Lack of submicroscopic rearrangements involving telomeres in reproductive failures

B. Benzacken1,5, L. Carbillon2, C. Dupont1, J.P. Siffroi4, F. Monier-Gavelle1, M. Bucourt3, M. Uzan2 and J.P. Wolf1

1 Service d'Histologie Embryologie Cytogénétique BDR, 2 Service de Gynécologie et d'Obstétrique and 3 Service d'anatomopathologie et Foetopathologie, Hôpital Jean Verdier, avenue du 14 Juillet, 93140 Bondy, 4 Service d'Histologie, Biologie de la Reproduction et Cytogénétique, Hôpital Tenon, 4 rue de la Chine 75020 Paris, France and Laboratoire de Cytologie Histologie, UFR Biomédicale, 45 rue des Saints-Pères, 75006, Paris, France


    Abstract
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 Abstract
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 Materials and methods
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BACKGROUND: It has been recognized that chromosomal abnormalities are one of the most important causes of the high mortality rate in human concepti. Among these abnormalities, the unbalanced transmission of a parental chromosomal rearrangement is frequently observed, and couples with a history of pregnancy losses are therefore referred for genetic counselling and to establish their karyotype. Unbalanced chromosomal rearrangements involving telomeres are emerging as an important cause of mental retardation and/or congenital malformations in humans. As suggested by several authors, they could also be responsible for recurrent miscarriages. The aim of this study was to screen cryptic chromosome abnormalities in couples referred to our laboratory for recurrent unexplained miscarriages. METHODS AND RESULTS: Karyotyping was performed in 57 couples (114 patients). A detectable chromosomal abnormality was diagnosed in seven cases, thus limiting the analysis of telomeres to only 100 patients. Two different protocols were used according to the number of metaphases on slides. No telomeric chromosome abnormality was detected in our study. CONCLUSION: The use of FISH telomeric probes is not of clinical interest in the systematic screening of couples with multiple miscarriages and should be performed only in those with a familial history of mental retardation and congenital malformations.

Key words: FISH/miscarriages/telomeric rearrangements


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In human embryos, frequency of chromosomal abnormalities is very high (30–50% of concepti) and represents the major cause of embryonic lethality (Boué et al., 1975Go; Plachot et al., 1989Go). However, the incidence of chromosomal anomalies decreases as the number of miscarriages increases (Ogasawara et al., 2000Go). Before 20 weeks of pregnancy, only 29% of spontaneous recurrent miscarriages may be due to fetal chromosomal anomalies, 94% of which are aneuploidies and only 6% are structural aberrations (Carp et al., 2001Go). Moreover, chromosomal abnormalities are known to be involved in 5–10% of birth defects or perinatal losses (Hook, 1982Go) and most of them are aneuploidies (Hassold et al., 1993Go; Angell et al., 1994Go). Among unbalanced structural chromosome rearrangements in aborti, about half are inherited and the other half appear as de novo (Boué et al., 1975Go).

Consequently, after three or more unexplained miscarriages, couples have to be investigated by both maternal and paternal karyotyping in order to check whether a balanced chromosomal rearrangement may be responsible for fetal losses (Boué and Boué, 1973;Go; Boué et al., 1973Go). Reciprocal or Robertsonian translocations represent the most common abnormal parental karyotypes diagnosed in these cases. Even if miscarriages can be considered as a consequence of the transmission of such a translocation in an unbalanced state, genetic counselling must take into account the various methods of segregation that can lead to the birth of a child with multiple malformations and/or mental retardation.

Because cytogenetic analysis at a 400–550 band resolution is the current mode of investigation for detecting chromosomal rearrangements in these patients, abnormalities involving chromosome segments of <5 megabases (Mb) are not detected in routine analysis (Knight and Flint, 2000Go). High resolution karyotyping at ~1000 bands may improve the level of chromosome analysis, but this technique is time-consuming and does not exclude absolutely the existence of an undetectable chromosomal rearrangement. Indeed, in children with mental retardation, several authors have emphasized the importance of cryptic cytogenetic abnormalities localized preferentially in subtelomeric regions that escape diagnosis by conventional cytogenetic methods (Flint et al., 1995Go; Knight et al., 1997Go). Using fluorescence in-situ hybridization (FISH) with specific telomeric probes, Knight and Flint showed that 8% of 287 children with severe mental retardation and dysmorphy carried an unbalanced cryptic telomeric rearrangement (Knight and Flint, 2000Go). The authors estimated the incidence of cryptic imbalance to be 1/5000, which raises this type of chromosomal pathology to be the second most frequent cause of mental retardation after trisomy 21. They also suggested that telomeric region rearrangements might be involved in other fields of clinical interest such as spontaneous recurrent miscarriages or infertility.

