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Phenylketonuria (PKU; also known as phenylalanine hydroxylase (PAH) deficiency) is an autosomal recessive disorder of phenylalanine metabolism, in which especially high phenylalanine concentrations cause brain dysfunction. If untreated, this brain dysfunction results in severe intellectual disability, epilepsy and behavioural problems. The prevalence varies worldwide, with an average of about 1:10,000 newborns. Early diagnosis is based on newborn screening, and if treatment is started early and continued, intelligence is within normal limits with, on average, some suboptimal neurocognitive function. Dietary restriction of phenylalanine has been the mainstay of treatment for over 60 years and has been highly successful, although outcomes are still suboptimal and patients can find the treatment difficult to adhere to. Pharmacological treatments are available, such as tetrahydrobiopterin, which is effective in only a minority of patients (usually those with milder PKU), and pegylated phenylalanine ammonia lyase, which requires daily subcutaneous injections and causes adverse immune responses. Given the drawbacks of these approaches, other treatments are in development, such as mRNA and gene therapy. Even though PAH deficiency is the most common defect of amino acid metabolism in humans, brain dysfunction in individuals with PKU is still not well understood and further research is needed to facilitate development of pathophysiology-driven treatments. Phenylketonuria is an inherited disorder of phenylalanine metabolism that can result in neurological dysfunction. This Primer reviews the epidemiology, aetiology, diagnosis and management of PKU as well as the impact on patients’ quality of life.

We propose a nosology for inborn errors of metabolism that builds on their recent redefinition. We established a strict definition of criteria to develop a self-consistent schema for inclusion of a disorder into the nosology. We identified 1015 well-characterized inborn errors of metabolism described in the literature. In addition, there are 111 less well-characterized conditions that may be inborn errors but do not meet strict criteria for inclusion in the current nosology. We provide a master list of all currently recognized inborn errors of metabolism grouped according to their pathophysiological basis, with the hope of setting a standard against which new errors should be defined, as well as to promote awareness and foster collaboration in the area. With the rapid advances in the field of genetics in recent years, it is likely that this nosology will need to be updated in the near future, a process that will benefit from broader input and collaboration of experts in the field to improve future versions of the proposed classification.

Phenylketonuria is the most prevalent disorder caused by an inborn error in aminoacid metabolism. It results from mutations in the phenylalanine hydroxylase gene. Phenotypes can vary from a very mild increase in blood phenylalanine concentrations to a severe classic phenotype with pronounced hyperphenylalaninaemia, which, if untreated, results in profound and irreversible mental disability. Neonatal screening programmes identify individuals with phenylketonuria. The initiation of a phenylalanine-restricted diet very soon after birth prevents most of the neuropsychological complications. However, the diet is difficult to maintain and compliance is often poor, especially in adolescents, young adults, and pregnant women. Tetrahydrobiopterin stimulates phenylalanine hydroxylase activity in about 20% of patients, and in those patients serves as a useful adjunct to the phenylalanine-restricted diet because it increases phenylalanine tolerance and allows some dietary freedom. Possible future treatments include enzyme substitution with phenylalanine ammonia lyase, which degrades phenylalanine, and gene therapy to restore phenylalanine hydroxylase activity.

This manual deals specifically with laboratory approaches to diagnosing inborn errors of metabolism. The key feature is that each chapter is sufficiently detailed so that any individual can adopt the described method into their own respective laboratory.

Tetrahydrobiopterin (BH4) deficiencies are a highly heterogeneous group of disorders with several hundred patients, and so far a total of 193 different mutant alleles or molecular lesions identified in the GTP cyclohydrolase I (GTPCH), 6‐pyruvoyl‐tetrahydropterin synthase (PTPS), sepiapterin reductase (SR), carbinolamine‐4a‐dehydratase (PCD), or dihydropteridine reductase (DHPR) genes. The spectrum of mutations causing a reduction in one of the three biosynthetic (GTPCH, PTPS, and SR) or the two regenerating enzymes (PCD and DHPR) is tabulated and reviewed. Furthermore, current genomic variations or SNPs are also compiled. Mutations in GCH1 are scattered over the entire gene, and only 5 out of 104 mutant alleles, present in a homozygous state, are reported to cause the autosomal recessive form of inheritable hyperphenylalaninemia (HPA) associated with monoamine neurotransmitter deficiency. Almost all other 99 different mutant alleles in GCH1 are observed together with a wild‐type allele and cause Dopa‐responsive dystonia (DRD, Segawa disease) in a dominant fashion with reduced penetrance. Compound heterozygous or homozygous mutations are spread over the entire genes for PTS with 44 mutant alleles, for PCBD with nine mutant alleles, and for QDPR with 29 mutant alleles. These mutations cause an autosomal recessive inherited form of HPA, mostly accompanied by a deficiency of the neurotransmitters dopamine and serotonin. Lack of sepiapterin reductase activity, an autosomal recessive variant of BH4 deficiency presenting without HPA, was diagnosed in patients with seven different mutant alleles in the SPR gene in exons 2 or 3 or in intron 2. Details on all mutations presented here are constantly updated in the BIOMDB database (www.bh4.org). Hum Mutat 27(9), 870–878, 2006. © 2006 Wiley‐Liss, Inc.

