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Turner Syndrome and Xp Deletions: Clinical and Molecular Studies in 47 Patients

Department of Pediatrics, Keio University School of Medicine, (T.O., K.M., N.M.), Tokyo 160-8582, Japan; Tokyo Electric Power Co. Hospital, (T.O., K.M.), Tokyo 160-0016, Japan; Tokai University School of Medicine (O.S.), Isehara 259-1193, Japan; Kyoto University School of Medicine (T.Y.), Kyoto 606-8507, Japan; Hiroshima Red-Cross Hospital (Y.N.), Horoshima 730-8619, Japan; Division of Endocrinology and Metabolism, Kiyose Children’s Hospital (Y.H.), Kiyose 204-0024, Japan; National Children’s Hospital (R.H.), Tokyo 154-8509, Japan; and Kanagawa Children’s Medical Center (K.T.), Yokohama 232-8555, 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

Abstract

Although clinical features of Turner syndrome have primarily been explained by the dosage effects of SHOX (short stature homeobox-containing gene) and the putative lymphogenic gene together with chromosomal effects leading to nonspecific features, several matters remain to be determined, including modifying factors for the effects of SHOX haploinsufficiency, chromosomal location of the lymphogenic gene, and genetic factors for miscellaneous features such as multiple pigmented nevi. To clarify such unresolved issues, we examined clinical findings in 47 patients with molecularly defined Xp deletion chromosomes accompanied by the breakpoints on Xp21–22 (group 1; n = 19), those accompanied by the breakpoints on Xp11 (group 2; n = 16), i(Xq) or idic(X)(p11) chromosomes (group 3; n = 8), and interstitial Xp deletion chromosomes (group 4; n = 4). The deletion size of each patient was determined by fluorescence in situ hybridization and microsatellite analyses for 38 Xp loci including SHOX, which was deleted in groups 1–3 and preserved in group 4. The mean GH-untreated adult height was -2.2 SD in group 1 and -2.7 SD in group 2 (GH-untreated adult heights were scanty in group 3). The prevalence of spontaneous breast development in patients aged 12.8 yr or more (mean ± 2 SD for B2 stage) was 11 of 11 in group 1, 7 of 12 in group 2, and 1 of 7 in group 3. The prevalence of wrist abnormality suggestive of Madelung deformity was 8 of 18 in group 1 and 2 of 23 in groups 2 and 3, and 9 of 18 in patients with spontaneous puberty and 1 of 23 in those without spontaneous puberty. The prevalence of short neck was 1 of 19 in group 1 and 7 of 24 in groups 2 and 3. Soft tissue and visceral anomalies were absent in group 1 preserving the region proximal to Duchenne muscular dystrophy and were often present in groups 2 and 3 missing the region distal to monoamine oxidase A (MAOA). Multiple pigmented nevi were observed in groups 1–3, with the prevalence of 0 of 7 in patients less than 10 yr of age and 15 of 36 in those 10 yr or older regardless of the presence or absence of spontaneous puberty. Turner phenotype was absent in group 4, including a fetus aborted at 21 wk gestation who preserved the region distal to MAOA.

The results provide further support for the idea that clinical features in X chromosome aberrations are primarily explained by haploinsufficiency of SHOX and the lymphogenic gene and by the extent of chromosome imbalance in mitotic cells and pairing failure in meiotic cells. Furthermore, it is suggested that 1) expressivity of SHOX haploinsufficiency in the limb and faciocervical regions is primarily influenced by gonadal function status and the presence or absence of the lymphogenic gene, respectively; 2) the lymphogenic gene for soft tissue and visceral stigmata is located between Duchenne muscular dystrophy and MAOA; and 3) multiple pigmented nevi may primarily be ascribed to cooperation between a hitherto unknown genetic factor and an age-dependent factor other than gonadal E.

Turner syndrome is a well defined sex chromosomal disorder characterized by short stature, gonadal dysgenesis, and somatic stigmata (1). Characteristic somatic stigmata can be classified into four groups: 1) skeletal anomalies, such as short metacarpals, cubitus valgus, Madelung deformity, high arched palate, and short neck; 2) soft tissue anomalies attributable to lymphatic obstruction, such as webbed neck, low posterior hairline, lymphedema, redundant skin, and nail dysplasia; 3) visceral anomalies, such as aortic coarctation and horseshoe kidney; and 4) miscellaneous anomalies, such as multiple pigmented nevi (1, 2).

