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Abstract Mucopolysaccharidosis type II (MPS II) is a neurometabolic disorder, due to the deficit of the lysosomal hydrolase iduronate 2-sulfatase (IDS). This leads to a severe clinical condition caused by a multi-organ accumulation of the glycosaminoglycans (GAGs/GAG) heparan- and dermatan-sulfate, whose elevated levels can be detected in body fluids. Since 2006, enzyme replacement therapy (ERT) has been clinically applied, showing efficacy in some peripheral districts. In addition to clinical monitoring, GAG dosage has been commonly used to evaluate ERT efficacy. However, a strict long-term monitoring of GAG content and composition in body fluids has been rarely performed. Here, we report the characterization of plasma and urine GAGs in Ids knock-out (Ids-ko) compared to wild-type (WT) mice, and their changes along a 24-week follow-up, with and without ERT. The concentration of heparan-sulfate (HS), chondroitin-sulfate (CS), and dermatan-sulfate (DS), and of the non-sulfated hyaluronic acid (HA), together with their differentially sulfated species, was quantified by capillary electrophoresis with laser-induced fluorescence. In untreated Ids-ko mice, HS and CS + DS were noticeably increased at all time points, while during ERT follow-up, a substantial decrease was evidenced for HS and, to a minor extent, for CS + DS. Moreover, several structural parameters were altered in untreated ko mice and reduced after ERT, however without reaching physiological values. Among these, disaccharide B and HS 2s disaccharide showed to be the most interesting candidates as biomarkers for MPS II. GAG chemical signature here defined provides potential biomarkers useful for an early diagnosis of MPS II, a more accurate follow-up of ERT, and efficacy evaluations of newly proposed therapies. Key messages Plasmatic and urinary GAGs are useful markers for MPS II early diagnosis and prognosis. CE-LIF allows GAG structural analysis and the quantification of 17 different disaccharides. Most GAG species increase and many structural features are altered in MPS II mouse model. GAG alterations tend to restore to wild-type levels following ERT administration. CS+DS/HS ratio, % 2,4dis CS+DS, and % HS 2s are potential markers for MPS II pathology and ERT efficacy.

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 o...

Elevated levels of oxidative nucleic acid modifications have been proposed to be associated with some of the clinical characteristics of Down syndrome. Oral intake of coenzyme Q10 improves oxidative status and shows a tendency toward protective effect on DNA oxidation in certain age groups of children with Down syndrome. Here, we demonstrate that long-term (i.e., 4 years) treatment with coenzyme Q10 (ubiquinone) at the dosage of 4 mg/kg/d does not affect whole body DNA and RNA oxidation.

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.

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.

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.

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.

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.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.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.Our results demonstrated that GAGs in human milk can be one of the important defensive factors against acute diarrheal infections in breast-fed infants.

Since 2005, the Pediatric Clinic of Maternal-Infantile Sciences Institute in Ancona, in collaboration with the Lega del Filo d'Oro in Osimo, has been taking care of 35 patients with clinical and molecular diagnosis of CHARGE syndrome. Our investigation is the largest Italian cohort study of CHARGE patients. CHARGE syndrome is a multiple malformation syndrome involving ocular coloboma, heart defects, choanal atresia, retardation of growth and\or development, genital anomalies and\or urinary and ear abnormalities which leads to visual-auditory disabilities, cognitive impairment and behavioral abnormalities. Our purpose is to expand the knowledge of this syndrome by reviewing this group of affected patients in order to delineate in detail the natural history of the disease, and in particular to define the cognitive and motor profiles using an Italian questionnaire called "Progress Guide". Our main results show that Italian CHARGE patients have more delayed development in their physical abilities or skills with respect to normal patients. In particular, the delay is statistically significant in regard to self-care skills (worse toileting, better washing) and the communication skill (language). On the other hand, the expressive skills are still preserved. When patients are considered according to their age (≤3 years) and (>3 years), the older ones have more delayed development than the younger ones when compared with healthy individuals of the same age.

Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high-voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2-aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on-capillary anionic borate-polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ∼50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.

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.

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 2–3 days 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 ~ 35 μg/mg creatinine was observed in 331 newborns versus 1.5 μg/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.

