ORIGINAL RESEARCH article

Front. Neurosci., 07 September 2022

Sec. Neurogenomics

Volume 16 - 2022 | https://doi.org/10.3389/fnins.2022.980000

Whole-genome sequencing combined RNA-sequencing analysis of patients with mutations in SET binding protein 1

  • 1. Department of Gynecology and Obstetrics, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China

  • 2. Institute of Rare Diseases, West China Hospital of Sichuan University, Chengdu, China

Abstract

SET binding protein 1 (SETBP1) is essential for human development, and pathogenic germline variants in SETBP1 lead to a recognizable developmental syndrome and variable clinical features. In this study, we assessed a patient with facial dysmorphism, intellectual disability and delayed motor development. Whole genome sequencing identified a novel de novo variation of the SETBP1 (c.2631C > A; p. S877R) gene, which is located in the SKI domain, as a likely pathogenic variant for the proband’s phenotype. RNA sequencing was performed to investigate the potential molecular mechanism of the novel variation in SETBP1. In total, 77 and 38 genes were identified with aberrant expression and splicing, respectively. Moreover, the biological functions of these genes were involved in DNA/protein binding, expression regulation, and the cell cycle, which may advance our understanding of the pathogenesis of SETBP1 in vivo.

Introduction

The SET binding protein 1 (SETBP1) gene is an oncogene located on the long (q) arm of chromosome 18 at position 12.3. The protein encoded by the SETBP1 gene contains several motifs and has been shown to bind the SET nuclear oncogene, which is associated with DNA replication and gene expression regulation (). Mutations in SETBP1 are involved in multiple diseases, leading to extremely complex genotype-phenotype correlations for the SETBP1 gene (). Somatic mutations of SETBP1 appear to be gain-of-function mutations and are associated with several hematological malignancies, such as myeloid leukemia (; ; ; ; ; ; ; ). In addition, germline loss-of-function mutations in the SETBP1 gene are correlated with developmental delay, which has a spectrum of symptoms, including absent speech/expressive language delays and mild-severe intellectual disability (). In contrast, germline gain-of-function mutations in the SETBP1 gene are linked with Schinzel–Giedion syndrome (SGS; OMIM 269150) (; ).

SGS is a rare genetic disorder characterized by characteristic facial features, multiple malformations, and neurological problems (; ; ). Germline de novo mutations in the SETBP1 gene cluster to a hotspot of 12 base pairs in exon 4 of the SETBP1 protein cause SGS (). This mutational hotspot is highly conserved and is part of a degron motif targeted by the SCF-βTrCP1 E3 ligase (). Previous studies have demonstrated that somatic mutations occurring in the hotspot region in the SETBP1 gene may cause a functional loss of the degron motif, resulting in accumulation of SETBP1 protein in cells and inhibition of the PP2A phosphatase through the SETBP1–SET–PP2A axis (; ; ). However, the understanding of the pathogenic mechanism of germline mutations in the SETBP1 gene is inadequate. Additional clinical and functional investigation is warranted to promote our understanding of the molecular mechanisms of SGS.

Recently, several studies have implemented total RNA sequencing integrated with whole-genome sequencing (WGS) to facilitate interpretation of the pathogenicity of variants by revealing expression and splicing outliers (; ; ; ). This approach provides an opportunity to explore the molecular mechanisms of germline mutations in the SETBP1 gene. In this study, we present the clinical characterization of a patient diagnosis as SGS and conducted WGS of parent-offspring trio. The results revealed a novel de novo mutation in SETBP1 that was predicted to be deleterious based on the concordance of generic damage prediction tools. Furthermore, RNA sequencing was performed in this family, and numerous aberrant expression/splicing genes provided supporting evidence for the role of SETBP1 and insight into the molecular mechanisms of germline mutations in the SETBP1 gene.

Materials and methods

Ethical compliance

Informed consent was obtained from the patient’s parents. This study was approved by the ethics committee of the Second Affiliated Hospital of Chongqing Medical University.

DNA isolation and whole genome sequencing

We sequenced the patient and her parents following the MGI-2000 protocol outsourced to BGI. Genomic DNA was isolated from peripheral blood using a blood genomic DNA extraction kit (Tiangen Biotech, Beijing, China) in accordance with the manufacturer’s protocol. One microgram of genomic DNA was randomly fragmented by Covaris, and the fragmented DNA was selected by an Agencourt AMPure XP-Medium kit to an average size of 200–400 bp, followed by adapter ligation and PCR amplification. The products were recovered by the AxyPrep Mag PCR clean up Kit. The double-stranded PCR products were heat-denatured and circularized by the splint oligo sequence. The single-strand circle DNA (ssCir DNA) was formatted as the final library and qualified by QC. WGS was performed on the MGI-2000 platform with an average depth of 30x, meaning that the entire genome was sequenced an average of 30 times.