The underlying factors for the development of Turner syndrome are being clarified by clinical and molecular studies. First, it has been shown that haploinsufficiency of SHOX (short stature homeobox-containing gene) (3), also known as PHOG (pseudoautosomal homeobox-containing osteogenic gene) (4), cloned from the short arm pseudoautosomal region causes not only short stature, but also characteristic skeletal features such as short metacarpals, cubitus valgus, Madelung deformity, high arched palate, and short neck (3, 5, 6, 7, 8, 9). In this regard, as limb skeletal features have occurred in a female-dominant and pubertal tempo-influenced fashion in normokaryotypic individuals with SHOX abnormalities and normal gonadal function, it has been suggested that gonadal E exerts a maturational effect on skeletal tissues that are susceptible to unbalanced premature fusion of growth plates because of SHOX haploinsufficiency, facilitating the development of limb skeletal lesions (7). Second, it has been proposed that gonadal dysgenesis is primarily ascribed to meiotic pairing failure of homologous chromosomes (2, 10). Indeed, the degree of gonadal dysfunction in sex chromosome aberrations is inexplicable by the dosage effect of genes on the sex chromosomes, but is well correlated with the extent of meiotic pairing failure (the size of unpaired region) (2, 10). Third, it has been suggested that lymphatic hypoplasia, a demonstrated malformation in Turner syndrome (11, 12), results in lymphatic distension and lymphedema because of lymph fluid stasis, leading to soft tissue and visceral anomalies by exerting deformational effects on tissues and/or organs adjacent to the lymphatic system (2, 13). As soft tissue and visceral stigmata are frequently observed in patients with Xp and Yp deletions (14), such stigmata have been explained as a malformation sequence initiated by haploinsufficiency of an Xp-Yp homologous lymphogenic gene(s) (2). Fourth, chromosome imbalance (quantitative alteration of euchromatic or noninactivated region) would also contribute to the development of Turner syndrome (2). It has been suggested that chromosome imbalance disturbs developmental homeostasis, resulting in global nonspecific defects such as growth failure and anomalous features shared by various aneuploidies (15, 16, 17). Thus, phenotype in Turner syndrome would be ascribed to both gene dosage effects responsible for characteristic features and chromosomal effects leading to nonspecific features shared by various aneuploidies. Furthermore, clinical consequences of several features would influence the development of other features (2). For example, as limb skeletal features in SHOX haploinsufficiency are facilitated by the skeletal-maturing effect of gonadal E in normokaryotypic patients (7, 18), gonadal function status would also influence the growth pattern and limb skeletal features in Turner patients missing SHOX. Similarly, the severity of faciocervical skeletal features such as short neck would also influence statural growth.

However, several matters remain to be determined for the underlying factors involved in the development of Turner syndrome. First, it remains to be clarified for SHOX haploinsufficiency whether limb skeletal features are actually influenced by gonadal function status in patients with X chromosome aberrations as well as in normokaryotypic patients, and whether faciocervical skeletal features are facilitated by some modifying factor(s). Second, the precise chromosomal location of the lympogenic gene has not been defined. Third, it is unknown whether miscellaneous features such as multiple pigmented nevi are explained by the previously proposed idea, such as the malformation sequence initiated by haploinsufficiency of the lymphogenic gene or ascribed to a hitherto unidentified genetic factor(s). Lastly, the hypothesis that clinical features in X chromosome aberrations are primarily explained by haploinsufficiency of SHOX and the lymphogenic gene and by extent of chromosome imbalance in mitotic cells and pairing failure in meiotic cells, which has primarily been based on the thorough literature analysis (2), should be evaluated by detailed studies in patients with X chromosome aberrations. Here, we report clinical and molecular findings in individuals with Xp deletions and discuss these unresolved issues.

