Growth failure is a problem for all girls with Turner syndrome (TS), with untreated individuals achieving an average adult stature 20 cm shorter than their peers¹⁾. Growth hormone (GH) treatment is now a standard of care for girls with TS²⁾, which ameliorates growth failure and improves final adult height (FAH).

Cochrane database review in 2007³⁾ reported that GH doses between 0.3 to 0.375 mg/kg/wk increased short-term growth in girls with TS by approximately 3 cm (2 cm) in the first (second) year of treatment, and that mean FAH in 61 GH treated women was 148 cm compared to 141 cm in 43 untreated women. Despite GH therapy, girls treated with GH are still substantially shorter than other women as adults.

In the literature, factors affecting FAH include young age and bone age (BA) delay at the start of GH treatment, GH dose, duration of GH treatment, maternal X chromosome origin, target height, and good first-year height response to GH treatment^4,5,6,7,8). Growth response is known to decrease over the years of GH treatment^8,9,10). Good first-year response to GH therapy is important in determining FAH. However, in order to achieve FAH above the third percentile of the general population and height compatible with one's genetic target height, good response to GH needs to be maintained. Increments in GH dose generate better short- and long-term growth response^7,8), thus identifying groups at risk of poor GH response and increasing doses accordingly is effective in terms of cost and safety.

Although one study reported that subjects with maternal X chromosome origin (Xm) had greater height gain during GH treatment than those with paternal X chromosome origin (Xp)⁶⁾, the issue as to whether X-linked imprinting affects the GH response remains controversial. The phenotypic difference between Xp and Xm groups may reflect sexual dimorphism, because 46, XY males have only Xm. However, the role of Y chromosome sequences (Yseq) needs to be considered to explain the sexual dimorphism. To our knowledge, there has not yet been studies comparing GH responses in TS patients with Y chromosome sequences (Xm_Yseq) with 45,Xp or 45,Xm patients.

We analyzed TS patients with 45,X and/or 46,X,mar(Y) karyo-types to evaluate whether parental origin of the intact X chromosome and/or the presence of Yseq affects GH response during 3 years of therapy and plays a role in decreased GH response.

This study was approved by the Seoul National University Hospital Ethics Committee. Informed consent was obtained from both the parents and children. All patients had confirmed karyotyping of 50 lymphocytes at the time of diagnosis of TS. Fifty-five TS patients (45,X, n=37; 45,X/46,X,+mar, n=18) were enrolled in genetic analyses to detect for Yseq and to determine parental origin of the intact X chromosome. Complete height data at the first, second and third year after start of GH therapy was available for analysis in a total of 33 patients (45,X, n=25; 45,X/46,X,+mar(Y), n=8). No patients took oral estrogen during these 3 years of GH therapy.

The mean baseline Z_Ht at the start of GH therapy was 1.26±1.04 (Z_Ht_Lyon) and -2.3±0.81 (Z_Ht_07Kor). The CA was 8.5±3.1 years and the BA was 7.5±3.2 years. The patients were started on GH treatments at a dose of 0.97±0.16 U/kg/wk (range, 0.47-1.4). The average GH dosage for all 3 years of treatment was 0.94±0.15 U/kg/wk (range, 0.54-1.34).

When baseline characteristics of the 3 groups (Xp, Xm, and, Xm_Yseq group) were analyzed, there were no differences in baseline CA or BA. The significant difference in baseline Z_Ht_Lyon between the 3 groups was found (0.75±0.61 vs. 1.08±0.74 vs. 2.21±1.39, P=0.047 by Welch's ANOVA, Table 1). While there was no significant difference in baseline Z_Ht_Lyon between the Xp and Xm groups, the Xm_Yseq group had a higher baseline Z_Ht_Lyon than the Xm group, after adjusting for BA delay and Z_MPH (P=0.04) (Fig. 1).

