Chromosomal instability in two siblings with gonad deficiency: Case report

J. Lespinasse1,7, P. Hoffmann2, A. Lauge3, D. Stoppa-Lyonnet3, F. Felmann4, J.C. Pons2 and G. Lesca5,6

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 Bernard–Lyon 1 University, Lyon, France

7 To whom correspondence should be addressed. Email: james.lespinasse{at}ch-chambery.rss.fr


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Non-random de novo autosomal chromosomal rearrangements have not been shown to cause exocrine or gonadal dysfunction. We report on two siblings, a brother and a sister, both with de novo chromosomal rearrangements and gonadal deficiency including premature ovarian failure. They had normal phenotypes without additional manifestations of known chromosomal breakage syndromes (except for the gonadal dysfunction) and normal alpha-fetoprotein dosage level. The association of sperm abnormalities in the brother and ovarian dysfunction in the sister suggested an increased spontaneous chromosomal instability. Since the co-occurrence of chromosomal anomalies and reproductive failures may not be coincidental, we performed repeated chromosomal analysis of peripheral blood lymphocytes prior to proposing ICSI for IVF (for the brother). In both sibs, infertility was associated with random and non-random de novo autosomal chromosomal abnormalities. We discuss the possible relationship between these unusual clinical and cytogenetic features and their potential links to ataxia–telangiectasia.

Key words: chromosomal rearrangement/gonadal deficiency/premature ovarian failure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Molecular biology techniques, such as in situ hybridization (FISH) coupled with conventional chromosomal analysis, have largely overcome the limitations of conventional banding in the accurate diagnosis and interpretation of subtle or complex chromosomal abnormalities. The use of various subtelomeric probes for all chromosome arms for FISH now allows the confirmation and identification of chromosome rearrangements. Nevertheless, the interpretation is often impaired by the lack of knowledge about genotype–phenotype correlation. As far as we know, non-random de novo autosomal chromosomal rearrangements are not known to cause exocrine or gonadal dysfunction whereas de novo aneuploïdy or chromosome abnormalities such as 45,X and t(X;A) are commonly found. We report here on two siblings who presented with the combination of gonadal deficiency, associated with a combination of sperm abnormalities in the brother and with premature ovarian failure (POF) in the sister. Both showed evidence of non-random de novo chromosomal rearrangements.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Family report
The 34 year old proband (II2) and his 30 year old wife (II3), non-consanguineous, consulted for secondary infertility (Figure 1). Patient II3 had two healthy children from a previous union. Their first common child (III1), conceived after a 1 year union, was born at 39 weeks of an uneventful pregnancy and was healthy. The couple consulted after attempting to conceive another child for 5 years without success. There was no familial history of congenital malformation nor of exposure to drugs, radiation or toxic environmental agents. Routine analyses showed no infectious cause for their infertility.



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Figure 1. Pedigree of the proband's family.

 
The medical history of patient II2 was unremarkable, but sperm analyses were abnormal (Table I). Patient II3's menarche occurred at the age of 12.5 years and her cycles were regular (28 days). The temperature curves were biphasic and normally shaped. Hysterosalpingography did not show any malformation. The Hühner test was negative, with appropriate mucus. According to the spermiogram results and the duration of infertility, an IVF with ICSI was programmed for this couple, after karyotyping of the proband and genetic counselling. The IVF attempt was unsuccessful, but they spontaneously conceived a few months later and gave birth to a healthy boy.


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Table I. Successive sperm analyses and spermocytograms (from two different laboratories)

 
The proband's sister (II1), aged 36 years, also suffered from infertility. Her menarche was delayed and occurred at 17 years, with oligomenorrhoea evolving to secondary amenorrhea after 1 year. She did not suffer from psychiatric illness. No abnormality was noticed on physical examination. She received combined (estrogen–progestogen) oral contraception for 7 years for ‘cycle control’. When she was 25 years old, she sought to conceive and underwent extensive testing. Hormone levels suggested a POF: gonadotrophin levels were high (FSH = 63 IU/l, LH = 2.5 IU/l) and estrogen levels low (estradiol = 15 pg/ml; n>50). Her karyotype was 46,XX on 38 mitoses. Laparoscopy showed a hypoplastic uterus with two rudimentary horns and streak-like gonads. Histological examination of bilateral ovarian biopsies showed fibrous albugineae and a fibrous ovarian stroma devoid of follicles and oocytes. Polycystic ovary Stein–Leventhal's syndrome was then mentioned as a possible cause.

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)Go 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., 1999Go). 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., 1996Go). 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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Current biology
Patient II2 had normal levels of gonadotrophins and androgens (FSH = 3.2 IU/l; LH = 2.5 IU/l; testosterone = 6.59 g/l, 3 < n<10.6) as well as normal AFP (2 µg/l) in serum.

