Introduction

Gonadotropin-releasing hormone (GnRH) is essential for the neuroendocrine control of reproduction. Previous studies of mouse embryos demonstrated that GnRH cells originate in the olfactory placode and then migrate with the olfactory nerves to reach their final destination in the hypothalamus (Wray et al. 1989; Schwanzel-Fukuda et al. 1992; Wray 2002). In the human, migrating GnRH-containing neurons play a critical role in the pathogenesis of Kallmann syndrome (KS), a disease with idiopathic hypogonadotropic hypogonadism (IHH) and anosmia/hyposmia (Waldstreicher et al. 1996; Seminara et al. 1998). One-third of IHH patients were familial, while the remaining patients were sporadic, and autosomal dominant, autosomal recessive, and X-linked forms of IHH have been reported (Waldstreicher et al. 1996). Protein of KAL1, the gene responsible for the X-linked KS (Legouis et al. 1991; Nagata et al. 2000), exhibits characteristics as a cell adhesion molecule and is involved in both migration of GnRH cells and olfactory neuronal cells. Failure of GnRH-cell migration has been confirmed in a KS fetus (Schwanzel-Fukuda et al. 1989; Hardelin 2001), and mutation in KAL1, DAX, and LHRHR account for 20% of patients with hypogonadotropic hypogonadism (Seminara et al. 1999; Oliveira et al. 2001; Beranova et al. 2001). Thus, KS patients might also be caused by other genes.

Recently, a novel mouse protein, Nelf (nasal embryonic LHRH factor), was isolated (Kramer and Wray 2000), and its gene (Nelf) is expressed in the olfactory sensory cells and GnRH cells during embryonic development. Nelf serves as a common guidance molecule for the olfactory axon and GnRH neurons across the nasal region (Kramer and Wray 2000). In addition, Nelf knocked-down mice inhibited the olfactory axon outgrowth and abnormal migration of the GnRH cell into the periphery of nasal explant (Kramer and Wray 2000). These findings suggest that the human homolog, NELF, is a functional candidate gene for KS. Here we report the characterization of NELF and a mutation screening in patients with IHH.

Materials and methods

Human NELF

We characterized NELF by assembling human EST clones, RACE, and RT-PCR on a human fetal brain cDNA library followed by sequence comparison with the human genomic data (http://www.ncbi.nih.gov/). Subcloned NELF cDNAs were digested to generate probes (exon 3 probe) for Northern blot hybridization. Digestion with RsaI (5 U/μg DNA) created a 304-bp fragment, corresponding to positions 304–607 within exon 3 of NELF. Radiolabeled exon 3 probe was hybridized to 2 μg poly (A)+ RNA according to the manufacturer’s instruction (Human Multiple Tissue Northern Blot, Clontech).

Screening for NELF mutations in patients with IHH

Sixty-five patients with IHH (33 patients with KS) who lacked KAL1 mutations were screened for mutations in NELF. Of the 65 patients, 35 (21 of 33 KS patients) had a family history of IHH other than that suggesting an X-linked inheritance. All subjects met the following criteria: (1) failure to completion of puberty by the age of 18 years; (2) low serum testosterone concentration (<100 ng/dl for men and estradiol <20 pg/ml for women); (3) low or normal levels of circulating gonadotropins (for normal values, see Waldstreicher et al. 1996; Kratz and Lewandrowski 1998); and (4) normal findings in the brain by magnetic resonance imaging. Genomic DNA was extracted from their peripheral blood leukocytes. Primers were designed to amplify each exon, including acceptor–donor splice sites, of NELF (Table 1). When a base-pair change in NELF was detected, 100 healthy control samples were examined for the change. This study was approved by the Subcommittee on Human Studies of the Massachusetts General Hospital, and all participants provided written informed consent.

