A research group at the Max Planck Institute
for Molecular Genetics in Berlin identified the gene defect
underlying a specific form of hereditary blindness, known as
retinitis pigmentosa (nature genetics, Vol. 19, No. 4, August
1998).
Retinitis pigmentosa (RD) is characterized by premature cell
death in the retina leading to a progressive contraction of
the visual field in affected patients. The retina lines the
back of the eye and contains different types of cells,
including the photoreceptors and neurons. There are two main
classes of photoreceptor cells: rods and cones. Rods are
responsible for vision under dim light conditions while cones
participate in fine and color vision. RP leads to a
preferential loss of the rod photoreceptor cells and night
blindness is one of the first clinical symptoms. During
disease progression, cones are affected as well and patients
become legally blind between the ages of 20 and 40.
The name retinitis pigmentosa reflects
observations with patients in whom ophthalmologists
frequently recognize an abnormal pigmentation in the back of
the eye as a consequence of the dying photoreceptor cells. So
far, 14 genes were identified which, when defective, lead to
the disease. Additional 14 loci were defined by genetic
linkage analysis in families affected by the disease. In
these cases, the genetic defect still has to be identified.
Most of the genes involved in retinitis pigmentosa encode
proteins from the so called phototransduction
cascade, a complex biochemical mechanism which
transforms the initial light stimulus to a chemical signal.
The latter accomplishes the communication of the
photoreceptor cells with the neurons of the retina.
Retinitis pigmentosa, due to a genetic defect, occurs with a
frequency of 1 in 4,000 individuals and about 15-25% of those
are caused by mutations in genes residing on the X
chromosome. Approximately one fifth of the X-chromosomal
cases is caused by mutations within the gene identified by
Uwe Schwahn and colleagues from the Max Planck Institute for
Molecular Genetics in Berlin (Dahlem). X-linked RP is
considered one of the most severe forms in terms of onset and
progression. The disease onset occurs by the time the patient
has turned 20 and progresses to legal blindness within 10-20
years.
The novel gene was identified by a molecular-biology strategy
known as positional cloning approach. The
isolation of genes by positional cloning is based on the
location of the gene in the human genome, without detailed
information on the biological function of the gene product.
The challenge was to find the needle of 3,800 base pairs
(which represents the protein coding portion of the RP2
gene) in a haystack of 4,000,000 base pairs of X-chromosomal
DNA where, according to data from family studies, the RP2
gene must be located. In order to get a hint where to look
first, the researchers applied the relatively new yeast
artificial chromosome (YAC) representation hybridization
technique. The first report on this technique was issued only
two years ago when they and their co-workers at the
University Hospital Nijmegen (The Netherlands) had narrowed
down the RP3 gene region and finally cloned the gene.
The technique essentially compares the DNA of a control to
that of patients. If there is a difference between them, this
shows up as aberrant pattern in the patient´s DNA.
The research group led by Wolfgang Berger identified such an
aberrant pattern in one out of 26 patients. The aberrant
hybridization pattern turned out to be caused by an insertion
of a mobile DNA element, called LINE1 (Long Interspersed
Nuclear Element). These genome free-riders have
the capacity to copy themselves occasionally from one
location to another. During evolution, they have colonized
the human DNA in a considerable number: about 60,000 copies
reside in the human genome. In this particular case, it
seemed that the insertion process had disrupted the function
of the RP2 gene.
Simple sequence determination around the integration site
failed to give a hint for a disrupted gene; also searching
physical and electronic libraries for transcribed sequences
(cDNA) around the integration site failed to succeed.
Therefore, lead author Uwe Schwahn and his co-workers decided
to use an alternative method called exon
trapping. This is an artificial transcription system
that identifies protein coding DNA stretches (exons) from a
genomic DNA source. The advantage now was that this approach
was independent of tissue type and transcription level of the
gene in question (libraries of transcribed sequences are
often made from a specific tissue and always represent only
the sufficiently transcribed genes).
Indeed, the breakthrough came from such a trapped exon that
showed significant homology to a transcribed sequence in the
database and finally identified the full length transcript of
the RP2 gene in a cDNA library. Seven out of 38
patients with X-chromosomal RP showed mutations in this new
gene, which has homology to cofactor C, a protein known to
play a role in beta-tubulin folding. Tubulins build up the
skeleton of cells, and assure internal cellular transport as
well as cell division. If this homology turns out to be of
functional relevance, then RP2 is the first example of
hereditary blindness where a malfunction of the cytoskeleton
forms the basis of premature cell death in the retina.
What does these findings mean for the patients and their
families? Although these results have no direct impact on
treatment of patients (currently there is no adequate
treatment for any form of retinitis pigmentosa), it helps to
identify the spectrum of DNA alterations in patients with the
disease as a prerequisite for a more efficient genetic
counselling. Additionally, the functional analysis of the
gene product will help to understand the molecular pathology
of retinitis pigmentosa and hopefully contribute to the
development of novel therapeutic strategies to cure this
severe form of blindness.
Journal
Nature Genetics