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Changes in hair morphology as a biomarker in gene expression-targeted isoflavone therapy for Sanfilippo disease
Gene 504 (2012) 292–295
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Changes in hair morphology as a biomarker in gene expression-targeted isoflavone therapy for Sanfilippo disease Magdalena Narajczyk a,⁎, Anna Tylki-Szymańska b, Grzegorz Węgrzyn c a b c
Laboratory of Electron Microscopy, University of Gdańsk, Kładki 24, 80–822 Gdańsk, Poland The Children's Memorial Health Institute, Al. Dzieci Polskich 20, 04–736 Warsaw, Poland Department of Molecular Biology, University of Gdańsk, Kładki 24, 80–822 Gdańsk, Poland
a r t i c l e
i n f o
Article history: Accepted 7 May 2012 Available online 22 May 2012 Keywords: Sanfilippo disease Mucopolysaccharidosis Gene expression-targeted isoflavone therapy Scanning electron microscopy Hair morphology
a b s t r a c t Lack of unequivocal markers for assessment of therapeutic effects of treatment procedures is a common problem, but it is especially pronounced in genetic diseases. One of them is Sanfilippo disease (mucopolysaccharidosis type III, or MPS III). Changes in hair morphology have already been reported as one of characteristic and easily detected symptoms of this disease and other mucopolysaccharidoses. This feature has been used previously to monitor efficacy of gene expression-targeted isoflavone therapy (GET IT) in pilot clinical studies performed with low number of patients. Here, we studied changes in hair morphology, observed by using scanning electron microscopy, in 35 patients subjected to GET IT for one year at doses of 5 and 15 mg/kg/day. We have observed statistically significant correction of hair morphology in both groups of patients, corroborating results of earlier studies with low number of patients that suggested easily observable improvement in this parameter during different therapies of MPS. Since the improvement was evident in both previous reports and this study, we propose that analysis of hair morphology may be considered as a non-invasive method in monitoring effects of treatment on somatic symptoms of Sanfilippo disease patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Treatment of genetic diseases is an extremely difficult task. In fact, although there are 6000 or so disorders caused by defects in single genes, specific therapies have been developed for only a very small fraction of them (Barranger and Cabrera-Salazar, 2007; Swarts, 2009). Another serious problem with such therapies is assessment of their efficacy. All genetic diseases are relatively rare, and symptoms developing in persons suffering from the same disease can vary significantly from one patient to another (Swarts, 2009). Therefore, it is often difficult to find specific parameters that should be monitored in order to assess efficacy of a therapy under study. An example of diseases that cause problems described above is mucopolysaccharidosis (MPS), and especially its type III, called Sanfilippo disease. In this disease, like in other MPS types, glycosaminoglycan (GAG) accumulation occurs in lysosomes, which is caused by dysfunction of genes coding for specific enzymes involved in degradation of these compounds (Neufeld and Muenzer, 2001). Lysosomal storage of heparan sulfate (one of GAGs) causes specific symptoms which consist of relatively mild somatic dysfunctions and severe neurological and behavioral changes (Valstar et al., 2008). Due to progressive nature of Sanfilippo
Abbreviations: MPS, Mucopolysaccharidosis; GET IT, gene expression-targeted isoflavone therapy; GAG, glycosaminoglycan. ⁎ Corresponding author. Tel.: + 48 58 523 6304; fax: + 48 58 523 6424. E-mail address: [email protected] (M. Narajczyk). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.05.006
disease, variability of symptoms and low number of patients (Valstar et al., 2010, 2011), it is extremely difficult to assess effects of any therapy for this disease. Such problems were met in recent clinical studies, in which gene expression-targeted isoflavone therapy (GET IT) was tested. A few pilot clinical studies testing GET IT for Sanfilippo disease were performed to date, but results of these studies are equivocal since opposite conclusions were drawn by different authors. Namely, some studies suggested an improvement in selected biochemical and somatic parameters, as well as cognitive abilities of patients after 1 year GET IT (Malinova et al., 2011; Piotrowska et al., 2008, 2011), while results of another trials were interpreted as a lack of improvement in patients’ disability (de Ruijter et al., 2011a; Delgadillo et al., 2011). Apparently, these discrepancies might be ascribed, at least partially, to different tests employed by different authors to monitor effects of the treatment. Although there are many somatic symptoms occurring in patients suffering from Sanfilippo disease, their severity is generally lower than in other MPS types (Valstar et al., 2008). Nevertheless, a spectrum of each of them is very broad. Therefore, choosing appropriate biomarkers to be assessed in the course of a clinical trial is problematic. On the other hand, although behavioral changes and cognitive disabilities are common in Sanfilippo disease, they are very difficult to be measured quantitatively. Therefore, it is a need for establishing a biomarker that could be used for assessment of efficacy of any therapy for MPS type III. It was demonstrated previously that morphology of hair is changed in patients suffering from some types of MPS, including Sanfilippo disease (Kloska et al., 2005; Malinowska et al., 2008). These changes can
