Identifizierung und Charakterisierung von ATP13A2-Varianten und ihre Bedeutung für neurodegenerative Erkrankungen

Das Kufor-Rakeb Syndrom (KRS) ist eine seltene, neurodegenerative Erkrankung, bei der die Patienten neben charakteristischen Kennzeichen des Parkinsonismus zusätzliche Symptome wie eine Spastik und eine Demenz entwickeln. Es handelt sich um eine autosomal rezessive Erkrankung, deren erste Symptome bereits im Jugendalter auftreten. Die genetische Ursache des KRS sind biallelische Mutationen im ATP13A2-Gen auf Chromosom 1p36. ATP13A2 kodiert für eine P-Typ Typ V ATPase mit zehn prädizierten Transmembrandomänen und einem Molekulargewicht von etwa 135 kDa, dessen Funktion und Substratspezifität allerdings bislang unbekannt sind. Das Ziel dieser Arbeit war die Charakterisierung beschriebener Genvarianten in ATP13A2 auf zellulärer Ebene zum besseren Verständnis der Pathogenese des KRS bzw. verwandter Formen des Parkinsonismus. Zur in vivo Analyse der motorischen und kognitiven Funktionsstörungen sollte zusätzlich eine Knock-Out Maus generiert werden, bei der Atp13a2 konstitutiv deletiert ist. Insgesamt wurden 15 verschiedene humane Mutationen in vitro untersucht, von denen acht bei Patienten mit typischer Parkinsonerkrankung (PD-Mutationen) und sieben bei KRS-Patienten identifiziert worden waren. Bei elf dieser Veränderungen handelte es sich um Missense-Veränderungen (P1-P8; K4-K6). Es gibt keinen Hinweis darauf, dass der jeweilige Aminosäurenaustausch einen Effekt auf die Konformation des Proteins hat. In zwei Fällen der bei KRS-Patienten identifizierten ATP13A2-Veränderungen war ein Nukleotid deletiert (K3 und K7). Dies führte jeweils zu einer Verschiebung des Leserasters und resultierte in einem präterminalen Stop der Translation und einer Trunkierung des Proteins. Ebenso führte die Insertion von 22 bp in Exon 16 (K2) aufgrund der Verschiebung des Lesenrasters zu einem trunkierten Protein. Schließlich wurde ein Basenaustausch in einer hochkonservierten Donorspleißstelle analysiert, der zu einer in frame Deletion des Exon 13 führte (K1). Die subzelluläre Lokalisation mittels Immunfluoreszenzanalyse ergab für das ATP13A2-Wildtypprotein und alle PD-Mutanten eine Co-Lokalisierung mit dem lysosomalen Membranprotein LAMP2. Dagegen zeigten alle KRS-Mutationen eine subzelluläre Co-Lokalisierung mit dem endoplasmatischen Reticulum (ER). Da das ER maßgeblich an wichtigen Prozessen wie Faltung und Degradierung von Proteinen beteiligt ist, ist diese Fehllokalisation sehr wahrscheinlich darauf zurückzuführen, dass diese Mutanten von dem Organell als defizitär erkennt werden. Unterschiede zwischen den verschiedenen Mutationen bzw. Mutationstypen ließen sich auch hinsichtlich der Proteinstabilität feststellen. Während die PD-Mutanten im Vergleich zum Wildtyp eine gleichbleibende Stabilität zeigten, waren die KRS-Mutanten weniger stabil. Zusätzlich konnte gezeigt werden, dass die KRS-Mutanten nicht posttranslational modifiziert wurden, während für ATP13A2-WT und die PD-Mutanten eine N-Glykosylierung festgestellt wurde. Für alle hier untersuchten Proteine konnte gezeigt werden, dass sie durch das Proteasom degradiert werden. Allerdings scheint dies auf einem Ubiquitin-unabhängigen Weg zu erfolgen, der erst für wenige Proteine beschrieben worden ist. Es wurden demnach ausschließlich für die KRS-Mutationen Veränderungen der Proteineigenschaften bzw. Proteinlokalisation nachgewiesen, die vermutlich eine Reduktion der ATPasen-Funktion zur Folge haben und somit höchstwahrscheinlich einen pathogenen Effekt besitzen. Bei den so genannten PD-Mutationen konnte dies nicht gezeigt werden. Daher handelt es sich bei ihnen eher um Genvarianten, die eine neutrale Wirkung auf das Protein haben und somit nicht ursächlich am Krankheitsgeschehen beteiligt sind. Die zentrale Aufgabe des Lysosoms ist die Degradierung von Proteinen und Organellen. Da funktionelle Störungen dieses Kompartiments mehrfach mit neurodegenerativen Erkrankungen in Verbindung gebracht worden sind, sollte die Bedeutung von ATP13A2 für die Autophagie anhand von Fibroblasten eines KRS-Patienten untersucht werden. Es konnte jedoch keine offensichtliche Beteiligung an diesem Abbaumechanismus gezeigt werden. Des Weiteren kann ein gestörter Lipidmetabolismus zum Entstehen einer neurodegenerativen Erkrankung beitragen. Zusätzlich sind die nah verwandten P-Typ Typ IV ATPasen am Lipidtransport beteiligt. Daher wurde das Lipidprofil der KRS-Fibroblasten mit dem von Kontrollzellen verglichen. Auch hierbei wurden keine signifikanten Unterschiede festgestellt. Neben genetischen Aspekten tragen auch Umweltfaktoren, die zum Zellstress führen, zur Entstehung des Parkinsonismus bei. Diesbezüglich konnte für ATP13A2-defiziente Zellen im Vergleich zu Kontrollfibroblasten eine statistisch signifikant erhöhte Sensitivität gegenüber verschiedenen Formen des Zellstresses gezeigt werden, die auf einen möglichen molekularen Zusammenhang zwischen Umwelt-bedingten und genetischen Risikofaktoren hinweisen könnte. Die in vivo Analyse des Atp13a2-Gendefekts hinsichtlich Funktion und Bedeutung für die Pathogenese des KRS war bislang nicht möglich, da die Keimbahntransmission des rekombinanten Allels in unabhängig verfolgten Strategien nicht gelang

