Advertisement

Ceramide Analysis in Combination With Genetic Testing May Provide a Precise Diagnosis for Self-Healing Collodion Babies

Open AccessPublished:November 01, 2022DOI:https://doi.org/10.1016/j.jlr.2022.100308

      Abstract

      Self-healing collodion baby (SHCB), also called “self-improving collodion baby”, is a rare mild variant of autosomal recessive congenital ichthyosis and is defined as a collodion baby who shows the nearly complete resolution of scaling within the first 3 months to 1 year of life. However, during the neonatal period, it is not easy to distinguish SHCB from other inflammatory forms of autosomal recessive congenital ichthyosis, such as congenital ichthyosiform erythroderma. Here, we report a case study of two Japanese SHCB patients with compound heterozygous mutations, c.235G>T (p.(Glu79∗))/ c.1189C>T (p.(Arg397Cys)) and c.1295A>G (p.(Tyr432Cys))/ c.1138delG (p.(Asp380Thrfs∗3)), in CYP4F22, which encodes cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22). Immunohistochemically, inflammation with the strong expression of IL-17C, IL-36γ, and TNF-α was seen in the skin at birth. CYP4F22 is an ultra-long-chain FA ω-hydroxylase responsible for ω-O-acylceramide (acylceramide) production. Among the epidermal ceramides, acylceramide is a key lipid in maintaining the epidermal permeability barrier function. We found that the levels of ceramides with ω-hydroxy FAs including acylceramides and the levels of protein-bound ceramides were much lower in stratum corneum samples obtained by tape stripping from SHCB patients than in those from their unaffected parents and individuals without SHCB. Additionally, our cell-based enzyme assay revealed that two mutants, p.(Glu79∗) and p.(Arg397Cys), had no enzyme activity. Our findings suggest that genetic testing coupled with noninvasive ceramide analyses using tape-stripped stratum corneum samples might be useful for the early and precise diagnosis of congenital ichthyoses, including SHCB.

      Supplementary key words

      Abbreviations:

