Biomedical subjects

M Fellous

Publications and source records attributed to M Fellous.

At least 37 records · Page 2Linked to original sources

PubMed2001

Expression of the human SRY protein during development in normal male gonadal and sex-reversed tissues.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells.

Adolescent↗

PubMed2001

Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype--phenotype correlation.

Mutations in FOXL2, a forkhead transcription factor gene, have recently been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder. In BPES type I a complex eyelid malformation is associated with premature ovarian failure (POF), whereas in BPES type II the eyelid defect occurs as an isolated entity. In this study, we describe the identification of novel mutations in the FOXL2 gene in BPES types I and II families, in sporadic BPES patients, and in BPES families where the type could not be established. In 67% of the patients studied, we identified a mutation in the FOXL2 gene. In total, 21 mutations (17 of which are novel) and one microdeletion were identified. Thirteen of these FOXL2 mutations are unique. In this study, we demonstrate that there is a genotype--phenotype correlation for either types of BPES by the finding that mutations predicted to result in a truncated protein either lacking or containing the forkhead domain lead to BPES type I. In contrast, duplications within or downstream of the forkhead domain, and a frameshift downstream of them, all predicted to result in an extended protein, cause BPES type II. In addition, in 30 unrelated patients with isolated POF no causal mutations were identified in FOXL2. Our study provides further evidence that FOXL2 haploinsufficiency may cause BPES types I and II by the effect of a null allele and a hypomorphic allele, respectively. Furthermore, we propose that in a fraction of the BPES patients the genetic defect does not reside within the coding region of the FOXL2 gene and may be caused by a position effect.

Alleles↗

PubMed2001

Testis determination in mammals: more questions than answers.

In humans, testis development depends on a regulated genetic hierarchy initiated by the Y-linked SRY gene. Failure of testicular determination results in the condition termed 46,XY gonadal dysgenesis (GD). Several components of the testis determining pathway have recently been identified though it has been difficult to articulate a cascade with the known elements of the system. It seems, however, that early gonadal development is the result of a network of interactions instead of the outcome of a linear cascade. Accumulating evidence shows that testis formation in man is sensitive to gene dosage. Haploinsufficiency of SF1, WT1 and SOX9 is responsible for 46,XY gonadal dysgenesis. Besides, data on SRY is consistent with possible dosage anomalies in certain cases of male to female sex reversal. 46,XY GD due to monosomy of distal 9p and 10q might also be associated with an insufficient gene dosage effect. Duplications of the locus DSS can lead to a failure of testicular development and a duplication of the region containing SOX9 has been implicated in XX sex reversal. Transgenic studies in mouse have shown, however, that this mammal is less sensitive to gene dosage than man. Here, we will try to put in place the known pieces of the jigsaw puzzle that is sex determination in mammals, as far as current knowledge obtained from man and animal models allows. We are certain that from this attempt more questions than answers will arise.

Animals↗

PubMed2001

Identification of the human KIF13A gene homologous to Drosophila kinesin-73 and candidate for schizophrenia.

Several studies have reported significant linkage for schizophrenia on 6p23, with a maximum lod score between D6S274 and D6S285. In this paper, we present a new human kinesin gene localized in this 2-cM interval. This gene, termed KIF13A, belongs to the unc-104/KIF1A kinesin subfamily and represents the orthologue of Drosophila kinesin-73. Several alternative transcripts are differentially expressed in human tissues, probably reflecting differences in cargo binding and transport of corresponding proteins. During early mouse development, its homologue (Kif13A) is expressed essentially in the central nervous system. In Caenorhabditis elegans, the unc-104 gene is involved in axonal anterograde transport, and null mutants present several behavioral defects. The putative function and genomic localization of KIF13A make this gene an interesting candidate for genetic predisposition to schizophrenia. We provide sequences of 20 single-nucleotide polymorphisms localized within KIF13A to test for association studies between this gene and schizophrenia.

Amino Acid Sequence↗

PubMed2001

Uncoordinated HLA-D gene expression in a RFXANK-defective patient with MHC class II deficiency.

