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Whole-exome sequencing in patients with ichthyosis reveals modifiers associated with increased IgE levels and allergic sensitizations

2014; Elsevier BV; Volume: 135; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2014.09.042

1097-6825

Dimitra Kiritsi, Manthoula Valari, Paola Fortugno, Ingrid Haußer, Lilia Lykopoulou, Giovanna Zambruno, Judith Fischer, Leena Bruckner‐Tuderman, Thilo Jakob, Cristina Has,

Contact Dermatitis and Allergies

The crucial role of the skin as a barrier against environmental insults is mainly dependent on terminally differentiated keratinocytes (ie, corneocytes), which are attached through corneodesmosomes and surrounded by a lipid-rich extracellular matrix. These well-adapted cells are dynamically and regularly desquamated, so that the stratum corneum becomes repopulated by new cells from the inner layers. Numerous molecular players govern these fine-tuned processes, including structural and transport proteins, proteases, and their inhibitors, as well as molecules with yet unknown functions. Mutations in the corresponding genes result in ichthyoses, rare disorders characterized by generalized skin scaling with or without extracutaneous features.1Oji V. Tadini G. Akiyama M. Blanchet Bardon C. Bodemer C. Bourrat E. et al.Revised nomenclature and classification of inherited ichthyoses: results of the First Ichthyosis Consensus Conference in Soreze 2009.J Am Acad Dermatol. 2010; 63: 607-641Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar Autosomal recessive congenital ichthyoses (ARCIs) comprise 2 main subtypes: lamellar ichthyoses, with pronounced scaling, and congenital erythrodermic ichthyoses, with marked erythema. Furthermore, genetic mutations or variants leading to defects in barrier function contribute to the pathogenesis of atopic dermatitis.2Sprecher E. Leung D.Y. Atopic dermatitis: scratching through the complexity of barrier dysfunction.J Allergy Clin Immunol. 2013; 132: 1130-1131Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Here we performed comprehensive whole-exome sequencing in a male patient with ichthyosis and multiple allergic sensitizations and took advantage of the genetic constellation of his family to illuminate the role of functional sequence variants as disease modifiers. Family members have similar genetic backgrounds and are exposed to similar environmental factors, thus providing good models to dissect genotype-phenotype correlations. The index case was the second child of healthy nonconsanguineous Albanian parents and had 2 siblings; no skin disorders or allergies were known in the family (see Table E1 in this article's Online Repository at www.jacionline.org). Soon after birth, skin dryness was noted, and at the age of 2 months, he had erythema and scaling of the skin. Although scaling persisted over the years, episodes of spontaneous regressions and aggravations of the erythema marked the disease course, which is rather unexpected in patients with ARCIs (Fig 1). At the age of 8 years, clinical examination revealed erythroderma with whitish-yellowish scales, keratoderma and fissuring of the palms and soles, and scaling of the scalp but normal hair and mild ectropion (Fig 1, A). There was dramatic amelioration with systemic steroids but no improvement after topical steroids or oral retinoids. In addition to mild perennial rhinitis, his history was negative for respiratory and food allergies and infections. At the age of 14 years, results of laboratory workup were within the normal range, except for increased serum IgE levels (8362 kU/L) and moderate-to-high levels of specific IgE for allergens from tree, grass, or weed pollen; cat dander; house dust mites; walnut; and wheat (ISAC-ImmunoCAP; Thermo Fisher Scientific, Uppsala, Sweden). Atopy patch testing with allergen extracts (Stallergenes, Antony, France) resulted in eczematous skin lesions for cat dander and house dust mite, suggesting a clinical relevance of the allergic sensitization (Fig 1, C). Moreover, within 1 week after testing, progressive exacerbation of the erythema was noted (Fig 1, D). Histopathology and electron microscopy of the patient's skin sample suggested abnormal cornification with incomplete keratinization and pronounced inflammation but were not characteristic for a specific ARCI type or genetic defect (Fig 1, E, and see Fig E1 in this article's Online Repository at www.jacionline.org). Whole-exome sequencing was used to elucidate the molecular basis of the cutaneous findings in the patient (see Table E2 in this article's Online Repository at www.jacionline.org). From 24,700 sequence variants identified, those located in genes associated with ichthyoses, atopic dermatitis, or high IgE levels were analyzed (see the Methods section and Table E3 in this article's Online Repository at www.jacionline.org). The homozygous mutation c.527C>A, p.A176D in NIPAL4, the gene encoding NIPAL4 (NIPA-like domain containing 4), or ichthyin emerged as disease causing, being reported in patients with ARCIs (see Table E4 in this article's Online Repository at www.jacionline.org). The precise role of ichthyin has not been elucidated; it has homologies to G protein–coupled receptors and magnesium transporter proteins and is proposed to be a receptor for trioxilins.3Li H. Lorie E.P. Fischer J. Vahlquist A. Torma H. The expression of epidermal lipoxygenases and transglutaminase-1 is perturbed by NIPAL4 mutations: indications of a common metabolic pathway essential for skin barrier homeostasis.J Invest Dermatol. 