An automated 13.5 hour system for scalable diagnosis and acute management guidance for genetic diseases.

While many genetic diseases have effective treatments, they frequently progress rapidly to severe morbidity or mortality if those treatments are not implemented immediately. Since front-line physicians frequently lack familiarity with these diseases, timely molecular diagnosis may not improve outcomes. Herein we describe Genome-to-Treatment, an automated, virtual system for genetic disease diagnosis and acute management guidance. Diagnosis is achieved in 13.5 h by expedited whole genome sequencing, with superior analytic performance for structural and copy number variants. An expert panel adjudicated the indications, contraindications, efficacy, and evidence-of-efficacy of 9911 drug, device, dietary, and surgical interventions for 563 severe, childhood, genetic diseases. The 421 (75%) diseases and 1527 (15%) effective interventions retained are integrated with 13 genetic disease information resources and appended to diagnostic reports ( https://gtrx.radygenomiclab.com ). This system provided correct diagnoses in four retrospectively and two prospectively tested infants. The Genome-to-Treatment system facilitates optimal outcomes in children with rapidly progressive genetic diseases.

Severe thrombocytopenia and intracranial hemorrhage in a newborn with Noonan syndrome and neonatal alloimmune thrombocytopenia.

Noonan syndrome (NS) is a genetic disorder with distinctive physical features and often multiple organ involvement. Bleeding disorders are reported in over half of patients with NS, including thrombocytopenia and platelet dysfunction. Neonatal alloimmune thrombocytopenia (NAIT) is an alloantigenic thrombocytopenia that can present with severe bleeding. Here, we present a case of intracranial hemorrhage and severe thrombocytopenia in a neonate found to have both NAIT and a de novo heterozygous pathogenic variant in PTPN11, consistent with Noonan syndrome.

Carriership of two copies of C9orf72 hexanucleotide repeat intermediate-length alleles is a risk factor for ALS in the Finnish population.

The hexanucleotide repeat expansion in intron 1 of the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In addition to the effects of the pathogenic expansion, a role of intermediate-length alleles has been suggested in ALS, corticobasal degeneration and Parkinson's disease. Due to the rarity of intermediate-length alleles with over 20 repeats and the geographical variability in their frequency, large studies that account for population stratification are needed to elucidate their effects. To this aim, we used repeat-primed PCR and confirmatory PCR assays to determine the C9orf72 repeat allele lengths in 705 ALS patients and 3958 controls from Finland. After exclusion of expansion carriers (25.5% of the ALS patients and 0.2% of the controls), we compared the frequency of intermediate-length allele carriers of 525 ALS cases and 3950 controls using several intermediate-length allele thresholds (7-45, 17-45, 21-45, 24-45 and 24-30). The carriership of an intermediate-length allele did not associate with ALS (Fisher's test, all p ≥ 0.15) nor was there any association with survival (p ≥ 0.33), when we divided our control group into three age groups (18-65, 66-84 and 85-105 years). Carriership of two intermediate-length alleles was associated with ALS, when the longer allele was ≥ 17 repeats (p = 0.002, OR 5.32 95% CI 2.02-14.05) or ≥ 21 repeats (p = 0.00016, OR 15.21 95% CI 3.79-61.0). Our results show that intermediate-length alleles are a risk factor of ALS when present in both alleles, whereas carrying just one intermediate-length allele was not associated with ALS or survival.

Mortality in a neonate with molybdenum cofactor deficiency illustrates the need for a comprehensive rapid precision medicine system.

Neonatal encephalopathy with seizures is a presentation in which rapid whole-genome sequencing (rWGS) has shown clinical utility and improved outcomes. We report a neonate who presented on the third day of life with seizures refractory to antiepileptic medications and neurologic and computerized tomographic findings consistent with severe generalized brain swelling. rWGS revealed compound heterozygous variants in the molybdenum cofactor synthesis gene, type 1A (MOCS1 c.*7 + 5G > A and c.377G > A); a provisional diagnosis of molybdenum cofactor deficiency on day of life 4. An emergency investigational new drug application for intravenous replacement of the MOCS1 product, cyclic pyranopterin monophosphate, was considered, but felt unsuitable in light of the severity of disease and delay in the start of treatment. The patient died on day of life 9 despite having a precise molecular diagnosis within the first week of life. This case illustrates that an rWGS-based molecular diagnosis within the first week of life may be insufficient to improve outcomes. However, it did inform clinical decision-making with regard to resuscitation and predicted long-term outcome. We suggest that to achieve optimal reductions in morbidity and mortality, rWGS must be implemented within a comprehensive rapid precision medicine system (CRPM). Akin to newborn screening (NBS), CRPM will have onboarding, diagnosis, and precision medicine implementation components developed in response to patient and parental needs. Education of health-care providers in a learning model in which ongoing data analyses informs system improvement will be essential for optimal effectiveness of CRPM.

