Whole-genome analysis of a putative rare and complex interchromosomal reciprocal insertion: thorough investigations for a straightforward interpretation.

RESEARCH QUESTION: Should whole-genome investigations be considered systematically before a complex chromosomal abnormality preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR) management is carried out using conventional cytogenetic techniques? DESIGN: A male carrying a putative rare interchromosomal reciprocal insertion (IRI) 46,XY,ins(14;?)(q11;?).ish der(14)ins(14;22)(q11.2;q11.2q11.2)(xcp14+,xcp22+,N25+,3'TRA/D+),der(22)ins(22;14)(q11.2;q11.2q11.2)(xcp22+,xcp14+,N25-,5'TRA/D+), and his partner were referred to our centre for preimplantation genetic testing analysis after three spontaneous miscarriages. Whole-genome sequencing was used to distinguish between the proposed IRI and an alternative explanation of reciprocal translocation. Fluorescence in-situ hybridization was used to detect all chromosome segments involved in this chromosomal rearrangement, to identify transferable normal and balanced embryos. RESULTS: Whole-genome sequencing allowed the determination of the number of chromosomal breakpoints involved in chromosomal rearrangement between chromosomes 14 and 22. Finally, only two breakpoints were identified instead of four in IRI rearrangements, which suggests a reciprocal translocation rearrangement. A probe strategy was established to highlight all chromosomal imbalances, whether IRI or reciprocal translocation, and preimplantation genetic testing cycles were achieved. CONCLUSION: Conventional cytogenetic techniques are not capable of identifying all complex chromosomal rearrangements, especially those involving centromeric regions and short arms of acrocentric chromosomes. The advent of new sequencing technologies has allowed for a better appreciation of genome complexity. In this study, whole-genome analysis provided additional information to explain the occurrence of genomic events and confirmed that the initial diagnosis of IRI identified by conventional cytogenetic techniques was, in fact, a simple reciprocal translocation. A reliable PGT-SR strategy was proposed for this couple to achieve their parental project.

Inheritance of imbalances in recurrent chromosomal translocation t(11;22): clarification by PGT-SR and sperm-FISH analysis.

RESEARCH QUESTION: To analyse why unbalanced viable offspring are derived mainly from the 3:1 segregation mode in t(11;22)(q23;q11.2) reciprocal translocation. DESIGN: Retrospective analysis of 24 pre-implantation genetic testing for chromosomal structural re-arrangements (PGT-SR) cycles was performed on seven male and five female carriers of t(11;22) translocation. Sperm analysis was performed on each male carrier. These patients were directed to the study centre after several years of miscarriages and/or abortions, primary infertility for male carriers or birth of an affected child. RESULTS: Twenty-four PGT-SR cycles were performed to exclude imbalances in both male and female carriers. The unbalanced embryos derived from the adjacent-1 segregation mode were the most represented in both male and female carriers (68.4% and 50%, respectively). These results were positively related with meiotic segregation analysis of reciprocal translocation in spermatozoa. A thorough analysis of the unbalanced embryo karyotypes determined that the expected viable +der22 karyotype resulting from 3:1 malsegregation was less represented at 5.3%. CONCLUSIONS: These findings highlight the divergence that may exist between meiotic segregation and post-zygotic selection. Post-zygotic selection would be responsible for the elimination of unbalanced embryos derived from the adjacent-1 segregation mode. The combined action of several factors occurs at the beginning of post-zygotic selection. Genetic counselling must consider the risk of a birth related to the adjacent-1 segregation mode, irrespective of the sex of the translocation carrier. These results will allow deeper understanding of the PGT results of t(11;22) carriers, which often include a high number of aneuploid embryos.

Is the resulting phenotype of an embryo with balanced X-autosome translocation, obtained by means of preimplantation genetic diagnosis, linked to the X inactivation pattern?

OBJECTIVE: To examine if a balanced female embryo with X-autosome translocation could, during its subsequent development, express an abnormal phenotype. DESIGN: Preimplantation genetic diagnosis (PGD) analysis on two female carriers with maternal inherited X-autosome translocations. SETTING: Infertility center and genetic laboratory in a public hospital. PATIENT(S): Two female patients carriers undergoing PGD for a balanced X-autosome translocations: patient 1 with 46,X,t(X;2)(q27;p15) and patient 2 with 46,X,t(X;22)(q28;q12.3). INTERVENTION(S): PGD for balanced X-autosome translocations. MAIN OUTCOME MEASURE(S): PGD outcomes, fluorescence in situ hybridization in biopsied embryos and meiotic segregation patterns analysis of embryos providing from X-autosome translocation carriers. RESULT(S): Controlled ovarian stimulation facilitated retrieval of a correct number of oocytes. One balanced embryo per patient was transferred and one developed, but the patient miscarried after 6 weeks of amenorrhea. In X-autosome translocation carriers, balanced Y-bearing embryos are most often phenotypically normal and viable. An ambiguous phenotype exists in balanced X-bearing embryos owing to the X inactivation mechanism. In 46,XX embryos issued from an alternate segregation, der(X) may be inactivated and partially spread transcriptional silencing into a translocated autosomal segment. Thus, the structural unbalanced genotype could be turned into a viable functional balanced one. It is relevant that a discontinuous silencing is observed with a partial and unpredictable inactivation of autosomal regions. Consequently, the resulting phenotype remains a mystery and is considered to be at risk of being an abnormal phenotype in the field of PGD. CONCLUSION(S): It is necessary to be cautious regarding to PGD management for this type of translocation, particularly in transferred female embryos.

