Fibroblasts from a male patient with compound heterozygous variants in the tyrosine hydroxylase gene (TH; OMIM: 191290; c.[385-C>T]; [692-G>C]/p.[R129*]; [R231P]), the rate-limiting enzyme for dopamine synthesis, were reprogrammed to iPSCs using episomal reprogramming delivering the reprogramming factors Oct3/4, Sox2, L-Myc, Lin28, Klf4 and p53 shRNA Okita et al. (2011). Pluripotency of TH-1 iPSC was verified by immunohistochemistry and RT-PCR analysis. Cells exhibited a normal karyotype and differentiated spontaneously into the 3 germ layers in vitro. TH-1 iPSC represents the first model system to study the pathomechanism of this rare metabolic disease and provides a useful tool for drug testing.

Whole exome sequencing (WES) is well established in research and is now being introduced into clinically indicated diagnostics (so-called clinical exomes). We evaluated the diagnostic yield and clinical implications of WES in 72 patients from 60 families with undiagnosed neurodevelopmental disorders (NDD), neurometabolic disorders, and dystonias. Pathogenic or likely pathogenic variants leading to a molecular diagnosis could be identified in 21 of the 60 families (overall 35%, in 36% of patients with NDD, in 43% of patients with neurometabolic disorders, in 25% of patients with dystonias). In one family two coexisting autosomal recessive diseases caused by homozygous pathogenic variants in two different genes were diagnosed. In another family, a homozygous frameshift variant in STRADA was found to cause a severe NDD with early onset epilepsy, brain anomalies, hypotonia, heart defect, nephrocalcinosis, macrocephaly and distinctive facies so far designated as PMSE (polyhydramnios, megalencephaly, symptomatic epilepsy) syndrome. In 7 of the 21 families with a molecular diagnosis the pathogenic variants were only identified by clinical follow-up, manual reevaluation of the literature, a change of filter setting, and/or reconsideration of inheritance pattern. Most importantly, clinical implications included management changes in 8 cases and impact on family planning in 20 families with a molecular diagnosis. This study shows that reevaluation and follow-up can improve the diagnostic rate and that WES results have important implications on medical management and family planning. Furthermore, we could confirm STRADA as a gene associated with syndromic ID but find it questionable if the current designation as PMSE depicts the most important clinical features.

Tetrahydrobiopterin (BH4) deficiencies comprise a group of six rare neurometabolic disorders characterized by insufficient synthesis of the monoamine neurotransmitters dopamine and serotonin due to a disturbance of BH4 biosynthesis or recycling. Hyperphenylalaninemia (HPA) is the first diagnostic hallmark for most BH4 deficiencies, apart from autosomal dominant guanosine triphosphate cyclohydrolase I deficiency and sepiapterin reductase deficiency. Early supplementation of neurotransmitter precursors and where appropriate, treatment of HPA results in significant improvement of motor and cognitive function. Management approaches differ across the world and therefore these guidelines have been developed aiming to harmonize and optimize patient care. Representatives of the International Working Group on Neurotransmitter related Disorders (iNTD) developed the guidelines according to the SIGN (Scottish Intercollegiate Guidelines Network) methodology by evaluating all available evidence for the diagnosis and treatment of BH4 deficiencies.

Aromatic l-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal recessive neurodevelopmental disorder characterized by impaired synthesis of dopamine, noradrenaline, adrenaline and serotonin, leading to a complex syndrome of motor, behavioral, and autonomic symptoms. This retrospective study assessed the symptoms and developmental outcome of a large international cohort of patients with AADCD via physician and/or caregiver responses to a detailed, standardized questionnaire. Sixty-three patients (60% female; ages 6 months-36 years, median 7 years; 58 living) from 23 individual countries participated. Common symptoms at onset (median age 3 months, range 0-12 months) were hypotonia, developmental delay, and/or oculogyric crises. Oculogyric crises were present in 97% of patients aged 2 to 12 years, occurred in the majority of patients in all age groups, and tended to be most severe during early childhood. Prominent non-motor symptoms were sleep disturbance, irritable mood, and feeding difficulties. The majority of subjects (70%) had profound motor impairment characterized by absent head control and minimal voluntary movement, while 17% had mild motor impairment and were able to walk independently. Dopamine agonists were the medications most likely to produce some symptomatic benefit, but were associated with dose-limiting side effects (dyskinesia, insomnia, irritability, vomiting) that led to discontinuation 25% of the time. The age distribution of our cohort (70% of subjects under age 13 years) and the observation of a greater proportion of patients with a more severe disease phenotype in the younger compared to the older patients, both suggest a significant mortality risk during childhood for patients with severe disease.

