Background: Short stature is a common pediatric clinical condition and one of the most frequent reasons for referral to pediatric endocrinologist. Despite comprehensive clinical and laboratory evaluation, in the majority of cases the underlying cause remains unidentified, a condition referred to as idiopathic short stature (ISS). As height is among the most heritable human traits, genetic factors play a pivotal role in its etiopathogenesis. Within this spectrum, rare monogenic variants in growth-related genes exert the most significant influence on growth. The objective of this doctoral dissertation was to implement an advanced genetic diagnostic evaluation for children and adolescents with idiopathic short stature through the comprehensive application of next-generation sequencing (NGS) technologies.
Methods: Study participants were selected upon defined inclusion and exclusion criteria. Inclusion criteria were: age between 3 and 20 years, height (Ht) ≤ −2 standard deviation score (SDS) and written informed consent for genetic testing. Exclusion criteria were: constitutional short stature, growth hormone deficiency, genetic syndromes and dysmorphic conditions, skeletal dysplasia, systemic, chronic or endocrine diseases and medications influencing growth (e.g., glucocorticoids). In patients with ISS, whole exome sequencing (WES) was employed as the primary laboratory method, while in patients with severe short stature whole genome sequencing (WGS) was additionally performed. Long-read sequencing was applied in selected cases where such an approach was deemed appropriate.
Results: Following systematic phenotyping, a total of 238 participants with ISS were selected for comprehensive genetic evaluation. WES identified likely pathogenic or pathogenic variants (LP/P) in 11% of probands (25/238), while an additional 13% (32/238) carried variants of uncertain significance. The most common genetic cause of ISS represented heterozygous LP/P variants in the ACAN gene, accounting for 3% of all ISS cases, followed by the heterozygous LP/P variants in the NPR2 gene in 2% of ISS cases. LP/P variants were detected in additional growth-related genes, i.e., GHSR, GHR, IGF1R, IGFALS, SHOX, IHH and PTPN11.
Among the study participants, we observed a broad spectrum of clinical presentations, with incomplete penetrance noted in the ACAN, NPR2, GHSR, and IGFALS genes. Novel phenotypic features were identified, including otosclerosis and very early-onset articular problems in patients with ACAN variants, as well as isolated short stature associated with the PTPN11 missense variant c.794G>A (p.Arg265Gln). In patients carrying ACAN and NPR2 variants, the efficacy of growth hormone therapy was evaluated. The findings demonstrated a modest response, particularly when treatment was initiated early.
By implementing a copy number variation (CNV) calling framework on WES data, intragenic and whole-gene CNVs were identified in ACAN, IGFALS, and SHOX, representing a minority of the detected variants in our study cohort. In cases of severe short stature with negative WES findings, WGS was subsequently performed, which did not provide a definitive molecular diagnosis in any of the selected participants.
In one selected family with an autosomal dominant pattern of short stature and the inversion on chromosome 2 (inv(2)(p11.2p25.2)), long-read sequencing successfully delineated the precise breakpoints of the inversion, spanning genomic coordinates 4,606,746 to 82,445,479 (GRCh37). The clinical significance of this structural variant remains to be elucidated.
Conclusion: This doctoral dissertation introduced a strategy that integrates systematic phenotyping with comprehensive genetic evaluation, both considered of equal importance, representing a significant advancement in the clinical approach to children and adolescents with ISS in Slovenia. Based on our findings, and in light of recent advances in genomic technologies, including CNV detection from whole exome sequencing and whole genome sequencing, we developed and proposed an updated genetic algorithm for the evaluation of children with short stature. The findings of the doctoral dissertation expanded the known genotypic and phenotypic spectrum of ISS, providing valuable information for clinicians and geneticists worldwide. Moreover, in the doctoral dissertation the response to growth hormone therapy in probands with ACAN and NPR2 variants was evaluated, revealing an overall moderate, but clinically meaningful treatment effect. These findings underscore the importance of a personalized therapeutic approach, as an individual’s genetic profile is expected to substantially influence both - therapy selection and the prediction of treatment efficacy in the future.
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