Insights into the expanding phenotypic spectrum of inherited disorders of biogenic amines

ARTICLE Insights into the expanding phenotypic spectrum of inherited disorders of biogenic amines Oya Kuseyri Hübschmann 1, Gabriella Horvath2, Elisenda Cortès-Saladelafont3,4,Yılmaz Yıldız5, Mario Mastrangelo6, Roser Pons7, Jennifer Friedman8, Saadet Mercimek-Andrews 9, Suet-Na Wong10, Toni S. Pearson 11, Dimitrios I. Zafeiriou12, Jan Kulhánek13, Manju A. Kurian14, Eduardo López-Laso15, Mari Oppebøen16, Sebile Kılavuz17, Tessa Wassenberg18,19, Helly Goez20, Sabine Scholl-Bürgi21, Francesco Porta22, TomášHonzík13, René Santer23, Alberto Burlina24, H. Serap Sivri 5, Vincenzo Leuzzi6, Georg F. Hoffmann1, Kathrin Jeltsch1, Daniel Hübschmann25,26,27,28, Sven F. Garbade 29, iNTD Registry Study Group*, Angeles García-Cazorla3& Thomas Opladen 1✉ Inherited disorders of neurotransmitter metabolism are rare neurodevelopmental diseases presenting with movement disorders and global developmental delay. This study presents the results of the first standardized deep phenotyping approach and describes the clinical and biochemical presentation at disease onset as well as diagnostic approaches of 275 patients from the registry of the International Working Group on Neurotransmitter related Disorders. The results reveal an increased rate of prematurity, a high risk for being small for gestational age and for congenital microcephaly in some disorders. Age at diagnosis and the diagnostic delay are influenced by the diagnostic methods applied and by disease-specific symptoms. The timepoint of investigation was also a significant factor: delay to diagnosis has decreased in recent years, possibly due to novel diagnostic approaches or raised awareness. Although each disorder has a specific biochemical pattern, we observed confounding exceptions to the rule. The data provide comprehensive insights into the phenotypic spectrum of neurotransmitter disorders. https://doi.org/10.1038/s41467-021-25515-5 OPEN A full list of author affiliations appears at the end of the paper. NATURE COMMUNICATIONS | (2021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5|www.nature.com/naturecommunications 1 1234567890():,; Inherited disorders of neurotransmitter metabolism represent a group of rare neurometabolic diseases. They are caused by impaired biosynthesis, breakdown or transport of neurotransmitters, or of their essential cofactors, such as tetrahydrobiopterin (BH 4 ). According to the chemical structure of the primarily affected metabolite they can be classified into distinct groups1(Table 1): (A) Disorders of biogenic amines (dopamine, serotonin, norepinephrine, epinephrine): (1) Primary disorders of biogenic amine metabolism: (i) Primary enzyme defects in biogenic amine biosynthesis (aromatic L-amino acid decarboxylase deficiency (AADCD), tyrosine hydroxylase deficiency (THD)); (ii) Disorders of biogenic amine catabolism (monoamine oxidase A deficiency (MAOAD), dopamine β-hydroxylase deficiency); (iii) Disorders of biogenic amine transport (vesicular monoamine transporter 2 deficiency, dopamine transporter deficiency (DATD)), (2) Disorders of tetrahydrobiopterin biosynthesis and recycling (autosomal dominant and recessive GTP-cyclohydrolase deficiency (ad/arGTPCHD), 6-pyruvoyl-tetrahydropterin synthase deficiency (PTPSD), sepiapterin reductase deficiency (SRD), dihydropteridine reductase deficiency (DHPRD), pterin-4a- carbinolamine dehydratase deficiency), (3) Co-chaperone associated disorders (DNAJC12 deficiency (DNAJC12D)) and (B) Disorders of amino acid neurotransmitters (glycine, glutamate, serine, γ-aminobutyric acid (GABA)). Manifestations of these disorders mainly involve the central nervous system but other organ systems such as autonomic nervous, hematological or cardiovascular can also be affected. The clinical phenotype consists of a broad spectrum of symptoms, ranging from mild hypotonia and late-onset movement disorders, to early-onset