Missense variants in the middle domain ofDNM1Lin cases of infantile encephalopathy alter peroxisomes and mitochondria when assayed inDrosophila

Abstract

Defects in organelle dynamics underlie a number of human degenerative disorders, and whole exome sequencing (WES) is a powerful tool for studying genetic changes that affect the cellular machinery. WES may uncover variants of unknown significance (VUS) that require functional validation. Previously, a pathogenic de novo variant in the middle domain of DNM1L (p.A395D) was identified in a single patient with a lethal defect of mitochondrial and peroxisomal fission. We identified two additional patients with infantile encephalopathy and partially overlapping clinical features, each with a novel VUS in the middle domain of DNM1L (p.G350R and p.E379K). To evaluate pathogenicity, we generated transgenic Drosophila expressing wild-type or variant DNM1L. We find that human wild-type DNM1L rescues the lethality as well as specific phenotypes associated with the loss of Drp1 in Drosophila. Neither the p.A395D variant nor the novel variant p.G350R rescue lethality or other phenotypes. Moreover, overexpression of p.A395D and p.G350R in Drosophila neurons, salivary gland and muscle strikingly altered peroxisomal and mitochondrial morphology. In contrast, the other novel variant (p.E379K) rescued lethality and did not affect organelle morphology, although it was associated with a subtle mitochondrial trafficking defect in an in vivo assay. Interestingly, the patient with the p.E379K variant also has a de novo VUS in pyruvate dehydrogenase 1 (PDHA1) affecting the same amino acid (G150) as another case of PDHA1 deficiency suggesting the PDHA1 variant may be pathogenic. In summary, detailed clinical evaluation and WES with functional studies in Drosophila can distinguish different functional consequences of newly-described DNM1L alleles.

Clinical-genetic features and peculiar muscle histopathology in infantileDNM1L-related mitochondrial epileptic encephalopathy

“…After the first description of Waterham et al (), there have been increasing reports in the last 2 years on patients, frequently sporadic, with de novo dominant‐negative DNM1L mutations who are affected by early onset encephalopathy with microcephaly and drug‐resistant seizures, progressive brain atrophy or abnormal brain development, optic atrophy, and occasionally persistent lactic acidemia. The degree of severity was broad, ranging from neonatal death to prolonged survival (Chao et al, ; Fahrner et al, ; Sheffer et al, ; Vanstone et al, ; Zaha et al, ). A similar spectrum of clinical presentations has been also reported for DNM1L ‐recessive mutations (Nasca et al, ; Yoon et al, ).…”

“…4 and 5), plasma phytanic and pristanic acids concentrations, typically increased in patients with peroxisomal disorders (ten Brink, et al, ), were normal. Excluding the 16 patients with mild nonsyndromic optic atrophy, 6 patients of the 12 reported in literature showing similar clinical manifestations died in early‐childhood (Chao et al, ; Waterham et al, ; Yoon et al, ; Zaha et al, ), and in four cases death occurred in the first year of age (Chao et al, ; Waterham et al, ; Yoon et al, ). It is not clear why some mutations give rise to premature death.…”

“…Histochemical and ultrastructure characterization of skeletal muscle biopsies of subjects harboring OPA1 mutations showed the presence of cytochrome c oxidase (COX)‐deficient fibers associated with multiple mitochondrial DNA deletions in the majority of patients, either affected by dominant optic atrophy plus variants or with isolated optic nerve involvement (Yu‐Wai‐Man et al, ). On the other hand, few papers have reported studies on muscle biopsy of patients with mutations in additional genes regulating mitochondrial morphology, and no abnormalities have been found (Fahrner et al, ; Koch et al, ; Nasca et al, ; Waterham et al, ; Yoon et al, ), although Chao et al () reported mitochondrial shape abnormalities at electron microscopy (EM).…”

A genetic screen for genes that impact peroxisomes in Drosophila identifies candidate genes for human disease

“…In this screen, we created homozygous mutant clones in the fat body of developing Drosophila larvae in an otherwise heterozygous animal and assayed for changes in the distribution pattern of a peroxisomal reporter, GFP-SKL ( Figure 1A). GFP-SKL is a GFP with a C-terminal peroxisomal localization signal, which we have previously shown to be an accurate marker for peroxisomal dynamics (CHAO et al 2016;WANGLER et al 2017a). We anticipated that our screen could uncover three major categories of peroxisomal impact: Category A-an increase in the number of peroxisomes, Category Ban increase in the size of peroxisomes, and Category C-decrease or loss of peroxisomes or the marker (Figure 1B).…”

“…We anticipated that our screen could uncover three major categories of peroxisomal impact: Category A-an increase in the number of peroxisomes, Category Ban increase in the size of peroxisomes, and Category C-decrease or loss of peroxisomes or the marker (Figure 1B). We have previously observed examples of Category B in mutants with peroxisomal fission defects (CHAO et al 2016) and Category C in biogenesis defects (WANGLER et al 2017a) in Drosophila. We screened 215 lethal mutant lines from the collection that was mapped to a complementation group or to a gene (Figure 1C, Supplemental Table 1).…”