Impact of theta transcranial alternating current stimulation on language production in adult classic galactosemia patients.

Patients with classic galactosemia (CG), an inborn error of galactose metabolism, suffer from impairments in cognition, including language processing. Potential causes are atypical brain oscillations. Recent electroencephalogram (EEG) showed differences in the P300 event-related-potential (ERP) and alterations in the alpha/theta-range during speech planning. This study investigated whether transcranial alternating current stimulation (tACS) at theta-frequency compared to sham can cause a normalization of the ERP post stimulation and improves language performance. Eleven CG patients and fourteen healthy controls participated in two tACS-sessions (theta 6.5 Hz/sham). They were engaged in an active language task, describing animated scenes at three moments, that is, pre/during/post stimulation. Pre and post stimulation, behavior (naming accuracy, voice-onset-times; VOT) and mean-amplitudes of ERP were compared, by means of a P300 time-window analysis and cluster-based-permutation testing during speech planning. The results showed that theta stimulation, not sham, significantly reduced naming error-percentage in patients, not in controls. Theta did not systematically speed up naming beyond a general learning effect, which was larger for the patients. The EEG analysis revealed a significant pre-post stimulation effect (P300/late positivity), in patients and during theta stimulation only. In conclusion, theta-tACS improved accuracy in language performance in CG patients compared to controls and altered the P300 and late positive ERP-amplitude, suggesting a lasting effect on neural oscillation and behavior.

Neuropsychological stability in classical galactosemia: A pilot study in 10 adult patients.

Classical galactosemia (CG) is an autosomal recessive disorder of galactose metabolism. Despite early initiation of a galactose-restricted diet, patients develop long-term complications including cognitive impairment. There is an ongoing debate whether the cognitive impairment in CG is stable throughout life or progresses with age. Earlier cross-sectional and longitudinal studies regarding intelligence suggest stability, but longitudinal neuropsychological studies focusing on specific cognitive functions are limited. Therefore, the aim of this study is to assess cognitive change over time in adult CG-patients. Ten adult patients with normal to borderline intelligence (mean age 33 years, range 22-49; IQ≥70 or independent work- or living situation) were assessed twice with a mean time interval of 3 years and 9 months (range 1023-1575 days). The neuropsychological assessments covered information processing speed, executive functioning, verbal fluency, and visuospatial functioning. Results showed that there was no significant decline or improvement in test scores on all neuropsychological measures except a decline on the Trail Making Test-A (p = 0.048). However, this group-level difference was subject to "regression to the mean" and was not endorsed by significant change in test scores measuring the same cognitive domain. Moreover, no specific pattern of reliable change (RCI > -1.96) was present on specific measures or within individual patients. This explorative study performed in 10 adult CG-patients with normal to borderline intelligence revealed no cognitive change on several cognitive domains. This implies that the subset of adults with a normal to borderline IQ in their early and middle adulthood are cognitively stable.

Brain function in classic galactosemia, a galactosemia network (GalNet) members review.

Classic galactosemia (CG, OMIM #230400, ORPHA: 79,239) is a hereditary disorder of galactose metabolism that, despite treatment with galactose restriction, affects brain function in 85% of the patients. Problems with cognitive function, neuropsychological/social emotional difficulties, neurological symptoms, and abnormalities in neuroimaging and electrophysiological assessments are frequently reported in this group of patients, with an enormous individual variability. In this review, we describe the role of impaired galactose metabolism on brain dysfunction based on state of the art knowledge. Several proposed disease mechanisms are discussed, as well as the time of damage and potential treatment options. Furthermore, we combine data from longitudinal, cross-sectional and retrospective studies with the observations of specialist teams treating this disease to depict the brain disease course over time. Based on current data and insights, the majority of patients do not exhibit cognitive decline. A subset of patients, often with early onset cerebral and cerebellar volume loss, can nevertheless experience neurological worsening. While a large number of patients with CG suffer from anxiety and depression, the increased complaints about memory loss, anxiety and depression at an older age are likely multifactorial in origin.

