Barnicoat, A

Drury, S

Harper, JI

Khan, A

Kinsler, VA

Lench, N

O'Shaughnessy, RF

Rukaite, G

Waelchli, R

PubMed

UCL Discovery

Research letterBJDBritish Journal of DermatologyA novel microdeletion in LOR causingautosomal dominant loricrin keratodermaDOI: 10.1111/bjd.13361DEAR EDITOR, The dramatic presentation of a collodion baby ismost commonly associated with a diagnosis of autosomalrecessive ichthyosis (ARCI). We investigated a family whereinthe father and all four children were collodion at birth, imply-ing an autosomal dominant inheritance pattern. Postnatally,the affected individuals presented with variable degrees ofgeneralized ichthyosis and palmoplantar keratoderma (PPK)(Fig. 1). These clinical features were more pronounced in thefather than in the children, and were reported to have becomemore prominent with age. The phenotype was of generalizednonerythematous xerosis and fine scaling, with adherent largerscale on the lower legs, and mild PPK that exhibited honey-comb patterning. The mother was unaffected and the parentswere nonconsanguineous. One of the children also had mod-erate neurodevelopmental delay and microcephaly, and thefather had asthma.With research ethics committee approval, blood was takenfrom one child and both parents, and DNA was extractedusing standard methods. Skin scrapings were obtained fromthe father and the child, with a 4-mm punch skin biopsytaken from the father. Next-generation sequencing of leuco-cyte DNA was performed using a Nextera Custom Enrich-ment Kit (Illumina, San Diego, CA, U.S.A.) targeting exonicregions of clinically relevant published dermatology genes,including known ichthyosis and PPK genes (Table S1; seeSupporting Information). A 50-ng DNA sample was simulta-neously fragmented using Illumina Enrichment Sample Prepand tagged with adaptors. Polymerase chain reaction (PCR)introduced sample-specific indexes to the library, and fol-lowing quantification this was hybridized to the custom-capture probes. Streptavidin beads captured the probes con-taining the targeted regions of interest and a series ofwashes removed nonspecific binding from the beads. Thisenrichment and capture was repeated once to enrich thetarget regions further. The enriched library was amplifiedby limited rounds of PCR, quantified, diluted and sequencedon the Illumina MiSeq. Analysis was using an in-housepipeline, where FASTQ files were aligned using Burrows–Wheeler Aligner1 and indexed with SAMtools (http://sam-tools.sourceforge.net/).2 Variant calling was performed withVarScan (http://varscan.sourceforge.net/),3,4 and VCF fileswere annotated with Variant Effect Predictor (http://www.ensembl.org). Annotated variants were then filtered inMicrosoft Excel, BAM files reviewed manually and mutationsconfirmed using Sanger sequencing.A novel heterozygous deletion inducing a frameshift wasfound in the loricrin gene Chr1 (GRCh37): g.153234085_153234086del, LOR c.660_661delGC, p.Gln222Alafs*113,RefProbandFatherMotherc.660_661delForRevRevRev(a) (b)(c) (d)(e)(f)Fig 1. (a) Transgradient palmar keratoderma, (b) honeycombpatterning on the palm and (c) sole, (d) ichthyosis on the lower leg,(e) microdeletion in LOR shown by next-generation sequencing and(f) confirmed by Sanger sequencing.© 2014 The Authors. British Journal of Dermatologypublished by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.262 British Journal of Dermatology (2015) 172, pp262–264This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use anddistribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.predicted in silico as possibly damaging (Polyphen score 0.799;http://genetics.bwh.harvard.edu/pph2/) (Fig. 1). This estab-lished a diagnosis of loricrin keratoderma (previously knownas Vohwinkel syndrome with ichthyosis, or Vohwinkel syn-drome variant), distinct from classical Vohwinkel syndromeby the co-occurrence of generalized ichthyosis and the absenceof hearing loss.5 The two conditions are genotypically distinct,with Vohwinkel syndrome due to mutations in GJB2 and Voh-winkel with ichthyosis due to mutations in LOR.6 The occur-rence of constrictive digital bands leading to autoamputationis variably reported to be a feature of the variant form, butwas not present in this family.Comparison of this novel mutation with previouslydescribed pathogenic mutations in this form of loricrin kerato-derma revealed a common mechanism of frameshift indelsaffecting the C terminus of the protein,7 with this being thefirst deletion reported in the literature. To characterize thismutation further we performed Western blotting for loricrinmonomers and dimers on skin scale and whole-skin lysate,and immunofluorescence for loricrin, interleukin (IL)-1a, ker-atin 1 and mouse double minute 2 homologue (MDM2) onwhole skin (Data S1; see Supporting Information). Monomericand dimeric loricrin were clearly detectable in the patient’sskin lysate compared with the normal skin lysate, where onlythe monomeric form was detected, There was weak expres-sion of dimer in patient skin scale, compared with no freeloricrin detectable in control scale (Fig. 2a). This defectiveincorporation of loricrin into the cornified envelope is analo-gous to that seen in ARCI.8 The domain of loricrin expressionwas expanded in patient hyperkeratotic skin, with nucleolarexpression of loricrin as previously reported with insertions(Fig. 2b).7 Histological analysis of patient