M. Narajczyk et al. / Gene 504 (2012) 292–295
be assessed using scanning electron microscopy. Enzyme replacement therapy of MPS type I resulted in correction of hair morphology, which suggested that this feature could be used as a biomarker in MPS treatment procedures (Kloska et al., 2005). In fact, hair morphology has been tested during pilot clinical studies with GET IT for Sanfilippo disease, however, either low number of patients was involved in those studies (Delgadillo et al., 2011; Malinova et al., 2011; Piotrowska et al., 2008) or patients were exposed to genistein treatment for a relatively short time (6 months) between microscopic analyses (de Ruijter et al., 2011a). Therefore, we assumed that studies on a larger group of patients, performed for at least 12 months, are required for testing whether hair morphology changes can be used as a biomarker in assessment of efficacy of a therapy for MPS type III. 2. Materials and methods 2.1. Patients 35 patients suffering from Sanfilippo A (17 patients), B (14 patients) and C (4 patients) subtypes were enrolled in this study. They were diagnosed on the basis of enzymatic and genetic tests, as described previously (Piotrowska et al., 2008, 2011).
293
extract, included in the product called Soyfem and provided by the manufacturer: Biofarm, Poznań, Poland; this product was described in details by Piotrowska et al., 2008). The doses corresponded to amount of genistein equal to either 5 or 15 mg/kg/day. No adverse effects were noted during the study. This treatment has been approved by the Independent Bioethics Committee of the Medical University of Gdańsk, Poland. 2.3. Hair morphology assessment Hair morphology was assessed by using scanning electron microscope according to Kloska et al. (2005). Hair samples were withdrawn at baseline and 12 months after the beginning of GET IT. Hair morphology changes were assessed according to a semiquantitative scale, described previously by Malinowska et al. (2008), in which value 0 corresponds to the normal morphology which value 5 indicates the most severe changes. Only a proximal part of each hair strand was considered, i.e. a fragment between hair root and 1 cm toward the distal hair end. 2.4. Statistical analysis The t-Student test has been used. Statistical significance was considered when p b 0.05.
250 x
800 x
0
1
2
3
4
5 Fig. 1. Examples of scanning electron micrographs of hair strands of Sanfilippo patients. Magnifications (250 and 800 times) are indicated above the micrographs. The values of hair dysmorphology, assessed according to the Malinowska's semiquantitative scale (Malinowska et al., 2008) are provided (left side of the figure).
3. Results Hair morphology was changed at baseline in most (28 out of 35) tested MPS III patients. The range of these changes, described in the semiquantitative scale (Malinowska et al., 2008), was from 1 (or 0 when including non-changed hair strands) to 4 (Fig. 1). After one year treatment of patients, improvement in hair morphology was noted in most of them (26 out of 35, or 26 out of 28 if excluding those with normal hair morphology at baseline), while no changes and worsening was observed in 8 (including 6 for which no changes were noted at baseline) and 1 patient(s), respectively (Fig. 2). All patients for which no improvement was detected belonged to the group receiving 5 mg/kg/day genistein, while hair morphology was corrected in all patients for which the dose of 15 mg/kg/day was used (Fig. 2).
hair dysmorphology score
Gene expression-targeted isoflavone therapy was performed for one year, with genistein-rich soy isoflavone extract (the SE-2000
hair dysmorphology score
2.2. Treatment
5 mg/kg/day 5 4 3 2 1 0
0 months
time
12 months
15 mg/kg/day
5 4 3 2 1 0
0 months
12 months
time
Fig. 2. Changes in hair morphology (assessed according to Malinowska's scale, see Fig. 1) in particular Sanfilippo patients during GET IT at the dose of genistein equal to 5 mg/kg/day (upper panel) or 15 mg/kg/day (lower panel). Hair morphology was assessed at baseline (0 months) and after one year of the treatment (12 months). Thickness of particular lines corresponds directly to number of patients responding in particular way to the treatment.