2012; 132: 2368-2375Crossref PubMed Scopus (26) Google Scholar NIPAL4 mutations have been associated with generalized scaling and palmoplantar keratoderma and distinct ultrastructural features4Dahlqvist J. Klar J. Hausser I. Anton-Lamprecht I. Pigg M.H. Gedde-Dahl Jr., T. et al.Congenital ichthyosis: mutations in ichthyin are associated with specific structural abnormalities in the granular layer of epidermis.J Med Genet. 2007; 44: 615-620Crossref PubMed Scopus (32) Google Scholar; by contrast, erythema and atopy were observed only in some patients (see Table E4). Therefore we hypothesized that in our case additional genetic variants could contribute to and superimpose the clinical and morphologic picture. Among all other analyzed genes (see Table E3), only the functional SNPs c.1103G>A, p.S368N and c.1258G>A, p.E420K in SPINK5, the gene encoding the lymphoepithelial Kazal type–related inhibitor (LEKTI), and c.1432C>T, p.P478S in FLG, the gene for filaggrin, found in a heterozygous state were previously associated with atopy (Fig 2, A, and see Tables E3 and E5 in this article's Online Repository at www.jacionline.org). A further incidental finding was the heterozygous mutation c.82delT in SLURP1, the gene associated with Mal de Meleda. All relevant variants were validated by means of Sanger sequencing in the patient and his family (Fig 2, A and B, and see Fig E2 in this article's Online Repository at www.jacionline.org). Importantly, although p.S368N and p.E420K have been associated with atopic dermatitis in large cohorts of patients,5Walley A.J. Chavanas S. Moffatt M.F. Esnouf R.M. Ubhi B. Lawrence R. et al.Gene polymorphism in Netherton and common atopic disease.Nat Genet. 2001; 29: 175-178Crossref PubMed Scopus (359) Google Scholar, 6Weidinger S. Baurecht H. Wagenpfeil S. Henderson J. Novak N. Sandilands A. et al.Analysis of the individual and aggregate genetic contributions of previously identified serine peptidase inhibitor Kazal type 5 (SPINK5), kallikrein-related peptidase 7 (KLK7), and filaggrin (FLG) polymorphisms to eczema risk.J Allergy Clin Immunol. 2008; 122: 560-568.e4Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 7Nishio Y. Noguchi E. Shibasaki M. Kamioka M. Ichikawa E. Ichikawa K. et al.Association between polymorphisms in the SPINK5 gene and atopic dermatitis in the Japanese.Genes Immun. 2003; 4: 515-517Crossref PubMed Scopus (108) Google Scholar their presence in either a homozygous or compound heterozygous state did not lead to cutaneous disease in the parents or siblings. In contrast, in the presence of the major cornification defect caused by the NIPAL4 mutation but under similar environmental life conditions, these variants seemed to act as genetic modifiers and modulate the disease, resulting in increased susceptibility to allergic sensitizations in the patient. Experimental evidence exists that the 420K variant alters LEKTI proteolytic activation8Fortugno P. Furio L. Teson M. Berretti M. El Hachem M. Zambruno G. et al.The 420K LEKTI variant alters LEKTI proteolytic activation and results in protease deregulation: implications for atopic dermatitis.Hum Mol Genet. 2012; 21: 4187-4200Crossref PubMed Scopus (15) Google Scholar and that it causes Netherton syndrome (NS) in patients with compound heterozygosity and a null mutation. The filaggrin variant p.P478S was associated with atopic dermatitis among Chinese subjects and was suggested to hamper protease cleavage.9Wang I.J. Lin T.J. Kuo C.F. Lin S.L. Lee Y.L. Chen P.C. Filaggrin polymorphism P478S, IgE level, and atopic phenotypes.Br J Dermatol. 2011; 164: 791-796Crossref PubMed Scopus (29) Google Scholar To get insights into the functional relevance of this mutation constellation, we performed in situ and in vitro studies (see this article's Methods section in the Online Repository). These showed an irregular distribution of LEKTI and filaggrin within the granular and upper spinous layers of the patient's skin, which contrasted with the well-demarcated localization seen in control skin (Fig 2, C and D). In situ zymographies demonstrated a moderate increase in protease activity in the patient's epidermis compared with the LEKTI420K/420K healthy control skin but less than in the LEKTI-negative skin of a patient with NS (Fig 2, E). In line with these results, immunoblotting and quantitative real-time PCR demonstrated a strong reduction of LEKTI protein and mRNA and FLG mRNA in the patient's keratinocytes compared with that seen in control cells (Fig 2, F and G). The decrease in LEKTI expression combined with the heterozygous variant p.E420K results in a strong reduction in the LEKTI proteolytic fragment (D6D9), which was associated with susceptibility to atopic dermatitis.8Fortugno P. Furio L. Teson M. Berretti M. El Hachem M. Zambruno G. et al.The 420K LEKTI variant alters LEKTI proteolytic activation and results in protease deregulation: implications for atopic dermatitis.Hum Mol Genet. 