PracticeUpdate Conference Series - SSIEM 2018

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SOCIETY FOR THE STUDY OF INBORN ERRORS OF METABOLISM ANNUAL SYMPOSIUM 2018 4–7 SEPTEMBER 2018 • ATHENS • GREECE

THE BEST OF SSIEM 2018 Cholesterol Ratios May Help Detect Risk of Insulin Resistance and Metabolic Syndrome in Patients With GSDI • Whole Exome Sequencing Is a Potent First-Line Diagnostic Tool for Neurometabolic Disorders • New Phenotype of Muscle Weakness in Classic Infantile Pompe Disease Has Emerged • NMR Analysis Helps Diagnose a Variety of Inborn Errors of Metabolism

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PracticeUpdate is guided by a world-renowned Editorial and Advisory Board that represents community practitioners and academic specialists with cross-disciplinary expertise. Editor-in-Chief David Rakel MD, FAAFP Associate Editors Tricia Elliott MD, FAAFP, Peter Lin MD, CCFP Advisory Board Robert Bonakdar MD, FAAFP, FACN , Dennis Butler PhD , Frederick Chen MD, MPH , Irene Hamrick MD, FAAFP, AGSF, Dipesh Navsaria MPH, MSLIS, MD , Jonathan Temte MD, PhD Editorial Contributors Michael Allen MD , Andrea Dotson MD

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Contents

SSIEM 2018 • 4–7 September 2018 • Athens, Greece BY THE PRACTICEUPDATE EDITORIAL TEAM

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2 Cholesterol Ratios May Help Detect 4 Though NGS Can Detect Genomic Deletions in Metabolic Disorders, Precise Characterization Is Lacking 6 Follow-Up of Newborns With Suspected Lysosomal Storage Diseases Can Be Reduced Significantly With Simultaneous MS/MS and Confirmatory Tests 8 Diagnosis of Mucopolysaccharidosis May Be Simplified Worldwide 9 Critical Care of Adults With MPS Involves Diverse Considerations 10 Targeted NGS Gene Panel Improves Diagnosis of Hypoglycemia Due to Inborn Errors of Metabolism Risk of Insulin Resistance and Metabolic Syndrome in Patients With GSDI

12 New Phenotype of Muscle Weakness in Classic Infantile Pompe Disease Has Emerged 14 Epicatechin Improves Several Measures of Friedreich’s Ataxia 15 Whole Exome Sequencing Is a Potent First-Line Diagnostic Tool for Neurometabolic Disorders 16 Genetic Analysis Essential for Diagnosis of Glucose Transporter-1 Deficiency Syndrome 18 NMR Analysis Helps Diagnose a Variety of Inborn Errors of Metabolism 19 New Cell Reporter Line Enables Drug Screening of NDST1 in Patients With Sanfilippo Lysosomal Storage Disorders 20 Sapropterin + Diet in Children With Phenylketonuria Raises and Maintains Phenylalanine Tolerance

ELSEVIER AUSTRALIA ABN 70 001 002 357 475 Victoria Avenue Chatswood NSW 2067 Australia Locked Bag 7500 Chatswood DC NSW 2067 Printed in Australia. EMCS101803

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Cholesterol Ratios May Help Detect Risk of Insulin Resistance and Metabolic Syndrome in Patients With GSDI Chronic hyperlipidemia and adipose tissue dysfunction may play a role in insulin resistance andmetabolic syndrome in patients with glycogen storage disease type Ia (GSDI).

N on-HDL cholesterol, triglyceride/ HDL ratio, and non-HDL cholesterol/ HDL cholesterol ratio may be useful tools for early detection of the risk of insulin resistance and metabolic syndrome in patients with GSDIa, reports an assessment of lipid markers in patients with GSDI. Alessandro Rossi, MD, of the Università Federico II in Naples, Italy, and colleagues set out to assess lipid markers in patients with GSDI. “We performed this study to unveil the metabolic risk of patients with GSDI,” Dr. Rossi told Elsevier’s PracticeUpdate . They enrolled 12 patients with GSDIa, 6 with GSDIb, 26 age-matched controls, and 12 sex-matched controls. They measured waist circumference, serum glucose and plasma cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and leptin.

Non-HDL cholesterol, triglyceride/ HDL cholesterol ratio, and non-HDL cholesterol/HDL cholesterol ratio were also assessed as well as serum insulin and markers of insulin resistance (Homeostatic Model Assessment of Insulin Resistance [HOMA-IR], Quantitative Insulin Sensitivity Check Index [QUICKI], insulin sensitivity index [ISI]). Patients with GSDIa exhibited higher waist circumference, cholesterol, triglyceride, and insulin levels (P < .001), non-HDL cholesterol (P < .01), triglyceride/HDL cholesterol ratio (P < .01), non-HDL cholesterol/HDL cholesterol ratio (P < .05), and HOMA-IR (P < .001) than controls. QUICKI and ISI were lower than in controls (P < .001). Glucose, HDL cholesterol, LDL cholesterol, and leptin did not differ significantly.

Patients with GSDIb demonstrated lower cholesterol, LDL cholesterol, and HDL cholesterol (P < .001). Waist circumference, glucose, non-HDL cholesterol, leptin, insulin, triglyceride/HDL cholesterol, non-HDL cholesterol/HDL ratio, HOMA-IR, QUICKI, and ISI did not differ significantly. In patients with GSDIa, non-HDL cholesterol and triglyceride/HDL cholesterol ratio correlated directly with glucose levels (P < .05). HDL cholesterol correlated inversely with waist circumference (P < .05). Leptin levels correlated directly with insulin and HOMA-IR (P < .05) and correlated inversely with QUICKI and ISI (P < .05). Dr. Rossi explained that a higher risk of developing insulin resistance and metabolic syndrome has been shown in patients with GSDIa. Hyperlipidemia is a typical feature of GSDIa.

