MOLECULAR CHARACTERISATION OF 85 INDIVIDUALS WITH PYRUVATE KINASE DEFICIENCY
(Abstract release date: 05/19/16)
EHA Library. Laas C. 06/09/16; 133020; E1471
Ms. Claire Laas
Contributions
Contributions
Abstract
Abstract: E1471
Type: Eposter Presentation
Background
Pyruvate kinase (PK) deficiency is the most common glycolytic cause of non-spherocytic haemolytic anaemia. It is inherited as an autosomal recessive condition with 50 individuals per million being affected in the Caucasian population. The PKLR gene codes for Pyruvate Kinase, a red cell specific isoenzyme that converts phosphoenolpyruvate to pyruvate which releases ATP. ATP is essential for red cell function and without it the half-life of the red cell is reduced, being cleared by the spleen and liver. There are 200 known mutations which can cause PK deficiency, which is diagnosed using an enzyme assay. KLF1 is a transcription factor known to bind to the promoter of the PKLR gene and drives expression. Variants in the KLF1 gene have previously been shown to produce a PK deficient phenotype.
Aims
We screened for genetic variants in individuals with PK enzyme activity <11 IU/g Hb to determine if a genetic cause could be identified in every case.
Methods
Individuals with reduced PK enzyme activity underwent PKLR gene sequencing. Cases without an affected genotype were screened for indels in the PKLR gene followed by sequencing of the KLF1 gene.
Results
Of the 85 cases, 21 were identified with genotypes consistent with PK deficiency with 27 cases left having identified only a single pathogenic variant. 35 Cases were left without a pathogenic variant (although 38% of samples were heterozygous for the c.-248delT polymorphism), all falling within the range of 8-11 IU/g Hb. This is consistent with other labs that use lower normal range. The sequencing analysis identified ten variants not previously described (c.319 A>G, c.508-57 G>A, c.508-2 A>G, c.602 G>T, c.627 G>C, c.822 C>G, c.959 T>C, c.965+2 T>C, c.1063 A>G, c.1299 C>A) of which seven are likely to be highly pathogenic. One individual had a deletion encompassing exon 11 in combination with a missense variant. One unknown variant (c.881 C>A) was identified in the KLF1 gene and in silico tools predicted this to be damaging. There were no PKLR variants found in this patient, yet they had a PK activity of 3.5 IU/g Hb.
Conclusion
Genetic investigations into the PKLR gene is not diagnostically informative in individuals with a PK enzyme activity of >8 IU/g Hb. Of the 39 individuals with an enzyme activity of <8 IU/g Hb, 46% had two affected alleles, 44% had one affected allele, and in 10% of the cases no pathogenic variants were identified. The enzyme activity range of the 10% group (n=3) was 7-7.9 IU/g Hb excluding the individual with a variant in KLF1. This indicates that the more severe the PK enzyme deficiency the more likely the assay is to identify a genetic cause. In those cases with a genetic cause most are non-synonymous base changes probably resulting in protein instability. Two splice site variants were identified affecting the core consensus motif and also a single case with a deletion, all predicted to be severe alleles. It is unknown if the KLF1 gene variant is influencing PKLR gene expression, and further work is necessary to understand this.
Session topic: E-poster
Keyword(s): Genomics, Kinase, PCR, Splenectomy
Type: Eposter Presentation
Background
Pyruvate kinase (PK) deficiency is the most common glycolytic cause of non-spherocytic haemolytic anaemia. It is inherited as an autosomal recessive condition with 50 individuals per million being affected in the Caucasian population. The PKLR gene codes for Pyruvate Kinase, a red cell specific isoenzyme that converts phosphoenolpyruvate to pyruvate which releases ATP. ATP is essential for red cell function and without it the half-life of the red cell is reduced, being cleared by the spleen and liver. There are 200 known mutations which can cause PK deficiency, which is diagnosed using an enzyme assay. KLF1 is a transcription factor known to bind to the promoter of the PKLR gene and drives expression. Variants in the KLF1 gene have previously been shown to produce a PK deficient phenotype.