M. Narajczyk et al. / Gene 504 (2012) 292–295
hair dysmorphology score
hair dysmorphology score
294
5 mg/kg/day 5 4 3 2 1 0
0 months
time
12 months
15 mg/kg/day 5 4 3 2 1 0
0 months
time
12 months
Fig. 3. Changes in hair morphology (assessed according to Malinowska's scale, see Fig. 1) in groups of Sanfilippo patients during GET IT at the dose of genistein equal to 5 mg/kg/day (upper panel) or 15 mg/kg/day (lower panel). Hair morphology was assessed at baseline (0 months) and after one year of the treatment (12 months). Mean values and SD (indicated as error bars) are shown. The differences between values measured at baseline and after one year of the treatment were statistically significant (pb 0.05) at both genistein doses.
Statistical analysis indicated significant changes between baseline and 12 months of GET IT in both groups, receiving either 5 or 15 mg/kg/day genistein (Fig. 3). However, no statistically significant changes were evident in the efficiency of hair morphology improvement between the groups of patients receiving different doses of genistein (data not shown). 4. Discussion Monitoring of therapeutic effects is crucial for newly developed procedures. This is, however, a real challenge in many diseases, particularly those in which symptoms are either variable from one patient to another or difficult to assess quantitatively or both. Such a problem is common to inherited metabolic diseases. One of them is Sanfilippo disease, a severe lysosomal storage disorder, for which no officially approved treatment is currently available. Nevertheless, novel therapeutic options have been proposed recently, and pilot clinical trials were conducted (de Ruijter et al., 2011b). Among these emerging therapies, there is gene expression-targeted isoflavone therapy (GET IT) (Jakóbkiewicz-Banecka et al., 2011; Węgrzyn et al., 2010). Although in vitro experiments (Jakóbkiewicz-Banecka et al., 2009; Kloska et al., 2011; Piotrowska et al., 2006, 2011) and studies with animal models (Malinowska et al., 2009, 2010) gave promising results, effects of pilot clinical trials were controversial, as some researchers reported an improvement in various parameters while others could not detect positive effects in tested patients (de Ruijter et al., 2011a; Delgadillo et al., 2011; Malinova et al., 2011; Piotrowska et al., 2008, 2011). Apparently, these differences might be ascribed, at least partially, to various tests used by different investigators and to high variability of symptoms among Sanfilippo patients. One of features of some MPS types, including MPS III (Sanfilippo disease) is changed hair morphology, evident when observed using scanning electron microscope (Malinowska et al., 2008). In fact, it was suggested that changes in hair morphology might be used as a biomarker in monitoring effects of treatment of MPS patients (Kloska et al., 2005). Improvement in this parameter was observed during either enzyme replacement therapy for MPS I (Kloska et al., 2005) and GET IT for MPS III (Malinova et al., 2011; Piotrowska et al., 2008, 2011). Moreover, correction of hair morphology was demonstrated during studies
on GET IT for MPS IIIB mice (Malinowska et al., 2009). However, clinical studies performed to date either included low number of patients or were performed for a relatively short time (6 months exposure to the drug). Therefore, in this work, hair samples from 35 MPS III patients, withdrawn at baseline and after 12 month GET IT (with the dose of soy isoflavone extract corresponding to 5 or 15 mg/kg/day genistein) were analyzed. The previously proposed semiquantitative scale of hair dysmorphology (Malinowska et al., 2008) was employed in assessment of the samples. Improvement in vast majority of patients, revealing hair dysmorphology at baseline, was detected. When considering the patients as a group, statistically significant changes in hair morphology were evident at both doses of the extract. Therefore, we suggest that estimation of hair morphology changes can be used as an assay for assessment of effects