A Molecular Vision Research Report
Fine Mapping of the Usher Syndrome Type IC to Chromosome 11p14
and Identification of Flanking Markers by Haplotype Analysis
R. Ayyagari1, Y.Li1, R.J.H.Smith2, M.Z. Pelias3, and J.F.Hejtmancik1*
1. National Eye Institute, National Institutes of Health
2. Department of Otolaryngology - Head and neck surgery,
University of Iowa
3. Department of Biometry and Genetics, Louisiana State
University Medical Center
*Corresponding author email:
f3h@helix.nih.gov
Purpose: To refine the map position of the Usher
syndrome type 1C
(USH1C) locus to 11p14-p15.1 in the French-Acadian population
settled in Louisiana.
Methods: Linkage and haplotype analysis of
Ush1C in the French-Acadian families from southwestern Louisiana
was carried out using additional markers known to map to the USH1C
interval. Markers localized to 11p were also mapped on the J1
somatic cell hybrid panel. This analysis also helped to localize
precisely the USH1C interval.
Results: New flanking markers for
USH1C have been identified, localizing the USH1C gene to a 1 cM
interval between markers D11S1397 and D11S1888. Markers D11S1890
and D11S1888 were placed within the USH1C interval. Analysis of
all the markers in the USH1C region flanked by D11S1397 and
D11S1888 on the J1 somatic cell hybrid panel localized USH1C to
the upper half of chromosome 11p14.
Conclusion: The Usher
Syndrome type 1C gene has been localized to a 1 cM interval between
the markers D11S1397 and D11S1888 on chromosome 11p14.
INTRODUCTION
The Usher syndromes (Ush) are a group of autosomal
recessive disorders characterized by congenital sensorineural hearing
impairment and retinitis pigmentosa. In the United States the
frequency of Ush has been estimated at 4.4 per 100,000, making Ush
the most common cause of deaf-blindness in developed countries (3).
Based on clinical symptoms, three distinct types of Ush have been
described (21).
Usher syndrome type I (Ush1) consists of profound congenital
hearing impairment usually associated with unintelligible speech,
early onset of retinitis pigmentosa, and vestibular dysfunction. In
Usher syndrome type II (Ush2), deafness is more moderate so that
speech is intelligible and the onset of retinitis pigmentosa tends
to be delayed to the second or third decade. Vestibular function is
normal in Ush2. In Usher syndrome type III (Ush3), both the
sensorineural hearing loss and retinitis pigmentosa are
progressive over time.
Genes causing Ush1, Ush2 and Ush3 have been mapped to five
different chromosomal loci (10, 12, 13, 15, 18, 20). The existence of
at least two additional loci for Ush type I and Ush type II is
suggested by the observation that Ush in some of the families
classified as type I and type II based on clinical symptoms have
been excluded >from the known Usher syndrome loci (2, 4). These
observations reflect the genetic heterogeneity of the various forms
of Usher syndrome.
There are three different known loci for Ush type I (10,20).
One of these, Usher syndrome type 1B on chromosome 11q, is myosin
VIIA (22). The gene causing Usher syndrome type I in the French-
Acadian population of Louisiana (USH1C) has been localized to a 5
cM interval between the markers D11S861 and D11S899 on the p arm of
chromosome 11(1,11), a locus for Ush so far not detected in any
other population. There is an unusually high incidence of Usher
syndrome in the French-Acadian population that was resettled in
Louisiana after France ceded Canada to Great Britain. A persuasive
case has been made that the high incidence of Ush resulting from a
unique locus in this population is due to a founder effect. If
that is the case, chromosomes bearing the USH1C mutation would be
expected to share a common haplotype in the USH1C region. Here we
use linkage mapping and haplotype analysis in Ush1 patients from
the French-Acadian population to localize the USH1C gene to a
region of approximately 1 cM interval of chromosome 11p14.
METHODS:
Clinical Studies: Seven Acadian families with a
history of Usher syndrome and individual affected patients with a history of Usher
syndrome in their families were studied in detail (20). Affected individuals and their family members underwent a detailed physical
examination including a battery of tests suggested for the clinical
diagnosis of Ush (19) including the Bruininks-Oseretsky subtests
of balance function, an otologic examination, and ice-water
calorics using Frenzel's glasses to prevent fixation and to
facilitate observation.
DNA and PCR analysis: Blood samples were collected from cooperative family members. DNA was isolated from blood or
transformed cell lines using phenol/chloroform extraction as
described previously (20). PCR and electrophoresis was performed
as described earlier (20). Alleles were scored by three independent
readers without reference to affectation status or pedigree, with
any disagreement resulting initially in reevaluation of those
observations. If that did not resolve the interpretation, the
marker was analyzed again.
