No association between T222P/LGR8 mutation and cryptorchidism in the Moroccan population

Original Paper

Horm Res 2008;70:236–239 DOI: 10.1159/000151596

No Association between T222P/LGR8 Mutation and Cryptorchidism in the Moroccan Population

Brahim El Houate

Hassan Rouba

Laila Imken

Hicham Sibai

Abdelaziz Chafik

Redouane Boulouiz

Elbakkay Chadli

Mohmmed Hassar

Ken McElreavey

Abdelhamid Barakat

a Human Genetic Laboratory, Pasteur Institute of Morocco, Casablanca , ^b Service of Visceral Surgery, CHU Ibn Rochd Morocco, Casablanca , ^c Laboratoire d’Anthropogénétique et Biostatistique, Université Chouaib Doukali Eljadida, Eljadida , Morocco; ^d Reproduction, Fertility and Population, Pasteur Institute of Paris, Paris , France

present in the Moroccan population, but an association be- tween cryptorchidism and the T222P mutation was not found. Copyright © 2008 S. Karger AG, Basel

Introduction

Cryptorchidism is the most common genital problem encountered in pediatrics. Cryptorchidism means hid- den testis and refers to an undescended or maldescended testis. Predisposing factors include prematurity, low birth weight, small size for gestational age, twinning, and ma- ternal exposure to estrogen during the first trimester [1] . Patients with cryptorchidism are at increased risk of tes- ticular tumor development and reduced fertility. Strong evidence demonstrates a genetic component to cryptor- chidism in mice [2–4] . Mutation of the genes encoding either

(also known as

nal phase of testicular descent, causing cryptorchidism.

The

scent via gubernacular development.

mRNA was

Cryptorchidism ⴢ LGR8 ⴢ T222P mutation

Abstract

Background: Cryptorchidism is the most common genital anomaly in men. The INSL3/LGR8 system is involved in tes- ticular descent via gubernacular development. INSL3 binds with high affinity to its receptor LGR8 and receptor activa- tion is associated with cAMP signaling. Analysis of human INSL3 and LGR8 mutations confirms that some cases of crypt- orchidism are caused by mutations in these genes. The T222P mutation is the only one within the LGR8 gene associ- ated with the cryptorchidism phenotype. A strong associa- tion of the T222P mutation with cryptorchidism was found in an Italian population. Due to the same mutation being found in patients within the Mediterranean area, a possible founder effect of this mutation is supposed. Methods: We screened 109 patients with cryptorchidism and 250 controls in a Moroccan population. Results: We found that 3 of the 109 patients tested carry the T222P mutation and 4 individu- als in the control group also carry the mutation. Conclu- sions: Our results show in fact that the same mutation is

Received: July 4, 2007 Accepted: January 10, 2008 Published online: September 5, 2008

HORMONE RESEARCH

Abdelhamid Barakat © 2008 S. Karger AG, Basel

T222P/LGR8 Mutation and Cryptorchidism in Morocco

Horm Res 2008;70:236–239 237

detected in human gubernaculums and treatment of gu- bernacular cells with Insl3 stimulated cAMP production and thymidine incorporation [5] . Insl3 activates only the Lgr8 receptor [6] .

A number of LGR8 splice variants are expressed in hu- man tissues besides the gubernaculum, including ovary, testis, uterus, brain and kidney [7, 8] . Transgenic mice with a deletion of

notype observed in

analysis of human

several allelic variants in the

sociated with maldescended testes, suggesting that some cases of human cryptorchidism are associated with ge- netic aberrations in INSL3/LGR8 signaling [9–11] .

The T222P mutation is the only mutation within the

type [10, 12] . It has been described in 5 patients: 1 French and the other 4 Italian. The mutation results in a trans- version of A to C at nucleotide 664 that is predicted to result in a missense substitution of tyrosine to proline.

This mutation has not been observed in northern Euro- pean populations, suggesting that it may be a founder mutation in the Mediterranean basin. Here, we investi- gated the relationship between the T222P mutation and cryptorchidism in 109 Moroccan patients with cryptor- chidism and a Moroccan control group.

Material and Methods

Cryptorchidism patients (n = 109) were recruited from the Service of Visceral Surgery at the Children’s Hospital CHU Ibn Rochd, Casablanca, Morocco. The control group comprised 250 men, including 127 donors with proven fertility and 123 without cryptorchidism. Informed consent was obtained from all subjects and from the parents in the case of children. All patients and con- trols were of Moroccan origin.

DNA was extracted from peripheral blood samples by stan- dard techniques. To screen for the T222P mutation, we used prim-

er-introduced restriction site (PIRS) and confirmation by se- quencing.

To amplify the sequence flanking the muted base in exon 8, we used the forward primer: 5 ⴕ -tttgtcagaattctagatgacaatccagta- 3ⴕ , which was modified in order to create a Mae III (New England BioLabs, Beverly, Mass., USA) restriction site, and the reverse primer was 5 ⴕ -tacatctttggtcaaccactgcaaa- 3ⴕ. This modified prim- er allows a RFLP between the muted allele and the normal allele.