Aims
We screened for genetic variants in individuals with PK enzyme activity <11 IU/g Hb to determine if a genetic cause could be identified in every case.
Methods
Individuals with reduced PK enzyme activity underwent PKLR gene sequencing. Cases without an affected genotype were screened for indels in the PKLR gene followed by sequencing of the KLF1 gene.
Results
Of the 85 cases, 21 were identified with genotypes consistent with PK deficiency with 27 cases left having identified only a single pathogenic variant. 35 Cases were left without a pathogenic variant (although 38% of samples were heterozygous for the c.-248delT polymorphism), all falling within the range of 8-11 IU/g Hb. This is consistent with other labs that use lower normal range. The sequencing analysis identified ten variants not previously described (c.319 A>G, c.508-57 G>A, c.508-2 A>G, c.602 G>T, c.627 G>C, c.822 C>G, c.959 T>C, c.965+2 T>C, c.1063 A>G, c.1299 C>A) of which seven are likely to be highly pathogenic. One individual had a deletion encompassing exon 11 in combination with a missense variant. One unknown variant (c.881 C>A) was identified in the KLF1 gene and in silico tools predicted this to be damaging. There were no PKLR variants found in this patient, yet they had a PK activity of 3.5 IU/g Hb.
Conclusion
Genetic investigations into the PKLR gene is not diagnostically informative in individuals with a PK enzyme activity of >8 IU/g Hb. Of the 39 individuals with an enzyme activity of <8 IU/g Hb, 46% had two affected alleles, 44% had one affected allele, and in 10% of the cases no pathogenic variants were identified. The enzyme activity range of the 10% group (n=3) was 7-7.9 IU/g Hb excluding the individual with a variant in KLF1. This indicates that the more severe the PK enzyme deficiency the more likely the assay is to identify a genetic cause. In those cases with a genetic cause most are non-synonymous base changes probably resulting in protein instability. Two splice site variants were identified affecting the core consensus motif and also a single case with a deletion, all predicted to be severe alleles. It is unknown if the KLF1 gene variant is influencing PKLR gene expression, and further work is necessary to understand this.
Session topic: E-poster
Keyword(s): Genomics, Kinase, PCR, Splenectomy
Abstract: E1471
Type: Eposter Presentation
Background
Pyruvate kinase (PK) deficiency is the most common glycolytic cause of non-spherocytic haemolytic anaemia. It is inherited as an autosomal recessive condition with 50 individuals per million being affected in the Caucasian population. The PKLR gene codes for Pyruvate Kinase, a red cell specific isoenzyme that converts phosphoenolpyruvate to pyruvate which releases ATP. ATP is essential for red cell function and without it the half-life of the red cell is reduced, being cleared by the spleen and liver. There are 200 known mutations which can cause PK deficiency, which is diagnosed using an enzyme assay. KLF1 is a transcription factor known to bind to the promoter of the PKLR gene and drives expression. Variants in the KLF1 gene have previously been shown to produce a PK deficient phenotype.
Aims
We screened for genetic variants in individuals with PK enzyme activity <11 IU/g Hb to determine if a genetic cause could be identified in every case.
Methods
Individuals with reduced PK enzyme activity underwent PKLR gene sequencing. Cases without an affected genotype were screened for indels in the PKLR gene followed by sequencing of the KLF1 gene.
Results
Of the 85 cases, 21 were identified with genotypes consistent with PK deficiency with 27 cases left having identified only a single pathogenic variant. 35 Cases were left without a pathogenic variant (although 38% of samples were heterozygous for the c.-248delT polymorphism), all falling within the range of 8-11 IU/g Hb. This is consistent with other labs that use lower normal range. The sequencing analysis identified ten variants not previously described (c.319 A>G, c.508-57 G>A, c.508-2 A>G, c.602 G>T, c.627 G>C, c.822 C>G, c.959 T>C, c.965+2 T>C, c.1063 A>G, c.1299 C>A) of which seven are likely to be highly pathogenic. One individual had a deletion encompassing exon 11 in combination with a missense variant. One unknown variant (c.881 C>A) was identified in the KLF1 gene and in silico tools predicted this to be damaging. There were no PKLR variants found in this patient, yet they had a PK activity of 3.5 IU/g Hb.
