Y chromosomal fertility factors kl-2 and kl-3 of Drosophila melanogaster encode dynein heavy chain polypeptides

The molecular identity and function of the Drosophila melanogaster Y-linked fertility factors have long eluded researchers. Although the D. melanogaster genome sequence was recently completed, the fertility factors still were not identified, in part because of low cloning efficiency of heterochromatic Y sequences. Here we report a method for iterative blast searching to assemble heterochromatic genes from shotgun assemblies, and we successfully identify kl-2 and kl-3 as 1β- and γ-dynein heavy chains, respectively. Our conclusions are supported by formal genetics with X-Y translocation lines. Reverse transcription–PCR was successful in linking together unmapped sequence fragments from the whole-genome shotgun assembly, although some sequences were missing altogether from the shotgun effort and had to be generated de novo. We also found a previously undescribed Y gene, polycystine-related (PRY). The closest paralogs of kl-2, kl-3, and PRY (and also of kl-5) are autosomal and not X-linked, suggesting that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes arising de novo from the autosomes.

Thymocyte-targeted overexpression of xiap transgene disrupts T lymphoid apoptosis and maturation

The X-linked inhibitor of apoptosis (XIAP) and other members of the inhibitor of apoptosis (IAP) family can suppress apoptosis induced by a diverse variety of triggers. Functional studies done to date have focused on tissue culture models and adenovirus overexpression of XIAP and other IAP proteins. Here we report the phenotype of an engineered transgenic mouse overexpressing a human IAP, as well as assessing the long-term consequence of IAP overexpression. We document the relative protein expression levels of the endogenous mouse homologue to XIAP, mouse inhibitor of apoptosis (MIAP 3), within thymocyte and T cell subpopulations. The consequence of lymphoid-targeted overexpression of XIAP in transgenic mice suggests a physiological role for the endogenous protein, MIAP3. Xiap-transgenic mice accumulated thymocytes and/or T cells in primary and secondary lymphoid tissue, T cell maturation was perturbed, and transgenic thymocytes resisted a variety of apoptotic triggers both in vitro and in vivo. These observations imply a possible key function for the intrinsic cellular inhibitor XIAP in maintaining the homeostasis of the immune system.

Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.

An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis.

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.

Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm).

Mutations in 12 genes regulating Drosophila melanogaster mushroom body (MB) development were each studied in two genetic backgrounds. In all cases, brain structure was qualitatively or quantitatively different after replacement of the "original" genetic background with that of the Canton Special wild-type strain. The mushroom body miniature gene (mbm) was investigated in detail. mbm supports the maintenance of MB Kenyon cell fibers in third instar larvae and their regrowth during metamorphosis. Adult mbm1 mutant females are lacking many or most Kenyon cell fibers and are impaired in MB-mediated associative odor learning. We show here that structural defects in mbm1 are apparent only in combination with an X-linked, dosage-dependent modifier (or modifiers). In the Canton Special genetic background, the mbm1 anatomical phenotype is suppressed, and MBs develop to a normal size. However, the olfactory learning phenotype is not fully restored, suggesting that submicroscopic defects persist in the MBs. Mutant mbm1 flies with full-sized MBs have normal retention but show a specific acquisition deficit that cannot be attributed to reductions in odor avoidance, shock reactivity, or locomotor behavior. We propose that polymorphic gene interactions (in addition to ontogenetic factors) determine MB size and, concomitantly, the ability to recognize and learn odors.

Single-copy transgenic mice with chosen-site integration.

We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.

Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).

Dynamic methylation adjustment and counting as part of imprinting mechanisms.

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.

Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with the most severe pathology in the T lymphocytes and platelets. The disease arises from mutations in the gene encoding the WAS protein. T lymphocytes of affected males with WAS exhibit a severe disturbance of the actin cytoskeleton, suggesting that the WAS protein could regulate its organization. We show here that WAS protein interacts with a member of the Rho family of GTPases, Cdc42. This interaction, which is guanosine 5'-triphosphate (GTP)-dependent, was detected in cell lysates, in transient transfections and with purified recombinant proteins. A weaker interaction was also detected with Rac1 using WAS protein from cell lysates. It was also found that different mutant WAS proteins from three affected males retained their ability to interact with Cdc42 and that the level of expression of the WAS protein in these mutants was only 2-5% of normal. Taken together these data suggest that the WAS protein might function as a signal transduction adaptor downstream of Cdc42, and in affected males, the cytoskeletal abnormalities may result from a defect in Cdc42 signaling.