2012; 21: 4187-4200Crossref PubMed Scopus (15) Google Scholar The interpretation of these observations is challenging, but they suggest that interactions between mutant ichthyin, LEKTI, and filaggrin exist. The atypical ultrastructure of the patient's skin, lacking the characteristic features confined by ichthyin mutations, strongly supports the modifying influence of the additional LEKTI and filaggrin variants (see Fig E1). The complexity of this molecular interplay cannot be fully addressed here. However, our global genetic analysis points to this "personalized" genetic constellation as the cause for high IgE levels and allergic sensitizations and suggests that SPINK5 and FLG functional variants become relevant in the presence of additional mutations, leading to clear keratinization defects, as seen with NIPAL4 in our patient. We thank Ioannis Athanasiou, Juna Leppert, and Kaethe Thoma for expert technical assistance. In particular, we thank the patient and his family. The contribution of the Center for Human Genetics Freiburg, led by Dr Jürgen Kohlhase as a sequencing facility, is acknowledged. After obtaining informed consent, EDTA blood samples were obtained from the patient and his family, as were skin biopsy specimens from the patient. The study was conducted according to the Declaration of Helsinki after obtaining approval from the Ethics Committee of the University of Freiburg. For immunohistochemical or immunofluorescence staining of the patient's and control subject's skin, formaldehyde-fixed 5-μm paraffin-embedded sections or cryosections were used, as were the primary antibodies against filaggrin (FLG01; Abcam, Cambridge, United Kingdom), desmoplakin (Progen Biotechnik, Heidelberg, Germany), and LEKTI (H-300; Santa Cruz, Heidelberg, Germany). The 3-amino-9-ethylcarbazole system (DAKO, Hamburg, Germany) was used, and the Alexa Fluor 488–conjugated goat anti-rabbit and anti-mouse IgG antibodies (Invitrogen, Karlsruhe, Germany) were used as secondary antibodies. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; Millipore, Temecula, Calif) or hematoxylin. DNA was extracted from EDTA blood with the Qiagen kit (Qiagen, Hilden, Germany). Exome capture was performed with the SeqCAP EZ Exome (Roche NimbleGen, Madison, Wis), and the captured material was sequenced by using Illumina's HiSeq technology (BaseClear, Leiden, The Netherlands; http://www.baseclear.com/). Annotation of exome variants was performed with ANNOVAR (http://www.openbioinformatics.org/annovar/). The sequence variants identified by means of whole-exome sequencing in NIPAL4 (NM_001099287.1), SPINK5 (NM_001127698), FLG (NM_002016), SLURP1 (NM_020427), LOR (NM_000427), STAT3 (NM_003150), and IL13 (NM_002188.2) were analyzed by using Sanger sequencing with the primers listed in Table E5. In addition, all coding exons and exon-intron boundaries of SPINK5 were analyzed to exclude additional mutations. The common FLG mutations in European subjects were also excluded. All PCR products were submitted to automated nucleotide sequencing in an ABI 3130XL genetic analyzer by using Big Dye Terminator Chemistry (Applied Biosystems, Darmstadt, Germany). The mutations were verified by means of sequencing in both directions and from an independent PCR reaction. In situ zymography was performed, as described previously.E1Fortugno P. Furio L. Teson M. Berretti M. El Hachem M. Zambruno G. et al.The 420K LEKTI variant alters LEKTI proteolytic activation and results in protease deregulation: implications for atopic dermatitis.Hum Mol Genet. 2012; 21: 4187-4200Crossref PubMed Scopus (78) Google Scholar Briefly, 5-μm frozen sections were rinsed with 2% Tween 20 in PBS and incubated at 37°C overnight with 100 μL of BODIPY FL casein (10 μg/mL; Invitrogen, Palo Alto, Calif) to assess total protease activity. Sections were rinsed with PBS solution and visualized with the Zeiss Axioskop 2 (Carl Zeiss, Jena, Germany). Frozen sections were photographed at equal time points and exposure times. Images were captured and analyzed with ImageJ software (http://rsb.info.nih.gov/ij). The intensity of the fluorescence signals was coded as a color gradient ranging from 0 (dark) to 255 (white). ISAC allergen chip data were obtained according to the manufacturer's instructions by using rIgE containing human serum (ISAC-ImmunoCAP). Atopy patch testing was performed with extracts containing the house dust mite Dermatophagoides pteronyssinus, cat dander, dog dander, grass, mugwort, and birch pollen allergens in a petroleum jelly vehicle (Stallergenes), as described previously.E2Darsow U. Laifaoui J. Kerschenlohr K. Wollenberg A. Przybilla B. Wuthrich B. et al.The prevalence of positive reactions in the atopy patch test with aeroallergens and food allergens in subjects with atopic eczema: a European multicenter study.Allergy. 