Molecular genetic analysis of candidate genes for glutaric aciduria type II in a cohort of patients from Queensland, Australia.

Glutaric aciduria type II (GAII) is a heterogeneous genetic disorder affecting mitochondrial fatty acid, amino acid and choline oxidation. Clinical manifestations vary across the lifespan and onset may occur at any time from the early neonatal period to advanced adulthood. Historically, some patients, in particular those with late onset disease, have experienced significant benefit from riboflavin supplementation. GAII has been considered an autosomal recessive condition caused by pathogenic variants in the gene encoding electron-transfer flavoprotein ubiquinone-oxidoreductase (ETFDH) or in the genes encoding electron-transfer flavoprotein subunits A and B (ETFA and ETFB respectively). Variants in genes involved in riboflavin metabolism have also been reported. However, in some patients, molecular analysis has failed to reveal diagnostic molecular results. In this study, we report the outcome of molecular analysis in 28 Australian patients across the lifespan, 10 paediatric and 18 adult, who had a diagnosis of glutaric aciduria type II based on both clinical and biochemical parameters. Whole genome sequencing was performed on 26 of the patients and two neonatal onset patients had targeted sequencing of candidate genes. The two patients who had targeted sequencing had biallelic pathogenic variants (in ETFA and ETFDH). None of the 26 patients whose whole genome was sequenced had biallelic variants in any of the primary candidate genes. Interestingly, nine of these patients (34.6%) had a monoallelic pathogenic or likely pathogenic variant in a single primary candidate gene and one patient (3.9%) had a monoallelic pathogenic or likely pathogenic variant in two separate genes within the same pathway. The frequencies of the damaging variants within ETFDH and FAD transporter gene SLC25A32 were significantly higher than expected when compared to the corresponding allele frequencies in the general population. The remaining 16 patients (61.5%) had no pathogenic or likely pathogenic variants in the candidate genes. Ten (56%) of the 18 adult patients were taking the selective serotonin reuptake inhibitor antidepressant sertraline, which has been shown to produce a GAII phenotype, and another two adults (11%) were taking a serotonin-norepinephrine reuptake inhibitor antidepressant, venlafaxine or duloxetine, which have a mechanism of action overlapping that of sertraline. Riboflavin deficiency can also mimic both the clinical and biochemical phenotype of GAII. Several patients on these antidepressants showed an initial response to riboflavin but then that response waned. These results suggest that the GAII phenotype can result from a complex interaction between monoallelic variants and the cellular environment. Whole genome or targeted gene panel analysis may not provide a clear molecular diagnosis.

Central Reading of Ulcerative Colitis Clinical Trial Videos Using Neural Networks.

BACKGROUND AND AIMS: Endoscopic disease activity scoring in ulcerative colitis (UC) is useful in clinical practice but done infrequently. It is required in clinical trials, where it is expensive and slow because human central readers are needed. A machine learning algorithm automating the process could elevate clinical care and facilitate clinical research. Prior work using single-institution databases and endoscopic still images has been promising. METHODS: Seven hundred and ninety-five full-length endoscopy videos were prospectively collected from a phase 2 trial of mirikizumab with 249 patients from 14 countries, totaling 19.5 million image frames. Expert central readers assigned each full-length endoscopy videos 1 endoscopic Mayo score (eMS) and 1 Ulcerative Colitis Endoscopic Index of Severity (UCEIS) score. Initially, video data were cleaned and abnormality features extracted using convolutional neural networks. Subsequently, a recurrent neural network was trained on the features to predict eMS and UCEIS from individual full-length endoscopy videos. RESULTS: The primary metric to assess the performance of the recurrent neural network model was quadratic weighted kappa (QWK) comparing the agreement of the machine-read endoscopy score with the human central reader score. QWK progressively penalizes disagreements that exceed 1 level. The model's agreement metric was excellent, with a QWK of 0.844 (95% confidence interval, 0.787-0.901) for eMS and 0.855 (95% confidence interval, 0.80-0.91) for UCEIS. CONCLUSIONS: We found that a deep learning algorithm can be trained to predict levels of UC severity from full-length endoscopy videos. Our data set was prospectively collected in a multinational clinical trial, videos rather than still images were used, UCEIS and eMS were reported, and machine learning algorithm performance metrics met or exceeded those previously published for UC severity scores.