F8 Inversions at Xq28 Causing Hemophilia A Are Associated With Specific Methylation Changes: Implication for Molecular Epigenetic Diagnosis

Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. For genomic inversions in particular, the disease causing mechanism may not be clear, especially if the inversion border does not cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, in a mainly epigenetic context, may provide additional information regarding sequence-specific regulation of gene expression in human diseases. Herein, we study one such inversion hotspot at Xq28, which leads to the disruption of F8 gene and results in hemophilia A phenotype. To determine the epigenetic consequence of this rearrangement, we evaluated DNA methylation levels of 12 CpG rich regions with the coverage of 550 kb by using bisulfite-pyrosequencing and next-generation sequencing (NGS)-based bisulfite re-sequencing enrichment assay. Our results show that this inversion prone area harbors widespread methylation changes at the studied regions. However, only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (two regions), intron 22 inversion (two regions) and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, two CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. While we found age to be an influencing factor on methylation levels at some regions, covariate analysis still confirms the differential methylation induced by inversion, regardless of age. The hemophilia A methylation inversion “HAMI” assay provides an advantage over conventional PCR-based methods, which may not detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in the F8 (Xq28) region are associated with detectable changes in methylation levels and can be used as an epigenetic diagnostic marker

CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT)

Background. Recently, we identified a microduplication in chromosomal band 1q21.1 encompassing the CHD1L/ALC1 gene encoding a chromatin-remodelling enzyme in congenital anomalies of the kidneys and urinary tract (CAKUT) patient. Methods. To explore the role of CHD1L in CAKUT, we screened 85 CAKUT patients for mutations in the CHD1L gene and performed functional analyses of the three heterozygous missense variants detected. In addition, we quantitatively determined CHD1L expression in multiple human fetal and adult tissues and analysed expression of CHD1L protein in human embryonal, adult and hydronephrotic kidney sections. Results. Two of three novel heterozygous missense variants identified in three patients were not found in >400 control chromosomes. All variants lead to amino acid substitutions in or near the CHD1L macro domain, a poly-ADP-ribose (PAR)-binding module interacting with PAR polymerase 1 (PARP1), and showed decreased interaction with PARP1 by pull-down assay of transfected cell lysates. Quantitative messenger RNA analysis demonstrated high CHD1L expression in human fetal kidneys, and levels were four times higher than in adult kidneys. In the human embryo at 7-11 weeks gestation, CHD1L immunolocalized in the early ureteric bud and the S- and comma-shaped bodies, critical stages of kidney development. In normal postnatal sections, CHD1L was expressed in the cytoplasm of tubular cells in all tubule segments. CHD1L expression appeared higher in the hydronephrotic kidney of one patient with a hypofunctional CHD1L variant than in normal kidneys, recapitulating high fetal levels. Conclusion. Our data suggest that CHD1L plays a role in kidney development and may be a new candidate gene for CAKU

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores

Background: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. Methods: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein-protein-interactions), were used for final prioritization. Conclusion: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.Funding information: The present study was supported by the German Research Foundation (DFG)-Grants BE 3828/8-1, LU 1944/2-1, MA 2546/5-1, and LU1944/3-1. ACKNOWLEDGMENTS: The authors thank all patients, relatives, and control individuals for their participation. We thank the German support group for individuals with cleft lip and/or palate (Wolfgang Rosenthal Gesellschaft) for assistance with recruitment.We acknowledge the invaluable assistance of all clinical, laboratory, and bioinformatic personnel. The authors thank the Next Generation Sequencing Core Facility of the Medical Faculty of the University of Bonn for sequencing the samples that were used in this study. DbGaP datasets were accessed through dbGaP accession number phs000094.v1.p1 (Supplemental Acknowledgments). Finally, the authors thank the Genome Aggregation Database (gnomAD), and all groups that provided exome and genome variant data to this resource. A full list of gnomAD contributors is provided in the gnomAD flagship paper (Karczewski et al., 2020). Open Access funding enabled and organized by Projekt DEAL

