Homozygous mutation within the conserved Ala-Phe-Asn-Glu-Thr motif of exon 7 of the LH receptor causes male pseudohermaphroditism

Abstract

Background: Human chorionic gonadotropin/luteinizing hormone (hCG/LH) function in the male is mediated by the LH receptor (LHR) and is crucial for the normal development of internal and external genitalia. We report a 46, XY patient who presented at the age of 16 with a female phenotype and delayed puberty. Gonads were located bilaterally in the inguinal canal, removed surgically and showed hypoplastic Leydig cells. Immunostaining for the LHR revealed that some Leydig cell progenitors were positive, while others were negative, reflecting different developmental stages of Leydig cell maturation. Methods and Results: Molecular analysis of the LHR was performed on DNA extracted from blood samples of the patient, her parents and sister. The 11 exons of the LHR gene were amplified by PCR and subjected to further single stranded conformation polymorphism (SSCP) analysis. Aberrant migration patterns were observed in exon 7. Upon sequencing, a homozygous T to G transversion was identified, resulting in a F194V substitution located in the extracellular domain. The parents and sister were heterozygous carriers of this mutation. Functional studies in transiently transfected COS-7 cells with the F194V LHR mutation showed the lack of cAMP production upon hCG stimulation, indicating complete inactivation of the receptor due to impaired trafficking of the receptor to the membrane. The mutation is located within a stretch of five amino acids Ala (A)-Phe (F)-Asn (N)-Gly (G)-Thr (T), highly conserved in glycoprotein hormone receptors. For the follicle-stimulating hormone (FSH) receptor (FSHR) loss-of-function mutations have been allocated to this region, a homozygous A189V mutation resulting in a resistant ovary syndrome and impaired spermatogenesis and a heterozygous N191I mutation with no apparent phenotype. Further mutational and functional analysis of the AFN region in the LHR and FSHR revealed that the integrity of this amino acid sequence is crucial for receptor function.

A splice site mutation combined with a novel missense mutation ofLHCGRcause male pseudohermaphroditism

“…Furthermore, the β-strands of LHCGR LRR 3 and 6 were identified as hCG/LH-selective determinants (Song et al, 2001a;Song et al, 2001b). In fact, Gromoll et al ( Gromoll et al, 2007) reported a homozygous p.Phe194Val mutation in a case of LCH, which further supports the conclusion of the AFNGT region encoded by the sequence of exon 7 as being crucial for receptor function. Collectively, these data suggest that the LHCGR lacking exon 7 caused by a mutation in the 3' splice site of intron 6 abolishes the function of the LHCGR gene.…”

“…Among them, p. Val144Phe and p.Phe194Val were observed to interfere with membrane insertion of the receptor (Richter-Unruh et al, 2004;Gromoll et al, 2007), while p.Ile114Phe in the LRR3 resulted in reduced ligand binding and signal transduction of the receptor, with less effect on membrane insertion of the receptor (Leung et al, 2006). The mutation of p. Ile152Thr located in the LRR5 was demonstrated to abolish the binding of the hormone and signal transduction activity.…”

“…Loss-of-function mutations of the LHCGR identified in LCH patients include missense mutations, nonsense mutations, insertions, and deletions spreading throughout the N-terminus, C-terminus and transmembrane domains of the receptor (Themmen et al, 2000;Kremer et al,1995;Laue et al, 1996;Latronico et al, 1996;Chan W 1998). To date, four missense mutations located in the leucine-rich repeats (LRRs) of extracellular domain, p.Ile114 Phe, p.Cys131Arg, p. Val144Phe and p.Phe194Val, have been observed in LCH patients (Leung et al, 2006;Misrahi et al, 1997;Richter-Unruh et al, 2004;Gromoll et al, 2007). However, while the functions of the deletions of exon 8 and 10 have come under discussion, no splice site mutation was reported (Laue et al, 1996;Gromoll et al, 2000).…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…Information condensed as follows: rhodopsin (Stojanovic and Hwa, 2002;Mendes et al, 2005;Tao, 2006); V2R (Bernier et al, 2004a,b;Fujiwara and Bichet, 2005;Bichet, 2006;Boson et al, 2006;Robben and Deen, 2007;Robben et al, 2006); GnRHR (Beranova et al, 2001;Janovick et al, 2003a;Ulloa-Aguirre et al, 2004a,b); CaR (Brown, 2007;Huang and Breitwieser, 2007); LHR (Gromoll et al, 2002;Martens et al, 2002;Huhtaniemi and Themmen, 2005;Piersma et al, 2007); FSHR (Rannikko et al, 2002;Meduri et al, 2003;Huhtaniemi and Themmen, 2005); TSHR (Biebermann et al, 1997;Costagliola et al, 1999;Tonacchera et al, 2000Tonacchera et al, , 2004Calebiro et al, 2005;Davies et al, 2005); E-BR (Tanaka et al, 1998;Fuchs et al, 2001;Chen et al, 2006;Tao, 2006); MC1R-4R (Beaumont et al, 2005;Clark et al, 2005;Govaerts et al, 2005;Tao, 2005Tao, , 2006Farooqi and O´Rahilly, 2006;Hinney et al, 2006;Lubrano-Berthelier et al, 2006;Alharbi et al, 2007;Lin et al, 2007); and CCR5 receptor (Lede...…”

“…There are other GPCRs in which mutations provoke loss of function of the receptor because of intracellular retention of the abnormal (and presumably misfolded and/or incompletely processed) receptor and, consequently, decreased or absent cell surface membrane expression. Some trafficking defective mutants of the glycoprotein hormone receptors [lutropin (LH), follitropin (FSH), and thyrotropin receptors] have been described in patients with Leydig cell hypoplasia, a rare autosomal recessive form of male pseudohermaphroditism (LH receptor) (Gromoll et al, 2002;Martens et al, 2002), in women with ovarian dysgenesis (FSH receptor) (Rannikko et al, 2002;Meduri et al, 2003), and in congenital hypothyroidism (thyrotropin receptor) (Biebermann et al, 1997;Costagliola et al, 1999;Tonacchera et al, 2000Tonacchera et al, , 2004. Loss-of-function mutations in the calcium-sensing receptor due to intracellular retention of the mutant receptor have been found in patients with familial hypocalciuric hypercalcemia (D'Souza-Li et al, 2002).…”