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MYO1F as a Candidate Gene for Nonsyndromic Deafness, DFNB15
Achih H. Chen, MD;
Dietrich A. Stephan, PhD;
Tama Hasson, MD;
Kunihiro Fukushima, MD;
Christiana M. Nelissen, BA;
Arthur F. Chen, BS;
Andrew I. Jun, MD;
Arabandi Ramesh, PhD;
Guy Van Camp, PhD;
Richard J. H. Smith, MD
Arch Otolaryngol Head Neck Surg. 2001;127:921-925.
ABSTRACT
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Background Earlier studies have mapped the autosomal recessive nonsyndromic deafness
locus, DFNB15, to chromosomes 3q21.3-q25.2 and 19p13.3-13.1,
identifying one of these chromosomal regions (or possibly both) as the site
of a deafness-causing gene. Mutations in unconventional myosins cause deafness
in mice and humans. One unconventional myosin, myosin 1F (MYO1F), is expressed in the cochlea and maps to chromosome 19p13.3-13.2.
Objective To evaluate MYO1F as a candidate gene for deafness
at the DFNB15 locus by determining its genomic structure
and screening each exon for deafness-causing mutations to identify possible
allele variants of MYO1F segregating in the DFNB15 family.
Methods We used radiation hybrid mapping to localize MYO1F on chromosome arm 19p. We next determined its genomic structure using
multiple long-range polymerase chain reaction experiments. Using these data,
we completed mutation screening using single-stranded conformational polymorphism
analysis and direct sequencing of affected and nonaffected persons in the
original DFNB15 family.
Results Radiation hybrid mapping placed MYO1F in the DFNB15 interval, establishing it as a positional candidate
gene. Its genomic structure consists of 24 coding exons. No mutations or genomic
rearrangements were found in the original DFNB15
family, making it unlikely that MYO1F is the disease-causing
gene in this kindred.
Conclusions Although we did not find MYO1F allele variants
in one family with autosomal recessive nonsyndromic hearing loss, the gene
remains an excellent candidate for hereditary hearing impairment. Given its
wide tissue expression, MYO1F might cause syndromic
deafness.
INTRODUCTION
PHENOTYPIC TRAITS are determined by the inheritance of genes. Diseases
caused by single genes (monogenic) that segregate in a family may be localized
to a specific chromosome by linkage analysis (see Ott,1
Jorde et al,2 Terwillinger and Ott,3 and Conneally and Rivas4
for a discussion of linkage analysis). However, because the linked chromosomal
region will contain many genes, identification of the specific disease-causing
gene can be difficult and time-consuming. Prioritizing genes in the linked
interval for further study is facilitated if their function is known. This
approach, the selection of a gene for in-depth analysis based on its chromosomal
location and purported function, is known as the "positional candidate gene"
approach to gene identification.
In general, if complementary DNA (cDNA) is available and the cDNA sequence
of a candidate gene is known, rapid mutation screening is possible. However,
because RNA is unstable and the presence of a given gene depends on the RNA
source, cDNA screening is not always possible. Genomic DNA has the benefit
of being much easier to isolate and very stable; however, mutation screening
requires knowledge of the candidate gene's genomic structure.
In an earlier study, Chen et al5 used
a small consanguineous family from India in which several children had prelingual
autosomal recessive nonsyndromic hearing loss to map the DFNB15 locus to chromosomes 3q21.3-q25.2 and 19p13.3-13.1 (Figure 1). Because the gene responsible for
deafness at this locus has not been identified, we have been using a candidate
gene approach to select genes for mutation screening. One interesting candidate
is MYO1F, an unconventional myosin that is expressed
in the cochlea and maps to chromosome 19p13.3-13.2.6-7
Unconventional myosins play a variety of roles necessary for cell locomotion,
phagocytosis, organelle transport, and mechanoregulation of membrane protein
function.8 Three unconventional myosin genes—MYO6, MYO7A, and MYO15—have
been demonstrated to be essential for normal hearing.9-12
In this article, we report the complete cDNA sequence of MYO1F, its genomic structure, and our mutation screening results.
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Figure 1. Pedigree of the DFNB15
family (see Chen et al5). Open square indicates
male; open circle, female; and solid symbols, affected.
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MATERIALS AND METHODS
MAPPING OF MYO1F
To verify that MYO1F is a positional DFNB15 candidate gene, we used primer pairs that amplified
exons 6, 13, and 21 and a radiation hybrid chromosome mapping panel (Genebridge
4; Research Genetics, Huntsville, Ala) to place MYO1F
on the framework map (Table 1).
Data were analyzed using RHMAPPER, provided by the Whitehead Institute for
Biomedical Research/MIT Center for Genome Research, Cambridge, Mass.
