Dominant optic atrophy: exclusion and fine genetic mapping of the
candidate gene, HRY
Anthony T. Moore,
Shom S. Bhattacharya
Department of Molecular Genetics, Institute of Ophthalmology, University College London, 11-43, Bath Street, London EC1V 9EL, UK
Moorfields Eye Hospital, City Road, London EC1V 2PD, UK
Received: 27 February 1998 / Accepted: 8 June 1998
Abstract. Autosomal dominant optic atrophy (OPA1) maps to
Chromosome (Chr) 3q28, and the disease interval has been refined
to within 1.4 cM, flanked by the markers D3S3669 and D3S3562.
HRY, the human homolog of the Drosophila segmentation gene,
hairy, maps by in situ hybridization to the chromosomal region
3q28-q29. We screened for mutations in HRY in 36 patients from
18 pedigrees with dominant optic atrophy and a group of normal
control individuals. Heteroduplex mutation analysis and direct se-
quencing of all four coding exons and one upstream putative un-
translated exon were performed. No disease-associated sequence
alterations were identified. A polymorphism in the untranslated
region of exon 2 was found, with four alleles. PCR amplification
of this part of exon 2 in four of the pedigrees affected by autosomal
dominant optic atrophy mapping to chromosome 3q, followed by
haplotype analysis, showed recombination between HRY and
OPA1 in one pedigree. This allows us to genetically position HRY
in relation to known microsatellite markers in the region, placing
HRY telomeric to marker D3S3562 and centromeric to D3S1305.
This is outside the published critical disease interval for dominant
optic atrophy. We have, therefore, excluded HRY as the gene for
dominant optic atrophy by sequence analysis, mapped it geneti-
cally, and identified a polymorphism in our population.
Autosomal dominant optic atrophy (McKusick No: 165500,
OPA1) is an inherited optic neuropathy causing loss of visual
acuity, color vision abnormalities, and visual field defects, char-
acterized by temporal optic nerve pallor. Both electrophysiology
(Elenius et al. 1991) and histopathology (Johnston et al. 1979; Kjer
1983) suggest the defect is in the ganglion cell layer.
The disease gene was mapped in 1994 (Eiberg et al. 1994) by
genetic linkage analysis to a 10-cM interval on Chr 3q28-qter. The
disease interval has been refined to within 1.4 cM (Jonasdottir et
al. 1997), flanked by the microsatellite markers D3S3669 and
D3S3562. There is evidence for the predominance of the Chr 3q
locus (Bonneau et al. 1995; Lunkes et al. 1995; Brown et al. 1997;
Johnston et al. 1997; Stoilova et al. 1997; Votruba et al. 1997),
although a family has recently been reported mapping to Chr
18q12.2-12.3 (Kerrison et al. 1998).
The human homolog HRY of the Drosophila segmentation
gene, hairy, has been cloned and sublocalized to Chr 3q28-q29 by
fluorescence in situ hybridization (Feder et al. 1994). The Dro-
sophila hairy gene encodes a basic helix-loop-helix protein that is
known to play a role in embryogenesis and segmentation (Rushlow
et al. 1989). Hes 1, the mouse homolog, suppresses neuronal dif-
ferentiation, maintains a proliferative state, and is rapidly induced
by growth factors known to influence neuronal differentiation
(Feder et al. 1993). It is expressed at high levels in neuroretinal
progenitors. The Hes 1 overexpression phenotype shows undiffer-
entiated retinal cells (Tomita et al. 1996). Mice lacking Hes 1
function show premature retinal differentiation and die at birth
from severe neural tube defects (Ishibashi et al. 1995). These fac-
tors make the gene HRY a good positional and functional candi-
date gene for dominant optic atrophy.
The coding region of the human gene comprises exons 2–5,
with an upstream putative untranslated exon 1. HRY has been
positioned on a YAC contig of the OPA1 region (Jonasdottir et al.
1997), mapping onto the CEPH YAC 975f4, which spans part of
the OPA1 critical interval and stretches beyond it. Unpublished
data (Jonasdottir et al. 1997) suggest that the sequence of exons 2,
3, and 4 is normal in individuals with dominant optic atrophy. The
largest exon 5, the 5Ј-untranslated region of exon 2, and the pu-
tative untranslated exon 1 were not evaluated by these authors,
who were, therefore, not able to conclusively exclude HRY as the
gene responsible for dominant optic atrophy.
We screened 36 individuals from 18 pedigrees from The Moor-
fields Eye Hospital Genetic Clinic database for mutations in the
human HRY gene. Blood samples (2 × 10 ml) were taken from two
affected individuals from each of 14 UK pedigrees and from 77
members of four further pedigrees [Pedigrees A, F, and L (Votruba
et al. 1997, 1998) and B, see Fig. 1] with dominant optic atrophy,
and DNA was extracted (Nucleon II DNA extraction kit, Scotlab,
Bioscience). The diagnosis of dominant optic atrophy was con-
firmed by a single examiner (MV) in all cases, and all the families
showed clear evidence of a dominant inheritance pattern. Nine of
the pedigrees had a Lod score of over 3 on linkage analysis to the
Chr 3q28-qter locus for dominant optic atrophy. Nine others were
from pedigrees that were too small for formal linkage to give a Lod
score of over 3, but none of them excluded the locus on linkage or
haplotype assessment (Votruba et al. 1998).
A 9CA repeat (bp178–195) in the genomic sequence of HRY
(Feder et al. 1994) (EMBL/GenBank accession No: L19314) was
identified in the putative untranslated exon 1 and evaluated for
potential polymorphism, by amplifying DNAs from 45 samples
from two of the unrelated families, A and L, by means of oligo-
nucleotide primers designed to flank the repeat sequence (Table 1).
The forward PCR primer was 5Ј-end-labeled with ␥
110 TBq/mmol; Amersham) and T4 polynucleotide kinase prior to
amplification. The PCR reaction mix of 10 L contained 200 ng of
genomic DNA, 0.25
of the forward and reverse primer, 50 m
KCl, 10 m
Tris-HCl (pH 9.0), 0.15% Triton-X-100, 1.5 m
of each dNTP, and 0.6 U of Taq DNA polymerase.
Amplification was performed with denaturation at 95°C for 5 min
and 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s.
The products were separated by electrophoresis on 6% polyacryl-
Correspondence to: M. Votruba
© Springer-Verlag New York Inc. 1998Mammalian Genome 9, 784–787 (1998).