Screening For Phenylketonuria Research Articles

Perspectives and Insights Into Phenylketonuria: Patient Narratives About the Early Years Following Newborn Screening.

Newborn screening for Phenylketonuria (PKU) began in 1963, and since then knowledge and treatment recommendations have evolved. In the decades following newborn screening for PKU, individual and family experiences varied widely. We present narratives by people living with PKU during these years, including individuals actively following in PKU clinic and those who have been out of PKU clinic for many years. These stories describe different individual experiences, including diet discontinuation in childhood, changing treatment guidelines, and new treatments that have become available.

Rapid Fingerprinting of Urinary Volatile Metabolites and Point-of-Care Diagnosis of Phenylketonuria on a Patterned Nanorod Sensor Array with Multiplexed Surface-Enhanced Raman Scattering Readouts.

Phenylketonuria (PKU) is one of the most common genetic metabolic diseases, especially among newborns. Traditional clinical examination of newborn blood samples for PKU is invasive, laborious, and limited to hospitals and healthcare facilities. We reported herein a SERS-based sensor array with three thiophenolic nanoreceptors built on a patterned nanorod vertical array for rapid and inexpensive detection of characteristic volatile biomarkers indicative of PKU in the urine and accurate classification of newborn baby patients all performed on a hand-held SERS spectrophotometer. The well-ordered array was generated from the volatility-driven assembly of gold nanorods (AuNRs) into an upright and closely packed hexagonal configuration. The uniformly distributed nanowells between AuNRs offered an intense and aspect-ratio-dependent plasmonic field for the molecular enhancement of SERS outputs. The SERS-based detector was integrated into a test chip for regular monitoring of volatile phenylketone bodies in the spiked solution or patients' urine within 5 min, allowing the quantification of a wide variety of normal or abnormal metabolites at their physiologically relevant concentration range. The detection limits for common biomarkers of PKU, including phenylpyruvic acid, 4-hydroxyphenylacetic acid, and phenylacetic acid, were at a few μM and well below the diagnostic thresholds. Moreover, the volatile headspace mixtures from a given urine sample could be fingerprinted by the sensor array and discriminated using machine-learning algorithms. Ultimately, the discrimination of baby patients among 26 cases of mild and classic PKU phenotypes and 17 cases of healthy volunteers could be realized with an overall accuracy of 97%. This hand-held SERS platform plays a pivotal role in advancing healthcare applications in quick screening of neonatal PKU through a facile urinary vapor test.

Delays in Newborn Screening for Phenylketonuria from Birth to Diagnosis and Factors Affecting This.

This study aims to evaluate the process of neonatal phenylketonuria (PKU) screening from birth to admission to the pediatric metabolism polyclinic, determining delays in the screening program and the factors influencing them. This study was conducted during 2021-2023. Blood collection dates, results, and probable parameters causing delays in the screening program were recorded. This study included 118 infants. Admission time to the polyclinic was (mean ± SD) 25.2 ± 12.6 days (min-max: 3.4-78.9 days). Admission time was significantly high for refugees, those whose parents were consanguineous, and those who had more heel-prick blood samples taken (p < 0.001, p = 0.005, and p < 0.001, respectively). The first heel-prick blood phenylalanine (phe) level was significantly negatively correlated with the admission time (p < 0.001). Patients' admission time whose first blood phe level < 240 μmol/L was statistically significantly higher than in those with ≥240 μmol/L (p < 0.001). We determined that there were delays in PKU screening from birth to admission to the polyclinic. Being a refugee, the presence of consanguineous marriages, the increase in the number of heel-prick tests, and blood phe levels at a range of 120-240 μmol/L were the factors that played a role in this delay. Taking steps to reduce the impact of these parameters can prevent delays in newborn PKU screening and increase the success of the screening program.

Newborn screening in France: news and perspectives

Newborn screening is a major public health concern. In France, it was established in 1972 with systematic screening for phenylketonuria. Subsequently, other screenings, including congenital hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis, and sickle cell disease, were added. The introduction of tandem mass spectrometry in screening laboratories in 2020 enabled the inclusion of eight additional inherited metabolic diseases: aminoacidopathies (tyrosinemia type I, maple syrup urine disease, and homocystinuria), organic acidurias (isovaleric and glutaric type I acidurias), and disorders of fatty acid metabolism (MCADD, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), and primary carnitine deficiency). We briefly present these newly added diseases, of which public awareness is still incomplete.

