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
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.