of tested medicines in Sanfilippo disese. The advantages of this assay are (i) its simplicity, (ii) non-invasive character and (iii) effects possible to be observed irrespective of the severity of symptoms at baseline (note that the improvement was observed in patients revealing both severe hair dysmorphology or minor changes at baseline). However, there are also some disadvantages, namely: (i) hair dysmorphology occurs in most but not all MPS III patients, (ii) a special equipment (scanning electron microscope) is required, and (iii) hair structure is of little clinical importance. Therefore, we propose that the assessment of hair dysmorphology should be considered as a biomarker suitable for rough assessment of effects of a tested therapy rather than as a clinical endpoint. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was supported by Ministry of Sciences and Higher Education of Poland (project grant no. N N301 668540 to GW), and was operated within the Foundation for Polish Science Team Programme co-financed by the EU European Regional Development Fund (grant no. TEAM/2008-2/7 to GW). A support from Polish Society for Mucopolysaccharidosis and Rare Diseases is greatly acknowledged. References Barranger, J.A., Cabrera-Salazar, M., 2007. Lysosomal Storage Disorders. Springer, BerlinHeidelberg. de Ruijter, J., Valstar, M.J., Wijburg, F.A., 2011. Mucopolysaccharidosis type III (Sanfilippo Syndrome): emerging treatment strategies. Curr. Pharm. Biotechnol. 12, 923–930. de Ruijter, J., Valstar, M.J., Narajczyk, M., Węgrzyn, G., Kulik, W., Ijlst, L., Wagemans, T., van der Wal, W.M., Wijburg, F.A., 2012. Genistein in Sanfilippo disease: a randomized controlled crossover trial. Ann. Neurol. 71, 110–120. Delgadillo, V., del Mar O'Callaghan, M., Artuch, R., Montero, R., Pineda, M., 2011. Genistein supplementation in patients affected by Sanfilippo disease. J. Inherit. Metab. Dis. 34, 1039–1044. Jakóbkiewicz-Banecka, J., Piotrowska, E., Narajczyk, M., Barańska, S., Węgrzyn, G., 2009. Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor-dependent pathway. J. Biomed. Sci. 16, 26. Jakóbkiewicz-Banecka, J., et al., 2011. Substrate reduction therapies for mucopolysaccharidoses. Curr. Pharm. Biotechnol. 12 (11), 1860–1865. Kloska, A., et al., 2005. Changes in hair morphologyof mucopolysaccharidosis I patients treated with recombinant human α-L-iduronidase (laronidase, Aldurazyme). Am. J. Med. Genet. A 139, 199–203. Kloska, A., Jakóbkiewicz-Banecka, J., Narajczyk, M., Banecka-Majkutewicz, Z., Węgrzyn, G., 2011. Effects of flavonoids on glycosaminoglycan synthesis: implications for substrate reduction therapy in Sanfilippo disease and other mucopolysaccharidoses. Metab. Brain Dis. 26, 1–8. Malinova, V., Węgrzyn, G., Narajczyk, M., 2011. The use of elevated doses of genisteinrich soy extract in the gene expression-targeted isoflavone therapy (GET IT) for Sanfilippo disease patients. J. Inherit. Metab. Dis. Rep., http://dx.doi.org/10.1007/ 8904_2011_87. Malinowska, M., et al., 2008. Abnormalities in the hair morphology of patients of same but not all types of mucopolysaccharidoses. Eur. J. Pediatr. 167, 203–209.
M. Narajczyk et al. / Gene 504 (2012) 292–295 Malinowska, M., et al., 2009. Genistein reduces lysosomal storage in peripheral tissues of mucopolysaccharide IIIB mice. Mol. Genet. Metab. 98, 235–242. Malinowska, M., et al., 2010. Genistein improves neuropathology and corrects behaviour in a mouse model of neurodegenerative metabolic disease. PLoS One 5, e14192. Neufeld, E.F., Muenzer, J., 2001. Mucopolysaccharidoses. In: Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D. (Eds.), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Co., New York, pp. 3421–3452. Piotrowska, E., et al., 2006. Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses. Eur. J. Hum. Genet. 14, 846–852. Piotrowska, E., et al., 2008. The use of genistein-rich isoflavone extract in substrate reduction therapy for Sanfilippo disease: open-label, pilot study in 10 pediatric patients. Curr. Ther. Res. Clin. Exp. 69, 166–179.
