2010; 16:178-183 <http://www.molvis.org/molvis/v16/a22>
Received 12 October 2009 | Accepted 5 February 2010 | Published 10 February 2010
1Department of Neurological, Neurosurgical and Behavioural Sciences, University of Siena, Siena, Italy; 2Italian Union of the Blind, Siracusa, Italy; 3Department of Pediatric, University of Catania, Catania, Italy
Correspondence to: Antonio Federico, Department of Neurological, Neurosurgical and Behavioural Sciences, University of Siena, Viale Bracci 2, 53100 Siena, Italy; Phone: 39-577-585763; FAX: 39-577-40327; email: firstname.lastname@example.org
Purpose: Autosomal dominant optic atrophy (ADOA) is the most common form of hereditary optic neuropathy caused by mutations in the optic atrophy 1 (OPA1) gene. It is characterized by insidious onset with a selective degeneration of retinal ganglion cells, variable loss of visual acuity, temporal optic nerve pallor, tritanopia, and development of central, paracentral, or cecocentral scotomas. Here we describe the clinical and molecular findings in a large Italian family with ADOA.
Methods: Routine ophthalmologic examination and direct sequencing of all coding regions of the OPA1 gene were performed. Further characterization of a new OPA1 gene insertion was performed by reverse transcription-PCR (RT–PCR) of RNA from patients and control subjects.
Results: We identified an Alu-element insertion located in intron 7 of OPA1 causing an in-frame deletion of exon 8 in 18 family members.
Conclusions: The predicted consequence of this mutation is the loss of the guanosine triphosphatase (GTPase) activity of OPA1. Alu insertions have been reported in the literature as causing human genetic disease. However, this is the first report of a pathogenic OPA1 gene mutation resulting from an Alu insertion.
Autosomal dominant optic atrophy (ADOA; OMIM 165500) is the most common form of hereditary optic neuropathy, with an estimated prevalence between 1:10,000 and 1:50,000 in different populations . It is characterized by insidious onset with a selective degeneration of retinal ganglion cells, variable loss of visual acuity, temporal optic nerve pallor, tritanopia, and development of central, paracentral, or cecocentral scotomas . ADOA is inherited in an autosomal dominant manner with high interfamilial and intrafamilial phenotypic variability and incomplete penetrance.
Mutations in the optic atrophy 1 (OPA1) gene (that consists of 30 coding exons) are responsible for approximately 90% of cases. OPA1 encodes a large guanosine triphosphatase (GTPase), implicated in the formation and maintenance of the mitochondrial network  and in protection against apoptosis by segregating cytochrome c inside the mitochondrial cristae . OPA1 comprises a highly basic N-terminus, a dynamin GTPase domain, and a C-terminus. More than 200 pathogenic mutations have been reported in the literature (see the eOPA1 database). No significant correlation has been observed between degree of visual impairment and location or type of mutation . Missense mutations in OPA1, however, leading to multiple mitochondrial DNA (mtDNA) deletions in skeletal muscle and a mosaic defect of cytochrome c oxidase, were recently found. In these cases the disorder presented with visual failure and optic atrophy in childhood, followed by Progressive External Ophthalmoplegia (PEO), ataxia, deafness, and sensory-motor neuropathy in adulthood. Moreover, some cases show additional neurologic symptoms, the so-called optic atrophy plus phenotypes .
Twenty-eight individuals of a single large Italian family were recruited from the Department of Neurologic, Neurosurgical and Behavioral Sciences of Siena, the Italian Union of the Blind of Siracusa and the Department of Pediatric of Catania. There were 15 females and 13 males ranging in age from 9 to 59 years. We use the term “family” to indicate descendants of a multigenerational pedigree of related individuals (Figure 1). Autosomal dominant inheritance was indicated by multiple affected individuals in each generation. All patients underwent routine ophthalmologic examination, including visual acuity, color vision, manual visual field, slit-lamp examination, intraocular pressure (IOP), and dilated fundoscopy. Clinical neurologic examination and genetic analysis was also performed in all patients.
After informed consent was obtained, 5 ml of blood from the proband, his family members, and healthy controls, was drawn into an ethylenediamine tetraacetic acid (EDTA) sample tube and preserved (frozen). Genomic DNA was extracted from blood samples using a QIAamp DNA blood kit (Qiagen, Limburg, Netherlands). All 30 coding exons (comprising intron–exon boundaries) were amplified by PCR using primers specific for OPA1 (Table 1). PCR reactions were performed in 25 μl reaction volumes containing 100 ng genomic DNA, 10 pmol forward and reverse primers, 100 mM dNTPs, 1U DNA polymerase (Eurotaq DNA polymerase; Euroclone, Milan, Italy), 1.5 mM MgSO4, 1× Reaction buffer, using a DNA thermal cycler (PTC-200; MJ Research, Waltham, MA). The PCR products of exon 8 were cloned into a PCR Cloning Vector pSC-A using the Strata Clone™ PCR Cloning Kit (Agilent Technologies, Stratagene Products Division, Santa Clara, CA). After purification of PCR products with QIAquick PCR Purification kits (Qiagen), sequencing was performed using the automated sequencer ABI 3730 (Applied Biosystems, Foster City, CA).
