these disorders are caused by mutations in genes coding for enzymes involved in degradation of glycosaminoglycans ( gags ) .
storage of gags in cells of patients results in progressive damage to the affected tissues , including the heart , respiratory system , bones , joints and , in most mps types and subtypes , the central nervous system ( cns ) .
mps are usually fatal diseases , with average expected life span of 1 or 2 decades , although patients with milder forms can survive into adulthood .
there are 11 known types and subtypes of mps , and each type is variable in severity of particular symptoms .
prediction of severity and clinical progression of mps is difficult , even when biochemical and genetic parameters are determined , but recent studies indicate that considering 2 or more parameters may give significantly better results than attempting to make conclusions based on a single biomarker .
mps type iii ( mps iii , called also sanfilippo disease ) is a group of 4 conditions ( mps iiia , b , c and d ) , revealing similar clinical symptoms and characterized by lysosomal storage of heparan sulfate ( hs ) , one of gags .
particular subtypes are classified according to gene coding for an enzyme involved in degradation of hs ( heparan n - sulfatase in mps iiia , -n - acetylglucosaminidase in mps iiib , acetyl - coa : -glycosaminide acetyltransferase in mps iiic and n - acetylglucosamine 6-sulfatase in mps iiid ) which is defective .
although other mps types are characterized by severe somatic symptoms and either some ( sometimes also severe ) or no neurological and behavioral problems , mps iii is specifically associated with severe learning difficulties and behavioral disturbances , accompanied with relatively mild somatic involvement . in most affected patients
the progressive nature of the disease leads to death in the second ( or rarely third ) decade of life . as this disorder primarily affects the brain and nervous system , attempts to cure patients have been especially difficult , and aside from experimental treatments ( see below ) , the best care that can be offered is palliative or symptomatic .
enzyme replacement therapy ( ert ) , based on an intravenous infusion of an active , recombinant form of a deficient enzyme , is currently the only officially approved treatment of mps , and is used in clinical practice for mps types i , ii and vi .
unfortunately , neurological symptoms due to hs accumulation in cns can not be managed by ert , due to an inefficient delivery of proteins , including recombinant enzymes , through the blood - brain barrier .
substrate reduction therapy ( srt ) , called also substrate deprivation therapy ( sdt ) , is an alternative approach for treatment of patients with lysosomal storage diseases .
this therapy is based on the action of small molecules that may , directly or indirectly , impair synthesis of compounds that can not be efficiently degraded in cells of patients . since such small molecules
may likely cross the blood - brain - barrier , it is hoped that they could be used to manage neurological symptoms of these diseases .
previous studies with rhodamine b ( [ 9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]-diethylammonium chloride ) and genistein ( 5 , 7-dihydroxy-3-(4-hydroxyphenyl ) -4h-1-benzopyran-4 - 1 ) revealed that each of these compounds can reduce the efficiency of gag synthesis in cells of various mps types .
importantly , these studies demonstrated not only a lack of further gags accumulation , but also a reduction in lysosomal storage of these compounds .
in fact , recently reported experiments indicated that rna interference - mediated silencing of genes coding for various gag synthetases causes a decrease in the amount of the storage material in cell lines of patients with sanfilippo disease .
tests on animal models of sanfilippo disease demonstrated efficacy of rhodamine b and genistein in vivo , including behavior of affected and treated mice .
although it is unlikely that rhodamine b can be used in clinical practice due to its potential toxicity to humans , genistein is a non - toxic and safe compound .
moreover , unlike rhodamine b , the mechanism of genistein - mediated inhibition of gag synthesis has been demonstrated .
this isoflavone inhibits phsophorylation of the epidermal growth factor receptor ( egfr ) , which in turn causes an impairment of expression of genes coding for enzymes necessary for gag synthesis .
gene expression - targeted isoflavone therapy ( abbreviated as get it ) , and an open - label , pilot study in 10 pediatric mps iii patients , treated with a genistein - rich isoflavone extract for 12 months , was performed .
after 1 year of treatment , statistically significant improvement in all measured parameters ( urinary gag excretion , hair morphology and cognitive functions ) was demonstrated .
