Patient care standards for primary mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society.

The purpose of this statement is to provide consensus-based recommendations for optimal management and care for patients with primary mitochondrial disease. This statement is intended for physicians who are engaged in the diagnosis and management of these patients. Working group members were appointed by the Mitochondrial Medicine Society. The panel included members with several different areas of expertise. The panel members utilized surveys and the Delphi method to reach consensus. We anticipate that this statement will need to be updated as the field continues to evolve. Consensus-based recommendations are provided for the routine care and management of patients with primary genetic mitochondrial disease.

Blood ammonia and glutamine as predictors of hyperammonemic crises in patients with urea cycle disorder.

PURPOSE: The aim of this study was to examine predictors of ammonia exposure and hyperammonemic crises in patients with urea cycle disorders. METHODS: The relationships between fasting ammonia, daily ammonia exposure, and hyperammonemic crises were analyzed in >100 patients with urea cycle disorders. RESULTS: Fasting ammonia correlated strongly with daily ammonia exposure (r = 0.764; P < 0.001). For patients with fasting ammonia concentrations <0.5 upper limit of normal (ULN), 0.5 to <1.0 ULN, and ≥1.0 ULN, the probability of a normal average daily ammonia value was 87, 60, and 39%, respectively, and 10.3, 14.1, and 37.0% of these patients, respectively, experienced ≥1 hyperammonemic crisis over 12 months. Time to first hyperammonemic crisis was shorter (P = 0.008) and relative risk (4.5×; P = 0.011) and rate (~5×, P = 0.006) of hyperammonemic crises were higher in patients with fasting ammonia ≥1.0 ULN vs. <0.5ULN; relative risk was even greater (20×; P = 0.009) in patients ≥6 years old. A 10- or 25-µmol/l increase in ammonia exposure increased the relative risk of a hyperammonemic crisis by 50 and >200% (P < 0.0001), respectively. The relationship between ammonia and hyperammonemic crisis risk seemed to be independent of treatment, age, urea cycle disorder subtype, dietary protein intake, or blood urea nitrogen. Fasting glutamine correlated weakly with daily ammonia exposure assessed as 24-hour area under the curve and was not a significant predictor of hyperammonemic crisis. CONCLUSION: Fasting ammonia correlates strongly and positively with daily ammonia exposure and with the risk and rate of hyperammonemic crises, suggesting that patients with urea cycle disorder may benefit from tight ammonia control.

Self-reported treatment-associated symptoms among patients with urea cycle disorders participating in glycerol phenylbutyrate clinical trials.

BACKGROUND: Health care outcomes have been increasingly assessed through health-related quality of life (HRQoL) measures. While the introduction of nitrogen-scavenging medications has improved survival in patients with urea cycle disorders (UCDs), they are often associated with side effects that may affect patient compliance and outcomes. METHODS: Symptoms commonly associated with nitrogen-scavenging medications were evaluated in 100 adult and pediatric participants using a non-validated UCD-specific questionnaire. Patients or their caregivers responded to a pre-defined list of symptoms known to be associated with the use of these medications. Responses were collected at baseline (while patients were receiving sodium phenylbutyrate [NaPBA]) and during treatment with glycerol phenylbutyrate (GPB). RESULTS: After 3 months of GPB dosing, there were significant reductions in the proportion of patients with treatment-associated symptoms (69% vs. 46%; p<0.0001), the number of symptoms per patient (2.5 vs. 1.1; p<0.0001), and frequency of the more commonly reported individual symptoms such as body odor, abdominal pain, nausea, burning sensation in mouth, vomiting, and heartburn (p<0.05). The reduction in symptoms was observed in both pediatric and adult patients. The presence or absence of symptoms or change in severity did not correlate with plasma ammonia levels or NaPBA dose. CONCLUSIONS: The reduction in symptoms following 3 months of open-label GPB dosing was similar in pediatric and adult patients and may be related to chemical structure and intrinsic characteristics of the product rather than its effect on ammonia control.

Ammonia control and neurocognitive outcome among urea cycle disorder patients treated with glycerol phenylbutyrate.

