# A Homozygous NDUFS6 Variant Associated with Neuropathy and Optic Atrophy

Andrea Gangfuß, Philipp Rating, Tomas Ferreira, Andreas Hentschel, Adela Della Marina, Heike Kölbel, Albert Sickmann, Angela Abicht, Florian Kraft, Tobias Ruck, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Andrea Gangfuß, Philipp Rating, Tomas Ferreira, Andreas Hentschel, Adela Della Marina, et al. A Homozygous NDUFS6 Variant Associated with Neuropathy and Optic Atrophy. Journal of Neuro-muscular Diseases, 2024, 11 (2), pp.485 - 491. 10.3233/jnd-230181. hal-04979591

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Research Report

# A Homozygous *NDUFS6* Variant Associated with Neuropathy and Optic Atrophy

Andrea Gangfuß<sup>a,*</sup>, Philipp Rating<sup>b</sup>, Tomas Ferreira<sup>c</sup>, Andreas Hentschel<sup>d</sup>, Adela Della Marina<sup>a</sup>, Heike Kölbel<sup>a</sup>, Albert Sickmann<sup>d</sup>, Angela Abicht<sup>e,f</sup>, Florian Kraft<sup>g</sup>, Tobias Ruck<sup>h</sup>, Johann Böhm<sup>i</sup>, Anne Schänzer<sup>j</sup>, Ulrike Schara-Schmidt<sup>a</sup>, Teresa M. Neuhann<sup>f,1</sup>, Rita Horvath<sup>c,1</sup>

and Andreas Roos<sup>a,k,1</sup>

Accepted 5 December 2023

Pre-press 8 January 2024

Published 5 March 2024

**Abstract.**

**Background:** The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (*NDUFS6*) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic *NDUFS6* variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS).

**Objective:** Here, we identified a homozygous variant (c.309 + 5 G > A) in *NDUFS6* in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability.

**Methods:** To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed.

**Results:** The analyses revealed loss of *NDUFS6* protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum.

<footnote><sup>a</sup>Department of Pediatric Neurology, Centre for Neuromuscular Disorders, Centre for Translational Neuro- and Behavioral Sciences, University Duisburg-Essen, Essen, Germany</footnote>

<footnote><sup>b</sup>Department of Ophthalmology, University Duisburg-Essen, Essen, Germany</footnote>

<footnote><sup>c</sup>Department of Clinical Neurosciences, John Van Geest Centre for Brain Repair, School of Clinical Medicine, University of Cambridge, Cambridge, UK</footnote>

<footnote><sup>d</sup>Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V. Dortmund, Germany</footnote>

<footnote><sup>e</sup>Department of Neurology, Friedrich-Baur Institute, Munich, Germany</footnote>

<footnote><sup>f</sup>MGZ - Medizinisch Genetisches Zentrum, Munich, Germany</footnote>

<footnote><sup>g</sup>Institute of Human Genetics und Genomic Medicine, RWTH-Aachen University, Aachen, Germany</footnote>

<footnote><sup>h</sup>Department of Neurology, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany</footnote>

<footnote><sup>i</sup>IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Inserm U1258, CNRS UMR7104, Université de Strasbourg, Illkirch, France</footnote>

<footnote><sup>j</sup>Institute of Neuropathology, Justus Liebig University, Giessen, Germany</footnote>

<footnote><sup>k</sup>Children's Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada</footnote>

<footnote n="1"><sup>1</sup>Shared last authors.</footnote>

<footnote>*Correspondence to: Dr. med. Andrea Gangfuß, Department of Pediatric Neurology, Centre for Neuromuscular Disorders, Centre for Translational Neuro- and Behavioral Sciences, Univer-</footnote>

<footnote>sity Duisburg-Essen, Hufelandstraße 55. 45147 Essen, Germany.
Tel.: +0049 201 723 2408; Fax: 0049 201 723 5389; E-mail: andrea.gangfuss@uk-essen.de.</footnote>
**Conclusions:** Hence, our combined clinical and biochemical data strengthen the concept of *NDUFS6* being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of *NDUFS6*-associated phenotypes.