Squalene Synthase Deficiency: Clinical, Biochemical, and Molecular Characterization of a Defect in Cholesterol Biosynthesis.

Mendelian disorders of cholesterol biosynthesis typically result in multi-system clinical phenotypes, underlining the importance of cholesterol in embryogenesis and development. FDFT1 encodes for an evolutionarily conserved enzyme, squalene synthase (SS, farnesyl-pyrophosphate farnesyl-transferase 1), which catalyzes the first committed step in cholesterol biosynthesis. We report three individuals with profound developmental delay, brain abnormalities, 2-3 syndactyly of the toes, and facial dysmorphisms, resembling Smith-Lemli-Opitz syndrome, the most common cholesterol biogenesis defect. The metabolite profile in plasma and urine suggested that their defect was at the level of squalene synthase. Whole-exome sequencing was used to identify recessive disease-causing variants in FDFT1. Functional characterization of one variant demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis/uptake in steady state.

Microdeletion of 9q22.3: A patient with minimal deletion size associated with a severe phenotype.

Basal cell nevus syndrome (also known as Gorlin Syndrome; MIM109400) is an autosomal dominant disorder characterized by recurrent pathological features such as basal cell carcinomas and odontogenic keratocysts as well as skeletal abnormalities. Most affected individuals have point mutations or small insertions or deletions within the PTCH1 gene on human chromosome 9, but there are some cases with more extensive deletion of the region, usually including the neighboring FANCC and/or ERCC6L2 genes. We report a 16-year-old patient with a deletion of approximately 400,000 bases which removes only PTCH1 and some non-coding RNA genes but leaves FANCC and ERCC6L2 intact. In spite of the small amount of DNA for which he is haploid, his phenotype is more extreme than many individuals with longer deletions in the region. This includes early presentation with a large number of basal cell nevi and other skin lesions, multiple jaw keratocysts, and macrosomia. We found that the deletion was in the paternal chromosome, in common with other macrosomia cases. Using public databases, we have examined possible interactions between sequences within and outside the deletion and speculate that a regulatory relationship exists with flanking genes, which is unbalanced by the deletion, resulting in abnormal activation or repression of the target genes and hence the severity of the phenotype.

Triple P for Parents of Children with Phenylketonuria: A Nonrandomized Trial.

OBJECTIVE: Families of children with phenylketonuria (PKU) report child emotional and behavioral problems, parenting stress, and parenting difficulties, which are associated with worse health-related quality of life. This study aimed to examine acceptability and feasibility of a brief, group-based parenting program (Healthy Living Triple P) for families of children with PKU. METHODS: An uncontrolled nonrandomized trial design was used. Families of children aged 2-12 years (N = 17) completed questionnaire measures assessing child behavior and impact of PKU on quality of life (primary outcomes), and parenting behavior, self-efficacy and stress, and children's behavioral and emotional adjustment (secondary outcomes). Routinely collected blood phenylalanine (Phe) levels were obtained from the treating team. Parents selected two child behaviors as targets for change. The intervention comprised two, 2-hr group sessions delivered face-to-face or online. Assessment was repeated at 4-week postintervention (T2) and 4-month follow-up (T3). RESULTS: Attrition was low and parent satisfaction with the intervention (face-to-face and online) was high. All families achieved success with one or both child behavior goals, and 75% of families achieved 100% success with both behavior goals by T3; however, there was no change in health-related quality of life. There were moderate improvements in parent-reported ineffective parenting (total score, d = 0.87, 95% CI -1.01 to 2.75) and laxness (d = 0.59, 95% CI -1.27 to 2.46), but no effects on parenting stress or children's adjustment. Phe levels improved by 6month post-intervention for children with elevated preintervention levels. CONCLUSIONS: Results support intervention acceptability and feasibility. A randomized controlled trial is warranted to establish intervention efficacy.

