A novel homozygous mutation disrupting the initiation

CED

Experimental dermatology • Concise report

Clinical and Experimental Dermatology

A novel homozygous mutation disrupting the initiation codon in the SLURP1 gene underlies mal de Meleda in a consanguineous family K. Shah,1 A. Nasir,1 Irfanullah,1 S. Shahzad,3 S.Khan2 and W. Ahmad1 1 Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University (QAU), Islamabad, Pakistan; 2Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology (KUST), Kohat 26000, Pakistan; and 3Department of Biotechnology, International Islamic University, Islamabad, Pakistan

doi:10.1111/ced.12864

Summary

Mal de Meleda (MDM) is a palmoplantar keratoderma (PPK), characterized by hyperkeratosis of the palms and soles, and keratotic skin lesions. Patients with MDM can develop perioral erythema, keratotic and lichenoid plaques over the joints (including the elbows and knees), nail abnormalities, joint contractures and stiffness, brachydactyly, sclerodactyly, pseudoainhum, and malodorous maceration. MDM is associated with mutations in the SLURP1 gene. We report a consanguineous family in which MDM was inherited in an autosomal recessive manner. Genotyping using microsatellite markers established linkage in the family to the SLURP1 gene, which has been mapped previously to chromosome 8q24.3. Sequence analysis revealed a homozygous missense mutation (c.2T>C, p.Met1Thr) in affected family members. Molecular docking studies using a ZDOCK server predicted disruption of binding of the mutant variant to its target a7-nAChR. This study further supports the previously reported findings that homozygous mutations in the SLURP1 gene cause MDM.

Mal de Meleda (MDM; MIM 248300) is an autosomal recessive form of palmoplantar keratoderma (PPK). The condition is characterized by a palmoplantar hyperkeratosis that extends from the palmar to the dorsal surfaces of the hands and feet. Patients with MDM may develop perioral erythema, keratotic and lichenoid plaques over the joints (including the elbows and knees), nail abnormalities, joint contractures and stiffness, brachydactyly, sclerodactyly, pseudoainhum, and malodorous maceration. Histological features of MDM include hyperkeratosis, hypergranulosis, acanthosis and mild to moderate perivascular inflammation. Sensing of touch is preserved, whereas sensing of Correspondence: Dr Wasim Ahmad, Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University (QAU) Islamabad, Pakistan E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. Accepted for publication 24 September 2015

ª 2016 British Association of Dermatologists

heat and pain is disturbed above thicker layers of keratosis in patients with MDM.1 The gene SLURP1 (MIM 606119), which is considered the possible cause of MDM, encodes a member of the lymphocyte antigen-6 (Ly-6) superfamily of proteins, which are homologous to frog cytotoxin and snake venom neurotoxins targeting neuromuscular acetylcholine receptors. Adeyo et al.2 reported presence of metabolic and neuromuscular abnormalities in addition to PPK in slurp1 knockout mice. The SLURP1 protein consists of a signal polypeptide chain, with three domains in its 3D structure and five disulphide bridges that are critical for its correct folding and function. SLURP1 has been reported to function as a late marker of epidermal differentiation,3 and may regulate keratinocyte growth, proliferation, differentiation and apoptosis.4 We report a consanguineous family in which MDM was inherited in an autosomal recessive manner. Genotyping using microsatellite markers established linkage in the family to the SLURP1 gene,


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A novel homozygous mutation in SLURP1 gene underlying MDM K. Shah et al.