      ARCI (autosomal recessive congenital ichthyosis), CLE (corneocyte lipid envelope), SHCB (self-healing collodion baby), ULC (ultra long chain), WES (whole-exome sequencing)
      The permeability barrier function of the stratum corneum (outermost layer of the epidermis) is achieved through the integration of lipids and proteins in the terminally differentiated keratinocytes (corneocytes); the cell membrane is replaced by corneocyte lipid envelope (CLE) mainly comprised of protein-bound ceramides, and the space between the cells is filled with multi-laminar lipid lamellae composed of ceramides, cholesterol, and free FAs (
      • Takeichi T.
      • Hirabayashi T.
      • Miyasaka Y.
      • Kawamoto A.
      • Okuno Y.
      • Taguchi S.
      • et al.
      SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation.
      ). Among the elements of the barrier structure in the stratum corneum of human skin, the lipid lamellae and CLE are of critical importance to skin barrier function (
      • Takeichi T.
      SDR9C7 plays an essential role in skin barrier function by dehydrogenating acylceramide for covalent attachment to proteins.
      ). The lipid lamellae and CLE are essential for the integrity of the permeability barrier, and these lacks are a major structural defect behind many diseases of barrier function (
      • Akiyama M.
      Corneocyte lipid envelope (CLE), the key structure for skin barrier function and ichthyosis pathogenesis.
      ).
      Each ceramide class is named using a combination of abbreviations for the constituent FAs (N, non-hydroxy FA; A, α-hydroxy FA; O, ω-hydroxy FA; EO, esterified ω-hydroxy FA; P–O, protein-bound ω-hydroxy FA) and long-chain bases (S, sphingosine; DS, dihydrosphingosine; P, phytosphingosine; H, 6-hydroxysphingosine; SD, 4,14-sphingadiene ) (supplemental Fig. S1) (
      • Kawana M.
      • Miyamoto M.
      • Ohno Y.
      • Kihara A.
      Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS.
      ). EO ceramides (EOS, EODS, EOP, EOH, and EOSD) are referred to as ω-O-acylceramides (acylceramides), and a linoleic acid is the predominant FA in the ω-position of these ceramides (
      • Kawana M.
      • Miyamoto M.
      • Ohno Y.
      • Kihara A.
      Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS.
      ). In humans, the major acylceramide classes are EOS, EOH, and EOP, in order of abundance, while EODS and EOSD are barely detectable (
      • Kawana M.
      • Miyamoto M.
      • Ohno Y.
      • Kihara A.
      Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS.
      ,
      • Suzuki M.
      • Ohno Y.
      • Kihara A.
      Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases.
      ). Acylceramides are essential for the formation and maintenance of lipid lamellae (
      • Sassa T.
      • Ohno Y.
      • Suzuki S.
      • Nomura T.
      • Nishioka C.
      • Kashiwagi T.
      • et al.
      Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain fatty acid production.
      ,
      • Yamamoto H.
      • Hattori M.
      • Chamulitrat W.
      • Ohno Y.
      • Kihara A.
      Skin permeability barrier formation by the ichthyosis-causative gene FATP4 through formation of the barrier lipid omega-O-acylceramide.
      ). Conventional ceramides (N and A ceramides) have FAs with a chain length (C) of C16–C24 in most tissues and C16–C28 in the epidermis, whereas acylceramides have much longer FAs (C30–C36) (
      • Kawana M.
      • Miyamoto M.
      • Ohno Y.
      • Kihara A.
      Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS.
      ,
      • Suzuki M.
      • Ohno Y.
      • Kihara A.
      Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases.
      ). FAs are classified into long-chain FAs (C11–C20), very-long-chain FAs (≥C21), and ultra-long-chain (ULC; ≥C26) FAs (
      • Kihara A.
      Very long-chain fatty acids: elongation, physiology and related disorders.
      ).
      A crucial step in acylceramide synthesis is the hydroxylation of the ULCFA at the ω-position by the ULCFA ω-hydroxylase CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) (
      • Ohno Y.
      • Nakamichi S.
      • Ohkuni A.
      • Kamiyama N.
      • Naoe A.
      • Tsujimura H.
      • et al.
      Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
      ,
      • Miyamoto M.
      • Itoh N.
      • Sawai M.
      • Sassa T.
      • Kihara A.
      Severe skin permeability barrier dysfunction in knockout mice deficient in a fatty acid omega-hydroxylase crucial to acylceramide production.
      ). CYP4F22 has been identified as a causative gene of autosomal recessive congenital ichthyosis (ARCI) (
      • Takeichi T.
      • Akiyama M.
      Inherited ichthyosis: non-syndromic forms.
      ). ARCI is an umbrella term used to describe a cutaneous phenotype of erythema and scaling over almost the entire body at birth (
      • Akiyama M.
      Corneocyte lipid envelope (CLE), the key structure for skin barrier function and ichthyosis pathogenesis.
      ). To date, 57 pathogenic mutations in CYP4F22 have been reported in ARCI, including those causing self-healing collodion baby (SHCB) (www.hgmd.cf.ac.uk, Human Gene Mutation Database Professional, as of 2021.4) (
      • Stenson P.D.
      • Mort M.
      • Ball E.V.
      • Chapman M.
      • Evans K.
      • Azevedo L.
      • et al.
      The Human Gene Mutation Database (HGMD((R))): optimizing its use in a clinical diagnostic or research setting.
      ). Of these, 17 missense mutations and one frameshift mutation were examined for their effect on activity of CYP4F22 (
      • Ohno Y.
      • Nakamichi S.
      • Ohkuni A.
      • Kamiyama N.
      • Naoe A.
      • Tsujimura H.
      • et al.
      Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
      ,
      • Nohara T.
      • Ohno Y.
      • Kihara A.
      Impaired production of the skin barrier lipid acylceramide by CYP4F22 ichthyosis mutations.
      ). CYP4F22 deficiency leads to defective acylceramide synthesis, resulting in reduced amounts of protein-bound ceramides and the malformation of the CLE in the stratum corneum. These ceramide abnormalities cause impaired stratum corneum barrier function in ARCI and SHCB.
      An SHCB, also called “a self-improving collodion baby”, is characterized as a collodion baby with the nearly complete resolution of scaling within the first three months to one year of life (
      • Takeichi T.
      • Akiyama M.
      Inherited ichthyosis: non-syndromic forms.
      ). Thus, SHCB seems to be a relatively mild type of ARCI. However, a quality-of-life survey by Hake et al. concluded that SHCB is a milder, underestimated, clinical variant of ARCI that includes distinct features such as brachydactyly and ear kinking (
      • Hake L.
      • Sussmuth K.
      • Komlosi K.
      • Kopp J.
      • Drerup C.
      • Metze D.
      • et al.
      Quality of life and clinical characteristics of self-improving congenital ichthyosis within the disease spectrum of autosomal-recessive congenital ichthyosis.
      ).
      Recently, Mohamad et al. reported 62 Middle Eastern families of various ethnic backgrounds with ARCI (
      • Mohamad J.
      • Samuelov L.
      • Malchin N.
      • Rabinowitz T.
      • Assaf S.
      • Malki L.
      • et al.
      Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.
      ). In their paper, pathogenic variants were identified by whole-exome sequencing (WES) in most ARCI-associated genes, including TGM1 (21%), CYP4F22 (18%), ALOX12B (14%), ABCA12 (10%), ALOXE3 (6%), NIPAL4 (5%), PNPLA1 (3%), LIPN (2%), and SDR9C7 (2%) (
      • Mohamad J.
      • Samuelov L.
      • Malchin N.
      • Rabinowitz T.
      • Assaf S.
      • Malki L.
      • et al.
      Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.
      ). In 19% of the ARCI cases, no mutation was identified (
      • Mohamad J.
      • Samuelov L.
      • Malchin N.
      • Rabinowitz T.
      • Assaf S.
      • Malki L.
      • et al.
      Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.
      ). Most of the CYP4F22 mutations in their cohort resulted in congenital ichthyosiform erythroderma (
      • Mohamad J.
      • Samuelov L.
      • Malchin N.
      • Rabinowitz T.
      • Assaf S.
      • Malki L.
      • et al.
      Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.
      ). Another recent study reported on genetic analyses performed using different sequencing methods, including Sanger sequencing or next-generation sequencing, for 68 patients with the clinical diagnosis of ARCI, including 16 SHCB patients (
      • Hake L.
      • Sussmuth K.
      • Komlosi K.
      • Kopp J.
      • Drerup C.
      • Metze D.
      • et al.
      Quality of life and clinical characteristics of self-improving congenital ichthyosis within the disease spectrum of autosomal-recessive congenital ichthyosis.
      ). Most of the causative mutations in the ARCI cohort were found in TGM1 (27.9%), followed by ALOX12B (16.2%), ALOXE3 (14.7%), NIPAL4 (13.2%), ABCA12 (13.2%), PNPLA1 (7.4%), CYP4F22 (5.9%), and SDR9C7 (1.5%) (
      • Hake L.
      • Sussmuth K.
      • Komlosi K.
      • Kopp J.
      • Drerup C.
      • Metze D.
      • et al.
      Quality of life and clinical characteristics of self-improving congenital ichthyosis within the disease spectrum of autosomal-recessive congenital ichthyosis.
      ). The genetically confirmed SHCB patients presented causative mutations in ALOXE3 (50.0%), ALOX12B (37.5%), PNPLA1 (6.3%), and CYP4F22 (6.3%) (
      • Hake L.
      • Sussmuth K.
      • Komlosi K.
      • Kopp J.
      • Drerup C.
      • Metze D.
      • et al.
      Quality of life and clinical characteristics of self-improving congenital ichthyosis within the disease spectrum of autosomal-recessive congenital ichthyosis.
      ).
      At birth, it cannot be determined whether collodion babies will become self-healing or will develop congenital ichthyosiform erythroderma, a severe form of ARCI. If the collodion baby phenotype is not self-healing and the patients develop congenital ichthyosiform erythroderma, then the itching, pain from fissures, and other symptoms are lifelong. Thus, it is very important for the families to know whether the phenotype is SHCB. Collodion babies with mutations in other genes causative of ARCI, such as ABCA12, are not expected to be self-healing. Therefore, detecting mutations in CYP4F22 and corresponding ceramide abnormalities would give us supportive data for the early diagnosis of SHCB. Here, we describe two Japanese SHCBs with compound heterozygous mutations in CYP4F22 from two independent families. The findings obtained in the present study suggest that genetic testing in combination with the analysis of ceramides from the stratum corneum in ARCI patients might be useful for the early and precise diagnosis of SHCB.

      Materials and Methods

      Ethics

      This study was approved by the ethics committees of Nagoya University Graduate School of Medicine (Permit nos.: 2013-0279 and 2016-0412) and Hokkaido University (Permit no.: 2020-002). Informed consent was obtained from all volunteers and the guardians of an SHCB patient, and the research was conducted in accordance with the principles expressed in the Declaration of Helsinki.

      Whole-exome sequencing

      Exonic DNA was captured using the Agilent SureSelect Human All Exon v5 target enrichment kit (Agilent Technologies, Santa Clara, CA), and sequencing was performed with paired-end 150-bp reads on the Illumina Hiseq 2500 (Illumina). About 150 M reads/individual were generated, resulting in approximately 100x coverage of the targeted exome. The Genome Analysis Toolkit (GATK v3.5) (Broad Institute) was used to perform variant discovery and genotyping. SNPs and indels were named according to GATK best practices. Variants were filtered under the assumption of a recessive inheritance model.

      Genotyping of ten reported FLG mutations in the Japanese population

      Real-time PCR-based genotyping of the Filaggrin (FLG) mutations was performed with the TaqMan probe genotyping assay, which we established in a previous study (
      • Kono M.
      • Akiyama M.
      • Inoue Y.
      • Nomura T.
      • Hata A.
      • Okamoto Y.
      • et al.
      Filaggrin gene mutations may influence the persistence of food allergies in Japanese primary school children.
      ).

