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| The INSL3-LGR8/GREAT ligand-receptor pair in human cryptorchidism |
| Testicular descent is a complex multistep embryonic process requiring the interaction between anatomical and hormonal factors. Failure in any of these steps results in cryptorchidism, the most frequent congenital anomaly of the urogenital tract in human males. Evidence for a genetic cause for cryptorchidism is numerous and supported by animal models. In particular, INSL3 and LGR8/GREAT proteins seem to act as ligand and receptor, respectively, and to have a role in gubernaculum development involved in testicular descent. In a cohort of 87 ex-cryptorchid patients and 80 controls, we looked for mutations in INSL3 and LGR8/GREAT genes by sequencing. Patients were classified on the basis of seminal, hormonal, and testicular cytological analyses. We found three mutations in the INSL3 gene in four patients and one LGR8/GREAT mutation in four patients (8 of 87, 9.2%). The eight patients show different phenotypes, ranging from normozoospermia to complete azoospermia, and from bilateral cryptorchidism to retractile testes. Furthermore, the endocrine function of the testis appears normal in all subjects. The findings of our study demonstrate that INSL3-LGR8/GREAT mutations are frequently associated with human cryptorchidism and are maternally inherited. The only clinical consequence of alterations of the INSL3-LGR8/GREAT system seems to be failure of the testis to normally descend in the scrotum during embryonic development, without affecting the spermatogenic and endocrine components of the testis itself. |
| Ferlin A, et al. J Clin Endocrinol Metab. 2003 Sep;88(9):4273-9 |
Insulin-like 3/relaxin-like factor gene mutations are associated with cryptorchidism |
| Cryptorchidism is a common anomaly of male sexual differentiation. Two phases of testicular descent are recognized, transabdominal and inguinoscrotal. With evidence that androgens and Mullerian inhibitory hormone were not completely responsible for testicular descent, the existence of a third testicular hormone mediating testicular descent was postulated. Insulin-like 3 (INSL3) [also known as relaxin-like factor (RLF) and Leydig insulin-like protein (LEY I-L)] is a member of the insulin/relaxin hormone superfamily that is highly expressed in Leydig cells. The phenotype of transgenic mice with targeted deletion of the Insl3 gene was bilateral cryptorchidism with morphological evidence of abnormal gubernacular development. With this implicit evidence that Insl3 mediates testicular descent in mice, we performed mutation detection analysis of the coding regions of the 2 exon INSL3 gene in genomic DNA samples obtained from 145 formerly cryptorchid patients and 36 adult male controls. Single-strand conformational polymorphism analysis was used for the mutation detection studies. Two mutations, R49X and P69L, and several polymorphisms were identified. Both mutations were located in the connecting peptide region of the protein. The frequency of INSL3/RLF gene mutations as a cause of cryptorchidism is low, because only 2 of 145 (1.4%) formerly cryptorchid patients were found to have mutations. |
| Tomboc M, et al. J Clin Endocrinol Metab. 2000 Nov;85(11):4013-8 |
A novel mutation of the insulin-like 3 gene in patients with cryptorchidism |
| Two independent studies demonstrated that transgenic mice with a targeted deletion of the insulin-like 3 ( INSL3) gene presented bilateral cryptorchidism. Studies in humans have investigated the possibility that mutations in the INSL3 gene are the cause of cryptorchidism. In the present study, genomic DNA was obtained from 150 patients with idiopathic cryptorchidism. DNA was amplified and the polymerase chain reaction products of both exons were sequenced. A previously unidentified missense mutation was found in only one of the patients studied. In exon 2, a heterozygous C/G substitution at nucleotide 2560, which turned asparagine into lysine at codon 86, was documented. The familial study revealed that the mother was a heterozygous carrier of the mutation and the father was a homozygote wild type. We also found three polymorphic changes, previously reported in exon 1. The Asn-into-Lys change is likely deleterious because it leads to a nonconservative amino acid substitution, changing a highly conserved residue. This mutation, located in the A-chain of the INSL3 protein, is the first mutation reported in this region. This finding provides new evidence that INSL3 is involved in testicular descent in humans; however, mutations of this gene are not a frequent cause of cryptorchidism. |
| Canto P, et al. J Hum Genet. 2003;48(2):86-90 |
Using SSCP analysis, we have identified
three variants involving the coding region and one involving
the intron in the INSL3/RLF gene. The 2450C T
mutation that changes codon 49 from arginine to a termination
codon is clearly a deleterious mutation because it leads to
a truncated protein product lacking the A chain. The 2511
CT
change that changes codon 69 from proline to leucine is
likely deleterious because it leads to a nonconservative
amino acid change, changes a highly conserved residue,
and occurs in less than 1% of the population (Fig. 5 ).
Both mutations occur in the connecting peptide region of the
INSL3/RLF protein. The intron variant seems to be a rare polymorphism
occurring preferentially in African-Americans. The linked
L18L;A36T variant seems to be a common polymorphism because
the frequency of the less common allele is high and is
similar among the patients and control subjects (32.8% and 29.2%,
respectively). Tomboc M, et al. J Clin Endocrinol Metab. 2000
Nov;85(11):4013-8 |
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| Comparison of protein sequences. Amino acid residues are indicated by the single universal letter code. The R residue at codon 49 and the P residue at position 69 are underlined. Tomboc M, et al. J Clin Endocrinol Metab. 2000 Nov;85(11):4013-8 |
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