Keywords: Charcot-Marie-Tooth disease, axonal neuropathy, *NDUFS6*, white blood cell proteomics

## INTRODUCTION

Since 2004, 12 patients have been described with pathogenic variants in NADH ubiquinone oxidoreductase subunit S6 encoded by the *NDUFS6* gene [1-7]. Dysfunction of this complex I subunit of the respiratory chain is one cause of mitochondrial complex I deficiency (MCID) [8], leading to a severe disease mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS) [1-5] with onset before 6 months of life and a progressive course [6]. The eight patients described by Kirby, Spiegel, Li and colleagues died aged less than 28 days due to central hypoventilation or ineffective treatment of severe lactic acidosis, respectively [1, 5, 7] (Supp. Tab. 1). Mitochondrial diseases often present with fatal infantile phenotypes but over the last years some authors reported isolated axonal neuropathy phenotypes, e.g. for *SCO2* [9] and *MTATP6* [10].

## MATERIALS AND METHODS

DNA was extracted from peripheral blood (3.5 ml EDTA). Whole exome enrichment was performed with the Twist Human Comprehensive Exome Kit (Twist Biosciences). Massively parallel sequencing was carried out on a Novaseq 6000 system (Illumina, San Diego, CA) as 150 bp paired-end runs using v1.5 SBS chemistry. Exome enrichment-based SNV, INDEL and CNV calling was conducted using varvis® 1.19.3 (Limbus Medical Technologies GmbH, Rostock). Only SNVs and small INDELs in the coding and flanking intronic regions 106 (±50 bp) were evaluated. The analysis of the parental samples was performed equally by whole exome enrichment and the Twist Human Comprehensive Exome Kit (Twist Biosciences) (see above). The evaluation was focused and targeted for the *NDUFS6* variant. Pre-screened genes via panel analyses for Leber Hereditary Optic Neuropathy (LHON): *MT-ND1* (m.3460 G < A), *MT-ND4* (m.11778 G > A), *MT-ND6* (m.14484T > C) and sequencing of *DNAJC30* (NM_032317) (hotspots). Gene panel for optic atrophy contained *ACO2*,

*AFG3L2*, *C12ORF65*, *CISD2*, *DNM1L*, *DNAJC30*, *ELOVL1*, *FDXR*, *MCAT*, *MFN2*, *MIEF1*, *MTPAP*, *NBAS*, *NDUFS2*, *NR2F1*, *OPA1*, *OPA3*, *PRPS1*, *RTN4IP1*, *SLC25A46*, *SPG7*, *SSBP1*, *TIMM8A*, *TMEM126A*, *WFS1*, *YME1L1*.

A three-millimeter skin punch biopsy was performed 10 cm above the lateral malleolus and fixated in Zamboni fixative. Quantification of intraepidermal nerve fiber density (IENFD) were performed at 50 µm sections with immunofluorescence staining with antibody against PGP9.5 to detect small intraepidermal nerve fiber as described previously [9, 11, 12].

Protein was extracted from white blood cells and analyzed by Western blotting. Samples were first lysed in RIPA buffer (Sigma-Aldrich) with cOmplet, Mini, EDTA-free Protease Inhibitor Cocktail (Roche). Twenty micrograms of protein was run per lane on NuPAGE 4-12% Bis-Tris Protein Gels (Invitrogen), transferred to a PVDF membrane and blocked with 5% milk (tris-buffered saline, skimmed milk powder, 0.1% Tween-20) for 1 h at room temperature. Primary antibodies were incubated in 5% milk overnight at 4°C, washed, and incubated with appropriate HRP secondary antibodies for 1 h at room temperature. Blots were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific). As primary antibody, we used anti-*NDUFS6*. Secondary antibody was goat anti-rat IgG (H+L) (ThermoFisher, 31470).

