Molecular Cytogenetic Classification of Down Syndrome and Screening of Somatic Aneuploidy in Mothers

Down Syndrome (DS) caused by trisomy 21 results in various congenital and developmental complications in children. It is crucial to cytogenetically diagnose the DS cases early for their proper health management and to reduce the risk of further DS childbirths in mothers. In this study, we performed a cytogenetic analysis of 436 suspected DS cases using karyotyping and fluorescent in situ hybridization. We detected free trisomies (95.3%), robertsonian translocations (2.4%), isochromosomes (0.6%), and mosaics (1.2%). We observed a slightly higher incidence of DS childbirth in younger mothers compared to mothers with advanced age. We compared the somatic aneuploidy in peripheral blood of mothers having DS children (MDS) and control mothers (CM) to identify biomarkers for predicting the risk for DS childbirths. No significant difference was observed. After induced demethylation in peripheral blood cells, we did not observe a significant difference in the frequency of aneuploidy between MDS and CM. In conclusion, free trisomy 21 is the most common type of chromosomal abnormality in DS. A small number of DS cases have translocations and mosaicism of chromosome 21. Additionally, somatic aneuploidy in the peripheral blood from the mother is not an effective marker to predict DS childbirths.

Karyotype Reorganization in Wheat–Rye Hybrids Obtained via Unreduced Gametes: Is There a Limit to the Chromosome Number in Triticale?

To date, few data have been accumulated on the contribution of meiotic restitution to the formation of Triticum aestivum hybrid karyotypes. In this study, based on FISH and C-banding, karyotype reorganization was observed in three groups of F5 wheat–rye hybrids 1R(1A) × R. Aberrations, including aneuploidy, telocentrics, and Robertsonian translocations, were detected in all groups. Some of the Group 1 plants and all of the Group 2 plants only had a 4R4R pair (in addition to 1R1R), which was either added or substituted for its homeolog in ABD subgenomes. In about 82% of meiocytes, 4R4R formed bivalents, which indicates its competitiveness. The rest of the Group 1 plants had 2R and 7R chromosomes in addition to 1R1R. Group 3 retained all their rye chromosomes, with a small aneuploidy on the wheat chromosomes. A feature of the meiosis in the Group 3 plants was asynchronous cell division and omission of the second division. Diploid gametes did not form because of the significant disturbances during gametogenesis. As a result, the frequency of occurrence of the formed dyads was negatively correlated (r = −0.73) with the seed sets. Thus, meiotic restitution in the 8n triticale does not contribute to fertility or increased ploidy in subsequent generations.

Chromosomal Aberrations in Cattle

Chromosomal aberrations and their mechanisms have been studied for many years in livestock. In cattle, chromosomal abnormalities are often associated with serious reproduction-related problems, such as infertility of carriers and early mortality of embryos. In the present work, we review the mechanisms and consequences of the most important bovine chromosomal aberrations: Robertsonian translocations and reciprocal translocations. We also discuss the application of bovine cell cultures in genotoxicity studies.

Analysis of sperm chromosomes in six carriers of rare and common Robertsonian translocations

Introduction: Robertsonian translocation (RobT) is the central fusion of the long arms of two acrocentric chromosomes, leading to 45 chromosomes in humans. The most common ones are rob(13;14) and rob(14;21) (91%). Other types of RobT are so-called rare cases. In the general population RobTs occur with a frequency of approximately 0.123%, but among men with reproductive failure this value rises 9-fold. Infertility in RobT carriers is associated with the formation of unbalanced spermatozoa resulting from segregation of the chromosomes involved in trivalent during the meiotic prophase. In spermatozoa of many RobT carriers an increased level of chromosomal aneuploidy is observed. Materials and Methods: We examined the hyperhaploidy level of chromosomes 7, 9, 18, 21, 22, X and Y in spermatozoa of 6 RobT unrelated carriers: two carriers with rare rob(13;15), one with rare rob(13;22), and three of the common rob(13;14). Results were compared with the control data from a group of 7 fertile men with a normal karyotype. Fluorescent in situ hybridization (FISH) was applied. Results: We found an increased level of sperm aneuploidy regarding at least one of the analyzed chromosomes in each of the carriers, while in rare RobTs interchromosomal effect (ICE) was observed. Meiotic segregation pattern of a rare rob(13;15) carrier revealed the 76% of normal /balanced spermatozoa. Disucussion: Due to the relatively high population frequency of RobTs, their influence on reproductive failure, hight risk of imbalancement in prenatal diagnosis (7%), and small amount of data for rare RobTs, each newly characterized case is valuable in genetic counseling.