Psychosocial functioning in children with phenylketonuria: Relationships between quality of life and parenting indicators.

OBJECTIVE: This study aimed to assess the impact of phenylketonuria (PKU) and its treatment on parent and child health-related quality of life (HRQoL) and to identify the parenting-related correlates of parent and child HRQoL, as well as metabolic control. METHODS: Eighteen mothers of 2- to 12-year-old children with PKU participated and completed a series of self-report questionnaires including the PKU Impact and Treatment Quality of Life Questionnaire (PKU-QOL). RESULTS: Mothers reported that the most significant impact of PKU on HRQoL was in relation to the impact of their child's anxiety during blood tests on their own HRQoL and guilt related to poor adherence to dietary restrictions and supplementation regimens. Higher reported intensity of child emotional and behavioural difficulties and parenting stress were associated with higher scores for PKU symptoms on the PKU-QOL, higher scores for emotional, social, and overall impact of PKU, and higher scores for the impact of dietary restriction. Where mothers reported greater use of overreactivity as a parenting strategy, children tended to have better lifetime phenylalanine levels; however, the overall impact of PKU and the impact of supplement administration on mothers' HRQoL were worse for these families. CONCLUSIONS: These findings have implications for a holistic family-centred approach to the care of children with PKU and their families.

The effect of height, weight and head circumference on gross motor development in achondroplasia.

PURPOSE: This study aimed to investigate whether height, weight, head circumference and/or relationships between these factors are associated with gross motor milestone acquisition in children with achondroplasia. METHOD: Population-based data regarding timing of major gross motor milestones up to 5 years were correlated with height, weight and head circumference at birth and 12 months in 48 children with achondroplasia born in Australia and New Zealand between 2000 and 2009. RESULTS: Although as a group children with achondroplasia showed delayed gross motor skill acquisition, within group differences in height, weight or head circumference did not appear to influence timing of gross motor skills before 5 years. The exception was lie to sit transitioning, which appears likely to occur earlier if the child is taller and heavier at 12 months, and later if the child has significant head-to-body disproportion. CONCLUSIONS: This is the first study to investigate the relationship between common musculoskeletal impairments associated with achondroplasia and timing of gross motor achievement. Identification of the musculoskeletal factors that exacerbate delays in transitioning from lying to sitting will assist clinicians to provide more proactive assessment, advice and intervention regarding motor skill acquisition for this population.

Mutations in ENPP1 are associated with 'idiopathic' infantile arterial calcification.

Idiopathic infantile arterial calcification (IIAC; OMIM 208000) is characterized by calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation. We analyzed affected individuals from 11 unrelated kindreds and found that IIAC was associated with mutations that inactivated ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). This cell surface enzyme generates inorganic pyrophosphate (PP(i)), a solute that regulates cell differentiation and serves as an essential physiologic inhibitor of calcification.

Study of LPIN1, LPIN2 and LPIN3 in rhabdomyolysis and exercise-induced myalgia.