Fast Label-Free Metabolic Profile Recognition Identifies Phenylketonuria and Subtypes.

Phenylketonuria (PKU) is the most common inherited metabolic disease in humans. Clinical screening of newborn heel blood samples for PKU is costly and time-consuming because it requires multiple procedures, like isotope labeling and derivatization, and PKU subtype identification requires an additional urine sample. Delayed diagnosis of PKU, or subtype identification can result in mental disability. Here, plasmonic silver nanoshells are used for laser desorption/ionization mass spectrometry (MS) detection of PKU with label-free assay by recognizing metabolic profile in dried blood spot (DBS) samples. A total of 1100 subjects are recruited and each DBS sample can be processed in seconds. This platform achieves PKU screening with a sensitivity of 0.985 and specificity of 0.995, which is comparable to existing clinical liquid chromatography MS (LC-MS) methods. This method can process 360 samples per hour, compared with the LC-MS method which processes only 30 samples per hour. Moreover, this assay enables precise identification of PKU subtypes without the need for a urine sample. It is demonstrated that this platform enables high-performance and fast, low-cost PKU screening and subtype identification. This approach might be suitable for the detection of other clinically relevant biomarkers in blood or other clinical samples.

Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria.

Phenylketonuria (PKU) is an autosomal genetic disorder caused by a deficiency of the phenylalanine hydroxylase (PAH) enzyme. The lack of PAH results in the inability of phenylalanine (PHE) to transform into tyrosine (TYR). Consequently, this leads to the accumulation of PHE in the blood samples of newborns causing metabolic diseases such as irreversible neurological problems. An analysis was required for determining the values of PHE and TYR in blood samples from newborn babies. In this study, therefore, we developed a derivatized method to monitor PHE and TYR in plasma samples using liquid phase chromatography linked with quadrupole mass spectrometry. Accessible formaldehyde isotopes and cyanoborohydride were used to react with PHE and TYR amino groups to generate h2-formaldehyde-modified PHE and TYR (as standards) and d2-formaldehyde-modified PHE and TYR (as internal standards). We used tandem mass spectrometry for multiple reaction monitoring. We demonstrated a derivatized method suitable for the PKU screening of newborns. The recoveries for PHE and TYR were 85% and 90%, respectively. Furthermore, we compared the values of PHE and TYR in different human plasma sample storage methods, including direct plasma and dried blood spots, and the results showed no significant difference.

Progress of newborn screening in China.

Newborn screening (NBS) plays a significant role in reducing the risk of birth defects. NBS in China began in the early 1980s. Under the protection of laws and regulations and the leadership of the national health administration, approved screening centers in public hospitals took the responsibility for publicity, screening, diagnosis, treatment, follow-up and management of birth defects. As of 2022, 31 provinces (autonomous regions and municipalities directly under the central government) have carried out NBS for phenylketonuria, congenital hypothyroidism, and hearing loss, 23 provinces have carried out screening for glucose-6-phosphate dehydrogenase (with a screening rate of 89.24%), and 24 provinces have carried out screening for congenital adrenal cortical hyperplasia (91.45% screening rate). Over the past four decades, screening techniques have evolved from bacterial inhibition, fluorescence analysis, and tandem mass spectrometry for the detection of biochemical markers to genetic testing, which has greatly contributed to the expansion of the types of diseases screened for. The combined use of metabolomics and genomics is currently being explored. Effective management and rigorous quality control of NBS are prerequisites for improving the quality and ensuring the accuracy of screening. The Quality Management System for Newborn Screening System Network (QMS-NBS), established by the National Center for Clinical Laboratories, covers all screening centers and related blood collection agencies. The operation of the QMS-NBS allows the quality and performance of screening to be transparent and measurable, ensuring the quality and efficiency of screening. This article provides an overview of the history of NBS, especially the evolution of policies for the NBS in China, the construction of screening institutions, the number of newborns screened, the incidence rates of screened diseases, the changes in screening technology, the expansion of new diseases screened for, and the quality control of NBS. Overall, the progress in NBS in China has not only benefited from the development and standardization at the technological level, but also benefited from the construction of policies, regulations and ethics.

