Pure and syndromic optic atrophy explained by deep intronic OPA1 mutations and an intralocus modifier

Abstract

The genetic diagnosis in inherited optic neuropathies often remains challenging, and the emergence of complex neurological phenotypes that involve optic neuropathy is puzzling. Here we unravel two novel principles of genetic mechanisms in optic neuropathies: deep intronic OPA1 mutations, which explain the disease in several so far unsolved cases; and an intralocus OPA1 modifier, which explains the emergence of syndromic 'optic atrophy plus' phenotypes in several families. First, we unravelled a deep intronic mutation 364 base pairs 3' of exon 4b in OPA1 by in-depth investigation of a family with severe optic atrophy plus syndrome in which conventional OPA1 diagnostics including gene dosage analyses were normal. The mutation creates a new splice acceptor site resulting in aberrant OPA1 transcripts with retained intronic sequence and subsequent translational frameshift as shown by complementary DNA analysis. In patient fibroblasts we demonstrate nonsense mediated messenger RNA decay, reduced levels of OPA1 protein, and impairment of mitochondrial dynamics. Subsequent site-specific screening of >360 subjects with unexplained inherited optic neuropathy revealed three additional families carrying this deep intronic mutation and a base exchange four nucleotides upstream, respectively, thus confirming the clinical significance of this mutational mechanism. Second, in all severely affected patients of the index family, the deep intronic mutation occurred in compound heterozygous state with an exonic OPA1 missense variant (p.I382M; NM_015560.2). The variant alone did not cause a phenotype, even in homozygous state indicating that this long debated OPA1 variant is not pathogenic per se, but acts as a phenotypic modifier if it encounters in trans with an OPA1 mutation. Subsequent screening of whole exomes from >600 index patients identified a second family with severe optic atrophy plus syndrome due to compound heterozygous p.I382M, thus confirming this mechanism. In summary, we provide genetic and functional evidence that deep intronic mutations in OPA1 can cause optic atrophy and explain disease in a substantial share of families with unsolved inherited optic neuropathies. Moreover, we show that an OPA1 modifier variant explains the emergence of optic atrophy plus phenotypes if combined in trans with another OPA1 mutation. Both mutational mechanisms identified in this study-deep intronic mutations and intragenic modifiers-might represent more generalizable mechanisms that could be found also in a wide range of other neurodegenerative and optic neuropathy diseases.

Keywords: ataxia; cryptic exon; deep intronic mutation; genetic modifier; mitochondrial network.

Figure 1

Funduscopy and optical coherence tomography (OCT) in affected and non-affected members of Family OAK 587. (A) Affected family members (Subjects II:1, II:2 and II:3): paleness of the optic nerve heads (white arrow) indicating atrophy of the optic nerve (left column), the optical coherence tomography scan demonstrates a thinning of the peripapillary retinal nerve fibre layer (right column). Both findings are most severe in the subject with the longest disease duration (Subject II:3). LE = left eye; RE = right eye. (B) Family members without clinical manifestation (Subjects II:4, I:2 and I:1): optical nerve heads showing the normal orange-pink appearance, thickness of the peripapillary retinal nerve fibre layer within normal limits.

Figure 2

MRI of the brain and cervical spinal cord of affected members of Family OAK 587. MRI of the cerebrum and the upper cervical spinal cord (A–C, sagittal T₁; D–F axial FLAIR; G–I coronal T₂) demonstrates cerebellar atrophy in all three affected subjects (long arrows), most severely in the subject with the longest disease duration and highest ataxia score (Subject II:3; A, D and G). All three subjects also show, to a variable degree, atrophy of the upper cervical spinal cord (short arrows in A–C).