RNA isolation and sequencing

Total RNA was extracted from peripheral blood and enriched by oligo-dT bead capture, and cDNA was synthesized according to the manufacturer’s protocol. cDNA libraries were constructed using the Illumina trueSeq stranded mRNA sample prep kit protocol (Illumina). Pooled samples were sequenced using a NovaSeq 6000 sequencing system.

Single-nucleotide variant/INDEL identification, annotation and interpretation

The raw data produced on the MGI-2000 platform were filtered and aligned against the human reference genome (hg19) using the Burrows–Wheeler Alignment tool () after evaluation according to Illumina Sequence Control Software (SCS). The single-nucleotide polymorphisms (SNPs) were called by using Genome Analysis ToolKit software ().

Variants were annotated using ANNOVAR (). The effects of single-nucleotide variants (SNVs) were predicted by the SIFT, Polyphen-2, and MutationTaster programs. Variants were filtered by a minor allele frequency (MAF) of < 0.1% in the gnomAD (), 1000 Genome (), ExAC () databases and the Exome Variant Server (EVS; NHLBI Exome Sequencing Project).

All variants were interpreted according to ACMG/AMP standards and categorized as pathogenic, likely pathogenic, variants of unknown clinical significance (VUS), likely benign and benign (). Variant validation was performed using Sanger sequencing (ABI 3730xl Genetic Analyzer).

Copy number variation identification and annotation

Copy number variations (CNVs) were detected by CNVnator, 100-bp bins and standard parameters were used to calculate the read-depth (RD) signal (). The CNVs identified were compared with CNVs from the Database of Genomic Variants1 to exclude previously reported polymorphisms. The non-polymorphic CNVs were compared with the entries in the DECIPHER,2 ISCA,3 ClinGen4, or ClinVar5 databases, evaluated against the literature for known syndromes and overlapping causal aberrations and further analyzed according to the type and size of aberration, function, and expression profile of genes.

Quality control for RNA-seq data

Fastp was used to filter low-quality reads from raw sequencing reads to obtain clean reads (). Then, FastQC and multiQC were used to evaluate the quality of sequencing data, and the average quality score for overall RNA sequences was > 30, indicating that a large percentage of the sequences were high quality (). DROP v1.2.1 was used to compute the evaluation metrics of mapping with sequencing depth, percentage of mapped reads, and the number of expressed genes (). The match between the RNA-seq sample and its annotated DNA sample was also determined by DROP with a cutoff of 0.8.

Detection of aberrant expression

Aberrant expression was fully detected based on DROP v1.2.1 (). The clean RNA-sequencing reads were mapped to the human reference genome (hg19) using STAR (2.7.8a) with the Gencode v29 annotation (). The summarize Overlaps function from the Genomic Alignments R package was used to count reads. To increase statistical power, we performed aberrant expression and splicing analysis by combining our data with 367 blood samples from GTEx data.6 Genes with a 95th percentile FPKM (Fragments Per Kilobase of transcript per Million mapped reads) < 1 were considered as lowly expressed in samples and were removed in downstream analysis. In total, nearly 10,000 genes were included. OUTRIDER was applied to identify expression outliers (). Technical and biological covariates, such as sex, age and sequencing batch, were automatically controlled by OUTRIDER, which used an autoencoder implementation. Genes were defined as having aberrant expression with a p < 0.01. Reverse transcription-quantitative PCR (RT-qPCR) was performed to validate candidate gene expression.

Detection of aberrant splicing

FRASER, which has been included in DROP, was used to obtain splicing outliers (). Exon–exon and exon–intron junctions with less than 20 reads in all samples were filtered out. In addition, junctions in which the total number of reads at the donor/acceptor splice site was 0 in more than 90% of the samples were also filtered out. Similar to OUTRIDER, FRASER also applies an autoencoder implementation to automatically control the technical and biological covariates. Splicing outlier genes were defined as genes with an adjusted p < 0.05. Outlier junctions were defined as those in splicing outlier genes, with an adjusted p < 0.05.

Pathway enrichment analysis

Functional enrichment of the aberrantly expressed and spliced genes was performed with KOBAS-i, a service that provides comprehensive pathway enrichment analysis using several databases, including GO, KEGG, Reactome, and GWAS catalogs (). An adjusted p < 0.1 was selected as the threshold for significant pathways.

