A synonymous mutation in SPINK5 exon 11 causes Netherton syndrome by altering exonic splicing regulatory elements

Abstract

Netherton syndrome (NS) is a rare, life-threatening ichthyosiform syndrome caused by recessive loss-of-function mutations in SPINK5 gene encoding lymphoepithelial Kazal-type-related inhibitor (LEKTI), a serine protease inhibitor expressed in the most differentiated epidermal layers and crucial for skin barrier function. We report the functional characterization of a previously unrecognized synonymous variant, c.891C4T (p.Cys297Cys), identified in the SPINK5 exon 11 of an NS patient. We demonstrated that the c.891C4T mutation is associated with abnormal pre-mRNA splicing and residual LEKTI expression in the patient's keratinocytes. Subsequent minigene splicing assays and in silico predictions confirmed the direct role of the synonymous mutation in inhibiting exon 11 inclusion by a mechanism that involves the activity of exonic regulatory sequences, namely splicing enhancer and silencer. However, this deleterious effect was not complete and a residual amount of normal mRNA and LEKTI protein could be detected, correlating with the relatively mild patient's phenotype. Our study represents the first identification of a disease-causing SPINK5 mutation that alters splicing without affecting canonical splice sites.

Exon-Specific U1s Correct SPINK5Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element

“…In SPINK5 gene, the relatively frequent synonymous c.891C>T transition induces E11 skipping causing NS. Its molecular mechanism, simply based on bioinformatics analysis was previously attributed to the disruption of an SRSF1-dependent ESE [Fortugno et al, 2012;Lacroix et al, 2012]. In this work, to better understand the pathological splicing event affecting E11 in the presence of the +9T mutation and to provide a mutation-specific correction strategy, we performed detailed binding and functional analysis of splicing factors and evaluated a novel ExSpe U1-based therapeutic approach for splicing correction.…”

“…More than 70 loss-of-function mutations have been described in SPINK5 [Lacroix et al, 2012]. One of these, the c.891C>T (p.Cys297Cys) synonymous variant within exon 11 (E11), is the most frequent mutation in NS patients of European origin [Fortugno et al, 2012;Lacroix et al, 2012]. This mutation leads to out-of-frame exon skipping and was suggested to disrupt an SRSF1dependent ESE [Lacroix et al, 2012].…”

Epidermolytic palmoplantar keratoderma caused by activation of a cryptic splice site inKRT9

“…All other point mutations in gene coding sequences are usually scored as missense, nonsense or silent mutations. However, exonic mutations altering mRNA splicing have been described in several genetic disorders including neurofibromatosis, type 1 (OMIM #162200), cystic fibrosis (OMIM #219700), ataxia telangiectasia (OMIM #208900), familial breast–ovarian cancer (OMIM #604370) and Netherton syndrome (OMIM #256500) . The present data further underline the importance of RNA analysis and full gene screening in the molecular analysis of typical phenotypes for which no mutations are found in routinely screened regions.…”

Is c.1431-12G>A A common European mutation of SPINK5? report of a patient with Netherton Syndrome

“…[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]. …”