Autosomal recessive pathogenic MSTO1 variants in hereditary optic atrophy

Abstract

Gerber et al report 2 autosomal recessive pathogenic Misato homolog 1 (MSTO1) variants causing hereditary optic atrophy and raise concerns about a previously identified dominant variant of MSTO1 by Gal et al (2017).

Figure 1. Family, muscular findings, MSTO1 and MSTO2P sequences alignment and variants analysis

(A) Pedigree of the family and segregation analysis of MSTO1 c.65C > A and c.220 + 5G > C variations. The proband (III‐2) and his 2 siblings (III‐1 and III‐3) developed early‐onset myopathy and severe optic neuropathy. Segregation analysis of MSTO1 variants using the MSTO1 Fw/MSTO1 Rev primer pair (specific to MSTO1; see panel G) shows heterozygosity for the c.65C > A substitution in unaffected subject IV‐4, demonstrating biallelism of the c.65C > A and c.220 + 5G > C variations in the affected trio III‐1, ‐2, and ‐3. (B–D) Muscle MRI. Axial T1‐weighted imaging on pelvis (B), upper thigh (C), and lower thigh (D) showing major fat infiltration of the thigh muscles. AB, adductor brevis muscle; AL, adductor longus muscle; AM, adductor magnus muscle; BF, biceps femoris muscle; GM, gluteus maximus muscle; GR, gracilis muscle; IL, ilio‐psoas muscle; PE, pectineus muscle; RF, rectus femoris muscle; SA, sartorius muscle; SM, semimembranosus muscle; ST, semitendinosus muscle; TFL, tensor fasciae latae; VL, vastus lateralis muscle; VM, vastus medialis muscle; VI, vastus intermedius muscle. (E) 5′ Donor Splice Site Prediction (SSP) by varSEAK SSP tool. The varSEAK Online splice site prediction tool was used to analyze the c.220 + 5G > C variant. The table shows a strong decrease in the score for authentic Splice Site (Pos 220 + 1, highlighted in purple in the table as well as on the graph on the right). The score corresponds to the likelihood for the variant to be functional (positive values) or not functional (negative values). The score column corresponds to the score of the splice site on the reference sequence (upper value) and on the variant sequence (lower value). The ΔScore corresponds to the difference between the two scores. There was a strong decrease in the score and ΔScore for the 5′ authentic Splice Site, thus predicting the loss of function and exon skipping of the variant. The overall prediction is given as a splice site prediction class ranging from 1 (No splicing effect) to 5 (Splicing effect). The c.220 + 5G > C variant was classified as affecting splicing (class 5). No cryptic splice sites were predicted to be activated. The MaxEntScan scoring algorithm also found a decrease in the score and negative values for the ΔMaxEntScan, thus predicting a deleterious effect. The sequence graph (on the right) depicts the reference and variant sequence and the 5′ authentic Splice Site. The variant is highlighted in red, the HGVS nomenclature is given above. (F) Sanger sequencing of the identified variants in the MSTO1 gene and RT–PCR analysis. Upper panel: Representative sequence chromatograms of PCR products generated using the M13F/M13R primers of the pCR™II‐TOPO^R Vector carrying the MSTO1 c.65A (mutant)—c.220 + 5G (wildtype) allele (Clones type 1) or the MSTO1 c.65C (wildtype)—c.220 + 5C (mutant) allele (Clones type 2), amplified from whole blood genomic DNA (gDNA) using the MSTO1 Fw/MSTO1 Rev primers (specific to MSTO1; see panel G). Middle panel: Representative sequence chromatograms of pCR™II‐TOPO^R Vector clones carrying MSTO2P alleles amplified from whole blood genomic DNA (gDNA) using MSTO2P Fw/MSTO2P Rev primer pair (specific to MSTO2P; see panel G) and showing wildtype exon 1 and exon2‐intron 2 junction sequences. Lower panel: Representative sequence chromatograms of reverse‐transcribed mRNA isolated from patient‐cultured fibroblasts, showing apparent homozygosity of the MSTO1‐mutated allele (A) at position c.65 (on the left). On the right, the chromatogram shows the patient sequence at the exon 2/exon 3 junction of MSTO1 cDNA. E1, E2, E3, and I2 denotes exons 1, 2, and 3 and intron 2, respectively. The bars above the chromatograms show the boundaries of exon 2 (blue), intron 2 (yellow), and/or exon 3 (green) and the authentic consensus splice donor site (purple). (G) Pairwise alignment of MSTO1 and MSTOP2 and position of primer pairs reported in [1] and specific to MSTO1 and MSTOP2 [this study]. A noncoding RNA MSTO1 pseudogene, misato homolog 2 pseudogene (MSTO2P), is predicted 133 kb downstream of MSTO1 (hg19_chr1:155579961–155584758 and chr1:155715559–155718322, respectively). Pairwise alignment (https://www.ebi.ac.uk/Tools/psa/lalign/) of the proximal 5′ region, exon 1, intron 1, and part of exon 2 of MSTO1 and MSTO2P shows 99.8% nucleotide identity; the few variable positions are shown in red. MSTO1 Ex1‐2 Fw and MSTO1 Ex1‐2 Rev primers reported in [1] and MSTO1 Fw/MSTO1 Rev and MSTO2P Fw/MSTO2P Rev primer pairs specific to MSTO1 and MSTO2P, respectively, are shown. The +1 indicates the position of the adenine of the initiation codon (ATG is underlined). The codon involving the MSTO1 c.22 nucleotide is squared. The MSTO1 c.65C > A and c.220 + 5G > C are shown. Exonic and intronic sequences are written in lower and uppercase letters, respectively. (H) Sanger sequencing analysis of the MSTO2P n.83G > A substitution using primers reported in [1] and primers specific to MSTO1 and MSTO2P, respectively. Representative Sanger sequencing electrophoregrams in one of the HON cases carrying the MSTO2P n.83G > A substitution (patient NOH‐11). Sequences were obtained from PCR products obtained using MSTO1 Ex1‐2 Fw/MSTO1 Ex1‐2 Rev [1] primers (upper panel) and from PCR products amplified using the M13F/M13R primers of the pCR™II‐TOPO^R Vector carrying MSTO1 or MSTO2P alleles amplified using the MSTO1 Fw / MSTO1 Rev (lower‐left panel) and MSTO2P Fw/MSTO2P Rev (lower right panel) primers, respectively.