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Several studies have demonstrated a high incidence of autistic spectrum features in individuals with Smith-Lemli-Opitz syndrome (SLOS). However, do these findings imply a converse relationship that has diagnostic utility? Is SLOS testing implicated when autism spectrum disorder (ASD) is the only clinical indication? AIM To determine if there is any correlation with a clinical indication of ASD and a biochemical diagnosis of SLOS, based on historical test request and assay data. METHODS Six years (2008-2013) of clinical test requests for 7-dehydrocholesterol (7-DHC) level were classified and summarised according to indication and final test result. RESULTS From the audit period, 988 valid test results from post-natal samples were identified. In plasma/serum, mean 7-DHC level was 264.7 μmol/L (normal range

The cause of neurodegeneration in MPS mouse models is the focus of much debate and what the underlying cause of disease pathology in MPS mice is. The timing of development of pathology and when this can be reversed or impacted is the key to developing suitable therapies in MPS. This study is the first of its kind to correlate the biochemical changes with the functional outcome as assessed using non-invasive behaviour testing across multiple mucopolysaccharidosis (MPS) mouse models. In the MPS brain, the primary lysosomal enzyme dysfunction leads to accumulation of primary glycosaminoglycans (GAGs) with gangliosides (G(M2) and G(M3)) being the major secondary storage products. With a focus on the neuropathology, a time course experiment was conducted in MPS I, MPS IIIA, MPS VII (severe and attenuated models) in order to understand the relative timing and level of GAG and ganglioside accumulation and how this correlates to behaviour deficits. Time course analysis from 1 to 6 months of age was conducted on brain samples to assess primary GAG (uronic acid), beta-hexosaminidase enzyme activity and levels of G(M2) and G(M3) gangliosides. This was compared to a battery of non-invasive behaviour tests including open field, inverted grid, rotarod and water cross maze were assessed to determine effects on motor function, activity and learning ability. The results show that the GAG and ganglioside accumulation begins prior to the onset of detectable changes in learning ability and behaviour. Interestingly, the highest levels of GAG and ganglioside accumulation was observed in the MPS IIIA mouse despite having 3% residual enzyme activity. Deficits in motor function were clearly observed in the severe Gus(mps/mps), which were significantly delayed in the attenuated Gus(tm(L175F)Sly) model despite their minimal increase in detectable enzyme activity. This suggests that genotype and residual enzyme activity are not indicative of severity of disease pathology in MPS disease and there exists a window when there are considerable storage products without detectable functional deficits which may allow an alteration to occur with therapy. usc Refereed/Peer-reviewed

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism characterized by a deficiency of the lysosomal enzyme, N‐acetylgalactosamine 6‐sulphatase (GALNS). Consequently, partially degraded GAG, chondroitin 6‐sulfate (CS) and keratan sulfate (KS), accumulate in the lysosomes of affected cells, primarily in cartilage resulting in skeletal disease. Excessive urinary excretion of these GAG is often used as the initial biochemical parameter to inform a laboratory diagnosis. Here we present the utility of a CS‐disaccharide with a non‐reducing 6‐sulfated N‐acetylgalactosamine residue (HNAc‐UA (1S))—the enzyme's substrate—for the diagnosis and biochemical monitoring of MPS IVA patients. Following implementation of this method into the diagnostic laboratory, we identified one MPS IVA patient over 3 years of MPS urine screening, with no false positive results from 2050 urines tested. Uniquely, urinary concentrations of HNAc‐UA (1S) are independent of age meaning that age‐related reference ranges are not required. Urinary HNAc‐UA (1S) was also able to identify two MPS IVA siblings who remained misdiagnosed with spondyloepiphyseal dysplasia for 5 years because of normal urinary GAG. HNAc‐UA (1S) could also be used as a biomarker for monitoring response to enzyme replacement therapy (ERT) as there was a drop in urinary concentration following the administration of ERT in all 12 patients and concentrations correlated with urinary KS (R 2 = 0.92). In conclusion, HNAc‐UA (1S) is a reliable, sensitive and specific biomarker for the diagnosis of MPS IVA and can be used to biochemically monitor the response to ERT.

Gaucher disease (GD) is an inherited metabolic disorder characterised by impaired catabolism of the glycosphingolipid, glucosylceramide. The deacetylated derivative, glucosylsphingosine (GluSph, lyso-Gb1) has materialised as a biomarker for GD. Further appraisal of the clinical utility of GluSph is required in terms of its prognostic power to inform disease course and pre-symptomatic testing. In this study, we show that plasma GluSph concentrations are significantly higher in GD patients with neuronopathic disease compared with non-neuronopathic disease, even in the neonatal period. A neonate diagnosed at 1 day of age (homozygous for N370S) due to an affected older sibling, returned GluSph of 70 nmol/L compared with 1070-2620 nmol/L for four neuronopathic patients diagnosed

Expanding treatments for the mucopolysaccharidoses—a family of genetic disorders—place unprecedented demands for accurate, timely diagnosis because best outcomes are seen with early initiation of appropriate therapies. Here we sought to improve the diagnostic odyssey by measuring specific glycosaminoglycan fragments with terminal residues complicit with the genetic defect resulting in precise diagnosis rather than the usual first-line, ambiguous total glycosaminoglycan determinations that return poor diagnostic yield. A derivatizing reagent was added to urine aliquots (0.5 μmol creatinine) before separation of the glycosaminoglycan fragments by liquid chromatography and quantification with electrospray ionization–tandem mass spectrometry using multiple reaction monitoring for 10 targeted fragments plus the internal standard. All 93 mucopolysaccharidosis patients were correctly identified as 1 of 10 subtypes from a total of 723 de-identified subjects—blinded to diagnosis—based on the presence of specific “signature” glycosaminoglycan fragments. Employing reference intervals calculated from 630 unaffected urines, with 99% confidence intervals, provided a laboratory test with 100% specificity and sensitivity. This novel urine assay allows diagnosis of 10 mucopolysaccharidosis subtypes in a single test. The precise quantification of unique glycosaminoglycan fragments also enables longitudinal biochemical monitoring following therapeutic interventions.