For mtDNA copy number quantification, DNA was obtained from white blood cells using the DNeasy Blood and Tissue Kit by Qiagen, and quantified using a nanodrop 2000 spectrophotometer (ThermoFisher). The samples were then diluted with nuclease-free water to achieve a final concentration of 2 ng/µL. The mtDNA copy number was quantified through a duplex TaqMan qPCR assay, amplifying both MT-ND1 (mitochondrial target) and B2M (nuclear target) using a CFX96™ Real-Time PC Detection System (Bio-Rad). The sequences for the probes and primers used can be found in supplementary table 3. The relative mtDNA copy number was calculated by subtracting the B2M cycle threshold (Ct) from the MT-ND1
Ct (ΔCt = CtB2M - Ct ND1) per sample. Sample mtDNA copy number calculated relative to a control ΔCt.

In addition, we evaluated the established mito-
chondrial biomarker Growth Differentiation Factor
15 (GDF15) using enzyme-linked immunosorbent
assay (ELISA). For the quantification of human
GDF15 (R&D, DGD150), we followed the manufac-
turer's instructions. Although GDF15 has not been
previously associated with nuclear-encoded complex
I respiratory chain complex defects, it has been impli-
cated as a biomarker in peripheral neuropathies [13].

Proteomic profiling on whole protein extracts derived from our index patient as well as his healthy family members (serving as controls) was carried out in a data-independent-acquisition mode and subsequent data analyses were carried out as described previously [14].

RESULTS

Here, we describe for the first time homozygosity
for a known pathogenic *NUDFS6* variant associ-
ated with a novel phenotype consisting of axonal
neuropathy, optic atrophy and borderline intellectual
disability. Of note, previously reported mutations in
*NDUFS6* were linked to severe lethal infantile phe-
notypes. Over the last years, some authors reported
purely axonal neuropathy associated with less severe
mutations in other “mitochondrial genes”, e.g. for
SCO2 [9] or *MTATP6* [10].

In the patient described here, the parents noticed an
abnormal gait with frequent falls at the age of 7 years.
Clinical examination at the age of 10 years showed
pronounced distal muscle weakness of the lower
limbs, high arched feet (left > right), and reduced
ability to lift both forefeet (Fig. 1A) resulting in
an abnormal gait with the inability to walk on the
heels. In contrast, calve muscles showed no atrophy
(Fig. 1A). Ankle jerk was absent, other deep ten-
don reflexes were normal. Vibration sensation was
reduced (feet 4/8, hands 5/8) but coordination was
normal. A standardised intelligence test (WISC-V)
revealed borderline intellectual disability (IQ 70).
Creatine kinase was normal, Blood lactate was mildly
elevated (2,5 mmol/l [normal range 0,5–1,6]). Neu-
rophysiologic studies showed results consistent with
pronounced axonal sensory and motor neuropathy
(reduced latency and amplitudes, mostly normal
conduction velocities). Ophthalmologic examination
(Fig. 1B) exhibited bilateral partial optic atrophy

leading to an reduced visual acuity (left = 0.05 and
right = 0.4) and altered visual evoked potentials (VEP,
P100 amplitudes left smaller right, latency left 102 ms
and right 105 ms). Cerebral Magnetic Resonance
Imaging (MRI) was normal.

Supplementary table 1 presents a detailed clinical
comparison of the patient described in this study and
patients with *NDUFS6* variants previously described
in the literature which highlights the difference in life
expectancy in our patient [1–7].

Leber Hereditary Optic Neuropathy (LHON) was excluded by genetic analysis of the common mtDNA variants and a gene panel covering nuclear optic atrophy-associated genes was also normal (for analysed genes see above). *PMP22* deletion or duplication was excluded by MLPA. Whole exome sequencing identified a homozygous splice site mutation c.309+5G>A in *NDUFS6* (NM_004553.6). The variant was assessed as “likely pathogenic” (ACMG class 4) and therefore compatible with the diagnosis “NDUFS6-associated mitochondrial diesease” in our patient. Next, segregation analysis of the healthy brother and both parents (cousins I°) was performed and revealed a heterozygous carrier status in all of them (Fig. 1C). Of note, Rouzier and colleagues described a patient showing compound heterozygosity for this variant in *NDUFS6* (c.309+5G>A and c.343T>C) and functionally demonstrated the pathogenicity of the intronic c.309+5G>A variant. Indeed, this variant strongly affects splicing, leads to exon 3 skipping, and results in a major decrease of the *NDUFS6* protein level [2]. Therefore, we suggest that the c.309+5G>A variant is causative for the patient’s symptoms.