PCRs were carried out in 25- ␮ l volumes containing 50 ng of ge- nomic DNA, 0.4 ␮ M for each primer, 200 ␮ M dNTPs, 1 ! buffer and 1 U of Taq DNA polymerase (Promega, Charbonnières-les- Bains, France). The reactions were then denaturated for 1 min at 95 ° C, and PCR amplification was subsequently carried out with 35 cycles at 95 ° C for 30 s, 58 ° C for 30 s, and 72 ° C for 50 s each step. The results is visualized in 4% gel agarose with 0.5% ethid- ium bromide (Sigma, St. Louis, Mo., USA). The muted DNA was then confirmed by direct sequencing. Using the reverse primer, direct sequencing of PCR products was performed with the ABI Prism Big Dye Terminator cycle sequencing Ready Reaction kit V 3.1 (ABI Prism/Applied Biosystem, Foster City, Calif., USA) and analyzed on an ABI Prism 3130 Genetic Analyzer (Applied Bio- systems).

To test if the mutation is within same haplo group described by Ferlin et al. [9] , we also looked for the three variants described by them, which define the associated haplotype with the T222P mutation. Sequence analysis was made up after amplification of the segment intron/exon 12 using forward primer: F1 5 ⴕ -ggatgat- gataattgtgtagg-3 ⴕ , and reverse primer R1 5 ⴕ -ctgaaatgcatgctcctgtg- 3 ⴕ . The ␹ ² test was used to assess the statistical significance be- tween groups. A p value of ! 0.05 was considered statistically sig- nificant.

Results

We screened 109 patients with cryptorchidism: 52 pa- tients with unilateral cryptorchidism; 38 patients with bi- lateral cryptorchidism, and 19 patients with cryptorchi- dism and hypospadias. We found 3 patients (3/109, 2.7%) with the T222P mutation: EC34, EC52, and EC75 ( ta- ble 1 ).

Table 1. Details of the Moroccan individuals with the T222P mutation

Individuals Phenotype Carrier in the family GA13 haplotype

EC34 Unilateral cryptorchidism Mother with T222P mutation +

EC52 Unilateral cryptorchidism – +

EC75 Bilateral cryptorchidism Father with T222P mutation +

SF14 Normal Father with T222P mutation +

SF16 Normal – +

MF12 Normal – +

RD13 Normal – +

In the control group (n = 250), we found 4 individuals (4/250, 1.6%) who carry the T222P mutation. There was no statistical difference between the 2 groups (p

0.05).

Details of all cases with the T222P mutation are sum- marized in table 1 . Family members were available for patients EC75 and EC34, and for control individual SF14.

Analyses of parents show that the mother was the carrier of the T222P mutation for patient EC34, whereas the mu- tation was transmitted to the EC75 patient and SF14 con- trol by their father, who did not have reported cryptor- chidism. Affected and unaffected individuals with crypt- orchidism, who carried the T222P mutation, were found to share the same haplotype GA13 found in LGR8 exon 12/intron 12.

Discussion

T222P-LGR8 is a unique mutation described in

gene associated with cryptorchidism [4] . It leads to a sub- stitution of tyrosine in proline at position 222. This mu- tation has drastic consequences on the function of the LGR8 receptor; it abolishes the binding of the INSL3 hor- mone to his receptor in in vitro studies [4] . The T222P mutation was described in 1 patient from southern France [4] and Ferlin et al. [9] found the same mutation in 4 pa- tients from Italy [9] . In addition, recent analysis of a large group of patients with cryptorchidism in the Italian pop- ulation, showed a significant association between the T222P mutation and cryptorchidism in a large popula- tion of patients [10] .

Analysis of the adjacent region lead to the identifica- tion of three polymorphisms A957G, G993A and inser- tion of polyA 9–13 that together form a haplotype associ- ated with the T222P mutation ( fig. 1 ). The GA13 haplo- type was found in all patients with the T222P mutation described [8] . This suggested a founder effect of the mu- tation in the Italian population [9] . Taking this observa- tion, we screened 109 Moroccan patients for the same

T222P mutation, and 250 healthy controls composed of men with no cryptorchidism and fertile men, all of Mo- roccan origin. We found that all carriers of the T222P mutation (patient and control) had the GA13 haplotype described by Ferlin et al. [9] in the Italian population, suggesting a founder mutation limited in geographic ex- tent to the Mediterranean basin.

However, we did not find an association of the T222P mutation with cryptorchidism in the Moroccan popula- tion. When we analyzed the family members of patient EC75 ( table 1 ), we found that the father had the T222P mutation but was not reported to have had cryptorchi- dism. Secondly, analysis of the control group showed 4 normal men without reported cryptorchidism who car- ried the T222P mutation. In addition, analysis of the par- ents of one of the control group showed that he inherited the mutated allele from his father, who did not have any sign of maldescended testis. Although the mutation abol- ishes the binding capacity of the hormone to the receptor ex vivo [9] , this does not result in cryptorchidism in some individuals. Bogatcheva et al. [10] detected the T222P mutation in 19 subjects (6 men with bilateral cryptorchi- dism, 12 men with unilateral cryptorchidism, and 1 pa- tient with bilateral cryptorchidism) in whom spontane- ous descent occurred during the first 5 years after birth.

The different clinical presentation seen in individuals carrying the same mutation suggests that the phenotypic variability observed in many individuals with cryptor- chidism may be the result of genetic or environmental modifiers interacting with the T222P mutation. Alterna- tively, the patients described in this study could carry al- leles that compensate for the effect of the mutation. Anal- ysis of the mutation in more populations may show the effects of population lineages.

T222P 957A 993G

A₉>A₁₃ 13

Fig. 1. Haplotype formed by polymorphism in the Exon 12/Intron 12 region associated with the T222P mutation.

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