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" …notably,

non-HDL cholesterol, triglyceride/HDL cholesterol ratio, non-HDL cholesterol/ HDL cholesterol ratio, and plasma leptin are reliable markers of insulin resistance and metabolic syndrome. "

Notably, non-HDL cholesterol, triglyc- eride/HDL cholesterol ratio, non-HDL cholesterol/HDL cholesterol ratio, and plasma leptin are reliable markers of insu- lin resistance and metabolic syndrome. GSDI, or von Gierke disease, is the most common of the glycogen storage diseases. This genetic disease results from a deficiency of the enzyme glucose- 6-phosphatase and has a US incidence of approximately one in 50,000 to 100,000 births. The most common forms of GSDI are designated GSDIa and GSDIb, the former accounting for over 80% of diagnosed cases and the latter for <20%. A few rarer forms have been described. The metabolic characteristics of GSDIa and Ib are similar. The deficiency impairs the ability of the liver to produce free glucose from glycogen and from gluconeogenesis.

Because these are the two principal metabolic mechanisms by which the liver supplies glucose to the rest of the body during periods of fasting, it causes severe hypoglycemia and results in increased glycogen storage in liver and kidneys. Both organs function normally in childhood but are susceptible to a variety of problems in adult years. Other metabolic derangements include lactic acidosis and hyperlipidemia. Frequent or continuous feedings of cornstarch or other carbohydrates are the principal treatment. Other therapeutic measures may be needed for associated problems. Dr. Rossi concluded that lipid markers are employed to assess metabolic risk in the general population. The present findings suggest that non-HDL cholesterol, triglyceride/HDL cholesterol

ratio, and non-HDL cholesterol/HDL cholesterol ratio may be useful tools for early detection of the risk of insulin resistance and metabolic syndrome in patients with GSDIa. Correlation data in GSDIa, together with normal lipid markers in patients with GSDIb, support the hypothesis that chronic hyperlipidemia and adipose tissue dysfunction may play a role in insulin resistance and metabolic syndrome in patients with GSDIa. “The results suggested that lipid markers might be helpful to detect risk for insulin resistance early in patients with GSD1a,” Dr. Rossi noted. “Further research should dissect the link between hyperlipidemia and insulin resistance,” he added.

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Though NGS Can Detect Genomic Deletions in Metabolic

Disorders, Precise Characterization Is Lacking

Next-generation sequencing (NGS) can detect genomic alterations but further methods are needed to characterize the alterations precisely.

I n a series of 15 genes sequenced by two NGS panels, 17 large deletions were identified. Nevertheless, 30% involved Alu sequences and two were not identified by either panel, reports an evaluation of two NGS panels.

The group of Belen Perez Gonzalez, PhD, of Centro de Diagnóstico de Enfermedades Moleculares, Universidad Autónoma de Madrid in Spain, evaluated the two NGS panels. Panel 1 was an in-house, targeted, customized exome sequencing panel to capture the exome of 120 genes involved inmetabolic disorders. The panel included the entire sequence of the PAH, ALDOB,

Belen Perez Gonzalez, PhD

Inborn errors of metabolism comprise heterogeneous, rare genetic diseases with a variety of overlapping or unspecific clinical phenotypes. Multiple proteins with enzymatic, transporter, regulatory, and other functions participate in the intricate complexity of metabolic pathways. A breakdown in normal function of some of these proteins may impair the metabolic state of an organism. These disruptions can generally be assessed biochemically by detecting metabolites in various biological fluids. Specificity and sensitivity of some of cases, the disease-causative gene could be involved in another pathway while yielding a similar phenotype. " " …metabolic diseases are highly heterogeneous. Overlapping phenotypes may confuse the clinical orientation. In these

OTC, SLC22A5, and PCCA genes. Panel 2 was an extended panel including all known disease- associated genes described in the Online Mendelian Inheritance in Man database (Mendeliome panel) as of 2013. Quantification of coverage and comparison between controls and cases revealed that the two panels were able to detect large deletions in 20 patients. The analysis detected 17 different large deletions in 15 genes ( SLC7A2, GK, ALG1, MLYCD, PHKA2, BCKDHA, IVD, ASS1, PAH, GLDC, ACADM, DBT, PDHX, and BCAT2 ). Of these, 10 are novel genomic deletions. Further characterization by long-range polymerase chain reaction was conducted. Results showed that 30% of deletions detected involved Alu sequences. Furthermore, two cases also exhibited an Alu insertion in SLC7A2 or PHKA2, which was not identified by either panel due to incorrect alignment of the inserted Alu with the reference genome. Dr. Perez Gonzalez explained that recent developments in high- throughput sequence capture have led to the feasibility of NGS for routine genetic diagnosis. Improvement of depth in coverage allows for detection of large genomic deletions in addition to single-nucleotide variants.

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these biomarkers, however, are not always high. Inborn errors of metabolism are, generally, severe diseases, and accurate identification of the molecular basis of these diseases is important for appropriate treatment and genetic counselling. The establishment of a diagnosis of an inborn error of metabolism is supported by clinical suspicion and biochemical investigations. The classic molecular studies previously conducted in clinical laboratories consisted of time-consuming and expensive genetic studies. Some genetic diseases are complex. For example, one gene can be associated with different phenotypes. Similar phenotypes may be caused by mutations in different genes. In addition, patterns of inheritance of some of these diseases are not necessarily characterized completely. NGS technology has arisen as an essential tool for rapid and effective diagnosis prior to complex functional studies (that is, cellular enzyme activity). To date, published NGS diagnostic applications have focused on specific disorders and overlapping phenotypes. The appearance of a clinical exome strategy, however, has facilitated simultaneous assessment of different phenotypes. With respect to inborn errors of metabolism, studies of hyperphenylalaninemia, phenylketonuria, cerebral creatine deficiency, glycogen storage diseases, and mitochondrial diseases have generally yielded good results.

