Expanded Targeted-Exome Sequencing and Functional Validation Improve Molecular Diagnosis and Refine Genotype-Phenotype Correlations in Congenital Hypogonadotropic Hypogonadism.

Introduction: Congenital Hypogonadotropic Hypogonadism (CHH) arises from defects in the synthesis, secretion, or action of gonadotropin-releasing hormone (GnRH), resulting in incomplete or absent pubertal development and various non-reproductive features. CHH is genetically heterogeneous, with over 50 genes implicated in its pathogenesis. This study aimed to elucidate the genetic variants of CHH in a cohort of patients from a single-center endocrinology unit.

Methods: We used a targeted next-generation sequencing panel to analyze 52 CHH-related genes in 35 patients. Functional studies validated two of the identified variants.

Results: Molecular etiology was identified in 20 of 35 patients (57%). Among these, 12 (39%) had variants in multiple CHH-related genes, with oligogenic inheritance confirmed in two cases. Novel pathogenic variants (both SNVs and CNVs) were identified, including ANOS1 p.Gln238* and c.318+2T>C, CHD7 p.Ser734Ilefs5 and p.Gln592Serfs16, FGFR1 p.Ala36Profs67, c.1663+5G>A, and p.Tyr210*, DMXL2 deletion (83.1 Kb), and SOX2 deletion (1.1 Mb). In total, 21 pathogenic or likely pathogenic variants across 15 CHH-related genes were detected, including ANOS1, CHD7, FGFR1, PROKR2, and others. A functional study confirmed the loss of function in the KISS1R p.Ala203Asp variant.

Conclusion: Our findings demonstrate the utility of a NGS panel in diagnosing genetically complex conditions like CHH and underscore the role of oligogenic inheritance in its phenotypic diversity. The inclusion of genes previously associated with syndromic CHH forms such as CHARGE in the exome panel may highlight possible shared mechanisms with neurodevelopmental disorders, aiding early diagnosis, genetic counseling, and treatment.