Diagnosis of Genetic White Matter Disorders by Singleton Whole-Exome and Genome Sequencing Using Interactome-Driven Prioritization.

BACKGROUND AND OBJECTIVES: Genetic white matter disorders (GWMD) are of heterogeneous origin, with >100 causal genes identified to date. Classic targeted approaches achieve a molecular diagnosis in only half of all patients. We aimed to determine the clinical utility of singleton whole-exome sequencing and whole-genome sequencing (sWES-WGS) interpreted with a phenotype- and interactome-driven prioritization algorithm to diagnose GWMD while identifying novel phenotypes and candidate genes. METHODS: A case series of patients of all ages with undiagnosed GWMD despite extensive standard-of-care paraclinical studies were recruited between April 2017 and December 2019 in a collaborative study at the Bellvitge Biomedical Research Institute (IDIBELL) and neurology units of tertiary Spanish hospitals. We ran sWES and WGS and applied our interactome-prioritization algorithm based on the network expansion of a seed group of GWMD-related genes derived from the Human Phenotype Ontology terms of each patient. RESULTS: We evaluated 126 patients (101 children and 25 adults) with ages ranging from 1 month to 74 years. We obtained a first molecular diagnosis by singleton WES in 59% of cases, which increased to 68% after annual reanalysis, and reached 72% after WGS was performed in 16 of the remaining negative cases. We identified variants in 57 different genes among 91 diagnosed cases, with the most frequent being RNASEH2B, EIF2B5, POLR3A, and PLP1, and a dual diagnosis underlying complex phenotypes in 6 families, underscoring the importance of genomic analysis to solve these cases. We discovered 9 candidate genes causing novel diseases and propose additional putative novel candidate genes for yet-to-be discovered GWMD. DISCUSSION: Our strategy enables a high diagnostic yield and is a good alternative to trio WES/WGS for GWMD. It shortens the time to diagnosis compared to the classical targeted approach, thus optimizing appropriate management. Furthermore, the interactome-driven prioritization pipeline enables the discovery of novel disease-causing genes and phenotypes, and predicts novel putative candidate genes, shedding light on etiopathogenic mechanisms that are pivotal for myelin generation and maintenance.

Delineating the neurological phenotype in children with defects in the ECHS1 or HIBCH gene.

The neurological phenotype of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) and short-chain enoyl-CoA hydratase (SCEH) defects is expanding and natural history studies are necessary to improve clinical management. From 42 patients with Leigh syndrome studied by massive parallel sequencing, we identified five patients with SCEH and HIBCH deficiency. Fourteen additional patients were recruited through collaborations with other centres. In total, we analysed the neurological features and mutation spectrum in 19 new SCEH/HIBCH patients. For natural history studies and phenotype to genotype associations we also included 70 previously reported patients. The 19 newly identified cases presented with Leigh syndrome (SCEH, n = 11; HIBCH, n = 6) and paroxysmal dystonia (SCEH, n = 2). Basal ganglia lesions (18 patients) were associated with small cysts in the putamen/pallidum in half of the cases, a characteristic hallmark for diagnosis. Eighteen pathogenic variants were identified, 11 were novel. Among all 89 cases, we observed a longer survival in HIBCH compared to SCEH patients, and in HIBCH patients carrying homozygous mutations on the protein surface compared to those with variants inside/near the catalytic region. The SCEH p.(Ala173Val) change was associated with a milder form of paroxysmal dystonia triggered by increased energy demands. In a child harbouring SCEH p.(Ala173Val) and the novel p.(Leu123Phe) change, an 83.6% reduction of the protein was observed in fibroblasts. The SCEH and HIBCH defects in the catabolic valine pathway were a frequent cause of Leigh syndrome in our cohort. We identified phenotype and genotype associations that may help predict outcome and improve clinical management.

Early and long-term effect of the treatment with pyridostigmine in patients with GMPPB-related congenital myasthenic syndrome.

GMPPB mutations cause congenital myasthenic syndromes (CMS) overlapping with muscular dystrophy. Treatment with pyridostigmine has been reported to be effective in those patients. Nevertheless, results of functional motor assessments to determine its precise impact on the short and long term were not available. We describe the response to treatment with pyridostigmine in three siblings with GMPPB-related CMS using functional motor scales performed regularly over a period of 40 months. The beneficial effect of the treatment was outstanding within the first hours, with all the scales showing a dramatic increase in only two days. This remarkable improvement remained steady during 12 months but a moderate decrease was subsequently detected in two of the three patients. Despite this decline in the scores of the scales at the end of follow up, the functional motor status of the patients was still significantly better than it was before starting treatment. The introduction of pyridostigmine at an early age of the disease in one of the patients, before the onset of scoliosis, may have had a protective effect on it.

Loss of the sphingolipid desaturase DEGS1 causes hypomyelinating leukodystrophy.

Sphingolipid imbalance is the culprit in a variety of neurological diseases, some affecting the myelin sheath. We have used whole-exome sequencing in patients with undetermined leukoencephalopathies to uncover the endoplasmic reticulum lipid desaturase DEGS1 as the causative gene in 19 patients from 13 unrelated families. Shared features among the cases include severe motor arrest, early nystagmus, dystonia, spasticity, and profound failure to thrive. MRI showed hypomyelination, thinning of the corpus callosum, and progressive thalamic and cerebellar atrophy, suggesting a critical role of DEGS1 in myelin development and maintenance. This enzyme converts dihydroceramide (DhCer) into ceramide (Cer) in the final step of the de novo biosynthesis pathway. We detected a marked increase of the substrate DhCer and DhCer/Cer ratios in patients' fibroblasts and muscle. Further, we used a knockdown approach for disease modeling in Danio rerio, followed by a preclinical test with the first-line treatment for multiple sclerosis, fingolimod (FTY720, Gilenya). The enzymatic inhibition of Cer synthase by fingolimod, 1 step prior to DEGS1 in the pathway, reduced the critical DhCer/Cer imbalance and the severe locomotor disability, increasing the number of myelinating oligodendrocytes in a zebrafish model. These proof-of-concept results pave the way to clinical translation.

Mitochondrial DNA depletion syndrome: new descriptions and the use of citrate synthase as a helpful tool to better characterise the patients.

Mitochondrial DNA depletion syndrome (MDS) is a clinically heterogeneous group of mitochondrial disorders characterised by a quantitative reduction of the mitochondrial DNA copy number. Three main clinical forms of MDS: myopathic, encephalomyopathic and hepatocerebral have been defined, although patients may present with other MDS associated clinical symptoms and signs that cover a wide spectrum of onset age and disease. We studied 52 paediatric individuals suspected to have MDS. These patients have been divided into three different groups, and the appropriate MDS genes have been screened according to their clinical and biochemical phenotypes. Mutational study of DGUOK, MPV17, SUCLA2, SUCLG1 and POLG allowed us to identify 3 novel mutations (c.1048G>A and c.1049G>T in SUCLA2 and c.531+4A>T in SUCLG1) and 7 already known mutations in 10 patients (8 families). Seventeen patients presented with mtDNA depletion in liver or muscle, but the cause of mtDNA depletion still remains unknown in 8 of them. When possible, we quantified mtDNA/nDNA and CS activity in the same tissue sample, providing an additional tool for the study of MDS. The ratio (mtDNA/nDNA)/CS has shed some light in the discrepant results between the mtDNA copy number and the enzymatic respiratory chain activities of some cases.