Hyperphenylalaninemia (HPA) and phenylketonuria (PKU) are variants of the same genetic condition whereby activity of liver enzyme, phenylalanine hydroxylase (PAH), is reduced.[[ 1]] This results in an inability to metabolise the amino acid phenylalanine (Phe).[[1]] Phe accumulates in the blood and tissues and becomes neurotoxic. In PKU, PAH activity is severely reduced or absent, whereas in HPA some activity is retained.[[ 1–2]]

When untreated, PKU results in intellectual disability, microcephaly, epilepsy, and psychiatric and behaviour problems.[[ 1–2]] Since the introduction of newborn screening and subsequent early treatment, this severe phenotype is rarely seen. However, even with treatment individuals with PKU have IQ scores 5–15 points lower than their unaffected siblings and parents (though within normal range) and show executive deficits, behavioural and psychological problems, and poorer quality of life (QoL).[[3–6]]

In contrast, individuals with HPA are thought to have normal neuropsychological function and typically receive no active follow-up.[[7–9]] However, early research supporting this approach focused on global intelligence, rather than the more subtle executive deficits seen in early treated PKU.[[3]] In a 2005 study comparing 35 children with HPA, 37 children with PKU, and 29 healthy controls, HPA participants produced significantly worse executive scores than controls.[[10]] Thus, further studies are still needed to safely exclude cognitive impairment and especially executive impairment in HPA, and to establish what Phe levels require dietary treatment. Considering cognition, executive function, and QoL, this study sought to add to the evidence by comparing children with HPA to age and gender matches with PKU and healthy controls.

Method

Participants

Three groups of children aged 6 to 16 years were recruited from across New Zealand—a group with HPA, a group with PKU, and a group of healthy controls. Groups were matched for age (+/-2 years), gender and ethnicity. In New Zealand, since the 1990s, infants with day two Phe levels exceeding 400umol/L at confirmatory testing (Guthrie card blood spot) have been classified as having PKU. Treatment consists of strict dietary protein restriction, amino acid supplementation, and regular monitoring of blood-Phe levels. Individuals with levels between 150 and 400umol/L at confirmatory testing are classified as having HPA and receive no long-term follow-up. Females are advised of the need for possible dietary treatment during pregnancy, where levels over 300umol/L can cause foetal damage. Of all cases of HPA identified between 2007 and 2014 in New Zealand, 19.2% were classified HPA (1:87,726 births), and 80.8% PKU (1:20,887 births).

Group demographic and metabolic characteristics appear in Table 1. All participants were aged between 6 and 15 and all were female. While we sought to recruit males and females aged 6 to 16 there were no males with HPA and no 16-year-olds eligible to complete the study. Within each group, five participants self-identified as New Zealand European/Pākehā, and one as Māori. Average blood Phe at birth was 281.50umol/L (SD=83.38umol/L) for the six HPA participants, and 1,248.00umol/L (SD=541.83umol/L) for the six PKU participants. As expected, levels were significantly higher amongst PKU participants. No significant demographic differences were found between groups.

Measures

Initial and historical blood Phe levels for HPA and PKU participants were obtained from clinical records. Phe was also measured on the day of testing. All participants completed a test battery assessing intelligence, achievement, executive functioning, and processing speed. Pen and paper questionnaires were completed by participants and a parent/guardian to produce a behavioural profile. Health-related QoL life was also examined, with PKU participants additionally completing a questionnaire about PKU-related QoL.

Cognitive tests

Wechsler Intelligence Scale for Children, 5th Edition, Australia and New Zealand (WISC-V)

The WISC-V assesses cognitive ability in children aged 6 to 16.11 Internal consistency for the subtests ranges from 0.74 to 0.95, and for the Primary Indices, from 0.86 to 0.96. Subtest scale scores and index scores, including the Full Scale Intelligence Quotient (FSIQ), were used in the analysis.

Wechsler Individual Achievement Test, 2nd Edition, Australian Abbreviated (WIAT-II-A)[[12]]

An abbreviated version of the WIAT-II ability measure, the WIAT-II-A Australian contains three subtests: Word Reading, Numerical Operations, and Spelling. It has demonstrated adequate reliability and validity. Subtest standard scores and the overall composite score were used in the analysis.