Results

Clinical features of the patient

A 3-year-old female was referred to our hospital with global developmental delay, hypertonia and facial dysmorphism. The patient was born after 39 weeks with a normal gestation history. Her parents had no medical history (Figure 1A). She was found to have a motor and language development delay at 2 years old. She had characteristic facial features, including microcephaly, a prominent forehead, midface hypoplasia, a high palatal arch and a protruding tongue (Figure 1B).

FIGURE 1

Brain MRI at 8 months of age showed delayed myelination of brain white matter and enlargement of the lateral ventricle, and the bilateral frontotemporal extracerebral space was significantly widened. Her karyotype analysis revealed normal results.

Whole genome sequencing analysis

An average of 102G sequencing data were acquired after WGS for the family member, and no pathogenic CNVs were detected in the proband’s WGS data (Supplementary Figure 1). After variant pathogenicity classification according to ACMG guidelines, one de novo missense variant located in the SETBP1 gene (PS2 + PM2_Supporting + PP3), NM_015559.2: g.42531936C > A, c.2631C > A (NM_000052.7), affected highly conserved residues in close proximity to the canonical region in the SKI domain (p. S877R shown in red in Figure 2), indicating a likely pathogenic variant for the proband’s phenotype. Sanger sequencing confirmed that the c.2631C > A variant was a novel de novo variant (Figure 1C and Supplementary Figure 2). In addition, this variant has never been reported in the ClinVar database, HGMD database, or gnomAD database before.

FIGURE 2

Transcriptome analysis

RNA sequencing was performed to investigate the potential molecular mechanism of the novel mutation in SETBP1 (c.2631C > A; p.S877R). Aberrant analysis results and RT-qPCR showed that the RNA expression of the SETBP1 gene in the patient and control was similar. In total, 77 and 38 genes were identified with aberrant expression and splicing in the patient, respectively (Supplementary Tables 1, 2 and Supplementary Figures 38). Several genes directly targeted by SETBP1 or associated with neurodevelopmental disorders (NDDs) have been identified as aberrant genes in patient. For example, the receptor for activated C kinase 1 (RACK1) gene and RUNX Family Transcription Factor 1 (RUNX1) gene. RACK1 was a part of the IRE1-RACK1-PP2A complex and was aberrantly spliced in the patient (Figure 3). The RACK1 gene can modulate neurodegeneration by promoting ERK degradation in Machado-Joseph disease (MJD) and Huntington’s disease (HD) models and participates in the process of neuronal differentiation by regulating SCN1A expression (; ; ). RUNX1 is a direct transcriptional target of SETBP1 and encodes a transcription factor involved in the generation of hematopoietic stem cells and their differentiation into myeloid and lymphoid lines (). Relative quantification of a subset of genes (SETBP1, WDFY3, AK1, RUNX1, and HLA-B) by means of RT-qPCR confirmed the accuracy of aberrant analysis with RNA-seq data (Figure 4; two sample t-tests; PSETBP1 = 0.485; PWDFY3 = 0.015; PAK1 = 0.037; PRUNX1 = 0.015 and PHLA–B = 0.004). We next performed enrichment analyses of the aberrantly expressed and spliced genes to delineate the most relevant biological pathways. Functional annotation demonstrated that the biological functions of these genes were involved in DNA/protein binding, expression regulation, and the cell cycle (Figure 5 and Supplementary Tables 3, 4).

FIGURE 3

FIGURE 4

FIGURE 5

Mutation pattern and genotype-phenotype correlations in the SKI domain of SETBP1

To evaluate the correlation between associated variants in the SKI domain and the phenotype of SETBP1, a systematic search of original papers was performed (; ; ; ; ; ; ; ; ; ; ; ; ; ; ). A total of 41 variants with SETBP1-related NDDs located in the SKI domain were included, including 28 with missense mutations and 13 with loss-of-function mutations (Supplementary Table 5). Overall, variants clustering to a hotspot of 12 base pairs coding for residues 868 to 871 of the SETBP1 protein are known to be associated with severe forms of SGS, possibly through a dominant negative effect (). In our study, a novel variant near this region was associated with a similar form of this disease (Figure 2).

