Absent Gene Research Articles

A novel Eyes Absent 2 protein is expressed in the human eye

The Drosophila eyes absent ( eya) gene has a role in regulating cell death and/or differentiation and is expressed throughout development. We evaluated the transcripts and proteins encoded by one of the human homologues of Drosophila eya coined Eyes Absent 2 ( EYA2). Interestingly, EYA2 was expressed in several neuroblastoma cell lines as four distinct transcripts having alternative 5′-ends, whereas only one EYA2 transcript was expressed in the normal human eye. Due to different translation start sites on the four unique transcripts, two isoforms of EYA2 protein (one previously identified and one novel) could be generated in neuroblastoma cells, but the sole EYA2 transcript expressed in the eye can only encode the novel isoform. Immunoblot analyses suggest that EYA2 may also be post-translationally modified. Finally, the alternative EYA2 transcripts have dissimilar numbers of upstream open reading frames in their 5′-untranslated regions. Therefore, in addition to encoding alternative isoforms of EYA2, regulation of EYA2 expression appears to involve both transcriptional and translational components.

Advances in the diagnosis of primary immunodeficiency disorders in childhood

Primary immunodeficiency disorders in childhood usually present as unusual, recurrent or severe infections, symptomatic infections with organisms of low pathogenicity, or as recognizable syndromes which are known to have associated immunological abnormalities. In many of the primary immunodeficiency disorders, there are known patterns of inheritance, and other family members may be affected.Some primary immunodeficiency disorders are relatively common, such as selective IgA deficiency, and often do not lead to major morbidity. Others, such as the severe combined immune deficiency syndromes, are relatively rare, and are fatal in early life if not recognized and treated early.Diagnosis of a primary immunodeficiency disorder depends on appropriate use of laboratory investigations. Often there will be abnormalities detected on a complete blood film and measurement of immunoglobulin isotypes. More complex investigations should be undertaken in conjunction with a paediatric immunology service.In recent years, many of the clinically defined primary immunodeficiency disorders have been shown to have associated specific gene defects. For some, this has led to the identification and characterization of defective or absent gene products. The consequences of this new knowledge are more accurate diagnosis, early diagnosis including antenatal diagnosis, detection of undiagnosed disease in other family members, and the potential for new therapies including gene or gene product therapy.

Yeast two-hybrid system identifies the ubiquitin-conjugating enzyme mUbc9 as a potential partner of mouse Dac

Using a yeast two hybrid system and pull-down assays we demonstrate that mouse Dac (mDac) specifically binds to mouse ubiquitin-conjugating enzyme mUbc9. In contrast to a direct interaction between Drosophila dachshund(dac) and eyes absent (eya)gene products, we cannot detect by the same methods that mDac binds to mEya2, a functional mouse homologue of the Drosophila Eya. Immunostaining of various cell lines that were transfected with mDac reveals that mDac protein is found predominantly in the nucleus but translocates to the cytoplasm and condensates along the nuclear membrane in a cell-cycle dependent manner. Deletion analysis of mDac show the intracellular localization and protein stability correlates with the binding to mUbc9. The C-terminal half of mDac, which associates with mUbc9, remains cytoplasmic and is degraded in proteasome whereas the non-interacting N-terminus is exclusively nuclear and more stable than the full-length mDac or its C-terminal portion. In situ hybridization on whole-mount embryos or tissue sections detects mUbc9 transcripts in complementary and overlapping areas with mDac expression, particularly in the proliferation zone of the limb buds, the spinal cord and forebrain. Mouse embryos stained with an anti-mDac antibody document that mDac is localized both in the nucleus and the cytoplasm with a cytoplasmic predominance in migrating neural crest cells. In the proliferation zone, visible nuclear envelopes are not formed and mDac is detected throughout the cells.

Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain.

The eyes absent (eya) gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other genes of eye development, sine oculis and dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, we have sequenced eya alleles. These mutations have revealed that loss of the entire Eya Domain is null for eya activity, but that alleles with truncations within the Eya Domain display partial function. We then extended the molecular genetic analysis to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminished the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it.

Gene therapy for ocular disease.

Gene therapy for ocular disease.

