Molecular and phenotypic variation in patients with severe Hunter syndrome
Molecular and phenotypic variation in patients with severe Hunter syndromeKirsten M. Timms*, Marie-Louise Bondeson1, M. Ali Ansari-Lari, Kristina Lagerstedt1, Donna M. Muzny, Shannon P. Dugan-Rocha, David L. Nelson, Ulf Pettersson1 and Richard A. Gibbs
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA and 1Department of Medical Genetics, Biomedical Centre, University of Uppsala, Uppsala, Sweden
Received November 11, 1996;Revised and Accepted December 6, 1996
Severe Hunter syndrome is a fatal X-linked lysosomal storage disorder caused by iduronate-2-sulphatase (IDS) deficiency. Patients with complete deletion of the IDS locus often have atypical phenotypes including ptosis, obstructive sleep apnoea, and the occurrence of seizures. We have used genomic DNA sequencing to identify several new genes in the IDS region. DNA deletion patients with atypical symptoms have been analysed to determine whether these atypical symptoms could be due to involvement of these other loci. The occurrence of seizures in two individuals correlated with a deletion extending proximal of IDS, up to and including part of the FMR2 locus. Other (non-seizure) symptoms were associated with distal deletions. In addition, a group of patients with no variant symptoms, and a characteristic rearrangement involving a recombination between the IDS gene and an adjacent IDS pseudogene (IDS[psi]), showed normal expression of loci distal to IDS. Together, these results identify FMR2 as a candidate gene for seizures, when mutated along with IDS.
Deficiency of the enzyme iduronate-2-sulphatase (IDS, EC3.1.6.13) results in Hunter syndrome (mucopolysaccharidosis type II, MPS-II), an X-linked recessive disorder. In the severe form of this disease abnormal storage of dermatan and heparan sulphate results in progressive mental retardation, physical disability and death before age 15 (1 ). The incidence of Hunter syndrome is ~1 in 132 000 live male births (2 ). The majority of cases are caused by point mutations and small deletions or insertions, with ~20% of cases being the result of major structural alterations, including large deletions and rearrangements (3 ). In between 6 and 8% (4 -8 ) of cases the disease is the result of complete deletion of the IDS gene. Patients with complete deletion of IDS have been described with additional symptoms not commonly associated with Hunter syndrome, including the occurrence of seizures (4 ,9 ). These variant phenotypes may be due to involvement of loci adjacent to the IDS gene (4 ,9 ).
In ~13% of patients with Hunter syndrome there has been an homologous recombination event between the IDS gene and an adjacent unexpressed IDS pseudogene (IDS[psi]), resulting in a disruption of the IDS gene in intron 7 and an inversion of the intervening DNA (10 ). These individuals have the severe MPS-II phenotype, with no variant symptoms. IDS gene transcripts in these patients contain novel sequences at their 3' end (11 ), suggesting that neighbouring loci may also be affected by the lesion in these individuals.
Two groups of patients with severe Hunter syndrome, with and without atypical symptoms, were analysed to determine whether the mutations in these individuals involved genes located in close proximity to the IDS gene. Mapping of deletion breakpoints demonstrated a correlation between the observed genotype and the atypical symptoms found in these patients. In individuals where the causative mutation was homologous recombination between IDS and IDS[psi], the mutation had no major effect on expression of other loci in this region. These phenotype-genotype correlations define a critical region and candidate gene for the seizures observed in some individuals with deletion of the IDS locus.
Sequencing of the IDS region has revealed that its organisation is very complex and that it is gene-rich (Fig. 1 ). Analysis of a 206 kb sequence contig (GenBank accession no. U66082) from the IDS region identified three genes (X, W and Y) in addition to IDS and IDS[psi]. Sequence from a second 73 kb contig (GenBank accession no. U66083) revealed that 27.5 kb of contiguous sequence, including all three newly identified genes, have been locally duplicated on Xq28. RT-PCR, cDNA isolation and sequencing, and computer analysis (33 ,35 ) were used to characterise these previously unknown genes. No similarity was found with known genes in the computer databases, and no potential functional roles have been assigned. Several factors including detectable levels of expression, tissue specific expression in the case of gene W (data not shown), and expression of both copies of duplicated genes would suggest that all these genes are functional.
Four patients with deletions spanning the IDS gene were analysed. PCRs or Southern hybridisations were carried out using STS markers and genes mapped from the boundary of Xq27.3-28. Earlier mapping studies carried out with these patients also gave some information on markers located proximal of and/or distal to our sequence contigs (7 ,12 ,13 ). All four of the patients were found to have deletions spanning IDS, IDS[psi], W and X. These data are summarised in Figure 2 .
