The International Working Group on Neurotransmitter related Disorders (iNTD): A worldwide research project focused on primary and secondary neurotransmitter disorders

INTRODUCTION: Neurotransmitters are chemical messengers that enable communication between the neurons in the synaptic cleft. Inborn errors of neurotransmitter biosynthesis, breakdown and transport are a group of very rare neurometabolic diseases resulting in neurological impairment at any age from newborn to adulthood. METHODS AND RESULTS: The International Working Group on Neurotransmitter related Disorders (iNTD) is the first international network focusing on the study of primary and secondary neurotransmitter disorders. It was founded with the aim to foster exchange and improve knowledge in the field of these rare diseases. The newly established iNTD patient registry for neurotransmitter related diseases collects longitudinal data on the natural disease course, approach to diagnosis, therapeutic strategies, and quality of life of affected patients. The registry forms the evidence base for the development of consensus guidelines for patients with neurotransmitter related disorders. CONCLUSION: The iNTD network and registry will improve knowledge and strengthen research capacities in the field of inborn neurotransmitter disorders. The evidence-based guidelines will facilitate standardized diagnostic procedures and treatment approaches

Succinic semialdehyde dehydrogenase deficiency: in vitro and in silico characterization of a novel pathogenic missense variant and analysis of the mutational spectrum of ALDH5A1

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter \u3b3-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 \u3b3-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

CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model

The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells - an in vitro model for PKU - corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the FokI-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases

Analysis of Catecholamines and Pterins in Inborn Errors of Monoamine Neurotransmitter Metabolism—From Past to Future

Inborn errors of monoamine neurotransmitter biosynthesis and degradation belong to the rare inborn errors of metabolism. They are caused by monogenic variants in the genes encoding the proteins involved in (1) neurotransmitter biosynthesis (like tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC)), (2) in tetrahydrobiopterin (BH4) cofactor biosynthesis (GTP cyclohydrolase 1 (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SPR)) and recycling (pterin-4a-carbinolamine dehydratase (PCD), dihydropteridine reductase (DHPR)), or (3) in co-chaperones (DNAJC12). Clinically, they present early during childhood with a lack of monoamine neurotransmitters, especially dopamine and its products norepinephrine and epinephrine. Classical symptoms include autonomous dysregulations, hypotonia, movement disorders, and developmental delay. Therapy is predominantly based on supplementation of missing cofactors or neurotransmitter precursors. However, diagnosis is difficult and is predominantly based on quantitative detection of neurotransmitters, cofactors, and precursors in cerebrospinal fluid (CSF), urine, and blood. This review aims at summarizing the diverse analytical tools routinely used for diagnosis to determine quantitatively the amounts of neurotransmitters and cofactors in the different types of samples used to identify patients suffering from these rare diseases

Generation of an iPSC line from a patient with GTP cyclohydrolase 1 (GCH1) deficiency: HDMC0061i-GCH1

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

Generation of 2 iPSC clones from a patient with DNAJC12 deficiency: DHMCi003-A and DHMCi003-B

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

Generation of an iPSC line from a patient with tyrosine hydroxylase (TH) deficiency: TH-1 iPSC

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

A large and diverse brain organoid dataset of 1,400 cross-laboratory images of 64 trackable brain organoids from four different clones

<p>This dataset is presented in the paper <strong><span>A large and diverse brain organoid dataset of 1,400 cross-laboratory images of 64 trackable brain organoids from four different clones</span></strong></p> <p> </p> <p>This dataset encompasses two sources of data:</p> <ol> <li>A comma-separated values ('CSV') file. This file serves as a key to our dataset with one image per row. Each image is represented by its image identifier ('img_id') with the format [org_id]_[clone]_d[imaging_day]_[imaging_lab]. For each image, the CSV file also specifies the organoid size for convenience. Alternatively, the organoid size can be calculated using the ground truth organoid segmentation (org_segGT). </li> <li>For each row of the CSV file, we provide the image and org_segGT. For Lab A, the images are in JPEG format. For lab B, the images are in TIF format. Org_segGT is a manually created binary 2D NumPy array with the same size as the image (1024 x 768 for lab A, 1388 x 1040 for lab B). A value of 1 in org_segGT at position (x, y) means that the same position (x, y) in the corresponding image is covered by the organoid. The image file and the org_segGT file have the following format: [img_id].[jpg|tif] and [img_id].npy. For day 12, organoids were imaged before and after embedding from 96-well plates in 12-well plates, allowing the investigation of well-specific optical properties.</li> </ol> <p>For segmentation and growth monitoring using this dataset, please see <a href="https://github.com/deiluca/robust_monitoring_organoid_growth">https://github.com/deiluca/robust_monitoring_organoid_growth</a>.</p&gt

A large and diverse brain organoid dataset of 1,400 cross-laboratory images of 64 trackable brain organoids

Abstract Brain organoids represent a useful tool for modeling of neurodevelopmental disorders and can recapitulate brain volume alterations such as microcephaly. To monitor organoid growth, brightfield microscopy images are frequently used and evaluated manually which is time-consuming and prone to observer-bias. Recent software applications for organoid evaluation address this issue using classical or AI-based methods. These pipelines have distinct strengths and weaknesses that are not evident to external observers. We provide a dataset of more than 1,400 images of 64 trackable brain organoids from four clones differentiated from healthy and diseased patients. This dataset is especially powerful to test and compare organoid analysis pipelines because of (1) trackable organoids (2) frequent imaging during development (3) clone diversity (4) distinct clone development (5) cross sample imaging by two different labs (6) common imaging distractors, and (6) pixel-level ground truth organoid annotations. Therefore, this dataset allows to perform differentiated analyses to delineate strengths, weaknesses, and generalizability of automated organoid analysis pipelines as well as analysis of clone diversity and similarity

Generation of an iPSC line from a patient with GTP cyclohydrolase 1 ( GCH1 ) deficiency: HDMC0061i-GCH1

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