since longer - term effects of get it for mps iii are not known , we performed a follow - up study with genistein - treated mps iii patients .
considering learning difficulties and behavioral disturbances to be the most severe clinical problems of sanfilippo disease , we focused on monitoring cognitive functions and general status of patients , including their behavior .
the patients were diagnosed for sanfilippo disease ( either mps iiia , mckusick s omim no . 252900 , or mps iiib , mckusick s omim
252920 ) by estimation of urinary gag levels ( highly elevated gag levels , observed in hemi - quantitative electrophoretic analysis , gave a preliminary indication for mps ) and measurement of activities of particular lysosomal hydrolases in leukocytes .
deficiency in activity of heparan n - sulfatase ( control value : 4.11.4 nmoles / mg of protein/18h ) or -n - aceltyl glucosaminidase ( control value : 9034 nmoles / mg of protein/42 h ) was considered as a diagnosis for mps iiia or mps iiib , respectively .
mutations in genes coding for the above - mentioned enzymes were determined for 5 patients , and 1 mutation was determined for 1 patient .
eight patients ( 6 with mps iiia and 2 with mps iiib ) were enrolled into the study .
all patients were of caucasian origin ; 2 males and 6 females . among the 8 patients ,
5 ( patients : iiia-2 , iiia-4 , iiia-5 , iiib-2 and iiib-5 ) were previously enrolled into the pilot study .
the mps iii patients characterized in table 1 were treated for 36 months with a genistein - rich soy isoflavone extract ( called se-2000 ) , provided by the manufacturer ( biofarm , pozna , poland ) in the form of tablets ( product name soyfem ) .
the extract consisted of genistin and genistein ( 26.90% ) , daidzin and daidzein ( 13.37% ) , glycitin and glycitein ( 1.98% ) , and soy proteins , carbohydrates and lipids ( remaining amount ) .
this extract was administered orally ( in the form of whole tablets or tablets crushed into powder ) at the dose corresponding to 5 mg of genistin and genistein ( genistin is a glycan that can be converted to genistien by either acid environment or intestinal bacterial flora ) per 1 kg of body weight daily .
the extract was administered twice a day , with equal amounts at morning and evening .
two types of evaluation ( the modified bae test and parents questionnaire , both described in the next subsections ) were performed at baseline and after 12 and 36 months of treatment .
the monitoring of adverse effects was based on reports of parents , who were appropriately instructed to signal any such effects immediately ( by either phone or email , with confirmation of receipt of the information ) , and who provided an assessment of such effects in writing every 3 months ( even if no adverse effects were observed ) .
this experimental treatment was approved by the independent bioethics committee of the medical university of gdask , poland ( approval no .
parents of the children involved in this study signed the informed consent form . to assess cognitive functions and behavioral changes , a modified version of the brief assessment examination  
this test estimated the following parameters : ( i ) alertness / activity , ( ii ) obeying commands , ( iii ) pointing at objects , ( iv ) pointing at colors , ( v ) matching shapes , ( vi ) speech , ( vii ) auditory digit span , and ( viii ) mathematics .
parents of patients were asked to evaluate 18 different parameters concerning changes in conditions of their children from a year before the onset of the treatment to the start of the therapy , from the onset of the treatment to a year after the start of the therapy , and from the onset of the treatment to 3 years after the start of therapy .
some parents reported they depended also on the opinion of other , uninitiated , people ( eg , teachers ) .
the patients were diagnosed for sanfilippo disease ( either mps iiia , mckusick s omim no . 252900 , or mps iiib , mckusick s omim
252920 ) by estimation of urinary gag levels ( highly elevated gag levels , observed in hemi - quantitative electrophoretic analysis , gave a preliminary indication for mps ) and measurement of activities of particular lysosomal hydrolases in leukocytes .
deficiency in activity of heparan n - sulfatase ( control value : 4.11.4 nmoles / mg of protein/18h ) or -n - aceltyl glucosaminidase ( control value : 9034 nmoles / mg of protein/42 h ) was considered as a diagnosis for mps iiia or mps iiib , respectively .
mutations in genes coding for the above - mentioned enzymes were determined for 5 patients , and 1 mutation was determined for 1 patient .