Report

DNA extraction and microsatellite-based genotyping was performed as described previously.5 Microsatellite markers mapped in the flanking regions of five genes (WNT10A, DSP, CTSC, SERPINB7 and SLURP1) were typed for all available affected (n = 5) and unaffected (n = 4) members of the family. Haplotype analysis established linkage in the family to the SLURP1 gene on chromosome 8q24.3 (Fig. 1a). Subsequently, the SLURP1 gene was sequenced (GenomeLab DTCS Quick Kit; Beckman Coulter Fullerton, CA, USA) in all affected and

The study was approved by the institutional review board, Quaid-i-Azam University, Islamabad, Pakistan, and all participants provided written informed consent. The studied patients were from a large consanguineous Pakistani family, who displayed autosomal recessive inheritance of MDM (Fig. 1a). All five affected patients showed the clinical features of MDM in the hands and feet described above. In addition, the fingernails of both hands had a parrot-beak-like appearance (Fig. 1b). (a)

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(f) (i)

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Figure 1 (a) Pedigree of a consanguineous Pakistani family segregating the autosomal recessive mal de Meleda (MDM) type of palmoplan-

tar keratoderma (PPK). Filled and unfilled symbols represent affected and unaffected individuals, respectively. For genotyped individuals, haplotypes of the closely linked microsatellite markers on chromosome 8q24.3 are shown beneath each symbol. Genetic distances (centimorgans; cM) are depicted according to the Rutgers combined-linkage physical map [build 36.2.38] (http://compgen.rutgers.edu/). (b–e) Clinical presentation of MDM in affected members in the family. (b,c) Affected individual IV-2 had (b) demarcated transgrediens hyperkeratosis on the hands with erythematous lesion and dystrophic nails and (c) transgrediens hyperkeratosis on the hand with restricted boundaries; (d) affected individual IV-1 had hyperkeratosis of the feet with dystrophic nails and fissures over the toes; and (e) affected member IV-4 had mild hyperkeratosis over the dorsa of the feet. (f–h) Nucleotide sequence of a missense mutation (c.2T>C) in the SLURP1 gene in (f) unaffected controls, (g) heterozygous carrier and (h) affected individual; arrows indicate positions of the mutation. (i) BsrD1 restriction enzyme analysis and PCR amplification of the resulting products of 427 bp showed three DNA bands (126, 301 and 427 bp) in carriers of this mutation, a single DNA band of 427 bp in individuals with MDM and two bands (126 and 301 bp) in normal controls. MWM, molecular weight marker.

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unaffected members of the family. Sequence variants were identified by the BioEdit sequence alignment tool (v6.0.7; http://www.mbio.ncsu.edu/BioEdit/bioedit. html). DNA sequence analysis detected a homozygous missense mutation (c.2T>C) in exon 1 of the SLURP1 gene in all five affected family members, and their parents were heterozygous for the same mutation (Fig. 1c). The mutation resulted in a change from methionine to threonine at amino acid position 1 (p.Meth1Thr) of the protein. The mutation abolished a restriction site for the restriction enzyme BsrD1. BsrD1 restriction analysis of products amplified by PCR of exon 1 revealed a single DNA band of 427 bp in affected family members, three DNA bands (126, 301 and 427 bp) in family members who were carriers of this mutation and two bands (126 and301 bp) in normal controls (Fig. 1d). The

nonpolymorphic nature of the sequence variant detected in the studied family was verified by BsrDI restriction enzyme analysis using genomic DNA of 250 ethnically matched controls. Using homology modelling, three-dimensional models of normal and mutated SLURP1 protein (p.Meth1Thr) were predicted (Fig. 2a,b) and evaluated by online structure analysis tools.6 Ramachandran plot indicated that approximately 91% of residues in the model lie in allowed regions of torsion angles of torsion angles. Analysis of the normal and mutant SLURP1 with ProSA plot showed Z scores of 0.74 and 3.35, respectively, indicating no significant deviation from the scores determined for proteins of similar size (Fig. 2c,d). Using molecular docking analysis, we detected binding of normal SLURP1 to neuronal nicotinic acetylcholine

(a)

(b)

