Feasibility of Targeted Next-Generation DNA Sequencing for Expanding Population Newborn Screening.

BACKGROUND: Newborn screening (NBS) is an effective public health intervention that reduces death and disability from treatable genetic diseases, but many conditions are not screened due to a lack of a suitable assay. Whole genome and whole exome sequencing can potentially expand NBS but there remain many technical challenges preventing their use in population NBS. We investigated if targeted gene sequencing (TGS) is a feasible methodology for expanding NBS. METHODS: We constructed a TGS panel of 164 genes which screens for a broad range of inherited conditions. We designed a high-volume, low-turnaround laboratory and bioinformatics workflow that avoids the technical and data interpretation challenges associated with whole genome and whole exome sequencing. A methods-based analytical validation of the assay was completed and test performance in 2552 newborns examined. We calculated annual birth estimates for each condition to assess cost-effectiveness. RESULTS: Assay analytical sensitivity was >99% and specificity was 100%. Of the newborns screened, 1.3% tested positive for a condition. On average, each individual had 225 variants to interpret and 1.8% were variants of uncertain significance (VUS). The turnaround time was 7 to 10 days. Maximum batch size was 1536 samples. CONCLUSIONS: We demonstrate that a TGS assay could be incorporated into an NBS program soon to increase the number of conditions screened. Additionally, we conclude that NBS using TGS may be cost-effective.

N-acetylglutamate synthase deficiency with associated 3-methylglutaconic aciduria: A case report.

N-acetylglutamate synthase (NAGS) deficiency is a rare autosomal recessive disorder, which results in the inability to activate the key urea cycle enzyme, carbamoylphosphate synthetase 1 (CPS1). Patients often suffer life-threatening episodes of hyperammonaemia, both in the neonatal period and also at subsequent times of catabolic stress. Because NAGS generates the cofactor for CPS1, these two disorders are difficult to distinguish biochemically. However, there have now been numerous case reports of 3-methylglutaconic aciduria (3-MGA), a marker seen in mitochondrial disorders, occurring in CPS1 deficiency. Previously, this had not been reported in NAGS deficiency. We report a four-day-old neonate who was noted to have 3-MGA at the time of significant hyperammonaemia and lactic acidosis. Low plasma citrulline and borderline orotic aciduria were additional findings that suggested a proximal urea cycle disorder. Subsequent molecular testing identified bi-allelic pathogenic variants in NAGS. The 3-MGA was present at the time of persistent lactic acidosis, but improved with normalization of serum lactate, suggesting that it may reflect secondary mitochondrial dysfunction. NAGS deficiency should therefore also be considered in patients with hyperammonaemia and 3-MGA. Studies in larger numbers of patients are required to determine whether it could be a biomarker for severe decompensations.

Hepatocellular carcinoma requiring liver transplantation in hereditary tyrosinemia type 1 despite nitisinone therapy and α1-fetoprotein normalization.

BACKGROUND: Hereditary tyrosinemia type 1 is a rare metabolic condition associated with an increased risk of hepatocellular carcinoma. Nitisinone (2-[2-nitro-4-trifluoromethylbenzoyl]-1,3-cyclohexanedione, NTBC) treatment has reduced but not eliminated the risk. The delayed initiation of nitisinone treatment, and persistently abnormal α1-fetoprotein (AFP) levels are recognized to be risk factors for late-onset hepatocellular carcinoma. We report three children diagnosed and treated with nitisinone since infancy who developed hepatocellular carcinoma despite long-term normalization of AFP. METHODS: A retrospective review of all patients with tyrosinemia on nitisinone managed at our center was undertaken. Patient demographics, age at diagnosis, duration of therapy, timing of AFP normalization, and radiographic imaging findings were noted. RESULTS: Three patients at our center with tyrosinemia type 1 developed hepatocellular carcinoma 9-13 years after diagnosis despite long-term nitisinone therapy and normalization of AFP. Two patients developed new nodules on imaging with an elevation of AFP leading to the diagnosis and subsequent liver transplant. The third patient proceeded with liver transplant because of a very nodular liver and increasing splenomegaly despite normal AFP and no change in surveillance gadoxetate magnetic resonance imaging. Early hepatocellular carcinoma was found in her liver explant. All three patients were cirrhotic at diagnosis. CONCLUSIONS: Patients with hereditary tyrosinemia type 1, especially those already cirrhotic at diagnosis, remain at high risk of developing hepatocellular carcinoma despite long-term nitisinone therapy and AFP normalization, and warrant close monitoring and surveillance.

