Diagnosis and management of children with McArdle Syndrome (GSD V) in New South Wales.

Glycogen storage type V (GSD V-McArdle Syndrome) is a rare neuromuscular disorder characterised by severe pain early after the onset of physical activity. A recent series indicated a diagnostic delay of 29 years; hence reports of children affected by the disorder are uncommon (Lucia et al., 2021, Neuromuscul Disord, 31, 1296-1310). This paper presents eight patients with a median onset age of 5.5 years and diagnosis of 9.5 years. Six patients had episodes of rhabdomyolysis with creatine kinase elevations >50 000 IU/L. Most episodes occurred in relation to eccentric non-predicted activities rather than regular exercise. One of the patients performed a non-ischaemic forearm test. One patient was diagnosed subsequent to a skeletal muscle biopsy, and all had confirmatory molecular genetic diagnosis. Three were homozygous for the common PYGM:c.148C > T (p.Arg50*) variant. All but one patient had truncating variants. All patients were managed with structured exercise testing to help them identify 'second-wind', and plan an exercise regimen. In addition all also had an exercise test with 25 g maltodextrin which had statistically significant effect on ameliorating ratings of perceived exertion. GSD V is under-recognised in paediatric practice. Genetic testing can readily diagnose the condition. Careful identification of second-wind symptomatology during exercise with the assistance of a multi-disciplinary team, allows children to manage activities and tolerate exercise. Maltodextrin can be used for structured exercise, but excessive utilisation may lead to weight gain. Early intervention and education may improve outcomes into adult life.

Treatment of HMG-CoA Lyase Deficiency-Longitudinal Data on Clinical and Nutritional Management of 10 Australian Cases.

3-Hydroxy-3-Methylglutaryl-CoA Lyase (HMGCL) deficiency can be a very severe disorder that typically presents with acute metabolic decompensation with features of hypoketotic hypoglycemia, hyperammonemia, and metabolic acidosis. A retrospective chart and literature review of Australian patients over their lifespan, incorporating acute and long-term dietary management, was performed. Data from 10 patients contributed to this study. The index case of this disorder was lost to follow-up, but there is 100% survival in the remainder of the cases despite several having experienced life-threatening episodes. In the acute setting, five of nine patients have used 900 mg/kg/day of sodium D,L 3-hydroxybutyrate in combination with intravenous dextrose-containing fluids (delivering glucose above estimated basal utilization requirements). All patients have been on long-term protein restriction, and those diagnosed more recently have had additional fat restriction. Most patients take L-carnitine. Three children and none of the adults take nocturnal uncooked cornstarch. Of the cohort, there were two patients that presented atypically-one with fulminant liver failure and the other with isolated developmental delay. Dietary management in patients with HMGCL deficiency is well tolerated, and rapid institution of acute supportive metabolic treatment is imperative to optimizing survival and improve outcomes in this disorder.

Intronic variants in inborn errors of metabolism: Beyond the exome.

Non-coding regions are areas of the genome that do not directly encode protein and were initially thought to be of little biological relevance. However, subsequent identification of pathogenic variants in these regions indicates there are exceptions to this assertion. With the increasing availability of next generation sequencing, variants in non-coding regions are often considered when no causative exonic changes have been identified. There is still a lack of understanding of normal human variation in non-coding areas. As a result, potentially pathogenic non-coding variants are initially classified as variants of uncertain significance or are even overlooked during genomic analysis. In most cases where the phenotype is non-specific, clinical suspicion is not sufficient to warrant further exploration of these changes, partly due to the magnitude of non-coding variants identified. In contrast, inborn errors of metabolism (IEMs) are one group of genetic disorders where there is often high phenotypic specificity. The clinical and biochemical features seen often result in a narrow list of diagnostic possibilities. In this context, there have been numerous cases in which suspicion of a particular IEM led to the discovery of a variant in a non-coding region. We present four patients with IEMs where the molecular aetiology was identified within non-coding regions. Confirmation of the molecular diagnosis is often aided by the clinical and biochemical specificity associated with IEMs. Whilst the clinical severity associated with a non-coding variant can be difficult to predict, obtaining a molecular diagnosis is crucial as it ends diagnostic odysseys and assists in management.

3-Methylglutaconyl-CoA hydratase deficiency: When ascertainment bias confounds a biochemical diagnosis.

