Genelica "8" ANNALES: DE GENETIQUE ELSEVIER “Annales de Gentigne 4S (2002) 17-20 ww eteviercomocue/angen Case Report del(X)(p22.1)/r(X)(p22.1q28) dynamic mosaicism in a Turner syndrome patient Melva Gutiérrez-Angulo ', Brissia Lazalde ', Ana I. Vasquez, Caridad Leal, Elisa Corral, Horacio Rivera * Division de Genética, Centro de Inesigaci6n Biomédica de Occidente, IMSS, Ap posal 1-3838, Guadalajara, Jalisco, Mexico Abstract ‘We report on a 16-year-old patient with Turner syndrome who presented a mos 46,X,del(%)(p22.1)135)45,X{19V46,X 1(X}(922.1q28) [6] GTG-band karyotype. The R-banding showed thatthe sbnormal X-chromosome was inactive in all 61 cells analyzed, Fluorescence in situ hybridization with a Xp/Yp subtelomeric probe revealed that both abnormal chromosomes lacked the complementary sequences, a fact consistent with a terminal deletion, Besides, the molecular analysis of the human androgen receptor gene showed thatthe rearranged ‘chromosome was paternal in origin, Since the deleted and the ring chromosomes had the same size and banding pattera, and because the former was the predominant cell lin, it was infeed that the Xp- formed a ring in some cells apparently without further loss of genstie material. However, the reverse sequence and even a simultaneous origin due to a complex intrachromosomal exchange are also conceivable. “The mild Turner syndrome phenotype i explained by the mosaicism and by the size of tie deleted segment. © 2002 Editions scientifiques et médicales Elsovier SAS, All rights reserved. ‘Kepuonds: Turner syndrome: Deleted X-chromowme; Ring Xcromosome; Dynamic mossicism 1. Introduction Tumer syndrome (TS) bas an incidence of 1:2500-1:3500 female livebirths, is characterized by short stature, gonadal dysgenesis and sometic stigmata and is, eaused by haploinsufficiency for cerlain genes on X-chromosome [1,11]. About 50% of cases ascertained postnatally have a 45,X Karyotype, while the rest are ‘mosaics or have structural X- or Y-chremosome abnormmali- ties [1]. In this paper we report a patient with TS phenotype who presented a dynamic mosaicism for a rearranged X-chromosome of paternal origin 2. Materials and methods 2.1. Proband ‘The proposita was the fourth child born to a 36-year-old mother and an unrelated father; pregnaney was uncompli- © Conesponding author. Email addres: hrivera@udgservcencar.adgmx (H. River) "These eutbors contributed equally to te werk (© 2002 Btions seienifques et mésicales Eever SAS, al igh reserved PI: $0003-3995(02)01109-7 cated and delivery was by cesarean section at the 40th week. Birth weight was 3000 g; lymphoedema of feet and mild bilateral dislocation of hip were noted at birth. Psychomotor development and scholarship have been unremarkable. She ‘was assessed at the age of 16 years because of short stature ‘and primary amenorrhea. The physical examination showed height of 147 cm (<3rd percentile), occipitofrontal circum- ference of 55cm, thick eyebrows, slant up palpebral fissures, ‘multiple nevi on face, short neck, broad thorax, and cubitus valgus. Sexual secondary development was incipient at 14 years but menarche did not occur; a pelvic ultrasound (US) showed 2 small uterus but reportedly normal ovaries At 16 years, hormonal studies documented a hypergona- dotrophic hypogonadism (FSH, 57.1 mIU mI"! and LH, 37.8 mIU mi") and thereafter a sequential hormonal treat- ment resulted in mammary development and menstruation. Several months later, the uterine size was normal as revealed by another US. 2.2. Cytogenetic studies Routine chromosome analysis of the proband and her mother was performed on GTG-banded metaphases from8 1M, Guirrer- Angulo eal. /Annales de Génétique 45 (2002) 17-20 eae eye) Fig, 1.) X-chromosomes fom two G-bended cll: normal X-chromoromet (left) and deleted or ring X-chromesome (sgh). t) A paral Rbanded ‘metaphase, The arom show the inttive deleted chromosome an th arosthead indicates the normal ative chromosome.) FISH witha XpV¥p probe revedled the absence of subzlomerie Sequences inte deleted chromosome (arrow) whereas the expected signal was presen! inthe normal homologue (aowhead) d) Analysis of the human androgen reeptor gene, Lanes 1 and 2: digested DNA fom the mother and the dausht recpectvely Lanes 3 and 4 non-igesed DNA from the mother and the deaghe, respectively. Note tht the intene allele doesnot comerpond to the alleles present ix the mother. PHA-stimulated peripheral blood lymphocytes. RBG- banding was also performed on metaphases of the proband. 