Balanced Translocation Carriers Research Articles

Do female translocations influence the ovarian response pattern to controlled ovarian stimulation in preimplantation genetic diagnosis?

Ovarian response in female translocation carriers is not well understood. We aimed to evaluate the impact of chromosomal autosomal balanced translocations on the ovarian response to controlled ovarian stimulation (COS) in female carriers undergoing IVF and PGD. In a retrospective study, we included all female translocation carriers who underwent PGD at our centre. We compared these patients to female patients from couples with male translocation carriers who underwent PGD. Results from 79 cycles of PGD from 33 female translocation carriers were compared with 116 cycles from 55 male translocation carriers. No difference was observed for patient characteristics: female age, anti-Müllerian hormone or antral follicle count. No difference in COS parameters was observed for the total dose of recombinant FSH, the number of retrieved oocytes and embryos on Day 3, for unaffected and transferred embryos. For the two groups, pregnancy rate was similar per cycle (12.7 versus 20.7%, P = 0.208). Multivariate analysis demonstrated that female translocation carriers had a significantly higher estradiol level on the day of hCG administration (+540 pg/ml, P = 0.05). This paper is the largest to report ovarian response of female translocation carriers. This study showed that the ovarian response to COS was not impaired by balanced translocation status, suggesting that female chromosomal structural abnormalities did not influence the results of COS in PGD. Thus, female carriers of balanced translocations could be considered normal responders and standard doses of gonadotrophins used for ovarian stimulation.

Karyotyping couples experiencing recurrent miscarriage really is worth the cost

Sir, We read with interest the retrospective study analysing the cost–benefit of karyotyping in recurrent miscarriage.1 We are concerned that the findings of a predicted cost of between £817,280 and £1,021,600 per unbalanced pregnancy detected will lead to a reduction in the karyotyping service for those experiencing recurrent miscarriage. As stated by the authors, they have not taken into account alternative pregnancy outcomes in those couples found to have a balanced translocation after testing for recurrent miscarriage. They state that owing to the large population tested, this is unlikely to have skewed the results. However, we beg to differ—they have not factored in the potentially large proportion of those who were found to be balanced translocation carriers who subsequently avoided pregnancy, had a miscarriage before prenatal diagnosis or became pregnant but declined prenatal diagnosis. Added to this, some carrier couples may well have chosen preimplantation genetic diagnosis or opted for ovum or sperm donation, none of which will have been captured by their data. If this is the case, the figure of between £817,280 and £1,021,600 per unbalanced pregnancy detected is inaccurate. Would it not have been more prudent to calculate the cost of karyotyping based on the 406 pregnancies where a balanced translocation was found? This would produce a figure of £8052–£10,065 per balanced translocation detected, which would better reflect the yield of positive result in karyotyping for those with recurrent miscarriage. It is important also to note that the finding of a normal or abnormal karyotype would act as a prognostic indicator in further attempted pregnancies, and if karyotyping were to be restricted according to the suggestions in the article, then such information would not be available to counsel regarding prognosis in future pregnancies. We would also point out that the emotional impact of knowing a couple do not carry a chromosomal abnormality far outweighs the cost of karyotyping in such a distressing condition as recurrent miscarriage, which is well-known to be associated with anxiety and depression.2 Therefore it would be wrong to conclude that karyotyping is only valuable in those pregnancies that yield a positive result—rather it is of great benefit to have reassurance from a finding of being chromosomally normal.

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier’s embryos- preliminary observations of two robertsonian translocation carrier families

Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

Rapid mapping of chromosomal breakpoints: from blood to BAC in 20 days.