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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Table II. Successive lymphocyte chromosomal analysis of patients II1 and II2

 
Fluorescence in situ hybridization (FISH)
FISH analysis confirmed the presence of abnormal cell lines with t(7;14) and inv(7) rearrangements and revealed various balanced or unbalanced reciprocal translocations in patients II1 and II2 (Table III and Figure 2a–). FISH analysis of the parents was normal.


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Table III. Various balanced or unbalanced reciprocal translocations by FISH analysis in patients II1 and II2

 


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Figure 2. FISH studies in patient II2. (A) Balanced reciprocal telomeric 7p and 7q translocations. (B) Derivative chromosome consisting of a presumed C group chromosome with additional subtelomeric extremity 2p. (C) Unidentified extra marker chromosome containing additional subtelomeric extremity 7p. (D) Presumed inverted duplication chromosome Y with one subtelomeric extremity Yq at each extremity. (E) Chromosome 7 with unknown additional material attached to subtelomeric extremity 7p.

 
Molecular biology
The Y chromosome markers used did not show any microdeletion in patient II2. None of the common mutations of the CFTR gene was found. Patient II1 had two CGG stretches of normal size at the FRAXA locus. No mutation of the ATM gene was identified with the technique used. The residual probability for patient II2 to be heterozygous for an ATM mutation was estimated to be ~30%.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the reported family, patients II1 and II2 presented with an unusual combination of random and non-random de novo chromosomal rearrangements and gonad deficiencies, i.e. combined sperm anomalies (II2) and POF (II1).

Semen abnormalities may result from multiple causes such as Y chromosome microdeletions and CFTR gene mutations (Foresta et al., 2001Go; Meng et al., 2001Go; Cruger et al., 2003Go), but these were not found in II2. POF occurs in 1% of women (Sherman, 2000Go). 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., 1998Go; Marozzi et al., 2000Go).

The cytogenetic features of these patients resemble those found in ataxia–telangiectasia (AT) and Nijmegen breakage syndrome (NBS). AT [MIM 208900] is a rare autosomal recessive disorder (Aurias and Dutrillaux, 1986Go). 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., 1996Go). 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 4–9% 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, 1985Go). 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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aurias A and Dutrillaux B (1986) Probable involvement of immunoglobulin superfamily genes in most recurrent chromosomal rearrangements from ataxia telangiectasia. Hum Genet 72, 210–214.[CrossRef][ISI][Medline]

Barlow C, Hirotsune S, Paylor R, Liyanage M, Eckhaus M, Collins F, Shiloh Y, Crawley JN, Ried T, Tagle D and Wynshaw-Boris A (1996) Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 86, 159–171.[ISI][Medline]

Biancalana V, Taine L, Bouix JC, Finck S, Chauvin A, De Verneuil H, Knight SJ, Stoll C, Lacombe D and Mandel JL (1996) Expansion and methylation status at FRAXE can be detected on EcoRI blots used for FRAXA diagnosis: analysis of four FRAXE families with mild mental retardation in males. Am J Hum Genet 59, 847–854.[ISI][Medline]

Conway GS, Payne NN, Webb J, Murray A and Jacobs PA (1998) Fragile X premutation screening in women with premature ovarian failure. Hum Reprod 13, 1184–1187.[Abstract]

Cruger DG, Agerholm I, Byriel L, Fedder J and Bruun-Petersen G (2003) Genetic analysis of males from intracytoplasmic sperm injection couples. Clin Genet 64, 198–203.[CrossRef][ISI][Medline]

Foresta C, Moro E and Ferlin A (2001) Y chromosome microdeletions and alterations of spermatogenesis. Endocr Rev 22, 226–239.[Abstract/Free Full Text]

ISCN 95 (1995) Mitelman F (ed.) An International System for Human Cytogenetic Nomenclature. Karger, Basel.

Marozzi A, Vegetti W, Manfredini E, Tibiletti MG, Testa G, Crosignani PG, Ginelli E, Meneveri R and Dalpra L (2000) Association between idiopathic premature ovarian failure and fragile X premutation. Hum Reprod 15, 197–202.[Abstract/Free Full Text]

Meng MV, Black LD, Cha I, Ljung BM, Pera RA and Turek PJ (2001) Impaired spermatogenesis in men with congenital absence of the vas deferens. Hum Reprod 16, 529–533.[Abstract/Free Full Text]

Reddy KS and Thomas IM (1985) Significance of acquired nonrandom 7/14 translocations. Am J Med Genet 22, 305–310.[ISI][Medline]

Sherman SL (2000) Premature ovarian failure in the fragile X syndrome. Am J Med Genet 97, 189–194.[CrossRef][ISI][Medline]

Simoni M, Bakker E, Eurlings MC, Matthijs G, Moro E, Muller CR and Vogt PH (1999) Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int J Androl 22, 292–299.[CrossRef][ISI][Medline]

Submitted on July 30, 2003; resubmitted on July 6, 2004; accepted on October 4, 2004.





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