Targeted next generation sequencing approach identifies eighteen new candidate genes in normosmic hypogonadotropic hypogonadism and Kallmann syndrome
Graphical abstract
Introduction
The development of reproductive function in humans is regulated by complex signaling interactions of the hypothalamic-pituitary-gonadal (HPG) axis. Disruption of any component of this system may result in delayed puberty and infertility (Layman, 2013a, Layman, 2013b). Central nervous system defects may impair GnRH action and function resulting in GnRH deficiency, also known as hypogonadotropic hypogonadism, which may be manifested clinically by low sex steroids and low or inappropriately normal gonadotropins. To date, two principle conditions constitute deficiency in GnRH signaling, namely normosmic hypogonadotropic hypogonadism (nHH) and Kallmann Syndrome (KS). Patients with KS present with hypogonadotropic hypogonadism and a lack of smell due to the impaired migration of GnRH and olfactory neurons. Apart from pubertal and reproductive disturbances, other associated anomalies such as renal agenesis, midfacial defects, neurologic defects, and cardiac anomalies have been reported (Layman, 2013a, Layman, 2013b, Bhagavath et al., 2006).
Even with improved knowledge of GnRH development and function, only 40% of all nHH/KS cases can be explained by reported mutations in more than 30 genes. Some mutations occur in ligand/receptors such as KAL1/FGF8/FGFR1/HS6ST1, LEP/LEPR, GNRH1/GNRHR, PROK2/PROKR2, KISS1/KISS1R, and TAC3/TACR3 (Layman, 2013a, Layman, 2013b). Other identified genes include NR0B1, CHD7, NELF, WDR11, SEMA3A, SOX10, FGF17, IL17RD, DUSP6, SPRY4, FLRT3, FEZF1, STUB1, HESX1, PCSK1, RNF216, and OTUD4 (Layman, 2013a, Layman, 2013b, Hanchate et al., 2012, Tornberg et al., 2011, Margolin et al., 2013). These causative genes were identified through linkage analysis, candidate gene approaches, and positional cloning (Layman, 2013a, Layman, 2013b, Kim et al., 2008a, Kim et al., 2008b, Kim et al., 2010). The inheritance pattern of nHH/KS includes X-linked recessive, autosomal dominant, autosomal recessive, sporadic, and at least several percent appear to be digenic/oligogenic (Quaynor et al., 2011, Sykiotis et al., 2010a, Sykiotis et al., 2010b). Molecular studies performed by both in vitro and in vivo analysis of the genes indicate that either the development/migration or signaling of GnRH is altered or impaired (Layman et al., 1998, Quaynor et al., 2013).
The majority of molecular causes of nHH/KS are yet to be characterized. This gap in our understanding may be narrowed with advancements in massively parallel deep resequencing methods, otherwise known as next generation sequencing (NGS), which includes targeted NGS, whole exome sequencing, and whole genome sequencing. More expedient, less expensive, and theoretically more accurate results should be able to be obtained using NGS (Metzker, 2010). The goal of the present study was to identify new nHH/KS candidate genes using targeted NGS of potentially relevant known genes that are involved in GnRH and olfactory neuron development, migration and signaling.
Section snippets
Genes selected for NGS
The list of 261 genes was gathered from the literature to identify important genes involved in hypothalamic or pituitary development, GnRH or olfactory neuron specification, migration, or regulation, nHH/KS known pathway genes, or genes located near derivative chromosome breakpoints in nHH/KS patients with chromosome translocations (genes shown in Table 1; references for gene selection shown in Supplemental Table 1). All known nHH/KS genes at the time of the study initiation in 2011 were also
Results
319 variants from 261 genes were identified by targeted NGS in 48 nHH/KS patients. Of the 319 variants identified, 56 were class 4 (frameshift, nonsense, and splice site) and 153 were class 3 (missense) variants. The remaining 110 variants were located in the 5′UTR, 3′UTR or >10 bp from splice sites in introns, and were not studied further. It should be noted that only true variants in genes identified by NGS that were confirmed by Sanger sequencing were considered important for nHH/KS (Table 2A
Discussion
The molecular basis of nHH/KS has been elucidated for 30–40% of all patients. When there is a family history of nHH/KS, particularly if there are associated anomalies, targeted sequencing of known genes may be warranted.(Layman, 2013a, Layman, 2013b). However, should results yield no positive findings, further analysis by targeted NGS could be considered. Despite false positive variants, the Sanger sequencing confirmed variants found by targeted NGS revealed meaningful results in both known and
Grants
Funding by NIH grant HD033004 & Medical College of Georgia Bridge Foundation (L.C.L.) (BFP00042) This work was presented in part at the Society for Reproductive Investigation in Florence, Italy, March 2014.
Disclosure statement
The authors have nothing to disclose. We would like to acknowledge students who contributed to this project– Luke V. Lee, Irene Falk, ME Jett, Julianne Jett, and Alli Falkenstrom.
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Samuel Quaynor and Maggie Bosley contributed equally.