Structural chromosome aberrations and associated segmental or chromosomal aneusomies are major causes of reproductive failure in humans. Despite the fact that carriers of reciprocal balanced translocation often have no other clinical symptoms or disease, impaired chromosome homologue pairing in meiosis and karyokinesis errors lead to over-representation of translocations carriers in the infertile population and in recurrent pregnancy loss patients. At present, clinicians have no means to select healthy germ cells or balanced zygotes in vivo, but in vitro fertilization (IVF) followed by preimplantation genetic diagnosis (PGD) offers translocation carriers a chance to select balanced or normal embryos for transfer. Although a combination of telomeric and centromeric probes can differentiate embryos that are unbalanced from normal or unbalanced ones, a seemingly random position of breakpoints in these IVF-patients poses a serious obstacle to differentiating between normal and balanced embryos, which for most translocation couples, is desirable. Using a carrier with reciprocal translocation t(4;13) as an example, we describe our state-of-the-art approach to the preparation of patient-specific DNA probes that span or 'extent' the breakpoints. With the techniques and resources described here, most breakpoints can be accurately mapped in a matter of days using carrier lymphocytes, and a few extra days are allowed for PGD-probe optimization. The optimized probes will then be suitable for interphase cell analysis, a prerequisite for PGD since blastomeres are biopsied from normally growing day 3--embryos regardless of their position in the mitotic cell cycle. Furthermore, routine application of these rapid methods should make PGD even more affordable for translocation carriers enrolled in IVF programs.

Cytogenetic analysis of 1483 patients with spontaneous abortion

Objective Conducting the genetic analysis of the patients with spontaneous abortion with karyotyping.Method In 2008, there were 4010 patients conducted karyotyping, and 1438 of them have the case history of spontaneous abortion. Result In the 1483 patients, we found 32balanced translo-cation carrier.Conclusion Conducting the karyotyping analysis of the patients with spontaneous abortion may give guidance and instruction to the eugenics,and can prevent the delivery of hereditary disease to the offspring. Key words: Genetics; Karyotyping; Spontaneous abortion; Balanced translocation carrier

A study of meiotic segregation of chromosomes in spermatozoa of translocation carriers using fluorescentin situhybridisation

In the infertile male population, there is a 2-20-time higher probability of having a structural chromosomal abnormality than in general population. Generally, these men have a normal phenotype but they can have sperm abnormalities. As they can produce a variable proportion of unbalanced gametes, it is important to evaluate the percentage of unbalanced chromosomal spermatozoa to assess the risk of injecting a chromosomally unbalanced gamete during ICSI procedure. We report here the meiotic segregation analysis of chromosomes in spermatozoa of 12 men with a balanced reciprocal translocation and 4 men with a Robertsonian translocation using a fluorescent in situ hybridisation analysis. The frequencies of normal or balanced spermatozoa ranged from 34.4% to 49.1% in balanced reciprocal translocation carriers. For Robertsonian translocation, the frequencies of normal or balanced spermatozoa ranged from 78.4% to 91.2%. These analyses allow us to define the orientation of genetic counselling according to the results of meiotic segregation obtained. As a last resort, it could then be discussed of the possibility of having recourse to donor spermatozoa or adoption.

Monozygotic multiple gestation after intracytoplasmic sperm injection and preimplantation genetic diagnosis

To report a possible association between intracytoplasmic sperm injection (ICSI)-preimplantation genetic diagnosis (PGD) and monozygotic multiple gestation. Small case series. In vitro fertilization unit in an academic medical center. Three patients were treated with ICSI-PGD for sexing as well as selection against a known translocation. Transfer of day 4 embryos to the uterus. Clinical pregnancy. Two pairs of monozygotic twins and a triplet pregnancy. Repeated manipulation of the zona pellucida as well as extended embryo culture during ICSI-PGD treatments may result in monozygotic twin and triplet pregnancies.

Inherited 14q duplication and 21q deletion: A rare adjacent‐2 segregation in multiple family members