A young woman with a history of previously undiagnosed episodic neurological deterioration since early childhood associated with viral illnesses developed fulminant encephalitis. Standard investigations gave no diagnosis. Next-generation sequencing identified a heterozygous mutation c.1754C>T (p.Thr585Met) in the RANBP2 gene, giving the diagnosis of recurrent acute necrotising encephalopathy type 1. This condition is probably underdiagnosed, especially in adults, and should be considered in patients with recurrent encephalopathy.

Maple syrup urine disease is caused by a deficiency of branched-chain alpha-ketoacid dehydrogenase, responsible for degradation of leucine, isoleucine, and valine. Biallelic pathogenic variants in BCKDHA, BCKDHB, or DBT genes result in enzyme deficiency. We report the case of a female infant who presented with mild gross motor delay at 4 months, and seizures with hypoglycaemia at 5 months. Newborn screening returned total leucine/isoleucine at the 99.5th centile of the population; however, as second-tier testing reported minimal alloisoleucine, the results were considered inconsistent with MSUD. Plasma amino acid and urine organic acid analyses at 5 months were, however, consistent with a diagnosis of MSUD. A brain MRI showed bilateral symmetrical T2 hyperintense signal abnormalities involving white matter, globus pallidus, thalamus, brainstem, and dentate nuclei with restricted diffusion. A repeat MRI 10 months post-dietary-intervention showed the resolution of these changes and progression in myelination. Her clinical phenotype, including protein tolerance, correlated with intermediate MSUD. Molecular analysis of all three genes identified two variants of uncertain significance, c.434-15_434-4del and c.365A>G (p. Tyr122Cys) in the DBT gene. The rate of leucine decarboxylation in fibroblasts was reduced, but not to the extent observed in classical MSUD patients, supporting an intermediate form of MSUD. Previously reported mRNA splicing studies supported a deleterious effect of the c.434-15_434-4del variant. This functional evidence and confirmation that the variants were in trans, permitted their reclassification as pathogenic and likely pathogenic, respectively, facilitating subsequent prenatal testing. This report highlights the challenges in identifying intermediate MSUD by newborn screening, reinforcing the importance of functional studies to confirm variant pathogenicity in this era of molecular diagnostics.

Context 4H or POLR3-related leukodystrophy is an autosomal recessive disorder typically characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism, caused by biallelic pathogenic variants in POLR3A, POLR3B, POLR1C, and POLR3K. The endocrine and growth abnormalities associated with this disorder have not been thoroughly investigated to date. Objective To systematically characterize endocrine abnormalities of patients with 4H leukodystrophy. Design An international cross-sectional study was performed on 150 patients with genetically confirmed 4H leukodystrophy between 2015 and 2016. Endocrine and growth abnormalities were evaluated, and neurological and other non-neurological features were reviewed. Potential genotype/phenotype associations were also investigated. Setting This was a multicenter retrospective study using information collected from 3 predominant centers. Patients A total of 150 patients with 4H leukodystrophy and pathogenic variants in POLR3A, POLR3B, or POLR1C were included. Main Outcome Measures Variables used to evaluate endocrine and growth abnormalities included pubertal history, hormone levels (estradiol, testosterone, stimulated LH and FSH, stimulated GH, IGF-I, prolactin, ACTH, cortisol, TSH, and T4), and height and head circumference charts. Results The most common endocrine abnormalities were delayed puberty (57/74; 77% overall, 64% in males, 89% in females) and short stature (57/93; 61%), when evaluated according to physician assessment. Abnormal thyroid function was reported in 22% (13/59) of patients. Conclusions Our results confirm pubertal abnormalities and short stature are the most common endocrine features seen in 4H leukodystrophy. However, we noted that endocrine abnormalities are typically underinvestigated in this patient population. A prospective study is required to formulate evidence-based recommendations for management of the endocrine manifestations of this disorder., Canadian Institutes of Health Research [201610PJT-377869, MOP-G2-341146-159133-BRIDG]; Fondation Les Amis d'Elliot; Leuco-Action; Fondation Lueur d'Espoir pour Ayden; Fondation le Tout pour Loo; Reseau de Medecine Genetique Appliquee of the Fonds de Recherche du Quebec-Sante; Compute Canada; Fonds de Recherche du Quebec en Sante (FRQS) Doctoral Scholarship; Fondation du Grand defi Pierre Lavoie Doctoral Scholarship; McGill Faculty of Medicine F. S.B. Miller Fellowship; Research Institute of the McGill University Health Centre Desjardins Studentship in Child Health Research; Directorate of Higher Education Overseas Scholarship-Dikti Scholarship, Ministry of National Education, Republic of Indonesia; CIHR [201603PJT-148695]; BC Children's Hospital Foundation through its intramural Investigator Grant Award Program (IGAP); National Institute for Neurological Disorders and Stroke [R01NS082094]; Jakob Kamens Chair in Translational Neurotherapeutics; Fonds de Recherche du Quebec-Sante (FRQS); Canadian Institutes of Health Research; European Reference Network for Rare Neurological Disorders (ERN-RND) [739510], This study was supported by grants from the Canadian Institutes of Health Research (201610PJT-377869, MOP-G2-341146-159133-BRIDG), Fondation Les Amis d'Elliot, Leuco-Action, Fondation Lueur d'Espoir pour Ayden, Fondation le Tout pour Loo, and Reseau de Medecine Genetique Appliquee of the Fonds de Recherche du Quebec-Sante to G. Bernard. This research was enabled in part by support provided by Compute Canada (www.computecanada.ca).S.Perrier is supported by the Fonds de Recherche du Quebec en Sante (FRQS) Doctoral Scholarship, the Fondation du Grand defi Pierre Lavoie Doctoral Scholarship, the McGill Faculty of Medicine F. S.B. Miller Fellowship, and the Research Institute of the McGill University Health Centre Desjardins Studentship in Child Health Research. F. K.C. is a recipient of the Directorate of Higher Education Overseas Scholarship-Dikti Scholarship, Ministry of National Education, Republic of Indonesia. W.T. G. received funding from the CIHR (201603PJT-148695) and is supported by the BC Children's Hospital Foundation through its intramural Investigator Grant Award Program (IGAP). B.L. F. was supported by the National Institute for Neurological Disorders and Stroke (R01NS082094). A.V. receives funding from the Jakob Kamens Chair in Translational Neurotherapeutics. G. Bernard has received a Research Scholar Junior 1 award from the Fonds de Recherche du Quebec-Sante (FRQS) (2012-2016) and the New Investigator Salary Award from the Canadian Institutes of Health Research (2017-2022). I.K.M., M. Synofzik, D. Tonduti, B.P.v.d.W., M.S. V.d.K., and N.I.W. are members of the European Reference Network for Rare Neurological Disorders (ERN-RND), project ID 739510.

