Maternal Phenylketonuria International Collaborative Study revisited: evaluation of maternal nutritional risk factors besides phenylalanine for fetal congenital heart defects

Abstract

Maternal phenylketonuria (MPKU) is known to affect fetal outcome, often being associated with microcephaly and congenital heart defects (CHD) if the maternal diet is not appropriately managed. We hypothesized that other nutrients aside from phenylalanine (Phe) may have significant effects on fetal outcome in MPKU pregnancies. The 416 pregnancies that resulted in live births reported in the Maternal PKU Collaborative Study (MPKUCS) were grouped according to whether or not the offspring were diagnosed with CHD. The groups were compared on first-trimester values of maternal data, including weight gain, plasma amino acids, protein and Phe intake, and red blood cell (RBC) folate. Patients were also grouped by first-trimester average blood Phe (≤910 μmol/L and >910 μmol/L) and then divided by total natural protein and medical food intake. The CHD group of 28 offspring had significantly higher blood Phe and lower proline, valine, methionine, isoleucine, leucine, lysine, arginine, and RBC folate. A significantly higher risk for CHD was found in the groups with lower natural protein and medical food intake, regardless of blood Phe levels. Insufficient natural protein and medical food product intake appears to be a risk factor for CHD independent of first-trimester plasma Phe levels. Low RBC folate and plasma methionine levels in the CHD group may suggest involvement of global DNA hypomethylation.

Introduction

Phenylketonuria (PKU), one of the most common inborn errors of metabolism, is due to defective phenylalanine (Phe) hydroxylase (PAH: EC 1.14.16.1) and causes intellectual disability if untreated or treated late (Blau et al. 2010). Maternal PKU (MPKU), which is known to affect fetal outcome often with microcephaly and congenital heart defects (CHD) if maternal diet is not appropriately managed, has been studied in the Maternal PKU Collaborative Study (MPKUCS), which was completed in 2002 (Koch et al. 2003). Pre- and periconceptional blood Phe controlled between 120 to 360 μmol/L is recommended to minimize the risk of fetal congenital malformations associated with MPKU (Koch et al. 2003). None of the study participants who had offspring with CHD had blood Phe controlled in the recommended range during the first 8–10 weeks of gestation (Rouse and Azen 2004). The mechanisms of developing CHD in embryos of MPKU individuals in poor dietary control are unknown. We reanalyzed data from the MPKUCS to investigate possible risk factors for fetal CHD, independent of blood Phe.

Methods

The 416 MPKUCS pregnancies resulting in live births were grouped according to whether or not the offspring were diagnosed with CHD. Patent ductus arteriosus was excluded from the CHD group in order to specify structural developmental abnormalities. The groups were compared on first trimester values of maternal data, including weight gain at 12 weeks of pregnancy, weekly blood Phe and monthly assessments of dietary intake of 19 nutrients, 16 plasma amino acids, RBC folate and blood or serum levels of vitamin B₁₂, trace elements, minerals, and ferritin using twosided Wilcoxon rank sum tests. Dietary analyses were calculated based on the available 3-day diet records of 218 study participants. All analytes were measured at commercial laboratories. The frequency of CHD between subgroups, created by dichotomizing continuous variables, was compared with Fisher’s exact test. Statistical tests were performed at a significance level of 0.05, using SAS/STAT™ v. 9.2. Multiple logistic regression was used to investigate factors associated with CHD after adjusting for blood Phe.

Results

Twenty-eight pregnancies resulted in newborns with CHD. Table 1 shows data that differed significantly between groups. The CHD group had significantly higher blood Phe levels and significantly lower levels of the plasma amino acids proline, valine, methionine, isoleucine, leucine, lysine, and arginine. RBC folate was also significantly lower in the CHD group. None of the other analytes investigated were significantly different between groups. Dietary protein intake, including natural and medical food, was lower in the group with CHD (p=0.0004), whereas Phe intake was higher (p=0.0434), meaning there was less use of medical food products. Multiple logistic regression showed protein intake to be a significant, independent predictor of CHD (p=0.0003) after controlling for blood Phe.

Table 1.

