A. A. Monavari

N-Glycan Abnormalities in Children with Galactosemia

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

Fatal presentation of ornithine transcarbamylase deficiency in a 62-year-old man and family studies

Summary: Ornithine transcarbamylase deficiency (OTCD) resulting from deficiency of the mitochondrial enzyme OTC shows extensive phenotypic heterogeneity influenced by allelic heterogeneity and modifying environmental influences such as protein intake. We report the fatal late-onset presentation of OTCD in a 62-year-old man with the V337L mutation, a previous presentation in his grandson and negative clinical and biochemical screening of the probands three daughters.

Should children with inherited metabolic disorders receive varicella vaccination

The aim was to determine the rate of varicella infection and complications in children with disorders of intermediary metabolism (IEM) between the ages of 1 and 16 years attending our national metabolic referral centre. Of 126 children identified, a response was received from 122. A history of previous varicella infection was identified in 64 cases (53%) and of varicella vaccination in 5 (4%). Fifty-three (43%) patients apparently did not have a history of clinical varicella infection. Of the 64 children with a history of varicella infection, five required hospitalisation for complications, including life-threatening lactic acidosis in one patient with mitochondrial disease and metabolic decompensation in four patients. In conclusion, varicella infection may cause an increased risk of metabolic decompensation in patients with IEMs. We propose that a trial of varicella vaccination be considered for this cohort of patients with monitoring of its safety and efficacy.

Diagnosing childhood-onset inborn errors of metabolism by next-generation sequencing

Background Inborn errors of metabolism (IEMs) underlie a substantial proportion of paediatric disease burden but their genetic diagnosis can be challenging using the traditional approaches. Methods We designed and validated a next-generation sequencing (NGS) panel of 226 IEM genes, created six overlapping phenotype-based subpanels and tested 102 individuals, who presented clinically with suspected childhood-onset IEMs. Results In 51/102 individuals, NGS fully or partially established the molecular cause or identified other actionable diagnoses. Causal mutations were identified significantly more frequently when the biochemical phenotype suggested a specific IEM or a group of IEMs (p<0.0001), demonstrating the pivotal role of prior biochemical testing in guiding NGS analysis. The NGS panel helped to avoid further invasive, hazardous, lengthy or expensive investigations in 69% individuals (p<0.0001). Additional functional testing due to novel or unexpected findings had to be undertaken in only 3% of subjects, demonstrating that the use of NGS does not significantly increase the burden of subsequent follow-up testing. Even where a molecular diagnosis could not be achieved, NGS-based approach assisted in the management and counselling by reducing the likelihood of a high-penetrant genetic cause. Conclusion NGS has significant clinical utility for the diagnosis of IEMs. Biochemical testing and NGS analysis play complementary roles in the diagnosis of IEMs. Incorporating NGS into the diagnostic algorithm of IEMs can improve the accuracy of diagnosis.

A recurrent mitochondrial p.Trp22Arg NDUFB3 variant causes a distinctive facial appearance, short stature and a mild biochemical and clinical phenotype

Background Isolated Complex I deficiency is the most common paediatric mitochondrial disease presentation, associated with poor prognosis and high mortality. Complex I comprises 44 structural subunits with at least 10 ancillary proteins; mutations in 29 of these have so far been associated with mitochondrial disease but there are limited genotype-phenotype correlations to guide clinicians to the correct genetic diagnosis. Methods Patients were analysed by whole-exome sequencing, targeted capture or candidate gene sequencing. Clinical phenotyping of affected individuals was performed. Results We identified a cohort of 10 patients from 8 families (7 families are of unrelated Irish ancestry) all of whom have short stature (<9th centile) and similar facial features including a prominent forehead, smooth philtrum and deep-set eyes associated with a recurrent homozygous c.64T>C, p.Trp22Arg NDUFB3 variant. Two sibs presented with primary short stature without obvious metabolic dysfunction. Analysis of skeletal muscle from three patients confirmed a defect in Complex I assembly. Conclusions Our report highlights that the long-term prognosis related to the p.Trp22Arg NDUFB3 mutation can be good, even for some patients presenting in acute metabolic crisis with evidence of an isolated Complex I deficiency in muscle. Recognition of the distinctive facial features—particularly when associated with markers of mitochondrial dysfunction and/or Irish ancestry—should suggest screening for the p.Trp22Arg NDUFB3 mutation to establish a genetic diagnosis, circumventing the requirement of muscle biopsy to direct genetic investigations.

G86(P) Clinical Phenotype associated with recessive mutations in LARS gene

Our study involves 8 individuals (0.3–34 yrs) from 6 Irish Traveller families with intermittent liver dysfunction; 4 presented with acute liver failure in infancy. Additional features include chronic microcytic anaemia, developmental delay, and poor growth. During intercurrent febrile illnesses, hepatic decompensation (7/8) and encephalopathy with complex seizures are observed. Two patients died following viral infections; the eldest patient’s worst decompensation was triggered by measles aged 4. The youngest patient was diagnosed at 4 weeks of age when he presented with poor tone, low birth weight, poor growth and a positive family history, hepatopathy subsequently developed at 2 months of age. Liver biopsies showed micro and macrosteatosis. Marrow erythroblasts have abnormal iron distribution. Extensive metabolic, genetic and mitochondrial investigations were negative. Exome sequencing has identified leucyl-t RNA synthetase (LARS) as the causative gene. Subsequent testing identified anothe Irish traveller patient who presented at 2 years with status epilepticus and a history of liver dysfunction and anaemia. He had acute Leigh like changes on MRI brain which resolved on follow up. Due to the multisystem nature of this disorder an underlying mitochondrial disorder had been suspected clinically, however knockdown of LARS in HEK293 cells did not impact on mitochondrial function sugesting that the hepatopathy is not primarily due to mitochondrial dysfunction. This in in keeping with the negative mitochondrial investigations. Recently, LARS was shown to activate mTORC1 which regulates autophagy, a process implicated in liver disease and host response to infections raising the possibilty that defective autophagy may be the underlying disease mechanism. Since our discovery, 2 further patients (non-Irish descent) with mutations in LARS have been identified at 2 US centres, reaffirming the cardinal clinical features of this disorder: poor infantile growth, microcytic anaemia, hepatopathy, decompensation and seizures with minor illness. In Ireland, LARS mutations should be suspected particularly in patients from the traveller population who present with liver dysfunction.