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.

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.

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.

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.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

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.

Current diagnosis and management of mucopolysaccharidosis VI in the Asia-Pacific region.

INTRODUCTION: Mucopolysaccharidosis (MPS) type VI (Maroteaux-Lamy syndrome) is a clinically heterogeneous lysosomal storage disorder. It presents significant diagnostic and treatment challenges due to the rarity of the disease and complexity of the phenotype. As information about MPS VI in Asia-Pacific countries is limited, a survey was conducted to assess current practices for diagnosis and management of MPS VI in this region. The participants were selected based on their experience in diagnosing and managing MPS patients. METHODS: The survey comprised 29 structured quantitative or qualitative questions. Follow-up consultations were undertaken to discuss the data further. RESULTS: Thirteen physicians from eight countries or regions (Australia, China, Hong Kong, Japan, Malaysia, Philippines, Taiwan and Thailand) were surveyed. At the time of the survey twenty-two patients with MPS VI were directly treated by the respondents and most (~80%) had rapidly progressing disease. A wide range of medical specialists are involved in managing patients with MPS VI, the most common being orthopedic surgeons, pediatricians and geneticists. The availability/accessibility of diagnostic tools, therapies and national insurance coverage vary greatly across the countries/regions and, in some cases, between different regions within the same country. Currently, there are national MPS management groups in Australia and Japan. Australia, Taiwan and Hong Kong have local guidelines for managing MPS and local MPS registries are available in Australia, Taiwan, and Japan. CONCLUSIONS: This survey highlights differences in the diagnosis and management of MPS VI between Asia-Pacific countries/regions. Important barriers to advancing the identification, understanding and treatment of MPS VI include the paucity of epidemiological information, limited access to laboratory diagnostics and therapies, low disease awareness, and a lack of monitoring and treatment guidelines. There is a clear need to facilitate communications between physicians and establish regional or national disease registries, a multidisciplinary referral network, and a centralized diagnostic and management framework.

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.

Enzyme replacement therapy "drug holiday": results from an unexpected shortage of an orphan drug supply in Australia.

The development of recombinantly manufactured enzyme replacement therapy (ERT) has revolutionised the management of some inherited disorders of metabolism. Gaucher disease was the first lysosomal storage disorder for which ERT became commercially available and ERT remains first-line treatment for affected individuals. In Australia, 70 patients with Gaucher disease are treated through a centrally administered Australian Government national program known as the Life Savings Drug Program (LSDP). Imiglucerase (Cerezyme), manufactured by Genzyme Corporation, is the only ERT currently registered in Australia for the treatment of Gaucher disease. In June 2009, Genzyme Corporation announced the detection of a virus in its Allston Landing manufacturing facility which resulted in inventories of imiglucerase being insufficient to meet projected global demand. The Australian Government sought advice from its Gaucher Disease Advisory Committee (GDAC) on recalculating patient doses in order to ration available imiglucerase to those most in need on a clinical severity basis. Management of this rationing process was urgent and required extensive investigation to develop a clinical severity hierarchy, to review available imiglucerase stock spread across multiple pharmacies, to implement a strategy for redistributing available imiglucerase according to specific patients' recalculated doses, to advise treating doctors and patients concerning these changed circumstances and to consider new monitoring schedules during the drug shortage phase. A cohort of 24 patients was withdrawn from therapy, 22 of whom had no discernable clinical adverse effect. This experience suggests that short-term studies of maintenance therapy without a no-treatment arm may lead to erroneous conclusions and that some patients may have treatment holidays or delayed infusions without short-term adverse outcomes.

The natural history and osteodystrophy of mucolipidosis types II and III.