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Mutation Screening Polymerase Chain Reaction Primers, Splice Donor
and Acceptor Sites, and Exon Sizes
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5' AND 3' RAPID AMPLIFICATION OF cDNA ENDS
We next determined the complete cDNA sequence of MYO1F using 5' and 3' rapid amplification of cDNA ends
(RACE) using the Marathon RACE Kit and, as template DNA, a fetal brain Marathon-ready
cDNA library (Clonetech Laboratories Inc, Palo Alto, Calif).
DETERMINATION OF INTRON-EXON BOUNDARIES
To determine the genomic structure of MYO1F,
we used exon-specific primers designed from the MYO1F
cDNA sequence (GenBank accession numbers X98411 and U57053). Possible splice
sites were determined by homology to consensus splice sites and by modeling
the genomic structure of MYO1F. Polymerase chain
reaction (PCR) amplified products longer than predicted by the cDNA sequence
were assumed to include intronic sequence.
Polymerase chain reactions were performed in a 25-µL reaction
mixture containing 50 ng of genomic DNA, 2.5 µL of 10x PCR buffer
(Bioline USA Inc, Reno, Nev), 2.25 µL of 50mM magnesium chloride (Bioline),
0.4 U of Biolase DNA Polymerase (Bioline), 1 µL of deoxynucleotide triphosphates
(dNTPs) (2.5mM each), 25pM forward and reverse primers, 2.5 µL of 50%
glycerol, and enough sterile double-distilled water to bring the volume to
25 µL. Polymerase chain reactions were completed in thermocyclers (Techne
Genius; Techne Inc, Princeton, NJ) using modifications of the following protocol:
94°C for 1 minute; 44 cycles at 94°C for 30 seconds, 55°C for
30 seconds, and 72°C for 35 seconds; and 72°C for 10 minutes. Long-range
PCRs were performed in 50-µL reactions using the Takara Long Range PCR
Kit (LA PCR Kit; Takara Shuzo Co, available through Panveria Corporation,
Madison, Wis), with 1.0 µg of genomic DNA as a template. Conditions
of PCR were described in the Takara touch down PCR directions. Products of
PCR were purified from 1% agarose gels using Amicon Ultrafree-DNA spin columns
(Millipore Corporation, Bedford, Mass). Each PCR product was bidirectionally
sequenced by dye primer using an automated sequencer (model 373; Applied Biosystems,
Norwalk, Conn).
MUTATION SCREENING
We screened the entire MYO1F coding region
and splice sites using single-stranded conformational polymorphism (SSCP)
and direct sequencing in the DFNB15 family. Labeled
PCRs for SSCP analysis were performed in a 10-µL reaction mixture containing
20 ng of genomic DNA; 1 µL of 10x PCR buffer (Bioline); 1 µL
of 50% glycerol; 0.9 µL of 50mM magnesium chloride (Bioline); 0.4 U
of Biolase polymerase (Bioline); 10 pmol of forward and reverse primers; 0.12
µL of [35S]deoxyadenosine triphosphate (dATP); 200 nmol each
of deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP),
and deoxycytidine triphosphate (dCTP); 2 nmol of cold dATP; and enough sterile
double-distilled water to bring the volume to 10 µL. Each reaction was
overlaid with mineral oil. Amplification was carried out for 44 cycles at
94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 35 seconds.
Each labeled PCR reaction was mixed with 2 µL of formamide loading dye
(95% formamide, 20mM EDTA, 0.05% bromophenyl blue, and 0.05% xylene cyanol).
Samples were denatured for 5 minutes at 94°C and electrophoresed at 20
W on a 6% polyacrylamide gel (49:1 acrylamide:bis, 5% glycerol, and 1x
Tris-EDTA (TE)). Total electrophoresis time was proportional to product size.
Constant temperature was maintained with a cooling fan. Gels were transferred
to 3M Whatman paper (Whatman International, Maidstone, England) and dried.
Kodak X-OMAT film (Kodak, Rochester, NY) was used for autoradiography. Band
shifts were assessed by visual inspection. Sequence data were compared with
published cDNA sequences for MYO1F using a software
package (Sequencer 3.1; Gene Codes Corp Inc, Ann Arbor, Mich).
RESULTS
Using the Genebridge 4 radiation hybrid screening panel (Research Genetics),
we mapped MYO1F to chromosome 19p13.3-13.2 within
the interval flanked by D19S216 and D19S221 (Figure 2). Mapping
data were concordant for primer pairs that amplified exons 6, 13, and 21 (Table 1).
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Figure 2. Mapping of MYO1F
within the DFNB15 interval on chromosome arm 19p.
