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
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Abstract |
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Key words: FISH/miscarriages/telomeric rearrangements
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Introduction |
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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;; Boué et al., 1973
). 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 400550 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, 2000). 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., 1995
; Knight et al., 1997
). 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, 2000
). 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.
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Materials and methods |
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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 1).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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Results |
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Discussion |
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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., 1995; Knight et al., 1997
). 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., 1980; Sachs et al., 1985
; Tharapel et al., 1985
) and females are more likely to carry a balanced chromosomal rearrangement than males (Lippman-Hand and Vekemans, 1983
) 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., 1996; Brackley et al., 1999
). 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., 1994
). 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.
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Notes |
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References |
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Submitted on July 23, 2001; accepted on December 10, 2001.