Chondroitin sulphate (CS) was purified for the first time from the Italian cheese Parmigiano-Reggiano and analysed to evaluate its structure and properties. Two main polysaccharides were identified as CS, ∼72%, and fast moving-heparin/heparan sulphate, ∼28%. Quantitative analyses yielded ∼1.5–3.0 μg of total GAGs per gram of Parmigiano-Reggiano (0.15–0.30%). By means of specific chondroitinases and HPLC separation of generated unsaturated disaccharides, CS was found to be composed of ∼9% of nonsulphated disaccharide, ∼26% of disaccharide monosulphated in 6 of the GalNAc, ∼65% of disaccharide monosulphated in 4 of the GalNAc, with a charge density of ∼0.91 and a 4/6-sulphated ratio of 2.45. The ratio of 4/6 sulphated residues was confirmed by 13C NMR experiments. Susceptibility to cleavage catalysed by chondroitinase B confirmed that the purified Parmigiano-Reggiano CS contains mainly GlcA (∼94%) as uronic acid. PAGE analysis showed a CS having a molecular mass with an average value of 15400.

Enzyme-replacement therapy (ERT) is a new option for the clinical management of MPS I. However, no detailed data are available on the structural characterization of glycosaminoglycans (GAGs) in the urine and plasma of patients before ERT and during treatment regimens. Before ERT and over a two-week period of enzyme infusion, GAGs in urine and plasma were analyzed in two patients with the Hurler-Scheie form of MPS I subjected to ERT for 6 years. In both patients before ERT, high amounts of a GAG were found in the urine, composed in particular of a high molecular mass polymer (approximately 13,000-13,500) consisting of approximately 75-78% iduronic acid and rich in 4-sulfated disaccharides (DeltaDi4s) and attributable to DS. Furthermore, a high amount of this GAG was directly detected in the blood. Plasma GAGs in MPS I patients subjected to ERT were found to be comparable to those of normal subjects with the absence of heparan sulfate and of DS. On the contrary, a polysaccharide possessing a high molecular mass, approximately 11,500-12,000, lower than the polymer extracted before ERT but slightly higher than the controls (approximately 11,000), was found in the urine of both patients. This macromolecule was characterized as a mixture of DS/chondroitin sulfate based on the high percentage of 4-sulfated disaccharide (4s/6s ratio of approximately 3.1) and iduronic acid ( approximately 60%). These results are indicative of the incapacity of ERT at the standard dose to definitively eliminate DS from the urine. Finally, a variable effect of ERT depending on each administration was also observed.

Mucopolysaccharidosis type I (MPS I) is a progressive and multisystemic disease, even in its attenuated Hurler-Scheie and Scheie forms. Clinical trials of enzyme-replacement therapy in MPS I have shown clinical benefit in patients with considerable preexisting disease, but no data exist on the effect of beginning enzyme replacement before the onset of significant clinical signs of disease. Here we present the 5-year follow-up of a boy with attenuated MPS I who had laronidase therapy initiated at the age of 5 months and compare his clinical course to that of his older sister, who began treatment at 5 years of age after she had developed typical signs of MPS I. After 5 years of treatment, the younger sibling has not developed any clinical manifestations of MPS I except for mild corneal clouding. In contrast, although many of the older sibling's clinical features have improved after 5 years of treatment, her dysostosis multiplex, cardiac valve involvement, and corneal clouding, although stabilized, have persisted. We suggest that early treatment of attenuated MPS I may significantly delay or prevent the onset of the major clinical signs, substantially modifying the natural history of the disease.

We report on a 4-year-old boy with mental retardation, facial and skeletal anomalies, cerebral angiomas, femoral nucleus necrosis, mild biochemical abnormalities. This complex of features resembles the Hall-Riggs syndrome but could represent a novel syndrome.

Down syndrome (DS) is a chromosomal abnormality (trisomy 21) associated with mental retardation and Alzheimer-like dementia, characteristic change of the individual's phenotype and premature ageing. Oxidative stress is known to play a major role in this pathology since a gene dose effect leads to elevated ratio of superoxide dismutase to catalase/glutathione peroxidase compared to controls in all age categories suggesting that oxidative imbalance contributes to the clinical manifestation of DS. Hyperuricemia is another feature of DS that has an interesting relationship with oxidative stress since uric acid represents an important free radical scavenger. However its formation is connected to the conversion of Xanthine dehydrogenase (XDH) to Xanthine oxidase (XO) which leads to concomitant production of free radicals. Here we report that plasma samples from DS patients in pediatric age, despite an increased total antioxidant capacity, largely due to elevated Uric acid content (UA), present significantly elevated markers of oxidative damage such as increased allantoin levels. Moreover DS plasma samples do not differ from healthy control ones in terms of Coenzyme Q10 and susceptibility to peroxidative stimuli. On the contrary, lymphocyte and platelet CoQ10 content was significantly lower in DS patients, a fact that might underlie oxidative imbalance at a cellular level.

