WGS and RNA-seq Combination: Multidisciplinary Case Studies in Diseases Diagnosis and Treatment

With the continuous development of life sciences, gene sequencing technology has undergone rapid innovation, and its sequencing costs have been significantly reduced, becoming the core driving force to promote the progress of clinical gene diagnosis. The profound changes "from local to panoramic" and "from single to combined" caused by this technological innovation are reshaping the pattern of clinical gene diagnosis in all directions, and pushing this field into a new stage of rapid development.

In recent years, multimethology combined with gene detection has gradually become the focus of scientific research and clinical application. Among these, the joint analysis of whole-genome sequencing (WGS) and transcriptome sequencing (RNA-Seq) has emerged. With their complementary advantages, the ability to interpret the variation function of non-coding regions has been greatly strengthened, and the efficiency of gene diagnosis has been significantly improved. This joint analysis model not only provides a more comprehensive and accurate perspective for researchers to deeply explore the pathogenesis of diseases but also lays a solid foundation for clinicians to formulate personalized diagnosis and treatment programs.

This paper discusses the application of WGS combined with RNA-seq in the study of ataxia, hepatoblastoma, congenital glycosylation disorder, autism and lung adenocarcinoma through several cases, and provides a new direction for disease diagnosis and treatment.

Application of WGS and RNA-seq in Molecular Diagnosis of Ataxia

Title: Integration of multi-omics technologies for molecular diagnosis inataxia patients

Publish Magazine: Front Genet

Impact Factors: 2.8

Publication Time: 2024.01.04

DOI: https://doi.org/10.3389/fgene.2023.1304711

Ataxia is a group of highly heterogeneous nervous system diseases with different genetic causes (such as single nucleotide polymorphism, structural variation, repeated expansion, etc.), which makes accurate molecular diagnosis challenging. Although the progress of genome sequencing technology has improved the success rate of partial diagnosis, there are still a large number of patients who have not been able to obtain a clear molecular cause. The purpose of this study is to integrate WGS, RNA-seq, and LRS techniques to explore and verify the potential pathogenic variation in complex ataxia cases.

Four of the eight patients (50%) were identified by multivariate analysis, and the potential molecular genetic mechanism of the disease was successfully revealed, which provided an important basis for the subsequent individualized diagnosis and treatment plan.

• SPG7: Two mutations (heterozygous mutations) located in different alleles were found, one missense mutation and the other nonsense mutation, which were related to recessive ataxia.
• ELOVL4: Intron variation near a splicing site was detected, which led to the jump of exons 4 and 5, and the splicing defect was further verified by LRS.
• PMPCB: It was identified that an intron mutation caused exon 9 to jump, and the gene expression decreased by more than 50%.
• ATXN2: It was found that CAG repeatedly expanded (32 repetitions) and reached the threshold of variable permeability, which was related to SCA2.

In ATXN2 carriers, a potential modifier mutation of ZFYVE26 was detected, which may have a modifying effect on the phenotype of patients.

Functional validation of individual #4 pathogenic genomic alterations (Audet et al., 2024)

This study highlights the great potential of multimethology in the molecular diagnosis of complex genetic diseases, especially in difficult cases where traditional detection methods are helpless. By organically integrating WGS, RNA-Seq, and LRS, the research team not only improved the success rate of molecular diagnosis of patients with paroxysmal ataxia to 50% but also found new pathogenic mechanisms such as splicing site variation in non-coding regions (such as ELOVL4 and PMPCB intron variation) and microsatellite repeat amplification (such as CAG32 of ATXN2). This multi-omics joint strategy provides a systematic framework for analyzing clinical phenotypic heterogeneity, and the genome-transcriptome association data generated by it also lays an important foundation for exploring the synergistic pathogenic effect of genetic modification factors (such as ZFYVE26 mutation).

Functional transcriptomic validation of genes with suspected alternative splicingvariants (Audet et al., 2024)

Reveal the Genetic Pathogenesis of HB Based on WGS and RNA-seq

Title: Whole-genome sequencing and RNA sequencing analysis reveals novel risk genes and differential expression patterns in hepatoblastoma

Publish Magazine: Gene

Impact Factors: 3.68

Publication Time: 2024.03.01

DOI: https://doi.org/10.1016/j.gene.2023.147991

Hepatoblastoma (HB) is a rare malignant liver cancer, which mainly affects infants and children. Its histological features are similar to those of fetal hepatocytes, mature hepatocytes, or bile duct cells. HB accounts for about 28% of all liver malignant tumors in children and adolescents, and its pathological mechanism is still unclear. This study reveals the genetic changes of HB by WGS and RNA-seq technology and finds out the potential pathogenic genes and related molecular mechanisms.