2.2.1. Fluorescence in situ hybridization “Metaphases of the patient were hybridized with a subte- lomeric Xp/Yp probe (SpectrumGreen TelVysion, Vysis) mixed with the X-alphoid repeat (Oncor) according to standard protocols. The alphoid signa’ was revealed by means of rhodamine-labelled antidigoxigenin whereas DAPI was used as counterstaining. Digital images were captured with a CCD camera (CoolSNAP-Pro, Media Cybernetics) coupled with an epi- ourescence microscope (Olympus AX 70). 23. DNA analysis ‘The human androgen receptor assay was used to deter- mine the parental origin of the inactive X-chromosome. The DNA of the patient and her mother was extracted from peripheral blood leukocytes by standard methods (the fa- her's DNA was not available). 250ng of DNA were digested with the methylation-sensitive enzyme Hpall. Both the digested and non-digested DNA samples were used for PCR with the primers ARAI 5.GCTGTGAAGGTTG- CTGITCCTCAT3' and ARA2 S-TCCAGAATCTGTT CCAGAGCGTGC:3' for 28 cycles of 45 s at 95 °C, 30s at 60°C and 30s at 72°C [2]. Ten microliters of the PCR product were added to 5 ul of loading solution. The ampli fied products were visualized in 6% polyacrilamide gels after staining with silver nitrate 3. Results 3.1. Cytogenetic studies In 60 G-banded metaphases, a three-cell line mosaicism was found: 46,X,del(X)(p22.1)135)/45,X119/46,X,00) (p22.1928) [6]. The ring(X) had the same G-banding pattern and size as the deleted chromosome (Fig. 1a). The Rebanding showed that the abnormal X-chromosome was inactivated in 61 suitable metaphases: 60 cells with the deletion and one cell with the ring chromosome (Fig. 1b). ‘The karyotype of the mother was normal 3.1.1. Fluorescence in situ hybridization analysis ‘The analysis with an Xp/Yp probe revealed the absence of the corresponding subtelomeric sequences in both the deleted and the annular X-chromosomes while the expected signal was oberved in the normal homologue (Fig. 1c) ‘The PCR with non-digested DNA showed two alleles in both the patient and her mother. The amplification of the digested DNA of the mother showed two alleles, while that cof the patient presented one rather intense allele and a second one barely perceptible (Fig. 1d). Since the intense allele did not correspond to the alleles present in the mother, it was deduced that such an inactive allele was paternal in origin 4, Discussion ‘The concurrence of two rearranged chromosomes (one linear and another annular) apparently composed of the same Xp22.1—sqter material indicates that the initial event ‘was a terminal Xp deletion with the deleted chromosome being stabilized by the acquisition of a (neo)telomere; in fact, constitutional terminal deletions are mostly stabilized by the synthesis of a neotelomere (3] and no deleted linear chromosomes lacking a telomere are known. Yet, if this stabilization was not achieved at once, the deletion could also result in the ring formation. Alternatively, te deletion could lead directly 10 the ring chromosome and this in turnM, Gwidres Angulo el. ‘Annales de Gduésiue 452002) 17-20 2 open to produce the deleted chromosome which was then stabilized. We favor the former sequence because there is no evidence of Xq deletion as the classical origin of a ring demands and because of the large proportion af cells with the deleted chromosome. Accordingly, Wwe consider that the Xp deletion occurred during the spermatogenesis and con- sequently the zygote started with such a deleted chromo- some and a normal maatemal X. During the first mitotic divisions the deleted chromosome formed a ring probably because of a somewhat dysfunctional Xp telomere as it has been documented in some ring chromosomes {10}. In tur, the ring instability led to the 43,X cell line, a fact in ‘agreement with the hypothesis thatthe large rings are more instable than the small ones (the apparent Tack of abnormal configurations secondary to the ring instability can be ‘ascribed to the