[Analysis of PAH gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria].

OBJECTIVE: To explore the characteristics of phenylalanine hydroxylase (PAH) gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria (PKU). METHODS: Forty three PKU pedigrees diagnosed at the First Affiliated Hospital of Zhengzhou University between 2019 and 2021 were selected as the study subjects. Variants of the PAH gene of the probands were screened by high-throughput sequencing, and candidate variants were verified by Sanger sequencing. Negative cases were further analyzed by multiplex ligation-dependent probe amplification (MLPA) to detect large fragment deletions and duplications of the PAH gene. For 43 women undergoing subsequent pregnancy, Sanger sequencing, MLPA, combined with short tandem repeats (STR) sequence-based linkage analysis, were carried out for prenatal diagnosis. RESULTS: Among the 86 alleles carried by the 43 probands, 78 nucleotide variants (90.70%) and 3 large deletions (3.49%) were found based on high-throughput sequencing and MLPA. The 81 mutant alleles had included 21 missense variants, 5 splice site variants, 4 nonsense variants, 2 microdeletions, 1 insertional variant and 2 large fragment deletions. Relatively common variants have included p.Arg243Gln (23.26%), p.Arg111Ter (8.14%), EX6-96A>G (6.98%), p.Val399Val (5.81%) and p.Arg413Pro (4.65%). Most of the variants were located in exons 7, 11, 3, 6 and 12. For the 43 families undergoing prenatal diagnosis, 9 fetuses (20.45%) were diagnosed with PKU, 20 (45.45%) were heterozygous carriers, and 15 (34.09%) did not carry the same pathogenic allele as the proband. All neonates were followed up till 6 months old, and the accuracy of prenatal diagnosis was 100%. CONCLUSION: The combination of high-throughput sequencing, Sanger sequencing, MLPA and linkage analysis can increase the diagnostic rate of PKU and attain accurate prenatal diagnosis.

Characterizing the mechanism of action for mRNA therapeutics for the treatment of propionic acidemia, methylmalonic acidemia, and phenylketonuria.

Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively.

Genetic Landscape and Clinical Features of Hyperphenylalaninemia in North Ossetia-Alania: High Frequency of P281L and P211T Genetic Variants in the PAH Gene.

This study, conducted in the Republic of North Ossetia-Alania (RNOA), aimed to explore the genetic landscape of hyperphenylalaninemia (HPA) and phenylketonuria (PKU) in the Ossetian population using data from newborn screening (NBS). Through comprehensive molecular genetic analysis of 29 patients with HPA from diverse ethnic backgrounds, two major genetic variants in the PAH gene, P281L and P211T, were identified, constituting 50% of all detected pathogenic alleles in Ossetian patients. Remarkably, these variants exhibited an exceptionally high frequency in the Ossetian population, surpassing global prevalence rates. This study unveiled a notable prevalence of mild forms of HPA (78%), underscoring the importance of genetic counseling for carriers of pathogenic variants in the PAH gene. Moreover, the findings emphasized the necessity for ongoing monitoring of patients with mild forms, as they may lack significant symptoms for diagnosis, potentially impacting offspring. Overall, this research offers valuable insights into the genetic landscape of HPA and PKU in the Ossetian population.

Unveiling the genetic tapestry: Rare disease genomics of spinal muscular atrophy and phenylketonuria proteins.

Rare diseases, defined by their low prevalence, present significant challenges, including delayed detection, expensive treatments, and limited research. This study delves into the genetic basis of two noteworthy rare diseases in Saudi Arabia: Phenylketonuria (PKU) and Spinal Muscular Atrophy (SMA). PKU, resulting from mutations in the phenylalanine hydroxylase (PAH) gene, exhibits geographical variability and impacts intellectual abilities. SMA, characterized by motor neuron loss, is linked to mutations in the survival of motor neuron 1 (SMN1) gene. Recognizing the importance of unveiling signature genomics in rare diseases, we conducted a quantitative study on PAH and SMN1 proteins of multiple organisms by employing various quantitative techniques to assess genetic variations. The derived signature-genomics contributes to a deeper understanding of these critical genes, paving the way for enhanced diagnostics for disorders associated with PAH and SMN1.