Trail Making Test for children (TMT)

The TMT is a simple, two-part “connect-the-dots” task.[[13]] Part A provides an indication of basic speed of processing, while Part B places demand on sequencing and ability to shift set. The children’s version published by Reitan shows good ecological validity and adequate reliability.[[13]] Z-scores were calculated using age-related normative data for analysis.[[14–15]]

Oral fluency

The oral fluency test is a short test of verbal, executive and speed of functioning. Both semantic (animals) and phonemic (FAS) fluency are assessed.[[16]] Test-retest correlations are high (>0.70) for both tasks, with the phonemic fluency task additionally showing good internal consistency.[[17]] Z-scores were calculated using age-related normative data for analysis.[[14,17–18]]

Contingency Naming Test (CNT)

The CNT measures processing speed, attention shift, and response inhibition in children.[[19]] Participants complete two baseline tasks, then two switching tasks evaluating rapid memory retrieval, inhibition and set shifting. The CNT is sensitive to brain maturation and damage, including that resulting from PKU. Z-scores for time taken and efficiency were included in the analysis.

Questionnaires

Conners Comprehensive Behavior Rating Scales (CBRS)

These assess a range of behavioural, emotional, social, and academic issues in school aged youth.[[20]] The CBRS demonstrates good test‐retest reliability, internal consistency and discriminative validity between different diagnoses.

Behavior Rating Inventory of Executive Function, 2nd Edition (BRIEF2)

These scales assess executive behaviours in children and adolescents.[[21]] The scales have shown good test-retest reliability.

Pediatric Quality of Life (PedsQL) Inventory

This measures functioning and health-related QoL in healthy children and those with health conditions across four domains: physical, emotional, social, and school.[[22]] The PedsQL has demonstrated fair reliability and construct validity.

Phenylketonuria-Quality of Life (PKU-QoL) Questionnaires

These assess the physical, emotional, and social impacts of PKU and its treatment on individuals with PKU and their families. Validity and reliability estimates are fair to good.[[23]]

Procedure

This study was approved by the Health and Disability Ethics Committee (HDEC; 17/CEN/272). Ten individuals with HPA were identified and contacted by the National Metabolic Service. One male was excluded due to severe autism, two had moved overseas, and another could not be contacted. The remaining six agreed to participate and were sent consent forms and questionnaires to complete prior to testing. Once an individual with HPA had completed the study, an age and gender matched individual with PKU was identified. For PKU participants, the questionnaire pack additionally included the PKU-QoL forms. Where possible, healthy controls were nominated by the family of participants, or otherwise recruited via word of mouth. Assessment of controls was the same, with the exception of not requiring a Phe level.

Testing took place at the hospital, university, outpatient metabolic clinic, or the child’s home or school. All tests were completed in a single session using a standardised procedure. Participants completed the WISC-V, followed by the executive tasks (oral fluency, TMT, CNT), and then the WIAT-II-A. Breaks were offered as and when required. The total duration of testing was between 85 and 160 minutes. Participants received a $40 supermarket voucher as a thank you for their participation. Parent report data were missing for two participants (one control, one PKU) whose parents did not respond to a third contact attempt.

All tests were scored in accordance with standardised procedures. Anonymised data was exported into SPSS 26.0 for analysis. Independent sample t-tests and Chi-squared tests were used to determine group differences in terms of demographic factors. Multivariate analyses of variance (MANOVA) were conducted to identify differences between the groups across outcome measures. Pearson’s bivariate correlations were generated to examine the relationship between performance and Phe levels for the HPA and PKU groups only.

Results

Cognitive functioning

Table 2 presents group performance on the WISC-V and WIAT-II-A. Mean scores for all three groups fell within the average range. Two MANOVAs were conducted with group (HPA, PKU, control) as the grouping variable and performance across subtests and indices of the WISC-V; and then the WIAT-II-A, as dependent variables. No significant effect of group on WISC-V [F(30,4)=2.495; p=0.327] or WIAT-II-A [F(8,24)=0.606; p=0.764] performance was identified.

