International Journal of Molecular Sciences

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Open AccessEditorial

State-of-the-Art Molecular Genetics and Genomics in Germany

by Jan Lukas

Int. J. Mol. Sci. 2023, 24(18), 14096; https://doi.org/10.3390/ijms241814096 - 14 Sep 2023

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Abstract

The Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany focuses on German researchers and their international peers, covering their recent advances in genetics, genomics, epigenetics, and cytogenetics/cytogenomics in relation to prokaryotic and eukaryotic multicellular to mammalian organisms in arras ranging from basic [...] Read more.

The Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany focuses on German researchers and their international peers, covering their recent advances in genetics, genomics, epigenetics, and cytogenetics/cytogenomics in relation to prokaryotic and eukaryotic multicellular to mammalian organisms in arras ranging from basic to medical research [...] Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

Research

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20 pages, 2716 KiB

Open AccessArticle

Sleeping Beauty Transposon Insertions into Nucleolar DNA by an Engineered Transposase Localized in the Nucleolus

by Adrian Kovač, Csaba Miskey and Zoltán Ivics

Int. J. Mol. Sci. 2023, 24(19), 14978; https://doi.org/10.3390/ijms241914978 - 7 Oct 2023

Viewed by 1442

Abstract

Transposons are nature’s gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into [...] Read more.

Transposons are nature’s gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration. Here, we evaluated an experimental strategy to manipulate SB’s target site distribution by preferentially compartmentalizing the SB transposase to the nucleolus, which contains repetitive ribosomal RNA (rRNA) genes. We generated a fusion protein composed of the nucleolar protein nucleophosmin (B23) and the SB100X transposase, which was found to retain almost full transposition activity as compared to unfused transposase and to be predominantly localized to nucleoli in transfected human cells. Analysis of transposon integration sites generated by B23-SB100X revealed a significant enrichment into the p-arms of chromosomes containing nucleolus organizing regions (NORs), with preferential integration into the p13 and p11.2 cytobands directly neighboring the NORs. This bias in the integration pattern was accompanied by an enrichment of insertions into nucleolus-associated chromatin domains (NADs) at the periphery of nucleolar DNA and into lamina-associated domains (LADs). Finally, sub-nuclear targeting of the transposase resulted in preferential integration into chromosomal domains associated with the Upstream Binding Transcription Factor (UBTF) that plays a critical role in the transcription of 47S rDNA gene repeats of the NORs by RNA Pol I. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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12 pages, 2779 KiB

Open AccessArticle

CRISISS: A Novel, Transcriptionally and Post-Translationally Inducible CRISPR/Cas9-Based Cellular Suicide Switch

by Maximilian Amberger, Esther Grueso and Zoltán Ivics

Int. J. Mol. Sci. 2023, 24(12), 9799; https://doi.org/10.3390/ijms24129799 - 6 Jun 2023

Cited by 1 | Viewed by 1693

Abstract

With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this [...] Read more.

With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this study, we present the CRISPR-induced suicide switch (CRISISS) as a tool to eliminate genetically modified cells in an inducible and highly efficient manner by targeting Cas9 to highly repetitive Alu retrotransposons in the human genome, causing irreparable genomic fragmentation by the Cas9 nuclease and resulting cell death. The suicide switch components, including expression cassettes for a transcriptionally and post-translationally inducible Cas9 and an Alu-specific single-guide RNA, were integrated into the genome of target cells via Sleeping-Beauty-mediated transposition. The resulting transgenic cells did not show signs of any impact on overall fitness when uninduced, as unintended background expression, background DNA damage response and background cell killing were not observed. When induced, however, a strong expression of Cas9, a strong DNA damage response and a rapid halt of cell proliferation coupled with near complete cell death within four days post-induction were seen. With this proof-of-concept study, we present a novel and promising approach for a robust suicide switch with potential utility for gene and cell therapy in the future. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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13 pages, 5171 KiB

Open AccessArticle

A Causal Treatment for X-Linked Hypohidrotic Ectodermal Dysplasia: Long-Term Results of Short-Term Perinatal Ectodysplasin A1 Replacement

by Holm Schneider, Christine Schweikl, Florian Faschingbauer, Smail Hadj-Rabia and Pascal Schneider

Int. J. Mol. Sci. 2023, 24(8), 7155; https://doi.org/10.3390/ijms24087155 - 12 Apr 2023

Cited by 6 | Viewed by 2447

Abstract

X-linked hypohidrotic ectodermal dysplasia (XLHED), caused by a genetic deficiency of ectodysplasin A1 (EDA1), is a rare developmental disorder of ectodermal derivatives such as hair, sweat glands, and teeth. The absence of sweat glands and perspiration can evoke life-threatening hyperthermia. As molecular genetic [...] Read more.