Newborn Screening for Neurodevelopmental Disorders May Exacerbate Health Disparities.

Newborn screening (NBS) began in the early 1960s with screening for phenylketonuria on blood collected on filter paper. The number of conditions included in NBS programs expanded significantly with the adoption of tandem mass spectrometry. The recommended uniform screening panel provides national guidance and has reduced state variability. Universality and uniformity have been supported to promote equity. Recently, a number of researchers have suggested expanding NBS to include genomic sequencing to identify all genetic disorders in newborns. This has been specifically suggested for genes that increase the risk for neurodevelopmental disorders (NDDs), with the presumption that early identification in the newborn period would reduce disabilities. We offer arguments to show that genomic sequencing of newborns for NDDs risks exacerbating disparities. First, the diagnosis of NDD requires clinical expertise, and both genetic and neurodevelopmental expertise are in short supply, leading to disparities in access to timely follow-up. Second, therapies for children with NDDs are insufficient to meet their needs. Increasing early identification for those at risk who may never manifest developmental delays could shift limited resources to those children whose parents are more poised to advocate, worsening disparities in access to services. Rather, we suggest an alternative: genomic sequencing of all children with diagnosed NDDs. This focused strategy would have the potential to target genomic sequencing at children who manifest NDDs across diverse populations which could better improve our understanding of contributory genes to NDDs.

Potential alteration enabled peptide elongation for direct serum detection of S100B in the early screening of phenylketonuria

DNA and aptamers have introduced new possibilities for utilizing synthetic probes in biosensing, effectively substituting antibodies. This substitution not only significantly cuts costs but also preserves analytical performance. However, in some cases, other biomacromolecules such as DNA processing enzymes remain indispensable for achieving signal amplification and high sensitivity. In this context, a novel bioassay has been developed, entirely eliminating the need for biomacromolecules. Instead, this approach employs fully synthesizable and modifiable peptides as the sensing probes. In lieu of enzymes, the strategy involves utilizing electrochemical potentials to guide peptide strand cleavage and the cross-coupling of peptide side chains at specific amino acids. This process mimics DNA-like chain elongation. The technique has already demonstrated success in detecting peripheral blood S100B levels, offering an early screening method for phenylketonuria in infants. These promising results suggest that the novel approach of utilizing electrochemical potentials to direct peptide bonding and cross-linking could find practical applications in clinical scenarios in the near future.

Genomic newborn screening: Are we entering a new era of screening?

Population newborn screening (NBS) for phenylketonuria began in the United States in 1963. In the 1990s electrospray ionization mass spectrometry permitted an array of pathognomonic metabolites to be identified simultaneously, enabling up to 60 disorders to be recognized with a single test. In response, differing approaches to the assessment of the harms and benefits of screening have resulted in variable screening panels worldwide. Thirty years on and another screening revolution has emerged with the potential for first line genomic testing extending the range of screening conditions recognized after birth to many hundreds. At the annual SSIEM conference in 2022 in Freiburg, Germany, an interactive plenary discussion on genomic screening strategies and their challenges and opportunities was conducted. The Genomics England Research project proposes the use of Whole Genome Sequencing to offer extended NBS to 100 000 babies for defined conditions with a clear benefit for the child. The European Organization for Rare Diseases seeks to include "actionable" conditions considering also other types of benefits. Hopkins Van Mil, a private UK research institute, determined the views of citizens and revealed as a precondition that families are provided with adequate information, qualified support, and that autonomy and data are protected. From an ethical standpoint, the benefits ascribed to screening and early treatment need to be considered in relation to asymptomatic, phenotypically mild or late-onset presentations, where presymptomatic treatment may not be required. The different perspectives and arguments demonstrate the unique burden of responsibility on those proposing new and far-reaching developments in NBS programs and the need to carefully consider both harms and benefits.

Comparison of Tandem Mass Spectrometry and the Fluorometric Method-Parallel Phenylalanine Measurement on a Large Fresh Sample Series and Implications for Newborn Screening for Phenylketonuria.

