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Endocrine Research: Francesca Nuti, Eliana Marinari, Edit Erdei, Manal El-Hamshari, Mirna Guadalupe Echavarria, Elisabet Ars, Giancarlo Balercia, Miklos Merksz, Claudia Giachini, Kamal Zaki Mahmoud Shaeer, Gianni Forti, Eduard Ruiz-Castané, and Csilla Krausz The Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 8 Gene T222P Mutation Does Not Cause Cryptorchidism J Clin Endocrinol Metab 2008; 93: 1072-1076 [Abstract] [Full text] [PDF]

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Csilla Krausz, Francesca Nuti and Gianni Forti   (11 February 2008)

The LGR8 gene T222P mutation is a risk factor for cryptorchidism

Carlo Foresta, Alberto Ferlin and Alexander I. Agoulnik   (12 January 2008)

Response to E-Letter 11 February 2008
Csilla Krausz, Faculty of Medicine University of Florence, Francesca Nuti and Gianni Forti Send letter to journal: Re: Response to E-Letter c.krausz{at}dfc.unifi.it Csilla Krausz, et al.	We read with interest and some dismay the letter from Foresta and colleagues. Our large multi-center study failed to confirm their previous conclusion that there is “an exclusive association between abnormal testis descent and T222P mutation” (1). Therefore, we believe that one should not propose genetic screening for this mutation. Instead of acknowledging this, they present their data on INSL3 and LGR8 mutations as a support for previous mouse and functional studies, and point out again that they have always found mutations only in affected individuals. While animal models are doubtless very useful, conclusion about the role of a gene in humans should be based on human studies. In this case, our human data has found unexpected differences from the mouse model, which is not surprising, especially if the penetrance of heterozygous Insl3 mice is already only partial. The statement by Foresta et al.’s letter that we “choose only men with history of orchidopexy” is incorrect. As clearly stated, we included men with a history of cryptorchidism (and not with orchidopexy), which means men with spontaneous descent after birth or descent after medical therapy. Moreover, the similar frequencies of the mutation in both the North Italian (4.2%) and Central Italian (5.0%) patients imply that the inclusion criteria were similar. Although Foresta et al. claim that the best group for controls should be newborn boys, all of their own previous publications were based on adults. Thus, our analysis was based on controls selected by the same criteria (i.e., self declaration of lack of history of testicular maldescent at birth and no retractile testis - by history or on examination). Given the clinical importance of our finding, before submitting the manuscript, we asked the control mutation carriers to confirm from their parents the lack of history of maldescent at birth; in all cases, this was done. Moreover, all mutations were double checked and confirmed in two different laboratories in a blinded manner. A new genetic finding requires validation in an independent series including affected and unaffected individuals. Our multiethnic study design confirmed the lack of a relationship between T222P mutations and testicular maldescent or retractile testis in Italians, Hungarians, and Spaniards. We are puzzled on why Foresta et al. did not find any mutation carrier in 650 controls selected by the same criteria, whereas we observed 5 carriers out of 275 Italian controls and 3 additional subjects in two other European countries. We also have some concern about considering T222P mutation as a risk factor for cryptorchidism. First of all, the preference for one tail p testing rather than the two tail chi-square probability is disputable. There is no direct evidence that heterozygous mutation should always lead to pathology and, consequently, our null hypothesis is that differences could be expected in both directions. We carefully checked all available published literature and found that the total number of controls reported has been 983, and only 353 were from France, Germany, the United States, Finland and Japan, with the remainder from North Italy. Reviewing these data, it is clear that geographic differences do exist, since no mutation was found either in controls or ex -cryptorchid men in Germany, the United States, Finland and Japan (a total of 149 patients). Moreover, in all non-Italian countries, including our own study, the cumulative mutation frequency is 6/366 (1.6%) in affected and 3/541 (0.5%) in control subjects, which is statistically not significant. The sharp contrast between data on the Central and North Italian population makes it even more difficult to combine data from different laboratories. Clearly, the highly significant p value calculated by Foresta et al. is due to their own data (27 mutation carriers in the affected and zero in the control group), reflecting a North Italian reality that may not mirror the rest of the world. In summary, our study provides clear evidence that there is not a strict cause effect relationship between T222P mutation and testicular maldescent. Given the moderate functional consequences of this mutation, it is possible that only homozygous mutation, which has not yet been found, is associated with the pathology. In our opinion, it is too early to conclude that T222P mutation is a risk factor for abnormal testis descent, especially if we expect a contribution from the ethnic and environmental background. Finally, we are highly confident of our results and believe that further studies in our laboratory and others’ will confirm our findings. Reference 1. Bogatcheva NV, Ferlin A, Feng S, Truong A, Gianesello L, Foresta C, Agoulnik AI 2007 T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane. Am J Physiol Endocrinol Metab 292:138-144
