Balanced complex chromosome rearrangement in male infertility: case report and literature review.

Complex chromosome rearrangements (CCRs) are structural rearrangements involving at least three chromosomes and three or more chromosome breakpoints. Generally, balanced CCR carriers have a normal phenotype but they are at a higher reproductive risk. Azoospermia was discovered in the male partner of a couple with primary infertility. Conventional cytogenetics identified a CCR refined by fluorescent in situ hybridisation. The CCR involved three chromosomes, four breakpoints and an insertion. A literature search identified 43 phenotypically normal males referred for reproductive problems presenting a CCR. More males were ascertained because of spermatogenesis failure or disturbances than because of repeated abortions and/or birth of a malformed child. Male carriers of CCR produce a high frequency of chromosomally abnormal spermatozoa due to the aberrant segregation of the rearranged chromosomes. The number of chromosomes and breakpoints involved in the rearrangement, the position of breakpoints, the relative size of the resultant chromosomes and the presence or absence of recombination inside the paired-rearranged segments are presumed to affect the fertility of the carrier. Testicular biopsy should not be performed in males with azoospermia. Intracytoplasmic sperm injection should not be proposed as a procedure for treating the infertility of CCR male carriers as a successful result is unlikely.

Aneuploidy and DNA fragmentation in sperm of carriers of a constitutional chromosomal abnormality.

Among various causes responsible for infertility, it has been admitted for a long time that male infertility can be due to impaired spermatogenesis and/or balanced structural chromosomal abnormalities. Sperm DNA fragmentation is also considered as another cause of infertility. Most of the studies on male infertility have concerned either aneuploidy in the sperm of carriers of constitutional chromosomal abnormalities or sperm DNA fragmentation. This review is aimed at analyzing these 2 parameters in the same patients. Furthermore, we present work on the study of these 2 parameters in the same gametes of 4 carriers of a balanced chromosomal abnormality. Meiotic segregation was analyzed by fluorescent in situ hybridization and DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. It was shown that aneuploidy and DNA fragmentation were increased in the sperm of carriers of a balanced chromosomal abnormality. For all 4 carriers of a balanced structural abnormality, there was a 2-5 times higher proportion of spermatozoa with unbalanced chromosomal content and fragmented DNA than among those with normal/balanced content. Moreover, we found a non-random distribution with more gametes with DNA fragmentation when these arose from a particular segregation mode. The mechanism which would tend to explain our results is abortive apoptosis. In conclusion, both meiotic segregation and DNA fragmentation studies should be integrated in the genetic exploration of male carriers of a chromosomal structural abnormality.

A study of meiotic segregation of chromosomes in spermatozoa of translocation carriers using fluorescent in situ hybridisation.

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.

Increased aneuploidy rates in spermatozoa of a male carrier of a trisomy 18 mosaicism.

Semen analysis of a 31-year-old infertile man showed a severe oligoteratozoospermia. Karyotyping of peripheral blood lymphocytes showed a 47,XY,+18[13]/46,XY[16] mosaicism. Cultured skin fibroblasts, right and left jugal smears showed 3, 50 and 65% trisomic cells respectively. The aim of the study was to evaluate the aneuploidy rates of chromosomes X, Y, 13, 18 and 21 and the diploidy rate in his spermatozoa by fluorescence in situ hybridization. The rate of disomy 18 was significantly increased in the spermatozoa of the patient (0.68%) compared to the control group (0.06%). A statistically significant difference in the rates of disomy for chromosome 13 (0.46% vs. 0.14%) and the gonosomes (0.78% vs. 0.24%) and diploidy (0.93% vs. 0.34%) was also found between the patient and the control group. However, no significant difference was observed for chromosome 21 (0.34% vs. 0.15%). Our results show evidence of a generalized perturbation of the meiotic mechanism that could lead to an increased risk for a mosaic trisomy 18 infertile male of producing offspring with aneuploidy that is not only on account of the father's mosaicism, but also more particularly because of severe oligoteratozoospermia.

Fluorescence in situ hybridization characterization of ider(20q) in myelodysplastic syndrome.

Isochromosome of the long arm of chromosome 20 with loss of interstitial material [ider(20q)] is a variant of deletion of chromosome 20q and a rare abnormality in myelodysplastic syndrome (MDS). We studied seven cases with an ider(20q) in MDS. Fluorescence in situ hybridization (FISH) studies showed all proximal breakpoints to be consistently located in 20q11.21 band whereas distal breakpoints were variable. Amplification of HCK, TNFRSF6B and DIDO1 genes included in retained regions associated with loss of tumour suppressor genes in deleted regions could explain cell tumour progression and possibly the less favourable prognosis of ider(20q) compared with del(20q).

Segregation of chromosomes in sperm of a t(X;18)(q11;p11.1) carrier inherited from his mother: case report.