Figure 3

Pedigrees of the Families OAK 587 and DUK2976 including the segregation of OPA1 mutations and coding SNPs. Upper pedigree: All affected siblings of Family OAK 587 inherited the same maternal OPA1 allele (mat.1), comprising the c.610+364G>A deep intronic mutation (red square), whereas all unaffected children inherited the non-mutant maternal OPA1 allele (mat.2). The father, Subject I:1, is homozygous for p.I437M (yellow square), therefore all children are heterozygous for this variant. Note that Subject I:1 is homozygous for all analysed variants in OPA1, most likely due to identity-by-descent. Coding SNPs in exon 4 and exon 21 were used to perform pyrosequencing for discrimination of allelic representation on complementary DNA level. Affected family members are indicated by black symbols and healthy family members by white symbols. Lower pedigree: In Family DUK2976 Subject III:2 is compound heterozygous for p.I437M and c.1316_1317insA/p.N440Kfs*14, and presents with a severe optic atrophy plus phenotype. In contrast, her parents—likely heterozygous for only one of the two OPA1 variants—present only with mild visual disturbances and no symptoms at all, respectively. However, the sister II:2 of the unaffected mother II:4 as well as her daughter III:1 present with non-syndromic optic atrophy (grey).

Figure 4

Unmasking of aberrant OPA1 transcripts in puromycin treated patient fibroblasts. Deep intronic mutations in OPA1 reverse transcriptase PCR amplification with primers located in exons 2 and 6 result in multiple cDNA products due to the alternation of constitutively (dark grey in scheme) and facultatively spliced exons (light grey) in this part of the OPA1 gene. The presence of a heterozygous coding SNP in exon 4 (rs7624750; depicted as A/G in the scheme) enabled to differentiate between maternal and paternal-allele derived transcripts in the analysed patient (see text). The pattern of reverse transcriptase PCR products obtained with RNA from untreated fibroblasts of Subject II:1 and the control is virtually identical. However treatment of the patient’s fibroblasts with puromycin before RNA isolation resulted in a drastically altered pattern with additional bands (arrow) and reduced amounts of regular fragments.

Figure 5

Novel splicing acceptor sites in intron 4b of OPA1 and resulting cryptic exon inclusion. Base transitions of G>A at positions 360 bp or 364 bp downstream of exon 4b create novel splice acceptor sites that exonize downstream sequences of 65 bp / 97 bp or 61 bp/93 bp. Top: Family OAK 587 (index) and Family OAK 302 share the c.610+364G>A mutation, whereas Families OAK 38 and OAK 344 share the mutation c.610+360G>A (centre). The cryptic exon is exclusively found in transcripts of the maternal allele (Family OAK 587) which can be determined e.g. by an SNP in exon 4 (rs7624750, bottom panel). Black bars = exons; dashed lines = introns; red bars = cryptic exons.

Figure 6

Reduced OPA1 protein amount in patients with deep intronic mutation. Semi-quantitative western blot immunodetection with antibodies against OPA1 (top), ACTB (centre) and TOMM40 (bottom) in whole fibroblast cell lysates from a patient with autosomal dominant optic atrophy with heterozygous c.868C>T/p.Arg290stop in OPA1 (nonsense-mediated messenger RNA decay control), the affected index patient of Family OAK 302, two affected patients (Subjects II:1 and II:2) as well as the parents of Family OAK 587, and a healthy control (wild-type) (from left to right). A clear reduction in OPA1 protein amount can be observed in all samples except the control and the unaffected father (only slight reduction).

Figure 7

Mitochondrial morphometry. (A) Mitochondrial network stained with MitoTracker Green FM(R) and visualized with a fluorescence microscope. Compared to an exemplary control (i), where mitochondria build a network of lengthy and closely connected mitochondrial alignments, the mitochondrial network is fragmented in all three affected siblings (ii–iv). Similarly, also mitochondria of the mother (vi, Subject I:2) show signs of fragmentation and, to a lesser extent, mitochondria of the father as well (v, Subject I:1). (B) Statistical analysis of mitochondrial morphometry in fibroblasts of the three affected siblings (dark red), the father (dark blue) and the mother (dark grey) of Family OAK 587 compared to age-matched controls. Four aspects of morphology were analysed: (i) surface; (ii) aspect ratio (major axis/minor axis); (iii) form factor (surface²/4 π volume); and (iv) volume. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not significant.