lethal encephalopathies. Initial symptoms can appear at any time from the perinatal period to adulthood. Since many clinical symptoms are unspecific or overlap with features seen in other neurological conditions, such as cerebral palsy, epileptic encephalopathies and hypoxic–ischemic encephalopathy, inherited neurotransmitter disorders are often underrecognized and misdiagnosed2. Within these disorders only a small group can be detected via newborn screening for phenylketonuria (PKU) while other diseases require selective diagnostic tests leading to prolonged diagnostic work-up and delayed treatment initiation3. The outcome depends on the underlying disorder, the timing of diagnosis, initiation and type of diseasespecific treatment, as well as long-term compliance to treatment4–10. Since inherited neurotransmitter disorders are rare disorders, the medical literature is comprised mainly of single case reports, small case series and retrospective cohort descriptions. The “International Working Group on Neurotransmitter Related Disorders (iNTD)”was founded in 2013 (www.intd-online.org), to overcome these limitations in clinical and scientific research11. Over the last few years it has steadily grown to include experts from 42 academic and clinical centers from 26 countries. In December 2014, iNTD set up the first international, longitudinal patient registry. This registry aims to improve our understanding of the natural history, epidemiology, genotype/phenotype correlations and clinical outcome, and to evaluate diagnostic and therapeutic strategies. In this work, we present the first standardized evaluation of the iNTD patient registry and report comprehensive insights into pre-, peri- and postnatal presentations of inherited disorders of biogenic amines, as well as effects of initial clinical and biochemical patterns on the diagnostic process. Results Description of the study population. Between January 1st 2015 and May 15th 2020, 429 patients were enrolled in the iNTD patient registry. Of these entries, 350 patients had a diagnosis of biogenic amine disorders. 75 patients who were transferred from the JAKE database on aromatic L-amino acid decarboxylase deficiency (http://www.biopku.org/home/jake.asp) were not analyzed in this study due to the high number of missing variables of interest. The remaining cohort of patients with disorders of biogenic amines consisted of 275 patients from 248 families (157 female (57%), from 42 countries: 196 patients born in Europe, 42 in North America, 34 in Asia, three in Central/South America and one in Africa). 109 patients had primary disorders of biogenic amine metabolism, 161 BH 4 deficiencies (BH 4 Ds) and five patients DNAJC12D (Tables 2and 3). All diagnoses were confirmed either by mutational analysis alone or by a combination of specific biochemical tests in CSF, urine and blood (Table 4). There were no patients with dopamine β-hydroxylase deficiency or vesicular monoamine transporter 2 deficiency. For a reliable explorative analysis, a minimum number of 6 patients was required. MAOAD, DATD and DNAJC12D were included only in the descriptive analysis. Pregnancy, delivery and peri- and postnatal outcome. Maternal health problems, medications taken during pregnancy and postnatal outcomes are depicted in Tables 2and 3and Supplementary Table 1. None of the patients were prenatally diagnosed. There was no difference in the mode of delivery between the different primary disorders of biogenic amine metabolism (Table 2). Both in AADCD and THD a high frequency of small for gestation age (SGA) babies was noted and a remarkably high number of patients with THD had birth length (BL) < 10th percentile (Table 2and Fig. 1). Table 1 Classification of inherited disorders of biogenic amines. Inherited disorders of biogenic amines Primary disorders of biogenic amine metabolism Disorders of tetrahydrobiopterin metabolism Co-chaperone associated disorders Biosynthesis Catabolism Transport