eight patients ( 6 with mps iiia and 2 with mps iiib ) were enrolled into the study .
all patients were of caucasian origin ; 2 males and 6 females . among the 8 patients ,
5 ( patients : iiia-2 , iiia-4 , iiia-5 , iiib-2 and iiib-5 ) were previously enrolled into the pilot study .
the mps iii patients characterized in table 1 were treated for 36 months with a genistein - rich soy isoflavone extract ( called se-2000 ) , provided by the manufacturer ( biofarm , pozna , poland ) in the form of tablets ( product name soyfem ) .
the extract consisted of genistin and genistein ( 26.90% ) , daidzin and daidzein ( 13.37% ) , glycitin and glycitein ( 1.98% ) , and soy proteins , carbohydrates and lipids ( remaining amount ) .
this extract was administered orally ( in the form of whole tablets or tablets crushed into powder ) at the dose corresponding to 5 mg of genistin and genistein ( genistin is a glycan that can be converted to genistien by either acid environment or intestinal bacterial flora ) per 1 kg of body weight daily .
the extract was administered twice a day , with equal amounts at morning and evening .
two types of evaluation ( the modified bae test and parents questionnaire , both described in the next subsections ) were performed at baseline and after 12 and 36 months of treatment .
the monitoring of adverse effects was based on reports of parents , who were appropriately instructed to signal any such effects immediately ( by either phone or email , with confirmation of receipt of the information ) , and who provided an assessment of such effects in writing every 3 months ( even if no adverse effects were observed ) .
this experimental treatment was approved by the independent bioethics committee of the medical university of gdask , poland ( approval no .
to assess cognitive functions and behavioral changes , a modified version of the brief assessment examination   was used .
this test estimated the following parameters : ( i ) alertness / activity , ( ii ) obeying commands , ( iii ) pointing at objects , ( iv ) pointing at colors , ( v ) matching shapes , ( vi ) speech , ( vii ) auditory digit span , and ( viii ) mathematics .
parents of patients were asked to evaluate 18 different parameters concerning changes in conditions of their children from a year before the onset of the treatment to the start of the therapy , from the onset of the treatment to a year after the start of the therapy , and from the onset of the treatment to 3 years after the start of therapy .
some parents reported they depended also on the opinion of other , uninitiated , people ( eg , teachers ) .
eight patients suffering from sanfilippo disease ( mps iiia and iiib ) were monitored during an experimental therapy consisting of a genistein - rich soy isoflavone extract ( soyfem ) at the dose of 5 mg / kg / day for 24 months .
five of these patients were enrolled in a 1-year pilot study ( patients iiia-2 , iiia-4 , iiia-5 , iiib-2 , iiib-5 ) , described previously , and the treatment ( called get it ) was continued for the next 2 years ( parents of these patients collaborated with researchers for the next 2 years and sent back their questionnaires on time ; although parents of the other 5 patients also agreed to collaborate , they failed to return completed questionnaires on time ) .
urinary gag levels and hair morphology are relatively simple assays that can be used for monitoring of mps treatment ; however , they do not reflect the behavioral and cognitive status of examined patients .
moreover , a statistically significant improvement in gag excretion and normalization of hair morphology were reported after 1 year of get it ; hence , one would not expect further improvement in these parameters during the next 24 months .
since cognitive and behavioral dysfunctions are the most severe symptoms in sanfilippo disease , we decided to monitor cognitive ability and behavior of patients by using a specific psychological test and a questionnaire for patients parents , as described previously .
this corroborates the conclusion that get it is a safe treatment , as described previously . to assess cognitive functions of the patients , a modified bae psychological test ( see appendix for details )
was performed at baseline , and at 12 and 36 months after the start of treatment . during the first year of the treatment , an improvement in the score in 7 patients and stabilization in 1 patient ( iiia-7 )
were observed ( table 2 ) , which corroborates previous results . after the third year
( 2-year follow - up ) further improvement ( relative to results after the first year ) was observed in 2 patients ( iiia-2 , iiia-5 ) , stabilization was observed in 3 patients ( iiia-4 , iiia-7 , iiib-5 ) , and deterioration was observed in 3 patients ( iiia-6 , iiia-8 , iiib-2 ) . in this last - mentioned group of patients ,
the scores after 36 months of treatment were equal or very similar to those achieved at baseline ( table 2 ) . to estimate the efficacy of the treatment ,
patients parents were asked to evaluate 18 different parameters concerning changes in conditions of their children from a year before the start of the treatment to the start of the therapy , from the onset of the treatment to a year after the start of the therapy , and from the start of the treatment to 3 years after the start of the therapy .
