John J. Mitchell

Phenylalanine hydroxylase deficiency

Phenylalanine hydroxylase deficiency is an autosomal recessive disorder that results in intolerance to the dietary intake of the essential amino acid phenylalanine. It occurs in approximately 1:15,000 individuals. Deficiency of this enzyme produces a spectrum of disorders including classic phenylketonuria, mild phenylketonuria, and mild hyperphenylalaninemia. Classic phenylketonuria is caused by a complete or near-complete deficiency of phenylalanine hydroxylase activity and without dietary restriction of phenylalanine most children will develop profound and irreversible intellectual disability. Mild phenylketonuria and mild hyperphenylalaninemia are associated with lower risk of impaired cognitive development in the absence of treatment. Phenylalanine hydroxylase deficiency can be diagnosed by newborn screening based on detection of the presence of hyperphenylalaninemia using the Guthrie microbial inhibition assay or other assays on a blood spot obtained from a heel prick. Since the introduction of newborn screening, the major neurologic consequences of hyperphenylalaninemia have been largely eradicated. Affected individuals can lead normal lives. However, recent data suggest that homeostasis is not fully restored with current therapy. Treated individuals have a higher incidence of neuropsychological problems. The mainstay of treatment for hyperphenylalaninemia involves a low-protein diet and use of a phenylalanine-free medical formula. This treatment must commence as soon as possible after birth and should continue for life. Regular monitoring of plasma phenylalanine and tyrosine concentrations is necessary. Targets of plasma phenylalanine of 120–360 μmol/L (2–6 mg/dL) in the first decade of life are essential for optimal outcome. Phenylalanine targets in adolescence and adulthood are less clear. A significant proportion of patients with phenylketonuria may benefit from adjuvant therapy with 6R-tetrahydrobiopterin stereoisomer. Special consideration must be given to adult women with hyperphenylalaninemia because of the teratogenic effects of phenylalanine. Women with phenylalanine hydroxylase deficiency considering pregnancy should follow special guidelines and assure adequate energy intake with the proper proportion of protein, fat, and carbohydrates to minimize risks to the developing fetus. Molecular genetic testing of the phenylalanine hydroxylase gene is available for genetic counseling purposes to determine carrier status of at-risk relatives and for prenatal testing.

CRTAP AND LEPRE1 MUTATIONS IN RECESSIVE OSTEOGENESIS IMPERFECTA

Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Recently, dysregulation of hydroxylation of a single proline residue at position 986 of both the triple‐helical domains of type I collagen α1(I) and type II collagen α1(II) chains has been implicated in the pathogenesis of recessive forms of OI. Two proteins, cartilage‐associated protein (CRTAP) and prolyl‐3‐hydroxylase‐1 (P3H1, encoded by the LEPRE1 gene) form a complex that performs the hydroxylation and brings the prolyl cis‐trans isomerase cyclophilin‐B (CYPB) to the unfolded collagen. In our screen of 78 subjects diagnosed with OI type II or III, we identified three probands with mutations in CRTAP and 16 with mutations in LEPRE1. The latter group includes a mutation in patients from the Irish Traveller population, a genetically isolated community with increased incidence of OI. The clinical features resulting from CRTAP or LEPRE1 loss of function mutations were difficult to distinguish at birth. Infants in both groups had multiple fractures, decreased bone modeling (affecting especially the femurs), and extremely low bone mineral density. Interestingly, “popcorn” epiphyses may reflect underlying cartilaginous and bone dysplasia in this form of OI. These results expand the range of CRTAP/LEPRE1 mutations that result in recessive OI and emphasize the importance of distinguishing recurrence of severe OI of recessive inheritance from those that result from parental germline mosaicism for COL1A1 or COL1A2 mutations. Hum Mutat 0, 1–8, 2008.