      Immunohistochemical analyses

      Immunohistochemical analysis of skin samples from one of the participants (case 1) was performed as described previously (
      • Takeichi T.
      • Sugiura K.
      • Muro Y.
      • Matsumoto K.
      • Ogawa Y.
      • Futamura K.
      • et al.
      Overexpression of LEDGF/DFS70 induces IL-6 via p38 activation in HaCaT cells, similar to that seen in the psoriatic condition.
      ), with slight modifications. Thin sections (4 μm) were cut from samples embedded in paraffin blocks. The sections were soaked for 20 min at room temperature in 0.3% H2O2/methanol to block endogenous peroxidase activity. After being washed in PBS with 0.01% Triton X-100, the sections were incubated for 30 min in PBS with 4% bovine serum albumin, followed by incubation overnight with the primary antibodies, polyclonal rabbit anti-IL-17C antibody (bs-2611R, Bioss, Woburn, MA; dilution 1:1000), anti-IL-36γ antibody (ab156783; Abcam, Cambridge, UK; dilution 1:1000), and anti-TNF-α antibody (bs-2081R; Bioss; dilution 1:1000) in PBS containing 1% bovine serum albumin. After being washed in PBS, the thin sections were stained with Dako EnVision+Single Reagents (HRP, rabbit) (Agilent Technologies, Santa Clara, CA) for 30 min at room temperature. An Olympus BX51 (Olympus Corporation, Tokyo, Japan) was used for photography.

      Tape stripping and lipid analysis by LC/MS/MS

      To examine the ceramide species present in the stratum corneum, tape stripping was performed by pressing and stripping an adhesive acrylic film (465#40; Teraoka Seisakusho, Tokyo, Japan) on the skin of the right leg of case 1, case 2, and both parents of case 1. Samples were also taken from the right leg of five normal children (around 2 years after birth) as controls. Five strips measuring 25 × 50 mm each were obtained from a single individual. The second strip was cut to 10 × 10 mm and used for lipid extraction. Unbound ceramides (A, N, O, and EO ceramides) and protein-bound ceramides (P–O ceramides) were extracted and their species with the C18 long-chain base were analyzed using an ultra-performance LC coupled with a triple quadrupole mass spectrometer Xevo TQ-S (Waters, Milford, MA) as described previously (
      • Suzuki M.
      • Ohno Y.
      • Kihara A.
      Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases.
      ). The top 100 unbound ceramide species (14 classes) and the top 30 protein-bound ceramide species (five classes), which covered more than 95% of the total amount for each of these categories, were quantified by calculating the ratio of the peak area for each ceramide species to that of the internal standard corresponding to each ceramide class (
      • Arai A.
      • Takeichi T.
      • Wakamoto H.
      • Sassa T.
      • Ito Y.
      • Murase Y.
      • et al.
      Ceramide profiling of stratum corneum in Sjogren-Larsson syndrome.
      ).

      Cell-based ULCFA ω-hydroxylase assay

      ULCFA ω-hydroxylase assay was performed as previously described (
      • Ohno Y.
      • Nakamichi S.
      • Ohkuni A.
      • Kamiyama N.
      • Naoe A.
      • Tsujimura H.
      • et al.
      Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
      ). The plasmids encoding human ELOVL4 (pCE-puro 3×FLAG-ELOVL4), CERS3 (pCE-puro 3×FLAG-CERS3), and CYP4F22 (pCE-puro 3×FLAG-CYP4F22) were used for the expression of N-terminal 3×FLAG-tagged proteins. Plasmids encoding CYP4F22 E79∗ ((p.(Glu79∗)) or R397C (p.(Arg397Cys)) were generated using the pCE-puro 3×FLAG-CYP4F22 plasmid as a template, appropriate primers (E79∗, 5′-CATGTACCTTCCAAATTAGGCGGGCCTTCAAG-3′ and 5′-CTTGAAGGCCCGCCTAATTTGGAAGGTACATG-3′; R397C, 5′-CATTAAGGAGAGCCTGTGCCAGTACCCACCTG-3′ and 5′-CAGGTGGGTACTGGCACAGGCTCTCCTTAATG-3′), and the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). HEK 293T cells were transfected with plasmids encoding 3×FLAG-ELOVL4, 3×FLAG-CERS3, and 3×FLAG-CYP4F22 (wild-type or mutant, E79∗ or R397C). Twenty-one hours after transfection, the cells were incubated for 30 min in a medium without fetal bovine serum and for another 3 h in a medium containing 1 μM seven deuterium (d7)-labeled sphingosine (Avanti Polar Lipids, Birmingham, AL) and 50 μM linoleic acid. The cells were washed twice with PBS and were collected in plastic tubes. After centrifugation (400 g, room temperature, 3 min), the cells were suspended in 100 μl of water and mixed with 375 μl of chloroform/methanol/12 M formic acid (100:200:1, vol/vol). As an internal standard, 2 pmol of N-(2′-(R)-hydroxypalmitoyl(d9))-D-erythro-sphingosine (d9-C16:0 AS, Avanti Polar Lipids) was added. Samples were then mixed with 125 μl of chloroform and 125 μl of water and centrifugated (20,400 g, room temperature, 3 min). The organic phase (lower phase) was collected, dried, and dissolved in 125 μl of chloroform/methanol (1:2, vol/vol). The products of CYP4F22, d7-ω-hydroxyceramides (d7-OS), were detected via LC/MS/MS (supplemental Table S1) and were quantified by calculating the ratio of the peak area of each d7-ω-hydroxyceramide species to that of the d9-C16:0 AS.

      Results

      Clinical features of the two SHCB cases

      Case 1 was the first child born to nonrelated parents without any family history of similar disorders. He was born at full term after an uneventful pregnancy, with a birth weight of 2,938 g. The Apgar Score was 8/10 and 9/10 at 1 and 5 min, respectively. On examination, at birth, he showed a collodion membrane over his entire body surface, with moderate fissuring at the joints (Fig. 1A). During the first 10 days of life, the thick scales gradually desquamated (Fig. 1B). Cultures for microorganisms from skin samples detected methicillin-resistant Staphylococcus aureus. Intravenous vancomycin decreased the skin redness and erosions. His hair, nails, and teeth were normal. He had neither neurological symptoms nor hearing loss.
      Figure thumbnail gr1
      Fig. 1Clinical and histological features of two patients with SHCB. A–D, case 1. A: At 6 days of age, there are diffuse erythematous lesions and erosions with large scales on the chest and abdomen. B: At 10 days of age, erythematous hyperkeratosis on the face and neck and an erosion on the upper chest are evident. C: At 2 years of age, mild whitish scales on the chest are observed. D: A biopsy sample from the ichthyotic skin of case 1 shows compact hyperkeratosis, which suggests a deficiency of the intercellular lipid layers in the stratum corneum. The granular layers seen in the epidermis of case 1 are thinner than those of a healthy skin sample. Scale bars = 100 μm. E, F: case 2. Mild hyperkeratosis with fine, whitish scales is seen on the extensor surfaces of the arms (E) and legs (F). SHCB, self-healing collodion baby.
      A skin biopsy specimen at 10 days after birth showed hyperkeratosis with thinned granular layers (Fig. 1D). Granular degeneration was not observed. Laboratory tests showed mild hyper-eosinophilia. At 2 years of age, he showed only extremely mild generalized ichthyosis and overlying mild fine scaling (Fig. 1C).
      Case 2 is a 11-year-old male and the third child of nonrelated healthy parents. At the age of 12 months, he was diagnosed with congenital ichthyosis and he received emollients. His aunt and cousin also have ichthyoses. Clinical examinations found very mild fine scales on the trunk and extremities (Fig. 1E, F). Case 1 and case 2 were from independent families.

