Whole-exome sequencing in patients with ichthyosis reveals modifiers associated with increased IgE levels and allergic sensitizations (2014)

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 were used to equal protein among was from differentiated keratinocytes by using according to the manufacturer's was into with and real-time PCR was on an ABI (Applied by using ABI (Applied and for SPINK5 FLG and were purchased from Applied or DNA gene expression was to the corresponding levels to for between of NIPAL4 SPINK5 exons and and FLG obtained by means of sequencing of the DNA extracted from the EDTA blood of the and of the patient. The containing mutations are marked by a and the sequence variant is in The is also in of genetic and allergy in the index patient and his at of of ISAC not high levels of p.A176D SPINK5 p.S368N p.E420K p.P478S of specific IgE to tree, grass, or weed pollen a 2, 1, and and p.A176D p.S368N p.E420K p.P478S of specific IgE to and of specific IgE and dog dander p.A176D p.S368N p.E420K p.P478S specific IgE patch test results positive for cat dander and house dust of erythema after exposure to house perennial p.A176D p.S368N p.E420K p.P478S high of specific IgE against or weed pollen and and a cat dander house dust mite 1 and 2, and 1 and to wheat a and a to and protein p.A176D p.S368N p.E420K p.P478S specific IgE index case is in −, at ISAC not high levels of in a new Table of the whole-exome sequencing analysis in the index sequence of of of of in a new Table identified in genes to ichthyosis and atopy with by means of Sanger inherited from the and and and in and is a variant in the gene encoding a by cells and an in the of allergic A at has been associated with as well as being to result in a distinct of with increased in a homozygous state in and nucleotide is a variant in the gene encoding a by cells and an in the of allergic A at has been associated with as well as being to result in a distinct of with increased S. Y. Baurecht H. N. L. et study of atopic dermatitis with on and Mol Genet. 2013; PubMed Scopus Google Scholar, R. a common variant associated with allergy and essential for human allergic Clin PubMed Scopus Google Scholar in a new Table features of patients reported to have ARCIs and the NIPAL4 mutation c.527C>A, at of membrane at IgE 14 over except from skin keratoderma and specific IgE to multiple 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 Google in or on the and in skin patients C. B. A. S. M. et in ichthyin a new gene on in a new of recessive congenital Mol Genet. 2004; PubMed Scopus Google in in some scaling on the and on the and keratoderma and and M. M. Y. is in the granular layer of human epidermis and in with recessive 2010; PubMed Scopus Google in in some patients have on the and generalized keratoderma and and clinical atopic dermatitis in some index case is in not at in a new Table used for Sanger sequencing in this in a new The index case is in +, −, nucleotide The index case is in not