Resolution of newborn screening results via targeted long-read sequencing
Laboratory Genetics and Genomics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction:
Newborn screening (NBS) programs frequently involve molecular techniques to confirm the diagnosis, help predict phenotype, and provide recurrence risk information. Molecular assays in clinical use for NBS confirmation include Sanger and short-read sequencing, which have limitations in their ability to detect structural variants such as copy number changes, non-coding variants, or variants within regions of low genomic complexity. For example, molecular diagnostic testing for Spinal Muscular Atrophy requires validation of a specialized assay such as multiplex ligation-dependent probe amplification (MLPA) because the causative gene, SMN1, shares 99% identity with its paralogous gene, SMN2. Additionally, because many of the conditions in NBS programs, such as Galactosemia and Pompe disease, are inherited in an autosomal recessive manner, testing of parents is often required to phase variants identified by Sanger or short-read sequencing approaches. We hypothesized long-read sequencing could streamline NBS confirmatory testing by capturing a broader spectrum of variants and phasing variants without the need for parental comparators.
Methods:
Residual DNA from peripheral blood was identified for eight individuals who had a positive NBS result for either Galactosemia or Pompe disease. Targeted long-read sequencing (LRS) was performed on an Oxford Nanopore PromethION using an R10.4.1 flow cell. Results of clinical Sanger sequencing of each proband and parents were compared to LRS data.
Results:
LRS identified all nonsense, missense, promoter, and copy number variants that had been reported clinically (n=24 for 8 cases). In each case, the gene was completely phased and the variants could be resolved onto one of two haplotypes. This phasing information was consistent with results of clinical testing of parental samples when available.
Conclusion:
We found that targeted LRS performed on DNA extracted from blood can be used to replicate the results of clinical molecular testing for eight individuals with positive NBS for either Galactosemia or Pompe. Advantages of this LRS-based approach include the ability to phase variants into haplotypes without the need for parental samples. We highlight here a case where two pathogenic GAA variants were found to be in trans with two benign variants. The need to coordinate parental testing lengthens the time to resolve potential false positive NBS results or may be impossible if one or both parents are unavailable. LRS approaches also have the potential to reduce the need for development and validation of specialized assays to capture the full spectrum of variants needed for conditions included on evolving NBS programs. Future benefits include the possibility of improved laboratory efficiency and faster, more complete results for patients and their families within this critical time period.
Newborn screening (NBS) programs frequently involve molecular techniques to confirm the diagnosis, help predict phenotype, and provide recurrence risk information. Molecular assays in clinical use for NBS confirmation include Sanger and short-read sequencing, which have limitations in their ability to detect structural variants such as copy number changes, non-coding variants, or variants within regions of low genomic complexity. For example, molecular diagnostic testing for Spinal Muscular Atrophy requires validation of a specialized assay such as multiplex ligation-dependent probe amplification (MLPA) because the causative gene, SMN1, shares 99% identity with its paralogous gene, SMN2. Additionally, because many of the conditions in NBS programs, such as Galactosemia and Pompe disease, are inherited in an autosomal recessive manner, testing of parents is often required to phase variants identified by Sanger or short-read sequencing approaches. We hypothesized long-read sequencing could streamline NBS confirmatory testing by capturing a broader spectrum of variants and phasing variants without the need for parental comparators.
Methods:
Residual DNA from peripheral blood was identified for eight individuals who had a positive NBS result for either Galactosemia or Pompe disease. Targeted long-read sequencing (LRS) was performed on an Oxford Nanopore PromethION using an R10.4.1 flow cell. Results of clinical Sanger sequencing of each proband and parents were compared to LRS data.
Results:
LRS identified all nonsense, missense, promoter, and copy number variants that had been reported clinically (n=24 for 8 cases). In each case, the gene was completely phased and the variants could be resolved onto one of two haplotypes. This phasing information was consistent with results of clinical testing of parental samples when available.
Conclusion:
We found that targeted LRS performed on DNA extracted from blood can be used to replicate the results of clinical molecular testing for eight individuals with positive NBS for either Galactosemia or Pompe. Advantages of this LRS-based approach include the ability to phase variants into haplotypes without the need for parental samples. We highlight here a case where two pathogenic GAA variants were found to be in trans with two benign variants. The need to coordinate parental testing lengthens the time to resolve potential false positive NBS results or may be impossible if one or both parents are unavailable. LRS approaches also have the potential to reduce the need for development and validation of specialized assays to capture the full spectrum of variants needed for conditions included on evolving NBS programs. Future benefits include the possibility of improved laboratory efficiency and faster, more complete results for patients and their families within this critical time period.