Long-term treatment of phenylketonuria with a new medical food containing large neutral amino acids.

This corrects the article DOI: 10.1038/ejcn.2016.166.

Long-term treatment of phenylketonuria with a new medical food containing large neutral amino acids.

BACKGROUND/OBJECTIVES: Phenylketonuria (PKU) is an autosomal recessive disease caused by deficient activity of phenylalanine hydroxylase. A low phenylalanine (Phe) diet is used to treat PKU. The diet is very restrictive, and dietary adherence tends to decrease as patients get older. Methods to improve dietary adherence and blood Phe control are continuously under investigation. SUBJECTS/METHODS: A new formula Phe-neutral amino acid (PheLNAA) has been tested in this study with the purpose of improving the compliance and lowering blood phenylalanine. The formula has been tested for nitrogen balance, and it is nutritionally complete. It is fortified with more nutritional additives that can be deficient in the PKU diet, such as B12, Biotin, DHA, Lutein and increased levels of large neutral amino acids to help lower blood Phe. The new formula has been tested on 12 patients with a loading test of 4 weeks. RESULTS: Fifty-eight percent of patients had a significant decline in blood Phe concentration from baseline throughout the study. The PheLNAA was well tolerated with excellent compliance and without illnesses during the study. CONCLUSIONS: In conclusion, the new formula is suitable for life-long treatment of PKU, and it offers the PKU clinic a new choice for treatment.

Double blind placebo control trial of large neutral amino acids in treatment of PKU: effect on blood phenylalanine.

Large neutral amino acids (LNAA) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In an open-label study using LNAA, a surprising decline of blood Phe concentration was found in patients with PKU in metabolic treatment centres in Russia, the Ukraine, and the United States. To validate the data obtained from this trial, a short-term double-blind placebo control study was done using LNAA in patients with PKU, with the participation of three additional metabolic centres--Milan, Padua and Rio de Janeiro. The results of the short trial showed significant lowering of blood Phe concentration by an average of 39% from baseline. The data from the double-blind placebo control are encouraging, establishing proof of principle of the role of orally administered LNAA in lowering blood Phe concentrations in patients with PKU. Long-term studies will be needed to validate the acceptability, efficacy and safety of such treatment.

Large neutral amino acids in the treatment of phenylketonuria (PKU).

Large neutral amino acids (LNAAs) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In earlier studies on mice with PKU (ENU(2)/ENU(2)), LNAAs were given and a surprising decline in blood Phe concentrations was observed. The formula used in the mouse experiment (PreKUnil) lacked lysine. Therefore, a new formulation of LNAAs (NeoPhe) was developed, introducing changes in the concentration of some amino acids and adding lysine, so that such a mixture could be used in humans. The new formula was found to be effective in reducing blood Phe concentration in mice by about 50% of the elevated levels. Patients with PKU were given LNAAs and blood Phe concentrations were determined in an open-label study. Three centers--in Russia, the Ukraine and the USA--took part in the study. NeoPhe was given at 0.5 g/kg per day in three divided doses to eight subjects with PKU and at 1.0 g/kg per day to three patients, for one week. The NeoPhe resulted in decrease of elevated blood Phe by 50% in both groups. The preliminary data from this study are encouraging and a double blind placebo-controlled trial will be required to show long-term efficacy and tolerance of LNAAs in the treatment of PKU.

Abnormal expression of genes associated with development and inflammation in the heart of mouse maternal phenylketonuria offspring.

This study describes gene expression in the fetus hearts obtained from mouse model for phenylketonuria. These hearts have cardiovascular disease (CVD). Therefore genes involved in CVD were examined. Several genes associated with heart development and inflammation were found to be altered. In order to investigate whether the abnormal gene expression alters transcription and translation, the levels of troponin mRNA and protein were determined. One step real time RT-PCR showed a reduction in cardiac troponin I, troponin T2 and ryanodine receptor 2. Determination of troponin I and T protein levels showed reduced levels of these proteins. Our results suggest that altered gene expression affects protein production. These changes are likely involved in the cardiovascular defects seen in the mouse.

Maternal Phenylketonuria Collaborative Study (MPKUCS)--the 'outliers'.