Variation of known pathogenic genes

• CTNNB1, AXIN2, and PARP1: Harmful mutations of these genes were detected, which supported their role in the pathogenesis of HB. These genes have been reported to be related to HB in previous studies.
• CTNNB1 is related to the Wnt/β-catenin signaling pathway, and its mutation may lead to uncontrolled cell proliferation.
• AXIN2 is a negative regulator of the Wnt signaling pathway, and its mutation may further destroy the pathway balance.

Circos plots of all somatic CNVs in HB tumor-normal samples called out with delly (Wang et al., 2024)

Newly discovered potential related genes

• BRCA2 and GPC3: Their roles in HB are not clear, but they may be involved in tumor development through DNA repair (BRCA2) or extracellular matrix regulation (GPC3).
• ABC C2: It was identified as a potential pathogenic gene by ACMG analysis, which may be related to HB risk.

Differential gene expression

• The candidate genes: IGF1R, METTL1, AXIN2, and TP53 were significantly abnormal in HB tumor tissues (P < 0.01).
• IGF1R: Insulin-like growth factor receptor, which is closely related to cell growth and proliferation.
• Mettl 1: RNA methylation-related enzyme, which may affect tumor progression through epigenetic mechanism.
• AXIN2: It not only involves the Wnt signaling pathway but also may play a role in tumor metabolism.
• TP53: A classical tumor suppressor gene, whose abnormality may destroy cell cycle regulation and DNA repair.

Bubble plots for GO analysis and KEGG analysis (Wang et al., 2024)

This study combined with WGS and RNA-seq technology provided a new insight for the etiological study of HB. The study not only verified the pathogenicity of known genes (such as CTNNB1 and AXIN2) but also revealed new potential genes (such as BRCA2, GPC3, and ABCC2). At the same time, the candidate genes (such as IGF1R, METTL1, and TP53) discovered by RNA-seq provide potential targets for HB personalized diagnosis and treatment.

WGS and RNA-seq Reveal Intron Variation Related to ATP6AP1 mRNA Processing

Title: Genome and RNA sequencing were essentialto reveal cryptic intronic variants associatedto defective ATP6AP1 mRNA processing

Publish Magazine: Molecular Genetics and Metabolism

Impact Factors: 3.7

Publication Time: 2024.07.06

DOI: https://doi.org/10.1016/j.ymgme.2024.108511

ATP6AP1-CDG belongs to X-linked glycosylation congenital disorder (CDG) pedigree disease, and its pathogenic mechanism is directly related to atp6ap1 gene variation. Up to now, 34 cases of confirmed patients have been reported in the world. The genotypic-phenotypic association study shows that missense mutation, frameshift mutation, and splicing site variation of the ATP6AP1 gene may trigger disease phenotype, but there are still a lot of unsolved fields in the specific pathogenesis.

RNA-seq and qPCR analyses in fibroblasts from P1 and P2 (Morales-Romero et al., 2024)

In this study, a combined analysis strategy of RNA sequencing (RNA-seq) and whole genome sequencing (WGS) was constructed. The abnormal splicing events at the transcript level were captured by RNA-seq, and the potential pathogenic factors such as structural variation and non-coding region mutation were analyzed by WGS at the whole genome scale. The disease-related genetic variation was systematically mined, and the pathogenic mechanism of ATP6AP1 gene variation was clarified through in vitro functional verification experiments, which provided a theoretical basis for accurate diagnosis and treatment of rare diseases.

ATP6AP1 cDNA analysis in fibroblasts from patients P1 and P2 (Morales-Romero et al., 2024)

Revealing the molecular mechanism of patients

P1 and P2: RNA-seq revealed the down-regulation of ATP6AP1 expression and abnormal splicing. Two deep intron variants were identified:

• P1：c.289-233C > T
• P2：c.289-289G > A
• P3: Trio-WGS found the same c.289-289G > A variation (somatic cell new development)

The c.289-233C > T and c.289-289G > A variants lead to altered ATP6AP1 mRNA processing (Morales-Romero et al., 2024)

RNA-seq and its functional effects

Abnormal expression: the level of ATP6AP1 mRNA decreased by about 80%, which was the lowest in the whole queue.