Selected genetic causes of miscarriages

Approximately 15–25% of pregnancies end in spontaneous abortion, which is an expulsion from the mother body of the fetus weighing less than 500 g or before the 20th week of gestation. Determining abortions etiology is difficult due to its multifactorial character. Chromosomal abnormalities cause 38.6–80% of miscarriages. The largest group (93%) of chromosomal aberrations found in miscarried fetuses are numerical changes – aneuploidies and polyploidies. Much rarer (7%) are unbalanced structural aberrations, which can arise de novo or can be inherited from a carrier parent. In couples with spontaneous abortions, reciprocal chromosomal translocations (RCT) occur the most frequently, next are Robertsonian translocations and inversions. More complex chromosome abnormalities, e.g. double aneuploidies are found in 3.8% of fetuses. Another group of causes responsible for abortions are monogenic diseases of embryo or fetus resulting from autosomal dominant, autosomal recessive or X-linked mutations. Among mutations which may contribute to pregnancy loss are factor V Leiden gene mutations (c.1601G>A, earlier 1691G>A) and prothrombin gene mutation (c.97G>A, earlier 20210G>A). The research on mutations in candidate genes, eg.: ALOX15, CR1, CYP1A1, CYP17, CYP2D6, FOXP3, HLA-G, IL-6, KHDC3L, NLRP7, NOS3, PLK4, SYCP3, TLR3, TNF, TP53 and VEGFA is still ongoing.

A Rapid NGS-Based Preimplantation Genetic Testing for Chromosomal Abnormalities in Day-3 Blastomere Biopsy Allows Embryo Transfer Within the Same Treatment Cycle

Nowadays, most of the preimplantation genetic testing (PGT) is performed with a strategy of comprehensive chromosome screening and trophectoderm biopsy. Nevertheless, patients with ovarian insufficiency may not have competent blastocysts. In the present study, we aimed to establish the value of multiple annealing and looping-based amplification cycle (MALBAC)-based next-generation sequencing (NGS) for PGT in day-3 embryos. A total of 94.3% (1168/1239) of embryos yielded informative results, and the overall embryo euploid rate was 21.9% (256/1168). Overall, 225 embryos were transferred in 169 cycles with a clinical pregnancy rate of 49.1% (83/169). The live birth and implantation rates were 47.3% (80/169) and 44.4% (100/225), respectively. Double embryos transfer showed higher clinical pregnancy and live birth rates compared with single embryo transfer, but the implantation rates were similar (44.2% vs. 44.6%, P > 0.05). The euploid rate for reciprocal translocations (16.1%) was significantly lower than that for Robertsonian translocations (28.0%, P < 0.01) and inversions (28.0%, P < 0.01). However, higher percentages of embryos with de novo abnormalities were observed with Robertsonian translocations (23.3%, P < 0.01) and inversions (30.5%, P < 0.01) than with reciprocal translocations (11.6%). We demonstrated that NGS for PGT on day-3 embryos is an effective clinical application, particularly for patients with a diminished ovarian reserve and limited embryos.

Meiotic Heterogeneity of Trivalent Structure and Interchromosomal Effect in Blastocysts With Robertsonian Translocations

BackgroundRobertsonian translocations are common structural rearrangements and confer an increased genetic reproductive risk due to the formation of trivalent structure during meiosis. Studies on trivalent structure show meiotic heterogeneity between different translocation carriers, although the factors causing heterogeneity have not been well elaborated in blastocysts. It is also not yet known whether interchromosomal effect (ICE) phenomenon occurs in comparison with suitable non-translocation control patients. Herein, we aimed to evaluate the factors that cause meiotic heterogeneity of trivalent structure and the ICE phenomenon.MethodsWe designed a retrospective study, comprising 217 Robertsonian translocation carriers and 134 patients with the risk of transmitting monogenic inherited disorders (RTMIDs) that underwent preimplantation genetic testing (PGT). Data was collected between March 2014 and December 2019. The segregation products of trivalent structure were analyzed based on the carrier’s gender, age and translocation type. In addition, to analyze ICE phenomenon, aneuploidy abnormalities of non-translocation chromosomes from Robertsonian translocation carriers were compared with those from patients with RTMIDs.ResultsWe found that the percentage of male carriers with alternate segregation pattern was significantly higher [P < 0.001, odds ratio (OR) = 2.95] than that in female carriers, while the percentage of adjacent segregation pattern was lower (P < 0.001, OR = 0.33). By contrast, no difference was observed between young and older carriers when performing stratified analysis by age. Furthermore, segregation pattern was associated with the D;G chromosomes involved in Robertsonian translocation: the rate of alternate segregation pattern in Rob(13;14) carriers was significantly higher (P = 0.010, OR = 1.74) than that in Rob(14;21) carriers, whereas the rate of adjacent segregation pattern was lower (P = 0.032, OR = 0.63). Moreover, the results revealed that the trivalent structure could significantly increase the frequencies of chromosome aneuploidies 1.30 times in Robertsonian translocation carriers compared with patients with RTMIDs (P = 0.026), especially for the male and young subgroups (P = 0.030, OR = 1.35 and P = 0.012, OR = 1.40), while the mosaic aneuploidy abnormalities presented no statistical difference.ConclusionsOur study demonstrated that meiotic segregation heterogeneity of trivalent structure is associated with the carrier’s gender and translocation type, and it is independent of carrier’s age. ICE phenomenon exists during meiosis and then increases the frequencies of additional chromosome abnormalities.

