Clinical and biochemical studies in mucopolysaccharidosis type II carriers

Transcription

1 J Inherit Metab Dis (2009) 32: DOI /s ORIGINAL ARTICLE Clinical and biochemical studies in mucopolysaccharidosis type II carriers I. V. D. Schwartz & L. L. C. Pinto & G. Breda & L. Lima & M. G. Ribeiro & J. G. Mota & A. X. Acosta & P. Correia & D. D. G. Horovitz & C. G. G. Porciuncula & E. Lipinski-Figueiredo & A. C. Fett-Conte & R. P. Oliveira Sobrinho & D. Y. J. Norato & A. C. Paula & C. A. Kim & A. R. Duarte & R. Boy & S. Leistner-Segal & M. G. Burin & R. Giugliani Received: 24 June 2008 /Submitted in revised form: 18 August 2009 /Accepted: 25 August 2009 /Published online: 10 October 2009 # SSIEM and Springer 2009 Summary The aim of the study was to characterize clinically and biochemically mucopolysaccharidosis type II (MPS II) heterozygotes. Fifty-two women at risk to be a carrier, with a mean age of 34.1 years (range years), were evaluated through pedigree analysis, medical history, physical examination, measurement of iduronate sulfatase (IDS) activities in Communicating editor: Ed Wraith Competing interests: None declared References to electronic databases. Mucopolysaccharidosis type II: OMIM Fabry disease: OMIM Iduronate sulfatase: EC I. V. D. Schwartz : R. Giugliani Department of Genetics, UFRGS, Porto Alegre, Brazil I. V. D. Schwartz (*) : L. L. C. Pinto : G. Breda : L. Lima : S. Leistner-Segal : M. G. Burin : R. Giugliani Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, Brazil ida.ez@terra.com.br L. L. C. Pinto : R. Giugliani Pediatrics Post Graduation Program, School of Medicine, UFRGS, Porto Alegre, Brazil M. G. Ribeiro Clinical Genetics Service, IPPMG, UFRJ, plasma and in leukocytes, quantification of glycosaminoglycans (GAGs) in urine, and analysis of the IDS gene. Eligibility criteria for the study also included being 16 years of age or older and being enrolled in a genetic counselling programme. The pedigree and DNA analyses allowed the identification of 40/52 carriers and 12/52 non-carriers. All women evaluated were clinically healthy, and their levels of urinary GAGs were within normal limits. Median plasma and leukocyte IDS activities found among carriers were significantly lower than the values found for noncarriers; there was, however, an overlap between carriers_ and non-carriers_ values. Our data suggests J. G. Mota Institute of Oncology of the South of Minas Gerais (ISMO), Pouso Alegre, Brazil A. X. Acosta Department of Pediatrics, School of Medicine of Bahia, UFBA, Salvador, Brazil P. Correia Post Graduation Program in Women and Children Health, Fernandes Figueira Institute, FIOCRUZ, D. D. G. Horovitz Department of Medical Genetics, Fernandes Figueira Institute, FIOCRUZ, C. G. G. Porciuncula : E. Lipinski-Figueiredo Clinical Genetics Service, University Hospital, UFAL, Maceió, Brazil

2 J Inherit Metab Dis (2009) 32: that MPS II carriers show lower plasma and leukocyte IDS activities but that this reduction is generally associated neither with changes in levels of urinary GAGs nor with the occurrence of clinical manifestations. Abbreviations a-gal A a-galactosidase A IDS iduronate-sulfatase GAGs glycosaminoglycans LSD lysosomal storage disorder MPS II mucopolysaccharidosis type II Introduction Mucopolysaccharidosis type II (OMIM ) (MPS II) is an X-linked lysosomal storage disease (LSD), caused by a deficiency of iduronate sulfatase (IDS; EC ) enzyme activity. IDS is one of the enzymes responsible for the degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. In MPS II, these partially degraded GAGs accumulate inside lysosomes and are excreted in high amounts in the urine. Estimates of the incidence of MPS II in European and Anglo-Saxon countries range from 1: to 1: male