Fabry disease (FD) results from a deficiency in the exoglycohydrolase, α-galactosidase A (AGA), an enzyme required for the sequential degradation of glycosphingolipids, which consequently accumulate in the lysosomes of affected cells. An X-linked inherited metabolic disorder, FD has a high incidence of a later onset phenotype that is under-diagnosed and under-recognised in adulthood despite the availability of specific treatment. As the first presenting feature in adults is often left ventricular hypertrophy (LVH), we hypothesized that testing patients with an attenuated echocardiographic phenotype of unexplained hypertrophic cardiomyopathy, might identify cases of undiagnosed FD. We employed a simple screening test by measuring AGA activity in dried blood spots collected from a finger-prick of blood in a cohort of 511 individuals aged between 18 and 75 with LVH between 1.2 and 1.5 cm. Two males were identified with AGA activity below the reference interval and subsequent molecular testing confirmed the commonly reported genetic variants, p.Ala143Thr in one individual and p.Asn215Ser, in the other. Additional biochemical measurement of plasma, lyso-Gb1 was normal in both patients. Of the 179 females screened, one individual returned AGA activity slightly below the reference interval but was lost to further follow-up. This pilot study suggests that screening patients with mild-to-moderate LVH of unknown aetiology does indeed identify undiagnosed cases of FD.

CBS deficient individuals undergoing betaine supplementation without sufficient dietary methionine restriction can develop severe hypermethioninemia and brain edema. Brain edema has also been observed in individuals with severe hypermethioninemia without concomitant betaine supplementation. We systematically evaluated reports from 11 published and 4 unpublished patients with CBS deficiency and from additional four cases of encephalopathy in association with elevated methionine. We conclude that, while betaine supplementation does greatly exacerbate methionine accumulation, the primary agent causing brain edema is methionine rather than betaine. Clinical signs of increased intracranial pressure have not been seen in patients with plasma methionine levels below 559 μmol/L but occurred in one patient whose levels did not knowingly exceed 972 μmol/L at the time of manifestation. While levels below 500 μmol/L can be deemed safe it appears that brain edema can develop with plasma methionine levels close to 1000 μmol/L. Patients with CBS deficiency on betaine supplementation need to be regularly monitored for concordance with their dietary plan and for plasma methionine concentrations. Recurrent methionine levels above 500 μmol/L should alert clinicians to check for clinical signs and symptoms of brain edema and review dietary methionine intake. Levels approaching 1000 μmol/L do increase the risk of complications and levels exceeding 1000 μmol/L, despite best dietetic efforts, should be acutely addressed by reducing the prescribed betaine dose.

We describe a sibling pair displaying an early infantile-onset, progressive neurodegenerative phenotype, with symptoms of developmental delay and epileptic encephalopathy developing from 12 to 14 months of age. Using whole exome sequencing, compound heterozygous variants were identified in SLC5A6, which encodes the sodium-dependent multivitamin transporter (SMVT) protein. SMVT is an important transporter of the B-group vitamins biotin, pantothenate, and lipoate. The protein is ubiquitously expressed and has major roles in vitamin uptake in the digestive system, as well as transport of these vitamins across the blood–brain barrier. Pathogenicity of the identified variants was demonstrated by impaired biotin uptake of mutant SMVT. Identification of this vitamin transporter as the genetic basis of this disorder guided targeted therapeutic intervention, resulting clinically in improvement of the patient’s neurocognitive and neuromotor function. This is the second report of biallelic mutations in SLC5A6 leading to a neurodegenerative disorder due to impaired biotin, pantothenate and lipoate uptake. The genetic and phenotypic overlap of these cases confirms mutations in SLC5A6 as the genetic cause of this disease phenotype. Recognition of the genetic disorder caused by SLC5A6 mutations is essential for early diagnosis and to facilitate timely intervention by triple vitamin (biotin, pantothenate, and lipoate) replacement therapy.

Fifty percent of inborn errors of metabolism are present in later childhood and adulthood, with crises commonly precipitated by minor viral illnesses or increased protein ingestion. Many physicians only consider IEM after more common conditions (such as sepsis) have been considered. In view of the large number of inborn errors, it might appear that their diagnosis requires precise knowledge of a large number of biochemical pathways and their interrelationship. As a matter of fact, an adequate diagnostic approach can be based on the proper use of only a few screening tests. A detailed history of antecedent events, together with these simple screening tests, can be diagnostic, leading to life-saving, targeted treatments for many disorders. Unrecognised, IEM can lead to significant mortality and morbidity. Advice is available 24/7 through the metabolic service based at the major paediatric hospital in each state and Starship Children's Health in New Zealand.

