Calling and Phasing of Single-Nucleotide and Structural Variants of the LDLR Gene Using Oxford Nanopore MinION.

The LDLR locus has clinical significance for lipid metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid metabolism-related diseases (coronary artery disease and Alzheimer's disease), but its intronic and structural variants are underinvestigated. The aim of this study was to design and validate a method for nearly complete sequencing of the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR amplicons from LDLR of three patients with compound heterozygous FH were analyzed. We used standard workflows of EPI2ME Labs for variant calling. All rare missense and small deletion variants detected previously by massively parallel sequencing and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion (exons 15 and 16) that was detected by ONT with precisely located breakpoints between AluY and AluSx1. Trans-heterozygous associations between mutation c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We demonstrated the ability of ONT to phase variants, thereby enabling haplotype assignment for LDLR with personalized resolution. The ONT-based method was able to detect exonic variants with the additional benefit of intronic analysis in one run. This method can serve as an efficient and cost-effective tool for diagnosing FH and conducting research on extended LDLR haplotype reconstruction.

The Precise Breakpoint Mapping in Paracentric Inversion 10q22.2q23.3 by Comprehensive Cytogenomic Analysis, Multicolor Banding, and Single-Copy Chromosome Sequencing.

Detection and precise genomic mapping of balanced chromosomal abnormalities in patients with impaired fertility or a clinical phenotype represent a challenge for current cytogenomics owing to difficulties with precise breakpoint localization in the regions enriched for DNA repeats and high genomic variation in such regions. Here, we present a comprehensive cytogenomic approach to breakpoint mapping in a rare paracentric inversion on 10q (in a patient with oligoasthenoteratozoospermia and necrozoospermia) that does not affect other phenotype traits. Multicolor banding, chromosomal microarray analysis, chromosome microdissection with reverse painting, and single-copy sequencing of the rearranged chromosome were performed to determine the length and position of the inverted region as well as to rule out a genetic imbalance at the breakpoints. As a result, a paracentric 19.251 Mbp inversion at 10q22.2q23.3 was described. The most probable location of the breakpoints was predicted using the hg38 assembly. The problems of genetic counseling associated with enrichment for repeats and high DNA variability of usual breakpoint regions were discussed. Possible approaches for cytogenomic assessment of couples with balanced chromosome rearrangements and problems like reproductive failures were considered and suggested as useful part of effective genetic counseling.

Induced pluripotent stem cell line ICGi038-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to compound heterozygous c.1246C > T/c.940 + 3_940 + 6del mutations in LDLR.

The development of cellular models for familial hypercholesterolemia (FH) is an important direction for creating new approaches to atherosclerosis treatment. Pathogenic mutations in the LDLR gene are the main FH source. We generated an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous c.1246C > T/c.940 + 3_940 + 6del LDLR mutation. The resulting iPSC line with confirmed patient-specific mutations maintains a normal karyotype and a typical undifferentiated state, including morphology, pluripotent gene expression, and in vitro differentiation potential. This iPSC line can be further differentiated toward relevant cells to better understand FH pathogenesis.

Induced pluripotent stem cell line ICGi037-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to heterozygous p.Trp443Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder increasing premature cardiovascular diseases risk due to atherosclerosis. Pathogenic mutations in the LDLR gene cause most FH cases. Available treatments are effective not for all LDLR mutations. Testing drugs on FH cell models help develop new efficient treatments. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with heterozygous p.Trp443Arg LDLR mutation. The iPSCs with confirmed patient-specific mutations express pluripotency markers, spontaneously differentiate into three germ layers and demonstrate normal karyotype. Patient-specific iPSCs-derived hepatocyte-like and endothelial cells are promising to develop new targeted therapies for FH.

Induced pluripotent stem cell line ICGi036-A generated by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia caused due to compound heterozygous p.Ser177Leu/p.Cys352Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is a monogenic disease, leading to atherosclerosis due to a high level of low-density lipoprotein cholesterol. Most cases of the disease are based on pathological variants in the LDLR gene. Hepatocyte-like and endothelial cells derived from individual iPSCs are a good model for developing new approaches to therapy. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous p.Ser177Leu/p.Cys352Arg mutation in LDLR using non-integrating vectors. The iPSCs with a confirmed patient-specific mutation demonstrate pluripotency markers, normal karyotype, and the ability to differentiate into derivatives of three germ layers.