Social cognition, emotion regulation and social competence in classical galactosemia patients without intellectual disability.

OBJECTIVE: Classical galactosemia (CG) is an inborn error of galactose metabolism. Many CG patients suffer from long-term complications including poor cognitive functioning. There are indications of social dysfunction but limited evidence in the literature. Therefore, this study aims to improve our understanding of social competence in CG by investigating social cognition, neurocognition and emotion regulation. METHODS: A comprehensive (neuro)psychological test battery, including self and proxy questionnaires, was administered to CG patients without intellectual disability. Social cognition was assessed by facial emotion recognition, Theory of Mind and self-reported empathy. Standardised results were compared to normative data of the general population. RESULTS: Data from 23 patients (aged 8-52) were included in the study. On a group level, CG patients reported satisfaction with social roles and no social dysfunction despite the self-report of lower social skills. They showed deficits in all aspects of social cognition on both performance tests (emotion recognition and Theory of Mind) and self-report questionnaires (empathy). Adults had a lower social participation than the general population. Parents reported lower social functioning, less adaptive emotion regulation and communication difficulties in their children. Individual differences in scores were present. CONCLUSION: This study shows that CG patients without intellectual disability are satisfied with their social competence, especially social functioning. Nevertheless, deficits in social cognition are present in a large proportion of CG patients. Due to the large variability in scores and discrepancies between self- and proxy-report, an individually tailored, comprehensive neuropsychological assessment including social cognition is advised in all CG patients. Treatment plans need to be customised to the individual patient.

Case report: Skin protective barrier cream interference in benzethonium chloride method for urine protein measurement in a 6-month-old girl.

This case report describes the positive interference of the commonly used skin protective barrier cream used together with urine collection bags on the benzethonium chloride method for urine protein measurements in a 6-month-old female baby, leading to falsely elevated results. The interference was identified by both artificially mixing urine samples with this cream and comparing the results obtained using the benzethonium chloride method with those obtained using the pyrogallol red method.

Optimizing the Dutch newborn screening for congenital hypothyroidism by incorporating amino acids and acylcarnitines in a machine learning-based model.

Objective: Congenital hypothyroidism (CH) is an inborn thyroid hormone (TH) deficiency mostly caused by thyroidal (primary CH) or hypothalamic/pituitary (central CH) disturbances. Most CH newborn screening (NBS) programs are thyroid-stimulating-hormone (TSH) based, thereby only detecting primary CH. The Dutch NBS is based on measuring total thyroxine (T4) from dried blood spots, aiming to detect primary and central CH at the cost of more false-positive referrals (FPRs) (positive predictive value (PPV) of 21% in 2007-2017). An artificial PPV of 26% was yielded when using a machine learning-based model on the adjusted dataset described based on the Dutch CH NBS. Recently, amino acids (AAs) and acylcarnitines (ACs) have been shown to be associated with TH concentration. We therefore aimed to investigate whether AAs and ACs measured during NBS can contribute to better performance of the CH screening in the Netherlands by using a revised machine learning-based model. Methods: Dutch NBS data between 2007 and 2017 (CH screening results, AAs and ACs) from 1079 FPRs, 515 newborns with primary (431) and central CH (84) and data from 1842 healthy controls were used. A random forest model including these data was developed. Results: The random forest model with an artificial sensitivity of 100% yielded a PPV of 48% and AUROC of 0.99. Besides T4 and TSH, tyrosine, and succinylacetone were the main parameters contributing to the model's performance. Conclusions: The PPV improved significantly (26-48%) by adding several AAs and ACs to our machine learning-based model, suggesting that adding these parameters benefits the current algorithm.

A Delphi Survey Study to Formulate Statements on the Treatability of Inherited Metabolic Disorders to Decide on Eligibility for Newborn Screening.

