Using Targeted Next Generation Sequencing to identify putative mutations in genes associated with GnRH development and hypogonadotropic hypogonadism

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 112-141-Hypothalamus-Pituitary Development & Biology
Basic/Clinical
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-132
Samuel D Quaynor*, Lynn P Chorich, Hyung-Goo Kim and Lawrence C Layman
Georgia Regents University, Augusta, GA
Coordination of the hypothalamic-pituitary-gonadal axis, which is governed by pulsatile release of gonadotropin-releasing hormone (GnRH), is essential for normal reproductive competence. GnRH is secreted from hypothalamic neurons that have migrated along with olfactory neurons from the nasal region to the arcuate nucleus during embryologic development. Disruption of GnRH neuron migration and/or interference with GnRH secretion results in hypogonadotropic hypogonadism, which may be normosmic (nHH) or anosmic/hyposmic Kallmann syndrome (KS). These patients with nHH/KS have in common a clinical presentation of absent puberty, low or inappropriately normal gonadotropins, and deficient sex steroids. The molecular basis of nHH/KS has been ascertained in 30-40% of patients, and may be attributed to mutations in one or more of 18 genes. The genetic basis of nHH/KS has been elucidated by a variety of techniques including candidate gene approaches, linkage analysis, and positional cloning using chromosomal deletions and/or rearrangements. However, since a majority of the molecular etiologies for nHH/KS have not yet been determined, next generation DNA sequencing represents a promising methodology for new gene discovery. Whole exome sequencing and whole genome sequencing have been used, but bioinformatic analysis may be challenging because of the large number of DNA sequence variants identified. We therefore employed a targeted sequencing approach. Utilizing in silico databases, pathway analysis, and relevant literature, 285 genes were selected to study in 48 nHH/KS patients without mutations in known genes. Candidate genes were arbitrarily categorized as being involved in hypothalamic development and patterning; olfactory development; GnRH neuron development, migration and signaling; and pituitary regulation of gonadotropin secretion. Known causative genes and additional candidates, as identified by position in nHH/KS patients with balanced chromosomal rearrangements, were also included. SureDesign Software (Agilent Technologies) was used to design probes for  the sequence of the 285 candidate genes. Coding exons were captured using the Agilent HaloPlex custom capture enrichment kit and DNA sequenced by Illumina Hiseq2000, which provided paired ends reads (2x 90) achieving 30-175 fold coverage of genomic sequence. Preliminary DNA sequence variants are being confirmed by Sanger sequencing and studied in family members and controls. Targeted next generation sequencing of relevant developmental genes may provide an additional useful approach to identify new genes involved in human pubertal development.

Nothing to Disclose: SDQ, LPC, HGK, LCL

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: NIH Grant HD33004 awarded to LCL