Analysis of functional variants in mitochondrial DNA of Finnish athletes.

BACKGROUND: We have previously reported on paucity of mitochondrial DNA (mtDNA) haplogroups J and K among Finnish endurance athletes. Here we aimed to further explore differences in mtDNA variants between elite endurance and sprint athletes. For this purpose, we determined the rate of functional variants and the mutational load in mtDNA of Finnish athletes (n = 141) and controls (n = 77) and determined the sequence variation in haplogroups. RESULTS: The distribution of rare and common functional variants differed between endurance athletes, sprint athletes and the controls (p = 0.04) so that rare variants occurred at a higher frequency among endurance athletes. Furthermore, the ratio between rare and common functional variants in haplogroups J and K was 0.42 of that in the remaining haplogroups (p = 0.0005). The subjects with haplogroup J and K also showed a higher mean level of nonsynonymous mutational load attributed to common variants than subjects with the other haplogroups. Interestingly, two of the rare variants detected in the sprint athletes were the disease-causing mutations m.3243A > G in MT-TL1 and m.1555A > G in MT-RNR1. CONCLUSIONS: We propose that endurance athletes harbor an excess of rare mtDNA variants that may be beneficial for oxidative phosphorylation, while sprint athletes may tolerate deleterious mtDNA variants that have detrimental effect on oxidative phosphorylation system. Some of the nonsynonymous mutations defining haplogroup J and K may produce an uncoupling effect on oxidative phosphorylation thus favoring sprint rather than endurance performance.

Near-Infrared Spectroscopy in the Diagnostic Evaluation of Mitochondrial Disorders: A Neonatal Intensive Care Unit Case Series.

We assessed the utility of near-infrared spectroscopy to evaluate neonates with mitochondrial disorders. We observed abnormally high cerebral oxygen saturation levels indicating insufficient tissue oxygen utilization. We propose that near-infrared spectroscopy may be an additional tool in the diagnostic evaluation of a suspected mitochondrial disorder.

Cinacalcet therapy in an infant with an R185Q calcium-sensing receptor mutation causing hyperparathyroidism: a case report and review of the literature.

Background Neonatal severe hyperparathyroidism (NSHPT) is commonly treated with either parathyroidectomy or pharmacologic agents with varying efficacy and numerous side effects. Reports of using cinacalcet for NSHPT have increased, however, the effective dose for pediatric patients from the onset of symptoms through infancy has not been established. Case presentation We describe the clinical course of a newborn with a de novo R185Q mutation in the calcium-sensing receptor (CASR) gene, causing NSHPT. The infant received cinacalcet from the first days of life until 1 year of age. Conclusions Cinacalcet therapy effectively controlled the patient's serum calcium, phosphorus, and parathyroid hormone (PTH) levels without side effects.

Pediatric Integrative Medicine.

The American Academy of Pediatrics is dedicated to optimizing the well-being of children and advancing family-centered health care. Related to this mission, the American Academy of Pediatrics recognizes the increasing use of complementary and integrative therapies for children and the subsequent need to provide reliable information and high-quality clinical resources to support pediatricians. This Clinical Report serves as an update to the original 2008 statement on complementary medicine. The range of complementary therapies is both extensive and diverse. Therefore, in-depth discussion of each therapy or product is beyond the scope of this report. Instead, our intentions are to define terms; describe epidemiology of use; outline common types of complementary therapies; review medicolegal, ethical, and research implications; review education and training for select providers of complementary therapies; provide educational resources; and suggest communication strategies for discussing complementary therapies with patients and families.

Review of Randomized Controlled Trials of Massage in Preterm Infants.

