Novel physiological RECQL4 alternative transcript disclosed by molecular characterisation of Rothmund–Thomson Syndrome sibs with mild phenotype

Abstract

Rothmund-Thomson syndrome is a rare genodermatosis caused by biallelic mutations of the RECQL4 gene and is characterised by poikiloderma, sparse hair, eyelashes and/or eyebrows, small stature, skeletal and dental abnormalities and cancer predisposition. Mutations predicted to result in the loss of RECQL4 protein have been associated with osteosarcoma risk, but mutation(s)-phenotype correlations are better addressed by combined DNA and RNA analyses. We describe two siblings with a mild phenotype, mainly restricted to the skin, who carry the unreported paternal c.2272C4T alteration in exon 14 and the previously reported maternal exon 15 c.2492_2493delAT, both predicted to result in premature termination codons (p.(Arg758*), p.(His831Argfs*52)). However real-time and transcript analysis showed, in the carrier father and affected daughter, increased levels of a novel RECQL4 physiological alternative transcript with partial in-frame skipping of exon 14, generated by increased usage of a weak cryptic splice site. This alternative transcript is expressed in all controls and tested tissues, its upregulation is specific to the paternal c.2272C4T mutation and depends on the abrogation of the binding motifs for SF2 and SRp55 serine/arginine-rich proteins with bypass of the mutation site located in the skipped exon 14 portion. Moreover, in the proband the increased levels of the alternative transcript, likely encoding a protein isoform with residual activity, may compensate for the dearth of the canonical transcript with the c.2492_2493delAT, accounting for the mild clinical phenotype of the siblings. Our results emphasise the value of RNA analysis to better predict the effects of RECQL4 mutations on the clinical phenotype. Keywords: Rothmund-Thomson; mild phenotype; RECQL4; alternative splicing; exonic splicing enhancer; hypomorphic mutation INTRODUCTION Rothmund-Thomson syndrome (RTS, MIM#268400) is a clinically and genetically heterogeneous autosomal recessive disorder caused by biallelic mutations of the RECQL4 gene (MIM*603780) 1 in up to 66% of patients. 2 To date, more than 60 different RECQL4 mutations, only a few recurrent, have been described in RECQL4-related diseases, 45 of which were in patients with RTS, defined as RTS type II. 3-6 RECQL4-negative patients are classified as RTS type I. 3 The relationship between RECQL4 genotype and syndromic phenotype depends on mutation type and intragenic position and the specific combination of two different mutations, which makes (with a few exceptions) each case incomparable to others previously described. It has been proposed that in RTS patients, 'deleterious' RECQL4 mutations, predicted to lead to truncated proteins, predispose to osteosarcoma 2 and skeletal malformations. 7 Until functional studies, currently available for a restricted number of RECQL4 mutations, 8 unravel possible genotype-phenotype correlations, the analysis of mutant alleles at the transcript level may complement DNA analysis in predicting the effect of mutations on the clinical phenotype, includi...

Rare presentation of Rothmund-Thomson syndrome with predominantly cutaneous findings

“…Genetic analysis confirmed RTS. Few cases of RTS have presented with mild symptoms, 5 but no reported case only had cutaneous symptoms. Several possible mechanism of this rare presentation include tissue differential expression of RECQL4 variants, incomplete penetrance of the gene, or presence of pathogenic variants in genes modulating RECQL4 gene function or expression.…”

Rothmund-Thomson Syndrome: Insights from New Patients on the Genetic Variability Underpinning Clinical Presentation and Cancer Outcome

“…Interestingly, the c.2412_2420del alteration of patient #38 falls in the region encoded by exon 14, which undergoes a physiological alternative splicing. An increased amount of the alternative in-frame transcript, encoding for a likely functional protein lacking 66 amino acids of the helicase domain, has been detected in two RTS siblings characterized by mild phenotype carrying the c.2272C>T mutation which is abolished by exon 14 alternative splicing [ 38 ]. Unfortunately, patient #38 RNA was not available to investigate an expression change of the physiological alternative transcript r.2266_2463del.…”

Rothmund–Thomson Syndrome: novel pathogenic mutations and frequencies of variants in the RECQL4 and USB1 (C16orf57) gene

“…This variant is worth testing on transcript level, as the RECQL4 gene is known to have a specific splicing pattern based on the short size of 13 of its introns (Colombo et al. ).…”