PNPLA1 knockdown inhibits esterification of γ-linolenic acid to ceramide 1 in differentiated keratinocytes.

Patatin-like phospholipase domain-containing 1 (PNPLA1) is crucial in the esterification of linoleic acid (LA; 18:2n-6) to ω-hydroxy fatty acids (FA) of ceramide 1 (Cer1), the major barrier lipid of the differentiated epidermis. We previously reported that γ-linolenic acid (GLA; 18:3n-6) as well as LA is esterified to Cer1 subspecies with sphingosine (d18:1) or eicosasphingosine (d20:1) amide-linked to two different ω-hydroxy FA (30wh:0; 32wh:1). Here, we further investigated whether PNPLA1 is also responsible for esterification of GLA to these Cer1 subspecies in normal human keratinocytes (NHK). As late/terminal differentiation was induced in NHK, PNPLA1 and differentiation markers were expressed, and LA-esterified Cer1 subspecies (18:2n-6/C30wh:0 or C32wh:0/d18:1; 18:2n-6/C32wh:0/d20:1) were detected, which were further increased with LA treatment. GLA-esterified Cer1 subspecies (18:3n-6/C30wh:0 or C32wh:0/d18:1; 18:3n-6/C32wh:0/d20:1) were detected only with GLA treatment. Specific small interfering RNA-mediated knockdown of PNPLA1 (KDP) in differentiated NHK decreased levels of these LA-esterified Cer1 subspecies overall and of involucrin (IVL), a terminal differentiation marker. Moreover, KDP resulted in lesser LA/GLA responses as characterized by more significant decreases in IVL and LA/GLA-esterified Cer1 subspecies overall and an accumulation of non-esterified ω-hydroxy ceramides, their putative precursors; the decrease of 18:3n-6/C32wh:0/d18:1, the predominant GLA-esterified Cer1 subspecies, specifically paralleled the increase of C32wh:0/d18:1, its corresponding precursor. PNPLA1 is responsible for NHK terminal differentiation and also for esterification of GLA to the ω-hydroxy FA of Cer1.

PNPLA1-Mediated Acylceramide Biosynthesis and Autosomal Recessive Congenital Ichthyosis.

The stratum corneum of the epidermis acts as a life-sustaining permeability barrier. Unique heterogeneous ceramides, especially ω-O-acylceramides, are key components for the formation of stable lamellar membrane structures in the stratum corneum and are essential for a vital epidermal permeability barrier. Several enzymes involved in acylceramide synthesis have been demonstrated to be associated with ichthyosis. The function of patatin-like phospholipase domain-containing protein 1 (PNPLA1) was a mystery until the finding that PNPLA1 gene mutations were involved in autosomal-recessive congenital ichthyosis (ARCI) patients, both humans and dogs. PNPLA1 plays an essential role in the biosynthesis of acylceramide as a CoA-independent transacylase. PNPLA1 gene mutations cause decreased acylceramide levels and impaired skin barrier function. More and more mutations in PNPLA1 genes have been identified in recent years. Herein, we describe the structural and functional specificity of PNPLA1, highlight its critical roles in acylceramide synthesis and skin barrier maintenance, and summarize the PNPLA1 mutations currently identified in ARCI patients.

ω-O-Acylceramides but not ω-hydroxy ceramides are required for healthy lamellar phase architecture of skin barrier lipids.

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1+/+ and Pnpla1-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.

Isotretinoin Treatment for Autosomal Recessive Congenital Ichthyosis in a Golden Retriever.

Ichthyoses are hereditary cornification disorders that manifest with abnormal differentiation and desquamation of keratinocytes in a form of generalized dry and scaly skin. In golden retriever dogs, autosomal recessive congenital ichthyosis (ARCI) has been associated with mutations in the PNPLA 1 gene. In human medicine, isotretinoin is frequently used to treat ARCIs. The aim of this study was to investigate the clinical and histological effects of isotretinoin on ARCI in a golden retriever dog with confirmed mutation in the PNPLA 1 gene. Clinical examination, blood analysis and histopathological examinations were conducted before and after 90 days of isotretinoin therapy. The clinical and histopathological findings indicate that treatment with oral isotretinoin was effective in improving ichthyosis without any side-effects.

Novel Pathogenic Mutation of PNPLA1 Identified in Autosomal Recessive Congenital Ichthyosis: A Case Report.

