[Spanish scientific output on thirty pathologies of genetic origin]. / Investigación científica española sobre treinta patologías de origen genético.

INTRODUCTION: The Spanish scientific output during the years 2000-2005 on 30 pathologies of genetic origin included in the OCDE survey on molecular genetic testing has been studied. RESULTS: A total of 105 articles were in MedLine journals on 20 of the pathologies considered. This represents 0.4% of the worldwide publications. However, the Spanish contribution is greater than 2% in some of the pathologies studied worldwide. A total of 266 articles, on 25 pathologies, were found in the Spanish databases IME/ISOC/ICYT. This result makes the total Spanish contribution equivalent to 1.4% of the worldwide output, a value lower than that observed for Spain in Biomedicine and Health Science (2.4%). The number of Spanish articles published on the pathologies studied is greater than those published by Portugal or Holland and lower than those from Italy and France, although Spanish publications are the most abundant for some pathologies. ANALYSIS: Out of the Spanish articles published in MedLine journals, 52% have been cited on an average of 12 times. These contain more basic research than those appearing in IME journals, the latter having a more applied character and being published more frequently in Pediatrics journals. Regarding the Spanish articles, 65% come from 97 laboratories in public hospitals, 10 of which are responsible for 43% of the articles in MedLine journals. These show a certain degree of specialization on at least five pathologies.

Fhx (Foxj2) expression is activated during spermatogenesis and very early in embryonic development.

FHX (FOXJ2) is a recently characterized human fork head transcriptional activator that binds DNA with a dual sequence specificity. We have cloned the cDNA for the mouse orthologue Foxj2 and characterized its expression in the gonads and along the early pre-implantation development of the mouse. In the testis, Foxj2 is expressed from pachytene spermatocytes to round spermatids, but not in spermatogonia. In addition to the germ lineage, only Sertoli cells of the testis showed expression of Foxj2. In the ovary, only granulosa cells of the follicles express the factor. Neither mature spermatozoa nor oocytes showed expression of Foxj2. Foxj2 expression is early activated in zygotic development, being detected since as early as 8-cell stage embryos. Both cell layers of the blastocyst: the trophectoderm (TE) and the inner cell mass (ICM), express Foxj2.

FHX.L and FHX.S, two isoforms of the human fork-head factor FHX (FOXJ2) with differential activity.

Many biological phenomena are dependent on mechanisms that fine-tune the expression levels of particular genes. This can be achieved by altering the relative activity of a single transcription factor, by post-translational modifications or by interaction with regulatory molecules. An alternative mechanism is based on competition between two or more differently active isoforms of the same transcription factor. We found that FHX, a recently characterized human fork-head transcriptional activator, may show such a mechanism for balancing its activity by expressing two differently sized isoforms, FHX.S and FHX.L, encoded by a single gene located on human chromosome 12. FHX. L and FHX.S showed different transcriptional capacities, the larger form, FHX.L, behaving as the more potent transactivator. A transactivation domain of the acidic type present only in FHX.L would account for this functional difference. The relative concentrations of these two FHX isoforms appear to vary in a number of cell types, a circumstance that may regulate the final activity of this transcription factor.

The Drosophila fl(2)d gene, required for female-specific splicing of Sxl and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich domain.

The Drosophila gene female-lethal(2)d [fl(2)d] interacts genetically with the master regulatory gene for sex determination, Sex-lethal. Both genes are required for the activation of female-specific patterns of alternative splicing on transformer and Sex-lethal pre-mRNAs. We have used P-element-mediated mutagenesis to identify the fl(2)d gene. The fl(2)d transcription unit generates two alternatively spliced mRNAs that can encode two protein isoforms differing at their amino terminus. The larger isoform contains a domain rich in histidine and glutamine but has no significant homology to proteins in databases. Several lines of evidence indicate that this protein is responsible for fl(2)d function. First, the P-element insertion that inactivates fl(2)d interrupts this ORF. Second, amino acid changes within this ORF have been identified in fl(2)d mutants, and the nature of the changes correlates with the severity of the mutations. Third, all of the phenotypes associated with fl(2)d mutations can be rescued by expression of this cDNA in transgenic flies. Fl(2)d protein can be detected in extracts from Drosophila cell lines, embryos, larvae, and adult animals, without apparent differences between sexes, as well as in adult ovaries. Consistent with a possible function in posttranscriptional regulation, Fl(2)d protein has nuclear localization and is enriched in nuclear extracts.

