Francesco Porta

Screening for later-onset Pompe's disease in patients with paucisymptomatic hyperCKemia.

BACKGROUND Pompes disease is an inherited metabolic myopathy caused by acid α-glucosidase deficiency. Early diagnosis optimizes the treatment effectiveness. METHODS One-hundred-thirty-seven consecutive patients with unexplained hyperCKemia underwent the assessment of acid α-glucosidase activity on dried blood spot. Second tier confirmatory testing in positive patients included the assessment of α-glucosidase activity on lymphocytes or muscle tissue and molecular analysis. RESULTS Three patients were diagnosed with later-onset Pompes disease, revealing 2.2% prevalence in asymptomatic hyperCKemia. Moreover, three patients positive to the screening revealed abnormal biochemical second tier testing, but were heterozygous for the common c.-32-13T>G mutation at molecular level. CONCLUSIONS The selective screening for later-onset Pompes disease in asymptomatic hyperCKemia allowed the identification of affected patients in a pre-clinical stage. Additionally, the identification of carriers with biochemical alterations related to Pompes disease extends the spectrum of its manifestations to heterozygous subjects.

Phenotypic variability, neurological outcome and genetics background of 6-pyruvoyl-tetrahydropterin synthase deficiency

Leuzzi V, Carducci Ca, Carducci Cl, Pozzessere S, Burlina A, Cerone R, Concolino D, Donati MA, Fiori L, Meli C, Ponzone A, Porta F, Strisciuglio P, Antonozzi I, Blau N. Phenotypic variability, neurological outcome and genetics background of 6‐pyruvoyl‐tetrahydropterin synthase deficiency.

TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation.

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.

Prospective bone ultrasound patterns during childhood acute lymphoblastic leukemia treatment

OBJECTIVE Bone impairment is a well-known complication in childhood acute lymphoblastic leukemia (ALL) survivors but less is known about bone dynamics during ALL therapy. We longitudinally assessed by Quantitative Ultrasound (QUS) skeletal modifications during this treatment. MATERIALS AND METHODS Forty-four newly diagnosed ALL children underwent bone measurement by QUS parameters BTT (Bone Transmission Time) and AD-SoS (Amplitude-Dependent Speed of Sound), mainly reliant on bone density and cortical thickness, respectively. Measurements were performed at diagnosis, and 6, 12, and 24 months thereafter. The occurrence of skeletal complications such as fractures, vertebral collapse, osteonecrosis, and osteopenia was related to measurement outcome. RESULTS A rapid deterioration of bone properties measured by BTT and AD-SoS was evident in the first semester of therapy (p<0.001). Subsequently, the next measurements were characterized by progressive uncoupling of the two QUS parameters (p<0.001). These were both significantly reduced at the end of therapy (p<0.001). Twelve subjects with in-treatment skeletal complications displayed an almost two-fold decrease of both parameters (p<0.001). BTT decreasing more than 1 Standard Deviation (SD) over 6 months of therapy was able to predict skeletal complication occurrence (p<0.001). CONCLUSION This report represents the largest longitudinal cohort systematically submitted to bone condition assessment from the beginning to the end of therapy for childhood ALL. Bone deterioration occurs early and persists throughout therapy, consistent with bone properties uncoupling. This pattern possibly reflects an initial impairment of both mineral density and cortical thickness with a subsequent recovery of this latter. QUS permits an early detection of bone deterioration and related skeletal complications in childhood ALL.

Phalangeal quantitative ultrasound in children with phenylketonuria: a pilot study.

Bone alterations in phenylketonuria (PKU) have been detected, especially with increasing age, in several studies by using different radiologic techniques. Quantitative ultrasound (QUS) assesses skeletal status by measuring the amplitude-dependent speed of sound (AD-SoS) and the bone transmission time (BTT), mainly dependent on mineral density and cortical thickness. Bone condition in 30 children and adolescents (mean age 15.1 +/- 6.4 y) affected by PKU was evaluated by phalangeal QUS, considering its relationship with their clinical, biochemical and therapeutic features. Measured AD-SoS Z-Score and BTT Z-Score were 0.27 +/- 1.42 and -0.26 +/- 1.21, respectively. In patients with previous fractures, the two QUS parameters were lower than in patients without history of fracture (p < 0.001 and p = 0.006, respectively). AD-SoS Z-Score and BTT Z-Score were negatively correlated with plasma phenylalanine (Phe) concentration in the year before QUS (p = 0.005 and p < 0.001, respectively) and with age (p < 0.001 for both parameters). These results parallel the previous findings obtained by different radiologic tools and suggest phalangeal QUS as an attractive option for the regular evaluation and longitudinal monitoring of bone condition in children and adolescents affected by PKU.