Peripheral blood, about 10 ml, was collected using vacutainer EDTA tubes (Becton-Dickinson, Franklin Lakes, NJ) from the proband (V-3, Figure 1), the proband’s mother (IV-8, Figure 1) and three healthy controls. Total RNA was extracted from peripheral blood leucocytes using the LeukoLOCK™ Total RNA Isolation System following the instruction supplied by the manufacturer (Ambion, Austin, TX). The amount of total RNA extracted from each blood sample was quantified using the NanoDrop ND-1000 photometer (NanoDrop Technologies, Wilmington, DE). For each sample, 1.0 μg of total RNA was reverse transcribed with Affinity Script (Agilent Technologies) using oligo dT primers. PCR amplifications were performed with primers AF1 (5′-GAC CAA CTT CAG GAA GAA CT-3′) and AR1 (5′-TGA GGA CCT TCA CTC AGA GT-3′). After the purification of cDNA products with QIAquick PCR Purification kits (Qiagen), sequencing was performed using the automated sequencer ABI 3730.
Neurologic examination was unremarkable (Table 2). Ophthalmologic examination revealed visual acuity ranging from 20/20 to less than 20/200. Color vision was normal in two subjects, the other 16 had different degrees of color sensitivity deficiency (eight of them showed generalized color sensitivity deficit). IOP with applanation was in the normal range in 11 patients (not exceeding 19–20 mmHg); four subjects had IOP over 20 mmHg. One patient had high IOP (40 mmHg). Optic nerve appearance was normal in two subjects, whereas nine showed temporal pallor and six diffuse neurorim pallor.
We sequenced the 30 coding exons of OPA1 in the propositus. We did not find any known pathogenic mutation, but amplification of exon 8 revealed a hemizygous product of about 604 bp (Figure 2, A). Cloning and sequencing of the longer band revealed an insertion of about 325 bp. Using BLAST and Repeat Masker  to elucidate the nature of the inserted fragment, we identified 289 bp of the insert as belonging to AluYb8 (99.65% homology with Alu Sb2). The Alu insert was composed of a 289-bp fragment and contained a 25-bp poly(A) tail at its 3′ end with perfect direct repeats of 17 bp flanking the insert (Figure 2B,C). In Figure 2C RNA polymerase III promoter (A and B boxes) and the consensus target site sequence are also indicated. The same mutation was found in 17 family members (Figure 1); in ten subjects the mutation was absent, and the analysis was not performed in the remaining family members. The Alu insertion was located in intron 7 of OPA1, 21 bp upstream of exon 8, near the acceptor site (C.784–21_22). The mutation caused the skipping of exon 8 in mRNA (Figure 2D,E).
In the case reported here, we found an unusual molecular defect in OPA1 causing ADOA in a large Italian family. The defect is the result of an Alu element insertion.
Alu sequences are the most frequent, short, interspersed elements in the human genome, numbering 1.1 million copies and more than 10% of the mass of the genome. Alu elements are sequences of approximately 300 nucleotides flanked by perfect direct repeats of the host sequence, consistent with insertion into staggered nicks in the target DNA . Alu and other repetitive elements have had great influence on the human genome and its evolution . They influence mRNA splicing, RNA editing, and protein translation . Not surprisingly, Alu insertions have been associated with several human genetic disorders. A total of 26 L1 trans-driven Alu insertions have so far been described as causes of diseases .
In our case the Alu element was inserted in the antisense direction with respect to OPA1. Alu element insertions in introns can have strong effects on gene expression, especially when inserted in the antisense direction. Five reports indicate that Alu insertions into intronic sequences in antisense orientation and close to the affected exon (19–50 nucleotides) cause the downstream exon to shift from constitutive splicing to full exon skipping or alternative splicing. Three have been found to cause skipping of downstream exons: one impairs correct recognition of the splice acceptor site (F8 gene) , while the other two may affect the branch site that is usually very close to the end of the intron (NF1 and TNFRSF6 genes) [13,14]. The orientation and position of the Alu in the upstream intron was recently studied. Lev-Maor et al. shed light on the positive and negative effects of Alu elements inserted in antisense orientation immediately upstream of the regulated exon .
In our case Alu insertion probably interferes with the branch site leading to production of an alternatively spliced mRNA lacking exon 8. Loss of this exon may be similar to that reported for Alu element insertion in NF1 and TNFRSF6 genes.
A consensus target site is probably generated in the antisense strand where integration of the retroposon begins: the first 5′-gtAAAA-3′ hexanucleotides (3′CA↓TTTT 5′) of the 17-bp target-site duplication flanking the Alu element matches one of the more frequent consensus sequences perfectly . The sense strand is nicked 17 bp from gtAAAA .
Phenotypic expression of this new mutation did not show particular peculiarities since ophthalmologic variability observed in our patients has also been described in other common OPA1 mutations. Moreover, the presence of the mutation in two unaffected family members suggests incomplete penetrance, as previously reported for other OPA1 gene mutations .
In conclusion, we describe an AluYb8  retrotransposition event in an intron of the OPA1 gene leading to exon skipping. The predicted consequence of this mutation is the loss of the GTPase activity of OPA1. Alu insertions have been reported in the literature as causing human genetic disease. However, this is the first report of a pathogenic OPA1 gene mutation resulting from an Alu insertion.
We thank Professor Jerzy Jurka for helpful suggestions on retroposon integration mechanisms, all patients, and Dr. Anna Rubegni for helping with the preparation of this manuscript. This work was supported by a grant from Regione Toscana and Monte dei Paschi Foundation to AF.