      Mutation detection

      To identify the underlying molecular genetic defects in both patients, we obtained blood samples from the two patients and their parents for genetic testing. Based on the mild phenotypes and high prevalence of common ichthyoses (
      • Takeichi T.
      • Akiyama M.
      Inherited ichthyosis: non-syndromic forms.
      ), we suspected that the diagnosis for the two patients was ichthyosis vulgaris caused by FLG mutations or recessive X-linked ichthyosis associated with STS (steroid sulfatase) deletions. Initial fluorescence in situ hybridization analysis for Xp22.3, which includes the region of STS on chromosome X, revealed no deletion of Xp22.3 in either patient. Next, the genotyping of the FLG mutations, all of which are loss-of-function mutations, was performed with the TaqMan probe genotyping assay. In neither patient did we identify any putative pathogenic mutation in FLG.
      Then, WES was performed for case 1, his parents, and case 2. The filtered rare variant list generated from the whole-exome data showed the two affected individuals to harbor rare or novel compound heterozygous mutations in CYP4F22. These mutations were verified by Sanger sequencing (Fig. 2A, B) and were confirmed to segregate with disease status in family members whose DNA was available. WES failed to reveal any pathogenic mutations in other genes known to be implicated in ichthyosis. Pathogenic variants of CYP4F22 are reported to be scattered throughout the CYP4F22 gene, and most of the pathogenic CYP4F22 mutations are located within the longest cytoplasmic domain of the CYP4F22 protein (www.hgmd.cf.ac.uk, Human Gene Mutation Database Professional, as of 2021.4) (
      • Nohara T.
      • Ohno Y.
      • Kihara A.
      Impaired production of the skin barrier lipid acylceramide by CYP4F22 ichthyosis mutations.
      ). The present four mutations lie in the CYP4F22 cytoplasmic domain (Fig. 2C). The mutation c.235G>T, p.(Glu79∗) has not been described in the gnomAD database (
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alfoldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      ) nor in the dbSNP database. The global allele frequencies of c.1189C>T, p.(Arg397Cys) (rs572771583) and c.1295A>G, p.(Tyr432Cys) (rs1430532183) are 0.000003976 (one heterozygous carrier was reported for each mutation in the gnomAD database (
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alfoldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      )). Several protein function prediction browsers (e. g., SIFT (
      • Ng P.C.
      • Henikoff S.
      SIFT: predicting amino acid changes that affect protein function.
      ), PolyPhen-2 (
      • Adzhubei I.A.
      • Schmidt S.
      • Peshkin L.
      • Ramensky V.E.
      • Gerasimova A.
      • Bork P.
      • et al.
      A method and server for predicting damaging missense mutations.
      ), MutationTaster (
      • Schwarz J.M.
      • Cooper D.N.
      • Schuelke M.
      • Seelow D.
      MutationTaster2: mutation prediction for the deep-sequencing age.
      )) attributed very high scores for likelihood of damage.
      Figure thumbnail gr2
      Fig. 2CYP4F22 mutations detected in the present two patients with SHCB and the domain structure of CYP4F22. A, B: Sanger sequencing confirms CYP4F22 mutations in genomic DNA. Chromatograms illustrate the four CYP4F22 mutations identified in this study. A: case 1 is compound heterozygous for the c.235G>T, p.(Glu79∗) and c.1189C>T, p.(Arg397Cys) mutations. B: case 2 is compound heterozygous for c.1295A>G, p.(Tyr432Cys) and c.1138delG, p.(Asp380Thrfs∗3). C: The CYP4F22 domain structure, with the mutations indicated. The mutations in case 1 and case 2 are marked by black arrows. SHCB, self-healing collodion baby.

      Immunohistochemical analysis of the skin specimen from case 1

      We conducted immunohistochemical analysis with anti-IL-17C, anti-IL-36γ, and anti-TNFα antibodies of a lesional skin sample from case 1 to assess whether cutaneous inflammation occurred. The staining intensities of IL-17C (Fig. 3A), IL-36γ (Fig. 3B), and TNF-α (Fig. 3C) were significantly greater in the patient’s skin than in the skin from a healthy control.
      Figure thumbnail gr3
      Fig. 3Expression of IL-17C, IL-36γ, and TNF-α in SHCB skin lesions and analysis of ceramide components in the patients’ stratum corneum. A–C: Skin samples from case 1 (right) and from healthy control donors (left) were stained with anti-IL-17C (A), anti-IL-36γ (B), and anti-TNF-α (C) antibodies. Scale bars: 100 μm. D: Amounts of ceramide from each class in the stratum corneum of both patients, the parents of case 1 and controls. SHCB, self-healing collodion baby.

