Targeted next generation sequencing approach identifies eighteen new candidate genes in normosmic hypogonadotropic hypogonadism and Kallmann syndrome

https://doi.org/10.1016/j.mce.2016.08.007Get rights and content

Highlights

  • Targeted and Sanger DNA sequencing of 261 genes was performed in 48 patients with hypogonadotropic hypogonadism.

  • Two novel FGFR1 likely pathogenic mutations were found.

  • Nineteen new candidate genes for hypogonadotropic hypogonadism were identified.

  • Eight possible digenic and one possible trigenic families were identified.

Abstract

The genetic basis is unknown for ∼60% of normosmic hypogonadotropic hypogonadism (nHH)/Kallmann syndrome (KS). DNAs from (17 male and 31 female) nHH/KS patients were analyzed by targeted next generation sequencing (NGS) of 261 genes involved in hypothalamic, pituitary, and/or olfactory pathways, or suggested by chromosome rearrangements. Selected variants were subjected to Sanger DNA sequencing, the gold standard. The frequency of Sanger-confirmed variants was determined using the ExAC database. Variants were classified as likely pathogenic (frameshift, nonsense, and splice site) or predicted pathogenic (nonsynonymous missense). Two novel FGFR1 mutations were identified, as were 18 new candidate genes including: AMN1, CCKBR, CRY1, CXCR4, FGF13, GAP43, GLI3, JAG1, NOS1, MASTL, NOTCH1, NRP2, PALM2, PDE3A, PLEKHA5, RD3, and TRAPPC9, and TSPAN11. Digenic and trigenic variants were found in 8/48 (16.7%) and 1/48 (2.1%) patients, respectively. NGS with confirmation by Sanger sequencing resulted in the identification of new causative FGFR1 gene mutations and suggested 18 new candidate genes in nHH/KS.

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.

References (48)

  • S. Richards et al.

    Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of medical genetics and Genomics and the association for molecular Pathology

    Genet. Med.

    (2015)
  • S.M. Strittmatter et al.

    Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43

    Cell

    (1995)
  • Y. Wang et al.

    Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling

    Cell

    (2003)
  • I.A. Adzhubei et al.

    A method and server for predicting damaging missense mutations

    Nat. Methods

    (2010)
  • C.W. Balmer et al.

    Loss of Gli3 and Shh function disrupts olfactory axon trajectories

    J. Comp. Neurol.

    (2004)
  • A. Cariboni et al.

    Defective gonadotropin-releasing hormone neuron migration in mice lacking SEMA3A signalling through NRP1 and NRP2: implications for the aetiology of hypogonadotropic hypogonadism

    Hum. Mol. Genet.

    (2011)
  • T.E. Dever

    Molecular biology. A new start for protein synthesis

    Science

    (2012)
  • S. Dowler et al.

    Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities

    Biochem. J.

    (2000)
  • G. Garrel et al.

    Sustained gonadotropin-releasing hormone stimulation mobilizes the cAMP/PKA pathway to induce nitric oxide synthase type 1 expression in rat pituitary cells in vitro and in vivo at proestrus

    Biol. Reprod.

    (2010)
  • Y. Gu et al.

    Steel factor controls primordial germ cell survival and motility from the time of their specification in the allantois, and provides a continuous niche throughout their migration

    Development

    (2009)
  • N.K. Hanchate et al.

    SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome

    PLoS Genet.

    (2012)
  • H.G. Kim et al.

    Clinical manifestations of impaired GnRH neuron development and function

    Neurosignals

    (2008)
  • L.C. Layman

    Clinical genetic testing for kallmann syndrome

    J. Clin. Endocrinol. Metab.

    (2013)
  • L.C. Layman et al.

    Delayed puberty and hypogonadism caused by mutations in the follicle-stimulating hormone beta-subunit gene

    N. Engl. J. Med.

    (1997)
  • Cited by (0)

    1

    Samuel Quaynor and Maggie Bosley contributed equally.

    View full text