Marta Llarena

Sanfilippo syndrome: Overall review.

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is a lysosomal storage disorder, caused by a deficiency in one of the four enzymes involved in the catabolism of glycosaminoglycan heparan sulfate. It is characterized by progressive cognitive decline and severe hyperactivity, with relatively mild somatic features. This review focuses on clinical features, diagnosis, treatment, and follow‐up of MPS III, and provides information about supplementary tests and differential diagnosis. Given that few reviews of MPS III have been published, several studies were compiled to establish diagnostic recommendations. Quantitative urinary glycosaminoglycan analysis is strongly recommended, and measurement of disaccharides, heparin cofactor II–thrombin complex and gangliosides is also used. Enzyme activity of the different enzymes in blood serum, leukocytes or fibroblasts, and mutational analysis for SGSH, NAGLU, HGSNAT or GNS genes are required to confirm diagnosis and differentiate four subtypes of MPS III. Although there is no global consensus for treatment, enzyme replacement therapy and gene therapy can provide appropriate results. In this regard, recent publications on treatment and follow‐up are discussed.

Tetrahydrobiopterin therapy vs phenylalanine-restricted diet: Impact on growth in PKU

BACKGROUND Treatment of phenylketonuria based upon strict vegetarian diets, with very low phenylalanine intake and supplemented by phenylalanine-free formula, has proven to be effective in preventing the development of long-term neurological sequelae due to phenylalanine accumulation. On the other hand, such diets have occasionally been reported to hinder normal development, some individuals presenting with growth retardation. Tetrahydrobiopterin therapy has opened up new treatment options for a significant proportion of phenylketonuric patients, enabling them to eat normal diets and be freed from the need to take synthetic supplements. However, little is known about how this therapy affects their physical development. METHODS We conducted a retrospective longitudinal study examining anthropometric characteristics (height, weight, body mass index and growth speed Z-scores) in a cohort of phenylketonuric patients on tetrahydrobiopterin therapy (38 subjects) comparing their characteristics with those of a group of phenylketonuric patients on phenylalanine-restricted diets (76 subjects). Nutritional issues were also considered, to further explore the possibility of higher natural protein intake being associated with better physical development. Data were collected every six months over two different periods of time (two or five years). RESULTS No improvement was observed in the aforementioned anthropometric variables in the cohort on tetrahydrobiopterin therapy, from prior to starting treatment to when they had been taking the drug for two or five years. Rather, in almost all cases there was a fall in the mean Z-score for the variables during these periods, although the changes were not significant in any case. Further, we found no statistically differences between the two groups at any considered time point. Growth impairment was also noted in the phenylketonuric patients on low-phenylalanine diets. Individuals on tetrahydrobiopterin therapy increased their natural protein intake and, in some instances, this treatment enabled individuals to eat normal diets, with protein intake meeting RDAs. No association was found, however, between higher protein intake and growth. CONCLUSION Our study identified growth impairment in patients with phenylketonuria on tetrahydrobiopterin, despite higher intakes of natural proteins. In fact, individuals undergoing long-term tetrahydrobiopterin treatment seemed to achieve similar developmental outcomes to those attained by individuals on more restricted diets.

Molecular epidemiology, genotype|[ndash]|phenotype correlation and BH4 responsiveness in Spanish patients with phenylketonuria

Phenylketonuria (PKU), the most common inborn error of amino acid metabolism, is caused by mutations in the phenylalanine-4-hydroxylase (PAH) gene. This study aimed to assess the genotype–phenotype correlation in the PKU Spanish population and the usefulness in establishing genotype-based predictions of BH4 responsiveness in our population. It involved the molecular characterization of 411 Spanish PKU patients: mild hyperphenylalaninemia non-treated (mild HPA-NT) (34%), mild HPA (8.8%), mild-moderate (20.7%) and classic (36.5%) PKU. BH4 responsiveness was evaluated using a 6R-BH4 loading test. We assessed genotype–phenotype associations and genotype–BH4 responsiveness in our population according to literature and classification of the mutations. The mutational spectrum analysis showed 116 distinct mutations, most missense (70.7%) and located in the catalytic domain (62.9%). The most prevalent mutations were c.1066-11G>A (9.7%), p.Val388Met (6.6%) and p.Arg261Gln (6.3%). Three novel mutations (c.61-13del9, p.Ile283Val and p.Gly148Val) were reported. Although good genotype–phenotype correlation was observed, there was no exact correlation for some genotypes. Among the patients monitored for the 6R-BH4 loading test: 102 were responders (87, carried either one or two BH4-responsive alleles) and 194 non-responders (50, had two non-responsive mutations). More discrepancies were observed in non-responders. Our data reveal a great genetic heterogeneity in our population. Genotype is quite a good predictor of phenotype and BH4 responsiveness, which is relevant for patient management, treatment and follow-up.

