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X-linked severe combined immunodeficiency (XSCID) is a hereditary disorder characterized by severe T cell lymphopenia and abnormal B cell function. The disease is caused by mutations in IL2RG, the gene encoding the interleukin-2 receptor common γ chain (γc) shared by several interleukin receptors. A Harvey retroviral bicistronic vector containing an IL2RG cDNA and cDNA encoding the multidrug transporter (MDR1) was constructed to investigate the correction of XSCID. Translation of the MDR1 cDNA is achieved from an internal ribosome entry site (IRES). Mouse fibroblasts transfected or transduced with the vector expressed both membrane proteins as detected with specific monoclonal antibodies by fluorescence activated cell sorting. Two human XSCID B cell lines were transduced using a filter concentration method in combination with phosphate depletion. Significant expression of both proteins was detected by Western blot analysis. This construct might be particularly useful if high expression of γc is required, as might be achievable through in vivo selection for drug resistance of recipient lymphocytes. [ABSTRACT FROM AUTHOR]

Recently, microdeletions in the azoospermic factor region of the Y chromosome, in addition to chromosomal anomalies, have been detected in men with azoospermia or severe oligozoospermia. In this study we evaluated the molecular and cytogenetic defects of infertile men. The frequency of Y microdeletions among 105 azoospermic, 28 oligozoospermic and 32 fertile men was tested on lymphocyte DNA using a series of 20 sequence-tagged sites. In addition, microdeletions were evaluated on testicular-derived DNA among 26 azoospermic patients who underwent testicular biopsy and in whom no sperm cells could be identified. Karyotype analysis was performed on 72 of the infertile patients. Deletions were detected in 6.7% azoospermic and 3.6% oligozoospermic men. No deletions were identified among the fertile men. Identical results were obtained with DNA derived either from lymphocytes or testicular tissue. The frequency of chromosomal aberrations in the 72 infertile patients tested (62 azoospermic, 10 oligozoospermic) was 16.6%, with a high percentage of gonosome anomalies. Additional andrological parameters (hormone values, cryptorchidism) failed to identify men at risk for having microdeletions before the test. Our findings support the recommendation to perform genetic defect screening among infertile men before their enrollment in an intracytoplasmic injection/in-vitro fertilization programme. [ABSTRACT FROM AUTHOR]

Background: The human X chromosome is enriched with testis-specific genes that may be crucial for male fertility. One is the ubiquitin-specific protease 26 (USP26). Five frequent mutations have been identified: 1737G>A, 1090C > T, 370-371insACA, 494T > C and 1423C>T (with the latter three usually detected in a cluster). Their role in infertility is still controversial. This study assesses the association of the most frequent USP26 mutations with male infertility and male infertility etiology factors.Methods: The study included 300 infertile and 287 fertile men. Data were collected on ethnicity (according to maternal origin) and family history of reproduction. Clinical records from 235 infertile and 62 fertile (sperm bank donors) men were available and summarized. The five mutations were investigated by bioinformatic tools and their frequencies were assessed by restriction analysis. The results were correlated with clinical findings. Segregation of the mutations in four families was analyzed.Results: The five analyzed mutations were detected in 44 men from both fertile and infertile groups. The cluster and the 1090C>T mutations showed the highest frequency among Arabs and Sephardic Jews of the infertile group, respectively. Inheritance studies showed that mutations were not always associated with the infertility trait. Mutations 1090C>T and 1737G>A were significantly associated with a history of inguinal hernia (P = 0.007 and P = 0.043, respectively). The prevalence of inguinal hernia among men with the 1090C > T mutation was 33.3% (5/15 men), higher than that reported in infertile men (6.7%).Conclusions: Mutation 1090C > T may be a new genetic risk factor for developing inguinal hernia which may be associated with impaired male fertility. [ABSTRACT FROM AUTHOR]

Background: The human X chromosome is enriched with testis-specific genes that may be crucial for male fertility. One is the ubiquitin-specific protease 26 (USP26). Five frequent mutations have been identified: 1737G>A, 1090C > T, 370-371insACA, 494T > C and 1423C>T (with the latter three usually detected in a cluster). Their role in infertility is still controversial. This study assesses the association of the most frequent USP26 mutations with male infertility and male infertility etiology factors., Methods: The study included 300 infertile and 287 fertile men. Data were collected on ethnicity (according to maternal origin) and family history of reproduction. Clinical records from 235 infertile and 62 fertile (sperm bank donors) men were available and summarized. The five mutations were investigated by bioinformatic tools and their frequencies were assessed by restriction analysis. The results were correlated with clinical findings. Segregation of the mutations in four families was analyzed., Results: The five analyzed mutations were detected in 44 men from both fertile and infertile groups. The cluster and the 1090C>T mutations showed the highest frequency among Arabs and Sephardic Jews of the infertile group, respectively. Inheritance studies showed that mutations were not always associated with the infertility trait. Mutations 1090C>T and 1737G>A were significantly associated with a history of inguinal hernia (P = 0.007 and P = 0.043, respectively). The prevalence of inguinal hernia among men with the 1090C > T mutation was 33.3% (5/15 men), higher than that reported in infertile men (6.7%)., Conclusions: Mutation 1090C > T may be a new genetic risk factor for developing inguinal hernia which may be associated with impaired male fertility.