To identify the novel 3' sequences observed in the IDS transcript from MPS-II patients with a rearrangement between IDS and IDS[psi], we carried out RT-PCR using primers in IDS and gene X (Fig. 3 ). Three products were isolated and sequenced (GenBank accession nos U66053, U66054 and U66055). The larger product contained exons from IDS, X and a previously unidentified exon located near the 5' end of gene W, but from the complementary strand. The smaller product contained only IDS and gene X exons. Transcripts containing this novel exon have also been detected in normal individuals where it is expressed from the other duplicated site as part of gene Z' (data not shown).
Large scale genomic sequencing from around the IDS locus has enabled us to carry out a comparison of the genotypes observed in patients suffering from variations of Hunter syndrome. STS markers from Xq28 and PCR primers from genes found within our sequence contigs were used to map the deletion breakpoints in four Hunter syndrome patients with large scale deletions spanning the IDS gene. All four individuals have symptoms which are considered atypical of Hunter syndrome, in addition to the severe form of the disorder. These atypical symptoms include ptosis, obstructive sleep apnoea, Hurler-like symptoms and seizures. In two of the patients the occurrence of seizures correlated with a deletion extending proximal of the IDS gene, towards, and probably including, part of the FMR2 locus. The phenotypes observed in other individuals (ptosis, Hurler-like symptoms) correlate with a larger distal deletion spanning the loci that we have identified in this region, including genes X and W.
In a second group of individuals the IDS locus is disrupted by homologous recombination with the neighbouring IDS pseudogene. IDS transcripts in these individuals contain exons from one other locus (X) and a novel exon located distal of gene W, in addition to IDS. These exons are normally found distal to the IDS gene in Xq28. RT-PCR based approaches were utilised to show that normal gene X and W transcripts are still expressed in all the patients examined, indicating that the recombination event has not disrupted normal expression of these two genes. The apparently normal functioning of these genes in a group of patients without any phenotypic variation, in addition to the close proximity of these loci to the IDS gene, indicates that they should be considered candidate genes for the unusual symptoms (ptosis, Hurler-like symptoms, but not seizures) associated with deletions extending distal of IDS in the deletion patients.
This homologous recombination event between the IDS gene and IDS[psi] is the most commonly observed mutation, accounting for ~13% of all cases of Hunter syndrome (10 ). A mechanistically similar homologous recombination event results in ~50% of cases of severe haemophilia A, where recombination occurs between the A gene located in exon 22 of the factor VIII gene and one of two A genes located ~500 kb distal (21 ,22 ). Both Hunter syndrome and haemophilia A are characterised by heterogeneous phenotypes ranging from mild to severe, and in both disorders the most common cause of the severe form of the disease is homologous recombination between duplicated, or partially duplicated, genes. A second rearrangement between the IDS gene and IDS[psi] has recently been described (23 ). In this patient interchromosomal recombination between the IDS gene and IDS[psi] is thought to have occurred during meiosis, resulting in two copies of the pseudogene on one chromosome and the loss of ~1.4 kb of the active IDS gene. The identification of an increasing number of cases of recombination between the IDS gene and its closely related pseudogene indicates that local duplications of genomic material are responsible for instability resulting in a large proportion of Hunter syndrome cases (10 ).
Expansion of the triplet repeat element at the FRAXE locus results in mild mental retardation in the affected individuals (15 -17 ). There have been no reports of the occurrence of seizures associated with this disorder, and it is difficult to predict how the loss of both the IDS locus and part of the FMR2 gene should manifest at the phenotypic level. It is thought that the distance between IDS and the FRAXE triplet repeat is ~750 kb (19 ). However, the FMR2 gene is very large and thought to span as much as 650 kb (18 ,19 ). It remains possible that there are unidentified loci located between our IDS sequence contig and the 3' end of the FMR2 gene.
In summary, analysis of mutations in patients with severe Hunter syndrome, with and without variant phenotypes, has identified a number of candidate genes for the observed atypical symptoms. Continued analysis of this region, and of additional MPS-II deletion patients, will be important for further elucidation of the genetic differences between subtle variants of Hunter syndrome.
All cosmids were derived from the human X chromosome flow-sorted library prepared at Lawrence Livermore National Laboratory (LLNL). The cosmids characterised in this study are identified using the LLNL nomenclature (e.g., U112G10); this name can be used to identify the original clones from the LLNL X-chromosome cosmid library. Cosmid DNA was isolated and purified using Qiagen Plasmid Maxi Kit columns and then further purified by caesium chloride banding. Cosmid walks to isolate minimally overlapping cosmids were performed as described previously (24 ).