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 were0.4 %, 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.

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.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.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.Our results indicate that the Holder pasteurisation does not significatively affect the concentration of the main human milk GAGs.

Background Twenty-five patients with Niemann Pick disease type C (age range: 7 months to 44 years) were enrolled in an Italian independent multicenter trial and treated with miglustat for periods from 48 to 96 months. Methods Based on the age at onset of neurological manifestations patients’ phenotypes were classified as: adult (n = 6), juvenile (n = 9), late infantile (n = 6), early infantile (n = 2). Two patients had an exclusively visceral phenotype. We clinically evaluated patients’ neurological involvement, giving a score of severity ranging from 0 (best) to 3 (worst) for gait abnormalities, dystonia, dysmetria, dysarthria, and developmental delay/cognitive impairment, and from 0 to 4 for dysphagia. We calculated a mean composite severity score transforming the original scores proportionally to range from 0 to 1 to summarize the clinical picture of patients and monitor their clinical course. Results We compared the results after 24 months of treatment in 23 patients showing neurological manifestations. Stabilization or improvement of all parameters was observed in the majority of patients. With the exception of developmental delay/cognitive impairment, these results persisted after 48–96 months in 41 – 55% of the patients (dystonia: 55%, dysarthria: 50%, gait abnormalities: 43%, dysmetria: 41%, respectively). After 24 months of therapy the majority of the evaluable patients (n = 20), demonstrated a stabilization or improvement in the ability to swallow four substances of different consistency (water: 65%, purée: 58%, little pasta: 60%, biscuit: 55%). These results persisted after 48–96 months in 40-50% of patients, with the exception of water swallowing. Stabilization or improvement of the composite severity score was detected in the majority (57%) of 7 patients who were treated early (within 3.5 years from onset) and rarely in patients who received treatment later. Conclusions The results of this study suggest that miglustat treatment can improve or stabilize neurological manifestations, at least for a period of time; the severity of clinical conditions at the beginning of treatment can influence the rate of disease progression. This conclusion applies particularly to patients with juvenile or adult onset of the disease. Trial registration EudraCT number 2006-005842-35 Electronic supplementary material The online version of this article (doi:10.1186/s13023-015-0240-y) contains supplementary material, which is available to authorized users.

TGF-beta-receptor 2 (TGFBR2) gene defects have been recently associated with Marfan syndrome (MFS) with prominent cardio-skeletal phenotype in patients with negative fibrillin-1 (FBN1) gene screening. Four mutations have been identified to date in five unrelated families. We screened TGFBR2 gene by direct automated sequencing in two adult patients diagnosed with MFS according to Ghent criteria, and in one girl clinically suspected as affected on the basis of a major cardiovascular criterion and skeletal involvement, all proven not to carry mutations in the exon-intron boundaries of FBN1 gene. We identified two novel and one known TGFBR2 gene mutations in the three unrelated probands. The D446N was identified in a 4-year-old girl with de novo disease characterized by severe cardiovascular disease and skeletal involvement. The M425V and R460H mutations were identified in two familial, autosomal dominant MFSs, both characterized by major cardio-skeletal signs and absence of major ocular signs. The mutation R460H has been recently reported in a family with thoracic aortic aneurysms and dissection. The three mutations are absent in 192 controls and affect evolutionarily conserved residues of the serine/threonine kinase domain (exon 5). Our data support the recently reported association between TGFBR2 gene and MFS without major ocular signs (MFS2). The number of genotyped cases however is too low to confirm that major ocular signs are characteristically absent in MFS2. Accordingly, all patients proven or suspected to be affected by MFS with negative FBN1 gene screening could benefit from rapid investigation of the TGFBR2 gene.

Single cell gel electrophoresis (SCGE), also known as comet assay is a widely used method to detect DNA damage. Its use is nonetheless subjected to some pitfalls, due to differences in experimental set-up, to operator-dependent variability and to quantification of the comets, which is usually accomplished by visual scoring or by image-analysis software. Biological variability in the extent of DNA damage must be taken into account particularly regarding in vivo studies. In the present paper we propose an improved methodology where major features are: a) cryopreservation of lymphocytes collected at different time points and simultaneous analysis in a single run; b) use of an internal control on each slide; c) development of a custom-made software with semi - automated image analysis in order to overcome operator dependent variability. Cryopreservation was accomplished by storing lymphocytes in liquid nitrogen in a solution commonly used for preserving vital cells to be reinfused. We found that this procedure did not alter DNA after 2 and 4 months of storage. The use of quality control from a batch of aliquoted lymphocytes from a healthy donor on each slide, enabled to highlight possible experimental anomalies as well as verify inter-experimental variability. Moreover, by using a newly developed software able to automatically recognise comets we minimised operator-dependent variability in the scoring process. This improved methodology is proposed for longitudinal in vivo studies and in the present work its application made it possible to assess a significant increase of DNA in pediatric Down Syndrome patients compared to healthy controls of the same age.