Short stature is a characteristic feature of most of the mucopolysaccharidoses, a group of inherited lysosomal storage disorders caused by a single enzyme deficiency. MPS patients present with progressive skeletal defects from an early age, including short stature due to impaired cartilage-to-bone conversion (endochondral ossification). The aim of this study was to determine which murine MPS model best reproduces the bone length reduction phenotype of human MPS and use this model to determine the earliest developmental stage when disrupted endochondral ossification first appears. Gusmps/mps mice representing severe MPS VII displayed the greatest reduction in bone elongation and were chosen for histopathological analysis. Tibial development was assessed from E12.5 to 6 months of age. Chondrocytes in the region of the future primary ossification center became hypertrophic at a similar age to normal in the MPS VII mouse fetus, but a delay in bone deposition was observed with an approximate 1 day delay in the formation of the primary ossification centre. Likewise, chondrocytes in the region of the future secondary ossification center also became hypertrophic at the same age as normal in the MPS VII early postnatal mouse. Bone deposition in the secondary ossification centre was delayed by two days in the MPS VII proximal tibia (observed at postnatal day 14 (P14) compared to P12 in normal). The thickness of the tibial growth plate was larger in MPS VII mice from P9 onwards. Abnormal endochondral ossification starts in utero in MPS VII and worsens with age. It is characterized by a normal timeframe for chondrocyte hypertrophy but a delay in the subsequent deposition of bone in both the primary and secondary ossification centres, accompanied by an increase in growth plate thickness. This suggests that the signals for vascular invasion and bone deposition, some of which are derived from hypertrophic chondrocytes, are altered in MPS VII. Refereed/Peer-reviewed

Antenatal screening for fetal anomalies has provided women and their partners with information to make reproductive choices based on the risk of serious chromosomal or structural defects since the 1990s. Alternative tests include first-trimester screening (combined ultrasound and maternal serum markers), second-trimester maternal serum markers and noninvasive cell-free DNA testing. The recent recommendations by the Royal Australian and New Zealand College of Obstetrics and Gynaecology and the Human Genetics Society of Australasia against second-trimester triple testing are based on unsound performance criteria, raise several contestable issues around access and equity and challenge the principles of governments providing affordable options.

In Australia, 58 patients with Gaucher disease were managed by a Gaucher Disease Advisory Committee (GDAC) through a centrally adminis-tered national programme, the Life Savings Drug Program (LSDP). In June 2009, Genzyme Corporation, which manufactures imiglucerase (Cerezyme), the only enzyme replacement therapy (ERT) registered for the treatment of Gaucher disease in Australia at that time, announced that due to a viral contamination problem there would be no further shipments of Cerezyme to Australia prior to the end of 2009. The GDAC allocated available drug supplies in order to maintain treatment to those most in need on a hierarchal clinical severity basis. A cohort of 24 patients with Type 1 Gaucher disease was withdrawn from therapy, 22 of whom had no discernible clinically significant adverse effects when reviewed off therapy for up to 6 months. In this paper, we review the course of 20 of the patients who have been on imiglucerase for periods of at least 24 months after the end of their 'drug holiday'. No patient experienced a bone crisis nor clinical nor magnetic resonance imaging evidence of new avascular necrosis events during this period. Two years after recommencing ERT after a 6-month drug holiday, no patient had developed an overt irreversible complication of their Gaucher disease, with the majority returning to their previous clinical status.

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.

IntroductionFabry disease is an X-linked disorder of glycosphingolipid metabolism arising from a deficiency of the enzyme, α-galactosidase A (AGA). In the absence of a family history, biochemical diagnosis is conferred by reduced activity in dried filter paper blood spots (DBS), elevations in globotriaosylsphingosine (lyso-Gb3) and/or identification of a mutation within the GLA gene.MethodsFrom 2008 to 2012, AGA determinations were performed on leucocytes and in 2012, DBS testing was introduced. A mass spectrometric method to quantify lyso-Gb3 in plasma was added to our repertoire of testing in 2016. Mutation analysis was performed using either Sanger or Next Generation Sequencing, the latter confirmed by sequencing.ResultsBetween 2008 and 2012, approximately 1000 samples were screened for Fabry disease using AGA determinations in leucocytes and 29 patients were confirmed positive (diagnosis rate of 2.9%). Since 2012, approximately 4000 samples have been screened from DBS, with 126 patients being confirmed positive (diagnosis rate of 3.2%). There were 24 patients with AGA deficiency and a GLA gene variant which did not have elevated lyso-Gb3 (approximately 20% of positives).DiscussionFabry disease is a relatively complicated disease to diagnose and requires significant interpretation of results from all three tests. As an X-linked disease, classically affected males are easier to identify, but females are more difficult to diagnose as their DBS can be normal, even when symptomatic. Further testing via lyso-Gb3 and mutation analysis is required to make a final biochemical diagnosis. The ease of submitting a DBS for testing has contributed to an increase in test requests.

IntroductionPompe disease is classified by age of onset and presentation. Late-onset Pompe disease (LOPD) generally presents with progressive limb-girdle and respiratory muscle weakness. Diagnosis is confirmed following dried blood spot (DBS) enzyme assay by a second method, either mutation or glucose tetra-saccharide analysis.MethodsFrom 2000 to 2016, acid α –glucosidase (GAA) activity in DBS was measured using the immuno-capture assay of . Umapathysivam et al. (2001) From 2016 testing used the multiplex tandem mass spectrometry method (6-PLEX LC-MSMS, Perkin Elmer), measuring six lysosomal storage disease enzymes from one DBS. Glucose tetra-saccharides were measured by mass spectrometry and mutation analysis of the GAA gene was performed using either Sanger or Next Generation Sequencing, the latter confirmed by sequencing.ResultsBetween 2000 and 2016, approximately 1500 samples were screened for Pompe disease with 77 positives, a diagnosis rate of 5.1%. Of these, 33 were infantile Pompe disease, with 44 LOPD. Since May 2016, a further 850 patients have been screened for Pompe disease using the MSMS method and of these, 28 patients have screened as positive, with 19 confirmed as having Pompe disease, either by molecular testing or urine tetra-saccharide measurement, a diagnosis rate of 2.2%. Of these 19 patients, 17 are LOPD, with only two infants being diagnosed in this time. Seven adults are still to be confirmed by a second test.ConclusionThe number of patients presenting for testing follows the recent Australian government funding for enzyme replacement therapy for adults with LOPD. The ease of submitting a dried blood spot sample for testing has contributed to an increase in test requests, although this may also reflect an increased recognition of this condition. These patients may have already been diagnosed clinically, but in the absence of a treatment, there was little benefit in a formal diagnosis.