Balanced reciprocal translocations are the most common structural abnormalities; most involve two autosomes while a few involve a gonosome (X or Y chromosome) and an autosome. These rearrangements are usually associated with infertility and/or a higher risk of chromosomal imbalances among offspring. This 26 years old man was first seen because of a 3-year history of primary infertility. He had been found to have a translocation, t(X;18)(q11;p11.1), inherited from his mother when he was 9 years old. Semen analysis showed a very severe oligoasthenoteratozoospermia (OAT). A total of 447 spermatozoa were analysed using three-colour fluorescent in situ hybridization (FISH). The alternate segregation pattern, leading to a normal or balanced chromosomal content, was found in 54.36% of the spermatozoa studied. The frequencies of Adjacent I, Adjacent II, 3:1 segregation and diploidy (or 4:0 segregation) were 8.28, 5.14, 22.37 and 2.01%, respectively. Balanced reciprocal translocations between an autosome and the X chromosome lead to important disruptions in human spermatogenesis. Almost all the males with an X-autosome translocation have azoospermia. The man reported here had very severe OAT and is the first in whom the meiotic segregation pattern was analysed. This case further emphasizes the interest in performing FISH studies in infertile males with a chromosomal translocation to provide them with a personalized imbalance risk.

Molecular cytogenetics of IGH rearrangements in non-Hodgkin B-cell lymphoma.

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin B-cell lymphomas (NHLs) and correlated to clinically relevant subgroups. However, the detection rate largely varied with the technique used. We analyzed the incidence of IGH rearrangements using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 96 patients with nodal NHL. An IGH rearrangement was identified in 71 cases (74%). A t(14;18)(q32;q21) was found in 37 of the 42 follicular lymphomas (88.1%) studied and a t(11;14)(q13;q32) in 12 of the 14 mantle cell lymphomas (85.7%). IGH rearrangements were identified in 21 of the 40 diffuse large B-cell lymphomas (52.5%), including seven t(14;18)(q32;q21) and four t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using 24-color FISH, chromosomal whole paints, telomeric probes and locus specific identifiers enabled us to characterize complex and/or masked IGH translocations in follicular lymphomas and mantle cell lymphomas and to identify all the chromosomal partners involved in IGH rearrangements 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 can be the first approach in NHL, after which chromosome painting and 24-color FISH can be used to identify the chromosomal partners involved in IGH rearrangements. The identification of these genes is of utmost importance for a better understanding of the molecular mechanisms involved in the genesis of lymphoma.

Characterisation of supernumerary chromosomal markers: a study of 13 cases.

Marker chromosomes are defined as 'structurally abnormal chromosomes in which no part can be identified' (ISCN 1995). Supernumerary marker chromosomes (SMC) are 'additional markers' whose origin and composition cannot be determined by conventional cytogenetics. Molecular cytogenetic methods are necessary to identify these additional chromosomal markers. In one third, the SMCs are clinically well-defined in the literature, the remaining two thirds present a major problem for genetic counselling in prenatal diagnosis. At present, different molecular cytogenetic methods are used to determine the origin of SMCs. In this work, we studied 13 SMCs detected by RHG-banding, completed by C-banding and/or NOR-staining. 24-color FISH was used as the primary technique when the chromosomal origin was unknown. Targeted FISH procedures with specific probes (whole chromosome painting, centromeric probe, locus-specific identifier, BAC, etc.) were then performed to confirm and/or specify the chromosomal material present in the SMC. Seven SMCs were found to be associated with phenotypic abnormalities. Five derived from autosomes and two from gonosomes; these are: der(12)t(4;12), dic(15), i(18p), r(19), der(22)t(11;22), r(X), and der(Y). Two markers, r(8) and idic(15), were identified during investigations of infertile couples. Three cases seemed to be phenotypically normal. Four were discovered prenatally: r(2) and r(19) referred for elevated maternal serum markers, der(13/21) referred for advanced maternal age. The fourth SMC, der(14/22), was found during familial investigation following the identification of the same marker in an infertile son. The precise characterisation of the SMCs is of utmost importance for genetic counselling, especially in prenatal diagnosis.

Prenatal diagnosis of a mosaic 45,X/46,X,r(X)46,XX with a small ring of the X chromosome.

We report the prenatal diagnosis of a mosaic 45,X/46,X,r(X)/46,XX foetus after amniocentesis for maternal second-trimester serum screening. Biparental contribution for the X chromosomes suggest the postzygotic formation of the X ring. The ring is tiny but contains the X-inactive-specific transcript gene (XIST). However, we could not determine whether XIST was correctly expressed or not. The foetal ultrasound examination at 21, 25, 31 weeks' gestation showed no physical abnormalities, prompting the parents to continue the pregnancy. Physical examination at one year-old revealed normal growth and psychomotor development. Only three cases exhibiting an identical 45,X/46,X,r(X)/46,XX mosaicism, but detected postnatally, are reported in the literature. This is the first reported case ofa mosaic 45,X/46,X,r(X)/46,XX identified in the prenatal period.

Contribution of fluorescence in situ hybridization analyses to the characterization of masked and complex Philadelphia chromosome translocations in chronic myelocytic leukemia.