Phenylketonuria Scientific Review Conference: State of the science and future research needs

New developments in the treatment and management of phenylketonuria (PKU) as well as advances in molecular testing have emerged since the National Institutes of Health 2000 PKU Consensus Statement was released. An NIH State-of-the-Science Conference was convened in 2012 to address new findings, particularly the use of the medication sapropterin to treat some individuals with PKU, and to develop a research agenda. Prior to the 2012 conference, five working groups of experts and public members met over a 1-year period. The working groups addressed the following: long-term outcomes and management across the lifespan; PKU and pregnancy; diet control and management; pharmacologic interventions; and molecular testing, new technologies, and epidemiologic considerations. In a parallel and independent activity, an Evidence-based Practice Center supported by the Agency for Healthcare Research and Quality conducted a systematic review of adjuvant treatments for PKU; its conclusions were presented at the conference. The conference included the findings of the working groups, panel discussions from industry and international perspectives, and presentations on topics such as emerging treatments for PKU, transitioning to adult care, and the U.S. Food and Drug Administration regulatory perspective. Over 85 experts participated in the conference through information gathering and/or as presenters during the conference, and they reached several important conclusions. The most serious neurological impairments in PKU are preventable with current dietary treatment approaches. However, a variety of more subtle physical, cognitive, and behavioral consequences of even well-controlled PKU are now recognized. The best outcomes in maternal PKU occur when blood phenylalanine (Phe) concentrations are maintained between 120 and 360 μmol/L before and during pregnancy. The dietary management treatment goal for individuals with PKU is a blood Phe concentration between 120 and 360 μmol/L. The use of genotype information in the newborn period may yield valuable insights about the severity of the condition for infants diagnosed before maximal Phe levels are achieved. While emerging and established genotype-phenotype correlations may transform our understanding of PKU, establishing correlations with intellectual outcomes is more challenging. Regarding the use of sapropterin in PKU, there are significant gaps in predicting response to treatment; at least half of those with PKU will have either minimal or no response. A coordinated approach to PKU treatment improves long-term outcomes for those with PKU and facilitates the conduct of research to improve diagnosis and treatment. New drugs that are safe, efficacious, and impact a larger proportion of individuals with PKU are needed. However, it is imperative that treatment guidelines and the decision processes for determining access to treatments be tied to a solid evidence base with rigorous standards for robust and consistent data collection. The process that preceded the PKU State-of-the-Science Conference, the conference itself, and the identification of a research agenda have facilitated the development of clinical practice guidelines by professional organizations and serve as a model for other inborn errors of metabolism.

The Morquio A Clinical Assessment Program: baseline results illustrating progressive, multisystemic clinical impairments in Morquio A subjects

OBJECTIVES The objectives of this study are to quantify endurance and respiratory function and better characterize spectrum of symptoms and biochemical abnormalities in mucopolysaccharidosis IVA subjects. METHODS MorCAP was a multicenter, multinational, cross sectional study amended to be longitudinal in 2011. Each study visit required collection of medical history, clinical assessments, and keratan sulfate (KS) levels. RESULTS Data from the first visit of 325 subjects (53% female) were available. Mean age was 14.5 years. Mean ± SD height z-scores were -5.6 ± 3.1 as determined by the CDC growth charts. Mean ± SD from the 6-minute-walk-test was 212.6 ± 152.2m, revealing limitations in functional endurance testing, and 30.0 ± 24.0 stairs/min for the 3-minute-stair-climb test. Respiratory function showed limitations comparable to MPS VI patients; mean ± SD was 1.2 ± 0.9l based on forced vital capacity and 34.8 ± 25.5l/min based on maximum voluntary ventilation. Mean urinary keratan sulfate (uKS) was elevated for all ages, and negatively correlated with age. Higher uKS correlated with greater clinical impairment based on height z-scores, endurance and respiratory function tests. The MPS Health Assessment Questionnaire reveals impairments in mobility and activities of daily living in comparison to an age-matched control population. CONCLUSIONS MPS IVA is a multisystem disorder with a continuum of clinical presentation. All affected individuals experience significant functional limitations and reduced quality of life. Older patients have more severe exercise and respiratory capacity limitations, and more frequent cardiac pathology illustrating the progressive nature of disease.

Effect of nitisinone (NTBC) treatment on the clinical course of hepatorenal tyrosinemia in Québec.