Preterm birth affects about 10% of infants born in the United States. Massage therapy is being used in some neonatal intensive care units for its potential beneficial effects on preterm infants. This article reviews published randomized controlled trials on the effects of massage in preterm infants. Most studies evaluating the effect of massage in weight gain in premature infants suggest a positive effect on weight gain. Increase in vagal tone has been reported in infants who receive massage and has been suggested as a possible mechanism for improved weight gain. More studies are needed on the underlying mechanisms of the effects of massage therapy on weight gain in preterm infants. While some trials suggest improvements in developmental scores, decreased stress behavior, positive effects on immune system, improved pain tolerance and earlier discharge from the hospital, the number of such studies is small and further evidence is needed. Further studies, including randomized controlled trials, are needed on the effects of massage in preterm infants.

Expanding the phenotype of hawkinsinuria: new insights from response to N-acetyl-L-cysteine.

Hawkinsinuria is a rare disorder of tyrosine metabolism that can manifest with metabolic acidosis and growth arrest around the time of weaning off breast milk, typically followed by spontaneous resolution of symptoms around 1 year of age. The urinary metabolites hawkinsin, quinolacetic acid, and pyroglutamic acid can aid in identifying this condition, although their relationship to the clinical manifestations is not known. Herein we describe clinical and laboratory findings in two fraternal twins with hawkinsinuria who presented with failure to thrive and metabolic acidosis. Close clinical follow-up and laboratory testing revealed previously unrecognized hypoglycemia, hypophosphatemia, combined hyperlipidemia, and anemia, along with the characteristic urinary metabolites, including massive pyroglutamic aciduria. Treatment with N-acetyl-L-cysteine (NAC) restored normal growth and normalized or improved most biochemical parameters. The dramatic response to NAC therapy supports the idea that glutathione depletion plays a key role in the pathogenesis of hawkinsinuria.

Impaired Health-Related Quality of Life in Children and Families Affected by Methylmalonic Acidemia.

An understanding of health related quality of life (HRQoL) in children and families affected by methylmalonic acidemia (MMA) is important in planning counseling and therapeutic intervention. Liver transplantation (LT) is used as a treatment for MMA; however, its risks and benefits continue to be investigated. The purpose of this study was twofold: (1) to measure HRQoL in children and families affected by MMA using the Pediatric Quality of Life Inventory (PedsQL™) parent version, and (2) to assess the impact of LT on HRQoL by comparing LT and non-LT patient scores and free responses. Parents/caregivers reported lower scores on the majority of the PedsQL™ scales as compared to samples of healthy children, children with solid organ transplants for indications other than MMA, and families affected by chronic conditions. Scores for children with MMA were lowest in school and social functioning and scores for families were lowest in worry and activity impairment. There were no significant differences in LT and non-LT patient scores on the PedsQL™ scales. Our results document the negative impact of MMA on HRQoL.

Treatment of methylmalonic acidemia by liver or combined liver-kidney transplantation.

OBJECTIVE: To assess biochemical, surgical, and long-term outcomes of liver (LT) or liver-kidney transplantation (LKT) for severe, early-onset methylmalonic acidemia/acid (MMA). STUDY DESIGN: A retrospective chart review (December 1997 to May 2012) of patients with MMA who underwent LT or LKT at Lucile Packard Children's Hospital at Stanford. RESULTS: Fourteen patients underwent LT (n = 6) or LKT (n = 8) at mean age 8.2 years (range 0.8-20.7). Eleven (79%) were diagnosed during the neonatal period, including 6 by newborn screening. All underwent deceased donor transplantation; 12 (86%) received a whole liver graft. Postoperative survival was 100%. At a mean follow-up of 3.25 ± 4.2 years, patient survival was 100%, liver allograft survival 93%, and kidney allograft survival 100%. One patient underwent liver re-transplantation because of hepatic artery thrombosis. After transplantation, there were no episodes of hyperammonemia, acidosis, or metabolic decompensation. The mean serum MMA at the time of transplantation was 1648 ± 1492 µmol/L (normal <0.3, range 99-4420). By 3 days, post-transplantation levels fell on average by 87% (mean 210 ± 154 µmol/L), and at 4 months, they were 83% below pre-transplantation levels (mean 305 ± 108 µmol/L). Developmental delay was present in 12 patients (86%) before transplantation. All patients maintained neurodevelopmental abilities or exhibited improvements in motor skills, learning abilities, and social functioning. CONCLUSIONS: LT or LKT for MMA eradicates episodes of hyperammonemia, results in excellent long-term survival, and suggests stabilization of neurocognitive development. Long-term follow-up is underway to evaluate whether patients who undergo early LT need kidney transplantation later in life.

