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Short Stature Homeobox-Containing Gene Duplication on the der(X) Chromosome in a Female with 45,X/46,X, der(X), Gonadal Dysgenesis, and Tall Stature¹

Department of Pediatrics, Keio University School of Medicine, (T.O., T.K.), Tokyo 160-8582; Department of Hygiene and Medical Genetics, Shinshu University School of Medicine (K.W., Y.F.), Matsumoto 390-8621; and Department of Obstetrics and Gynecology, Hiroshima University School of Medicine (M.Y., N.M.), Hiroshima 734-0037, Japan

Address all correspondence and requests for reprints to: Dr. Tsutomu Ogata, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: t-ogata{at}po.iijnet.or.jp

	Abstract

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We report on a Japanese female with 45,X[40]/46,X, der(X)[60], primary amenorrhea, and tall stature. She was confirmed to have complete gonadal dysgenesis at 19 yr of age and was placed on hormone replacement therapy. Growth assessment revealed that she had a low normal height until her early teens, but continued to grow with a nearly constant height velocity in her late teens, attaining a final height of 172 cm (+2.9 SD), which surpassed her target height range. Fluorescence in situ hybridization analysis for 10 loci/regions on the X-chromosome together with the whole X-chromosome and the Xp-specific and Xq-specific paintings showed that the der(X) chromosome was associated with duplication of roughly distal half of Xp, including SHOX (short stature homeobox-containing gene), and deletion of most of Xq. Microsatellite analysis for eight loci at Xp22 and nine loci at Xq26–28 indicated that the normal X-chromosome was of maternal origin, and the der(X) chromosome was of paternal origin.

The results, in conjunction with the adult height data in 47,XXX, 46,XX gonadal dysgenesis, 47,XXY, 46,XY gonadal dysgenesis, and 46,X, idic(Xq-), suggest that the tall stature of this female is caused by the combined effects of SHOX duplication on the der(X) chromosome and gonadal estrogen deficiency. Furthermore, the similarity in the growth pattern between this female and patients with estrogen resistance or aromatase deficiency implies that the association of an extra copy of SHOX with gonadal estrogen deficiency may represent the further clinical entity for tall stature resulting from continued growth in late teens or into adulthood.

	Introduction

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IN 1997, RAO et al. (1) cloned a novel gene from the short stature critical region in the short arm pseudoautosomal region (PAR1) of the human sex chromosomes and named it SHOX (short stature homeobox-containing gene). This gene was also identified by Ellison et al. (2) and termed PHOG (pseudoautosomal homeobox-containing osteogenic gene). SHOX is most strongly expressed in bone marrow fibroblasts, suggesting that SHOX is relevant to skeletal growth and development (1). In addition, SHOX is expressed from an inactive X-chromosome as well as active X- and Y-chromosomes, implying that SHOX escapes X inactivation and exerts a dosage effect in sex chromosome aberrations (1). Furthermore, Rao et al. (1) identified a heterozygous SHOX nonsense mutation cosegregating with short stature phenotype in a German family, providing compelling evidence for SHOX being the growth gene for short stature.

Subsequently, haploinsufficiency of SHOX has also been shown to cause Turner skeletal features such as short metacarpals, cubitus valgus, and Léri-Weill dyschondrosteosis (3, 4, 5). The development of skeletal features and resultant growth deficiency tend to be more severe in females than in males and become more obvious with puberty in individuals with the combination of SHOX haploinsufficiency and normal gonadal function; males with that combination usually have borderline short stature alone before puberty and often show mild skeletal features in addition to borderline short stature from puberty, whereas females with that combination usually have borderline short stature and mild skeletal features before puberty and frequently exhibit severe skeletal features, such as Léri-Weill dyschondrosteosis and growth deficiency, from puberty (5). It has been suggested, therefore, that gonadal estrogens exert a maturational effect on skeletal tissues that are susceptible to unbalanced premature fusion of growth plates because of SHOX haploinsufficiency, facilitating skeletal lesions in a female-dominant and pubertal tempo-influenced fashion (5). This idea postulates that the association of SHOX haploinsufficiency with gonadal estrogens exerts a deleterious effect on skeletal growth and development, especially in the pubertal period.