as expected , parents reported deterioration of patients in many parameters in the period from 1 year before the treatment to the start of the trial , which confirmed that the children were in the clinically progressive stage of the disease ( table 3 ) . to estimate status of the patients , we calculated a general score for each patient on the basis of answers to the questionnaires .
this general score was calculated by summing all the answers and giving the following weights to particular types of answers : ( 2 ) considerably deteriorated , ( 1 ) somewhat deteriorated , ( 0 ) no changes , ( + 1 ) somewhat improved , and ( + 2 ) considerably improved .
the results of questionnaires revealed improvement in all patients after the first year of treatment , further improvement in 5 patients ( iiia-2 , iiia-5 , iiia-8 , iiib-2 , iiib-5 ) , and deterioration in 3 patients ( iiia-4 , iiia-6 , iiia-7 ) , during the next 2 years ( table 2 ) .
after unsuccessful trials with bone marrow transplantation and with treatments employing small molecules , either miglustat or glucosamine , to our knowledge , this pilot study on a specific substrate deprivation therapy , called get it , demonstrates the first treatment of patients with sanfilippo disease that demonstrates a statistically significant improvement in biochemical and morphological parameters , as well as improvement in clinical status of patients . since get it appeared to be a promising treatment , in the absence of information on clinical effects of longer treatment , a follow - up of treated patients was necessary . in this report
we present a 2-year follow - up of sanfilippo disease patients treated with a genistein - rich soy isoflavone extract ( soyfem ) at the dose corresponding to the amount of genistein equal to 5 mg / kg / day . when monitoring cognitive abilities and general status of the patients after the third year of the treatment , we observed either further improvement / stabilization of the patients , or their slow deterioration .
this is in contrast to rapid deterioration of untreated mps iii patients presented in the literature   and evident from questionnaire responses in this report ( table 3 ) .
interestingly , such a picture of the clinical manifestation of symptoms in treated mps iii patients is similar to that observed during ert of mps i , although only effects on somatic tissues could be considered in the latter case .
namely , mps i patients treated with laronidase improve relatively rapidly during the first several months of the treatment , and then slow improvement , stabilization or slow deterioration occurs .
it is plausible that during the first period of both ert and get it , the reversible effects of diseases are corrected , and thereafter it is only possible to either prevent further progression or to slow the irreversible changes .
the main mechanism of genistein - mediated impairment of gag synthesis is inhibition of phosphorylation of egfr .
thus , one might predict that when using higher doses of genistein it should be possible to slow the gag production even more effectively .
the dose of the se-2000 extract used in both the previous trial   and this study corresponded to the amount of genistein equal to 5 mg / kg / day .
since it was recently reported that a dose as high as 160 mg / kg / day is safe ( with no adverse effects ) for mps iiib mice , it is likely that significantly ( at least 200% ) higher doses than that used in this study may be safe for humans .
therefore , there is great need for a clinical trial evaluating get it for sanfilippo disease .
oxidative stress , inflammation , cytotoxicity and apoptosis may be involved in the mechanism of neurodegeneration in sanfilippo disease [ 1820 ] . moreover ,
accumulation of hyperphosphorylated tau protein ( p - tau ) , which forms aggregates characteristic of alzheimer s disease , has been detected in brains of mps iiib mice .
thus , it is intriguing that genistein has been described as a factor attenuating oxidative stress in the brain , having a neuroprotective effect against beta amyloid - induced neurotoxicity , inhibiting apoptosis in primary neuronal cell cultures , preventing alzheimer s disease - associated inflammation , and revealing a general neuroprotective effect .
genistein , apart from its action as an indirect inhibitor of gag synthesis , may also be beneficial for mps iii patients due to its neuroprotective functions .