Most studies have utilized whole exome sequencing, but customized NGS approaches are being implemented in clinical practice. Additionally, comprehensive genetic testing of Mendelian childhood diseases through NGS is cost-saving. Biochemical diagnosis must remain a key part of the diagnostic algorithm to ensure that selected genetic variants are the real cause of the observed phenotype, because most of these workflows are still biased by personal experience and lack of specificity. Unsolved cases can be explained by various causes. First, the causative genemight not be included in the panel design. Genes may encode proteins involved in altering a biochemical marker that are currently unknown or not related with human disease. Second, metabolic diseases are highly heterogeneous. Overlapping phenotypes may confuse the clinical orientation. In these cases, the disease-causative gene could be involved in another pathway while yielding a similar phenotype. Dr Perez Gonzales concluded that NGS is capable of detecting genomic deletions in metabolic disorders.

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Follow-Up of Newborns With Suspected Lysosomal Storage Diseases Can Be Reduced Significantly With Simultaneous MS/MS and Confirmatory Tests Unneeded follow-up of these infants can beminimized.

S imultaneous determination of multiple enzyme activities by tandem mass spectrometry, adding specific biochemical markers as confirmatory tests, can significantly reduce fol- low-up of newborns with pseudodeficiency enzyme activities or carrier status, reports a tandemmass spectrometry investigation. AlbertoBurlina,MD, of theUniversitàdi Padova, Italy, andcolleagues set out to determine the activities of acid β-glucocerebrosidase (ABG; Gaucher disease), acid α-glucosidase (GAA, Pompe disease), acid α-galactosidase (GLA, Fabry disease), and acid α-L-iduronidase (IDUA, mucopolysaccharidosis I) in dried blood spots in a single assay at age of 48 h by multiplex tandem mass spectrometry using the NeoLSD® kit (Perkin Elmer). After methanolysis, biochemical confirmatory tests including assays of the following biomarkers were considered in patients who screened positive: Since 2015, 84,631 newborns have been screened. A total of 94 neonates (0.11%) have been recalled to donate a second dried blood spot. Low enzyme activities have been confirmed in 43 patients, who subsequently underwent the following confirmatory tests: ƒ ƒ LysoGb1 for Gaucher disease ƒ ƒ Glc4 for Pompe disease ƒ ƒ LysoGb3 for Fabry disease ƒ ƒ Urinary GAGs for mucopolysaccharidosis I Of these 43 patients, 15 exhibited abnormal biomarker values and mutational analyses confirmed disease presence. In 28 of the 43 patients, biomarkers were normal and mutational analysis showed pseudodeficiency or carrier status. Of note, the incidences of Pompe and Gaucher diseases were 1 in 21,158; of Fabry disease, 1 in 14,105; and of mucopolysaccharidosis, 1 in 42,316. The combined incidence of the four disorders was 1 in 5289 births. Dr. Burlina explained that the increasing availability of treatments and the importance of early intervention have stimulated newborn screening for lysosomal storage diseases. Early diagnosis of lysosomal storage diseases at the asymptomatic/presymptomatic stage is recognized as a valid ƒ ƒ Plasma glucosylsphingosine (LysoGb1) ƒ ƒ Lyso-globotriaosylceramide (LysoGb3) ƒ ƒ Urinary tetraglucoside (Glc4) ƒ ƒ Urinary glycosaminoglycans (GAGs)

public health objective. Inclusion of lysosomal storage diseases in newborn screening protocols is becoming more widespread internationally. Screening methods that provide a robust differentiation between affected and nonaffected individuals can help minimize false positives and avoid unnecessary treatment. Of the lysosomal storage diseases, Pompe disease and mucopolysaccharidoses I and II are the most favored for inclusion in screening programs. Fabry and Gaucher diseases are somewhat less favored. Krabbe disease is the least favored for newborn screening.

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Dr. Burlina concluded that simultaneous determination of multiple enzyme activities by tandemmass spectrometry, adding specific biochemical markers as confirmatory tests, significantly reduces the need for unnecessary follow-up of newborns with pseudodeficiency enzyme activities or carrier status. The NeoLSDMS/MS assay system for dried blood spot screening proved effective in identifying neonates at risk of lysosomal storage diseases in this study. Establishing cut-off values before starting a screening program is essential to avoid a high number of false positives, which are a source of needless anxiety and unnecessary medical interventions. Long-term follow-up of affected infants may provide important information about the natural history of the disease and their specific mutations, and should allow for optimized treatment outcomes. It has been demonstrated recently that the analytical range of mass spectrometry methods for screening is wider than that of fluorometric methods. Mass spectrometry provides a more accurate value of enzymatic activity, especially for very low values. This feature allows for better differentiation between patients with pathogenic mutations, pseudodeficiency alleles, and/or benign variants at the time of screening. This superior differentiation may, in turn, reduce the number of false positives. “The number of positive patients with the four lysosomal diseases screened was higher than we thought before starting the project,” Dr. Burlina told Elsevier’s PracticeUpdate.