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Piotrowska, E., et al., 2011. Two-year follow-up of Sanfilippo Disease patients treated with a genistein-rich isoflavone extract: assessment of effects on cognitive functions and general status of patients. Med. Sci. Monit. 17, CR196-202. Swarts, K., 2009. Genetic Disorders. Greenhaven Press, Farmington Hills, MI. Valstar, M.J., Ruijter, G.J., van Diggelen, O.P., Poorthuis, B.J., Wijburg, F.A., 2008. Sanfilippo syndrome: a mini-review. J. Inherit. Metab. Dis. 31, 240–252. Valstar, M.J., et al., 2010. Mucopolysaccharidosis type IIIA: clinical spectrum and genotype-phenotype correlations. Ann. Neurol. 68, 876–887. Valstar, M.J., Marchal, J.P., Grootenhuis, M., Colland, V., Wijburg, F.A., 2011. Cognitive development in patients with Mucopolysaccharidosis type III (Sanfilippo syndrome). Orphanet J. Rare Dis. 6, 43. Węgrzyn, G., et al., 2010. Genistein: a natural isoflavone with a potential for treatment of genetic diseases. Biochem. Soc. Trans. 38, 695–701.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Changes in hair morphology as a biomarker in gene expression-targeted isoflavone therapy for Sanfilippo disease Magdalena Narajczyk a,⁎, Anna Tylki-Szymańska b, Grzegorz Węgrzyn c a b c
Laboratory of Electron Microscopy, University of Gdańsk, Kładki 24, 80–822 Gdańsk, Poland The Children's Memorial Health Institute, Al. Dzieci Polskich 20, 04–736 Warsaw, Poland Department of Molecular Biology, University of Gdańsk, Kładki 24, 80–822 Gdańsk, Poland
a r t i c l e
i n f o
Article history: Accepted 7 May 2012 Available online 22 May 2012 Keywords: Sanfilippo disease Mucopolysaccharidosis Gene expression-targeted isoflavone therapy Scanning electron microscopy Hair morphology
a b s t r a c t Lack of unequivocal markers for assessment of therapeutic effects of treatment procedures is a common problem, but it is especially pronounced in genetic diseases. One of them is Sanfilippo disease (mucopolysaccharidosis type III, or MPS III). Changes in hair morphology have already been reported as one of characteristic and easily detected symptoms of this disease and other mucopolysaccharidoses. This feature has been used previously to monitor efficacy of gene expression-targeted isoflavone therapy (GET IT) in pilot clinical studies performed with low number of patients. Here, we studied changes in hair morphology, observed by using scanning electron microscopy, in 35 patients subjected to GET IT for one year at doses of 5 and 15 mg/kg/day. We have observed statistically significant correction of hair morphology in both groups of patients, corroborating results of earlier studies with low number of patients that suggested easily observable improvement in this parameter during different therapies of MPS. Since the improvement was evident in both previous reports and this study, we propose that analysis of hair morphology may be considered as a non-invasive method in monitoring effects of treatment on somatic symptoms of Sanfilippo disease patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Treatment of genetic diseases is an extremely difficult task. In fact, although there are 6000 or so disorders caused by defects in single genes, specific therapies have been developed for only a very small fraction of them (Barranger and Cabrera-Salazar, 2007; Swarts, 2009). Another serious problem with such therapies is assessment of their efficacy. All genetic diseases are relatively rare, and symptoms developing in persons suffering from the same disease can vary significantly from one patient to another (Swarts, 2009). Therefore, it is often difficult to find specific parameters that should be monitored in order to assess efficacy of a therapy under study. An example of diseases that cause problems described above is mucopolysaccharidosis (MPS), and especially its type III, called Sanfilippo disease. In this disease, like in other MPS types, glycosaminoglycan (GAG) accumulation occurs in lysosomes, which is caused by dysfunction of genes coding for specific enzymes involved in degradation of these compounds (Neufeld and Muenzer, 2001). Lysosomal storage of heparan sulfate (one of GAGs) causes specific symptoms which consist of relatively mild somatic dysfunctions and severe neurological and behavioral changes (Valstar et al., 2008). Due to progressive nature of Sanfilippo
Abbreviations: MPS, Mucopolysaccharidosis; GET IT, gene expression-targeted isoflavone therapy; GAG, glycosaminoglycan. ⁎ Corresponding author. Tel.: + 48 58 523 6304; fax: + 48 58 523 6424. E-mail address: [email protected] (M. Narajczyk). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.05.006
