Gregory M. Rice

Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, phase 1 dose-escalation trial

BACKGROUND Phenylketonuria is an inherited disease caused by impaired activity of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine, leading to accumulation of phenylalanine and subsequent neurocognitive dysfunction. Phenylalanine ammonia lyase is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. We aimed to assess the safety, tolerability, pharmacokinetic characteristics, and efficacy of recombinant Anabaena variabilis phenylalanine ammonia lyase (produced in Escherichia coli) conjugated with polyethylene glycol (rAvPAL-PEG) in reducing phenylalanine concentrations in adult patients with phenylketonuria. METHODS In this open-label, phase 1, multicentre trial, single subcutaneous injections of rAvPAL-PEG were given in escalating doses (0·001, 0·003, 0·010, 0·030, and 0·100 mg/kg) to adults with phenylketonuria. Participants aged 18 years or older with blood phenylalanine concentrations of 600 μmol/L or higher were recruited from among patients attending metabolic disease clinics in the USA. The primary endpoints were safety and tolerability of rAvPAL-PEG. Secondary endpoints were the pharmacokinetic characteristics of the drug and its effect on concentrations of phenylalanine. Participants and investigators were not masked to assigned dose group. This study is registered with ClinicalTrials.gov, number NCT00925054. FINDINGS 25 participants were recruited from seven centres between May 6, 2008, and April 15, 2009, with five participants assigned to each escalating dose group. All participants were included in the safety population. The most frequently reported adverse events were injection-site reactions and dizziness, which were self-limited and without sequelae. Two participants had serious adverse reactions to intramuscular medroxyprogesterone acetate, a drug that contains polyethylene glycol as an excipient. Three of five participants given the highest dose of rAvPAL-PEG (0·100 mg/kg) developed a generalised skin rash. By the end of the study, all participants had developed antibodies against polyethylene glycol, and some against phenylalanine ammonia lyase as well. Drug concentrations peaked about 89-106 h after administration of the highest dose. Treatment seemed to be effective at reducing blood phenylalanine in all five participants who received the highest dose (mean reduction of 54·2% from baseline), with a nadir about 6 days after injection and an inverse correlation between drug and phenylalanine concentrations in plasma. Phenylalanine returned to near-baseline concentrations about 21 days after the injection. INTERPRETATION Subcutaneous administration of rAvPAL-PEG in a single dose of up to 0·100 mg/kg was fairly safe and well tolerated in adult patients with phenylketonuria. At the highest dose tested, rAvPAL-PEG reduced blood phenylalanine concentrations. In view of the development of antibodies against polyethylene glycol (and in some cases against phenylalanine ammonia lyase), future studies are needed to assess the effect of repeat dosing. FUNDING BioMarin Pharmaceutical.

The Role of Molecular Testing and Enzyme Analysis in the Management of Hypomorphic Citrullinemia

Expanded newborn screening detects patients with modest elevations in citrulline; however it is currently unclear how to treat these patients and how to counsel their parents. In order to begin to address these issues, we compared the clinical, biochemical, and molecular features of 10 patients with mildly elevated citrulline levels. Three patients presented with clinical illness whereas seven came to attention as a result of expanded newborn screening. One patient presented during pregnancy and responded promptly to IV sodium phenylacetate/sodium benzoate and arginine therapy with no long‐term adverse effects on mother or fetus. Two children presented with neurocognitive dysfunction, one of these responded dramatically to dietary protein reduction. ASS enzyme activity was not deficient in all patients with biallelic mutations suggesting this test cannot exclude the ASS1 locus in patients with mildly elevated plasma citrulline. Conversely, all symptomatic patients who were tested had deficient activity. We describe four unreported mutations (p.Y291S, p.R272H, p.F72L, and p.L88I), as well as the common p.W179R mutation. In silico algorithms were inconsistent in predicting the pathogenicity of mutations. The cognitive benefit in one patient of protein restriction and the lack of adverse outcome in seven others restricted from birth, suggest a role for protein restriction and continued monitoring to prevent neurocognitive dysfunction.

Glycomacropeptide for nutritional management of phenylketonuria: a randomized, controlled, crossover trial