Autosomal recessive congenital ichthyosis (ARCI) is characterized by being born as collodion babies, hyperkeratosis, and skin scaling. We described a collodion baby at birth with mild ectropion, eclabium, and syndactyly. Whole exome sequencing showed a compound heterozygous variant c.[56C>A], p.(Ser19X) and c.[100G>A], p.(Ala34Thr) in the PNPLA1 gene [NM_001145717; exon 1]. The protein encoded by PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. The variant was located in the patatin core domain of PNPLA1 and resulted in a truncated protein which could disrupt the function of the protein. This case report highlights a novel compound heterozygous mutation in PNPLA1 identified in a Chinese child.

Mutation analysis of the PNPLA1 gene in three families with congenital ichthyosiform erythroderma / 中华皮肤科杂志

Objective:To detect gene mutations in 3 Chinese families with congenital ichthyosiform erythroderma.Methods:Exome sequencing of peripheral blood DNA was performed for 3 probands clinically diagnosed with congenital ichthyosiform erythroderma by using a gene panel targeting hereditary skin diseases to identify mutation sites. Primers were designed according to the mutation sites for PCR amplification, and Sanger sequencing was performed to verify the mutations in probands and other family members in order to identify the cause of the disease.Results:The probands 1 and 2 presented with generalized skin dryness and scaling, and polygonal dark brown scales on the extensor aspect of the lower limbs; the proband 3 mainly presented with well-circumscribed erythema, papules and scales scattered on the trunk and extremities. All probands denied family history of similar diseases. Genetic testing showed that the proband 1 carried compound heterozygous mutations c.100G>A and c.377G>A in the PNPLA1 gene, which were inherited from her mother and father respectively; the proband 2 carried compound heterozygous mutations c.320T>A and c.434T>C in the PNPLA1 gene, which were inherited from her mother and father respectively; a homozygous mutation c.1300delG was identified in the PNPLA1 gene in the proband 3. The mutations co-segregated with the disease phenotypes in the two families with compound heterozygous mutations. Among the 5 identified mutations, the two missense mutations (c.377G>A and c.320T>A) were firstly reported.Conclusion:Biallelic mutations in the PNPLA1 gene are the causative mutations responsible for autosomal recessive congenital ichthyosis in the three probands, and the newly reported mutations expand the mutation spectrum in the disease.

Novel Pathogenic Mutation of PNPLA1 Identified in Autosomal Recessive Congenital Ichthyosis: A Case Report / 中国医学科学杂志(英文版)

Autosomal recessive congenital ichthyosis (ARCI) is characterized by being born as collodion babies, hyperkeratosis, and skin scaling. We described a collodion baby at birth with mild ectropion, eclabium, and syndactyly. Whole exome sequencing showed a compound heterozygous variant c.[56C>A], p.(Ser19X) and c.[100G>A], p.(Ala34Thr) in the PNPLA1 gene [NM_001145717; exon 1]. The protein encoded by PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. The variant was located in the patatin core domain of PNPLA1 and resulted in a truncated protein which could disrupt the function of the protein. This case report highlights a novel compound heterozygous mutation in PNPLA1 identified in a Chinese child.

Variants in the PNPLA1 Gene in Families with Autosomal Recessive Congenital Ichthyosis Reveal Clinical Significance.

The term autosomal recessive congenital ichthyosis (ARCI) is the subgroup of ichthyosis, which describes a highly heterogeneous group of genetic disorders of the skin characterized by cornification and defective keratinocytes differentiation associated with mutations in at least 14 genes including PNPLA1. To study the molecular basis of the Pakistani kindreds (A and B) affected by ARCI, whole-exome sequencing (WES) in the DNA samples of affected members was performed followed by Sanger sequencing of the candidate gene to hunt down the disease-causing sequence variant/s. WES data analysis led to the identification of a novel nonsense sequence variant (c.892C>T; p.Arg298*, family A) and a recurrent missense variant (c.102C>A; p.Asp34Glu, family B) in PNPLA1 mapped to the ARCI locus in chromosome 6p21.31. Validation and cosegregation analysis of the variants in the remaining family members of the respective families were confirmed by Sanger sequencing. The current investigation expands the spectrum of PNPLA1 mutations and helps establish the proper clinico-genetic diagnosis and correct genotype-phenotype correlation.