Biosynthesis Recycling Aromatic L-amino acid decarboxylase deficiency (AADCD) Monoamine oxidase A deficiency (MAOAD) Vesicular monoamine transporter 2 deficiency (VMAT2D) Autosomal dominant and recessive GTP-cyclohydrolase deficiency (ad/arGTPCHD) Dihydropteridine reductase deficiency (DHPRD) DNAJC12 deficiency (DNAJC12D) Tyrosine hydroxylase deficiency (THD) Dopamine βhydroxylase deficiency (DßHD) Dopamine transporter deficiency (DATD) 6-pyruvoyl-tetrahydropterin synthase deficiency (PTPSD) Pterin-4a- carbinolamine dehydratase deficiency (PCDD) Sepiapterin reductase deficiency (SRD) ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 2NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications Table 2 Anthropometrical parameters, mode of delivery and postnatal outcomes in primary disorders of biogenic amines. Disease name Total number of patients (families) Gestational age Birth weight Birth length Birth head circumference sIUGR Mode of delivery Postnatal outcome in mature newborn Postnatal outcome in prematurity nnMean Prematurity (%) n<10% perc (SGA) (%) n<10% perc (%) n<10% perc (%) <3% perc (microcephaly) (%) (%) nn AADCD 56 (52) 51 38.5 18 (n=9) range: 32nd –36th GW 48 33 29 21 19 5 5 0 21 SVD, 28 C- section, 2 IVD, 2 VE, 3 NA 28 Feeding problems (n=16), hyperbilirubinemia (n=10), temperature instability (n=4), hypoglycemia (n=4), respiratory distress syndrome (n=2), each once: need for mechanical ventilation, meconium aspiration, hypotonia, sleepiness, hypokinesia, tongue fasciculation, reflux, constipation, nasal congestion, tremor, jitteriness 7 Feeding problems, temperature instability, bradycardia, hypoglycemia, neonatal distress THD 44 (44) 41 38.8 7 (n=3) range: 33rd –36th GW 37 49 23 39 16 31 0 10 22 SVD, 17 C- section, 3 IVD, 1 VE, 1 NA 13 Hyperbilirubinemia (n=5), feeding problems (n=3), each once: need for mechanical ventilation, sepsis, hemorrhage, sleepiness, umbilical sepsis, club feet 3 Feeding problems, temperature instability, bradycardia, hypoglycemia, neonatal distress MAOAD 4 (3) 4 40 0 4 0 NA NA 0 NA NA NA 2 SVD, 2 VE 1 Feeding problems, hypotonia, excessive sedation NA DATD 5 (5) 5 39 0 4 25 2 50 2 0 0 0 2 SVD, 3 C-section 3 Feeding problems (n=2), hyperbilirubinemia, temperature instability, resuscitation, need for mechanical ventilation NA C-section cesarean section, GW gestational week, IVD induced vaginal delivery, nnumber reported, NA not available, perc percentile, SGA small for gestational age, sIUGR symmetrical intrauterine growth restriction, SVD spontaneous vaginal delivery, VE vacuum extraction. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 ARTICLE NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications 3 Table 3 Anthropometrical parameters, mode of delivery and postnatal outcomes in BH 4 deficiencies and DNAJC12 deficiency. Disease name Total number of patients (families) Gestational age Birth weight Birth length Birth head circumference sIUGR Mode of delivery Postnatal outcome in mature newborn Postnatal outcome in prematurity nnMean Prematurity (%) n<10% perc (SGA) (%) n<10% perc (%) n<10% perc (%) <3% perc (microcephaly) (%) (%) nn arGTPCHD 18 (17) 17 40 0 15 33 8 25 7 29 0 8 9 SVD, 8 C- section, 1 NA 8 Jitteriness (n =2), hyperbilirubinemia (n =2), each once: tremor, irritability, feeding problems, vomiting, temperature instability NA adGTPCHD 36 (25) 33 39.4 3 (n=1) 36th GW 27 11 15 13 8 0 0 0 29 SVD, 5 C- section, 1 IVD,1 VE 3 Hypoglycemia, congenital torticollis, tachypnoea due to a small ventricular septal defect 1 None PTPSD 56 (53) 49 38.2 10 (n=5) range: 33rd-36th GW 45 56 36 33 29 34 24 18 20 SVD, 24 C-section, 4 IVD, 1 VE, 7 NA 17 Hyperbilirubinemia (n=8), feeding problems (n=6), hypoglycemia (n=5), each once: sepsis, hypothermia, floppiness, respiratory distress 2 Hypoglycemia, hyperbilirubinemia, feeding problems, temperature instability DHPRD 37 (34) 36 38.7 11 (n=4) range: 31st-36th GW 29 24 16 6 15 27 7 4 21 SVD, 11 C- section, 