Background: To prevent cognitive impairment, phenylketonuria requires lifelong management of blood phenylalanine (Phe) concentration with a low-Phe diet. The diet restricts intake of Phe from natural proteins in combination with traditional amino acid medical foods (AA-MFs) or glycomacropeptide medical foods (GMP-MFs) that contain primarily intact protein and a small amount of Phe. Objective: We investigated the efficacy and safety of a low-Phe diet combined with GMP-MFs or AA-MFs providing the same quantity of protein equivalents in free-living subjects with phenylketonuria. Design: This 2-stage, randomized crossover trial included 30 early-treated phenylketonuria subjects (aged 15–49 y), 20 with classical and 10 with variant phenylketonuria. Subjects consumed, in random order for 3 wk each, their usual low-Phe diet combined with AA-MFs or GMP-MFs. The treatments were separated by a 3-wk washout with AA-MFs. Fasting plasma amino acid profiles, blood Phe concentrations, food records, and neuropsychological tests were obtained. Results: The frequency of medical food intake was higher with GMP-MFs than with AA-MFs. Subjects rated GMP-MFs as more acceptable than AA-MFs and noted improved gastrointestinal symptoms and less hunger with GMP-MFs. ANCOVA indicated no significant mean ± SE increase in plasma Phe (62 ± 40 μmol/L, P = 0.136), despite a significant increase in Phe intake from GMP-MFs (88 ± 6 mg Phe/d, P = 0.026). AA-MFs decreased plasma Phe (−85 ± 40 μmol/L, P = 0.044) with stable Phe intake. Blood concentrations of Phe across time were not significantly different (AA-MFs = 444 ± 34 μmol/L, GMP-MFs = 497 ± 34 μmol/L), suggesting similar Phe control. Results of the Behavior Rating Inventory of Executive Function were not significantly different. Conclusions: GMP-MFs provide a safe and acceptable option for the nutritional management of phenylketonuria. The greater acceptability and fewer side effects noted with GMP-MFs than with AA-MFs may enhance dietary adherence for individuals with phenylketonuria. This trial was registered at www.clinicaltrials.gov as NCT01428258.

TBC1D24 genotype–phenotype correlation: Epilepsies and other neurologic features

Objective: To evaluate the phenotypic spectrum associated with mutations in TBC1D24. Methods: We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. TBC1D24 mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). Results: Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some TBC1D24 mutations, associated with the most severe TBC1D24-associated disorders, are not necessarily the most disruptive to this gene function. Conclusions: TBC1D24-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, TBC1D24 mutation screening is indicated in a wide variety of epilepsies. A TBC1D24 consortium was formed to develop further research on this gene and its associated phenotypes.

Microdissection-based high-resolution genomic array analysis of two patients with cytogenetically identical interstitial deletions of chromosome 1q but distinct clinical phenotypes.

We describe two boys with cytogenetically identical interstitial deletions in the q42.11‐q42.13 region of the long arm of chromosome 1 detected by high‐resolution G‐banding analysis. These children share some phenotypic features but also exhibit distinct morphologic differences. We further characterized the deletions using a new technical strategy—microdissection‐based high‐resolution genomic array (MHGA) analysis—to define the breakpoints, genomic sizes, and gene contents of the deletions. This showed that the patients had distinguishable deletions that were adjacent but did not overlap, thus explaining the observed phenotypic differences. These results were surprising because we expected at least some degree of overlap to explain the features that were shared. MHGA can quickly give precise and detailed information about any rearrangement in the genome using as little material as a single cell. This novel strategy provides unique advantages for both clinical diagnosis and genomic research.

Clinical and Molecular Characterization of Overlapping Interstitial Xp21-p22 Duplications in Two Unrelated Individuals

Development and implementation of high‐density DNA arrays demonstrated the important role of copy number changes on the X chromosome in the etiology of developmental delay and mental retardation (MR). We describe two unrelated patients with developmental delay due to similar interstitial duplications at Xp21‐p22. The first patient is a 6‐month‐old male with multiple affected family members including many females. The second patient is a 5‐year‐old adopted female. In both patients, chromosome analysis and array comparative genomic hybridization (aCGH) showed duplications of overlapping regions at Xp21‐p22. The duplicated segments contain numerous genes associated with MR, including AP1S2, NHS, CDKL5, RPS6KA3, SMS, and ARX. Except for developmental delay, there is little phenotypic overlap between the male and the female patient. Additionally, the female patient and affected female relatives of the male patient have variable severities of cognitive impairment, likely due to different X‐inactivation patterns and effects of other, nonduplicated genes important for normal development. These cases illustrate that increased gene dosage of X‐linked MR genes lead to cognitive impairment. Precise delineation of chromosome rearrangements by aCGH and identification of genes within duplicated segments helped in establishing genotype–phenotype correlations for each of our patients, in comparing them to each other, as well as with previously reported cases of Xp21‐p22 duplications. However, we show that even with detailed molecular characterization, phenotype prediction remains challenging in patients with structural abnormalities of the X chromosome.