Successful management of severe coronoid process hyperplasia in a patient with mucopolysaccharidosis VI: a case report.

Mucopolysaccharidoses are a group of rare lysosomal storage diseases caused by a deficiency of enzymes, which breakdown glycosaminoglycans, with consequent dysfunction of affected tissues. Mandibular coronoid hyperplasia, with associated trismus, has been recently described as a feature of the craniofacial abnormalities seen in these patients. However, the details of the surgical and post-operative management of these patients have not been previously documented. This case describes the successful management of severe trismus from coronoid process hyperplasia in a 14-year-old male, utilising an extra-oral approach for bilateral coronoidectomies and removal of exophytic zygomatic bone, followed by immediate and long-term physiotherapy. An improvement of mandibular opening from 8 to 45 mm has been maintained at 18 months post operation.

Safety and Efficacy of Elosulfase Alfa in Australian Patients with Morquio a Syndrome: A Phase 3b Study

Abstract The safety and efficacy of elosulfase alfa were evaluated in a multicenter, open-label, phase 3b study in Australian Morquio A patients, consisting of a 49-week initial phase and an extension phase until elosulfase alfa was government funded. Thirteen patients (1-27 years) were enrolled. No new safety concerns were identified over 138 weeks. Most drug-related adverse events were mild or moderate in severity; none led to study discontinuation. After 49 weeks of treatment, median improvements from baseline were seen in the 6-minute walk test (+41.0 m), 3-minute stair climb test (+14.0 stairs/min), forced vital capacity (+16.4%), forced expiratory volume in 1 second (+14.1%), urine keratan sulfate (-7.1 µg/mg creatinine), and pain intensity. Growth, cardiac function, sleep, and quality of life results were mixed or stable. These results provide further evidence of the acceptable safety/tolerability profile of elosulfase alfa. The improvements in endurance, pulmonary function, and pain support findings from previous studies.

Recommendations for the management of MPS IVA: systematic evidence- and consensus-based guidance.

INTRODUCTION: Mucopolysaccharidosis (MPS) IVA or Morquio A syndrome is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of the N-acetylgalactosamine-6-sulfatase (GALNS) enzyme, which impairs lysosomal degradation of keratan sulphate and chondroitin-6-sulphate. The multiple clinical manifestations of MPS IVA present numerous challenges for management and necessitate the need for individualised treatment. Although treatment guidelines are available, the methodology used to develop this guidance has come under increased scrutiny. This programme was conducted to provide evidence-based, expert-agreed recommendations to optimise management of MPS IVA. METHODS: Twenty six international healthcare professionals across multiple disciplines, with expertise in managing MPS IVA, and three patient advocates formed the Steering Committee (SC) and contributed to the development of this guidance. Representatives from six Patient Advocacy Groups (PAGs) were interviewed to gain insights on patient perspectives. A modified-Delphi methodology was used to demonstrate consensus among a wider group of healthcare professionals with experience managing patients with MPS IVA and the manuscript was evaluated against the validated Appraisal of Guidelines for Research and Evaluation (AGREE II) instrument by three independent reviewers. RESULTS: A total of 87 guidance statements were developed covering five domains: (1) general management principles; (2) recommended routine monitoring and assessments; (3) disease-modifying interventions (enzyme replacement therapy [ERT] and haematopoietic stem cell transplantation [HSCT]); (4) interventions to support respiratory and sleep disorders; (5) anaesthetics and surgical interventions (including spinal, limb, ophthalmic, cardio-thoracic and ear-nose-throat [ENT] surgeries). Consensus was reached on all statements after two rounds of voting. The overall guideline AGREE II assessment score obtained for the development of the guidance was 5.3/7 (where 1 represents the lowest quality and 7 represents the highest quality of guidance). CONCLUSION: This manuscript provides evidence- and consensus-based recommendations for the management of patients with MPS IVA and is for use by healthcare professionals that manage the holistic care of patients with the intention to improve clinical- and patient-reported outcomes and enhance patient quality of life. It is recognised that the guidance provided represents a point in time and further research is required to address current knowledge and evidence gaps.

Recommendations for the management of MPS VI: systematic evidence- and consensus-based guidance.