[Analysis of pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase gene].

OBJECTIVE: To explore the pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase (PAH) gene among patients with PAH deficiency. METHODS: Thirty seven children diagnosed with PAH deficiency at the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University between July 2016 and June 2021 were selected as the study subjects. Clinical data and results of genetic testing were retrospectively analyzed. RESULTS: Among the 37 patients, mild hyperphenylalaninemia (HPA) was observed in 34 cases, two PAH variants (including c.158G>A), which formed a compound heterozygous mutation genotype, were detected in 33 patients, and the remainder one was found to harbor three PAH variants, including homozygous c.158G>A variants and a heterozygous c.842+2T>A variant. Classical phenylketonuria (PKU) was observed in 3 patients, and three PAH variants were detected in each of them, including two with c.[158G>A,842+2T>A]/c.728G>A and c.[158G>A,842+2T>A]/c.611A>G, respectively, and one with c.[158G>A, c.722G>A]/c.728G>A. The c.158G>A variant has a minimal influence on the PAH activity and is associated with a mild HPA phenotype. The variant should thereby be classified as likely benign. CONCLUSION: When the c.158G>A variant and other pathogenic variants are arranged in cis position, the ultimate phenotype will be determined by the pathogenicity of other variants.

[Genetic analysis of eighteen patients from Gansu Province with Tetrahydrobiopterin deficiency].

OBJECTIVE: To explore the genetic basis of eighteen patients with Tetrahydrobiopterin deficiency (BH4D) from Gansu Province. METHODS: Eighteen patients diagnosed with BH4D at Gansu Provincial Maternal and Child Health Care Hospital from January 2018 to December 2021 were selected as the study subjects. Whole exome sequencing was carried out, and candidate variants were verified by Sanger sequencing. RESULTS: All of the thirty-six alleles of the eighteen patients were successfully determined by molecular genetic testing. Sixteen patients were found to harbor variants of the PTS gene, and two had harbored variants of the QDPR gene. Ten variants were detected in the PTS gene, with the most common ones being c.259C>T (34.38%) and c.286G>A (15.63%). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.259C>T was classified as a pathogenic variant, whilst the c.286G>A, c.166G>A, c.200C>T, c.272A>G, c.402A>C, c.421G>T, c.84_291A>G and c.317C>T were classified as likely pathogenic variants. A novel c.289_290insCTT variant was classified as likely pathogenic (PM1+PM2_Supporting+PM3+PP3+PP4). The two variants (c.478C>T and c.665C>T) detected in the QDPR gene were both classified as variants of uncertain significance (PM1+PM2_Supporting+PP3+PP4). CONCLUSION: Genetic testing has clarified the pathogenic variants in these BH4D patients, which has enabled timely and accurate clinical intervention and treatment, and provided a reference for genetic counseling and reproductive guidance for their families.

An unusual case of trisomy 8 mosaicism complicated by coexistence of phenylketonuria.

Trisomy 8 syndrome, also known as " Warkany syndrome type 2 ", was first reported in 1971. Complete trisomy 8 are mostly aborted spontaneouslyinthe first trimester. Trisomy 8 mosaicism (T8M), predominated in the current cases reported. Itisahighlyheterogeneous Chromosome disorder. We know little about its effects on fertility. In this case, a patient with T8M combined with phenylketonuria was diagnosed. She's mentally retarded. After evaluating the anatomy and function of the reproductive system, the patient conceived through preimplantationgenetictesting-intracytoplasmicsperminjection-embryotransfer (PGT-ICSI-ET) and obtained a healthy fetus, which is the first report. The study focuses on the maintenance of fertility in patients with T8M, the effects of phenylketonuria and genetic counseling.

Tetrahydrobiopterin (BH4) treatment stabilizes tyrosine hydroxylase: Rescue of tyrosine hydroxylase deficiency phenotypes in human neurons and in a knock-in mouse model.

