Compound Heterozygous Mutations Involving Splicing Mutations Cause Rothmund-Thom

Chao-Lan Pan,Qiao-Yu Cao,Yue Li,Jia Zhang,Zhen Zhang,Yu-Meng Wang,Fu-Ying Chen,Ru-Hong Cheng,Xiao-Xiao Wang,Zhi-Rong Yao,Zhi-Yong Lu,*,Ming Li,*

1Department of Dermatology,Xinhua Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200092,China;

2Institute of Dermatology,Shanghai Jiaotong University School of Medicine,Shanghai 200092,China.

Abstract

Keywords:exon-skipping,genodermatosis,RECQL4 variants,Rothmund-Thomson syndrome,splice-site mutation

Introduction

Rothmund-Thomson syndrome(RTS)(OMIM#268400)is a rare recessive disorder that was identified through the combination of research by Rothmund1and Thomson.2A characteristic facial rash(poikiloderma)was initially the classic phenotype of this disease.3Patients with RTS thereafter exhibited strong phenotypic and genotypic heterogeneity,presenting with poikiloderma,growth retardation,rapidly progressive bilateral juvenile cataracts,sparse scalp hair and eyelashes,and/or eyebrows,skeletal abnormalities,radial ray defects,premature aging,and a predisposition to cancers.Based on clinical and genetic molecular analyses,RTS has been classified into two types:type I is characterized by classic phenotypes and biallelic alterations in the Aanaphase promoting complex subunit gene and has been identified in ten patients,4and type II is characterized by an increased risk of cancers and a positive result of a RecQ like helicase(RECQL4)mutation scan.5

RecQL4,the gene product that is defective in patients with RTS,belongs to the RecQ helicase family,which also includes RecQL1,Werner,Bloom,and RecQL5 in mammals.Several syndromes are associated with the RecQ helicase gene family,including Werner syndrome,Bloom syndrome,and RTS,which are associated with pathogenic variants in the WRN,BLM,and RECQL4 genes,respectively.6-8Mutations in RECQL4 have been identified not only in the majority of patients with RTS but also in patients with RAPADILINO syndrome.9These helicases participate in diverse DNA metabolic activities such as DNA replication,transcription,recombination,and repair through their DNA-unwinding function.In particular,RECQL4 plays multifaceted roles in maintaining the genomic stability of telomeres and the nuclear and mitochondrial integrity of normal cells and cancer cells.10

Intriguingly,RecQL4 consists of 1,208 amino acids and is characterized by the highly conserved RecQ helicase domain(encoded by exons 8-14),while the N-terminus and C-terminus are unique among the RecQ family.The Sld2-like N-terminus(1-388 amino acid residues)of the protein is necessary for the initiation of DNA replication.10A nuclear targeting signal and a mitochondrial localization sequence have been found within the N-terminus.The Cterminus with a RecQ4-Zn2+-binding domain(836-1,045 amino acid residues)lacks structural resemblance to the conserved RecQ C-terminal(RQC)domain,yet it is functionally comparable with the other RecQ helicases as an essential part of the catalytic core.11-12

We herein report on two unrelated families with RTS-IIaffected members,highlighting the mild clinical manifestation depending on the strength of the underlying pathogenic variants and their heterozygous combination.We also herein characterize the causative RECQL4 genetic lesions and explore their pathogenic effect by transcripts analysis to address mutation-phenotype correlations.

Materials and methods

Patients

Two patients participated in the study after their parents provided appropriate informed consent for performance of the genetic tests.This study was approved by the ethics committee of Xinhua Hospital,Shanghai JiaotongUniversity School of Medicine(Approval No.XHEC-D-2020-140).

DNA isolation,RNA isolation,and complementary DNA(cDNA)synthesis

Genomic DNA was extracted from peripheral blood samples from the families using the QIAamp DNA Mini kit(Qiagen,Hilden,Germany)according to manufacturer’s instructions.The DNA concentration was measured using a Qubit 3.0 Fluorometer(Invitrogen,Carlsbad,CA,USA).The RNAiso Plus kit(Takara Bio-engineering Co.Ltd.,Dalian,China)was used to isolate RNA from fresh tissues of the patients following the manufacturer’s protocols.Samples were reverse-transcribed using a reverse transcription kit(Takara Bio-engineering Co.Ltd.).Different fragments of RECQL4 cDNA(exons 14-17,exons 16-17,and exons 17-19)were amplified and sequenced by Sanger sequencing.Nucleotide sequences were compared with the major RECQL4 transcript reference sequence(GenBank:NM_004260.3).NG_016430.1 was used as reference for exon numbering.

Multi-gene panel sequencing(next-generation sequencing)and data analysis and interpretation

Next-generation sequencing and data analysis and interpretation were performed as previously described.13The genomic DNA was analyzed using a 596-gene panel associated with monogenic hereditary diseases.The frequencies of identified variants were confirmed to be 0 or＜1% in the Asian populations in all used databases,including the ExAC database(https://gnomad.broadinsti tute.org/)and the 1,000 Genomes database(http://www.internationalgenome.org/data-portal/sample).All identified variants were evaluated against databases such as National Center for Biotechnology Information Database for Single Nucleotide Polymorphisms(http://www.ncbi.nlm.nih.gov/SNP/),Online Mendelian Inheritance in Man(http://www.omim.org/),Human Gene Mutation Database(http://www.hgmd.cf.ac.uk/ac/index.php),and National Center for Biotechnology Information ClinVar(https://www.ncbi.nlm.nih.gov/clinvar/).