INTRODUCTION: Mucopolysaccharidosis (MPS) VI or Maroteaux-Lamy syndrome (253200) is an autosomal recessive lysosomal storage disorder caused by deficiency in N-acetylgalactosamine-4-sulfatase (arylsulfatase B). The heterogeneity and progressive nature of MPS VI necessitates a multidisciplinary team approach and there is a need for robust guidance to achieve optimal management. This programme was convened to develop evidence-based, expert-agreed recommendations for the general principles of management, routine monitoring requirements and the use of medical and surgical interventions in patients with MPS VI. METHODS: 26 international healthcare professionals from various disciplines, all with expertise in managing MPS VI, and three patient advocates formed the Steering Committee group (SC) and contributed to the development of this guidance. Members from six Patient Advocacy Groups (PAGs) acted as advisors and attended interviews to ensure representation of the patient perspective. A modified-Delphi methodology was used to demonstrate consensus among a wider group of healthcare professionals with expertise and experience managing patients with MPS VI and the manuscript has been evaluated against the validated Appraisal of Guidelines for Research and Evaluation (AGREE II) instrument by three independent reviewers. RESULTS: A total of 93 guidance statements were developed covering five domains: (1) general management principles; (2) recommended routine monitoring and assessments; (3) enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT); (4) interventions to support respiratory and sleep disorders; (5) anaesthetics and surgical interventions. Consensus was reached on all statements after two rounds of voting. The greatest challenges faced by patients as relayed by consultation with PAGs were deficits in endurance, dexterity, hearing, vision and respiratory function. The overall guideline AGREE II assessment score obtained for the development of the guidance was 5.3/7 (where 1 represents the lowest quality and 7 represents the highest quality of guidance). CONCLUSION: This manuscript provides evidence- and consensus-based recommendations for the management of patients with MPS VI and is for use by healthcare professionals that manage the holistic care of patients with the intention to improve clinical- and patient-reported outcomes and enhance patient quality of life. It is recognised that the guidance provided represents a point in time and further research is required to address current knowledge and evidence gaps.

Nutrition process improvements for adult inpatients with inborn errors of metabolism using the i-PARIHS framework.

AIM: This project aimed to implement consensus recommendations and innovations that improve dietetic services to promote timely referral to optimise nutritional management for adult inpatients with inborn errors of metabolism (IEM). METHODS: The i-PARIHS framework was used to identify service gaps, implement innovations and evaluate the innovations within this single-site study. The constructs of this framework are: (i) review of the evidence; (ii) recognising patients and staff knowledge and attitudes; (iii) acknowledging the local context; and (iv) the facilitators role. This included a literature review and metabolic centre service comparisons to investigate dietetic referral and foodservice processes to inform the innovation. A 12-month chart audit (6 months retrospective and prospective of implemented innovation, respectively) to evaluate newly established dietetic referral and IEM nutrition provision procedures was also completed. RESULTS: The innovations implemented encompassed a clinical alert triggering urgent referral, nutrition sick day plans and metabolic diet and formula prescription via an 'alert' tab in electronic records. Eleven metabolic protein-restricted diets and nine formula recipes were introduced. Prior to the innovations, only 53% (n = 19/36) of inpatients with IEM were assessed by the dietitian and received appropriate nutrition within 24 hours. Following implementation of the innovations, 100% (n = 11/11) of inpatients with IEM received timely dietetic assessment and therapeutic nutrition. CONCLUSIONS: Implementation of innovations developed using the i-PARIHS framework is effective in timely notification of the metabolic dietitian of referrals. This ensures optimal nutritional management during admissions which is required in this group of high-risk patients.

Lysosomal storage disorders: A review of the musculoskeletal features.

The lysosomal storage disorders are a collection of progressive, multisystem disorders that frequently present in childhood. Their timely diagnosis is paramount as they are becoming increasingly treatable. Musculoskeletal manifestations often occur early in the disease course, hence are useful as diagnostics clues. Non-inflammatory joint stiffness or pain, carpal tunnel syndrome, trigger fingers, unexplained pain crises and short stature should all prompt consideration of a lysosomal storage disorder. Recurrent ENT infections, hepatosplenomegaly, recurrent hernias and visual/hearing impairment - especially when clustered together - are important extra-skeletal features. As diagnostic and therapeutic options continue to evolve, children with lysosomal storage disorders and their families are facing more sophisticated options for screening and treatment. The aim of this article is to highlight the paediatric presentations of lysosomal storage disorders, with an emphasis on the musculoskeletal features.