First-trimester variables differing between maternal phenylketonuria (MPKU) pregnancies with and without congenital heart defects (CHD). First-trimester variables differing between MPKU pregnancies with and without CHD in offspring compared by Wilcoxon rank sum tests. P values for association with CHD, after adjusting for blood phenylalanine (Phe) using multiple logistic regression

		No CHD (n=388)			CHD (n=28)
Variable	Number	Median	(IQR)	n	Median	(IQR)	Wilcoxon P value	Adjusted* P value
Blood Phe weeks 4–8	387	556.27	(326.92–967.07)	28	1227.3	(987.60–1502.05)	<0.0001	n/a
Protein Intake (g)**	206	67.92	(55.70–77.24)	12	38.6	(29.43–56.47)	0.0004	0.0003
Phe Intake (mg)	206	543	(399–804)	11	802	(597.18–1204)	0.0434	0.0916
Plasma amino acid (uM)
Proline	197	140.02	(110.05–176.98)	11	100.76	(87.03–124.99)	0.0028	0.0118
Valine	211	196.31	(165.27–230.89)	11	152.28	(134.71–181.30)	0.0023	0.0537
Methionine	211	20.98	(16.42–26.51)	11	16.76	(14.48–19.97)	0.0183	0.0093
Isoleucine	215	52	(43.00–63.01)	11	40.03	(37.97–52.00)	0.0178	0.0185
Leucine	215	99.03	(84.01–119.50)	11	78.49	(69.03–84.01)	0.003	0.0144
Phenylalanine	218	486.03	(300.52–790.56)	12	825.3	(513.53–1071.00)	0.0285	0.3208
Lysine	210	149.45	(127.20–180.22)	11	113.68	(97.13–151.21)	0.0053	0.0075
Arginine	93	59.01	(46.49–76.00)	7	38	(23.99–52.49)	0.0226	0.0665
RBC folate (ng/ml)	149	420	(281–605)	9	271	(257–391)	0.0363	0.0437

IQR interquartile range, CHD not including patent ductus arteriosus

^*Association with CHD, adjusted for blood Phe, by multiple logistic regression

^**Protein Intake includes protein from natural food and medical food.

Additional significant risk factors, independent of blood Phe, included the amino acids proline (p=0.012), methionine (p=0.0093), isoleucine (p=0.019), leucine (p=0.014), and lysine (p=0.0075), as well as RBC folate (p=0.0437). Maternal weight gain at 12 weeks of pregnancy was not statistically different between the two groups (p=0.40, data not shown). Table 2 shows the frequency and percent of offspring with CHD for pregnancies classified by blood Phe (at or below vs. above 910 μmol/L) and total protein intake, including natural and medical food (at or below vs. above 50 g). For each blood Phe group, CHD was significantly higher in the group with lower dietary protein intake.

Table 2.

Frequency of offspring with congenital heart defects (CHD) in maternal phenylketonuria (MPKU) pregnancies: classified by blood phenylalanine (Phe) and protein intake. Number and percent of MPKU pregnancies with CHD in offspring classified by mean blood Phe during 4–8 weeks of gestation and mean first-trimester protein intake compared by Fisher’s exact test within blood Phe strata. Higher protein intake with lower Phe intake indicates use of PKU formula

Blood Phe	Total protein intake (natural+medical food)	Number	Phe intake* (mg)	CHD	Percent	P value
Low (≤910 μmol/L)	High (>50 g)	134	543(401–845)	1	0.8%	0.006
	Low (≤ 50 g)	33	662(465–963)	4	12.1%
High (>910 μmol/L)	High (>50 g)	41	523(392–615)	3	7.3%	0.02
	Low (≤ 50 g)	10	1044(541–1333)	4	40.0%

CHD not including patent ductus arteriosus, IQR interquartile range

^*median (IQR)

Discussion

Managing MPKU has primarily focused on controlling high blood Phe levels, with little attention given to other factors. The large cohort studies showed associations between blood Phe levels and neonatal sequelae, including small for gestational age, microcephaly, intellectual disability, and facial dysmorphism (Prick et al. 2012). Teissier et al. (2012) recently reported an increased risk of intrauterine growth retardation in MPKU individuals with low Phe levels, especially in the second and third trimesters. Their study suggests that high maternal blood Phe was not the only risk factor and that other maternal nutritional factors were involved in fetal development and growth.