To further prove the pathogenicity of the homozy-
gous *NDUFS6* variant identified in our index
patient, we studied small nerve fibers and performed
functional studies including determination of mito-
chondrial copy number in addition to proteomic
studies on white blood cells and ELISA based quan-
tification of GDF15 has been undertaken as described
previously [13].

Quantitiave analysis of IENFD confirmed an
involvement of small intraepidermal nerve fibers. The
IENFD of 3.8 fibers/mm was significantly decreased
in our patient compared to the published normative
values for adults (6.1–10.9 fibers/mm) [11]. More-
over, IENFD is expected to be even higher in younger
children [12]. Reduced density of small fibers thus
indicates the presence of axonal sensorimotor neu-
ropathy (Fig. 1D).
<figure><img src="image_3.png" /><figcaption>Fig. 1. (A) Photographs of the index patient at the age of 9 years illustrate high arched feet (left > right) and reduced ability to lift both forefeet. (B) Ocular fundus showing bilateral partial optic atrophy (left > right). (C) Pedigree chart of the family described in this study. (D) Significant decrease of intraepidermal nerve fiber stain with PGP9.5 (arrows).</figcaption></figure>

Quantitative PCR based determination of mito-
chondrial copy number revealed no differences
compared to the heterozygous healthy family mem-
bers (Fig 2A). Immunoblot studies on whole protein
extracts of white blood cells of the index patient
showed absence of the NDUFS6 protein and it was
also reduced in the heterozygous father. A faint

smaller band may represent a mis-spliced NDUFS6
however we cannot fully confirm it (Fig. 2B).

Proteomic profiling holds the potential to identify
pathophysiological processes in an unbiased man-
ner by monitoring thousands of proteins in one
experiment [14]. Recently, we successfully applied
proteomic profiling on white blood cells to address
<figure><img src="image_4.png" /><figcaption>Fig. 2. (Continued)</figcaption></figure>
the pathogenicity of a new *SCO2* variant associ-
ated with neuropathy and obtain new insights into
the underlying pathophysiology [9]. After confirm-
ing the loss of expression of *NDUFS6* in white
blood cells via immunoblotting (see above), we per-
formed proteomic profiling on whole protein extracts
derived from white blood cells of the index case
in addition to the healthy brother and father (serv-
ing as controls) as described previously. Based on
the applied data-independent approach, we quanti-
fied 3538 proteins (Fig 2C). Setting the boundaries
for relevant protein increases and decreases to > 2.0
and < 0.5 fold of regular abundance, respectively, a
total of 36 proteins (1.02% of the quantified proteins)
showed a significant dysregulation: 10 proteins were
decreased, whereas 26 were increased (Fig. 2D &
Supp. Tab. 2). A Gene Ontology (GO)-term based in
silico analysis (Fig. 2E) aimed to pinpoint pathophys-
iological processes upon presence of the homozygous
*NDUFS6* variant. Notably, decreased proteins
include three mitochondrial NADH dehydrogenase
subunit/assembly proteins (NDUFA12, NDUFS4
and NDUFV1) indicative of altered mitochondrial
respiratory chain complex I assembly (Fig. 2E),
thus suggesting a pathophysiological effect of the
homozygous *NDUFS6* variant. The neuropathy-
related mitochondrial biomarker GDF15 did not
show a significant change in our patient (Fig. 2F).

DISCUSSION

Here, we describe a patient carrying a homozy-
gous splice variant in *NDUFS6* leading to a milder
phenotype than previously reported patients with
*NDUFS6* variants: Our patient presented with a pro-
nounced peripheral axonal neuropathy accompanied
by involvement of small sensory nerve fibers, optic
atrophy and borderline intellectual disability, how-

ever brain MRI was normal. Immunoblot studies
revealed loss of the protein and proteomic findings
further supported the pathogenicity of the identi-
fied splice-site variant by highlighting a concomitant
decrease of three further mitochondrial NADH dehy-
drogenase subunit/assembly proteins (NDUFA12,
NDUFS4 and NDUFV1), which are reflecting the
dysfunction of the NDUFS6 protein.