Aim To assess the natural history and impact of the secondary bone disease observed in patients with Mucolipidosis II and III.

OBJECTIVE: Tandem mass spectrometry is widely applied to routine newborn screening but there are no long-term studies of outcome. We studied the clinical outcome at six years of age in Australia. METHODS: In a cohort study, we analyzed the outcome at 6 years for patients detected by screening or by clinical diagnosis among >2 million infants born from 1994 to 1998 (1 017 800, all unscreened) and 1998 to 2002 (461 500 screened, 533 400 unscreened) recording intellectual and physical condition, school placement, other medical problems, growth, treatment, diet, and hospital admissions. Results were analyzed separately for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and other disorders, and grouped patients as those who presented clinically or died in the first 5 days of life; patients presented later or diagnosed by screening, and those with substantially benign disorders. RESULTS: Inborn errors, excluding phenylketonuria, were diagnosed in 116 of 1 551 200 unscreened infants (7.5/100 000 births) and 70 of 461 500 screened infants (15.2/100 000 births). Excluding MCADD, 21 unscreened patients with metabolic disorders diagnosed after 5 days of life died or had a significant intellectual or physical handicap (1.35/100 000 population) compared with 2 of the screened cohort (0.43/100 000; odds ratio: 3.1 [95% CI: 0.73–13.32]). Considering the likely morbidity or mortality among the expected number of never-diagnosed unscreened patients, there would be a significant difference. Growth distribution was normal in all cohorts. CONCLUSION: Screening by tandem mass spectrometry provides a better outcome for patients at 6 years of age, with fewer deaths and fewer clinically significant disabilities.

Hyperammonaemic encephalopathy has been infrequently reported after both standard and high-dose chemotherapy for solid and haematological malignancies. It is important to consider this diagnosis for patients with unexplained behaviour changes or encephalopathy and this case report emphasizes this condition and the importance of early diagnosis and is the first reported in association with rituximab-containing chemotherapy.

The Fabry Registry is a global observational research platform established to define outcome data on the natural and treated course of this rare disorder. Participating physicians submit structured longitudinal data to a centralized, confidential database. This report describes the baseline demographic and clinical characteristics of the first 1765 patients (54% males (16% aged < 20 years) and 46% females (13% < 20 years)) enrolled in the Fabry Registry. The median ages at symptom onset and diagnosis were 9 and 23 years (males) and 13 and 32 years (females), respectively, indicating diagnostic delays in both sexes. Frequent presenting symptoms in males included neurological pain (62%), skin signs (31%), gastroenterological symptoms (19%), renal signs (unspecified) (17%), and ophthalmological signs (11%). First symptoms in females included neurological pain (41%), gastroenterological symptoms (13%), ophthalmological (12%), and skin signs (12%). For those patients reporting renal progression, the median age at occurrence was 38 years for both sexes, but onset of cerebrovascular and cardiovascular events was later in females (median 43 and 47 years, respectively) than in males (38 and 41 years, respectively). This paper demonstrates that in spite of the considerable burden of disease in both sexes that begins to manifest in childhood or adolescence, the recognition of the underlying diagnosis is delayed by 14 years in males and 19 years in females. The Fabry Registry provides data that can increase awareness of common symptoms in all age groups, as well as insight into treated and untreated disease course, leading to improved recognition and earlier treatment, and possibly to improved outcomes for affected individuals.

Summary 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.

We present two siblings with a previously undescribed congenital disorder of glycosylation (CDG). The first child died in utero with severe hydrops fetalis and the second presented following preterm delivery with respiratory insufficiency, generalised edema and a protein-losing enteropathy. Both had a similar pattern of facial dysmorphism and joint contractures. The diagnosis of CDG-I was made following the birth of the second child based on the serum transferrin isoform pattern. CDG-Ia and -Ib were excluded by specific enzyme analysis. Joint contractures are a relatively uncommon finding in CDG, although fetal hydrops (CDG-Ia) and protein-losing enteropathy (CDG-Ib) are well recognized. CDG must be considered in the differential diagnosis of hydrops fetalis, congenital hypoproteinemia and death in early infancy, particularly when associated with dysmorphic features.

Idiopathic hyperammonemic encephalopathy is an uncommon but frequently fatal complication of chemotherapy. It is characterised by abrupt alteration in mental status with markedly elevated plasma ammonia levels in the absence of obvious liver disease or any other identifiable cause, and frequently results in intractable coma and death. It usually occurs in patients with haematologic malignancies during the period of neutropenia following cytoreductive therapy or bone marrow transplantation, and in solid organ malignancies treated with 5-fluorouracil. Although the aetiology of this syndrome is yet to be determined, it appears to be multi-factorial in nature. Optimal management remains to be formally established, and the critical step is increased awareness of the syndrome by measurement of plasma ammonium levels in patients with neurological symptoms, leading to early diagnosis and the prompt implementation of therapy.

