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1.  Molecular diagnosis of putative Stargardt disease probands by exome sequencing 
BMC Medical Genetics  2012;13:67.
The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases.
We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis.
Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified.
Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.
PMCID: PMC3459799  PMID: 22863181
Stargardt Disease; Macular Degeneration; Exome; Mutation Screening; Molecular Diagnostics; ABCA4; PRPH2
2.  Association of the Asn306Ser variant of the SP4 transcription factor and an intronic variant in the β-subunit of transducin with digenic disease 
Molecular Vision  2007;13:287-292.
SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations.
270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants.
Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the α-, β- and γ-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the β-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant.
If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.
PMCID: PMC2633482  PMID: 17356515

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