Dihydropyrimidinase deficiency via the DPYS Gene
- Summary and Pricing
- Clinical Features and Genetics
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In the largest studied cohort of patients suspected of dihydropyrimidinase deficiency (metabolic profile and clinical features of DHP deficiency), all 17 (100%) patients were found to be homozygous or compound heterozygous for pathogenic variants (van Kuilenburg et al. 2010). Analytical sensitivity should be close to 100% because all reported pathogenic variants are detectable by sequencing.
Dihydropyrimidinase (DHP) deficiency is an inherited error in pyrimidine metabolism that results in dihydropyrimidinuria. Dihydropyrimidinase is the second enzyme in the 3-step catabolic pathway of uracil and thymine. It catalyzes the hydrolytic cleavages of dihydropyrimidines (5,6-dihydrouracil and 5,6-dihydrothymine) which creates intermediate metabolites that are processed further to β-alanine and β-aminoisobutyric acid. Patients may have elevated uracil, thymine, dihydrouracil, and dihydrothymine while β-alanine and β-aminoisobutyric acid will be low or undetectable. Most patients with complete DHP deficiency have neurological symptoms ranging from developmental delay, intellectual disability, seizures, hypotonia, and autism (van Kuilenburg et al. 2007; van Kuilenburg et al. 2010; Yeung et al. 2013). Other clinical features include dysmorphic features, failure to thrive, and gastrointestinal problems. Phenotypic variability is common, and some patients with DHP deficiency may even by asymptomatic (van Kuilenburg et al. 2010).
Irrespective of manifestation of symptoms, DHP-deficient patients are vulnerable to a potentially life-threatening toxic reaction to the fluoropyrimidine type of anti-cancer agents such as 5-fluorouracil (5-FU). Dihydropyrimidinase (DHP) is the second enzyme in the catabolism of 5-fluorouracil (5FU). Symptoms included stomatitis, leukopenia, thrombocytopenia, hair loss, diarrhea, fever, marked weight loss, cerebellar ataxia, and neurologic symptoms (Fidlerova et al. 2012). Although DHP deficiency is inherited in an autosomal recessive manner, 5-FU toxicity has been widely found in individuals with one mutated allele of the DYPS gene (Fidlerova et al. 2010; van Kuilenburg et al. 2003; Thomas et al. 2007). Since genotype-phenotype correlations are yet unclear, routine DPH testing to anticipate 5-FU associated toxicities is still lacking in consensus (Ciccolini et al. 2010; van Kuilenburg 2006; Yen and McLeod 2007). Therefore, this testing is NOT for complete risk evaluation of DPYS-associated fluoropyrimidine toxicities.
DHP deficiency is an autosomal recessive disorder caused by pathogenic variants in the DPYS gene, which has 9 coding exons that encode the dihydropyrimidinase protein. The native enzyme is a tetramer with molecular weight of 217 kDa consisting of four subunits of 54 kDa each (Webster et al. 2014). DPH is a metalloenzyme containing one zinc atom in each subunit. In the largest study of DHP deficient patients, ~70% of pathogenic variants were located in exons 5-8 (van Kuilenburg et al. 2010). The majority of pathogenic variants are missense; however, nonsense and small deletions/insertions have also been reported (Human Gene Mutation Database).
Testing involves PCR amplification from genomic DNA and bidirectional Sanger sequencing of the 9 coding exons in the DPYS gene and ~20bp of adjacent noncoding sequences. This testing strategy will reveal coding sequence changes, splice site mutations and small insertions or deletions in the DPYS gene, but will not detect large duplications or deletions of the DPYS locus. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Indications for Test
Candidates for this test are patients with elevated levels of dihydropyrimidines in urine, plasma, and CSF. Testing is also indicated for family members of patients who have known DPYS pathogenic variants.
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- Genetic Counselor Team - firstname.lastname@example.org
- McKenna Kyriss, PhD - email@example.com
- Ciccolini J, Gross E, Dahan L, Lacarelle B, Mercier C. 2010. Routine dihydropyrimidine dehydrogenase testing for anticipating 5-fluorouracil-related severe toxicities: hype or hope? Clin Colorectal Cancer 9: 224–228. PubMed ID: 20920994
- Fidlerova J, Kleiblova P, Bilek M, Kormunda S, Formankova Z, Novotny J, Kleibl Z. 2010. Contribution of dihydropyrimidinase gene alterations to the development of serious toxicity in fluoropyrimidine-treated cancer patients. Cancer Chemother. Pharmacol. 65: 661–669. PubMed ID: 19649633
- Fidlerova J, Kleiblova P, Kormunda S, Novotny J, Kleibl Z. 2012. Contribution of the β-ureidopropionase (UPB1) gene alterations to the development of fluoropyrimidine-related toxicity. Pharmacol Rep 64: 1234–1242. PubMed ID: 23238479
- Human Gene Mutation Database (Bio-base).
- Thomas HR, Ezzeldin HH, Guarcello V, Mattison LK, Fridley BL, Diasio RB. 2007. Genetic regulation of dihydropyrimidinase and its possible implication in altered uracil catabolism. Pharmacogenet. Genomics 17: 973–987. PubMed ID: 18075467
- van Kuilenburg ABP, Dobritzsch D, Meijer J, Meinsma R, Benoist J-F, Assmann B, Schubert S, Hoffmann GF, Duran M, Vries van MC de, Kurlemann G, Eyskens FJM, Greed L, Sass JO, Schwab KO, Sewell AC, Walter J, Hahn A, Zoetekouw L, Ribes A, Lind S, Hennekam RC. 2010. Dihydropyrimidinase deficiency: Phenotype, genotype and structural consequences in 17 patients. Biochim. Biophys. Acta 1802: 639–648. PubMed ID: 20362666
- van Kuilenburg ABP, Meijer J, Dobritzsch D, Meinsma R, Duran M, Lohkamp B, Zoetekouw L, Abeling NGGM, Tinteren HLG van, Bosch AM. 2007. Clinical, biochemical and genetic findings in two siblings with a dihydropyrimidinase deficiency. Mol. Genet. Metab. 91: 157–164. PubMed ID: 17383919
- van Kuilenburg ABP, Meinsma R, Zonnenberg BA, Zoetekouw L, Baas F, Matsuda K, Tamaki N, Gennip AH van. 2003. Dihydropyrimidinase Deficiency and Severe 5-Fluorouracil Toxicity. Clin Cancer Res 9: 4363–4367. PubMed ID: 14555507
- van Kuilenburg ABP. 2006. Screening for dihydropyrimidine dehydrogenase deficiency: to do or not to do, that’s the question. Cancer Invest. 24: 215–217. PubMed ID: 16537192
- Webster, Dianne R., et al. "Hereditary Orotic Aciduria and Other Disorders of Pyrimidine Metabolism." . Eds. David Valle, et al. New York, NY: McGraw-Hill, 2014. n. pag. OMMBID. Web. 26 May 2015
- Yen JL, McLeod HL. 2007. Should DPD analysis be required prior to prescribing fluoropyrimidines? Eur. J. Cancer 43: 1011-1016. PubMed ID: 17350823
- Yeung CW, Yau MM, Ma CK, Siu TS, Tam S, Lam CW. 2013. Diagnosis of dihydropyrimidinase deficiency in a Chinese boy with dihydropyrimidinuria. Hong Kong Med J 19: 272–275. PubMed ID: 23732435
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
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- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.