4 IVD, 1 NA 12 Hyperbilirubinemia (n=6), feeding problems (n=6), hypoglycemia (n=2), each once: sepsis, hypoxic ischemic encephalopathy, oculogyric crisis, hypertonia, meconium aspiration, dysplastic left kidney 3 Neonatal distress, tachycardia, ascites SRD 14 (11) 14 38.6 21 (n=3) range: 33rd-36th GW 10 30 7 29 5 40 0 11 8 SVD, 3 C- section,3 IVD 4 Hyperbilirubinemia (n=2), feeding problems, perinatal hypoxia 3 Hyperbilirubinemia, feeding problems, need for mechanical ventilation DNAJC12D 5 (4) 5 37.8 20 (n=1) 36th GW 5 0 5 20 5 0 0 0 3 SVD, 2 C-section 2 Hyperbilirubinemia 1 None C-section cesarean section, GW gestational week, IVD induced vaginal delivery, nnumber reported, NA not available, perc percentile, SGA small for gestational age, sIUGR symmetrical intrauterine growth restriction, SVD spontaneous vaginal delivery, VE vacuum extraction. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 4NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications Table 4 Age at initial symptoms and diagnosis as well as diagnostic methods. Disease name Total number of patients Age at initial symptoms (months) Age at diagnosis (months) Diagnostic work-up nnmean min max nmean min max Type of diagnostic screening Results of NBS for PKU Confirmatory methods (number of available results) AADCD 56 49 2 0 12 51 41.2 0.9 389.3 54 SS, 1 HRFs, 1 NA NA mutational analysis (46/ 56) ± biogenic amines in CSF (42/56) / AADC enzyme activity in plasma (23/56) THD 44 41 6.6 0 60 43 64.6 2 474.5 43 SS, 1 HRFs NA mutational analysis (41/ 44) ± biogenic amines in CSF (32/44) MAOAD 4 3 10.3 1 24 4 177.5 6.5 447 4 SS NA mutational analysis (3/ 4) ± biogenic amines in CSF (1/4) DATD 5 4 3 1 5 5 62.1 9 194.7 5 SS NA mutational analysis (3/ 5) ± biogenic amines in CSF (3/5) arGTPCHD 18 16 23 0 204 16 31 0.7 170.3 15 SS, 2 NBS*, 1 HRFs 7 normal, 8 HPA (2 NBS, 6 SS), 3 not done mutational analysis (14/ 18) ± biogenic amines in CSF (10/18)/pterins in CSF (8/18)/pterins in urine (7/18) adGTPCHD 36 31 80 6 516 35 155.6 18 632.7 30 SS, 5 HRFs, 1 NA 23 normal, 6 not done mutational analysis (36/ 36) PTPSD 56 33 10.7 0 192 54 19.6 0.1 401.5 15 SS, 37 NBS*, 2 HRFs, 2 NA 43 HPA (37 NBS, 4 SS, 2 HRFs), 11 not done mutational analysis (39/ 56) ± biogenic amines in CSF (25/56)/pterins in CSF (20/56)/pterins in urine (31/56) DHPRD 37 24 10.3 1 72 37 21.4 0.4 255.5 22 SS, 13 NBS*, 2 HRFs 1 normal, 30 HPA (13 NBS, 17 SS), 5 not done, 1 NA DHPR enzyme activity in blood (23/37) ± mutational analysis (24/ 37)/biogenic amines in CSF(18/37)/pterins in CSF (16/37)/pterins in urine (10/37) SRD 14 12 3.1 0 6 14 114.1 7 316.3 13 SS, 1 HRFs 10 normal, 4 not done mutational analysis (13/ 14) ± biogenic amines in CSF (9/14)/pterins in CSF (5/14) DNAJC12D 5 5 21.6 6 48 5 111.2 73 170.3 2 SS, 2 NBS*, 1 HRFs 2 normal, 3 HPA mutational analysis (5/5) *indicates that diagnosis was established immediately following HPA on NBS. CSF cerebrospinal fluid, HRFs high-risk family screening, nnumber reported, NA not available, NBS newborn screening, PKU phenylketonuria, SS selective screening. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 ARTICLE NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications 5 In BH 4 Ds, 27 pregnancies with maternal health issues were reported. Among four mothers affected with adGTPCHD in this group, two mothers did not require any drug treatment despite having intermittent dystonia. There was no difference with regard to the mode of delivery between arGTPCHD and PTPSD while spontaneous vaginal delivery (SVD) was the most frequent mode of delivery in adGTPCHD and DHPRD (Table 3). Interestingly, newborns with PTPSD showed the highest rate of symmetrical intrauterine growth restriction (sIUGR) and SGA (Table 3and Fig. 1). Initial clinical presentation. The most common initial symptoms in AADCD and THD were developmental delay and truncal hypotonia (Fig. 2a). Thermoregulation disorders and oculogyric crises were prominent in AADCD and