Phenylalanine Hydroxylase Deficiency via the PAH Gene

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.

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).

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.

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).