IS c.1431-12G>A A COMMON EUROPEAN MUTATION OF SPINK5? REPORT OF A PATIENT WITH NETHERTON SYNDROME
Śmigiel R, Królak-Olejnik B, Śniegórska D, Rozensztrauch A, Szafrańska A, Sasiadek MM, Wertheim-Tysarowska K
*Corresponding Author: Robert Śmigiel, M.D., Ph.D., Department of Pediatrics, Wroclaw Medical University, Bartla 5, PL 51-618 Wroclaw, Poland. Tel: +48-71-784-13-26. Fax: +48-71-784-00-63. E-mail: robert.smigiel@ umed.wroc.pl
page: 81

DISCUSSION

LEKTI1 is expressed in epithelial, thymus, tonsils, parathyroid glands, hair follicles and the trachea [3], but the vast majority of data concerning its biology and functions comes from epithelium research. The spectrum of LEKTI1 activity as a serine protease inhibitor is wide and it has been shown that LEKTI1 is not only expressed as full-length protein (145 kDa), but also as two other isoforms (a short one: 125 kDa, and a long one: 148 kDa). Furthermore, full length isoform in addition to its own activity, undergoes several proteolytic cleavages, resulting in generation of various LEKTI1 domains with different inhibitory specificities [2]. Various LEKTI1 derived poly-peptides inhibit KLK5, KLK7 and KLK14 (kallikrein-related peptidases) and it is hypothesized that deficient inhibition of those proteins is one of the major causes of epidermal symptoms observed in NS [1,4]. Hovnanian [2] proposed that as a result of LEKTI1 deficiency, KLK5, KLK7 and ELA2 (elastase 2) are improperly activated leading to abnormal skin desquamation, development of proinflammatory response together with abnormalities of lipids and improper profilaggrin processing. This mechanism triggers further actions, which, together with higher penetration of microbes and allergens, aggravate proin-flammatory response leading to development of NS symptoms [2,6,7]. According to Human Gene Mutation Database (HGMD) data, 72 distinct mutations were reported to cause NS, of which only two are missenses and others result in premature termination codon (PTC), frameshifts or splicing disruptions [8]. Similarly, the mutations identified in our patient are predicted to affect splicing. According to bioinformatics analyzes, the c.1431-12G>A introduces novel acceptor splicing site 10 nucleotides upstream of exon 16, while unreported before c.1816_1820+21delinsCT abolishes donor splicing site in intron 19, but also leads to a frameshift. The c.1431-12G>A mutation was reported by Raghunath et al. [9], who found it in two independent patients originating from Bosnia (homozygote) and Austria (heterozygote). Both parents of the Bosnian child were carriers of this mutation and were reported not to be consanguineous. Raghunath et al. [9] did not detect the LEKTI1 protein by immunohistochemical staining of skin biopsies and/or immunoblot analysis of plucked hair roots from these patients. These results and our data indicate that the majority of SPINK5 mRNA is truncated in our patient. Importantly, they do not rule out the possibility that small amount of full length mRNA may also be produced. According to Lacroix et al. [10], some deep intronic mutations activating cryptic splicing sites, may not completely abolish the natural splicing sites, allowing low level production of full length protein. This phenomena was observed in the case of mutations c.1820+ 53G>A and c.283-12T>A, and recognized to possibly be linked with a milder phenotype. This was also supported by analysis of residual expression of LEKTI1 in cultures of NS patients’ (genotyped as c.1820+53G>A/p. Cys297 Cys and c.283-12T>A/p.Cys297Cys) keratinocytes, which proved that LEKTI1 fragments could be detected. The p.Cys297Cys is an exonic silent variant that causes disruption of exonic splicing enhancers and also introduces a cryptic splicing site. Although Lacroix et al. [10] hypothesized that p.Cys297Cys is fully penetrant, Fortugno et al. [11] showed that p.Cys297Cys also does not cause complete abolishing of natural splice sites and partial expression of LEKTI1 is preserved [11]. Moreover, Bitoun et al. [12] suggest that certain PTCs in SPINK5 do not lead to mRNA degradation and may be used for truncated protein production. Primary structure of full length LEKTI1 consists of 15 different serine proteases inhibitory domains (D1-D15). As stated before, several isoforms of LEKTI1 with distinct inhibitory activities are expressed and/or subsequently generated. Thus, if truncated proteins (due to PTC) are produced, some domains would potentially be generated and functional. Komatsu et al. [13], despite unsuccessful attempts to identify truncated proteins in patients’ tissues, performed a series of experiments aimed at differentiation of the profile of inhibitory activity of LEKTI1 in five NS patients. They showed that there is an adverse correlation between the number of retained LEKTI1 domains and clinical severity of NS. The more domains preserved, the less severe clinical features such as skin lesions, growth retardation, skin infections were noticed. Results of some biochemical tests also supported these findings. With regard to this theory, our patient may hypothetically have 1-7 and 1-9 domains retained. Although it is impossible to speculate about the potential effect of their preservation, the Komatsu et al. [13] results of an in vitro assay with recombinant polypeptides containing different LEKTI1 domains, suggest that D6- D9 seem to be responsible for dose-dependent inhibition of trypsin, such as Phe-Ser-Arg, and chymotrypsin-like activity. Despite a growing number of findings explaining the molecular basis of phenotypic differences in NS patients, we still do not understand them. The LEKTI1 is involved in interaction with several proteins that play an important role in epidermal biology. Although the NS phenotype seems to be at least partially dependent on modifier genes and environmental factors [2], the hypothesis concerning preserved activity of LEKTI1 cannot be neglected. Correlation studies need to be performed on a larger cohort of patients that is always difficult in the case of orphan diseases. Thus, we are convinced that the case presented by us will provide further data for future studies. Furthermore, our patient is the third proband reported to carry the c.1431-12G>A mutation, thus our results provide evidence that c.1431-12G>A can be referred to as a frequent central- south European mutation of SPINK5. The majority of mutations in this gene is family-specific, however, several recurrent mutations have also been identified in different populations. With only a few exceptions, these mutations, similar to c.1431-12G>A, were mostly identified in two, three or four unrelated families [10,12,14,15].



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