Two small deletion mutations of the HEXB gene are present in DNA from a patient with infantile Sandhoff disease.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (alpha beta) and B (beta beta). The alpha subunit is encoded by the HEXA gene and the beta subunit by HEXB gene. Defects in the alpha or beta subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, AGA/GTC, to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect beta-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity.     

Characterization of the human HEXB gene encoding lysosomal beta-hexosaminidase

The lysosomal enzyme beta-hexosaminidase A contains alpha- and beta-subunits that are encoded by the HEXA and HEXB genes, respectively. The human HEXB gene has been isolated and characterized. It is 45 kb long and is split into 14 exons. Of the 13 introns, 12 interrupt the coding sequences at homologous positions in the HEXA and HEXB genes. The 5' flanking region contains the functional HEXB gene promoter. While a fine-structure analysis has yet to be done, we note that the sequence is GC rich and has several GC boxes and one CAAT box. There are also sequences related or identical to a progesterone response element and an AP-1 binding motif.     

A second mutation associated with apparent beta-hexosaminidase A pseudodeficiency: identification and frequency estimation.

Deficient activity of beta-hexosaminidase A (Hex A), resulting from mutations in the HEXA gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e., individuals who are pseudodeficient) have been described. Recently, an apparently benign C739-to-T (Arg247Trp) mutation was found among individuals with Hex A levels indistinguishable from those of carriers of Tay-Sachs disease. This allele, when in compound heterozygosity with a second "disease-causing" allele, results in Hex A pseudodeficiency. We examined the HEXA gene of a healthy 42-year-old who was Hex A deficient but did not have the C739-to-T mutation. The HEXA exons were PCR amplified, and the products were analyzed for mutations by using restriction-enzyme digestion or single-strand gel electrophoresis. A G805-to-A (Gly269Ser) mutation associated with adult-onset GM2 gangliosidosis was found on one chromosome. A new mutation, C745-to-T (Arg249Trp), was identified on the second chromosome. This mutation was detected in an additional 4/63 (6%) non-Jewish and 0/218 Ashkenazi Jewish enzyme-defined carriers. Although the Arg249Trp change may result in a late-onset form of GM2 gangliosidosis, any phenotype must be very mild. This new mutation and the benign C739-to-T mutation together account for approximately 38% of non-Jewish enzyme-defined carriers. Because carriers of the C739-to-T and C745-to-T mutations cannot be differentiated from carriers of disease-causing alleles by using the classical biochemical screening approaches, DNA-based analyses for these mutations should be offered for non-Jewish enzyme-defined heterozygotes, before definitive counseling is provided.     

Metabolic correction in microglia derived from Sandhoff disease model mice

Sandhoff disease is an autosomal recessive lysosomal storage disease caused by a defect of the beta-subunit gene (HEXB) associated with simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), and excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylglucosamine (GlcNAc) residues at their non-reducing termini. Recent studies have shown the involvement of microglial activation in neuroinflammation and neurodegeneration of this disease. We isolated primary microglial cells from the neonatal brains of Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). The cells expressed microglial cell-specific ionized calcium binding adaptor molecule 1 (Iba1)-immunoreactivity (IR) and antigen recognized by Ricinus communis agglutinin lectin-120 (RCA120), but not glial fibrillary acidic protein (GFAP)-IR specific for astrocytes. They also demonstrated significant intracellular accumulation of GM2 and GlcNAc-oligosaccharides. We produced a lentiviral vector encoding for the murine Hex beta-subunit and transduced it into the microglia from SD mice with the recombinant lentivirus, causing elimination of the intracellularly accumulated GM2 and GlcNAc-oligosaccharides and secretion of Hex isozyme activities from the transduced SD microglial cells. Recomibinant HexA isozyme isolated from the conditioned medium of a Chinese hamster ovary (CHO) cell line simultaneously expressing the human HEXA (alpha-subunit) and HEXB genes was also found to be incorporated into the SD microglia via cell surface cation-independent mannose 6-phosphate receptor and mannose receptor to degrade the intracellularly accumulated GM2 and GlcNAc-oligosaccharides. These results suggest the therapeutic potential of recombinant lentivirus encoding the murine Hex beta-subunit and the human HexA isozyme (alphabeta heterodimer) for metabolic cross-correction in microglial cells involved in progressive neurodegeneration in SD mice.     

