A new LH receptor splice mutation responsible for male hypogonadism with subnormal sperm production in the propositus, and infertility with regular cycles in an affected sister

Abstract

Background: Inactivating LH receptor (LHR) mutations have been described so far in men as well as in women. Phenotypes in men have been variable with in nearly all cases impairment of sex differentiation or azoospermia. We report a milder reproductive phenotype both in a male patient and his sister.

Methods and results: We describe a family that carries a homozygous mutation G-->A at position -1 at the intron 10-exon 11 boundary of the LHR gene. The male patient presented with delayed puberty, micropenis and oligospermia. Two of his sisters were homozygous for the same mutation and were infertile. Surprisingly, one of them was found to have had regular ovarian cycles for years and showed normal LH values (6.5 and 10.6 mIU/ml for LH and FSH, respectively). In vitro analysis showed that this altered splicing resulted in an LHR from which eight amino acids are deleted from the extracellular domain (Delta Tyr(317)-Ser(324)). In vitro expression has shown that the receptor was expressed and capable of LH-induced signaling, albeit with reduced potency (P < 0.001).

Conclusions: LHR mutations may represent an underestimated cause of infertility in women, in addition to being responsible for male hypogonadism with reduced spermatogenesis.

Figure 1:

Family pedigree of the propositus, indicating the mutation in intron 10 (−1) G→A. Propositus II-7 is indicated by an arrow.

Figure 2:

In vitro splicing of WT and patient minigenes. (A) Schematic overview of the wild type (WT) and patient minigenes derived from pSPL3. For clarity, only sequence from pSPL3 splice donor (SD) to splice acceptor (SA) is represented. To allow splicing to exon 11, part of pSPL3 SA was deleted. Location of oligonucleotides forward Fw_exon10 and reverse Rv_exon11 is depicted as arrows. In the patient minigene, mutation G(−1)→A at intron 10–exon 11 boundary is present. (B) RT–PCR on complementary DNA (cDNA) derived from in vitro spliced minigenes using oligonucleotides Fw_exon10–Rv_exon11. (C) Reconstruction of in vitro splicing using WT and patient minigenes, based on sequencing of in vitro splice products.

Figure 3:

Allele-specific amplification of alternative splice products. Design of general (Fw_exon10) and WT (Fw_WT) or patient (Fw_patient) specific oligonucleotides that, in combination with Rv_exon11, should result in an allele-discriminating PCR (A). The most 3′ base of the specific oligonucleotides and corresponding sequence in WT and patient cDNA are boxed. General and allele-specific amplification of WT (B) or patient (C) cDNA (obtained after in vitro splicing). Parallel PCR on expression plasmids (1 ng) is used for validation of PCR selectivity. The size of the expected PCR products is depicted below the gel pictures. As a control for cDNA synthesis, the housekeeping enzyme hypoxanthine–guanine phosphoribosyl transferase (HPRT) is amplified (D), this control is also shown for non-transfected (mock) cells, pSPL3-transfected cells, as well as samples without reverse transcriptase (-RT).

Figure 4:

Pharmacological characterization of WT and patient LHRs. Dose–response curves of hCG (A) or LH (B) for WT (▪), patient (○) or Δexon 10 (▴)* LHRs or mock transfected controls (▿), as measured by a CAMP response element (CRE) luc-driven reporter gene assay. A representative experiment is shown. A parallel enzyme-linked immunosorbent assay, targeting an N-terminal HA-tag is used to evaluate receptor expression (C). Potencies (EC₅₀) of LH for WT and patient LHRs after transfecting 7, 3.5 or 1.75 µg expression plasmids as measured by a CRE luc-driven reporter gene assay (D). Total amount of transfected plasmid is kept constant by supplementing empty pSG5. (*Gromoll et al., 2000; Müller et al., 2003).