BACKGROUND Hepatorenal tyrosinemia (HT1, fumarylacetoacetate hydrolase deficiency, MIM 276700) can cause severe hepatic, renal and peripheral nerve damage. In Québec, HT1 is frequent and neonatal HT1 screening is practiced. Nitisinone (NTBC, Orfadin ®) inhibits tyrosine degradation prior to the formation of toxic metabolites like succinylacetone and has been offered to HT1 patients in Québec since 1994. METHODS We recorded the clinical course of 78 Québec HT1 patients born between 1984 and 2004. There were three groups: those who never received nitisinone (28 patients), those who were first treated after 1 month of age (26 patients) and those treated before 1 month (24 patients). Retrospective chart review was performed for events before 1994, when nitisinone treatment began, and prospective data collection thereafter. FINDINGS No hospitalizations for acute complications of HT1 occurred during 5731 months of nitisinone treatment, versus 184 during 1312 months without treatment (p<0.001). Liver transplantation was performed in 20 non-nitisinone-treated patients (71%) at a median age of 26 months, versus 7 late-treated patients (26%, p<0.001), and no early-treated patient (p<0.001). No early-treated patient has developed detectable liver disease after more than 5 years. Ten deaths occurred in non-nitisinone treated patients versus two in treated patients (p<0.01). Both of the latter deaths were from complications of transplantation unrelated to HT1. One probable nitisinone-related event occurred, transient corneal crystals with photophobia. INTERPRETATION Nitisinone treatment abolishes the acute complications of HT1. Some patients with established liver disease before nitisinone treatment eventually require hepatic transplantation. Patients who receive nitisinone treatment before 1 month had no detectable liver disease after more than 5 years.

Maternal riboflavin deficiency, resulting in transient neonatal-onset glutaric aciduria Type 2, is caused by a microdeletion in the riboflavin transporter gene GPR172B.

Riboflavin, or vitamin B2, is a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) molecules, required in biological oxidation‐reduction reactions. We previously reported a case of a newborn female who had clinical and biochemical features of multiple acyl‐CoA dehydrogenation deficiency (MADD), which was corrected by riboflavin supplementation. The mother was then found to be persistently riboflavin deficient, suggesting that a possible genetic defect in riboflavin transport in the mother was the cause of the transient MADD seen in the infant. Two recently‐identified riboflavin transporters G protein‐coupled receptor 172B (GPR172B or RFT1) and riboflavin transporter 2 (C20orf54 or RFT2) were screened for mutations. Two missense sequence variations, c.209A>G [p.Q70R] and c.886G>A [p.V296M] were found in GPR172B. In vitro functional studies of both missense variations showed that riboflavin transport was unaffected by these variations. Quantitative real‐time PCR revealed a de novo deletion in GPR172B spanning exons 2 and 3 in one allele from the mother. We postulate that haploinsufficiency of this riboflavin transporter causes mild riboflavin deficiency, and when coupled with nutritional riboflavin deficiency in pregnancy, resulted in the transient riboflavin‐responsive disease seen in her newborn infant. This is the first report of a genetic defect in riboflavin transport in humans.

Whole-exome sequencing identifies novel ECHS1 mutations in Leigh syndrome

Leigh syndrome (LS) is a rare heterogeneous progressive neurodegenerative disorder usually presenting in infancy or early childhood. Clinical presentation is variable and includes psychomotor delay or regression, acute neurological or acidotic episodes, hypotonia, ataxia, spasticity, movement disorders, and corresponding anomalies of the basal ganglia and brain stem on magnetic resonance imaging. To date, 35 genes have been associated with LS, mostly involved in mitochondrial respiratory chain function and encoded in either nuclear or mitochondrial DNA. We used whole-exome sequencing to identify disease-causing variants in four patients with basal ganglia abnormalities and clinical presentations consistent with LS. Compound heterozygote variants in ECHS1, encoding the enzyme enoyl-CoA hydratase were identified. One missense variant (p.Thr180Ala) was common to all four patients and the haplotype surrounding this variant was also shared, suggesting a common ancestor of French-Canadian origin. Rare mutations in ECHS1 as well as in HIBCH, the enzyme downstream in the valine degradation pathway, have been associated with LS or LS-like disorders. A clear clinical overlap is observed between our patients and the reported cases with ECHS1 or HIBCH deficiency. The main clinical features observed in our cohort are T2-hyperintense signal in the globus pallidus and putamen, failure to thrive, developmental delay or regression, and nystagmus. Respiratory chain studies are not strikingly abnormal in our patients: one patient had a mild reduction of complex I and III and another of complex IV. The identification of four additional patients with mutations in ECHS1 highlights the emerging importance of this pathway in LS.