Molecular heterogeneity in the infantile and juvenile forms of Sandhoff disease (O-variant GM2 gangliosidosis)

There are two major beta-hexosaminidase, EC 3.2.1.52, isozymes in normal human tissues. They exist as active dimers of alpha- and/or beta-subunits. A defect of their beta-subunit results in Sandhoff disease (O-variant GM2 gangliosidosis), an inherited, clinically heterogeneous, lysosomal storage disease. The status of the HEXB gene, pre beta-polypeptide chain mRNA, and residual beta-hexosaminidase activities were examined in a clinically and ethnically diverse collection of 16 fibroblast cell lines from patients with Sandhoff disease. Differentiation of the two major clinical types, infantile and juvenile onset, could be made by the determination of the activity of the residual beta-hexosaminidase eluting in the same pH range as hexosaminidase A. All the juvenile lines were found to have normal or reduced levels of pre beta-chain mRNA and no gross abnormalities in the HEXB gene. Of the 11 infantile type cell lines examined, four were found to contain no detectable pre beta-chain mRNA. Two cell lines in this group contained partial gene deletions localized to the 5' end of the HEXB gene. One of these cell lines has previously been assigned to the single complementation group in Sandhoff disease, conclusively demonstrating that the primary gene defect in the majority of Sandhoff cases is in the HEXB gene itself. These data suggest that each clinical group is made up of a collection of different HEXB mutations.     

The X-ray crystal structure of human beta-hexosaminidase B provides new insights into Sandhoff disease

Human lysosomal beta-hexosaminidases are dimeric enzymes composed of alpha and beta-chains, encoded by the genes HEXA and HEXB. They occur in three isoforms, the homodimeric hexosaminidases B (betabeta) and S (alphaalpha), and the heterodimeric hexosaminidase A (alphabeta), where dimerization is required for catalytic activity. Allelic variations in the HEXA and HEXB genes cause the fatal inborn errors of metabolism Tay-Sachs disease and Sandhoff disease, respectively. Here, we present the crystal structure of a complex of human beta-hexosaminidase B with a transition state analogue inhibitor at 2.3A resolution (pdb 1o7a). On the basis of this structure and previous studies on related enzymes, a retaining double-displacement mechanism for glycosyl hydrolysis by beta-hexosaminidase B is proposed. In the dimer structure, which is derived from an analysis of crystal packing, most of the mutations causing late-onset Sandhoff disease reside near the dimer interface and are proposed to interfere with correct dimer formation. The structure reported here is a valid template also for the dimeric structures of beta-hexosaminidase A and S.     

LIS1 missense mutations: variable phenotypes result from unpredictable alterations in biochemical and cellular properties

Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH alpha1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes.     

Knock‐down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC‐derived dendritic cells

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β‐N‐acetyl‐hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay‐Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock‐down immunogenic DCs (i‐DCs) from CD34⁺‐haemopoietic stem cells (CD34⁺‐HSCs), thus mimicking the Tay‐Sachs (HEXA?/?) and Sandhoff (HEXB?/?) cells. We showed that the absence of β‐N‐acetyl‐hexosaminidase activity does not alter the differentiation of i‐DCs from HSCs, but it is critical for the activation of CD4⁺T cells because knock‐down of HEXA or HEXB gene causes a loss of function of i‐DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β‐N‐acetyl‐hexosaminidase A isoenzyme within this mechanism. Copyright © 2011 John Wiley & Sons, Ltd.     

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.     

An alpha-subunit loop structure is required for GM2 activator protein binding by beta-hexosaminidase A

The alpha- and/or beta-subunits of human beta-hexosaminidase A (alphabeta) and B (betabeta) are approximately 60% identical. In vivo only beta-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the alpha-subunit. A model for this interaction suggests that two loop structures, present only in the alpha-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-beta-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant alpha-subunits demonstrate that only the site that is removed from the beta-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events.     