The Sanfilippo type A syndrome, one of the most frequent forms of mucopolysaccharidosis III, is characterized by severe mental retardation, progressive neurological degeneration, and mild somatic changes. It is due to a deficiency of heparan-N-sulfatase (sulfamidase) activity and consequent excretion of heparan sulfate in the urine. The disease is transmitted through an autosomal recessive mechanism, and more than 60 gene mutations have been identified. Up to now, only 10 cases of attenuated form of Sanfilippo type A syndrome have been described, and the specific mutation has been identified only in two of them. We report here on a female patient, 20 years old, with Sanfilippo type A syndrome presenting with a mild clinical phenotype characterized essentially by a moderate nonevolving mental retardation. The genetic analysis demonstrated that the patient is homozygous for mutation R206P; presence of polymorphism R456H was also found. This study places R206P as a mild mutation underlying Sanfilippo type A disease.

The aim of this study was to evaluate the frequency of callosal abnormalities and white matter alterations in syndromic patients. The authors report on the cerebral magnetic resonance imaging (MRI) morphologic analysis of the corpus callosum and white matter in 73 normal subjects and 61 syndromic patients. The study of the corpus callosum was carried out by MRI using different morphometric methods: measurement of the dimensions of length and thickness of genu, body, and splenium; measurement of angles obtained using the sagittal plane; and application of the proportional grid of Talairach. The evaluation of the white matter was carried out by applying a subjective grading scale. Abnormalities of the corpus callosum were found in about 50% of the syndromic subjects; in half of these cases, the abnormalities were associated with white matter alterations. In five syndromic patients (8.2%), the white matter alterations were not associated with corpus callosum abnormalities. This study shows that corpus callosum abnormalities are frequent in syndromology regardless of the syndrome type. (J Child Neurol 2002;17:164-168).

The characteristic composition of the intestinal microbiota of breast-fed neonates is due to the presence of particular substances in human milk. This chapter aims to identify the components that are able to promote the prebiotic effect in human milk, and is oriented towards modifying the composition of infant formulas in order to obtain an intestinal microbiota similar to that of breast-fed babies. It discusses history, synthesis, structure, metabolism of human milk oligosaccharides (HMO). No natural substances have the same biochemical composition as that of HMO, nor can they be synthesized in large quantities at acceptable prices. To overcome these problems, the industry has focused on the production of several carbohydrates, so-called nondigestible oligosaccharides (NDO), which, although having compositions different from those of HMO, are able to selectively stimulate the growth of bifidobacteria and lactobacilli in the colon, reproducing the prebiotic effects of HMO. In conclusion, data related to the use of NDO in infant formulas clearly show the efficacy of galactooligosaccharides (GOS)/inulin mixtures, the capacity of GOS to induce a bifidogenic effect, even if in only a small number of studies, and the positive effects of FOS at a concentration of 4.0 g/liter on the intestinal microbiota. It has been demonstrated that, due to the presence of resistant bonds in their molecules, NDO are able to exert a prebiotic effect. This further confirms that, because of their peculiar structure, HMO have a very significant role in modulating the intestinal microbiota of neonates.

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).

