6-Pyruvoyltetrahydropterin Syntase (PTPS) Deficiency via the PTS Gene
- Summary and Pricing
- Clinical Features and Genetics
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In a total of 19 patients from different families with a clinical diagnosis of 6-pyruvoyltetrahydropterin syntase (PTPS) deficiency, pathogenic variants were detected by sequencing on 35 out of 38 alleles, suggesting an overall detection rate of ~92% (Leuzzi et al. 2010). Analytical sensitivity should also be high because nearly all reported pathogenic variants thus far are detectable by sequencing.
Hyperphenylalaninemias due to tetrahydrobiopterin (BH4) deficiency are a result of a disruption in phenylalanine homeostasis and dopamine and serotonin biosynthesis. These disorders are caused by pathogenic variants in genes encoding enzymes involved in the biosynthesis or regeneration of BH4. The phenylalanine, tyrosine, and tryptophan hydroxylases all require BH4 as a cofactor, and lack of this cofactor results in secondary hyperphenylalaninemia and depletion of the neurotransmitters dopamine and serotonin. Early detection and treatment can reduce or prevent neurologic symptoms (Blau et al. 2014).
The most common of the tetrahydrobiopterin deficiencies is 6-pyruvoyltetrahydropterin syntase (PTPS) deficiency, which accounts for approximately 60% of all BH4 deficiencies (Blau et al. 2014). The phenotype of PTPS deficient patients is classified as severe or mild. The severe form is observed in approximately 80% of patients, and such patients develop neurologic symptoms including psychomotor retardation, delayed development, tonal abnormalities, seizures, and dystonia. Patients may also present with abnormal thermogenesis, microcephaly, swallowing difficulties and hypersalivation (Blau et al. 2014). Biochemically, patients classified with severe PTPS deficiency are found to have abnormal levels of CSF neurotransmitters (Leuzzi et al. 2010; Blau et al. 2014). PTPS deficient patients diagnosed with the mild form are generally found to be neurologically normal and have normal neurotransmitter levels, although transient tonal abnormalities, sleeping difficulties and other neurological problems are occasionally observed (Leuzzi et al. 2010; Blau et al. 2014).
PTPS deficient patients are usually detected via newborn screening due to hyperphenylalaninemia. To distinguish PTPS deficiency from PAH deficiency and other causes of hyperphenylalaninemia, additional studies must be performed, such as a urinary pterin profile, measurement of DHPR enzyme activity, a BH4 loading test, analysis of pterins, folates and neurotransmitters in CSF, and direct PTPS enzyme activity measurement (Ye et al. 2013; Blau et al. 2014). Early diagnosis and treatment is imperative. The neurological outcome, particularly in patients with severe PTPS deficiency, has been shown to be greatly improved if treatment is started before the second month of life (Leuzzi et al. 2010). A phenylalanine-restricted diet is not sufficient to control symptoms in these patients, and many must also be treated with BH4 and neurotransmitter precursor supplementation, although this does not help all patients (Oppliger et al. 1997; Blau et al. 2014).
6-Pyruvoyltetrahydropterin syntase deficiency is inherited in an autosomal recessive manner. The PTS gene (chromosome 11q22.3, 6 exons) encodes the PTPS enzyme which is involved in the de novo biosynthesis of tetrahydrobiopterin. More specifically, PTPS converts dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin (Blau et al. 2014). To date, over 80 pathogenic variants have been reported in the PTS gene. The majority of causative variants are missense, although nonsense, splicing, small deletions and insertions, and one gross deletion have all been reported (Human Gene Mutation Database). Pathogenic variants are spread evenly along the coding sequence. Other inborn errors of BH4 metabolism can present with a similar clinical course and involve dihydropteridine reductase (QDPR gene), GTP cyclohydrolase I (GCH1 gene), and pterin-4α-carbinalamine dehydratase (PCBD1 gene) (Blau et al. 2014, Trujillano et al. 2014).
This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the PTS gene plus ~20 bp of flanking non-coding DNA on each side. 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
Newborn screening indicative of hyperpheylalaninemia. Patients with decreased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) in CSF, increased urinary neopterin and decreased biopterin, and a high neopterin/biopterin ratio are good candidates for this test, as are individuals that exhibit clinical symptoms of tetrahydrobiopterin deficiency and family members of patients known to have PTS variants. We will also sequence the PTS gene to determine carrier status.
|Official Gene Symbol||OMIM ID|
|Dihydropteridine Reductase (DHPR) Deficiency via the QDPR Gene|
|Hyperphenylalaninemia Sequencing Panel|
|Pterin-4 alpha-Carbinolamine Dehydratase (PCD) Deficiency via the PCBD1 Gene|
|Sepiapterin Reductase (SR) Deficiency via the SPR Gene|
- Genetic Counselor Team - firstname.lastname@example.org
- McKenna Kyriss, PhD - email@example.com
- Blau N.et al. 2014. Disorders of Tetrahydrobiopterin and Related Biogenic Amines. Online Metabolic & Molecular Bases of Inherited Disease, New York, NY: McGraw-Hill.
- Human Gene Mutation Database (Bio-base).
- Leuzzi V. et al. 2010. Clinical Genetics. 77: 249-57. PubMed ID: 20059486
- Oppliger T. et al. 1997. Human Mutation. 10: 25-35. PubMed ID: 9222757
- Trujillano D. et al. 2014. European Journal of Human Genetics : Ejhg. 22: 528-34. PubMed ID: 23942198
- Ye J. et al. 2013. Journal of Inherited Metabolic Disease. 36: 893-901. PubMed ID: 23138986
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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- 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.
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(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.
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- Culture and send at least two T25 flasks of confluent cells.
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- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
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- We strongly recommend maintaining a local back-up culture. We do not culture cells.