A pseudodeficiency allele common in non-Jewish Tay-Sachs carriers: Implications for carrier screening

Deficiency of beta-hexosaminidase A (Hex A) activity typically results in Tay-Sachs disease. However, healthy subjects found to be deficient in Hex A activity (i.e., pseudodeficient) by means of in vitro biochemical tests have been described. We analyzed the HEXA gene of one pseudodeficient subject and identified both a C739-to-T substitution that changes Arg247----Trp on one allele and a previously identified Tay-Sachs disease mutation on the second allele. Six additional pseudodeficient subjects were found to have the C739-to-T mutation. This allele accounted for 32% (20/62) of non-Jewish enzyme-defined Tay-Sachs disease carriers but for none of 36 Jewish enzyme-defined carriers who did not have one of three known mutations common to this group. The C739-to-T allele, together with a "true" Tay-Sachs disease allele, causes Hex A pseudodeficiency. Given both the large proportion of non-Jewish carriers with this allele and that standard biochemical screening cannot differentiate between heterozygotes for the C739-to-T mutations and Tay-Sachs disease carriers, DNA testing for this mutation in at-risk couples is essential. This could prevent unnecessary or incorrect prenatal diagnoses.     

Characterization of two HEXB gene mutations in Argentinean patients with Sandhoff disease.

Beta-hexosaminidase A (beta-N-acetyl-D-hexosaminidase, EC 3.2.1.5.2) is a lysosomal hydrolase composed of an alpha- and a beta-subunit. It is responsible for the degradation of GM2 ganglioside. Mutations in the HEXB gene encoded beta-subunit cause a form of GM2 gangliosidosis known as Sandhoff disease. Although this is a rare disease in the general population, several geographically isolated groups have a high carrier frequency. Most notably, a 1 in 16-29 carrier frequency has been reported for an Argentinean population living in an area contained within a 375-km radius from Córdoba. Analysis of the genomic DNA of two patients from this region revealed that one was homozygous for a G to A substitution at the 5' donor splice site of intron 2. This mutation completely abolishes normal mRNA splicing. The other patient was a compared of the intron 2 G-->A substitution and a second allele due to a 4-bp deletion in exon 7. The beta-subunit mRNA of this allele is unstable, presumably as a result of an early stop codon introduced by the deletion. Two novel PCR-based assays were developed to detect these mutations. We suggest that one of these assays could be modified and used as a rapid screening procedure for 5' donor splice site defects in other genes. These results provide a further example of the genetic heterogeneity that can exist even in a small geographically isolated population.     

Identification and rapid detection of three Tay-Sachs mutations in the Moroccan Jewish population.

Infantile Tay-Sachs disease (TSD) is caused by mutations in the HEXA gene that result in the complete absence of beta-hexosaminidase A activity. It is well known that an elevated frequency of TSD mutations exists among Ashkenazi Jews. More recently it has become apparent that elevated carrier frequencies for TSD also occur in several other ethnic groups, including Moroccan Jews, a subgroup of Sephardic Jews. Elsewhere we reported an in-frame deletion of one of the two adjacent phenylalanine codons at position 304 or 305 (delta F304/305) in one HEXA allele of a Moroccan Jewish TSD patient and in three obligate carriers from six unrelated Moroccan Jewish families. We have now identified two additional mutations within exon 5 of the HEXA gene that account for the remaining TSD alleles in the patient and carriers. One of the mutations is a novel C-to-G transversion, resulting in a replacement of Tyr180 by a stop codon. The other mutation is a G-to-A transition resulting in an Arg170-to-Gln substitution. This mutation is at a CpG site in a Japanese infant with Tay-Sachs disease and was described elsewhere. Analysis of nine obligate carriers from seven unrelated families showed that four harbor the delta F304/305 mutation, two the Arg170----Gln mutation, and one the Tyr180----Stop mutation. We also have developed rapid, nonradioactive assays for the detection of each mutation, which should be helpful for carrier screening.     

In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits,Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.     

