Nasal embryonic LHRH factor (NELF) mutations in patients with normosmic hypogonadotropic hypogonadism and Kallmann syndrome

Abstract

Objective: To determine if mutations in NELF, a gene isolated from migratory GnRH neurons, cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS).

Design: Molecular analysis correlated with phenotype.

Setting: Academic medical center.

Patient(s): A total of 168 IHH/KS patients as well as unrelated control subjects were studied for NELF mutations.

Intervention(s): NELF coding regions/splice junctions were subjected to polymerase chain reaction (PCR)-based DNA sequencing. Eleven additional IHH/KS genes were sequenced in three patients with NELF mutations.

Main outcome measure(s): Mutations were confirmed by sorting intolerant from tolerant, reverse-transcription (RT)-PCR, and Western blot analysis.

Result(s): Three novel NELF mutations absent in 372 ethnically matched control subjects were identified in 3/168 (1.8%) IHH/KS patients. One IHH patient had compound heterozygous NELF mutations (c.629-21G>C and c.629-23C>G), and he did not have mutations in 11 other known IHH/KS genes. Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/KAL1 (c.757G>A; p.Ala253Thr of NELF and c.488_490delGTT; p.Cys163del of KAL1) and NELF/TACR3 (c.1160-13C>T of NELF and c.824G>A; p.Trp275X of TACR3). In vitro evidence of these NELF mutations included reduced protein expression and splicing defects.

Conclusion(s): Our findings suggest that NELF is associated with normosmic IHH and KS, either singly or in combination with a mutation in another gene.

Figure 1. Location and conservation of NELF, KAL1, and TACR3 mutations

(A) Exon and intron structure of the 58 kb NELF gene (NM_015537) with locations of human missense and intronic mutations identified in sporadic IHH and KS patients. Important exons shown as blue rectangles to scale are numbered along with their size in basepairs (bp). The locations of start and stop codons are to scale, but the sizes of introns are not to scale. (B,C) Graphical views of KAL1-encoded anosmin-1 (NP_000207) and TACR3 (NP_001050) proteins with known domains. Shown is the single AA cysteine in frame KAL1 deletion in the whey-acidic-protein domain (WAP) in B—although the annotation (c.487_489delTGT) was incorrect in our previous report (17). Also shown is the TACR3 truncation mutation p.Trp275X within a cytoplasmic domain between the 5^th and 6^th transmembrane domains in C). The relative size between anosmin-1 and TACR3 proteins and the size and locations of depicted domains are to scale. Abbreviations include S=signal peptide; Cys-rich (cysteine-rich), FN-III=fibronectin III; H=histidine; T1-T7=transmembrane 1-7.

Figure 2. Functional protein analysis of human NELF mutations

Western blot analysis is shown using the anti-NELF antibody (27), which recognizes the ~63 kDa NELF protein in lymphoblastoid cell lines extracts. β-actin was used as internal loading control (~42kDa). Lane 1 represents a normal male control, whereas lanes 2-3 represent individuals with NELF mutations. Additional controls and other mutations did not alter protein expression, and are not shown. The westerns were performed three times, and the results were consistently the same. Protein expression for patient C68 was not altered (not shown).

Figure 3. NELF Splice Mutant

Subcloning and sequencing analysis of RT-PCR products of NELF exons 8-12 from patient and control lymphoblastoid RNA confirms that the human NELF c.1160-13C>T mutation causes aberrant exon 10 skipping. Splicing patterns are compared between a normal control and the mutant by cloned cDNA sequencing (the WT or mutant band is a cloned fragment). An expected NELF product of 280 bp including exons 9-10 is observed in the control (lane 2) whereas an abnormal product of 246 bp skipping exon 10 (34 bp) is observed in a KS patient with c.1160-13C>T (lane 3). Exon 11 nucleotides and out-of-frame novel AA sequence are depicted in blue. A 123 bp DNA marker is shown in the first lane.