Recurrent Muscle Weakness with Rhabdomyolysis, Metabolic Crises, and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations.

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3-9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3-9. Additionally, a homozygous exons 4-6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3-9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.

Executive functioning of 4 children with hyperphenylalaninemia from childhood to adolescence.

Hyperphenylalaninemia is a variant of phenylketonuria, and debate remains as to what, if any, active management of this condition is required to preserve cognitive function and psychological well-being. This study is the first to examine longitudinally the executive function (EF) in adolescents with hyperphenylalaninemia. Two sibling pairs with mild hyperphenylalaninemia underwent neuropsychological examination in early childhood and again in adolescence using EF tests that were highly sensitive to phenylalanine exposure. By early adolescence, none of the 4 children demonstrated EF impairment. The children demonstrated a typical developmental trajectory of EF from childhood to adolescence, given phenylalanine exposure consistent with their condition.

Identifying the need for a multidisciplinary approach for early recognition of mucopolysaccharidosis VI (MPS VI).

Mucopolysaccharidosis VI (MPS VI, Maroteaux-Lamy syndrome) is caused by deficient activity of the enzyme, N-acetylgalactosamine-4-sulfatase, resulting in impaired degradation of the glycosaminoglycan dermatan sulfate. Patients experience a range of manifestations including joint contractures, short stature, dysostosis multiplex, coarse facial features, decreased pulmonary function, cardiac abnormalities, corneal clouding and shortened life span. Recently, clinicians from institutions in the Asia-Pacific region met to discuss the occurrence and implications of delayed diagnosis and misdiagnosis of MPS VI in the patients they have managed. Eighteen patients (44% female) were diagnosed. The most common sign presented by the patients was bone deformities in 11 patients (65%). Delays to diagnosis occurred due to the lack of or distance to diagnostic facilities for four patients (31%), alternative diagnoses for two patients (15%), and misleading symptoms experienced by two patients (15%). Several patients experienced manifestations that were subtler than would be expected and were subsequently overlooked. Several cases highlighted the unique challenges associated with diagnosing MPS VI from the perspective of different specialties and provide insights into how these patients initially present, which may help to elucidate strategies to improve the diagnosis of MPS VI.

Rare presentation of a treatable disorder: glutaric aciduria type 1.

A 32-year-old female patient presented with migraine and a bipolar disorder with frontal lobe dysfunction and bilateral pyramidal tract signs on examination. MRI brain revealed confluent bilateral symmetric white matter signal abnormality on T2 and FLAIR images with mild cerebral atrophy. Classic widening of Sylvian fissures and CSF space anterior to temporal lobes was seen. In view of the clinical and radiologic findings suggestive of a leukodystrophy, she was investigated for the same. Her investigations revealed an high level of urinary glutaric acid 857 mmol/mol creatinine (normal <4mmol/mol creatinine) and 3-hydroxyglutaric acid 44 mmol/mol creatinine (normal <1 mmol/mol creatinine) and plasma glutaryl carnitine 1.2 micromol/L; (normal <0.34 micromol/L). This was diagnostic of glutaric aciduria type 1. She was started on L-carnitine with which she showed clinical improvement. Testing for urinary organic acids is important when looking for treatable metabolic disorders (such as glutaric aciduria type I) in patients with leukodystrophy.

Overcoming the barriers to diagnosis of Morquio A syndrome.

BACKGROUND: Morquio A syndrome is an autosomal recessive lysosomal storage disease often resulting in life-threatening complications. Early recognition and proficient diagnosis is imperative to facilitate prompt treatment and prevention of clinical complications. METHODS: Experts in Asia Pacific reviewed medical records focusing on presenting signs and symptoms leading to a diagnosis of Morquio A syndrome. RESULTS: Eighteen patients (77% female) had a mean (median; min, max) age of 77.1 (42.0; 0.0, 540.0) months at symptom onset, 78.9 (42.0; 4.5, 540.0) months at presentation and 113.8 (60.0; 7.0, 540.0) months at diagnosis. Orthopedic surgeons and pediatricians were most frequently consulted pre-diagnosis while clinical geneticists/metabolic specialists most frequently made the diagnosis. Delayed diagnoses were due to atypical symptoms for 5 patients (28%), while 4 patients (22%) experienced each of subtle symptoms, symptoms commonly associated with other diseases, or false-negative urine glycosaminoglycan analysis. Two patients (11%) each experienced overgrowth within the first year of life. Two patients with Morquio A syndrome (11%) were diagnosed with craniosynostosis and 1 (6%) for each of Legg-Calvé-Perthes disease, Leri-Weill syndrome, and pseudoachondroplasia. Early radiographic features of Morquio A syndrome led to more efficient diagnosis. CONCLUSIONS: Increased awareness of clinical symptomology overlapping with Morquio A syndrome is essential. Clinicians encountering patients with certain skeletal dysplasia should consider Morquio A syndrome in their differential diagnosis. Atypical or subtle symptoms should not eliminate Morquio A syndrome from the differential diagnosis, especially for patients who may have non-classical phenotype of Morquio A syndrome.