Silver-Russell syndrome (SRS) is an imprinting disorder which is characterised by severe primordial growth retardation, relative macrocephaly and a typical facial gestalt. The clinical heterogeneity of SRS is reflected by a broad spectrum of molecular changes with hypomethylation in 11p15 and maternal uniparental disomy of chromosome 7 (upd(7)mat) as the most frequent findings. Monogenetic causes are rare, but a clinical overlap with numerous other disorders has been reported. However, a comprehensive overview on the contribution of mutations in differential diagnostic genes to phenotypes reminiscent to SRS is missing due to the lack of appropriate tests. With the implementation of next generation sequencing (NGS) tools this limitation can now be circumvented.

TUBB2A tubulinopathy is a rare neurodevelopmental disorder with developmental delay, epilepsy, and less frequent malformations of cortical development compared to other tubulinopathies. Peripheral blood mononuclear cells (PBMCs) from a male subject harboring the heterozygous de novo TUBB2A variant c.[743C>T] (p.[Ala248Val]) were reprogrammed to induced pluripotent stem cells (iPSCs) using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Generated iPSCs showed a normal karyotype, expression of pluripotency markers, spontaneous in vitro differentiation in all three germ layers, and are a suitable human disease model to analyze pathomechanisms underlying TUBB2A tubulinopathy and potential therapeutic targets.

Inherited methylation disorders are a group of rarely reported, probably largely underdiagnosed disorders affecting transmethylation processes in the metabolic pathway between methionine and homocysteine. These are methionine adenosyltransferase I/III, glycine N-methyltransferase, S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. This paper provides the first consensus recommendations for the diagnosis and management of methylation disorders. Following search of the literature and evaluation according to the SIGN-methodology of all reported patients with methylation defects, graded recommendations are provided in a structured way comprising diagnosis (clinical presentation, biochemical abnormalities, differential diagnosis, newborn screening, prenatal diagnosis), therapy and follow-up. Methylation disorders predominantly affect the liver, central nervous system and muscles, but clinical presentation can vary considerably between and within disorders. Although isolated hypermethioninemia is the biochemical hallmark of this group of disorders, it is not always present, especially in early infancy. Plasma S-adenosylmethionine and S-adenosylhomocysteine are key metabolites for the biochemical clarification of isolated hypermethioninemia. Mild hyperhomocysteinemia can be present in all methylation disorders. Methylation disorders do not qualify as primary targets of newborn screening. A low-methionine diet can be beneficial in patients with methionine adenosyltransferase I/III deficiency if plasma methionine concentrations exceed 800 μmol/L. There is some evidence that this diet may also be beneficial in patients with S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. S-adenosylmethionine supplementation may be useful in patients with methionine adenosyltransferase I/III deficiency. Recommendations given in this article are based on general principles and in practice should be adjusted individually according to patient's age, severity of the disease, clinical and laboratory findings.

Variants in different neuronal tubulin isotypes cause severe neurodevelopmental disorders with cerebral malformations accompanied by developmental delay, motor impairment, and epilepsy, known as tubulinopathies. Induced pluripotent stem cells were generated from peripheral blood mononuclear cells from a female subject carrying the heterozygous de novo variant c.[521C > T] (p.[Ala174Val]) in the TUBA1A gene. PBMCs were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) and showed a normal karyotype, expression of pluripotency markers, and spontaneous in vitro differentiation into all three germ layers. The generated iPSCs represent a useful tool to study the pathophysiology of TUBA1A tubulinopathy.

Fibroblasts from a female patient carrying a heterozygous variation in GTP cyclohydrolase 1 (GCH1; OMIM: 600225; HGNC: 4193; c.235_240del/p.(L79_S80del)), the rate-limiting enzyme of tetrahydrobiopterin (BH4) synthesis, were reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) delivering the four reprogramming factors Oct3/4, Sox2, c-Myc and Klf4. Pluripotency of HDMC0061i-GCH1 was verified using immunohistochemistry and RT-PCR analysis. Cells differentiated spontaneously into the 3 germ layers in vitro and presented a normal karyotype. HDMC0061i-GCH1 represents the first model system to elucidate the pathomechanism underlying this rare metabolic disease and a useful tool for drug testing.

