Are Carriers Unaffected? A Literature Review of Metabolic and Health Outcomes among Genetic Carriers of Phenylketonuria

Phenylketonuria (PKU) is a rare metabolic genetic condition that results in reduced enzymatic functioning of the phenylalanine hydroxylase (PAH) pathway, which is involved in the metabolism of phenylalanine (Phe) into tyrosine (Tyr) [1]. Without interventions, individuals with PKU can develop Phe toxicity, causing severe neurological impairment and other health risks [2, 3]. Diagnosis of PKU is achieved through neonatal testing [3]. Until modern treatment options, foods that are high in protein such as eggs, meat, and milk needed to be highly restricted in the diet [4]. Other foods including bread and pasta have modified versions that are specifically low in Phe, allowing individuals with PKU to consume these altered foods [4]. Due to the strict food restrictions, PKU patients need to consume Phe-free formula that provides amino acids (especially Tyr), vitamins, and minerals so that these individuals are not lacking other required nutrients [4]. If individuals with PKU do not follow this strict diet, they are at risk of severe developmental delay, neurological deficits, and psychiatric symptoms among others [3, 5]. More recently, pharmacological treatments have allowed individuals with PKU more lenience from this strict diet [6, 7]. For example, sapropterin dihydrochloride has been approved by the Food and Drug Administration and is a synthetic form of tetrahydrobiopterin (BH₄) and has been shown to decrease Phe levels in individuals with PKU [6]. BH₄ is a cofactor required for proper function of the PAH enzyme [6]. Sapropterin thus augments the effects of BH₄ acting as a cofactor to the PAH enzyme and increasing its activity, subsequently lowering Phe levels [6]. Individuals with less severe forms of PKU and therefore more PAH enzymatic activity present tend to benefit more from this treatment option [6]. Pegvaliase is a newer pharmaceutical drug that acts as a substitute for the deficient PAH enzyme [8]. This treatment is recommended for patients with PKU who are already trying to manage their condition through dietary restrictions but still have elevated Phe levels [8].

PKU is an autosomal recessive condition; therefore, both parents of a child with PKU must carry a mutation of the PAH gene [1]. While being diagnosed with PKU is rare, affecting around 1 in every 10,000 individuals, carriers of PKU are more common, occurring in approximately 1 in every 50 individuals [5]. Individuals may have different genetic variants, which differ in the ability to tolerate and metabolize Phe [3]. PKU diagnosis is defined on a continuum; the most common and severe form is classical PAH deficiency which is diagnosed when blood Phe levels are greater than 1,200 μmol/L (20 mg/dL), less common is mild PAH deficiency defined by blood Phe levels between 600 and 1,200 μmol/L (10–20 mg/dL), and the least severe being hyperphenylalaninemia (HPA) with blood Phe levels falling between 120 and 600 μmol/L (2–10 mg/dL) [9, 10]. Those who are diagnosed with PKU are homozygous or compound heterozygous for a pathogenic variant, meaning they possess two pathogenic PAH variants [11]. The PAH gene is found on chromosome 12 and encodes the PAH protein [12]. The most common genetic variants include I65T, R261Q, G272*, R252W, R261*, R408W, IVS12 + 1G > A, Y414C, and IVS10-11G > A [12]. As recently as 2022, approximately 1,500 different variants of the PAH gene have been identified [12]. Individuals who only have one pathogenic variant are by definition heterozygotes and classified as carriers of PKU [11]. As such, they typically do not know they have a single pathogenic variant unless they have a biological child or parent with PKU or have previously undergone genetic testing [11].

While PKU is a relatively thoroughly studied disease, literature regarding PKU carriers is limited. Studies have found that carriers tend to have an elevated ratio of Phe to Tyr (tend to have higher Phe levels and lower Tyr levels than noncarriers) [13]. Relatedly, PKU carriers have demonstrated reduced PAH enzymatic activity compared to the general population [14, 15]. One study stated a 30% reduction in activity, while another study found that carriers may only have 7.3–10% residual PAH enzymatic activity (i.e., 90–92.7% reduction in activity) [14, 15]. While these studies were conducted in the 1970s, using older technology, the findings suggest that carriers show an intermediate level of PAH enzymatic activity (i.e., functioning somewhere between that of individuals with PKU and non-PKU carriers). It is possible that this could manifest clinically, highlighting the importance of further studying potential neurological or psychological impacts that PKU carriers may be experiencing. However, more current literature with updated protocols are required to confirm these results as these studies had major limitations including small sample sizes. The purpose of this review was to summarize the research conducted to date in order to gauge the possible metabolic and health impacts of being a carrier of PKU, while making recommendations for future research in this population.