disease, variability of symptoms and low number of patients (Valstar et al., 2010, 2011), it is extremely difficult to assess effects of any therapy for this disease. Such problems were met in recent clinical studies, in which gene expression-targeted isoflavone therapy (GET IT) was tested. A few pilot clinical studies testing GET IT for Sanfilippo disease were performed to date, but results of these studies are equivocal since opposite conclusions were drawn by different authors. Namely, some studies suggested an improvement in selected biochemical and somatic parameters, as well as cognitive abilities of patients after 1 year GET IT (Malinova et al., 2011; Piotrowska et al., 2008, 2011), while results of another trials were interpreted as a lack of improvement in patients’ disability (de Ruijter et al., 2011a; Delgadillo et al., 2011). Apparently, these discrepancies might be ascribed, at least partially, to different tests employed by different authors to monitor effects of the treatment. Although there are many somatic symptoms occurring in patients suffering from Sanfilippo disease, their severity is generally lower than in other MPS types (Valstar et al., 2008). Nevertheless, a spectrum of each of them is very broad. Therefore, choosing appropriate biomarkers to be assessed in the course of a clinical trial is problematic. On the other hand, although behavioral changes and cognitive disabilities are common in Sanfilippo disease, they are very difficult to be measured quantitatively. Therefore, it is a need for establishing a biomarker that could be used for assessment of efficacy of any therapy for MPS type III. It was demonstrated previously that morphology of hair is changed in patients suffering from some types of MPS, including Sanfilippo disease (Kloska et al., 2005; Malinowska et al., 2008). These changes can
M. Narajczyk et al. / Gene 504 (2012) 292–295
be assessed using scanning electron microscopy. Enzyme replacement therapy of MPS type I resulted in correction of hair morphology, which suggested that this feature could be used as a biomarker in MPS treatment procedures (Kloska et al., 2005). In fact, hair morphology has been tested during pilot clinical studies with GET IT for Sanfilippo disease, however, either low number of patients was involved in those studies (Delgadillo et al., 2011; Malinova et al., 2011; Piotrowska et al., 2008) or patients were exposed to genistein treatment for a relatively short time (6 months) between microscopic analyses (de Ruijter et al., 2011a). Therefore, we assumed that studies on a larger group of patients, performed for at least 12 months, are required for testing whether hair morphology changes can be used as a biomarker in assessment of efficacy of a therapy for MPS type III. 2. Materials and methods 2.1. Patients 35 patients suffering from Sanfilippo A (17 patients), B (14 patients) and C (4 patients) subtypes were enrolled in this study. They were diagnosed on the basis of enzymatic and genetic tests, as described previously (Piotrowska et al., 2008, 2011).
293
extract, included in the product called Soyfem and provided by the manufacturer: Biofarm, Poznań, Poland; this product was described in details by Piotrowska et al., 2008). The doses corresponded to amount of genistein equal to either 5 or 15 mg/kg/day. No adverse effects were noted during the study. This treatment has been approved by the Independent Bioethics Committee of the Medical University of Gdańsk, Poland. 2.3. Hair morphology assessment Hair morphology was assessed by using scanning electron microscope according to Kloska et al. (2005). Hair samples were withdrawn at baseline and 12 months after the beginning of GET IT. Hair morphology changes were assessed according to a semiquantitative scale, described previously by Malinowska et al. (2008), in which value 0 corresponds to the normal morphology which value 5 indicates the most severe changes. Only a proximal part of each hair strand was considered, i.e. a fragment between hair root and 1 cm toward the distal hair end. 2.4. Statistical analysis The t-Student test has been used. Statistical significance was considered when p b 0.05.
250 x
800 x
0
1
2
3
4
5 Fig. 1. Examples of scanning electron micrographs of hair strands of Sanfilippo patients. Magnifications (250 and 800 times) are indicated above the micrographs. The values of hair dysmorphology, assessed according to the Malinowska's semiquantitative scale (Malinowska et al., 2008) are provided (left side of the figure).