although some of these functions were observed in vitro at genistein concentrations as low as 100 nm , in most in vivo studies some effects were evident only at 1015 mg / kg [ 2227 ] .
these facts strengthen the need for results of clinical studies in which patients with sanfilippo disease are treated with genistein at doses of 1015 mg / kg / day or higher .
results of recent studies on animals suggest that get it may be effective in treatment of other types of mps . since improvement in cns was observed in genistein - treated mice with mps ii , an x chromosome - linked disorder , get it could be considered as a potential treatment for mps ii - associated mental retardation .
the treatment of sanfilippo patients with a genistein - rich soy isoflavone extract ( get it ) , at the dose corresponding to 5 mg / kg / day , may be effective in either inhibition ( in some patients ) or slowing ( in other patients ) of the cognitive dysfunction , behavioral problems and general deterioration caused by the disease , over a period of 3 years .
an increase in the dose of genistein is suggested to improve the efficacy of the treatment .
general excitement 0 points short - term goal directed behaviour 2 points short - term goal directed activity 4 points repeated goal directed activity 6 points full adequate activity 8 points fulfilling simple commands none fulfilled 0 points none pointed 0 points none pointed 0 points none matched 0 points individual echoic words 2 points individual words 4 points simple sentences 6 points expanded statements 8 points none reproduced 0 points 1 reproduced 4 points 2 reproduced 6 points 3 reproduced 8 points does not count 0 points counts to 10 8 points
general excitement  kinetic excitement , no goal - oriented activity , lack of even short - term concentration on a single stimulus .
short - term goal directed behaviour  temporary concentration , e.g. watching somebody or something .
short - term goal directed activity  conscious , purposeful manipulation of an item , visual examination , intentional attempts to socialize .
full adequate activity  goal directed activity dominates in child s behaviour . fulfilling simple commands commands like  give mammy the toy  ,  put the book on the shelve  ,  put the ball into the bag  may be supported by the gesture but without presentation .
pointing as a response to a verbal request , e.g.  show me , where the flower is 
pointing items in a named colour , e.g. show the blue block ( among several basic colours : red , yellow , blue , green or white ) . matching the shapes in a big puzzle  into the holes in a desk :  
individual echoic words : any attempts to direct towards anybody vocal communication , any repeated voice reactions to the stimuli , etc .
repeating by heart digits in a line of two or three elements ( e.g. 74 , 582 ) .
does not count , has no idea of numbers counts to 5 : counts items in the limit of five counts to 10 : counts items in the limit of 10 .
 summarybackgroundmucopolysaccharidoses ( mps ) are inherited metabolic disorders caused by deficiencies in enzymes involved in degradation of glycosaminoglycans . 
 mps type iii ( sanfilippo disease ) is clinically characterized mainly by progressive and severe behavioral disturbances and cognitive dysfunction . 
 recent 1-year experimental treatment of 10 patients with a genistein ( 4 , 5 , 
 7-trihydroxyisoflavone)-rich extract resulted in improvement of tested parameters , including cognitive and behavioral functions.material/methodseight pediatric patients with sanfilippo disease were enrolled into the study . 
 the modified version of the brief assessment examination was used to assess cognitive functions . 
 moreover , 18 different parameters concerning changes in conditions of patients were assessed by their parents.resultsduring the first year of the treatment , an improvement of cognitive functions in 7 patients and stabilization in 1 patient were assessed , while after the third year ( 2-year follow - up ) further improvement was observed in 2 patients , stabilization in 3 patients and some deterioration in 3 patients . 
 monitoring of general and behavioral symptoms revealed improvement in all patients after the first year of the treatment , further improvement in 5 patients , and deterioration in 3 patients during the next 2 years.conclusionswe conclude that the treatment of sanfilippo patients with a genistein - rich soy isoflavone extract ( called gene expression - targeted isoflavone therapy [ get it ] ) may be effective in either inhibition ( in some patients ) or slowing down ( in other patients ) of behavioral and cognitive problems over a longer period . 
 an increased dose of genistein may improve the efficacy of the treatment . 
Background
Material and Methods
Patients
Treatment
The modified BAE test
Parents observations
Results
Discussion
Conclusions
A. The point scale
B. Description of the scale