Functional Analysis of an Eye Enhancer of the Drosophila eyes absent Gene: Differential Regulation by Eye Specification Genes

Genes involved in eye development are highly conserved between vertebrates and Drosophila. Given the complex genetic network controlling early eye development, identification of regulatory sequences controlling gene expression will provide valuable insights toward understanding central events of early eye specification. We have focused on defining regulatory elements critical for Drosophila eyes absent (eya) expression. Although eya has a complex expression pattern during development, analysis of eye-specific mutations in the gene revealed a region selectively deleted in the eye-specific alleles. Here we have performed detailed analysis of the region deleted in the eye-specific eya2 allele. This analysis shows that this region can direct earlyeya gene expression in a pattern consistent with that of normal eya in eye progenitor cells. Functional studies indicate that this element will restore appropriate eya transcript expression to rescue the eye-specific allele. We have examined regulation of this element during eye specification, both in normal eye development and in ectopic eye formation. These studies demonstrate that the element was activated upon ectopic expression of the eye specification genes eyeless and dachshund, but does not respond to ectopic expression of eya or sine oculis. The differential regulation of this element by genes involved during early retinal formation reveals new aspects of the genetic hierarchy of eye development.

Mutations of a human homologue of the Drosophila eyes absent gene (EYA1) detected in patients with congenital cataracts and ocular anterior segment anomalies.

The Drosophila eyes absent gene ( eya ) is involved in the formation of compound eyes. Flies with loss-of-function mutations of this gene develop no eyes and form the ectopic eye in the antennae and the ventral zone of the head on target expression. A highly conserved homo-logous gene in various invertebrates and vertebrates has been shown to function in the formation of the eye. In contrast, a human homologue, EYA1, has been identified by positional cloning as a candidate gene for branchio-oto-renal (BOR) syndrome, in which phenotypic manifestations are restricted to the areas of branchial arch, ear and kidney, with usually no anomalies in the eye. We have examined genomic DNA isolated from patients with various types of developmental eye anomaly for EYA1 mutations by the use of polymerase chain reaction-single-strand conformation polymorphism and sequencing. We identified three novel missense mutations in patients who had con-genital cataracts and ocular anterior segment anomalies. One of the patients had clinical features of BOR syndrome as well. This result implies that the human EYA1 gene is also involved in eye morphogenesis, and that a wide variety of clinical manifestations may be caused by EYA1 mutations.

Evidence in favour of ancient octaploidy in the vertebrate genome.

Vertebrate genomes are larger than invertebrates and show evidence of extensive gene duplication, including many collinear chromosomal segments. On the basis of this intra-genomic synteny, it has been proposed that two rounds of whole genome duplication (octaploidy) occurred early in the vertebrate lineage. Recently, this early vertebrate octaploidy has been challenged on the basis of gene trees. We report new linkage groups encompassing the matrilin (MATN), syndecan (SDC), Eyes Absent (EYA), HCK kinase and SRC kinase paralogous gene quartets. In contrast to other studies, the sequence trees are weakly supportive of ancient octaploidy. It is concluded that there is no strong evidence against the octaploidy, provided that consecutive genome duplication was rapid.

Molecular genetic analysis of Drosophila eyes absent mutants reveals an eye enhancer element.

The eyes absent (eya) gene is critical for normal eye development in Drosophila and is highly conserved to vertebrates. To define regions of the gene critical for eye function, we have defined the mutations in the four viable eya alleles. Two of these mutations are eye specific and undergo transvection with other mutations in the gene. These were found to be deletion mutations that remove regulatory sequence critical for eye cell expression of the gene. Two other viable alleles cause a reduced eye phenotype and affect the function of the gene in additional tissues, such as the ocelli. These mutations were found to be insertion mutations of different transposable elements within the 5' UTR of the transcript. Detailed analysis of one of these revealed that the transposable element has become subject to regulation by eye enhancer sequences of the eya gene, disrupting normal expression of EYA in the eye. More extended analysis of the deletion region in the eye-specific alleles indicated that the deleted region defines an enhancer that activates gene expression in eye progenitor cells. This enhancer is responsive to ectopic expression of the eyeless gene. This analysis has defined a critical regulatory region required for proper eye expression of the eya gene.