Bone marrow samples from 112 patients with chronic myelocytic leukemia were investigated using cytogenetic methods. Fluorescent in situ hybridization (FISH) with whole-chromosome paints and BCR-ABL probes was used to confirm and/or complete the banding findings when a variant or a masked Philadelphia chromosome (Ph) translocation was found. Eight variant Ph translocations were identified. Three-way Ph translocations were found in seven patients. Chromosome 4 was involved in two of these cases and chromosomes 3, 11, 14, 17, and 16 in one case each; in the patient with chromosome 16 involvement, a ring of the translocated chromosome 9 was identified, that is r(9)t(9;16;22). The eighth patient had a five-way Ph translocation: t(2;9;16;22;22). The BCR-ABL fusion gene was detected on the Ph chromosome in all eight cases; two cases presented also a deletion of the 5' ABL region on the derivative chromosome 9. In the five-way translocation, the 3' DNA sequence of the ABL oncogene was fused with the 5' DNA sequence of the BCR gene on the Ph chromosome and the 5' end of ABL was inserted into the other chromosome 22. A masked Ph chromosome was identified in one of the 112 patients; it involved the insertion of the 3' ABL into BCR on an apparently normal chromosome 22, resulting in the BCR-ABL fusion gene. In conclusion, FISH analyses allowed not only a more accurate characterization of complex Ph translocations with subtle abnormalities and the identification of cryptic rearrangements, but also the recognition of deletion of the 5' ABL region, which could carry with it a poor prognosis.

In vitro study of translocation t(4;11) acute leukemia. Sequential range of phenotype.

The nature of translocation t(4;11) acute leukemia cells has been widely discussed over the past few years. Many authors report their phenotypic heterogeneity, ranging from apparently common acute lymphoblastic leukemia antigen-positive to monoblastic leukemia, through "promiscuous" phenotype. We studied in vitro phenotypic modulation of three typical cases after induction by 12-O-tetradecanoyl phorbol 13 acetate. The initial phenotype was different in each case, but all of them exhibited changes in morphologic shape, cytochemistry, and immunophenotype; common features appeared after induction by 12-O-tetradecanoyl phorbol 13 acetate, including esterase activity, expression of CD-9, CD-15, and CD-18 surface antigens, and monocyte/macrophage morphology. In all cases a B-associated surface antigen, CD-19, persisted. During clinical evolution, some previously reported cases have shown a karyotypic and phenotypic transformation. In one of our cases this phenomenon correlated with in vitro phenotypic modulation of initial blast population. Furthermore, clinical relapses and in vitro modulation always seem to evolve toward a more "mature" phenotype. Those results support the "promiscuous lineage" hypothesis, and point out the usefulness of in vitro studies to express the myeloid potential of this category of acute leukemia, which can be regarded as a model of early hematopoietic differentiation.

[Cystic hygroma of the neck. Antenatal diagnosis, prognostic factors, management. 42 cases]. / Hygroma kystique du cou. Diagnostic anténatal, facteurs pronostiques, contduite à tenir. A propos de 42 cas.

The authors report a series of 42 cases of cystic hygroma of the fetal neck diagnosed antenatally. Cystic hygroma is one of the signs suggestive of chromosomal or congenital abnormalities that occur very early and are very specific. A diagnosis can be made from the ninth week of amenorrhoea onwards by vaginal ultrasound. 73% of the karyotypes that were obtained were abnormal. The large majority (54% = have Turner's Syndrome, but there are some of the karyotypes that are normal. Our figures correspond with those in the literature. Several factors were analysed to show the influence of this pathology on the prognosis which is overall awful (only 11.5% of infants were born alive an only 7.5% survived). Factors for a good prognosis would be a normal karyotype and the spontaneous resolution of cystic hygroma in the second trimester of the pregnancy. Hydrops is a factor of poor prognosis and it occurs in 60% of cases of cystic hygroma but unfortunately 30% of cases where the karyotype is normal have severe malformations (bone, kidney and digestive tract). The resolution of the cystic hygroma in the second trimester of pregnancy does not exclude an abnormal karyotype or a severe congenital malformation associated with the condition. As cases do recur in the same family there is an indication that a suspicion that the condition can be an autosomic, recessive or even dominant condition. The authors advise that the diagnosis should be made early and thoroughly in order to carry out chorionic villus sampling to determine the karyotype early before the very important sign for abnormality disappears as it may.

[Strategies to identify supernumerary chromosomal markers in constitutional cytogenetics]. / Stratégies d'identification des marqueurs chromosomiques surnuméraires en cytogénétique constitutionnelle.

Supernumerary marker chromosomes (SMCs) are defined as extrastructurally abnormal chromosomes which origin and composition cannot be determined by conventional cytogenetics. SMCs are an heterogeneous group of abnormalities concerning all chromosomes with variable structure and size and are associated with phenotypic heterogeneity. The characterisation of SMCs is of utmost importance for genetic counselling. Different molecular techniques are used to identify chromosomal material present in markers such as 24-colour FISH (MFISH, SKY), centromere specific multicolour FISH (cenMFISH) and derivatives (acroMFISH, subcenMFISH), comparative genomic hybridisation (CGH), arrayCGH, and targeted FISH techniques (banding techniques, whole chromosome painting...). Based on the morphology of SMC with conventional cytogenetic and clinical data, we tried to set up different molecular strategies with all available techniques.