Proteostatic regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, is crucial for maintaining proper brain neurotransmitter homeostasis. Variants of the TH gene are associated with tyrosine hydroxylase deficiency (THD), a rare disorder with a wide phenotypic spectrum and variable response to treatment, which affects protein stability and may lead to accelerated degradation, loss of TH function and catecholamine deficiency. In this study, we investigated the effects of the TH cofactor tetrahydrobiopterin (BH4) on the stability of TH in isolated protein and in DAn- differentiated from iPSCs from a human healthy subject, as well as from THD patients with the R233H variant in homozygosity (THDA) and R328W and T399M variants in heterozygosity (THDB). We report an increase in TH and dopamine levels, and an increase in the number of TH+ cells in control and THDA cells. To translate this in vitro effect, we treated with BH4 a knock-in THD mouse model with Th variant corresponding to R233H in patients. Importantly, treatment with BH4 significantly improved motor function in these mice, as demonstrated by increased latency on the rotarod test and improved horizontal activity (catalepsy). In conclusion, our study demonstrates the stabilizing effects of BH4 on TH protein levels and function in THD neurons and mice, rescuing disease phenotypes and improving motor outcomes. These findings highlight the therapeutic potential of BH4 as a treatment option for THDA patients with specific variants and provide insights into the modulation of TH stability and its implications for THD management.

State-of-the-art 2023 on gene therapy for phenylketonuria.

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.

A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant.

The c.1222C>T (p.Arg408Trp) phenylalanine hydroxylase (PAH) variant is the most frequent cause of phenylketonuria (PKU), an autosomal recessive disorder characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Here we devised a therapeutic base editing strategy to correct the variant, using prime-edited hepatocyte cell lines engineered with the c.1222C>T variant to screen a variety of adenine base editors and guide RNAs in vitro, followed by assessment in c.1222C>T humanized mice in vivo. We found that upon delivery of a selected adenine base editor mRNA/guide RNA combination into mice via lipid nanoparticles (LNPs), there was sufficient PAH editing in the liver to fully normalize blood Phe levels within 48 h. This work establishes the viability of a base editing strategy to correct the most common pathogenic variant found in individuals with the most common inborn error of metabolism, albeit with potential limitations compared with other genome editing approaches.

DNAJC12 in Monoamine Metabolism, Neurodevelopment, and Neurodegeneration.

Recent studies show that pathogenic variants in DNAJC12, a co-chaperone for monoamine synthesis, may cause mild hyperphenylalaninemia with infantile dystonia, young-onset parkinsonism, developmental delay and cognitive deficits. DNAJC12 has been included in newborn screening, most revealingly in Spain, and those results highlight the importance of genetic diagnosis and early intervention in combating human disease. However, practitioners may be unaware of these advances and it is probable that many patients, especially adults, have yet to receive molecular testing for DNAJC12. Hence, this review summarizes genotype-phenotype relationships and treatment paradigms for patients with pathogenic variants in DNAJC12. It provides an overview of the structure of DNAJC12 protein, known genetic variants, domains, and binding partners, and elaborates on its role in monoamine synthesis, disease etiology, and pathogenesis. © 2023 International Parkinson and Movement Disorder Society.

Genotypic variants of the tetrahydrobiopterin (BH4) biosynthesis genes in patients with hyperphenylalaninemia from different regions of Iran.

BACKGROUND: Hyperphenylalaninemia (HPA) is a metabolic disorder classified into phenylalanine-4-hydroxylase (PAH) and non-PAH deficiency. The latter is produced by mutations in genes involved in the tetrahydrobiopterin (BH4) biosynthesis pathway and DNAJC12 pathogenetic variants. The BH4 metabolism, including de novo biosynthesis involved genes (i.e., guanosine 5'-triphosphate cyclohydrolase I (GTPCH/GCH1), sepiapterin reductase (SR/SPR), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS)), and two genes that play roles in cofactor regeneration pathway (i.e., dihydropteridine reductase (DHPR/QDPR) and pterin-4α-carbinolamine dehydratase (PCD/PCBD1)). The subsequent systemic hyperphenylalaninemia and monoamine neurotransmitter deficiency lead to neurological consequences. The high rate of consanguineous marriages in Iran substantially increases the incidence of BH4 deficiency. METHODS: We utilized the Sanger sequencing technique in this study to investigate 14 Iranian patients with non-PAH deficiency. All affected subjects in this study had HPA and no mutation was detected in their PAH gene. RESULTS: We successfully identified six mutant alleles in BH4-deficiency-associated genes, including three novel mutations: one in QDPR, one in PTS, and one in the PCBD1 gene, thus giving a definite diagnosis to these patients. CONCLUSION: In this light, appropriate patient management may follow. The clinical effect of reported variants is essential for genetic counseling and prenatal diagnosis in the patients' families and significant for the improvement of precision medicine.

