IDS Gene Research Articles

Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II

Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation.

AB082. Phenotype and genotype of Vietnamese patients with mucopolysaccharidosis II: first case series report

Background and objective Mucopolysaccharidosis II (MPS II, Hunter syndrome, OMIM 309900) is an X-linked lysosomal storage disorder. MPS II is caused by a deficiency in the enzyme iduronate-2 sulfatase (I2S), leading to the accumulation of the glycosaminoglycans (GAGs) dermatan sulfate and heparan sulfate in lysosomes. Excessive storage of these GAGs causes a variety of clinical manifestations: coarse facies, hearing loss, cardiac valve disease, restrictive and obstructive airway disease, hepatosplenomegaly, skeletal abnormalities, joint contractures, short stature. The study aims to describe clinical characteristics and to identify mutations in the IDS gene in Vietnamese patients with MPS II.

Focal Reactivation of X-Chromosome in Female Patients with PV and ET

First and second authors contributed equally.During early female embryogenesis, the most of genes in either the maternal or paternal X-chromosomes are randomly inactivated; this remains remarkably constant in the progeny of these cells throughout life. The X-inactive specific transcript gene (XIST), which encodes long non-coding RNA, is expressed only from the inactive X-chromosome and plays a crucial role in this process. X-chromosome inactivation has been used to define clonality of malignant and premalignant disorders such polycythemia vera (PV) and essential thrombocythemia (ET). Using a quantitative, transcriptional clonality assay based on polymorphisms on 5 X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD), we found that the allelic usage ratio of these genes varies among normal females, and from tissue to tissue, but is the same in all blood cell lineages, including T-cells, and remained constant over 3 years of study (Prchal, J Exp Med, 1996). Further, in females heterozygous for more than 1 of these genes, the allelic usage ratio is the same for each marker. Over more than a decade, we analyzed over 150 informative PV and JAK2V617F-positive ET females and all were clonal. However, we recently encountered 4 exceptional cases. Two PV (P1 and P3) and 1 ET (P2) females appeared polyclonal using an IDS marker, and one female (P4), using a G6PD marker, in platelets and/or granulocytes, whereas all of these females appeared clonal using at least 1 other X-chromosome marker in these cells. Further, T-cell IDS allelic ratios were different from those in platelets and granulocytes; T-cell X-chromosome usage ratio is, in any female, a marker of normal hematopoiesis prior to a clonal event.It was recently reported that the conditional deletion of Xist in hematopoietic stem cells in female mice leads to complete penetrance of a highly aggressive PV/ET-like syndrome that progresses to invariably fatal lymphoma and or acute leukemia. Further, reactivation of X-chromosome has been reported in ovarian and breast cancers. We propose that reactivation of inactive IDS and G6PD in these females, and perhaps other X-chromosome genes, contributes to the PV/ET phenotype.In ongoing studies, we performed additional experiments on 2 of these females (P1 and P4). We analyzed their X-chromosome transcripts using clonogenic assays of early erythroid progenitors, which formed erythroid colonies (burst-forming units-erythroid, BFU-Es) in vitro when cultured in methylcellulose in the presence of EPO. In controls, each BFU-E expressed only a single X-chromosome transcript; however, in both of these females we observed at least 1 clone which expressed both X-chromosome genes. We then investigated the epigenetic status of the IDS gene in P1. We performed chromatin immunoprecipitation of granulocyte DNA from P1 using histone H3 lysine 27 trimethylation (H3K27m3) antibodies to assess the level of polycomb silencing in the immunoprecipitated granulocyte DNA fragments. In this analysis, the IDS gene was ~4 times less enriched for H3K27m3 compared to normal controls (see Figure). Ongoing analysis of the XIST gene revealed normal mRNA levels in P1 and P4 and inexplicably increased XIST gene transcript in P2 and P3. Analysis of XIST transcripts in platelets, erythroid progenitors and stem cells is ongoing, as well as sequencing of the XIST gene. Preliminary DNA methylation analysis of granulocytes and CD34+ cells from P1 revealed focal hypomethylation of the inactivated X-chromosome, including promoters of SRPL3, PIM2, PHKA1, PHKA2, and RPS6KA3 genes; in all four subjects, platelet and granulocyte transcripts of 2 of these genes, PIM2 and PHKA1, were overexpressed compared to normal controls.These results suggest that reactivation of some X-chromosome genes in PV and ET clones present in some female patients plays an important role in the diversity of PV and ET phenotypes and may also play a role in the demonstrated differences in PV and ET phenotypes between males and females (Spivak JL, N Engl J Med, 2013). [Display omitted] DisclosuresIssa:Astex Pharmaceuticals, Inc.: Consultancy, Research Funding.

NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene

The mucopolysaccharidoses (MPSs) are a group of rare, inherited lysosomal storage disorders that are clinically characterized by abnormalities in multiple organ systems and reduced life expectancy. Whereas the lysosome is essential to the functioning of the immune system, some authors suggest that the MPS patients have abnormalities in the immune system similar to the patients with primary immunodeficiency.In this study, we evaluated 8 male MPS type II patients of the same family with novel mutation in the IDS gene. We found in this MPS family a quantitative deficiency of NK and B cells with normal values of IgG, IgM and IgA serum antibodies and normal response to polysaccharide antigens. Interestingly, abnormalities found in these patients were not observed in other MPS patients, suggesting that the type of mutation found in the IDS gene can be implicated in the immunodeficiency.

Mutation analysis of mucopolysaccharidosis type II and prenatal diagnosis

To analyze the mutations of IDS gene in a mucopolysaccharidosis type II (MPSII) family and to make prenatal diagnosis on the high-risk fetus which has been pregnant for eleven weeks. IDS gene was analyzed by bidirectional DNA sequencing in 2 patients and their mother, and 5 unaffected individuals. Prenatal diagnosis for the high-risk fetus was performed by chorionic villus sampling after the genotypes was identified. The mutation c.344delA (N115fsX15) was detected in the two patients, and the mother of patients carried the heterozygous c.344delA (N115fsX15) mutation. None of the mutant was detected in the 5 unaffected subjects. The fetus carried c.344delA (N115fsX15) heterozygous mutation and was a carrier. The deletion mutation c.344delA (N115fsX15) is causative to the pedigree of MPSII, and prenatal diagnosis is the efficient method to avoid defect birth.

Effects of idursulfase enzyme replacement therapy for Mucopolysaccharidosis type II when started in early infancy: Comparison in two siblings

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder that is progressive and involves multiple organs and tissues. While enzyme replacement therapy (ERT) with idursulfase has been shown to improve many somatic features of the disease, some such as dysostosis multiplex and cardiac valve disease appear irreversible once established, and little is known about the preventative effects of ERT in pre-symptomatic patients. We report on two siblings with severe MPS II caused by an inversion mutation with recombination breakpoints located within the IDS gene and its adjacent pseudogene, IDS-2. The siblings initiated treatment with idursulfase at 3.0years (older brother) and 4months (younger brother) of age, and we compared their outcomes following 2years of treatment. At the start of treatment, the older brother showed typical features of MPS II, including intellectual disability. After 34months of ERT, his somatic disease was stable or improved, but he continued to decline cognitively. By comparison, after 32months of ERT his younger brother remained free from most of the somatic features that had already appeared in his brother at the same age, manifesting only exudative otitis media. Skeletal X-rays revealed characteristic signs of dysostosis multiplex in the older brother at the initiation of treatment that were unchanged two years later, whereas the younger brother showed only slight findings of dysostosis multiplex throughout the treatment period. The younger brother's developmental quotient trended downward over time to just below the normal range. These findings suggest that pre-symptomatic initiation of ERT may prevent or attenuate progression of the somatic features of MPS II. Follow-up in a larger number of patients is required to confirm the additive long-term benefits of ERT in pre-symptomatic patients.

Important aspects in the molecular diagnosis of mucopolysaccharidoses

None.

A novel mutation of IDS gene in a Chinese patient with mucopolysaccharidosis II by next-generation sequencing

BackgroundMucopolysaccharidosis (MPS) is induced by the absence or malfunctioning of lysosomal enzymes. MPS I and MPS II are similar in phenotypes but they are different in genotypes, which are caused by the deficiencies of alpha-L-iduronidase gene (IDUA) and iduronate 2-sulfatase gene (IDS) respectively. In this work, a 5-year-old Chinese young male with manifestations of MPS in a family with unaffected parents was described. Methods12kb of all the targeted exon sequences plus flanking sequences chromosomal DNA of IDS and IDUA genes from the proband and 20 other case-unrelated controls were captured and sequenced by using next-generation sequencing technology. ResultsOne single-nucleotide deletion variant (c.1270delG) resulting in frameshift and premature truncation of I2S enzyme was detected, out of 20 controls, only in the proband, and which was further verified by Sanger sequencing. The proband's mother was also proved carrying c.1270delG by Sanger method but not for his father. ConclusionsThe novel variant (c.1270delG) is a candidate disease-causing mutation predicted to affect the normal structure and function of the enzyme. Target sequence capture and next-generation sequencing technology can be effective for the gene testing of MPS II disorder.