BACKGROUND: Recessive LPIN1 mutations were identified as a cause of severe rhabdomyolysis in pediatric patients. The human lipin family includes two other closely related members, lipin-2 and 3, which share strong homology and similar activity. The study aimed to determine the involvement of the LPIN family genes in a cohort of pediatric and adult patients (n = 171) presenting with muscular symptoms, ranging from severe (CK >10 000 UI/L) or moderate (CK <10 000 UI/L) rhabdomyolysis (n = 141) to exercise-induced myalgia (n = 30), and to report the clinical findings in patients harboring mutations. METHODS: Coding regions of LPIN1, LPIN2 and LPIN3 genes were sequenced using genomic or complementary DNAs. RESULTS: Eighteen patients harbored two LPIN1 mutations, including a frequent intragenic deletion. All presented with severe episodes of rhabdomyolysis, starting before age 6 years except two (8 and 42 years). Few patients also suffered from permanent muscle symptoms, including the eldest ones (≥ 40 years). Around 3/4 of muscle biopsies showed accumulation of lipid droplets. At least 40% of heterozygous relatives presented muscular myalgia. Nine heterozygous SNPs in LPIN family genes were identified in milder phenotypes (mild rhabdomyolysis or myalgia). These variants were non-functional in yeast complementation assay based on respiratory activity, except the LPIN3-P24L variant. CONCLUSION: LPIN1-related myolysis constitutes a major cause of early-onset rhabdomyolysis and occasionally in adults. Heterozygous LPIN1 mutations may cause mild muscular symptoms. No major defects of LPIN2 or LPIN3 genes were associated with muscular manifestations.

Recent developments in the diagnosis of Marfan syndrome and related disorders.

Marfan syndrome is a multisystem disorder of connective tissue that is inherited in an autosomal dominant fashion, and results from mutation of the FBN1 gene on human chromosome 15. There are a number of conditions of the connective tissue with a similar phenotype that can be confused with Marfan syndrome. Modifications of the diagnostic criteria have recently been published, facilitating the differentiation of Marfan syndrome from these conditions. It is still difficult to use modern genetic testing for diagnosis because Marfan syndrome can be caused by many different mutations in FBN1, a large gene with 65 coding segments, while mutations in other genes can cause overlapping phenotypes. Several clinical trials of drug therapy, including the antihypertensive drug losartan, are in progress.

Development in children with achondroplasia: a prospective clinical cohort study.

AIM: Achondroplasia is characterized by delays in the development of communication and motor skills. While previously reported developmental profiles exist across gross motor, fine motor, feeding, and communication skills, there has been no prospective study of development across multiple areas simultaneously. METHOD: This Australasian population-based study utilized a prospective questionnaire to quantify developmental data for skills in children born from 2000 to 2009. Forty-eight families from Australia and New Zealand were asked to report every 3 months on their child's attainment of 41 milestones. Results include reference to previously available prospective information. RESULTS: Information from questionnaires was used to develop an achondroplasia-specific developmental recording form. The 25th, 50th, 75th, and 90th centiles were plotted to offer clear guidelines for development across gross motor, fine motor, feeding, and communication skills in children with achondroplasia. INTERPRETATIONS: Consistent with results from previous research, children with achondroplasia are delayed in development of gross motor and ambulatory skills. Young children with achondroplasia demonstrate a number of unique movement strategies that appear compensatory for the biomechanical changes. While delays were seen in development of later communication items, there were fewer delays seen across development of early communication, fine motor, and feeding skills.

Medical management of children with achondroplasia: evaluation of an Australasian cohort aged 0-5 years.

AIMS: Achondroplasia is the most common form of osteochondrodysplasia and is associated with a number of life-threatening complications. The complexity of the condition led to the development of Heath Supervision Guidelines published by the American Academy of Pediatrics in 1995 and revised in 2005. There remains limited population-based information on utilisation of medical and therapy services for children with achondroplasia. Increased information regarding use of these services will assist in future service development. METHODS: Data regarding frequency and timing of medical and allied health consultations, investigations and interventions were collected from 53 Australasian families via questionnaire, based on recommendations of the Health Supervision Guidelines, an expert reference group and literature review. RESULTS: Rates varied with age for medical consultations (geneticist, paediatric rehabilitation physician/paediatrician, respiratory physician, orthopaedic consultant, neurologist, neurosurgeon), medical investigations (sleep study, magnetic resonance imaging/computed tomography), operative procedures (brain-stem decompression, tonsillectomy/adenoidectomy, shunt insertion, shunt revision and insertion of grommets) and allied health consultations (physiotherapist, occupational therapist, speech pathologist, dietician and orthotist). CONCLUSIONS: Access to geneticists and paediatricians within the first year is high as recommended by the 2005 American Academy of Pediatrics guidelines. Utilisation of craniocervical magnetic resonance imaging/computed tomography, polysomnography studies and formal speech review appears low, reflecting more emphasis on clinical monitoring for cervical cord compression and disordered sleep breathing as well as possible difficulties in accessing services for polysomnography and speech pathology. Grommet insertion, tonsillectomy/adenoidectomy and cervicomedullary decompression rates are similar to results reported previously. Over half of the children accessed physiotherapy and/or occupational therapy services, warranting consideration of these professionals in future guideline recommendations.

