A modular microfluidic platform to enable complex and customisable in vitro models for neuroscience.

Disorders of the central nervous system (CNS) represent a global health challenge and an increased understanding of the CNS in both physiological and pathophysiological states is essential to tackle the problem. Modelling CNS conditions is difficult, as traditional in vitro models fail to recapitulate precise microenvironments and animal models of complex disease often have limited translational validity. Microfluidic and organ-on-chip technologies offer an opportunity to develop more physiologically relevant and complex in vitro models of the CNS. They can be developed to allow precise cellular patterning and enhanced experimental capabilities to study neuronal function and dysfunction. To improve ease-of-use of the technology and create new opportunities for novel in vitro studies, we introduce a modular platform consisting of multiple, individual microfluidic units that can be combined in several configurations to create bespoke culture environments. Here, we report proof-of-concept experiments creating complex in vitro models and performing functional analysis of neuronal activity across modular interfaces. This platform technology presents an opportunity to increase our understanding of CNS disease mechanisms and ultimately aid the development of novel therapies.

Male patients affected by mosaic PCDH19 mutations: five new cases.

Pathogenic variants in the PCDH19 gene are associated with epilepsy, intellectual disability (ID) and behavioural disturbances. Only heterozygous females and mosaic males are affected, likely due to a disease mechanism named cellular interference. Until now, only four affected mosaic male patients have been described in literature. Here, we report five additional male patients, of which four are older than the oldest patient reported so far. All reported patients were selected for genetic testing because of developmental delay and/or epilepsy. Custom-targeted next generation sequencing gene panels for epilepsy genes were used. Clinical data were collected from medical records. All patients were mosaic in blood for likely pathogenic variants in the PCDH19 gene. In most, clinical features were very similar to the female phenotype, with normal development before seizure onset, which occurred between 5 and 10 months of age, clustering of seizures and sensitivity to fever. Four out of five patients had mild to severe ID and behavioural problems. We reaffirm the similarity between male and female PCDH19-related phenotypes, now also in a later phase of the disorder (ages 10-14 years).

Implementing Pharmacogenomics in Europe: Design and Implementation Strategy of the Ubiquitous Pharmacogenomics Consortium.

Despite scientific and clinical advances in the field of pharmacogenomics (PGx), application into routine care remains limited. Opportunely, several implementation studies and programs have been initiated over recent years. This article presents an overview of these studies and identifies current research gaps. Importantly, one such gap is the undetermined collective clinical utility of implementing a panel of PGx-markers into routine care, because the evidence base is currently limited to specific, individual drug-gene pairs. The Ubiquitous Pharmacogenomics (U-PGx) Consortium, which has been funded by the European Commission's Horizon-2020 program, aims to address this unmet need. In a prospective, block-randomized, controlled clinical study (PREemptive Pharmacogenomic testing for prevention of Adverse drug REactions [PREPARE]), pre-emptive genotyping of a panel of clinically relevant PGx-markers, for which guidelines are available, will be implemented across healthcare institutions in seven European countries. The impact on patient outcomes and cost-effectiveness will be investigated. The program is unique in its multicenter, multigene, multidrug, multi-ethnic, and multihealthcare system approach.

Xq28 duplications including MECP2 in five females: Expanding the phenotype to severe mental retardation.

Duplications leading to functional disomy of chromosome Xq28, including MECP2 as the critical dosage-sensitive gene, are associated with a distinct clinical phenotype in males, characterized by severe mental retardation, infantile hypotonia, progressive neurologic impairment, recurrent infections, bladder dysfunction, and absent speech. Female patients with Xq duplications including MECP2 are rare. Only recently submicroscopic duplications of this region on Xq28 have been recognized in four females, and a triplication in a fifth, all in combination with random X-chromosome inactivation (XCI). Based on this small series, it was concluded that in females with MECP2 duplication and random XCI, the typical symptoms of affected boys are not present. We present clinical and molecular data on a series of five females with an Xq28 duplication including the MECP2 gene, both isolated and as the result of a translocation, and compare them with the previously reported cases of small duplications in females. The collected data indicate that the associated phenotype in females is distinct from males with similar duplications, but the clinical effects may be as severe as seen in males.

Methods to detect CNVs in the human genome.

The detection of quantitative changes in genomic DNA, i.e. deletions and duplications or Copy Number Variants (CNVs), has recently gained considerable interest. First, detailed analysis of the human genome showed a surprising amount of CNVs, involving thousands of genes. Second, it was realised that the detection of CNVs as a cause of genetic disease was often neglected, but should be an essential part of a complete screening strategy. In both cases new efficient CNV screening methods, covering the entire range from specific loci to genome-wide, were behind these developments. This paper will briefly review the methods that are available to detect CNVs, discuss their strong and weak points, show some new developments and look ahead. Methods covered include microscopy, fluorescence in situ hybridization (including fiber-FISH), Southern blotting, PCR-based methods (including MLPA), array technology and massive parallel sequencing. In addition, we will show some new developments, including a 1400-plex CNV bead assay, fast-MLPA (from DNA to result in approximately 6 h) and a simple Melting Curve Analysis assay to confirm potential CNVs. Using the 1400-plex CNV bead assay, targeting selected chromosomal regions only, we detected confirmed rearrangements in 9% of 320 mental retardation patients studied.

