Herma C. van der Linde

Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly

Preaxial polydactyly (PPD) is a common limb malformation in human. A number of polydactylous mouse mutants indicate that misexpression of Shh is a common requirement for generating extra digits. Here we identify a translocation breakpoint in a PPD patient and a transgenic insertion site in the polydactylous mouse mutant sasquatch (Ssq). The genetic lesions in both lie within the same respective intron of the LMBR1/Lmbr1 gene, which resides ≈1 Mb away from Shh. Genetic analysis of Ssq reveals that the Lmbr1 gene is incidental to the phenotype and that the mutation directly interrupts a cis-acting regulator of Shh. This regulator is most likely the target for generating PPD mutations in human.

Rescue of behavioral phenotype and neuronal protrusion morphology in Fmr1 KO mice

Lack of fragile X mental retardation protein (FMRP) causes Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA-binding protein and is a component of messenger ribonucleoprotein complexes, associated with brain polyribosomes, including dendritic polysomes. FMRP is therefore thought to be involved in translational control of specific mRNAs at synaptic sites. In mice lacking FMRP, protein synthesis-dependent synaptic plasticity is altered and structural malformations of dendritic protrusions occur. One hypothesized cause of the disease mechanism is based on exaggerated group I mGluR receptor activation. In this study, we examined the effect of the mGluR5 antagonist MPEP on Fragile X related behavior in Fmr1 KO mice. Our results demonstrate a clear defect in prepulse inhibition of startle in Fmr1 KO mice, that could be rescued by MPEP. Moreover, we show for the first time a structural rescue of Fragile X related protrusion morphology with two independent mGluR5 antagonists.

AFQ056, a new mGluR5 antagonist for treatment of fragile X syndrome.

Fragile X syndrome, the most common form of inherited intellectual disability, is caused by a lack of FMRP, which is the product of the Fmr1 gene. FMRP is an RNA-binding protein and a component of RNA-granules found in the dendrites of neurons. At the synapse, FMRP is involved in regulation of translation of specific target mRNAs upon stimulation of mGluR5 receptors. In this study, we test the effects of a new mGluR5 antagonist (AFQ056) on the prepulse inhibition of startle response in mice. We show that Fmr1 KO mice have a deficit in inhibition of the startle response after a prepulse and that AFQ056 can rescue this phenotype. We also studied the effect of AFQ056 on cultured Fmr1 KO hippocampal neurons; untreated neurons showed elongated spines and treatment resulted in shortened spines. These results suggest that AFQ056 might be a potent mGluR5 antagonist to rescue various aspects of the fragile X phenotype.

Locomotor activity assay in zebrafish larvae: Influence of age, strain and ethanol ☆

Several characteristics warrant the zebrafish a refining animal model for toxicity testing in rodents, thereby contributing to the 3R principles (Replacement, Reduction, and Refinement) in animal testing, e.g. its small size, ease of obtaining a high number of progeny, external fertilization, transparency and rapid development of the embryo, and a basic understanding of its gene function and physiology. In this context we explored the motor activity pattern of zebrafish larvae, using a 96-well microtiter plate and a video-tracking system. Effects of induced light and darkness on locomotion of zebrafish larvae of different wild-type strains and ages (AB and TL, 5, 6 and 7 dpf; n=25/group) were studied. Locomotion was also measured in zebrafish larvae after exposure to different concentrations of ethanol (0; 0.5; 1; 2 and 4%) (AB and TL strain, 6 dpf; n=19/group). Zebrafish larvae showed a relatively high swimming activity in darkness when compared to the activity in light. Small differences were found between wild-type strains and/or age. Ethanol exposure resulted in hyperactivity (0.5-2%) and in hypo-activity (4%). In addition, the limitations and/or relevance of the parameters distance moved, duration of movements and velocity are exemplified and discussed. Together, the results support the suggestion that zebrafish may act as an animal refining alternative for toxicity testing in rodents provided internal and external environmental stimuli are controlled. As such, light, age and strain differences must be taken into account.

Novel parkin mutations detected in patients with early-onset Parkinson's disease

A multiethnic series of patients with early‐onset Parkinsons disease (EOP) was studied to assess the frequency and nature of parkin/PARK2 gene mutations and to investigate phenotype–genotype relationships. Forty‐six EOP probands with an onset age of <45 years, and 14 affected relatives were ascertained from Italy, Brazil, Cuba, and Turkey. The genetic screening included direct sequencing and exon dosage using a new, cost‐effective, real‐time polymerase chain reaction method. Mutations were found in 33% of the indexes overall, and in 53% of those with family history compatible with autosomal recessive inheritance. Fifteen parkin alterations (10 exon deletions and five point mutations) were identified, including four novel mutations: Arg402Cys, Cys418Arg, IVS11‐3C>G, and exon 8‐9‐10 deletion. Homozygous mutations, two heterozygous mutations, and a single heterozygous mutation were found in 8, 6, and 1 patient, respectively. Heterozygous exon deletions represented 28% of the mutant alleles. The patients with parkin mutations showed significantly earlier onset, longer disease duration, more frequently symmetric onset, and slower disease progression than the patients without mutations, in agreement with previous studies. This study confirms the frequent involvement of parkin and the importance of genetic testing in the diagnostic work‐up of EOP.

