large number of cytosolic lipid droplets containing triacylglycerol (TG) in various tissues such as the skin, liver, and leukocytes. Mutations in the CGI-58 (also called ABHD5) gene encoding a 39-kDa protein of the alpha/beta hydrolase domain subfamily have been shown to be responsible for this disorder. In adipocytes, CGI-58 is involved in TG degradation on lipid droplets; in doing so, it coordinates with several lipolytic factors including perilipin, a member of the PAT protein family, and ATGL, a putative rate-limiting lipase in adipocytes. In quiescent adipocytes, CGI-58 interacts with perilipin on the surfaces of lipid droplets. Upon hormonal stimulation, CGI-58 facilitates massive lipolysis by activating ATGL. Some CGI-58 mutations found in CDS patients cancel the ability to interact with perilipin or activate ATGL, indicating that the loss of these interactions is physiologically important. However, based on the tissue distributions of these lipolytic factors, there are likely multiple molecular targets of CGI-58 actions. This in turn gives rise to the multiple phenotypes of CDS, such as ichthyosis, liver steatosis, or neurosensory diseases.

CGI-58 is an alpha/beta-hydrolase within lipid transporting lamellar granules of differentiated keratinocytes.

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.

Plasminogen activator inhibitor-2 is expressed in different types of congenital ichthyosis: in vivo evidence for its cross-linking into the cornified cell envelope by transglutaminase-1.

BACKGROUND: Plasminogen activator inhibitor-2 (PAI-2), a regulatory serpin of the plasminogen activator (PA) system, has been described as a potential component of the cornified cell envelope (CE). Protease inhibitors are essential for skin homeostasis and in particular for the regulation of the desquamation process. Therefore, an aberrant expression of PAI-2 could be involved in the pathogenesis of certain cornification disorders. OBJECTIVES: Evaluation of the expression of PAI-2 in different types of congenital ichthyosis, especially in lamellar ichthyosis/nonbullous congenital ichthyosiform erythroderma (LI/NCIE) and in Netherton syndrome (NTS). Demonstration of the functional relationship between PAI-2 and transglutaminase (TGase)-1. PATIENTS AND METHODS: Using immunohistochemistry we evaluated cryosections from individuals suffering from LI/NCIE (n=67), NTS (n=6), ichthyosis-follicularis-atrichia-photophobia syndrome (n=2) and Harlequin ichthyosis (n=1) in comparison with psoriasis vulgaris and healthy skin. Moreover, we assessed the respective TGase-1 activity and the presence of TGase-1 protein. A functional assay was developed to elucidate whether PAI-2 is a substrate for TGase-1. RESULTS: PAI-2 is expressed in different types of congenital ichthyosis and there is a strong correlation between TGase-1 activity and PAI-2 protein signal. Double staining revealed a strong colocalization of TGase-1 activity and PAI-2 protein. The epidermal incorporation of the specific PAI-2 peptide containing a TGase binding site revealed a strong pericellular staining in the stratum granulosum in healthy skin. In contrast, TGase-1-deficient skin showed only a lamellar staining in the stratum corneum. CONCLUSIONS: We provide in vivo evidence that PAI-2 is a substrate of TGase-1. The normal expression of PAI-2 in a large group of TGase-1-proficient LI/NCIE patients makes it rather unlikely that PAI-2 alone is a primary molecular cause of LI/NCIE.

Mutations in a new cytochrome P450 gene in lamellar ichthyosis type 3.

We report the identification of mutations in a non-syndromic autosomal recessive congenital ichthyosis (ARCI) in a new gene mapping within a previously identified locus on chromosome 19p12-q12, which has been defined as LI3 in the OMIM database (MIM 604777). The phenotype usually presents as lamellar ichthyosis and hyperlinearity of palms and soles. Seven homozygous mutations including five missense mutations and two deletions were identified in a new gene, FLJ39501, on chromosome 19p12 in 21 patients from 12 consanguineous families from Algeria, France, Italy and Lebanon. FLJ39501 encodes a protein which was found to be a cytochrome P450, family 4, subfamily F, polypeptide 2 homolog of the leukotriene B4-omega-hydroxylase (CYP4F2) and could catalyze the 20-hydroxylation of trioxilin A3 from the 12(R)-lipoxygenase pathway. Further oxidation of this substrate by the fatty alcohol:nicotinamide-adenine dinucleotide oxidoreductase (FAO) enzyme complex, in which one component, ALDH3A2, is known to be mutated in Sjögren-Larsson syndrome (characterized by ichthyosis and spastic paraplegia), would lead to 20-carboxy-(R)-trioxilin A3. This compound could be involved in skin hydration and would be the essential missing product in most forms of ARCI. Its chiral homolog, 20-carboxy-(S)-trioxilin A3, could be implicated in spastic paraplegia and in the maintenance of neuronal integrity.