“For Pompe disease and mucopolysaccharidosis I,” he said, “the possibility of diagnosing patients in their first week of life allowed us to start treatment (enzyme replacement therapy for the infant with Pompe disease and enzyme replacement therapy + bone marrow transplantation for the infant with muco- polysaccharidosis I). This early treatment changed the outcome of the diseases.” Dr. Burlina said that the multiplexed NeoLSD assay system for Pompe, Fabry, Gaucher, and mucopolysaccharidosis I has been integrated into the routine workflow of his newborn screening laboratory and has performed well for dried blood spot screening. He added, “We plan to continue our project and evaluation, and to add other diseases, in particular, mucopolysaccharidoses II and VI.”

www.practiceupdate.com/c/73404 " For Pompe disease and mucopolysaccharidosis I, the

possibility of diagnosing patients in their first week of life allowed us to start treatment. This early treatment changed the outcome of the diseases. "

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Diagnosis of Mucopolysaccharidosis May Be Simplified Worldwide Expansion of dried blood spot testing allows for use of a seven-enzymemucopolysaccharidosis panel

E xpansion of dried blood spot testing allows for use of a seven-enzyme mucopolysaccharidosis panel that can simplify the diagnosis of muco- polysaccharidosis (MPS) worldwide. This conclusion, based on results of a description of a tandem mass spectrometry evaluation, was presented at SSIEM 2018. T i m o t h y C .

however. Such analysis also requires a relatively large volume of blood. These requirements are problematic in international shipments of specimens and in testing infants. Adaptation of these enzyme assays for use in dried blood spots has ameliorated these issues in a number of lysosomal storage disorders. The development of tandem mass spectrometry substrates has allowed multiple enzyme reactions to be combined in a single assay, increasing efficiency. Enzyme deficiencies result in the accumulation of mucopolysaccharides (glycosaminoglycans) in lysosomes of various tissues and in the excessive excretion of partially degraded glycos- aminoglycans in urine. Specific degradative lysosomal enzyme deficiencies have been identified for all the MPS disorders. Glycosaminoglycans stored and excreted in the urine of the various MPS are dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin 4/6 sulfates. " The development of tandemmass spectrometry substrates has allowed multiple enzyme reactions to be combined in a single assay, increasing efficiency. " Clinical manifestations of the MPS depend on the specific enzyme deficiency, the end organ affected, and the accumulation of glycosaminoglycans in the affected organs. In diseases in which the brain is not involved, no mental retardation occurs. On the other hand, if the brain is affected and other somatic manifestations are minimal, features characteristic of MPS are not as prominent.

Patients suffer from a wide spectrum of symptoms including skeletal dysplasia (with short stature, dysostosis multiplex, and degenerative joint disease), cardiac valvulopathies, impaired pulmonary function, hepatosplenomegaly, sinusitis, otitis media, hearing loss, sleep apnea, corneal clouding, inguinal or umbilical hernia, and neurologic complications. Management of MPS includes sympto- matic treatment, enzyme replacement therapy (laronidase for MPS I, idursulfase for MPS II, galsulfase for MPS VI), and hematopoietic stem cell transplantation for MPS I and VI. Patients with MPS can suffer from compression of the cervical spinal cord caused by glycosaminoglycan infiltration of the dura. Such compression can lead to spastic paresis if not corrected by neurosurgical intervention. In MPS VI, intellectual deficit is generally absent. Patients may present with cervical spinal cord compression caused by cervical spinal instability, meningeal thickening, or bony stenosis, as well as communicating hydrocephalus and optic nerve atrophy. Carpal tunnel syndrome is a common feature of MPS I, II, and VI. Communicating hydrocephalus is common in severe MPS I and typically progresses slowly over months to years. Progressive cognitive impairment is characteristic of severe MPS, whereas cognition is gener- ally normal in attenuated MPS I. Patients with the severe form of MPS II suffer from behavioral disorders and early psychomotor regression, leading to intellectual deficit, as well as seizures. Dr. Wood concluded that his laboratory can now analyze 18 enzymes in dried blood spots to diagnose 20 different lysosomal storage disorders. Molecular testing from the original dried blood spot is available, limiting the need for additional sampling. Expansion of dried blood spot testing allows for use of a seven-enzyme MPS panel. The expansion may potentially simplify the diagnosis of MPS worldwide.

Wood, PhD, of the Greenwood Genetic Center in Greenwood, South Carolina, and col- leagues reported validation results of a new six-plex assay for diag- nosis of five MPS

Timothy C. Wood, PhD

disorders (MPS II, IIIB, IVA, VI, and VII) and neuronal ceroid lipofuscinosis type 2 in dried blood spots using ultraperformance liquid chromatography-tandem mass spectrometry. Analysis of substrates employed a Waters triple quadrupole ultraperformance liquid chromatography-tandem mass spectrometer. Overall, 99 samples with a confirmed diagnosis of one of the six disorders were used to evaluate clinical sensitivity and specificity. To develop normal ranges, 256 blood spots were obtained from unaffected controls or patients with a confirmed alternative diagnosis. A clear separation between normal and affected patients was noted for all six enzymes, providing 100% clinical sensitivity. Intra- and interday precision was acceptable (<20% coefficient of variation). Additionally, by analyzing multiple enzymes, mucolipidosis types II and III and multiple sulfatase deficiency were shown to be detectable. Dr. Wood explained that enzyme analysis is the gold standard for diagnosis of lysosomal storage disorders. Enzyme analysis in leukocytes requires that whole blood samples arrive at the testing laboratory within 24–48 h of collection,