Much evidence has been obtained that several human milk glycans (glycoproteins, glycolipids, and especially oligosaccharides), behaving as cell surface receptor homologs, are able to inhibit binding of pathogens and thus protect the newborn against several enteric infections. Moreover, recent studies have focused attention on human milk complex sulfated polysaccharides, the glycosaminoglycans (GAGs), as well as their structure and their possible biological roles. However, no data are yet available regarding the metabolic fate of GAGs and their possible presence in the feces of breastfed newborns or regarding their composition and structure, which would be useful to shed light on their metabolism. Following written parental consent, morning fecal samples were obtained from 12 term breastfed newborns (six females, six males) between days 5 and 33 postpartum (mean– SD, 13.92– 7.89 days). Samples were immediately stored at 20 C and then lyophilized. GAGs were were extracted and purified, and disaccharide mapping was determined according to previous methodology to obtain GAG structural characterization. We determined the presence of chondroitin sulfate (CS) (approximately 67%) and dermatan sulfate (approximately 4%) for a total content of approximately 32 mg/100 g of feces, and the total amount of heparan sulfate (HS)/heparin (Hep) (approximately 29%) was approximately 13 mg/100 g of feces. Disaccharide evaluation of CS showed the presence of a large percentage of disaccharide sulfated in position 4 with a lower content of disaccharide sulfated in position 6 and of the nonsulfated disaccharide for a charge density value of approximately 0.81 ( T1c Table 1). The nonsulfated disaccharide 4,5-unsaturated uronic acidN-acetylglucosamine, typical of HS, was found to constitute the main species (approximately 28%) present in feces along with the three main monosulfated (approximately 23%), the three main disulfated (approximately 35%), and the trisulfated (approximately 14%)disaccharide typical ofHep (Table 1). Because of this peculiar disaccharide composition, the HS extractedfromhumannewbornfeceswasfoundtohaveacharge density value (approximately 1.40) similar to the polymer characterized in human milk showing a high content of disulfated and trisulfated disaccharides and a high charge density. Our results demonstrate the presence in the feces of breastfed newborns of a highly sulfated HS molecule rather similar in structure and disaccharide composition to the polyanion characterized in human milk. However, the CS structure was found to be different, in particular because of a higher content of 4-sulfated disaccharide and a lower contribution of the nonsulfated species for a generally high charge density, with respect to CS from human milk (0.81 vs. 0.36 of human milk). This primary structure of feces CS may be the result of the partial absorption of its chains, in particular those lacking in sulfate groups as demonstrated in previous studies. In fact, the partial (and preferential) absorption of CS chains/oligomers may be the result of its degradation by enzymes produced by intestinal bacteria, such as chondroitinases produced by Bacteroides strains from the human colon able to degrade CS of various structures exhibiting broad specificity. The presence of Bacteroides species from the human intestine suggests that the catabolism of human milk GAGs might partially take place in newborns. However, we still found the presence in newborns’ feces of CS and, in particular, HS/Hep structurally similar to those evaluated in human milk, demonstrating that a part of these polymers could be excreted as unmodified macromolecules. This is new information enabling us to understand more fully the role and the presence of these complex macromolecules in human milk and their relationship with the health of newborns. In fact, Newburg et al. have demonstrated that GAGs in human milk are able to inhibit the binding of the human immunodeficiency virus envelope glycoprotein gp120 to the cellular CD4 receptor. From these preliminary data, we can suppose that human milk GAGs, in particular HS/Hep, pass partially undigested through the entire digestive system of newborns, acting as possible soluble receptors able to inhibit the binding of different pathogens to the intestinal mucosa, thus protecting the infant from infections. However, although feces HS/Hep seems likely to be derived from ingested milk, the origin or potential modification

The priming activity on polymorphonuclear leukocytes (PMLs) of nasal lavage fluid obtained from children with seasonal allergic rhinitis after specific allergic challenge, before and after a 14-day treatment period with orally administered oxatomide 12.5 mg twice daily, was investigated by means of chemiluminescence. Luminol-amplified chemiluminescence of PMLs activated with N-formylmethionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA) was evaluated in the presence or absence of nasal lavage fluid. A significant increase in the chemiluminescence response of fMLP-stimulated PMLs was induced by nasal lavage fluid. No significant change was observed in PMA-stimulated PMLs. Oxatomide treatment significantly reduced the effect of nasal lavage fluid on PMLs. These data suggest a priming potency on PMLs of nasal lavage fluid obtained from patients with seasonal allergic rhinitis after specific challenge, probably because of the presence of various inflammatory mediators. Oxatomide treatment significantly reduced this priming capacity. This effect may be attributed to the inhibition of cellular recruitment or to the reduction of mediator release at the inflammation site.

Objective: To evaluate the anomalies of the central nervous system (CNS) by magnetic resonance imaging (MRI) in normal subjects and in syndromic patients. Methods and material: Seventy-three normal subjects and 50 different syndromic patients with mental retardation (from 3 months to 16 years) were studied utilizing several morphometric parameters (degree of myelination of the white matter, evaluation of liquoral spaces, septo-caudate distance, Evans index, Aboulezz method, and length, width and angles of corpus callosum). Results: A high frequency of anomalies of the corpus callosum, the Chiari anomaly and alterations either of the white matter or of the ventricular and periencephalic system have been observed. Conclusion: The authors point out the importance of cerebral MRI in the study of CNS in patients with malformation syndromes. The present research, carried out on a large number of both normal subjects and patients with malformation syndromes, represents one of the first systematic studies in this field.