Tall stature and duplication of the insulin-like growth factor I receptor gene.

Trisomy of 15q26-qter is frequently associated with tall stature and mental retardation. Here we describe a patient with such trisomy, without a partial monosomy of another chromosome. The tall stature in this patient is most probably caused by duplication of the IGF1R gene. A duplication of the IGF1R gene is not a frequent finding in patients with tall stature. In 38 patients with features of Sotos syndrome without NSD1 alterations, a duplication was found only once. This patient was already known to have an unbalanced 2;15 translocation. Looking for a duplication of the 15qter region is still worth consideration in patients with tall stature and features of Sotos syndrome without an NSD1 alteration, especially when there is craniosynostosis or marked speech delay.

Cerebral white matter abnormalities in 6p25 deletion syndrome.

Submicroscopic deletion of the terminal part of the short arm of chromosome 6, including 6p25, leads to developmental retardation, hearing impairment, ocular dysgenesis, and dysmorphic features. We diagnosed 3 patients referred because of white matter abnormalities of unknown origin. MR imaging showed multifocal areas of abnormal signal and enlarged perivascular spaces in the cerebral white matter that were stable during follow-up. Multifocal white matter abnormalities are most commonly seen in static, nonmetabolic encephalopathies, including chromosomal abnormalities.

Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents.

BACKGROUND: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. OBJECTIVE AND METHODS: Array-CGH with approximately 3500 large insert clones spaced at approximately 1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. RESULTS: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. CONCLUSIONS: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

Genomic imbalances in mental retardation.

INTRODUCTION: It has been estimated that cytogenetically visible rearrangements are present in approximately 1% of newborns. These chromosomal changes can cause a wide range of deleterious developmental effects, including mental retardation (MR). It is assumed that many other cases exist where the cause is a submicroscopic deletion or duplication. To facilitate the detection of such cases, different techniques have been developed, which have differing efficiency as to the number of loci and patients that can be tested. METHODS: We implemented multiplex amplifiable probe hybridisation (MAPH) to test areas known to be rearranged in MR patients (for example, subtelomeric/pericentromeric regions and those affected in microdeletion syndromes) and to look for new regions that might be related to MR. RESULTS: In this study, over 30 000 screens for duplications and deletions were carried out; 162 different loci tested in each of 188 developmentally delayed patients. The analysis resulted in the detection of 19 rearrangements, of which approximately 65% would not have been detected by conventional cytogenetic analysis. A significant fraction (46%) of the rearrangements found were interstitial, despite the fact that only a limited number of these loci have so far been tested. DISCUSSION: Our results strengthen the arguments for whole genome screening within this population, as it can be assumed that many more interstitial rearrangements would be detected. The strengths of MAPH for this analysis are the simplicity, the high throughput potential, and the high resolution of analysis. This combination should help in the future identification of the specific genes that are responsible for MR.

Decreased mortality of ischaemic heart disease among carriers of haemophilia.

BACKGROUND: Coagulation plays an important part in ischaemic cardiovascular disease. Results of studies have shown that extremes in hypocoagulability protect against ischaemic cardiovascular disease. We have investigated overall mortality and death from cardiovascular causes in carriers of haemophilia, who in most cases have mildly decreased coagulability without clinical signs. METHODS: We followed-up a cohort of 1012 mothers of all known people with haemophilia in the Netherlands from birth to death, or the end-of-study date (41984 person years of follow-up). We obtained vital status and causes of death, if deceased, and compared overall and cause-specific mortality in our cohort with that in the general Dutch female population adjusted for age and calendar period by calculating the standardised mortality ratio (SMR). FINDINGS: Overall mortality was reduced by 22% (261 observed deaths, 333.74 expected; SMR 0.78 [95% CI 0.69-0.88]). Deaths from ischaemic heart disease were reduced by 36% (39 observed deaths, 60.53 expected; SMR 0.64 [0.47-0.88]). We did not note decreased mortality for cerebral stroke (ischaemic and haemorrhagic combined) (28 observed deaths, 36.82 expected; SMR 0.76 [0.53-1.10]). A separate analysis of these two types of stroke was not possible. Women in our cohort had an increased risk of death from extracranial haemorrhage (5 observed deaths, 0.18 expected; SMR 27.78 [8.49-58.18]); however, the number of deaths from this cause was much lower than that for ischaemic heart disease. CONCLUSION: The results show that a mild decrease in coagulability has a protective effect against fatal ischaemic heart disease.

Mitochondrial function and free fatty acid levels in rats after portacaval shunt.

The effects of portacaval shunting on the oxidative phosphorylation process of mitochondria isolated from rat liver and skeletal muscle were evaluated and correlated with mitochondria free fatty acid (FFA) contents. ADP/O ratios, respiratory control index and QO2 values were significantly depressed in liver mitochondria from portacaval-shunted rats; these changes were associated with decreased mitochondrial FFA contents. The mitochondrial function of skeletal muscle was unaltered.