Contributions
Abstract: EP711
Type: E-Poster Presentation
Session title: Enzymopathies, membranopathies and other anemias
Background
Pyruvate kinase deficiency (PKD) is a genetic blood disorder typically caused by mutations in the PKLR gene, leading to low levels of pyruvate kinase enzyme, which is required by red blood cells to make energy. PKD is inherited in an autosomal recessive fashion but in some individuals only one pathogenic variant can be identified after sequencing gene exons. Patient genotypes have been associated with severity of disease, with patients carrying a non-missense mutation having a more severe presentation. Therefore, understanding the underlying genetic defect in individuals is important as it can inform patient management. On rare occasions, PKD can arise from pathogenic variants in the KLF1 gene, a transcription factor that controls PKLR gene expression.
We have developed a small NGS panel to sequence the entire footprint of the PKLR gene and exons of KLF1. Bloodspots have also been investigated as a sample source. They do not need expensive packaging like liquid blood samples, and can be sent through the post. This small change could help countries refer samples for analysis if they do not have access to PKLR gene sequencing.
Aims
To develop a targeted NGS assay capable of using DNA from liquid blood samples and from bloodspots to detect all known types of genetic variants that cause PKD (SNVs, small indels and deletions).
To cover the entire footprint of the PKLR gene to detect deep intronic variants which may contribute to the phenotype.
Methods
Residual clinical samples were used as positive controls from patients previously diagnosed with PKD. These individuals had undergone Sanger sequencing of the PKLR gene as part of their diagnostic work up. Bloodspots were created from liquid blood samples and DNA extracted from both for comparison.
Qiagen Qiaseq was used to target and amplify genomic regions of interest in all samples. This uses unique molecular indices to label each amplicon enabling duplicate reads to be discarded and CNV detection possible. All genetic variation was assed using the ACMG guidelines.
Results
Comparing the sequence between the new method and the existing Sanger sequencing method there was 100% concordance in the genetic findings. A minimum of 10ng of input DNA is required for the new methodology which can be extracted from a bloodspot. Liquid blood and bloodspot samples from the same primary sample identified the same genetic variants, indicating that bloodspots are a suitable sample for diagnosis.
Sequencing 80 patient cases (mostly liquid blood), identified 73 different genetic variants in total (19 class 1, 7 class 2, 22 class 3, 11 class 4 and 14 class 5). Most of the common variants were present in all cases. Six of the class 3 variants warrant further investigation, four of these are deep intronic variants which may affect PKLR gene splicing. These have been identified in individuals with PKD but with only one known pathogenic variant identified to date.
Conclusion
Our data show that bloodspots are a good source of DNA and are suitable for clinical diagnosis. The assay and the bioinformatic pipeline is able to identify all variant types which are known to cause PKD. The approach to sequence the entire PKLR gene may be clinically beneficial, but it does identify more variants of unknown clinical significance compared to exon only sequencing. Functional studies are required in order to investigate these further, to build the knowledge base and to make this data clinically useful.
Keyword(s): Diagnosis, Genetic, Pyruvate kinase deficiency
Abstract: EP711
Type: E-Poster Presentation
Session title: Enzymopathies, membranopathies and other anemias
Background
Pyruvate kinase deficiency (PKD) is a genetic blood disorder typically caused by mutations in the PKLR gene, leading to low levels of pyruvate kinase enzyme, which is required by red blood cells to make energy. PKD is inherited in an autosomal recessive fashion but in some individuals only one pathogenic variant can be identified after sequencing gene exons. Patient genotypes have been associated with severity of disease, with patients carrying a non-missense mutation having a more severe presentation. Therefore, understanding the underlying genetic defect in individuals is important as it can inform patient management. On rare occasions, PKD can arise from pathogenic variants in the KLF1 gene, a transcription factor that controls PKLR gene expression.
We have developed a small NGS panel to sequence the entire footprint of the PKLR gene and exons of KLF1. Bloodspots have also been investigated as a sample source. They do not need expensive packaging like liquid blood samples, and can be sent through the post. This small change could help countries refer samples for analysis if they do not have access to PKLR gene sequencing.
Aims
To develop a targeted NGS assay capable of using DNA from liquid blood samples and from bloodspots to detect all known types of genetic variants that cause PKD (SNVs, small indels and deletions).
To cover the entire footprint of the PKLR gene to detect deep intronic variants which may contribute to the phenotype.
Methods
Residual clinical samples were used as positive controls from patients previously diagnosed with PKD. These individuals had undergone Sanger sequencing of the PKLR gene as part of their diagnostic work up. Bloodspots were created from liquid blood samples and DNA extracted from both for comparison.
Qiagen Qiaseq was used to target and amplify genomic regions of interest in all samples. This uses unique molecular indices to label each amplicon enabling duplicate reads to be discarded and CNV detection possible. All genetic variation was assed using the ACMG guidelines.
Results
Comparing the sequence between the new method and the existing Sanger sequencing method there was 100% concordance in the genetic findings. A minimum of 10ng of input DNA is required for the new methodology which can be extracted from a bloodspot. Liquid blood and bloodspot samples from the same primary sample identified the same genetic variants, indicating that bloodspots are a suitable sample for diagnosis.
Sequencing 80 patient cases (mostly liquid blood), identified 73 different genetic variants in total (19 class 1, 7 class 2, 22 class 3, 11 class 4 and 14 class 5). Most of the common variants were present in all cases. Six of the class 3 variants warrant further investigation, four of these are deep intronic variants which may affect PKLR gene splicing. These have been identified in individuals with PKD but with only one known pathogenic variant identified to date.
Conclusion
Our data show that bloodspots are a good source of DNA and are suitable for clinical diagnosis. The assay and the bioinformatic pipeline is able to identify all variant types which are known to cause PKD. The approach to sequence the entire PKLR gene may be clinically beneficial, but it does identify more variants of unknown clinical significance compared to exon only sequencing. Functional studies are required in order to investigate these further, to build the knowledge base and to make this data clinically useful.
Keyword(s): Diagnosis, Genetic, Pyruvate kinase deficiency