Morquio A syndrome (Mucopolysaccharidosis type IVA) (MPS IVA) is a rare inherited metabolic disorder characterized by the defective degradation of keratan sulfate and chondroitin-6-sulfate. Classically, MPS IVA patients present with severe multisystemic involvement and have a short life expectancy. Attenuated forms with clinical features limited to minor skeletal abnormalities and short stature have also been described, sometimes associated to an early-onset osteoporotic phenotype. No treatment with allogenic bone marrow transplantation or gene therapy is currently available for Morquio A syndrome, and enzyme replacement therapy is under evaluation. We report a case of MPS IVA, who manifested tardily attenuated phenotype and significant bone mass reduction, which was treated with a bisphosphonate (BPN), resulting in an improvement of X-ray skeletal aspects and functional bone performance. We suggest that the use of bisphosphonates may be an interesting supportive therapeutic option for Morquio A patients with osteoporotic phenotype, but further studies involving more patients are necessary to confirm our findings.

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant disorder characterized by high penetrance, widely variable expressivity and occurrence of specific skeletal changes such as tibial osteopathy (TO). We collected data on patients referred to the Italian Neurofibromatosis Study Group in order to compare clinical features between 49 NF1 patients with TO, and 98 age-matched NF1 patients without TO, and to determine whether the presence of TO is associated with a different risk of developing the typical NF1 complications. We assessed both groups for: age at diagnosis of NF1, gender distribution, family history, gender inheritance, presence of scoliosis, sphenoid wing osteopathy, other skeletal abnormalities, macrocrania, hydrocephalus, plexiform neurofibromas, tumors, optic pathway gliomas, T2H (high-signal intensity areas on T2 weighted brain MRI), epilepsy, headache, mental retardation, cardiovascular malformations, and Noonan phenotype. Patients of both groups were subdivided by gender and re-evaluated for these items. Statistical comparison was carried out between the two groups of patients for each feature. We collected data on type of treatment and on the clinical conditions of NF1-TO patients after follow-up. Patient's age at NF1 diagnosis was significantly younger in NF1-TO subjects compared with NF1 subjects without TO, and the incidence of T2H was significantly reduced in NF1-TO males compared with NF1 males without TO. The presence of TO does not imply that there is an increased risk of developing typical complications of NF1 (e.g., optic pathway glioma, plexiform neurofibroma, etc.), however, it does allow us to make an earlier diagnosis of NF1. © 2013 Wiley Periodicals, Inc.

Objectives We report the case of a 28-year-old female subject affected by the attenuated phenotype of mucopolysaccharidosis type IIIA characterized by moderate slowly evolving mental retardation in which the urinary content of heparan sulfate was demonstrated as being substantially low compared to that found in patients with the severe phenotype. Design and methods The specific evaluation of macromolecular heparan sulfate by electrophoresis and the determination of related glucosamine in the urine were performed. Results In our patient, the urinary macromolecular heparan sulfate content (4.2 μg/mg creatinine) was ~ 7.5-times higher than in healthy subjects (0.56 μg/mg creatinine ± 0.9 SD) while it was ~ 28-times lower compared to the severe mucopolysaccharidosis IIIA group (117 μg/mg creatinine ± 44.8 SD). Furthermore, the urinary glucosamine (86.4 μg/mg creatinine) was ~ 2.4-times greater than in healthy subjects (36.0 μg/mg creatinine ± 18.2 SD) but ~ 2.4-times lower than in severe subjects (208.1 μg/mg creatinine ± 55.0 SD). Conclusions The above data could reflect the reduced heparan sulfate storage in her tissues and organs, and in particular in the brain, consequently explaining her moderate mental retardation. Furthermore, the clinical presentation of patients with an attenuated form of MPS III confirms the need for a specific evaluation of urinary GAGs in all young and adult subjects showing a not well-defined or not particularly severe mental retardation, along with an early MPS diagnosis. Such investigation should also be associated with a more specific characterization of heparan sulfate.

