|Molecular Vision 2007;
Received 8 June 2007 | Accepted 30 September 2007 | Published 2 October 2007
Analysis of MYO7A in a Moroccan family with Usher syndrome type 1B: novel loss-of-function mutation and non-pathogenicity of p.Y1719C
Redouane Boulouiz,1,2 Yun Li,3,4 Omar Abidi,1
Hanno Bolz,4 Abdelaziz Chafik,2 Christian
Kubisch,3,4,5 Hassan Rouba,1
Bernd Wollnik,3,4 Abdelhamid
1Department of Genetics, Institute Pasteur du Maroc, Casablanca, Morocco, 2Faculté des Sciences Université Chouaib Doukkali, El-Jadida, Morocco, 3Center for Molecular Medicine Cologne, 4Institute of Human Genetics, 5Institute for Genetics, University of Cologne, Cologne, Germany.
Correspondence to: Bernd Wollnik, Center for Molecular Medicine Cologne and Institute of Human Genetics, Kerpener Str. 34, 50931 Cologne, Germany; Phone: +49-221-478-86817; FAX: +49-221-478-86812; email: firstname.lastname@example.org
Purpose: Mutations in the MYO7A gene are responsible for Usher syndrome type 1B (USH1B), the most common USH1 subtype, which accounts for the largest proportion of USH1 cases in most populations. Molecular genetic diagnosis in Usher syndrome is well established and identification of the underlying mutations in Usher patients is important for confirmation of the clinical diagnosis and genetic counseling.
Methods: We analyzed a large consanguineous USH1 family from Morocco and linked the disease in this family to the MYO7A/USH1B locus.
Results: We identified the frequently described missense change p.Y1719C. In addition, we found the homozygous c.1687G>A mutation in the last nucleotide of exon 14, which is predicted to result in aberrant splicing and may lead to loss of MYO7A transcript. We further showed that p.Y1719C is not disease-causing but does represent a frequent polymorphism in the Moroccan population, with an estimated carrier frequency of 0.07.
Conclusions: This finding has an important impact for molecular diagnosis and genetic counseling in USH1B families.
Usher syndrome (USH) is an autosomal recessive disorder characterized by bilateral sensorineural hearing loss, retinitis pigmentosa, and, occasionally in subtypes 1 and 3, vestibular dysfunction [1,2]. USH accounts for up to 50% of deaf-blind individuals . There are three different subtypes, USH1, USH2, and USH3, that can be distinguished according to the clinical presentation, onset, and severity of symptoms. USH1 is characterized by profound congenital hearing loss, pre-pubertal onset of retinitis pigmentosa, and vestibular areflexia [1,2]. USH1 is genetically heterogeneous with six loci (USH1B-USH1G) and five causative genes identified to date (Hereditary hearing loss homepage). Mutations in the MYO7A gene on chromosome 11q13, encoding the myosin VIIA protein, are responsible for USH1B , the most common USH1 subtype, which accounts for approximately 45-70% of USH1 cases [5,6]. MYO7A mutations also underlie non-syndromic autosomal recessive deafness, DFNB2, and autosomal dominant hearing loss, DFNA11 [7,8].
The MYO7A gene (accession number NM_000260.2) consists of 49 exons and encodes a 2,175 amino acid unconventional myosin (NP_000251) that is required for sensory hair cell development and maintenance. MYO7A is expressed in inner and outer hair cells of the organ of corti, retinal pigment epithelium, and photoreceptor cells, and plays different functional roles in differentiation and organization of hair cell stereocilia .
More than 80 USH1-related mutations have been identified in MYO7A including nonsense/missense mutations, small deletions and insertions, gross deletions, and splice-site mutations that are spread all over the gene (Human Gene Mutation Database). Molecular genetic diagnosis of Usher families and patients and identification of the underlying mutations in these patients is important for both, confirmation of the clinical diagnosis and individual genetic counseling of the patient. Therefore, highest accuracy of diagnostic testing and interpretation of identified alterations is crucial.
In this paper, we provide evidence that the previously described p.Y1719C alteration in MYO7A is not disease-causing in the large USH1B family described herein, but represents a frequent polymorphism in the Moroccan population. In the same family, we identified the novel homozygous c.1687G>A mutation that is predicted to result in aberrant splicing and putative loss of MYO7A transcript.