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Figure 2 (a,b) Modelled structure of (a)

normal and (b) mutant SLURP1 protein; orange, purple and grey represent helix, beta-sheet and coil, respectively. (c,d) Protein model quality scores of (c) normal and (d) mutant SLURP1; the Z scores of both are represented in the plots by the large black dots on the left of the plots. Z scores of all proteins in protein data bank as determined by lighter bluegrey colour ight blue indicateds determined by X-ray crystallography (light blue-grey) and nuclear magnetic resonance spectroscopy (dark blue-grey) are shown. (e,f) Surface view of (e) the interacting region ‘e’ and (f) the interacting region ‘f’ of the complex [also marked by the same letters in (g)]. Interacting residues are shown in atomic representations, and H bonds are shown by black lines with the distances indicated in angstroms ( A). (g) The a-7 nicotinic acetylcholine receptor (nAChR) and normal SLURP1 interface; the nAChR–SLURP1 complex is represented as a ribbon diagram, with nAChR and SLURP1 shown in cyan and green, respectively.


(e)

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receptor (nAChR) through several residues, including Trp5, Val7, Leu10, Leu11, Val12, Trp15, Met51, Phe63, Ile85, Leu90, Ile91, Phe92 and Phe95. To date, 18 disease-causing mutations have been identified in the SLURP1 gene in various populations around the world. The most probable effect of the mutation p.Met1Thr identified in this study is the use of ATG at codon position 17, resulting in the truncated protein lacking the first 16 amino acids. It is highly likely that this truncated protein would result in inaccurate peptide targeting.7 PolyPhen software also predicted that replacement of Met by Thr can disrupt protein function. Mutations involving the initiation codon and resulting in defective mRNA translation have been documented as underlying several different genetic disorders; these include mutation p.Met1Leu in SLURP1, p.Met1Val in PAX9 and p.Met1Arg in XP, resulting in MDM, oligodontia and xeroderma pigmentosum, respectively.8–10 SlURP1 has been localized to human skin, exocervix, gums, stomach and esophagus, with the highest levels found in keratinocytes particularly in the palms and soles.2,3 The precise function performed by SLURP1 is poorly understood, but it is hypothesized that SLURP1 preferentially modulates nAChR, or binds to a7 and/or some other nAChR subtypes.4 The nAChR family is a superfamily of ligand-gated ion channel proteins that are involved in keratinocyte growth, proliferation, differentiation and apoptosis.3 The truncated SLURP1 protein resulting from the mutation p.Meth1Thr, identified in our family, is likely to be deficient in seven residues (Trp5, Val7, Leu10, Leu11, Val12, Trp15) involved in binding to a7-nAChR, and will thus lose its target. Previous studies suggest that SLURP1 binding to a7-nAChR leads to increased acetylcholine signalling, which in turn leads to enhanced calcium signalling in keratinocytes, affecting kinase expression. Presence of inflamed phenotypes in affected patients can be ascribed to a role of SLURP1 in immune system regulation.4

Acknowledgements We are grateful to the family members for their participation in this study. This work was supported by Higher Education Commission (HEC) Islamabad, Pakistan. We thank Dr A. Wadood, Department of Biochemistry, Abdul Wali Khan University Mardan, for providing the MOE software.

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Learning points • The primary cutaneous feature of MDM is

transgrediens hyperkeratosis of palms and soles. • The histological features of MDM are hyperker-

atosis, hypergranulosis, acanthosis and mild to moderate perivascular inflammation. • SLURP1 is the causative gene of MDM. • We identified a mutation in SLURP1 in five members of a consanguineous family in Pakistan, who had MDM. • Identification of this mutation supports the previous reports of homozygous variants in the SLURP1 gene causing MDM. • Molecular analysis showed that the mutated SLURP1 protein is deficient in several residues involved in binding to the a7-nAChR protein, and will therefore lose its target, a7-nAChR.

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10 Khan SG, Kyu-Seon Oh, Steffen E et al. XPC initiation codon mutation in xeroderma pigmentosum patients with and without neurological symptoms. DNA Repair 2009; 8: 114–25.


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