Description of a large kindred with autosomal dominant inheritance of branchial arch anomalies, hearing loss, and ear pits, and exclusion of the branchio‐oto‐renal (BOR) syndrome gene locus (chromosome 8q13.3)

It has been suggested that branchio-oculo-facial (BOF) syndrome, deafness with ear pits, and associated conditions [MIM nos. 125100, 120502], and branchio-oto-renal (BOR) [MIM no. 113650] or Melnick-Fraser syndrome represent phenotypic variants of the BOR syndrome, which is inherited in an autosomal dominant (AD) manner and has variable clinical expression. Recently, the BOR gene was mapped to chromosome region 8q13.3 and its sequence was identified as the human homolog of the Drosophila eyes absent (EYA1) gene. We studied an extended family with AD inheritance of branchial arch anomalies (BAA), hearing loss, and ear pits, whose phenotype differed from that of patients with BOR in that none of the affected members had renal abnormalities or lacrimal duct stenosis. Fifteen affected members were studied; ear pits were present in all of them, whereas hearing loss and other BAA were present in 40 and 20%, respectively. Blood was collected from 31 patients; DNA was extracted by standard methods and amplified using primers from microsatellite sequences flanking the BOR locus on chromosome 8q13.3 (D8S1807, D8S530, and D8S543). Linkage analysis was performed under two models of AD inheritance with different penetrance: 100% and 80%. In both cases, the logarithm of odds(LOD) scores produced were significantly less than −2; exclusion of the 8q13.3 locus was also confirmed by multipoint LOD score analysis. We conclude that, in one large family with AD inheritance of BAA, hearing loss and ear pits, the BOR locus was excluded. This represents the first documentation of heterogeneity in branchio-oto anomalies, syndromes with phenotypes similar to BOR syndrome. Am. J. Med. Genet. 79:209–214, 1998. © 1998 Wiley-Liss, Inc.

Description of a large kindred with autosomal dominant inheritance of branchial arch anomalies, hearing loss, and ear pits, and exclusion of the branchio-oto-renal (BOR) syndrome gene locus (chromosome 8q13.3)

It has been suggested that branchio-oculo-facial (BOF) syndrome, deafness with ear pits, and associated conditions [MIM nos. 125100, 120502], and branchio-oto-renal (BOR) [MIM no. 113650] or Melnick-Fraser syndrome represent phenotypic variants of the BOR syndrome, which is inherited in an autosomal dominant (AD) manner and has variable clinical expression. Recently, the BOR gene was mapped to chromosome region 8q13.3 and its sequence was identified as the human homolog of the Drosophila eyes absent (EYA1) gene. We studied an extended family with AD inheritance of branchial arch anomalies (BAA), hearing loss, and ear pits, whose phenotype differed from that of patients with BOR in that none of the affected members had renal abnormalities or lacrimal duct stenosis. Fifteen affected members were studied; ear pits were present in all of them, whereas hearing loss and other BAA were present in 40 and 20%, respectively. Blood was collected from 31 patients; DNA was extracted by standard methods and amplified using primers from microsatellite sequences flanking the BOR locus on chromosome 8q13.3 (D8S1807, D8S530, and D8S543). Linkage analysis was performed under two models of AD inheritance with different penetrance: 100% and 80%. In both cases, the logarithm of odds (LOD) scores produced were significantly less than -2; exclusion of the 8q13.3 locus was also confirmed by multipoint LOD score analysis. We conclude that, in one large family with AD inheritance of BAA, hearing loss and ear pits, the BOR locus was excluded. This represents the first documentation of heterogeneity in branchio-oto anomalies, syndromes with phenotypes similar to BOR syndrome.

Correction of Abnormal T-Cell Receptor Repertoire During Interferon-α Therapy in Patients With Hairy Cell Leukemia

Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-α (IFN-α) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-α in the past, at initiation, and at several intervals up to 6 years of IFN-α treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+cells; 600 to 700/μL whole blood), which decreased during IFN-α therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex–unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-α can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.

Correction of Abnormal T-Cell Receptor Repertoire During Interferon-α Therapy in Patients With Hairy Cell Leukemia

AbstractPatients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-α (IFN-α) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-α in the past, at initiation, and at several intervals up to 6 years of IFN-α treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+cells; 600 to 700/μL whole blood), which decreased during IFN-α therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex–unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-α can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.