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Critical Care of AdultsWithMPS Involves Diverse Considerations Diverse considerations need to be factored into coordinated care plans of adults with mucopolysaccharidosis. E veryadultwithmucopolysaccharidosis (MPS) requires consideration of his or her surgical and medical needs,

system involvement and only minimal somatic involvement; coarse hair, hirsutism, mild hepatosplenomegaly, and an enlarged head; occasionally, mild dysostosis multiplex and joint stiffness; eventually, by age 8–10 years, profound retardation with severely disturbed social behaviour. ƒ ƒ Morquio syndrome (MPS IV). Findings on examination in the severe form include orthopedic involvement (for example, spondyloepiphyseal dysplasia) as the primary finding; preservation of intelligence; genu valgum, short stature, spinal curvature, odontoid hypoplasia, ligamentous laxity, and atlantoaxial instability. The mild form exhibits much slower progression of skeletal dysplasia. ƒ ƒ Maroteaux-Lamy syndrome (MPS VI). Features are very similar to those of MPS IH. ƒ ƒ Sly syndrome (MPS VII). Features are similar to those of MPS IH. Nonemergency surgical care for specific MPS may include ventriculoperitoneal shunting for hydrocephalus; corneal transplantation for corneal clouding; valve replacement for cardiovascular disease; tracheostomy for obstructive airway disease; carpal tunnel release for orthopedic conditions; soft tissue procedures to release hip, knee, and ankle contractures; hip containment surgeries; corrective osteotomy for progressive valgus deformity at the knee; and posterior spinal fusion. Dr. Stepien concluded that these cases show the diverse considerations that need to be factored into coordinated care plans of adults with MPS. When adult care clinicians possess limited practical skills in managing patients with the disorder, expertise can be sought from more experienced pediatric teams. Standardized approaches, including input from pediatric and adult experts familiar with the surgical and medical needs of patients with MPS as well as individualized support, should be considered for every adult with the disorder.

MPS involves the defective activity of the lysosomal enzymes, which blocks degradation of mucopolysaccharides and leads to abnormal accumulation of heparan sulfate, dermatan sulfate, and keratan sulfate. Subclassifications are: ƒ ƒ Hurler syndrome (MPS IH). Findings on examination may include corneal clouding, hepatosplenomegaly, skeletal deformities (dysostosis multiplex), coarse facial features, large tongue, prominent forehead, joint stiffness, and short stature; upper airway obstruction, recurrent ear infections, noisy breathing, and persistent nasal discharge; hirsutism, hearing loss, hydrocephalus, and mental retardation.

including support from pediatric teams, results of a case report of nine patients show. K.M. Stepien, MD, of the Salford Royal National Health Services Trust in Salford, UK, presented a series of critical care cases to highlight challenges and practical solutions to optimize care of adult patients with MPS. A total of 9 cases (MPS I, II, IVa, and VI) in patients age 21–37 years were provided by four leading inherited metabolic disease centers in Europe. Critical care situations included surgical procedures (spinal decompression, cardiac valve or

" When adult care clinicians possess limited practical skills in managing patients with the disorder, expertise can be sought frommore experienced pediatric teams. "

corneal replacement, and tracheostomy), pregnancy, and a thrombus in a port- a-cath. Major surgical challenges included managing complex cardiac and respiratory dysfunction, and short stature, in the context of limited clinical expertise in MPS. These challenges were resolved involving pediatric and adult care specialists with expertise in the disorder, from external centers, when needed. Medical challenges included disrupted enzyme replacement therapy during pregnancy, which resulted in an increased risk of infection. Regular follow-up and antibiotics were used to address this disruption. A thrombus in a port-a-cath was resolved by inserting a Hickman line. Sterile methods and patient support were applied in the insertion. Management of MPS is evolving as patients increasingly survive to adulthood due to earlier diagnosis, treatment availability, and multidisciplinary care. MPS can lead to a range of critical clinical situations, however, in adult patients.

ƒ ƒ Hurler-Scheie syndrome (MPS I-H/S). Findings on examination may include milder features; normal intelligence and micrognathia; corneal clouding, joint stiffness, and heart disease. ƒ ƒ Scheie syndrome (MPS IS). Findings on examination may include aortic valve disease, corneal clouding, and joint stiffness; normal intelligence and stature. ƒ ƒ Hunter syndrome (MPS II). Findings on examination in the severe form include pebbly ivory skin lesions on the back, arms, and thighs; coarse facial features, skeletal deformities, and joint stiffness; retinal degeneration with clear cornea and hydrocephalus, mental retardation, and aggressive behavior. Findings in the mild form include similar features, but with much slower progression; normal intelligence and no hydrocephalus; hearing impairment and loss of hand function. ƒ ƒ Sanfilippo syndrome (MPS III). This most common mucopolysaccharidosis disorder may include findings on examination of severe central nervous

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Targeted NGS Gene Panel Improves Diagnosis of Hypoglycemia Due to Inborn Errors of Metabolism In childrenwith persistent hypoglycemia, diagnosis of inborn errors of metabolismcan be improved using a targeted next-generation sequencing (NGS) gene panel.

N GS is cost-effective compared with Sanger sequencing of multiple genes and represents a powerful tool for diagnosis of inborn errors of metabolism that present with persistent hypoglycemia, reports an evaluation of NGS sequencing in 64 children. Alfonso M. Maiorana, MD, of the Bambino Gesu Children’s Hospital in Rome, Italy, and colleagues set out to evaluate the impact of NGS in the genetic diagnosis of 64 pediatric patients with persistent hypoglycemia due to inborn errors of metabolism. The 64 children underwent extensive work-up. They were divided into three diagnostic classes: 1. Single candidate gene (n=9) 2. Multiple candidate genes (n=43) 3. No candidate gene (n=12) Subsequently, they were tested via a custom gene panel of 65 targeted genes, which included five disease categories: 1. Hyperinsulinemic hypoglycemia 2. Fatty acid oxidation and ketogenesis defects 3. Ketolysis defects 4. Glycogen storage diseases and other disorders of carbohydrate metabolism 5. Mitochondrial disorders Molecular data were compared with clinical and biochemical data. A proven diagnosis was obtained in 78% of patients suspected of harboring a single candidate gene, in 49% with multiple candidate genes, and in 33% with no candidate genes. The diagnostic yield was 48% for hyperinsulinemic hypoglycemia, 66% per fatty acid oxidation and ketogenesis defect, 59% for glycogen storage diseases and other carbohydrate disorders, and 67% for mitochondrial disorders. Remarkably, a molecular diagnosis was established in 33% of patients belonging to the no candidate gene class.