Group mean Z-scores across executive measures generally fell between -1 and 1 (oral fluency being the exception; see Table 3). All three groups performed similarly on the CNT simple naming (Trial 1), however, as demand on shifting capacity increased (Trials 3–4), more frequent errors and slower speed of processing were observed in HPA and PKU groups relative to controls. The HPA group produced time and efficiency Z-scores more than 1 SD below the normative mean for Trial 4 and for the total score (Trials 1–4). A MANOVA with time and efficiency scores as dependent variables did not identify any significant effect of group. Similarly, for the TMT, HPA and PKU participants were slower to complete both Parts A and B than were controls. Group mean scores for phonemic fluency (FAS) were generally equivalent (+/-2 responses). On semantic fluency, however, the HPA group produced fewer correct responses than the control and PKU groups. A second MANOVA with scores on the phonemic and semantic fluency tasks, and Parts A and B of the TMT as dependent variables similarly did not identify any significant effect of group.

Behavioural functioning

Conners CBRS

Group mean scores on the CBRS are presented in Table 4. Rates of behavioural and emotional difficulties were highest amongst the PKU group, with mean scores falling in the clinically elevated range on four of the parent-reported scales. The control group produced the lowest scores (with the exception of the Math scale), with the HPA group scoring somewhere in the middle. Given the large number of scores produced by the CBRS, content and symptom scales for the self- and parent-report forms were analysed using separate MANOVA. These analyses did not identify any significant effect of group.

A frequency table was produced to examine the presence of clinically significant scores within the sample (see Table 5). Parents of PKU children endorsed “Very Elevated” difficulties on 15.6% of the total subscale scores, exceeding that expected based on normative data (i.e., expected in 2% of normal population). The frequency of “Very Elevated” scores amongst HPA participants was much lower, but still double that expected (4.2%), while control participants rates (2.5%) were proportionate to the normal population.

BRIEF-2

Table 6 shows mean group scale and index scores for the BRIEF-2. Reports of executive difficulties were most frequent amongst PKU participants and least frequent amongst controls. A MANOVA with index scores as dependent variables revealed a significant effect of group [F(2,12)=32.24; p=0.030], with self-reported Shift and Emotion Regulation Index scores contributing significantly to the difference. Post hoc analyses indicate that PKU participants scored significantly higher than controls (p=0.044) on the shift scale; and significantly higher than both control (p=0.003) and HPA groups (p=0.008) on the Emotion Regulation Index.

Quality of life

PedsQL

Table 7 presents group mean QoL scores on self- and parent-reported PedsQL. The PKU group generally reported the lowest quality of life, with differences in school functioning being most pronounced. A MANOVA examining the impact of group on the PedsQL fell short of statistical significance [F(16,10)=1.97; p=0.140].

PKU-QoL

PKU participants self-reported severe tiredness, stomach aches, concentration difficulties and moodiness, as well as moderately severe headaches, slowed thinking and irritability. Parents endorsed similar difficulties, but less severely than participants themselves. Participants and parents emphasised the emotional burden of treatment. Participants reported strongly disliking the taste of supplements, low food enjoyment, extreme food temptation, difficulties with adherence, and guilt for non-adherence. Parents endorsed being greatly impacted by their child’s anxiety related to blood testing and elevated Phe levels, difficulties ensuring adherence, and guilt related to non-adherence. Adherence to supplements was identified as more challenging than adherence to the low protein diet.

Correlations with Phe levels

Due to the small sample size, the following findings need to be viewed with caution. Bivariate correlations were generated between all outcomes and Phe levels (birth and current). There was only one significant correlation with the cognitive measures; birth Phe was negatively correlated with WIAT-II-A numerical operations performance (r=-0.58; p=0.05).

In regards QoL, self-reported school functioning was significantly correlated to birth and current Phe (r=-0.64, p=0.04; and r=-0.62, p=0.04, respectively) as was parent-reported school functioning and current Phe (r=-0.70; p=0.02). Correlations with the PKU-QoL were also conducted but should be interpreted with additional caution given the measures were administered only to PKU participants. Birth Phe was significantly positively correlated with self-reported tiredness, lack of concentration, and overall health status. Higher current Phe was significantly positively correlated with parent reported aggression, social impact, and difficulties with adherence related to the strict diet and taste of formula.