Mutations associated with a congenital form of ichthyosis (NCIE) inactivate the epidermal lipoxygenases 12R-LOX and eLOX3.

Non-bullous congenital ichthyosiform erythroderma (NCIE) is one of the main clinical forms of ichthyosis. Genetic studies indicated that 12R-lipoxygenase (12R-LOX) or epidermal lipoxygenase-3 (eLOX3) was mutated in six families affected by NCIE [F. Jobard, C. Lefevre, A. Karaduman, C. Blanchet-Bardon, S. Emre, J. Weissenbach, M. Ozguc, M. Lathrop, J.F. Prud'homme, J. Fischer, Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1, Hum. Mol. Genet. 11 (2002) 107-113.], but the impact of these mutations on LOX function has not been defined. To explore this, we overexpressed the wild-type or mutated enzymes in E. coli and COS7 cells and then analyzed the essential catalytic properties. We showed recently that human eLOX3 is a hydroperoxide isomerase (hepoxilin synthase) that converts the product of 12R-LOX, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) to a specific epoxyalcohol. Using incubations with [(14)C]-labeled substrates and HPLC analyses, we found that the naturally occurring mutations totally eliminate the lipoxygenase activity of 12R-LOX and the hydroperoxide isomerase activity of eLOX3. We further demonstrate that the 12R-LOX/eLOX3-derived 8R-hydroxy-11R,12R-epoxide is converted by an epoxide hydrolase in COS7 cells and in human keratinocytes to a single isomer of 8,11,12-trihydroxyeicosa-5,9,14-trienoic acid. Taken together, the results support the hypothesis that 12R-LOX, eLOX3, and perhaps an epoxide hydrolase function together in the normal process of skin differentiation, and that the loss of function mutations are the basis of the LOX-dependent form of NCIE.

Cross-linked envelopes in nail plate in lamellar ichthyosis.

BACKGROUND: Corneocytes of the nail plate, like those of the stratum corneum, generate cornified envelopes (CEs) of cross-linked protein that can be visualized readily after removal of non-cross-linked protein by detergent extraction. Defective CE formation occurs in epidermal scale and hair in transglutaminase 1 (TGM1)-negative lamellar ichthyosis (LI) and has been proposed as a diagnostic aid for this syndrome. OBJECTIVES: (i) To ascertain whether TGM1 is important for CE formation in nail; (ii) to characterize CE abnormalities occurring in LI that may be distinguished from other types of inherited ichthyosis when nail samples are subjected to detergent extraction; and (iii) to evaluate the utility of nails as a diagnostic aid for LI. METHODS: Nail samples were provided by nine patients previously classified as having TGM1-negative LI, four with other types of ichthyotic conditions and six normal controls. Samples were extracted extensively in sodium dodecyl sulphate under reducing conditions and examined by light and electron microscopy. RESULTS: After extraction, defective CE cross-linking was visualized in epidermal corneocytes from seven of nine patients exhibiting TGM1-negative LI, whereas nail samples from patients with the other syndromes were normal. The defects in CE structure resembled those recently reported for LI scale, although in some cases residual CE and CE-associated structures were present. CONCLUSIONS: Despite the paucity of clinical nail symptoms in LI, TGM1 activity is important for generation of normal CE in nail plate, consistent with its importance in protein cross-linking in interfollicular epidermis and hair. Lack of this activity leads to a strikingly aberrant appearance of CE in LI nail after detergent extraction that is evident ultrastructurally in a large majority of cases. Nail envelopes therefore could provide a useful diagnostic tool in distinguishing LI from other ichthyoses with overlapping clinical features.