occurred more often than in THD (Fig. 3a). Dystonia was similarly frequent in both disease groups. Sleep disturbances were only observed in AADCD. The following additional symptoms (not listed in Figs. 2a and 3a) were reported in AADCD: gastrointestinal symptoms (n=7, 12.5%), nasal congestion, (n=6, 11%), tremor (n=3, 5%) and hypokinetic rigid syndrome (n=3, 5%). In THD, tremor (n=9, 20.5%), hypokinetic rigid syndrome (n=3, 7%), gastrointestinal symptoms (n=3, 7%) and suspected seizures (n=3, 7%) were reported. Developmental delay was reported in two out of four patients with MAOAD, epileptic seizures and encephalopathy in one and sleep disorders the other patient. Developmental delay, hypomimia and drooling were reported in one patient with DATD and dystonia and hypokinesia in another patient. Microcephaly and irritability each were the initial symptoms in other two patients with DATD. The mean age at onset of first symptoms was 4.3 months across all disorders in this group (range 0–60 months, Table 4). Patients with AADCD were already symptomatic in the neonatal period −3 −2 −1 0 1 Birth weight SDS a AADCD (n=56) THD (n=44) MAOAD (n=4) DATD (n=5) −2 0246 Birth length SDS b AADCD (n=29) THD (n=23) DATD (n=2) −2 −1 0 1 2 3 4 Birth head circumference SDS c AADCD (n=19) THD (n=16) DATD (n=2) −4 −2 0 2 Birth weight SDS Disorder d arGTPCHD (n=15) adGTPCHD (n=27) PTPSD (n=45) DHPRD (n=29) SRD (n=10) −3 −2 −1 0 1 2 3 Birth length SDS Disorder e arGTPCHD (n=8) adGTPCHD (n=15) PTPSD (n=36) DHPRD (n=16) SRD (n=7) −5 −4 −3 −2 −1 0 1 2 Birth head cirmumference SDS Disorder f arGTPCHD (n=7) adGTPCHD (n=8) PTPSD (n=29) DHPRD (n=15) SRD (n=5) Fig. 1 Birth metrics. Distribution of birth metrics in primary disorders of biogenic amine metabolism (a–c) and BH deficiencies (d–f). White spot: median, black bars: interquartile range (25% and 75%), n: number reported. Percent of patients 0 1020304050 dyskinesia hypoglycemic episodes microcephaly epileptic seizures encephalopathy sleep disorders thermoregulation disorders dystonia upper limb tone abnormality oculogyric crisis lower limb tone abnormality truncal hypotonia developmental delay 2.75% 2.75% 3.67% 4.59% 5.5% 8.26% 11.01% 16.51% 24.77% 26.61% 26.61% 33.03% 41.28% a Percent of patients 0 5 10 15 20 25 thermoregulation disorders dyskinesia encephalopathy hypoglycemic episodes microcephaly sleep disorders epileptic seizures oculogyric crisis upper limb tone abnormality lower limb tone abnormality truncal hypotonia developmental delay dystonia 0% 1.24% 1.24% 1.86% 3.11% 3.11% 4.35% 4.97% 12.42% 13.66% 19.25% 20.5% 21.74% b Fig. 2 Symptoms at disease onset. Initial symptoms in primary disorders of biogenic amine metabolism (a) and BH deficiencies (b), age independent analysis. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 6NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications and in early infancy while the majority of patients with THD became symptomatic during infancy (between 1 and 15 months, Fig. 3a). Only three out of 33 patients with THD were symptomatic neonatally with developmental delay and oculogyric crises while two patients showed initial symptoms in childhood (aged 3 and 5 years), one with hypotonia of extremities, dyskinesia and dystonia, and the other with gait disorder and speech difficulties. The most common initial symptoms among BH 4 Ds were dystonia, developmental delay and truncal hypotonia (Fig. 2b). Symptom onset occurred most frequently during the neonatal period or infancy in all patients with BH 4 Ds except for those with adGTPCHD (Table 4and Fig. 3b). This neonatal/ infantile initial presentation was typically characterized by developmental delay and (truncal) hypotonia. In contrast, dystonia (61%) and lower extremity hypertonia (13%) were the most frequent initial symptoms in adGTPCHD, together with gait disorders (n=6, 16%), orthopedic problems (n=5, 14%), tremor (n=3, 8%) and toe walking (n=3, 8%, not listed in Figs. 2b and 