We describe the analysis of a patient, JER, presenting classical immunological features of MHC class II deficiency. Unexpectedly, some HLA transcripts (HLA-DRA, HLA-DQA, and HLA-DMA) were found to be expressed in the JER cell line at nearly wild-type levels, while HLA-DPA and the HLA-D beta-chain transcripts were not detected. Gene reporter experiments confirmed the differential transcriptional activities driven by the HLA-D promoters in the JER cells. A defect in RFXANK was first suggested by genetic complementation analyses, then assessed with the demonstration of a homozygous mutation affecting a splice donor site downstream exon 4 of RFXANK. Because the severe deletion of the resulting protein cannot account for the expression of certain HLA-D genes, minor alternative transcripts of the RFXANK gene were analyzed. We thereby showed the existence of a transcript lacking exon 4, encoding a 28-aa-deleted protein that retains a transcriptional activity. Altogether, we characterize a new type of mutation in the RFXANK gene in a MHC class II-defective patient leading to an uncoordinated expression of the HLA-D genes, and propose that this phenotype is ensured by severely limited amounts of an active, although truncated RFXANK protein.

Alternative Splicing↗

PubMed2001

Transduction of the human gene FAM8A1 by endogenous retrovirus during primate evolution.

Capture of cellular mRNA by mobile elements has been an evolutionary catalyst for the spread of genes and a cause of cancer development. Here we present evidence that an orphan gene, FAM8A1 (family with sequence similarity 8), was captured by a retrovirus, followed by multiple retrotransposition events, during primate evolution between 45 and 58 million years ago. This represents the first record of cellular mRNA transduction in humans. The human gene is localized on chromosome 6p23 with five related pseudogenes (FAM8A2P-A6P), each inserted within a human endogenous retrovirus (HERV). Only the functional FAM8A1 gene is expressed and displays a ubiquitous mRNA and a testis-specific transcript present in the haploid phase of spermatogenesis. The structural features of the FAM8A1 pseudogenes include two short sequences of similarity between the FAM8A1 mRNA and the HERV sequences at both the 5' and 3' integration sites. These hallmarks suggest an alternative model to account for the capture of FAM8A1 cellular mRNA by HERV-K, involving illegitimate recombination events at the two sites of sequence similarity during reverse transcription. Unlike previous models, which assume at least one step of retroviral integration in the genome, our model is consistent with in vitro observations showing that multiple template switches occur among packaged viral transcripts. This leads to the speculation that, in some cases, cellular mRNAs may have been captured through similar processes involved in the retroviral life cycle.

Amino Acid Sequence↗

PubMed2001

[Origin and evolution of mammalian sex chromosomes].

Mammals present an XX/XY system of chromosomal sex determination, males being the heterogametic sex. Comparative studies of the gene content of sex chromosomes from the major groups of mammals reveal that most Y genes have X-linked homologues and that X and Y share homologous pseudoautosomal regions. These observations, together with the presence of the two homologous regions (pseudoautosomal regions) at the tips of the sex chromosomes, suggest that these chromosomes began as an ordinary pair of homologous autosomes. Birds present a ZW/ZZ system of chromosomal sex determination where females are the heterogametic sex. In this case, avian sex chromosomes are derived from different pairs of autosomes than mammals. The evolutionary pathway from the autosomal homomorphic departure to the present-day heteromorphic sex chromosomes in mammals includes suppression of X-Y recombination, differentiation of the nascent non-recombining regions, and progressive autosomal addition and attrition of the sex chromosomes. Recent results indicate that the event marking the beginning of the differentiation between the extant X and Y chromosomes occurred about 300 million years ago.

Animals↗

PubMed2001

NSPc1, a novel mammalian Polycomb gene, is expressed in neural crest-derived structures of the peripheral nervous system.

Anterior-posterior (A-P) patterning is a key element in early embryonic development. Polycomb group (PcG) genes act as transcriptional repressors to regulate A-P patterning by either directly or indirectly controlling the coordinated expression of the HOM/Hox homeobox (Curr. Opin. Genet. Dev. 7 (1997) 488; Trends Genet. 13 (1997) 167). We describe the isolation and characterization of a novel mammalian PcG gene, termed Nervous System Polycomb-1 (NSPc1). Human and mouse NSPc1 genes encode proteins with an N-terminal RING finger domain and share homology with Drosophila melanogaster lethal(3)73Ah and the mammalian Mel18 and Bmi1 genes. Transcripts are observed at 10 dpc in the otic vesicle, urogenital bud and dorsal root ganglia. At 11.5 dpc, transcripts are present in a subset of neural crest cell derivatives of the peripheral nervous system, and in the neural tube. NSPc1 expression is ubiquitous in adult tissue.