The aim of this prospective study was to screen cryptic chromosome abnormalities in 114 patients (57 couples) referred to our laboratory for recurrent unexplained miscarriages. The interest of this work, in terms of genetic counselling and prenatal diagnosis possibilities, is discussed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Couples who had had three or more miscarriages, defined as a loss of pregnancy before 20 weeks, were studied. Only those who had an apparently normal karyotype, after conventional methods, were included in the study of telomeres by FISH.

Karyotype
Chromosome analysis was performed on peripheral blood lymphocytes after cell culture and classical cytogenetic techniques. Slides were stained by Giemsa after G and R banding.

FISH
According to the number of metaphases on chromosome preparations, two different protocols (A and B) were used. Therefore, it was necessary to examine carefully the cell density before each hybridization experiment.

Protocol A
This protocol was chosen in slides with a high density of mitosis and was carried out using the Chromoprobe Multiprobe T system® (Cytocell Ltd, UK) (Figure 1Go).This system identifies 41 out of the 46 human telomeres by labelling both arm tips of each chromosome pair with different colours. Indeed, probes are labelled by nick translation with biotin-16 dUTP [short arm (p) probes] or digoxigenin-11-dUTP [long arm (q) probes] (Boehringer Mannheim, Mannheim, Germany) and are distributed in little square sections under coverslip devices which are hybridized onto chromosome preparation slides (National Institutes of Health, 1996). The five telomeres that are not represented are the short arm telomeres of the acrocentric chromosomes 13, 14, 15, 21 and 22. Each square of the coverslip was hybridized according to the manufacturer's protocol.



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Figure 1. Illustrations of telomeric probe hybridizations and their usefulness for the diagnosis of cryptic chromosomal rearrangements.(A) Labelling of both chromosome 5 short arm (p) (green signal) and long arm (q) (red signal) using protocol A in a normal metaphase.(B) Multiple FISH with mixture 2 using protocol B (for details, see Table IIGo) in a normal mitosis. (C) Duplication of the chromosome 5p diagnosed after protocol A. (D) Diagnosis of a balanced reciprocal translocation between chromosome 2p and 22q using protocol A.

 
Protocol B
When metaphase density was too low, protocol B was chosen using the Multi-color DNA probe Mixtures® (Adgenix), which consists of a total of 62 DNA FISH probes. The mixtures include various combinations of telomeric, centromeric (CEP: X, 17 and 18) and locus specific (LSI: 13,14,15,21 and 22) probes (Table IGo). Each chromosome preparation slide was examined using phase contrast microscopy to identify regions that contained at least five metaphases appropriate for FISH. For hybridizing the whole 15 probe mixtures, a minimum of three slides were used for delineating five target areas per slide. Metaphase preparations for FISH were prepared using standard cytogenetic techniques and hybridized according to the manufacturer's procedure.


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Table I. Description of the multi-colour DNA FISH probe mixtures
 
Microscopic analysis
FISH analysis was performed using a Zeiss Axiophot microscope equipped with filters to detect DAPI, spectrum green and spectrum orange separately and with a triple band pass filter to detect all signals simultaneously. For each chromosome pair, the presence or absence of telomere probe signals, as well as their displacement from the chromosome of origin to another one in reciprocal translocations, was evaluated in approximately five metaphases in the corresponding square. A telomeric deletion was expected to give a lack of fluorescent signal at one of both appropriate chromosome end, and duplication by the presence of three signals in the appropriate square.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Karyotype analysis
Of the 57 couples, seven were excluded because an abnormal karyotype was diagnosed in either the male or the female partner (five women and two men; Table IIGo). The following abnormalities were observed for women: three mosaicisms for numerical sex chromosome aberrations, two reciprocal translocations: t(8;20) (p10;q10) and t(8;14) (q21.1;q31); and for men: one Robertsonian translocation t(13;14) (q10;q10) and one double Y, 47,XYY.