The causes of negative genetic results include the possibility of detecting variants of unknown significance that have not been definitely linked to the disease, the presence of insertion/deletion of few nucleotides, mutations in novel disease- causing genes as well as mutations in noncoding regions, and the possibility of variant(s) in known disease genes not included on the panel for whom whole exome/genome sequencing approaches would improve the diagnostic yield. In 45% of patients in whom clinical and laboratory evaluation did not allow identification of a single candidate gene, the targeted NGS approach provided a rapid diagnosis, overtaking genetic heterogeneity and clinical variability. A further advantage of NGS is its cost-effectiveness versus traditional Sanger sequencing. Single- gene testing, the traditional approach for genetic diagnosis, is appropriate in selected cases that present with specific clinical and biochemical features. Use of a gene panel eliminates the hurdle of having to prioritize multiple candidate genes for stepwise sequencing. NGS analysis also confers better accuracy and higher sensitivity in mosaicism detection than Sanger sequencing. The approach also helps avoid invasive, inaccurate, or expensive procedures, such as liver biopsy and enzyme assay.

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hyperinsulinemic hypoglycemia; fatty acid oxidation defects; ketogenesis/ketolysis defects, glycogen storage diseases; disorders of carbohydrate metabolism (for example, fructose-1,6-biphosphatase deficiency; hereditary fructose intolerance); mitochondrial disorders; along with some congenital glycosylation defects; and some aminoacidopathies (eg, maple syrup urine disease). The establishment of a precise diagnosis allows targeting of specific nutritional and pharmacologic therapies. The clinical and biochemical pattern may suggest a specific disease category. Genetic heterogeneity of these conditions makes identification of a single candidate gene difficult, however. Classic molecular gene- by-gene analysis is time-consuming and expensive for the number and size of involved genes. Dr. Maiorana concluded that NGS provided a diagnosis in approximately 50% of patients in whom clinical and laboratory evaluation did not allow for identification of a single candidate gene.

NGS will not replace metabolic testing, central to establish biochemical phenotypeandguidepatient management and follow-up. NGS represents a tool to provide information on genotype- phenotype correlation. It offers diagnostic confirmation and facilitates diagnosis of complex disorders by reducing the number of tests or procedures. It facilitates decision making and fills the gaps in laboratory diagnosis. Furthermore, NGS allows genetic counseling for the family and provides recurrence risk and prenatal diagnosis in future pregnancies. Hypoglycemia is an important cause of morbidity in the pediatric population and is associated with a large number of inborn errors of metabolism. Multiple proteins with different functions (enzymes, transporters, transcription factors, and others) participate in the complex machinery of glucose homeostasis. Mutations in many genes lead to impairment of glucose homeostasis. These disorders comprise heterogeneous and rare genetic diseases with a variety of overlapping and often unspecific clinical and biochemical phenotypes, including

" NGS provided a diagnosis in

approximately 50% of patients in whom clinical and laboratory evaluation did not allow for identification of a single candidate gene. "

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SSIEM 2018 • PRACTICEUPDATE CONFERENCE SERIES 11

New Phenotype of Muscle Weakness in Classic Infantile Pompe Disease Has Emerged Etiology of this new phenotype is unclear.

O ver a median of 6.0 years, patients with classic infantile Pompe disease who were treated with enzyme replacement therapy, despite improving in proximal muscle strength and function, developed moderate to severe weakness of the dorsiflexors of the feet at an early age. This outcome of a long-term assessment of qualitative motor development and function in 15 patients with classic infantile Pompe disease was reported at SSIEM 2018. E. Poelman, MD, of Erasmus Medical College in Rotterdam, The Netherlands, and colleagues focused the assessment on the development of distal muscle weakness in relation to general motor development. Patients with classic infantile Pompe disease with a follow-up duration of ≥3.0 years who had learned to walk were included. A comprehensive exam- ination of motor function was conducted every 3 months and recorded on video. Qualitative motor development and function were assessed independently by three reviewers using these recordings. Achievement of motor milestones was scored during regular outpatient visits. Median follow-up duration was 6.0 (3.1–18.2) years in 15 patients. Median age at which motor milestones were achieved included the following: ƒ ƒ Sitting: 10.6 (8.3–13.7) months Overall, 5 patients lost the ability to walk and stand, all of whomexhibited proximal weakness of the legs. Early weakness of the foot dorsiflexors was found in 14 patients, which led to foot drop during walking. Weakness of the hands leading to loss of pincer grasp was found in 4 patients, only in patients with general muscle weakness. Dr. Poelman explained that the introduction of enzyme replacement therapy has improved the prospects for patients with classic infantile Pompe disease significantly. The clinical phenotype of patients who survive with enzyme replacement therapy is now ascertained. ƒ ƒ Standing: 12.2 (9.2–16.5) months ƒ ƒ Walking: 17.2 (14.0–21.3) months