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The complete coding sequence of mouse Myo1f,
as well as the 3' and part of the 5' untranslated regions (UTRs),
has been reported by Crozet et al.6 The encoded
protein is 1099 amino acids in length and structurally similar to other unconventional
myosins. However, the cDNA sequence of the human orthologue, MYO1F, has been reported only in part (GenBank accession numbers U57053
and X98411).6-7 We determined
the complete 5' coding region of MYO1F using
5' RACE to identify a start codon (ATG) that corresponds with that described
in murine Myo1f. We then used 3' RACE to determine
the unreported portion of the 3' UTR and the poly-A tail (Figure 3). In most respects, MYO1F shows
good homology to Myo1f, but the head domain of MYO1F is lengthened by 52 amino acids encoded by a 156–base
pair (bp) stretch of cDNA sequence that is not homologous to Myo1f.
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Figure 3. Human myosin 1F (MYO1F)
and mouse Myo1f complementary DNA (cDNA). A, Dashed area on the
mouse cDNA represents a 156–base pair interval present in human MYO1F but not in mouse Myo1f. B, Previously unpublished
5' and 3' cDNA sequences and the interval of cDNA that is present
in human MYO1F but absent in mouse Myo1f. RACE indicates
rapid amplification of cDNA ends; ATP, adenosine triphosphate.
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The genomic structure of MYO1F was determined
by a series of conventional and long-range PCR reactions using exon-specific
primers generated from a combination of published MYO1F cDNA sequences (GenBank accession numbers X98411 and U57053) and cDNA
sequences determined using 3' and 5' RACE. Polymerase chain reaction
primers were chosen using homology to consensus splice site sequences and
modeling of MYO1F genomic structure. The PCR product
sizes were compared with the sizes predicted by the cDNA sequence. Products
that were longer than predicted were assumed to contain an intron and were
bidirectionally sequenced to determine exon-intron boundaries. The genomic
structure of MYO1F was found to span 25.45 kilobase
(kb) and to consist of 24 exons. The first exon contained the 5' UTR,
and the 3' UTR was within exon 24 (Figure 4).
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Figure 4. Genomic structure of MYO1F with intron and exon sizes (kb indicates kilobase).
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Each exon was screened for mutations using DFNB15-affected family members 3 and 5 and an obligate carrier, as well as
an unrelated normal-hearing individual. Mutation screening was completed first
using SSCP and then using direct sequencing of each exon using the primer
pairs listed in Table 1. The primers
were designed to include splice donor and acceptor sites. All coding and splice
site regions were screened. No mutations were found, making it unlikely that MYO1F is the cause of DFNB15-related
hearing impairment.
COMMENT
Severe hearing impairment affects approximately 1 in 1000 newborns,
making it the most common sensory disorder.13-14
Hearing impairment might be inherited with a constellation of other phenotypic
features as a recognizable syndrome or in isolation as nonsyndromic hearing
loss. Nonsyndromic hearing loss can be inherited in an autosomal dominant,
autosomal recessive, or X-linked fashion. By convention, DFNA denotes a nonsyndromic, dominant deafness locus, with the numerical
suffix reflecting the order of locus discovery (ie, DFNA1,
DFNA2, etc). Autosomal recessive nonsyndromic deafness loci and X-linked
deafness loci are designated DFNB and DFN, respectively.
The most common form of prelingual inherited hearing impairment is autosomal
recessive nonsyndromic hearing loss. It is highly heterogeneous and almost
exclusively monogenic. In a previous study, Chen et al5
mapped DFNB15 to chromosomes 3q21.3-q25.2 and 19p13.3-13.1,
identifying one or possibly both regions as the site of a deafness gene. In
this study, using a radiation hybrid mapping panel, we placed MYO1F, an unconventional myosin gene expressed in the cochlea, within
the DFNB15 interval.
Unconventional myosins differ from conventional myosins. They do not
form bipolar filaments but instead function as intracellular motors that move
along actin filaments, generating force through the hydrolysis of adenosine
triphosphate. They share a conserved head domain that contains adenosine triphosphate
and actin binding sites, a neck regulatory domain that can contain 1 or more
IQ motifs that bind calmodulin or calmodulin-like light chains,15
and a tail domain that varies from one unconventional myosin to another. It
is this last domain that determines the function of each unconventional myosin.