Sanger sequencing

Direct Sanger sequencing was applied to verify the identified variants using an ABI PRISM 3730XL automated sequencer(Applied Biosystems,Foster City,CA,USA).All primers involved in the Sanger sequencing are listed in Table 1.

Table 1 Primers used in the Sanger sequencing.

Results

Clinical reports of patients with RTS Family A

The proband was a 4-year-old girl.She was the only child of non-consanguineous parents.Since the age of 8 months,the child had shown erythema on the cheeks that subsequently spread to both ears.At the age of 4 years,poikiloderma,mostly restricted to the face,was clearly apparent.However,no similar lesion was detected in other parts of the skin,including sun-exposed areas.In addition,photosensitivity and heat sensitivity were noted starting at an early age.The patient showed no abnormalities in growth parameters(height,100cm;weight,15.2kg),skin annexes,eyes,or hematologic function.The orthopedic doctors found no obvious skeletal defects,but tiny skeletal deformities could not be excluded unless a comprehensive computed tomography examination had been performed.The onset of poikiloderma on the face and ears was this patient’s unique clinical phenotype(Fig.1A).

Family B

The propositus was a young girl,the second child of healthy unrelated parents.Persistent erythema on her cheeks had been present since the age of 5 months,accompanied by sensitivity to sun and heat exposure.By the age of 21 months,which was the time of clinical diagnosis and molecular diagnosis of RTS,the malar erythema had developed to true poikiloderma and spread to her distal arms and legs,exhibiting a pattern of reticulated hypo/hyperpigmentation,thin desquamation,mild atrophy,and telangiectases(Fig.1B).Conversely,she showed no hair defects,nail or teeth abnormalities,growth retardation,or other dysfunctions.The patient’s clinical signs were mainly restricted to the skin.The first child and parents in the family B are normal.

Molecular characterization of study subjects

Biallelic alterations in the RECQL4 gene were found in both patients by employing a multi-gene panel strategy.

In Family A,the RECQL4 molecular study showed that the proband was compound heterozygous for the paternally inherited c.2885+1G＞A in intervening sequence(IVS)16 and the maternally inherited c.2272C＞T in exon 14(Fig.1A).The maternally inherited mutation is a transition predicted to result in a stop codon at residue 758(p.R758X),which has been previously reported.13Amplification of the cDNA portion of c.2885+1G＞A in the small IVS16(72 nucleotides)allowed retrieval of two different RECQL4 amplicons:the expected transcript with exon 15-16 junction and an aberrant transcript resulting from exons 16 and 17 skipping(Fig.2A).The skipped transcript carried the in-frame exon 16-17 deletion and,if translated,would encode a protein lacking 100 amino acids(p.A919Gdel100).

In the proband of Family B,the paternally inherited classical splicing mutation c.2886-1G＞A was identified in IVS16 of RECQL4,and the maternally inherited nonsense mutation c.2752G＞T was identified in exon 15(Fig.1B).The maternally inherited mutation was a transition predicted to result in a stop codon at residue 918(p.E918X).Sequencing of the amplification of the cDNA portion of c.2886-1G＞A revealed the insertion of the whole IVS16 between the sequences of exon 15 and 16(Fig.2B).The altered mRNA would be translated into a truncated protein lacking 246 amino acids.At the time of this writing,neither variant had been reported in the literature in individuals with RECQL4-related diseases.

Discussion

RTS is a rare autosomal recessive disorder with clinical features consisting of poikiloderma,skeletal abnormalities,sparse hair,absent or scanty eyelashes and eyebrows,short stature,and a high risk of developing osteosarcoma and other cancers.However,the two patients described herein presented with a phenotype mainly restricted to cutaneous alterations;therefore,they were considered to have the mild type of RTS.Nevertheless,careful follow-up of these patients is needed because of the early stage of their disease.

We reviewed the reported mutations from previous literature,including missense/nonsense mutations,splicing mutations,small deletions and insertions,small indels,and gross deletions.The structure of RECQL4 has been long known to render this gene prone to mis-splicing.We found 21 different splice mutations in RECQL4.

In this study,we identified four pathogenic RECQL4 variants in two Chinese patients with clinically suspected RTS from two unrelated families.As previously described,the c.2272C＞T alteration could lead to the specific upregulation of a novel RECQL4 physiological alternative transcript,likely encoding a protein isoform with residual activity;this may offset the dearth of the canonical transcript and account for the mild clinical phenotype.14The novel nonsense mutation c.2752G＞T is expected to result in premature termination of protein translation(p.E918X).