Subjects and Methods

Patients

This study consisted of 47 Japanese female patients with cytogenetically recognizable X chromosome abnormalities involving Xp. Appropriate informed consent was obtained from each patient or her parents. The G-banding karyotype and age of each case are shown in Table 1. The karyotype has been described according to the International System for Human Cytogenetic Nomenclature (19). For example, del(X)(p11.2) denotes a terminal Xp deletion with breakage at Xp11.2 (deletion of a region distal to Xp11.2), del(X)(p11.2p11.4) indicates an interstitial Xp deletion with breakage and reunion at Xp11.2 and Xp11.4 (deletion of a region between Xp11.2 and Xp11.4), i(Xq) shows an isochromosome for Xq with breakage around the centromeric region (deletion of nearly entire Xp and duplication of whole Xq), psu idic(X)(p11.2) represents an isodicentric chromosome with a single active centromere resulting from breakage and reunion at Xp11.2 (deletion of a region distal to Xp11.2 and duplication of a region from Xp11.2 to Xq telomere), der(X)t(X;Y)(p22.3;q11) depicts a derivative X chromosome resulting from unbalanced translocation between Xp22.3 and Yq11 (deletion of a region distal to Xp22.3 and addition of a region distal to Yq11), der(X)t(X;2)(p22;p21) denotes a derivative X chromosome resulting from unbalanced translocation between Xp22 and 2p21 (deletion of a region distal to Xp22 and addition of a region distal to 2p21), and der(X) represents a derivative X chromosome caused by complex rearrangement within an X chromosome (in this study, deletion of a partial Xp region accompanied by other X chromosomal rearrangement).

Clinical assessment

Clinical assessment was performed for statural growth, gonadal function, and somatic stigmata. Statural growth was evaluated by the longitudinal growth standard for the Japanese females (25). When possible, target height (TH) was obtained from the equation of Ogata et al. (26) (a modified Tanner’s equation for the Japanese with a positive height secular trend; TH is not obtained in patients with familial Xp deletions, because it predicts the adult height of a child born to normal parents). Gonadal function was assessed for breast development, menarchial age, and fertility. Breast development and menarchial age were evaluated by those of normal Japanese girls (Tanner stage: B2, 10.0 ± 1.4; B3, 11.6 ± 1.5; B4, 13.3 ± 1.5; B5, 14.2 ± 1.2 yr; menarchial age: 12.25 ± 1.25 yr; mean ± SD) (Ref. 27 and our unpublished observation). Somatic stigmata were primarily assessed by physical examinations. In addition, radiographs of the hands and wrists obtained for bone age determination were used for the assessment of short metacarpals and phalanges and for wrist abnormalities characteristic of Madelung deformity, such as decreased carpal angle, metaphyseal lucency and/or epiphyseal hypoplasia at the ulnar side of the distal radius, and angulation of the distal radius and/or ulna (18, 28). Furthermore, ultrasound studies were performed for the evaluation of cardiac and renal anomalies together with standard examinations such as auscultation, electrocardiograms, and routine laboratory tests. In case 44, an autopsy was carried out after abortion at 21 wk gestation.

Statistical analysis was performed for patient populations consisting of five or more cases. The results for distributions are expressed as the mean ± SD. The statistical significance of the mean was examined by t test, and that of the prevalence was analyzed by Fisher’s exact probability test. P 0.05 was considered significant.

Deletion analysis

Deletion maps were constructed by FISH and microsatellite analyses for 38 loci shown in Fig. 1. For FISH analysis, metaphase spreads were prepared from lymphocytes or lymphoblastoid cell lines in cases 1–43 and 45–47 and from skin fibroblasts in case 44 and were hybridized with probes defining 13 loci from the Xp telomere region (Xptel) to the X centromere region (DXZ1). In all of the FISH studies, a probe for DXZ1 or the Xq telomere region (Xqtel) was concomitantly hybridized to metaphase spreads as an internal signal control. The probes for Xptel, Xqtel, and DXZ1 were purchased from Vysis (http://www.vysis.com/) and were detected according to the manufacturer’s protocol. The remaining probes were prepared by Mitsubishi Kagaku Bioclinical Laboratories (Tokyo, Japan) and were labeled with digoxigenin and detected by rhodamine antidigoxigenin or were labeled with biotin and detected by avidin conjugated to fluorescein isothiocyanate.