Perinatal features of the RASopathies: Noonan syndrome, cardiofaciocutaneous syndrome and Costello syndrome.

The RASopathies are a family of developmental disorders caused by heritable defects of the RAS/MAPK signaling pathway. While the postnatal presentation of this group of disorders is well known, the prenatal and neonatal findings are less widely recognized. We report on the perinatal presentation of 10 patients with Noonan syndrome (NS), nine with Cardiofaciocutaneous syndrome (CFCS) and three with Costello syndrome (CS), in conjunction with the results of a comprehensive literature review. The majority of perinatal findings in NS, CS, and CFCS are shared: polyhydramnios; prematurity; lymphatic dysplasia; macrosomia; relative macrocephaly; respiratory distress; hypotonia, as well as cardiac and renal anomalies. In contrast, fetal arrhythmia and neonatal hypoglycemia are relatively specific to CS. NS, CS, and CFCS should all be considered as a possible diagnosis in pregnancies with a normal karyotype and ultrasound findings of a RASopathy. Recognition of the common perinatal findings of these disorders should facilitate both their prenatal and neonatal diagnosis.

Degree of glutathione deficiency and redox imbalance depend on subtype of mitochondrial disease and clinical status.

Mitochondrial disorders are associated with decreased energy production and redox imbalance. Glutathione plays a central role in redox signaling and protecting cells from oxidative damage. In order to understand the consequences of mitochondrial dysfunction on in vivo redox status, and to determine how this varies by mitochondrial disease subtype and clinical severity, we used a sensitive tandem mass spectrometry assay to precisely quantify whole blood reduced (GSH) and oxidized (GSSG) glutathione levels in a large cohort of mitochondrial disorder patients. Glutathione redox potential was calculated using the Nernst equation. Compared to healthy controls (n = 59), mitochondrial disease patients (n = 58) as a group showed significant redox imbalance (redox potential -251 mV ± 9.7, p<0.0001) with an increased level of oxidation by ∼ 9 mV compared to controls (-260 mV ± 6.4). Underlying this abnormality were significantly lower whole blood GSH levels (p = 0.0008) and GSH/GSSG ratio (p = 0.0002), and significantly higher GSSG levels (p<0.0001) in mitochondrial disease patients compared to controls. Redox potential was significantly more oxidized in all mitochondrial disease subgroups including Leigh syndrome (n = 15), electron transport chain abnormalities (n = 10), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (n = 8), mtDNA deletion syndrome (n = 7), mtDNA depletion syndrome (n = 7), and miscellaneous other mitochondrial disorders (n = 11). Patients hospitalized in metabolic crisis (n = 7) showed the greatest degree of redox imbalance at -242 mV ± 7. Peripheral whole blood GSH and GSSG levels are promising biomarkers of mitochondrial dysfunction, and may give insights into the contribution of oxidative stress to the pathophysiology of the various mitochondrial disorders. In particular, evaluation of redox potential may be useful in monitoring of clinical status or response to redox-modulating therapies in clinical trials.

Abnormal hepatocellular mitochondria in methylmalonic acidemia.

Methylmalonic acidemia (MMA) is one of the most frequently encountered forms of branched-chain organic acidemias. Biochemical abnormalities seen in some MMA patients, such as lactic acidemia and increased tricarboxylic acid cycle intermediate excretion, suggest mitochondrial dysfunction. In order to investigate the possibility of mitochondrial involvement in MMA, we examined liver tissue for evidence of mitochondrial ultrastructural abnormalities. Five explanted livers obtained from MMA mut(0) patients undergoing liver transplantation were biopsied. All patients had previous episodes of metabolic acidosis, lactic acidemia, ketonuria, and hyperammonemia. All biopsies revealed a striking mitochondriopathy by electron microscopy. Mitochondria were markedly variable in size, shape, and conformation of cristae. The inner matrix appeared to be greatly expanded and the cristae were diminutive and disconnected. No crystalloid inclusions were noted. This series clearly documents extensive mitochondrial ultrastructure abnormalities in liver samples from MMA patients undergoing transplantation, providing pathological evidence for mitochondrial dysfunction in the pathophysiology of MMA mut(0). Considering the trend to abnormally large mitochondria, the metabolic effects of MMA may restrict mitochondrial fission or promote fusion. The correlation between mitochondrial dysfunction and morphological abnormalities in MMA may provide insights for better understanding and monitoring of optimized or novel therapeutic strategies.