Conclusion
Genetic investigations into the PKLR gene is not diagnostically informative in individuals with a PK enzyme activity of >8 IU/g Hb. Of the 39 individuals with an enzyme activity of <8 IU/g Hb, 46% had two affected alleles, 44% had one affected allele, and in 10% of the cases no pathogenic variants were identified. The enzyme activity range of the 10% group (n=3) was 7-7.9 IU/g Hb excluding the individual with a variant in KLF1. This indicates that the more severe the PK enzyme deficiency the more likely the assay is to identify a genetic cause. In those cases with a genetic cause most are non-synonymous base changes probably resulting in protein instability. Two splice site variants were identified affecting the core consensus motif and also a single case with a deletion, all predicted to be severe alleles. It is unknown if the KLF1 gene variant is influencing PKLR gene expression, and further work is necessary to understand this.
Session topic: E-poster
Keyword(s): Genomics, Kinase, PCR, Splenectomy
Type: Eposter Presentation
Background
Pyruvate kinase (PK) deficiency is the most common glycolytic cause of non-spherocytic haemolytic anaemia. It is inherited as an autosomal recessive condition with 50 individuals per million being affected in the Caucasian population. The PKLR gene codes for Pyruvate Kinase, a red cell specific isoenzyme that converts phosphoenolpyruvate to pyruvate which releases ATP. ATP is essential for red cell function and without it the half-life of the red cell is reduced, being cleared by the spleen and liver. There are 200 known mutations which can cause PK deficiency, which is diagnosed using an enzyme assay. KLF1 is a transcription factor known to bind to the promoter of the PKLR gene and drives expression. Variants in the KLF1 gene have previously been shown to produce a PK deficient phenotype.
Aims
We screened for genetic variants in individuals with PK enzyme activity <11 IU/g Hb to determine if a genetic cause could be identified in every case.
Methods
Individuals with reduced PK enzyme activity underwent PKLR gene sequencing. Cases without an affected genotype were screened for indels in the PKLR gene followed by sequencing of the KLF1 gene.
Results
Of the 85 cases, 21 were identified with genotypes consistent with PK deficiency with 27 cases left having identified only a single pathogenic variant. 35 Cases were left without a pathogenic variant (although 38% of samples were heterozygous for the c.-248delT polymorphism), all falling within the range of 8-11 IU/g Hb. This is consistent with other labs that use lower normal range. The sequencing analysis identified ten variants not previously described (c.319 A>G, c.508-57 G>A, c.508-2 A>G, c.602 G>T, c.627 G>C, c.822 C>G, c.959 T>C, c.965+2 T>C, c.1063 A>G, c.1299 C>A) of which seven are likely to be highly pathogenic. One individual had a deletion encompassing exon 11 in combination with a missense variant. One unknown variant (c.881 C>A) was identified in the KLF1 gene and in silico tools predicted this to be damaging. There were no PKLR variants found in this patient, yet they had a PK activity of 3.5 IU/g Hb.
Conclusion
Genetic investigations into the PKLR gene is not diagnostically informative in individuals with a PK enzyme activity of >8 IU/g Hb. Of the 39 individuals with an enzyme activity of <8 IU/g Hb, 46% had two affected alleles, 44% had one affected allele, and in 10% of the cases no pathogenic variants were identified. The enzyme activity range of the 10% group (n=3) was 7-7.9 IU/g Hb excluding the individual with a variant in KLF1. This indicates that the more severe the PK enzyme deficiency the more likely the assay is to identify a genetic cause. In those cases with a genetic cause most are non-synonymous base changes probably resulting in protein instability. Two splice site variants were identified affecting the core consensus motif and also a single case with a deletion, all predicted to be severe alleles. It is unknown if the KLF1 gene variant is influencing PKLR gene expression, and further work is necessary to understand this.
Session topic: E-poster
Keyword(s): Genomics, Kinase, PCR, Splenectomy
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