2004; 59: 1318-1325Crossref PubMed Scopus (245) Google Scholar Test substances were applied on back skin, which displayed minimal scaling because of the underlying ichthyosis in the index patient. The reactions were evaluated after 24, 48, and 72 hours. Grading of positive reactions was similar to the criteria used in conventional contact allergy patch testing: −, negative result; ?, only erythema, questionable; +, erythema, infiltration; ++, erythema, few papules (≤3); +++, erythema, papules from 4 to many; ++++, erythema, many or spreading papules; and +++++, erythema, vesicles. A patch test with the pure vehicle, petroleum jelly, served as a negative control. Primary keratinocytes were grown in Keratinocyte Growth Medium (Invitrogen) containing 0.15 mmol/L Ca++ until confluence to analyze LEKTI expression. Cell differentiation was then induced by culturing confluent cells for 5 days in Keratinocyte Growth Medium containing 1.2 mmol/L Ca++. Conditioned medium was recovered, and cell debris was removed by means of centrifugation at 13,000g and 4°C for 15 minutes. Proteins were concentrated by means of acetone precipitation. Cultured cells were lysed in RIPA buffer containing cOmplete Protease Inhibitor (Roche Applied Science, Mannheim, Germany). Lysates were clarified by means of centrifugation at 13,000g and 4°C for 15 minutes. The affinity-purified rabbit polyclonal antibody directed to LEKTI region D7D12 was previously described.E3Fortugno P. Bresciani A. Paolini C. Pazzagli C. El Hachem M. D'Alessio M. et al.Proteolytic activation cascade of the Netherton syndrome-defective protein, LEKTI, in the epidermis: implications for skin homeostasis.J Invest Dermatol. 2011; 131: 2223-2232Crossref PubMed Scopus (61) Google Scholar Anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (F1-335) was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). For LEKTI fragment analysis, conditioned media (400 μL or 1.2 mL) or cell extracts of differentiated keratinocytes were separated on 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ). Membranes were incubated with anti–LEKTI-D7D12 polyclonal antibody. Ponceau-S staining (Sigma-Aldrich, St Louis, Mo) or GAPDH immunostaining were used to ensure equal protein loading among samples. Total RNA was isolated from differentiated keratinocytes by using TRIzol, according to the manufacturer's guidelines (Invitrogen). RNA was reverse transcribed into cDNA with Super Script III and random hexamers (Invitrogen). Quantitative real-time PCR was realized on an ABI Prism 7500 Sequence Detection System (Applied Biosystems) by using ABI TaqMan Gene Expression Master Mix (Applied Biosystems). Primers and FAM-conjugated probes for SPINK5 (Hs00928578_m1), FLG (Hs00863478_g1), and HPRT1 (Hs.PT.51.2145446) were purchased from Applied Biosystems or Integrated DNA Technologies (IDT, Coralville, Iowa). Target gene expression was normalized to the corresponding HPRT1 levels to allow for comparisons between samples.Fig E2Partial sequences of NIPAL4 exon 4, SPINK5 exons 13 and 14, and FLG exon 3 obtained by means of sequencing of the DNA extracted from the EDTA blood of the father and mother of the patient. The codons containing mutations are marked by a line, and the minor sequence variant is indicated in red. The amino acid substitution is also in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Summary of genetic and allergy investigations in the index patient and his familySubject ID [age (y)]∗Age at last examination.History of allergiesGenetic backgroundIgE levelsResults of ImmunoCAP ISAC screening†ISAC standardized units [ISU]: not detectable ( 15 ISU) levels of sIgE.I.1 (49)NoNIPAL4 p.A176D +/− SPINK5 p.S368N −/−SPINK5 p.E420K +/+FLG p.P478S +/−46 kU/LLow titers of specific IgE to walnut (Jug r 2); plane tree, grass, or weed pollen (Pla a 2, Cyn d 1, and Phl p 4); and wasp venom (Pol d 5)I.2 (42)NoNIPAL4 p.A176D +/−SPINK5 p.S368N +/+SPINK5 p.E420K −/−FLG p.P478S −/−83.1 kU/LLow titers of specific IgE to buckwheat (Fag e 2) and kiwi (Act d 8)Moderate titers of specific IgE toBlomia tropicalis (Blo t 5) and dog dander (Can f 3)II.1 (18)NoNIPAL4 p.A176D +/−SPINK5 p.S368N −/+SPINK5 p.E420K +/−FLG p.P478S −/−167 kU/LNo specific IgE detectedII.2 (14)Atopy patch test results positive for cat dander and house dust miteExacerbation of erythema after exposure to house dustMild perennial rhinitisNIPAL4 p.A176D +/+SPINK5 p.S368N −/+SPINK5 p.E420K +/−FLG p.P478S +/−8362 kU/LVery high titers of specific IgE against grass or weed pollen (Cyn d 1; Phl p 4, 5b, 6, and 11; and Che a 1), cat dander (Fel d 1), house dust mite (Der p 1 and Der f 1), walnut (Jug r 2), profilins (Bet v 2, Hev b 8, and Mer 1 a) and CCD (MuxF3)Moderate-to-high sIgE titers to wheat (Tri a aA TI) and plane tree (Pla a 2)Low sIgE titers to kiwi (Act d 1), Polistes species venom (Pol d 5), Vespula species venom (Ves v 5), cockroach (Bla g 7), and PR-10 protein (Api g 1)II.3 (4)NoNIPAL4 p.A176D −/−SPINK5 p.S368N −/+SPINK5 p.E420K +/−FLG p.P478S +/−17.7 kU/LNo specific IgE detectedThe index case is shown in boldface.