      Ceramide profiles in the stratum corneum of the patients

      The levels of unbound and protein-bound ceramides in the tape-stripped skin samples from the right leg were examined by LC/MS/MS. Although the overall effects of the bi-allelic mutations in CYP4F22 on the levels of total ceramides were small, it is notable that the levels of acylceramides (EOS, EOH, and EOP) and protein-bound ceramides (P-OS and P-OH) were much lower in the stratum corneum of the patients than in the unaffected parents and normal controls (Fig. 3D and Table 1, Table 2, Table 3). Additionally, the amounts of NP and AP were lower in the stratum corneum of the patients than in the controls. In contrast, the levels of NS and AS were elevated in the patients’ samples. Regarding FA composition, shortening of nonacylated ceramides (e. g., NH) was observed (Table 4). In patients, NH species with ≥C26 FAs were reduced compared to healthy subjects, but instead C16–C22 species were increased.
      Table 1Amount of each ceramid class in the stratum corneum (pmol/mg protein)
      Ceramide ClassesAverage of Controls (n = 5)Father of Case 1Mother of Case 1Case 1Case 2
      NDS481516474404398
      NS249453309648684
      NH910185515573201317
      NP96419062030209510
      ADS3651751248
      AS241368294798691
      AH568786837405888
      AP21031144989168
      EOS2151035942
      EOH1331057794
      EOP35454511
      OS27191344
      OH137720
      OP86552
      Total40896530623029104718
      Table 2Percentages of ceramide classes in the stratum corneum
      Ceramide ClassesAverages of Controls (n = 5)Father of Case 1Mother of Case 1Case 1Case 2
      NDS127.97.613.98.4
      NS6.36.9522.314.5
      NH21.628.4251127.9
      NP23.729.232.67.210.8
      ADS0.90.81.20.41
      AS6.15.64.727.414.7
      AH13.31213.413.918.8
      AP5.34.87.23.13.6
      EOS5.41.60.90.10.1
      EOH3.31.61.20.30.1
      EOP0.90.70.700
      OS0.70.30.20.20.1
      OH0.30.10.10.10
      OP0.20.10.10.20.1
      Table 3Amount of protein-bound ceramide in the stratum corneum (pmol/mg protein)
      Ceramide ClassesAverage of Controls (n = 5)Father of Case 1Mother of Case 1Case 1Case 2
      P-OS11393165037
      P-ODS00.10.800
      P-OH31478805
      P-OP23.86.700.1
      P-OSD222.500.5
      Table 4Quantities of total NH and each NH species and percentages of each NH species
      NH Species of Different Carbon LengthAverage of Controls (n = 5)Father of Case 1Mother of Case 1Case 1Case 2
      pmol/mg ProteinPercentage (%)pmol/mg ProteinPercentage (%)pmol/mg ProteinPercentage (%)pmol/mg ProteinPercentage (%)pmol/mg ProteinPercentage (%)
      C16:0111.413.21.221.70.8175.371.45.4
      C18:09.214.50.47.70.322.57433.3
      C20:06.60.85.30.350.313.64.2221.7
      C22:0192.117.8119.41.123.17.2634.8
      C23:020.72.320.11.324.31.314.54.538.22.9
      C24:0213.122.8308.819.3358.919.810131.5299.522.7
      C25:086.89.2170.610.3190.611288.795.77.3
      C26:031834.8652.239.7737.241.973.523362.927.5
      C27:052.45.686.66.3116.85.63.3159.34.5
      C28:0137.515.7217.415.8292.51413.84.3181.713.8
      C29:016.61.924.71.732.11.64.51.425.72
      C30:019.32.335.82.6492.35.51.754.74.2
      Total910.21001556.91001855.1100320.21001317.3100

      CYP4F22 mutations impair enzymatic activity

      To assess the effect of the mutations on CYP4F22 enzymatic activity, a cell-based assay was conducted, where wild type or mutants (p.Glu79∗ and p.Arg397Cys) of CYP4F22 were overproduced, together with the FA elongase ELOVL4 and the ceramide synthase CERS3 (all tagged with 3×FLAG). Expression of these proteins was confirmed by immunoblotting (Fig. 4A). Quantification of the CYP4F22 products ω-hydroxy ceramides (OS) by LC/MS/MS revealed that the OS levels in cells expressing either mutant was comparable to those in the vector-transfected cells (Fig. 4B), indicating that both mutants have deficient enzyme activity.
      Figure thumbnail gr4
      Fig. 4Mutations, p.(Glu79∗) and p.(Arg397Cys), in CYP4F22 impair enzymatic activity. HEK 293T cells were transfected with the pCE-puro 3×FLAG-1 (vector) or pCE-puro 3×FLAG-CYP4F22 (wild type [WT], E79∗, or R397C mutant) plasmid, together with pCE-puro 3×FLAG-ELOVL4 and pCE-puro 3×FLAG-CERS3 plasmids and were incubated with 1 μM d7-sphingosine and 50 μM linoleic acid for 3 h. A: Total cell lysates (5 μg) were separated by SDS-PAGE and subjected to immunoblotting using anti-FLAG antibody (upper panel) or anti-GAPDH antibody (lower panel, loading control). E79∗, p.(Glu79∗); R397C, p.(Arg397Cys). B: Amounts of d7-ω-hydroxyceramide species with saturated and monounsaturated FAs (C30–36) were quantified by LC/MS/MS analyses. Values represent the mean + standard deviations of three independent experiments (n = 3, ∗∗ P < 0.01; Dunnett’s test; vs. WT). IB, immunoblotting.

