Haematologica 2002; 87:(08)ECR30
[Medline] [prev] [index] [next]Haemophilia A and mucopolysaccharidosis I-H (Hurler syndrome): a case report
Majda Benedik-Dolnicar,1 Lana Strmecki,2 Eduard Paschke,3 Cordula Steglich,4 Olga Kranjc,5 Radovan Komel2
1Haemophilia Centre, Paediatric Clinic, University Clinical Centre, SI-1000 Ljubljana, Slovenia. 2Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia. 3Laboratory of Metabolic Diseases, Department of Paediatrics, University of Graz, A-8036 Graz, Austria. 4Institut fur Humangenetik, Universitatsklinikum Hamburg-Eppendorf, Butenfeld 42, D-22529 Hamburg, Germany. 5Department of Paediatrics, Maribor Teaching Hospital, SI- 2000 Maribor, Slovenia. Correspondence: Majda Benedik-Dolnicar, Haemophilia Centre, Pediatric Clinic, SI-1000 Ljubljana, Slovenia.Phone +386 1 2324298; fax +386 1 5229294). E-mail: Majda.Benedik-Dolnicar@mf.uni-lj.si
We report the case of a boy with combined severe hemophilia A and mucopolysaccharidosis I-H (Hurler syndrome). The underlying mutations for both disorders have been elucidated. Hemophilia A in the patient is caused by the proximal intrachromosomal inversion of FVIII (type A inversion) whereas mucopolysaccharidosis I-H is due to the IDUA gene stop mutations Q70X and W402X. Hemophilia A and mucopolysaccharidosis type I Hurler (MPS I-H) are both well characterized hereditary disorders. Hemophilia A is a recessive chromosome X-linked bleeding disorder affecting 1 in 5-10,000 males in all populations with approximately one-third of the occurrences due to spontaneous genetic mutations.1 Factor VIII (FVIII) is a plasma glycoprotein essential for normal hemostasis. Even though the disease is caused by a large number of distinct FVIII gene mutations, the intrachromosomal inversion defect is implicated in 40-48% of severe hemophilia A cases.2 Hemophilia A is classified according to the plasma concentration of factor VIII clotting activity (FVIII:C) as severe (FVIII:C <0.01 E/mL), moderate (FVIII:C 0.01-0.05 E/mL) or mild (FVIII:C 0.05-0.30 E/mL). Patients affected with the severe form of the disease suffer from spontaneous bleedings into joints and muscles although this bleeding can be prevented or arrested by the administration of factor VIII concentrate. A serious complication arising as a result of this therapy is the development of inhibitory antibodies directed against factor VIII.3,4 MPS I is a mucopolysaccharidosis leading to a wide spectrum of clinical phenotypes. The most severe form, Hurler syndrome (MPS I-H), is characterized by dysostosis multiplex, hepatospenomegaly, severe mental retardation and death within the first decade of life, while a mild phenotype, Scheie syndrome (MPS I-S), exhibits normal intelligence, few somatic features (joint stiffness, aortic valve disease and corneal clouding) and normal life expectancy. In addition numerous cases with intermediate severity ('Hurler-Scheie compounds') have been described. MPS I is inherited in the autosomal recessive mode. At the cellular level, excessive accumulation of heparan and dermatan sulphate within lysosomes due to the inactivity of the lysosomal enzyme alpha-iduronidase is observed. Numerous mutations in its structural gene iduronidase (IDUA) have been reported. In patients of central and southern European descent with MPS I-H, two common mutations, Q70X and W402X, cause about 40% of cases.5 The disease can be alleviated by allogenic bone marrow transplantation which restores normal activity of alpha-L-iduronidase.6-8 A novel protocol of enzyme replacement therapy is currently under evaluation.9
Materials and Methods. The patient. Five years ago a male child was born at full term to unrelated parents of Slovenian descent. The mother developed pre-eclampsia at 16 weeks gestation. At 26 weeks she was given dexamethasone prophylaxis to enhance fetal lung development due to her increased risk of pre-term delivery. During the last 3 months of pregnancy she developed facial and ankle edema. The child was delivered after 12 hours labour with normal weight, height and head circumference. Presentation was breech and no instruments were used. His Apgar score was 9/9 at delivery. Hypospadias and right inguinal hernia were found. The baby was discharged from hospital at day 5 post delivery with diagnoses of indirect hyperbilirubinemia secondary to a neck hematoma with a complication of left facial nerve paresis. The baby was re-hospitalized after 1 month for acute respiratory distress. On neurological examination evidence of flaccidity of the upper extremities, developmental delay with diminished motor reflexes