Infantile onset encephalomyopathy, retinopathy, optic atrophy, and mitochondrial DNA depletion associated with a novel pathogenic DHX16 variant

We studied a patient with mitochondrial DNA depletion in skeletal muscle and a multiorgan phenotype, including fatal encephalomyopathy, retinopathy, optic atrophy, and sensorineural hearing loss. Instead of pathogenic variants in the mitochondrial maintenance genes, we identified previously unpublished variant in DHX16 gene, a de novo heterozygous c.1360C>T (p. Arg454Trp). Variants in DHX16 encoding for DEAH‐box RNA helicase have previously been reported only in five patients with a phenotype called as neuromuscular oculoauditory syndrome including developmental delay, neuromuscular symptoms, and ocular or auditory defects with or without seizures. We performed functional studies on patient‐derived fibroblasts and skeletal muscle revealing, that the DHX16 expression was decreased. Clinical features together with functional data suggest, that our patient's disease is associated with a novel pathogenic DHX16 variant, and mtDNA depletion could be a secondary manifestation of the disease.


| INTRODUCTION
Mitochondrial DNA depletion syndromes (MDDS) are a heterogeneous group of diseases with a severe reduction of mtDNA content. 1 In our previous study, we identified a female patient with infantile onset encephalomyopathy, optic atrophy, pigmentary retinopathy, and sensorineural hearing impairment.The disease course was progressive, leading to death at the age of 4 years 8 months.The muscle mtDNA content was only 0.20 relative to age-matched controls, but wholeexome sequencing (WES) did not reveal pathogenic variants in any of the known mtDNA maintenance genes (the index patient no.2). 2 In the current work, we repeated WES analysis, and performed functional studies on candidate gene variants by using patient-derived fibroblasts and tissue samples.Furthermore, we compared phenotypes of our patient and previously published cases with similar clinical manifestations.

| MATERIALS AND METHODS
All the available clinical and laboratory data on the index patient were collected from the medical files of Oulu University Hospital, Oulu, Finland.As part of the diagnostic protocol, skin and muscle biopsies had previously been taken and these samples were available for functional studies.The detailed methods of genetic and functional studies are described in the Supplementary Material S1.
The fundi in both eyes were pale, and there was brown-gray pigmentation and depigmentation in a wide belt shape.
At the age of 3 years, the patient could move her hands and head but could not sit without support.Failure to thrive persisted, and progressive growth retardation was noted (length decreased to a level of-3.2SD).Furthermore, progressive spasticity was identified.Even-  1B and Supplemental Figure 1A) showing the DHX16 variant being a de novo finding, and present also in the mRNA level.The DHX16 variant p.Arg454Trp is in a highly conserved region (Figure 1C), and it is located in the known functional domain responsible for ATP binding (Figure 1D). 3,4The CHRNB2 variant segregated recessively parents being heterozygous carriers (Supplemental Figure 1A).

| Comparison of WES and clinical data
Comparison of the genotype and phenotype of our patient with previously published cases (Table 1) revealed, that de novo variants in DHX16 have recently been reported in association with a neuromuscular oculoauditory syndrome (NMOAS, OMIM #618733) including developmental delay, neuromuscular symptoms, ocular or auditory defects, and with or without seizures. 4Fjaer et al. described two siblings with epilepsy and brain calcifications carrying a heterozygous CHRNB2 p.Arg460Gly variant which they inherited from a healthy mother.However, they also had a heterozygous SLC20A2 variant reported to cause idiopathic familial brain calcifications (Table 1). 5The phenotype of the index patient differed from previously published patients with dominant CHRNB2 variants causing nocturnal frontal lobe epilepsy-3 (ENFL3, OMIM #605375), and no patients with recessive variants have been reported. 6

| Functional studies
The role of DHX16 and CHRNB2 variants was examined further at the protein level by using patient-derived fibroblasts and tissue samples.and MitoTracker CMXRos staining, and they did not show clear difference compared to control (Supplemental Figure 2D, E).

DHX16 antibody staining of the patient-derived fibroblasts
showed that the localization of the protein was preserved in the nucleus.Signal intensity appeared to be weaker in the patient-derived cell line, supporting the finding of the Western blot analysis (Figure 2D).TEM analysis of the skeletal muscle revealed a disordered myofibrillar structure in places with a vast amount of extracellular collagen fibers, glycogen accumulation, and changes in endoplasmic reticulum structures (Figure 2E).

| DISCUSSION
Here we describe a pediatric patient with a progressive, fatal encephalomyopathy associated with retinopathy, optic atrophy, sensorineural hearing loss, and mitochondrial DNA depletion.WES revealed a heterozygous DHX16 variant p.Arg454Trp and a homozygous variant in CHRNB2 p.Arg460Gly.Neither of these variants has previously been associated with mtDNA depletion.Heterozygous de novo variants in DHX16 have been connected to NMOAS but to our knowledge, no functional data from patient-derived samples has been published. 4,8munoblotting showed a statistically significant decrease in DHX16 The CHRNB2 variants have previously been associated ENFL3, but our patient did not present with any recognizable seizures. 6RNB2 is widely found in the brain and eyes but its expression in skin and muscle is very low according to Human Protein Atlas. 9Based on previously published clinical phenotypes associated with variants in DHX16 and CHRNB2 and our functional studies on patient-derived sample material we suggest that the primary cause of disease is dominant p. Arg454Trp variant in DHX16.However, without further studies we cannot completely exclude even minor contributing role of recessive CHRNB2 p. Arg460Gly variant to the phenotype of the patient.
The DHX16 gene encodes the DEAH-box RNA helicase, a group of RNA binding proteins with ATPase activity, that participate in the unwinding of RNA secondary structures and the remodeling of ribonucleoprotein. 10,11In the GTEx database, it is almost ubiquitously distributed throughout the body. 9It has been shown that DHX16 is a pre-mRNA splicing factor and pathogenic variants in DHX16 lead to unspliced transcripts that accumulate in the nucleus, some of those genes have been linked to neurodevelopment.1). 4,8Interestingly, a muscle biopsy of one previously described patient also revealed myopathy and increased glycogen and fatty vacuole corresponding to the findings in our patient (Table 1). 4 MtDNA analysis on skeletal muscle biopsy revealed mtDNA depletion and patients with MDDS have several clinical features similar to our patient, including neuromuscular symptoms, and ocular and auditory involvement. 12A few RNA helicases are also located in mitochondria affecting mitochondrial function and cells energy metabolism. 13,14However, there are also studies showing mtDNA depletion as a secondary finding of for example neurogenic muscle atrophy, suggesting an unspecific role for mtDNA depletion in different muscle-affecting pathologies. 15