3-Methylglutaconyl-CoA hydratase deficiency (MGA1) is a defect in leucine catabolism, which causes the accumulation of urinary 3-methylglutaconate, with or without 3-hydroxyisovalerate and 3-methylglutarate. It is an ultra-rare condition, with <30 cases published in the literature. It is unclear whether the clinical features seen in reported patients are caused by the biochemical abnormalities, or whether they simply represent an ascertainment bias in patients that come to clinical attention. We reviewed the collective Australian experience of patients with confirmed MGA1, four of whom were diagnosed when asymptomatic through newborn screening (NBS). When our cohort is considered alongside the broader literature, there is no clear evidence of a specific childhood-onset clinical phenotype associated with this disorder. Some patients have non-specific clinical features (such as autism spectrum disorder [ASD]); however, there are also other family members with ASD in the absence of MGA1, suggesting a multifactorial aetiology. Importantly, all four patients diagnosed through NBS (including three with over 18 years of clinical follow-up) remain asymptomatic in the absence of treatment. Based on the available literature, we suggest that MGA1 represents a biochemical phenotype, with an absence of a childhood clinical phenotype. The burdens of sustained treatment (particularly with intensive dietary leucine restriction) in asymptomatic individuals may be of little benefit, and likely to result in poor compliance. Longer-term follow-up of patients detected via NBS (or biochemical screening of large cohorts of asymptomatic adult individuals) will be required to conclusively prove or disprove the association with adult-onset leukoencephalopathy.

A narrative review of metabolomics in the era of "-omics": integration into clinical practice for inborn errors of metabolism.

Background and Objective: Traditional targeted metabolomic investigations identify a pre-defined list of analytes in samples and have been widely used for decades in the diagnosis and monitoring of inborn errors of metabolism (IEMs). Recent technological advances have resulted in the development and maturation of untargeted metabolomics: a holistic, unbiased, analytical approach to detecting metabolic disturbances in human disease. We aim to provide a summary of untargeted metabolomics [focusing on tandem mass spectrometry (MS-MS)] and its application in the field of IEMs. Methods: Data for this review was identified through a literature search using PubMed, Google Scholar, and personal repositories of articles collected by the authors. Findings are presented within several sections describing the metabolome, the current use of targeted metabolomics in the diagnostic pathway of patients with IEMs, the more recent integration of untargeted metabolomics into clinical care, and the limitations of this newly employed analytical technique. Key Content and Findings: Untargeted metabolomic investigations are increasingly utilized in screening for rare disorders, improving understanding of cellular and subcellular physiology, discovering novel biomarkers, monitoring therapy, and functionally validating genomic variants. Although the untargeted metabolomic approach has some limitations, this "next generation metabolic screening" platform is becoming increasingly affordable and accessible. Conclusions: When used in conjunction with genomics and the other promising "-omic" technologies, untargeted metabolomics has the potential to revolutionize the diagnostics of IEMs (and other rare disorders), improving both clinical and health economic outcomes.

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.

The relationship between beta-ureidopropionase deficiency due to UPB1 variants and human phenotypes is uncertain.

BACKGROUND: Beta-ureidopropionase deficiency, caused by variants in UPB1, has been reported in association with various neurodevelopmental phenotypes including intellectual disability, seizures and autism. AIM: We aimed to reassess the relationship between variants in UPB1 and a clinical phenotype. METHODS: Literature review, calculation of carrier frequencies from population databases, long-term follow-up of a previously published case and reporting of additional cases. RESULTS: Fifty-three published cases were identified, and two additional cases are reported here. Of these, 14 were asymptomatic and four had transient neurological features; clinical features in the remainder were variable and included non-neurological presentations. Several of the variants previously reported as pathogenic are present in population databases at frequencies higher than expected for a rare condition. In particular, the variant most frequently reported as pathogenic, p.Arg326Gln, is very common among East Asians, with a carrier frequency of 1 in 19 and 1 in 907 being homozygous for the variant in gnomAD v2.1.1. CONCLUSION: Pending the availability of further evidence, UPB1 should be considered a 'gene of uncertain clinical significance'. Caution should be used in ascribing clinical significance to biochemical features of beta-ureidopropionase deficiency and/or UPB1 variants in patients with neurodevelopmental phenotypes. UPB1 is not currently suitable for inclusion in gene panels for reproductive genetic carrier screening. SYNOPSIS: The relationship between beta-ureidopropionase deficiency due to UPB1 variants and clinical phenotypes is uncertain.