• Splicing defect: The sequence of intron 2 is abnormally preserved, resulting in hidden exons (1CE and 2CE), which leads to a frameshift mutation and nonsense-mediated decay (NMD). ​
• Minigene verification: mutation leads to abnormal splicing of ATP6AP1, resulting in abnormal transcripts consistent with patients.

Biochemical phenotype

• Transferrin: P1/P2 showed a CDG II pattern (mono/trisialic acid type increased), and P3 was accompanied by an O-glycosylation defect. ​
• LAMP2 protein: Low glycosylation bands appeared in patients' fibroblasts, which confirmed glycosylation disorder.

In this study, three male patients with clinically suspected glycosylation disorder but undiagnosed by whole exon sequencing (WES) were studied. By RNA-seq and WGS, the deep intron variation of ATP6AP1 gene (P1 is c.289-233C>T, P2 and P3 are c.289-289G>A) was found, which led to abnormal splicing and mRNA degradation, and then triggered ATP6AP1-CDG. Functional experiments confirmed that mutation can activate hidden exons and affect mRNA processing. The study emphasized the importance of multi-group technology combined with functional verification in the diagnosis of rare diseases.

Glycosylation studies in patients (Morales-Romero et al., 2024)

Identified New Genes of Autism According to WGS and RNA-seq

Title: Whole-genome sequencing identifies novelgenes for autism in Chinese trios

Publish Magazine: Sci China Life Sci

Impact Factors: 8.0

Publication Time: 2024.08.07

DOI: https://doi.org/10.1007/s11427-023-2564-8

The research team carried out a genome-wide sequencing study on autism and identified 146 new mutations in the coding region and 60 genetic mutations in the coding region. Among them, four genes including CTNND1 found multiple mutation sites, and the rest found one mutation site. A total of 33 potential ASD genes were obtained by integrating these genetic variations with TADA. Compared with the research results of WES, WGS, and GWAS in the world and the genes in SFARI Gene, it is found that these genes include many known ASD genes, including CHD8, SCN2A, SHANK3, NF1, etc., as well as many new genes, as well as genes DBT and INTS1.

De novo variants and inherited variants in coding regions (Chang et al., 2024)

The clinical data of patients with these genes and patients with these gene variants in SPARK, SSC, and other databases were analyzed, and it was found that patients had high comorbidity, including attention deficit hyperactivity disorder, mental retardation, language delay, and so on.

In addition, the study made a variety of functional annotations to SNV in non-coding regions and found that the number of newborn mutations was positively correlated with the father's age at birth. At the same time, 456 short structural variations with a length of less than 500bp and 295 long structural variations were identified by four tools for structural variation analysis. Finally, the study integrated the RNA-seq data of patients and analyzed the expression level of mutation-related genes in patients with the mutation. It was found that the newborn mutation located in the coding region had a greater impact on the gene expression level.

Correlation of de novo variants with parental ages, gender and expression of corresponding genes (Chang et al., 2024)

Identify Potential Therapeutic Targets for Pancreatic Cancer

Title: Multi-omics and clustering analyses reveal the mechanismsunderlying unmet needs for patients with lung adenocarcinoma andidentify potential therapeutic targets

Publish Magazine: Mol Cancer

Impact Factors: 41.4

Publication Time: 2024.09.02

DOI: https://doi.org/10.1186/s12943-024-02093-w

In this study, the integrated analysis of multi-omics data was used to reveal the unknown carcinogenesis mechanism and potential therapeutic targets of lung adenocarcinoma (LUAD) in non-small cell lung cancer (NSCLC). Emphasis is placed on patients with "non-CAGAs". Through WGS, RNA-seq, ChIP-seq, and other technologies, new molecular mechanisms are discovered, and new methods of patient stratification and treatment are proposed.