newborns (Applegarth et al. 2000; Baehner et al. 2005; Machill et al. 1991; Meikle et al. 1999; Nelson 1997; Nelson et al. 2003; Poorthuis et al. 1999; Young and Harper 1982). MPS II seems to be more frequent in Israel (estimated incidence: 1: male newborns) (Schaap and Bach 1980). It is the most common type of MPS in Taiwan and northern Asia (Lin et al. 2006, 2009). The severe type of MPS II is characterized by coarse facial features, short stature, skeletal deformities, joint A. C. Fett-Conte Department of Molecular Biology, FAMERP, São José do Rio Preto, Brazil R. P. Oliveira Sobrinho : D. Y. J. Norato Department of Medical Genetics, UNICAMP, Campinas, Brazil A. C. Paula : C. A. Kim Genetics Unit, IC-HC-USP, São Paulo, Brazil A. R. Duarte Medical Genetics Service, IMIP, Recife, Brazil R. Boy Pediatrics Department, UERJ, stiffness and mental retardation, while the attenuated form is characterized by preservation of intelligence, survival into adulthood, somatic features similar to those seen in severely affected patients (although usually in a milder degree) and hypoacusia (Neufeld and Muenzer 2001; Schwartz et al. 2007). The occurrence of a pebbly, ivory-coloured skin lesion is almost unique to MPS II (Neufeld and Muenzer 2001). Full expression of MPS II in females is very rare (to our knowledge, about 10 cases have been reported to date) and is secondary to the presence of either pathogenic mutations in both alleles of the IDS gene (Cudry et al. 2000) or non-random inactivation of the X chromosome in heterozygotes or in carriers of structural X-chromosome abnormality (Broadhead et al. 1986; Clarke et al. 1990; Cudry et al. 2000; Mossman et al. 1983; Neufeld et al. 1977; Sukegawa et al. 1997, 1998; Tuschl et al. 2005; Winchester et al. 1992). Although the majority of MPS II carriers are considered asymptomatic (Clarke et al. 1993; Whitley 1993; Winchester et al. 1992), we have not found in the literature studies aiming to identify the presence of subtle clinical manifestations of MPS II in these women and to characterize their excretion of urinary GAGs. We present here the results of a clinical and biochemical investigation of 52 women who were at risk to be MPS II carriers (Fpossible MPS II carriers_). Patients and methods This study was approved by the local Institutional Review Board and by the Brazilian National Research Ethics Committee, and any procedure related to it was performed only after the signing of an Informed Consent Form. Possible MPS II carriers (mothers and sisters of patients with a confirmed diagnosis of MPS II, as well as their maternal grandmothers, aunts, and cousins), aged 16 years or older, were invited to take part in this study, provided they were engaged in a genetic counselling programme. Study procedures included: pedigree analysis, investigation of medical history, physical examination, and collection of blood samples (for measurement of plasma and leukocyte IDS activities and analysis of the IDS gene) and of 24-hour urine samples (for GAG quantification). The measurement of plasma and leukocyte IDS activities was performed according to the fluorimetric method described by Voznyi and colleagues (2001); in our laboratory, reference values for the assay in plasma are nmol/4 h per ml (mean 260, SD 92) (n=49) and for the assay in leukocytes nmol/4 h per mg protein (mean 61.6, SD 28.1) (n=20). The quantifica-