A serendipitous journey to a promoter variant: The c.-106C>A variant and its role in late-onset ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked urea cycle disorder characterised by reduced or absent OTC enzyme activity, resulting in the accumulation of neurotoxic ammonia. Approximately 80%-90% of the causative variants are identified by Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA) of the OTC gene. A 23-year-old male with biochemical evidence of OTCD was referred for molecular analysis. Initial Sanger sequencing yielded no pathogenic variants. MLPA testing raised suspicion of a mosaic deletion of exon 1; however, high-resolution microarray did not identify a copy number variant on the X chromosome. Sequencing over the suspected breakpoint detected a hemizygous likely pathogenic promoter variant, c.-106C > A, which was located within the MLPA probe binding site. Subsequently, historical patients referred to our centre, without a molecular aetiology for their OTCD, were re-sequenced with these primers and this variant was also identified in two additional unrelated males. All three patients described in this case series have the late-onset disease. Two presented at 5 years of age with vomiting, whilst the other was managed from birth based on a family history of late-onset OTCD. One patient required liver transplantation due to recurrent decompensations; the other two are managed with a protein-restricted diet. All three patients have not sustained any significant neurological insults and are functioning well as adults. These cases support screening of the promoter region within the OTC gene, particularly if a molecular basis has not been elucidated by MLPA or sequencing of the coding regions.

FGF21 outperforms GDF15 as a diagnostic biomarker of mitochondrial disease in children.

Several studies have shown serum fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) levels are elevated in patients with mitochondrial disease (MD) where myopathy is a feature. In this study we investigated the utility of FGF21 and GDF15 as biomarkers for MD in a phenotypically and genotypically diverse pediatric cohort with suspected MD against a panel of healthy controls and non-mitochondrial disease controls with some overlapping clinical features. Serum was collected from 56 children with MD, 104 children with non-mitochondrial disease (27 neuromuscular, 26 cardiac, 21 hepatic, 30 renal) and 30 pediatric controls. Serum FGF21 and GDF15 concentrations were measured using ELISA, and their ability to detect MD was determined. Median FGF21 and GDF15 serum concentrations were elevated 17-fold and 3-fold respectively in pediatric MD patients compared to the healthy control group. Non-mitochondrial disease controls had elevated serum GDF15 concentrations while FGF21 concentrations were in the normal range. Elevation of GDF15 in a range of non-mitochondrial pediatric disorders limits its use as a MD biomarker. FGF21 was elevated in MD patients with a spectrum of clinical phenotypes, including those without myopathy. Serum FGF21 had an area under the receiver operating characteristic curve of 0.87, indicating good ability to discriminate between pediatric MD and healthy and non-mitochondrial disease controls. Triaging of pediatric MD patients by clinical phenotyping and serum FGF21 testing, followed by massively parallel sequencing, may enable more rapid diagnosis of pediatric MD.

Mitochondrial iron-sulfur cluster biogenesis and neurological disorders.

Iron-sulfur clusters (ISCs) are highly conserved moieties embedded into numerous crucial proteins in almost all bacteria, plants and mammals. As such, ISC biosynthesis is critical to cellular function. The pathway was first characterized in bacteria by the late 1990s, and over the subsequent 20 years there has been increasing understanding of its components in humans. Defects in the ISC pathway are now associated with many different human disease states, such as Friedreich ataxia and ISCU myopathy. Whilst the disorders have variable clinical features, most involve neurological phenotypes. There are common biochemical signatures in most of these conditions, as a lack of ISCs causes deficiencies of target proteins including Complex I, II and III, aconitase and lipoic acid. This review focuses on the disorders of ISC biogenesis that have been described in the literature to-date. Key clinical, biochemical and neuroradiological features will be discussed, providing a reference point for clinicians diagnosing and managing these patients. Therapies are mostly supportive at this stage. However, the improved understanding of the pathophysiology of these conditions could pave the way for disease-modifying therapies in the near future.

Paediatric genomic testing: Navigating genomic reports for the general paediatrician.