LCR-initiated rearrangements at the IDS locus, completed with Alu-mediated recombination or non-homologous end joining

Mucopolysaccharidosis type II (MPS II) is caused by mutations in the IDS gene, which encodes the lysosomal enzyme iduronate-2-sulfatase. In ∼20% of MPS II patients the disorder is caused by gross IDS structural rearrangements. We identified two male cases harboring complex rearrangements involving the IDS gene and the nearby pseudogene, IDSP1, which has been annotated as a low-copy repeat (LCR). In both cases the rearrangement included a partial deletion of IDS and an inverted insertion of the neighboring region. In silico analyses revealed the presence of repetitive elements as well as LCRs at the junctions of rearrangements. Our models illustrate two alternative consequences of rearrangements initiated by non-allelic homologous recombination of LCRs: resolution by a second recombination event (that is, Alu-mediated recombination), or resolution by non-homologous end joining repair. These complex rearrangements have the potential to be recurrent and may be present among those MSP II cases with previously uncharacterized aberrations involving IDS.

Identification of a novel mutation of <I>IDS</I> gene from a Chi-nese pedigree with MPS II

The purpose of this study was to understand the molecular genetic mechanism of mucopolysaccharidosis type II (MPS II) and to provide a prerequisite for future prenatal gene diagnosis. A preliminary diagnosis was made by qualitative detection of Urinary Glycosaminoglycans of the suspected MPS II proband. Then, mutation detection was performed on the proband and his family members with PCR and direct sequencing of PCR products. After the novel mutation of c.876 del 2 in IDS gene was detected, sequence analysis was performed on exon 6 of IDS gene of the 135 cases, which consisted of 120 randomly selected normal controls, and other 15 patients with MPS I, IV, and VI other than MPS II. Besides, the patho-genicity of the novel mutation was analyzed with the following 2 methods: conservative analysis of the sequence of muta-tion spots of different species and the direct test of the IDS enzyme activity of the patient and his relative family members. The result of uroscopy of the proband was strong positive (GAGs +++). There was a novel deletion mutation of c.876-877 del TC in the coding region of exon 6 of IDS gene, which was a hemizygous mutation. However, the mutation of his mother and sister was a heterozygous mutation. Detection of the exon 6 of IDS gene showed that the mutation was not found among normal controls and other patients with MPS I, IV, and VI other than MPS II. Homology comparison of amino acid sequences from different species showed that the phenylalanine (F) glutamine (Q) of the mutation site of c.876-877 del TC located in p.292-293 was highly conserved. The activity of IDS enzyme of the proband was only 2.3 nmol/4 h/mL, which was much lower than normal; but the activity of IDS enzyme of his father, mother and sister was 641.9 nmol/4 h/mL, 95.8 nmol/4h/mL and 103.2 nmol/4h/mL, respectively. These results illustrated that the deletion and frame-shift mutation of c.876-877 del TC detected was a novel pathologic mutation, which was the underlying cause of MPS II of this patient.

90 Molecular investigations of a novel mutation in the IDS gene and an iduronate-2-sulfatase mutant without the first 33 amino acids

90 Molecular investigations of a novel mutation in the IDS gene and an iduronate-2-sulfatase mutant without the first 33 amino acids

Clinical and molecular cytogenetic characterization of two patients with non‐mutational aberrations of the FMR2 gene

We report on two patients; a female having mild mental retardation (MR) with a balanced translocation, 46,XX,t(X;15)(q28;p11.2), and a male diagnosed as having mucopolysaccharidosis type II (MPS II or Hunter syndrome) with atypical early-onset MR and a normal male karyotype. Molecular cytogenetic analyses, including fluorescence in situ hybridization and array-based comparative genomic hybridization using an in-house X-tiling array, revealed that first patient to have a breakpoint at Xq28 lying within the FMR2 gene and the second to have a small deletion at Xq28 including part of FMR2 together with the IDS gene responsible for MPS II. In Patient 1, X-chromosome inactivation predominantly occurred in the normal X in her lymphocytes, suggesting that her MR might be explained by a disruption of the FMR2 gene on der(X) t(X;15) concomitant with the predominant inactivation of the intact FMR2 gene in another allele. We compared phenotypes of Patient 2 with those of MPS II cases with deletion of the IDS gene alone reported previously, suggesting that the early-onset MR might be affected by the additional deletion of FMR2.

Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a defect in the iduronate-2-sulfatase gene (IDS). Alternative splicing of the IDS gene can occur and the underlying regulatory mechanism may be rather complex. Nevertheless, little information is available on the role of variations at the IDS locus in the splicing process. Here we report that splice mutations at the IDS locus are an important source of MPS II pathogenicity, accounting for almost 56% of Portuguese cases. Among 16 unrelated Portuguese MPS II patients, 15 different mutations were identified: six intronic splice mutations (c.104-2AG, c.241-2A>G, c.241-1G>A, c.418+1G>A, c.880-8AG and c.1181-1G>C); two exonic splice mutations (c.1006G>lC and c.1122C>T); five missense mutations (D269V, D69V, D148N, R88C and P86L); one nonsense mutation (Q465Ter); one total IDS gene deletion; and one rearrangement involving a IDS gene inversion. Furthermore, nine of the 15 detected mutations affected the usual splicing pattern at the locus. Some of them are responsible for dramatic changes in the splicing mechanism. For example, the substitution mutation, c.418+1G>A, revealed the presence of an exonic sequence inside intron 3. Our study provides evidence that the IDS locus is prone to splicing mutations and that such susceptibility is particularly high in exon 3 and neighbouring regions. Consequently, mutation screening of the IDS gene cannot be restricted to gDNA examination. Unless cDNA analysis is also conducted, misclassifications as silent or missense mutations can be produced and even uncharacteristic splice-site mutations can be misinterpreted as classic splicing defects that may generate severe, unconventional splicing alterations.

TGF-β1-induced cell growth arrest and partial differentiation is related to the suppression of Id1 in human hepatoma cells

Transforming growth factor beta 1 (TGF-beta1) is a proposed regulator of Ids (inhibitors of DNA binding/differentiation) gene expression in epithelial cells. We previously reported that Id proteins are variously expressed in human hepatocellular carcinomas (HCC). However, the mechanism of regulation of Ids in HCC remains obscure. Here, we examined the relationship between Id1 and TGF-beta1 in four HCC cell lines, and studied the changes in cell proliferation, cell cycle and differentiation. The four HCC cell lines expressed Id1, TGF-beta1 and their receptors at various levels. TGF-beta1 strongly inhibited the growth of HuH7 cells, while the growth inhibition was moderate in PLC/PRF/5, and was not observed in HLE and HLF cell lines. TGF-beta1-induced growth inhibition in HuH7 cells was associated with cell accumulation in the G1 phase and partial induction of differentiation (with reduction of AFP and AFP-L3). Induction by TGF-beta1 dose-dependently suppressed Id1 expression in HuH7 cells; 1 ng/ml TGF-beta1 inhibited Id1 by 84.0 and 78.6% that of the untreated control at transcriptional and protein levels, respectively. HLE and HLF cells, which did not exhibit a TGF-beta1 growth inhibitory effect, lacked TGF-beta receptors and Id1 expression was not altered. In PLC/PRF/5 cells, Id1 augmentation was not observed in response to TGF-beta1, indicating that TGF-beta1-induced growth inhibition was not related to Id1 in this cell line. Our results suggest that, in some HCC cells, the pathway of suppression of Id1 by TGF-beta1 may be important in TGF-beta1-induced growth inhibition and partial differentiation.

Mitochondrial Ferritin Expression and Clonality of Hematopoiesis in Patients with Refractory Anemia with Ringed Sideroblasts.