The Wilson and Jungner (W&J) and Andermann criteria are meant to help select diseases eligible for population-based screening. With the introduction of next-generation sequencing (NGS) methods for newborn screening (NBS), more inherited metabolic diseases (IMDs) can technically be included, and a revision of the criteria was attempted. This study aimed to formulate statements and investigate whether those statements could elaborate on the criterion of treatability for IMDs to decide on eligibility for NBS. An online Delphi study was started among a panel of Dutch IMD experts (EPs). EPs evaluated, amended, and approved statements on treatability that were subsequently applied to 10 IMDs. After two rounds of Delphi, consensus was reached on 10 statements. Application of these statements selected 5 out of 10 IMDs proposed for this study as eligible for NBS, including 3 IMDs in the current Dutch NBS. The statement: 'The expected benefit/burden ratio of early treatment is positive and results in a significant health outcome' contributed most to decision-making. Our Delphi study resulted in 10 statements that can help to decide on eligibility for inclusion in NBS based on treatability, also showing that other criteria could be handled in a comparable way. Validation of the statements is required before these can be applied as guidance to authorities.

The challenges of classical galactosemia: HRQoL in pediatric and adult patients.

BACKGROUND: Classical galactosemia (CG), an inborn error of galactose metabolism, results in long-term complications including cognitive impairment and movement disorders, despite early diagnosis and dietary treatment. Two decades ago, lower motor-, cognitive- and social health related quality of life (HRQoL) was demonstrated in pediatric and adult patients. Since then, the diet has been relaxed, newborn screening was implemented and new international guidelines resulted in major changes in follow-up. The aim of this study was to assess HRQoL of CG by means of online self- and/or proxy-HRQoL-questionnaires focusing on the main areas of concern of CG (i.e. anxiety, depression, cognition, fatigue, social- and upper extremity function) within the patient-reported outcomes measurement information system (PROMIS®) and generic HRQoL-questionnaires (TAPQOL, TACQOL, TAAQOL). RESULTS: Data of 61 Dutch patients (aged 1-52 years) were collected and compared to available Dutch or US reference populations. On the PROMIS-questionnaires, children reported more fatigue (P = 0.044), lower function in upper extremities (P = 0.021), more cognitive difficulties (P = 0.055, d = 0.56) and higher anxiety (P = 0.063, d = 0.52) than reference children although the latter findings remained non-significant. Parents of CG patients reported lower quality of peer relationships of their children (P < 0.001). Both children and parents reported lower cognitive functioning (P = 0.005, P = 0.010) on the TACQOL. Adults reported on PROMIS domains lower cognitive functioning (P = 0.030), higher anxiety (P = 0.004) and more fatigue (P = 0.026). Cognitive difficulties were reported on the TAAQOL by adults (P < 0.001), as well as physical-, sleeping and social difficulties. CONCLUSIONS: CG remains to impact the HRQoL of pediatric and adult patients negatively on several domains including cognition, anxiety, motor function and fatigue. A lower social health was mainly reported by parents, and not by patients themselves. The Covid-19 pandemic might have amplified the results on anxiety although higher levels of anxiety fit pre-pandemic findings. The reported fatigue is a new finding in CG. Because the effect of lockdown fatigue could not be eliminated and fatigue is a frequent finding in patients with chronic disorders, future studies are warranted. Clinicians and researchers should be attentive to both pediatric and adult patients, and the age-dependent difficulties they might encounter.

Machine learning to improve false-positive results in the Dutch newborn screening for congenital hypothyroidism.

