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The cell line MLTC-1 was established in 1982 as a transplantable Leydig cell tumor from a C57BL/6 mouse. The cell line has already been applied in >100 studies: still, the only information about its genetic content is given in the first description: MLTC-1 initially had a polyploid karyotype. Here, a molecular karyotyping and multicolor banding-based molecular cytogenetic study was done to provide the first chromosomal/ (molecular) cytogenetic characterization of MLTC-1. A hexaploid karyotype with 72 to 105 chromosomes was hereby characterized. Besides polyploidy, unbalanced two- and three-way translocations, dicentrics and one neocentric derivative were identified. Also, no Y-chromosome could be detected in this clearly male derived cell line. Overall, a completely unbalanced genome is present in MLTC-1 with ~20 regions being subject to copy number losses or gains. After translating these imbalances into the human genome in a standardized way, a 40% match of imbalances with human Leydig cell tumors was evident; however, about the same rate of concordance was detectable for human spermatocytic seminomas and non-seminomas as well as testicular germ cell tumor. Accordingly, MLTC-1 is better suited as an advanced testicular germ cell tumor model in general, rather than specifically for human Leydig cell tumors.

Variants in size of the acrocentric short arms (acro-ps) are normally not reported and considered as chromosomal heteromorphisms (CHMs) without any influence on the carrier’s phenotype. However, if acro-ps are translocated to ends of A-chromosomes (i.e. human chromosomes 1-22 and X or Y), those rearrangements are studied in more detail. The aim of the study: Here we characterized 11 healthy carriers of a non-acrocentric satellited chromosomes der(A)t(A;acro)(pter or qter;p1?1.2) to determine the frequency of chromosome 15p and 22p in such rearrangements. Materials and methods: 11 carriers of one (10 cases) or two (1 case) der(A)t(A;acro) were identified during routine cytogenetic analyses. They were originally referred due to infertility or due to a mentally retarded child with otherwise abnormal karyotype. Here derivative chromosomes were studied by fluorescence in situ hybridization applying probes D15Z1 (specific for 15p11.2) and D22Z4 (specific for 22p11.2). As there are no DNA-sequences available for 13p11.2, 14p11.2 and 21p11.2 these regions could not be tested. Results: D15Z1 sequences were identified in 1 out of 12 derivatives der(A)t(A;acro). D22Z1 could not be detected in any of the 11 remainder derivatives. However, only 3 of the 12 der(A)t(A;acro) had acro-ps large enough to potentially comprise sub-band p11.2. Conclusion: In contrast to der(Y)t(Y;acro)(q12;p1?1.2), where in at least 65% of the cases the acro-p part contains D15Z1 sequences, here it could be shown that in der(A)t(A;acro) 15p involvement can be substantiated much less frequently. Also, in none of the two groups D22Z4-sequences were detected in acro-p-parts yet. Besides, breakpoint of acro-pparts in der(A)t(A;acro) seem to be in ~75% of the cases distal from p11.2.

Interphase or metaphase nuclei can be accessed in molecular cytogenetic analyses. Metaphase spreads are routinely studied by fluorescence in situ hybridization (FISH) to answer clinical genetic questions. Even though metaphase quality is essential for FISH studies, there is limited ability in clinical cases to improve the quality of cytogenetic preparations. However, the quality of preps is important for the exact localization of FISH signals, which is necessary to identify individual chromosomes and chromosomal sub-regions using inverted DAPI banding. Here we present an efficient and easy-to-perform variant of standard slide pretreatment before a normal FISH procedure. This method reproducibly leads to solid, "steel," nonfuzzy, and well-DAPI-banded metaphases. This protocol works in blood lymphocyte and amniotic fluid-derived fibroblasts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Slide pretreatment for high-quality metaphases for molecular cytogenetics.

Human epithelial stem cells (ESCs) are characterized by long-term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly-ectodermal dysplasia-clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (gamma-secretase inhibitor; N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine T-butyl ester). All cells were able to differentiate in vitro but exhibited variable self-renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up- or down-regulated during their lifetime, thus allowing to identify druggable gene networks and off-the-shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells., Journal of Cellular and Molecular Medicine, 26 (14), ISSN:1582-1838, ISSN:1582-4934

The murine tumor cell line LA-4 (also known as LA4 or LA 4) has been extensively used in ~70 studies from 1975 to present. However, the genetic characteristics have not been comprehensively delineated, apart from a single solid-stain cytogenetic study, which reported an average of 116 chromosomes per cell. LA-4 was created via urethane induction in an A/He mouse and demonstrated characteristic features of lung adenoma cells. In the present study, multicolor banding-based molecular cytogenetics was combined with molecular karyotyping to characterize ploidy, copy number alterations and chromosomal breakpoints of LA-4. A hyper-tetraploid karyotype with 85-93 chromosomes per cell, three distinct (pseudo-) dicentric derivatives, two neocentrics, four unbalanced translocations, two chromosomes with terminal deletions and one chromosome with a balanced inversion were detected. The results were translated into the human genome and a comparison with the literature revealed that LA-4 is well-suited as a murine model for human squamous cell lung cancer.