Generation and Characterization of Fmr1 Knockout Zebrafish

Fragile X syndrome (FXS) is one of the most common known causes of inherited mental retardation. The gene mutated in FXS is named FMR1, and is well conserved from human to Drosophila. In order to generate a genetic tool to study FMR1 function during vertebrate development, we generated two mutant alleles of the fmr1 gene in zebrafish. Both alleles produce no detectable Fmr protein, and produce viable and fertile progeny with lack of obvious phenotypic features. This is in sharp contrast to published results based on morpholino mediated knock-down of fmr1, reporting defects in craniofacial development and neuronal branching in embryos. These phenotypes we specifically addressed in our knock-out animals, revealing no significant deviations from wild-type animals, suggesting that the published morpholino based fmr1 phenotypes are potential experimental artifacts. Therefore, their relation to fmr1 biology is questionable and morpholino induced fmr1 phenotypes should be avoided in screens for potential drugs suitable for the treatment of FXS. Importantly, a true genetic zebrafish model is now available which can be used to study FXS and to derive potential drugs for FXS treatment.

Reduction in fragile X related 1 protein causes cardiomyopathy and muscular dystrophy in zebrafish.

SUMMARY Lack of the FMR1 gene product causes fragile X syndrome, the commonest inherited cause of mental impairment. We know little of the roles that fragile X related (FXR) gene family members (FMR1, FXR2 and FXR1) play during embryonic development. Although all are expressed in the brain and testis, FXR1 is the principal member found in striated and cardiac muscle. The Fxr1 knockout mice display a striated muscle phenotype but it is not known why they die shortly after birth; however, a cardiac cause is possible. The zebrafish is an ideal model to investigate the role of fxr1 during development of the heart. We have carried out morpholino knockdown of fxr1 and have demonstrated abnormalities of striated muscle development and abnormal development of the zebrafish heart, including failure of looping and snapping of the atrium from its venous pole. In addition, we have measured cardiac function using high-speed video microscopy and demonstrated a significant reduction in cardiac function. This cardiac phenotype has not been previously described and suggests that fxr1 is essential for normal cardiac form and function.

Exome sequencing and functional analyses suggest that SIX6 is a gene involved in an altered proliferation-differentiation balance early in life and optic nerve degeneration at old age.

Primary open-angle glaucoma (POAG) is a hereditary neurodegenerative disease, characterized by optic nerve changes including increased excavation, notching and optic disc hemorrhages. The excavation can be described by the vertical cup-disc ratio (VCDR). Previously, genome-wide significant evidence for the association of rs10483727 in SIX1-SIX6 locus with VCDR and subsequent POAG was found. Using 1000 genomes-based imputation of four independent population-based cohorts in the Netherlands, we identified a missense variant rs33912345 (His141Asn) in SIX6 associated with VCDR (Pmeta = 7.74 × 10(-7), n = 11 473) and POAG (Pmeta = 6.09 × 10(-3), n = 292). Exome sequencing analysis revealed another missense variant rs146737847 (Glu129Lys) also in SIX6 associated with VCDR (P = 5.09 × 10(-3), n = 1208). These two findings point to SIX6 as the responsible gene for the previously reported association signal. Functional characterization of SIX6 in zebrafish revealed that knockdown of six6b led to a small eye phenotype. Histological analysis showed retinal lamination, implying an apparent normal development of the eye, but an underdeveloped lens, and reduced optic nerve diameter. Expression analysis of morphants at 3 dpf showed a 5.5-fold up-regulation of cdkn2b, a cyclin-dependent kinase inhibitor, involved in cell cycle regulation and previously associated with VCDR and POAG in genome-wide association studies (GWASs). Since both six6b and cdkn2b play a key role in cell proliferation, we assessed the proliferative activity in the eye of morphants and found an alteration in the proliferative pattern of retinal cells. Our findings in humans and zebrafish suggest a functional involvement of six6b in early eye development, and open new insights into the genetic architecture of POAG.

New insights into the genetics of primary open-angle glaucoma based on meta-analyses of intraocular pressure and optic disc characteristics

Primary open-angle glaucoma (POAG), the most common optic neuropathy, is a heritable disease. Siblings of POAG cases have a ten-fold increased risk of developing the disease. Intraocular pressure (IOP) and optic nerve head characteristics are used clinically to predict POAG risk. We conducted a genome-wide association meta-analysis of IOP and optic disc parameters and validated our findings in multiple sets of POAG cases and controls. Using imputation to the 1000 genomes (1000G) reference set, we identified 9 new genomic regions associated with vertical cup-disc ratio (VCDR) and 1 new region associated with IOP. Additionally, we found 5 novel loci for optic nerve cup area and 6 for disc area. Previously it was assumed that genetic variation influenced POAG either through IOP or via changes to the optic nerve head; here we present evidence that some genomic regions affect both IOP and the disc parameters. We characterized the effect of the novel loci through pathway analysis and found that pathways involved are not entirely distinct as assumed so far. Further, we identified a novel association between CDKN1A and POAG. Using a zebrafish model we show that six6b (associated with POAG and optic nerve head variation) alters the expression of cdkn1a. In summary, we have identified several novel genes influencing the major clinical risk predictors of POAG and showed that genetic variation in CDKN1A is important in POAG risk.

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.

BackgroundHirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human.ResultsWe performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS.ConclusionsOur data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.