+, Present; −, absent; CCD, cross-reactive carbohydrate determinants; PR-10, pathogenesis-related class 10.∗ Age at last examination.† ISAC standardized units [ISU]: not detectable ( 15 ISU) levels of sIgE. Open table in a new tab Table E2Details of the whole-exome sequencing analysis in the index patientTotal sequence (raw reads)Matched readsMean coverage target regionPercentage of target >1×Percentage of target >5×Percentage of target >10×Percentage of target >20×92,486,49686,285,73473.7597.04%94.56%91.34%83.67% Open table in a new tab Table E3Variants identified in genes related to ichthyosis and atopy with WESFunctionGeneExonic functionAA change1000G_ALLdbSNP132ChromosomeStartEndZygosityAllele variationsFrequenciesCountsCoverageRemarksValidation by means of Sanger sequencingExonicFLGNonsynonymousNM_002016:c.C1432T:p.P478S0.50rs115843401152285930152285930HeterozygousG/A56.9/43.182/62144YesExonicNIPAL4NonsynonymousNM_001099287:c.C527A:p.A176D5156895736156895736HomozygousA96.68689YesExonicSLURP1Frameshift deletionNM_020427:c.82delT:p.C28fs8143823317143823317HeterozygousA/−50.6/45.539/3577Yes, inherited from the motherExonicSPINK5NonsynonymousNM_001127698:c.G1103A:p.S368N0.49rs23030635147480027147480027HeterozygousG/A63.3/36.738/2260YesExonicSPINK5NonsynonymousNM_001127698:c.A1258G:p.K420E0.51rs23030675147480955147480955HeterozygousA/G52.9/47.137/3370YesExonicABCA12Synonymous SNVNM_015657:c.G4446A:p.T1482T0.23rs168530222215839570215839570HeterozygousC/T50.0/50.051/51102ExonicABCA12Synonymous SNVNM_015657:c.T3172C:p.L1058L0.34rs343519342215851303215851303HeterozygousG/A60.0/40.027/1845ExonicABCA12Nonsynonymous SNVNM_015657:c.T1375A:p.S459T1.00rs75600082215876166215876166HomozygousT1003737ExonicABCA12Synonymous SNVNM_015657:c.A1170G:p.A390A0.51rs101980642215876371215876371HeterozygousT/C51.9/47.154/49104ExonicABCA12Synonymous SNVNM_173076:c.G888A:p.V296V0.34rs175018372215901774215901774HeterozygousC/T52.0/48.026/2450ExonicALDH3A2Synonymous SNVNM_000382:c.A1446T:p.A482A0.84rs7216171957887319578873HeterozygousT/A60.8/39.276/49125ExonicALOXE3Synonymous SNVNM_001165960:c.C2520T:p.S840S0.59rs38098811779999577999957HeterozygousG/A60.4/39.667/44111ExonicCDSNSynonymous SNVNM_001264:c.C1317G:p.S439S0.55rs313098263108407531084075HomozygousC1002727ExonicCDSNNonsynonymous SNVNM_001264:c.C1302A:p.S434R0.00298rs15084615863108409031084090HeterozygousG/T54.8/45.217/1431ExonicCDSNNonsynonymous SNVNM_001264:c.T1229C:p.L410S0.52rs313255463108416331084163HomozygousG1004141ExonicCDSNNonsynonymous SNVNM_001264:c.T1222G:p.S408A0.19rs104212763108417031084170HomozygousC92.93942ExonicCDSNSynonymous SNVNM_001264:c.A1104G:p.A368A0.52rs104212663108428831084288HomozygousC10088ExonicCDSNSynonymous SNVNM_001264:c.G753A:p.R251R0.19rs471343663108463931084639HomozygousT90910ExonicCDSNSynonymous SNVNM_001264:c.C708T:p.S236S0.52rs309421563108468431084684HomozygousA1001717ExonicCDSNSynonymous SNVNM_001264:c.G600A:p.Q200Q0.57rs313098363108479231084792HomozygousT1004040ExonicCERS3Nonsynonymous SNVNM_178842:c.A1108G:p.R370G1.00rs243992815100942962100942962HomozygousC999596ExonicCERS3Synonymous SNVNM_178842:c.A159G:p.R53R0.84rs135433215101041896101041896HomozygousC1003131ExonicCLDN1Synonymous SNVNM_021101:c.T369C:p.G123G0.83rs98692633190030680190030680HomozygousG1008585ExonicCYP4F22Synonymous SNVNM_173483:c.G582A:p.A194A0.09rs11666601191564871515648715HeterozygousG/A58.3/41.756/4096ExonicEBPSynonymous SNVNM_006579:c.G15T:p.A5A0.34rs3048X4838217448382174HomozygousT91.14145ExonicFLGNonsynonymous SNVNM_002016:c.G11213A:p.R3738H0.47rs774228311152276149152276149HeterozygousC/T51.1/48.971/68139ExonicFLGNonsynonymous SNVNM_002016:c.G10903A:p.D3635Nrs754481551152276459152276459HeterozygousC/T69.4/30.684/37121Low frequencyExonicFLGNonsynonymous SNVNM_002016:c.G10590T:p.R3530S0.48rs726970001152276772152276772HeterozygousC/A55.9/44.195/75170High coverageExonicFLGSynonymous SNVNM_002016:c.T10491C:p.D3497D0.43rs31260661152276871152276871HeterozygousA/G52.5/47.5106/96202High coverageExonicFLGSynonymous SNVNM_002016:c.T10473C:p.N3491N0.59rs31260671152276889152276889HeterozygousA/G55.4/44.6113/91204High coverageExonicFLGNonsynonymous SNVNM_002016:c.G10307C:p.G3436A0.48rs20659551152277055152277055HeterozygousC/G68.6/31.4109/50159High