We present a family with multiple carriers of a subtle balanced translocation t(14;21)(q21.2;q21.2) and three patients with a resultant adjacent-2 malsegregation containing a +der(14)t(14;21)(q21.2;q21.2),-21 in their chromosome complement. The initial study was performed when a 2-month-old female was referred to genetics clinic for evaluation of developmental delay, growth retardation, and failure to thrive. Physical findings included prominent eyes, micrognathia, prominent and simple external ears, camptodactyly, contractures of the wrists, ankles, and hips, hypoplasia of the corpus callosum, prominent atria and occipital horns, cerebellopontine hypoplasia; and small atrial septal defect. High resolution chromosomes showed an extra band on the proximal 21q and fluorescence in situ hybridization (FISH) demonstrated only one signal for the centromere of 21. Karyotypes of the parents and grandparents revealed that the mother and maternal grandfather were carriers of a balanced translocation, and the propositus contained an unbalanced chromosome complement with partial duplication of proximal 14q and partial deletion of proximal 21q. Investigations performed on an institutionalized maternal aunt revealed identical karyotypic abnormalities as in the propositus. More recently, array comparative genomic hybridization (aCGH) on a subsequent child with multiple congenital anomalies further out in the extended family allowed for more accurate identification of the breakpoints. Our investigation includes analysis on a total of 11 family members spanning three generations. Among those investigated, there were no living members with other possible consequential unbalanced translocations or with adjacent-2 segregation resulting in -14,+der(21). Chromosome rearrangements require FISH and aCGH studies for accurate identification and elucidation of the abnormality and breakpoints.

Development and validation of comprehensive triple factor PGD: simultaneous 24 chromosome aneuploidy, translocation derivative, and single gene disorder screening from a single biopsy

OBJECTIVE: A patient with a balanced translocation and Alagille syndrome 46,XX, t(2:20)(q21;p12.2) presented for PGD. She had a 2.36 MB deletion including the JAG1 locus near the translocation. PGD for this case needs to distinguish normal embryos from those with balanced translocations since the latter would still have Alagille syndrome. The goal of this study was to develop a methodology which allows simultaneous screening for translocation derivatives, the deletion related to the single gene disorder, and 24 chromosome aneuploidy screening (AS). DESIGN: Prospective. MATERIALS AND METHODS: Microarray (MA) based 24-chromosome AS was done using a validated protocol. Using this methodology, 121 discarded embryos from 10 patients with various reciprocal balanced translocations were studied and compared to FISH balanced translocation screening results. The ability to accurately determine single gene disorder inheritance was established by evaluating single cells from cell lines and affected embryos from individuals with single gene disorders. Following validation, triple factor PGD was applied to the patient with Alagille syndrome. RESULTS: Trophectoderm (TE) biopsies were obtained from 6 blasts. Half of the TE biopsy was evaluated by FISH for the microdeletion, translocations, and aneuploidy of chromosome 18, X, and Y. The remaining TE was evaluated by Q-PCR for the microdeletion, and by MA for the microdeletion, translocations, and 24-chromosome aneuploidy. 2 of the blastocysts were diagnosed as carriers of the maternal balanced translocation and affected by Alagille syndrome, and 4 were unbalanced or had aneuploidy for one or more chromosomes. Q- PCR, MA, and FISH based microdeletion, translocation, and aneuploidy analyses were 100% consistent. CONCLUSIONS: Despite a lack of unaffected euploid embryos available for transfer, the patient has initiated another treatment cycle. This technology represents the only validated method available for comprehensive triple factor PGD.

A new assay system for distinction between a balanced translocation and a normal karyotype in preimplantation genetic diagnosis (PGD)