Z_Ht_Lyon significantly increased during 3 years of GH treatment (P<0.001) (Fig. 2A, Table 2). While Z_Ht_Lyon significantly increased by 0.69 in the first year (P<0.001) and by 0.38 in the second year of GH treatment (P=0.003), the third-year increase of 0.11 was not statistically significant (P=0.99). The average dose (IU/kg/wk) of GH was 0.97±0.16 (0.5-1.4), 0.92±0.17 (0.48-1.27), and 0.93±0.17 (0.64-1.35) during the first, second and third years of treatment, respectively. However, there was no difference between the 3 groups (Xp, Xm, and Xm_Yseq) as to the overall pattern of change in Z_Ht_Lyon with GH treatment (P=0.536) (Fig. 2A, Table 2). The differences in the mean Z_Ht_Lyon values during the 3 years (Xp vs. Xm_Yseq, P=0.004; Xm vs. Xm_Yseq, P=0.013) were no longer significant, after adjusting for baseline Z_Ht_Lyon, CA at the start of GH, BA delay, GH dose and Z_MPH (P=0.715) (Fig. 2A).

The proportion of poor responders (△Z_Ht_Lyon<0.1) increased progressively from the first, second and third years of treatment by 9.1%, 27.3%, and 51.5% respectively (Fig. 2B). The percentage of short patients (Z_Ht_07Kor<-1.88) declined successively from 69.7% at the time of GH commencement to 54.6% and 48.4% during the first and second years, but increased to 76.7% during the third year of treatment (Fig. 2C). These 25 short patients (76.7%) after 3 years of GH treatment included 8 (53.3%) of 15 patients who were consistently good responders for all 3 years, 12 of 13 patients (92.3%) who became poor responders in the third year of treatment, and all 5 patients (100%) who had remained poor responders for all 3 years (P=0.011, chi-square test for trend).

To identify groups at risk of poor GH response, factors affecting the third year response to GH (third year △Z_Ht_Lyon) were analyzed. In simple linear regression analysis, the third year △Z_Ht_Lyon was negatively related to the second year △Z_Ht_Lyon (P=0.040), and positively associated with the △second and third year GH dose (P=0.011), respectively (Table 3). However, the origin of the intact X and/or the presence of Yseq did not have a significant effect on the third year △Z_Ht_Lyon (Xm vs. Xp, P=0.98; Xm vs. Xm_Yseq, P=0.69) (Table 3). No significant correlation between the second year △Z_Ht_Lyon and the △second year and third-year GH dose was observed (P=0.51). The modifying effect of the second-year △Z_Ht_Lyon on the association of △second year and third-year GH dose with the third-year △Z_Ht_Lyon was not significant (P for interaction=0.15). In multivariate linear regression analysis, the only factor found to contribute to the third-year △Z_Ht_Lyon was the △second year and third-year GH dose (P=0.017) (Fig. 3).

The mean baseline Z_Ht did not differ according to Xp or Xm origin, however the mean baseline Z_Ht was higher in the Xm_Yseq group than in Xm group, after adjusting for BA delay and Z_MPH. When this difference in baseline Z_Ht was accounted for, there was no difference between the 3 groups in the response to 3 years of GH therapy. The height response to GH therapy decreased progressively each year, such that the increase in Z_Ht was not significant during the third year of therapy. This third-year decrease in treatment response was unaffected by parental origin of X chromosome and/or presence of Yseq. The response to GH treatment during the third year was positively correlated with the increase in GH dose from the second to third year.

In contrast to several autosomal genes identified in humans and X-linked imprinted genes in mice, an X-linked imprinted gene has not been identified in humans. Phenotypic differences between TS patients with Xm and those with Xp suggest the presence of X-linked imprinted genes in humans. Zinn et al.¹⁵⁾ suggested that a stature-determining critical region can be mapped between Xp22.1 and Xp11.2, which is located more proximally than short stature homeobox (SHOX) gene. Candidate imprinted regions on the X chromosome may be located in genes showing extensively variable expression between individual inactivated X chromosomes¹⁶⁾ rather than in pseudoautosomal regions. Previous studies^6,17) have been published on the contribution of the parental origin of the intact X chromosome to the response to GH treatment. Hamelin et al.⁶⁾ first reported a significantly greater gain in height during a 5- to 6-year period of GH therapy in Xm than in Xp TS females. A report in patients with Klinefelter syndrome showed that growth parameters were greater in an extra Xp group than in an extra Xm group¹⁸⁾. However, the imprinting effect in patients with TS or Klinefelter syndrome has not been replicated convincingly, and additional studies are needed to confirm or exclude X chromosomal imprinting. In our study, we included only 45,X patients without hidden X or Y chromosome sequences (pure Xp or Xm) to exclude the effect of different karyotype on the growth of TS patients. Our results show no difference in baseline Z_Ht of Xp and Xm groups as well as no differences in the response to 3 years of GH therapy.