Introduction In this study, we aimed to evaluate whether fasting plasma citrulline concentration predicts subsequent glucose absorption in critically ill patients. Methods In a prospective observational study involving 15 healthy and 20 critically ill subjects, fasting plasma citrulline concentrations were assayed in blood samples immediately prior to the administration of a liquid test meal (1 kcal/ml; containing 3 g of 3-O-methylglucose (3-OMG)) that was infused directly into the small intestine. Serum 3-OMG concentrations were measured over the following 4 hours, with the area under the 3-OMG concentration curve (AUC) calculated as an index of glucose absorption. Results The groups were well matched in terms of age, sex and body mass index (BMI) (healthy subjects versus patients, mean (range) values: age, 47 (18 to 88) versus 49 (21 to 77) years; sex ratio, 60% versus 80% male; BMI, 25.2 (18.8 to 30.0) versus 25.5 (19.4 to 32.2) kg/m2). Compared to the healthy subjects, patients who were critically ill had reduced fasting citrulline concentration (26.5 (13.9 to 43.0) versus 15.2 (5.7 to 28.6) μmol/L; P

Blood was obtained by cordocentesis from a fetus with non-immune hydrops demonstrated by ultrasound scanning at 27 weeks' gestation. Abnormalities of serum transferrin isoelectric focussing (IEF) were identified, characteristic of a congenital disorder of glycosylation type I (CDG-Ia). A diagnosis of CDG-Ia was confirmed by enzyme analysis of cultured amniocytes. This is the first report of CDG-Ia diagnosed by serum analysis in a fetus. Previous reports have warned that diagnostic abnormalities do not appear in serum until several weeks after birth. The sensitivity of cordocentesis transferrin IEF is unknown but is less than 100% effective because cases have been diagnosed postnatally after normal prenatal or neonatal studies. Enzyme analysis or mutation analysis is required for diagnosis of congenital disorder of glycosylation (CDGs) regardless of whether a diagnostic transferrin pattern is identified prenatally. The analysis of a small sample of serum, from cordocentesis, performed to check for fetal anemia, simplified the investigation, diagnosis, and genetic counselling of a case of non-immune hydrops detected at 27 weeks' gestation. This might be a useful test for other cases in these circumstances, as fetal blood is usually collected to check for anemia.

Aim: To study the effectiveness of a specific national programme of enzyme replacement therapy (ERT) for patients with severe forms of Gaucher disease, a disorder of sphingolipid metabolism resulting from an inherited deficiency of the lysosomal enzyme β-Glucocerebrosidase. Methods: Prospective analysis of data submitted at entry and every 6 months on therapy. The responses of haemoglobin (Hb) and platelet (plt) concentrations, liver and spleen volumes were assessed. Patients: Forty-eight patients were treated with ERT for a minimum of 6 months. Forty patients had Type 1 disease and eight had Type 3B. The age range was 1–70 years (median 24 years). Duration of therapy at the time of analysis was 6–114 months. Results: Thirty-six per cent of patients started with a normal Hb increasing to 76% after 6 months. The mean improvement in Hb from baseline to the end of study period was 20 g/L, when the Hb was normal in 85% (41 patients). Thirty per cent of patients had a normal plt count at the start of therapy, with a more gradual increase in the count at 6 monthly intervals of 50, 91, 108 and 142% of starting value. Seventy-five per cent of patients had a normal plt count at the end of study. Spleen volumes reduced by a mean of 56% in 33 evaluable patients, and the liver by 27% in 30 of 38 evaluable patients. Eight patients had an increase in liver volume of 28%. Conclusion: Enzyme replacement therapy produced a spectrum of beneficial responses in patients with Gaucher disease, but all had some evidence of reversal of haematological complications and/or reduction in visceromegaly. Future analyses will examine the effect of therapy on bone disease, prepubertal growth and quality of life. (Intern Med J 2005; 35: 156–161)

Objectives: To determine the proportion of urine amino and organic acids screening tests (UMS) undertaken for patients referred with developmental delay or intellectual disability (DD/ID), and within the group with DD/ID, to determine the diagnostic yield, the proportion of diagnoses with a therapy and the associated recurrence risks. Methods: A retrospective review of request forms and results of UMS, in individuals older than 28 days, referred to the Women's and Children's Hospital, North Adelaide, between 1 January 1992 and 31 December 1998 was carried out. Urine was analysed by ion exchange chromatography (amino acids), gas chromatography/mass spectrometry (organic acids), colorimetric assay (orotic acid) and stable isotope−dilution mass spectrometry (trimethylamine). Results: A total of 3316 samples were received, 1447 being from patients with DD/ID. A diagnosis was determined for 1.8% of all referrals. For patients with DD/ID, the diagnostic yield was 1.1%, with a similar yield for isolated DD/ID and DD/ID with other features (9/828 vs 7/619; χ2 = 0.006; P = 0.93). Specific therapies were available for 69% of diagnoses associated with DD/ID and 87.5% had known Mendelian or mitochondrial inheritance. Conclusion: Urine metabolic screening is an important part of the evaluation of children with DD/ID as it can enable families to make reproductive decisions and children to receive appropriate therapy early.

Neonatal screening is an essential preventative public health program, and is the standard practice of care world– wide. India is yet to start any publicly funded program despite this having been established in many countries for over 50 years. The purpose of neonatal screening is to reduce morbidity and mortality in the newborn. Detection must have a clear benefit for the baby and be cost effective when compared to the cost associated with delayed treatment. These criteria are clearly satisfied for screening a baby at birth for congenital hypothyroidism (CHT). Though systematic neonatal screening for congenital hypothyroidism was introduced in the early 1970 in many countries, in India an estimated 10,000 babies are born with congenital hypothyroidism every year, yet there is no screening program for this. We present the details of our study, which reveals that CHT in India has a high incidence and is an URGENT high priority for public screening.

We report a patient with late-onset nonketotic hyperglycinaemia managed with a sequential approach to drug therapy in placebo-controlled therapeutic trials. Partial response to low-protein diet and sodium benzoate and dramatic response to imipramine are demonstrated, with parental scores on the Developmental Behavioural Checklist falling from the 86th centile before treatment to normal with combined benzoate and imipramine therapy.

