Enhancing Genetic Diagnostics: The Critical Role of Enzyme Activity Assays and RNA-Sequencing as Primary Multi-Omics Screening Tools
Laboratory Genetics and Genomics
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Primary Categories:
- Clinical Genetics
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Secondary Categories:
- Clinical Genetics
Introduction:
Conventional genetic diagnostic protocols rely on DNA sequencing as the primary screening method. When potential causative variants are identified, secondary non-DNA omics approaches are sometimes employed to validate or refute their pathogenicity. An alternative strategy—utilizing non-DNA omics as the primary screening tool, followed by secondary DNA sequencing of candidate genes or regions—has remained largely unexplored.
Methods:
We re-analyzed a case that had yielded no significant findings through a conventional ‘gene panel sequencing as the primary step’ approach. We first considered the patient’s phenotype to define a list of candidate genes. We then tested the activity of the encoded enzymes and analyzed corresponding transcripts in blood-derived RNA sequencing data by visual inspection in the integrated genome viewer. We finally Sanger-sequenced a suggestive region and used in silico prediction to interpret the identified variant. All wet lab analyses were done from dried blood spot filter cards.
Results:
The clinical information (11-year-old boy presenting with short stature, joint stiffness, dysostosis multiplex, a coarse face, umbilical and inguinal hernia, hypertrophic adenoids, and recurrent infections) led us to suspect mucopolysaccharidosis (MPS) or a related metabolic disorder. Enzyme activities were normal for ARSB, GUSB, IDUA, GALNS, GLB1, MAN2B1, but below the level of detection for IDS. The transcripts for most candidate genes showed no abnormalities, but insertion of an intron 6-derived stretch of 46 nucleotides was observed in the IDS transcript. DNA sequencing of this intronic region (46 +/- 80 nucleotides) revealed a hemizygous IDS variant (c.880-1180A>T; NM_000202.5), which is predicted to create a strong splice donor. Together with a nearby splice acceptor from a rarely used alternative exon, this motif suggests that the 46-nucleotide inclusion is due to aberrant splicing. The variant was classified as likely pathogenic based on ACMG/AMP/ClinGen guidelines for observed impact on splicing.
Conclusion:
Our study demonstrates the significant potential of non-DNA primary screening approaches, particularly enzyme activity assays and RNA-sequencing, in improving the efficiency and cost-effectiveness of genetic diagnostics for rare (metabolic) disorders. It also suggests that such a multi-omics concept can be based on dried blood spot filter cards as the single sample type. To establish the utility of our approach in routine clinical settings, further research involving systematic, automated protocols and large-scale validation studies is necessary.
Conventional genetic diagnostic protocols rely on DNA sequencing as the primary screening method. When potential causative variants are identified, secondary non-DNA omics approaches are sometimes employed to validate or refute their pathogenicity. An alternative strategy—utilizing non-DNA omics as the primary screening tool, followed by secondary DNA sequencing of candidate genes or regions—has remained largely unexplored.
Methods:
We re-analyzed a case that had yielded no significant findings through a conventional ‘gene panel sequencing as the primary step’ approach. We first considered the patient’s phenotype to define a list of candidate genes. We then tested the activity of the encoded enzymes and analyzed corresponding transcripts in blood-derived RNA sequencing data by visual inspection in the integrated genome viewer. We finally Sanger-sequenced a suggestive region and used in silico prediction to interpret the identified variant. All wet lab analyses were done from dried blood spot filter cards.
Results:
The clinical information (11-year-old boy presenting with short stature, joint stiffness, dysostosis multiplex, a coarse face, umbilical and inguinal hernia, hypertrophic adenoids, and recurrent infections) led us to suspect mucopolysaccharidosis (MPS) or a related metabolic disorder. Enzyme activities were normal for ARSB, GUSB, IDUA, GALNS, GLB1, MAN2B1, but below the level of detection for IDS. The transcripts for most candidate genes showed no abnormalities, but insertion of an intron 6-derived stretch of 46 nucleotides was observed in the IDS transcript. DNA sequencing of this intronic region (46 +/- 80 nucleotides) revealed a hemizygous IDS variant (c.880-1180A>T; NM_000202.5), which is predicted to create a strong splice donor. Together with a nearby splice acceptor from a rarely used alternative exon, this motif suggests that the 46-nucleotide inclusion is due to aberrant splicing. The variant was classified as likely pathogenic based on ACMG/AMP/ClinGen guidelines for observed impact on splicing.
Conclusion:
Our study demonstrates the significant potential of non-DNA primary screening approaches, particularly enzyme activity assays and RNA-sequencing, in improving the efficiency and cost-effectiveness of genetic diagnostics for rare (metabolic) disorders. It also suggests that such a multi-omics concept can be based on dried blood spot filter cards as the single sample type. To establish the utility of our approach in routine clinical settings, further research involving systematic, automated protocols and large-scale validation studies is necessary.