Défice do transportador de creatina : até onde investigar a causa de um atraso do desenvolvimento?

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Bases moléculaires et cellulaires d’un trouble neurodéveloppemental causé par l’haploinsuffisance de SYNGAP1

La déficience intellectuelle est la cause d’handicap la plus fréquente chez l’enfant. De nombreuses évidences convergent vers l’idée selon laquelle des altérations dans les gènes synaptiques puissent expliquer une fraction significative des affections neurodéveloppementales telles que la déficience intellectuelle ou encore l’autisme. Jusqu’à récemment, la majorité des mutations associées à la déficience intellectuelle a été liée au chromosome X ou à la transmission autosomique récessive. D’un autre côté, plusieurs études récentes suggèrent que des mutations de novo dans des gènes à transmission autosomique dominante, requis dans les processus de la plasticité synaptique peuvent être à la source d’une importante fraction des cas de déficience intellectuelle non syndromique. Par des techniques permettant la capture de l’exome et le séquençage de l’ADN génomique, notre laboratoire a précédemment reporté les premières mutations pathogéniques dans le gène à transmission autosomique dominante SYNGAP1. Ces dernières ont été associées à des troubles comportementaux tels que la déficience intellectuelle, l’inattention, des problèmes d’humeur, d’impulsivité et d’agressions physiques. D’autres patients sont diagnostiqués avec des troubles autistiques et/ou des formes particulières d’épilepsie généralisée. Chez la souris, le knock-out constitutif de Syngap1 (souris Syngap1+/-) résulte en des déficits comme l’hyperactivité locomotrice, une réduction du comportement associée à l’anxiété, une augmentation du réflexe de sursaut, une propension à l’isolation, des problèmes dans le conditionnement à la peur, des troubles dans les mémoires de travail, de référence et social. Ainsi, la souris Syngap1+/- représente un modèle approprié pour l’étude des effets délétères causés par l’haploinsuffisance de SYNGAP1 sur le développement de circuits neuronaux. D’autre part, il est de première importance de statuer si les mutations humaines aboutissent à l’haploinsuffisance de la protéine. SYNGAP1 encode pour une protéine à activité GTPase pour Ras. Son haploinsuffisance entraîne l’augmentation des niveaux d’activité de Ras, de phosphorylation de ERK, cause une morphogenèse anormale des épines dendritiques et un excès dans la concentration des récepteurs AMPA à la membrane postsynaptique des neurones excitateurs. Plusieurs études suggèrent que l’augmentation précoce de l’insertion des récepteurs AMPA au sein des synapses glutamatergiques contribue à certains phénotypes observés chez la souris Syngap1+/-. En revanche, les conséquences de l’haploinsuffisance de SYNGAP1 sur les circuits neuronaux GABAergiques restent inconnues. Les enjeux de mon projet de PhD sont: 1) d’identifier l’impact de mutations humaines dans la fonction de SYNGAP1; 2) de déterminer si SYNGAP1 contribue au développement et à la fonction des circuits GABAergiques; 3) de révéler comment l’haploinsuffisance de Syngap1 restreinte aux circuits GABAergiques affecte le comportement et la cognition. Nous avons publié les premières mutations humaines de type faux-sens dans le gène SYNGAP1 (c.1084T>C [p.W362R]; c.1685C>T [p.P562L]) ainsi que deux nouvelles mutations tronquantes (c.2212_2213del [p.S738X]; c.283dupC [p.H95PfsX5]). Ces dernières sont toutes de novo à l’exception de c.283dupC, héritée d’un père mosaïque pour la même mutation. Dans cette étude, nous avons confirmé que les patients pourvus de mutations dans SYNGAP1 présentent, entre autre, des phénotypes associés à des troubles comportementaux relatifs à la déficience intellectuelle. En culture organotypique, la transfection biolistique de l’ADNc de Syngap1 wild-type dans des cellules pyramidales corticales réduit significativement les niveaux de pERK, en fonction de l’activité neuronale. Au contraire les constructions