Background: The Y-chromosome AZF regions include genes whose functions and specific roles in spermatogenesis have not been fully clarified. This study investigated the expression of several AZF (USP9Y, DDX3Y/DDX3Yt1, EIF1AY and PRY) and USP9X transcripts in testicular biopsies of 89 azoospermic men who had been classified by histology and cytology assessments., Methods: Expression was analysed by RT-PCR, and some biopsies were evaluated by multiplex RT-PCR. Quantitative PCR was performed in some biopsies to determine the ratio of the testis-specific transcript DDX3Yt1 to the total DDX3Y transcription., Results: The expression of USP9Y, USP9X and DDX3Y was found in all the specimens tested, whereas DDX3Yt1 expression was diminished or undetectable in several biopsies with impaired spermatogenesis. EIF1AY was detected in all except two of the specimens. Noteworthy, PRY expression was detected mainly in biopsies with germ cells, and this association was significant (P < 0.001). An identical expression profile was obtained by either single or multiplex RT-PCR., Conclusions: These findings suggest that PRY is usually expressed in germ cells, whereas the other transcripts are also expressed in testicular somatic cells. The absence of EIF1AY expression might sporadically contribute to azoospermia. The decreased ratio of DDX3Yt1/DDX3Y transcript in impaired spermatogenesis suggests that the DDX3Yt1 transcript is under-expressed in impaired spermatogenesis. The findings contribute to the search and selection of the most valuable gene markers potentially useful as additional tools for predicting complete spermatogenesis by multiplex expression analysis.

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.

Sperm cells can be retrieved directly from the testis (testicular sperm extraction [TESE] procedure) and used for intracytoplasmic sperm injection (ICSI), circumventing underlying spermatogenetic defects. Thus, it is important that added information be available on the genetic defects in men undergoing TESE for the ICSI procedure and on the transmission of genetic factors associated with infertility to the offspring. We report a three-generation genetic analysis of a family with a case of male factor infertility. The proband, previously diagnosed as infertile, was physically examined and laboratory tested for gonadotrophic hormones, semen analysis, karyotype and Y-chromosome microdeletion screening in the blood and testis. The Y-chromosome microdeletion screening was performed by multiplex polymerase chain reaction with 20 Y-chromosome sequenced, tagged sites located at the Y chromosome. A microdeletion including the AZF-c region was detected in the azoospermic patient. His father, four brothers, and three offspring born after ICSI also underwent Y-chromosome microdeletion screening. The genetic analysis of the male members of the patient's family did not reveal similar microdeletions. The newborn male was found to bear a Y-chromosome microdeletion similar to that of his father. The fertilization capacity of the proband testicular microdeleted spermatozoa by the ICSI procedure is described. The transfer of the genetic defect raises the possibility that the son will have the same fertility problem as his father.

Optimization of retroviral gene transfer into hematopoietic cells of the dog will facilitate gene therapy of canine X-linked severe combined immunodeficiency (XSCID) and in turn advance similar efforts to treat human XSCID. Both canine and human XSCID are caused by defects in the common gamma chain, gammac, of receptors for interleukin-2 and other cytokines. In this study, normal dogs were given retrovirally transduced bone marrow cells with and without preharvest mobilization by the canine growth factors granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Harvey sarcoma virus and Moloney murine leukemia virus constructs were used, both containing cDNA encoding human gammac. The Harvey-based vector transduced into cytokine-primed marrow yielded persistent detectable provirus in bone marrow and blood and expression of human gammac on peripheral lymphocytes. In three dogs, human gammac expression disappeared after 19 to 34 weeks but reappeared and was sustained, in one dog beyond 16 months posttransplantation, upon immunosuppression with cyclosporin A and prednisone, with up to 25% of lymphocytes expressing human gammac. The long-term expression of human gammac in a high proportion of normal canine lymphocytes predicts that retrovirus-mediated gene correction of hematopoietic cells may prove to be of clinical benefit in humans affected with this XSCID. This is a US government work. There are no restrictions on its use.