Lamellar ichthyosis associated with pseudoainhum of the toes and eye changes.

Classic lamellar ichthyosis is a rare, autosomal recessive, genetically heterogeneous skin disease caused by mutations in the transglutaminase 1 gene. Pseudoainhum is characterized by the appearance of a constricting band around a digit which may lead to spontaneous amputation. We describe a 41-year-old man with classic lamellar ichthyosis with unusual eye changes and pseudoainhum of both the fifth and the third right toes. Eye abnormalities included bilateral ectropion of the lower eyelids, chronic blepharitis, and nuclear cataract. A radiometric assay revealed greatly reduced skin transglutaminase activity. To the best of our knowledge, this report is unique as classic lamellar ichthyosis with deficient transglutaminase activity has never been associated with pseudoainhum of the toes and the early development of nuclear cataract.

A novel homozygous mutation 371delA in TGM1 leads to a classic lamellar ichthyosis phenotype.

Malformation of the cornified cell envelope (CCE) arising from mutations of the transglutaminase (TGase) 1 gene (TGM1) is the cause of some cases of lamellar ichthyosis (LI). However, genotype/phenotype correlation in TGM1 mutations has not yet been fully clarified. We report a typical case of LI caused by a novel mutation in TGM1. The patient, a 33-year-old woman, showed thick, lamellar scales on the entire body surface. Immunofluorescence labelling with anti-TGase 1 antibodies was negative in the patient's epidermis. In situ TGase activity assay detected markedly reduced TGase activity in granular layers of the patient's epidermis. Electron microscopy revealed incomplete thickening of the CCE during keratinization in the epidermis. Sequencing of the entire exons and exon-intron borders of TGM1 revealed that the patient was a homozygote for a novel deletion mutation 371delA in exon 3. This mutation leads to a frameshift resulting in a premature termination codon 43 bp downstream from the mutation site. According to the protein modelling of TGase 1, the truncated protein from this mutated allele loses the entire catalytic core domain of TGase 1. Thus, the present homozygous mutation is expected to cause total loss of TGase 1 activity, resulting in large, dark, lamellar scales on the entire body, the classic phenotype of LI, in this patient.

Lamellar ichthyosis: response to etretinate with transglutaminase 1 recovery.

We studied a case of typical lamellar ichthyosis before and after etretinate treatment for the expression of transglutaminase 1 (TGK) and the presence or absence of the marginal band. Before the treatment, TGK was undetectable, although two other components of the marginal band, loricrin and involucrin, were detected by immunostaining in a normal pattern. The marginal band of the corneocytes was either thin and irregular or completely absent by electron microscopic study. After therapy with etretinate, 50 mg/day, the patient' s skin improved, and biopsies were taken at 4 and 8 months. Transglutaminase 1 became detectable by immunostaining. The marginal band was still absent in most corneocytes.

Haplotype association and mutation analysis of the transglutaminase 1 gene for prenatal exclusion of lamellar ichthyosis.

Lamellar ichthyosis (LI) is an autosomal recessive keratinization disorder of the skin. Genetic heterogeneity has been shown for the disease and there is evidence for the involvement of the transglutaminase 1 (TGM1) gene on chromosome 14q11. We have previously identified chromosome 14q11 haplotypes associated with ichthyosis in the Norwegian population. In this paper we describe antenatal exclusion of ichthyosis in two Norwegian families by chromosome 14q11 haplotype association and direct mutation analysis. In one pregnancy, the 11-week old fetus at risk for LI was found to share only one disease-associated haplotype. A subsequent mutation analysis of the TGM1 gene in fetal DNA revealed that the fetus carried a novel 3795A-->T transversion. The affected proband was compound heterozygous for the mutations 3795A-->T and 3239G-->C resulting in an Asp430Val and a Val379Leu, respectively. In another LI family, the 11-week old fetus was found to be heterozygous for the 14q11 haplotype associated with the disease. Subsequent mutation analysis revealed that the fetus was heterozygous for the 2526A-->G transition in the splice site of intron 5 whereas the proband was homozygous for the same mutation. Our results show that haplotyping can be a useful tool for prenatal diagnosis in diseases with genetic heterogeneity.