The genetic mechanism of the two novel splice-site mutations(c.2885+1G＞A and c.2886-1G＞A)in the same intron of RECQL4 is described as follows.Analysis of the transcripts of the novel splice-site mutation(c.2885+1G＞A)found in a 4-year-old Chinese girl showed the product of a two-exon skip.To the best of our knowledge,this is the first demonstration of multiple-exon skipping as the pathogenic basis for RTS.It is conceivable that the c.2885+1G＞A mutation might lead to aberrant splicing due to abolishment of the splice site.However,the mechanism by which the mutation leads to skipping of both exons in the transcript remains unknown.There is strong evidence to support the theory that the order and speed of splicing are important determinants of mRNA transcripts with multiple-exon skipping.15-17However,it is difficult to apply the argument to this splicing.Additionally,the poor fit of the splice-donor site and the donor-site consensus sequence cannot explain the skip.18-19However,increasing evidence shows that the RNA-specific secondary structure influences the splicing machinery and plays an important role in exon definition for particular transcripts.20Therefore,the fact that the splice mutation skipped exons 16 and 17 might be explained by the change in the splice direction(which may have been caused by the alteration of the specific secondary RNA structure).

Figure 1.Clinical manifestation and genetical variants analysis of the probands with Rothmund-Thomson syndrome type II.(A)The pedigree illustrates the inheritance pattern of the RTS nuclear Family A.Clinical images(pictured at 4 years of age)and DNA sequencing of the proband identified the paternally inherited c.2885+1G＞A(I:1)and maternally inherited c.2272C＞T variants(I:2).(B)The pedigree illustrates the inheritance pattern of the RTS nuclear Family B.Clinical images(pictured at 2 years of age)and DNA sequencing of the proband identified the paternally inherited c.2886-1G＞A(I:1)and maternally inherited c.2752G＞T variants(I:2).Squares represent males and circles represents females.Halffilled shapes indicate heterozygous parents while black shapes indicate affected patients.(C)Scheme of the RECQL4 gene mutations.Exons are indicated by boxes,and introns are indicated by lines.Deep blue identifies the exons that encode the RecQ helicase domain.Yellow boxes and arrows indicate mutations detected in Family A,and green boxes and arrows indicate mutations detected in Family B.Red characters indicate missense/nonsense mutations,blue characters indicate splicing mutations,purple characters indicate insertion mutations,green characters indicate deletion mutations,and black characters indicate indel mutations.RECQL4 GenBank NM_004260.3.

Figure 2.Variants inherited from parents with Rothmund-Thomson syndrome type II and cDNA sequencing of patients and schematic representation of splicing.(A and B)DNA sequencing of parents of Family A and Family B.(C)cDNA sequencing and sketch maps of Patient A show the expected wild-type allele and a non-canonical exon 15-18 junction resulting from exon 16 and 17 deletion.(D)cDNA sequencing and sketch maps of Patient B reveal an insertion of the whole IVS16 between the sequences of exon 15 and 16,resulting in premature termination codons.cDNA:complementary DNA.

The other splice-site variant,c.2886-1G＞A,caused insertion of the whole IVS16,which caused premature translation that led to the production of a truncated protein.This was caused by the mutation within a donor splice site,which then“weakened”this splice site in some way and led to the insertion of IVS16 in mRNA.21

Two of these mutations are localized in the conserved helicase domain(exons 8-14)shared by the other RecQ helicases and are thought to destroy or disturb the DNA helicase activity of RecQL4.However,the c.2272C＞T alteration could initiate a novel transcript to offset the loss of the core function,and the location of c.2752G＞T on the tail end of the domain may have a minor influence on its function(Fig.1C).

Both splice-site mutations impact the RQC domain,which comprises a zinc-binding domain and a winged helix domain involved in protein-protein interactions and regulation of helicase activity.12The fact that our patients presented with an overall mild phenotype allows us to infer that their splicing variants,although much less deleterious than truncating mutations,were able to reproduce all the main cutaneous findings of RTS,likely because of the defective function of this RQC terminal region.Moreover,we also speculate that the putative residual helicase activity of the proteins encoded by the observed aberrant transcripts might have contributed to the patients’mild phenotype.

This study has two limitations:First,there is a lack of experiments to verify the existence and rescue effect of non-classical transcripts.Second,the clinical phenotype of patients requires long-term follow-up visits and observation.Only a prolonged and careful follow-up of patients will allow validation of the suggested correlation between their genotype at the RECQL4 locus and their mild clinical presentation.Further studies such as functional experiments are needed to characterize the effect of these RECQL4 mutations.

Reports on mutations located outside the coding regions and associated with human diseases are rapidly growing.In this study,we reported on two Chinese girls with RTS and confirmed the presence of compound heterozygous variants.We identified three novel pathogenic RECQL4 variants related to RTS,which expanded the mutational spectrum of the RECQL4 gene and explored their pathogenic effect by transcripts analysis to address mutation-phenotype correlations.

Acknowledgments

The authors thank the patients and their families for their cooperation in this study.The authors also appreciate all related fellows contributing this work.

Source of founding

This study was supported by the National Nature Science Foundation of China(Nos.82073422 and 81874239),Shanghai Health System Excellent Academic Leader Training Project(No.2018BR22),and Pujiang Talents Program(No.18PJ1407300).