X-linked hypohidrotic ectodermal dysplasia (XLHED), caused by a genetic deficiency of ectodysplasin A1 (EDA1), is a rare developmental disorder of ectodermal derivatives such as hair, sweat glands, and teeth. The absence of sweat glands and perspiration can evoke life-threatening hyperthermia. As molecular genetic findings are not always conclusive, the concentrations of circulating EDA1 may help to distinguish between total and partial EDA1 deficiencies. We previously treated nine male patients with obvious signs of XLHED with a recombinant EDA1 replacement protein, Fc-EDA, either shortly after birth (n = 3) or by prenatal administration in gestational week 26 and beyond (n = 6). Here, we present the long-term follow-up for up to six years. In patients who had received Fc-EDA after birth, neither sweat glands nor sweating ability were detected at the age of 12–60 months. In contrast, prenatal EDA1 replacement resulted in ample sweat gland development and pilocarpine-inducible sweating in all treated subjects, who also attained more permanent teeth than their untreated affected relatives. Normal perspiration has persisted for six years in the two oldest boys treated repeatedly with Fc-EDA in utero. When they had a sauna, adequate thermoregulation was evidenced. Lower sweat production after single prenatal dosing may indicate a dose–response relationship. The absence of circulating EDA1 in five prenatally treated subjects proved that these children would have been unable to perspire if they had been left untreated. The sixth infant was shown to produce an EDA1 molecule that, albeit interacting with its cognate receptor, cannot activate EDA1 signaling. In conclusion, a causal treatment of XLHED before birth is feasible. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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17 pages, 1161 KiB

Open AccessArticle

Cost-Effective Next Generation Sequencing-Based STR Typing with Improved Analysis of Minor, Degraded and Inhibitor-Containing DNA Samples

by Sara-Sophie Poethe, Julia Holtel, Jan-Philip Biermann, Trine Riemer, Melanie Grabmüller, Burkhard Madea, Ralf Thiele and Richard Jäger

Int. J. Mol. Sci. 2023, 24(4), 3382; https://doi.org/10.3390/ijms24043382 - 8 Feb 2023

Cited by 1 | Viewed by 2914

Abstract

Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons [...] Read more.

Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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14 pages, 2428 KiB

Open AccessArticle

Cas9-Mediated Nanopore Sequencing Enables Precise Characterization of Structural Variants in CCM Genes

by Dariush Skowronek, Robin A. Pilz, Loisa Bonde, Ole J. Schamuhn, Janne L. Feldmann, Sabine Hoffjan, Christiane D. Much, Ute Felbor and Matthias Rath

Int. J. Mol. Sci. 2022, 23(24), 15639; https://doi.org/10.3390/ijms232415639 - 9 Dec 2022

Cited by 3 | Viewed by 2590

Abstract

Deletions in the CCM1, CCM2, and CCM3 genes are a common cause of familial cerebral cavernous malformations (CCMs). In current molecular genetic laboratories, targeted next-generation sequencing or multiplex ligation-dependent probe amplification are mostly used to identify copy number variants (CNVs). However, [...] Read more.

Deletions in the CCM1, CCM2, and CCM3 genes are a common cause of familial cerebral cavernous malformations (CCMs). In current molecular genetic laboratories, targeted next-generation sequencing or multiplex ligation-dependent probe amplification are mostly used to identify copy number variants (CNVs). However, both techniques are limited in their ability to specify the breakpoints of CNVs and identify complex structural variants (SVs). To overcome these constraints, we established a targeted Cas9-mediated nanopore sequencing approach for CNV detection with single nucleotide resolution. Using a MinION device, we achieved complete coverage for the CCM genes and determined the exact size of CNVs in positive controls. Long-read sequencing for a CCM1 and CCM2 CNV revealed that the adjacent ANKIB1 and NACAD genes were also partially or completely deleted. In addition, an interchromosomal insertion and an inversion in CCM2 were reliably re-identified by long-read sequencing. The refinement of CNV breakpoints by long-read sequencing enabled fast and inexpensive PCR-based variant confirmation, which is highly desirable to reduce costs in subsequent family analyses. In conclusion, Cas9-mediated nanopore sequencing is a cost-effective and flexible tool for molecular genetic diagnostics which can be easily adapted to various target regions. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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18 pages, 1382 KiB