While carriers of autosomal recessive inborn errors of metabolism are typically described as “unaffected,” it is hypothesized that a more accurate descriptor for some carriers of PKU may be “intermediately affected” (shown in Fig. 1) [16]. This condition is autosomal recessive whereby individuals with biallelic variants in the PAH gene who have PKU exhibit profound loss of PAH enzymatic functioning, but intriguingly, heterozygotes also exhibit loss of PAH enzymatic functioning – to a lesser extent than those with biallelic variants who have PKU [14]. An early study, published in 1975, analyzed liver biopsies and found that both individuals with PKU and heterozygotes for the PAH gene variant had less enzymatic PAH functioning compared to noncarriers [14]. This study found that individuals with PKU only had up to 5% of PAH enzymatic function [14]. It was compelling however to observe that in this study, heterozygotes or “unaffected” carriers only had about 7.3–10% residual PAH activity (compared to unaffected individuals with no PAH variants for PKU having 100% enzymatic functioning) [14]. This vastly defies the level of residual enzymatic activity that one might anticipate among carriers with only a single pathogenic variant (i.e., closer to 50%), but it is important to note that this study was published nearly 50 years ago and newer research with more robust methods has not been completed to confirm or refute this extreme observed decrease in enzymatic activity [17]. However, this work provided some of the earliest evidence that PKU carriers could be experiencing an intermediate phenotype (i.e., a phenotype that falls between that observed in noncarriers and that observed in individuals with PKU), at least metabolically, of reduced PAH enzymatic activity. Later, in 1982, another research team that studied diagnosed individuals with PKU additionally sampled two parents of the PKU children (carriers) [18]. It was found using needle liver biopsies that one of the parents had only 27% PAH enzyme activity, while the other had only 42% [18]. In addition, a breath test methodology has been established for in vivo PAH activity estimation, and a preliminary study using this methodology also found significantly lower PAH activity (specifically, significantly reduced conversion of L-Phe to carbon dioxide) in PKU carriers compared to healthy controls after Phe loading [19]. Thus, decreased PAH activity has repeatedly been found in PKU carriers; however, the degree of activity loss may vary depending on the individual [18]. Due to the presence of a null allele in carriers, up to 50% of normal enzymatic activity could be expected [17]. However the results of these studies demonstrated that an even greater impact on PAH enzymatic activity is being recorded in some individuals, compared to this 50% expected reduction [14, 18]. It is indeed plausible that different PAH variants could be impacting the severity of residual PAH enzyme activity. Of note, these studies were conducted over 40–50 years ago. Thus, the older methodologies used in this work may not be as reliable, and the lack of genetic sequencing could not confirm the carrier status of the participants (or the variants at play), further stressing the need for updated literature. Current research neglects this metabolic effect that has been documented in carriers, and to date an explanation for this greater than 50% reduction of enzymatic activity has not been thoroughly investigated. Further metabolic research in PKU carriers is needed to better understand these results.

Not long after these findings, a research team observed a decreased IQ in carriers compared to noncarriers (further described in neurological outcomes section of this review) and evaluated intracellular levels of Phe and Tyr in lymphocytes as an attempt to find a metabolic explanation [20]. Without any intervention, these researchers found significantly higher intracellular Phe (10.06 compared to 8.16 nmol/10⁶ cells in carriers and noncarriers, respectively) and Tyr (10.26 compared to 7.00 nmol/10⁶ cells in carriers and noncarriers, respectively) levels in lymphocytes of carriers compared to noncarriers [20]. Notably, the concentrations described are intracellular levels of Phe and Tyr, whereas most literature and clinical concentrations are based on blood plasma levels, explaining the discrepancy between these values and other reported concentrations. They hypothesized that plasma Phe levels would always be comparable between carriers and noncarriers, which was their justification for monitoring intracellular levels [21]. However, since these preliminary studies, Phe-loading trials conducted in the 1990s showed significantly higher plasma Phe levels in PKU carriers compared to noncarriers following Phe consumption [22‒24].