and torticolis to the right was found. While hospitalized he developed another hematoma, this time occuring at a venepuncture site on the dorsum of the right hand, which was surgically removed (dimensions unknown). Coagulation assays were performed and showed an APTT value of 144 s (normal value (n.v.) 26-56 s) and severe hemophilia A was diagnosed. At 9 months the child was extremely hypotonic, had problems with deglutition, was macrocephalic, had a saddle nose, thoracic kyphosis and gingival hyperplasia. At ten months high response FVIII antibodies were detected. This was after he had received several FVIII concentrates (Octavi, Hemate P, Hemofil M) in a total dose of 14 000 IE administered over 7 days of replacement therapy. At 13 months a computerized tomography scan showed generalized cerebral atrophy. At 17 months he developed a large intracranial hemorrhage, and subsequently had another intracranial hemorrhage at 21 months with a simultaneous large right pulmonary hemorrhage. He died at 25 months of age. FVIII:C. Coagulation assays were performed using the modified APTT method (Activated Partial Thromboplastin Time Method, IL TEST KIT Cat.No. 97570-10, IL). FVIII:inhibitor. Factor VIII inhibitor antibodies were assayed using the Bethesda assay.10 Mucopolysaccharides. Mucopolysaccharides were assayed in the urine using the modified uronic carbazole reaction.11 The mucopolysaccharide fractions were characterized by high-resolution electrophoresis.12 Activity of alpha-L-iduronidase. The activity of alpha-L-iduronidase was measured in isolated leukocytes and cultured fibroblasts as described elsewhere.13 DNA extraction. Samples of peripheral blood were collected into test tubes containing EDTA. DNA was isolated with the standard salting out procedure.14 IDUA gene screening. Sequence alterations in the IDUA cDNA were screened by single strand conformation polymorphysm analysis.15 The presence of sequence alterations was further confirmed by DNA sequencing and restriction enzyme analysis. Mutation Q70X leads to the loss of an AvaII restriction site in exon 2, while mutation W402X is seen as gain of a MaeI restriction site in exon 9. Southern blot. Ten milligrams of DNA were digested with 60 units of BclI overnight at 50°C and electrophoresed on 0.7%, 1 cm thick agarose gels at 30V for 20 hours. The gel was depurinated for 10 min with 0.25M HCl, denatured in 0.5M NaOH, 1.5M NaCl for 20 min, neutralized in 0.5M TrisHCl pH 7.5, 3M NaCl for 30 min and blotted overnight onto Hybond N+ membrane (Amersham) using 10xSSC buffer. The membrane was prehybridized for 1 h and hybridized overnight at 42°C in 50% formamide, with the 0.9 kb EcoRI/SacI fragment from plasmid p482.6, radiolabeled with 32(P)a-dCTP using random hexamers (Amersham). The membrane was washed for 20 min in 2xSSC, 0.1% SDS at room temperature, then in 0.5xSSC, 0.05% SSC at 50°C for an additional 35 min and finally washed in 2xSSC. The membranes were autoradiographed at -70°C for 1-3 days.16
Results. The patient presented with severe hemophilia A with plasma concentration of factor VIII clotting activity, FVIII:C < 0.01E/mL. The highest titer of inhibitory antibodies ever developed was 95 BE. Southern blot assay could not be performed on the patient's DNA sample but analysis of a sample from his mother (HA269) detected a FVIII gene inversion. This assay revealed a normal BclI digest pattern of 21.5 kb, 16 kb, and 14 kb DNA fragments in addition to 20 kb, 16 kb, and 15.5 kb fragments. This shows the mother to be heterozygous for the proximal FVIII inversion (inversion type A) and thus a carrier of hemophilia A (Figure 1). The concentration of uronic acid in urine was 524.2 mg/mg creatinine (n.v. 15-55 mg uronic acid/mg creatinine). Dermatan sulphate-1 was present in 36%, heparan sulphate in 22%, dermatan sulphate-2 in 23% and chondroitin sulphate in 19% of the total glycosaminoglycans. The activity of alpha-L-iduronidase was not detectable in leukocytes (n.v. 0.215-0.448 mU/min/mg) or cultured fibroblasts (n.v. 1.4-15.45 mU/min/mg) while beta-hexosaminidase and iduronate-2-S sulphatase were within normal values. Analysis of the IDUA gene by SSCP and subsequent DNA sequencing of exons 2 and 9 revealed the patient to be a compound heterozygote for the stop mutations Q70X and W402X. The Q70X mutation was inherited from the father and is already present in the paternal grandmother, whereas W402X was passed on from the mother and was also detected in the maternal grandfather (data not shown). The same mutations were confirmed by restriction analysis of PCR-derived amplicons (Figure 2).