Phenylketonuria (PKU) was the first disease to be identified by the newborn screening (NBS) program. Currently, there are various methods for determining phenylalanine (Phe) values, with tandem mass spectrometry (MS/MS) being the most widely used method worldwide. We aimed to compare the MS/MS method with the fluorometric method (FM) for measuring Phe in the dried blood spot (DBS) and the efficacy of both methods in the NBS program. The FM was performed using a neonatal phenylalanine kit and a VICTOR2TM D fluorometer. The MS/MS method was performed using a NeoBaseTM 2 kit and a Waters Xevo TQD mass spectrometer. The Phe values measured with the MS/MS method were compared to those determined by the FM. The cut-off value for the NBS program was set at 120 µmol/L for FM and 85 µmol/L for MS/MS. We analyzed 54,934 DBS. The measured Phe values varied from 12 to 664 µmol/L, with a median of 46 µmol/L for the MS/MS method and from 10 to 710 µmol/L, with a median of 70 µmol/L for the FM. The Bland-Altman analysis indicated a bias of -38.9% (-23.61 µmol/L) with an SD of 21.3% (13.89 µmol/L) when comparing the MS/MS method to the FM. The Phe value exceeded the cut-off in 187 samples measured with FM and 112 samples measured with MS/MS. The FM had 181 false positives, while the MS/MS method had 106 false positives. Our study showed that the MS/MS method gives lower results compared to the FM. Despite that, none of the true positives would be missed, and the number of false-positive results would be significantly lower compared to the FM.

Mutation Characteristics of Phenylalanine Hydroxylase Gene in Children with Phenylketonuria in Yinchuan City.

Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive disorder affecting phenylalanine (Phe) metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene. It has a complex phenotype with many variants and genotypes in various populations. This study sets out to analyze the screening results of children with phenylketonuria (PKU) in Yinchuan City and characterize the mutation variants of the PAH gene. Phenylketonuria screening results were retrospectively analyzed in 398,605 neonates (207,361 males and 191,244 females) born in different maternity hospitals in Yinchuan City between January 2017 and December 2021. Screening for genetic metabolic diseases was performed with parental consent at their own expense. A comprehensive diagnosis was performed by integrating tandem mass spectrometry (MS/MS) findings with clinical presentations. High-throughput sequencing (HTS) was used to detect genetic and metabolic disease-associated genes in children with PKU who were clinically diagnosed and voluntarily tested. The identified loci were validated through Sanger sequencing and parental verification. Among the screened newborns, 45 (11.3/100,000) PKU cases were diagnosed. In the 38 cases that underwent self-financed PAH sequencing, 56 mutations were detected in 76 chromosomes, with an overall detection rate of 73.7%. All patients harbored mutant genes, and the 56 mutations detected identified represented 14 variants, including 8 missense mutations, 2 splicing mutations, 2 nonsense mutations, and 2 silent mutations. The mutations were primarily distributed in exons 2, 3, 6, 7, 9, 11, and intron 4, with the highest frequency observed in exon 7 (25 [44.7%]), followed by exon 11 (15 [26.7%]). The most prevalent mutations were exon 7-p.R252W (10 [17.9%]) and exon 7-p.R261Q (8 [14.3%]). The PAH gene mutations in children with PKU in Yinchuan City are predominantly concentrated in exons 6, 7, and 11, with the highest detection rates observed for p.R252W and p.R261Q mutations.

Optimization of Phenylalanine Cut-Off Value in Newborn Screening Based on Blood Sampling Time.

The aim of this study was to optimize the cut-off value of phenylalanine (Phe) for phenylketonuria (PKU) screening in Xinjiang Uygur Autonomous Region based on the time of blood sampling. In this study, 110,806 neonates born in 91 obstetrics and gynecology hospitals of Xinjiang Uygur Autonomous Region between June 2017 and December 2019 were divided into two groups (i.e., groups 1 and 2) based on the sampling time. The concentration of Phe was determined using fluorimetric method. The optimization of the Phe cut-off value was conducted using the receiver operating characteristic curve from the treating set involving 80,354 neonates. Then, the diagnostic values of the optimized Phe cut-off value were evaluated using validation set involving 30,452 neonates, based on the comparison of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) obtained from conventional cut-off value. A range of cut-off values was used for preliminary Phe concentrations in the two groups to analyze the sensitivity, specificity, PPV, and NPV. The optimized cut-off value of Phe in group 1 was 2.0, while that in the group 2 was 2.21. A comparison was given to PPV, NPV, sensitivity, and specificity generated by the optimized cut-off value and the conventional cut-off value, which yielded similar sensitivity, specificity, and PPV, and less recalled number of samples. The optimization of cut-off value of Phe based on sampling time is feasible for PKU screening in Xinjiang Uygur Autonomous Region. In addition, the false positive rate was significantly reduced, which may save more efforts in sample recalling process. · The optimization of Phe cut-off value for Xinjiang Region.. · The optimized cut-off value reduced the recalling samples.. · Our cut-off value is feasible for PKU screening in Xinjiang..