coverageExonicFLGSynonymous SNVNM_002016:c.T10194C:p.S3398S0.42rs30912761152277168152277168HeterozygousA/G66.7/33.386/43129Low frequencyExonicFLGSynonymous SNVNM_002016:c.G10017A:p.Q3339Q0.47rs20659561152277345152277345HeterozygousC/T56.6/43.456/4399ExonicFLGSynonymous SNVNM_002016:c.A9966G:p.Q3322Q0.49rs66814331152277396152277396HeterozygousC/T50.0/50.064/64128ExonicFLGNonsynonymous SNVNM_002016:c.A8807G:p.D2936G0.25rs802213061152278555152278555HeterozygousT/C59.1/40.926/1844ExonicFLGSynonymous SNVNM_002016:c.G8673T:p.V2891V0.49rs576721671152278689152278689HeterozygousA/C66.4/33.699/50149High coverageExonicFLGNonsynonymous SNVNM_002016:c.A8506C:p.S2836R0.27rs115820871152278856152278856HeterozygousT/G74.3/25.7199/69268High coverageExonicFLGNonsynonymous SNVNM_002016:c.A7956C:p.E2652D0.221152279406152279406HomozygousG10055Low coverageExonicFLGNonsynonymous SNVNM_002016:c.G7633A:p.G2545R0.47rs31260721152279729152279729HeterozygousC/T61.9/37.378/47126ExonicFLGNonsynonymous SNVNM_002016:c.C7521G:p.H2507Q0.44rs31260741152279841152279841HeterozygousG/C56.3/43.7103/80183High coverageExonicFLGNonsynonymous SNVNM_002016:c.T7442C:p.L2481S0.48rs556503661152279920152279920HeterozygousA/G52.4/47.689/81170High coverageExonicFLGNonsynonymous SNVNM_002016:c.A7330G:p.K2444E0.42rs716252001152280032152280032HeterozygousT/C57.3/42.7106/79185High coverageExonicFLGNonsynonymous SNVNM_002016:c.G7192C:p.E2398Q0.43rs716252011152280170152280170HeterozygousC/G60.3/39.744/2973ExonicFLGNonsynonymous SNVNM_002016:c.G7097C:p.S2366T0.43rs716252021152280265152280265HeterozygousC/G75.0/25.018/624Low-frequency and low-coverage variantExonicFLGNonsynonymous SNVNM_002016:c.G6891C:p.E2297Drs781798351152280471152280471HeterozygousC/G72.2/27.8117/45162High coverageExonicFLGNonsynonymous SNVNM_002016:c.A6626G:p.H2209Rrs669772401152280736152280736HeterozygousT/C73.6/26.4153/55208High coverageExonicFLGSynonymous SNVNM_002016:c.T6603C:p.D2201D0.38rs23385541152280759152280759HeterozygousA/G71.8/28.2148/58206High coverageExonicFLGNonsynonymous SNVNM_002016:c.T6580C:p.Y2194Hrs21849531152280782152280782HeterozygousG/A62.0/37.5134/81216High coverageExonicFLGNonsynonymous SNVNM_002016:c.A6574C:p.K2192Qrs669543531152280788152280788HeterozygousT/G71.7/28.3152/60212High coverageExonicFLGSynonymous SNVNM_002016:c.T6498C:p.S2166S0.50rs21849541152280864152280864HeterozygousG/A54.2/45.865/55120ExonicFLGNonsynonymous SNVNM_002016:c.A6462C:p.Q2154Hrs741294521152280900152280900HeterozygousT/G61.4/38.689/56145ExonicFLGNonsynonymous SNVNM_002016:c.T6355C:p.Y2119Hrs75125531152281007152281007HeterozygousA/G69.6/30.4144/63207High coverageExonicFLGSynonymous SNVNM_002016:c.T6354C:p.H2118Hrs75125541152281008152281008HeterozygousA/G69.8/30.2143/62205High coverageExonicFLGNonsynonymous SNVNM_002016:c.C6323T:p.A2108V0.34rs75229251152281039152281039HeterozygousG/A70.5/29.0141/58200High coverageExonicFLGNonsynonymous SNVNM_002016:c.G6134C:p.S2045T0.24rs75461861152281228152281228HeterozygousC/G70.5/29.579/33112Low frequencyExonicFLGNonsynonymous SNVNM_002016:c.C5883A:p.H1961Q0.60rs31260791152281479152281479HeterozygousT/G62.0/38.093/57150High coverageExonicFLGNonsynonymous SNVNM_002016:c.G5672A:p.R1891Q0.42rs124077481152281690152281690HeterozygousC/T52.9/46.692/81174High coverageExonicFLGNonsynonymous SNVNM_002016:c.C5414T:p.A1805V0.45rs124052411152281948152281948HeterozygousG/A56.2/43.873/57130ExonicFLGNonsynonymous SNVNM_002016:c.C5095T:p.R1699C0.49rs124052781152282267152282267HeterozygousG/A53.4/46.631/2758ExonicFLGNonsynonymous SNVNM_002016:c.G5051A:p.R1684H0.48rs124078071152282311152282311HeterozygousT/C50.8/49.231/3061ExonicFLGNonsynonymous SNVNM_002016:c.C4445A:p.S1482Y0.49rs112049781152282917152282917HeterozygousT/G50.5/49.597/95192High coverageExonicFLGNonsynonymous SNVNM_002016:c.A4126G:p.R1376G0.44rs115814331152283236152283236HeterozygousT/C73.2/26.852/1971Low frequencyExonicFLGNonsynonymous SNVNM_002016:c.G4079A:p.R1360H0.43rs115866311152283283152283283HeterozygousC/T74.7/25.374/2599Low frequencyExonicFLGNonsynonymous SNVNM_002016:c.C3500G:p.A1167G0.43rs580010941152283862152283862HeterozygousG/C65.4/34.6104/55159High coverageExonicFLGSynonymous SNVNM_002016:c.T3387C:p.S1129S0.47rs668316741152283975152283975HeterozygousA/G52.9/47.163/56119ExonicFLGSynonymous SNVNM_002016:c.T2508C:p.D836D0.471152284854152284854HeterozygousA/G65.9/33.5108/55164High coverageExonicFLGNonsynonymous SNVNM_002016:c.G2263A:p.E755K0.47rs741294611152285099152285099HeterozygousC/T54.1/45.953/4598ExonicFLGNonsynonymous SNVNM_002016:c.A1360G:p.T454A0.53rs20113311152286002152286002HeterozygousT/C60.2/39.865/43108ExonicFLGNonsynonymous SNVNM_002016:c.G1330A:p.G444R0.48rs115881701152286032152286032HeterozygousC/T64.9/35.163/3497ExonicFLGNonsynonymous SNVNM_002016:c.G995T:p.G332V0.51rs412671541152286367152286367HeterozygousC/A51.0/49.053/51104ExonicKRT1Nonsynonymous SNVNM_006121:c.A1898G:p.K633R0.48rs14024125306901453069014HeterozygousC/T66.7/33.38/412ExonicKRT1Synonymous