Quantification of arginine and its methylated derivatives in healthy children by liquid chromatography-tandem mass spectrometry.

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of nitric oxide synthase, which is responsible for most of the vascular nitric oxide (NO) produced. NO is an important physiological mediator of vascular tone and structure in normally functioning endothelial cells. We report the optimization of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of arginine (Arg) and its derivatives in biological samples. Chromatographic separation and mass detection were performed by reverse phase chromatography coupled with tandem mass spectrometry. For sample preparation, plasma proteins were removed by centrifugal filters. Positive electrospray ionization was performed and analytes were detected by multiple reaction monitoring. Inter- and intra-day repeatability, accuracy, recovery, and limits of detection and quantification were evaluated to validate the method. Plasma and urine levels were measured in healthy children to establish control values: 52.2-124.7 µM for Arg, 0.06-0.16 µM for MMA, 0.42-1.10 µM for ADMA and 0.41-0.96 µM for SDMA in plasma. Quantification of Arg and its methylated derivatives by LC-MS/MS can be carried out without the need of organic solvents for sample preparation, and be used as a valuable tool in research on endothelial dysfunction.

Profile of sodium phenylbutyrate granules for the treatment of urea-cycle disorders: patient perspectives

Urea-cycle disorders are a group of rare hereditary metabolic diseases characterized by deficiencies of one of the enzymes and transporters involved in the urea cycle, which is necessary for the removal of nitrogen produced from protein breakdown. These hereditary metabolic diseases are characterized by hyperammonemia and life-threatening hyperammonemic crises. Pharmacological treatment of urea-cycle disorders involves alternative nitrogen-scavenging pathways. Sodium benzoate combines with glycine and phenylacetate/phenylbutyrate with glutamine, forming, respectively, hippuric acid and phenylacetylglutamine, which are eliminated in the urine. Among the ammonia-scavenging drugs, sodium phenylbutyrate is a well-known long-term treatment of urea-cycle disorders. It has been used since 1987 as an investigational new drug, and was approved for marketing in the US in 1996 and the EU in 1999. However, sodium phenylbutyrate has an aversive odor and taste, which may compromise patients’ compliance, and many patients have reported difficulty in taking this drug. Sodium phenylbutyrate granules are a new tasteless and odor-free formulation of sodium phenylbutyrate, which is indicated in the treatment of urea-cycle disorders. This recently developed taste-masked formulation of sodium phenylbutyrate granules was designed to overcome the considerable issues that taste has on adherence to therapy. Several studies have reported the clinical experience of patients with urea-cycle disorders treated with this new tasteless formulation of sodium phenylbutyrate. Analysis of the data indicated that this taste-masked formulation of sodium phenylbutyrate granules improved quality of life for urea-cycle disorder patients. Furthermore, a postmarketing report on the use of the product has confirmed the previous observations of improved compliance, efficacy, and safety with this taste-masked formulation of sodium phenylbutyrate.