Families and patients
Two affected females and two affected males of the Moroccan SF11 family were born to consanguineous parents. The age of affected individuals ranged between 35 and 44 years of age. Five additional healthy siblings and the parents were also recruited. Family members underwent general otological examinations, pure-tone audiometry, and vestibular evaluation. The two affected family members IV-6 and IV-7 underwent visual examination including funduscopic tests and angiography. Informed consent was obtained from each family member participating in the study before blood was sampled. Additionally, 113 healthy and unrelated individuals from different geographic areas of Morocco with no familial history of hearing problems were enrolled as control group after written informed consent was signed. Age and gender of the control group was not matched to the family. The study was approved by the institutional ethics committee.
Genomic was extracted from 5 ml whole-cell blood of each individual of the family following a standard protocol: 5 ml of EDTA-blood were added up with lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0,1 mM EDTA in aqua dest., pH 7,4) to a total volume of 40 ml and incubated on ice for 15 min. After centrifugation (15 min, 1.500 rpm, 4 °C), pellets were resuspended in 5 ml nucleus lysis buffer (10 mm Tris, 400 mM NaCl, 2 mM EDTA in H2O, pH 8,2). After addidition of 330 μl 10% SDS and 250 μl proteinase K (20 mg/ml in H2O), the solution was incubated as 37 °C over night. After addition of 1,66 ml saturated NaCl (about 6 M), supernatants were centrifuged twice (10 min, 2,683 xg, room temperature). DNA was precipitated by adding 6 ml isopropanol to the supernatant, washed in 70% ethanol and solved in 500 μl TE. We performed linkage analysis with polymorphic microsatellite markers covering all known loci for USH. For each locus, three markers were genotyped. PCR reactions for marker analysis were carried out in 25 μl volumes containing 50 ng of genomic DNA, 0.4 μM for each primer, 200 μM dNTPs, 1x buffer, and 1 U of Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA). The reactions were denaturated for 1 min at 95 °C, and PCR-amplification was subsequently carried out with 35 cycles at 95 °C for 30 s, 52-59 °C for 30 s, and 72 °C for 50 s each step. PCR products were run on 6% polyacrylamide gels, transferred onto N+ hybond membranes (Amersham Bioscience, Piscataway, NJ) and hybridized with a poly-AC probe labeled with 32P dCTP. All 49 coding exons and exon-intron junctions of the MYO7A gene were amplified and sequenced in the index patient. Direct sequencing of PCR products was performed with the ABI prism Big Dye Terminator cycle sequencing Ready Reaction kit V 3.1 (ABI Prism/Apllied Biosystems, Foster City, CA) and analyzed on an ABI Prism 3100 Genetic Analyser (Applied Biosystem). We also tested for the identified MYO7A alterations in DNA drawn from 113 healthy control individuals from Morocco.
We obtained 5 ml peripheral blood samples in EDTA tubes from the affected index patient (Figure 1A, IV-6). Total RNA was isolated from 200 μl of the proband's blood sample (QIAamp RNA kit, Qiagen, Hilden, Germany), and reverse transcription from total RNA was carried out with reverse transcriptase (Fermentas, St. Leon-Rot, Germany) using oligo-dT primers. cDNA-specific PCR-amplification for evaluation of a putative effect of the c.1687G>A mutation on splicing was carried out using forward primer 5'-GCT CTG CAT CAA CTT CGC CAT T-3' (exon 13) and reverse primer 5'-GCA TCA GCA GCT CCA GTG ACC-3' (exon 16), which amplified a 517 bp fragment in the wildtype. An independent control cDNA amplification of a 631 bp fragment from exon 6 to 10 of the LMNA gene using primer pair 5'-GCA GCA GCA GCT GGA CGA GTA-3' and 5'-GGA GCA GGT CAT CTC CAT CCT-3' was done in order to test the quality of cDNA templates used for the analysis. The amplifications of MOY7A and LMNA were done in three independent replicates.
We identified a family, in which four affected siblings presented with progressive retinitis pigmentosa and vestibular dysfunction in addition to the profound congenital sensorineural hearing loss (Figure 1A). The diagnosis of USH-1 was given accordingly. Six additional siblings of this family did not show any clinical symptoms. The healthy parents were first degree cousins and originated from the eastern part of Morocco (Figure 1A).