3b). Dystonia was more frequently seen in arGTPCHD (22%) than in PTPSD (11%), DHPRD (5%) and SRD (7%) in an age-independent analysis (Fig. 3b, age-dependent analysis). Seizures were reported only in PTPSD (5%) and DHPRD (11%) among all BH 4 Ds. As additional initial symptoms, hypokinetic rigid syndrome was reported in PTPSD (n=3, 5%) and DHPRD (n=3, 8%) and failure to thrive in PTPSD (n=3, 5%). Of note is the higher frequency of oculogyric crises (21%), and sleep disorders (14%) in SRD compared to other BH 4 Ds (0–5% and 0–6% respectively; age-independent analysis). Developmental delay alone was reported in one patient with DNAJC12D, together with oculogyric crises in another patient and behavioral problems in two patients. Diagnostic work-up and diagnostic delay. The latency to diagnosis was comparably long for AADCD (mean =41 months, range 0–386 months) and THD (mean =45 months, range −6–361 months) (Fig. 4a). Patients with AADCD born before 2009 had a statistically significant longer latency to diagnosis (mean 68.1 months) than those born after 2009 (mean 7.3 months, p=0.00041, WMW-test) (Fig. 4b). In the group of patients with THD, this pattern was also found for patients born before and after 2005, respectively (mean diagnostic delay of 89.8 months vs. 14.4 months, p=0.00041, WMW-test). Investigating the effect of symptoms on age at diagnosis or on the diagnostic delay, we found that presentation with hypotonia, seizures, encephalopathy, microcephaly, sleep disturbances or thermoregulation disorders was associated with earlier age at diagnosis (3.2 years) and with less diagnostic delay (30 months), than presentation with dystonia, dyskinesia, hypoglycemia or developmental delay (diagnosis age 4.9 years, t(88.14) =−1.68; p=0.1; diagnostic delay 45 months, t(175.43) =−1.89; p=0.06; Fig. 5a and b). Since disorders presenting with hyperphenylalaninemia (HPA), arGTPCHD, PTPSD and DHPRD, can be detected by newborn screening (NBS) for phenylketonuria, they would be expected to be diagnosed at a younger age and with less delay than those without HPA. In our study cohort, diagnostic work-up as well as diagnostic delay varied strongly depending on the occurrence of HPA (Table 4 and Fig. 4a). In patients with arGTPCHD, PTPSD and DHPRD, HPA on NBS led to significantly shorter diagnostic delay than in cases without HPA or without any NBS performed (mean 3.1 vs. 39.6 months, t(24.05) =2.99; p=0.006, Fig. 4c). There was no significant difference in diagnostic delay between the latter group vs. those BH 4 Ds without HPA and AADCD and THD (mean 39.6 vs. 76.7 vs. 51 months, t(2) =1.89; p=0.15). Disorders without HPA (adGTPCHD and SRD) were mainly diagnosed by selective screening after onset of symptoms or by high-risk family screening (HRFs). In our study, the majority of patients with adGTPCHD were symptomatic in childhood and had a mean diagnostic delay of 60 months (range 1–245 months). Most patients with SRD were symptomatic in the first 6 months of life, however, this group in particular showed a prominently prolonged latency to diagnosis with an average duration of 112 months (range 12–316 months, Table 4, Fig. 4a). SRD patients born after 2009 had a significantly shorter diagnostic delay than those born before (mean 140 months, vs. 29.9 months, sleep disorders thermoregulation disorders oculogyric crisis dystonia lower limb tone abnormality upper limb tone abnormality truncal hypotonia developmental delay Number of patients 0 5 10 15 20 25 16.3% 22.4% 42.9% 14.3% 24.5% 24.5% 34.7% 42.9% a. AADCD dystonia lower limb tone abnormality upper limb tone abnormality truncal hypotonia developmental delay Number of patients 02468 25% 31.2% 31.2% 31.2% 37.5% b. arGTPCHD dystonia lower limb tone abnormality upper limb tone abnormality truncal hypotonia developmental delay Number of patients 0 5 10 15 20 18.2% 21.2% 24.2% 39.4% 33.3% PTPSD oculogyric crisis truncal hypotonia developmental delay Number of patients 012345 25% 33.3% 