      Discussion

      We previously reported that IL-17C and IL-36 family cytokines were upregulated in the skin of an adult ARCI patient with a NIPAL4 mutation (
      • Murase Y.
      • Takeichi T.
      • Kawamoto A.
      • Tanahashi K.
      • Okuno Y.
      • Takama H.
      • et al.
      Reduced stratum corneum acylceramides in autosomal recessive congenital ichthyosis with a NIPAL4 mutation.
      ). In the literature, the significant upregulation of IL-17/TNF-α-related genes and psoriasis hallmark genes has been reported in various ARCI patients (
      • Malik K.
      • He H.
      • Huynh T.N.
      • Tran G.
      • Mueller K.
      • Doytcheva K.
      • et al.
      Ichthyosis molecular fingerprinting shows profound TH17 skewing and a unique barrier genomic signature.
      ). Malik et al. reported that IL-17-associated markers in ichthyotic skin and serum IL-17A levels cluster tightly with disease severity (ichthyosis area and severity index–erythema), whereas epidermal dysfunction (transepidermal water loss) correlate most closely with EREG and IL36B mRNA expression levels (
      • Malik K.
      • He H.
      • Huynh T.N.
      • Tran G.
      • Mueller K.
      • Doytcheva K.
      • et al.
      Ichthyosis molecular fingerprinting shows profound TH17 skewing and a unique barrier genomic signature.
      ). However, patients with SHCB caused by CYP4F22 mutations were not included in their study. Our immunohistochemical staining results indicate that there was certain cutaneous inflammation with the strong expression of IL-17C, IL-36γ, and TNF-α in case 1 at birth (Fig. 3). Although skin samples from SHCB patients after self-healing were unavailable, the initial inflammation in the SHCB patients due to barrier dysfunction is expected to abate with age. Thus, during the neonatal period, the immune profiles might be shared between SHCB and other ARCIs, and it is not easy to distinguish SHCB from other forms of ARCI, such as CIE.
      LC/MS/MS measurement in this study revealed decreases in the amounts of acylceramides (EO ceramides), protein-bound ceramides (P-O ceramides), and P-type ceramides (NP and AP) in the SHCB patients (Fig. 3D and Table 1, Table 2, Table 3). Acylceramides are thought to stabilize lipid lamellae by interconnecting the layers of the lamellae (
      • Lundborg M.
      • Narangifard A.
      • Wennberg C.L.
      • Lindahl E.
      • Daneholt B.
      • Norlen L.
      Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.
      ). Protein-bound ceramides (CLE) may function to connect lipid lamellae and corneocytes. The 4-hydroxyl group in the long-chain base moiety of P-type ceramides enhances the lipid-lipid interaction in lipid lamellae through hydrogen bonding. NP levels are decreased in patients with atopic dermatitis and are inversely correlated with the values of transepidermal water loss in healthy subjects in addition to the patients (
      • Ishikawa J.
      • Narita H.
      • Kondo N.
      • Hotta M.
      • Takagi Y.
      • Masukawa Y.
      • et al.
      Changes in the ceramide profile of atopic dermatitis patients.
      ,
      • Yokose U.
      • Ishikawa J.
      • Morokuma Y.
      • Naoe A.
      • Inoue Y.
      • Yasuda Y.
      • et al.
      The ceramide [NP]/[NS] ratio in the stratum corneum is a potential marker for skin properties and epidermal differentiation.
      ). Thus, all of the ceramide classes reduced in the SHCB patients in this study are important for lipid lamella or CLE formation. The increased amounts of AS and NS would be primarily due to compensation for the decrease in P-type ceramides.
      Interestingly, although very low levels of acylceramides and protein-bound ceramides were found in the stratum corneum, both patients showed mild to moderate phenotypes of congenital ichthyosis. We speculate that these rather mild phenotypes from CYP4F22 mutations might be caused by mild inflammation in the skin. Clinically, the cutaneous inflammation in patients with KDSR (3-Ketodihydrosphingosine Reductase) mutations, PHGDH (Phosphoglycerate Dehydrogenase) mutations, and NIPAL4 mutations is more severe than in patients with SDR9C7 mutations (
      • Takeichi T.
      SDR9C7 plays an essential role in skin barrier function by dehydrogenating acylceramide for covalent attachment to proteins.
      ,
      • Murase Y.
      • Takeichi T.
      • Kawamoto A.
      • Tanahashi K.
      • Okuno Y.
      • Takama H.
      • et al.
      Reduced stratum corneum acylceramides in autosomal recessive congenital ichthyosis with a NIPAL4 mutation.
      ,
      • Takeichi T.
      • Torrelo A.
      • Lee J.Y.W.
      • Ohno Y.
      • Lozano M.L.
      • Kihara A.
      • et al.
      Biallelic mutations in KDSR disrupt ceramide synthesis and result in a spectrum of keratinization disorders associated with thrombocytopenia.
      ,
      • Takeichi T.
      • Okuno Y.
      • Kawamoto A.
      • Inoue T.
      • Nagamoto E.
      • Murase C.
      • et al.
      Reduction of stratum corneum ceramides in Neu-Laxova syndrome caused by phosphoglycerate dehydrogenase deficiency.
      ). This difference is also histologically confirmed in skin biopsy samples from the patients. Additionally, microarray gene expression profiling by using Sdr9c7 knockout mice indicated that the gene expression changes of skin-associated immune responses in the skin from Sdr9c7 knockout mice were very limited compare with those changes in wild-type mice (
      • Takeichi T.
      • Hirabayashi T.
      • Miyasaka Y.
      • Kawamoto A.
      • Okuno Y.
      • Taguchi S.
      • et al.
      SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation.
      ). The severity of inflammation modifies the phenotype in each patient with ichthyosis and, clinically, anti-inflammatory therapies (e. g., anti-TNF-α, anti-IL-17, and anti-IL-4/IL-13 antibodies) have been reported as useful treatments for several types of inherited ichthyoses (
      • Roda A.
      • Mendonca-Sanches M.
      • Travassos A.R.
      • Soares-de-Almeida L.
      • Metze D.
      Infliximab therapy for Netherton syndrome: a case report.
      ,
      • Murase C.
      • Takeichi T.
      • Taki T.
      • Yoshikawa T.
      • Suzuki A.
      • Ogi T.
      • et al.
      Successful dupilumab treatment for ichthyotic and atopic features of Netherton syndrome.
      ).
      The present study revealed a novel mutation in CYP4F22, c.1295A>G, p.(Tyr432Cys), in case 2, and three previously reported mutations: c.235G>T/p.(Glu79∗), c.1189C>T/p.(Arg397Cys), and c.1138delG/p.(Asp380Thrfs∗3) (
      • Ohno Y.
      • Nakamichi S.
      • Ohkuni A.
      • Kamiyama N.
      • Naoe A.
      • Tsujimura H.
      • et al.
      Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
      ,
      • Mohamad J.
      • Samuelov L.
      • Malchin N.
      • Rabinowitz T.
      • Assaf S.
      • Malki L.
      • et al.
      Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.
      ,
      • Zhao L.
      • Wang C.
      • Zhang Y.
      • Li J.
      • Liu H.
      • Feng D.
      Whole-exome sequencing identified a novel pathogenic mutation of the CYP4F22 gene in a Chinese patient with autosomal recessive congenital ichthyosis and in vitro study of the mutant CYP4F22 protein.
      ) (Fig. 2). The nonsense mutation p.(Glu79∗) causes the complete loss of enzyme activity for CYP4F22 (Fig. 4B). The frameshift mutation c.1138delG in CYP4F22 is predicted to cause truncation of the CYP4F22 p.(Asp380Thrfs∗3) protein and to lead loss of enzyme activity. The missense mutation p.(Arg397Cys) changes a basic amino acid into a neutral, polar one in the cytoplasmic domain of the molecule. As for p.(Tyr432Cys), both tyrosine and cysteine are neutral, polar amino acids, but unlike cysteine, tyrosine is an aromatic acid. Several protein function prediction browsers including SIFT, PolyPhen-2, and MutationTaster attribute very high scores for likelihood of damage to both substitutions. Indeed, we confirm that p.(Arg397Cys) loses activity (Fig. 4B).
      The present probands showed SHCB, a relatively mild phenotype of ARCI. At present, genotype/phenotype correlations in ARCI that are associated with CYP4F22 mutation are uncertain. Both of the present patients are compound heterozygous for missense and truncating mutations in CYP4F22. However, there are a few differences in the stratum corneum ceramide profiles between the present two SHCB patients. In the literature, there are several reports of homozygous or compound heterozygous missense mutations in CYP4F22 leading not only to SHCB but also to lamellar ichthyosis, a nonimproving ARCI phenotype (
      • Noguera-Morel L.
      • Feito-Rodriguez M.
      • Maldonado-Cid P.
      • Garcia-Minaur S.
      • Kamsteeg E.J.
      • Gonzalez-Sarmiento R.
      • et al.
      Two cases of autosomal recessive congenital ichthyosis due to CYP4F22 mutations: expanding the genotype of self-healing collodion baby.
      ,
      • Esperon-Moldes U.
      • Ginarte-Val M.
      • Rodriguez-Pazos L.
      • Fachal L.
      • Martin-Santiago A.
      • Vicente A.
      • et al.
      Novel CYP4F22 mutations associated with autosomal recessive congenital ichthyosis (ARCI). Study of the CYP4F22 c.1303C>T founder mutation.
      ). There are no obvious differences in the results of the present cell-based ω-hydroxylase assay between missense and truncating mutations in CYP4F22. Thus, whether causative mutations are missense or truncating might not be a definitive factor for the SHCB phenotype in patients with ARCI from CYP4F22 mutations. Concerning the mechanism of “self-healing” in the present patients, several possibilities are conceivable, including the normalization of ceramide profiles with improvements in the patient’s phenotype and compensation for the role of CYP4F22, ω-hydroxylation of the ceramides by another cytochrome p450 enzyme. However, in our present results, the ceramide profile did not normalize with improvements in the patients. Furthermore, we previously reported that the production of ω-hydroxy ceramides is below detectable levels in HEK293T cells that overexpress ELOVL4, CERS3, and CYP4F11 (
      • Ohno Y.
      • Nakamichi S.
      • Ohkuni A.
      • Kamiyama N.
      • Naoe A.
      • Tsujimura H.
      • et al.
      Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
      ). Thus, it is unlikely that CYP4F11 would take over the role of ω-hydroxylating the ceramides. We would like to accumulate and analyze additional SHCB patients with CYP4F22 mutations in order to clarify the mechanism of self-healing.
      There are unique and characteristic features of the ceramide composition in the stratum corneum of patients with syndromic or nonsyndromic ichthyosis, depending on the causative genes. Genetic diagnosis using next-generation sequencing, such as WES, sometimes detects many variants of unknown significance and it is very difficult to determine the truly pathogenic variants. Thus, well-established in vitro or in vivo functional studies are frequently needed to determine the pathogenicity of novel mutations (
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ). The work involved, such as a cell-based assay using mutant plasmids (
      • Zhao L.
      • Wang C.
      • Zhang Y.
      • Li J.
      • Liu H.
      • Feng D.
      Whole-exome sequencing identified a novel pathogenic mutation of the CYP4F22 gene in a Chinese patient with autosomal recessive congenital ichthyosis and in vitro study of the mutant CYP4F22 protein.
      ), is often time-consuming, labor-intensive, and expensive. In this context, detailed analyses of ceramides in the stratum corneum of patients with ichthyosis might provide valuable clues for detecting causative genes in each patient. WES coupled with noninvasive ceramide analyses using stratum corneum samples obtained by tape stripping is useful for clinical diagnosis, especially for patients with ichthyosis in early infancy. In the Middle-Eastern population, higher prevalences of CYP4F22 and ABCA12 pathogenic variants and lower prevalences of TGM1 and NIPAL4 variants have been reported, as compared with data obtained in other regions of the world (
      • Mohamad J.
      • Samuelov L.
      • Malchin N.
      • Rabinowitz T.
      • Assaf S.
      • Malki L.
      • et al.
      Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.
      ). Perhaps the present results from noninvasive ceramide analyses are more valuable for patients with ARCI in the Middle-Eastern population.
      In conclusion, our findings, in combination with previous reports, suggest that WES coupled with noninvasive ceramide analyses using stratum corneum samples obtained by tape stripping might be useful for the early and precise diagnosis of congenital ichthyosis.