UNLABELLED: Glycerol phenylbutyrate is under development for treatment of urea cycle disorders (UCDs), rare inherited metabolic disorders manifested by hyperammonemia and neurological impairment. We report the results of a pivotal Phase 3, randomized, double-blind, crossover trial comparing ammonia control, assessed as 24-hour area under the curve (NH3 -AUC0-24hr ), and pharmacokinetics during treatment with glycerol phenylbutyrate versus sodium phenylbutyrate (NaPBA) in adult UCD patients and the combined results of four studies involving short- and long-term glycerol phenylbutyrate treatment of UCD patients ages 6 and above. Glycerol phenylbutyrate was noninferior to NaPBA with respect to ammonia control in the pivotal study, with mean (standard deviation, SD) NH3 -AUC0-24hr of 866 (661) versus 977 (865) µmol·h/L for glycerol phenylbutyrate and NaPBA, respectively. Among 65 adult and pediatric patients completing three similarly designed short-term comparisons of glycerol phenylbutyrate versus NaPBA, NH3 -AUC0-24hr was directionally lower on glycerol phenylbutyrate in each study, similar among all subgroups, and significantly lower (P < 0.05) in the pooled analysis, as was plasma glutamine. The 24-hour ammonia profiles were consistent with the slow-release behavior of glycerol phenylbutyrate and better overnight ammonia control. During 12 months of open-label glycerol phenylbutyrate treatment, average ammonia was normal in adult and pediatric patients and executive function among pediatric patients, including behavioral regulation, goal setting, planning, and self-monitoring, was significantly improved. CONCLUSION: Glycerol phenylbutyrate exhibits favorable pharmacokinetics and ammonia control relative to NaPBA in UCD patients, and long-term glycerol phenylbutyrate treatment in pediatric UCD patients was associated with improved executive function (ClinicalTrials.gov NCT00551200, NCT00947544, NCT00992459, NCT00947297). (HEPATOLOGY 2012).

Methylmalonic acidemia: a megamitochondrial disorder affecting the kidney.

BACKGROUND: Classical (or isolated) methylmalonic acidemia (MMA) is a heterogeneous inborn error of metabolism most typically caused by mutations in the vitamin B12-dependent enzyme methylmalonyl-CoA mutase (MUT). With the improved survival of individuals with MMA, chronic kidney disease has become recognized as part of the disorder. The precise description of renal pathology in MMA remains uncertain. METHODS: Light microscopy, histochemical, and ultrastructural studies were performed on the native kidney obtained from a 19-year-old patient with mut MMA who developed end stage renal disease and underwent a combined liver-kidney transplantation. RESULTS: The light microscopy study of the renal parenchyma in the MMA kidney revealed extensive interstitial fibrosis, chronic inflammation, and tubular atrophy. Intact proximal tubules were distinguished by the widespread formation of large, circular, pale mitochondria with diminished cristae. Histochemical preparations showed a reduction of cytochrome c oxidase and NADH activities, and the electron microscopy analysis demonstrated loss of cytochrome c enzyme activity in these enlarged mitochondria. CONCLUSIONS: Our results demonstrate that the renal pathology of MMA is characterized by megamitochondria formation in the proximal tubules in concert with electron transport chain dysfunction. Our findings suggest therapies that target mitochondrial function as a treatment for the chronic kidney disease of MMA.

Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes.

BACKGROUND: Methylmalonic acidemia (MMA), a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition. METHODS: To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction. RESULTS: Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes. CONCLUSION: These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

Recommendations for the Management of Strokelike Episodes in Patients With Mitochondrial Encephalomyopathy, Lactic Acidosis, and Strokelike Episodes.

IMPORTANCE: Strokelike episodes are a cardinal feature of several mitochondrial syndromes, including mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS). Recent advances in the understanding of the pathophysiologic mechanisms of strokelike episodes in MELAS have led to improved treatment options. OBSERVATIONS: Current understanding of the cause of strokelike episodes in MELAS and present recommendations to assist in the identification and treatment of patients with MELAS who present with stroke are presented. Mounting evidence points toward a benefit of the nitric oxide precursors, arginine, to both prevent and reduce the severity of strokes in patients with MELAS. CONCLUSIONS AND RELEVANCE: Although much information is still needed regarding the appropriate dosing and timing of arginine therapy in patients with MELAS, urgent administration of nitric oxide precursors in patients with MELAS ameliorates the clinical symptoms associated with strokelike episodes.

Four cases with hypoplastic thumbs and encephaloceles.

We report on four infants with hypoplastic thumbs and occipital encephaloceles. None had either a chromosome abnormality or a family history of any major malformation. The literature and database were searched intensively. No similar cases were reported previously, suggesting that the constellations might represent a new genetic syndrome.

Rod photoreceptor function in children with mitochondrial disorders.

