Irenaeus F. M. de Coo

Introduction: Structural neuroimaging relies on T1-weighted (T1w) magnetic resonance imaging (MRI) for brain morphometry, yet at 7 Tesla (7 T) transmit field (B ⁺ ) inhomogeneity remains a major source of bias. Although Magnetization Prepared 2 Rapid Acquisition Gradient Echoes (MP2RAGE) improves the tissue contrast, residual B ⁺ effects may persist and may be exacerbated in aging or clinical populations, where anatomical and physiological factors further challenge image quality and preprocessing. The impact of B ⁺ inhomogeneity on automated quality assessment and morphometric statistical inference remains insufficiently understood. Methods: Submillimeter 7 T MP2RAGE brain acquisitions from carriers of a mitochondrial gene mutation (m.3243A>G) and controls were retrieved from previous studies. Image quality before and after B ⁺ inhomogeneity correction was assessed by multiple automated pipelines. Case-control morphometric studies, including regional volume and mean cortical thickness, were analyzed in both registration-based and deep learning-based segmentation frameworks. Changes in image quality metrics (IQMs) and morphometric statistical significance were evaluated to determine the impact of B ⁺ inhomogeneity correction. Results: Overall image quality rating and metrics sensitive to intensity non-uniformity and topological integrity consistently improved after B ⁺ inhomogeneity correction. However, its impact on morphometric statistical inferences was strongly method-dependent. Some pipelines showed redistribution of significant regions, whereas others predominantly demonstrated increased effects in sensitivity. Across methods, B ⁺ inhomogeneity correction altered the findings of morphometric analyses, particularly in cortical regions. Conclusion: Residual B ⁺ inhomogeneity at 7 T substantially influences both image quality control and morphometric evaluations. Current automated quality control approaches can hardly capture these effects reliably. B ⁺ inhomogeneity correction will not only improve intensity uniformity, but also change sensitivity of morphometric statistical inferences. To establish reliable morphometric biomarkers at UHF strengths, explicit B ⁺ inhomogeneity correction and carefully chosen preprocessing strategies are practically necessary and highly recommended.

Context: Monocarboxylate transporter (MCT) 8 facilitates thyroid hormone (TH) transport across the blood-brain barrier. Pathogenic variants in SLC16A2 cause MCT8 deficiency (Allan-Herndon-Dudley syndrome), characterized by intellectual and motor disability and abnormal thyroid function tests. MCT8 deficiency typically affects males due to its X-linked inheritance. Objective: Here, we report 8 female patients with heterozygous pathogenic variants in SLC16A2 who presented with variable neurocognitive impairment, behavioral problems, and TH function abnormalities. Methods: We performed X-chromosome inactivation studies in female patients in whom heterozygous pathogenic variants in SLC16A2 were identified. The effect of SLC16A2 variants on TH transport was assessed in transfected cells and patient-derived fibroblasts. Results: In all patients (mean age 8.6 years; range, 2.3-25 years) routine care genetic analyses identified heterozygous variants in SLC16A2 (p.(R445C), p.(N193I), p.(G276R), t(X;20), resulting in a breakpoint in intron 1, t(X;19), resulting in a breakpoint in SLC16A2, p.(I562Sfs566*), p.(G221R)). All missense variants showed substantially reduced MCT8-mediated TH uptake in transiently transfected cells. X-chromosome inactivation studies in patient cells showed skewed X-inactivation in all 7 evaluated individuals. In 5 out of 7 evaluated cases, MCT8-mediated 3,5,3′-triiodothyronine (T3) uptake in patient-derived fibroblasts was impaired to a similar degree as in fibroblasts derived from male patients with MCT8 deficiency. Conclusion: Female patients with heterozygous pathogenic variants in SLC16A2 and skewed X-chromosome inactivation may present with variable neuro(psycho)logical, behavioral, and thyroid function test abnormalities. Female patients presenting with neurocognitive impairment and abnormal TH function tests (low free thyroxine and/or high total T3 concentrations) should be tested for genetic variants in SLC16A2.

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.

Mutations of the mitochondrial DNA are an important cause of inherited diseases that can severely affect the tissue’s homeostasis and integrity. The m.3243A > G mutation is the most commonly observed across mitochondrial disorders and is linked to multisystemic complications, including cognitive deficits. In line with in vitro experiments demonstrating the m.3243A > G’s negative impact on neuronal energy production and integrity, m.3243A > G patients show cerebral grey matter tissue changes. However, its impact on the most neuron dense, and therefore energy-consuming brain structure—the cerebellum—remains elusive. In this work, we used high-resolution structural and functional data acquired using 7 T MRI to characterize the neurodegenerative and functional signatures of the cerebellar cortex in m.3243A > G patients. Our results reveal altered tissue integrity within distinct clusters across the cerebellar cortex, apparent by their significantly reduced volume and longitudinal relaxation rate compared with healthy controls, indicating macroscopic atrophy and microstructural pathology. Spatial characterization reveals that these changes occur especially in regions related to the frontoparietal brain network that is involved in information processing and selective attention. In addition, based on resting-state functional MRI data, these clusters exhibit reduced functional connectivity to frontal and parietal cortical regions, especially in patients characterized by (i) a severe disease phenotype and (ii) reduced information-processing speed and attention control. Combined with our previous work, these results provide insight into the neuropathological changes and a solid base to guide longitudinal studies aimed to track disease progression.