OBJECTIVE: The Dutch Congenital hypothyroidism (CH) Newborn Screening (NBS) algorithm for thyroidal and central congenital hypothyroidism (CH-T and CH-C, respectively) is primarily based on determination of thyroxine (T4) concentrations in dried blood spots, followed by thyroid-stimulating hormone (TSH) and thyroxine-binding globulin (TBG) measurements enabling detection of both CH-T and CH-C, with a positive predictive value (PPV) of 21%. A calculated T4/TBG ratio serves as an indirect measure for free T4. The aim of this study is to investigate whether machine learning techniques can help to improve the PPV of the algorithm without missing the positive cases that should have been detected with the current algorithm. DESIGN & METHODS: NBS data and parameters of CH patients and false-positive referrals in the period 2007-2017 and of a healthy reference population were included in the study. A random forest model was trained and tested using a stratified split and improved using synthetic minority oversampling technique (SMOTE). NBS data of 4668 newborns were included, containing 458 CH-T and 82 CH-C patients, 2332 false-positive referrals and 1670 healthy newborns. RESULTS: Variables determining identification of CH were (in order of importance) TSH, T4/TBG ratio, gestational age, TBG, T4 and age at NBS sampling. In a Receiver-Operating Characteristic (ROC) analysis on the test set, current sensitivity could be maintained, while increasing the PPV to 26%. CONCLUSIONS: Machine learning techniques have the potential to improve the PPV of the Dutch CH NBS. However, improved detection of currently missed cases is only possible with new, better predictors of especially CH-C and a better registration and inclusion of these cases in future models.

Development of medicines for rare diseases and inborn errors of metabolism: Toward novel public-private partnerships.

Medicine development for rare diseases, including inborn errors of metabolism (IEMs) is challenging. Many academic innovations fail to reach the patient, either by stranding in the translational stage or due to suboptimal patient access related to pricing or uncertain effectiveness. Expanding and solidifying the role of the academic in public-private partnerships (PPPs) may present an innovative solution to help overcome these complexities. This narrative review explores the literature on traditional and novel collaborative approaches to medicine development for rare diseases and analyzes examples of PPPs, with a specific focus on IEMs. Several academic institutions have introduced guidelines for socially responsible licensing of innovations for private development. The PPP model offers a more integrative approach toward academic involvement of medicine development. By sharing risks and rewards, failures in the translational stage can be mutually absorbed. If socially responsible terms are not included, however, high pricing can impede patient access. Therefore, we propose a framework for socially responsible PPPs aimed at medicine development for metabolic disorders. This socially responsible PPP framework could stimulate successful and accessible medicine development for IEMs as well as other rare diseases if the establishment of such collaborations includes terms securing joint data ownership and evidence generation, fast access, and socially responsible pricing.

Multi-omics in classical galactosemia: Evidence for the involvement of multiple metabolic pathways.

Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.

A multinational study of acute and long-term outcomes of Type 1 galactosemia patients who carry the S135L (c.404C > T) variant of GALT.

Patients with galactosemia who carry the S135L (c.404C > T) variant of galactose-1-P uridylyltransferase (GALT), documented to encode low-level residual GALT activity, have been under-represented in most prior studies of outcomes in Type 1 galactosemia. What is known about the acute and long-term outcomes of these patients, therefore, is based on very limited data. Here, we present a study comparing acute and long-term outcomes of 12 patients homozygous for S135L, 25 patients compound heterozygous for S135L, and 105 patients homozygous for two GALT-null (G) alleles. This is the largest cohort of S135L patients characterized to date. Acute disease following milk exposure in the newborn period was common among patients in all 3 comparison groups in our study, as were long-term complications in the domains of speech, cognition, and motor outcomes. In contrast, while at least 80% of both GALT-null and S135L compound heterozygous girls and women showed evidence of an adverse ovarian outcome, prevalence was only 25% among S135L homozygotes. Further, all young women in this study with even one copy of S135L achieved spontaneous menarche; this is true for only about 33% of women with classic galactosemia. Overall, we observed that while most long-term outcomes trended milder among groups of patients with even one copy of S135L, many individual patients, either homozygous or compound heterozygous for S135L, nonetheless experienced long-term outcomes that were not mild. This was true despite detection by newborn screening and both early and life-long dietary restriction of galactose. This information should empower more evidence-based counseling for galactosemia patients with S135L.