Cost-Effectiveness of Newborn Screening for Phenylketonuria and Congenital Hypothyroidism

To evaluate the long-term costs and health effects of the Swedish newborn screening program for classic phenylketonuria (PKU) alone and in combination with congenital hypothyroidism compared with no screening. A decision-analytic model was developed to estimate and compare the long-term (80years) costs and health effects of newborn screening for PKU and congenital hypothyroidism. Data were obtained from the literature and translated to Swedish conditions. A societal perspective was taken, including costs falling on health care providers, municipal care and services, as well as production loss due to morbidity. Screening 100 000 newborns for PKU resulted in 73 gained quality-adjusted life-years (QALYs) compared with no screening. When adding congenital hypothyroidism, the number of gained QALYs was 232 compared with PKU alone, adding up to a total of 305 QALYs gained. Corresponding cost estimates were $80.8, $70.3, and $10.05 million USD for no screening, PKU screening, and PKU plus congenital hypothyroidism screening, respectively, indicating that screening for PKU plus congenital hypothyroidism was more effective and less costly compared with the other strategies. The majority of cost savings with PKU plus congenital hypothyroidism screening was due to reductions in productivity losses and municipal care and services costs. The Swedish newborn screening program for PKU and congenital hypothyroidism saves substantial costs for society while generating additional QALYs, emphasizing the importance of public investments in early diagnosis and treatment.

Random forest classifier improving phenylketonuria screening performance in two Chinese populations.

Phenylketonuria (PKU) is a genetic disorder with amino acid metabolic defect, which does great harms to the development of newborns and children. Early diagnosis and treatment can effectively prevent the disease progression. Here we developed a PKU screening model using random forest classifier (RFC) to improve PKU screening performance with excellent sensitivity, false positive rate (FPR) and positive predictive value (PPV) in all the validation dataset and two testing Chinese populations. RFC represented outstanding advantages comparing several different classification models based on machine learning and the traditional logistic regression model. RFC is promising to be applied to neonatal PKU screening.

Epidemiological aspects in phenylketonuria patients from a region in northwestern Romania