View larger version (46K): [in this window] [in a new window]   Figure 1. Chromosomal location of examined loci on Xp. The left side ideogram shows the G-banding pattern of the X chromosome short arm. The chromosomal position of loci examined by FISH analysis is based on the report of the Sixth International X Chromosome Workshop (29 ), and the order of loci investigated by microsatellite analysis is based on the LDB map (http://cedar.genetics.soton.ac.uk/pub/chromX/map.html). Xptel, SHOX, and MIC2 reside in the short arm pseudoautosomal region, and the remaining loci lie in the X-differential region.

Results

Clinical assessment

Clinical features in each case are summarized in Table 1. Statural growth was variable among cases 1–43. The SD score ranged from -0.9 to -5.6 for actual height (AH) in patients of various ages and for adult height in patients older than 20 yr of age or confirmed to stop growing (growth rate, <0.5 cm/yr). The difference between the SD score for AH and that for TH ranged from -0.9 to -5.1 in patients of various ages and from -0.9 to -4.4 in patients attaining the adult height. GH therapy had been performed in 20 of 37 cases (6 of the 43 cases had attained the adult height before GH therapy became widely available). In GH-untreated patients, the AH SD score was similar between groups 1 and 2 [-2.5 ± 0.9 (n = 12) vs. -2.7 ± 1.4 (n = 5)] as was adult height SD score [-2.2 ± 1.0 (n = 6) vs. -2.7 ± 1.4 (n = 5); GH-untreated patients were scanty in group 3].

Gonadal function was diverse in cases 1–43. In patients older than 12.8 yr (mean ± 2 SD for B2 stage), the prevalence of spontaneous breast development (B2–B5) was more frequent in group 1 than in group 2 (11 of 11 vs. 7 of 12; P < 0.05) and, though not statistically significant, tended to be higher in group 2 than in group 3 (7 of 12 vs. 1 of 7; P = 0.08). In patients older than 14.6 yr (mean ± 2 SD for B3 stage), the prevalence of spontaneous breast development (B3–B5) was similar between group 1 and group 2 (8 of 8 vs. 4 of 5) and was more frequent in group 2 than in group 3 (4 of 5 vs. 0 of 5; P < 0.05). In patients older than 14.75 yr (mean ± 2 SD for menarchial age), the prevalence of spontaneous menarche was 8 of 8 in group 1 (in addition, 2 cases younger than 14.75 yr also experienced menarche), 5 of 9 in group 2, and 0 of 4 in group 3, with statistical significance between groups 1 and 2 (P = 0.05). Menarchial age SD score was +0.9 ± 1.5 for 15 cases with menarche in groups 1 and 2 and was similar between groups 1 and 2 [+0.9 ± 1.6 (n = 8) or +1.0 ± 1.4 (n = 10) vs. +0.6 ± 1.8 (n = 5)]. Fertility was confirmed in four cases in group 1 and 1 case in group 2. Case 24 had secondary amenorrhea. Hormone replacement therapy had been started in 7 cases.

Somatic features were variable in cases 1–43. Limb skeletal anomalies were exhibited by 30 cases in groups 1–3, and 20 of them had plural limb skeletal features. Short metacarpals and phalanges were severe in case 28, and wrist abnormalities suggestive of Madelung deformity were indicated in 10 cases (Fig. 2); however, overt bayonet sign or mesomelic appearance was absent in groups 1–3. Faciocervical skeletal features were manifested by 14 cases in groups 1–3, and 5 of them had plural faciocervical skeletal features. Both limb and faciocervical skeletal features were identified in 12 cases. Soft tissue anomalies were found in 14 cases in groups 2 and 3, and 9 of them had plural soft tissue stigmata. The expressivity was mild, especially for webbed neck, which appeared obvious only in 3 cases and mild to borderline in 9 cases. Visceral anomalies were detected in only 2 cases with soft tissue anomalies (renal hypoplasia in case 27 and horseshoe kidney in case 42). Multiple pigmented nevi were found in 15 cases of groups 1–3 regardless of the presence or absence of skeletal or soft tissue anomalies.