Dual functions of the Drosophila eyes absent gene in the eye and embryo

In eyes absent (eya) mutants, eye progenitor cells undergo cell death early in development. Whereas the phenotype of eya1 is limited to the eye, other mutations are lethal. Genetic and molecular analysis reveals that mutations in one region of the gene cause embryonic lethality, whereas mutations throughout the gene cause defects in eye development. Mosaic analysis indicates that the eya requirement is cell autonomous. In eye-specific mutants, expression in the eye disc is lacking while embryonic expression is normal. Both the type I and type II transcripts are expressed in the developing eye, and expression of either can rescue the eye phenotype. These data indicate a specific requirement for eya function in eye progenitor cells that is normally fulfilled by both transcripts.

Multiple Roles of theeyes absentGene inDrosophila

Theeyes absent(eya) gene plays an essential role in the events that lead to formation of theDrosophilaeye; without expression ofeyain retinal progenitor cells, they undergo programmed cell death just prior to the morphogenetic furrow, leading to an eyeless or reduced eye phenotype. Theeyagene has recently been found to be highly conserved to humans, defining a new gene family. Insights into the gene's function in the fly, therefore, are likely to be relevant to the role of its homologs in vertebrates. Detailed studies at the subcellular level indicate that the Eya protein is localized to the nucleoplasm, suggesting a role in control of nuclear events. Theeyagene shows expression and roles in tissues other than the eye, including subsets of cells of the adult visual system, brain, and ovary, as well as an elaborate expression pattern in the embryo. Various mutations in theeyagene cause loss of ocelli, female sterility, or lethality. Analysis of the embryonic lethal phenotype indicates that mutant alleles show defects in head morphogenesis. These data indicate thateyahas critical roles in morphogenesis of a number of tissues in the animal, in addition to its role in early eye formation. Despite multiple roles at multiple stages of development of the fly, both the type I and type II forms of the protein, when expressed ectopically during larval development, can direct eye formation.

The Drosophila eyes absent gene directs ectopic eye formation in a pathway conserved between flies and vertebrates.

The fly eyes absent (eya) gene which is essential for compound eye development in Drosophila, was shown to be functionally replaceable in eye development by a vertebrate Eya homolog. The relationship between eya and that of the eyeless gene, a Pax-6 homolog, critical for eye formation in both flies and man, was defined: eya was found to be essential for eye formation by eyeless. Moreover, eya could itself direct ectopic eye formation, indicating that eya has the capacity to function as a master control gene for eye formation. Finally, we show that eya and eyeless together were more effective in eye formation than either gene alone. These data indicate conservation of the pathway of eya function between flies and vertebrates; they suggest a model whereby eya/Eya gene function is essential for eye formation by eyeless/Pax-6, and that eya/Eya can in turn mediate, via a regulatory loop, the activity of eyeless/Pax-6 in eye formation.

Clustering of mutations responsible for branchio-oto-renal (BOR) syndrome in the eyes absent homologous region (eyaHR) of EYA1.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder, characterised by the association of branchial, otic and renal anomalies with variable degrees of severity. We have recently identified EYA1 , a human homologue of the Drosophila eyes absent gene, as the gene underlying this syndrome. The products of both genes share a highly conserved 271 amino acid C-terminal region (eyaHR). The eyaHR was also found in the products of two other human genes (EYA2 and EYA3), demonstrating the existence of a novel gene family. We report here on the complete genomic structure of EYA1. This gene consists of 16 coding exons and extends over 156 kb. It encodes various alternatively spliced transcripts differing only in their 5' regions. Sequence analysis of the entire EYA1 coding region was performed for 20 unrelated patients affected by BOR syndrome, and six novel mutations were identified. Among these mutations, two are missense mutations, highlighting amino acid residues essential for the function of the EYA1 protein, and one mutation comprises a de novo Alu insertion into an exon. This insertion presumably occurs by retrotransposition, and the mobile Alu element has a poly(A) tail that is unstable throughout generations. To date, 14 mutations have been detected in BOR patients, all of which are different. However, all the mutations are located within or in the immediate vicinity of the eyaHR; the significance of this clustering is discussed.

Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function.