      Data Availability

      All data are contained within the article.

      Supplemental Data

      This article contains supplemental data.

      Conflicts of Interest

      The authors declare that we have no conflicts of interest.

      Acknowledgments

      The authors thank Ms. Haruka Ozeki and Ms. Yuka Terashita for their technical help in analyzing CYP4F22. The patients and their guardians in this article have given written informed consent for the publication of their case details.

      Author Contributions

      T. T. and M. A. conceptualization; T. T., T. O., A. K., and M. A. funding acquisition; T. T., Y. O., K. T., and Y. I. investigation; T. T. and Y. O. methodology; T. T., K. Shiraishi, R. U., S. Y., K. I., H. N., S. M., and K. Sayama, resources; T. T. and Y. O. writing–original draft; T. O. data curation; T. O. formal analysis; Y. M., A. K., and M. A. writing–review and editing; M. A. project administration; M. A. supervision.

      Funding and Additional Information

      This research was supported by a Health and Labor Sciences Research Grant for Research on Intractable Diseases ( 20FC1052 ) from the Ministry of Health , Labor and Welfare of Japan to M. A. This work was supported by a JST FOREST Program grant (Number JPMJFR210R ) to T. T. This study was supported in part by grant-in-aid JSPS KAKENHI Grants Number JP21H02941 to M. A., JP22H04986 to A. K., and JP20K08648 to T. T. and by Japan Agency for Medical Research and Development under Grant Number JP20ek0109488 to T. T, T. O., and M. A.