Monogenic rare disorders contribute significantly to paediatric morbidity and mortality, and elucidation of the underlying genetic cause may have benefits for patients, families and clinicians. Advances in genomic technology have enabled diagnostic yields of up to 50% in some paediatric cohorts. This has led to an increase in the uptake of genetic testing across paediatric disciplines. This can place an increased burden on paediatricians, who may now be responsible for interpreting and explaining test results to patients. However, genomic results can be complex, and sometimes inconclusive for the ordering paediatrician. Results may also cause uncertainty and anxiety for patients and their families. The paediatrician's genetic literacy and knowledge of genetic principles are therefore critical to inform discussions with families and guide ongoing patient care. Here, we present four hypothetical case vignettes where genomic testing is undertaken, and discuss possible results and their implications for paediatricians and families. We also provide a list of key terms for paediatricians.

Effectiveness of early hematopoietic stem cell transplantation in preventing neurocognitive decline in aspartylglucosaminuria: A case series.

Aspartylglucosaminuria (AGU) (OMIM #208400) is a recessively inherited disorder of glycoprotein catabolism, a subset of the lysosomal storage disorders (LSDs). Deficiency of the enzyme glycosylasparaginase (E.C. 3.5.1.26) leads to accumulation of aspartylglucosamine in various organs and its excretion in the urine. The disease is characterized by an initial period of normal development in infancy, a plateau in childhood, and subsequent regression in adolescence and adulthood. No curative treatments are currently available, leading to a protracted period of significant disability prior to early death. Hematopoietic stem cell transplantation (HSCT) has demonstrated efficacy in other LSDs, by providing enzyme replacement therapy in somatic viscera and decreasing substrate accumulation. Moreover, donor-derived monocytes cross the blood-brain barrier, differentiate into microglia, and secrete enzyme in the central nervous system (CNS). This has been shown to improve neurocognitive outcomes in other LSDs. The evidence to date for HSCT in AGU is varied, with marked improvement in glycosylasparaginase enzyme activity in the CNS in mice models, but varying neurocognitive outcomes in humans. We present a case series of four children with AGU who underwent HSCT at different ages (9 years, 5 years, 5 months, and 7 months of age), with long-term follow-up post-transplant (over 10 years). These cases demonstrate similar neurodevelopmental heterogeneity based on formal developmental assessments. The third case, transplanted prior to the onset of neurocognitive involvement, is developing normally despite a severe phenotype in other family members. This suggests that further research should examine the role of early HSCT in management of AGU.

Clinically Responsive Genomic Analysis Pipelines: Elements to Improve Detection Rate and Efficiency.

Massively parallel sequencing has markedly improved mendelian diagnostic rates. This study assessed the effects of custom alterations to a diagnostic genomic bioinformatic pipeline in response to clinical need and derived practice recommendations relative to diagnostic rates and efficiency. The Genomic Annotation and Interpretation Application (GAIA) bioinformatics pipeline was designed to detect panel, exome, and genome sample integrity and prioritize gene variants in mendelian disorders. Reanalysis of selected negative cases was performed after improvements to the pipeline. GAIA improvements and their effect on sensitivity are described, including addition of a PubMed search for gene-disease associations not in the Online Mendelian Inheritance of Man database, inclusion of a process for calling low-quality variants (known as QPatch), and gene symbol nomenclature consistency checking. The new pipeline increased the diagnostic rate and reduced staff costs, resulting in a saving of US$844.34 per additional diagnosis. Recommendations for genomic analysis pipeline requirements are summarized. Clinically responsive bioinformatics pipeline improvements increase diagnostic sensitivity and increase cost-effectiveness.

CDH1 Mutation Distribution and Type Suggests Genetic Differences between the Etiology of Orofacial Clefting and Gastric Cancer.

Pathogenic variants in CDH1, encoding epithelial cadherin (E-cadherin), have been implicated in hereditary diffuse gastric cancer (HDGC), lobular breast cancer, and both syndromic and non-syndromic cleft lip/palate (CL/P). Despite the large number of CDH1 mutations described, the nature of the phenotypic consequence of such mutations is currently not able to be predicted, creating significant challenges for genetic counselling. This study collates the phenotype and molecular data for available CDH1 variants that have been classified, using the American College of Medical Genetics and Genomics criteria, as at least 'likely pathogenic', and correlates their molecular and structural characteristics to phenotype. We demonstrate that CDH1 variant type and location differ between HDGC and CL/P, and that there is clustering of CL/P variants within linker regions between the extracellular domains of the cadherin protein. While these differences do not provide for exact prediction of the phenotype for a given mutation, they may contribute to more accurate assessments of risk for HDGC or CL/P for individuals with specific CDH1 variants.