FHX, a novel fork head factor with a dual DNA binding specificity.

The HNF3/fork head family includes a large number of transcription factors that share a structurally related DNA binding domain. Fork head factors have been shown to play important roles both during development and in the adult. We now describe the cloning of a novel mammalian fork head factor that we have named FHX (fork head homologous X (FHX), which is expressed in many adult tissues. In the embryo, FHX expression showed a very early onset during the cleavage stages of preimplantation development. Polymerase chain reaction-assisted site selection experiments showed that FHX bound DNA with a dual sequence specificity. Sites recognized by FHX could be classified into two different types according to their sequences. Binding of FHX to sequences of each type appeared to occur independently. Our data suggest that either different regions of the fork head domain or different molecular forms of this domain could be involved in binding of FHX to each type of site. In transfection assays, FHX was capable of activating transcription from promoters containing FHX sites of either type.

Genomic cloning and characterization of the human homeobox gene SIX6 reveals a cluster of SIX genes in chromosome 14 and associates SIX6 hemizygosity with bilateral anophthalmia and pituitary anomalies.

The Drosophila gene sine oculis (so), a nuclear homeoprotein that is required for eye development, has several homologues in vertebrates (the SIX gene family). Among them, SIX3 is considered to be the functional orthologue of so because it is strongly expressed in the developing eye. However, embryonic SIX3 expression is not limited to the eye field, and SIX3 has been found to be mutated in some patients with holoprosencephaly type 2 (HPE2), suggesting that SIX3 has wide implications in head development. We report here the cloning and characterization of SIX6, a novel human SIX gene that is the homologue of the chick Six6(Optx2) gene. SIX6 is closely related to SIX3 and is expressed in the developing and adult human retina. Data from chick and mouse suggest that the human SIX6 gene is also expressed in the hypothalamic and the pituitary regions. SIX6 spans 2567 bp of genomic DNA and is split in two exons that are transcribed into a 1393-nucleotide-long mRNA. Chromosomal mapping of SIX6 revealed that it is closely linked to SIX1 and SIX4 in human chromosome 14q22.3-q23, which provides clues about the origin and evolution of the vertebrate SIX family. Recently three independent reports have associated interstitial deletions at 14q22.3-q23 with bilateral anophthalmia and pituitary anomalies. Genomic analyses of one of these cases demonstrated SIX6 hemizygosity, strongly suggesting that SIX6 haploinsufficiency is responsible for these developmental disorders.

Genomic cloning, structure, expression pattern, and chromosomal location of the human SIX3 gene.

The Drosophila gene sine oculis (so) is a nuclear homeoprotein that is required for eye development. Homologous genes to so, denoted SIX genes, have been found in vertebrates. Among the SIX genes, SIX3 is considered to be the functional homologue of so. To provide insight into the potential implications of SIX3 in human ocular malformations, we have cloned and characterized the human SIX3 gene. In human eye, SIX3 produces a 3-kb transcript that codes for a 332-amino-acid polypeptide that is virtually identical to its mouse and chick homologues. Expression of SIX3 was detected in human embryos as early as 5-7 weeks of gestation and found to be maintained in the eye throughout the entire period of fetal development. At 20 weeks of gestation, expression of SIX3 in the human retina was detected in the ganglion cells and in cells of the inner nuclear layer. The human SIX3 gene spans 4.4 kb of genomic DNA and is split in two exons separated by a 1659-bp intron. SIX3 was mapped to human chromosome 2p16-p21, between the genetic markers D2S119 and D2S288. Interestingly, the map position of human SIX3 overlaps the locations of two dominant disorders with ocular phenotypes that have been assigned to this chromosomal region, holoprosencephaly type 2 and Malattia Leventinese.

Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.

Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal.

The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF65 to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF65 can explain tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing.

The human homogentisate 1,2-dioxygenase (HGO) gene.

