First experience of combined enzyme replacement therapy and hematopoietic stem cell transplantation in alpha-mannosidosis.

We describe the first case of bridge therapy in alpha-mannosidosis (AM) in an infant diagnosed at only 5 months of life who underwent enzyme replacement therapy (ERT) in the pre- and peri-transplant phases. Eight ERT infusions were administered before hematopoietic stem cell transplantation (HSCT) and continued for additional 90 days until complete engraftment. The clinical and laboratory data after 3 years post-HSCT show that the early combined intervention may reduce the disease progression and the urine and plasma content of mannosyl-oligosaccharides (OS) monitored by liquid chromatography tandem mass spectrometry (LC-MS/MS). This report highlights that early diagnosis and prompt initiation of such treatments in AM are the best chance to minimize the progression of symptoms.

18-year follow-up of enzyme-replacement therapy in two siblings with attenuated mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase and characterized by a progressive course with multisystem involvement. Clinically, MPS I is divided into two forms: (1) severe (Hurler syndrome), which presents in infancy and is characterized by rapid progressive neurological involvement; (2) attenuated (Hurler/Scheie and Scheie syndromes), which displays a slower progression and absent to mild nervous system involvement. The specific treatment for attenuated MPS I consists of enzyme-replacement therapy with laronidase (human recombinant α-L-iduronidase, Aldurazyme). We present updated data after 18 years of laronidase treatment in two siblings affected by the attenuated form of MPS I who started therapy at 5 months and 5 years of age, respectively. Clinical and laboratory data of the siblings show that long-term enzyme replacement therapy may improve/stabilize many symptoms already present at the time of the diagnosis and reduce the disease progression. This study confirms that early diagnosis and early initiation of enzyme-replacement therapy are essential to modify positively the natural history of the attenuated form of MPS I.

Cardiac involvement in MPS patients: incidence and response to therapy in an Italian multicentre study.

BACKGROUND: Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders caused by the deficit of lysosomal hydrolases involved in the degradation of glycosaminoglycans (GAGs). The course is chronic and progressive, with multisystemic involvement that often leads to cardiovascular disease. We describe the overall incidence and type of cardiac damage in a cohort of Italian MPS patients, and their progression over time, also with reference to treatment efficacy in patients under Enzyme Replacement Therapy (ERT). Moreover, we report a possible association between genetic variants and cardiac phenotype in homozygous and hemizygous patients to understand whether a more aggressive clinical phenotype would predict a greater cardiac damage. RESULTS: Our findings confirm that cardiac involvement is very common, already at diagnosis, in MPS VI (85.7% of our cohort), and in MPS II (68% of our cohort) followed by MPS I subjects (55% of our cohort). The most frequent heart defect observed in each MPS and at any time-point of evaluation was mitral insufficiency; 37% of our patients had mitral insufficiency already at diagnosis, and 60% at post-ERT follow-up. After at least six years of treatment, we observed in some cases (including 6 MPS II, 2 MPS IV and 2 MPS VI) a total regression or improvement of some signs of the cardiac pathology, including some valve defects, though excluding aortic insufficiency, the only valvulopathy for which no regression was found despite ERT. The general clinical phenotype proved not to be strictly correlated with the cardiac one, in fact in some cases patients with an attenuated phenotype developed more severe heart damage than patients with severe phenotype. CONCLUSIONS: In conclusion, our analysis confirms the wide presence of cardiopathies, at different extent, in the MPS population. Since cardiac pathology is the main cause of death in many MPS subtypes, it is necessary to raise awareness among cardiologists about early cardiac morpho-structural abnormalities. The encouraging data regarding the long-term effects of ERT, also on heart damage, underlines the importance of an early diagnosis and timely start of ERT.

Early biochemical effects of velmanase alfa in a 7-month-old infant with alpha-mannosidosis.