Functional performance in young Australian children with achondroplasia.

AIM: The aim of this study was to determine population-specific developmental milestones for independence in self-care, mobility, and social cognitive skills in children with achondroplasia, the most common skeletal dysplasia. METHODS: Population-based recruitment from October 2008 to October 2010 identified 44 Australian children with achondroplasia aged 3 to 7 years. Consenting parents of 35 children (16 males, 19 females 14 aged 3y; 12 aged 5y; nine aged 7y) reported their child's self-care, mobility, and social cognition function using the Functional Independence Measure for Children (WeeFIM-II) at the ages of 3 (n=14), 5 (n=12), or 7 (n=9) years. Children were excluded from the study if they had an additional neurological or musculoskeletal condition. RESULTS: Functioning improved in children with achondroplasia between the ages of 3 and 5 years, but not subsequently. Milestones in the achondroplasia group were delayed across all ages and domains compared with normative reference data. Children with achondroplasia required greater caregiver assistance for self-care and mobility skills than typically developing children based on normative data. Social cognition appeared to be an area of relative strength. INTERPRETATION: Children up to 7 years of age with achondroplasia show delayed milestone acquisition and a greater need for caregiver assistance for all domains. As functional delays are likely to be related to common musculoskeletal impairments associated with achondroplasia, access to physiotherapists, occupational therapists, and speech and language pathologists skilled in achondroplasia management may assist children and families to become more independent, particularly around the time of starting school.

Tyrosine monitoring in children with early and continuously treated phenylketonuria: results of an international practice survey.

Investigations into the biochemical markers associated with executive function (EF) impairment in children with early and continuously treated phenylketonuria (ECT-PKU) remain largely phenylalanine-only focused, despite experimental data showing that a high phenylalanine:tyrosine (phe:tyr) ratio is more strongly associated with EF deficit than phe alone. A high phe:tyr ratio is hypothesized to lead to a reduction in dopamine synthesis within the brain, which in turn results in the development of EF impairment. This paper provides a snapshot of current practice in the monitoring and/or treatment of tyrosine levels in children with PKU, across 12 countries from Australasia, North America and Europe. Tyrosine monitoring in this population has increased over the last 5 years, with over 80% of clinics surveyed reporting routine monitoring of tyrosine levels in infancy alongside phe levels. Twenty-five percent of clinics surveyed reported actively treating/managing tyrosine levels (with supplemental tyrosine above that contained in PKU formulas) to ensure tyrosine levels remain within normal ranges. Anecdotally, supplemental tyrosine has been reported to ameliorate symptoms of both attention deficit hyperactivity disorder and depression in this population. EF assessment of children with ECT-PKU was likewise highly variable, with 50% of clinics surveyed reporting routine assessments of intellectual function. However when function was assessed, test instruments chosen tended towards global measures of IQ prior to school entry, rather than specific assessment of EF development. Further investigation of the role of tyrosine and its relationship with phe and EF development is needed to establish whether routine tyrosine monitoring and increased supplementation is recommended.

Uniting Discovery and Care: The Role of Pharmaceutical Companies in Research, Clinical Studies, and Patient Care.

In an era of increased complexity of clinical research, a demand for personalized medicine, an increasing value of diversity, a focus on digital health, and a call for patient centricity, the discovery and development of new medicines, more than ever, is dependent on collaboration between multiple stakeholders.