For example, in a study where researchers administered 33 mg/kg of Phe to each participant, 2 h after supplementation, plasma Phe levels were significantly higher in heterozygotes (220 μmol/L) compared to noncarriers (140 μmol/L) (shown in Fig. 2) [24]. While both the carriers and noncarriers surpassed the HPA diagnostic cutoff of 120 μmol/L, Phe levels in carriers were notably above this diagnostic criteria (noting however that diagnoses are not typically made following Phe loading) [10, 24]. For context, a 33 mg/kg meal for a 70 kg (154 lb) individual is approximately equivalent to the amount of Phe in a meal consisting of one, 175 g, chicken breast, 100 g of French fries, and a small side salad (shown in Fig. 2). Additionally, this study along with others have found that after Phe loading, heterozygotes (carriers) had significantly higher ratios of Phe:Tyr [22, 24]. More specifically, the control groups (noncarriers) tended to have higher Tyr levels following Phe consumption, indicating more efficient Phe metabolism compared to heterozygotes [22, 24]. Silva and colleagues gave participants a 100 mg/kg aspartame dose (high in the amino acid Phe) and evaluated plasma Phe and Tyr levels in the fasted state and 30 min after supplementation, and Phe levels in carriers were 102.71 μmol/L fasted and 110.91 μmol/L following Phe intake, while levels were 70.40 μmol/L and 80.83 μmol/L, respectively, in noncarriers [22]. In addition to the findings stated previously, they also found that in the fasted state (with no supplementation) PKU heterozygotes had higher Phe, lower Tyr, and thus higher ratio of Phe:Tyr than the noncarriers (ratio of 0.33 in carriers and 0.15 in noncarriers) [22]. Another study that investigated the effect of Phe consumption on metabolism, found that Phe levels increased in both carriers and noncarriers after consuming a high-protein meal with 85 μmol/kg body weight of aspartame rather than the high-protein meal alone [23]. Both the carriers and noncarriers experienced increased Phe levels after the high-protein meal alone and the high-protein meal with aspartame [23]. However, the PKU carriers had higher plasma Phe levels than the noncarriers at 1 h (approximately 140 μmol/L in carriers and 95 μmol/L in noncarriers) and 3-h postprandial (approximately 155 μmol/L in carriers and 90 μmol/L in noncarriers) [23]. While both the carriers and noncarriers experienced increases in Phe levels, the carriers notably surpassed the lower end of the HPA diagnostic criteria of 120 μmol/L [10, 23]. Aspartame is metabolized into aspartic acid, Phe, and ethanol (among other metabolites) [24]. Since Phe is a building block of aspartame as well as a byproduct of aspartame metabolism, this explains why higher Phe levels are observed following aspartame consumption in the carriers [24]. It is important to note that these studies were performed over 20 years ago. However, more recently, in 2015, similar results were discovered after administration of a 25 mg/kg dose of Phe to PKU carriers and noncarriers [25]. Once again, the carriers exhibited higher Phe levels, above that of HPA diagnosis criteria (208.84 μmol/L in carriers and 131.96 μmol/L in noncarriers 90 min after Phe dose), as well as lower Tyr levels and higher Phe/Tyr ratio than the noncarrier control group [25]. Andrade et al. [25] have demonstrated that even with newer and more robust methodologies, results of older literature are being replicated when investigating PKU carriers, specifically in terms of metabolite levels. It is important to note that HPA diagnosis criteria are assessed in a fasted state, generally in newborns or young children; thus, the Phe levels reported in these studies cannot be directly compared to diagnostic cutoffs. However, overall PKU carriers consistently have significantly higher Phe levels than noncarriers.