Alpha mannosidosis is an ultrarare pathology with variable phenotypic manifestations, characterized by the deficiency of lysosomal alpha mannosidase which causes accumulation of neutral oligosaccharides. Until recently, the hematopoietic stem cell transplantation was the only clinical feasible therapeutic option. Only in 2018, the European Medicines Agency's committee approved the recombinant enzyme velmanase alfa for long-term treatment of non-neurological manifestations in mild and moderate forms of alpha-mannosidosis. In this study, the very early biochemical effects of enzyme replacement therapy in in a 7-month-old patient with alpha-mannosidosis were described. Velmanase alpha was administered as supporting therapy awaiting for hematopoietic stem cell transplantation, the treatment chosen for the patient because of the early onset form. The results showed that the enzyme replacement therapy was able to reduce the content of three different mannosyl-oligosaccharides monitored by tandem mass spectrometry after 2 months of treatment. In particular, the mean relative changes from baseline values were -67% in urine and -53% in serum at the latest observation. The study also showed that the enzymatic activity detected in serum 1 week after the first infusion was four times higher than the normal values and constant in the following points of observation. These findings led us to assume that velmanase alfa might be biologically active in this young patient.

Substrate reduction therapy with Miglustat in pediatric patients with GM1 type 2 gangliosidosis delays neurological involvement: A multicenter experience.

BACKGROUND: In GM1 gangliosidosis the lack of function of ß-galactosidase results in an accumulation of GM1 ganglioside and related glycoconjugates in visceral organs, and particularly in the central nervous system, leading to severe disability and premature death. In the type 2 form of the disease, early intervention would be important to avoid precocious complications. To date, there are no effective therapeutic options in preventing progressive neurological deterioration. Substrate reduction therapy with Miglustat, a N-alkylated sugar that inhibits the enzyme glucosylceramide synthase, has been proposed for the treatment of several lysosomal storage disorders such as Gaucher type 1 and Niemann Pick Type C diseases. However, data on Miglustat therapy in patients with GM1 gangliosidosis are still scarce. METHODS: We report here the results of Miglustat administration in four Italian children (average age: 55 months, range 20-125) affected by GM1 gangliosidosis type 2 treated in three different Italian pediatric hospitals specialized in metabolic diseases. CONCLUSION: This treatment was safe and relatively well tolerated by all patients, with stabilization and/or slowing down of the neurological progression in three subjects.

Human milk glycosaminoglycan composition from women of different countries: a pilot study.

Objective: In this pilot study, we report the composition, structure and properties of glycosaminoglycans (GAG) present in milk samples of various countries and ethnicities.Methods: Fifty samples of human milk were analyzed, 10 from east Europe, 10 from North Africa, 10 from Central Africa, 10 from South America and 10 from Asia. Moreover, 30 samples were obtained during the first week and 20 between 8 to 30 days of life.Results: Overall, no significant differences were observed for the qualitative composition of GAGs, mainly chondroitin sulfate, heparan sulfate and hyaluronic acid, comparing the mothers from the various countries and between the 30 milks obtained during the first week and the 20 samples collected thereafter. Moreover, no significant differences in human milk GAGs within the different groups analyzed belonging to various counties and ethnicities were observed.Conclusions: These results may be of useful, as in the case of pilot studies with infant formulas enriched with chondroitin sulfate (CS) and/or heparan sulfate (HS) necessary to verify their possible positive effects on newborns feeding in countries at high risk of infection and/or infestation.

Breast milk oligosaccharides: effects of 2'-fucosyllactose and 6'-sialyllactose on the adhesion of Escherichia coli and Salmonella fyris to Caco-2 cells.