Analysis of outcome data from 305 of the 414 offspring from the Maternal Phenylketonuria Collaborative Study (MPKUCS), plus 70 control offspring, revealed significant deficits in the IQ (intelligence quotient), as measured by the Wechsler Intelligence Scale for Children--Revised (WISC-R), when maternal metabolic control during pregnancy was delayed and/or inadequate. There were, however, 23 'outliers' (7.5% of the 305) in which the offspring's intellectual IQ was worse (n =10) or better (n =13) than expected. The aim of this study was to determine whether collection parameters were incomplete or whether these subjects were true biological variants influenced by other undetected factors or, perhaps, by modifier genes. Among the 10 offspring whose intellectual functioning was worse than expected, additional complications were uncovered that could explain the poor outcome. Four of the 13 offspring with higher than expected IQ had mothers with mild variants of PKU in which the insult to the fetus would not be expected to be as profound. For the other nine offspring whose intellectual performance was better than expected, there was no explanation, based on the parameters studied. We hypothesize that modifier genes will, at times, protect the fetus despite high maternal concentrations of phenylalanine. Not all offspring from the same (untreated) PKU mother may be similarly affected. Finding the source of these modifiers might effect the treatment of MPKU.

Mental retardation and hypotonia seen in the knock out mouse for Canavan disease is not due to succinate semialdehyde dehydrogenase deficiency.

Canavan disease (CD) is an autosomal recessive disorder caused by aspartoacylase deficiency leading to accumulation of N-acetylaspartic acid and spongy degeneration of the brain. The mouse model for CD showed low levels of glutamate and gamma-aminobutyric acid (GABA) in the brain. Whether the low levels of glutamate and GABA observed in the CD mouse brain lead to abnormal production of glutamate-GABA associated enzymes and resulting succinate production is not obvious. While glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase complex activities are lower in the cerebellum and brain stem of the CD mouse, alanine aminotransferase and succinate semialdehyde dehydrogenase (SSADH) activities and succinate level are similar to the levels observed in the wild type. Deficiency of SSADH has been suggested to be associated with mental retardation and hypotonia, similar to the clinical features of CD. The normal SSADH activity in the CD mouse brain suggests that mental retardation and hypotonia seen in the CD mouse is not due to SSADH activity and if documented also in patients with CD.

Intake of major nutrients by women in the Maternal Phenylketonuria (MPKU) Study and effects on plasma phenylalanine concentrations.

BACKGROUND: Women with untreated phenylketonuria (PKU) often have poor reproductive outcomes. OBJECTIVE: We assessed the effects of intakes of major nutrients on plasma phenylalanine concentrations and we measured phenylalanine hydroxylase activity and phenylalanine intakes in pregnant women with PKU. DESIGN: Dietary intakes and plasma phenylalanine concentrations were compared in 4 subject groups defined on the basis of plasma phenylalanine concentrations: group 1 (n = 23), <360 micromol/L by 10 wk gestation and 120-360 micromol/L throughout the remainder of pregnancy; group 2 (n = 46), <600 micromol/L but not <360 micromol/L by 10 wk gestation and 120-600 micromol/L throughout the remainder of pregnancy; group 3 (n = 24), <600 micromol/L by 10 wk gestation but >600 micromol/L at least once thereafter; group 4 (n = 147), never <600 micromol/L. RESULTS: Except in the first trimester, mean intakes of phenylalanine, energy, and fat tended to be greater in group 1 than in the other groups. The mean protein intake of group 1 tended to be greater than that of the other groups. Intakes of protein (P < 0.0001), fat (P < 0.0001), and energy (P < 0.007) were negatively correlated with maternal plasma phenylalanine concentrations. It appeared that genotype did not affect phenylalanine tolerance. CONCLUSIONS: Maternal genotype appeared to have little influence on phenylalanine requirements during the first trimester. Early decline and maintenance of maternal plasma phenylalanine concentrations at <360 micromol/L and mean protein intake greater than the recommended dietary allowance (RDA) with mean energy intake near the RDA resulted in the best reproductive outcomes. Inadequate intakes of protein, fat, and energy may result in elevated plasma phenylalanine concentrations and may contribute to poor reproductive outcomes.

The International Collaborative Study of Maternal Phenylketonuria: status report 1998.

