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

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.

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 (BH4). 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 under-recognized 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 disease-specific treatment, as well as long-term compliance to

treatment4,5,6,7,8,9,10.

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.

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 BH4

deficiencies (BH4Ds) and five patients DNAJC12D (Tables 2 and 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.

Maternal health problems, medications taken during pregnancy and postnatal outcomes are depicted in Tables 2 and 3 and 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)  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. 4a are explained by an early diagnosis via NBS or HRFs before onset of

symptoms. The lowest values for the mean diagnostic delay (8 months) and for the maximum diagnostic delay (86 months) were recorded in DHPRD.

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 BH4Ds, 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 BH4Ds.

Diagnostically relevant and disease-specific constellations of biochemical parameters are presented in Fig. 6. 

Disease-specific changes of biochemical parameters in plasma (P), dried blood spot (DBS), urine (U) and cerebrospinal fluid (CSF) before treatment.

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 and neopterin in CSF were observed in some AADCD patients (Fig. 6) Urinary vanillactic acid was not reported as an initial

diagnostic parameter in our AADCD cohort. In THD, HVA and the ratio HVA/5-HIAA were decreased in almost all samples, while 5-HTP and 5-HIAA were typically normal. Prolactin in plasma showed

high variability in both diseases (Fig. 6).

Phenylalanine (Phe) was normal or high in CSF and plasma in arGTPCHD while it was predominantly normal in both blood and CSF in adGTPCHD (Fig. 6). Phe was high in both CSF and in plasma in

PTPSD and DHPRD. In SRD, Phe was increased in CSF while being normal in plasma, in line with previous reports12. Pterin disturbances were reported in CSF and in urine for adGTPCHD. Results

on pterins in dried blood spots (DBS) in adGTPCHD were not available. 7-8-dihydrobiopterin in CSF was determined rarely but three measurements in DHPRD and two in SRD were high while being

always normal in other BH4 disorders. HVA and 5-HIAA were more frequently decreased in PTPSD, DHPRD and SRD than in ad/arGTPCHD. In PTPSD and DHPRD prolactin was elevated in 44% and 82% of

cases, respectively, while it was normal in almost all other BH4Ds. Decreased 5-methyltetrahydrofolate in CSF was reported only in DHPRD, except in one case with arGTPCHD and three cases

with PTPSD.

Three patients with AADCD and one with THD died during the study period. Death occurred at 2.4, 2.6 and 19.8 years of life in the AADCD patients. One patient died of pneumonia while in the

remaining two cases the cause was unknown. The THD patient died at 13 years of age because of an acute lower respiratory tract infection.

The evaluation of 275 patients with disorders of biogenic amines (224 new and 51 previously published cases) that were analyzed using a standardized longitudinal approach revealed new

phenotypic aspects of the initial clinical and biochemical presentation, peri- and postnatal courses as well as diagnostic work-up. We present an increased incidence of prematurity in AADCD

and of SGA in THD and in PTPSD. Patients with PTPSD were also prone to sIUGR and congenital microcephaly. We report one patient with DHPRD without HPA on NBS. We confirm the significant

impact of HPA detection on NBS on the diagnostic work-up in a group of BH4Ds. Furthermore, we present the association of specific symptoms, such as oculogyric crises, dystonia, sleep and

thermoregulation disorders, with age at diagnosis and diagnostic delay.

Pregnancies in both main disease groups were rarely complicated by medical problems. The issues described were most likely due to the pregnancy itself and not to the fetal disease.

Exceptions were those cases in which mothers were affected by adGTPCHD, consistent with previous literature13. First symptoms of disorders of biogenic amines typically occur in the neonatal

period or in infancy. Our data on the anthropometrical values at birth raise questions about prenatal disease manifestation. AADCD (18%) and SRD (21%) showed an increased rate of prematurity

in our study compared to the global incidence of prematurity that is estimated as 9.6% ranging from 6.2% in Europe to 9.1 % in Asia, 10.6 % in North America and 11.9% in Africa14. Various

causal factors such as fetal or maternal health conditions along with genetic, environmental, behavioral and socioeconomic factors as well as the differences in availability of preventive

interventions between developed and developing countries influence the estimated rates. Since most of the patients in this study were born in Europe, North America or Asia, the background

preterm birth rate may be expected to be between 6.2% and 10.6%. While the rate in SRD should be critically interpreted due the small number of cases, our data document an increased rate of

prematurity in AADCD.

Additionally, the risk of SGA at birth is higher in PTPSD and THD (56% and 49%, respectively) compared to the considerable variation in the prevalence of infants born SGA, ranging from

4.6–15.3 % across Europe and 5.3% in east Asia to 41.5% in south Asia15,16. AADCD, arGTPCHD, DHPRD and SRD also show elevated but not as high SGA rates. While neonates with PTPSD are also

prone to sIUGR and congenital microcephaly (24%), we could not observe any trend towards microcephaly in DHPRD in contrast to the reported 25% rate in a historical cohort17. The observation

that PTPSD patients were at high risk for prematurity could not be confirmed in our cohort but the detection of SGA and sIUGR are in line with the previously reported tendency to have very

low birth weight (BW