The LGR8 gene T222P mutation is a risk factor for cryptorchidism 12 January 2008
Carlo Foresta, Professor of Clinical Pathology University of Padova, Dept Histology, Microbiology and Medical Biotechnologies, Alberto Ferlin and Alexander I. Agoulnik Send letter to journal: Re: The LGR8 gene T222P mutation is a risk factor for cryptorchidism carlo.foresta{at}unipd.it Carlo Foresta, et al.	We read with much interest the article by Nuti et al. (1) regarding the screening for the T222P mutation in the LGR8 gene in men with history of cryptorchidism from four different countries. Although the molecular screening is adequate, we have a number of concerns regarding the selection of studied subjects, statistical analysis, and ultimately with the main conclusion of the paper stating that the mutation cannot be considered either causative or a risk factor for cryptorchidism. It was reported, contrary to what was stated by Nuti et al., that 75% of heterozygous Insl3 mice had unilateral or bilateral cryptorchidism at birth (with delayed gubernacula regression) with subsequent spontaneous descent (2). This finding perfectly agrees with the human phenotypes associated with LGR8 and INSL3 mutations. In the largest series of men screened for mutations in these genes (3, 4), we found that they all were associated with different degrees of testicular maldescent, such as bilateral to unilateral cryptorchidism requiring orchidopexy, spontaneous descent after birth, retractile testes or ascending testis (review in ref. 5). Molecular analysis showed that the T222P mutation reduced receptor surface expression and hence abolished signal transduction (4) supporting proposed haploinsufficiency as the underlying mechanism. Thus it is very important that an accurate medical history and physical exam be done to identify all possible forms of testicular maldescent. In this regards, it is not clear why Nuti et al. (1) choose only men with history of orchidopexy, therefore excluding patients with retractile testes, spontaneous descent after birth and testes descended with medical therapy. Furthermore, cases and controls recruited were adults, thus precluding detailed clinical evaluation at birth. Screening of newborns should be performed before concluding that the mutation is not associated with the phenotype. We have recently completed a screening of almost 300 cryptorchid boys at birth and found 1.5% of T222P prevalence, whereas no mutation was found in a comparable number of non-cryptorchid boys. Variable penetrance and expressivity of the T222P mutation does speak to the possible significance of genetic background and/or environmental factors on mutant phenotype development. Therefore it is important to compare the frequency of the mutation within specific populations. Indeed, the data presented in Nuti et al. (1) reveals that there is a clear trend, albeit statistically insignificant between ex-cryptorchid (8/159, 5.0%) and control (5/275, 1.8%) Italian men with the mutation, with a two tail chi-square probability value of 0.058 (one tail P = 0.029). Comparison for the other studied populations is more difficult because of the lesser number of subjects (about 70 in Hungary and Spain) or absence of the control group (Egypt). More importantly, published data reveal that the T222P mutation has been found in 28/979 (2.9%) cryptorchid or ex-cryptorchid subjects and in 0/1097 controls, as reported by seven studies analyzing populations from France, Germany, United States, Finland, Italy and Japan (review in ref. 5). Therefore, with the data of Nuti et al. (1) the cumulative frequency of mutation is 3.1% in cases (41/1338) and 0.5% (8/1560) in controls, a value that is highly statistically significant (P < 0.0001, relative risk 5.97, 95% CI 2.86-12.51). Thus, the T222P does appear to be associated with maldescent of the testis and a risk factor for human cryptorchidism. References 1. Nuti F, Marinari E, Erdei E, El-Hamshari M, Echavarria MG, Ars E, Balercia G, Merksz M, Giachini C, Shaeer KZ, Forti G, Ruiz-Castané E, Krausz C 2007 The LGR8 gene T222P mutation does not cause cryptorchidism. J Clin Endocrinol Metab Dec 11; [Epub ahead of print] 2. Nef S, Parada LF 1999 Cryptorchidism in mice mutant for Insl3. Nat Genet 22:295-299 3. Ferlin A, Bogatcheva NV, Gianesello L, Pepe A, Vinanzi C, Agoulnik AI, Foresta C 2006 Insulin-like factor 3 gene mutations in testicular dysgenesis syndrome: clinical and functional characterization. Mol Hum Reprod 12:401-406 4. Bogatcheva NV, Ferlin A, Feng S, Truong A, Gianesello L, Foresta C, Agoulnik AI 2007 T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane. Am J Physiol Endocrinol Metab 292:E138-144 5. Ferlin A, Zuccarello D, Garolla A, Selice R, Foresta C 2007 Hormonal and genetic control of testicular descent. Reprod Biomed Online 15:659-665