Systems Biology and Inborn Error of Metabolism: Analytical Strategy in Investigating Different Biochemical/Genetic Parameters.

Inborn errors of metabolism (IEM) are a group of about 500 rare genetic diseases with large diversity and complexity due to number of metabolic pathways involved in. Establishing a correct diagnosis and identifying the specific clinical phenotype is consequently a difficult task. However, an inclusive diagnosis able in capturing the different clinical phenotypes is mandatory for successful treatment. However, in contrast with Garrod's basic assumption "one-gene one-disease," no "simple" correlation between genotype-phenotype can be vindicated in IEMs. An illustrative example of IEM is Phenylketonuria (PKU), an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism, ascribed to variants of the phenylalanine hydroxylase (PAH) gene encoding for the enzyme complex phenylalanine-hydroxylase. Blood values of Phe allow classifying PKU into different clinical phenotypes, albeit the participation of other genetic/biochemical pathways in the pathogenetic mechanisms remains elusive. Indeed, it has been shown that the most serious complications, such as cognitive impairment, are not only related to the gene dysfunction but also to the patient's background and the participation of several nongenetic factors.Therefore, a Systems Biology-based strategy is required in addressing IEM complexity, and in identifying the interplay between different pathways in shaping the clinical phenotype. Such an approach should entail the concerted investigation of genomic, transcriptomics, proteomics, metabolomics profiles altogether with phenylalanine and amino acids metabolism. Noticeably, this "omic" perspective could be instrumental in planning personalized treatment, tailored accordingly to the disease profile and prognosis.

Results of neonatal screening for congenital hypothyroidism and hyperphenylalaninemia in Zhejiang province from 1999 to 2022. / 浙江省1200ä¸‡ä¾‹æ–°ç”Ÿå„¿å ˆå¤©æ€§ç”²çŠ¶è ºåŠŸèƒ½å‡é€€ç—‡ã€é«˜è‹¯ä¸™æ°¨é ¸è¡€ç—‡ç­›æŸ¥ç»“æžœåˆ†æž.

OBJECTIVES: To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) in Zhejiang province from 1999 to 2022. METHODS: A total of 11 922 318 newborns were screened from September 1999 and December 2022 in Zhejiang province. The blood thyroid stimulating hormone (TSH) levels were measured by a 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. RESULTS: The overall neonatal screening rate during 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 1/21 176. 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 hotspot mutations. There were 81 PAH gene variants detected in a total of 250 cases of cPKU, and c728G>A (p.R243Q) (24.4%), c.721C>T (p.R241C) (15.0%) were the hotspot mutations. 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 cases of BH4D, and c.259C>T (p.P87S) (31.9%) was the hotspot mutation. CONCLUSIONS: The incident of CH has increased from 1999 to 2022 in Zhejiang province, and it is higher than that of national and global levels; while the incidence of HPA is similar to the national average. DUOX2 gene variation is the most common in CH patients; c.728G>A (p.R243Q) is the hotspot mutation in cPKU patients, while c.259C>T (p.P87S) is the hotspot mutation in BH4D patients.

Analysis of gene variation and long-term follow-up in children with phenylalanine hydroxylase deficiency diagnosed by newborn screening. / æ–°ç”Ÿå„¿ç­›æŸ¥ç¡®è¯Šè‹¯ä¸™æ°¨é ¸ç¾ŸåŒ–é ¶ç¼ºä¹ç—‡æ‚£è€ åŸºå› å˜å¼‚åŠé•¿æœŸéšè®¿åˆ†æž.