OBJECTIVE: To test the hypothesis that function of the rod photoreceptors is abnormal in pediatric patients with mitochondrial disorders. METHODS: Patients (n = 22; median age, 5 years) with a deficiency of 1 or more of the mitochondrial enzyme complexes, or a mutation in mitochondrial DNA, were studied by means of scotopic, full-field electroretinography (ERG). The conditions of ERG testing allowed derivation of the parameters of the activation of rod phototransduction from the ERG a-wave, and postreceptoral function from b-wave and P(2) stimulus-response functions. The deactivation of phototransduction was studied in 5 patients. The patients' ERG responses were compared with those of healthy control subjects (n = 25). RESULTS: Responses from 19 patients were sufficient for analysis of rod photoreceptor and postreceptoral function. Saturated amplitudes of the rod photoresponse and b-wave sensitivity were significantly depressed in the patients. Saturated amplitudes of rod cell and P(2) responses were correlated. The kinetics of deactivation of phototransduction were slowed even if the kinetics of activation were normal. CONCLUSIONS: In patients with mitochondrial disorders, some abnormalities of the scotopic ERG responses originate in the rod photoreceptors, but postreceptoral processes may also be abnormal. From a practical perspective, ERG testing can contribute to diagnosis of mitochondrial disorders.

Newborn screening for hepatorenal tyrosinemia by tandem mass spectrometry: analysis of succinylacetone extracted from dried blood spots.

OBJECTIVES: To develop a method for the determination of succinylacetone (SA) in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). METHODS: SA was extracted from DBS with an acetonitrile and water solution (80:20 by volume) containing formic acid and hydrazine hydrate (both at 0.1% by volume), and analyzed by MS/MS with a total run time per sample under 2 min. The reference range for SA in newborns was determined by analyzing a control group of 3199 DBS. SA was also measured in stored newborn specimens from three patients diagnosed clinically with hepatorenal tyrosinemia (HT). RESULTS: The within-run precision was

Determination of phenylalanine and tyrosine in dried blood specimens by ion-exchange chromatography using the Hitachi L-8800 analyzer.

OBJECTIVES: The treatment for phenylketonuria (PKU) includes monitoring blood phenylalanine (Phe) levels on a regular basis. To reduce inconvenience to the patient and family, blood specimens on filter paper can be obtained at home and mailed to the clinic or analytical laboratory. For this reason, we validated an 8-min isothermal and isocratic HPLC method using the Hitachi L-8800 analyzer for quantitation of Phe and tyrosine (Tyr) from dried blood specimens (DBS). DESIGN AND METHODS: The method was worked out using DBS fortified with Phe and Tyr. For method comparison, blood samples from 31 PKU patients and 5 non-PKU volunteers were analyzed as DBS by HPLC using the Hitachi L-8800 analyzer, and compared both to plasma analyzed by HPLC and DBS analyzed using tandem mass spectrometry (MS/MS). RESULTS: For HPLC analysis of DBS, the within-run precision for Phe and Tyr was < or = 5.1% and < or = 4.5%, respectively, and total precision measured over a 3-month period was < or = 7.2% and < or = 8.7%, respectively. Correlation analysis was performed using results from fresh plasma analyzed by HPLC (r = 0.988 for Phe, r = 0.964 for Tyr) and from DBS analyzed by MS/MS (r = 0.960 for Phe, r = 0.942 for Tyr). Difference plots revealed good agreement between the HPLC and MS/MS methods. CONCLUSIONS: Determination of Phe and Tyr in DBS using this HPLC technique compares well with other methods. This technique with its short analytical time is convenient for monitoring patients with PKU and might be particularly useful in centers following many patients.

Effect of expanded newborn screening for biochemical genetic disorders on child outcomes and parental stress.