Our objective was to review the Australasian experience of X-linked adrenoleukodystrophy (ALD), to compare the spectrum of disease seen in Australasia with previously published data from elsewhere, and to assess the reliability of carrier testing. Study design was a retrospective review of records collected over a 15-year period, the setting was an international referral laboratory for the study of metabolic disease, and the subjects were all known cases of ALD diagnosed in Australia and New Zealand between 1981 and 1996 and their families. We estimate that the combined incidence of ALD and its variants in Australasia is at least 1.6 per 100,000. Of 95 affected males, 51 had cerebral adrenoleukodystrophy, 24 had adrenomyeloneuropathy, 15 had Addison's disease only, and 5 remained asymptomatic when last examined. However, the distribution of phenotypes among newly diagnosed patients has changed substantially over the last 15 years, with cerebral forms of the disease forming a decreasing proportion of new diagnoses. The measurement of plasma very long chain fatty acids (VLCFAs) alone detects 93% of women who can be proven to be carriers. The addition of genetic linkage studies or assay of VLCFAs in cultured fibroblasts improved this detection rate to the point that there were no obligate carriers who could not be detected using a combination of two or more techniques.

The interruption of the manufacturing process of agalsidase beta has led to a worldwide shortage of this drug. In the EU, nearly all patients initially reduced their agalsidase beta dose, and many of these switched to agalsidase alfa (Replagal Shire HGT). The clinical consequences of this period of drug shortage need to be further evaluated. A gradual increase of agalsidase beta supply is now expected. This implies that patients could resume or even commence agalsidase beta treatment. Guidance for prioritization of patients is needed to support equitable distribution of agalsidase beta to EU member states. To achieve this, in absence of level I clinical evidence, a draft consensus proposal was initiated and distributed. No full consensus was achieved, as there is disagreement regarding the indications for switching patients from agalsidase alfa to agalsidase beta. Some physicians support the concept that the 1.0 mg/kg EOW dose of agalsidase beta is more effective than agalsidase alfa at 0.2 mg/kg EOW, while others believe that at recommended dose, the preparations are equivalent. In light of these difficulties and the uncertainties with respect to supply of agalsidase beta, recommendations were agreed upon by a subgroup of physicians. These current recommendations focus on prioritization of criteria indicative of disease progression.

Genomic testing using gene panels and whole-exome sequence data are becoming increasingly attractive options to support the clinical management of patients. Whole-exome sequencing to diagnose rare genetic disorders in paediatrics offers a cost-effective and efficient first screen for cases where there is no obvious clinical diagnosis, thus fundamentally shifting medical service provision paradigms. In other areas of medicine, the implementation of targeted gene panels using next generation sequencing is associated with improved diagnosis rate and faster time to diagnosis. Successful integration of genomic testing into the patient management pathway requires updating of the traditional models of test provision. Important factors to be considered include: who should qualify for what genomic test, payment for testing, the appropriate referral mechanism, and how to effectively communicate the results and limitations of genomic tests to patients and clinicians. We discuss strategies and give examples from implementing gene panels and whole-exome sequencing in clinical diagnostics to support the management of patients in South Australia. The next few years are likely to be an exciting and challenging time in genomic test provision in Australia, as these new platforms establish themselves as part of routine medical care and different stakeholders (local, international, direct-to-consumer) define their share of the clinical sequencing market.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in sulphamidase (NS), a lysosomal enzyme required for the degradation of heparan sulphate glycosaminoglycans (gags). The MPS IIIA mouse is a naturally occurring model that accurately reflects the human pathology and disease course. It displays primarily central nervous system pathology accompanied by widespread accumulation of gag in somatic tissues. MPS IIIA mice exhibit greater bodyweight gain than normal littermates and attain a higher mature bodyweight. In this study, gastrointestinal morphology and function was characterised in the IIIA mouse. Stomach and duodenum weight increased in MPS IIIA mice and duodenum length also increased. An increased submucosal thickness was observed in MPS IIIA intestine compared to normal mice and lysosomal storage of gag was observed in this region. Storage was also observed in the lamina propria of the villus tip. All other morphometric measurements including villus height and crypt depth fell within the normal range. The gastric emptying half-life of solid and liquid meals decreased with age in normal mice whereas the T½ of solid meals did not alter with age in MPS IIA mice such that they were elevated above normal by 38 weeks of age. Sucrase activity was higher than normal in MPS IIIA at all ages tested. These abnormalities in GI structure and function observed in MPS IIIA may contribute to weight gain in this disorder. Refereed/Peer-reviewed

For over two decades gene therapy has been actively pursued as a treatment modality for the inherited diseases that affect the paediatric population, however, it is still to make a real impact in the clinic. There are many reasons for this including inadequate technology and a lack of understanding of the biological complexities that impact on the efficiency of gene delivery and its outcomes, both positive and negative. However, recent progress is now addressing these issues and indicates that these problems can be overcome, and that gene therapy will play a significant role in the treatment of at least some of these disorders. This review will first give a short overview of relevant gene delivery technologies, what strategies can be used and which diseases are potential targets for gene therapy, and then illustrate several specific diseases for which gene therapy is actively being developed.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a specific lysosomal storage disorder caused by an enzyme deficiency in sulphamidase, which is required for the degradation of heparan sulphate glycosaminoglycan (gag). This deficiency results in widespread gag storage and leads to severe CNS degeneration and mild somatic pathology. We have developed substrate deprivation as a therapy (SDT) for MPS disorders to reduce the initial production of gag substrate for the deficient enzyme, using the compound rhodamine B as an inhibitor of gag biosynthesis. This should restore the balance between gag level and residual enzyme activity towards normal and improve patient outcome. To determine if SDT improved CNS function, MPS IIIA mice were treated for 6 months with weekly, intravenous 1 mg/kg rhodamine B and then tested in a 4-arm water cross maze, which measures spatial learning and memory. MPS IIIA untreated mice were unable to perform to the same level as normal littermates, having increased escape latency, increased incorrect entries and decreased correct entries. Rhodamine B treatment improved MPS IIIA performance towards normal with treated mice having decreased escape latency, decreased incorrect entries and increased correct entries when compared to MPS IIIA untreated littermates. This provides the first report of SDT resulting in a beneficial effect on CNS function in an MPS disorder and SDT targeting gag synthesis may be a viable treatment option for children with MPS.