OBJECTIVE: Using the widely accepted present FISH method for PGD is impossible to distinguish completely between a normal karyotype and a balanced translocation. Therefore, theoretically half of the offspring inherit the translocation from their infertile parent. In this study, we developed a new assay system for PGD of the carriers of balanced translocations.DESIGN: Cytogenetic analysis of biopsied blastomeres at the metaphase after electrical fusion with human IVM oocytes.MATERIALS AND METHODS: After providing informed consent, morphologically excellent 3-day-old human embryos (8-cell stage after IVF), along with immature GV oocytes, were donated by our clinic's patients for this study. The immature GV oocytes were cultured overnight until the first polarbody was extruded. The 3-day-old embryo and IVM oocytes were freed from the zona-pellucida with a 0.3% tripsin solution. The embryos were then placed in PBS (without Ca and Mg ions) to separate the blastomeres. Each separated blastomere and IVM oocyte were bonded together in 0.03% phytohemagglutinin-P solution and then electrically stimulated (AC 15V, 2 sec and DC 1000V/cm, 50 μsec) in 295mM D-mannitol containing PVP, Ca+ and Mg+. One hour after stimulation, each hybrid cell was treated with hypotonic solution (30% fetal bovine serum) for 20 min, and then affixed to a chromosome slide using the gradual fixation-air drying method. Chromosome analysis was performed after 2% Giemsa solution staining.RESULTS: 22 human blastomeres and 22 IVM oocytes successfully combined through electrical stimulation to create 20 hybrid cells. Of these 20 hybrid cells 3 were unsuccessful, 13 contained 1 complete set of clearly arranged meiotic chromosomes (n=23) that were derived from the oocyte.CONCLUSIONS: This assay system has the advantage that it only takes 2 hours from biopsy to chromosome analysis. In this study, the success rate for chromosome analysis was as high as 65.0% (13/20) when the hybrid cells were prepared for chromosome slides one hour after electro-fusion. OBJECTIVE: Using the widely accepted present FISH method for PGD is impossible to distinguish completely between a normal karyotype and a balanced translocation. Therefore, theoretically half of the offspring inherit the translocation from their infertile parent. In this study, we developed a new assay system for PGD of the carriers of balanced translocations. DESIGN: Cytogenetic analysis of biopsied blastomeres at the metaphase after electrical fusion with human IVM oocytes. MATERIALS AND METHODS: After providing informed consent, morphologically excellent 3-day-old human embryos (8-cell stage after IVF), along with immature GV oocytes, were donated by our clinic's patients for this study. The immature GV oocytes were cultured overnight until the first polarbody was extruded. The 3-day-old embryo and IVM oocytes were freed from the zona-pellucida with a 0.3% tripsin solution. The embryos were then placed in PBS (without Ca and Mg ions) to separate the blastomeres. Each separated blastomere and IVM oocyte were bonded together in 0.03% phytohemagglutinin-P solution and then electrically stimulated (AC 15V, 2 sec and DC 1000V/cm, 50 μsec) in 295mM D-mannitol containing PVP, Ca+ and Mg+. One hour after stimulation, each hybrid cell was treated with hypotonic solution (30% fetal bovine serum) for 20 min, and then affixed to a chromosome slide using the gradual fixation-air drying method. Chromosome analysis was performed after 2% Giemsa solution staining. RESULTS: 22 human blastomeres and 22 IVM oocytes successfully combined through electrical stimulation to create 20 hybrid cells. Of these 20 hybrid cells 3 were unsuccessful, 13 contained 1 complete set of clearly arranged meiotic chromosomes (n=23) that were derived from the oocyte. CONCLUSIONS: This assay system has the advantage that it only takes 2 hours from biopsy to chromosome analysis. In this study, the success rate for chromosome analysis was as high as 65.0% (13/20) when the hybrid cells were prepared for chromosome slides one hour after electro-fusion.

Cardiac Tamponade: An Alternative Procedure for Late Feticide

Improvements in equipment and diagnostic skills mean that more abnormalities can now be detected antenatally, thus increasing the demand for pregnancy termination at later stages of gestation. Potassium chloride injected into the fetal circulation is the most frequently used procedure. In this study, we propose a new method of feticide using injection of normal saline into the fetal pericardial space to induce cardiac tamponade, resulting in late fetal reduction with minimal maternal risk. A 32-year-old, gravida 6, woman was a carrier of a balanced translocation 45,XX,der(2),t(2,22), and had a poor obstetric history. Chorionic villus sampling was performed, and the results revealed the fetus to be a balanced translocation carrier, like the mother. Microcephaly was detected during the third trimester and amniocentesis was performed. The results showed a 45,XX,der(2)t(2;22)(q37,q11.2)mat,-22 karyotype. After counseling, the mother elected to undergo pregnancy termination. Feticide was performed before labor induction. However, she experienced discomfort when a minimal amount of potassium chloride (3 mEq) was injected into the fetal heart, without inducing fetal asystole. We, therefore, induced cardiac tamponade using 10 mL of normal saline instilled into the fetal pericardial space. Fetal asystole was noted. The whole procedure was performed without incident, and termination was achieved by hysterotomy after informed consent was given. Many methods of feticide have been used, including injection of potassium chloride, lidocaine, digoxin or hyperosmolar urea into the fetal circulation. However, these methods all use medications or compounds with some potential for maternal toxicity or side effects. We provide a relatively safe and effective method of feticide for use in late termination of pregnancy.