Targeted regions sequencing identified four novel PNPLA1 mutations in two Chinese families with autosomal recessive congenital ichthyosis.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) is a rare genetically heterogeneous cutaneous disease predominantly characterized by erythroderma, generalized abnormal scaling of the whole body and a collodion membrane at birth. Numerous causative genes have been demonstrated to be responsible for ARCI including PNPLA1 which can cause ARCI type 10. The objectives of this study are to describe clinical features of three ARCI patients from two Chinese unrelated families and to identify the underlying causative mutations. METHODS: Genomic DNA was extracted from peripheral venous blood obtained from the two Chinese ARCI families in Shandong province. Subsequently, targeted regions sequencing (TRS) followed by Sanger sequencing was conducted to identify and validate the likely pathogenic mutations of the ARCI families. RESULTS: Genetic analyses revealed four novel PNPLA1 variants that are predicted to be probably to lead to ARCI in three patients of two families. Patient 1 in one family was in compound heterozygous status for c.604delC/p.Arg202Glyfs*27 and c.820dupC/p.Arg274Profs*15, whereas c.738_742delinsCCCACAGATCCTGC/ p.Gly247_Tyr248delinsProGlnIleLeuHis, and c.816dupC/p.Arg274Profs*15 were found in patient 2 and 3 of the other family. In addition, these variants cosegregate in the two pedigrees and are all within highly conserved regions of the PNPLA1 protein, which indicate that the four mutations are likely pathogenic. CONCLUSION: Our findings not only broaden the mutational spectrum of PNPLA1, but also contribute to establishing genotype-phenotype correlations for different forms of ARCI.

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.

Impairment of lipophagy by PNPLA1 mutations causes lipid droplet accumulation in primary fibroblasts of Autosomal Recessive Congenital Ichthyosis patients.

BACKGROUND: Autosomal Recessive Congenital Ichthyosis (ARCI) is a group of epidermal keratinization disorders. One of the disease-associated proteins, patatin-like phospholipase domain-containing protein-1 (PNPLA1), plays a key role in the epidermal omega-O-acylceramide synthesis and localizes on the surface of lipid droplets (LDs). OBJECTIVE: Previously, routine clinical test results showed abnormal LD accumulation in blood smear samples of our ARCI patients with PNPLA1 mutations. To investigate the abnormal accumulation of LDs, we analyzed primary fibroblast cells of ARCI patients with PNPLA1 mutations (p.Y245del and p.D172N). We hypothesized that PNPLA1 mutations might affect lipophagy-mediated regulation of LDs and cause intracellular lipid accumulation in ARCI patients. METHODS: LD accumulation was analyzed by fluorescence staining with BODIPY®493/503 in the fibroblasts of patient cells and PNPLA1 siRNA transfected control fibroblast cells. The expression of PNPLA1 and its effects on the lipophagy-mediated degradation of LDs were analyzed by immunocytochemistry and immunoblotting. RESULTS: Our results showed that mutant or downregulated PNPLA1 protein causes abnormal intracellular LD accumulation. We found that PNPLA1 mutations affect neither the cellular localization nor the expression levels of the protein in fibroblast cells. When we analyzed lipophagic degradation process, LC3 expression and the number of autophagosomes were significantly decreased in fibroblast cells of the patients. In addition, co-localization of LDs with autophagosomes and lysosomes were markedly less than that of the control group. CONCLUSION: PNPLA1 mutations caused disturbances in both autophagosome formation and fusion of autophagosomes with lysosomes. Our results indicate a possible role for PNPLA1 protein in LD regulation via lipophagy-mediated degradation.

The role of PNPLA1 in ω-O-acylceramide synthesis and skin barrier function.

The human genome encodes nine enzymes belonging to the patatin-like phospholipase domain-containing lipase (PNPLA)/Ca2+-independent phospholipase A2 (iPLA2) family. Although most PNPLA/iPLA2 enzymes are widely distributed and act on phospholipids or neutral lipids as (phospho)lipases to play homeostatic roles in lipid metabolism, the function of PNPLA1 remained a mystery until a few years ago. However, the recent finding that mutations in the human PNPLA1 gene are linked to autosomal recessive congenital ichthyosis (ARCI), as well as evidence obtained from biochemical and gene knockout studies, has shed light on the function of this enzyme in skin-specific sphingolipid metabolism rather than glycerophospholipid metabolism. PNPLA1 is specifically expressed in differentiated keratinocytes and plays a crucial role in the biosynthesis of ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide. In this review, we overview the biosynthetic route and biological role of epidermal ω-O-acylceramide, highlight the function of PNPLA1 as a bona fide acylceramide synthase required for proper skin barrier function and keratinocyte differentiation, and summarize the mutations of PNPLA1 currently identified in ARCI patients. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.

Prevalence of PNPLA1 Gene Mutation in 48 Breeding Golden Retriever Dogs.