33.3% SRD oculogyric crisis dystonia lower limb tone abnormality upper limb tone abnormality truncal hypotonia developmental delay Number of patients 0 5 10 15 20 25 17.1% 22% 39% 36.6% 41.5% 46.3% THD dystonia lower limb tone abnormality Number of patients 0 5 10 15 20 25 71% 16.1% adGTPCHD epileptic sei ures lower limb tone abnormality upper limb tone abnormality truncal hypotonia developmental delay Number of patients 0246810 16% 12% 16% 28% 20% DHRPD neonatal infancy childhood adolescence adulthood Fig. 3 Age dependency of initial symptoms. Age-dependent onset of initial symptoms in primary disorders of biogenic amine metabolism (a) and BH deficiencies (b). Initial symptoms reported in minimum 5 patients––except in arGTPCHD, DHPRD and SRD in minimum 3 patients. The percentages correlate with available data about both occurrence of the symptom and age at onset of disease and not to the whole disease group. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 ARTICLE NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications 7 p=0.021, WMW-test). For adGTPCHD no significantly discriminating date was found (Fig. 4b). Seven patients with arGTPCHD had normal NBS results without HPA. Eight patients presented with HPA on NBS but in six of these eight cases the diagnostic work-up was initiated later (Table 4). In patients with arGTPCHD who were diagnosed via selective screening, the mean diagnostic delay in the group with HPA on NBS (7.8 months) differed from the other group with normal results on NBS (21.5 months) without reaching statistical significance (t(7.39) =−1.78; p=0.11, Fig. 4d). Only in the PTPSD group did all the available NBS results demonstrate HPA (Table 4). While the vast majority of the patients with DHPRD (30/37) had HPA on NBS, one patient surprisingly had a normal NBS and presented normal phenylalanine levels in plasma but high levels in CSF on repeated measurements. This patient was homozygous for a new variant in the QDPR gene (NM_000320.3, c.466 G > A, p. Ala156Thr). There were 17 DHPRD patients whose diagnosis was established late despite having HPA on NBS and who had a longer mean diagnostic delay (6 months) than those who were diagnosed by specific work-up immediately following the detection of HPA on NBS (-4.5 months, t(16.44) =−1.87; p=0.08, Fig. 4e). Negative values for the diagnostic delay in PTPSD and DHPRD in Fig. 4aareexplainedbyanearly diagnosisviaNBSorHRFsbeforeonsetofsymptoms.The lowest values for the mean diagnostic delay (8 months) and for the maximum diagnostic delay (86 months) were recorded in DHPRD. 0 100 200 300 400 DATD (n=4) MAOAD (n=3) THD (n=40) AADCD (n=45) SRD (n=12) DHPRD (n=25) PTPSD (n=33) adGTPCHD (n=30) arGTPCHD (n=14) Diagnostic delay [months] a THD DHPRD SRD AADCD arGTPCHD adGTPCHD PTPSD 1980 2000 2020 1980 2000 2020 1980 2000 2020 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 Year of birth Diagnostic delay [months] b HPA in NBS Diagnostic delay [log] no/not done yes 1d 5d 1m 5m 10m 50m 100m 200m c HPA in NBS normal NBS 0 10 20 30 40 50 Selective screening Diagnostic delay [months] d NBS work−up selective screening 40 30 20 10 0 10 20 HPA in NBS Diagnostic delay [months] e Fig. 4 Effects on diagnostic delay in different diseases. a Diagnostic delay in disorders of biogenic amine metabolism and BH deficiencies. White spot: median, black bars: interquartile range (25% and 75%), n: number reported. bEffect of date of birth on diagnostic delay; bold line: significant, two-sided WMW-test, p< 0.05, cDiagnostic delay in arGTPCHD, PTPSD and DHPRD in patients without any or normal newborn screening (NBS) results (n=24) vs. patients with hyperphenylalaninemia (HPA) on NBS (n=47); two-sided t-test, p=0.006. dDiagnostic delay in arGTPCHD regarding HPA on NBS (n=5) vs. normal NBS (n=6); two-sided t-test, p=0.11. eDiagnostic delay in DHPRD in patients having HPA on NBS diagnosed directly after NBS (n= 6) vs. via selective screening (n=14); two-sided t-test, p=0.08. Diamond: arithmetic mean, bold line: median, boxes present interquartile range (25% and 75%). ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 8NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications We identified the birth year 1993 as the most strongly discriminating and statistically significant time point regarding changes in the latency to diagnosis for PTPSD (mean 86.6 months, SD 88.6 months vs. mean 3.4 months, SD 17.3 months, p =0.028, WMW-test, Fig. 4b). In the remaining BH 4 Ds, we could detect a trend around the years 1999 for arGTPCHD and 2012 for DHPRD but these dates did not reach statistical significance. Truncal hypotonia, upper limb hyper-/or hypotonia, developmental delay, epilepsy, encephalopathy, microcephaly, thermoregulation disorders, oculogyric crises, dyskinesia or hypoglycemia were associated with earlier age at diagnosis (2.7 years) than lower limb hypo-/or hypertonia (4.7 years) or dystonia and sleep disorders (8.8 years; ANOVA; F(2,165) =14.89; p=0.16 for 2.7 vs 4.7 years; p=0.0000005 for 2.7 vs 8.8 years; p=0.02 for 4.7 vs. 8.8 years). Other than in primary disorders of biogenic amine metabolism, developmental delay, tone abnormalities in upper limb and trunk as well as epilepsy were associated with a shorter diagnostic delay (28 months) than oculogyric crises, dystonia, lower limb tone abnormalities and sleep problems (47 months, t (117.73) =−2.15; p=0.03, Fig. 5c, d) in BH 4 Ds. Initial biochemical presentation. Diagnostically relevant and disease-specific constellations of biochemical parameters are presented in Fig. 6. Along with typical changes in biogenic amines (i.e. reduced homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA), elevated 3-O-methyl-Dopa (3-OMD), levodopa (L-Dopa) and 5-hydroxytryptophan (5-HTP)), abnormalities of tetrahydrobiopterin Age at diagnosis [years] 0 4 8 1216202428323640 developmental delay (n=43) dyskinesia (n=3) dystonia (n=18) encephalopathy (n=5) epileptic seizures (n=5) hypoglycemic episodes (n=3) lower limb tone abnormality (n=27) microcephaly (n=4) oculogyric crisis (n=28) sleep disorders (n=9) thermoregulation disorders (n=11) truncal hypotonia (n=33) upper limb tone abnormality (n=25) a (Primary disorders of biogenic amine metabolism) Age at diagnosis [years] 0 4 8 1216202428323640 developmental delay (n=32) dyskinesia (n=1) dystonia (n=33) encephalopathy (n=2) epileptic seizures (n=7) hypoglycemic episodes (n=3) lower limb tone abnormality (n=22) microcephaly (n=5) oculogyric crisis (n=8) sleep disorders (n=4) truncal hypotonia (n=31) upper limb tone abnormality (n=20) c (Disorders of BH4 metabolism) Diagnostic delay [months] 0 50 100 150 200 250 300 350 400 developmental delay (n=40) dyskinesia (n=3) dystonia (n=17) encephalopathy (n=5) epileptic seizures (n=5) hypoglycemic episodes (n=3) lower limb tone abnormality (n=26) microcephaly (n=4) oculogyric crisis (n=27) sleep disorders (n=9) thermoregulation disorders (n=11) truncal hypotonia (n=31) upper limb tone abnormality (n=25) b (Primary disorders of biogenic amine metabolism) Diagnostic delay [months] 50 0 50 100 150 200 250 developmental delay (n=27) dystonia (n=33) encephalopathy (n=2) epileptic seizures (n=7) hypoglycemic episodes (n=3) lower limb tone abnormality (n=21) microcephaly (n=4) oculogyric crisis (n=8) sleep disorders (n=3) truncal hypotonia (n=29) upper limb tone abnormality (n=19) d (Disorders of BH4 metabolism) Fig. 5 Association between initial symptoms and age at diagnosis vs. diagnostic delay. Association between initial symptoms and age at diagnosis vs. diagnostic delay in disorders of primary biogenic amine metabolism (a,b) and BH metabolism (c,d). Diamond: arithmetic mean, bold line: median, boxes present interquartile range (25% and 75%). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25515-5 ARTICLE NATURE COMMUNICATIONS | (2 021) 12:5529 | https://doi.org/10.1038/s41467-021-25515-5 | www.nature.com/naturecommunications 9