3. Results Hair morphology was changed at baseline in most (28 out of 35) tested MPS III patients. The range of these changes, described in the semiquantitative scale (Malinowska et al., 2008), was from 1 (or 0 when including non-changed hair strands) to 4 (Fig. 1). After one year treatment of patients, improvement in hair morphology was noted in most of them (26 out of 35, or 26 out of 28 if excluding those with normal hair morphology at baseline), while no changes and worsening was observed in 8 (including 6 for which no changes were noted at baseline) and 1 patient(s), respectively (Fig. 2). All patients for which no improvement was detected belonged to the group receiving 5 mg/kg/day genistein, while hair morphology was corrected in all patients for which the dose of 15 mg/kg/day was used (Fig. 2).
hair dysmorphology score
Gene expression-targeted isoflavone therapy was performed for one year, with genistein-rich soy isoflavone extract (the SE-2000
hair dysmorphology score
2.2. Treatment
5 mg/kg/day 5 4 3 2 1 0
0 months
time
12 months
15 mg/kg/day
5 4 3 2 1 0
0 months
12 months
time
Fig. 2. Changes in hair morphology (assessed according to Malinowska's scale, see Fig. 1) in particular Sanfilippo patients during GET IT at the dose of genistein equal to 5 mg/kg/day (upper panel) or 15 mg/kg/day (lower panel). Hair morphology was assessed at baseline (0 months) and after one year of the treatment (12 months). Thickness of particular lines corresponds directly to number of patients responding in particular way to the treatment.
M. Narajczyk et al. / Gene 504 (2012) 292–295
hair dysmorphology score
hair dysmorphology score
294
5 mg/kg/day 5 4 3 2 1 0
0 months
time
12 months
15 mg/kg/day 5 4 3 2 1 0
0 months
time
12 months
Fig. 3. Changes in hair morphology (assessed according to Malinowska's scale, see Fig. 1) in groups of Sanfilippo patients during GET IT at the dose of genistein equal to 5 mg/kg/day (upper panel) or 15 mg/kg/day (lower panel). Hair morphology was assessed at baseline (0 months) and after one year of the treatment (12 months). Mean values and SD (indicated as error bars) are shown. The differences between values measured at baseline and after one year of the treatment were statistically significant (pb 0.05) at both genistein doses.
Statistical analysis indicated significant changes between baseline and 12 months of GET IT in both groups, receiving either 5 or 15 mg/kg/day genistein (Fig. 3). However, no statistically significant changes were evident in the efficiency of hair morphology improvement between the groups of patients receiving different doses of genistein (data not shown). 4. Discussion Monitoring of therapeutic effects is crucial for newly developed procedures. This is, however, a real challenge in many diseases, particularly those in which symptoms are either variable from one patient to another or difficult to assess quantitatively or both. Such a problem is common to inherited metabolic diseases. One of them is Sanfilippo disease, a severe lysosomal storage disorder, for which no officially approved treatment is currently available. Nevertheless, novel therapeutic options have been proposed recently, and pilot clinical trials were conducted (de Ruijter et al., 2011b). Among these emerging therapies, there is gene expression-targeted isoflavone therapy (GET IT) (Jakóbkiewicz-Banecka et al., 2011; Węgrzyn et al., 2010). Although in vitro experiments (Jakóbkiewicz-Banecka et al., 2009; Kloska et al., 2011; Piotrowska et al., 2006, 2011) and studies with animal models (Malinowska et al., 2009, 2010) gave promising results, effects of pilot clinical trials were controversial, as some researchers reported an improvement in various parameters while others could not detect positive effects in tested patients (de Ruijter et al., 2011a; Delgadillo et al., 2011; Malinova et al., 2011; Piotrowska et al., 2008, 2011). Apparently, these differences might be ascribed, at least partially, to various tests used by different investigators and to high variability of symptoms among Sanfilippo patients. One of features of some MPS types, including MPS III (Sanfilippo disease) is changed hair morphology, evident when observed using scanning electron microscope (Malinowska et al., 2008). In fact, it was suggested that changes in hair morphology might be used as a biomarker in monitoring effects of treatment of MPS patients (Kloska et al., 2005). Improvement in this parameter was observed during either enzyme replacement therapy for MPS I (Kloska et al., 2005) and GET IT for MPS III (Malinova et al., 2011; Piotrowska et al., 2008, 2011). Moreover, correction of hair morphology was demonstrated during studies