Introduction. Phenylketonuria (PKU), a genetic disease with autosomal dominant transmission, is the most frequent inborn error in aminoacidic metabolism. The variations in phenylalanine-hydroxylase (PAH) gene lead to a lowered enzymatic activity causing hyperphenylalaninemia. PKU has a mean European prevalence of 1:10,000 newborns, with a large variation in different ethnicities and geographic regions. The large genetic variability (over 1200 genetic variants known) as well as other factors determines a wide spectrum of metabolic phenotypes. Untreated, PKU leads to irreversible intellectual disability, low stature, hypopigmentation, motor deficits, seizures, but the early diagnosis and treatment enables almost normal somatic and mental development. Aim. The aim of this study is the determination of the impact of non-genetic factors over the clinical phenotype of PKU patients in a region of north-west Romania. Material and method. The study group is formed from 44 patients diagnosed with phenylketonuria in the 1981 – 2021 period, found in the database of Bihor Regional Center for Medical Genetics, Emergency Clinical County Hospital, Oradea, Romania. The collected data was referring to the age, sex and domicile of the patients, the age of the diagnosis and the beginning of the treatment, also the metabolic control over the years, the metabolic phenotype of the patients and its impact on the clinical phenotype (IQ, the presence or absence of intellectual disability or the existence of a specific clinical phenotype). Results. The majority of patients (66%) were diagnosticated with phenylketonuria in the first 4 months of life, although there were cases with a late diagnosis, 20.5% of the patients were diagnosticated after the age of 1 year. Based on the pre-treatment plasmatic levels of phenylalanine, the majority of cases (72.7%) had a severe metabolic phenotype (classic PKU - cPKU), 20.5% of cases had a milder form of PKU (mPKU) and 6.8% of patients were found with a mild hyperphenylalaninemia (HPA). In the case of 23 patients, an optimal metabolic control was not obtained. The specific phenotype (blonde hair, light skin, blue eyes) was found in 22.7% of cases, 77.3% not having these features. At 68.2% of cases intellectual disability was found, with different levels of severity: 5 patients (11.5%) had liminal intellect, 9 patients (20.5%) had mild mental retardation, 6 patients (13.6%) had moderate mental retardation, 9 cases (20.5%) were with severe mental retardation and 1 patient (2.3%) had profound mental retardation; 31.8% of cases had normal intellect. The prevalence in Bihor county is 1:7,843 newborns. Discussions. A partial or, in rare cases, total lack of dietetic treatment was observed in all patients over 20 years old (current age). The delay in treatment initiation or an insufficient treatment, with a suboptimal metabolic control, will affect patient’s intellect, regardless of metabolic phenotype. If in 20 years old patients, or older, the main reason for mental retardation is the lack of dietetic treatment availability in the first years of life, for the younger patients the reason for mental retardation is usually a lack of compliance with the treatment. The majority of metabolic phenotypes is cPKU, in concordance with the literature data; the mild phenotype (HPA) was observed in a small percentage of patients, smaller than the data reported in the literature. In the first two studied decades the mild phenotypes were seldom observed. In the absence of screening tests or suggestive clinical manifestation it can be assumed that HPA patients remained undiagnosed, which would explain the small HPA percentage in the study group. A significant improvement in metabolic control in younger patients compared with older ones was observed, which denotes a better access to specific alimentation on one side, and o the other side, a better understanding of the disease from the patients and their families. Also, this study confirms a known fact that the diet in PKU is of great importance in the disease evolution. In this study there were included patients with severe metabolic phenotype with good metabolic control which reached adulthood without intellectual deficits, with higher education, social integrated and also patients with mild metabolic phenotype but with a poor metabolic control which developed intellectual deficiency. Conclusions. The PKU prevalence in Bihor county is higher than the estimated national value. The late diagnosis and treatment or the poor metabolic control led to intellectual disability, regardless of the metabolic phenotype. PKU screening and the better access to treatment allows younger generations of patients to enjoy a superior quality of life than the patients from the first two studied decades.

EP001: Newborn screening experience for very long chain Acyl-CoA Dehydrogenase (VLCAD) deficiency in Kuwait

Among the various inborn errors of fatty acid oxidation disorders, very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common disorder in Kuwaiti population, which has been noticed especially following the launch of the expanded newborn screening program in Kuwait in October 2014. VLCAD deficiency is a rare autosomal recessive disorder with a worldwide incidence of 1:30,000 to 1:100,000 births. It is caused by deficiency of VLCAD coenzyme, encoded by ACADVL gene which converts very-long-chain fatty acids into energy. In October 2014, the Kuwait Ministry of Health has started a publicly funded expanded newborn screening program meeting the highest international standards to screen for a wide range of metabolic and endocrine disorders including a total of 22 disorders via testing dried blood spots and thus replacing the old, limited newborn screening for congenital hypothyroidism and phenylketonuria that was introduced in 2005. A retrospective analysis of the data registry for the newborn screening over the 6-year period between January 2015 and December 2020 in Kuwait has been conducted after obtaining consent from the newborn screening program. This data included newborns delivered in hospitals all over Kuwait. Data on metabolite concentrations in dried blood spots at the time of screening obtained from all newborns were reviewed and only dried blood spots detecting elevated blood C14:1 (cutoff 0.29 mmol/L) and C14:1/C2 ratio (cutoff 0.03) via tandem mass spectrometry (MS/MS) were included in this study. The positive initial screening is followed by a confirmatory plasma or dried blood acylcarnitine analysis (cutoff ≥ 1 mmol/L) with or without a follow-up genetic analysis of ACADVL gene, either targeted variant testing using PCR amplification followed by Sanger sequencing, or through next generation sequencing technology. Total of 36 cases (19 male/17 female) out of 304,086 screened newborns have been identified and confirmed to have VLCAD deficiency with an incidence of ∼1:8300. The diagnosis was based on the detection of elevated blood C14:1 and C14:1/C2 ratio in the initial dried blood spots in the newborn screening, followed by a confirmatory plasma or dried blood acylcarnitines profile for VLCAD deficiency with or without a follow-up genetic analysis, except for three babies who had positive initial screen but have died before obtaining confirmatory acylcarnitine or genetic analyses. Out of the 36 individuals, there were 3 cases from Kingdom of Saudi Arabia, 2 cases from India and the rest were Kuwaiti. Molecular testing of 24 of them has revealed a founder truncating pathogenic variant in exon 2 of the ACADVL gene, * c.65C>A; p.(Ser22Ter); the reference transcript is NM_001178008.2(hg19/GRCh3). We have identified three genetically confirmed cases with VLCAD deficiency following a positive initial screen but negative confirmatory acylcarnitine analysis. Furthermore, we identified four heterozygous individuals for VLCAD deficiency via molecular testing, after a positive initial newborn screening and normal confirmatory acylcarnitine profile. This is the first study to review the Kuwaiti newborn screening experience of VLCAD deficiency since its launch in October 2014. We recommend including molecular genetic testing for ACADVL gene as part of the newborn screening for VLCAD deficiency particularly for the cases with negative confirmatory acylcarnitine profile. Our study provides evidence that the expanded newborn screening in Kuwait has led to the early detection of VLCAD deficiency cases and thus the initiation of the adequate management plan for these individuals aiming to prevent death and disability.