This finding has also been described in another study by a research team that administered an 8-oz beverage either containing aspartame or an unsweetened beverage (no aspartame) every hour over the course of 8 h to PKU carriers [26]. The carriers consuming aspartame had significantly higher Phe levels than the carriers drinking the unsweetened beverage [26]. More specifically, carriers consuming aspartame had increasing Phe levels after every beverage (every hour) until the 5th hour when the Phe levels plateaued. The highest mean value of Phe levels peaked at 16.5 ± 3.44 μmol/dL (165 ± 34.4 μmol/L) in carriers [26]. Overall, no matter the format of Phe delivery (through a high-protein meal, aspartame, or a pure Phe supplement), PKU carriers appear to consistently present with higher Phe levels than noncarriers during these acute-Phe-loading studies. To our knowledge, research has yet to evaluate the impact of chronic Phe consumption (e.g., from following specific dietary patterns such as a high-protein diet) among carriers or PKU. Future research should explore this topic.

Evidence that has been published more recently, which integrated whole-genome sequencing and metabolomics analyses, found that 33% of individuals with a single pathogenic variant in the PAH gene (PKU carriers) had increased Phe levels compared to standard Phe levels [27]. This finding further illustrates the variable pathogenicity of specific genetic variants and the probability that certain PKU carriers (perhaps those carrying variants for classic PKU) may experience an intermediate phenotype [27]. Higher Phe levels in adult PKU carriers have been continuously depicted in the research described above, most of which was conducted several decades ago in adult populations. However, in 2022, these results were also found in newborns [28]. It was found that newborns without a pathogenic variant of the PAH gene had blood Phe levels, collected through a heel prick, of 0.89 ± 0.31 mg/dL (53.4 ± 18.6 μmol/L) [28]. Whereas the newborns with one PAH variant (carriers) had blood Phe levels of 1.15 ± 0.34 mg/dL (69 ± 20.4 μmol/L) [28]. A blood Phe level of >2 mg/dL (>120 μmol/L) is required to be diagnosed with HPA or PKU; thus, PKU carriers generally go undetected even though they still express higher Phe levels than those who are unaffected [10, 28]. This is intriguing, especially considering that newborn screening (NBS) is typically completed within the first day of life, when nutritional intake is typically very limited, though neonates are catabolic in the first few days of life and Phe from muscle breakdown will contribute to Phe levels detected via NBS [29]. Current North American recommendations for individuals with PKU suggest that safe blood Phe levels should be between 120 and 360 μmol/L (for children and adults), and while PKU carriers seem to fall in this range, it is important to note that individuals with PKU still present with chronic health and neurological risks even when their PKU is well controlled [10, 13, 30‒32]. Overall, from a metabolic standpoint, PKU carriers consistently present with decreased enzymatic activity in the PAH pathway and altered Phe and Tyr levels.

Given the evidence of metabolic perturbations among carriers, researchers have hypothesized that these “unaffected” carriers may actually have unique cognitive and health risks compared to noncarriers. For example, early research has demonstrated that PKU carriers tend to have lower verbal IQs than unaffected noncarriers [21]. However, while this finding is interesting, it is not clear why the differences were only restricted to the verbal IQ scale, when patients with early-treated PKU do not tend to present with verbal perturbations [2, 3, 33]; further research in this area is needed. Thalhammer and colleagues connected this phenomenon to their metabolic findings discussed above [20]. They hypothesized that these results were perhaps due to an intracellular Phe threshold and that once PKU heterozygotes or well-treated homozygotes (with PKU) reached this threshold, they may start to experience minor neurological effects that result in decreased IQ [20]. Additionally, they hypothesized that the high intracellular Phe observed in carriers could be directly causing toxic effects on ganglion cells, resulting in cell injury and clinically leading to decreased IQ [20]. These hypotheses were discussed based on of the results of the study and were simply plausible explanations, which still needed to be confirmed in future research.