Background: It is well known that human milk oligosaccharides play an important role as prebiotics, anti-inflammatory, and anti-infective agents. In the last few years, several studies have been performed using specific oligosaccharides, such as 2'-fucosyllactose and 6'-sialylactose, to evaluate their biological functions. Objectives: The aim of the present study is to evaluate the anti-adhesive effect of the above oligosaccharides on Escherichia coli and Salmonella fyris. Methods: Adhesion experiments were performed in the presence of 2'-fucosyllactose and 6'-sialyllactose as potential inhibitors of Escherichia coli and Salmonella fyris adhesion to Caco-2 cells. The oligosaccharides were used at different concentrations and the adhesion experiments were performed in triplicate and repeated at least three times. Results: A significant reduction of Escherichia coli adhesion was observed in the presence of 2'-fucosyllactose and 6'-sialyllactose at the human milk concentration. On the contrary, no positive effects were observed in both oligosaccharides on Salmonella firis. Conclusions: Our results suggest that the supplementation in infant formulas of 2'-fucosyllactose and 6'-sialyllactose, actually commercially available and absent in cow milk, could play positive effects in artificially fed infants.

False positive screen test for mucopolysaccharidoses in healthy female newborns.

BACKGROUND: In total, 930 urine samples obtained on 2nd and 3rd day from birth have been analyzed for the early diagnosis of Mucopolysaccharidoses. METHODS: Dimethylmethylene blue (DMB) assay and one-dimensional electrophoresis were performed in all urine samples. Agarose gel electrophoresis, before and after treatment with chondroitinase ABC and heparinases, was used for a comprehensive characterization. RESULTS: Out of 930 urine samples 7 showed anomalous electrophoretic pattern; 5 of them had high GAG levels by DMB test. Atypical samples (n = 7) were analyzed by agarose gel electrophoresis. After enzymatic digestion, some slow bands were still visible. A second urine sample of the above 7 newborns was analyzed at the age of 1 month, demonstrating both a normal pattern and normal GAG levels. Additional urine and vaginal mucus samples from 10 term neonates with vaginal bleeding showed the same electrophoretic pattern observed in the 7 false positive samples. CONCLUSIONS: The altered electrophoretic pattern may be due to the presence of glycoproteins and not to specific GAGs, due to high levels of maternal hormones exposure during pregnancy. To our knowledge, this is the first time maternal estrogen hormones are proposed as a likely cause of false-positive urinary glycosaminoglycan screen test in healthy newborns.

Composition and structure of glycosaminoglycans in DBS from 2-3-day-old newborns for the diagnosis of mucopolysaccharidosis.

Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 µg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types.

Age-related modulation of plasmatic beta-Galactosidase activity in healthy subjects and in patients affected by T2DM.

ß-Galactosidase (ß-Gal) activity has been the most extensively utilized biomarker for the detection of cellular senescence. It can be measured also in plasma, and few recent evidence showed an altered plasmatic ß-Gal activity in patients affected by some age-related diseases (ARDs). Since T2DM is one of the most common ARDs, we aimed to investigate if plasmatic ß-Gal activity is modulated in T2DM patients and if "age" could affect such modulation. To gain mechanistic insights we paralleled this investigation with the evaluation of ß-Gal activity in young and senescent endothelial cells (HUVECs) cultured in normo- and hyper-glycaemic environment. A significant age-related increase of plasmatic ß-Gal activity was observed in healthy subjects (n. 230; 55-87 years), whereas the enzymatic activity was significantly reduced in T2DM patients (n. 230; 55-96 years) compared to healthy subjects. ß-Gal activity detectable both in cells and in the culture medium was significantly increased in senescent cells compared to the younger ones, both under normo- and hyper-glycaemic condition. However, the hyper-glycaemic condition was not associated with an increased ß-Gal activity in milieu compared to normo-glycaemic condition. Overall our data reinforce the notion that plasmatic ß-Gal activity could be a systemic biomarker of aging, whereas T2DM patients are characterized by a different age-releated trend.

Importance of the combined urinary procedure for the diagnosis of Mucopolysaccharidoses.

BACKGROUND: Mucopolysaccharidoses are characterized by the accumulation of undegraded glycosaminoglycans in lysosomes in multiple organs and by their excretion in high amounts in urine. The aim of this study is to determine if this simple, reliable and reproducible method is useful for the diagnosis of Mucopolysaccharidoses. METHODS: The study included 2154 normal urine samples and 210 samples from 73 patients affected by different types of Mucopolysaccharidoses. The glycosaminoglycans were quantified by a dimethylmethylene blue method and size-fractionated by a modified one-dimensional electrophoresis method. RESULTS: The combination of the two methods allowed to identify all the patients affected by the different types of Mucopolysaccharidosis with 100% sensitivity and specificity. CONCLUSION: This combined approach gives fast diagnostic orientation about the different types of Mucopolysaccharidoses, offering an important tool for a better understanding of diagnosis and patient management.