UNLABELLED: The Maternal Phenylketonuria Study began in 1984 and during the intervening years, 572 pregnancies in hyperphenylalaninemic women and 99 controls and their outcomes have been evaluated. Among hyperphenylalaninemic women who delivered a live infant, only 15.9% were treated and in metabolic control preconceptually, however, another 18.4% were in control by 10 weeks. Compared to the results reported by Lenke and Levy in 1980, there is a marked improvement in outcome with treatment. Microcephaly was unusual in preconceptually treated pregnancies with well controlled phenylalanine restricted diets. Even in pregnancies that established control after conception but before the 8th week, congenital heart disease did not occur in the offspring, however, it did occur in 12% of pregnancies not achieving control until after 10 weeks of pregnancy. CONCLUSION: The recommended level of blood phenylalanine during pregnancy is 120-360 mumol/l. Best results were obtained by close cooperation between the attending obstetrician and a metabolic team experienced in the care of persons with phenylketonuria.

Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system.

BACKGROUND: Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N-acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock-out mouse for Canavan disease that can be used for gene transfer. METHODS: Genomic library lambda knock-out shuttle (lambdaKOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid with 10 base pair (bp) deletion of exon four of the murine aspa. Following linearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and confirmed by Southern analysis. Chimeras were generated by injection of ES cells to blastocysts. Germ line transmission was achieved by the birth of heterozygous mice as confirmed by Southern analysis. RESULTS: Heterozygous mice born following these experiments have no overt phenotype. The homozygous mice display neurological impairment, macrocephaly, generalized white matter disease, deficient ASPA activity and high levels of NAA in urine. Magnetic resonance imaging (MRI) and spectroscopy (MRS) of the brain of the homozygous mice show white matter changes characteristic of Canavan disease and elevated NAA levels. CONCLUSION: The newly created ASPA deficient mouse establishes an important animal model of Canavan disease. This model should be useful for developing gene transfer vectors to treat Canavan disease. Vectors for the central nervous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data generated from this animal model will be useful for developing strategies for gene therapy in other neurodegenerative diseases.

Spongy degeneration of the brain, Canavan disease: biochemical and molecular findings.

Canavan disease is a severe progressive leukodystrophy characterized by swelling and spongy degeneration of the white matter of the brain. It is an autosomal recessive disease found more frequently among Ashkenazi Jews. The clinical features are those of severe mental retardation with inability to gain developmental milestones. Hypotonia, head lag and macrocephaly are characteristic of Canavan disease and become apparent after 5-6 months of age. Massive excretion in the urine of N-acetylaspartic acid is the biochemical marker for Canavan disease, which is caused by deficiency of the enzyme aspartoacylase. This discovery allowed for accurate diagnosis of Canavan disease, while prior to that, a brain biopsy was needed. The gene for aspartoacylase has been cloned and two mutations predominate among Ashkenazi Jewish individuals with Canavan disease and account for more than 98% of the Ashkenazi Jewish patients. The mutations among other ethnic groups are more diverse. The carrier frequency for the two common mutations among Ashkenazi Jews was found to be surprisingly high, 1:37. Screening for carriers is now common practice for this population. A knock-out mouse for Canavan disease is being genetically engineered in our laboratory. The mouse model will allow for development of strategies for gene therapy.

Biochemistry and molecular biology of Canavan disease.

Canavan in 1931 described spongy degeneration of the brain in a child who was thought to have had Schilder's disease. Since that classic histological description, Canavan disease has become a distinct clinical entity, with the recognition by Van Bogaert and Bertrand that this is an autosomal recessive disease prevalant among children of Jewish extraction. Recent advances in the understanding of the biochemical defect led to an increase in awareness and ease in diagnosis, and indeed the disease is not as rare as initially thought. Exploring the molecular aspects of Canavan disease has led to exciting new developments in carrier detection and prevention of Canavan disease. Work is underway in our laboratory to develop a knock-out mouse for Canavan disease for understanding of the pathophysiology of this disease and formulating gene therapy.

Recent advances in Canavan disease.

More studies are needed to elucidate the pathophysiology of Canavan disease and how the inability to hydrolyze NAA leads to spongy degeneration. The creation of an animal model would be helpful in the understanding of the disease and the formulation of gene therapy.

Maternal mild hyperphenylalaninemia: results of treated and untreated pregnancies in two sisters.

The outcomes of mild hyperphenylalaninemia (MHP) in three children of two sisters were compared. The IQ of the child from an untreated pregnancy was 105; the developmental quotients of the two infant offspring from treated and untreated pregnancies were 122 and 114, respectively. The IQ of the sister with untreated MHP was 101; that of the sister who received dietary treatment for MHP during infancy was 90. Thus MHP and maternal MHP appear to have been clinically inconsequential in this family.