Current Scenario and Future Direction of Newborn Screening and Management Program for Phenylketonuria in Bangladesh

Phenylketonuria (PKU) is a correctable inborn error of metabolism which causes lethal intellectual delay and neurobehavioral anomalies. A screening package, especially for early recognition can support to regulate the PKU process of most patients. New-born screening program in any country focuses at the earliest detection of inheritance deficiency disorders in order to avoid the most severe repercussion by appropriate medication. This screening program needs a concomitant diagnosis and involves additional clinical research. Strategies from developed countries recommend that new-born screening should be done as soon as possible after birth before hospital/clinic discharge because if detected later, it conveys to significantly increase in disability as well as morbidity. Although exact protocol differs among different countries, testing procedures for PKU should be followed universally recognized in the developed world. Unfortunately, new-born screening program in Bangladesh is in lying-in room or possibly in pilot study in particular hospital, because the health-care system is classically targeted mortality (like childbirth complications) and transmittable morbidities (such as COVID-19) but not inborn frailties. Although policies and management of childbirth complications have been successfully lowered infant and mother mortality rates, the number of disabled babies increased tremendously. The study aims to investigate the current status of new-born screening (NBS) program of PKU in the Rajshahi Division Bangladesh, and focus on future plans to manage with life-long treatment. The primary challenges such as financial support for newborn screening, publicity, should be identified and implemented for national PKU-NBS policy as a role model of Bangladesh for developing countries.

Quantitation of Phenylalanine in Dried Blood Spot Using Liquid Chromatography Tandem Mass Spectrometry for Monitoring of Patients with Phenylketonuria (PKU).

Newborn screening for phenylketonuria (PKU) is performed by analysis of phenylalanine in dried blood spot (DBS). Once diagnosed by a definitive method, a patient's dietary control is performed by repeated analysis of phenylalanine in venous blood or DBS. Since venipuncture is time consuming, painful, and may often be difficult to achieve in newborns, the use of DBS for analysis of phenylalanine is becoming a preferred method for dietary monitoring of patients with PKU. Using a lancet, patients or their guardians collect finger capillary blood on an approved filter paper. Once collected, the filter paper with DBS is sent to the laboratory for phenylalanine analysis. In the laboratory, phenylalanine is extracted from the DBS using organic solvents. Here, we describe an LC-MS/MS method for the analysis of phenylalanine from DBS with an approximation to serum levels.

Диференціальна діагностика фенілкетонурії і порушень обміну птеринів у хворих із гіперфенілаланінемією

