The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on ...Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.
•We examined changes in neural reward responsivity to food cues upon RYGB.•Food preferences shifted away from high-fat/sweet, towards low-energy foods after RYGB.•RYGB leads to changed responsivity ...of a frontoparietal control network.•Neural changes correlated to the shift in food preferences, but not to hormone levels.
After Roux-en-Y gastric bypass (RYGB) surgery, patients report a shift in food preferences away from high-energy foods.
We aimed to elucidate the potential mechanisms underlying this shift in food preferences by assessing changes in neural responses to food pictures and odors before and after RYGB. Additionally, we investigated whether altered neural responsivity was associated with changes in plasma endocannabinoid and ghrelin concentrations.
19 RYGB patients (4 men; age 41 ± 10 years; BMI 41 ± 1 kg/m2 before; BMI 36 ± 1 kg/m2 after) participated in this study. Before and two months after RYGB surgery, they rated their food preferences using the Macronutrient and Taste Preference Ranking Task and BOLD fMRI responses towards pictures and odors of high-, and low-energy foods and non-food items were measured. Blood samples were taken to determine plasma endocannabinoid and ghrelin concentrations pre- and post-surgery.
Patients demonstrated a shift in food preferences away from high-fat/sweet and towards low-energy/savory food products, which correlated with decreased superior parietal lobule responsivity to high-energy food odor and a reduced difference in precuneus responsivity to high-energy versus low-energy food pictures. In the anteroventral prefrontal cortex (superior frontal gyrus) the difference in deactivation towards high-energy versus non-food odors reduced. The precuneus was less deactivated in response to all cues. Plasma concentrations of anandamide were higher after surgery, while plasma concentrations of other endocannabinoids and ghrelin did not change. Alterations in appetite-related hormone concentrations did not correlate with changes in neural responsivity.
RYGB leads to changed responsivity of the frontoparietal control network that orchestrates top-down control to high-energy food compared to low-energy food and non-food cues, rather than in reward related brain regions, in a satiated state. Together with correlations with the shift in food preference from high- to low-energy foods this indicates a possible role in new food preference formation.
Structural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is ...expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs.
Optical genome mapping was performed to improve SV detection in short-read genome sequencing−negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria.
In a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses.
Critical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.
CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish ...genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3-8% of alleles, and 30-45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.
The identification of pathogenic variants in monogenic diseases has been of interest to researchers and clinicians for several decades. However, for inherited diseases with extremely high genetic ...heterogeneity, such as hearing loss and retinal dystrophies, establishing a molecular diagnosis requires an enormous effort. In this review, we use these two genetic conditions as examples to describe the initial molecular genetic identification approaches, as performed since the early 90s, and subsequent improvements and refinements introduced over the years. Next, the history of DNA sequencing from conventional Sanger sequencing to high-throughput massive parallel sequencing, a.k.a. next-generation sequencing, is outlined, including their advantages and limitations and their impact on identifying the remaining genetic defects. Moreover, the development of recent technologies, also coined "third-generation" sequencing, is reviewed, which holds the promise to overcome these limitations. Furthermore, we outline the importance and complexity of variant interpretation in clinical diagnostic settings concerning the massive number of different variants identified by these methods. Finally, we briefly mention the development of novel approaches such as optical mapping and multiomics, which can help to further identify genetic defects in the near future.
Anophthalmia and microphthalmia (A/M) are among the most severe congenital developmental eye disorders. Despite the advancements in genome screening technologies, more than half of A/M patients do ...not receive a molecular diagnosis. We included seven consanguineous families affected with A/M from Pakistani cohort and an unknown molecular basis. Single gene testing of FOXE3 was performed, followed by genome sequencing for unsolved probands in order to establish a genetic diagnosis for these families. All seven families were provided with a genetic diagnosis. The identified variants were all homozygous, classified as (likely) pathogenic and present in an A/M-associated gene. Targeted FOXE3 sequencing revealed two previously reported pathogenic FOXE3 variants in four families. In the remaining families, genome sequencing revealed a known pathogenic PXDN variant, a novel 13bp deletion in VSX2, and one novel deep intronic splice variant in PXDN. An in vitro splice assay was performed for the PXDN splice variant which revealed a severe splicing defect. Our study confirmed the utility of genome sequencing as a diagnostic tool for A/M-affected individuals. Furthermore, the identification of a novel deep intronic pathogenic variant in PXDN highlights the role of non-coding variants in A/M-disorders and the value of genome sequencing for the identification of this type of variants.
Introduction:
Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (
ABCA4
) gene is one of these genes and has been ...linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone–rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported
ABCA4
variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants.
Methods:
Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both
in silico
and
in vitro
. The
in silico
approach was based on the deep-learning tool SpliceAI. For the
in vitro
approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing
ABCA4
exons 46 to 48.
Results:
Through the analysis of WGS data, we identified two candidate variants in
ABCA4
in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in
trans
. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases.
Conclusion:
A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.
Inherited retinal dystrophies (IRDs) are characterized by photoreceptor dysfunction or degeneration. Clinical and phenotypic overlap between IRDs makes the genetic diagnosis very challenging and ...comprehensive genomic approaches for accurate diagnosis are frequently required. While there are previous studies on IRDs in Pakistan, causative genes and variants are still unknown for a significant portion of patients. Therefore, there is a need to expand the knowledge of the genetic spectrum of IRDs in Pakistan. Here, we recruited 52 affected and 53 normal individuals from 15 consanguineous Pakistani families presenting non-syndromic and syndromic forms of IRDs. We employed single molecule Molecular Inversion Probes (smMIPs) based panel sequencing and whole genome sequencing to identify the probable disease-causing variants in these families. Using this approach, we obtained a 93% genetic solve rate and identified 16 (likely) causative variants in 14 families, of which seven novel variants were identified in ATOH7, COL18A1, MERTK, NDP, PROM1, PRPF8 and USH2A while nine recurrent variants were identified in CNGA3, CNGB1, HGSNAT, NMNAT1, SIX6 and TULP1. The novel MERTK variant and one recurrent TULP1 variant explained the intra-familial locus heterogeneity in one of the screened families while two recurrent CNGA3 variants explained compound heterozygosity in another family. The identification of variants in known disease-associated genes emphasizes the utilization of time and cost-effective screening approaches for rapid diagnosis. The timely genetic diagnosis will not only identify any associated systemic issues in case of syndromic IRDs, but will also aid in the acceleration of personalized medicine for patients affected with IRDs.
•SmMIPs based targeted sequencing is a cost-effective genetic diagnostic tool.•Whole genome sequencing provided a genetic diagnosis in 5 from 6 families.•Genetic diagnosis provided phenotype correction and identified syndromic IRDs.
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