Analysis using fish of sperm and embryos from two carriers of rare rob(13;21) and rob(15;22) robertsonian translocation undergoing PGD.

The majority of fluorescence in situ hybridization (FISH) studies on the meiotic segregation of Robertsonian translocations focus on the most common types, rob(13; 14) and rob(14; 21). Here we report the first study for carriers of rare Robertsonian translocations rob(13; 21) and rob(15; 22) combining analysis of meiotic segregation in sperm and blastomeres following pre-implantation genetic diagnosis (PGD). Dual-colour FISH was applied to nuclei from spermatozoa and blastomeres from PGD embryos using two subterminal contig probes for each translocation, and a second round with probes for chromosomes 16 and 18. Patient 1 had a rob(13; 21) and patient 2 had a rob(15; 22), and 86.3% and 87.5% of gametes respectively were consistent with meiotic segregation resulting in a normal or balanced chromosome complement. Analysis of embryos showed that for patient 1 and 2 respectively, 25% and 46% were balanced, and of the unbalanced embryos, 50% and 31% were mosaic or chaotic. Our patients with a rob(13; 21) and rob(15; 22) were found to have a similar meiotic segregation pattern to that for male carriers of the common Robertsonian translocations. The observed rate in unbalanced embryos being mosaic or chaotic may result in an increased risk of chromosomal abnormalities. Our results may help to improve the genetic counseling for carriers of rare Robertsonian translocations.

Characterization of IGH rearrangements in non-Hodgkin's B-cell lymphomas by fluorescence in situ hybridization.

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin's B-cell lymphomas (NHL) and correlated to clinical relevant subgroups. However, the detection rate varied greatly with the technique used. The incidence of IGH rearrangements was analyzed using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 57 patients with nodal NHL. An IGH rearrangement was identified in 42 cases (73.7%). A t(14;18)(q32;q21) was found in 17 of the 20 follicular lymphomas (85%) studied and a t(11;14)(q13;q32) in 10 of the 11 mantle cell lymphomas (91%). IGH rearrangements were identified in 12 of the 26 diffuse large B-cell lymphomas (46%), including 5 t(14;18)(q32;q21) and 2 t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using Multi-FISH and/or chromosomal whole paint enabled the characterization of complex IGH translocations in follicular lymphomas and mantle cell lymphomas and the identification of all the chromosomal partners involved in the IGH rearrangement in diffuse large B-cell lymphomas. This study shows the interest of using metaphase FISH in addition to conventional cytogenetics. Following banding techniques, FISH with the IGH dual color probe could be the first approach in NHL, after which chromosome painting and M-FISH could be used to identify the chromosomal partner involved in the IGH rearrangement.

An increased incidence of autosomal aneuploidies in spermatozoa from a patient with Klinefelter's syndrome.

OBJECTIVE: To determine whether there is an increased incidence of autosomal aneuploidies in spermatozoa from a subject with nonmosaic Klinefelter's syndrome. DESIGN: Analysis of sperm nuclei by fluorescence in situ hybridization. SETTINGS: Hospital-based laboratory for reproductive biology. PATIENT(S): A patient with Klinefelter's syndrome and a 46,XY fertile man were analyzed. INTERVENTION(S): The sperm samples were prepared for fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): The disomy frequencies for chromosomes 7, 9, 13, 18, and 21 and sex chromosomes were determined using fluorescence in situ hybridization. RESULT(S): Significant differences were found in the frequency of disomy for chromosomes 13, 18, and 21 between the patient and a normospermic control. No significant differences were observed for chromosomes 7 and 9. The frequencies of gonosomal abnormalities and diploid spermatozoa were also significantly increased in the patient. CONCLUSION(S): Our results indicate that there is an increased incidence of autosomal aneuploidies for chromosomes 13, 18, and 21 in the spermatozoa from the patient with Klinefelter's syndrome than in the general population. Furthermore, most of the studies have also shown an increased frequency of autosomal aneuploidy in the spermatozoa from 46,XY males with oligozoospermia or oligoasthenoteratozoospermia. Thus, the offspring of the patients with Klinefelter's born by intracytoplasmic sperm injection may be at higher risk of autosomal aneuploidy due to oligoasthenoteratozoospermia.