In regards behavioural difficulties, birth Phe was significantly positively correlated with self-reported conduct disorder and parent reported perfectionistic and compulsive behaviour. Current Phe was significantly positively correlated with parent reported upsetting thoughts, OCD, ADHD –predominantly inattentive, major depressive episode, and manic episode. There was only one significant relationship with the BRIEF-2, whereby participants with higher current Phe self-reported greater self-monitor difficulties (r=0.86; p=0.03).

View Table 1–7.

Discussion

All three groups performed in the normal range across the ability and achievement measures indicating intelligence was not significantly compromised in HPA or PKU groups compared to population norms or matched controls. This is consistent with the early literature on HPA, but in contrast with more recent findings.[[9–10,24]] Previous research on PKU has generally found IQ within the average range, but marginally lower than the general population.[[6,25–26]] In this study, the PKU group obtained a mean IQ score one point higher than the mean for controls, and four points higher than the HPA group. We hypothesised that working memory and processing speed difficulties would be present in both HPA and PKU groups, given previous studies of PKU and the vulnerability of these functions to neurological insult.[[4,26–27]] However, this was not supported.

Though caution is needed given the small size of the sample, our findings do support the effectiveness of the existing programme in preventing cognitive deficits. For HPA participants, we did not find any evidence of deficits warranting active management, and for early treated PKU participants, we observed a normal cognitive profile.

In regards behaviour, while group mean scores did not differ significantly, the proportion of “Very Elevated” scores was much higher in the PKU group, occurring at a rate nearly eight times that of the normal population, especially with regards to externalising behaviour and anxiety. Executive behaviour difficulties were also more commonly self-reported by the PKU group, including significantly higher rates of problems shifting set compared to controls and poorer emotional regulation compared to both HPA and controls. Higher Phe levels were linked with conduct disordered behaviour, attention and self-monitoring difficulties, upsetting thoughts, and poorer school-related QoL.

Few studies have assessed QoL in HPA, whilst studies of PKU participants have produced contradictory, yet modest results. Though not significantly different, QoL was poorest amongst PKU participants and best amongst controls and was likely related to the burden of dietary treatment.[[28–29]] The weighing and monitoring of dietary protein is time intensive, and difficulty explaining the diet to others limits supports for parents as well as child social inclusion (e.g., invitations to sleepovers). Further, complaints related to the acrid taste of formula, hunger, food refusal, and temptation to eat restricted foods increase parent–child conflict, guilt and emotional strain.

Strengths and limitations

This is the first study to assess New Zealanders diagnosed with HPA. Assessment was broad, including cognition, executive abilities, behaviour and QoL, and spanned both self and parent reports. This study adds to the literature on QoL assessed using the PKU-QoL. Data from the PKU-QoL elucidated the link between strict dietary adherence and parental burden.

As with any rare disease, the biggest challenge was sample size. Small sample size greatly compromised power and increased the risk of type I error. Use of the CBRS, which has a minimum administration age (11 years), further reduced sample size. Males were not represented in this study (only two males were diagnosed with HPA within the period). Gender has not been found to impact outcomes in HPA.[[24–25]]

Clinical implications

While the findings suggest cognition is preserved for both HPA and PKU participants within the current model of care, further examination of the impact of PKU, and especially PKU treatment, on behavioural and QoL outcomes is warranted.

With PKU, it is recognised that there is no “one size fits all” target Phe level. While there are international guidelines re recommended Phe levels, due to the complexity and difficulty of the diet these may not be easily achieved in some patients. The younger developing brain is more vulnerable than the older brain, and so a more intensive diet is desired for younger children to ensure Phe levels remain in a safe range. However, Phe targets may be slightly relaxed for older children, especially those who struggle with adherence due to the adverse impacts of this on individual and family QoL.

With HPA, less rigorous follow-up including monitoring of Phe levels and clinical review may be indicated, without routine treatment as per PKU. Formalising a pathway for routine monitoring would also benefit identification and appropriate treatment of women at risk of maternal HPA.[[31]]

Conclusion

At this stage, there is not sufficient evidence to warrant dietary treatment of HPA except during pregnancy but there is also insufficient evidence to safely exclude the presence of cognitive impairment. Establishing conclusive, evidence-based recommendations for the management of HPA will likely require an international consensus statement regarding HPA as well as collaborative research strategies that pool resources and provide shared knowledge platforms.[[16]]