Dopamine agonists in 6-pyruvoyl tetrahydropterin synthase deficiency

Objective: To report the efficacy, tolerability, and safety of the dopamine agonist pramipexole in a series of 5 patients affected by inherited 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency and needing l-3,4 dihydroxyphenylalanine (l-dopa) therapy. Methods: Patients included 4 males and 1 female with ages ranging from 2 to 26 years. Their medication included tetrahydrobiopterin (BH4), 5-hydroxytryptophan, l-dopa, carbidopa, selegiline, and entacapone. All experienced residual symptoms of dopamine deficiency, movement and behavioral disability, and complications of l-dopa therapy, associated with fluctuating hyperprolactinemia. Patients had full assessment of clinical and biochemical condition, including evaluation by an adapted Unified Parkinsons Disease Rating Scale (UPDRS) and measurement of plasma prolactin (PRL) and catecholamines, before and after a 6-week trial with pramipexole. Pramipexole was administered twice daily as an adjunct to l-dopa therapy in dosages upwardly titrated, with a concurrent reduction of l-dopa dosage. Clinical follow-up went on for 1 year. Results: Pramipexole was well tolerated by all patients, with marked improvement and stabilization of their clinical picture. The mean improvement on the total UPDRS score was 43% (range 33.3%–55.6%) from baseline. Diurnal profiles of plasma PRL normalized and plasma catecholamine levels lasted unchanged. The daily administrations of l-dopa were curtailed from 3 or 4 to 2, and the l-dopa dosage was reduced up to 40%. Conclusions: The addition of pramipexole to the treatment of 6-pyruvoyl tetrahydropterin synthase deficiency improves the results of l-3,4 dihydroxyphenylalanine therapy. Similar benefits may be expected in other forms of inherited tetrahydrobiopterin deficiency.

Impact of Neonatal Protein Metabolism and Nutrition on Screening for Phenylketonuria

Objectives: Early blood phenylalanine (Phe) elevation after birth enables screening for and anticipation of the diagnosis of phenylketonuria. The differential impact of factors involved in this phenomenon, however, has not been elucidated. To solve this question, phenotype, genotype, dietary Phe intake, timing of blood collection, and Phe metabolism were retrospectively analyzed in 21 phenylketonuria newborns and prospectively in 1. Patients and Methods: Patients were assigned to 1 of 4 classes of phenylalanine hydroxylase (PAH) deficiency (severe, moderate, mild, and benign) on the basis of their Phe tolerance. Phe ingested, tolerated, and released from endogenous catabolism was assessed. Results: From birth to screening test, the amount of Phe tolerated ranged from 704 to 1620 mg, according to the class of PAH deficiency. The amount of Phe ingested ranged only from 204 to 405 mg, whereas the endogenous Phe breakdown ranged from 812 to 1534 mg, resulting in a rate of Phe catabolism ranging from 262 to 341 mg/day, regardless of the class of PAH deficiency. Conclusions: The high rate of protein catabolism is the main determinant of neonatal hyperphenylalaninemia. It is sufficient to turn to positive the screening test in severe and moderate PAH deficiency. In mild and benign PAH deficiency, the outcome of screening procedures can be substantially altered by the concurrence of genetic and peristatic factors. These results imply that the value of blood Phe at the screening test is not fully predictive of the phenylketonuria phenotype, and strengthen concerns regarding the reliability of early screening procedures.

Increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells in phenylketonuria

SummaryPhenylketonuria (PKU) is commonly complicated by a progressive bone impairment of uncertain aetiology. The therapeutic phenylalanine (Phe)-restricted diet and the possible noxious effects of high plasma Phe concentrations on bone have previously been suggested as possible determinant factors. Since osteoclasts are involved in bone reabsorption, they could play a role in determining bone damage in PKU. The reported increased excretion of bone resorption markers in PKU patients is consistent with this hypothesis. Although different diseases characterized by bone loss have been related to increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs), to date there is no evidence of increased osteoclast formation in PKU. In this study, we compared the spontaneous osteoclastogenesis from PBMCs in 20 patients affected by PKU with that observed in age- and sex-matched healthy subjects. Phenylketonuric patients showed the number of osteoclasts to be almost double that observed in controls (159.9 ± 79.5 and 87.8 ± 44.7, respectively; p = 0.001). Moreover, a strict direct correlation between the spontaneous osteoclastogenesis in PKU patients and the mean blood Phe concentrations in the preceding year was observed (r = 0.576; p = 0.010). An imbalance between bone formation and bone resorption might explain, at least in part, the pathogenesis of bone loss in this disease. These findings could provide new insights into the biological mechanisms underlying bone damage in PKU.

Unresponsiveness to tetrahydrobiopterin of phenylalanine hydroxylase deficiency

Conflicting results have been reported concerning the efficacy of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase, for reducing phenylalanine (Phe) concentration in phenylketonuria (PKU). We aimed to test quantitatively the effects of BH4 in PKU patients. Seven fully characterized patients were selected among a population of 130 PKU subjects as harboring PKU mutations predicted as BH4 responsive and previously considered responsive to a cofactor challenge. They received a simple Phe (100 mg/kg) and 2 combined Phe (100 mg/kg) and BH4 (20 mg/kg) oral loading tests. Cofactor was administered either before or after the amino acid. The concentrations of Phe, tyrosine (Tyr), and biopterin were measured over 24 hours after loading. The comparative analysis of the loading tests showed that in all patients plasma Phe concentrations peaked within 3 hours, and fell within 24 hours by about 50% in benign, 20% in mild, and 15% in severe phenylalanine hydroxylase deficiency regardless of BH4 administration. A consistent or moderate increase of plasma Tyr, again independent of the cofactor challenge, was observed only in the less severe forms of PAH deficiency. Mean blood biopterin concentration increased 6 times after simple Phe and 34 to 39 times after combined loading tests. The administration of BH4 does not alter Phe and Tyr metabolism in PKU patients. The clearance of plasma Phe after oral loading and, as well as Tyr production, is not related to cofactor challenge but to patients phenotype. The assessment of BH4 responsiveness by the methods so far used is not reliable, and the occurrence of BH4-responsive forms of PKU still has to be definitely proven.

Impact of metabolic control on bone quality in phenylketonuria and mild hyperphenylalaninemia.

Objectives: A reduction of bone mineral density of unknown etiology has been reported in phenylketonuria (PKU) by radiological techniques, whereas no data on bone density in mild hyperphenylalaninemia (HPA) are available. We aimed to assess bone condition in PKU and HPA by quantitative ultrasound (QUS), taking into account patients’ clinical and biochemical features. Patients and Methods: Phalangeal QUS has been used for bone assessment in 78 patients affected by PKU (n = 42) or mild HPA (n = 36). For each patient, blood phenylalanine concentrations in the 2 years before the study have been recorded and related to bone assessment. Results: Overall normal bone quality has been observed in the whole study group (AD-SoS standard deviation score [SDS] 0.25 ± 1.29; BTT SDS −0.13 ± 1.08). PKU adolescents (age older than 15 years, AD-SoS SDS −0.54 ± 1.33; BTT SDS −0.85 ± 1.21) and patients with poor compliance with treatment (blood phenylalanine >10 mg/dL, AD-SoS SDS −0.47 ± 1.39; BTT SDS −0.97 ± 1.14) showed lower BTT SDS with respect to normal population (P = 0.003 and P < 0.001, respectively). Patients with PKU with good compliance with treatment (blood phenylalanine < 10 mg/dL, AD-SoS SDS 0.65 ± 1.33; BTT SDS 0.15 ± 0.94) and patients with mild HPA (AD-SoS SDS 0.44 ± 1.06 and BTT SDS 0.19 ± 0.85) showed normal bone mineral density and cortical thickness. Conclusions: Good compliance with treatment in PKU during adolescence and adulthood is desirable because diet discontinuation is associated with bone loss. Mild HPA seems not to be complicated by bone damage.