Mutation spectrum of non-CAGA LUAD patients

• Mutations are mainly concentrated in chromosomes 1, 5, 7, and 8, and some patients show copy number gain.
• Some patients have a loss of copy number neutral heterozygosity (LOH), and the mutation of TP53 is significantly increased in the high-risk subgroup (72%).

Discovery of key gene MAML2

• In non-CAGA patients, MAML2 enhancer activity was significantly inhibited, accompanied by down-regulation of gene expression.
• H3k27ac signal analysis showed that the methylation level of the MAML2 gene region increased, which was negatively correlated with its low expression.
• Mamml2 regulates Notch and Wnt/β-catenin signaling pathways, and its downstream genes (such as BCL2 and CCND3) are also suppressed.

Genetic and epigenetic analysis in non-CAGA lung adenocarcinoma samples (Asada et al., 2024)

Prognostic markers and potential therapeutic targets

• Fat 4, HMCN1, CD302, UTRN, and FOXN3 were identified as significant prognostic markers, and the overall survival rate of patients with decreased expression was poor.
• Deg analysis showed that PLK1, UBE2C, etc. were significantly up-regulated in high-risk subgroups, which may be potential therapeutic targets.

Improvement of layering strategy

• The second-level clustering combined with multi-gene expression can significantly improve the prognosis prediction ability of patients with stratification.

Functional enrichment analysis

• Up-regulated genes in high-risk groups are enriched in cell cycle and angiogenesis pathways, while down-regulated genes are involved in tissue structure development and morphogenesis.

It is revealed for the first time that the mutation in the non-coding region of MAML2 inhibits the expression through apparent regulation (H3K27ac deletion+hypermethylation), which affects the Notch/Wnt pathway and drives tumorigenesis. The patients were stratified by secondary cluster analysis, and the prognostic-related genes such as FAT4, HMCN1, CD302, UTRN, and FOXN3 were identified. It was found that the high-risk group was rich in TP53 mutation and high tumor mutation load (TMB). Finally, the potential therapeutic targets such as PLK1 and UBE2C were determined by differential expression analysis and functional enrichment, which provided a new direction for the mechanism analysis and precise treatment of non-CAGAs LUAD.

Second-level cluster analysis to improve patient stratification (Asada et al., 2024)

Conclusion

To sum up, the combination of WGS and RNA-seq has shown excellent application value in the field of disease research. In the diagnosis of rare diseases, it broke through the limitations of traditional methods and successfully excavated the pathogenic variation that was difficult to find before. In tumor research, we can not only reveal the key genetic changes of tumor occurrence and development, but also analyze the interaction between immune cells and tumor cells in tumor microenvironment.

Looking forward to the future, with the continuous innovation of sequencing technology and continuous optimization of analysis algorithm, the combination technology of WGS and RNA-seq will develop in the direction of more Qualcomm, more accurate and more intelligent. Its application in disease diagnosis will be more popular, and it is expected to become a routine clinical detection method, further improving the early diagnosis rate and accurate treatment level of diseases.

References:

1. Audet S, Triassi V, Gelinas M, et al. "Integration of multi-omics technologies for molecular diagnosis in ataxia patients." Front Genet. 2024 14: 1304711 https://doi.org/10.3389/fgene.2023.1304711
2. Wang W, Zhang N, Chen L, et al. "Whole-genome sequencing and RNA sequencing analysis reveals novel risk genes and differential expression patterns in hepatoblastoma." Gene. 2024 897: 147991 https://doi.org/10.1016/j.gene.2023.147991
3. Morales-Romero B, Muñoz-Pujol G, Artuch R, et al. "Genome and RNA sequencing were essential to reveal cryptic intronic variants associated to defective ATP6AP1 mRNA processing." Mol Genet Metab. 2024 142(3): 108511 https://doi.org/10.1016/j.ymgme.2024.108511
4. Chang S, Liu JJ, Zhao Y, et al. "Whole-genome sequencing identifies novel genes for autism in Chinese trios." Sci China Life Sci. 2024 67(11): 2368-2381 https://doi.org/10.1007/s11427-023-2564-8
5. Asada K, Kaneko S, Takasawa K, et al. "Multi-omics and clustering analyses reveal the mechanisms underlying unmet needs for patients with lung adenocarcinoma and identify potential therapeutic targets." Mol Cancer. 2024 23(1): 182 https://doi.org/10.1186/s12943-024-02093-w