Phenylalanine Hydroxylase Deficiency via the PAH Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
530 PAH$810.00 81406 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Based on the literature, we estimate that sequencing will detect at least one likely causative variant in >99% of hyperphenylalaninemia patients and two likely causative variants in >90% of patients (Mitchell 2013). Also, up to 2% of cases of hyperphenylalaninemia are due not to PAH Deficiency, but rather to defects in tetrahydrobiopterin metabolism (Mitchell 2013; Blau 2016).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PAH$990.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Kozak et al. (2006) reported on 59 out of a total of 1,042 biochemically diagnosed PAH Deficient patients that were found to harbor zero or one pathogenic allele identified by sequencing. Out of these 59 incompletely genetically diagnosed individuals, 31 exonic deletions were identified (Kozak et al. 2006). Similarly, Gable et al. (2003) reported on 38 incompletely genetically diagnosed individuals out of a total of 1,010 biochemically diagnosed PAH Deficient patients. In their study, exonic deletions or duplications were detected on 9 alleles. Overall, these results suggest that gross deletions or duplications may account for up to ~3% of PAH pathogenic variants.

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Clinical Features

Phenylalanine Hydroxylase (PAH) Deficiency is a defect in the enzymatic conversion of phenylalanine to tyrosine. If uncorrected by diet, PAH Deficiency results in decreased dietary tolerance of phenylalanine and increased blood phenylalanine levels. There are several classifications of PAH deficiency, each defined by pre-treatment blood Phe levels (Guldberg et al. 1998; Mitchell 2013; Camp et al. 2014). The current classification system divides PAH Deficiency into five groups ranging from classical phenylketonuria (PKU), which is the most severe, to hyperphenlyalaninemia (HPA), which is the most mild. Categories are defined based on patient blood concentration levels of phenylalanine prior to treatment (Camp et al. 2014): 

        - CLASSICAL PHENYLKETONURIA (PKU): >1200 μmol phenylalanine/L 

        - MODERATE PKU: 900-1200 μmol phenylalanine/L

        - MILD PKU: 600-900 μmol phenylalanine/L

        - MILD HPA-GRAY ZONE: 360-600 μmol phenylalanine/L

        - MILD HPA-NT*: 120-360 μmol phenylalanine/L

        * NT = not requiring treatment 

Treatment of individuals with PAH deficiency is generally done by restricting dietary phenylalanine intake. Some individuals are also found to be responsive to supplementation with tetrahydrobiopterin (BH4) (Mitchell 2013; Camp et al. 2014). In untreated individuals classified with mild, moderate or classic PKU, the high levels of phenylalanine interfere with normal brain development and can lead to profound mental retardation, microcephaly, epilepsy and behavioral issues (Mitchell 2013). There is some disagreement about whether or not to treat individuals that reside in the mild HPA-gray zone and mild HPA-NT groups (Camp et al. 2014; Vockley et al. 2014). It is critically important, though, that women with PAH Deficiency carefully control their phenylalanine levels in the months before and during pregnancy, maintaining a blood Phe level of <360μmol phenylalanine/L (Camp et al. 2014; Donlon et al. 2014).  

Since the 1960’s, nearly all cases of PAH Deficiency in America, Canada and Western Europe have been detected by routine neonatal screening with Guthrie Cards, although modern detection is typically done via tandem mass spectrometry (MS/MS) (Mitchell 2013; Vockley et al. 2014). Incidence varies quite a bit based on the population, with a range of approximately ~1/2,600 in the Turkish population to ~1/200,000 in the Ashkenazi Jewish and Finnish populations (Mitchell 2013). PAH Deficiency is particularly common in the caucasian population, with an overall occurrence of roughly 1/10,000 live births (Vockley et al. 2014).


PAH Deficiency exhibits autosomal recessive inheritance, with genetic and non-genetic modifying factors. To date, nearly 1000 PAH causative variants have been reported (Human Gene Mutation Database; PAHvdb: Phenylalanine Hydroxylase Gene Locus-Specific Database). Causative variants are ~60% missense, ~15-20% frameshift, ~15% splicing, and ~5% nonsense. The remainder are gross deletions and duplications (Mitchell 2013; Human Gene Mutation Database). Causative variants are located throughout the length of the gene. Approximately three-fourths of patients reported in the PAHvdb are compound heterozygous for two pathogenic variants (Blau 2016).

Some correlations have been made between genotype and phenotype (Kayaalp et al. 1997). In general, null mutations and others that result in little to no residual protein activity are associated with more severe forms of PAH Deficiency, and are often less likely to be responsive to BH4 treatment (Zurflüh et al. 2008; Camp et al. 2014). Certain pathogenic variants have been reported to be more common in particular populations (Blau 2016); in a study including patients of several different nationalities, the most commonly reported variants were the missense variants R261Q, A403V, R408W, Y414C, and the splice variants c.1066-11G>A and c.1315+1G>A (Zurflüh et al. 2008).

Hyperphenylalaninemia can also be caused by defects in the tetrahydrobiopterin synthetic pathway. Such defects would be due to variants in the GCH1, PCBD1, PTS or QDPR genes. Tetrahydrobiopterin deficiencies are a rare cause of hyperphenylalaninemia, accounting for only approximately 2% of HPA cases (Mitchell 2013). More detail about these disorders can be found on the individual gene test pages.

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the PAH gene plus ~10 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

All individuals with PAH Deficiency or even modest hyperphenylalaninemia are candidates for this test. Individuals that exhibit clinical symptoms of PAH Deficiency, particularly if newborn screening was not performed for them, and family members of patients known to have PAH variants are also good candidates. We will also sequence the PAH gene to determine carrier status.


Official Gene Symbol OMIM ID
PAH 612349
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Phenylketonuria AR 261600


Genetic Counselors
  • Blau N. 2016. Human Mutation. 37: 508-15. PubMed ID: 26919687
  • Camp K.M. et al. 2014. Molecular Genetics and Metabolism. 112: 87-122. PubMed ID: 24667081
  • Donlon J. et al. 2014. Hyperphenylalaninemia: Phenylalanine Hydroxylase Deficiency. In: Valle D, Beaudet A.L., Vogelstein B, et al., editors. New York, NY: McGraw-Hill. OMMBID.
  • Gable M. et al. 2003. Human Mutation. 21: 379-86. PubMed ID: 12655547
  • Guldberg P. et al. 1998. American Journal of Human Genetics. 63: 71-9. PubMed ID: 9634518
  • Human Gene Mutation Database (Bio-base).
  • Kayaalp E. et al. 1997. American Journal of Human Genetics. 61: 1309-17. PubMed ID: 9399896
  • Kozak L. et al. 2006. Molecular Genetics and Metabolism. 89: 300-9. PubMed ID: 16931086
  • Mitchell J.J. 2013. Phenylalanine Hydroxylase Deficiency. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301677
  • PAHvdb: Phenylalanine Hydroxylase Gene Locus-Specific Database (
  • Vockley J. et al. 2014. Genetics in Medicine. 16: 188-200. PubMed ID: 24385074
  • Zurflüh M.R. et al. 2008. Human Mutation. 29: 167-75. PubMed ID: 17935162
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  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 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 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 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

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).

Analytical Limitations

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 10 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.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • 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.
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