Evidence of redox imbalance in a patient with succinic semialdehyde dehydrogenase deficiency.

The pathophysiology of succinic semialdehyde dehydrogenase (SSADH) deficiency is not completely understood. Oxidative stress, mitochondrial pathology, and low reduced glutathione levels have been demonstrated in mice, but no studies have been reported in humans. We report on a patient with SSADH deficiency in whom we found low levels of blood reduced glutathione (GSH), and elevations of dicarboxylic acids in urine, suggestive of possible redox imbalance and/or mitochondrial dysfunction. Thus, targeting the oxidative stress axis may be a potential therapeutic approach if our findings are confirmed in other patients.

A new LC-MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood.

Reduced levels of glutathione (γ-glutamylcysteinylglycine, GSH) and the ratio of GSH to glutathione disulfide (GSSG) can serve as important indicators of oxidative stress and disease risk. Measured concentrations of GSH and GSSG vary widely between laboratories, largely due to the instability of GSH during sample handling and variables arising from different analytical methods. We have developed a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring whole blood GSH and GSSG that minimizes preanalytic and analytic variability, reliably eliminates interference from ion suppression, and can easily be implemented in clinical laboratories. Samples were deproteinized with sulfosalicylic acid (SSA) and derivatized with N-ethylmaleimide (NEM) in a single preparative step, and the resulting supernatants combined with stable-isotope internal standards (GSH-(13)C, (15)N-NEM and GSSG-(13)C,(15)N), subjected to chromatographic separation using a Hypercarb column, and analyzed by MS/MS in the positive-ion mode. Results showed excellent linearity for both GSH and GSSG over the ranges of physiologic normal, with inter- and intra-assay CV's of 3.1-4.3% and accuracy between 95% and 101%. The lower limits of detection (LLOD) were 0.4µM for GSH and 0.1µM for GSSG and the lower limits of quantitation (LLOQ) were 1.5µM for GSH and 0.1µM for GSSG. Derivatized samples are stable for at least 3 years when stored at -80°C, and underivatized samples for at least 24h at either 4°C or room temperature. Reference intervals were determined for 59 control samples, and were (mean±SD): GSH 900±140µM; GSSG 1.17±0.43µM; GSH/GSSG 880±370.

Liver transplantation for urea cycle disorders in pediatric patients: a single-center experience.

LT has emerged as a surgical treatment for UCDs. We hypothesize that LT can be safely and broadly utilized in the pediatric population to effectively prevent hyperammonemic crises and potentially improve neurocognitive outcomes. To determine the long-term outcomes of LT for UCDs, charts of children with UCD who underwent LT were retrospectively reviewed at an academic institution between July 2001 and May 2012. A total of 23 patients with UCD underwent LT at a mean age of 3.4 yr. Fifteen (65%) patients received a whole-liver graft, seven patients (30%) received a reduced-size graft, and one patient received a living donor graft. Mean five-yr patient survival was 100%, and allograft survival was 96%. Mean peak blood ammonia (NH(3) ) at presentation was 772 µmol/L (median 500, range 178-2969, normal <30-50). After transplantation, there were no episodes of hyperammonemia. Eleven patients were diagnosed with some degree of developmental delay before transplantation, which remained stable or improved after transplantation. Patients without developmental delay before transplantation maintained their cognitive abilities at long-term follow-up. LT was associated with the eradication of hyperammonemia, removal of dietary restrictions, and potentially improved neurocognitive development. Long-term follow-up is underway to evaluate whether LT at an early age (<1 yr) will attain improved neurodevelopmental outcomes.