The phenotypic difference between Xp, Xm and Xm_Yseq groups may reflect sexual dimorphism. There have not been previous studies that compared phenotypic differences in 3 groups of TS patients. In our study, changes in height of the 3 groups without missing data during 3 years of GH therapy were analyzed. Although Xm_Yseq patients had greater baseline Z_Ht than Xm and Xp patients, there was no significant difference in the response to GH therapy. Despite the fact that height is a multifactorial trait affected by genetic and environmental factors, the greater baseline height of Xm_Yseq over Xm suggests that the Y chromosome harbors genes that affect height. This hypothesis is also supported by the observations that XY females are generally taller than XX females, that XY males are taller than XX males¹⁹⁾, and that patients with pure XX or XY gonadal dysgenesis show height differences despite having gonadal steroid deficiency²⁰⁾. Correlations between breakpoints on Yq and the height of respective patients assigned the growth controlling gene on the Y chromosome (GCY) locus to the pericentromeric region of Yq11²¹⁾. Kirsch et al.²²⁾ confirmed the proximal interval between markers DYZ3 and DYS11 as the GCY critical interval on Yq using detailed Fluorescence in situ hybridization and high density sequence-tagged sites (STS) analysis. DYS 11 is located between DYS 270 and DYS 280, which STS were included in our analysis. The marker chromosome of the Xm_Yseq patients in this study contained the Y chromosome short arm (Yp) and the DYS 270 locus, making it likely that the GCY critical interval is also included. Previous reports indicate GH-deficient men are more responsive to GH therapy than are women²³⁾. Thangavel and Shapiro²⁴⁾ proposed that the cellular actions of GH, normally mediated by activation of the Jak2/Stat5B pathway, are suppressed in female cells compared with male cells. However, there was no difference in response to 3 years of GH therapy between the Xp, Xm and Xm_Yseq groups when adjusted for differences in baseline Z_Ht in our study.

When the response to GH therapy during the third year was assessed, there was no significant increase in Z_Ht during this period with more than 50% of patients being poor responders. The percentage of short patients smaller than the third percentile (Z_Ht_07Kor<-1.88) increased from 69.6% at the commencement of GH therapy to 76.7% during the third year. Starting in the year 2000, the insurance coverage guidelines of the Korean Ministry of Health and Welfare recommended GH doses of 1.0 U/kg/wk up to a height of 150 cm. An additional provision to increase this dosage to a maximum of 1.4 U/kg/wk was added in the year 2010 for those patients with poor response up to a height of 150 cm. The patients in this study received GH according to the guidelines before 2010, so there was a restriction in the range of GH increments. Although there was a general tendency to increase the GH dosage with poor response during the previous year, the incremental range was too small to show a significant correlation between previous response to GH therapy and next year GH dose increment. The response to GH therapy progressively declined so that there was no significant response during the third year of therapy. The decrease in response did not differ between Xp, Xm, and Xm_Yseq groups. The change in GH dosage from the second to third year of treatment was the only significant predictor influencing the third-year growth response. A recent study proposed that the dose increments in the second and third years of treatment in patients with poor response during the previous year attenuated the normal decline in GH response⁸⁾. A study of step-up dosage regimes published by van Pareren et al.¹⁰⁾ showed that dose increments had a significant effect on final height, although the effect was discernible only after 4-7 years. GH dosage needs to be increased at the third year without regard to the response during the previous year or parental origin of X chromosome and/or Yseq.

This study included only those TS patients with pure 45,X and 45,X/46,X,+mar(Y) karyotypes who had complete height data throughout 3 years of GH therapy and is therefore limited by the small sample size. Additionally, the effect of GH therapy on FAH was not included in this study. Once patients reached a height of 150 cm, Korean national insurance guidelines did not provide GH coverage. GH treatments were usually discontinued even though FAH was not attained, making analysis of the GH response in determining FAH difficult. Despite limitations, this is the first study to assess three year GH response in TS patients including pure Xp, pure Xm homogenous subjects and in those with Xm_Yseq karyotypes.

In conclusion, there was no evidence of X-linked imprinted genes and/or Yseq affecting height response to three years of GH therapy. Without regard to the parental origin of intact X chromosome and/or presence of Yseq and GH response during the previous year, increasing GH dosages may help attenuate the decrease of the third year response to GH therapy in TS patients with 45,X and/or 46,X/mar(Y).