Our data suggest that some cases present with a milder phenotype than these otherwise severe childhood onset mitochondrial diseases, possibly due to residual protein levels, however may be influenced by other genetic, epigenetic or environmental factors. Along this line, peripheral neuropathy as the leading symptom has already been reported in patients with mutations in some other primary mitochondrial disease genes (e.g. *SCO2*, *SURF1*) which are commonly associated with much more severe clinical presentations or even early death. An unbiased exome and genome sequencing approach is superior to targeted panel testing, as it enables the genetic diagnosis of these clinically unusual patients. However until this approach will become the standard diagnostic test we recommend that *NDUFS6* together with other mitochondrial genes (including *SCO2* and *SURF1*) should be included in panels for molecular genetic testing of neuropathies, intellectual disability and optic atrophy.

ACKNOWLEDGMENTS

The authors are grateful to the family for cooper-
ation and for the permission to publish the data and
photographs. We thank Mrs. Swantje Hertel and Mrs.
Nancy Meyer for their expert technical assistance.
Some of the authors of this publication are members
of the European Reference Network for Neuromus-
cular Diseases-Project ID N° 870177.

Fig. 2. Molecular studies on white blood cells derived from our index patient and family members. (A) Quantitative PCR based determination of mitochondrial copy numbers did not reveal significant differences between the index and its family members. (B) Immunoblot studies of NUDFS6 revealed an absence of NDUFS6 (13 kDa) in protein extracts of white blood cells derived from the index, and decreased level of NDUFS6 in the heterozygous father compared to the level detected in control human fibroblasts. A smaller band on the Western blot may potentially represent an aberrantly spliced transcript. The 18 kDa band is unspecific. Coomassie staining was performed to demonstrate equal protein loading. (C) Abundance plot showing the dynamic range of all proteins identified in proteins extracts of white blood cells via liquid chromatography coupled to tandem mass spectrometry based on their relative quantification using always the 3 highest abundant peptides for each protein, allowing protein comparison within an experiment. All identified proteins of the controls (black) are sorted with decreasing abundance while the patient (red) was plotted in the same order to directly compare the different abundances. All identified proteins cover a dynamic range of eight orders of magnitude. (D) Volcano plot highlighting statistically significant increased proteins (purple dots) as well as decreased proteins (yellow dots). (E) Gene Ontology (GO) enrichment analyses for increased (left panel) and decreased (right panel) proteins separately. Decreased proteins clearly indicate a mitochondrial vulnerability upon reduced NDUFS6 level, as exemplified by our immunoblot studies. MF: molecular function of affected proteins, CC: cellular components affected by dysregulated proteins and BP: biological processes affected by dysregulated proteins.
## FUNDING

A.R. received funding from the Deutsche Gesellschaft für Muskelkranke (DGM) as well as from the French Muscular Dystrophy Association (AFM-Téléthon; grant 21644). A.G. received funding from the University of Duisburg-Essen. Parts of the study were funded through the European Regional Development Fund (ERDF; project: NME-GPS). R.H. is a Wellcome Trust Investigator (109915/Z/15/Z), who receives support from the Medical Research Council (UK) (MR/V009346/1), the Addenbrooke's Charitable Trust (G100142), the Evelyn Trust, the Stoneygate Trust, the Lily Foundation, Action for AT and an MRC strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) MR/S005021/1. This research was supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. We gratefully acknowledge the financial support by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen and the Bundesministerium für Bildung und Forschung.

## CONFLICTS OF INTEREST

The authors declare that there is no conflict of interest regarding the publication of this article.

## DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request. Proteomic data have been uploaded to ProteomeXchange (identifier: PXD026283).

## SUPPLEMENTARY MATERIAL

The supplementary material is available in the electronic version of this article: https://dx.doi.org/10.3233/JND-230181.

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