Patients with SHOX mutations present a broad phenotypic variability. SHOX mutations are very frequent in LWD (89%), in opposition to ISS (3.2%) in our cohort. The use of SH/H SDS as a selection criterion increases the frequency of SHOX mutation detection to 22% and should be used for selecting ISS children to undergo SHOX mutation molecular studies.

SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA). Forty-four patients (8 LWD and 36 DSS) were analyzed. The cosmid LLNOYCO3'M'34F5 was used as a probe for the FISH analysis and microsatellite analysis were performed using three intragenic microsatellite markers. MLPA was performed using commercial kits. Twelve patients (8 LWD and 4 DSS) had deletions in SHOX area detected by MLPA and 2 patients generated discordant results with the other methodologies. In the first case, the deletion was not detected by FISH. In the second case, both FISH and microsatellite analyses were unable to identify the intragenic deletion. In conclusion, MLPA was more sensitive, less expensive and less laborious; therefore, it should be used as the initial molecular method for the detection of SHOX gene deletion.

The homozygous mutation S226I in WSXWS-like motif of GHR causes GH insensitivity. The decrease in IGF-I and IGFBP-3 levels after GnRHa therapy, which was not reversed with DHT administration, suggests that sex steroids have, through oestradiol, a GH-independent action on IGF-I and IGFBP-3 levels. A direct effect of GnRHa on GH secretion cannot be excluded.

Considering that no mutations were found in the present cohort with NS-related signs, it is unlikely that mutations would be found in unselected ISS children. The van der Burgt et al. criteria are adequate in attaining NS diagnosis and selecting patients for molecular studies. Mutations in the PTPN11 gene are commonly involved in the pathogenesis of NS but are not a common cause of ISS.

The IGF-I generation test has been proposed to select patients with GH insensitivity. Studies have shown that children with idiopathic short stature and GH deficiency, who were expected to be sensitive to GH, presented absent IGF-I and IGF binding protein (IGFBP)-3 responses and/or discordant results at IGF-I and IGFBP-3 generation test for unknown reasons. To assess the reproducibility of the generation test, we studied a group of 12 prepubertal children with short stature and normal GH secretion in whom defects in coding region of GH receptor gene were ruled out. All patients underwent the test twice. Discordant responses between the first and second test were found in five and six patients for IGF-I and IGFBP-3, respectively. When the results of the generation tests were compared, one notices that IGF-I generation has more concordant positive results than IGFBP-3. In eight generation tests that showed discordant results between IGF-I and IGFBP-3, seven presented a positive IGF-I with a negative IGFBP-3 response. Taking both tests into account, all children presented a normal IGF-I generation. Our findings showed that IGF-I and IGFBP-3 generation test was not reproducible in children that should have responded to GH stimulation. We suggest that, when IGF-I and IGFBP-3 levels fail to respond in the generation test, another test should be performed to confirm GH insensitivity.