View larger version (70K): [in this window] [in a new window]   Figure 2. Representative radiographs of the hand and wrists. A, Left hand of case 28 at 14 yr of age, showing short metacarpals and phalanges. In particular, the fifth metacarpal is severely affected. B, Left hand of case 30 at 14 yr of age, showing decreased carpal angle, epiphyseal hypoplasia at the ulnar side of distal radius, and angulation of the distal radius and ulna.

Deletion maps

The Xp deletion maps of the abnormal X chromosomes are shown in Fig. 3, and representative FISH and microsatellite results are shown in Figs. 4 and 5, respectively. As a whole, the deletion sizes were small in group 1, as expected, and similar in groups 2 and 3. SHOX was deleted from abnormal X chromosomes in groups 1–3 and preserved in group 4. The region proximal to Duchenne muscular dystrophy (DMD) was preserved in all cases in group 1 without soft tissue or visceral stigmata, whereas the region distal to monoamine oxidase A (MAOA) was deleted in all cases with soft tissue and visceral stigmata from groups 2 and 3. The region distal to DAX-1 was lost in all cases with multiple pigmented nevi from groups 1–3. The region distal to SMCX was the largest deletion size identified in cases with normal breast development and menarche.

View larger version (115K): [in this window] [in a new window]   Figure 3. Xp deletion maps of the rearranged X chromosomes in the 47 patients. The case numbers correspond to those in Table 1. The black segments show definitely positive loci (positive FISH signals or 2 heterozygous microsatellite peaks). The striped segments denote presumably positive loci inferred from interpolation (single, probably homozygous, microsatellite peaks with no parental microsatellite results). The stippled segments indicate unexamined, but presumably positive, loci inferred from interpolation. The open segments indicate presumably negative loci inferred from interpolation (single, probably hemizygous, microsatellite peaks with no parental microsatellite results). ..., Definitely negative loci (negative FISH signals or single hemizygous microsatellite peaks confirmed by informative parental microsatellite results); · · ·, unexamined, but presumably negative, loci inferred from interpolation; ?, loci unknown for the presence or absence (unexamined or single microsatellite peaks with no informative parental microsatellite results).

View larger version (95K): [in this window] [in a new window]   Figure 4. Representative FISH results in case 20. A, MAOB (arrows) and DXZ1 (arrowheads). MAOB is present on the deleted X chromosome as well as on the normal X chromosome. B, OTC (arrow) and DXZ1 (arrowheads). OTC is lost from the deleted X chromosome and is preserved on the normal X chromosome.

View larger version (25K): [in this window] [in a new window]   Figure 5. Representative microsatellite results in case 44. A, DXS8054. The paternal marker is not inherited by the patient, and the maternal marker alone is transmitted to the patient, demonstrating hemizygosity of this locus in the patient. Peak size: 158 bp for the father, 162 bp for the patient and the mother. B, ALAS2. The patient is heterozygous with the paternally and maternally derived peaks. Peak size: 157 bp for the father, 157 and 161 bp for the patient, and 161 bp for the mother.

Statural growth

Statural growth was variable among cases 1–43 with SHOX haploinsufficiency. This would be consistent with stature in females with Xp deletions being subject to multiple genetic and environmental factors, including 1) gonadal function status influencing the expressivity of SHOX haploinsufficiency and the pubertal growth pattern (7, 30), 2) degree of growth disadvantage caused by chromosome imbalance (17), 3) original growth potential as represented by parental height (31), 4) presence or absence of GH treatment and, in patients treated with GH, height and age at the beginning of GH therapy and dosage and duration of treatment (32) (in this connection, there may be a bias that GH is administrated more preferentially to patients with severe short stature than to those with mild short stature), and 5) in patients receiving E replacement therapy, height and age at the beginning of therapy and the dosage of treatment (14). This implies that adequate assessment is difficult for statural growth in cases 1–43 with high heterogeneity for such growth-related factors. In this regard, as the effects of such factors other than the presence or absence of GH therapy are variable in GH-untreated patients, appropriate assessment is difficult for the similarity in the GH-untreated stature between groups 1 and 2. In addition, patient number appears to be insufficient to permit adequate statistical assessment. Thus, further studies are necessary for the evaluation of statural growth in females with Xp deletions.