Prevalence and Phenotypic Impact of Robertsonian Translocations

Robertsonian translocations (RTs) result from fusion of 2 acrocentric chromosomes (e.g., 13, 14, 15, 21, 22) and consequential losses of segments of the p arms containing 47S rDNA clusters and transcription factor binding sites. Depending on the position of the breakpoints, the size of these losses vary considerably between types of RTs. The prevalence of RTs in the general population is estimated to be around 1 per 800 individuals, making RTs the most common chromosomal rearrangement in healthy individuals. Based on their prevalence, RTs are classified as “common,” rob(13;14) and rob(14;21), or “rare” (the 8 remaining nonhomologous combinations). Carriers of RTs are at an increased risk for offspring with chromosomal imbalances or with uniparental disomy. RTs are generally regarded as phenotypically neutral, although, due to RTs formation, 2 of the 10 ribosomal rDNA gene clusters, several long noncoding RNAs, and in the case of RTs involving chromosome 21, several mRNA encoding genes are lost. Nevertheless, recent evidence indicates that RTs may have a significant phenotypic impact. In particular, rob(13;14) carriers have a significantly elevated risk for breast cancer. While RTs are easily spotted by routine karyotyping, they may go unnoticed if only array-CGH and NextGen sequencing methods are applied. This review first discusses possible molecular mechanisms underlying the particularly high rates of RT formation and their incidence in the general population, and second, likely causes for the elevated cancer risk of some RTs will be examined.

New Recommendations for Robertsonian Translocation Management After Preimplantation Genetic Testing of Structural Rearrangement (PGT-SR) Attempts: What Should Be Done With Mosaic Embryos?

Robertsonian translocation (RT) carriers are phenotypically normal, but they are known to be at increased risk of repeated miscarriages compared with the general population estimated at about 15% of pregnancies, and also resulting in the birth of a child with a mental retardation or congenital anomalies. Preimplantation Genetic Testing (PGT) is therefore a solution for RT carriers. An appropriate probe strategy allows to differentiate balanced embryos, unbalanced embryos, and mosaic embryos. We performed the first comparative analysis between two or three probes FISH strategies to analyze if a probe strategy choice for PGT-SR studies of Robertsonian translocations (RT) influences the fate of embryos? Our investigations present 13 years of experience of PGT for Robertsonian translocation carriers to improve the accuracy of abnormality detections. A deeper analysis of 283 PGT-SR attempts by comparing two strategies of probes highlighted the irrelevance of using a third probe for FISH diagnosis and above all a significant difference of mosaic embryo rates between probe strategies. These findings could be used as new recommendations of Robertsonian translocation management in many laboratories to improve their practices. It could be readily run, less expensive, reliable and accurate. Furthermore, the propounded strategy of mosaic embryo transfer should be considered after a detailed genetic counseling.

TBP-Related Factor 2 as a Trigger for Robertsonian Translocations and Speciation

Robertsonian (centric-fusion) translocation is the form of chromosomal translocation in which two long arms of acrocentric chromosomes are fused to form one metacentric. These translocations reduce the number of chromosomes while preserving existing genes and are considered to contribute to speciation. We asked whether hypomorphic mutations in genes that disrupt the formation of pericentromeric regions could lead to centric fusion. TBP-related factor 2 (Trf2) encodes an alternative general transcription factor. A decrease of TRF2 expression disrupts the structure of the pericentromeric regions and prevents their association into chromocenter. We revealed several centric fusions in two lines of Drosophila melanogaster with weak Trf2 alleles in genetic experiments. We performed an RNAi-mediated knock-down of Trf2 in Drosophila and S2 cells and demonstrated that Trf2 upregulates expression of D1—one of the major genes responsible for chromocenter formation and nuclear integrity in Drosophila. Our data, for the first time, indicate that Trf2 may be involved in transcription program responsible for structuring of pericentromeric regions and may contribute to new karyotypes formation in particular by promoting centric fusion. Insight into the molecular mechanisms of Trf2 function and its new targets in different tissues will contribute to our understanding of its phenomenon.