Enzyme replacement therapy (ERT) is the worldwide standard of care for a number of mucopolysaccharidosis (MPS) diseases. We report a kinetic study of plasmatic dermatan sulfate (DS) in a 3-year-old subject affected by a severe form of MPS II during the first 10 months of ERT with Idursulfase. A strong increase in the DS plasmatic concentration was measured immediately after the first enzyme infusion, with a maximum after 3 h, followed by a continuous decrease in the 8–15 days following the beginning of treatment. After this, a constant plasmatic content of DS concentration was observed. Overall, during the 10-month treatment period, ERT reduced the plasmatic concentration of DS up to ~80–85 %, but it was unable to totally remove it from the blood. We can suppose that immediately after the first enzyme administrations, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and thereafter only minimal changes are observed. The persistency of the residual amounts of DS with the actually recommended dosage in our Patient may suggest the opportunity to promote further studies with increased enzyme dosages to completely remove the accumulation of lysosomal DS.

The evaluation of plasmatic galactosaminoglycans, dermatan sulfate (DS) and chondroitin sulfate (CS) can be helpful in the early identification of MPS patients, also considering that primary storage of one type of GAG can lead to secondary accumulation of other lysosomal substrates. We explore the possibility to determine plasmatic DS and CS in numerous healthy pediatric (and sometimes adult) subjects depending on age and in patients affected by various forms of MPS. A highly sensitive HPLC separation and fluorescence detection was applied for plasma/serum DS and CS determination after a specific enzymatic treatment able to release their constituent disaccharides. DS and CS content decrease significantly with age in controls having high values in the first year (∼8 μg/mL). A highly significant decrease was observed for 1–5-year-old (∼−33%) and 5–10-year-old (∼−65%) healthy subgroups. No further decrease was determined showing a stabilization after 5 years of age. MPS I Scheie and Hurler patients showed rather similar DS and CS content significantly higher than controls matched for age. Similarly, MPS II, III and IV subjects all presented significantly higher plasmatic DS and CS content compared to healthy subjects matched for age. The same trend was determined for the only patient affected by MPS VI. Plasmatic DS and CS analyzed by the present procedure may be a useful diagnostic and screening marker for various forms of MPS.

Recently the first complete characterization and quantitative evaluation of glycosaminoglycans (GAGs) in both human and bovine milk have been reported. Chondroitin sulfate and heparin represent, respectively, approximately 55% and approximately 42% of the total human milk GAGs, whereas dermatan sulfate and hyaluronic acid make up the remaining 3%. In contrast, in bovine milk, which represents the starting material for infant formulas, dermatan sulfate constituted the main component (approximately 40%). Moreover, the total amount of GAGs in bovine milk is approximately seven times lower compared with human milk (60.2 mg/L vs. 416.2 mg/L, respectively). In a subsequent study, the GAG content in the milk from mothers delivering term or preterm newborns during the first month of lactation has been evaluated. A progressive decrease of GAG concentration was observed in both milks, with absolute amounts being consistently significantly higher in preterm milk. The highest values were found in colostrum (9.3 and 3.8 g/L in preterm and term milk, respectively, at Day 4 of lactation), followed by a progressive decrease up to Day 30 (4.3 and 0.4 g/L, respectively). From the above data it can be deduced that breastfed infants daily ingest consistent amounts of GAGs. Structurally, milk GAGs are sulfated linear polysaccharides synthesized in alveolar cells of the mammary gland by the sequential action of specific glycosyl transferases. Because of the absence of specific glycosidases and sulfatases secreted in the pancreatic juice or present on the intestinal wall, we can suppose that, once ingested, human milk GAGs can reach the small intestine in intact form. Accumulated evidence indicates that many microorganisms (pathogenic bacteria, viruses, and parasites) bind GAG chains of enterocytes for efficient invasion and propagation. As a consequence, the free GAGs, acting as soluble receptor analogs, could play an important role in preventing and protecting breastfed infants from intestinal infections. Moreover, it has been reported that breastfed newborns develop less oxidative stress than formula-fed ones. As chondroitin sulfate (and other GAGs) are well-known antioxidant and anti-inflammatory agents that act by activating the protein kinase C/phosphoinositide 3-kinase/Akt signaling pathway able to induce antioxidant enzymes, these macromolecules can contribute to the antioxidant properties of human milk, which are particularly important during the neonatal period. Finally, as bifidobacteria possess specific enzymes involved in the metabolism of complex carbohydrates, we can speculate that the undigested GAGs reaching the colon may behave as prebiotics to contribute to the development of bifidogenic flora, typical of breastfed infants (studies are in progress to evaluate this possible capacity of human milk GAGs).