Shared genetic risk between eating disorder- and substance-use-related phenotypes:Evidence from genome-wide association studies

First published: 16 February 202

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Dynamical reconstruction of the global ocean state during the Last Glacial Maximum

The global ocean state for the modern age and for the Last Glacial Maximum (LGM) was dynamically reconstructed with a sophisticated data assimilation technique. A substantial amount of data including global seawater temperature, salinity (only for the modern estimate), and the isotopic composition of oxygen and carbon (only in the Atlantic for the LGM) were integrated into an ocean general circulation model with the help of the adjoint method, thereby the model was optimized to reconstruct plausible continuous fields of tracers, overturning circulation and water mass distribution. The adjoint‐based LGM state estimation of this study represents the state of the art in terms of the length of forward model runs, the number of observations assimilated, and the model domain. Compared to the modern state, the reconstructed continuous sea‐surface temperature field for the LGM shows a global‐mean cooling of 2.2 K, and the reconstructed LGM ocean has a more vigorous Atlantic meridional overturning circulation, shallower North Atlantic Deep Water (NADW) equivalent, stronger stratification, and more saline deep water

The serotonin receptor 3E variant is a risk factor for female IBS-D

Irritable bowel syndrome (IBS) is a gut-brain disorder of multifactorial origin. Evidence of disturbed serotonergic function in IBS accumulated for the 5-HT receptor family. 5-HT Rs are encoded by HTR3 genes and control GI function, and peristalsis and secretion, in particular. Moreover, 5-HT R antagonists are beneficial in the treatment of diarrhea predominant IBS (IBS-D). We previously reported on functionally relevant SNPs in HTR3A c.-42C > T (rs1062613), HTR3C p.N163K (rs6766410), and HTR3E c.*76G > A (rs56109847 = rs62625044) being associated with IBS-D, and the HTR3B variant p.Y129S (rs1176744) was also described within the context of IBS. We performed a multi-center study to validate previous results and provide further evidence for the relevance of HTR3 genes in IBS pathogenesis. Therefore, genotype data of 2682 IBS patients and 9650 controls from 14 cohorts (Chile, Germany (2), Greece, Ireland, Spain, Sweden (2), the UK (3), and the USA (3)) were taken into account. Subsequent meta-analysis confirmed HTR3E c.*76G > A (rs56109847 = rs62625044) to be associated with female IBS-D (OR = 1.58; 95% CI (1.18, 2.12)). Complementary expression studies of four GI regions (jejunum, ileum, colon, sigmoid colon) of 66 IBS patients and 42 controls revealed only HTR3E to be robustly expressed. On top, HTR3E transcript levels were significantly reduced in the sigma of IBS patients (p = 0.0187); more specifically, in those diagnosed with IBS-D (p = 0.0145). In conclusion, meta-analysis confirmed rs56109847 = rs62625044 as a risk factor for female IBS-D. Expression analysis revealed reduced HTR3E levels in the sigmoid colon of IBS-D patients, which underlines the relevance of HTR3E in the pathogenesis of IBS-D. [Abstract copyright: © 2022. The Author(s).

Bipolar multiplex families have an increased burden of common risk variants for psychiatric disorders.

Multiplex families with a high prevalence of a psychiatric disorder are often examined to identify rare genetic variants with large effect sizes. In the present study, we analysed whether the risk for bipolar disorder (BD) in BD multiplex families is influenced by common genetic variants. Furthermore, we investigated whether this risk is conferred mainly by BD-specific risk variants or by variants also associated with the susceptibility to schizophrenia or major depression. In total, 395 individuals from 33 Andalusian BD multiplex families (166 BD, 78 major depressive disorder, 151 unaffected) as well as 438 subjects from an independent, BD case/control cohort (161 unrelated BD, 277 unrelated controls) were analysed. Polygenic risk scores (PRS) for BD, schizophrenia (SCZ), and major depression were calculated and compared between the cohorts. Both the familial BD cases and unaffected family members had higher PRS for all three psychiatric disorders than the independent controls, with BD and SCZ being significant after correction for multiple testing, suggesting a high baseline risk for several psychiatric disorders in the families. Moreover, familial BD cases showed significantly higher BD PRS than unaffected family members and unrelated BD cases. A plausible hypothesis is that, in multiplex families with a general increase in risk for psychiatric disease, BD development is attributable to a high burden of common variants that confer a specific risk for BD. The present analyses demonstrated that common genetic risk variants for psychiatric disorders are likely to contribute to the high incidence of affective psychiatric disorders in the multiplex families. However, the PRS explained only part of the observed phenotypic variance, and rare variants might have also contributed to disease development