Small supernumerary marker chromosomes (sSMCs) are additional derivative chromosomes present in an otherwise numerically and structurally normal karyotype. They may derive from each of the 24 human chromosomes, and most contain a normal centromeric region with an alphoid sequence from a single chromosome. The majority of human chromosomes have a unique centromeric DNA-sequence enabling their indubitable characterization. However, chromosomes 14 and 22 share a common centromeric sequence D14/22Z1, and sSMCs with this DNA-stretch can derive from either chromosome. Euchromatin-carrying sSMCs(14 or 22) may be further characterized by molecular cytogenetics. However, in most diagnostic laboratories, heterochromatic sSMCs cannot be differentiated between chromosomes 14 or 22 derivation and are often reported as der(14 or 22). Still, heterochromatic sSMC(14 or 22) can be distinguished from each other using the D22Z4 probe (non-commercial) localized to 22p11.2. Herein, 355 sSMC(14 or 22) analyzed in the authors’ laboratory during the last ~ 20 years are summarized to address the questions: (1) What are the true frequencies of chromosome 14- and chromosome 22- derived sSMCs within D14/22Z1-positive cases? (2) Does sub-characterization of sSMC(14) and sSMC(22) make a difference in routine diagnostics? These questions could be answered as follows: (ad 1) within the studied group of sSMCs ~ 40% are derived from chromosome 14 and ~ 60% from chromosome 22; (ad 2) the knowledge on exact sSMC origin can help to save costs in routine diagnostics; i.e. in a clinically abnormal person with sSMC(14) a test for uniparental disomy is indicated, which is not necessary if a chromosome 22 origin for the sSMC was determined.

Cryptic balanced chromosomal aberrations can be an underlying cause of infertility. In 2003 Cockwell and coworkers highlighted the relevance of euchromatic pericentric regions of acrocentric chromosomes that may be a yet ignored genomic region hosting cryptic rearrangements. Here we offer the first follow-up study to further explore this idea. Two specific molecular cytogenetic probe sets were established to elucidate such cryptic rearrangements together with chromosomal heteromorphisms of acrocentric centromeres. In 28 infertile couples and 20 controls, the rate of centromeric heteromorphisms was almost comparable in both groups and one heteromorphism was noted in ~30% of the cases, and two heteromorphisms in ~15% and three heteromorphisms 5%. However, none of the studied groups revealed any cryptic euchromatic pericentromeric abnormalities of the acrocentrics. Nonetheless, in parallel an infertile case with an inv(13)(p12q12.1?2) was uncovered, being not part of the systematically studied group of infertile. As unbalanced products of meiosis with such or similar karyotypes can potentially contribute to abortions, the existence of rare, cryptic pericentromeric euchromatic abnormalities in the acrocentrics thus needs to be still expected in banding cytogenetic diagnostics. Accordingly, this study reflected that suspicious acrocentric short arms in infertile need special attention and further characterization by fluorescence in situ hybridization.

Background Copy number variants (CNVs) are the genetic bases for microdeletion/ microduplication syndromes (MMSs). Couples with an affected child and desire to have further children are routinely tested for a potential parental origin of a specific CNV either by molecular karyotyping or by two color fluorescence in situ hybridization (FISH), yet. In the latter case a critical region probe (CRP) is combined with a control probe for identification of the chromosome in question. However, CNVs can arise also due to other reasons, like a recombination-event based on a submicroscopic, cryptic inversion in one of the parents. Results Seventy-four patients with different MMSs and overall 81 CNVs were studied here by a novel three color FISH approach. The way how three locus-specific probes are selected (one is the CRP and two are flanking it in a distance of 5-10 Mb) enables to detect or exclude two possible parental conditions as origins of the CNV seen in the index: (i) direct parental origin of the CNV (deletion or duplication) or (ii) a parental cryptic inversion. Thus, for overall 51/81 CNVs (63%) a parental origin could be determined. 36/51 (70.5%) inherited the CNV directly from one of the parents, but 15/51 (29.5%) were due to an exclusively by three color FISH detectable parental inversion. A 2:1 ratio of maternal versus paternal inheritance was found. Also almost two times more male than female were among the index patients. Conclusion The new, here suggested three color FISH approach is suited for more comprehensive parental studies of patients with MMS. The detection rate for parental origin was increased by 140% in this study. Still, for 30/81 cases (37%) no reason for the ‘de novo’ MMS in the affected index patient could be found by the here suggested FISH-probe set. Electronic supplementary material The online version of this article (10.1186/s13039-018-0369-1) contains supplementary material, which is available to authorized users.