Alkaptonuria (AKU; McKusick No. 203500), a rare hereditary disorder of the phenylalanine catabolism, was the first disease to be interpreted as an inborn error of metabolism (A. E. Garrod, 1902, Lancet 2: 1616-1620). AKU patients are deficient for homogentisate 1,2-dioxygenase (HGO; EC 1.13.11.5). This enzymatic deficiency causes homogentisic aciduria, ochronosis, and arthritis. Recently we cloned the human HGO gene and showed that AKU patients carry two copies of a loss-of-function HGO allele. Here we describe the complete nucleotide sequence of the human HGO gene and the identification of its promoter region. The human HGO gene spans 54,363 bp and codes for a 1715-nt-long transcript that is split into 14 exons ranging from 35 to 360 bp. The HGO introns, 605 to 17,687 bp in length, contain representatives of the major classes of repetitive elements, including several simple sequence repeats (SSR). Two of these SSRs, a (CT)n repeat in intron 4 and a (CA)n repeat in intron 13, were found to be polymorphic in a Spanish population sample. The HGO transcription start site was determined by primer extension. We report that sequences from -1074 to +89 bp (relative to the HGO transcription start site) are sufficient to promote transcription of a CAT reporter gene in human liver cells and that this fragment contains putative binding sites for liver-enriched transcription factors that might be involved in the regulation of HGO expression in liver.

Regulation of the gene Sex-lethal: a comparative analysis of Drosophila melanogaster and Drosophila subobscura.

The Drosophila gene Sex-lethal (Sxl) controls the processes of sex determination and dosage compensation. A Drosophila subobscura genomic fragment containing all the exons and the late and early promotors in the Sxl gene of D. melanogaster was isolated. Early Sxl expression in D. subobscura seems to be controlled at the transcriptional level, possibly by the X:A signal. In the region upstream of the early Sxl transcription initiation site are two conserved regions suggested to be involved in the early activation of Sxl. Late Sxl expression in D. subobscura produces four transcripts in adult females and males. In males, the transcripts have an additional exon which contains three translational stop codons so that a truncated, presumably nonfunctional Sxl protein is produced. The Sxl pre-mRNA of D. subobscura lacks the poly-U sequence presented at the polypirimidine tract of the 3' splice site of the male-specific exon present in D. melanogaster. Introns 2 and 3 contain the Sxl-binding poly-U stretches, whose localization in intron 2 varies but in intron 3 is conserved. The Sxl protein is fully conserved at the amino acid level in both species.

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster.

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2) d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.

Cloning and characterization of the scute (sc) gene of Drosophila subobscura.

The scute (sc) gene, a member of the achaete-scute complex of Drosophila melanogaster, has dual functions: sisterless (sis-b) function required for sex determination and dosage compensation and scute function, which is involved in neurogenesis. The sc homologue of D. subobscura was cloned. It lacks introns and encodes a single 1.7-kb transcript slightly larger than that of D. melanogaster (1.6 kb). The sc protein of D. subobscura is slightly larger than that of D. melanogaster (382 vs. 345 amino acids). Sequence comparisons between both species show the Sc protein to have a highly conserved bHLH domain. Outside this domain, amino acid replacements are not randomly distributed. Two additional conserved domains, of 20 and 36 amino acids, are present near the C-terminal end. They may represent domains confering specificity upon the Sc protein with respect to other proteins of the achaete-scute complex. In its 3' untranslated region, Sc RNA contains uridine stretches, putative Sxl protein DNA-binding sites. The D. subobscura Sc protein can cooperate with other D. melanogaster bHLH proteins because D. subobscura sc supplies sis-b function when introduced into D. melanogaster transgenic flies mutant for sc.

The molecular basis of alkaptonuria.

Alkaptonuria (AKU) occupies a unique place in the history of human genetics because it was the first disease to be interpreted as a mendelian recessive trait by Garrod in 1902. Alkaptonuria is a rare metabolic disorder resulting from loss of homogentisate 1,2 dioxygenase (HGO) activity. Affected individuals accumulate large quantities of homogentisic acid, an intermediary product of the catabolism of tyrosine and phenylalanine, which darkens the urine and deposits in connective tissues causing a debilitating arthritis. Here we report the cloning of the human HGO gene and establish that it is the AKU gene. We show that HGO maps to the same location described for AKU, illustrate that HGO harbours missense mutations that cosegregate with the disease, and provide biochemical evidence that at least one of these missense mutations is a loss-of-function mutation.