The availability of therapies for lysosomal storage diseases (LSDs) and clear documentation from animal studies that optimal therapy depends on early diagnosis have set the scene for newborn screening for LSDs. The combined incidence of this group of conditions is approximately 1 in 7000, well within the feasible range for newborn screening programmes. The availability of multiplex technology has facilitated the technical aspects of initial screening. The scientific challenge is to predict disease severity early enough to influence choice of therapy. LSD screening is discussed from the point of view of the scientists, the families affected by these conditions, the community and clinicians.

To study the effectiveness of a specific national programme of enzyme replacement therapy (ERT) for patients with severe forms of Gaucher disease, a disorder of sphingolipid metabolism resulting from an inherited deficiency of the lysosomal enzyme beta-Glucocerebrosidase.Prospective analysis of data submitted at entry and every 6 months on therapy. The responses of haemoglobin (Hb) and platelet (plt) concentrations, liver and spleen volumes were assessed.Forty-eight patients were treated with ERT for a minimum of 6 months. Forty patients had Type 1 disease and eight had Type 3B. The age range was 1-70 years (median 24 years). Duration of therapy at the time of analysis was 6-114 months.Thirty-six per cent of patients started with a normal Hb increasing to 76% after 6 months. The mean improvement in Hb from baseline to the end of study period was 20 g/L, when the Hb was normal in 85% (41 patients). Thirty per cent of patients had a normal plt count at the start of therapy, with a more gradual increase in the count at 6 monthly intervals of 50, 91, 108 and 142% of starting value. Seventy-five per cent of patients had a normal plt count at the end of study. Spleen volumes reduced by a mean of 56% in 33 evaluable patients, and the liver by 27% in 30 of 38 evaluable patients. Eight patients had an increase in liver volume of 28%.Enzyme replacement therapy produced a spectrum of beneficial responses in patients with Gaucher disease, but all had some evidence of reversal of haematological complications and/or reduction in visceromegaly. Future analyses will examine the effect of therapy on bone disease, prepubertal growth and quality of life.

Study Smith-Lemli-Opitz (SLO) syndrome is an autosomal recessive disorder of cholesterol synthesis due to deficiency in 7-dehydrocholesterol (7-DHC) reductase. SLO is associated with specific dysmorphology, microcephaly, syndactyly, cleft palate, growth retardation, genital ambiguity and intellectual disability with autistic features. The autistic features respond to cholesterol supplementation. Increased levels of 7-DHC are a marker for SLO and routinely used for testing of autistic children. Methods A total of 5 years of requests for 7-DHC testing at S A Pathology were audited (2008-2012). Data were broadly classified according to clinical indications, including isolated autism, developmental delay, seizure, dysmorphism, neurological, anatomic, genital or metabolic anomalies. Data were sub-classified as single or multiple clinical indication/s or no clinical information supplied. Results 809 tests were performed and 16 cases of SLO were detected. No cases of SLO were diagnosed based on an isolated clinical indication of autism or where no clinical information was supplied. Discussion Experience of several years of 7-DHC testing at SA Pathology indicates that autism as an isolated clinical trait is not associated with SLO. 7-DHC measurement should not be included in the testing panel when autism spectrum disorders are the only clinical indication.

Background: Arginosuccinic acid synthetase (ASA) (EC 6.3.4.5) deficiency (citrullinaemia) (McKusick 215700) is a well-recognized cause of neonatal hyperammonaemic coma with poor long-term intellectual function, despite good medical management. Methods: Cadaveric hepatic transplantation was performed in a 12-year-old boy with citrullinaemia under poor biochemical control. Subsequent development of fulminant hepatic failure necessitated a second cadaveric transplant. Psychometric assessments before and after transplantation were performed using a variety of age-appropriate tests. Results: Normalization of plasma ammonium in our patient post transplantation has resulted in dramatic improvement in mental functioning and well-being and he now enjoys a normal diet. Psychometric assessment confirmed decline in his abilities prior to transplantation with particular post-transplantation improvement in perceptual organization and visuospatial abilities; these did not, however, return to normal. His family report considerable reduction in stress associated with the unpredictable nature of previous hyperammonaemic crises and recurrent hospitalization. Conclusions: Liver transplantation should be considered as an early therapeutic option in children with citrullinaemia to prevent ongoing cerebral insult associated with hyperammonaemia.

We report a family who experienced an unexplained neonatal death. Twelve years after the death, we retrospectively diagnosed multiple acyl-coenzyme A dehydrogenase deficiency by demonstrating an abnormal acyl-carnitine profile in the child's archived newborn screening card, using tandem mass spectrometry.

A. Poole, A. Deane, J. Magarey, S. O’Connor, S. Millington, J. Fletcher, A. Zaknic, M. Summers, M. Chapman

Aim To evaluate the usefulness of microarray in fetal autopsy cases with or without malformations. Introduction Microarrays were performed as part of autopsy investigation on 90 cases, including stillbirths, fetuses that died in utero or were terminated due to abnormal ultrasound findings between February 2009 and September 2011. Methods Microarray was performed using the BlueGnome Cyto-chip oligo ISCA 60K array platform. Microarray results and autopsy reports of all cases were reviewed to determine the presence of any fetal malformations and to categorise the positive microarray findings as causative, of unknown clinical significance or not causative. The number of causative findings identified by microarray in cases with at least one malformation was compared to the number of causative findings identified in cases without any malformations. Results Five microarrays failed. Of the remaining 85 cases with microarray results, 53 cases had at least one malformation and 32 cases had no malformation. Four cases (7.5%) with one or more malformations had a causative finding on microarray, compared to no cases without any malformations. Conclusions Microarray is a valuable investigation in fetal autopsy cases with at least one malformation, but not for those without any malformation.