Male pseudohermaphroditism, or Leydig cell hypoplasia (LCH), is an autosomal recessive disorder in individuals with a 46,XY karyotype, characterized by a predominantly female phenotype, a blind-ending vagina, absence of breast development, primary amenorrhea, and the presence of testicular structures. It is caused by mutations in the luteinizing hormone/chorionic gonadotropin receptor gene (LHCGR), which impair either LH/CG binding or signal transduction. However, molecular analysis has revealed that the LHCGR is apparently normal in about 50% of patients with the full clinical phenotype of LCH. We therefore searched the LHCGR for novel genomic elements causative for LCH.
Methods and Findings
In the present study we have identified a novel, primate-specific bona fide exon (exon 6A) within the LHCGR gene. It displays composite characteristics of an internal/terminal exon and possesses stop codons triggering nonsense-mediated mRNA decay (NMD) in LHCGR. Transcripts including exon 6A are physiologically highly expressed in human testes and granulosa cells, and result in an intracellular, truncated LHCGR protein of 209 amino acids. We sequenced exon 6A in 16 patients with unexplained LCH and detected mutations in three patients. Functional studies revealed a dramatic increase in the expression of the mutated internal exon 6A transcripts, indicating aberrant NMD. These altered ratios of LHCGR transcripts result in the generation of predominantly nonfunctional LHCGR isoforms, thereby preventing proper expression and functioning.
The identification and characterization of this novel exon not only identifies a new regulatory element within the genomic organization of LHCGR, but also points toward a complex network of receptor regulation, including events at the transcriptional level. These findings add to the molecular diagnostic tools for LCH and extend our understanding of the endocrine regulation of sexual differentiation.
Joerg Gromoll and colleagues describe the identification and characterization of a novel exon that appears to be a new regulatory element within the luteinizing hormone/chorionic gonadotropin receptor gene of three individuals with Leydig cell hypoplasia.
A person's sex is determined by their complement of X and Y (sex) chromosomes. Someone who has two X chromosomes is genetically female and usually has ovaries and female external sex organs. Someone who has an X and a Y chromosome is genetically male and has testes and male external sex organs. Sometimes, though, the development of the reproductive organs proceeds abnormally, resulting in a person with an “intersex” condition whose chromosomes, gonads (ovaries or testes), and external sex organs do not correspond. Leydig cell hypoplasia (LCH; also called male pseudohermaphroditism or a disorder of sex development) is an XY female intersex condition. People with this inherited condition develop testes but also have a vagina (which is not connected to a womb), and they do not develop breasts or have periods. This mixture of sexual characteristics arises because the Leydig cells in the testes are underdeveloped. Leydig cells normally secrete testosterone, the hormone that promotes the development and maintenance of male sex characteristics. Before birth, chorionic gonadotropin (CG; a hormone made by the placenta) stimulates Leydig cell development and testosterone production; after birth, luteinizing hormone (LH), which is made by the pituitary gland, stimulates testosterone production. Both hormones bind to the LH/CG receptor, a protein on the surface of Leydig cells. In LCH, this receptor either does not bind CG and LH or fails to tell the Leydig cells to make testosterone.
Why Was This Study Done?
The gene that encodes the LH/CG receptor is called LHCGR. Several mutations (genetic changes) that inactivate the LC/CG receptor have been identified in people with LCH. However, the LHCGR gene is apparently normal in 50% of people with this intersex condition. In this study, the researchers examine the LHCGR gene in detail to try to find the underlying genetic defect in these individuals.
What Did the Researchers Do and Find?
The researchers used several molecular biology techniques to identify a new exon—exon 6A—within the human LHCGR gene. (Exons are DNA sequences that contain the information for making proteins; introns are DNA sequences that interrupt the coding sequence of a gene. Both introns and exons are transcribed into messenger RNA [mRNA] and the exons are then “spliced” together to make the mature mRNA, which is translated into protein.) The researchers identify several differently spliced LHCGR mRNA transcripts that contain exon 6A—a terminal exon 6A mRNA that contains exons 1–6 and exon 6A, and two internal exon 6A mRNAs that also contain exons 7–11. The researchers report that human testes express high levels of the terminal exon 6A transcript, which is translated into a short version of LHCGR protein that remains within the cell (full-length LHCGR moves to the cell surface). By contrast, testes contain low levels of the internal exon 6A mRNAs. This is because exon 6A contains two premature stop codons (DNA sequences that mark the end of a protein), which trigger “nonsense-mediated decay” (NMD), a cellular surveillance mechanism that regulates protein synthesis by degrading mRNAs that contain internal stop codons. When the researchers screened 16 people with LCH but without known mutations in the LHCGR gene, three had mutations in exon 6A. Laboratory experiments show that these mutations greatly increased the amounts of the internal exon 6A transcripts present in cells and interfered with the cells' normal response to chorionic gonadotropin.
What Do These Findings Mean?
These findings identify a new, functional exon in the LHCGR gene and show that mutations in this exon cause some cases of LCH. This is the first time that a human disease has been associated with mutations in an exon that is a target for NMD. In addition, these findings provide important insights into how the LHCGR is regulated. The researchers speculate that a complex network that involves the exon 6A-containing transcripts and NMD normally tightly regulates the production of functional LHCGR already at the transcriptional level. When mutations are present in exon 6A, they suggest, NMD is the predominant pathway for all the exon 6A-containing transcripts, thereby drastically decreasing the amount of functional LHCGR.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050088.
The MedlinePlus Encyclopedia has a page on intersex conditions (in English and Spanish)
Wikipedia has pages on intersexuality and on the LH/CG receptor (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Intersex Society of North America provides information and support for the parents of children with intersex conditions
The Androgen Insensitivity Syndrome Support Group also provides some general information about intersex conditions, including information about LCH and other XY female conditions (in several languages)
Sequence-Structure-Function-Analysis (SSFA), run by a group of researchers in Germany (Leibniz-Institut für Molekulare Pharmakologie; Humboldt-Universitätzu Berlin), is a database dealing the sequence, structure, and function of glycoprotein hormone receptors
Glycoprotein-hormone Receptors Information System (GRIS), from Université Libre de Bruxelles and Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, is a database giving structural information on the LHCGR