Linkage analysis: The LINKAGE program package version 5.1 was used
for linkage analysis (14) with MLINK and ILINK used for two point
analysis. USH1C was assumed to be a fully penetrant autosomal
recessive trait with an affected allele frequency of 0.001. Marker
allele frequencies were taken from the Genome Data Base at the
Welch Library, Johns Hopkins University.
Chromosomal sublocalization of microsatellite markers: Seven
somatic cell hybrids (J1, J11, 4B, J8, J9, J23, J24) containing
parts of human chromosome 11 (8) were used for the regional
localization of markers on chromosome 11. Genomic DNA isolated
>from chromosome 11 hybrid cell lines was used as template in the
PCR reaction carried out with the primers for microsatellite
markers described in the Genome Data Base.
RESULTS
Localization of microsatellite markers:
Markers D11S1397, D11S902, D11S921, D11S1890, D11S1888 and
D11S1310 were localized to the 'g' segment of chromosome 11p, which
spans the upper half of the 11p14 region. D11S861 and D11S419 were
localized to the 'f' segment which covers 11p15.1 as described by
Glasser et al. (8).
Linkage Analysis:
Lod scores from the two-point analysis of chromosome 11p
microsatellite markers with USH1C in French-Acadian families are
shown in Table 1. All the markers in this region show maximum lod
scores greater than 1.26 without recombination (Table 1). The
highest lod scores obtained were with markers D11S902 (Zmax = 6.44
at theta = 0.00) and D11S1888 (Zmax = 6.40 at theta = 0.0).
Table 1: Two point lod scores of 11p microsatellite markers versus USH1C in French-Acadian families. Marker order corresponds to Figure 1.
| MARKER |
0.00 |
0.01 |
0.02 |
0.03 |
0.04 |
0.05 |
0.10 |
0.20 |
0.30 |
Z max |
Theta |
| D11S861 |
omega |
3.22 |
3.39 |
3.43 |
3.41 |
3.37 |
2.99 |
1.97 |
0.88 |
3.42 |
0.03 |
| D11S419 |
2.95 |
2.83 |
2.77 |
2.71 |
2.65 |
2.58 |
2.25 |
1.54 |
0.82 |
2.95 |
0.00 |
| D11S1397 |
1.26 |
1.22 |
1.18 |
1.14 |
1.10 |
1.06 |
0.87 |
0.50 |
0.21 |
1.26 |
0.00 |
| D11S902 |
6.44 |
6.26 |
6.09 |
5.92 |
5.75 |
5.57 |
4.71 |
3.02 |
1.50 |
6.44 |
0.00 |
| D11S921 |
3.31 |
3.24 |
3.16 |
3.08 |
3.00 |
2.92 |
2.51 |
1.67 |
0.86 |
3.31 |
0.00 |
| D11S1890 |
2.26 |
2.20 |
2.13 |
2.07 |
2.01 |
1.95 |
1.64 |
1.06 |
0.54 |
2.26 |
0.00 |
| D11S1888 |
6.40 |
6.23 |
6.05 |
5.88 |
5.71 |
5.53 |
4.66 |
2.97 |
1.47 |
6.40 |
0.00 |
| D11S1310 |
3.84 |
3.74 |
3.63 |
3.53 |
3.43 |
3.32 |
2.81 |
1.79 |
0.88 |
3.84 |
0.00 |
| D11S899 |
5.46 |
5.31 |
5.16 |
5.01 |
4.85 |
4.70 |
3.95 |
2.51 |
1.23 |
5.46 |
0.00 |
| D11S928 |
omega |
1.58 |
1.81 |
1.91 |
1.97 |
1.99 |
1.89 |
1.34 |
0.70 |
1.99 |
0.05 |
Haplotype analysis in Acadian Families:
In order to localize the USH1C gene more precisely, haplotypes
of chromosomes with the USH1C and normal alleles were determined.
Haplotypes were constructed for markers in the USH1C region in the
following order, as determined by linkage analysis and physical
mapping: tel - D11S861 - D11S419 - D11S1397 - D11S902 - D11S921 -D11S1890 - D11S1888 - D11S1310 - cen (6,16,17). Haplotypes from 70 chromosomes
with the USH1C mutation and 13 non-USH1C chromosomes from the
French-Acadian population were determined. Individuals belonging
to the Acadian USH1C families used in the original localization of
the USH1C gene to chromosome 11(20) were used for haplotype
analysis. Additional individuals not in families 1-7 were
analyzed only if they were affected and then were assumed to have
two chromosomes carrying the USH1C allele, consistent with
autosomal recessive inheritance of Usher syndrome type 1C.