Discussion. The predicted frequencies for hereditary disorders calculated in the western European population are 150:10000 for autosomal dominant, 75:10000 for autosomal recessive, and 15:10000 for chromosome-X linked disorders. These numbers can be significantly higher within isolated populations and ethnic groups, especially for the autosomal recessive traits. Mutation rates differ between the sexes and rise exponentially with age.17 Our patient was a sporadic case of severe hemophilia A. As severe disease is implicated by the presence of an intrachromosomal inversion event in approximately 50% of cases, we performed Southern analysis to elucidate the underlying FVIII mutation. In Slovenia the FVIII inversion is present in 48% (19/39) of severe hemophilia A cases studied.18 We were able to show the presence of the proximal inversion of FVIII (inversion type A) by Southern blot analysis in the DNA sample of the patient's mother (HA269), showing her to be heterozygous for the defect (Figure 1). We were not able to detect any bands in the patient's DNA, presumably due to low DNA concentration of the sample. We deduced that the patient must have inherited this FVIII change from his carrier mother. It has been shown that in 83% of sporadic cases of hemophilia A the mother is the carrier of the mutation.19 A serious complication of FVIII replacement therapy in severe hemophilia A is the development of inhibitory antibodies directed against it, which occurs in 20-35% of patients with severe disease.20 The highest titer of inhibitory antibodies ever developed in our patient was 95 BE. The amount of factor eliciting an immune response varies considerably between patients. In some cases antibodies manifest early, while in other cases antibodies only arise after prolonged replacement therapy. It has been proposed that the presence of antibodies in severe hemophiliacs was correlated with the presence of the FVIII inversion.21 HLA-haplotype correlation studies performed so far have been unable to show any real immunological basis for inhibitor formation.22,23 The prevalence of inhibitors in patients with mild or moderate severity hemophilia A has been estimated to be between 3% and 13%.20 The annual incidence of inhibitors reported in the UK, was 3.5 per 1000 patients registered with severe hemophilia A and 0.84 per 1,000 patients registered with mild and moderately severe hemophilia A.24 It is assumed that tolerance in these patients is due to at least some form of endogenous FVIII being present and the infrequent use of FVIII concentrates.21 The manifestation of antibodies is probably linked to a number of factors, both genetic (HLA-haplotype) and non-genetic (type of therapy and FVIII concentrate used, immunocompromised status). This has been demonstrated by studies on homozygous twins with hemophilia A: two separate cases22 reported that antibodies were only detected in one of the twins. The patient also showed severe symptoms of MPS I-H which is the most severe form of mucopolysaccharidoses I.15 Screening of the IDUA gene as well as restriction analysis revealed that the patient carried stop mutations Q70X and W402X. A combined case of these two hereditary disorders has not been reported to date. Amongst the populations studied the incidence of MPS I-H is variable. It has been calculated to be ~1:76000 for Northern Ireland,25 ~1:160000 in Germany,26 ~1:84000 in the Netherlands27 and Austria.5 A combined incidence of MPS I-H and MPS I-S was reported to be ~1:111000 for Australia.28 MPS I-H combined with hemophilia A would statistically occur rarely, with an approximate incidence of 1:107. A combination of hereditary disorders can occur as a contiguous gene syndrome or as two separate unlinked genetic disorders. Cases of combined chromosome X-linked disorders such as Duchenne muscular dystrophy and hemophilia A29 in Slovenia (unpublished results), or retinitis pigmentosa and hemophilia A30 have been reported. Pulmonary valvular stenosis and hemophilia A linked to distinct chromosomes has also been described in three unrelated patients.31-33References