Clinical and molecular cytogenetic characterization of four patients with unbalanced translocation der(1)t(1;22)(p36;q13)

Deletion of chromosome 1p36 is the most commonly observed terminal deletion in humans with a frequency of 1 in 5,000 in the general population. In contrast, 22q13 duplications are rare and only a few cases have been reported. Unbalanced translocations resulting in monosomy 1p36 and a trisomy of 22q13.3 are, thus far, unreported in the literature. Here we present the clinical data and the results of array CGH and FISH analysis of four patients with unbalanced translocations t(1;22)(p36;q13) inherited from unrelated balanced translocation carrier parents. The sizes of the imbalances ranged from 0.12 Mb to nearly 10 Mb. One balanced translocation carrier parent had disruption of the period homolog 3 (PER3) gene and reported sleep disturbances. Overall, patients tended to have more features consistent with deletion of 1p36 than duplication of 22q.

Unexpected complexity at breakpoint junctions in phenotypically normal individuals and mechanisms involved in generating balanced translocations t(1;22)(p36;q13)

Approximately one in 500 individuals carries a reciprocal translocation. Balanced translocations are usually associated with a normal phenotype unless the translocation breakpoints disrupt a gene(s) or cause a position effect. We investigated breakpoint junctions at the sequence level in phenotypically normal balanced translocation carriers. Eight breakpoint junctions derived from four nonrelated subjects with apparently balanced translocation t(1;22)(p36;q13) were examined. Additions of nucleotides, deletions, duplications, and a triplication identified at the breakpoints demonstrate high complexity at the breakpoint junctions and indicate involvement of multiple mechanisms in the DNA breakage and repair process during translocation formation. Possible detailed nonhomologous end-joining scenarios for t(1;22) cases are presented. We propose that cryptic imbalances in phenotypically normal, balanced translocation carriers may be more common than currently appreciated.

Breakpoint Mapping and Array CGH in Translocations: Comparison of a Phenotypically Normal and an Abnormal Cohort

We report the analyses of breakpoints in 31 phenotypically normal and 14 abnormal carriers of balanced translocations. Our study assesses the differences between balanced translocations in normal carriers and those in abnormal carriers, focusing on the presence of genomic imbalances at the breakpoints or elsewhere in the genome, presence of cryptic chromosome rearrangements, and gene disruption. Our hypothesis is that all four features will be associated with phenotypic abnormalities and absent or much less frequent in a normal population. In the normal cohort, we identified neither genomic imbalances at the breakpoints or elsewhere in the genome nor cryptic chromosome rearrangements. In contrast, we identified candidate disease-causing imbalances in 4/14 abnormal patients. These were three breakpoint associated deletions and three deletions unrelated to the breakpoints. All six de novo deletions originated on the paternally inherited chromosome. Additional complexity was also present in one of these cases. Gene disruption by the breakpoints was present in 16/31 phenotypically normal individuals and in 5/14 phenotypically abnormal patients. Our results show that translocations in phenotypically abnormal patients are molecularly distinct from those in normal individuals: the former are more likely to be associated with genomic imbalances at the breakpoints or elsewhere and with chromosomal complexity, whereas the frequency of gene disruption is similar in both normal and abnormal translocation carriers.