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.

Following the broad application of new analytical methods, more and more pathophysiological processes in previously unknown diseases have been elucidated. The spectrum of clinical presentation of rare inherited metabolic diseases (IMDs) is broad and ranges from single organ involvement to multisystemic diseases. With the aim of overcoming the limited knowledge about the natural course, current diagnostic and therapeutic approaches, the project has established the first unified patient registry for IMDs that fully meets the requirements of the European Infrastructure for Rare Diseases (ERDRI).

Dihydropteridine reductase (QDPR) catalyzes the recycling of tetrahydrobiopterin (BH4), a cofactor in dopamine, serotonin, and phenylalanine metabolism. QDPR-deficient patients develop neurological symptoms including hypokinesia, truncal hypotonia, intellectual disability and seizures. The underlying pathomechanisms are poorly understood. We established a zebrafish model for QDPR deficiency and analyzed the expression as well as function of all zebrafish QDPR homologues during embryonic development. The homologues qdpra is essential for pigmentation and phenylalanine metabolism. Qdprb1 is expressed in the proliferative zones of the optic tectum and eye. Knockdown of qdprb1 leads to up-regulation of pro-proliferative genes and increased number of phospho-histone3 positive mitotic cells. Expression of neuronal and astroglial marker genes is concomitantly decreased. Qdprb1 hypomorphic embryos develop microcephaly and reduced eye size indicating a role for qdprb1 in the transition from cell proliferation to differentiation. Glutamine accumulation biochemically accompanies the developmental changes. Our findings provide novel insights into the neuropathogenesis of QDPR deficiency.

To elucidate the genetic cause of an Egyptian family with dopa-responsive dystonia (DRD), a childhood-onset dystonia, responding therapeutically to levodopa, which is caused by mutations in various genes.

Metabolic breakdown of valproate (VPA), carbamazepine (CBZ) and phenytoin (PHT) by the cytochrome P450 pathway generates toxic drug intermediates and reactive oxygen species (ROS). This mechanism has been suspected to play a role in the pathogenesis of secondary cerebral folate deficiency (CFD). Using KB-cell cultures, highly expressing the folate receptor 1 (FOLR1), the effect of antiepileptic drugs (AEDs) and reactive oxygen species (ROS) on the FOLR1 dependent 5-methyltetrahydrofolate (MTHF) uptake was studied. MTHF uptake is time and concentration dependent and shows saturation kinetics. At physiological MTHF concentrations the high-affinity FOLR1 represents the predominant mechanism for cellular incorporation, while at high MTHF concentrations other transport mechanisms participate in folate uptake. Exposure to PHT for more than 8h led to a higher MTHF uptake and decreased cell count, whereas MTHF uptake remained unaltered by VPA and CBZ. However, exposure to superoxide and hydrogen peroxide radicals significantly decreased cellular MTHF uptake. By specific elimination and downregulation of FOLR1 using phosphatidyl-inositol-specific phospholipase C (PIPLC) and siRNA silencing, it was shown that ROS not only inhibited FOLR1 mediated MTHF uptake but also affected all other mechanisms of membrane-mediated MTHF uptake. Generation of ROS with the use of AED might therefore provide an additional explanation for the disturbed folate transfer across the blood-CSF barrier in patients with CFD.