CONTEXT: Tandem mass spectrometry now allows newborn screening for more than 20 biochemical genetic disorders. Questions about the effectiveness and risks of expanded newborn screening for biochemical genetic disorders need to be answered prior to its widespread acceptance as a state-mandated program. OBJECTIVES: To compare newborn identification by expanded screening with clinical identification of biochemical genetic disorders and to assess the impact on families of a false-positive screening result compared with a normal result in the expanded newborn screening program. DESIGN: Prospective study involving an inception cohort of newly diagnosed children. SETTING: Massachusetts, Maine, and a private laboratory in Pennsylvania with expanded newborn screening; other New England states with limited screening. PARTICIPANTS: Families of 50 affected children identified through expanded newborn screening (82% of eligible cases); 33 affected children identified clinically (97% of eligible cases); 94 screened children with false-positive results (75% of eligible cases); and 81 screened children with normal results (63% of eligible cases). MAIN OUTCOME MEASURES: Child's health and development and the Parental Stress Index. RESULTS: Within the first 6 months of life, 28% of children identified by newborn screening compared with 55% of clinically identified children required hospitalization (P =.02). One child identified by newborn screening compared with 8 (42%) identified clinically performed in the range of mental retardation (P<.001). Mothers in the screened group reported lower overall stress on the Parental Stress Index than mothers in the clinically identified group (z = 3.38, P<.001). Children with false-positive results compared with children with normal results were twice as likely to experience hospitalization (21% [n = 20] vs 10% [n = 8], respectively; P =.06). Mothers of children in the false-positive group compared with mothers of children with normal screening results attained higher scores on the Parental Stress Index (z = 4.25, P<.001) and the Parent-Child Dysfunction subscale (z = 5.30, P<.001). CONCLUSIONS: Expanded newborn screening may lead to improved health outcomes for affected children and lower stress for their parents. However, false-positive screening results may place families at risk for increased stress and parent-child dysfunction.

Practice patterns of mitochondrial disease physicians in North America. Part 1: diagnostic and clinical challenges.

Mitochondrial medicine is a young subspecialty. Clinicians have a limited evidence base on which to formulate clinical decisions regarding diagnosis, treatment and patient management. Mitochondrial medicine specialists have cobbled together an informal set of rules and paradigms for preventive care and management based in part on anecdotal experience. The Mitochondrial Medicine Society (MMS) assessed the current state of clinical practice from diagnosis, to preventive care and treatment, as provided by various mitochondrial disease specialists in North America. We hope that by obtaining this information we can begin moving towards formulating a set of consensus criteria and establishing standards of care.

Metabolic profiling of total homocysteine and related compounds in hyperhomocysteinemia: utility and limitations in diagnosing the cause of puzzling thrombophilia in a family.

We describe a family illustrating the diagnostic difficulties occurring when pyridoxine-responsive cystathionine beta-synthase (CBS) deficiency presents with thrombotic disease without associated ocular, skeletal, or CNS abnormalities, a situation increasingly recognized. This family had several thromboembolic episodes in two generations with apparently inconstant elevations of plasma total homocysteine (tHcy). When taking (sometimes even low amounts) of pyridoxine, the affected family members had low-normal tHcy and normal values for cystathionine, methionine, and cysteine. Withdrawal of vitamin therapy was necessary before lower cystathionine, elevated methionine, and decreased cysteine became apparent, a pattern suggestive of CBS deficiency, leading to the finding that the affected members were each compound heterozygotes for CBS p.G307S and p.P49L. To assist more accurate diagnosis of adults presenting with thrombophilia found to have elevated tHcy, the patterns of methionine-related metabolites in CBS-deficient patients are compared in this article to those in patients with homocysteine remethylation defects, including inborn errors of folate or cobalamin metabolism, and untreated severe cobalamin or folate deficiency. Usually serum cystathionine is low in subjects with CBS deficiency and elevated in those with remethylation defects. S-Adenosylmethionine and S-adenosylhomocysteine are often markedly elevated in CBS deficiency when tHcy is above 100 umol/L. We conclude that there are likely other undiagnosed, highly B6-responsive adult patients with CBS deficiency, and that additional testing of cystathionine, total cysteine, methionine, and S-adenosylmethionine will be helpful in diagnosing them correctly and distinguishing CBS deficiency from remethylation defects.

Maple syrup urine disease in Cypriot families: identification of three novel mutations and biochemical characterization of the p.Thr211Met mutation in the E1alpha subunit.

We report five mutations, three of them novel, responsible for maple syrup urine disease in four unrelated Cypriot families. The five children studied are the first cases of classic maple syrup urine disease to be reported among Cypriots. The first novel mutation identified is a single-base deletion in exon 6 of the Elalpha gene (c.718delG), which leads to a frameshift after Ala240 and to a stop codon 89 residues further downstream. The other two novel mutations identified are in the Elbeta subunit: a two-base deletion in exon 6, c.662_663delCC, which leads to a frameshift after Ala221 and creates a stop codon 17 residues further downstream, as well as a splice mutation, IVS3[+3]delA, which results in the skipping of exon 3. The two known mutations identified are in the Elalpha gene: the G > C transversion at the 3'-splice acceptor site, (IVS5-1G > C), which results in the deletion of the entire exon 6, and the missense mutation in exon 5 (c.632C > T), which corresponds to a p.Thr211Met substitution. The p.Thr211Met substitution is located in a potassium-ion pocket in the E1 component required for stability of the bound cofactor thiamine diphosphate. The mutant E1 protein harboring the p.Thr211Met substitution was shown unable to bind thiamine diphosphate, leading to undetectable E1 activity.