Of the 70 affected chromosomes, 56 had haplotype A as shown in
Table 2. In contrast, haplotype A was not seen on any of the 13
chromosomes with the unaffected allele. One affected chromosome
had haplotype B, which differs from haplotype A only by having
allele 4 rather than 3 at the D11S861 locus. No chromosomes with
the unaffected allele showed haplotype B. Seven additional
chromosomes with the USH1C allele had haplotype C, which differs
>from haplotype A at D11S1397, having allele 3 rather than allele 1.
No chromosomes with the unaffected allele showed haplotype C. Four
chromosomes carrying the USH1C mutation had haplotype D, which
differs from haplotype A by having allele 2 rather than allele 4
at D11S1310. None of the chromosomes with the unaffected allele
showed haplotype D. Two USH1C chromosome showed haplotype E, which
differs from the consensus haplotype A by having allele 1 rather
than 4 at the D11S1888 locus. In addition, the 2 chromosomes with
haplotype E also have allele 3 at D11S1310. One chromosome with the
USH1C mutation had haplotype F, which differs from haplotype A by
having allele 1 at D11S130 locus rather than allele 4. None of the
chromosomes with the unaffected allele carry haplotype E or F.
Table 2. Association of haplotypes with the USH1C mutation in the French-Acadian population of Louisiana. Alleles are named in a fashion consistent with that used in the CEPH database.
|
|
|
|
Markers |
|
|
|
|
Chromosomes |
| Haplotype |
D11S861 |
D11S419 |
D11S1397 |
D11S902 |
D11S921 |
D11S1890 |
D11S1888 |
D11S1310 |
USH(%) |
Normal(%) |
| A |
3 |
2 |
1 |
4 |
3 |
2 |
4 |
4 |
55 (78.0) |
0 |
| B |
4 |
2 |
1 |
4 |
3 |
2 |
4 |
4 |
1 (1.43) |
0 |
| C |
3 |
2 |
3 |
4 |
3 |
2 |
4 |
4 |
7 (10.0) |
0 |
| D |
3 |
2 |
1 |
4 |
3 |
2 |
4 |
2 |
4 (5.71) |
0 |
| E |
3 |
2 |
1 |
4 |
3 |
2 |
1 |
3 |
2 (2.80) |
0 |
| F |
3 |
2 |
1 |
4 |
3 |
2 |
4 |
1 |
1 (1.43) |
0 |
|
2 |
2 |
3 |
3 |
3 |
1 |
1 |
3 |
0 |
1 (0.08) |
|
1 |
2 |
3 |
2 |
2 |
4 |
2 |
4 |
0 |
1 (0.08) |
|
4 |
2 |
1 |
2 |
3 |
2 |
2 |
3 |
0 |
1 (0.08) |
|
2 |
1 |
1 |
2 |
1 |
5 |
1 |
4 |
0 |
1 (0.08) |
|
5 |
2 |
1 |
3 |
3 |
1 |
3 |
4 |
0 |
1 (0.08) |
|
1 |
1 |
1 |
2 |
2 |
2 |
1 |
1 |
0 |
1 (0.08) |
|
1 |
1 |
3 |
2 |
3 |
2 |
2 |
1 |
0 |
1 (0.08) |
|
4 |
1 |
1 |
3 |
1 |
2 |
1 |
2 |
0 |
1 (0.08) |
|
1 |
2 |
1 |
2 |
1 |
3 |
1 |
2 |
0 |
1 (0.08) |
|
1 |
1 |
1 |
2 |
3 |
2 |
2 |
5 |
0 |
1 (0.08) |
|
1 |
1 |
1 |
1 |
3 |
3 |
3 |
3 |
0 |
1 (0.08) |
|
2 |
2 |
3 |
3 |
1 |
2 |
1 |
3 |
0 |
1 (0.08) |
|
6 |
1 |
2 |
2 |
3 |
2 |
2 |
2 |
0 |
1 (0.08) |
|
|
|
|
|
|
|
|
|
___________ |
___________ |
|
|
|
|
|
|
|
|
|
70 |
13 |
Most affected individuals were homozygous for all markers in
the USH1C region, having two copies of the consensus haplotype A
(Table 2; Example: individual 5 of pedigree 101, see Fig. 1). A small number
showed a single allele change from the consensus haplotype A
indicating one copy of haplotype A and one copy of an alternative
haplotype B-E differing by one allele (Examples: individual 11 of
pedigree 102; individual 3 of pedigree 105, see Fig. 1). Finally, an affected individual
not in families 1-7 was homozygous for all markers between D11S861
and D11S1310, but with two copies of haplotype C.