OBJECTIVES: To retrospectively analyze the variation and characteristics of phenylalanine hydroxylase (PAH) gene, and to observe the long-term treatment effect and follow-up of newborns with PAH deficiency. METHODS: Clinical data, treatment and follow-up results of 198 patients with PAH deficiency diagnosed by newborn screening in Jinan from 1996 to 2021 were collected. The genetic analysis of 55 patients with PAH deficiency diagnosed by newborn screening in Jinan and 213 patients referred from the surrounding areas of Jinan were summarized. Gene variations were checked by a customized Panel gene detection method. Blood phenylalanine-concentration and physical development indicators including height and weight were regularly monitored. Intellectual development was assessed using a neuropsychological development scale for patients aged 0-6 years and academic performance, and brain injury in patients was assessed using brain magnetic resonance imaging. RESULTS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. The genotype of c.158G>A variation is compound heterozygous variation, with mainly a mild hyperpheny-lalaninemia. 168 patients with PAH deficiency who were followed-up regularly had normal physical development without dwarfism or malnutrition. Among the 33 preschool patients who underwent mental development assessment, 2 were mentally retarded and the initial treatment age was older than 6 months. Nine patients with an average age of (17.13±2.42) years completed brain magnetic resonance imaging, one case was normal, and 8 cases were abnormal. There were patchy or patchy hyperintense foci near the bilateral lateral ventricles on T2WI, and the intellectual development was normal. Compared with the other eight patients, the blood phenylalanine concentration of the normal child was better and stably controlled within the ideal range. CONCLUSIONS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. After standardized treatment, most patients 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.

Preliminary analysis of oral and gut microbiome of an elderly patient with late-diagnosed phenylketonuria.

Phenylketonuria (PKU) is a metabolic and genetic disorder caused by a phenylalanine hydroxylase (PAH) gene deficiency that raises Phe levels in organs. Dietary therapy involves an elimination diet and Phe-free items, which may alter microbiota. The study examined the oral and intestinal microbiomes of a 63-year-old PKU patient and a control man, living in rural areas. iSeq100 (Illumina) sequenced the stool and oral 16S rRNA gene V3-V4 region. PKU guts had more Firmicutes and fewer Bacteroidetes than control. Clostridia predominated in PKU, while Bacteroidia dominated in control. Oral Bacteroidetes. Firmicutes, Proteobacteria, and Fusobacteria phyla were similar in both men. The microbiome may differ from those fed a Phe-free diet from birth due to late diagnosis and treatment of PKU. Due to the age of the 63-year-old patient's and late therapy, the results differ from earlier studies. No study has compared an older PKU patient's gut and oral microbiomes.

Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need.

The c.1222C>T (p.Arg408Trp) variant in the phenylalanine hydroxylase gene (PAH) is the most frequent cause of phenylketonuria (PKU), the most common inborn error of metabolism. This autosomal-recessive disorder is characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Using real-world data, we observed that despite dietary and medical interventions, most PKU individuals harboring at least one c.1222C>T variant experience chronic, severe Phe elevations and do not comply with Phe monitoring guidelines. Motivated by these findings, we generated an edited c.1222C>T hepatocyte cell line and humanized c.1222C>T mouse models, with which we demonstrated efficient in vitro and in vivo correction of the variant with prime editing. Delivery via adeno-associated viral (AAV) vectors reproducibly achieved complete normalization of blood Phe levels in PKU mice, with up to 52% whole-liver corrective PAH editing. These studies validate a strategy involving prime editing as a potential treatment for a large proportion of individuals with PKU.

Predictors of eventual requirement of phenylalanine-restricted diet in young infants with phenylalanine hydroxylase deficiency initially managed with sapropterin monotherapy.

