Periodic Report Summary - 5-FU PGX (Prevalence of various genetic and epigenetic changes in genes in patients with severe toxicity, healthy donors and various Israeli populations)
The purpose of the project was to develop and manufacture a clinical diagnostic kit for easy screening of Dihydropyrimidine dehydrogenase (DPYD) gene known and novel mutations that cause for the toxicity of 5-FU. We started by screening 195 patients either prior to or after 5FU treatment, for the most common mutation in DPYD gene (NM_000110.3) by direct sequencing of exon 14. Six patients (3 %) were found to carry the well characterised mutation in intron 14: IVS14 (c.1905+1G>A).
Of those patients who were negative for the IVS14 mutation we further screened 23 patients for variants / mutations in DPYD gene by sequencing of exons: 2, 13, 14 and 22. 11 of the 23 patients screened had a known variant in the DPYD gene. In one patient we found a new mutation in exon 13, which results in a premature stop codon: c.1681C>T p.561R>X. This mutation was not reported so far in the literature.
To facilitate the testing of the common DPYD mutation by clinical laboratories, Pronto has developed an easy to use and inexpensive clinical diagnostic kit for screening of IVS14 mutation in DPYD gene. The 5FURisk kit is based on the patented Pronto technology. This technology forms the basis for one of the first commercial Single nucleotide polymorphism (SNP) assays for clinical diagnostics that utilises a single-nucleotide primer extension Enzyme-linked immunosorbent assay (ELISA) assay.
The kit requires five steps:
a) genomic Deoxyribonucleic acid (DNA) extraction;
b) amplification of DNA fragment by Polymerase chain reaction (PCR);
c) inactivation of dNTPs - post PCR;
d) prontoTM - single nucleotide primer extension;
e) detection of results by ELISA.
To develop a simple, robust and well-standardised diagnostic kit that will be used for clinical use, it is necessary to first establish the optimal conditions to execute the test and to evaluate the robustness of the kit. The capabilities and sensitivities to changes in manufacturing and working conditions were studied by several validations steps (we used as a positive control a DNA sample ordered from Coriell):
1. Validations for amplification mix:
- different batches of primers in amplification mix;
- different DNA extraction procedures;
- use of different Taq polymerases in PCR step;
- minimum and maximum genomic DNA concentrations allowed;
- ± 50 % of Taq DNA polymerase;
- sensitivity of amplification to ± 2 degrees of Celsius variation in thermocycler temperature;
- prolonged incubation at room temperature after addition of Taq polymerase.
2. Validations of post amplification / detection:
variation in amount of post PCR reagents;
- performance without taking out amplified DNA for gel electrophoresis;
- variation of post amplified DNA taken to the pronto plate.
3. Validations of Pronto plate / complete kit:
- test of different batches of primers and nucleotides in Pronto plate;
- ± 2 degrees of Celsius error of thermocycler in Pronto.
4. Test batch: 20 samples were tested by two different conditions (lab workers, thermocyclers, reagents lots etc.) using the prototype kit. We validated the kit results by another gold standard method: sequencing.
Production of a prototype kit and QC steps:
- preparation of manufacturing and QC procedures - Standard operating procedure (SOP)s;
- preparation of package inserts and labels;
- controlled production of the first batch of the kit;
- the kit is produced in laboratories that have the ISO 13285 certification for production;
- QC: 20 samples were tested using the prototype kit by two different lab workers.
Of those patients who were negative for the IVS14 mutation we further screened 23 patients for variants / mutations in DPYD gene by sequencing of exons: 2, 13, 14 and 22. 11 of the 23 patients screened had a known variant in the DPYD gene. In one patient we found a new mutation in exon 13, which results in a premature stop codon: c.1681C>T p.561R>X. This mutation was not reported so far in the literature.
To facilitate the testing of the common DPYD mutation by clinical laboratories, Pronto has developed an easy to use and inexpensive clinical diagnostic kit for screening of IVS14 mutation in DPYD gene. The 5FURisk kit is based on the patented Pronto technology. This technology forms the basis for one of the first commercial Single nucleotide polymorphism (SNP) assays for clinical diagnostics that utilises a single-nucleotide primer extension Enzyme-linked immunosorbent assay (ELISA) assay.
The kit requires five steps:
a) genomic Deoxyribonucleic acid (DNA) extraction;
b) amplification of DNA fragment by Polymerase chain reaction (PCR);
c) inactivation of dNTPs - post PCR;
d) prontoTM - single nucleotide primer extension;
e) detection of results by ELISA.
To develop a simple, robust and well-standardised diagnostic kit that will be used for clinical use, it is necessary to first establish the optimal conditions to execute the test and to evaluate the robustness of the kit. The capabilities and sensitivities to changes in manufacturing and working conditions were studied by several validations steps (we used as a positive control a DNA sample ordered from Coriell):
1. Validations for amplification mix:
- different batches of primers in amplification mix;
- different DNA extraction procedures;
- use of different Taq polymerases in PCR step;
- minimum and maximum genomic DNA concentrations allowed;
- ± 50 % of Taq DNA polymerase;
- sensitivity of amplification to ± 2 degrees of Celsius variation in thermocycler temperature;
- prolonged incubation at room temperature after addition of Taq polymerase.
2. Validations of post amplification / detection:
variation in amount of post PCR reagents;
- performance without taking out amplified DNA for gel electrophoresis;
- variation of post amplified DNA taken to the pronto plate.
3. Validations of Pronto plate / complete kit:
- test of different batches of primers and nucleotides in Pronto plate;
- ± 2 degrees of Celsius error of thermocycler in Pronto.
4. Test batch: 20 samples were tested by two different conditions (lab workers, thermocyclers, reagents lots etc.) using the prototype kit. We validated the kit results by another gold standard method: sequencing.
Production of a prototype kit and QC steps:
- preparation of manufacturing and QC procedures - Standard operating procedure (SOP)s;
- preparation of package inserts and labels;
- controlled production of the first batch of the kit;
- the kit is produced in laboratories that have the ISO 13285 certification for production;
- QC: 20 samples were tested using the prototype kit by two different lab workers.