on GET IT for MPS IIIB mice (Malinowska et al., 2009). However, clinical studies performed to date either included low number of patients or were performed for a relatively short time (6 months exposure to the drug). Therefore, in this work, hair samples from 35 MPS III patients, withdrawn at baseline and after 12 month GET IT (with the dose of soy isoflavone extract corresponding to 5 or 15 mg/kg/day genistein) were analyzed. The previously proposed semiquantitative scale of hair dysmorphology (Malinowska et al., 2008) was employed in assessment of the samples. Improvement in vast majority of patients, revealing hair dysmorphology at baseline, was detected. When considering the patients as a group, statistically significant changes in hair morphology were evident at both doses of the extract. Therefore, we suggest that estimation of hair morphology changes can be used as an assay for assessment of effects of tested medicines in Sanfilippo disese. The advantages of this assay are (i) its simplicity, (ii) non-invasive character and (iii) effects possible to be observed irrespective of the severity of symptoms at baseline (note that the improvement was observed in patients revealing both severe hair dysmorphology or minor changes at baseline). However, there are also some disadvantages, namely: (i) hair dysmorphology occurs in most but not all MPS III patients, (ii) a special equipment (scanning electron microscope) is required, and (iii) hair structure is of little clinical importance. Therefore, we propose that the assessment of hair dysmorphology should be considered as a biomarker suitable for rough assessment of effects of a tested therapy rather than as a clinical endpoint. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was supported by Ministry of Sciences and Higher Education of Poland (project grant no. N N301 668540 to GW), and was operated within the Foundation for Polish Science Team Programme co-financed by the EU European Regional Development Fund (grant no. TEAM/2008-2/7 to GW). A support from Polish Society for Mucopolysaccharidosis and Rare Diseases is greatly acknowledged. References Barranger, J.A., Cabrera-Salazar, M., 2007. Lysosomal Storage Disorders. Springer, BerlinHeidelberg. de Ruijter, J., Valstar, M.J., Wijburg, F.A., 2011. Mucopolysaccharidosis type III (Sanfilippo Syndrome): emerging treatment strategies. Curr. Pharm. Biotechnol. 12, 923–930. de Ruijter, J., Valstar, M.J., Narajczyk, M., Węgrzyn, G., Kulik, W., Ijlst, L., Wagemans, T., van der Wal, W.M., Wijburg, F.A., 2012. Genistein in Sanfilippo disease: a randomized controlled crossover trial. Ann. Neurol. 71, 110–120. Delgadillo, V., del Mar O'Callaghan, M., Artuch, R., Montero, R., Pineda, M., 2011. Genistein supplementation in patients affected by Sanfilippo disease. J. Inherit. Metab. Dis. 34, 1039–1044. Jakóbkiewicz-Banecka, J., Piotrowska, E., Narajczyk, M., Barańska, S., Węgrzyn, G., 2009. Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor-dependent pathway. J. Biomed. Sci. 16, 26. Jakóbkiewicz-Banecka, J., et al., 2011. Substrate reduction therapies for mucopolysaccharidoses. Curr. Pharm. Biotechnol. 12 (11), 1860–1865. Kloska, A., et al., 2005. Changes in hair morphologyof mucopolysaccharidosis I patients treated with recombinant human α-L-iduronidase (laronidase, Aldurazyme). Am. J. Med. Genet. A 139, 199–203. Kloska, A., Jakóbkiewicz-Banecka, J., Narajczyk, M., Banecka-Majkutewicz, Z., Węgrzyn, G., 2011. Effects of flavonoids on glycosaminoglycan synthesis: implications for substrate reduction therapy in Sanfilippo disease and other mucopolysaccharidoses. Metab. Brain Dis. 26, 1–8. Malinova, V., Węgrzyn, G., Narajczyk, M., 2011. The use of elevated doses of genisteinrich soy extract in the gene expression-targeted isoflavone therapy (GET IT) for Sanfilippo disease patients. J. Inherit. Metab. Dis. Rep., http://dx.doi.org/10.1007/ 8904_2011_87. Malinowska, M., et al., 2008. Abnormalities in the hair morphology of patients of same but not all types of mucopolysaccharidoses. Eur. J. Pediatr. 167, 203–209.
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