Amino Acid Sequence↗

PubMed2001

Y chromosome microsatellite variation in three populations of Jerba Island (Tunisia).

Six Y-chromosome linked microsatellites were typed in a sample of 135 unrelated males representing three different ethnic groups: Arabs, Berbers and Blacks of Jerba Island (Tunisia). Analysis of variation at the six Y chromosome STRs showed significant differences in allele distributions between the Black group and the two other Islander groups. The Black group revealed the highest level of genetic diversity as compared to Arabs and Berbers, while the latter group was the most homogeneous. Allele frequencies obtained for the three islander groups analysed were compared to data available for some European, Mediterranean and African populations. Principal-coordinate analyses showed genetic differentiation between the three geographically closed groups of Jerba. The absence of the YAP insertion marker and the position of Arabs and Jerban Blacks near the European cluster would suggest their relative 'admixture' with European populations.

Alleles↗

PubMed2001

A 11.7-kb deletion triggers intersexuality and polledness in goats.

Mammalian sex determination is governed by the presence of the sex determining region Y gene (SRY) on the Y chromosome. Familial cases of SRY-negative XX sex reversal are rare in humans, often hampering the discovery of new sex-determining genes. The mouse model is also insufficient to correctly apprehend the sex-determination cascade, as the human pathway is much more sensitive to gene dosage. Other species might therefore be considered in this respect. In goats, the polled intersex syndrome (PIS) mutation associates polledness and intersexuality. The sex reversal affects exclusively the XX individuals in a recessive manner, whereas the absence of horns is dominant in both sexes. The syndrome is caused by an autosomal gene located at chromosome band 1q43 (ref. 9), shown to be homologous to human chromosome band 3q23 (ref. 10). Through a positional cloning approach, we demonstrate that the mutation underlying PIS is the deletion of a critical 11.7-kb DNA element containing mainly repetitive sequences. This deletion affects the transcription of at least two genes: PISRT1, encoding a 1.5-kb mRNA devoid of open reading frame (ORF), and FOXL2, recently shown to be responsible for blepharophimosis ptosis epicanthus inversus syndrome (BPES) in humans. These two genes are located 20 and 200 kb telomeric from the deletion, respectively.

Animals↗

PubMed2001

Absence of mutations involving the LIM homeobox domain gene LHX9 in 46,XY gonadal agenesis and dysgenesis.

The etiology of most cases of 46,XY gonadal dysgenesis in the absence of extragenital anomalies is not accounted for by mutations in the genes known to date to be involved in sex determination. We have investigated the possibility that mutations in the gene LHX9, whose murine ortholog causes isolated gonadal agenesis when inactivated, might be responsible for gonadal dysgenesis and agenesis in humans. We isolated a human LHX9 complementary DNA (cDNA), mapped the gene to the long arm of human chromosome 1, and determined its genomic structure. We found that LHX9 is highly conserved between species, sharing in particular over 98% amino acid identity. A mutational screen was performed in a sample of patients with a range of gonadal maldevelopment, including bilateral gonadal agenesis in two sisters with an opposite sex karyotype. We did not detect mutations in the open reading frame of LHX9 in the patients studied. However, the extent of between-species structural conservation suggests that LHX9 deserves further consideration as a determinant of gonadal function in humans.

Amino Acid Sequence↗

PubMed2000

The human doublesex-related gene, DMRT2, is homologous to a gene involved in somitogenesis and encodes a potential bicistronic transcript.