Indirect evidence of alteration in the expression of the rDNA genes in interspecific hybrids between Drosophila melanogaster and Drosophila simulans.

Crosses between Drosophila melanogaster females and D. simulans males produce viable hybrid females, while males are lethal. These males are rescued if they carry the D. simulans Lhr gene. This paper reports that females of the wild-type D. melanogaster population Staket do not produce viable hybrid males when crossed with D. simulans Lhr males, a phenomenon which we designate as the Staket phenotype. The agent responsible for this phenomenon was found to be the Staket X chromosome (Xmel, Stk). Analysis of the Staket phenotype showed that it is suppressed by extra copies of D. melanogaster rDNA genes and that the Xmel, Stk chromosome manifests a weak bobbed phenotype in D. melanogaster Xmel, Stk/0 males. The numbers of functional rDNA genes in Xmel, Stk and Xmel, y w (control) chromosomes were found not to differ significantly. Thus a reduction in rDNA gene number cannot account for the weak bobbed Xmel, Stk phenotype let alone the Staket phenotype. The rRNA precursor molecules transcribed from the Xmel, Stk rDNA genes seem to be correctly processed in both intraspecific (melanogaster) and interspecific (melanogaster-simulans) conditions. It is therefore suggested that the Xmel, Stk rDNA genes are inefficiently transcribed in the melanogaster-simulans hybrids.

Dosage compensation in sciarids is achieved by hypertranscription of the single X chromosome in males.

Dosage compensation refers to the process whereby females and males with different doses of sex chromosomes have similar amounts of products from sex chromosome-linked genes. We analyzed the process of dosage compensation in Sciara ocellaris, Diptera of the suborder Nematocera. By autoradiography and measurements of X-linked rRNA in females (XX) and males (XO), we found that the rate of transcription of the single X chromosome in males is similar to that of the two X chromosomes in females. This, together with the bloated appearance of the X chromosome in males, support the idea that in sciarids dosage compensation is accomplished by hypertranscription of the X chromosome in males.

Sex determination in Drosophila melanogaster: X-linked genes involved in the initial step of sex-lethal activation.

Sex determination is the commitment of an embryo to either the female or the male developmental pathway. The ratio of X chromosomes to sets of autosomes is the primary genetic signal that determines sex in Drosophila, by triggering the functional state of the gene Sex-lethal: in females (2X;2A) Sxl will be ON, whereas in males (X;2A) Sxl will be OFF. Genetic and molecular studies have defined a set of genes involved in the formation of the X:A signal, as well as other genes, with either maternal or zygotic effects, which are also involved in regulating the initial step of Sex-lethal activation. We review these data and present new data on two more regions of the X chromosome that define other genes needed for Sxl activation. In addition, we report on the interaction between some of the genes regulating Sxl activation.

Sex determination in the germ line of Drosophila melanogaster: activation of the gene Sex-lethal.

The germ line exhibits sexual dimorphism as do the somatic tissues. Cells with the 2X;2A chromosome constitution will follow the oogenic pathway and X;2A cells will develop into sperm. In both somatic and germ-line tissues, the sexual pathway chosen by the cells depends on the gene Sex-lethal (Sxl), whose function is continuously needed for female development. In the soma, the sex of the cells is autonomously determined by the X:A signal while, in the germ line, the sex is determined by cell autonomous (the X:A signal) and somatic inductive signals. Three X-linked genes have been identified, scute (sc), sisterless-a (sis-a) and runt (run), that determine the initial functional state of Sxl in the soma. Using pole cell transplantation, we have tested whether these genes are also needed to activate Sxl in the germ line. We found that germ cells simultaneously heterozygous for sc, sis-a, run and a deficiency for Sxl transplanted into wild-type female hosts develop into functional oocytes. We conclude that the genes sc, sis-a and run needed to activate Sxl in the soma seem not to be required to activate this gene in the germ line; therefore, the X:A signal would be made up by different genes in somatic and germ-line tissues. The Sxlf7M1/Sxlfc females do not have developed ovaries. We have shown that germ cells of this genotype transplanted into wild-type female hosts produce functional oocytes. We conclude that the somatic component of the gonads in Sxlf7M1/Sxlfc females is affected, and consequently germ cells do not develop.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of a dual function in fl(2)d, a gene needed for Sex-lethal expression in Drosophila melanogaster.