A non-epidermolytic ichthyosis has been identified in Golden Retrievers due to a variant in the PNPLA1 gene, and a genetic test is available to detect wild-type, heterozygous and homozygous dogs. The aims of this study were to investigate the prevalence of the PNPLA1 gene variant in Golden Retrievers used for breeding and to provide more information to breeders in order to restrict the spread of this disease. Clinical examination and assessment of the PNPLA1 genotype using PCR testing of oral swabs were performed in 48 breeding Golden Retrievers. Wild-type, heterozygous or homozygous variants of the PNPLA1 gene were demonstrated in 10 (21%), 23 (48%), and 15 (31%) of the 48 dogs, respectively. In only 3 of the 48 dogs were clinical signs suggestive of ichthyosis identified. Data collected agreed with data reported in the literature. The high prevalence of homozygous and heterozygous variants makes the exclusion of mutated dogs from breeding impractical. Furthermore, the reliability of the PNPLA1 mutation in prediction of clinical signs of ichthyosis is unclear. Additional studies are needed to investigate if PNPLA1 is the only gene involved or if other genes and environmental factors have a role in the development of ichthyosis in Golden Retrievers.

Identification of two novel PNPLA1 mutations in Turkish families with autosomal recessive congenital ichthyosis.

Dökmeci-Emre S, Taskiran ZE, Yüzbasioglu A, Önal G, Akarsu AN, Karaduman A, Özgüç M. Identification of two novel PNPLA1 mutations in Turkish families with autosomal recessive congenital ichthyosis. Turk J Pediatr 2017; 59: 475-482. Autosomal recessive congenital ichthyosis (ARCI) is a group of inherited keratinization disorders that are characterized by abnormal epidermal keratinization. ARCI patients generally represent serious symptoms including collodion baby phenotype accompanied by dehydration, heat loss, electrolytic imbalance, and sepsis. ARCI shows high degree of clinical and genetic heterogeneity. To date, nine genes were shown to be responsible for ARCI phenotype. One of these genes, patatin-like phospholipase domain containing protein-1 (PNPLA1) was suggested to be involved in the synthesis of ω-O-acylceramides related to epidermal cornified lipid envelope organization. In addition to previously reported PNPLA1 mutations, we report two novel PNPLA1 mutations including one novel missense mutation c.335C > A (p.Ser112Tyr) and one novel deletion mutation c.733_735delTAC (p.Tyr245del) in Turkish ARCI patients from unrelated consanguineous families. We also report previously reported missense mutation c.514G > A (p.Asp172Asn) in Turkish ARCI patients. Novel PNPLA1 mutations were shown to be located in the catalytic patatin domain of PNPLA1 gene. Identification of novel mutations in PNPLA1 gene expands the mutational spectrum in the causative gene. Increase in the total number of cases has high diagnostic value in terms of genotype-phenotype correlation in ARCI patients.

Mutation analysis of the PNPLA1 gene in a family with autosomal recessive congenital ichthyosis / 中华皮肤科杂志

Objective To identify a causative gene of autosomal recessive congenital ichthyosis (ARCI) in a Chinese family,and to analyze the genotype-phenotype correlation.Methods Peripheral blood samples were collected from the proband,his elder brother and parents,and genomic DNA was extracted from these blood samples.Genome-wide exome sequencing was conducted to determine the mutation site in the proband,and then allele-specific oligonucleotide primers were designed based on the mutation site.PCR was performed to detect the mutation site to further identify the causative gene of ARCI in the family.Results A new homozygous missense mutation was identified in exon 4 in 1 allele of the PNPLA1 gene in the proband,which led to a codon change from cytosine (C) to thymine (T) at position 700 (c.700C ＞ T) and resulted in the substitution of proline by serine (p.pro234ser).The same mutation was also detected in the proband's brother,and his parents were the mutation carriers.No mutations were found in unrelated healthy Chinese individuals.Conclusion The missense mutation in the PNPLA1 gene (p.pro234ser) is associated with clinical symptoms of the patient with ARCI.

Identification mouse patatin-like phospholipase domain containing protein 1 as a skin-specific and membrane-associated protein.

Patatin-like phospholipase domain containing protein 1 (PNPLA1) mutations have been identified to be associated with autosomal recessive congenital ichthyosis (ARCI) in recent years. However, its molecular characters have not been achieved until now. In the current study, the full length coding cDNA sequence of mouse PNPLA1 (mPNPLA1) was identified firstly. There were several putative transmembrane domains (TMDs) in mPNPLA1 by bioinformation analysis. mPNPLA1 was further found to be expressed exclusively in the membrane fraction in mammalian cells. However, it did not colocalized with the endoplasmic reticulum (ER) or lipid droplets (LDs). Moreover, the mRNA levels of mPNPLA1 was detected to be highly expressed in the skin, while very weak or even less in other mouse tissues by quantitative PCR. In addition, based on experiments with inhibitors and inducer of protein degradation pathways, mPNPLA1 was demonstrated to be degraded by macroautophagy, but not by the proteasome. These results indicated PNPLA1 was a skin-specific and membrane-associated protein for the first time, suggesting that it may mainly play a role in the skin.