      Supplemental data

      References

        • Takeichi T.
        • Hirabayashi T.
        • Miyasaka Y.
        • Kawamoto A.
        • Okuno Y.
        • Taguchi S.
        • et al.
        SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation.
        J. Clin. Invest. 2020; 130: 890-903
        • Takeichi T.
        SDR9C7 plays an essential role in skin barrier function by dehydrogenating acylceramide for covalent attachment to proteins.
        J. Dermatol. Sci. 2020; 98: 82-87
        • Akiyama M.
        Corneocyte lipid envelope (CLE), the key structure for skin barrier function and ichthyosis pathogenesis.
        J. Dermatol. Sci. 2017; 88: 3-9
        • Kawana M.
        • Miyamoto M.
        • Ohno Y.
        • Kihara A.
        Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS.
        J. Lipid Res. 2020; 61: 884-895
        • Suzuki M.
        • Ohno Y.
        • Kihara A.
        Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases.
        J. Lipid Res. 2022; 63100235
        • Sassa T.
        • Ohno Y.
        • Suzuki S.
        • Nomura T.
        • Nishioka C.
        • Kashiwagi T.
        • et al.
        Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain fatty acid production.
        Mol. Cell. Biol. 2013; 33: 2787-2796
        • Yamamoto H.
        • Hattori M.
        • Chamulitrat W.
        • Ohno Y.
        • Kihara A.
        Skin permeability barrier formation by the ichthyosis-causative gene FATP4 through formation of the barrier lipid omega-O-acylceramide.
        Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 2914-2922
        • Kihara A.
        Very long-chain fatty acids: elongation, physiology and related disorders.
        J. Biochem. 2012; 152: 387-395
        • Ohno Y.
        • Nakamichi S.
        • Ohkuni A.
        • Kamiyama N.
        • Naoe A.
        • Tsujimura H.
        • et al.
        Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
        Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 7707-7712
        • Miyamoto M.
        • Itoh N.
        • Sawai M.
        • Sassa T.
        • Kihara A.
        Severe skin permeability barrier dysfunction in knockout mice deficient in a fatty acid omega-hydroxylase crucial to acylceramide production.
        J. Invest. Dermatol. 2020; 140: 319-326.e314
        • Takeichi T.
        • Akiyama M.
        Inherited ichthyosis: non-syndromic forms.
        J. Dermatol. 2016; 43: 242-251
        • Stenson P.D.
        • Mort M.
        • Ball E.V.
        • Chapman M.
        • Evans K.
        • Azevedo L.
        • et al.
        The Human Gene Mutation Database (HGMD((R))): optimizing its use in a clinical diagnostic or research setting.
        Hum. Genet. 2020; 139: 1197-1207
        • Nohara T.
        • Ohno Y.
        • Kihara A.
        Impaired production of the skin barrier lipid acylceramide by CYP4F22 ichthyosis mutations.
        J. Dermatol. Sci. 2021; 101: 69-71
        • Hake L.
        • Sussmuth K.
        • Komlosi K.
        • Kopp J.
        • Drerup C.
        • Metze D.
        • et al.
        Quality of life and clinical characteristics of self-improving congenital ichthyosis within the disease spectrum of autosomal-recessive congenital ichthyosis.
        J. Eur. Acad. Dermatol. Venereol. 2021; 36: 582-591
        • Mohamad J.
        • Samuelov L.
        • Malchin N.
        • Rabinowitz T.
        • Assaf S.
        • Malki L.
        • et al.
        Molecular epidemiology of non-syndromic autosomal recessive congenital ichthyosis in a Middle-Eastern population.
        Exp. Dermatol. 2021; 30: 1290-1297
        • Kono M.
        • Akiyama M.
        • Inoue Y.
        • Nomura T.
        • Hata A.
        • Okamoto Y.
        • et al.
        Filaggrin gene mutations may influence the persistence of food allergies in Japanese primary school children.
        Br. J. Dermatol. 2018; 179: 190-191
        • Takeichi T.
        • Sugiura K.
        • Muro Y.
        • Matsumoto K.
        • Ogawa Y.
        • Futamura K.
        • et al.
        Overexpression of LEDGF/DFS70 induces IL-6 via p38 activation in HaCaT cells, similar to that seen in the psoriatic condition.
        J. Invest. Dermatol. 2010; 130: 2760-2767
        • Arai A.
        • Takeichi T.
        • Wakamoto H.
        • Sassa T.
        • Ito Y.
        • Murase Y.
        • et al.
        Ceramide profiling of stratum corneum in Sjogren-Larsson syndrome.
        J. Dermatol. Sci. 2022; 107: 114-122
        • Karczewski K.J.
        • Francioli L.C.
        • Tiao G.
        • Cummings B.B.
        • Alfoldi J.
        • Wang Q.
        • et al.
        The mutational constraint spectrum quantified from variation in 141,456 humans.
        Nature. 2020; 581: 434-443
        • Ng P.C.
        • Henikoff S.
        SIFT: predicting amino acid changes that affect protein function.
        Nucl. Acids Res. 2003; 31: 3812-3814
        • Adzhubei I.A.
        • Schmidt S.
        • Peshkin L.
        • Ramensky V.E.
        • Gerasimova A.
        • Bork P.
        • et al.
        A method and server for predicting damaging missense mutations.
        Nat. Met. 2010; 7: 248-249
        • Schwarz J.M.
        • Cooper D.N.
        • Schuelke M.
        • Seelow D.
        MutationTaster2: mutation prediction for the deep-sequencing age.
        Nat. Met. 2014; 11: 361-362
        • Murase Y.
        • Takeichi T.
        • Kawamoto A.
        • Tanahashi K.
        • Okuno Y.
        • Takama H.
        • et al.
        Reduced stratum corneum acylceramides in autosomal recessive congenital ichthyosis with a NIPAL4 mutation.
        J. Dermatol. Sci. 2020; 97: 50-56
        • Malik K.
        • He H.
        • Huynh T.N.
        • Tran G.
        • Mueller K.
        • Doytcheva K.
        • et al.
        Ichthyosis molecular fingerprinting shows profound TH17 skewing and a unique barrier genomic signature.
        J. Allergy Clin. Immunol. 2019; 143: 604-618
        • Lundborg M.
        • Narangifard A.
        • Wennberg C.L.
        • Lindahl E.
        • Daneholt B.
        • Norlen L.
        Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.
        J. Struct. Biol. 2018; 203: 149-161
        • Ishikawa J.
        • Narita H.
        • Kondo N.
        • Hotta M.
        • Takagi Y.
        • Masukawa Y.
        • et al.
        Changes in the ceramide profile of atopic dermatitis patients.
        J. Invest. Dermatol. 2010; 130: 2511-2514
        • Yokose U.
        • Ishikawa J.
        • Morokuma Y.
        • Naoe A.
        • Inoue Y.
        • Yasuda Y.
        • et al.
        The ceramide [NP]/[NS] ratio in the stratum corneum is a potential marker for skin properties and epidermal differentiation.
        BMC Dermatol. 2020; 20: 6
        • Takeichi T.
        • Torrelo A.
        • Lee J.Y.W.
        • Ohno Y.
        • Lozano M.L.
        • Kihara A.
        • et al.
        Biallelic mutations in KDSR disrupt ceramide synthesis and result in a spectrum of keratinization disorders associated with thrombocytopenia.
        J. Invest. Dermatol. 2017; 137: 2344-2353
        • Takeichi T.
        • Okuno Y.
        • Kawamoto A.
        • Inoue T.
        • Nagamoto E.
        • Murase C.
        • et al.
        Reduction of stratum corneum ceramides in Neu-Laxova syndrome caused by phosphoglycerate dehydrogenase deficiency.
        J. Lipid Res. 2018; 59: 2413-2420
        • Roda A.
        • Mendonca-Sanches M.
        • Travassos A.R.
        • Soares-de-Almeida L.
        • Metze D.
        Infliximab therapy for Netherton syndrome: a case report.
        JAAD Case Rep. 2017; 3: 550-552
        • Murase C.
        • Takeichi T.
        • Taki T.
        • Yoshikawa T.
        • Suzuki A.
        • Ogi T.
        • et al.
        Successful dupilumab treatment for ichthyotic and atopic features of Netherton syndrome.
        J. Dermatol. Sci. 2021; 102: 126-129
        • Zhao L.
        • Wang C.
        • Zhang Y.
        • Li J.
        • Liu H.
        • Feng D.
        Whole-exome sequencing identified a novel pathogenic mutation of the CYP4F22 gene in a Chinese patient with autosomal recessive congenital ichthyosis and in vitro study of the mutant CYP4F22 protein.
        J. Dermatol. 2022; 49: 550-555
        • Noguera-Morel L.
        • Feito-Rodriguez M.
        • Maldonado-Cid P.
        • Garcia-Minaur S.
        • Kamsteeg E.J.
        • Gonzalez-Sarmiento R.
        • et al.
        Two cases of autosomal recessive congenital ichthyosis due to CYP4F22 mutations: expanding the genotype of self-healing collodion baby.
        Pediatr. Dermatol. 2016; 33: e48-e51
        • Esperon-Moldes U.
        • Ginarte-Val M.
        • Rodriguez-Pazos L.
        • Fachal L.
        • Martin-Santiago A.
        • Vicente A.
        • et al.
        Novel CYP4F22 mutations associated with autosomal recessive congenital ichthyosis (ARCI). Study of the CYP4F22 c.1303C>T founder mutation.
        PLoS One. 2020; 15e0229025
        • Richards S.
        • Aziz N.
        • Bale S.
        • Bick D.
        • Das S.
        • Gastier-Foster J.
        • et al.
        Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
        Genet. Med. 2015; 17: 405-424