3 734 J Inherit Metab Dis (2009) 32: tion of GAGs in an aliquot of a 24-hour urine sample was performed according to de Jong and colleagues (1992); at our laboratory, the upper limit for normality above 16 years of age is 5.1 mg/mmol creatinine (n=20, of whom 10 were women). After the mutation present in the proband was identified (data not shown), the IDS gene of the possible carrier was assessed for the presence or absence of the specific mutation (data not shown). According to family pedigree analysis, we classified as obligate heterozygotes the women who had either two or more children affected by MPS II or one affected child and another family member also affected by this disease. Carriers were compared with non-carriers in relation to age, plasma and leukocyte IDS activities, levels of urinary GAGs and clinical features. The Student t-test and the Mann Whitney test were used, respectively, for the analysis of continuous variables with normal and asymmetric distribution. Analysis of the categories of variables was conducted by c 2 test or Fisher_s exact test. Correlation between the continuous variables was made by Spearman_s correlation coefficients and the partial correlation coefficients (the latter for adjustments for age). The level of statistical significance adopted was 5% (p=0.05). Results Fifty-two possible carriers were included in the study (39 mothers, 6 sisters, 5 aunts, 1 grandmother, and 1 cousin). Mean age at evaluation was 34.1 years (range 16 57). Analysis of the IDS gene was performed in 46/ 52 possible carriers; it confirmed the diagnosis of carrier status in 34/46 and excluded the diagnosis in 12/46 women. Another 6 women were identified as obligate heterozygotes through the analysis of the pedigree. Carriers (n=40/52) and non-carriers (n=12/ 52) did not differ in relation to age (Table 1). The distribution of the plasma IDS activity in normal controls (n=49) and in MPS II patients diagnosed in our laboratory (n=112), as well as in carriers (n=40) and non-carriers (n=12) in this study, is shown in Fig. 1. Median plasma IDS activity found in carriers was nmol/4 h per ml (Table 1); this value differs from those found for normal controls (p<0.001) and for non-carriers (p=0.001) (Table 1). Median plasma IDS activity did not differ between noncarriers and normal controls (p=0.07). Values found for carriers and normal controls/non-carriers overlap clearly (Fig. 1). The distribution of the leukocyte IDS activity in normal controls (n=20) and in MPS II patients diagnosed in our laboratory (n=22/112), as well as in carriers (n=28/40) and non-carriers (n=6/12), is shown in Fig. 2. Median leukocyte IDS activity found in carriers was 24.5 nmol/4 h per mg protein (Table 1); this value differs from the values found for normal controls (p<0.001) and for non-carriers (p=0.017) (Table 1). The median leukocyte IDS activity found in non-carriers does not differ from the value found for normal controls in our laboratory (p=0.78). There was also a wide overlap between values found for carriers and normal controls/non-carriers (Fig. 2). None of the women studied exhibited increased excretion of urinary GAGs (Table 1). In non-carriers, age showed a significant positive correlation with the levels of urinary GAGs (r=0.812, p=0.002). Plasma IDS activity and levels of urinary GAGs also showed a significant negative correlation adjusted for age in non-carriers (r=j0.776; p=0.008); however, a nonsignificant correlation in carriers was observed Table 1 Age, plasma and leukocyte IDS activities and levels of urinary glycosaminoglycans found in MPS II carriers and non-carriers Carriers Non-carriers p Age (years) n 40/40 12/ Mean T SD 35.5T T10.7 Plasma IDS activity (nmol/4 h per ml) n 40/40 12/ Median (interquartile range 25th 75th) a ( ) ( ) Leukocyte IDS activity (nmol/4 h per mg protein) n 28/40 6/ Median (interquartile range 25th 75th) 24.5 ( ) 49 ( ) Urinary glycosaminoglycans (mg/mmol creatinine) n 28/40 11/ Mean T SD 2.71T T1.12 a When median is shown, the variable is asymmetric, and the Mann Whitney test was used to calculate p (see Patients and Methods).