Sibling studies corroborate the above findings of cognitive alterations among carriers. A biological sibling of someone with PKU has a fifty percent chance of inheriting one PAH variant from either parent and being a PKU carrier (shown in Fig. 1) [11]. One study found that siblings of individuals with PKU scored significantly lower on IQ tests than the general population [34]. However, another sibling study appeared to have contradictory findings when they compared biological siblings (without PKU) of individuals with PKU to the general Caucasian population and found that the non-PKU siblings had a mean IQ that was significantly higher than the general population [32]. However, these researchers further explored sibling pairs and found that in the majority of the pairs, one sibling had a significantly higher IQ than the other sibling, and this discordance of IQ was significantly larger than would be expected among siblings in the general population [32]. Based on previously stated evidence that carriers were found to have lower IQs and metabolic perturbations, it is plausible that the sibling with the lower IQ was a PKU carrier while the siblings with higher IQ had no pathogenic variants in the PAH gene, leading to a significant difference between sibling pairs. Further investigating this neurological effect is an important area of future research. It is important to note that studies investigating IQ in PKU carriers were performed around 40 years ago and had notable limitations including small sample sizes and, at times, overly extensive extrapolation of results [21, 32, 34]. However, the trends that were repeatedly found in these studies may warrant future research with more robust methodologies to find more valid, reliable, and current results. At this time, we cannot conclude with confidence if the IQ of PKU carriers is lower than noncarriers.

Beyond IQ outcomes, possible psychological impacts among PKU carriers may be worth exploring further. For example, an abstract highlighted that carriers have an increased risk of experiencing greater brain irritability and late-onset schizophrenia with depression [35]. However, it does not appear that a full manuscript further describing this study was ever published, thus rendering these findings less reliable [35]. Schizophrenia is a complex multifactorial disorder; therefore, more robust research is needed. Other studies conducted in the 1970s found no significant difference between bipolar manic-depressive patients and controls for Phe blood level after an L-Phe-loading test [36]. When understanding the relationship between PKU carriers and mental health, burden of illness should also be considered. As previously discussed, most carriers are only aware of their carrier status because they likely have a child with PKU. Being a primary caregiver and raising a child with a condition with a significant caregiver burden, such as PKU, can impact quality of life, in turn having a toll on their mental health [37]. Overall, the association between PKU carriers and mental health outcomes has not been investigated thoroughly and is an important area of future research, especially given our improved understanding and validation of screening and diagnostic methods that can be used for evaluating mental health outcomes.

Furthermore, cognition has also been investigated. A study looking at early-treated adults with PKU found that these individuals had impairments in executive functioning compared to individuals without PKU [30]. The early-treated PKU participants performed worse than unaffected participants in terms of executive functioning, processing speed, motor skills, and visuospatial skills [30]. Their impaired performance was also correlated with higher Phe levels [30]. These findings have similarly been described in PKU carriers. In a study published in 2018, it was discovered that parents of PKU children (carriers) performed worse on cognitive test than noncarriers [31]. Specifically, carriers had worse executive functioning, processing speed, and inhibitory control than the noncarriers [31]. Carriers also tested worse than noncarriers in terms of delayed and immediate memory as well as word reading and color naming tasks, indicating reduced processing speed [31]. These findings demonstrate diminished executive functioning and cognition among carriers as compared to noncarriers, highlighting the importance of further studying cognitive functioning in this population [31]. In a study described earlier, Thalhammer and colleagues also found that IQ results were similar between carriers and well-treated PKU patients [21]. Overall, the findings of these two studies indicate that well-treated PKU patients may experience similar cognitive impairments that have been found in PKU carriers [21, 30]. Investigating cognitive effects in PKU carriers has been widely underexplored and is an intriguing area of future research.