From the pedigree shown in Fig. 1, it was possible to
determine the chromosomal haplotypes of 13 phenotypically normal
parents of individuals affected by USH1C unambiguously. The
haplotypes of the parental chromosomes not transmitted to the
affected offspring were assumed not to contain the USH1C mutation
and were taken as control haplotypes for the Acadian population.
There was no consensus haplotype among these chromosomes and none
of these 13 non-USH1C chromosomes resembled any of the USH1C
associated haplotypes (Table 2).
[Figure 1]
[Pedigree 101]
[Pedigree 102]
[Pedigree 103]
[Pedigree 104]
[Pedigree 105]
[Pedigree 106]
[Pedigree 107]
Note for Fig.1 and Pedigrees: Distinct haplotypes are represented by unique colors in pedigree figures. Non-recombinant affected chromosomes are blue.
DISCUSSION
Usher Syndrome type I C was initially localized to a 5.5
cM interval on the p arm of chromosome 11 bounded by the markers
D11S861 and D11S899 (1, 11) using linkage analysis. Later the USH1C
critical region was futher narrowed to the region between the
markers D11S1397 and D11S1310 (16). Two new microsatellite markers,
D11S1890 and D11S1888 have since been localized to the region
between D11S902 and D11S1310 based on radiation hybrid
mapping (5, 9). However the precise location of these markers with
respect to the other markers on the existing linkage maps is not
known. Preliminary evidence based on construction of a YAC contig
across this region suggests that the likely order is Tel - D11S861 -
D11S419 - D11S1397 - D11S902 - D11S921 - D11S1890 - D11S1888 - D11S1310 -
D11S899 - Cen (17)
Two point linkage analysis of USH1C families with markers
D11S1890 and D11S1888 gave lod scores of 2.26 and 6.40 respectively
without recombination. These results localize the markers
D11S1890 and D11S1888 close to the USH1C locus. Since obligate
recombination events were observed with markers D11S861 and D11S928
in USH1C families from the French-Acadian population in Louisiana
and the markers D11S1890 and D11S1888 showed no recombination,
these two markers could be localized to the region flanked by the
markers D11S861 and D11S928.
The reported order of the markers in this region on linkage
maps was Tel - D11S861 - D11S419 - D11S1397 - D11S902 - D11S921 - D11S1310
-
D11S899 - Cen(7, 16). The markers D11S1890 and D11S1888 map to the g
segment of the J1 hybrid panel, as do the markers D11S1397,
D11S902, D11S921, D11S1301 and D11S899. The markers D11S861 and
D11S419 map to the f segment of chromosome 11p. This suggests that
D11S1890 and D11S1888 are located between D11S419 and D11S899.
These results are in agreement with the reported location of these
to the region bounded by D11S902 and D11S1310 (5, 9). Thus, the
available linkage data, results of the J1 hybrid panel, and our
physical mapping efforts are all consistent with the order:Tel - D11S861 -
D11S419 - D11S1397 - D11S902 - D11S921 - D11S1890 - D11S1888 - D11S1310 -
Cen.
Haplotype analysis of 70 USH1C chromosomes carrying the
disease allele for USH1C and 13 chromosomes carrying normal allele
>from the French-Acadian population was carried out with the map:
Tel - D11S861 - D11S419 - D11S1397 - D11S902 - D11S921 - D11S1890 -
D11S1888 - D11S1310 - Cen. Seventy percent of the affected chromosomes
possess
a common haplotype for markers between D11S1397 to D11S1888.
However, the occurrence of variant haplotypes in some chromosomes
with the USH1C allele restricts the USH1C to the approximately 1cM
interval bounded by D11S1397 and D11S188. These results also
localize the USH1C locus to the upper one third of 11p14 based on
the analysis of these flanking markers on the J1 hybrid panel. The
precise localization of USH1C locus to a smaller region helps in
construction of a physical map of the region encompassing the USH1C
locus and isolation of the gene responsible for Usher Syndrome type
I in the French-Acadian population.
Acknowledgements
We thank Dr. Carol Jones for kindly providing the chromosome
11,
J1 somatic cell hybrid panel lines.
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Received xx September 1995 | Revised 19 October 1995 | Accepted 24 October 1995 | Uploaded 25 October 1995
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