plasmidiques exprimant les mutations W362R, P562L, ou celle précédemment répertoriée R579X, n’engendre aucun effet significatif sur les niveaux de pERK. Ces résultats suggèrent que ces mutations faux-sens et tronquante résultent en la perte de la fonction de SYNGAP1 ayant fort probablement pour conséquences d’affecter la régulation du développement cérébral. Plusieurs études publiées suggèrent que les déficits cognitifs associés à l’haploinsuffisance de SYNGAP1 peuvent émerger d’altérations dans le développement des neurones excitateurs glutamatergiques. Toutefois, si, et auquel cas, de quelle manière ces mutations affectent le développement des interneurones GABAergiques résultant en un déséquilibre entre l’excitation et l’inhibition et aux déficits cognitifs restent sujet de controverses. Par conséquent, nous avons examiné la contribution de Syngap1 dans le développement des circuits GABAergiques. A cette fin, nous avons généré une souris mutante knockout conditionnelle dans laquelle un allèle de Syngap1 est spécifiquement excisé dans les interneurones GABAergiques issus de l’éminence ganglionnaire médiale (souris Tg(Nkx2.1-Cre);Syngap1flox/+). En culture organotypique, nous avons démontré que la réduction de Syngap1 restreinte aux interneurones inhibiteurs résulte en des altérations au niveau de leur arborisation axonale et dans leur densité synaptique. De plus, réalisés sur des coupes de cerveau de souris Tg(Nkx2.1-Cre);Syngap1flox/+, les enregistrements des courants inhibiteurs postsynaptiques miniatures (mIPSC) ou encore de ceux évoqués au moyen de l’optogénétique (oIPSC) dévoilent une réduction significative de la neurotransmission inhibitrice corticale. Enfin, nous avons comparé les performances de souris jeunes adultes Syngap1+/-, Tg(Nkx2.1-Cre);Syngap1flox/+ à celles de leurs congénères contrôles dans une batterie de tests comportementaux. À l’inverse des souris Syngap1+/-, les souris Tg(Nkx2.1-Cre);Syngap1flox/+ ne présentent pas d’hyperactivité locomotrice, ni de comportement associé à l’anxiété. Cependant, elles démontrent des déficits similaires dans la mémoire de travail et de reconnaissance sociale, suggérant que l’haploinsuffisance de Syngap1 restreinte aux interneurones GABAergiques dérivés de l’éminence ganglionnaire médiale récapitule en partie certains des phénotypes cognitifs observés chez la souris Syngap1+/-. Mes travaux de PhD établissent pour la première fois que les mutations humaines dans le gène SYNGAP1 associés à la déficience intellectuelle causent la perte de fonction de la protéine. Mes études dévoilent, également pour la première fois, l’influence significative de ce gène dans la régulation du développement et de la fonction des interneurones. D’admettre l’atteinte des cellules GABAergiques illustre plus réalistement la complexité de la déficience intellectuelle non syndromique causée par l’haploinsuffisance de SYNGAP1. Ainsi, seule une compréhension raffinée de cette condition neurodéveloppementale pourra mener à une approche thérapeutique adéquate.

An investigation of the 5-HT2C receptor gene as a migraine candidate gene

Migraine is a common complex disorder, currently classified into two main subtypes, migraine with aura (MA) and migraine without aura (MO). The strong preponderance of females to males suggests an X-linked genetic component. Recent studies have identified an X chromosomal susceptibility region (Xq24-q28) in two typical migraine pedigrees. This region harbours a potential candidate gene for the disorder, the serotonin receptor 2C (5-HT2C) gene. This study involved a linkage and association approach to investigate two single nucleotide variants in the 5-HT2C gene. In addition, exonic coding regions of the 5-HT2C gene were also sequenced for mutations in X-linked migraine pedigrees. Results of this study did not detect any linkage or association, and no disease causing mutations were identified. Hence, results for this study do not support a significant role of the 5-HT2C gene in migraine predisposition. (C) 2003 Wiley-Liss, Inc.

