Reinforcement of a minor alternative splicing event in MYO7A due to a missense mutation results in a mild form of retinopathy and deafness

Abstract

Purpose: Recessive mutations of the myosin VIIA (MYO7A) gene are reported to be responsible for both a deaf-blindness syndrome (Usher type 1B [USH1B] and atypical Usher syndrome) and nonsyndromic hearing loss (HL; Deafness, Neurosensory, Autosomal Recessive 2 [DFNB2]). The existence of DFNB2 is controversial, and often there is no relationship between the type and location of the MYO7A mutations corresponding to the USH1B and DFNB2 phenotype. We investigated the molecular determinant of a mild form of retinopathy in association with a subtle splicing modulation of MYO7A mRNA.

Methods: Affected members underwent detailed audiologic and ocular characterization. DNA samples from family members were genotyped with polymorphic microsatellite markers. Sequencing of MYO7A was performed. Endogenous lymphoid RNA analysis and a splicing minigene assay were used to study the effect of the c.1935G>A mutation.

Results: Funduscopy showed mild retinitis pigmentosa in adults with HL. Microsatellite analysis showed linkage to markers in the region on chromosome 11q13.5. Sequencing of MYO7A revealed a mutation in the last nucleotide of exon 16 (c.1935G>A), which corresponds to a substitution of a methionine to an isoleucine residue at amino acid 645 of the myosin VIIA. However, structural prediction of the molecular model of myosin VIIA shows that this amino acid replacement induces only minor structural changes in the immediate environment of the mutation and thus does not alter the overall native structure. We found that, although predominantly included in mature mRNA, exon 16 is in fact alternatively spliced in control cells and that the mutation at the very last position is associated with a switch toward a predominant exclusion of that exon. This observation was further supported using a splicing minigene transfection assay; the c.1935G>A mutation was found to trigger a partial impairment of the adjacent donor splice site, suggesting that the unique change at the last position of the exon is responsible for the enhanced exon exclusion in this family.

Conclusions: This study shows how an exonic mutation that weakens the 5' splice site enhances a minor alternative splicing without abolishing a complete exclusion of the exon and therefore causes a less severe retinitis pigmentosa than the USH1B-associated alleles. It would be interesting to examine a possible correlation between intrafamilial phenotypic variability and the subtle variation in exon 16 inclusion, probably related to genetic background specificities.

Figure 1

Pedigree and analysis of a Tunisian family segregating autosomal recessive hearing loss with variability in the diagnosis of retinitis pigmentosa. A: Pedigree of a Tunisian family and haplotypes of polymorphic markers in the MYO7A region are shown. In pedigree, the square symbol indicates male, the circle symbol denotes female and black symbol represents affected individuals. B: DNA chromatogram from a control individual (bottom) and patient MB9 (top) are shown. Mutation consists on a homozygous mutation (G-A) at nucleotide 1935 located in exon 16, resulting in a Met to Ile substitution at amino acid 645 of myosin VIIA. Asterisk indicates the position of the homozygous mutation. C: Fundus photographs in 40-year-old (P1: MB9) and 47-year-old (P2: MB60) patients showed variability in the severity of retinitis pigmentosa (RP). MB9 had a mild peripheral RP, while MB60 had only some pigments. D: Visual field test results obtained on the right (RE) and the left (LE) eye of patient MB9 at the age of 40 years are shown. A series of random lights of different intensities are flashed in the peripheral field of vision of the patient. When the patient perceives the computer generated light suddenly appearing in his or her field of view, the patient presses a button to indicate a response. In the picture of the visual field a lighter gray spot is assigned. If the patient is unable to see the light in an appropriate portion of the field of view, then we see on the computer a darker gray spot (Dot don’t see) indicating vision loss. Visual field loss was severe in this patient. In fact, the nasal and temporal fields were not preserved and only the central field was maintained. Ganzfeld electroretinogram (ERG) and visual-evoked potentials (VEP) of RE and LE of patient MB9 are shown. The ERG and the VEP test the function of the visual pathway from the retina (ERG) to the occipital cortex (VEP). These tests were conducted by placing a standard ERG device attached to the skin 2 mm above the orbit. VEPs were recorded simultaneously from an electrode attached to the occipital scalp 2 mm above the region on the midsagittal plane. An electrode placed on the forehead provided a ground. The results can be directly related to the part of a visual field that might be defective. This is based on the anatomic relationship of the retinal images and the visual field. After dark adaptation for 30 min, the doctor places anesthetic drops in the patient's eye and places a contact lens on the surface of the eye. Once the contact lens is in place, a series of blue, red, and white lights is shown to the patient. The VEP is an evoked electrophysiological potential that can be extracted, using signal averaging, from the electroencephalographic activity recorded at the scalp. Both the ERG and VEP were differentially amplified band pass filtered (0, 1, 30 Hz), recorded over 300 ms epochs, and signal averaged. The visual evoked potential to flash stimulation consists of a series of negative and positive waves. The earliest detectable response has a peak latency of approximately 30 ms post stimulus. For the flash VEP, the most robust components are the negative peak N2 and positive peak P2 peaks. Measurements of the P2 amplitude should be made from the positive P2 peak at around 207.3 ms. The ERG recorded in MB9 showed an absence of responses. While the VEP showed a normal response in both eyes. These traces confirm the evidence of a significant bilateral global retinal degeneration. Only cone flicker responses of less than 15% of the normal mean were recordable under photopic conditions, while all other responses were below noise level, a typical finding for patients with RP.

Figure 2

Homology model of human myosin VIIA showing the exon 16-encoded peptide in green in the middle of the motor domain, which is colored in orange. This exon encodes the HW helix [22], the third strand of the central seven-stranded β-sheet, and the associated loops. Wild-type methionine and mutant isoleucine 645 residues before the SH2 helix (in orange) are represented as sticks and highlighted in green and magenta, respectively. Structure visualization was done using Pymol software.

Figure 3

Exon 16 splicing pattern associated with the c.1935G>A mutation. A: Analysis of endogenous RNA from lymphoid cells. We amplified a 346-bp fragment from exon 14 to exon 17 of MYO7A in a healthy control individual. An additional faint band appeared in the control; it corresponded to an mRNA species missing exon 16 (lane 2). In patient MB9, the splicing pattern showed a predominant exclusion of exon 16 (lane 1). Lane 3 refers to negative control and M indicates size marker. Direct sequencing of these two different transcripts revealed that a shorter band of 246 bp corresponds to an abnormal transcript without exon 16, whereas the other band of 374 bp corresponds to a normal transcript that contains exon 16. B: Ex vivo splicing assays were performed. Wild-type and mutant constructs were stably transfected in HeLa cells, and the exon 16 splicing pattern was analyzed by reverse transcription-polymerase chain reaction. Consistent with data presented in A, exon 16 is predominantly included from the wild-type construct (lane 1). The c.1935G>A mutation at the end of exon 16 resulted in massive skipping of the exon (lane 2). Lane 3 refers to negative control and M indicates size marker. UE and DE refer to upstream and downstream exons of the cassette, respectively.