Beyond metabolic and neurological studies, researchers are investigating other health outcomes among PKU carriers compared to noncarriers as well. In 2018, researchers found an association between melanoma risk and PKU carrier status [13]. The PAH pathway is involved in melanogenesis as Tyr is needed as the primary precursor for melanin production [13]. It has been found that high Phe concentrations can inhibit melanin production and increase the activation of the mitogen-activated protein kinase pathway which is involved with melanoma [13]. In a study that examined public melanoma databases, it was found that there was a carrier frequency of 3.56% in the melanoma cohorts [13]. This frequency is a twofold increase (p = 3.4 × 10⁻⁵) compared to the general Caucasian population, which has a PKU carrier frequency of approximately 2% [13]. This significant carrier frequency enrichment observed in the melanoma population indicates an association between PKU carriers and increased melanoma risk [13]. PKU is also more commonly found in Northern European populations than all Caucasians, seeming to be more prevalent in populations with less melanin [1]. A study conducted to compare comorbid conditions with PKU patients also found a significant difference between the prevalence of benign neoplasms of the skin in PKU patients compared to the general population [38]. In terms of melanoma, there was a prevalence of 0.11% in PKU patients compared to the general population which reported a prevalence of 0.04%; however, these differences were not statistically significant [38]. Other skin conditions such as dermatitis, eczema, and alopecia are also more prevalent in individuals with PKU compared to those without PKU [38]. Arbesman et al. [13] only studied carriers and noncarriers in the Caucasian population comparatively, and Burton et al. [38] studied individuals from a variety of geographical locations and races. Therefore, it is possible that ethnicity could play a role in this correlation. Arbesman et al. [13] have been the only group to report any findings regarding the correlation between melanoma and PKU carriers. In addition, genome-wide association studies have not found associations between melanoma and PAH genetic variation, though research in this field is still growing [39]. Despite this, with the increased prevalence of various skin conditions in patients with PKU, PAH pathway implications related to melanogenesis, and the findings of Arbesman et al. [13], this preliminary correlation may warrant further exploration [38].

In addition to BH₄, PAH is an iron-dependent enzyme required for proper functionality. Preliminary research has demonstrated that iron deficiency leads to reduced in vivo PAH activity [40]. Specifically, in this study, 10 patients with iron deficiency demonstrated PAH activity of only 56%; this compares to 100% in healthy controls and 37% in PKU carriers [40]. When the patients with iron deficiency normalized their iron status, PAH activity returned to normal [40]. Given this, it would be beneficial to further study iron-related outcomes among patients with PKU as well as PKU carriers.

Other research has found that parents (considered “unaffected” carriers) of children with HPA had a 1/83 chance of having undiagnosed mild HPA or mild PKU (mild PAH deficiency) [3]. This study was done specifically in France, so depending on the frequency of PKU in a certain population, and this result would likely vary [3]. These researchers highlight that even though screening for PKU is very well established, there are undiagnosed cases of mild PKU and HPA still occurring [3]. We suggest that HPA and mild PKU cases may perhaps be missed as PKU screening typically occurs within the first few days after birth, when feeding may not yet be well established; thus, Phe levels may not yet be elevated, particularly in individuals who still have some residual PAH activity such as PKU carriers.

Another interesting health outcome that has been evaluated is birth weight. A study performed in 1977 found that non-PKU individuals, both carriers and noncarriers, born from PKU heterozygotes had significantly larger birth weights than the average weight of newborns [41]. This finding suggests that PKU heterozygotes may be at higher risk of experiencing complications when giving birth, due to the child’s large birth weight (independent of the child’s carrier status) [41]. However, this research warrants further investigation, using more robust research methods, to confirm this possible finding, particularly considering the conflicting research in research conducted in individuals with HPA or PKU. For example, in patients with HPA, it was found that there was no correlation between birth weight and the severity of their condition [42]. Other studies found that infants born from PKU mothers (the infants being PKU carriers) were born with the same expectation for growth and development as those born to non-PKU mothers as long as the PKU mothers maintained healthy Phe levels throughout pregnancy [43, 44]. Thus, the PKU carrier status of the infant and the diagnosis of the mother do not seem to impact birth weight and later development [43, 44]. Overall, current literature on the effect of birth weight among babies of PKU carriers is limited. Diving further into the impact of maternal PKU on a developing fetus, if a mother with PKU does not strictly control her Phe intake during pregnancy, her offspring is at significantly higher risk of many health risks [45]. These risks include microcephaly, developmental delays, congenital heart defects, and abnormal neurologic findings [45]. When further studying this population, we should also keep in mind that all offspring of mothers with PKU or HPA are, at minimum, obligate carriers.