Detection and Quantification of Mosaic Mutations in Disease Genes by Next-Generation Sequencing

The identification of mosaicism is important in establishing a disease diagnosis, assessing recurrence risk, and genetic counseling. Next-generation sequencing (NGS) with deep sequence coverage enhances sensitivity and allows for accurate quantification of the level of mosaicism. NGS identifies low-level mosaicism that would be undetectable by conventional Sanger sequencing. A customized DNA probe library was used for capturing targeted genes, followed by deep NGS analysis. The mean coverage depth per base was approximately 800×. The NGS sequence data were analyzed for single-nucleotide variants and copy number variations. Mosaic mutations in 10 cases/families were detected and confirmed by NGS analysis. Mosaicism was identified for autosomal dominant (JAG1, COL3A1), autosomal recessive (PYGM), and X-linked (PHKA2, PDHA1, OTC, and SLC6A8) disorders. The mosaicism was identified either in one or more tissues from the probands or in a parent of an affected child. When analyzing data from patients with unusual testing results or inheritance patterns, it is important to further evaluate the possibility of mosaicism. Deep NGS analysis not only provides insights into the spectrum of mosaic mutations but also underlines the importance of the detection of mosaicism as an integral part of clinical molecular diagnosis and genetic counseling.

Longitudinal analysis of endurance and respiratory function from a natural history study of Morquio A syndrome

OBJECTIVES Baseline data from the Morquio A Clinical Assessment Program (MorCAP) revealed that individuals with Morquio A syndrome show substantial impairment in multiple domains including endurance and respiratory function (Harmatz et al., Mol Genet Metab, 2013). Here, 1- and 2-year longitudinal endurance and respiratory function data are presented. METHODS Endurance was assessed using the 6-minute walk test (6MWT) and the 3-minute stair climb test (3MSCT). Respiratory function was evaluated by measuring forced vital capacity (FVC) and maximum voluntary ventilation (MVV). Data were analyzed using repeated measures ANCOVA models. Annualized estimates of change were determined using model estimates and interpolation. RESULTS 353, 184, and 78 subjects were assessed at Year 0 (baseline), Year 1, and Year 2, respectively. The overall annualized estimate of change (SE) in 6MWT distance was -4.86±3.25m; a larger decline of -6.84±5.38m was observed in the subset of subjects meeting the inclusion/exclusion criteria of the Phase 3 clinical trial of elosulfase alfa (≥5years of age with baseline 6MWT distance ≥30 and ≤325m). In contrast, little change (-0.14±0.60stairs/min) was observed in 3MSCT. Annualized changes (SE) in FVC and MVV were 2.44±0.68% and 1.01±2.38%, respectively. FVC and MVV increased in patients aged ≤14years, but decreased in older patients. CONCLUSIONS The natural history of Morquio A syndrome is characterized by progressive impairment of endurance as measured by the 6MWT. Longitudinal trends in FVC and MVV showing increase in younger patients, but decrease in older patients, are likely to be influenced by growth. Changes in 6MWT may represent a sensitive measure of disease progression in ambulatory Morquio A patients.

Safety and physiological effects of two different doses of elosulfase alfa in patients with morquio a syndrome: A randomized, double‐blind, pilot study

The primary treatment outcomes of a phase 2, randomized, double‐blind, pilot study evaluating safety, physiological, and pharmacological effects of elosulfase alfa in patients with Morquio A syndrome are herewith presented. Patients aged ≥7 years and able to walk ≥200 m in the 6‐min walk test (6MWT) were randomized to elosulfase alfa 2.0 or 4.0 mg/kg/week for 27 weeks. The primary objective was to evaluate the safety of both doses. Secondary objectives were to evaluate effects on endurance (6MWT and 3‐min stair climb test [3MSCT]), exercise capacity (cardio‐pulmonary exercise test [CPET]), respiratory function, muscle strength, cardiac function, pain, and urine keratan sulfate (uKS) levels, and to determine pharmacokinetic parameters. Twenty‐five patients were enrolled (15 randomized to 2.0 mg/kg/week and 10 to 4.0 mg/kg/week). No new or unexpected safety signals were observed. After 24 weeks, there were no improvements versus baseline in the 6MWT, yet numerical improvements were seen in the 3MSCT with 4.0 mg/kg/week. uKS and pharmacokinetic data suggested no linear relationship over the 2.0–4.0 mg/kg dose range. Overall, an abnormal exercise capacity (evaluated in 10 and 5 patients in the 2.0 and 4.0 mg/kg/week groups, respectively), impaired muscle strength, and considerable pain were observed at baseline, and there were trends towards improvements in all domains after treatment. In conclusion, preliminary data of this small study in a Morquio A population with relatively good endurance confirmed the acceptable safety profile of elosulfase alfa and showed a trend of increased exercise capacity and muscle strength and decreased pain.