Genetic susceptibility to optic neuropathy in patients with alcohol use disorder – Journal of Translational Medicine

Ethics statement

This retrospective study was conducted in accordance with the ethical recommendations of the Declaration of Helsinki. It received a favorable opinion from the Ethics Committee of the University Hospital of Angers, France, under reference 2020/149. Data from the routine care of patients followed for alcohol intoxication were collected and analyzed retrospectively. Genetic analyses, performed as part of the routine etiologic diagnosis of optic neuropathy, were performed after informed consent was obtained from the patients.

Study participants

We collected health data from 102 patients consecutively enrolled over six months (between May and November 2019), in a dedicated AUD unit at the Department of Psychiatry and Addiction of the University Hospital Center of Angers, France. All included patients (age 21.562.3 years) were diagnosed with AUD according to the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5). None of the included patients reported visual symptoms. However, their optic nerve structure was systematically assessed, using OCT to detect retinal neuronal and axonal loss, suggestive of an infraclinical optic neuropathy. Patients with known or previously associated retinopathy or optic neuropathy were excluded from the study.

Ophthalmic investigations used OCT images focused on the optic discs (Topcon DRI Triton Swept-Source OCT), aiming to measure the thickness of the ganglion cell complex (GCC) and peripapillary retinal fiber layer (RNFL). Since age leads to a decrease in the amount of GCC, an adjustment was made by the device using linear regression. Depending on the OCT findings, the included patients were divided into two groups: (1) a group of patients with retinal neuron loss, defined as thinning of the average GCC in at least one eye, compared to normative data; and (2) a group of patients without retinal neuron loss, if OCT results were within normal limits in both eyes. All patients in the optic neuropathy group underwent a complete clinical ophthalmic examination, including assessment of best-corrected visual acuity, intraocular pressure, and a formal visual field. Color vision was assessed using a 15 Hue desaturated color vision test. All patients with retinal neuron loss underwent extensive workup aimed at excluding other causes of optic neuropathies (eg, compressive, inflammatory, infectious, glaucomatous, etc.). The included patients underwent measurements of plasma levels of vitamins B9, B12, B1 and B6.

Molecular analyses

The search for genetic susceptibility variants was performed at the Department of Biochemistry and Molecular Biology of the University Hospital Center of Angers, France. Genetic analysis includes complete mitochondrial DNA sequencing and a panel of 87 nuclear genes, routinely used for the diagnosis of hereditary optic neuropathies. [17].

Genomic DNA of patients was extracted from peripheral blood using a DNA blood array on an EZ1 apparatus (Qiagen, Courtaboeuf, France). The presence of LHON mutations and other pathogenic variants in mitochondrial DNA was assessed using complete mtDNA sequencing, as described previously [18]. The panel of 87 nuclear genes included the following known and candidate genes involved in hereditary optic neuropathies: ACO2, ACOX1, AFG3L2, AGXT, ALG3, ANTXR1, AOX1, ASPA, ATAD3A, ATP1A3, ATXN1, ATXN7, AUH, BOLA3, BTD, C12orf65, C19orf12, CCD1,CDN1,CDB1,C4P1,C4B1,DNP1, C4B1 9, DNM1L, DNMT1, DPYD, FA2H, FDXR, FH, FIS1, HSD17B10, IBA57, KLC2, LOXL1, LTBP2, MECR, MFF, MFN1, MFN2, MIEF1, MIEF2, MMP19, MTPAP, MYOC, NDUFS2, NEFMAT31. , NDUFS2, NEFPMA1, NDUFS2, NEFPMA OPN1SW, OPTN, PCLO, PDHX, PDSS1, PLAA, PLP1, PMPCA, POLR3A, PRPS1, RAB3GAP1, RPIA, RTN4IP1, SIX6, SLC19A2, SLC25A46, SLC52A2, SPG TIMM8 , TMEM126A, TRAPPC12, TSFM, TUBB4A, UCHL1, VPS33A, WARS2, WDR36, WDR73, WFS1, YME1L1,AND ZNHIT3.

The capture panel was designed with SureDesign of Agilent Technologies, including exon and exon/intron boundaries. Libraries were constructed using SureSelect DNA target enrichment probes according to the manufacturers’ recommendations (Agilent Technologies France, les Ullis, France). gDNA samples were fragmented using the Agilent SureSelect Enzymatic Fragmentation Library. After molecular adapters and barcode ligation, the libraries were amplified and hybridized with target-specific probes of the gene panel. Probe/DNA complexes were captured with streptavidin-coated beads and amplified. Final library concentrations were determined using the Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Twelve libraries were pooled at equimolar concentration (150 pM) and sequenced on an Ion Proton apparatus (Ion Torrent Technology, Thermo Fisher Scientific, Waltham, MA, USA).

Variant calling, annotation and prioritization of genetic variants were performed using our in-house pipeline. Variant calling was based on three caller variants: deep variant, strelka and gatkHC. Annotation and prioritization steps were performed using the NCBI variant reporter and ANNOVAR. Classification of nuclear gene variants was determined using Varsome [19] and Franklin’s algorithms [20] based on American College of Medical Genetics and Genomics (ACMG) standards and guidelines [21]. Variants consistent with clinical presentation and mode of inheritance of classes 5 (Pathogenic), 4 (Possibly pathogenic) and 3 (VUS) strongly predicted as pathogenic by the algorithms were retained. The involvement of pathogenic mtDNA variants was determined using the Mitomap database [22].

Statistical analyses

Comparisons between two groups of patients (affected vs unaffected) were performed using a Wilcoxon rank sum test with continuity correction in R version 4.3.0 (R Foundation for Statistical Computing, Vienna, Austria; https://www .R-project .org/). Only the measurements of the right eyes were included in the OCT statistical analysis. Adjustment for age, sex, drug use, smoking status, and duration of alcohol intoxication was performed in multivariate analysis using Statistical Analysis System version 9.4 (SAS Institute Inc., Cary, NC, USA). or p-value (p) less than 0.05 was considered statistically significant.

Availability of data

Anonymized data from this study are available from the corresponding author upon reasonable request.