The clinical phenotype of the derivative (8)t(7;8)(q22;p23.3) in two siblings

We report on 2 siblings with a partial trisomy of 7q (7q22→qter) and concomitant partial monosomy of 8p (8p23.3→pter), which were shown by FISH using probes located at the telomere region of each chromosome. All the balanced translocation carriers (father and a sister) in this family had a normal phenotype. The 2 siblings with the same abnormal karyotype had similar multiple congenital anomalies and dysmorphic features. During the follow-up, the first male patient died in the neonatal period, but the female sibling is still alive at 2 years and 6 months of age.

Forty-eight new cases with infertility due to balanced chromosomal rearrangements: Detailed molecular cytogenetic analysis of the 90 involved breakpoints

A molecular cytogenetic study was performed on 48 infertile patients who were identified as carriers of balanced translocations (40 cases), inversions (6 cases) or insertions (2 cases) by means of banding cytogenetics. Cases with a Robertsonian translocation or pericentric inversion 2 or 9 were not included. In summary, 100 break-events occurred in these patients, and 90 different chromosomal regions were involved. Thus, this study confirmed the presence of abnormal karyotypes in a subgroup of patients seeking infertility treatment. Breaks were demonstrated to appear preferentially in GTG-light bands in these patients. Furthermore, the observed breakpoints were associated with genomic regions prone to instability due to the presence of segmental duplications. Nonetheless, further detailed molecular analysis will be necessary in the future to characterize the mechanisms and genetic basis for this phenomenon.

Identification of cryptic imbalance in phenotypically normal and abnormal translocation carriers

Approximately one in 500 individuals carries a reciprocal translocation. Of the 121 monosomy 1p36 subjects ascertained by our laboratory, three independent cases involved unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34). These derivative chromosomes were inherited from balanced translocation carrier parents. To understand better the causes and consequences of chromosome breakage and rearrangement in the human genome, we characterized each derivative chromosome at the DNA sequence level and identified the junctions between 1p36 and 9q34. The breakpoint regions were unique in all individuals. Insertions and duplications were identified in two balanced translocation carrier parents and their unbalanced offspring. Sequence analyses revealed that the translocation breakpoints disrupted genes. This study demonstrates that apparently balanced reciprocal translocations in phenotypically normal carriers may have cryptic imbalance at the breakpoints. Because disrupted genes were identified in the phenotypically normal translocation carriers, caution should be exercised when interpreting data on phenotypically abnormal carriers with apparently balanced rearrangements that disrupt putative candidate genes.

Systematic re-examination of carriers of balanced reciprocal translocations: a strategy to search for candidate regions for common and complex diseases

Balanced reciprocal translocations associated with genetic disorders have facilitated the identification of a variety of genes for early-onset monogenic disorders, but only rarely the genes associated with common and complex disorders. To assess the potential of chromosomal breakpoints associated with common/ complex disorders, we investigated the full spectrum of diseases in 731 carriers of balanced reciprocal translocations without known early-onset disorders in a nation-wide questionnaire-based re-examination. In 42 families, one of the breakpoints at the cytogenetic level concurred with known linkage data and/or the translocation co-segregated with the reported phenotype, for example, we found a significant linkage (lod score=2.1) of dyslexia and a co-segregating translocation with a breakpoint in a previously confirmed locus for dyslexia. Furthermore, we identified 441 instances of at least two unrelated carriers with concordant breakpoints and traits. If applied to other populations, re-examination of translocation carriers may identify additional genotype-phenotype associations, some of which may be novel and others that may coincide with and provide additional support of data presented here.

The Importance of Family History Risk Assessment in the Infertility Setting

The Importance of Family History Risk Assessment in the Infertility Setting

Robertsonian and Reciprocal Translocations: Is Preimplantation Genetic Diagnosis (PGD) a Realistic Treatment Option?

In recent years, preimplantation genetic diagnosis (PGD) has been offered to carriers of balanced translocations as an alternative to prenatal diagnosis and pregnancy termination of unbalanced foetuses. However, the rate of expected unbalanced embryos is high in translocations: approximatively 60% for robertsonian translocations (ROT) and 70% for reciprocal translocations (RET). We report the results of PDG cycles for translocations at our institution, in order to determine whether or not the prognosis was acceptable to initiate a particularly expensive and heavy treatment for couples.