Prenatal Diagnosis of Small Supernumerary Marker Chromosome 10 by Array-Based Comparative Genomic Hybridization and Microdissected Chromosome Sequencing.

Interpreting the clinical significance of small supernumerary marker chromosomes (sSMCs) in prenatal diagnosis is still an urgent problem in genetic counselling regarding the fate of a pregnancy. We present a case of prenatal diagnosis of mosaic sSMC(10) in a foetus with a normal phenotype. Comprehensive cytogenomic analyses by array-based comparative genomic hybridization (aCGH), sSMC microdissection with next-generation sequencing (NGS) of microdissected library, fluorescence in situ hybridization (FISH) with locus-specific and telomere-specific DNA probes and quantitative real-time PCR revealed that sSMC(10) had a ring structure and was derived from the pericentromeric region of chromosome 10 with involvement of the 10p11.21-p11.1 and 10q11.21-q11.23 at 1.243 Mb and 7.173 Mb in size, respectively. We observed a difference in the length of sSMC(10) between NGS data of the DNA library derived from a single copy of sSMC(10), and aCGH results that may indicate instability and structural mosaicism for ring chromosomes in foetal cells. The presence of a 9 Mb euchromatin region in the analysed sSMC(10) did not lead to clinical manifestations, and a healthy girl was born at term. We suggest that the ring structure of sSMCs could influence sSMC manifestations and should be taken into account in genetic counselling during prenatal diagnosis.

Impact of Xist RNA on chromatin modifications and transcriptional silencing maintenance at different stages of imprinted X chromosome inactivation in vole Microtus levis.

In vole Microtus levis, cells of preimplantation embryo and extraembryonic tissues undergo imprinted X chromosome inactivation (iXCI) which is triggered by a long non-coding nuclear RNA, Xist. At early stages of iXCI, chromatin of vole inactive X chromosome is enriched with the HP1 heterochromatin-specific protein, trimethylated H3K9 and H4K20 attributable to constitutive heterochromatin. In the study, using vole trophoblast stem (TS) cells as a model of iXCI, we further investigated chromatin of the inactive X chromosome of M. levis and tried to find out the role of Xist RNA. We demonstrated that chromatin of the inactive X chromosome in vole TS cells also contained the SETDB1 histone methyltransferase and KAP1 protein. In addition, we observed that Xist RNA did not contribute significantly to maintenance of X chromosome inactive state during iXCI in vole TS cells. Xist repression affected neither transcriptional silencing caused by iXCI nor maintenance of trimethylated H3K9 and H4K20 as well as HP1, KAP1, and SETDB1 on the inactive X chromosome. Moreover, the unique repertoire of chromatin modifications on the inactive X chromosome in vole TS cells could be disrupted by a chemical compound, DZNep, and then restored even in the absence of Xist RNA. However, Xist transcript was necessary for recruitment of an additional repressive histone modification, trimethylated H3K27, to the inactive X chromosome during vole TS cell differentiation.

A regulatory potential of the Xist gene promoter in vole M. rossiaemeridionalis.

X chromosome inactivation takes place in the early development of female mammals and depends on the Xist gene expression. The mechanisms of Xist expression regulation have not been well understood so far. In this work, we compared Xist promoter region of vole Microtus rossiaemeridionalis and other mammalian species. We observed three conserved regions which were characterized by computational analysis, DNaseI in vitro footprinting, and reporter construct assay. Regulatory factors potentially involved in Xist activation and repression in voles were determined. The role of CpG methylation in vole Xist expression regulation was established. A CTCF binding site was found in the 5' flanking region of the Xist promoter on the active X chromosome in both males and females. We suggest that CTCF acts as an insulator which defines an inactive Xist domain on the active X chromosome in voles.

Variability of sequence surrounding the Xist gene in rodents suggests taxon-specific regulation of X chromosome inactivation.

One of the two X chromosomes in female mammalian cells is subject to inactivation (XCI) initiated by the Xist gene. In this study, we examined in rodents (voles and rat) the conservation of the microsatellite region DXPas34, the Tsix gene (antisense counterpart of Xist), and enhancer Xite that have been shown to flank Xist and regulate XCI in mouse. We have found that mouse regions of the Tsix gene major promoter and minisatellite repeat DXPas34 are conserved among rodents. We have also shown that in voles and rat the region homologous to the mouse Tsix major promoter, initiates antisense to Xist transcription and terminates around the Xist gene start site as is observed with mouse Tsix. A conservation of Tsix expression pattern in voles, rat and mice suggests a crucial role of the antisense transcription in regulation of Xist and XIC in rodents. Most surprisingly, we have found that voles lack the regions homologous to the regulatory element Xite, which is instead replaced with the Slc7a3 gene that is unassociated with the X-inactivation centre in any other eutherians studied. Furthermore, we have not identified any transcription that could have the same functions as murine Xite in voles. Overall, our data show that not all the functional elements surrounding Xist in mice are well conserved even within rodents, thereby suggesting that the regulation of XCI may be at least partially taxon-specific.