Mutations of the mitochondrial DNA (mtDNA) are an important cause of inherited diseases that can severely affect the tissue’s homeostasis and integrity. The m.3243A>G mutation is the most commonly observed across mitochondrial disorders and is linked to multisystemic complications, including cognitive deficits. In line with in vitro experiments demonstrating the m.3243A>G’s negative impact on neuronal energy production and integrity, m.3243A>G patients show cerebral gray matter tissue changes. However, its impact on the most neuron-dense, and therefore energy-consuming brain structure – the cerebellum – remains elusive. In this work, we used high resolution structural and functional data acquired using 7 Tesla MRI to characterize the neurodegenerative and functional signatures of the cerebellar cortex in m.3243A>G patients. Our results reveal altered tissue integrity within distinct clusters across the cerebellar cortex, apparent by their significantly reduced volume and longitudinal relaxation rate compared to healthy controls, indicating macroscopic atrophy and microstructural pathology. Spatial characterization reveals that these changes occur especially in regions related to the frontoparietal brain network that is involved in information processing and selective attention. In addition, based on resting-state fMRI data, these clusters exhibit reduced functional connectivity to frontal and parietal cortical regions, especially in patients characterized by (i) a severe disease phenotype and (ii) reduced information processing speed and attention control. Combined with our previous work, these results provide crucial insights into the neuropathological changes and a solid base to guide longitudinal studies aimed to track disease progression.

Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system, with an incidence of 1 in 5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle–specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in 8 individuals variably associating HSCR, CIPO, peripheral neuropathy, and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis, and intestinal smooth muscle abnormalities. The cell type–specific ErbB3 and ErbB2 function was further analyzed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using quantitative real-time PCR (RT-qPCR) on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing WT or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies, including intestinal dysmotility.

Objective: The aim of this study was to evaluate if urinary sediment cells offered a robust alternative to muscle biopsy for the diagnosis of single mtDNA deletions.Methods: Eleven adult patients with progressive external ophthalmoplegia and a known single mtDNA deletion were investigated. Urinary sediment cells were used to isolate DNA, which was then subjected to long‐range polymerase chain reaction. Where available, the patient`s muscle DNA was studied in parallel. Breakpoint and thus deletion size were identified using both Sanger sequencing and next generation sequencing. The level of heteroplasmy was determined using quantitative polymerase chain reaction.Results: We identified the deletion in urine in 9 of 11 cases giving a sensitivity of 80%. Breakpoints and deletion size were readily detectable in DNA extracted from urine. Mean heteroplasmy level in urine was 38% ± 26 (range 8 ‐ 84%), and 57% ± 28 (range 12 – 94%) in muscle. While the heteroplasmy level in urinary sediment cells differed from that in muscle, we did find a statistically significant correlation between these two levels (R = 0.714, P = 0.031(Pearson correlation)).Interpretation: Our findings suggest that urine can be used to screen patients suspected clinically of having a single mtDNA deletion. Based on our data, the use of urine could considerably reduce the need for muscle biopsy in this patient group.

Summary: Background: Variants in POLG are one of the most common causes of inherited mitochondrial disease. Phenotypic classification of POLG disease has evolved haphazardly making it complicated and difficult to implement in everyday clinical practise. The aim of our study was to simplify the classification and facilitate better clinical recognition.Methods: A multinational, retrospective study using data from 155 patients with POLG variants recruited from seven European countries.Results: We describe the spectrum of clinical features associated with POLG variants in the largest known cohort of patients. While clinical features clearly form a continuum, stratifying patients simply according to age of onset—onset prior to age 12 years; onset between 12 and 40 years and onset after the age of 40 years, permitted us to identify clear phenotypic and prognostic differences. Prior to 12 years of age, liver involvement (87%), seizures (84%), and feeding difficulties (84%) were the major features. For those with onset between 12 and 40 years, ataxia (90%), peripheral neuropathy (84%), and seizures (71%) predominated, while for those with onset over 40 years, ptosis (95%), progressive external ophthalmoplegia (89%), and ataxia (58%) were the major clinical features. The earlier the onset the worse the prognosis. Patients with epilepsy and those with compound heterozygous variants carried significantly worse prognosis.Conclusion: Based on our data, we propose a simplified POLG disease classification, which can be used to guide diagnostic investigations and predict disease course.

Background: Myopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy.Methods: We assessed the heteroplasmy level of patient-derived mesoangioblasts, isolated from skeletal muscle of multiple carriers of different mtDNA point-mutations (n = 25). Mesoangioblast cultures with < 10% mtDNA mutation were further analyzed with respect to immunophenotype, proliferation capacity, in vitro myogenic differentiation potential, mitochondrial function, and mtDNA quantity.Results: This study demonstrated that mesoangioblasts in half of the patients contained no or a very low mutation load (< 10%), despite a much higher mutation load in their skeletal muscle. Moreover, none of the large-scale mtDNA deletion carriers displayed the deletion in mesoangioblasts, despite high percentages in skeletal muscle. The mesoangioblasts with no or a very low mutation load (< 10%) displayed normal mitochondrial function, proliferative capacity, and myogenic differentiation capacity.Conclusions: Our data demonstrates that in half of the mtDNA mutation carriers, their mesoangioblasts are (nearly) mutation free and can potentially be used as source for autologous cell therapy for generation of new muscle fibers without mtDNA mutation and normal mitochondrial function.