N-carbamylglutamate markedly enhances ureagenesis in N-acetylglutamate deficiency and propionic acidemia as measured by isotopic incorporation and blood biomarkers.

N-acetylglutamate (NAG) is an endogenous essential cofactor for conversion of ammonia to urea in the liver. Deficiency of NAG causes hyperammonemia and occurs because of inherited deficiency of its producing enzyme, NAG synthase (NAGS), or interference with its function by short fatty acid derivatives. N-carbamylglutamate (NCG) can ameliorate hyperammonemia from NAGS deficiency and propionic and methylmalonic acidemia. We developed a stable isotope (13)C tracer method to measure ureagenesis and to evaluate the effect of NCG in humans. Seventeen healthy adults were investigated for the incorporation of (13)C label into urea. [(13)C]urea appeared in the blood within minutes, reaching maximum by 100 min, whereas breath (13)CO(2) reached a maximum by 60 min. A patient with NAGS deficiency showed very little urea labeling before treatment with NCG and normal labeling thereafter. Correspondingly, plasma levels of ammonia and glutamine decreased markedly and urea tripled after NCG treatment. Similarly, in a patient with propionic acidemia, NCG treatment resulted in a marked increase in urea labeling and decrease in glutamine, alanine, and glycine. These results provide a reliable method for measuring the effect of NCG on nitrogen metabolism and strongly suggest that NCG could be an effective treatment for inherited and secondary NAGS deficiency.

The importance of gut motility in the metabolic control of propionic acidemia.

We hypothesized that gut motility likely plays a critical role in the metabolic stability in propionic acidemia (PA). Therefore, 4 known patients with PA (aged 47 months to 185 months) were prospectively studied over 7 days in the Clinical Research Center at Children's Hospital, Boston. Determinations of ammonia, bicarbonate, and amino acids in blood; organic acids and propionylglycine in urine; and a lactulose breath test were conducted under two study conditions: on regular therapy (for 4 days) and on regular therapy plus Senekot (Purdue Frederick Company, Norwalk, Conn), an intestinal motility agent (for 3 days). The total gastrointestinal transit time was calculated using 20 nonabsorbable, inert, radio-opaque markers. The addition of an intestinal motility agent resulted in a significant decrease in blood ammonia, urinary excretion of propionylglycine, and a rise in the ratio of free to total carnitine over baseline. We concluded that enhancement of gut motility can improve metabolic stability in patients with PA.

Improved growth and nutrition status in children with methylmalonic or propionic acidemia fed an elemental medical food.

BACKGROUND: Failure-to-thrive (FTT) has been described in patients with organic acidemias treated with low protein diets. OBJECTIVE: To determine if patients with methylmalonic (MMA) or propionic acidemia (PA) can achieve normal growth and nutrition status. METHODS: A 6-month multicenter outpatient study was conducted with infants and toddlers treated with Propimex-1 Amino Acid-Modified Medical Food With Iron (Ross Products Division, Abbott Laboratories, Columbus, OH). Main outcome measures were anthropometrics, protein status indices, plasma retinol, and alpha-tocopherol. RESULTS: Sixteen patients completed the study. Mean baseline age was 0.54 +/- 0.02 years (range 0.03-3.00 years). By study end, mean National Center for Health Statistics (NCHS) weight centile increased from 26 to 49%; mean crown-heel length centile from 25 to 33%; and mean head circumference centile from 43 to 54%. Mean (+/- SE) protein and energy intakes by <6-month-old, 6<12-month-old, and 1<4-year-old patients were 15.3 +/- 0.9 g and 645 +/- 10 kcal; 18.3 +/- 1.1 g and 741 +/- 92 kcal; and 25.1 +/- 2.46 g and 1062 +/- 100 kcal, respectively. Plasma glycine concentrations were significantly and negatively correlated with energy intake (r=-0.77, p<0.0005). No correlation was found between dietary protein intakes and plasma ammonia concentrations. Protein status indices, retinol and alpha-tocopherol concentrations were within reference ranges at study end. CONCLUSIONS: Propimex-1 improved growth and nutrition status in patients with MMA or PA in just 6 months when fed in sufficient amounts. Providing energy and protein for patients with FTT at intakes recommended for catch-up growth may have resulted in even better growth.