BackgroundAutosomal recessive mutations in DNAJC12, encoding a cochaperone of HSP70 with hitherto unknown function, were recently described to lead to hyperphenylalaninemia, central monoamine neurotransmitter (dopamine and serotonin) deficiency, dystonia and intellectual disability in six subjects affected by homozygous variants.ObjectivePatients exhibiting hyperphenylalaninemia in whom deficiencies in hepatic phenylalanine hydroxylase and tetrahydrobiopterin cofactor metabolism had been excluded were subsequently analysed for DNAJC12 variants.MethodsTo analyse DNAJC12, genomic DNA from peripheral blood (Sanger sequencing), as well as quantitative messenger RNA (Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)) and protein expression (Western blot) from primary skin fibroblasts were performed.ResultsWe describe five additional patients from three unrelated families with homozygosity/compound heterozygosity in DNAJC12 with three novel variants: c.85delC/p.Gln29Lysfs*38, c.596G>T/p.*199Leuext*42 and c.214C>T/p.(Arg72*). In contrast to previously reported DNAJC12-deficient patients, all five cases showed a very mild neurological phenotype. In two subjects, cerebrospinal fluid and primary skin fibroblasts were analysed showing similarly low 5-hydroxyindolacetic acid and homovanillic acid concentrations but more reduced expressions of mRNA and DNAJC12 compared with previously described patients. All patients responded to tetrahydrobiopterin challenge by lowering blood phenylalanine levels.ConclusionsDNAJC12 deficiency appears to result in a more heterogeneous neurological phenotype than originally described. While early identification and institution of treatment with tetrahydrobiopterin and neurotransmitter precursors is crucial to ensure optimal neurological outcome in DNAJC12-deficient patients with a severe phenotype, optimal treatment for patients with a milder phenotype remains to be defined.

The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the Fok I endonuclease ( Fok I-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase ( PAH ) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a Fok I-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH _c.1222C>T COS-7 cells – an in vitro model for PKU – corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the Fok I-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases.

Childhood cerebral folate deficiency is a disabling neurologic disorder in which folate is reduced in the cerebrospinal fluid but not in the blood. The usual signs of folate deficiency are therefore absent. This study showed that children with cerebral folate deficiency produce autoantibodies that block the binding of folate to the folate receptor. Very high doses of folate resulted in clinical improvement in some children. Children with cerebral folate deficiency produce autoantibodies that block the binding of folate to the folate receptor. This report widens the scope of knowledge about receptor-binding autoantibodies and introduces provocative ideas about certain brain disorders of childhood. Cerebral folate deficiency can be defined as any neuropsychiatric condition associated with low levels of 5-methyltetrahydrofolate (5MTHF), the active folate metabolite in the cerebrospinal fluid, in association with normal folate metabolism outside the central nervous system, as reflected by normal hematologic values, normal serum homocysteine levels, and normal levels of folate in serum and erythrocytes. Infantile-onset cerebral folate deficiency is a neurologic syndrome that develops four to six months after birth. Its major manifestations are marked irritability, slow head growth, psychomotor retardation, cerebellar ataxia, pyramidal tract signs in the legs, dyskinesias (e.g., choreoathetosis and ballismus), and in some cases, seizures. . . .

In cerebral folate deficiency syndrome, the presence of autoantibodies against the folate receptor (FR) explains decreased folate transport to the central nervous system and the clinical response to folinic acid. Autoantibody crossreactivity with milk FR from different species prompted us to test the effect of a milk‐free diet. Intervention with a milkfree diet in 12 children (nine males, three females; mean age 6y [SD 4y 11mo], range 1‐19y), decreased autoantibody titer significantly from 2.08pmol of FR blocked per ml of serum (SD 2.1; range 0.24‐8.35) to 0.35pmol (SD 0.49; range 0‐1.32; p=0.012) over 3 to 13 months, whereas FR autoantibody titer increased significantly to 6.53 (SD 6.08; range 0.54‐14.07; p=0.013) in nine children who were reexposed to milk for 6 to 14 weeks. In 12 children on a normal diet (eight males, four females; mean age 5y 5mo [SD 4y 1mo], range 1y 6mo‐16y 4mo), the antibody titer increased significantly from 0.84pmol of FR blocked per ml (SD 0.39; range 0.24‐1.44) to 3.04pmol (SD 1.42; range 0.84‐6.01; p=0.001) over 10 to 24 months. Decreasing the autoantibody titer with a milk‐free diet in conjunction with folinic acid therapy may be advocated for these patients.

Monitoring phenylalanine (Phe) concentrations is critical for the management of phenylketonuria (PKU). This can be done in dried blood spots (DBS) or in EDTA plasma derived from capillary or venous blood. Different techniques are used to measure Phe, the most common being flow-injection analysis tandem mass spectrometry (FIA-MS-MS) and ion exchange chromatography (IEC). Significant differences have been reported between Phe concentrations in various sample types measured by different techniques, the cause of which is not yet understood. We measured Phe concentrations in 240 venous blood samples from 199 patients with hyperphenylalaninemia in dried blood spots, EDTA plasma and erythrocytes by FIA-MS-MS and IEC. Phe concentrations were significantly lower in erythrocytes than in plasma leading to about 19% lower Phe DBS concentrations compared with plasma independent from the method used for quantification. As most therapy recommendations for PKU patients are based on plasma concentrations reliable conversion of DBS into plasma concentrations is necessary. Variances of Phe concentrations in plasma and DBS are not linear but increases with higher concentrations indicating heteroscedasticity. We therefore suggest the slope of the 75th percentile from quantile regression as a correction factor.