In Drosophila melanogaster, the female sexual development of the soma and the germline requires the activity of the gene Sxl. The somatic cells need the function of the gene fl(2)d to follow the female developmental pathway, due to its involvement in the female-specific splicing of Sxl RNA. Here we report the analysis of both fl(2)d1 and fl(2)d2 mutations: (1) fl(2)d1 is a temperature-sensitive mutation lethal in females and semilethal in males; (2) fl(2)d2 is lethal in both sexes; (3) the fl(2)d1/fl(2)d2 constitution is temperature-sensitive and lethal in females, while semilethal in males. The temperature-sensitive period of fl(2)d1 in females expands the whole development. SxlM1 partially suppresses the lethality of fl(2)d1 homozygous females and that of fl(2)d1/fl(2)d2 constitution, whereas it does not suppress the lethality of fl(2)d2 homozygous females. The addition of extra Sxl+ copies does not increase the suppression effect of SxlM1. The fl(2)d1 mutation in homozygosis and the fl(2)d1/fl(2)d2 constitution, but not the fl(2)d2 in homozygosis, partially suppress the lethality of SxlM1 males. This suppression is not prevented by the addition of extra Sxl+ copies. The semilethality of both fl(2)d1 and fl(2)d1/fl(2)d2 males, and the lethality of fl(2)d2 males, is independent of Sxl function. There is no female synergistic lethality between mutations at fl(2)d and neither at sc or da. However, the female synergistic lethality between mutations at Sxl and either sc or da is increased by fl(2)d mutations. We have analyzed the effect of the fl(2)d mutations on the germline development of both females and males. For that purpose, we carried out the clonal analysis of fl(2)d1 in the germline. In addition, pole cells homozygous for fl(2)d2 were transplanted into wild-type host embryos, and we checked whether the mutant pole cells were capable of forming functional gametes. The results indicated that fl(2)d mutant germ cells cannot give rise to functional oocytes, while they can form functional sperm. Moreover, SxlM1 suppresses the sterility of the fl(2)d1 homozygous females developing at the permissive temperature. Thus, with respect to the development of the germline the fl(2)d mutations mimic the behavior of loss-of-function mutations at the gene Sxl. Females double heterozygous for fl(2)d and snf1621 are fully viable and fertile. fl(2)d2 in heterozygosis partially suppresses the phenotype of female germ cells homozygous for snf1621; however, this is not the case with the fl(2)d1 mutation. The fl(2)d mutations partially suppress the phenotype of the female germ cells homozygous for ovoDIrSI.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic and molecular analysis of new female-specific lethal mutations at the gene Sxl of Drosophila melanogaster.

We have isolated three female-specific lethal mutations at the gene Sex-lethal (Sxl): Sxlfb, Sxlfc and Sxlfd. We have carried out the complementation analysis between these mutations and other previously reported Sxlf mutations. It is possible to classify the alleles tested in this report into two complementation groups: the bc group defined by Sxlfb, and Sxlfc, and the LS group defined by SxlfLS. The other alleles tested affect both complementation groups albeit with different degrees. Contrary to what happens with mutations at the LS group, mutations at the bc group do not affect sex determination, nor late dosage compensation nor oogenesis. Both Sxlfb and Sxlfc present a DNA insertion of at least 5 kb between position -10 and -11 on the molecular map, within the fourth intron. On the contrary, Sxlfd, a strong mutation affecting all Sxl functions, is not associated to any detectable DNA alteration in Southern blots, so that it seems to be a "point" mutation. In agreement with their phenotypes, both Sxlfc/SxlfLS and Sxlfc homozygous female larvae express only the late Sxl transcripts characteristic of females, while females homozygous for SxlfLS express only the late Sxl transcripts characteristic of males. Moreover, Sxlfc presents a lethal synergistic interaction with mutations at either da or the X:A ratio, two signals that define the initial activity state of Sxl, while SxlfLS do not. These data suggest that the two complementation groups are related to the two sets of early and late Sxl transcripts, which are responsible for the early and late Sxl functions, respectively: Sxlfb and Sxlfc would affect the early functions and SxlfLS would affect the late Sxl functions.