John Gottsch and S. Amer Riazuddin, The Johns Hopkins University; and J. Fielding Hejtmancik, National Eye Institute
Tayyab Husnain, Centre of Excellence in Molecular Biology; Nadeem Butt and Haiba Kaul, Allama Iqbal Medical College; and Z.A. Qazi, Layton Rehmatullah Benevolent Trust Hospital
Pakistani Funding (HEC): $236,801
US Funding (Department of State): $270,326
Project Dates: November 15, 2010 - November 14, 2013 (Extended through November 14, 2014)
Blindness affects all ages and societies worldwide and has enormous personal, social, and economic costs, limiting the education and life choices of otherwise healthy people and placing a significant burden on their families and communities. Every year millions lose their eye sight, mostly through preventable causes, and Pakistan has a particularly high incidence of such cases. While many socioeconomic factors play a role, one of the most important is familial, arising from intra-family marriages, which are socially preferred and widely practiced throughout Pakistan. This project involves a collaborative effort to establish a facility to screen families afflicted with familial forms of cataract and corneal dystrophies and counsel these families to preclude future inheritable cases of blindness. In addition, the research team will investigate the genetic basis of these anomalies in the Pakistani population to enhance the reliability of screening tests. Finally, the researchers will train personnel to confront similar problems beyond the life of this project.
Earlier this year, the US team identified causal mutations in FOXE3 in two familial cases. Later the team further established that the causal mutations do not affect the expression pattern of the FOXE3 protein. During the last 12 months, the team successfully identified the transcriptome (the total transcripts present in cell) and proteome (total protein present in cell) of human cells expressing mutant FOXE3 protein. Next year, the US team will focus on analyzing the data and expect to identify the physiologically relevant molecular targets that are up- and/or down- regulated either at RNA level and/or at the protein levels at the site of pathology in ocular tissue. This will help the team better understand the pathomechanism of cataract development leading to better clinical treatments and therapeutic approaches. The team is also pleased to report that they have successfully completed an animal model for FYCO1, a gene that accounts significantly to the total genetic load of congenital cataracts in the Pakistani population. They are now back-crossing these mice to generate homogenous background to prepare for the histological analysis of the FYCO1 KO lenses utilizing different microscopic techniques.
On the Pakistani side, researchers are continuing to collect samples, clinical data, and extract DNA for analysis. Within the past nine months, a total of 27 cases of congenital cataracts were identified at the Allama Iqbal Medical College and the Layton Rahmatulla Benevolent Trust (LRBT) hospital. The Pakistani team has prepared two manuscripts reporting the results of exclusion analyses completed at CEMB and will be submitting the manuscripts to peer reviewed journals for publication in the coming month.
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2011 Show summary || Hide summary || Download full report
The Pakistani partners have so far identified about 70 cases of congenital cataracts and have enrolled in their study 21 familial cases with multiple affected individuals, with efforts under way to enroll the more recently identified patients. An additional 10 cases of primary congenital glaucoma were identified and enrolled in the study on Pakistani side. Most of the cases identified so far have been in Punjab province, so Pakistani co-PI Dr. Nadeem Butt has established a network of ophthalmologists based in Sindh and Khyber Pakhtunkhwa provinces to help recruit additional study participants in those areas. Ten junior researchers with undergraduate degrees are involved in the project on the Pakistani side (eight of whom are female), and four of them will be beginning their PhD studies next year. On the US side, the researchers report that they have recently identified a gene mutation responsible for 10-12 percent of the total genetic load of recessive cataracts in the Pakistani population, and their article
on this work was published in June 2011 by the American Journal of Human Genetics
. They have completed whole exome-sequencing on the genomic DNA of two individuals affected with congenital cataracts and are applying a whole-genome sequencing approach to analyzing the genomic DNA of five other individuals. The results should be ready for preliminary analysis in the fall of 2011.
2012 Show summary || Hide summary || Download full report
Drs. Husnain and Butt organized the second workshop on this project on November 22, 2011. Held at the Center Excellence in Molecular Biology (CEMB) in Lahore, the workshop was primarily focused on providing theoretical and practical training in molecular and clinical aspects of ophthalmic genetics. US PIs Dr. Gottsch and Dr. Hejtmancik and Dr. Allen Eghrari of the Wilmer Eye Institute delivered lectures via conference call, and Dr. Riazuddin and Dr. Butt made their presentations in person. During his visit to CEMB in connection with the workshop, Dr. Riazuddin also worked with his counterparts on planning future sample collection and data analysis on the project. On the US side, the team has completed next-generation whole genome sequencing on genomic DNA from six cases congenital cateracts and is currently conducting further study of the possible role of selected genes previously localized to several different chromosomes. The researchers are also working to design an experiment involving knockout mice aimed at identifying certain genetic mutations associated with one type of congenital cateract. This work will continue in the coming months; meanwhile, a manuscript on results from work completed earlier in the project is expected to be submitted in May 2012. On the Pakistani side the researchers are continuing to collect samples and extract DNA for analysis. Pakistani co-PI Dr. Nadeem Butt is also working to expand his network pf ophthalmologists based in Sindh and Khyber Pakhtunkhwa provinces to help recruit additional study participants in those areas. Ten junior researchers with undergraduate degrees are involved in the project on the Pakistani side (eight of whom are female).
During this quarter, a total of two cases of congenital cataracts were identified at the Allama Iqbal Medical College and two cases were identified at the Layton Rahmatulla Benevolent Trust hospital. The identification and recruitment of familial cases of congenital cataracts and will continue in the next few months. Initiated by Dr. Butt in the end of 2011, a network of anterior segment ophthalmologist that identifies and recruits familial cases presented in clinics and/or hospitals in the Sindh and Khyber Pakhtunkhwa provinces has been established, with the logistics of recruiting familial cases remaining to be developed.
On the US side, the research team reported the identification of three new genes that harbor causal mutations responsible for the disease phenotype in three of the six families (samples collected in earlier study). The team is currently investigating the functional significance of these genetic variations. Also, to further their study on FYCO1, the team is breeding four heterozygous Fyco1 conditional knock-out (KO) male mice they recently received in order to produce heterozygous Fyco1 conditional KO females. These heterozygous animals will be inbred to obtain homozygous KO animals (expected in September or October 2012), which will provide an in vivo model for studying the role of Fyco1 in the development and maintenance of the ocular lens. Due to technical difficulties, the team was not able to make substantial progress on the project component initiated to develop a model system to establish the role of FYCO1 in chaperon mediated autophagy. In the next few months, the team will be constructing an human lens epithelial cell line that will express the tetracycline repressor protein in order to overcome the technical challenges they have faced.
2013 Show summary || Hide summary || Download full report
On the US side, the team is conducting further studies of the selected genes previously localized to several different chromosomes. The team previously identified FOXE3 mutations in two familial cases and using genetic sequencing and in cultured cells to understand the functional significance of the mutations that likely interfere with the development of the ocular lens. They are using genetically engineered “knock-out” mice as animal model to identifying certain genetic mutations associated with one type of congenital cataract. The researchers signed a MTA with the European Mouse Mutant Archive to provide these mice.
On the Pakistani side, researchers are continuing to collect samples, clinical data, and extract DNA for analysis. Within the first half of 2013, a total of 16 cases of congenital cataracts were identified at the Allama Iqbal Medical College and 9 cases were identified at The Layton Rahmatulla Benevolent Trust (LRBT) hospital. The Pakistani team is drafting a manuscript describing the first report of associating LIM2 with autosomal recessive congenital cataract (arCC) in Pakistan that will be submitted for publication later this summer.