Total and single species of uronic acid-bearing glycosaminoglycans in urine of newborns of 2-3days of age for early diagnosis application.

BACKGROUND: Urine are easily accessible and relatively simple to process and uronic acid-bearing glycosaminoglycans (UA-GAGs) may serve as biomarkers for several diseases, like for mucopolysaccharidosis. METHODS: We report a study from a large cohort of healthy newborns of 2-3days to have a basic profile of total content of urinary UA-GAGs, their composition and structural signatures utilizing a rapid extractive method and sensitive separation of enzymatic released disaccharides by capillary electrophoresis-light induced fluorescence. Results were also compared with those obtained from normal adult subjects. RESULTS: A total of UA-GAGs content of ~35µg/mg creatinine was observed in 331 newborns versus 1.5µg/mg creatinine of adult urine composed of ~90% chondroitin sulfate (CS), ~7% heparan sulfate (HS) and ~3% hyaluronic acid (HA). No significant differences were observed with adults. Specific ratios between the main CS disaccharides were informative of a significant greater 4-sulfation and charge density for newborn compared to adults. The HS from newborn urine was mainly composed by the non-sulfated (~64%) and mono-sulfated (~28%) disaccharides. No significant differences were observed versus adult urine. CONCLUSIONS: The present method is able to measure changes in UA-GAG composition and their structure independently of the age of subjects and rapidly applicable to the newborn diagnosis without necessity to have creatinine levels. Moreover, modifications in charge density values as well as the presence of sulfate groups in specific positions may be indicative of altered conditions.

12 year follow up of enzyme-replacement therapy in two siblings with attenuated mucopolysaccharidosis I: the important role of early treatment.

BACKGROUND: Mucopolysaccharidosis type I is an autosomal recessive disorder caused by deficiency of α-L-iduronidase and characterized by a progressive course with multisystem involvement. Clinically, Mucopolysaccharidosis type I is classified into two forms: severe (Hurler syndrome), which presents in infancy and is characterized by rapid progressive neurological involvement and attenuated (Hurler/Scheie and Scheie syndromes), which presents with slower progression and absent to mild nervous system involvement. The specific treatment for attenuated Mucopolysaccharidosis type I consists of enzyme-replacement therapy with laronidase (human recombinant α-L-iduronidase, Aldurazyme). We present here the clinical and laboratory results in an 12-year-old patient affected by the attenuated form of Mucopolysaccharidosis type I treated by enzyme-replacement therapy from the age of 5 months, compared with his 17 year old affected sister, who started therapy at 5 years of age. CASE PRESENTATION: Clinical evaluation of these siblings shows that initiation of therapy prior of the onset of clinically detectable disease resulted in considerable improvement in outcome in the young sibling. After 12 years of enzyme-replacement therapy, facial appearance, linear growth rate, and liver and spleen volumes were normal; moreover, the degree of joint disease, vertebral, and cardiac valvular involvement were only minimal compared with those of his sister. CONCLUSION: This study demonstrates that early diagnosis and early initiation of enzyme-replacement therapy substantially modify the natural history of the attenuated form of Mucopolysaccharidosis type I.

Metabolic fate of milk glycosaminoglycans in breastfed and formula fed newborns.