Gender diagnosticity and androgen receptor gene CAG repeat sequence

The gender diagnosticity (GD) approach of Lippa (1995) was used to evaluate the relationship of within-sex differences in psychological masculinity-femininity to a genetic characteristic, the length of a repeated CAG sequence in the X-linked androgen receptor (AR) gene. Previously assessed adult samples in Australia and Sweden were used for this purpose. A weak relationship (correlations in the range .11 to .14) was obtained in both countries. Additional data from adolescent twins from Australia (12-, 14-, 16-year-olds) did not confirm such a relationship at those ages, especially for males. The fact that this sample consisted of twins permitted two kinds of within-pair comparisons: (1) Did the dizygotic twin who had the longer AR sequence have the higher GD score? (2) Was one twin's GD score more highly correlated with the other twin's AR score in MZ than in DZ pairs? The answer in both cases was negative. Clarification of these relationships will require large samples and measurements at additional ages.

Polymorphic CAG repeat length in the androgen receptor gene and association with neurodegeneration in a heterozygous female carrier of Kennedy's disease

Kennedy's disease (spinobulbar muscular atrophy) is an X-linked form of motor neuron disease affecting adult males carrying a CAG trinucleotide repeat expansion within the androgen receptor gene. While expression of Kennedy's disease is thought to be confined to males carrying the causative mutation, subclinical manifestations have been reported in a few female carriers of the disease. The reasons that females are protected from the disease are not clear, especially given that all other diseases caused by CAG expansions display dominant expression. In the current study, we report the identification of a heterozygote female carrying the Kennedy's disease mutation who was clinically diagnosed with motor neuron disease. We describe analysis of CAG repeat number in this individual as well as 33 relatives within the pedigree, including two male carriers of the Kennedy's mutation. The female heterozygote carried one expanded allele of the androgen receptor gene with CAG repeats numbering in the Kennedy's disease range (44 CAGs), with the normal allele numbering in the upper-normal range (28 CAGs). The subject has two sons, one of whom carries the mutant allele of the gene and has been clinically diagnosed with Kennedy's disease, whilst the other son carries the second allele of the gene with CAGs numbering in the upper normal range and displays a normal phenotype. This coexistence of motor neuron disease and the presence of one expanded allele and one allele at the upper limit of the normal range may be a coincidence. However, we hypothesize that the expression of the Kennedy's disease mutation combined with a second allele with a large but normal CAG repeat sequence may have contributed to the motor neuron degeneration displayed in the heterozygote female and discuss the possible reasons for phenotypic expression in particular individuals.

Lesch-Nyhan disease in a 20-year-old man incorrectly described as developing 'cerebral palsy' after general anaesthesia in infancy

Lesch–Nyhan disease (LND) is a rare X-linked recessive genetic disorder caused by a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The classic clinical condition is characterized by cognitive impairment, hypotonia at rest, choreoathetosis, hyperuricaemia and the hallmark symptom of severe and involuntary self-mutilation. We describe a man with LND who was initially thought to have suffered from a dyskinetic cerebral palsy after an uncomplicated inguinal herniorrhaphy under general anaesthesia at 5 1/2 months of age. In the absence of overt self-injurious behaviour, the diagnosis was not considered for nearly two decades. The diagnosis of LND was established at 20 years of age through clinical review, biochemical examinations and molecular analysis. HPRT haemolysate activity was 7.6% of the normal control, suggesting that he had a milder variant of the disease. Mutation analysis of the HPRT gene revealed a novel missense mutation, c.449T > G in exon 6 (p.V150G). Cascade testing of family members revealed that the mother was heterozygous for the mutation but two siblings (a brother and a sister) did not carry the sequence mutation. Whether the onset of neurological abnormalities in this particular case can be attributed to the general anaesthesia is discussed.