AIM: To assess the natural history and impact of the secondary bone disease observed in patients with mucolipidosis (ML) II and III. METHODS: Affected children and adults were ascertained from clinical genetics units around Australia and New Zealand. Diagnoses were confirmed by the National Referral Laboratory in Adelaide. The study encompassed all patients ascertained between 1975 and 2005. Data focussing on biochemical parameters at diagnosis, and longitudinal radiographic findings were sought for each patient. Where feasible, patients underwent clinical review and examination. Examinations included skeletal survey, bone densitometry, and measurement of serum and urine markers of bone metabolism. In a subset of patients, functional assessment using the Pediatric Evaluation and Disability Inventory (PEDI) and molecular analysis of GNPTAB were performed. RESULTS: Twenty-five patients with mucolipidosis were ascertained over a 30-year period. Morbidity and functional outcomes on living patients were described. Serum calcium and phosphate were normal. All, but one patient, had normal alkaline phosphatase. Serum osteocalcin and urine deoxypyridinoline/creatinine were elevated. Two radiological patterns were observed (i) transient neonatal hyperparathyroidism in infants with ML II and (ii) progressive osteodystrophy in patients with ML intermediate and ML III. Molecular analyses of GNPTAB in nine subjects are reported. CONCLUSION: ML is characterised by a progressive bone and mineral disorder which we describe as the 'osteodystrophy of mucolipidosis'. The clinical and radiographic features of this osteodystrophy are consistent with a syndrome of 'pseudohyperparathyroidism'. Much of the progressive skeletal and joint pathology is attributable to this bone disorder.

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.

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.

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.

Outcome of neonatal screening for medium-chain acyl-CoA dehydrogenase deficiency in Australia: a cohort study.

BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the disorder thought most to justify neonatal screening by tandem-mass spectrometry because, without screening, there seems to be substantial morbidity and mortality. Our aim was to assess the overall effectiveness of neonatal screening for MCAD deficiency in Australia. METHODS: We identified MCAD-deficient patients from a total population of 2,495,000 Australian neonates (810,000 screened) born between April 1, 1994, and March 31, 2004. Those from a cohort of 1,995,000 (460,000 screened) were followed up for at least 4 years, and we recorded number of deaths and severe episodes, medical and neuropsychological outcome, and hospital admissions within the screened and unscreened groups. FINDINGS: In cohorts aged at least 4 years there were 35 MCAD-deficient patients in those not screened (2.28 per 100,000 total population) and 24 in the screened population (5.2 per 100,000). We estimated that patients with this disorder in the unscreened cohort remained undiagnosed. Before 4 years of age, three screened patients had an episode of severe decompensation (including one neonatal death) versus 23 unscreened patients (including five deaths). At the most conservative estimate, relative risk of an adverse event was 0.44 (95% CI 0.13-1.45). In the larger cohort the relative risk (screened vs unscreened) of an adverse event by age 2 years was 0.26 (95% CI 0.07-0.97), also a conservative estimate. 38 of 52 living patients had neuropsychological testing, with no suggestions of significant differences in general cognitive outcome between the groups. INTERPRETATION: Screening is effective in patients with MCAD deficiency since early diagnosis reduces deaths and severe adverse events in children up to the age of 4 years.

Acute hyperammonemic encephalopathy in adult onset ornithine transcarbamylase deficiency.

OBJECTIVES: To report the clinical manifestations of acute hyperammonemic encephalopathy in adult onset ornithine transcarbamylase deficiency (OTCD). DESIGN: Case report. SETTING: Intensive care unit of a tertiary medical centre. PATIENT: A 48-year-old Caucasian male body builder who developed acute loss of consciousness after a febrile illness. INTERVENTIONS: The patient was immediately started on hemodia-filtration, protein elimination and ammonia scavenging medications. MEASUREMENTS AND RESULTS: Serum ammonium was elevated and plasma and urine amino acids had a pattern indicative of a urea cycle defect. DNA studies revealed a mutation of the urea cycle enzyme, ornithine transcarbamylase. The encephalopathy resolved and the patient slowly recovered though with some cognitive impairment. CONCLUSIONS: Adult presentation of OTCD is rare and the mortality and morbidity rates are high. However, survival is possible with rapid correction of hyperammonemia. As the clinical manifestations are non-specific, a high index of suspicion is necessary for the correct diagnosis and management.