skin showed notonly hyperkeratosis, but also focal areas of parakeratosis(Fig. 2c)(a)(b)(c)Fig 2. Western blots (WB) and immunofluorescence of the proband reveal features common to both loricrin keratoderma and autosomal recessiveichthyosis (ARCI). (a) Western blot of whole-cell lysates and scale from the proband and an unaffected control. Note the monomeric and dimericloricrin in the proband skin lysate. No free loricrin is detectable in the control scale, while weak expression of loricrin dimer is detected in theproband scale. These data specifically indicate the change in expression of wild-type loricrin as a result of the presence of the mutant loricrin. (b)Immunofluorescence of loricrin in the proband and control skin. The domain of loricrin expression is expanded in proband skin. (c) Histologyand immunofluorescence of interleukin (IL)-1A, keratin 1 and mouse double minute 2 homologue (MDM2) in control skin, proband skin andrepresentative samples of ARCI skin.9 Focal parakeratosis is indicated in the proband skin. Expression of both IL-1A and MDM2 is increased in thespinous and basal layers of both proband and ARCI skin, while keratin 1 increase is seen only in the ARCI skin. Bar 50 lm (b, both top andbottom panels, and c).© 2014 The Authors. British Journal of Dermatologypublished by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.British Journal of Dermatology (2015) 172, pp262–264Research letter 263The upregulation of IL-1a and MDM2 occurs in all lamel-lar-type ARCIs irrespective of genotype,9 and is consistentwith the hyperkeratosis observed in MDM2-overexpressingmice.10 The upregulation of these proteins in patient skin pro-vides further support for a common mechanism for hyperker-atosis.8,9 Interestingly keratin 1 expression was not increasedin affected skin, further refining the hyperkeratosis signaturecommon to ichthyoses due to different genetic defects.These results describe a novel mutation and the first dele-tion leading to loricrin keratoderma unusually presenting withcollodion membrane, described only once previously,11 andprovide further evidence of a common, MDM2-mediated geneexpression pathway leading to ichthyosis and collodion mem-brane formation. This diagnosis should be considered as a rarecause of the clinical presentation of collodion baby.V .A . K IN S L E R1,2S . DRURY3A . KHAN2R . WAELCHL I 2G. RUKA I T E 3A . BARN I COAT4N. LENCH3J . I . HARP ER5R .F .L . O’SHAUGHNE S S Y5 , 61Genetics and Genomic Medicine,5Livingstone Skin Research Centreand 6Immunobiology and Dermatology, UCLInstitute of Child Health, 30 Guilford St,London WC1N 1EJ, U.K.2Paediatric Dermatology,3North Thames Regional Genetics Laboratoryand 4Clinical Genetics, Great Ormond StHospital for Children, London, U.KCorrespondence: Veronica KinslerE-mail: v.kinsler@ucl.ac.ukReferences1 Li H, Durbin R. Fast and accurate short read alignment withBurrows-Wheeler Transform. Bioinformatics 2009; 25:1754–60.2 Li H, Handsaker B, Wysoker A et al. The Sequence Alignment/Mapformat and SAMtools. Bioinformatics 2009; 25:2078–9.3 Koboldt DC, Chen K, Wylie T et al. VarScan: variant detection inmassively parallel sequencing of individual and pooled samples.Bioinformatics 2009; 25:2283–5.4 Koboldt D, Zhang Q, Larson D et al. VarScan 2: somatic mutationand copy number alteration discovery in cancer by exomesequencing. Genome Res 2012; 22:568–76.5 Camisa C, Rossana C. Variant of keratoderma hereditaria mutilans(Vohwinkel’s syndrome). Treatment with orally administered iso-tretinoin. Arch Dermatol 1984; 120:1323–8.6 Maestrini E, Monaco AP, McGrath JA et al. A molecular defect inloricrin, the major component of the cornified cell envelope,underlies Vohwinkel’s syndrome. Nat Genet 1996; 13:70–7.7 Ishida-Yamamoto A, Kato H, Kiyama H et al. Mutant loricrin is notcrosslinked into the cornified cell envelope but is translocated intothe nucleus in loricrin keratoderma. J Invest Dermatol 2000;115:1088–94.8 O’Shaughnessy RF, Choudhary I, Harper JI. Interleukin-1 alphablockade prevents hyperkeratosis in an in vitro model of lamellarichthyosis. Hum Mol Genet 2010; 19:2594–605.9 Youssef G, Ono M, Brown SJ et al. Identifying a hyperkeratosis sig-nature in autosomal recessive congenital ichthyosis: Mdm2 inhibi-tion prevents hyperkeratosis in a rat ARCI model. J Invest Dermatol2014; 134:858–61.10 Alkhalaf M, Ganguli G, Messaddeq N et al. MDM2 overexpressiongenerates a skin phenotype in both wild type and p53 null mice.Oncogene 1999; 18:1419–34.11 Matsumoto K, Muto M, Seki S et al. Loricrin keratoderma: a causeof congenital ichthyosiform erythroderma and collodion baby.Br J Dermatol 2001; 145:657–60.Supporting InformationAdditional Supporting Information may be found in the onlineversion of this article at the publisher’s website:Data S1. Supplementary methods.Table S1. Ichthyosis and palmoplantar keratoderma genescovered by the custom-designed next-generation sequencingpanel at the time of investigation.© 2014 The Authors. British Journal of Dermatologypublished by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.British Journal of Dermatology (2015) 172, pp262–264264 Research letter

A novel microdeletion in LOR causing autosomal dominant loricrin keratoderma

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A novel microdeletion in LOR causing autosomal dominant loricrin keratoderma

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