A high-resolution normal-phase high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and structural characterization of the main oligosaccharides along with lactose from human milk samples is described. A total of 22 commercially available oligosaccharides were fluorotagged with 2-aminoacridone and separated on an amide column and identified on the basis of their retention times and mass spectra. Derivatized species having mass lower than approximately 800 to 900 exhibited mainly [M-H](-1) anions, oligomers with mass up to approximately 1000 to 1100 were represented by both [M-H](-1) and [M-2H](-2) anions, and oligomers greater than approximately 1200 to 1300 were characterized by a charge state of -3. Furthermore, the retention times were directly related to the glycans' molecular mass. Human milk samples from the four groups of donors (Se±/Le±) were analyzed for their composition and amount of free oligosaccharides after rapid and simple prepurification and derivatization steps also in the presence of lactose in high content. This analytical approach enabled us to perform the determination of species not detected by traditional techniques, such as sialic acid, as well as of species present in low content easily mistaken with other peaks. Finally, labeled human milk oligosaccharides were analyzed without any interference from excess fluorophore or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid.

Background: In a recent study, we performed a complete structural characterization of glycosaminoglycans (GAGs) in human mature milk. However, no data are available on the total content of GAGs in human milk from healthy mothers having delivered term or preterm newborns. Objectives: In this study, we evaluated the total content of GAGs in pooled milk from healthy mothers having delivered term or preterm newborns during the first month of lactation. Methods: Highly specific and sensitive analytical approaches were used to quantify human milk total GAGs. Results: Highest GAG values are present at day 4 (9.3 and 3.8 g/l in preterm and term milk, respectively), followed by a progressive decrease up to day 30 (4.3 and 0.4 g/l). The more remarkable differences are related to the first phases of lactation in which a strong decrease in GAGs was observed between days 4 and 10 (about –73% in term and –50% in preterm newborns). Conclusions: During the first month of lactation, the absolute amount of polysaccharides was constantly and significantly higher in preterm than in term milk, with a similar behavior in the decrease. These data further indicate that human milk GAGs may have an active role in protecting newborns during the first phases of lactation.

We report the structural characterization of plasmatic and urinary GAGs in a patient affected by MPS II (Hunter syndrome) before and during the first 10 months of enzyme-replacement therapy (ERT). Plasmatic GAGs before ERT were rich in pathological DS consisting of iduronic acid (IdoA) and composed of ~90% ΔDi4s and trace amounts of disulfated disaccharides. DS was also characterized as the main (~90%) urinary GAG mainly composed of ~90% ΔDi4s with minor percentages of monosulfated and disulfated disaccharides, in particular ΔDi2,4dis. After 300 days of ERT, plasmatic DS strongly decreased but ~14% of IdoA-rich ΔDi4s was still detected. Similarly, urinary galactosaminoglycans were mainly composed of 78% ΔDi4s, ~11% ΔDi6s and ~4% ΔDi0s with the persistence of ΔDi2,4dis (~4%). About 40% of IdoA-formed ΔDi4s were also calculated, thus confirming that pathological DS is still present in excreted urinary GAGs during ERT. By considering the % of IdoA, we observed rather similar kinetics of excretion in fluids from the beginning of the treatment. Immediately after the first enzyme infusion, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and this process lasts for about 2 weeks. After this, the percentage of IdoA-rich material present in biological fluids remains fairly constant over the following 9 months of treatment. To date, these are the first data regarding plasmatic and urinary kinetics directly measured on products released by the activity of the recombinant enzyme Idursulfase, iduronate-2-sulfatase, evaluated using specific and sensitive analytical procedures.

We report on a male infant with oral, facial, digital, and skeletal anomalies in association with severe psychomotor delay. This may represent a new oral-facial-digital syndrome. © 1994 Wiley-Liss, Inc.

In 1990, the Italian Study Group for Turner's Syndrome (ISGTS) undertook a nationwide survey, involving the retrospective collection of cross-sectional data and longitudinal growth profiles of 772 girls with Turner's syndrome born between 1950 and 1990. The study was carried out in 29 pediatric endocrinological centers. In this first report, the familial characteristics and neonatal data of Turner girls are described, compared to those of the general population, and related to postnatal somatic development. Furthermore, charts for birth weight and growth standards for height and weight from infancy to adulthood are presented (these are the first charts based on a large sample from the Mediterranean area). The main findings were: (1) incidence of Turner births increases with parental age or parity; (2) most of the neonates are small for dates; (3) girls with normal birth weight tend to be both taller and heavier than girls with low birth weight during the whole growth period; and (4) a 10-cm difference in midparental height leads to a 6.5-cm difference in adult stature.