SNVNM_006121:c.A1665C:p.G555G125306924753069247HeterozygousT/G75.0/25.03/14Low coverageExonicKRT1Synonymous SNVNM_006121:c.A1413C:p.T471T0.78rs698170125307012153070121HomozygousG1006161ExonicKRT1Synonymous SNVNM_006121:c.C1389T:p.R463R0.42rs936958125307014553070145HeterozygousA/G60.4/39.632/2153ExonicKRT10Nonsynonymous SNVNM_000421:c.T302G:p.I101S1.00rs4261597173897853638978536HomozygousC1003232ExonicKRT10Nonsynonymous SNVNM_000421:c.C125T:p.S42F0.0005rs142050024173897871338978713HeterozygousG/A51.5/48.517/1633ExonicKRT2Synonymous SNVNM_000423:c.C150T:p.G50G0.48rs11835758125304577753045777HeterozygousA/G69.2/30.89/413ExonicKRT9Synonymous SNVNM_000226:c.A1407T:p.G469G0.85rs3890472173972399039723990HeterozygousT/A57.9/42.111/819ExonicKRT9Synonymous SNVNM_000226:c.A429C:p.G143G0.80rs8075921173972781639727816HeterozygousT/G50.0/48.946/4592ExonicKRT9Synonymous SNVNM_000226:c.C195T:p.G65G0.94rs8070680173972805039728050HeterozygousA/G63.2/34.224/1338ExonicLIPNNonsynonymous SNVNM_001102469:c.C395T:p.S132L0.02rs41284088109052433590524335HeterozygousC/T55.6/44.435/2863ExonicLORSynonymous SNVNM_000427:c.C81T:p.G27G1153233506153233506HeterozygousC/T66.7/33.34/26Low coverageNoExonicLORNonsynonymous SNVNM_000427:c.A85G:p.S29G0.17rs66616011153233510153233510HeterozygousA/G71.4/28.65/27Low-frequency and low-coverage variantNoExonicLORNonsynonymous SNVNM_000427:c.C887A:p.P296Q1153234312153234312HeterozygousC/A75.0/25.03/14Low coverageNoExonicMBTPS2Synonymous SNVNM_015884:c.A222G:p.Q74Q0.53rs3213451X2186143421861434HomozygousG96.27679ExonicNIPAL4Nonsynonymous SNVNM_001172292:c.A580G:p.R194G0.60rs68605075156898690156898690HomozygousG97.74344ExonicNIPAL4Synonymous SNVNM_001172292:c.T1245C:p.V415V0.96rs47048705156899869156899869HomozygousC1001616ExonicNSDHLSynonymous SNVNM_015922:c.T132G:p.G44G0.89rs5969919X152018832152018832HomozygousG1005151ExonicPNPLA1Nonsynonymous SNVNM_001145716:c.C1010A:p.P337H0.47rs1219958063627013036270130HeterozygousA/C53.1/46.934/3064ExonicSPINK5Synonymous SNVNM_001127698:c.T1188C:p.H396H0.49rs23030655147480112147480112HeterozygousC/T51.6/48.447/4491ExonicSPINK5Synonymous SNVNM_001127698:c.A1389G:p.G463G0.49rs68963035147481430147481430HeterozygousG/A54.1/45.933/2861ExonicSPINK5Synonymous SNVNM_001127698:c.C1557A:p.G519G0.50rs8806875147486677147486677HeterozygousA/C54.7/45.335/2964ExonicSPINK5Synonymous SNVNM_001127698:c.C1659T:p.V553V0.53rs23030715147488367147488367HeterozygousC/T52.6/47.430/2757ExonicSPINK5Nonsynonymous SNVNM_001127698:c.G2132A:p.R711Q0.57rs37771345147498019147498019HeterozygousG/A57.8/42.237/2764ExonicSPINK5Synonymous SNVNM_001127698:c.C2358T:p.L786L0.57rs177049085147499616147499616HeterozygousC/T50.0/50.020/2040ExonicSPINK5Synonymous SNVNM_001127698:c.C2412T:p.G804G0.57rs339203975147499670147499670HeterozygousC/T60.0/40.024/1640ExonicSPINK5Synonymous SNVNM_001127698:c.G2769A:p.A923A0.57rs37649305147505116147505116HeterozygousG/A70.0/30.028/1240Low frequencyExonicSPINK5Synonymous SNVNM_006846:c.T3009C:p.G1003G0.56rs24004785147510866147510866HeterozygousT/C52.7/47.389/80169High coverageExonicST14Synonymous SNVNM_021978:c.C1215T:p.N405N0.68rs47610611130066335130066335HeterozygousT/C63.6/36.414/822ExonicST14Synonymous SNVNM_021978:c.G1659C:p.G553G0.15rs1182792411130068491130068491HeterozygousG/C62.5/37.510/616Low-coverage variantExonicSUMF1Synonymous SNVNM_001164674:c.T1041C:p.T347T0.61rs2633852344038374403837HeterozygousA/G52.4/47.611/1021ExonicSUMF1Nonsynonymous SNVNM_001164674:c.G188A:p.S63N0.34rs2819590345087424508742HeterozygousT/C75.0/25.03/14Low coverageExonicTGM1Synonymous SNVNM_000359:c.C1146A:p.G382G0.21rs1126432142472829424728294HeterozygousG/T57.1/42.920/1535ExonicDOCK8Nonsynonymous SNVNM_001190458:c.C85A:p.P29T0.63rs5292089286593286593HomozygousA9697101ExonicDOCK8Synonymous SNVNM_001190458:c.T495C:p.N165N0.17rs20390459312124312124HomozygousC96.72930ExonicDOCK8Synonymous SNVNM_001190458:c.A1608G:p.K536K0.23rs9137039370244370244HomozygousG97.37375ExonicDOCK8Synonymous SNVNM_001190458:c.G2136C:p.L712L0.24rs108144319377111377111HomozygousC95.82324ExonicDOCK8Synonymous SNVNM_001190458:c.C2616T:p.T872T0.24rs22970759390512390512HomozygousT98.7227230High coverageExonicDOCK8Synonymous SNVNM_001190458:c.C3807G:p.L1269L0.56rs22970799421032421032HomozygousG93.83032ExonicDOCK8Synonymous SNVNM_001190458:c.T4191C:p.F1397F0.99rs78540359429719429719HomozygousC1009191ExonicDOCK8Synonymous SNVNM_001190458:c.G5133A:p.E1711E0.84rs18879579441952441952HomozygousA1005757ExonicDSG1Synonymous SNVNM_001942:c.C2115T:p.Y705Y0.01182893427428934274HeterozygousC/T51.4/48.618/1735ExonicIL13Nonsynonymous SNVNM_002188:c.A431G:p.Q144R0.73rs205415131995964131995964HomozygousG985051ExonicIL6RSynonymous SNVNM_000565:c.G93A:p.A31A0.16rs81922821154401679154401679HeterozygousG/A52.0/48.013/1225ExonicIL6RNonsynonymous SNVNM_000565:c.C1328G:p.S443W1154437777154437777HeterozygousC/G75.0/25.018/624Low-frequency and low-coverage