Vertebrate limb tendons are derived from connective cells of the lateral plate mesoderm. Some of the developmental steps leading to the formation of vertebrate limb tendons have been previously identified; however, the molecular mechanisms responsible for tendinous patterning and maintenance during embryogenesis are largely unknown. The eyes absent (eya) gene of Drosophila encodes a novel nuclear protein of unknown molecular function. Here we show that Eya1 and Eya2, two mouse homologues of Drosophila eya, are expressed initially during limb development in connective tissue precursor cells. Later in limb development, Eya1 and Eya2 expression is associated with cell condensations that form different sets of limb tendons. Eya1 expression is largely restricted to flexor tendons, while Eya2 is expressed in the extensor tendons and ligaments of the phalangeal elements of the limb. These data suggest that Eya genes participate in the patterning of the distal tendons of the limb. To investigate the molecular functions of the Eya gene products, we have analyzed whether the highly divergent PST (proline-serine-threonine)-rich N-terminal regions of Eya1-3 function as transactivation domains. Our results demonstrate that Eya gene products can act as transcriptional activators, and they support a role for this molecular function in connective tissue patterning.

Inhibition of IL-10 Protein Synthesis Induces Major Histocompatibility Complex Class II Gene Expression in Class II-Deficient Patients

Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency, characterized by a lack of constitutive expression of the human leukocyte antigen (HLA) class II genes. The patients investigated in this study are histoidentical twin brothers with a new phenotype in MHC class II deficiency. Examination of HLA-D locus genes in their fractionated peripheral mononuclear cells (MNCs) revealed an unusual and uncoordinated mRNA pattern. Here we analyzed the distribution of pro- and anti-inflammatory cytokines expressed in these patients' adherent and nonadherent MNCs. We show that gene expression of IL-1α, IL-1β, IL-6, granulocyte–colony-stimulating factor, and IL-10 was induced in both cell fractions, whereas increased mRNA levels of interferon-γ and the inducible nitric oxide synthase were exclusively detected in the patients' nonadherent MNCs. As IL-10 is known to be able to downregulate transcription of MHC class II and expression of IL-10 in the patients' MNCs was increased, we investigated the regulatory function of this cytokine. Interestingly, inhibition of IL-10 protein synthesis with IL-10-specific antisense oligonucleotide DNA (IL-10-AS-ODN) induced HLA-D locus genes in these MHC class II-deficient patients. Exposure of the nonadherent cell fraction to IL-10-AS-ODN resulted in a profound induction of a previously absent DRβ1and DPα gene expression. HLA-DQβ mRNA levels, however, were increased in both the adherent and the nonadherent MNC population. Albeit expression of HLA-D locus genes was inducible via inhibition of IL-10 translation, surface expression of HLA class II antigens on the patients' MNCs was essentially negative. The data presented support the concept of a coordinated network of pro- and anti-inflammatory cytokine regulation and this network obviously has a significant role in the cell-type-specific regulation of MHC class II expression.

Eyes absent: a gene family found in several metazoan phyla.

Genes related to the Drosophila eyes absent gene were identified in vertebrates (mouse and human), mollusks (squid), and nematodes (C. elegans). Proteins encoded by these genes consist of conserved C-terminal and variable N-terminal domains. In the conserved 271-amino acid C-terminal region, Drosophila and vertebrate proteins are 65-67% identical. A vertebrate homolog of eyes absent, designated Eya2, was mapped to Chromosome (Chr) 2 in the mouse and to Chr 20q13.1 in human. Eya2 shows a dynamic pattern of expression during development. In the mouse, expression of Eya2 was first detected in 8.5-day embryos in the region of head ectoderm fated to become the forebrain. At later stages of development, Eya2 is expressed in the olfactory placode and in a variety of neural crest derivatives. In the eye, expression of Eya2 was first detected after formation of the lens vesicle. At day 17.5, the highest level of Eya2 mRNA was observed in primary lens fibers. Low levels of Eya2 expression was detected in retina, sclera, and cornea. By postnatal day 10, Eya2 was expressed in secondary lens fibers, cornea, and retina. Although Eya2 is expressed relatively late in eye development, it belongs to the growing list of factors that may be essential for eye development across metazoan phyla. Like members of the Pax-6 gene family, eyes absent gene family members were probably first involved in functions not related to vision, with recruitment for visual system formation and function occurring later.