GTP cyclohydrolase 1, encoded by the GCH1 gene, is an essential enzyme for dopamine production in nigrostriatal cells. Loss-of-function mutations in GCH1 result in severe reduction of dopamine synthesis in nigrostriatal cells and are the most common cause of DOPA-responsive dystonia, a rare disease that classically presents in childhood with generalized dystonia and a dramatic long-lasting response to levodopa. We describe clinical, genetic and nigrostriatal dopaminergic imaging ([(123)I]N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) tropane single photon computed tomography) findings of four unrelated pedigrees with DOPA-responsive dystonia in which pathogenic GCH1 variants were identified in family members with adult-onset parkinsonism. Dopamine transporter imaging was abnormal in all parkinsonian patients, indicating Parkinson's disease-like nigrostriatal dopaminergic denervation. We subsequently explored the possibility that pathogenic GCH1 variants could contribute to the risk of developing Parkinson's disease, even in the absence of a family history for DOPA-responsive dystonia. The frequency of GCH1 variants was evaluated in whole-exome sequencing data of 1318 cases with Parkinson's disease and 5935 control subjects. Combining cases and controls, we identified a total of 11 different heterozygous GCH1 variants, all at low frequency. This list includes four pathogenic variants previously associated with DOPA-responsive dystonia (Q110X, V204I, K224R and M230I) and seven of undetermined clinical relevance (Q110E, T112A, A120S, D134G, I154V, R198Q and G217V). The frequency of GCH1 variants was significantly higher (Fisher's exact test P-value 0.0001) in cases (10/1318 = 0.75%) than in controls (6/5935 = 0.1%; odds ratio 7.5; 95% confidence interval 2.4-25.3). Our results show that rare GCH1 variants are associated with an increased risk for Parkinson's disease. These findings expand the clinical and biological relevance of GTP cycloydrolase 1 deficiency, suggesting that it not only leads to biochemical striatal dopamine depletion and DOPA-responsive dystonia, but also predisposes to nigrostriatal cell loss. Further insight into GCH1-associated pathogenetic mechanisms will shed light on the role of dopamine metabolism in nigral degeneration and Parkinson's disease.

Skin fibroblasts were isolated from a male patient with DNAJC12 deficiency and reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Two clones, DHMCi003-A and DHMCi003-B, were characterized for expression of pluripotency marker genes (Oct4, Nanog, Lin28, SSEA-4, TRA-1-60) and differentiated into all three germ layers using embryoid body (EB) formation. Karyotype of both clones was normal and presence of the homozygous mutation in the DNAJC12 gene was verified by PCR and Sanger sequencing. Both clones represent a useful tool to study the pathomechanisms underlying the deficiency.

Melatonin is synthesized from serotonin and it is excreted as sulphatoxymelatonin in urine. We aim to evaluate urinary sulphatoxymelatonin as a biomarker of brain serotonin status in a cohort of patients with mutations in genes related to serotonin biosynthesis. We analized urinary sulphatoxymelatonin from 65 healthy subjects and from 28 patients with genetic defects. A total of 18 patients were studied: 14 with autosomal dominant and recessive guanosine triphosphate cyclohydrolase-I deficiency; 3 with sepiapterin reductase deficiency; and 1 with aromatic L-amino acid decarboxylase deficiency. Further 11 patients were studied after receiving serotoninergic treatment (serotonin precursors, monoamine oxidase inhibitors, selective serotonin re-uptake inhibitors): 5 with aromatic L-amino acid decarboxylase deficiency; 1 with sepiapterin reductase deficiency; 3 with dihydropteridine reductase deficiency; and 2 with 6-pyruvoyltetrahydropterin synthase deficiency. Among the patients without therapy, 6 presented low urinary sulphatoxymelatonin values, while most of the patients with guanosine triphosphate cyclohydrolase-I deficiency showed normal values. 5 of 11 patients under treatment presented low urine sulphatoxymelatonin values. Thus, decreased excretion of sulphatoxymelatonin is frequently observed in cases with severe genetic disorders affecting serotonin biosynthesis. In conclusion, sulphatoxymelatonin can be a good biomarker to estimate serotonin status in the brain, especially for treatment monitoring purposes.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.

Cerebral organoids recapitulate the structure and function of the developing human brain in vitro, offering a large potential for personalized therapeutic strategies. The enormous growth of this research area over the past decade with its capability for clinical translation makes a non-invasive, automated analysis pipeline of organoids highly desirable. This work presents a novel non-invasive approach to monitor and analyze cerebral organoids over time using high-field magnetic resonance imaging and state-of-the-art tools for automated image analysis. Three specific objectives are addressed, (I) organoid segmentation to investigate organoid development over time, (II) global cysticity classification and (III) local cyst segmentation for organoid quality assessment. We show that organoid growth can be monitored reliably over time and cystic and non-cystic organoids can be separated with high accuracy, with on par or better performance compared to state-of-the-art tools applied to brightfield imaging. Local cyst segmentation is feasible but could be further improved in the future. Overall, these results highlight the potential of the pipeline for clinical application to larger-scale comparative organoid analysis.