4 J Inherit Metab Dis (2009) 32: Plasma IDS (nml/4h per ml) Normal controls Carriers Non-carriers Patients Fig. 1 Activity of iduronate sulfatase (IDS) in plasma (nmol/4 h per ml) (normal controls, n=49; carriers, n=40; non-carriers, n=12; MPS II patients, n=112). Reference values: normal controls nmol/4 h per ml (r=j0.239; p=0.272). No significant correlation was found between plasma IDS activity and age of carriers (r=j0.163; p=0.348) or non-carriers (r=j0.413; p=0.182), nor between the age of carriers and the level of urinary GAGs (r=0.221, p=0.3). No significant correlation adjusted for age was found between the levels of leukocyte IDS activity and the levels of urinary GAGs in carriers (r=j0.243; p=0.33) or noncarriers (r=0.373; p=0.536), nor between those levels and the age of carriers (r=j0.082; p=0.698) or noncarriers (r=j0.486; p=0.329). None of the women studied presented symptomatology compatible with MPS II (Table 2). In 13/40 carriers and in 6/12 non-carriers some abnormal findings in the review of systems, past medical history, and/or physical examination were present but all were non-specific and showed no significant differences between the two groups (Table 2). Moreover, no significant difference was found between the age of women presenting some alteration in anamnesis/physical examination and the age of women whose anamnesis/physical examination was normal (p=0.363). Discussion We investigated 52 women at risk of being carriers for MPS II who were eventually identified as carriers (n=40) and non-carriers (n=12). Of the non-carriers, 6 were mothers of isolated cases. Although the possibility of germline mosaicism must be considered, these women were considered non-carriers for the purpose of this study. We should point out that the occurrence of germline and somatic mosaicism in MPS II carriers has been reported only once in the literature and that there is no report of isolated gonadal mosaicism for this disease (Froissart et al. 1997). Our study confirms that the heterozygotes for MPS II are in general well and present neither subtle manifestations of MPS II nor increased excretion of GAGs. Until the advent of molecular techniques, these individuals could be identified by pedigree analysis and by methods based on the phenomenon of lyonization (cloning analysis of fibroblasts; the method based on preferential survival of the enzymedeficient cells in contrast to the normal cells by repeated freezing and subcultivation; hair root analysis; use of fructose 1-phosphate to inhibit the uptake of IDS by the deficient cultured fibroblasts; measurement of the IDS activity in serum and lymphocytes) (Archer et al. 1981, 1983; Capobianchi and Romeo 1976; Danes and Bearn 1965; Donnelly and Di Ferrante 1975; Nwokoro and Neufeld 1979; Tonnesen, 1984; Tonnesen et al. 1982; Zlogotora and Bach, 1984). In our study, the measurement of plasma and/or leukocyte IDS activities did not discriminate adequately between carriers and non-carriers. Other authors have obtained better results using assays different from ours in serum only (Archer et al. 1981) or in serum and lymphocytes (Zlogotora and Bach 1984). It is expected that at least 5 10% of normal females will present extreme skewing of X inactivation. In heterozygotes for X-linked disorders, the degree of skewing is expected to be correlated with the degree to Leukocyte IDS (nmoles/4h per mg protein) Normal controls Carriers Non-carriers Patients Fig. 2 Activity of iduronate sulfatase (IDS) in leukocytes (nmol/ 4 h per mg protein) (normal controls, n=20; carriers, n=28; noncarriers, n=6; MPS II patients, n=22). Reference values: normal controls nmol/4 h per mg protein 2 3 4

5 736 J Inherit Metab Dis (2009) 32: Table 2 Abnormal findings in review of systems, past medical history and/or physical examination of MPS II carriers and non-carriers Carriers Noncarriers n=40 n=12 Women presenting at least one abnormal 13 6 finding in anamnesis/physical examination a Prematurity 2 0 Seizures during childhood 1 0 Myopia and/or astigmatism 5 1 Hypothyroidism 1 1 Sleep obstructive apnoea syndrome (mild) 0 1 Hypoacusia 0 1 Tumors b 2 0 Prolapse of mitral valve 1 0 Interventricular communication 1 0 Short stature 0 0 Macrocephaly 0 0 Palpebral ptosis 0 0 Facial asymmetry 0 1 Joint contractures 0 0 Pebbly skin lesion 0 0 Café au lait spots 2 1 Hepatosplenomegaly 0 0 Kyphosis 1 0 Stiff joints 0 0 Developmental delay (mild) 1 0 Learning problems 1 1 Depression 1 1 Psychiatric disorder 0 1 a p=0.316; b non-malignancies. which females present symptoms of