variantNoExonicKLK5Nonsynonymous SNVNM_001077492:c.A457G:p.N153D1.00rs183854195145225051452250HomozygousC1006464ExonicKLK7Nonsynonymous SNVNM_001243126:c.T161C:p.L54P0.91rs2659067195148562251485622HomozygousG1002626Exonic; splicingSTAT3Synonymous SNVNM_003150:c.C2100T:p.G700G174046924140469241HeterozygousG/A52.6/47.440/3676Yes, present in mother and sisterExonicTMEM79Nonsynonymous SNVNM_032323:c.G439A:p.V147M0.24rs66845141156255456156255456HeterozygousG/A54.5/45.56/511Low-coverage variantExonicIL13∗rs20541 is a missense variant in the gene encoding IL-13, a cytokine predominantly secreted by TH2 cells and an important factor in the regulation of allergic inflammation. Allele A (encoding Q at amino acid position 144) has been repeatedly associated with asthma, as well as being shown to result in a distinct isoform of IL-13 with increased biological activity.E4,E5Nonsynonymous SNVNM_002188:c.A431G:p.Q144R0.73rs205415131995964131995964HomozygousG985051Yes, present in a homozygous state in father and sisterSNV, Single nucleotide variant.∗ rs20541 is a missense variant in the gene encoding IL-13, a cytokine predominantly secreted by TH2 cells and an important factor in the regulation of allergic inflammation. Allele A (encoding Q at amino acid position 144) has been repeatedly associated with asthma, as well as being shown to result in a distinct isoform of IL-13 with increased biological activity.E4Weidinger S. Willis-Owen S.A. Kamatani Y. Baurecht H. Morar N. Liang L. et al.A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis.Hum Mol Genet. 2013; 22: 4841-4856Crossref PubMed Scopus (164) Google Scholar, E5Vladich F.D. Brazille S.M. Stern D. Peck M.L. Ghittoni R. Vercelli D. IL-13 R130Q, a common variant associated with allergy and asthma, enhances effector mechanisms essential for human allergic inflammation.J Clin Invest. 2005; 115: 747-754Crossref PubMed Scopus (160) Google Scholar Open table in a new tab Table E4Clinical features of patients reported to have ARCIs and the NIPAL4 mutation c.527C>A, p.A176DAffected subject ID; age∗Age at last examination.; originAge of onsetCollodion membrane at birthErythema/dermatitisScalingPalmoplantar keratodermaAtopy, IgE levelsOne patient; 14 y; Albania (this study)2 moNoGeneralizedLarge, white-to-yellow scales over whole integument, except from skin foldsMild keratoderma and fissuresIgE >8000 kU/L, specific IgE to multiple allergens22 patients; 1-73 y; ScandinaviaE6Dahlqvist J. Klar J. Hausser I. Anton-Lamprecht I. Pigg M.H. Gedde-Dahl Jr., T. et al.Congenital ichthyosis: mutations in ichthyin are associated with specific structural abnormalities in the granular layer of epidermis.J Med Genet. 2007; 44: 615-620Crossref PubMed Scopus (57) Google ScholarNAPresent in 1/22 patientsNo or mildScaling on the trunk and in skin foldsIn most patients plantar keratodermaNo10 patients; NA; Colombia, Turkey, AlgeriaE7Lefevre C. Bouadjar B. Karaduman A. Jobard F. Saker S. Ozguc M. et al.Mutations in ichthyin a new gene on chromosome 5q33 in a new form of autosomal recessive congenital ichthyosis.Hum Mol Genet. 2004; 13: 2473-2482Crossref PubMed Scopus (151) Google ScholarNAPresent in 4/10 patientsGeneralized in some casesFine whitish scaling on the face and trunk; larger brownish scales on the neck, buttocks, and legspalmoplantar keratoderma and fissuresNo15 patients; children and adults; PakistanE8Wajid M. Kurban M. Shimomura Y. Christiano A.M. NIPAL4/ichthyin is expressed in the granular layer of human epidermis and mutated in two Pakistani families with autosomal recessive ichthyosis.Dermatology. 2010; 220: 8-14Crossref PubMed Scopus (25) Google ScholarAt birthPresent in most patientsPresent in some adultsSome patients have fine whitish scales on the face and trunk; others show generalized brownish reticulated scalesPalmoplantar keratoderma and fissuresSevere itching and clinical picture resembling atopic dermatitis in some affected subjectsThe index case is shown in boldface.NA, Information not available.∗ Age at last examination. Open table in a new tab Table E5Primers used for Sanger sequencing in this studyGene, primer nameSequence 5′-3′NIPAL4, exon 4FTCTGGGATTCTCCAGCTTGTNIPAL4, exon 4RGCAAACATTCCCAGGGTCTASPINK5, exon 14FTGCAATTGTGAGGATTTCACAGSPINK5, exon 14RCCTGAACATGATCTGTGGATCSPINK5, exon 13FGAGATGTAACATTAGTTTCTGCSPINK5, exon 13RATGTCTCCAATCAGACAGTTTCTCFLG, exon 3FCACGGAAAGGCTGGGCTFLG, exon 3RACCTGAGTGTCCAGACCTATTSLURP1, exon FCCTTGAAAGATGTCAGCGAGSLURP1, exon RGTGTGGCAGCCTGTTCTGLOR, exon 2FAGCGGCTGCATCATCAGTLOR, exon 2RCAAACCTCGGGTAGCATCATSTAT3, exon 22-23FTTCCTCCAGCTCTGCTTACTGSTAT3, exon 22-23RATCACACAAAGGGGACCAACIL13-FCAAGGGGAAGGCTGAGGTCIL13-RTGAAGGGAAGCTGGCTGAAT Open table in a new tab The index case is shown in boldface. +, Present; −, absent; CCD, cross-reactive carbohydrate determinants; PR-10, pathogenesis-related class 10. SNV, Single nucleotide variant. The index case is shown in boldface. NA, Information not available.