Reversion of the biochemical defects in murine embryonic Sandhoff neurons using a bicistronic lentiviral vector encoding hexosaminidase alpha and beta

Sandhoff disease, a neurodegenerative disorder characterized by the intracellular accumulation of GM2 ganglioside, is caused by mutations in the hexosaminidase beta-chain gene resulting in a hexosaminidase A (alphabeta) and B (betabeta) deficiency. A bicistronic lentiviral vector encoding both the hexosaminidase alpha and beta chains (SIV.ASB) has previously been shown to correct the beta-hexosaminidase deficiency and to reduce GM2 levels both in transduced and cross-corrected human Sandhoff fibroblasts. Recent advances in determining the neuropathophysiological mechanisms in Sandhoff disease have shown a mechanistic link between GM2 accumulation, neuronal cell death, reduction of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity, and axonal outgrowth. To examine the ability of the SIV.ASB vector to reverse these pathophysiological events, hippocampal neurons from embryonic Sandhoff mice were transduced with the lentivector. Normal axonal growth rates were restored, as was the rate of Ca(2+) uptake via the SERCA and the sensitivity of the neurons to thapsigargin-induced cell death, concomitant with a decrease in GM2 and GA2 levels. Thus, we have demonstrated that the bicistronic vector can reverse the biochemical defects and down-stream consequences in Sandhoff neurons, reinforcing its potential for Sandhoff disease in vivo gene therapy.     

The GM2 activator protein, a novel inhibitor of platelet-activating factor.

The GM2 activator protein is required as a substrate-specific cofactor for beta-hexosaminidase A to hydrolyze GM2 ganglioside. The GM2 activator protein reversibly binds and solubilizes individual GM2 ganglioside molecules, making them available as substrate. Although GM2 ganglioside is the strongest binding ligand for the activator protein, it can also bind and transport between membranes a series of other glycolipids, even at neutral pH. Biosynthetic studies have shown that a large portion of newly synthesized GM2 activator molecules are not targeted to the lysosome, but are secreted and can then be recaptured by other cells through a carbohydrate independent mechanism. Thus, the GM2 activator protein may have other in vivo functions. We found that the GM2 activator protein can inhibit, through specific binding, the ability of platelet activating factor (PAF) to stimulate the release of intracellular Ca2+ pools by human neutrophils. PAF is a biologically potent phosphoacylglycerol. Inhibitors for PAF's role in the pathogenesis of inflammatory bowel disease and asthma have been sought as potential therapeutic agents. The inherent stability and protease resistance of the small, monomeric GM2 activator protein, coupled with the ability to produce large quantities of the functional protein in transformed bacteria, suggest it may serve as such an agent.     

Expression and characterization of 14 GLB1 mutant alleles found in GM1-gangliosidosis and Morquio B patients

GM1-gangliosidosis and Morquio B disease are lysosomal storage disorders caused by beta-galactosidase deficiency attributable to mutations in the GLB1 gene. On reaching the endosomal-lysosomal compartment, the beta-galactosidase protein associates with the protective protein/cathepsin A (PPCA) and neuraminidase proteins to form the lysosomal multienzyme complex (LMC). The correct interaction of these proteins in the complex is essential for their activity. More than 100 mutations have been described in GM1-gangliosidosis and Morquio B patients, but few have been further characterized. We expressed 12 mutations suspected to be pathogenic, one known polymorphic change (p.S532G), and a variant described as either a pathogenic or a polymorphic change (p.R521C). Ten of them had not been expressed before. The expression analysis confirmed the pathogenicity of the 12 mutations, whereas the relatively high activity of p.S532G is consistent with its definition as a polymorphism. The results for p.R521C suggest that this change is a low-penetrant disease-causing allele. Furthermore, the effect of these beta-galactosidase changes on the LMC was also studied by coimmunoprecipitations and Western blotting. The alteration of neuraminidase and PPCA patterns in several of the Western blotting analyses performed on patient protein extracts indicated that the LMC is affected in at least some GM1-gangliosidosis and Morquio B patients.     