BACKGROUND: Phenylalanine (Phe)-restricted diet is associated with lower quality of life for patients with phenylketonuria (PKU), and a concern for caregivers of recently-diagnosed infants. Sapropterin is an oral drug used as an alternative or adjunct to dietary treatment. We have observed that some of the young infants initially managed successfully with sapropterin monotherapy have required dietary treatment in long-term follow-up. We aimed to determine the baseline factors associated with future initiation of dietary treatment in these patients. METHODS: Data were obtained retrospectively from the medical records of 80 PKU patients started on sapropterin monotherapy before 3 months of age between 2011 and 2021. RESULTS: The patients were followed for a median of 3.9 years (Q1-Q3: 2.5-5.75 years). Sapropterin was tapered down and discontinued in 5 patients (6.3%) as their Phe levels remained below 360 µmol/L without treatment. Sapropterin monotherapy was sufficient in 62 patients (77.5%), while 13 (16.2%) required dietary treatment. Phe and tyrosine (Tyr) levels, and Phe:Tyr ratios differed significantly among the patients maintained on sapropterin monotherapy and those started on dietary treatment, but the Phe:Tyr ratio at diagnosis was the most important independent baseline variable (OR: 1.61, 95% CI: 1.15-2.27, p = 0.006), with Phe:Tyr ratio at diagnosis >5.25 associated with dietary treatment (sensitivity: 90.0%, specificity: 81.8%). Genotypic phenotype value (GPV), unavailable at baseline, was also associated with dietary treatment (median GPV 9.2 vs. 3.8, p = 0.006), but some genotypes were not specific to the final treatment modality. DISCUSSION: We propose that the Phe:Tyr ratio at diagnosis is an important indicator to predict dietary requirement in young infants initially managed with sapropterin monotherapy.

[Development of analytics in newborn screening-from the Guthrie card to genetics]. / Entwicklung der Analytik im Neugeborenen-Screening ­ Von der Guthrie-Karte zur Genetik.

For more than five decades, all newborns in Germany have been offered a screening examination for the early detection of congenital treatable diseases. Since its inception, about 35 million children have been screened in this way.Originally, screening exams only included early detection of phenylketonuria, which, without timely treatment, would lead to mental retardation that could no longer be corrected. The bacteriological Guthrie test allowed the detection of elevated concentrations of phenylalanine. The methods used today are the result of decades of development. They have been expanded to include tests to determine enzyme activities, immunoassays for the early detection of important hormonal disorders such as congenital hypothyroidism, and high-pressure liquid chromatography for the diagnosis of pathologic hemoglobins. The very sophisticated tandem mass spectrometry enables the simultaneous detection of amino acid and fatty acid compounds. Steroids can also be identified. The specificity can be further increased by combining tandem mass spectrometry with chromatographic pre-separation. In recent years, chemical-analytical analyses have been supplemented by genetic diagnostic methods such as quantitative or qualitative polymerase chain reaction (PCR).The current state of laboratory technology is by no means final. Both classical analytics and especially genetic methods are facing further rapid development. Although the expansion of screening is also a consequence of technical development, the inclusion of further congenital diseases is fundamentally dependent on the given therapy. But it is precisely here that many innovations are currently being investigated. Gene therapy is at the forefront of interest.

Carriers of autosomal recessive conditions: are they really 'unaffected?'

Mendel's Law of Dominance suggests that recessive disease expression requires the inheritance of two mutated alleles as the dominant, wildtype allele suppresses disease presentation leading to the expression of physiological normal phenotypes. However, there is existing evidence that challenges this school of thought. Here, we summarise existing literature evaluating metabolic and health impacts among carriers of autosomal recessive conditions, focusing on phenylketonuria (PKU), classical homocystinuria, galactosemia and Usher syndrome as examples. Our findings suggest that carriers, often described as 'unaffected', may actually display attenuated symptoms for the recessive disease they are carrying. For instance, PKU is an inborn error of metabolism characterised by the build-up of plasma phenylalanine attributed to the deficiency of the phenylalanine hydroxylase (PAH) enzyme. While less severe, PKU carriers also exhibit this impaired enzymatic activity, leading to elevated plasma phenylalanine levels, especially after phenylalanine consumption. Related to these metabolic alterations in the PAH pathway, there is early evidence to suggest that PKU carriers may have compromised cognitive and mental health outcomes. Overall, research on the health and metabolic impacts of PKU carriers is sparse, with most studies conducted several decades ago. However, early evidence suggests that intermediate phenotypes among carriers of autosomal recessive conditions are plausible. The illustrated possible intermediate phenotypes observed among carriers necessitates future research to determine possible clinical implications among this population.