Intense efforts are currently being pursued to identify autosomal genes associated with 46,XY male-to-female sex reversal. The genes DMRT1 and 2 are located on distal 9p, a region deleted in 46,XY sex-reversed patients. They are considered excellent candidates because of their homology to regulators of sex development in invertebrates. We present the genomic structure of DMRT2, showing that it generates several transcripts with distinct coding potential. In addition to the previously reported 226-amino-acid protein-encoding transcript, we describe other mRNA isoforms that are potentially bicistronic and are predicted to encode an additional 328-amino-acid polypeptide. Finally, a stop codon-containing exon (exon 4) can be skipped by alternative splicing and can generate a transcript that is predicted to encode a fusion protein. The latter shares 58% amino acid identity with a gene recently described in fish, termed terra. Differences in expression pattern exist for DMRT2 mRNA isoforms among the human adult tissues tested, between adult tissues and human embryos, and between DMRT2 and DMRT1 during embryonic development. We failed to detect mutations by sequencing of DMRT2 in a sample of 46,XY female patients. The interesting structure of DMRT2 coupled to preliminary functional studies in fish showing that terra is involved in somitogenesis suggests that validation or exclusion of this gene as a cause of sex reversal will require more in-depth investigations.

Adult↗

PubMed2000

The region on 9p associated with 46,XY sex reversal contains several transcripts expressed in the urogenital system and a novel doublesex-related domain.

Deletions of 9p have been associated with 46,XY gonadal dysgenesis, and the smallest region of overlap has been mapped to the tip of chromosome 9. Two candidate genes (DMRT1 and 2) have been found in the region. Despite intensive mutation searches, no mutations have been detected in these genes. To gain insights into the genomics of the region and to isolate other candidate genes for the phenotype, we have constructed a P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC) contig spanning over 500 kb and covering the consensus critical region. We have analyzed the expression pattern of several ESTs mapped or sublocalized within the framework of the contig. In addition, a sample shotgun sequencing of a PAC containing the mentioned DM genes led to the detection of novel transcripts displaying an expression pattern specific to testis and kidney, consistent with a role in the development of the urogenital system. One of them, expressed in adult testis and human embryos aged 4-5 weeks, encodes a potential polypeptide and is located immediately downstream of a sequence capable of encoding a novel DM domain. The region was partially screened for mutations in sex-reversed patients by Southern blot, sequencing, and FISH. No mutations were found. Our results suggest that the critical region on 9p involved in male-to-female sex reversal displays greater gene density and genomic complexity than previously anticipated. Future investigations will include functional and mutational studies of the novel transcripts mapped or sublocalized within the critical region by this study as well as cloning efforts to isolate additional candidate genes.

Adult↗

PubMed2000

A study of the coregulation and tissue specificity of XG and MIC2 gene expression in eukaryotic cells.

CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative polymorphism coregulated with the polymorphism of the XG blood group gene. As a preliminary study of this phenomenon, human XG and CD99 recombinant proteins were expressed in murine RAG cells and analyzed by flow cytometry. Both proteins were expressed independently and at a similar level in single and double transfectants. Immunoprecipitation and Western blot analysis, using the murine monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd (CD99), respectively. A putative 28-kd intracellular precursor of CD99 was also detected, as was a 26-kd species after neuraminidase treatment of CD99-expressing cells. No evidence of association or complex formation between XG and CD99 proteins could be proven, either on transfected RAG cells or on human erythrocytes. These results were confirmed using somatic hybrids between single transfectants. These findings suggest that the phenotypic relationship between XG and CD99 is mostly regulated at the transcriptional level, but they do not formally exclude some posttranscriptional effect. Studies on the tissue specificity of XG expression showed that surface expression of the XG protein could not be restored in somatic hybrids between B-lymphoblastoid cell lines from Xg(a+) persons and fibroblasts (RAG) or erythroid (MEL) cells. RT-PCR analysis of the transcripts revealed the existence of an XG mRNA in each cell line, suggesting that the tissue-specific regulation of cell surface XG expression occurs either at a quantitative transcriptional level or is a posttranscriptional event. By Northern blot analysis, XG transcripts were detected in erythroid tissues and several nonerythroid tissues. (Blood. 2000;95:1819-1826)

12E7 Antigen↗

PubMed2000

WT1 splice-site mutations are rarely associated with primary steroid-resistant focal and segmental glomerulosclerosis.