We genotyped polymorphic markers located at the published USH1B to USH1G loci and found compatibility with linkage with markers D11S906, D11S911, and D11S1789 corresponding to the USH1B locus. Markers from other USH1 loci were informative in the family and clearly excluded these loci. Both affected individuals from whom DNA was available showed a homozygous haplotype that was present in heterozygous form in the parents. None of the healthy siblings was homozygous for the putative disease haplotype (Figure 1A). Sequencing of all 49 coding exons of MYO7A revealed two homozygous alterations that co-segregated with the disease in the family: the previously described c.5156G>A (p.Y1719C) alteration in exon 37 ; and the novel c.1687G>A alteration in exon 14, which predicts a putative amino acid substitution from glycine to serine at position 563 of the protein (p.G563S; Figure 1B). Screening for both alterations in 113 healthy control individuals from Morocco identified 13 individuals with heterozygosity and one who was homozygous for p.Y1719C. There was no history of hearing problem or a family history of hearing loss in this control individual homozygous for p.Y1719C. These data demonstrate that p.Y1719C is a polymorphism with an estimated carrier frequency of 0.07 in the Moroccan population. In contrast, we did not find the c.1687G>A alteration in our control group, suggesting that this is the causative mutation in the family.
As the c.1687G>A mutation affects the last nucleotide of exon 14 (Figure 1B), we used several splice-site prediction programs (Neural Network Splice Site Prediction Tool; Spliceview; SPL Search Potential) to evaluate the quality of the altered splice-site. The wild type sequence resulted in predicted scores of 0.94, 0.81, 0.85, respectively for the three programmes. In contrast, the donor site was not recognized anymore in case of the c.1687G>A mutation. No alternative donor site was predicted in close proximity using FGENESH_GC programme at SPL. In conclusion, all programmes predicted a loss of exon 14 donor site caused by the c.1687G>A mutation. In order to investigate this putative splice effect further, we amplified a 517 bp fragment from exon 13 to 16 of MYO7A on cDNA from blood of a healthy control individual and verified the amplification product by sequencing (Figure 1C). In contrast to control cDNA, we could not amplify a MYO7A-specific fragment from the patient's cDNA (Figure 1D). We tested the quality of cDNA by amplification of a 631 bp control fragment of the LMNA gene and and could show amplification in both, control and patient cDNA both, control and patient cDNA (Figure 1D), suggesting that the MYO7A transcript is unstable in the affected individual and possibly degraded by nonsense-mediated RNA decay. RNA samples from heterozygous mutation carriers were not available for further analysis.
We have identified the novel c.1687G>A mutation in exon 14 of the MYO7A gene in a Moroccan family with USH1. The mutation co-segregated with the disease in the family members and was not found in 113 control individuals. The c.1687G nucleotide is the last exonic nucleotide before the invariable intronic GT motif of the donor splice site adjacent to exon 14. Computer-assisted analysis suggested that the G>A exchange disrupts the recognition of this donor splice site, which could lead to skipping of exon 14 or the usage of an alternative donor site. To test this, we analyzed the MYO7A transcripts of the index patient in blood and observed that the transcript could not be amplified. LMNA transcript amplification was used as a control and proved the integrity of RNA. Although we can not completely role out an unpredicted splicing event that might affect exon 13 (in which one primer for cDNA amplification was located), and thereby mimick loss of MYO7A transcript, a predicted aberrant splicing of exon 14 and subsequent loss of MYO7A transcript by RNA decay is a likely mechanism caused by the c.1687G>A mutation. Unfortunately, RNA from a heterozygous carrier of the family was not available for analysis; it is likely that it could have shown the loss of the mutated transcript in the presence of the wild-type transcript. In addition, we found a second homozygous alteration, p.Y1719C, which has been described as a disease-causing mutation in USH1 patients in several studies [10-12]. Although Najera et al. reported the identification of three heterozygous p.Y1719C alleles in a studied control group, the nature of this change remained uncertain and it is still regarded as putative mutation or alteration with unknown effect . We were able to definitely prove that p.Y1719C is a rather frequent polymorphism in the Moroccan population with an estimated carrier frequency of 0.07. This high carrier frequency clearly argues against a disease causing nature of p.Y1719C. Furthermore, one individual without obvious hearing problems of the control cohort was homozygous for p.Y1719C. This finding has an important impact for molecular diagnosis and genetic counseling in USH1B families. Molecular diagnosis for families in which this change has been identified should be critically re-evaluated.
The authors appreciate the kind participation of patients and their families. We thank Dr. Hammadi Ayadi, Laboratoire de Genetique Moleculaire Humaine, Sfax, Tunisia, and Dr. Mohamed Benajiba, Center Transfusion Oujda, Morocco, for their contributions, and Maria Langen for technical assistance. This work was supported by the FP6 Integrated Project EUROHEAR (LSHG-CT-20054-512063 to C.K. and B.W).
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