Mind the B2: Life-Threatening Neonatal Complications of a Strict Vegan Diet during Pregnancy.

An increasing number of women of reproductive age follow vegan diets. Because vegan diets are deficient in a number of essential nutrients, guidelines address the necessity of supplementations such as iron, zinc, and vitamin B12. However, the risk of riboflavin (vitamin B2) deficiency is not properly addressed. We report a case of a male neonate with a life-threatening hypoglycaemia and lactic acidosis due to severe riboflavin deficiency. The mother followed a strict vegan diet with intermittent use of supplements (folic acid, vitamin B12, vitamin D, omega 3). This case highlights the importance of adequate counselling of all pregnant women adhering to vegan diets to ensure sufficient intake of essential nutrients and vitamins, including riboflavin.

Neurocognitive outcome and mental health in children with tyrosinemia type 1 and phenylketonuria: A comparison between two genetic disorders affecting the same metabolic pathway.

Tyrosinemia type 1 (TT1) and phenylketonuria (PKU) are both inborn errors of phenylalanine-tyrosine metabolism. Neurocognitive and behavioral outcomes have always featured in PKU research but received less attention in TT1 research. This study aimed to investigate and compare neurocognitive, behavioral, and social outcomes of treated TT1 and PKU patients. We included 33 TT1 patients (mean age 11.24 years; 16 male), 31 PKU patients (mean age 10.84; 14 male), and 58 age- and gender-matched healthy controls (mean age 10.82 years; 29 male). IQ (Wechsler-subtests), executive functioning (the Behavioral Rating Inventory of Executive Functioning), mental health (the Achenbach-scales), and social functioning (the Social Skills Rating System) were assessed. Results of TT1 patients, PKU patients, and healthy controls were compared using Kruskal-Wallis tests with post-hoc Mann-Whitney U tests. TT1 patients showed a lower IQ and poorer executive functioning, mental health, and social functioning compared to healthy controls and PKU patients. PKU patients did not differ from healthy controls regarding these outcome measures. Relatively poor outcomes for TT1 patients were particularly evident for verbal IQ, BRIEF dimensions "working memory", "plan and organize" and "monitor", ASEBA dimensions "social problems" and "attention problems", and for the SSRS "assertiveness" scale (all p values <0.001). To conclude, TT1 patients showed cognitive impairments on all domains studied, and appeared to be significantly more affected than PKU patients. More attention should be paid to investigating and monitoring neurocognitive outcome in TT1 and research should focus on explaining the underlying pathophysiological mechanism.

Genetic, biochemical, and clinical spectrum of patients with mitochondrial trifunctional protein deficiency identified after the introduction of newborn screening in the Netherlands.

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is included in many newborn screening (NBS) programs. Acylcarnitine-based NBS for LCHADD not only identifies LCHADD, but also the other deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid ß-oxidation. Besides LCHAD, MTP harbors two additional enzyme activities: long-chain enoyl-CoA hydratase (LCEH) and long-chain ketoacyl-CoA thiolase (LCKAT). Deficiency of one or more MTP activities causes generalized MTP deficiency (MTPD), LCHADD, LCEH deficiency (not yet reported), or LCKAT deficiency (LCKATD). To gain insight in the outcomes of MTP-deficient patients diagnosed after the introduction of NBS for LCHADD in the Netherlands, a retrospective evaluation of genetic, biochemical, and clinical characteristics of MTP-deficient patients, identified since 2007, was carried out. Thirteen patients were identified: seven with LCHADD, five with MTPD, and one with LCKATD. All LCHADD patients (one missed by NBS, clinical diagnosis) and one MTPD patient (clinical diagnosis) were alive. Four MTPD patients and one LCKATD patient developed cardiomyopathy and died within 1 month and 13 months of life, respectively. Surviving patients did not develop symptomatic hypoglycemia, but experienced reversible cardiomyopathy and rhabdomyolysis. Five LCHADD patients developed subclinical neuropathy and/or retinopathy. In conclusion, patient outcomes were highly variable, stressing the need for accurate classification of and discrimination between the MTP deficiencies to improve insight in the yield of NBS for LCHADD. NBS allowed the prevention of symptomatic hypoglycemia, but current treatment options failed to treat cardiomyopathy and prevent long-term complications. Moreover, milder patients, who might benefit from NBS, were missed due to normal acylcarnitine profiles.