6R-tetrahydrobiopterin treated PKU patients below 4 years of age: Physical outcomes, nutrition and genotype

BACKGROUND AND AIMS Phenylalanine-restricted diets have proven effective in treating phenylketonuria. However, such diets have occasionally been reported to hinder normal development. Our study aimed to assess whether treating 0-4-year-old phenylketonuric patients with 6R-tetrahydrobiopterin might prevent growth retardation later in life. METHODS We conducted a longitudinal retrospective study which examined anthropometric characteristics of phenylketonuric patients on 6R-tetrahydrobiopterin therapy (22 subjects), and compared them with a group of phenylketonuric patients on protein-restricted diets (44 subjects). Nutritional issues were also considered. We further explored possible relationships between mutations in the PAH gene, BH4 responsiveness and growth outcome. RESULTS No significant growth improvements were observed in either the group on 6R-tetrahydrobiopterin treatment (height Z-score: initial= -0.57 ± 1.54; final=-0.52 ± 1.29; BMI Z-score: initial=0.17 ± 1.05; final=0.18 ± 1.00) or the diet-only group (height Z-score: initial=-0.92 ± 0.96; final= -0.78 ± 1.08; BMI Z-score: initial=0.17 ± 0.97; final=-0.07 ± 1.03) over the 1-year observation period. Furthermore, we found no significant differences (p>0.05) between the two groups at any of the time points considered (0, 6 and 12 months). Patients on 6R-tetrahydrobiopterin increased their phenylalanine intake (from 49.1 [25.6-60.3] to 56.5 [39.8-68.3] mgkg(-1)day(-1)) and natural protein intake (from 1.0 [0.8-1.7] to 1.5 [1.0-1.8] g kg(-1)day(-1)), and some patients managed to adopt normal diets. Higher phenylalanine and natural protein intakes were positively correlated with better physical outcomes in the diet-only group (p<0.05). No correlation was found between patient genotype and physical outcomes, results being similar regardless of the nutritional approach used. We did not detect any side effects due to 6R-tetrahydrobiopterin administration. CONCLUSIONS Our study indicates that treating 0-4-year-old phenylketonuric patients with 6R-tetrahydrobiopterin is safe. However, poor developmental outcomes were observed, despite increasing the intake of natural proteins. Genotype could be a valid predictor of tetrahydrobiopterin-responsiveness, since patients who carried the same genotype responded similarly to the 6R-tetrahydrobiopterin loading test. On the other hand, harbouring 6R-tetrahydrobiopterin responsive genotypes did not predispose patients to better physical outcomes.

Influence of phenylketonuria's diet on dimethylated arginines and methylation cycle.

Abstract Phenylketonurias (PKU) treatment based on low natural protein diet may affect homocysteine (Hcys) metabolic pathway. Hcys alteration may be related to the methylation of arginine to asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which both modify nitric oxide production. The aim of this work is to evaluate the status of Hcys formation methylation cycle and ADMA and SDMA levels in patients with PKU in order to establish a potential relationship. Forty-two early diagnosed PKU patients under dietary treatment and good adherence to their diets were enrolled in this cross-sectional study. Their nutritional and biochemical profile, as well as Hcys synthesis status, ADMA and SDMA levels were analyzed and compared with a control group of 40 healthy volunteers. ADMA and SDMA were determined by high-performance liquid chromatography system coupled to triple quadrupole mass spectrometer. In this study, 23 classic PKU, 16 moderate PKU, and 3 mild HPA were enrolled. The median age was 10 years old. Median ADMA, SDMA, and Hcys concentration levels (5.1 &mgr;M [2.3–25.7], 0.35 &mgr;M [0.18–0.57], 0.43 &mgr;M [0.26–0.61], respectively) were lower in patients with PKU (P < .001 for ADMA and SDMA) whereas vitamin B12 and folate levels (616 pg/mL [218–1943] and 21 ng/mL [5–51], respectively) were higher comparing with controls. Statistically significant correlations were found between ADMA, and Phe (r = −0.504, P = .001) and Hcys (r = −0.458, P = .037) levels. Several nutrition biomarkers, such as prealbumin, 25-hydroxy vitamin D, selenium, and zinc, were below the normal range. Our study suggests that patients with PKU suffer from poor methylation capacity. Restriction of natural proteins in addition to high intake of vitamin B12 and folic acid supplementation in the dietary products, produce an impairment of methylation cycle that leads to low Hcys and ADMA levels. As a result, methylated compounds compete for methyl groups, and there is an impairment of methylation cycle due to low Hcys levels, which is related to the lack of protein quality, despite of elevated concentrations of cofactors.