NBS is conducted in infants to provide early diagnosis and treatment of various genetic conditions [46]. This test has now been routine in many countries around the world since the 1960s, while advancements have been implemented as technology and knowledge progressed [46, 47]. NBS has been shown to be extremely effective in preventing death or disability as well as improving quality of life for those with diseases that are screened [46]. NBS is routinely done between the first 24–48 h of life [48]. However, if tested too early, before the first 24 h of life when feeding has not yet been well established, levels of Phe may not raise due to overall limited dietary intake of Phe, and thus many cases of PKU have potential to be missed through NBS [49]. Specifically, it has been reported that approximately 1/3 cases of PKU may be missed if the sample is taken within the first 12 h of life, and 10% may be missed within the second 12 h [49]. More recently, a study comparing early screening (before 24 h of life) and standard screening (between 24 and 48 h) observed altered metabolite levels during early detection [48]. There are certain situations in which repeat testing is done, such as if the screening card is not properly filled out, if there is not enough blood collected, if something goes wrong during transportation, or if it is caught that screening is done too early [50]. Repeat testing can thus catch some of the early screening cases [50]. However, it should also be noted that neonates are catabolic in their first few days of life, and muscle breakdown can contribute to higher Phe levels, thus potentially, leading to inaccuracies in test results depending on the timing of NBS [29]. Furthermore, neonates who specifically receive packed red blood cell transfusions prior to NBS may require repeat testing [50]. Given this, in conjunction with our recently improved knowledge of genetic implications, the addition of genetic testing to NBS practices could mitigate the issue of PKU and HPA false negatives as well as decrease the need for repeat testing, and it is a practice worth implementing into standard NBS practices. Overall, while NBS for PKU (using heel prick testing to analyze Phe levels) is routine practice in many countries and shown to improve the clinical course of the disease, genetic testing could be useful to identify cases that might be missed on NBS [3, 10]. There are multiple genetic variants that impact PAH functioning [12], and different variants are associated with varying severity of PKU [3]. Depending on the individual’s severity of PKU, or HPA, their tolerance for Phe intake will be variable [3, 28]. If individuals with PKU have varying severity of PKU depending on their genotype, it could be suggested that PKU carriers may also have a varying severity of Phe tolerance [3, 28].

To date, researchers have discovered over 1,500 pathogenic variants associated with the PAH gene [12]. A study investigating population genetics found that many variants in the Quebec population are like that seen in European populations showing tracers of historical demography [51, 52]. Another study looked at the restricted fragment length polymorphism haplotype of the PAH gene and found that different haplotypes were associated with different variants depending on the population [52]. These findings mean that when predicting disease outcomes, the haplotype, variant, and population all need to be considered [51, 52].

More recently, using whole-exome sequencing, novel variants are being discovered outside of the PAH gene. For example, variants in DNAJC12 were recently found to be associated with HPA [53]. The DNAJC12 gene codes for a chaperone protein that is involved in the proper folding for proteins associated with the PAH pathway [53]. With increasing knowledge of the human genome, it is possible that more variants relevant to PKU beyond those identified in the PAH gene will be discovered.

Overall, this review provides thought-provoking evidence for a possible intermediate phenotype of PKU among PKU carriers, which warrants further exploration. Ultimately, there is a need for further studies evaluating the health and metabolic impacts among PKU carriers. A small and outdated body of research has established that carriers likely have impaired metabolism, specifically related to decreased PAH enzymatic functioning and increased Phe levels, especially following Phe consumption. However, whether or not these metabolic implications result in clinical perturbations has yet to be thoroughly and robustly explored. Preliminary research, mostly conducted several years ago, indicates there may be a possible correlation between cognition, mental health, and other health risks, such as melanoma, associated with PKU carrier status, though we reiterate that more research is needed before we can confirm or refute such clinical implications. Given the many recent technological advancements including the integration of multi-omics technologies into health research, it is now possible to more robustly and comprehensively study this topic further. In addition, methods for screening and diagnosing health conditions and neurological outcomes including those related to mental health and cognition have improved dramatically over the past several decades. With these recent advancements, we have the potential to better understand the plausible metabolic and health impacts in the 1 in 50 individuals who are genetic carriers of PKU. Further research in this area would be beneficial in order to improve our understanding of this population.

The authors would like to thank Amber Hames for her contributions to developing the article figures.

The authors have no conflicts of interest to declare.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Sophia M. Khan was a major contributor in writing the manuscript. Justine R. Keathley was involved in writing and editing the manuscript. Robyn R. Heister was involved in editing the manuscript. All authors have seen and approved the study submitted.