1 Cytogenetic Laboratory, General Hospital, BP 1125, 73011 Chambéry cedex, 2 Gynecology, Obstetric and Reproductive Medicine, University Hospital of Grenoble, BP 217, 38015 Grenoble Cedex, 3 Department of Oncology Genetics, Institut Curie, Paris, 4 Laboratory of Genetics, Regional Hospital, Place St Jacques, 25030 Besançon, 5 Laboratory of Genetics, E.Herriot Hospital, Place d'Arsonval, 69003 Lyon and 6 Claude BernardLyon 1 University, Lyon, France
7 To whom correspondence should be addressed. Email: james.lespinasse{at}ch-chambery.rss.fr
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Abstract |
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Key words: chromosomal rearrangement/gonadal deficiency/premature ovarian failure
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Introduction |
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Materials and methods |
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Since it appeared that both siblings had gonadal deficiency, they underwent another karyotype with complementary cytogenetic analysis.
Methods
Current biology
Alpha-fetoprotein (AFP) was measured by nephelometry and turbidity on blood sample from patient II2, and hormonal as well as selective immunoglobulin (using jellified gel medium precipitation) dosages were assayed.
Conventional cytogenetic analysis
Chromosome analyses were repeated on peripheral blood lymphocytes from patients II1 and II2 as well as their parents (I1 and I2). Studies of metaphase chromosomes used standard (450 bands) and high-resolution (800 bands) procedures. The karyotypes were described according to the ISCN 95 (1995) nomenclature.
Fluorescence in situ hybridization (FISH)
We performed FISH of the all-telomeric sequence (TTAGGG)n. Commercial probes were used for all the chromosomes. Specific probes for the short arms were either labelled with Digoxigenin and detected with an FITC-conjugated amplification system, or labelled with biotin and detected with Cy3-conjugated antibodies for long arms. Chromosomes were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Chromosome denaturation, hybridization and signal detection were carried out according to the instruction manual supplied by the manufacturer. Slides were analysed on a Zeiss (Germany) epifluorescence microscope equipped with appropriate filters and a Vysis (USA) image analysis system.
Molecular biology
Microdeletions of the Y chromosome were sought in patient II2 using PCR amplification of the following DNA markers: ZFY, SRY, sY84 and sY86 (AZFa), sY114, sY127 and sY134 (AZFb), sY254 and sY255 (AZFc), following the guidelines of the European Association of Andrology (Simoni et al., 1999). The size of the FRAXA CGG trinucleotide repeat in patient II3 was evaluated by the Southern method using double enzymatic digestion by EcoRI and EagI and the Stb12.3 DNA probe, as described previously (Biancalana et al., 1996
). The most frequent CFTR gene mutations in patient II2 were checked with a CF-OLA commercial kit (Abbott Diagnostics). The coding sequence of the ATM gene was sequenced in patient II2 as follows: mRNA was extracted from a lymphoblastoid cell line and cDNA was obtained by random priming and reverse transcription and then sequenced through eight overlapping fragments (primers available on request), using the BigDye Terminator Cycle Sequencing V1.1 Ready Reaction kit (Applied Biosystems), followed by electrophoresis. Data were analysed with the ABI PRISM 3100 Genetic (Applied Biosystems).
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Results |
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Cytogenetic features
RHG-banding and GTG-banding revealed abnormal karyotypes in patients II1 and II2 and normal karyotypes in their parents and in patient II3 (100 mitoses analysed, data not shown). Cytogenetic features of patients II1 and II2 are described in Table II.
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Discussion |
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Semen abnormalities may result from multiple causes such as Y chromosome microdeletions and CFTR gene mutations (Foresta et al., 2001; Meng et al., 2001
; Cruger et al., 2003
), but these were not found in II2. POF occurs in 1% of women (Sherman, 2000
). In patient II1, complete lack of follicles on the ovarian biopsy samples suggested a gonadal dysgenesis. She had no FRAXA premutation, previously shown to be significantly increased among women with POF (Conway et al., 1998
; Marozzi et al., 2000
).
The cytogenetic features of these patients resemble those found in ataxiatelangiectasia (AT) and Nijmegen breakage syndrome (NBS). AT [MIM 208900] is a rare autosomal recessive disorder (Aurias and Dutrillaux, 1986). Onset usually occurs in infancy, and the clinical features include progressive cerebellar degeneration, ocular and cutaneous telangiectasia, immune deficiencies, and lymphoreticular malignancies. Cytogenetic features include non-random chromosomal rearrangements with preferential breakpoints in bands 14q11, 14q32, 7q35, 7p14, 2p11 and 22q11, involving T/B-cell receptors and immunoglobulin genes. ATM is expressed in spermatogonia and Atm-mutated mice display infertility with azoospermia (Barlow et al., 1996
). NBS [OMIM 251260] is a rarer autosomal recessive disease characterized by growth and mental retardation, craniofacial dysmorphy, ovarian failure, immune deficiencies, lymphoid malignancies, chromosome instability, and radiosensitivity. Recurrent chromosomal rearrangements are similar to those of AT.
Patient II2 had a t(7;14) in 49% of the mitoses from peripheral blood. The breakpoints involved band 14q11 in 4.25% of the mitoses and band 14q32 in 1%. Despite the facts that patients II1 and II2 had no clinical feature of AT (or NBS) and that no mutation was found in the ATM gene, we cannot exclude a moderate expression of the disease. Acquired non-random 7/14 translocations were previously found in a few metaphases of patients without clinical features of these two disorders (Reddy and Thomas, 1985). Although the cytogenetic findings in the present family may be artefacts due to the lymphocyte stimulation by PHA, their occurrence in two siblings is puzzling.
In conclusion, the association of gonadal deficiency and multiple de novo chromosomal rearrangements in two siblings is uncommon. Although the clinical and cytogenetic features in the present family may be a rare coincidence, we can raise the hypothesis that they might result from a common mechanism, i.e. an uncommonly moderate expression of AT or another genetic condition. A better understanding would be brought by studying other families.
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References |
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Submitted on July 30, 2003; resubmitted on July 6, 2004; accepted on October 4, 2004.
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