Discussion

In this study, we reported a patient of SGS with severe intellectual disability, developmental delay, epilepsy, hypertonia and distinctive facial dysmorphism. WGS and Sanger validation revealed that these phenotypes may be caused by a novel de novo germline missense mutation of the SETBP1 gene (NM_015559.2: c.2631C > A), which can cause the amino acid change p. S877R (serine - arginine). Due to the extremely low prevalence and great phenotypic heterogeneity of SGS, it is difficult to recognize by clinicians and is usually diagnosed based on the reminiscent clinical features (). In this case, an accurate genetic diagnosis significantly improved the management of the patient and reproduction of this family.

Our results revealed that germline de novo heterozygous missense variants adjacent to the mutation hotspot of the SETBP1 gene tend to cause atypical SGS. Previous studies have shown that germline de novo mutations in the SETBP1 gene cluster to residues 868–871 of the SETBP1 protein, which are associated with severe forms of SGS (). In this study, the de novo variant identified in this patient is located in residue 877, which is close to the mutation hotspot of the SETBP1 gene. Some phenotypes in this patient were mild relative to typical SGS patients, including ventriculomegaly, skeletal abnormalities, and hydronephrosis. In addition, several individuals with atypical SGS carrying heterozygous missense variants outside the mutation hotspot have been reported. identified four individuals carrying SETBP1 variants in close proximity to the canonical mutation hotspot, including p.(Glu862Lys), p.(Ser867Arg), and p.(Thr873Ile), who showed a milder developmental phenotype with clinical characteristics that partially overlapped with classical SGS. Moreover, individuals with variants located further from the mutation hotspot showed a variable clinical phenotype ranging from mild to severe intellectual disability (; ). These findings highlight that the variable severity of broad clinical features depends on the proximity of variants to the mutation hotspot.

Integrative analyses identified that the missense variant reported in this study likely disrupts SETBP1 protein functions via mechanisms including DNA/protein binding, transcription and the cell cycle. While the transcription of the SETBP1 gene was not affected, it is possible that this missense SETBP1 mutation has a subtle but distinct effect on the regulation, since 77 aberrantly expressed and 38 spliced genes have been identified in the patient. Consistent findings have been reported by other researchers. In a recent paper, Wong et al. revealed through cellular experiments that classical SGS variants located in the mutation hotspot showed increased protein stability and higher SETBP1 protein levels, while SETBP1 variants outside the mutation hotspot disrupt DNA binding and transcription independent of protein abundance (). Future studies that delineate the structural impact of SETBP1 variants and how they affect interactions with other genes will contribute to the understanding of their impacts on protein functions and thus etiology.

Taken together, our findings expand the current understanding of the genetics and clinical spectrum of SETBP1 variants. In addition, by integrating WGS and RNA-seq analyses, we provide insight into the pathogenicity of a germline de novo SETBP1 variant in a patient diagnosed with atypical SGS.

Statements

Data availability statement

The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

Ethics statement

The studies involving human participants were reviewed and approved by the Ethics Committee at The Second Affiliated Hospital of Chongqing Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

Author contributions

LL performed the experiments and wrote the manuscript. XF and SL performed RNA-seq analysis and wrote the manuscript. YZ and XD collected the clinical information of the patient and her parents. HY and BT designed and supervised the study and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Joint International Research Lab for Reproduction and Development, Ministry of Education, China, Program for Youth Innovation in Future Medicine, Chongqing Medical University (W0122), “Kuanren talents” project of the Second Affiliated Hospital of Chongqing Medical University (13-003-003), Nan’an District Science and Health Joint Medical Scientific Research Project (2020-01), and the Scientific and Technological Research Program of Chongqing Municipal Education Commission (Grant No. 81).

Acknowledgments

We thank the patient and the family members for their participation in this study.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins.2022.980000/full#supplementary-material

References

Summary

Keywords

RNA-seq, de novo, missense variant, SETBP1, clinical diagnosis

Citation

Liu L, Feng X, Liu S, Zhou Y, Dong X, Yao H and Tan B (2022) Whole-genome sequencing combined RNA-sequencing analysis of patients with mutations in SET binding protein 1. Front. Neurosci. 16:980000. doi: 10.3389/fnins.2022.980000

Received

28 June 2022

Accepted

11 August 2022

Published

07 September 2022

Volume

16 - 2022

Edited by

Bo Xiong, Huazhong University of Science and Technology, China

Reviewed by

Yi Zhang, Central South University, China; Yan Wang, Beijing Institutes of Life Science (CAS), China

Updates

Copyright

*Correspondence: Hong Yao, Bo Tan,

†These authors have contributed equally to this work and share first authorship

This article was submitted to Neurogenomics, a section of the journal Frontiers in Neuroscience

Disclaimer

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Outline

Figures

Cite article

Copy to clipboard


Export citation file


Share article

Article metrics