Актуальність. Гіперфенілаланінемія (HPA), або підвищення концентрації фенілаланіну (Phe) в крові, є наслідком порушення перетворення фенілаланіну в тирозин. Яскравим прикладом цього клінічного стану є класична фенілкетонурія (PKU). У 1970-х роках стало зрозуміло, що в частини хворих із PKU, незважаючи на ефективну дієтотерапію, розвивалися тяжкі неврологічні порушення. Ці випадки верифікували як атипові, або злоякісні, варіанти PKU, що, як було досліджено, спричинялось мутаціями в генах, які регулюють синтез та регенерацію тетрагідробіоптерину (ВН4). Порушення метаболізму тетрагідробіоптерину, або ВН4-дефіцитні синдроми, — гетерогенна група спадкових захворювань ЦНС, спричинених дефіцитом моноамінів-нейротрансмітерів, які в низці випадків супроводжуються гіперфенілаланінемією. ВН4-дефіцитні синдроми, які є рідкісними вродженими порушеннями метаболізму, спричиненими недостатністю нейротрансмітерів, за відсутності своєчасного лікування призводять до прогресуючої, нерідко летальної, енцефалопатії. Упровадження в медичну практику ранньої диференціальної клінічної та лабораторної діагностики гіперфенілаланінемій дозволить запобігти розвитку летальних енцефалопатій та суттєво підвищити ефективність їх лікування. Матеріали та методи. Були обстежені 59 пацієнтів із діагнозом PKU віком від 2 до 16 років. Гіперфенілаланінемія була загальним симптомом і встановлена в усіх обстежених пацієнтів. Усім хворим було проведено молекулярно-генетичне обстеження на панель мутацій у гені РАН (R408W, Y414C (12-й екзон), R158Q (5-й екзон), Ivs10nt546 (5-й інтрон), Ivs12nt1 (12-й інтрон), R252W, R261W, R261Q, R261X, G272X, P281L, S273F (7-й екзон). Усі хворі були обстежені на спектр птеринів (біоптерин та неоптерин) в крові та сечі. У 4 пацієнтів із вираженими неврологічними симптомами додатково визначали активність дигідроптеринредуктази в сухих краплях крові. Двом пацієнтам проводилося вимірювання концентрації гомованілової кислоти та 5-гідроксііндолоцтової кислоти в спинномозковій рідині методом високоефективної рідинної хроматографії. Усі пацієнти лікувались за допомогою дієтотерапії з використанням продуктів спеціалізованого лікувального харчування. Результати. 59 пацієнтів із гіперфенілаланінемією були обстежені на птериновий профіль. У 55 пацієнтів був установлений діагноз PKU, у 4 пацієнтів із прогресуючим ураженням нервової системи та порушенням птеринового профілю було встановлено діагноз ВН4-дефіцитних синдромів: 3 пацієнти — із діагнозом «PTPS-синдром», 1 пацієнт — «DHPR-синдром». Основні клінічні прояви ВН4-дефіцитних синдромів у виявлених пацієнтів були такими: затримка розумового розвитку, судоми, прогресування екстрапірамідних симптомів, порушення тонусу м’язів і пози, ковтання, гіперсалівація. Перші симптоми виникали частіше протягом перших місяців життя дитини. Головним біохімічним маркером на початку захворювання була гіперфенілаланінемія. При застосуванні дієтотерапії рівень Phe швидко досягав рекомендованих безпечних для нервової системи значень, але хвороба прогресувала. Висновки. Гіперфенілаланінемія — основний прояв гетерогенної групи рідкісних нейрометаболічних захворювань, які вимагають подальшої діагностичної верифікації. У новонароджених, у яких під час проведення масового неонатального скринінгу на фенілкетонурію виявлений підвищений рівень фенілаланіну, необхідно застосовувати додатковий діагностичний протокол (визначення птеринового профілю в крові та сечі, обов’язкове дослідження активності дигідроптеринредуктази у сухій краплі крові). Дослідження птеринів необхідне в пацієнтів із раніше встановленим діагнозом «фенілкетонурія» за неефективності дієтотерапії, прогресування когнітивних і неврологічних порушень, зниження тонусу м’язів, виникнення судомного синдрому, наявності екстрапірамідних симптомів. Рання діагностика ВН4-дефіцитних синдромів та своєчасно розпочате лікування дозволяють суттєво покращити стан хворих. Для деяких ВН4-дефіцитних синдромів гіперфенілаланінемія не характерна, що унеможливлює раннє виявлення таких пацієнтів під час неонатального скринінгу. Тому виявлення прогресуючих неврологічних симптомів у пацієнтів віком до 1 року й у ранньому дитинстві, які супроводжуються втратою когнітивних функцій на тлі м’язової гіпотонії, дистонічних порушень і судом, зумовлює необхідність дослідження птеринового профілю в крові й сечі.