Though Pompe disease is known to cause proximal muscle weakness predominantly. Substantial distal weakness was noted to develop in these patients. Dr. Poelman concluded that over a median of 6.0 years, patients with classic infantile Pompe disease treated with enzyme replacement therapy, despite improvement of proximal muscle strength and function, developed moderate to severe weakness of the dorsiflexors of the feet at an early age. A typical phenotype emerged with distal as well as proximal weakness, which differs from patients with nonclassic Pompe disease. The etiology of this new phenotype of muscle weakness is unclear. Further investigation is required. In a related study, Sehnaz Basaran, MD, of Çukurova University in Adana, Turkey, and colleagues evaluated the motor function of 17 patients with infantile Pompe disease. The following tests were performed on 17 patients from a group of 19 mobile patients, as well as on healthy children and patients with Duchenne

muscular dystrophy: ƒ ƒ 6-minute walk test ƒ ƒ Timed 10-m walk test

PRACTICEUPDATE CONFERENCE SERIES • SSIEM 2018 12

" A typical phenotype emerged with distal as well as proximal weakness, which differs from patients with nonclassic Pompe disease. The etiology of this new phenotype of muscle weakness is unclear. "

and male patients with infantile Pompe disease. The tonus and elasticity of vastus medialis, and elasticity of gastrocnemius muscles, and hand-held strength were significantly higher in healthy than in patients with infantile Pompe disease (P < .005). Distal muscles of the lower extremities and biceps brachii muscles in patients with infantile Pompe disease were affected less. Dr. Basaran explained that though enzyme replacement therapy is a lifesaving therapy, the effectiveness of muscle function on mobility has not been evaluated in detail. Dr. Basaran concluded that enzyme replacement therapy has extended survival in patientswith infantile Pompe diseasewith respect to cardiomyopathy. Data are sparse, however, on ambulatory patients. The quick motor function test is valid for nonclassic Pompe disease, though few reports in limited numbers of patients with infantile Pompe disease are available.

ƒ ƒ Quick motor function test by myotonometry and MicroFET3 dynamometry of elasticity, bicep, vastus medialis, gastrocnemius, and tibialis anterior muscle tonus and stiffness ƒ ƒ 3-minute stair climb test ƒ ƒ Muscle strength by hand-held dynamometry Of 39 patients, 24 were alive, 2 mechanically ventilated, and 3 younger than 18 months of age. A total of 10 girls and 7 boys began enzyme replacement therapy at 3.5 ± 3.1 months of age. Ages were 57.6 ± 25.9 months, and mean duration of enzyme replacement therapy was 53.2 ± 27.8 months. P.L299P (c.896T>C) was the most common mutation. A significant difference in 6-minute walk test and 10-m walk test was observed between healthy children and those with infantile Pompe disease (P < .005). Six-minute and 10-m walk test were nearly similar for Duchenne muscular dystrophy

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SSIEM 2018 • PRACTICEUPDATE CONFERENCE SERIES 13

Epicatechin Improves Several Measures of Friedreich’s Ataxia Treatment with epicatechinwas safe and tolerable over 24 weeks and resulted in neurological improvement.

T reatment with epicatechin was safe and tolerable over 24 weeks and resulted in neurological improve- ment of total Friedreich's Ataxia Rating Scale score, 8-m timed walk, nine-hole peg test, and a reduction in left ventricu- lar myocardial mass index in a subset of patients. This outcome of a phase II open-label, single-center trial was reported at SSIEM 2018. Marc Patterson, MD, of the Mayo Clinic in Rochester, Minnesota, and colleagues evaluated the safety and efficacy of epicatechin for 24 weeks in 10 subjects with Friedreich’s ataxia. Participants 10 to 50 years of age with a confirmed diagnosis of Friedreich’s ataxia, disease duration ≤7 years, and affected cardiac/neurological systems took epicatechin 75 mg daily orally. The dose was escalated to 150 g daily after 3 months in subjects not showing improvement. Primary endpoints were safety and efficacy according to neurological, cardiac, and metabolic evaluations before and after treatment. Secondary endpoints included frataxin upregulation and urine F2-isoprostane reduction before and after treatment. A total of 10 patients completed all safety and efficacy measurements, except for 1 patient who did not complete the Friedreich's Ataxia Rating Scale and 8-m time walk after 24 weeks. Epicatechin was safe and tolerable, without clinically significant side effects but with a transient increase in the frequency of migraine. No subjects discontinued the study. Improvement in Friedreich's Ataxia Rating Scale score was observed in

50% of subjects, by a mean of 5.5 (range 2–8) points and in the 8-m walk in 30% of subjects by a mean of 2.2 s (range 2.2–2.3). In the nine-hole peg test, improvement was seen after 24 weeks in 50% of subjects by a mean of 4.2 s (range 1–9) in the right hand and 5.4 s (range 3–7) in the left hand. A reduction was seen in MRI-derived left ventricular myocardial mass index by >10% in 6 subjects at 12 weeks and in one additional subject at 24 weeks (70%). Left ventricular myocardial mass index showed slight worsening after 24 weeks in the 6 subjects. No improvement in secondary endpoints was observed. Dr. Pa t t e r son exp l a i ned t ha t Friedreich’s ataxia is a rare, progressive neurodegenerative disease caused by mutations in the gene encoding for the frataxin protein, important for normal functioning of mitochondria. Friedreich’s ataxia is clinically characterized by progressive ataxia (lack of voluntary muscle coordination), dysarthria (speech impairment), areflexia, and progressive motor weakness. Patients also develop hypertrophic cardiomyopathy and diabetes. Symptoms appear in childhood and worsen progressively through adulthood. Friedreich’s ataxia commonly fatal in mid-adulthood. Neuropathologically, Friedreich’s ataxia involves degeneration of the dentate nuclei in the cerebellum and sensory and motor tracts of the spinal cord. No therapies are available for Friedreich’s ataxia. Medications can only treat symptoms and address disease- associated cardiovascular, neurological, or motor dysfunction. Epicatechin is a flavonoid that confers similar health benefits to exercise.