In this study, the content, structure and residual percentages of glycosaminoglycans (GAGs) in the feces of seven breastfed newborns after ingesting a known amount of milk were studied. A comparison was made with five newborns fed with formula milk. Characterization of GAGs from milk and feces samples was performed according to previous methodology. Compared to the ingested GAGs present in milk, residual feces GAGs of breastfed newborns were <0.4 %, contrary to formula milk fed children, where the residues were ~4 %. As a consequence, >99 % of human milk GAGs are utilized as opposed to ~96 % of formula milk. Hyaluronic acid utilization was found to be fairly similar contrary to chondroitin sulfate/dermatan sulfate and heparan sulfate, which were found to be ~10-18 times lower in formula milk fed children. Our new results further demonstrate that the elevated content of human milk GAGs passes undigested through the entire digestive system of newborns, possibly protecting the infant from infections. In the distal gastrointestinal tract, these complex macromolecules are catabolized by a cohort of bacterial enzymes and constituent monosaccharides/oligosaccharides utilized for further metabolic purposes potentially useful for bacteria metabolism or internalized by intestinal cells. Thanks to their elevated structural heterogeneity, milk GAGs are used differently depending on their distinct primary structure. Finally, a different utilization and availability was observed for human milk GAGs compared to formula milk due to their various composition and structural heterogeneity.

Human milk glycosaminoglycans inhibit in vitro the adhesion of Escherichia coli and Salmonella fyris to human intestinal cells.

BACKGROUND: Breast-fed infants have a lower incidence of acute gastroenteritis due to the presence of several anti-infective factors in human milk. The aim of this work is to study the capacity of human milk glycosaminoglycans (GAGs) to inhibit the adhesion of some common pathogenic bacteria. METHODS: GAGs were isolated from a pool of milk samples collected from different mothers during the first month of lactation. Experiments were carried out to study the ability of GAGs to inhibit the adhesion of two intestinal micro-organisms (enteropathogenic Escherichia coli serotype 0119 and Salmonella fyris) to Caco-2 and Int-407 cell lines. RESULTS: The study showed that the GAGs had an anti-adhesive effect on the two pathogenic strains studied with different degrees of inhibition. In particular, in the presence of human milk GAGs, the adhesion of S. fyris to Caco-2 cells and to Int-407 cells of both tested strains was significantly reduced. CONCLUSION: Our results demonstrated that GAGs in human milk can be one of the important defensive factors against acute diarrheal infections in breast-fed infants.

Effect of holder pasteurisation on human milk glycosaminoglycans.

OBJECTIVES: The benefits of human milk for preterm infants are mainly the result of its nutritional characteristics and the presence of biologically active compounds. Among these compounds, glycosaminoglycans (GAGs) play an emerging leading role. When mother's milk is unavailable or in short supply, pasteurised donor milk represents an important nutritional alternative. The aim of this study was to evaluate the effect of Holder pasteurisation on the concentration of different GAGs in preterm human milk. METHODS: Milk samples collected from 9 mothers having delivered preterm were divided into 2 parts. One part of each sample was immediately frozen (-80°C), whereas the other part was pasteurised with the Holder method before being frozen at -80°C. Specific analytical procedures were applied to evaluate the amount, composition, and structure of main human milk GAGs. RESULTS: No significative differences were measured between not-treated and pasteurised samples for total GAGs content, relative percentages of chondroitin sulfate and heparan sulfate, and main parameters related to galactosaminoglycans structure, even if a slight decrease of total GAGs content of ∼18% was observed in treated samples. CONCLUSIONS: Our results indicate that the Holder pasteurisation does not significatively affect the concentration of the main human milk GAGs.

Multiple sulfatase deficiency with neonatal manifestation.

Multiple Sulfatase Deficiency (MSD; OMIM 272200) is a rare autosomal recessive inborn error of metabolism caused by mutations in the sulfatase modifying factor 1 gene, encoding the formylglycine-generating enzyme (FGE), and resulting in tissue accumulation of sulfatides, sulphated glycosaminoglycans, sphingolipids and steroid sulfates. Less than 50 cases have been published so far. We report a new case of MSD presenting in the newborn period with hypotonia, apnoea, cyanosis and rolling eyes, hepato-splenomegaly and deafness. This patient was compound heterozygous for two so far undescribed SUMF1 mutations (c.191C > A; p.S64X and c.818A > G; p.D273G).