A systematic review of the prevalence of Morquio A syndrome: challenges for study reporting in rare diseases.

BACKGROUND: Morquio A (MPS IVA) is a rare disease characterised by a deficiency of N-acetylgalactosamine-6 sulfatase (GALNS) and presenting with short stature, abnormal gait, cervical spine instability and shortened lifespan. PURPOSE: To prepare a systematic review of the prevalence of Morquio A in multiple countries and suggest recommendations for reporting rare diseases. METHODS: Medline, Medline In-Process, Medline Daily Update, Embase, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, Database of Abstracts of Reviews of Effects, Health Technology Assessment Database and PROSPERO were searched from inception to October 2013 to identify relevant information on the epidemiology of Morquio A. Forty Patient Organisation Representatives (POR) and Key Opinion Leaders (KOL) across 24 countries were contacted for data. Observational studies were included and case reports were excluded. Searches were performed without date or language restriction. Two researchers independently screened and extracted data. Quality of study reporting was assessed using a checklist adapted from STROBE (STrengthening the Reporting of OBservational studies in Epidemiology). Point or birth prevalence was stratified according to diagnostic method and discussed narratively. RESULTS: In total 9,074 records were retrieved from searching and 25 studies were included for data extraction. Twenty out of 40 KOL and POR responded (50%) and 9 provided data (23%). Point prevalence of Morquio A was 1 per 926,000 in Australia, 1 per 1,872,000 in Malaysia and 1 per 599,000 in UK and Morquio (unclassified) was 1 per 323, 000 in Denmark. Birth prevalence of Morquio A (using recommended diagnostic methods) ranged from 1 per 71,000 in UAE to 1 per 500,000 in Japan. All results were compromised by poor study reporting and internal validity. CONCLUSIONS: The review highlighted that there is a misunderstanding of the definitions for prevalence and incidence in the field; that studies were poorly reported (diagnostic methods and patient characteristics) and that no suitable quality assessment tool exists. Overestimation and underestimation of prevalence data can occur. Bespoke reporting guidelines and a quality assessment tool specifically for prevalence of rare diseases are recommended.

Depression, anxiety, and stress in young adults with phenylketonuria: associations with biochemistry.

OBJECTIVE: Increasing concurrent phenylalanine to "off-diet" levels in adults with phenylketonuria (PKU) has been shown to lead to mood disturbances. However, the impact of controlled phenylalanine exposure across the developmental life span and mood stability remains questionable. The aim of this study was to investigate correlations between lifetime and concurrent phenylalanine (Phe) and tyrosine (Tyr) with levels of depression, anxiety, and stress in young adults maintaining a continuously treated diet for PKU. METHOD: Eight young adults (6 females, 2 males) aged 15 to 25 years (mean = 19.37, SD = 3.62) recruited through the Royal Children's Hospital Brisbane, with early and continuously treated PKU completed the Depression Anxiety Stress Scale, which was correlated with lifetime and concurrent measures of Phe, Tyr, and Phe-Tyr ratios. RESULTS: There was a strong significant correlation between lifetime Phe and levels of anxiety and stress (p < .01). Greater Phe-to-Tyr blood ratios were indicative of increased depression and anxiety levels (p < .01). CONCLUSIONS: An interaction between biochemical regulation and depression, anxiety, and stress levels was present in young adults with continuously treated PKU who were actively attempting to maintain dietary control. Strong associations were present with lifetime levels, suggesting a developmental impact of PKU-related biochemical exposure and the emergence of mood disturbances.