Gonadal function

Gonadal function was well preserved in group 1, moderate in group 2, and severely affected in group 3. The results are consistent with the idea that the degree of gonadal dysfunction is correlated with the extent of pairing failure (2, 10), because the size of the unpaired region should be small in group 1, moderate in group 2, and large in group 3. For example, in 46,X,del(X)(p22) of group 1, X chromosome pairing would take place in the Xqtel–Xp22 region between the normal X and the del(Xp) chromosomes, leaving the Xp22–Xptel region of the normal X chromosome unpaired; in 46,X,del(X)(p11) of group 2, X chromosome pairing would occur in the Xqtel–Xp11 region between the normal X and the del(Xp) chromosomes, leaving the Xp11–Xptel region of the normal X chromosome unpaired; and in 46,X,i(Xq) of group 3, Xq pairing would take place between the normal X and the i(Xq) chromosomes, leaving the Xp of the normal X chromosome and the Xq of the i(Xq) chromosome unpaired, or the i(Xq) chromosome would form a self-pairing, leaving the whole normal X chromosome unpaired (2, 10) (thus, cases 9 and 11 in group 1 would have gonadal dysfunction in later age because of obviously severe pairing failure). By contrast, loss of X-linked genes appears to be irrelevant to gonadal dysfunction. Although the sizes of Xp deletions and corresponding loss of Xp genes are more severe in group 2 than in group 1, they are similar in groups 2 and 3. In addition, severe gonadal dysfunction in patients with apparently nonmosaic 46,X,idic(Xq–) accompanied by duplication of all the Xp genes would also argue against the relevance of loss of Xp genes to gonadal dysfunction (2, 10).

Menarche took place at relatively late age in most cases with spontaneous puberty, and secondary amenorrhea was present in case 24. This suggests that gonadal function is mildly affected even in patients with spontaneous menarche, because delayed puberty and premature ovarian failure are often manifested by patients with disorders of primary ovarian dysfunction, including sex chromosome aberrations (33).

Skeletal features

Skeletal features were variable among cases 1–43 with SHOX haploinsufficiency, and some patients exhibited plural features, whereas others had no features. In this regard human embryo studies have shown that SHOX is exclusively expressed in the developing limbs and in the first and second pharyngeal arches where Turner skeletal features are recognized (8). Thus, the diversity of skeletal features would be explained by assuming that SHOX haploinsufficiency results in various skeletal features when severely manifested, whereas it leads to no feature when subclinically manifested.

Limb skeletal anomalies, especially wrist abnormalities suggestive of Madelung deformity, were predominantly exhibited by patients with spontaneous puberty. This provides further support for the idea that gonadal E worsen skeletal anomalies by promoting unbalanced premature fusion caused by SHOX haploinsufficiency (7). In this context, the absence of overt Madelung deformity such as bayonet sign or mesomelic appearance would be ascribed to relatively late maturation in patients with Xp deletions, because Madelung deformity tends to be severe in early maturing girls who are exposed to gonadal E from a relatively early age (7). In addition, the presence of wrist abnormality in a prepubertal patient (case 3) implies that some factor(s) other than gonadal E is also operating as a modifier in the development of skeletal lesions (7). For limb skeletal features, although the prevalence was higher in patients 10 yr of age and older, and wrist abnormality was primarily manifested by patients with small Xp deletions (group 1), this would be due to the fact that pubertal development usually proceeds in teens and is more frequent in patients with small Xp deletions (Ref. 2 and this study).