Table 1 PCR primers used for amplification of the coding sequence and sequence variations of human NELF. T m indicates annealing temperatures for PCR conditions. Freq indicates allele frequency

Results and discussion

Characterization of human NELF

RT-PCR of NELF using the human fetal brain cDNA library revealed five alternatively spliced variants. NELF-v1 (GenBank accession no. AY255128), corresponding to the full-length cDNA and composed of 16 exons and 15 introns with a 1,590-bp ORF, is predicted to encode a protein of 530 amino acids. NELF-v2 (GenBank accession no. AY255129) lacks 6 bp comprising the entire sequence of exon 5. NELF-v3 (GenBank accession no. AY255130) has the sequence missing 69 bp that corresponds to exon 6. NELF-v4 (GenBank accession no. AY255131) lacks 90 bp for exon 8. NELF-v5 (GenBank accession no. AY255132) includes 236 bp from intron 9. Size of the five transcripts excluding the poly (A) tail is 2,994, 2,988, 2,925, 2,904, and 3,230 bp, respectively. NELF-v1 shows 93 and 94% identity at the amino acid level to mouse Nelf (GenBank accession no. BAC25597) and rat Nelf (GenBank accession no. CAC20866), respectively. All these transcripts isolated are highly conserved among the human, mouse, and rat. Using the public database (BLASTP and Swiss-prot), no genes or proteins homologous to human NELF-v1 were identified. Although human genome browser suggests the existence of two ESTs with possible novel splice sites (GA/AG in exon 15, GenBank accession no. AI871403; and GC/GG in exon 16, GenBank accession no. AK074602), RT-PCR in the present study never detected such spliced transcripts. The result suggested their very low expression in the human fetal brain.

Multiple-tissue Northern blot analysis revealed 4.9 and 3.0-kb mRNA in all the tissues (Fig. 1). The 3.0-kb transcript with the highest expression level appeared in the adult and fetal brain, testis (fetal ovary not tested), and kidney, indicating that NELF may play an important role in the function of these tissues. In the 3′ RACE and database search, we found only one poly (A) signal (GenBank accession no. AY255128) located 1,147 bp downstream to the stop codon and generating the 3.0-kb transcript. However, the usage of either another poly (A) signal located in the gap sequence downstream to NELF or a >2.0-kb poly (A) tail cannot be excluded. This may lead to the 4.9-kb transcript. The arguments are supported by the fact that the mouse Nelf uses two different poly (A) signals generating 2,924-bp and 4,607-bp mRNA, respectively. An additional 3.2-kb transcript from human NELF was also found in the adult and fetal brain, raising the possibility of a tissue-specific splice variant.

Fig. 1
figure 1

Northern blot analysis of human NELF: upper pane shows the NELF expression, and lower pane the beta-actin expression as a control. A 4.9-kb major transcript is expressed in all tissues examined, and a 3.2-kb transcript is seen in the brain only, whereas a 3.0-kb transcript identical in size to the NELF consensus mRNA is highly expressed in the brain, kidney, and testis

Mutation analysis

We detected one heterozygous missense mutation (1438A>G, T480A) at the donor-splice site in exon 15 of NELF (GenBank accession no. AI871403) in a sporadic case of IHH (Fig. 2). This amino acid substitution switches from a polar side chain in threonine to a nonpolar side chain in alanine. Neither the 100 normal control individuals had an ApaI site that is newly created by the A→G change, nor was any such substitution found in the public SNP database. These findings, together with the fact that Thr480 is highly conserved among the mouse, rat, and human, suggest that T480A is associated with the pathogenesis of IHH. Alternatively, the 1438A>G substitution may be a rare polymorphism (SNP). However, as samples from the patient were no longer available, it remains to be seen whether the mutation leads to dysfunction of the gene. Four novel base substitutions (102C>T and 1029C>T within the coding region and IVS14+47C>T and IVS15+41G>A) were also identified in NELF (Table 1). They will be useful in further studies to determine a possible relationship between NELF and any reproduction and olfaction disorders.

Fig. 2a,b
figure 2

Analysis for 1438A>G substitution. a An ApaI site created by the mutation. b Electropherogram of ApaI fragments from sibs (lanes 1 and 2), the patient (lane 3), a control (lane 4), and size marker (lane 5)