Difference between random and imprinted X inactivation in common voles.

During early development in female mammals, most genes on one of the two X-chromosomes undergo transcriptional silencing. In the extraembryonic lineages of some eutherian species, imprinted X-inactivation of the paternal X-chromosome occurs. In the cells of the embryo proper, the choice of the future inactive X-chromosome is random. We mapped several genes on the X-chromosomes of five common vole species and compared their expression and methylation patterns in somatic and extraembryonic tissues, where random and imprinted X-inactivation occurs, respectively. In extraembryonic tissues, more genes were expressed on the inactive X-chromosome than in somatic tissues. We also found that the methylation status of the X-linked genes was always in accordance with their expression pattern in somatic, but not in extraembryonic tissues. The data provide new evidence that imprinted X-inactivation is less complete and/or stable than the random form and DNA methylation contributes less to its maintenance.

FGF4 independent derivation of trophoblast stem cells from the common vole.

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells.

A dual origin of the Xist gene from a protein-coding gene and a set of transposable elements.

X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding genes. In this study we show that the key XIC gene Xist, which displays fragmentary homology to a protein-coding gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The Xist gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 gene. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into Xist accompanies the overall evolution of the gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the Xist gene and is found in other XIC genes producing non-coding nuclear RNA.

DNA content of the B chromosomes in grasshopper Podisma kanoi Storozh. (Orthoptera, Acrididae).

A DNA library derived from the B chromosome of Podisma kanoi was obtained by chromosome microdissection. A total of 153 DNA clones were isolated from the microdissected DNA library. Twenty of them were sequenced. A comparison of B chromosome DNA sequences with sequences of other species from the DDBJ/GenBank/EMBL database ( http://www.ddbj.nig.ac.jp/ ) was performed. Different patterns of signals were observed after FISH with labeled cloned DNA fragments. FISH signals with cloned DNA fragments painted either whole Bs or their different regions. Some clones also gave signals in pericentromeric regions of A chromosomes. Other cloned DNA fragments gave only background-like signals on A and B chromosomes. Comparative FISH analysis of B chromosomes in Podisma kanoi and P. sapporensis with DNA probes derived from the Bs of these species revealed homologous DNA that was confined within pericentromeric and telemetric regions of the B chromosome in P. kanoi. In contrast to the B chromosomes in P. sapporensis containing large regions enriched with rDNA, only a small cluster of rDNA was detected in one of the examined B chromosomes in P. kanoi. The data strongly suggest an independent origin of B chromosomes in two closely related Podisma species.

Genes flanking Xist in mouse and human are separated on the X chromosome in American marsupials.

X inactivation, the transcriptional silencing of one of the two X chromosomes in female mammals, achieves dosage compensation of X-linked genes relative to XY males. In eutherian mammals X inactivation is regulated by the X-inactive specific transcript (Xist), a cis-acting non-coding RNA that triggers silencing of the chromosome from which it is transcribed. Marsupial mammals also undergo X inactivation but the mechanism is relatively poorly understood. We set out to analyse the X chromosome in Monodelphis domestica and Didelphis virginiana, focusing on characterizing the interval defined by the Chic1 and Slc16a2 genes that in eutherians flank the Xist locus. The synteny of this region is retained on chicken chromosome 4 where other loci belonging to the evolutionarily ancient stratum of the human X chromosome, the so-called X conserved region (XCR), are also located. We show that in both M. domestica and D. virginiana an evolutionary breakpoint has separated the Chic1 and Slc16a2 loci. Detailed analysis of opossum genomic sequences revealed linkage of Chic1 with the Lnx3 gene, recently proposed to be the evolutionary precursor of Xist, and Fip1, the evolutionary precursor of Tsx, a gene located immediately downstream of Xist in eutherians. We discuss these findings in relation to the evolution of Xist and X inactivation in mammals.