Open AccessArticle

Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP)

by Renzo J. M. Riemens, Gunter Kenis, Jennifer Nolz, Sonia C. Susano Chaves, Diane Duroux, Ehsan Pishva, Diego Mastroeni, Kristel Van Steen, Thomas Haaf and Daniël L. A. van den Hove

Int. J. Mol. Sci. 2022, 23(24), 15571; https://doi.org/10.3390/ijms232415571 - 8 Dec 2022

Cited by 1 | Viewed by 1349

Abstract

A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using [...] Read more.

A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using laser capture microdissection (LCM). For this purpose, we established a novel approach for targeted DNA methylation profiling of individual genes that relies on a combination of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). Using this approach, we determined cytosine-phosphate-guanine (CpG) methylation rates of single alleles derived from 50 neurons that were isolated from unfixed post-mortem brain tissue. In the present manuscript, we describe the general workflow and, as a showcase, demonstrate how targeted methylation analysis of various genes, in this case, RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be performed simultaneously. By doing so, we describe an adapted data analysis pipeline for LDBSP, allowing one to include and correct CpG methylation rates derived from multi-allele reactions. In addition, we show that the efficiency of LDBSP on DNA derived from LCM neurons is similar to the efficiency obtained in previously published studies using this technique on other cell types. Overall, the method described here provides the user with a more accurate estimation of the DNA methylation status of each target gene in the analyzed cell pools, thereby adding further validity to this approach. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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13 pages, 1860 KiB

Open AccessArticle

Abnormal Pre-mRNA Splicing in Exonic Fabry Disease-Causing GLA Mutations

by Franziska Alfen, Elena Putscher, Michael Hecker, Uwe Klaus Zettl, Andreas Hermann and Jan Lukas

Int. J. Mol. Sci. 2022, 23(23), 15261; https://doi.org/10.3390/ijms232315261 - 3 Dec 2022

Cited by 3 | Viewed by 2085

Abstract

Fabry disease (FD) is a rare X-linked disease due to a multiverse of disrupting mutations within the GLA gene encoding lysosomal α-galactosidase A (AGAL). Absent AGAL activity causes the accumulation of complex glycosphingolipids inside of lysosomes in a variety of cell types and [...] Read more.

Fabry disease (FD) is a rare X-linked disease due to a multiverse of disrupting mutations within the GLA gene encoding lysosomal α-galactosidase A (AGAL). Absent AGAL activity causes the accumulation of complex glycosphingolipids inside of lysosomes in a variety of cell types and results in a progressive multisystem disease. Known disease-associated point mutations in protein-coding gene regions usually cause translational perturbations and result in premature chain termination, punctual amino acid sequence alterations or overall altered sequence alterations downstream of the mutation site. However, nucleotide exchanges at the border between introns and exons can affect splicing behavior and lead to abnormal pre-mRNA processing. Prediction with the Human Splicing Finder (HSF) revealed an indication of a significant change in splicing-relevant information for some known FD-associated GLA mutations. To experimentally determine the extent of the change, we made use of a minigene reporter assay and verified alternative splicing events for the exonic mutations c.194G>T and c.358C>G, which led to the usage of alternative donor splice sites at exon 1 and exon 2, respectively. In addition, the mutations c.548G>T and c.638A>T led to significant exon 4 skipping. We conclude that splicing phenotype analysis should be employed in the in vitro analysis of exonic GLA gene mutations, since abnormal splicing may result in a reduction of enzyme activity and alter the amenability for treatment with pharmacological chaperone (PC). Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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17 pages, 898 KiB

Open AccessArticle

Evolution of Transcriptomes in Early-Generation Hybrids of the Apomictic Ranunculus auricomus Complex (Ranunculaceae)

by Claudia Paetzold, Birthe H. Barke and Elvira Hörandl

Int. J. Mol. Sci. 2022, 23(22), 13881; https://doi.org/10.3390/ijms232213881 - 10 Nov 2022

Cited by 4 | Viewed by 1194

Abstract

Hybridisation in plants may cause a shift from sexual to asexual seed formation (apomixis). Indeed, natural apomictic plants are usually hybrids, but it is still unclear how hybridisation could trigger the shift to apomixis. The genome evolution of older apomictic lineages is influenced [...] Read more.