Abnormal N-glycan fucosylation, galactosylation, and sialylation of IgG in adults with classical galactosemia, influence of dietary galactose intake.

BACKGROUND: Classical galactosemia (CG) (OMIM #230400) is a rare disorder of carbohydrate metabolism, due to deficiency of galactose-1-phosphate uridyltransferase (EC 2.7.7.12). The pathophysiology of the long-term complications, mainly cognitive, neurological, and female infertility remains poorly understood. OBJECTIVES: This study investigated (a) the association between specific IgG N-glycosylation biomarkers (glycan peaks and grouped traits) and CG patients (n = 95) identified from the GalNet Network, using hydrophilic interaction ultraperformance liquid chromatography and (b) a further analysis of a GALT c.563A-G/p.Gln188Arg homozygous cohort (n = 49) with correlation with glycan features with patient Full Scale Intelligence Quotient (FSIQ), and (c) with galactose intake. RESULTS: A very significant decrease in galactosylation and sialylation and an increase in core fucosylation was noted in CG patients vs controls (P < .005). Bisected glycans were decreased in the severe GALT c.563A-G/p.Gln188Arg homozygous cohort (n = 49) (P < .05). Logistic regression models incorporating IgG glycan traits distinguished CG patients from controls. Incremental dietary galactose intake correlated positively with FSIQ for the p.Gln188Arg homozygous CG cohort (P < .005) for a dietary galactose intake of 500 to 1000 mg/d. Significant improvements in profiles with increased galactose intake were noted for monosialylated, monogalactosylated, and monoantennary glycans. CONCLUSION: These results suggest that N-glycosylation abnormalities persist in CG patients on dietary galactose restriction which may be modifiable to a degree by dietary galactose intake.

Improving the Dutch Newborn Screening for Central Congenital Hypothyroidism by Using 95% Reference Intervals for Thyroxine-Binding Globulin.

INTRODUCTION: Newborn screening (NBS) for congenital hypothyroidism (CH) in the Netherlands consists of thyroxine (T4), thyroid-stimulating hormone (TSH), and T4-binding globulin (TBG) measurements to detect thyroidal CH and central CH (CH-C). CH-C is detected by T4 or a calculated T4/TBG ratio, which serves as an indirect measure of free T4. TSH and TBG are only measured in the lowest 20 and 5% of daily T4 values, respectively. A recent evaluation of the Dutch NBS for CH showed that the T4 and T4/TBG ratio contribute to the detection of CH-C but also lead to a low positive predictive value (PPV). Dried blood spot (DBS) reference intervals (RIs) are currently unknown and may contribute to improvement of our NBS algorithm. MATERIALS AND METHODS: RIs of T4, TSH, TBG, and the T4/TBG ratio were determined according to Clinical & Laboratory Standards Institute guidelines in heel puncture cards from routine NBS in both sexes and at the common NBS sampling ages. Scatter plots were used to compare the healthy reference population to previously published data of CH-C patients and false positives. RESULTS: Analyses of 1,670 heel puncture cards showed small differences between subgroups and led to the formulation of total sample DBS RIs for T4 (56-118 nmol/L), TSH (<2.6 mIU/L), TBG (116-271 nmol/L), and the T4/TBG ratio (>20). 46% of false-positive referrals based on T4 alone had a TBG below the RI, indicating preventable referral due to partial TBG deficiency. One case of CH-C also had partial TBG deficiency (TBG 59 and T4 12 nmol/L blood). DISCUSSION/CONCLUSION: Established DBS RIs provided possibilities to improve the PPV of the Dutch CH NBS algorithm. We conclude that by taking partial TBG deficiency into account, approximately half of T4 false-positive referrals may be prevented while maintaining NBS sensitivity at the current level.

