RNA processing defects of the helicase gene RECQL4 in a compound heterozygous Rothmund–Thomson patient

Abstract

Rothmund-Thomson syndrome (RTS) (OMIM 268400) is an autosomal recessive genodermatosis associated with genomic instability and increased risk of mesenchymal cancers. Mutations in the RECQL4 gene, encoding a protein of the family of Werner (WRN) and Bloom (BLM) helicases, have been identified in a subset of RTS patients. Apart from congenital poikiloderma, the clinical presentation of RTS is widely variable, raising the question of the possible existence of a second locus. Results herein reported on a sporadic Caucasian patient emphasize the concept that mutation analyses at both DNA and RNA level complement the genetic defect suggested by clinical and cytogenetic signs. The patient presented with typical congenital poikiloderma and bone defects and exhibited significant genomic instability in the peripheral blood karyotype. By RECQL4 DNA mutation analysis, he was found to carry a 1473delT (mut 5) on one allele and an AG to AC change at the 3'-splice site of exon 13 (a variant of mut 4) on the second allele. RT-PCR analysis of RECQL4 cDNA encompassing the entire helicase domain showed diffuse splicing defects indicating that the loss of a single 3'-splice signal motif disregulates the correct splice-site selection and affects the overall RNA processing. The presence of an unstable minisatellite which ends at 3'-splice site of IVS12 may enhance the mutation at this site. This genomic feature together with a number of short introns in the RECQL4 gene may account for the common missplicing of RECQL4 mRNA. While it is possible that defects of RECQL4 mRNA processing might account for part of the clinical variability observed for this syndrome, only a thorough analysis at both genomic and RNA level may allow a genotype-phenotype correlation in RTS patients, restricting the search of a second RTS locus to the specific patients.

Clinical utility gene card for: Rothmund–Thomson syndrome

“…5,6,7 1.7 Analytical validation Bidirectional sequencing results are confirmed by sequencing using different sets of primers. Pathogenicity of novel missense alterations must be verified by testing a set of at least 100 control chromosomes of the same ethnic origin and by in silico prediction methods.…”

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

“…This RECQL4 isoform is a fraction of the physiological RECQL4 transcripts (Figure 2b and Supplementary Figure 1), but is markedly increased in the compound heterozygous RTS proband (II-1) and her father (I-1) (Figure 2b). The enhancement of the physiological AltTr appears to be dependent on the paternal c.2272 C4T mutation, as its expression is not affected by other RECQL4 mutations, such as that of the proband's mother or those carried by a previously characterised RTS individual 9 (Figure 3a and b). Indeed, the c.2272 C4T mutation, abolishing the binding sites for SF2 and SRp55, enhances the use of a weak cryptic splice site lying 7 nt upstream to the detriment of the canonical IVS14 donor splice site (Figure 2c).…”

“…Indeed, the mother showed reduced levels of the canonical full-length exon 14 transcript but levels of the AltTr were comparable to those observed in controls (Figure 3a and b). The same quantitative analysis of RECQL4 transcripts was performed in another compound heterozygous RTS patient with two different RECQL4 mutations, one affecting the 3 0 splice site of exon 13, 9 and hence close to the paternally inherited mutation of our siblings. This analysis highlighted decreased levels of both the full-length and the partially skipped exon 14 RECQL4 isoforms compared with controls and family members, suggesting that enhancement of AS leading to a partially skipped exon 14 is a mutation-specific mechanism.…”

“…According to our Figure 3 Real-time expression profile of RECQL4 isoforms. (a) RECQL4 partially skipped exon 14 and (b) RECQL4 full-length exon 14 mRNA expression in case II-1, her healthy parents and an RTS patient 9 with different RECQL4 mutations compared with five controls (CTRL). Samples were run in triplicate and data are expressed as mean ± SD.…”

The versatile RECQL4

“…9 This peculiar gene structure predisposes mutated RECQL4 mRNA to mis-splicing alterations resulting from the mutation in the primary gene transcript. 10 Consequently, intronic size constraint and point mutations in the introns of the RECQL4 gene have been shown to play a role in the development of RTS in addition to exonic defects. 10 -12 Human RECQL4 encodes a 133 kDa protein of 1,208 amino acids that contains characteristic sequences of the RecQ-family's helicase domain.…”