Here, we propose that the opposite combination of an extra copy of SHOX and gonadal estrogen deficiency may lead to tall stature, which becomes discernible at pubertal age in normal children.

	Subjects and Methods

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Case report

This Japanese female was born to healthy nonconsanguineous parents after an uncomplicated term pregnancy. Allegedly her birth size was normal, and her postnatal course was uneventful until late teens.

At 19 yr of age, she presented with primary amenorrhea. Physical examination showed mild webbed neck and poor pubertal development (breast, Tanner stage 1; pubic hair, Tanner stage 2), and endocrine studies showed hypergonadotropic hypogonadism (basal serum FSH, >30 IU/L). Laparoscopic exploration revealed hypoplastic uterus and streak gonads, and histological examination showed complete gonadal dysgenesis. Unfortunately, bone age and serum values for GH, insulin-like growth factor I, and thyroid hormones were not obtained, but she was not acromegaloid or hyperthyroid clinically. She received hormone replace therapy with conjugated estrogen (1.25 mg/day, from the 1st through the 21st day of the month) and medroxyprogesterone acetate (5 mg/day, from the 12th through the 21st day of the month) and thereafter had breast development and regular menses. At present, she is 29 yr old and shows full breast development.

Growth assessment

The height of the patient was assessed by the longitudinal growth curve for normal Japanese girls (6). Target height (TH) and target range (TR) were obtained from the following equations for Japanese girls, which allow for a positive height secular trend in Japan: TH = [(PH - 13) + MH]/2 + 2 cm and TR = TH ± 8 cm, where PH is paternal height, and MH is maternal height (7). Parental heights were assessed by age-matched Japanese standards.

Conventional and molecular cytogenetic studies

Chromosome analysis was performed on peripheral lymphocytes of the patient and the mother using G-banding. Fluorescence in situ hybridization (FISH) analysis was performed for lymphocyte metaphase spreads, using probes for the Xp telomeric region (8), SHOX on PAR1 (5), KAL on Xp22.3, DMD on Xp21, OTC on Xp11.4, MAOB on Xp11.3, DXZ1 on the centromere, XIST on Xq13, CD40 ligand on Xq26, and the Xq telomeric region (8). In addition, a whole X-chromosome-painting probe, an Xp-specific painting probe, and an Xq-specific painting probe were hybridized to metaphase spreads. Probes were labeled with either digoxigenin or biotin; digoxigenin-labeled probes were detected by rhodamine and antidigoxigenin, and biotin-labeled probes were detected by avidin and fluorescein isothiocyanate.

Microsatellite analysis

Genomic DNA was extracted from peripheral blood leukocytes of the patient and the mother and was amplified by PCR for eight loci at Xp22 and nine loci at Xq26–28 (Table 1). Amplification was performed in a reaction volume of 50 µL containing 0.3 µg genomic DNA, 20 pmol fluorescently labeled forward primer, 20 pmol unlabeled reverse primer, 0.2 mmol/L deoxy-NTPs, and 2 U Taq polymerase. The primer sequences and the PCR conditions have been reported in the Genome database. The PCR products were determined for the product size on an Autosequencer (ABI PRISM 310, PE Applied Biosystems, Perkin-Elmer Corp., Norwalk, CT) using GeneScan.

	Results

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The patient’s growth pattern is shown in Fig. 1, together with TH/TR, PH, and MH. She had a low normal height until her early teens, but continued to grow with a nearly constant height velocity in her late teens, attaining tall stature. She ceased to grow shortly after initiation of hormone replacement therapy. On the last examination, her height was 172 cm (+2.9 SD), which surpassed her TH/TR.