Data from MPKUCS revealed a high Phe level and lower levels of seven amino acids in the CHD group. A high blood Phe and a low methionine level likely results in even lower intracellular methionine concentration due to competitive transport of the amino acids through the common large neutral amino acid (LNAA) transporter LAT-1 (Pietz et al. 1999). Phe has the highest affinity with LAT-1 among the amino acids transported through LAT-1 (Pietz et al. 1999). LAT-1 expression has been studied in mice and is known to be expressed in all stages of preimplantation embryo development (Chrostowski et al. 2009). Lower maternal plasma methionine, i.e., methyl donor, may contribute to global DNA hypomethylation, which likely affects embryonal organ development (Cooney et al. 2001). Low folate levels might also contribute to lower plasma methionine.

Chowdhury et al. (2011) recently reported that women whose pregnancies were affected by nonsyndromic CHD had higher mean concentrations of plasma homocysteine and s-adenosylhomocysteine (SAH) and lower mean concentrations of plasma methionine and s-adenosylmethionine (SAM) compared with controls. The SAM/SAH ratio was found to be significantly lower in the group with nonsyndromic CHD compared with controls. Maternal global DNA hypomethylation was found through the study, with long interspersed nucleotide elements-1 (LINE-1) DNA hypomethylation in the group with nonsyndromic CHD (Chowdhury et al. 2011). It has been reported that patients with Down syndrome and CHD have a low SAM/SAH ratio, suggesting involvement of global DNA hypomethylation (Van Direl et al. 2008). An association of low methionine and SAM levels has been reported in brain tissue of PKU mice (Vogel et al. 2012). Although SAM and SAH were not measured in MPKUCS, low methionine and high Phe levels in maternal blood are suggestive of low methionine and SAM levels in the embryonic tissue. This might further suggest involvement of global hypomethylation as a contributing factor causing CHD in MPKU pregnancies. We measured serum SAM levels in 23 PKU adult individuals who were not treated with medical food products and nine adult controls with the analytical method described by Inoue-Choi et al. (2003). Although serum SAM levels were not statistically different between these two groups (data not shown), cellular concentrations of methionine and SAM are likely lower due to transport of LNAA through LAT-1 based on the observation in PKU mice (Vogel et al. 2012). Increases in LAT-1 activities in response to amino acid depletion, specific for arginine with changes in LAT-1 messenger RNA (mRNA) levels, in cultured rat hepatocytes has been reported (Campbell et al. 2000). This suggests that depletion of amino acids may increase cellular uptake of Phe relative to the other LNAA through LAT-1 due to the high affinity of Phe with LAT-1. Consequently, this may lead to a deficiency of intracellular LNAA, including methionine. Protein synthesis is also known to be decreased by increased Phe levels and decreased levels of other large neutral amino acids (Surtees and Blau 2000).

Blood arginine was found to be lower in the CHD group. Arginine is required as a substrate for endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO). Low concentrations of arginine may result in decreased embryonal endothelial NO production. In support of this notion, it has been shown that CHD occurs in transgenic mice that are deficient in eNOS (Feng et al. 2002).

Low concentrations of multiple amino acids strongly suggest insufficient protein intake in the first trimester in the CHD group. Table 2 further provides supportive evidence that insufficient intake of protein, including medical food products, is a contributing factor for CHD. Higher protein intake with lower Phe intake indicates use of a larger amount of medical food products. Lower protein intake with higher Phe intake indicates restriction of natural protein with less use of medical foods. The subgroups of higher protein intake (>50 g) consumed approximately 10 g of natural protein, based on Phe intake, with the rest of the protein originating from medical food products (Weetch and Macdonald 2006). It appears that approximately 40 g of protein intake from medical food products is associated with decreased risk of fetal CHD.

Managing maternal PKU should address adequacy of protein intake, particularly the amount of medical food products, and—in addition to monitoring blood Phe—plasma amino acids, homocysteine, and RBC folate should be evaluated before conception and during the early first trimester to minimize the risk of CHD.