Impaired elastic-fiber assembly by fibroblasts from patients with either Morquio B disease or infantile GM1-gangliosidosis is linked to deficiency in the 67-kD spliced variant of beta-galactosidase

We have previously shown that intracellular trafficking and extracellular assembly of tropoelastin into elastic fibers is facilitated by the 67-kD elastin-binding protein identical to an enzymatically inactive, alternatively spliced variant of beta-galactosidase (S-Gal). In the present study, we investigated elastic-fiber assembly in cultures of dermal fibroblasts from patients with either Morquio B disease or GM1-gangliosidosis who bore different mutations of the beta-galactosidase gene. We found that fibroblasts taken from patients with an adult form of GM1-gangliosidosis and from patients with an infantile form, carrying a missense mutations in the beta-galactosidase gene-mutations that caused deficiency in lysosomal beta-galactosidase but not in S-Gal-assembled normal elastic fibers. In contrast, fibroblasts from two cases of infantile GM1-gangliosidosis that bear nonsense mutations of the beta-galactosidase gene, as well as fibroblasts from four patients with Morquio B who had mutations causing deficiency in both forms of beta-galactosidase, did not assemble elastic fibers. We also demonstrated that S-Gal-deficient fibroblasts from patients with either GM1-gangliosidosis or Morquio B can acquire the S-Gal protein, produced by coculturing of Chinese hamster ovary cells permanently transected with S-Gal cDNA, resulting in improved deposition of elastic fibers. The present study provides a novel and natural model validating functional roles of S-Gal in elastogenesis and elucidates an association between impaired elastogenesis and the development of connective-tissue disorders in patients with Morquio B disease and in patients with an infantile form of GM1-gangliosidosis.     

Identification and expression profiling of Ceratitis capitata genes coding for β-hexosaminidases

The goal of this study was to identify the genes coding for β-N-acetylhexosaminidases in the Mediterranean fruit fly (medfly) Ceratitis capitata, one of the most destructive agricultural pests, belonging to the Tephritidae family, order Diptera. Two dimeric β-N-acetylhexosaminidases, HEXA and HEXB, have been recently identified on Drosophila sperm. These enzymes are involved in egg binding through interactions with complementary carbohydrates on the surface of the egg shell. Three genes, Hexosaminidase 1 (Hexo1), Hexosaminidase 2 (Hexo2) and fused lobes (fdl), encode for HEXA and HEXB subunits. The availability of C. capitata EST libraries derived from embryos and adult heads allowed us to identify three sequences homologous to the D. melanogaster Hexo1, Hexo2 and fdl genes. Here, we report the expression profile analysis of CcHexo1, CcHexo2 and Ccfdld in several tissues, organs and stages. Ccfdl expression was highest in heads of both sexes and in whole adult females. In the testis and ovary the three genes showed distinct spatial and temporal expression patterns. All the mRNAs were detectable in early stages of spermatogenesis; CcHexo2 and Ccfdl were also expressed in early elongating spermatid cysts. All three genes are expressed in the ovarian nurse cells. CcHexo1 and Ccfdl are stage specific, since they have been observed in stages 12 and 13 during oocyte growth, when programmed cell death occurs in nurse cells. The expression pattern of the three genes in medfly gonads suggests that, as their Drosophila counterparts, they may encode for proteins involved in gametogenesis and fertilization.     

Molecular analysis of HEXA gene in Argentinean patients affected with Tay-Sachs disease: possible common origin of the prevalent c.459+5A>G mutation

Tay-Sachs disease (TSD) is a recessively inherited disorder caused by the deficient activity of hexosaminidase A due to mutations in the HEXA gene. Up to date there is no information regarding the molecular genetics of TSD in Argentinean patients. In the present study we have studied 17 Argentinean families affected by TSD, including 20 patients with the acute infantile form and 3 with the sub-acute form. Overall, we identified 14 different mutations accounting for 100% of the studied alleles. Eight mutations were novel: 5 were single base changes leading to drastic residue changes or truncated proteins, 2 were small deletions and one was an intronic mutation that may cause a splicing defect. Although the spectrum of mutations was highly heterogeneous, a high frequency of the c.459+5G>A mutation, previously described in different populations was found among the studied cohort. Haplotype analysis suggested that in these families the c.459+5G>A mutation might have arisen by a single mutational event.