View larger version (30K): [in this window] [in a new window]   Figure 1. The growth curve of the female depicted on the standard growth curves for Japanese females (mean, ±1 SD, and ±2 SD). HRT, Hormone replacement therapy. TH is shown together with TR, indicated by the vertical bars.

The karyotype of the patient was initially interpreted as 45,X[40]/46,X, del(X)(q24)[60]. However, FISH analysis showed that the Xp telomeric region, SHOX, KAL, and DMD were present in two copies on both of the distal parts of the der(X) chromosome (Fig. 2A). OTC, MAOB, DXZ1, and XIST were present in a single copy on the middle part of the der(X) chromosome, and CD40 ligand and the Xq telomeric region were absent from the der(X) chromosome. The whole X-painting probe homogeneously stained the entire der(X) chromosome. The Xp-painting probe stained most of the der(X) chromosome, except for its middle part, which was stained with the Xq-painting probe. Thus, it was shown that the roughly distal half of Xp was duplicated and attached to the proximal Xq to form the der(X) chromosome (Fig. 2B). The karyotype of the patient was finally determined as 45,X[40%]/46,X, der(X) (pterq13 or q21::p11.4 or p21.1pter)[60%]. The maternal karyotype was 46,XX, and FISH analysis showed normal findings.

View larger version (53K): [in this window] [in a new window]   Figure 2. A, FISH analysis for a 46,X, der(X) lymphocyte. SHOX is detected on both ends of the der(X) chromosome and one end of the normal X-chromosome (arrows), whereas the Xq telomere region is identified on the normal X-chromosome alone (arrowhead). The SHOX probe was labeled with digoxigenin and was detected by rhodamine antidigoxigenin, and the Xq telomere probe was labeled with biotin and was detected by avidin conjugated to fluorescein isothiocyanate. B, A schematic representation of the normal X-chromosome (left) and the der(X) chromosome (right) of the female. The chromosomal locations of 10 loci/regions examined by FISH analysis are shown. The white areas represent the short arm and the long arm pseudoautosomal regions, striped areas indicate the centromeric region, the densely stippled areas denote the X-differential regions of Xp, and the coarsely stippled areas depict the X-differential regions of Xq.

The results are summarized in Table 1. For DXYS233, DXS996, DXS7108, DXS1224, and DXS987 at Xp22, both maternally derived and nonmaternally (paternally) derived markers were detected in the patient. For the remaining three loci at Xp22 and for all nine loci at Xq26–28, single markers only were detected in the patient, and the comparison of the PCR product sizes between the patient and the mother was consistent with the single markers being derived from the mother. Thus, it was indicated that the normal X-chromosome was of maternal origin, and the der(X) chromosome was of paternal origin.

	Discussion

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Structural analysis was performed for the der(X) chromosome of this female with primary amenorrhea and tall stature, showing duplication of roughly the distal half of Xp and deletion of most of Xq. It is likely that the der(X) chromosome was formed by a de novo aberrant Xp-Xq interchange during paternal meiosis. To our knowledge, there has been only a single report describing such a rearranged X-chromosome. Lepping et al. (10) identified an apparently nonmosaic 46,X, der(X)(pterq13::p11.4pter) karyotype in a 17-yr-old female with primary amenorrhea and relatively tall stature [171.9 cm, +1.6 SD]. As the phenotypes were similar in the two females, this may suggest a causal relationship between the der(X) chromosome and clinical features.

