In a groundbreaking study published in the Journal of Child Neurology, our research team demonstrated that whole genome sequencing (WGS) combined with RNA sequencing (RNA-seq) successfully diagnosed an additional 25% of patients with complex neurological phenotypes who had previously received negative whole exome sequencing (WES) results.
The Diagnostic Gap: When Exome Sequencing Isn't Enough
Whole exome sequencing revolutionized genetic diagnosis by efficiently sequencing all protein-coding regions of the genome. However, exomes represent only about 2% of the total genome, and many patients with clearly genetic conditions remain undiagnosed after WES testing.
This study asked a critical question: What can we find when we look beyond the exome?
Study Design and Patient Population
The research examined 22 patients from 20 families, all presenting with complex neurological phenotypes including:
- Developmental delays and intellectual disability
- Progressive neurological deterioration
- Movement disorders and ataxia
- Seizure disorders
- Muscle weakness and myopathy
All patients had undergone comprehensive WES testing without obtaining a molecular diagnosis, representing the most challenging diagnostic cases.
The Power of Dual Technologies
By applying both WGS and RNA-seq to these unsolved cases, we leveraged complementary strengths:
Whole Genome Sequencing Advantages
- Intronic Variants: Detection of pathogenic variants in non-coding regions that affect splicing or gene regulation
- Structural Variants: Identification of deletions, duplications, and rearrangements invisible to exome sequencing
- Regulatory Elements: Coverage of promoters, enhancers, and other control regions
- Repeat Expansions: Better resolution of repetitive sequences
RNA Sequencing Benefits
- Functional Validation: Direct observation of whether variants affect gene expression
- Aberrant Splicing: Detection of splice defects not predicted from DNA sequence
- Allele-Specific Expression: Identification of imbalanced gene expression suggesting regulatory variants
- Novel Transcripts: Discovery of unexpected fusion transcripts or alternative splicing
Key Findings: Diagnostic Success
The results demonstrated the transformative potential of comprehensive genomic analysis:
25% Diagnostic Rate:
Five of 20 families (25%) received definitive molecular diagnoses through WGS and RNA-seq after negative WES results.
60% Were WES-Invisible Variants:
Remarkably, 60% of the newly diagnosed cases involved variants that WES simply could not detect—intronic variants affecting splicing, deep intronic mutations, and structural rearrangements outside coding regions.
Additional Candidate Variants:
An additional 5% of cases yielded strong candidate variants requiring functional studies for definitive confirmation.
Clinical Case Examples
Deep Intronic Splice Variant
One patient harbored a pathogenic variant deep within an intron that created a cryptic splice site, causing aberrant mRNA processing. WES would never sequence this region, but WGS detected the variant and RNA-seq confirmed its functional impact by showing the abnormal transcript.
Regulatory Region Mutation
Another case involved a variant in a gene's promoter region that dramatically reduced expression levels. DNA sequencing alone suggested the variant might be pathogenic, but RNA-seq provided definitive proof by demonstrating near-complete loss of gene expression from the affected allele.
Complex Structural Rearrangement
A third patient had a complex genomic rearrangement involving multiple exons that appeared as ambiguous results on microarray but was fully characterized through WGS, with RNA-seq confirming loss of normal transcript production.
Implications for Clinical Practice
This study has profound implications for genetic diagnostic strategies:
WES Should Not Be the Endpoint:
For patients with strong clinical evidence of genetic disease, negative WES results should prompt comprehensive WGS and RNA-seq rather than ending the diagnostic odyssey.
Upfront Comprehensive Testing:
In many cases, starting with WGS plus RNA-seq may be more efficient than sequential testing approaches, particularly for complex phenotypes.
RNA-seq as Functional Validation:
The ability to directly observe variant consequences through RNA analysis transforms variant interpretation from prediction to demonstration.
Our Diagnostic Philosophy
This research directly shaped the diagnostic approach at Praxis Genomics. We recognized that solving the most challenging cases requires:
- Comprehensive genome coverage through WGS
- Structural variant detection via optical genome mapping
- Functional validation through transcriptome analysis
By integrating these technologies from the outset, we maximize diagnostic success rates and minimize the time families spend searching for answers.
Continuing to Push Boundaries
Since this publication, we've continued to refine and expand our multi-platform approach, adding long-read sequencing for repeat expansion disorders and enhancing our bioinformatic analysis pipelines. The core principle remains unchanged: comprehensive testing yields comprehensive answers.
Read the full publication: Journal of Child Neurology
Have you received negative or inconclusive exome sequencing results? Learn about our comprehensive WGS and RNA-seq testing or consult with our genetic counselors.