Sideroblastic anemias are a heterogeneous group of disorders that have in common the presence of erythroblasts with iron-loaded mitochondria defined as ringed sideroblasts. We have previously demonstrated that mitochondrial iron deposition in these disorders is in the form of mitochondrial ferritin (MtF), suggesting that this latter may be a specific marker of sideroblastic anemia (Cazzola et al, Blood 2003; 101:1996–2000). The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts, which is generally associated with a relatively benign clinical course. In the present work, we studied the relationship between MtF expression and clonality of hematopoiesis in 55 consecutive female patients with low-risk MDS, including 20 cases with ringed sideroblasts and 35 cases without ringed sideroblasts. The expression of MtF, as well as that of cytosolic ferritin (H and L subunits) and of transferrin receptor (CD71), was evaluated by flow cytometry in bone marrow erythroid cells; in selected cases, these immunophenotypic investigations were also performed on liquid cultures of purified CD34-positive cells. X-chromosome inactivation patterns (XCIPs) were assessed in peripheral blood granulocytes and in bone marrow CD34-positive cells by analysis of both DNA methylation at the HUMARA and PGK loci and of IDS gene expression. Within informative females, 11 out of 12 patients with ringed sideroblasts displayed clonal XCIPs in granulocytes; by contrast, only 9 out of 22 patients without ringed sideroblasts displayed clonal XCIPs. Purified CD34-positive cells showed clonal XCIPs in 6 out of 7 patients with ringed sideroblasts but had polyclonal XCIPs in 4 out of 5 individuals without ringed sideroblasts. Flow cytometry evaluation of bone marrow erythroid cells showed that MtF expression was restricted to MDS patients with ringed sideroblasts. A close positive relationship was found between MtF and CD71 expression (r=.49, P=.001); this association may reflect the cytosolic iron deprivation induced by MtF overexpression. Analysis of cultured erythroid progenitor cells showed that MtF was detectable at a very early stage of differentiation from CD34-positive cells. Addition of erythropoietin to the culture system sustained the appearance of a polyclonal erythroid population in 2 out of 4 patients with ringed sideroblasts and clonal CD34-positive cells. These observations suggest that refractory anemia with ringed sideroblasts is a truly clonal stem cell disorder, while more than 50% of patients with refractory anemia without ringed sideroblasts have evidence of polyclonal hematopoiesis. The clonal pattern of CD34-positive cells and the early appearance of MtF during erythroid differentiation suggest that - despite be benign natural history of refractory anemia with ringed sideroblasts - the initial pathogenetic event in this condition occurs in multipotent stem cells. Although the mechanisms responsible for overexpression of MtF are still unclear, flow cytometry evaluation of this protein is a useful diagnostic tool that also provides helpful prognostic information.

Evidence of Polyclonal Hematopoiesis in a Large Proportion of Patients with Low-Risk Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are usually defined as clonal proliferations of pluripotent stem cells, which retain their capacity to differentiate but do so in an inefficient manner, so that mature cells in the peripheral blood are variably altered and reduced. Various approaches can be used to prove the existence of a clonal population of cells. Analysis of X inactivation-dependent methylation patterns in double heterozygous females has been used in MDS, and this approach showed clonal hematopoietic progenitors capable of granulocytic-monocytic differentiation in most cases. However, in a recent study (Blood 2003; 102:1211–6) clonality, defined as a clonal population accounting for 35% or more of total granulocytes, was confirmed in only one third of patients with refractory anemia. We studied X-chromosome inactivation patterns (XCIP) in 50 consecutive female patients with MDS. These included 18 RA, 6 RARS, 12 RCMD, 4 RCMD-RS, 2 MDS with del(5q), 4 RAEB-1 and 4 RAEB-2. The XCIP was established by analysis of the IDS gene expression and of DNA methylation at the HUMARA and PGK loci. In order to assess the clonality status of single hematopoietic lineages, the relative expression of HUMARA alleles in reticulocytes, granulocytes and platelets was evaluated by RT-PCR. Finally, telomere length, as a major determinant of the replicative lifespan of hematopoietic cells, was analyzed on peripheral blood granulocytes by flow-FISH (Dako Telomere PNA kit/FITC). XCIP and telomere length analyses were performed at the diagnosis (21 cases) or during the follow-up (29 cases, median time from diagnosis 10 months, range 1–230). XCIP were informative in 43 patients: 20 displayed clonal or ambiguous XCIP (46.5%), while 23 showed polyclonal XCIP (53.5%). Polyclonal XCIP were found in 20 of 36 informative MDS patients without excess blasts (55.5%), and 3 of 7 (42.9%) with excess blasts. Among MDS patients with polyclonal hematopoiesis, clonal cytogenetic abnormalities were found in 9 out of 17 informative cases, all of them showing a chimeric karyotype with normal and abnormal metaphases. Telomere length was significantly shorter in patients with monoclonal patterns than in patients with polyclonal hematopoiesis (P=.01). A preliminary study of XCIP by expression of HUMARA alleles showed that 6 patients with polyclonal XCIP on PMN had clonal XCIP on both reticulocytes and platelets. In brief, these data provide evidence that more than 50% of patients with low risk MDS had some polyclonal hematopoiesis. However, detection of karyotypic abnormalities in these patients confirmed the clonal nature of the disease, the myelodysplastic clones coexisting with residual normal hematopoiesis. Preliminary data indicate that the myelodysplastic clone may originate in a common erythroid-megakaryocytic progenitor. Finally, the telomere data suggest that monoclonal hematopoiesis is associated with telomere shortening.