Successful management of a neonate with OTC deficiency presenting with hyperammonemia and severe cardiac dysfunction with extracorporeal membrane oxygenation support and continuous renal replacement therapy.

Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder which-in severe form-results in rapid accumulation of ammonia and glutamine with subsequent irreversible brain injury. We present a case of severe left ventricular dysfunction with hyperammonemic crisis caused by OTC deficiency which was managed with veno-arterial extracorporeal membrane oxygenation support combined with continuous renal replacement therapy. Aggressive treatment led to normalization of ammonia and full left ventricular recovery.

Safety, efficacy, and pharmacokinetic overview of low-dose daily administration of tadalafil.

INTRODUCTION: Several phosphodiesterase type 5 (PDE5) inhibitors are commercially available for the treatment of erectile dysfunction (ED). Development of the first once-daily alternative dosing regimen with a PDE5 inhibitor was motivated by the behavioral complexities associated with sexual intimacy. AIM: To provide an alternative dosing option for certain men who may benefit from the removal of the temporal linkage between administration of an ED therapy and sexual intimacy or for men and their partners who anticipate at least twice-weekly sexual activity. METHODS: Pharmacokinetic predictions of tadalafil plasma concentrations were generated based upon empirical data following 20-mg, single-dose administration coupled with tadalafil usage patterns from as-needed clinical trials. To support the pharmacokinetic simulations and pharmacodynamic assumptions, clinical trials were conducted to demonstrate the efficacy and safety of once-daily, low-dose tadalafil 2.5 and 5 mg. MAIN OUTCOME MEASURES: Simulated tadalafil plasma concentrations and comparison with safety and efficacy measures from clinical trials. RESULTS: Based upon pharmacodynamic and pharmacokinetic data, once-daily doses of tadalafil 5 mg were predicted to provide therapeutic concentrations that would be maintained throughout the 24-hour dosing interval. Additionally, for a subgroup of men who anticipate at least twice-weekly sexual activity and are currently taking tadalafil 20 mg, a reduction in daily tadalafil exposure was predicted. To support the hypothesis that low-dose, once-daily tadalafil may be a safe and effective treatment alternative, clinical trials were conducted to demonstrate the safety and efficacy of once-daily tadalafil 2.5 and 5 mg. These results were similar to those of historical as-needed studies evaluating tadalafil 10 and 20 mg. CONCLUSIONS: Consistent with pharmacokinetic predictions, data from clinical trials indicate that once-daily use of low-dose tadalafil is a safe and effective treatment for men with ED.

Retinal hemorrhages associated with meningitis in a child with a congenital disorder of glycosylation.

A 9-month old infant presented in a state of shock to a district hospital. She was subsequently referred to the regional tertiary hospital. On admission, bruises were noted on the vertex of the skull. Retinal hemorrhages were present on ophthalmological examination. CT scan of the brain showed poor grey-white matter differentiation with apparent frontoparietal fractures of the skull. Her case was subsequently referred to the relevant authorities as it was suspicious for nonaccident injury (NAI). Her condition deteriorated and she died the next day. Postmortem examination showed that the bruises on the vertex were caused by rapid widening of the sutures of the skull, caused by rising intracranial pressure. There was no skull fracture or evidence of trauma. Histological examination of the brain showed meningitis which had extended to the optic nerve sheath. Hemorrhages were noted in the retinas as well as the optic nerve sheath. An incidental congenital disorder of glycosylation (CDG) was diagnosed on brain histology and confirmed by metabolic tests. Retinal hemorrhages are known to occur in head injuries especially in association with NAI. In this case, suspicion of NAI was further augmented by the presence of apparent bruises on the head. The full postmortem examination showed no evidence of injuries and instead showed that the child was suffering from meningitis. Blood culture grew Group A Streptococcus pyogenes. The underlying mechanisms for such a presentation and the association with CDG are discussed.