Unconventional myosins play a variety of roles necessary for cell locomotion,
phagocytosis, organelle transport, and mechanoregulation of membrane protein
function.8 They are important constituents
of the cytoskeletal framework of the inner ear, the integrity of which is
essential for cochlear mechanoelectrical transduction. Three unconventional
myosins—myosins VI, VIIA, and XV—have been demonstrated to be
essential for normal hearing.9-12
Mutations in murine Myo6 are found in the Snell waltzer
mutant, which has a phenotype characterized by deafness and vestibular dysfunction;
the protein is concentrated in the cuticular plate.10
Mutations of Myo7a are found in the shaker-1 mutant,
which also presents with hearing impairment and vestibular dysfunction.9 Myosin VIIA has been implicated in formation of the
cytoskeletal network and in intracellular vesicular transport.16
It is expressed in the inner ear and retina and is the cause of nonsyndromic
deafness (DFNB2 and DFNA11)
and syndromic deafness (USH1B—characterized
by hearing loss, vestibular dysfunction, and retinitis pigmentosa).17-19 Mutations in Myo15 give rise to the shaker-2 mutant, which, similar
to the other 2 mouse mutants, has auditory and vestibular impairment20; mutations in MYO15 result
in DFNB3.21
Based on these findings and the map location of MYO1F, we considered MYO1F an attractive functional
and positional DFNB15-causing candidate gene. Genomic
DNA was used for mutation screening because there was no readily available
source of RNA. To determine the genomic structure of MYO1F for mutation screening, we used a series of conventional and long-range
PCR experiments.
MYO1F was found to span 25.45 kb and to consist
of 24 exons. The average size of a human nuclear gene, including introns,
is approximately 10 kb,22 although there is
enormous variability, with gene size ranging from a few hundred nucleotides
to several megabases. The average number of exons per gene also has wide variability
but roughly correlates with gene size. Small genes might have only 1 exon,
whereas large genes have numerous exons. MYO1F follows
these trends; however, with an average size of 176 bp, MYO1F exons are slightly smaller than the average nuclear gene exon
size of 200 bp. Exon size is independent of gene size, whereas intron size
is directly correlative. The largest MYO1F intron
measures 2 kb. Although we found no mutations using SSCP or direct sequencing
of the exons, because approximately 15% of disease-causing mutations lie outside
the coding region, there remains the possibility that a mutation in MYO1F within the 5' or 3' UTRs or the promoter
might be the cause of DFNB15.
Determining the genomic structure of an attractive candidate gene is
often time and labor intensive, and previous knowledge of its structure would
facilitate mutation screening. Completion of sequencing of the human genome
should make this goal possible. Currently, the government's Human Genome Project
and P. E. Celera Genomics Inc (Norwalk, Conn) are racing toward completion
of the entire genome sequence. Celera, a for-profit corporation, has been
sequencing the human genome using the shotgun sequencing approach. Recently,
Celera reported that they completed a rough draft of the human genome; however,
a subscription fee might be charged to use their data. In contrast, the Human
Genome Project, a government-funded, multilaboratory effort, will provide
freely accessible sequence data. The Human Genome Project projected that they
will have a final version of the human genome in 2003, 2 years ahead of the
originally predicted completion date. Irrespective of the data source or which
group completes sequencing of the human genome first, completion of sequencing
will make the candidate gene approach to discovering disease-causing genes
faster and easier.
CONCLUSIONS
The candidate gene approach to determining the disease-causing gene
for a specific hereditary condition is only one method among many. However,
selection of a putative disease-causing gene for mutation screening based
on chromosome location, tissue expression, and purported function will become
an even more important approach as sequencing of the human genome is completed.
Although we did not find MYO1F allele variants
in one family with autosomal recessive nonsyndromic hearing loss, the gene
remains an excellent candidate for hereditary hearing impairment. MYO1F is expressed in liver, kidney, spleen, eye, brain, lung, and
small intestine (Crozet et al6). Given its
wide range of tissue expression, we believe that MYO1F
might be a candidate for a syndromic hearing loss.
AUTHOR INFORMATION
Accepted for publication March 17, 2001.
Corresponding author and reprints: Richard J. H. Smith, MD, Department
of Otolaryngology–Head and Neck Surgery, University of Iowa Hospital
and Clinics, Iowa City, IA 52242 (e-mail: richard-smith{at}uiowa.edu).
From the Departments of Otolaryngology and Pediatrics, University of
Iowa, Iowa City (Drs A. H. Chen, Jun, and Smith, Ms Nelissen, and Mr A. F.
Chen); the Cancer Genetics Branch, National Human Genome Research Institute/National
Institutes of Health, Bethesda, Md (Dr Stephan); the Department of Biology,
Yale University, New Haven, Conn (Dr Hasson); the Department of Otolaryngology,
Okayama University Medical School, Okayama, Japan (Dr Fukushima); the Department
of Genetics, University of Madras, Madras, India (Dr Ramesh); and the Department
of Medical Genetics, University of Antwerp, Antwerp, Belgium (Dr Van Camp).
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