M13 shotgun libraries were prepared from physically sheared cosmid DNA using one of two adapter-based strategies (25 ,26 ). Sequence template was prepared and sequenced as described (27 ,28 ). Reverse sequencing template was prepared using a modified asymmetric PCR protocol (29 ). Dye primer sequencing using sequence-specific primers and M13 templates was performed for gap closure. Reagent kits were provided by Applied Biosystems and reactions were performed on PEC 9600 thermocyclers. Sequence reactions were electrophoresed on ABI 373 or 377 DNA sequencers.
Sequence reads were edited using the software SEQPREP developed by the Molecular Biology Computational Resource Centre at Baylor College of Medicine. After editing sequence was assembled using the Staden XDAP and XGAP software (30 ). Gap closure was performed as described previously (29 ,31 ), and by utilising an M13 nested deletion protocol for gaps where there were no available clones in the shotgun sequencing library. Clones which spanned these gaps were selected and then a nested deletion protocol carried out as described previously (32 ).
Computer analysis of sequence was via a suite of programs assembled by the Baylor College of Medicine genome informatics group. They include BLAST (33 ), GRAIL2 (34 ), BEAUTY (35 ), CENSOR (36 ), FASTA (37 ) and GCG (sequence analysis software package, v. 8, Genetics Computer Group, Madison, WI). Most of these programs can be accessed from the Baylor College of Medicine genome informatics group World Wide Web page at http://kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.html
Standard procedures for Southern hybridisation were utilised (38 ), except that transfers were carried out using alkaline conditions. In addition, hybridisation buffer contained 1 M NaHPO4 (pH 7.0) and 7% SDS, and wash buffer contained 0.08-0.2 M NaHPO4 (pH 7.0) and 1% SDS. Probes were generated using Rediprime kits supplied by Amersham, and purified using NICK columns from Pharmacia. Oligodeoxynucleotide probes were labelled using calf alkaline phosphatase as described previously (38 ). The labelled oligodeoxynucleotide was purified using NICK (>20mer) or NAP-25 (<20mer) columns from Pharmacia.
Total RNA was isolated from human fibroblasts, HT1080 human fibrosarcoma cells or white blood cells using guanidinium thiocyanate extraction and caesium chloride centrifugation as described previously (38 ). Single-strand DNA was synthesised using oligo (dT15) for use in RT-PCR or modified LA-PCR (20 ) reactions. For 3'-RACE (39 ), oligo (dT15) containing a universal tail was used. In some cases a nested PCR approach was used when performing LA-PCR or 3'-RACE. Primers used in RT-PCR, LA-PCR and 3'-RACE are shown in Table 1 .
PCR was performed using AmpliTaq or AmpliTaq Gold DNA polymerase in a PEC 4800 thermocycler. Each 50 [mu]l reaction contained 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 25 mM each dNTP, 20 pmol each primer and 2.5 U AmpliTaq. DMSO (10% vol/vol) was included in reactions where template DNA had high GC content. For template, ~10 ng of cosmid DNA, 100 ng of genomic DNA or 2 [mu]l of reverse transcriptase reaction was used. PCR conditions were 94oC/3 min, followed by 30 reaction cycles of 94oC/30 s, 55oC/30 s, 68oC/2 min. After completion of the cycle, samples were held at 68oC/7 min and then cooled to 4oC. Primers used in PCRs are shown in Table 1 .
Library plating and screening was carried out as described previously (38 ). Rediprime kits (Amersham) were used to label the probes, which were purified using Pharmacia NICK columns. Single positive plaques were identified after tertiary screening and phagemid was isolated using standard procedures (38 ).
Products from RT-PCR, LA-PCR, 3'-RACE and PCR were cloned into pGEM-T vector supplied by Promega. Dye-primer universal and reverse sequence reads were performed on each clone. Complete sequence was obtained using sequence specific primers and dye-terminator chemistry. For cDNAs >1.5 kb in length a concatenated cDNA library construction procedure was used to generate an M13 shotgun library of the original cDNAs (40 ). Shotgun sequencing using dye-primer chemistry was used to sequence these clones.
We thank C. C. Lee and P. Sideras for cDNA libraries; B.-M. Carlberg for excellent technical assistance; J. J. Hopwood, W. J. Kleijer, and T. Tönnesen for providing patient samples; the Baylor College of Medicine Human Genome Sequencing Centre personnel (W. Lee, J. Ding, T. Malley, S. Odom, W. Liu and S. Brown) for genomic sequencing; J. Lu and Y. Shen for sequence assembly. This work was supported in part by grants RO1 HG00823 and P30 HG00210 from the National Centre for Human Genome Research, and from the Medical Research Council of Sweden, the Beijer Foundation, and Marcus Bórgström's Foundation.
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Recent sequence data generated in our laboratory (cosmid Gu-3C3) confirms that marker DXS295 is within one of the introns of the coding region of FMR2.
*To whom correspondence should be addressed
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