A polymorphism of the X-linked gene IDS increases the number of females informative for transcriptional clonality assays.

Studies of clonality have been essential for understanding the hierarchy of hematopoiesis and the biology of malignancies. Most clonality assays are based on the X chromosome inactivation phenomenon in females; these assays detect protein polymorphisms, differences in DNA methylation, or transcripts of the active X chromosome. Assays based on protein polymorphisms or DNA methylation have significant shortcomings. The major disadvantage of transcriptional assays is their limited applicability since only approximately half of females are informative for these studies. We have developed a new transcriptional assay based on an exonic polymorphism of the X-chromosome gene IDS. This gene is located in the same X-chromosome region (Xq28) as G6PD and p55, two genes with exonic polymorphisms for which we previously developed transcriptional assays. We developed non-radioactive PCR-based assays for rapid screening of genotype and determination of clonality. We also report reaction conditions for a quantitative ligase detection assay of IDS allelic transcripts. The frequency of the IDS polymorphism is 46% in Caucasian females and 39% in African-American females; in combination with G6PD and p55, 76% of Caucasian females and 62% of African-American females are informative for these assays. While this gene is highly polymorphic in Caucasian and African-American females, it is not informative in Oriental females. We established that the IDS gene is in linkage equilibrium with G6PD and p55. Unlike methylation-based assays, this assay is suitable for studying clonality in non-nucleated cells such as platelets and reticulocytes. With the discovery of exonic polymorphisms of other X-chromosome genes, all females should eventually be suitable for X-chromosome transcriptional clonality analysis.

Hunter disease in the Spanish population: molecular analysis in 31 families.

Mucopolysaccharidosis type II (Hunter disease) is an X-linked disorder due to deficiency of the lysosomal enzyme iduronate 2-sulphatase. Here we report an update of molecular studies in 31 Spanish families with Hunter disease. We found a total of 22 novel small mutations (7 reported previously by our group), and 4 large deletions or rearrangements. Particularly relevant are two mutations, one showing an alternatively spliced product although the normal splice site is conserved; the other mutation results in an amino acid change that most likely modifies regulation of expression of the IDS gene. Except for large gene alterations and for the G374sp mutation already described, we could not establish a clear phenotype-genotype correlation. Mutation G374sp is the point mutation most frequent in our population (10%) and is always associated with mild phenotype. Our molecular analyses carried out in a relatively large series of patients with Hunter disease contribute to the identification of new mutations and reinforce the conclusions drawn in other populations about the genotype-phenotype correlation and the gene distribution of mutations.

Mutation analysis in the iduronate-2-sulphatase gene in 43 Japanese patients with mucopolysaccharidosis type II (Hunter disease).

Our series of studies on Hunter disease in Japanese patients showed allelic heterogeneity of IDS gene mutations, genotype/phenotype correlation and racial differences in distribution of mutations. Twenty-five different small mutations have been characterized. Small mutations in the Japanese population are widely distributed through the IDS gene, although some mutations were unevenly concentrated on exon 5 (28%) and on exon 9 (24%). Mutations were seen at the same codon 468 in exon 9 in 5 patients. These findings are in good agreement with data on other ethnic groups. Two unique mutations linked to a severe phenotype were apparently associated with aberrant splicings; one was a point mutation within exon 3 (P86L), partially activating a cryptic splice acceptor site at 28 bp downstream from the mutation site within exon 3 and producing a 44-base truncated mRNA, and the other was a point mutation at the consensus sequence of the splice donor site of intron 2, causing exon 2 skipping.

DNA deletion confined to the iduronate‐2‐sulfatase promoter abolishes IDS gene expression

DNA deletion confined to the iduronate‐2‐sulfatase promoter abolishes IDS gene expression