A new case of maternal phenylketonuria treated with sapropterin dihydrochloride (6R-BH4)

Abstract Purpose: A woman with phenylketonuria (PKU) was diagnosed through neonatal screening, her PAH mutation was p.V388M/p.I65T, for which she received treatment with phenylalanine restriction, and was administered oral sapropterin dihydrochloride (6R-BH4) from the age of thirty. The purpose of this article is to describe the treatment with BH4 during her pregnancy and to evaluate a plan for its use. Methods: The patient had an unplanned pregnancy at 34 years of age, for which she received a phenylalanine-free supplement enriched with essential fatty acids, vitamins and trace elements. Results: The dose of 6R-BH4 was reduced from 500 mg/day to 100 mg/day until its suspension in the 28th week of gestation, and was well tolerated. Blood phenylalanine control was easily accomplished during this pregnancy, and no nutritional deficiency was seen. Conclusion: The pregnancy had a normal outcome, and so we consider that adaptation of the dose of 6R-BH4 to the prenatal periods aided a greater efficiency and a lower risk in the treatment of maternal PKU. Chinese abstract 目的：一名女性在新生儿筛查时被诊断患有苯丙酮尿症（PKU），她的PAH突变为p.V388M/p.I65T，因此接受苯丙氨酸限制疗法，自30岁起开始口服沙丙蝶呤二盐酸盐（6R-BH4）。本文目的在于描述患者在妊娠过程中的BH4治疗方法，评估其使用方案。 方法：该患者在34岁时意外妊娠，故其添加了不含苯丙氨酸的物质，包含必需脂肪酸、维生素及微量元素。 结果：6R-BH4的剂量由500mg/天减至100mg/天，至妊娠28周中止，患者耐受性良好。妊娠期血中苯丙氨酸被顺利控制，且未出现营养素缺乏状况。 结论：该患者妊娠结局正常，所以我们认为在产前期调整6R-BH4的剂量有助于提高其有效性，且降低母体PKU的治疗风险。

A Metabolomics Approach to Metabolic Diseases

Metabolomics, defined as the comprehensive analysis of compounds in a biological specimen, is an emerging technology that helps several pathologies to inform about new biomarkers. Metabolic diseases comprise a group of rare conditions that in total represent an important health problem. Historically, small numbers of metabolites have been used to diagnose complex metabolic diseases such as diabetes or metabolic diseases. Metabolomic methodology, due to the evolution of clinical chemistry technologies, could detect thousands of organic compounds. In this way, metabolomic approach gives information of metabolic pathways describing physiopathology that underlies disease, including the possibility of discovery of new markers that could be used to diagnose or check the efficacy of the treatments. Diabetes, classic inborn error of metabolism as methylmalonic aciduria, lysosomal diseases and rare optic neuropathy affecting adults are discussed in this chapter.

Dimethylarginines as biomarkers for the kidney transplant management in methylmalonic aciduria.

Methylmalonic aciduria (MMA) is an inborn error of metabolism associated with many complications despite treatment. Chronic renal failure is the most common problem, and patients may eventually require kidney transplant. Therefore, it is worth investigating whether living donor kidney transplant offers a better option than deceased kidney donors; and the value of novel vascular risk biomarkers in the assessment of transplanted MMA patients. We report a case of a 26‐year‐old man with MMA, who progressed to end‐stage renal disease and received kidney transplant from a heterozygous next‐of‐kin living donor at 20 years of age. Although post‐transplant urinary levels of methylmalonic acid decreased, this reduction was lower than previously reported for deceased donors. No episodes of metabolic decompensation were observed after transplantation. During his clinical progress, vascular complications appeared; and finally, pancreatitis was the cause of death. After kidney transplant, we evaluated novel vascular risk factors, such as asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which were used as early biomarkers of progression and metabolic management for this transplanted patient. This case report illustrates the disadvantage of transplantation with an allograft from a heterozygous living donor, and the utility of vascular risk biomarkers in renal transplant assessment.