Hyperphenylalaninemia

To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) from 1999 to 2022 in Zhejiang province.

A total of 11 922 318 newborns were screened from September 2009 and December 2022 in Zhejiang province. The blood thyroid stimulating hormone (TSH) levels were measured by fluorescence method and blood phenylalanine (Phe) levels were measured by fluorescence method or tandem mass spectrometry. TSH≥9 μIU/mL was considered positive for CH; while Phe>120 μmol/L and/or Phe/Tyr ratio>2.0 were considered positive for HPA. The positive newborns in screening were recalled, and the gene variations were detected by high-throughput sequencing and MassARRAY tests.

The overall neonatal screening rate druing 1999-2022 was 89.41% (11 922 318/13 333 929) and the screening rate was increased from 6.46% in 1999 to 100.0% in 2022. A total of 8924 cases of CH were diagnosed among screened newborns with an incidence rate of 1/1336. A total of 563 cases of HPA were diagnosed, including 508 cases of classic phenylketonuria (cPKU) and 55 cases of tetrahydrobiopterin deficiency (BH4D), with an incidence rate of 1/21176. Ninety seven out of 8924 cases of CH underwent genetic analysis. Gene mutations were detected in 9 CH related genes, the highest frequency mutations were found in DUOX2 gene (69.0%) with c.3329G>A (p.R1110Q) (18.2%) and c.1588A>T (p.K530X) (17.3%) as the hotpot variants. There were 81 PAH gene variants detected in 250 cPKU patients; and c728G>A (p.R243Q)(24.4%), c.721C>T (p.R241C) (15.0%) were the hotpot variants. Meanwhile 7 novel variants in PAH gene were detected: c.107C>A (p.S36*), c.137G>T (p.G46V), c.148A>G(p.K50E), c.285C>T (p.I95I), c.843-10delTTCC, exon4-7del and c.1066-2A>G. There were 12 PTS gene variants detected in 36 BH4D patients, and c.259C>T (p.P87S) (31.9%)was the hotpot variants.

The results of neonatal screening shows that the incident of CH has increased from 1999 to 2022 in Zhejiang province and it is higher than than of national and global levels; while the incidence of HPA was similar to the national average. DUOX2 gene is the most common gene in CH patients with; c.728G>A (p.R243Q) is the hot spot variant in cPKU patients, while c.259C>T (p.P87S) is the hot spot variant in BH4D patients.

To retrospectively analyze the variation characteristics of phenylalanine hydroxylase (PAH) gene, and to observe the long-term treatment effect and follow-up of newborns with PAH deficiency.

The clinical data, treatment and follow-up results of 198 children with PAH deficiency, which diagnosed by newborn screening in Jinan from 1996 to 2021 were collected. The genetic analysis results of 55 children with PAH deficiency diagnosed by newborn screening in Jinan and 213 children referred from the surrounding areas of Jinan were summarized. Gene mutations were checked by a customized Panel gene detection method. As also as blood phenylalanine-concentration, physical development indicators include height and weight were regularly monitored. Intellectual development was assessed using the neuropsychological development scale for children aged 0-6 years and academic performance, and brain injury in children was assessed using brain magnetic resonance imaging.

c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G mutations were common in PAH gene. The genotype of c.158G>A mutation is compound heterozygous mutation, mainly mild hyperphenylalaninemia, but it is also found in both classic phenylketo-nuriaand mild phenylketonuria. 168 children with PAH deficiency who were followed up regularly had normal physical development without dwarfism or malnutrition.Among the 33 preschool children who underwent mental development assessment, 2 were mentally retarded and the initial treatment age was older than 6 months. Nine children with an average age of (17.13±2.42) years completed brain magnetic resonance imaging, 1 case was normal and 8 cases were abnormal. Brain magnetic resonance imaging results showed different differences. There were patchy or patchy hyperintense foci near the bilateral lateral ventricles on T2WI, and the intellectual development was normal. The blood phenylalanine concentration of one normal child was better and stably controlled within the ideal range than that of the other eight children.

c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G mutations were common in PAH gene. After standardized treatment, most children with PAH deficiency diagnosed by screening can obtain normal growth and intellectual development in adolescence, but there are different degrees of organic lesions in the cerebral white matter.

The cornerstone treatment of hyperphenylalaninemia (HPA) and phenylketonuria (PKU) is a lifelong low-protein diet with phenylalanine (Phe) free L-amino acid supplements. However, the PKU diet has significant shortcomings, and there is a clinically unmet need for new therapeutics to improve patient outcomes. CDX-6114 is a modified phenylalanine ammonia-lyase (PAL) enzyme obtained by a mutation in the Anabaena variabilis PAL sequence. CodeEvolver® protein engineering technology has been applied to improve the degradation resistance of the enzyme. In our first phase I trial, 19 patients were given a single oral dose of CDX-6114 at 7.5 g, 2.5 g, 0.7 g, or placebo in a cross-over design. After an overnight fast, patients received a standardised breakfast of 20 g of protein, thus exceeding the dietary recommendations for a single meal in patients with PKU. Plasma levels of Phe and cinnamic acid (CA) were measured over a 5-h period following CDX-6114 dosing. During the development of CDX-6114, a stability assessment using reverse-phase high-performance liquid chromatography (HPLC) assay revealed two peaks. The second peak was identified as CA. It was not previously known that as part of the mechanism of action, the CA remained associated with the protein following the conversion of Phe. Thus, recalculating the historical PAL enzyme amounts in CDX-6114 bulk substance was necessary. An updated extinction coefficient was achieved by applying a correction factor of 0.771 to previously reported doses. Postprandial plasma levels of Phe increased in all dose cohorts over time between 10% and 30% from baseline, although the actual peak of Phe levels was not achieved within the 5-h observation. When accounting for the interquartile ranges, these concentrations were similar to the placebo. As plasma levels of Phe were no longer a reliable marker for pharmacodynamics, the consistently detectable amount of CA seen in all patients who received CDX-6114 provided proof of the enzymatic activity of CDX-6114 in metabolising gastrointestinal Phe. Peak levels of CA were seen shortly after CDX-6114 intake, with a rapid decline, and remained low compared with the plasma Phe levels. This pattern indicates a short half-life, possibly due to the liquid formulation or the inability to withstand the lower pH in the human stomach compared with animal models in earlier studies. This was the first trial in patients with PKU to establish the safety and tolerability of CDX-6114. A single dose of CDX-6114 was safe and well tolerated, with no serious adverse events or presence of anti-drug antibodies detected. Efficacy will be explored in future trials using an optimised formulation.