Hybridisation in plants may cause a shift from sexual to asexual seed formation (apomixis). Indeed, natural apomictic plants are usually hybrids, but it is still unclear how hybridisation could trigger the shift to apomixis. The genome evolution of older apomictic lineages is influenced by diverse processes such as polyploidy, mutation accumulation, and allelic sequence divergence. To disentangle the effects of hybridisation from these other factors, we analysed the transcriptomes of flowering buds from artificially produced, diploid F2 hybrids of the Ranunculus auricomus complex. The hybrids exhibited unreduced embryo sac formation (apospory) as one important component of apomixis, whereas their parental species were sexual. We revealed 2915 annotated single-copy genes that were mostly under purifying selection according to dN/dS ratios. However, pairwise comparisons revealed, after rigorous filtering, 79 genes under diversifying selection between hybrids and parents, whereby gene annotation assigned ten of them to reproductive processes. Four genes belong to the meiosis-sporogenesis phase (ASY1, APC1, MSP1, and XRI1) and represent, according to literature records, candidate genes for apospory. We conclude that hybridisation could combine novel (or existing) mutations in key developmental genes in certain hybrid lineages, and establish (together with altered gene expression profiles, as observed in other studies) a heritable regulatory mechanism for aposporous development. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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20 pages, 3916 KiB

Open AccessArticle

PTEN Loss Enhances Error-Prone DSB Processing and Tumor Cell Radiosensitivity by Suppressing RAD51 Expression and Homologous Recombination

by Xile Pei, Emil Mladenov, Aashish Soni, Fanghua Li, Martin Stuschke and George Iliakis

Int. J. Mol. Sci. 2022, 23(21), 12876; https://doi.org/10.3390/ijms232112876 - 25 Oct 2022

Cited by 5 | Viewed by 1918

Abstract

PTEN has been implicated in the repair of DNA double-strand breaks (DSBs), particularly through homologous recombination (HR). However, other data fail to demonstrate a direct role of PTEN in DSB repair. Therefore, here, we report experiments designed to further investigate the role of [...] Read more.

PTEN has been implicated in the repair of DNA double-strand breaks (DSBs), particularly through homologous recombination (HR). However, other data fail to demonstrate a direct role of PTEN in DSB repair. Therefore, here, we report experiments designed to further investigate the role of PTEN in DSB repair. We emphasize the consequences of PTEN loss in the engagement of the four DSB repair pathways—classical non-homologous end-joining (c-NHEJ), HR, alternative end-joining (alt-EJ) and single strand annealing (SSA)—and analyze the resulting dynamic changes in their utilization. We quantitate the effect of PTEN knockdown on cell radiosensitivity to killing, as well as checkpoint responses in normal and tumor cell lines. We find that disruption of PTEN sensitizes cells to ionizing radiation (IR). This radiosensitization is associated with a reduction in RAD51 expression that compromises HR and causes a marked increase in SSA engagement, an error-prone DSB repair pathway, while alt-EJ and c-NHEJ remain unchanged after PTEN knockdown. The G₂-checkpoint is partially suppressed after PTEN knockdown, corroborating the associated HR suppression. Notably, PTEN deficiency radiosensitizes cells to PARP inhibitors, Olaparib and BMN673. The results show the crucial role of PTEN in DSB repair and show a molecular link between PTEN and HR through the regulation of RAD51 expression. The expected benefit from combination treatment with Olaparib or BMN673 and IR shows that PTEN status may also be useful for patient stratification in clinical treatment protocols combining IR with PARP inhibitors. Full article

(This article belongs to the Special Issue State-of-the-Art Molecular Genetics and Genomics in Germany)

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