Clinical relevance of the discrepancy in phenylalanine concentrations analyzed using tandem mass spectrometry compared with ion-exchange chromatography in phenylketonuria

Introduction Metabolic control of phenylketonuria (PKU) and compliance with the low-phenylalanine (phe) diet are frequently assessed by measuring blood phe concentrations in dried blood spots (DBS) collected by patients instead of plasma phe concentrations. Objective Our objective was to investigate the difference in blood phe concentrations in DBS collected by subjects and analyzed using either a validated newborn screening tandem mass spectrometry (MS/MS) protocol or ion-exchange chromatography (IEC) compared to plasma phe concentrations obtained simultaneously and analyzed using IEC. Design Three to four fasting blood samples were obtained from 29 subjects with PKU, ages 15–49 years. Capillary blood was spotted on filter paper by each subject and the DBS analyzed using both MS/MS and IEC. Plasma was isolated from venous blood and analyzed using IEC. Results Blood phe concentrations in DBS analyzed using MS/MS are 28% ± 1% (n = 110, p < 0.0001) lower than plasma phe concentrations analyzed using IEC resulting in a blood phe concentration of 514 ± 23 μmol/L and a plasma phe concentration of 731 ± 32 μmol/L (mean ± SEM). This discrepancy is larger when plasma phe is > 600 μmol/L. Due to the large variability across subjects of 13.2%, a calibration factor to adjust blood phe concentrations is not recommended. Analysis of DBS using IEC reduced the discrepancy to 15 ± 2% lower phe concentrations compared to plasma analyzed using IEC (n = 38, p = 0.0001). This suggests that a major contributor to the discrepancy in phe concentrations is the analytical method. Conclusion Use of DBS analyzed using MS/MS to monitor blood phe concentrations in individuals with PKU yields significantly lower phe levels compared to plasma phe levels analyzed using IEC. Optimization of current testing methodologies for measuring phe in DBS, along with patient education regarding the appropriate technique for spotting blood on filter paper is needed to improve the accuracy of using DBS to measure phe concentrations in PKU management.

Inborn Errors of Metabolism (Metabolic Disorders).

By their very nature, rare inborn errors of metabolism challenge the generation and application of evidence-based medicine. • On the basis of limited research evidence as well as consensus, newborn screening for select metabolic disorders, including phenylketonuria, medium-chain acyl CoA dehydrogenase deficiency, and glutaric acidemia type I, may improve long-term outcomes for affected children. • On the basis of primarily consensus, due to lack of relevant clinical studies, inborn errors due to defects in the metabolism of energy sources (protein, fatty acids, and carbohydrates) may present in infancy with overwhelming metabolic decompensation, and initial laboratory evaluations may reveal hyperammonemia, nonketotic hypoglycemia, or a metabolic acidosis with an elevated anion gap, depending on the disorder. • On the basis of primarily consensus, due to lack of relevant clinical studies, specific laboratory testing for inborn errors of metabolism should include plasma amino acids, urine organic acids, plasma carnitine, and plasma acylcarnitine profile. • On the basis of primarily consensus, due to lack of relevant clinical studies, disorders of cellular organelles, such as lysosomal and peroxisomal disorders, may present with progressive organomegaly, developmental regression, dysmorphic facial characteristics. or sensory loss.

A case of congenitally absent left internal carotid artery: vascular malformations in 22q11.2 deletion syndrome.

Our report is on a Hispanic boy for whom, shortly after birth, clinical suspicion of 22q11.2 deletion syndrome (22q11.2DS) was raised as a result of his characteristic features, including facial dysmorphisms and hypotonia. The 22q11.2DS was confirmed by fluorescence in situ hybridization (FISH), noting a 22q11.2 deletion. Further evaluation revealed complete congenital absence of the left internal carotid artery and focal pachygyria of the left hemisphere. Multiple cardiac and vascular anomalies have been previously described in 22q11 deletion syndrome, but congenital absence of the internal carotid has not been previously reported in the literature. We present a clinical case report in detail of this unique 22q11.2 deletion syndrome associated finding.

Use of Gastrostomy Tube to Prevent Maternal PKU Syndrome

Maternal Phenylketonuria Syndrome (MPKU) can occur in infants born to mothers with PKU with poor metabolic control during pregnancy. Elevated phenylalanine (phe) acts as a teratogen to the developing fetus with consequences including intellectual disability, microcephaly, facial dysmorphism, growth retardation, and congenital heart disease. MPKU can be prevented if metabolic control is achieved by 8-10 weeks gestation. If control is not achieved, there is a significant risk for MPKU. Therefore, in women with poor metabolic control at time of pregnancy, establishing metabolic control quickly is important.Clinically, establishing metabolic control in women with PKU can present challenges. Social issues, psychological issues, and insufficient education about PKU play an important role in a patients inability to reinstitute this challenging diet. Maintaining phe levels within a range to allow for infant growth, while preventing toxicity, is challenging, particularly for those women who no longer follow the PKU diet. Gastrostomy tube placement is an option to deliver medical formula to women who are unable to restart diet due to severe nausea or palatability issues.Here we discuss two pregnancies in which a gastrostomy tube was placed to achieve metabolic control after other measures failed to reduce phe concentrations into the recommended range. For these two pregnancies, placement of the gastrostomy tube led to improvement in phe levels with normal infant outcomes including normal growth, head circumference, and heart structure.