| CONCLUSIONS
We report on a patient with infantile onset encephalomyopathy,

3
months, she was referred to a pediatric neurologist due to hypotonia, poor weight gain, and a lack of eye contact.Severe encephalomyopathy was diagnosed.Brain MRI, heart ultrasound, and electroencephalogram were normal.Creatine kinase (CK) showed progressive elevation rising to 1371 U/L (normal 35-210 U/L) by the age of 7 months.Blood pyruvate and lactate were unremarkable.The patient was diagnosed with moderate to severe sensorineural hearing impairment and brain auditory evoked potentials showed no response.The amplitudes of visual evoked potential were low, and no response was reached in electroretinogram.At the age F I G U R E 1 (A)) Pedigree showing that the patient was the only affected family member.(B) Electropherogram of Sanger sequencing presenting de novo heterozygous variant c.1360C>T, p.Arg454Trp in patient DNA and mRNA.(C) Sequence alignment of DHX16 homologs reveals that the gene is highly conserved between different species.(D) Protein schematic representation of DHX16 (Uniprot ID: O60231) showing known functional domains and previously published pathogenic DHX16 variants together with p.Arg454Trp (highlighted). 3,4,8Functional domains have been named as follows.DEXHc: domain containing the ATP-binding region.DEAH: DEAH-box motif.HelicC: The helicase C-terminal domain, catalyzes the separation of double-stranded nucleic acids.HA2: The domain is found in multiple RNA helicases; the precise function is unknown.OB_NTP_bind: central to the regulation of helicase activity through its binding of both RNA and G-patch domain proteins.[Colour figure can be viewed at wileyonlinelibrary.com]T A B L E 1 Overview of the clinical phenotypes of all reported patients with DHX16 variants and siblings with CHRNB2 c.1378C>G p. Arg460Gly variant.
Figure 1B, C).Cellular and mitochondrial ultrastructure of patient's fibroblasts were examined by transmission electron microscopy (TEM) protein expression, and the clinical phenotype resembled previously published patients with NMOAS syndrome.Based on our results, DHX16 p. Arg454Trp variant can be classified as pathogenic.F I G U R E 2 (A)) DHX16 Immunoblotting in patient-derived fibroblasts and control cell lines in technical triplicates shows a slight difference (expression 67% compared to the control, p = 0.057).(B) Immunoblotting of patient-derived muscle samples (two technical samples marked 1 and 2) and three pediatric control samples (marked C1, C2, C3) with DHX16 antibody show significantly lower expression (11% compared to control, p = 0.032).(C) Numerical presentation of the immunoblotting data by ImageJ analysis.*p < 0.05.(D) Patient-derived fibroblast and normal human dermal fibroblast control cell line stained with DHX16 antibodyshows that the localization of the protein is preserved at the nucleus, and intensity seems to be lowered compared to control.Actin cytoskeleton is stained with phalloidin (green) and nucleus with Hoechst (blue).Magnification 63Â.(E) 1. Transmission electron microscopy of the patient's muscle tissue shows a disordered myofibrillar structure in places (square) with a vast amount of extracellular collagen fibers (collagen " †", nerve " ⁋"). 2. Accumulation of glycogen is seen beneath the sarcolemma membrane and between myofibres (glycogen arrow).3. The shape and structure of mitochondria are intact (mitochondria "*").The endoplasmic reticulum forms pathological structures resembling enlarged cisterns (endoplasmic reticulum "#").4. Peripheral nerves are seen and have normal myelin sheaths (nerve " ⁋").[Colour figure can be viewed at wileyonlinelibrary.com] retinopathy, optic atrophy, and sensorineural hearing loss associated with a novel, heterozygous DHX16 (p.Arg454Trp) variant and mitochondrial DNA depletion in skeletal muscle.Clinical features resembling recently published patients with NMOAS, together with functional data, suggest that our patient's disease is associated with a novel pathogenic DHX16 variant, and mtDNA depletion in skeletal muscle could be a secondary manifestation.Further studies are needed to study the role of DHX16 in cellular function, including studies on mitochondrial function and maintenance.
she died peacefully at the age of 4 years 8 months due a pro- encoding for neuronal acetylcholine receptor subunit beta-2.Additional variants identified in the WES are shown in Supplementary Table1.Variants were verified by Sanger sequencing (Figure 4,10DEAH-box family members have been suggested to have a role also in cellular signaling, cell fate, and survival.
81Only recently, Paine et al. described four patients with DHX16 variants associated with the disease of NMOAS and de novo heterozygous variants that resided within established functional domains of the protein.4In 20 Archana et al. reported a fifth patient with a dominant DHX16 variant.8All the published patients presented with developmental delay, hypotonia, muscle weakness, retinopathy, and hearing loss (Table