BACKGROUND: Donor splice-site de novo heterozygous mutations in intron 9 of the Wilms' tumor gene (WT1) have been reported in Frasier syndrome, which is defined by the association of focal and segmental glomerulosclerosis (FSGS), male pseudohermaphroditism, and gonadoblastoma. These splice-site mutations alter the WT1 alternative splicing leading to two WT1 isoforms, with (+) or without (-) three amino acids, lysine-threonine-serine (KTS), between zinc fingers 3 and 4. The aim of this work was to investigate the possibility that some cases of primary steroid-resistant nephrotic syndrome associated with FSGS may be caused by WT1 splice-site mutations. METHODS: We analyzed WT1 exons 8 and 9 and the surrounding exon/intron boundary DNA sequences in 37 children with nonfamilial primary steroid-resistant nephrotic syndrome. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the relative ratio of +KTS/-KTS transcripts from immortalized lymphocyte RNA. RESULTS: One boy with FSGS and associated pathologies (diaphragmatic hernia, proximal hypospadias, and unilateral testicular ectopia) was found to carry the heterozygous 1228 +4 C-->T splice-site mutation. RT-PCR quantitation of the +KTS/-KTS transcripts from immortalized lymphocyte RNA of this patient showed a diminution of the +KTS/-KTS isoform ratio (0.43), which is identical to that reported in patients with Frasier syndrome. Using the same approach, healthy control subjects have +KTS/-KTS ratios ranging from 1.50 to 2.00. CONCLUSIONS: This study expands the range of the phenotypic presentation of the intron 9 splice-site WT1 mutations and adds to the already reported heterogeneity of primary steroid-resistant nephrotic syndromes. We suggest that these mutations are not likely to be a common cause of isolated steroid-resistant nephrotic syndrome, and recommend a WT1 exon 9/intron 9 splice-site study in children with primary steroid-resistant nephrotic syndrome if genital or diaphragmatic anomalies are associated. The identification of such WT1 mutations has practical implications for the management of these patients.

Adolescent↗

PubMed2000

Sex chromosome mosaicism in males carrying Y chromosome long arm deletions.

Microdeletions of the long arm of the Y chromosome (Yq) are a common cause of male infertility. Since large structural rearrangements of the Y chromosome are commonly associated with a 45,XO/46,XY chromosomal mosaicism, we studied whether submicroscopic Yq deletions could also be associated with the development of 45,XO cell lines. We studied blood samples from 14 infertile men carrying a Yq microdeletion as revealed by polymerase chain reaction (PCR). Patients were divided into two groups: group 1 (n = 6), in which karyotype analysis demonstrated a 45,X/46,XY mosaicism, and group 2 (n = 8) with apparently a normal 46,XY karyotype. 45,XO cells were identified by fluorescence in-situ hybridization (FISH) using X and Y centromeric probes. Lymphocytes from 11 fertile men were studied as controls. In addition, sperm cells were studied in three oligozoospermic patients in group 2. Our results showed that large and submicroscopic Yq deletions were associated with significantly increased percentages of 45,XO cells in lymphocytes and of sperm cells nullisomic for gonosomes, especially for the Y chromosome. Moreover, two isodicentric Y chromosomes, classified as normal by cytogenetic methods, were detected. Therefore, Yq microdeletions may be associated with Y chromosomal instability leading to the formation of 45,XO cell lines.

Adult↗

PubMed2000

Absence of mutations involving the INSL3 gene in human idiopathic cryptorchidism.

The aetiology of cryptorchidism is for the most part unknown and appears to be multifactorial. Recently, a product of Leydig cells termed Leydig insulin-like hormone (INSL3) has been proposed as a putative trophic hormone of the first part of descent. Absence of Insl3 in male mice results in bilateral cryptorchidism and mutations involving this gene may be a cause of cryptorchidism in man. We sequenced both exons of the human INSL3 gene in 31 men who presented with idiopathic unilateral or bilateral cryptorchidism. The only sequence variant was an amino acid substitution in the C-peptide of the molecule. This change was also found in a control group of normal fertile men indicating that it is a polymorphism unrelated to the phenotype. These results suggest that mutations involving the human INSL3 gene are not a common cause of cryptorchidism in man.

Amino Acid Sequence↗

PubMed2000

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal.

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein.

DNA-Binding Proteins↗