Ornithine Transcarbamylase Deficiency Presenting as Acute Liver Failure in Girls: A Paediatric Case Series.

Ornithine transcarbamylase deficiency (OTCD) is the most common of the urea cycle disorders and follows an X-linked inheritance pattern. The classical form in male infants causes vomiting and lethargy in the neonatal period; if untreated the severe hyperammonaemia can cause acute neurotoxic complications and permanent disability. OTCD may also occur in heterozygote female individuals, though the manifestations are variable. We report 2 cases of female paediatric patients with OTCD, who presented with acute liver failure. Both patients had limited oral intake at the time of presentation, causing an absence of orotic aciduria, which delayed the diagnosis. These cases demonstrate the need to consider urea cycle disorders in children presenting with acute liver failure, and that repeating the urine metabolic screen at the time of an unrestricted diet is warranted if there is a high clinical suspicion.

User Acceptability of Whole Exome Reproductive Carrier Testing for Consanguineous Couples in Australia.

The study aimed to explore with consanguineous couples in Australia the acceptability and perceived utility of whole exome reproductive carrier screening for autosomal recessive and X-linked recessive conditions. Semi-structured interviews with 21 consanguineous couples were conducted prior to the offer of screening. Interviews were coded, and thematic analysis was informed by an inductive approach. Three major themes were identified: experiences and attitudes of Australian consanguineous couples, childhood genetic conditions and beliefs, and the perceived utility of genomic screening. All but one couple had previously sought genetic advice, and a large majority of couples were aware of childhood conditions within their family or community. Thirteen couples perceived consanguinity as increasing the risk of having affected children. Nine spoke of premarital screening programs routinely conducted in their countries of origin. All supported the concept and availability of genomic reproductive carrier screening. Hypothetically, if found to be carriers of a severe childhood disorder, 13 couples reported they would test a pregnancy, and 12 of whom would consider termination of pregnancy or pre-implantation genetic diagnosis. Four couples would not test a pregnancy and two were unsure. A majority of couples would communicate potential at-risk status to family members, although there were some caveats. Fourteen couples chose to have exome screening and reported that they would utilize the results with the goal of preventing childhood conditions. Of these couples, nine (64%) had an affected child but were aware that testing may reveal they were at risk for a child with a different condition and five (71%) without an affected child. While from diverse ethnic and backgrounds, all couples practiced a religion and all but one couple were recruited from the same clinical genetics unit, with a likely higher genetic literacy and bias towards accepting genetic testing. However, the choice made by all couples was reportedly made with consideration of their personal values, their current family situation, and exome testing issues, including fear of incidental findings and concerns about test reliability.

Beyond the panel: preconception screening in consanguineous couples using the TruSight One "clinical exome".

PURPOSE: To provide proof of concept by broadening preconception screening beyond targeted testing to inform reproductive risk in consanguineous couples. METHODS: Consanguineous couples were screened for autosomal recessive and X-linked disorders using the TruSight One panel of 4,813 genes associated with human disease. RESULTS: We recruited 22 couples, of whom 15 elected to have sequencing. We found four couples to be at risk of autosomal recessive disorders, including one with a child affected by Poretti-Boltshauser syndrome (a diagnosis not made prior to the study) and another previously known to carry a ß-globin variant. Two couples were found to carry variants unrelated to known family history. These variants were in the genes C5orf42 (associated with Joubert syndrome and orofaciodigital syndrome) and GYS2 (associated with glycogen synthase deficiency). One known variant was not detected-a single exon deletion in FAM20C. We would not expect to identify this variant with the methodology employed. Of the four variants identified, only the ß-globin variant would have been found using available commercial preconception screening panels. CONCLUSION: Preconception screening of consanguineous couples for recessive and X-linked disorders using genomic sequencing is practicable, and is likely to detect many more at-risk couples than any targeted panel could achieve. A couples-based approach greatly reduces the associated analysis and counselling burden.