| Περίληψη: | Isolated Gonadotropin Releasing Hormone (GnRH) Deficiency (IGD) is a rare disease with a wide spectrum of reproductive and non- reproductive clinical characteristics. Apart from the phenotypic heterogeneity, IGD is also a genetically enriched disease with >35 genes being implicated in its pathophysiology. When IGD is associated with anosmia it is referred as Kallmann Sydnrome (KS), whereas its normosmic phenotype is known as normosmic idiopathic hypogonadotropic hypogonadism (nIHH). Apart from those two rare forms of the disease, GnRH deficiency spans a wide spectrum of other disorders such as constitutional delay of puberty (CDP), hypothalamic amenorrhea (HA) as well as the adult onset of hypogonadotropic hypogonadism (AHH). Non- reproductive features of the disease include unilateral renal agenesis, synkinesia, cleft lip and palate, dental agenesis, eye and heart defects and many more. Genes associated with IGD can either affect the neurodevelopmental GnRH pathway, i.e. the migration of the GnRH neurons along the olfactory neurons to the hypothalamus, or the neuroendocrine pathway of the GnRH secretion and/or action. Despite the large number of genes that are associated with the disease, ~60% of the patients remain genetically uncharacterized. Different strategies have been utilized for the gene discovery including analysis of chromosomal rearrangements, sequencing of syndromic cases, candidate- gene approach and studies of endogamous families/ populations. Utilizing next- generation –sequencing (NGS) techniques the number of genes and variants being identified is increasing significantly and more genes are now emerging as candidate IGD genes. Thus, before the field moves forward we would need to pause and look how the “known” IGD genes have been discovered and if genetic enrichment exists in IGD subpopulations such as the Greek IGD cohort.
Scope of the study: In the first part of the study we performed a retrospective review of important genes that have been associated with IGD. We used the recently published criteria by MacArthur et al to test the candidacy of the “known” IGD genes based on the gene disruption, expression in relevant tissues, biochemical function, protein- protein interactions, animal studies, rescue experiments and burden testing. In the second part of our study we analyzed the largest cohort of Greek patients with IGD ever been studied and performed detailed phenotyping and genotyping, with the goal to estimate the prevalence of genetic variation in this sub-population. We used both a “traditional” approach, i.e. Sanger sequencing, and whole exome sequencing (WES) to determine if application of NGS has any effect on the estimation of prevalence of genetic variation and oligogenicity in the cohort studied.
Methods: For the first part of our analysis we performed a systematic review of the literature for all studies published on 24 “known” IGD genes. For the second part of our analysis we performed detailed phenotyping and genetic screening utilizing Sanger sequencing in 81 probands looking for variation in 14 genes vs. WES in 87 probands, searching for genetic variation in 37 genes. The pathogenicity of the detected RSVs was estimated based on segregation data, conservational data, in vitro and in silico analysis.
Results: Even though all known IGD genes seem to fulfill the criteria for gene disruption, biochemical function, protein- protein interactions and tissue expression, data are more limited when it comes to animal studies, rescue experiments and burden testing. However, NSG will enable the sequencing of large IGD and control cohort making statistical studies more feasible in the future. With regards to analysis of the Greek cohort detailed phenotyping revealed a variety of phenotypic characteristics with renal agenesis and synkinesia being enriched in patients with KS. Sanger sequencing detected a prevalence of genetic variation in 26% of the IGD patients sequenced in 7/14 IGD genes without any evidence of oligegenicity. WES revealed that 31% of IGD patients carrying a rare genetic change in a total of 15 genes with 4,5% of the IGD cases being oligogenic.
Discussion: Tremendous efforts have been undertaken by many researches to discover novel IGD genes and several studies have attempted to establish pathogenicity of the genes and/ or variants detected, including animal studies and other experiments. NGS has yielded many genes and variants in diseased and control cohorts, making burden testing a crucial step in gene validation. In our study we used NGS in a large IGD cohort of Greek patients showing prevalence of genetic variation in 1/3 of the cohort. In addition, we were able to detect oligogenicity, suggesting that NGS techniques can discover previously undetected variation and will soon become the standardized method for screening patients with rare and/ or more common disorders.
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