Faciocervical skeletal anomalies, especially short neck, were more frequent in patients with relatively large Xp deletions (groups 2 and 3). This may suggest that cystic hygroma and facial edema exert a compressive effect on skeletal tissues primarily in fetal life, facilitating the development of faciocervical skeletal features under SHOX haploinsufficiency, because the putative lymphogenic gene is considered to be preserved in group 1 and deleted in groups 2 and 3 (see below; in this context, limb skeletal features in groups 2 and 3 may also be contributed by peripheral lymphedema). Although faciocervical skeletal anomalies were not necessarily associated with soft tissue anomalies, this would not pose a major problem. It is known that haploinsufficiency of genes involved in human development, such as SHOX and the lymphogenic gene, usually shows a wide range of expressivity and penetrance (34), and soft tissue stigmata may have been present at a subclinical level at the time of investigation because of resolution of lymphatic flow with age (2, 35, 36). In addition, chromosome imbalance might have raised the susceptibility to skeletal features by decreasing the developmental buffering effect against genetic and environmental forces (15, 16). In support of both possibilities, faciocervical skeletal features occur in 30–40% of 45,X Turner patients (1), but are rarely described in normokaryotypic patients with SHOX abnormalities (5, 6, 7, 8, 9). For faciocervical skeletal features, although the prevalence was higher in patients 10–20 yr of age and older, this appears to be incidental, because the prevalence was low in patients less than 10 yr of age and in those 20 yr or older.

Soft tissue stigmata and the lymphogenic gene

Soft tissue stigmata were variable, and some patients had plural stigmata, whereas other patients had no stigmata. This appears to be compatible with the idea that soft tissue stigmata are explained as the deformational consequences resulting from haploinsufficiency of the lymphogenic gene (2). It is assumed that haploinsufficiency of the lymphogenic gene causes plural soft tissue features when severely manifested, whereas it yields no feature when subclinically manifested.

Soft tissue stigmata were exclusively identified in groups 2 and 3. This implies that the lymphogenic gene resides in the proximal part of Xp (see below). Although the expressivity was low in most manifesting patients, this would primarily be due to spontaneous amelioration of lymphatic flow beginning from fetal life (35, 36). Indeed, soft tissue stigmata are relatively obvious during infancy and tend to resolve with age (1, 2). In this context, although the prevalence was not higher in patients less than 10 yr of age in the present study, the youngest patient in groups 2 and 3 was 8.5 yr old (case 22), so that lymphatic flow would have ameliorated at the time of examinations in most patients. In addition, as the prevalence of soft tissue stigmata is higher in patients with 45,X than in those with 46,X,del(X)(p11) (2, 37, 38), it may be possible that genes for lymphatic development exist on both Xp and Xq, so that loss of Xp gene alone causes relatively mild soft tissue stigmata. It may also be possible that chromosome imbalance contributes to the development of soft tissue stigmata by disturbing the developmental buffering effect against genetic and environmental insults (15, 16). For soft tissue stigmata, although the prevalence was higher in patients without spontaneous puberty, this would be due to the frequent association of gonadal dysfunction with large Xp deletions (Ref. 2 and this study).

The present study is informative for the localization of the lymphogenic gene on Xp. First, the region proximal to DMD was preserved in all patients in group 1 lacking soft tissue stigmata. Second, the region distal to MAOA was deleted in all patients with soft tissue stigmata from groups 2 and 3. Third, patients in group 4 had no soft tissue stigmata; especially, case 44, preserving the region distal to MAOA, had no soft tissue stigmata at 21 wk gestation when lymphatic obstruction stigmata should be the most prominent (35, 36). Collectively, these findings suggest that the lymphogenic gene resides between MAOA and DMD, is deleted in groups 2 and 3, and is preserved in groups 1 and 4. In addition, it has been reported that a female with an interstitial Xp deletion involving a region from adrenal hypoplasia congenita (AHC) to ornithine transcarbamylase (OTC) is free from soft tissue stigmata (39, 40). If the lymphogenic gene is preserved in this female, the critical region for the lymphogenic gene would further be narrowed to the region between MAOA and OTC in distal Xp11. In this context, it is noteworthy that the Xp11 region is one of the early replicating segments on the inactive X chromosome and contains multiple genes escaping X inactivation (41, 42). This regional property is consistent with the idea that the lymphogenic gene is an Xp-Yp homologous gene escaping X inactivation (2). According to the report of the sixth international X chromosome workshop (29), the size is estimated to be roughly 9 megabase for the region between MAOA and DMD and approximately 3 megabase for the region between MAOA and OTC. Thus, in conjunction with the previous localization of the lymphogenic gene on Yp (43), it is likely that the lymphogenic gene is a homologous gene shared by the Xp region from DMD to MAOA and the Yp region from PABY and DYS255.