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Table II. Characteristics of the seven couples with abnormal karyotypes found in this study
 
FISH analysis
Subtelomeric FISH analysis was performed in a total of 100 patients. The method was efficient in 100% of cases. In 65% of cases the mitosis density was high, and protocol A was performed. In 35% of cases, protocol B was carried out. However, no abnormality was detected in our study. Using protocol A, three patients presented an absence of orange signal in the 2 q ter region. Because Cytocell Ltd notified users of a polymorphism for this probe, verification with the Adgenix probe for chromosome 2 was carried out for each of the three patients. No deletion of this region was found with this second method.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of telomeric probe sets as a diagnostic tool is a new and powerful cytogenetic method that allows the examination of all subtelomeric regions involved in chromosomal pathology. The first commercially available probe set (Chromoprobe Multiprobe T system®; Cytocell Ltd) was studied in order to analyse 41 different telomeres in a single microscope slide. However, this technique requires a very good quality of chromosome preparations with a high density of metaphases, which was possible in only 65% of our patients. Moreover, a number of polymorphisms or cross-reactions have been observed using these probes, particularly at the 2q ter level, which was apparently deleted in some patients. In these cases, the use of a second set of telomeric probes (Multi-color DNA probe Mixtures®; Adgenix) was necessary and allowed us to rectify the diagnosis. This second set was also useful for examining poor quality chromosome preparations or those with a low density of mitosis.

Taken as a whole, human chromosome telomeres can now be accessible to cytogenetic analysis by either one or both of these commercial telomeric probe sets with a 100% success rate. Such a possibility is of great clinical importance because rearrangements involving subtelomeric regions are more likely to have phenotypic consequences than rearrangements in any other parts of the genome. Several genetic diseases involving telomeric rearrangements have already been documented, especially those found in children with mental retardation and/or dysmorphic features (Flint et al., 1995Go; Knight et al., 1997Go). However, the apparent enrichment for chromosome rearrangements in telomeres suggests that cryptic telomeric abnormalities might be responsible for additional human genetic diseases like infertility or recurrent miscarriages. To our knowledge, this study is the first attempt to screen submicroscopic chromosome aberrations in couples affected by numerous spontaneous fetal losses.

In most cases, repetitive spontaneous miscarriages remain idiopathic which makes genetic counselling particularly difficult. Even if a cytogenetic cause is strongly suspected, due to the lack of any other anatomical or biological problem in the mother, fetal loss is considered to be the consequence of an aneuploidy rather than the missegregation of a parental chromosomal rearrangement. Indeed, karyotypes in parents are abnormal in <10% of couples when analysed by conventional techniques (Antich et al., 1980Go; Sachs et al., 1985Go; Tharapel et al., 1985Go) and females are more likely to carry a balanced chromosomal rearrangement than males (Lippman-Hand and Vekemans, 1983Go) or to be affected by sex chromosome mosaicism. However, such frequencies have been established before the occurrence of FISH in cytogenetic practice and the question arises whether subtle chromosomal abnormalities may have been misdiagnosed in these studies.

Our results showed 6.1% of detectable chromosome aberrations in 57 couples analysed, which is in agreement with data in the literature, but failed to detect any cryptic rearrangement. Some authors have already described cryptic parental translocations in couples referred for recurrent miscarriages, but only after the birth of a child with multiple congenital malformations (Shaffer et al., 1996Go; Brackley et al., 1999Go). According to the various types of chromosome segregation in reciprocal translocations, the co-existence of both fetal losses and the birth of an abnormal child from a carrier parent is not surprising. Indeed, viability thresholds in chromosomal imbalances have been estimated at 5% of haploid autosomal length for pure trisomies and 3% for pure monosomies, although when imbalances are in combination, no value exceeds 3.6% for trisomy and 0.6% for monosomy (Cohen et al., 1994Go). Therefore, because of the tiny size of chromosomal segments involved in cryptic translocations, these latter are unlikely to give isolated recurrent miscarriages but instead mixed familial histories of fetal losses and congenital malformations.

Although parental karyotyping still remains an obligatory step in biological investigations proposed in couples with spontaneous miscarriages (Salat-Baroux, 1998), our results lead to the conclusion that the screening of telomeric cryptic tranlocations by FISH should be offered only after the conception of a fetus and/or the birth of a child carrying chromosomal syndrome features. Such a diagnostic procedure could allow the detection of couples at risk and the possibility of a prenatal diagnosis, thus avoiding the birth of a second affected child.


    Notes
 
5 To whom correspondence should be addressed. E-mail: brigitte.benzacken{at}jvr.ap-hop-paris.fr Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on July 23, 2001; accepted on December 10, 2001.





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