the X-linked disease (Willard 2001). Therefore, partial expression of MPS II should not be as rare in MPS II carriers as it appears to be. The absence of clinical manifestations and the presence of normal urinary levels of the partially degraded substrate are findings that differentiate MPS II carriers from carriers of the other X- linked LSDs, i.e. Fabry disease (OMIM ), which is caused by the deficient activity of enzyme a- galactosidase A (a-gal A). When compared with the values found for hemizygotes and normal controls, the heterozygotes for Fabry disease usually exhibit intermediate levels of activity of a-gal A and of plasma and urine ceramide trihexoside (substrate of the deficient enzyme) (Desnick et al. 1973; Hozumi et al. 1990). Heterozygotes for Fabry disease may present all the clinical manifestations commonly described in hemizygotes, although in most cases less severely. Approximately 30% show few isolated angiokeratomas; fewer than 10% show acroparaesthesias; and about 70% show corneal dystrophy (Desnick et al. 2001). However, recent reports suggest that the prevalence of symptoms in females is much higher in Fabry disease than has been described previously (Deegan et al. 2006). Cardiological and cerebrovascular abnormalities are common, especially in older women (Deegan et al. 2006; Kampmann et al. 2002a,b; MacDermot et al. 2001; Wang et al. 2007). There is no consensus in the literature on whether the clinical heterogeneity of heterozygotes for Fabry disease is or is not secondary to the phenomenon of X inactivation (MacDermot et al. 2001; Maier et al. 2006; Whybra et al. 2001a,b). According to Dobyns and colleagues (2004), MPS II would belong to the group of X-linked disorders whose gene product is non-autonomous, that is, disorders in which the enzyme involved would be exchanged between cells and, therefore, taken up by cells in which the X chromosome without the mutation is inactivated. However, recent studies suggest that the transfer of the a-gal A to the mutant cells is not so efficient as to substitute the enzymatic deficiency (Mehta et al. 2004). Thus, differences between MPS II and Fabry carriers in relation to the expression of the disease could be the result of different uptake rates of IDS and a-gal A by cells that do not produce them, e.g. due to differences in enzymes properties such as water solubility. However, we cannot rule out that other factors are in play; for instance, in some cells of MPS II carriers, such as chondrocytes or hepatocytes, the X chromosome carrying the mutant allele could be preferentially inactivated or reduce the viability of cells. Additional studies should be undertaken to clarify the reasons for the different clinical expressions of X-linked diseases in women. It is important to point out that if we had analysed MPS II carriers by means of more sensitive clinical investigation (such as radiography or abdominal CT/MRI scans), or if the mean age of our sample had been higher, perhaps we would have found symptomatic women. It is known that the proportion of females showing a highly skewed pattern of X inactivation increases markedly with age (Willard 2001). Archer and colleagues (1983), for example, have found that IDS levels in normal serum increase significantly with age from the age of 18 years. However, we did not find a significant correlation between plasma IDS and age of carriers or between leukocyte IDS and age of carriers, and these findings do not support the occurrence of age-related modification in the expression of the IDS gene. This interpretation is complicated by the fact that we have studied the enzyme activity only in plasma and leukocytes, not in the cells that are relevant to the progression of the disease.

6 J Inherit Metab Dis (2009) 32: Another interesting finding in our study was that there seems to be a positive correlation between age and levels of urinary GAGs and a negative correlation between levels of urinary GAGs and IDS plasma activity in non-carriers. Although we should consider these data with caution, owing to the small sample of non-carriers, the study by Gallegos-Arreola and colleagues (2000) found a non-significant positive trend (r=0.35, p=0.1) when correlating levels of urinary GAGs and age in healthy individuals of years of age. On the other hand, Larking and colleagues (1987) did find a highly significant increase in the levels of urinary GAG in women after menopause. In our study, however, only one of the non-carriers was postmenopausal. 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