Observations suggest a signaling mechanism that may induce mitochondrial biogenesis in mitochondria-depleted muscle fibers and neurons as well as induction of antioxidant enzymes. Epicatechin has been shown to improve mitochondrial function in various models of cardiac and neuromuscular diseases. A study reported a reduction in oxidative stress and some restoration of new mitochondria formation of in senile mice. On the basis of these promising results in mice, an initial, proof-of-concept study was implemented in humans. Following 7 days of epicatechin treatment, significant positive effects were observed in circulating follistatin/myostatin plasma levels and grip strength. Thus, epicatechin is the first compound ever noted, to favorably modulate both regulators of muscle growth and suggest increases in strength. In a small phase I study in 2014, epicatechin was found to help improve mitochondrial enzyme function and plasma levels of frataxin. These changes, in turn, may help reduce the risk of cardiovascular and neurodegenerative disease and improve skeletal muscle structure and strength. Dr. Patterson concluded that treatment with epicatechin was safe and tolerable over 24 weeks and resulted in neurological improvement of total Friedreich's Ataxia Rating Scale score, 8-m timed walk, nine-hole peg test, and a reduction in left ventricular myocardial mass index in a subset of patients. Further studies of epicatechin are warranted for Friedreich’s ataxia. www.practiceupdate.com/c/73327 Epicatechin is not registered in Australia by the TGA therefore the efficacy and safety of this product has not been established.

PRACTICEUPDATE CONFERENCE SERIES • SSIEM 2018 14

Whole Exome Sequencing Is a Potent First-Line Diagnostic Tool for NeurometabolicDisorders Covering a wide phenotypic spectrum, whole exome sequencing facilitates a definite diagnosis in 46% and possible diagnosis in 30% of cases. W hole exome sequencing has been demonstrated to be a potent first-line diagnostic tool (approximately 50%; >3000) are expected to be uncovered in the next decade.

Dr. Buchert explained that molecular diagnoses of inherited neurometabolic disorders are challenging due to genetic and clinical heterogeneity. Especially in inborn errors of metabolism, early identification is crucial for treatment decisions. Neurometabolic disorders are a subgroup of inborn errors of metabolism with neurological manifestations. They are the largest group of genetic intellectual developmental disorders amenable to causal therapy targeting pathophysiology, such as medical diets, supplements, medications, hematopoietic stem cell transplants, and gene therapy. Early diagnosis is critical for timely initiation of treatment and prevention of irreversible brain damage. Traditional observational epidemiology is inefficient in elucidating rare and clinically and genetically heterogeneous Mendelian diseases such as neurometabolic disorders. Despite extensive, expensive, and often invasive testing, diagnostic delays are common and many patients remain undiagnosed. These concerning challenges have begun to be addressed with the advent of genome-wide screening approaches based on next- generation sequencing (NGS). An alternative to whole genome sequencing is sequencing a portion of the genome, either the entire set of protein-coding sequences (whole exome sequencing) or a subset (gene panels). Due to its lower cost, the whole exome sequencing has been used widely to identify the genetic basis of rare Mendelian disorders. Whole exome sequencing has skyrocketed the pace at which new gene-disease associations are established. With further advances in NGS, the majority of unknown gene-disease associations

In addition, NGS has facilitated detection of new gene-phenotype associations for genes that have been implicated in Mendelian conditions either by establishing causative relationships with different known or novel conditions or by expanding the clinical spectrum of established Mendelian conditions. These new discoveries of rare diseases genetics enhance the ability to diagnose patients. In 2016, the diagnostic efficiency of exome- wide and large gene panel sequencing in children with complex neurometabolic phenotypes was established. This success further supported the assertion that next-generation sequencing + exquisite data interpretation is the most time-efficient path to definite diagnosis in these rare heterogeneous conditions. Given the limitations of the exome- sequencing technology combined with slow updating of included genes within DNA capture technologies, the more promising approach is an up-to-date virtual panel of known disease genes applied to whole genome sequencing data. This combination allows clinical restriction to a subset of genetic information, but allows future exploration of the entire genome for undiagnosed cases. Though NGS-based genetic testing is established in Europe, researchers and clinicians need to promote the potential benefit of whole exome/genome sequencing as a first-line diagnostic tool. Dr. Buchert concluded that whole exome sequencing is a potent first-line diagnostic tool for neurometabolic disorders. Though the phenotypic spectrum is wide and variable, whole exome sequencing facilitated a definite diagnosis in 46% and a possible diagnosis pending follow-up in 30% of cases.

for neurometabolic disorders. Amid a wide phenotypic spectrum, whole exome sequencing facilitated a definite diagnosis in 46% and possible diagnosis pending follow-up in 30% of cases, reports a whole exome sequencing evaluation. Rebecca Buchert, MD, of the University of Tuebingen in Germany, reported results of whole exome sequencing-based diagnostics in a cohort of 97 index cases to evaluate the diagnostic utility of such sequencing in inherited neurometabolic disorders. Inclusion criteria were suspected neurometabolic disorders based on biochemical or radiological findings. Coding genomic regions were enriched for sequencing as 2×125 bp/2×100 bp paired-end reads on an Illumina HiSeq2500/NovaSeq6000 system. Generated sequences were analyzed using the megSAP pipeline. Clinical variant prioritization included filtering steps according to an in-house operating procedure. In 44 (46%) cases, likely pathogenic or pathogenic variants were identified in genes associated with neurometabolic disorders (n=135) or other neurodevelop- mental disorders (n=9). Targeted therapy was available in 36% of these. In 33%, established diagnoses implicated a modification of clinical management. In 31% of cases, however, disorders were not amenable to targeted therapy. In 29 cases (30%) variants of unknown significance were identified with pending follow-up studies for functional analyses. Moreover, five candidate genes were newly identified. A total of 18 cases (19%), however, remain unsolved. Ongoing investigations of the latter in a research setting include whole genome and transcriptome sequencing.

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SSIEM 2018 • PRACTICEUPDATE CONFERENCE SERIES 15