reduced proportion of cells with the ring) Such an instability is thought 0 result ftom sister chromatid exchanges that occur in the ting and generate cells with Gouble-sized dicentric rings and cells without the ring, This process of créating new genetic unbalauces fom an already sthnormal cell is termed ‘dynamic. mosaicism’, a phenom- ‘enon well known in ring chromosomes but also observed in some marker and deleted chromosomes [5,7] Besides these alternative sequences, @ simukaneous ori- gin of both rearranged chromosomes can be envisaged. In describing a boy with a 46,XY¥s(2)(p28.2432.246,XY, dup(Zq)del(2p\iqrer4q33.2::p25.2-qter) mosaicism, ‘Wyandk et al. [12}. postulated that both abnormal configu- tations arose simultaneously from a complex intrachtomo- somal exchange involving an isochromatid 2p25.2 break and a single chromatid 2933.2 break, events that led 0 the following outcome: the chromatid with broken ends re- joined to form the ring whereas the other chromatid also broken at 2925.2 rejoined with the q segment distal 10 the single chromatid break at 2433.2 and thereby resulted in the up/del chromosome (the segment 2p25.2-spter was lost) Then, the present patient's rearranged chromosomes may hhave resulted from an isochromatid break at Xp22.1 with « chromatid forming the Xp22.1-sqter ring and the other being stabilized as a deleted chromasome via the acquisition of a (neo}telomere. According to Wyandt et al. [12], the lack of a normal cell line indicates that the intrachromosomal exchauge must have been initiated, atthe iateet, during G, of the ftst zygotic division, or even in G, in the gamete or in the pronucleus, ‘There are two reports of a similar mosaicism involving ttroe cel Hines: 45,X/46,%,del(X)46,X,c(X), Ogata etal. {9} described a patient without TS phenotype but with the nicrophthalmia with linear skin defects syndrome (MLS) who presented « karyotype mos 45,Xid6,X,nlp22q21V46,X, del(XY(p22) in peripheral lymphocytes. The MLS pheno- type was thought to result from the functional absence ofthe MLS gene, which may be provoked by inactivation of the normal X-chromosome. On the other hand, Migeon etal [8] reported on a female with a 45,X/46,X del(X\@q21.3)/46, X.x(X) karyotype in skin ang food eels vino showed TS igmata and other characteristics usually not associated ‘with TS; noticeably, the s(X) was smaller than the deleted X and was described as ‘tiny’. The fact that in the present patient the ting chromosome had the same G-banding pattern and size as the deleted homologue suggests that n0 further loss of genetic material has occurred in the interven- ing process. In this sonse, therefore, her karyotype differs from those of the previous patients (8,9) in whom the the ‘ing was considerably smaller than the deloted chromosome. Altogether, the cytogenetic and molecular data in this patient indicate tat dhe eamanged X-chromasome fad & paternal origin and exhibited a preferential inactivation, The inferred paternal origin of the rearranged X-chromosome agrees with the findings of Jacobs et al, (6] who reported that the majority of deleted and ring X-chromosomes were paternal in origin; however, no differences in the parental ‘origin were found by Tsezou etal. (L1J- Although it fas been proposed tha some characteristics of TS phenciype such as cardiovascular anomalies and neck webbing result from retsining the maternal X and therefore may be influenced by ‘imprinting (4,6), our patient lacked such anomalies. The presence of shor stature can be duc 1 haploinsufficieney of the SHOX gene which is localized in the Xp pseudoauto- somal region {23} whereas the mild TS phenotype is explained by both the mosaicism and the size ofthe deleted segment, Acknowledgements Melva Gvtiéwez-Angvlo and Brissia Lazalde are sup- ported by CONACYT scholarships (numbers 117466 and 117471, respectively). We thank to Dr, Lourdes Ramirez- Duefias for editing the FISH picture. References UM. Adsehi, K. Tachibana, Y. Asakura, K, Moroya, T, Ogata Deilkp01.)) i a mother 2nd ovo daughters: genotype-phenotype cenrelation of Turner features, Hum. Genet. 106 2000) 306-310 2} RC Allen, FLY. Zoghb, A.B. Mosley, HM. Rosenblat, 1. 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