Hyperphenylalaninemia/Phenylalanine Hydroxylase Deficiency via the PAH Gene

Newborn screening (NBS) tests are performed soon after birth with the goal of identifying individuals that may be affected by certain disorders before disease-related disability or death occurs. Appropriate medical management beginning early in life can prevent all or many symptoms in the affected individuals (Watson et al. 2006. PubMed ID: 16783161; https://www.cdc.gov/newbornscreening/). While NBS is required within all states and territories in the United States, individual state or territory public health departments determine which conditions are included on the NBS panel (https://www.babysfirsttest.org/newborn-screening/states). NBS protocols outside of the United States vary from country to country. At a minimum, all core conditions on the Recommended Uniform Screening Panel (RUSP) should be included on NBS panels within the United States. In addition, NBS testing may also include secondary conditions, which are disorders that can be detected as part of the differential diagnosis of a core condition (https://www.hrsa.gov/advisory-committees/heritable-disorders/rusp). Following an abnormal NBS result, follow up diagnostic testing is indicated. Such testing may include biochemical methodologies (for example, urine organic acid analysis or plasma acylcarnitine analysis), enzyme assays, and/or molecular genetic testing.

This test, or the hyperphenylalaninemia panel, are appropriate for individuals with NBS results showing elevated phenylalanine which can indicate a deficiency of the phenylalanine hydroxylase (PAH) enzyme. PAH deficiency, or phenylketonuria, is a core condition on the RUSP. 

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 phenylalanine levels (Guldberg et al. PubMed ID: 9634518; Regier and Greene 2017. PubMed ID: 2030167; Camp et al. 2014. PubMed ID: 24667081). 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. PubMed ID: 24667081):

- CLASSICAL 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) (Regier and Greene 2017. PubMed ID: 2030167; Camp et al. 2014. PubMed ID: 24667081). In untreated individuals classified with mild, moderate, or classical PKU, the high levels of phenylalanine interfere with normal brain development and can lead to profound intellectual disability, microcephaly, epilepsy, and behavioral issues (Regier and Greene 2017. PubMed ID: 2030167). 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. PubMed ID: 24667081; Vockley et al. 2014. PubMed ID: 24385074). 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 phenylalanine level of <360μmol phenylalanine/L (Camp et al. 2014. PubMed ID: 24667081; 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) (Regier and Greene 2017. PubMed ID: 2030167; Vockley et al. 2014. PubMed ID: 24385074). 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 (Regier and Greene 2017. PubMed ID: 2030167). PAH deficiency is particularly common in the White population, with an overall occurrence of roughly 1/10,000 live births (Vockley et al. 2014. PubMed ID: 24385074).

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 (Regier and Greene 2017. PubMed ID: 2030167; 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. PubMed ID: 26919687).

Some correlations have been made between genotype and phenotype (Kayaalp et al. 1997. PubMed ID: 9399896). In general, null variants 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 (Zurfluh et al. 2008. PubMed ID: 17935162; Camp et al. 2014. PubMed ID: 24667081). Certain pathogenic variants have been reported to be more common in particular populations (Blau. 2016. PubMed ID: 26919687). In a study including patients of several different nationalities, the most commonly reported variants were the missense variants p.Arg261Gln, p.Ala403Val, p.Arg408Trp, p.Tyr414Cys, and the splice variants c.1066-11G>A and c.1315+1G>A (Zurfluh et al. 2008. PubMed ID: 17935162).

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 approximately 2% of HPA cases (Regier and Greene 2017. PubMed ID: 2030167). 

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 (Regier and Greene 2017. PubMed ID: 2030167; Hillert et al. 2020. PubMed ID: 32668217). Literature results suggest that gross deletions or duplications may account for up to ~3% of PAH pathogenic variants (Gable et al. 2003. PubMed ID: 12655547; Kozak et al. 2006. PubMed ID: 16931086). In addition, up to 2% of cases of hyperphenylalaninemia are due not to PAH deficiency but rather to defects in tetrahydrobiopterin metabolism (Regier and Greene 2017. PubMed ID: 2030167; Blau. 2016. PubMed ID: 26919687).

This test provides full coverage of all coding exons of the PAH gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as >20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).