Second-tier Testing for 21-Hydroxylase Deficiency in the Netherlands: A Newborn Screening Pilot Study.

CONTEXT: Newborn screening (NBS) for classic congenital adrenal hyperplasia (CAH) consists of 17-hydroxyprogesterone (17-OHP) measurement with gestational age-adjusted cutoffs. A second heel puncture (HP) is performed in newborns with inconclusive results to reduce false positives. OBJECTIVE: We assessed the accuracy and turnaround time of the current CAH NBS algorithm in comparison with alternative algorithms by performing a second-tier 21-deoxycortisol (21-DF) pilot study. METHODS: Dried blood spots (DBS) of newborns with inconclusive and positive 17-OHP (immunoassay) first HP results were sent from regional NBS laboratories to the Amsterdam UMC Endocrine Laboratory. In 2017-2019, 21-DF concentrations were analyzed by LC-MS/MS in parallel with routine NBS. Diagnoses were confirmed by mutation analysis. RESULTS: A total of 328 DBS were analyzed; 37 newborns had confirmed classic CAH, 33 were false-positive and 258 were categorized as negative in the second HP following the current algorithm. With second-tier testing, all 37 confirmed CAH had elevated 21-DF, while all 33 false positives and 253/258 second-HP negatives had undetectable 21-DF. The elevated 21-DF of the other 5 newborns may be NBS false negatives or second-tier false positives. Adding the second-tier results to inconclusive first HPs reduced the number of false positives to 11 and prevented all 286 second HPs. Adding the second tier to both positive and inconclusive first HPs eliminated all false positives but delayed referral for 31 CAH patients (1-4 days). CONCLUSION: Application of the second-tier 21-DF measurement to inconclusive first HPs improved our CAH NBS by reducing false positives, abolishing the second HP, and thereby shortening referral time.

Phenylketonuria.

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.

Monitoring phenylalanine concentrations in the follow-up of phenylketonuria patients: An inventory of pre-analytical and analytical variation.

BACKGROUND: Reliable measurement of phenylalanine (Phe) is a prerequisite for adequate follow-up of phenylketonuria (PKU) patients. However, previous studies have raised concerns on the intercomparability of plasma and dried blood spot (DBS) Phe results. In this study, we made an inventory of differences in (pre-)analytical methodology used for Phe determination across Dutch laboratories, and compared DBS and plasma results. METHODS: Through an online questionnaire, we assessed (pre-)analytical Phe measurement procedures of seven Dutch metabolic laboratories. To investigate the difference between plasma and DBS Phe, participating laboratories received simultaneously collected plasma-DBS sets from 23 PKU patients. In parallel, 40 sample sets of DBS spotted from either venous blood or capillary fingerprick were analyzed. RESULTS: Our data show that there is no consistency on standard operating procedures for Phe measurement. The association of DBS to plasma Phe concentration exhibits substantial inter-laboratory variation, ranging from a mean difference of -15.5% to +30.6% between plasma and DBS Phe concentrations. In addition, we found a mean difference of +5.8% in Phe concentration between capillary DBS and DBS prepared from venous blood. CONCLUSIONS: The results of our study point to substantial (pre-)analytical variation in Phe measurements, implicating that bloodspot Phe results should be interpreted with caution, especially when no correction factor is applied. To minimize variation, we advocate pre-analytical standardization and analytical harmonization of Phe measurements, including consensus on application of a correction factor to adjust DBS Phe to plasma concentrations.