The salient feature of this female is tall adult height surpassing her TH/TR. Two factors are noteworthy for the tall stature. One is the SHOX duplication on the der(X) chromosome, because SHOX has the dosage effect on the adult height (11). The other is gonadal dysgenesis, because gonadal estrogen deficiency permits a prolonged growth period. In this regard, comparison of the mean adult height among Caucasian females with 47,XXX accompanied by three copies of SHOX and relatively well preserved estrogen production (167.9 ± 7.7 cm; n = 19) (11), those with 46,XX gonadal dysgenesis accompanied by two copies of SHOX and gonadal estrogen deficiency (164.3 ± 7.7 cm; n = 22) (12), and normal Caucasian females accompanied by two copies of SHOX and gonadal estrogen production (162.2 ± 6.0 cm; the British standard) (13) suggests that each factor alone is insufficient to explain the tall stature of this female. This idea would also be supported by comparison of the mean adult height among Caucasian males with 47,XXY accompanied by three copies of SHOX and relatively well preserved androgen (and resultant estrogen) production (178.3 ± 7.2 cm; n = 85) (11), those with 46,XY gonadal dysgenesis accompanied by two copies of SHOX and gonadal steroid deficiency (172.0 ± 7.0 cm; n = 24) (12), and normal Caucasian males accompanied by two copies of SHOX and gonadal androgen (and resultant estrogen) production (174.7 ± 6.7 cm; the British standard) (13). By contrast, Caucasian females with apparently nonmosaic 46,X, idic(Xq-) and primary amenorrhea untreated until their late teens (>17 yr of age) exhibit tall stature with probably three copies of SHOX and gonadal estrogen deficiency (174.8 ± 4.6 cm; n = 5) (14, 15, 16, 17, 18). Thus, the tall stature of this female appears to be due to the combined effects of the two factors.

It should be pointed out, however, that another growth-related gene(s) escaping X-inactivation might be present on Xp (11, 19). If such a gene(s) may have been duplicated on the der(X) chromosome, this would also be relevant to the tall stature of this female. This possibility awaits further investigations.

Her growth pattern is also noteworthy. She continued to grow with a nearly constant height velocity from childhood through the pubertal age of normal children. The unique growth pattern is reminiscent of that in estrogen resistance or aromatase deficiency, which lacks biological effects of both gonadal and extragonadal estrogens (20, 21). This may suggest that an extra copy of SHOX has a potential to override the growth-suppressing effect of extragonadal estrogens. In support of the possible counteraction between SHOX and estrogens, it has been reported that Turner skeletal features caused by SHOX haploinsufficiency are often associated with radiologically discernible premature fusion of growth plates (22, 23). This implies that SHOX functions as a repressor for growth plate fusion, in contrast to estrogens with a skeletal maturing effect. The combination of an extra copy of SHOX and gonadal estrogen deficiency may, therefore, represent the further clinical entity for tall stature resulting from continued growth in late teens or into adulthood.

For the statural growth, the mosaicism should also be considered. As a 45,X karyotype was detected in 40% of lymphocytes, this raises the question of why she had tall stature under such a mosaicism. However, the mosaic cell ratio in lymphocytes would not reflect that in other tissues such as skeletal tissues (24). As the 45,X cell lineage is attributable to mitotic instability of the der(X) chromosome, it would be more frequent in rapidly dividing cells such as lymphocytes than in slowly dividing cells such as bone marrow fibroblasts. Thus, it is possible that cells with the der(X) chromosome account for a fairly large portion of the skeletal tissues of this female. As an extreme example of this, Mirzayants and Baranovskaya (25) have reported a 23-yr-old female with 45,X/46,X, idic(X)(q27), primary amenorrhea, and relatively tall stature [173 cm, 1.8 SD] in whom a 45,X karyotype has been detected in 80% of lymphocytes.

In summary, although several important data, such as bone age and serum GH and insulin-like growth factor I levels, were not available in this female, the results suggest that the combination of an extra copy of SHOX and gonadal estrogen deficiency leads to tall stature because of continued growth during the pubertal age of normal children. Further studies in similarly affected individuals will serve to clarify the statural effect of an extra copy of SHOX with gonadal estrogen deficiency.

	Acknowledgments

 
We thank Mr. Hiroshi Terasaki, Mitsubishi Kagaku Bioclinical Laboratories, for technical assistance.

	Footnotes

 
¹ This work was supported in part by the Keio University Medical Science Fund and the Pharmacia & Upjohn, Inc. Fund for Growth and Development Research.

Received February 10, 2000.

Revised May 17, 2000.

Accepted May 17, 2000.

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