Visceral anomalies

Visceral anomalies were found in two cases with soft tissue stigmata. This would be consistent with the concept that visceral stigmata are also caused by haploinsufficiency of the lymphogenic gene (2). The rarity of visceral anomalies is compatible with the previous reports that cardiovascular and renal anomalies are infrequent in patients with Xp deletions (2, 33, 38). Thus, it is likely that visceral organs are more resistant to the deformational effect initiated by lymphatic obstruction than soft tissues. In addition, as the prevalence of visceral stigmata is higher in 45,X patients than in those with large Xp deletions (2), this might also suggest the presence of another lymphogenic gene on Xq and/or the relevance of chromosome imbalance to the development of visceral anomalies.

Other stigmata

Multiple pigmented nevi were observed in groups 1–3 and were more frequent in patients 10 yr of age or older in the presence or absence of spontaneous puberty. This may suggest that a genetic factor other than the lymphogenic gene is cooperating with an age-dependent factor other than gonadal E in the development of multiple pigmented nevi. The relevance of the lymphogeneic gene would further be refuted by the finding that soft tissue stigmata usually resolve after infancy, whereas pigmented nevi usually appear from childhood (1, 2). The genetic factor remains unknown, but gene dosage effect or chromosome imbalance might be involved. In this context, if a gene(s) for pigmented nevi is present on the X chromosome, it would be assigned distal to DAX-1 by the findings of the present study. The age-dependent factor also remains unknown, but a factor common to normal populations, such as a somatic mutation (44), may be involved, because pigmented nevi also sometimes begin to appear from childhood or puberty in normal individuals (45).

Remarks and conclusion

Several points should be made with regard to the present study. First, the number of analyzed patients is still insufficient to draw a definite conclusion. Second, a selection bias may exist, because cytogenetic studies would be preferentially performed for patients with some Turner features. Third, several patients other than cases 19, 28, 36, 39, and 40 with demonstrable mosaicism may also have cryptic or tissue-specific mosaicism. Fourth, there might be undetected complex chromosomal abnormalities. Fifth, assessment of somatic stigmata is subjective, so that some features may be overlooked, and other features may be overestimated. Sixth, several features, such as micrognathia, shield chest, otitis media, autoimmune diseases, and cognitive dysfunction, remained unexamined. Lastly, the parent of origin effect also remained unexamined, although such an epigenetic imprinting effect is unlikely for growth failure, gonadal dysgenesis, and somatic stigmata (46, 47).

Despite the above caveats, the present study provides further support for the idea that clinical features in X chromosome aberrations are primarily explained by haploinsufficiency of SHOX and the putative lymphogenic gene and by the extent of chromosome imbalance in mitotic cells and pairing failure in meiotic cells. Furthermore, it is suggested that 1) expressivity of SHOX haploinsufficiency in the limb and faciocervical regions is primarily influenced by gonadal function status and the presence or absence of the lymphogenic gene, respectively; 2) the lymphogenic gene for soft tissue and visceral stigmata is located between DMD and MAOA on Xp; and 3) multiple pigmented nevi may primarily be ascribed to cooperation between a hitherto unknown genetic factor and an age-dependent factor other than gonadal E. These ideas await further studies, such as investigation of a large number of patients with Xp deletions and cloning of the lymphogenic gene.

We thank following clinicians for kindly providing us with the blood samples and clinical data: T. Hasegawa, E. Ogawa, Y. Nakagomi, M. Minagawa, N. Igarashi, O. Nose, K. Hanew, M. Ogawa, T. Aikawa, S. Uehara, M. Kamitomo, K. Aizu, and M. Adachi. We also thank Mr. M. Saito for technical assistance.

Footnotes

This work was supported in part by a grant for Pediatric Research from the Ministry of Health and Welfare; a grant-in-aid from the Ministry of Education, Science, Sports, and Culture; Keio University Medical Science Fund; and Pharmacia Fund for Growth and Development Research.

Abbreviations: AH, Actual height; AHC, adrenal hypoplasia congenita; DMD, Duchenne muscular dystrophy; FISH, fluorescence in situ hybridization; MAOA, monoamine oxidase A; OTC, ornithine transcarbamylase; TH, target height.

Received February 2, 2001.

Accepted August 7, 2001.

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