Simultaneous mutation detection in 90 retinal disease genes in multiple patients using a custom-designed 300-kb retinal resequencing chip.

PURPOSE: To develop a high-throughput, cost-effective diagnostic strategy for the identification of known and new mutations in 90 retinal disease genes. DESIGN: Evidence-based study. PARTICIPANTS: Sixty patients with a variety of retinal disorders, including Leber's congenital amaurosis, ocular albinism, pseudoxanthoma elasticum, retinitis pigmentosa, and Stargardt's disease. METHODS: We designed a custom 300-kb resequencing chip. Polymerase chain reaction (PCR) amplification, DNA fragmentation, and chip hybridization were performed according to Affymetrix recommendations. Hybridization signals were analyzed using Sequence pilot module seq-C mutation detection software (2009). This resequencing approach was validated by Sanger sequence technology. MAIN OUTCOME MEASURES: Disease-causing sequence changes. RESULTS: We developed a retinal resequencing chip that covers all exons of 90 retinal disease genes. We developed and tested multiplex primer sets for 1445 amplicons representing the genes included on the chip. We validated our approach by screening 87 exons from 25 retinal disease genes containing 87 known sequence changes previously identified in our patient group using Sanger sequencing. Call rates for successfully hybridized amplicons were 98% to 100%. Of the known single nucleotide changes, 99% could be detected on the chip. As expected, deletions could not be detected reliably. CONCLUSIONS: We designed a custom resequencing chip that can detect known and new sequence changes in 90 retinal disease genes using a new high-throughput strategy with a high sensitivity and specificity for one tenth of the cost of conventional direct sequencing. The developed amplification strategy allows for the pooling of multiple patients with non-overlapping phenotypes, enabling many patients to be analyzed simultaneously in a fast and cost-effective manner.

Course of visual decline in relation to the Best1 genotype in vitelliform macular dystrophy.

PURPOSE: To describe the disease course in patients with vitelliform macular dystrophy (VMD) with a Best1 mutation and to determine the association between Best1 genotype and visual prognosis. DESIGN: Consecutive case series. PARTICIPANTS: Fifty-three patients with VMD with Best1 mutations from 27 Dutch families, aged 11 to 87 years. METHODS: Best-corrected visual acuity (VA), fundus appearance, and Arden ratio on the electro-oculogram (EOG) during clinical follow-up were assessed from medical records. Mutation analysis of the Best1 gene was performed on DNA samples using denaturing high-pressure liquid chromatography and direct sequencing. MAIN OUTCOME MEASURES: Cumulative lifetime risk of visual decline below 0.5, 0.3, and 0.1 for the entire group and stratified for genotype. RESULTS: Median age of onset of visual symptoms was 33 years (range: 2-78). The cumulative risk of VA below 0.5 (20/40) was 50% at 55 years and 75% at 66 years. The cumulative risk of decline less than 0.3 (20/63) was 50% by age 66 years and 75% by age 74 years. Two patients progressed to VA less than 0.1 (20/200). Fourteen different mutations were found. Most patients (96%) had missense mutations; the Thr6Pro, Ala10Val, and Tyr227Asn mutations were most common. Visual decline was significantly faster in patients with an Ala10Val mutation than either the Thr6Pro or the Tyr227Asn mutation (P=0.001). CONCLUSIONS: Age of onset of visual symptoms varies greatly among patients with VMD. All patients show a gradual decrease in VA, and most progress to visual impairment at a relatively late age. Our data suggest a phenotype-genotype correlation, because the Ala10Val mutation has a more rapid disease progression than other common mutations. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Complement component C3 and risk of age-related macular degeneration.

OBJECTIVE: To explore the association between polymorphisms in the complement component 3 (C3) gene and age-related macular degeneration (AMD), and to investigate the modifying effect of complement factor H (CFH) Y402H, LOC387715 A69S and smoking. DESIGN: Pooled data from the prospective, population-based Rotterdam Study (enrolment between 1990 and 1993, and 3 follow-up examinations between September 1, 1993, and December 31, 2004) and an independent case-control study from the Netherlands. PARTICIPANTS: The Rotterdam Study comprised a total of 6418 persons aged >or=55 years who had gradable fundus photographs. The case-control study consisted of 357 unrelated AMD patients and 173 control individuals aged >or=55 years. METHODS: The variants R102G and P314L of the C3 gene, CFH Y402H and LOC387715 A69S, were genotyped in all study participants. Information on cigarette smoking was obtained by interview at baseline. MAIN OUTCOME MEASURES: Early and late stages of prevalent and incident AMD, graded according to the international classification and grading system for AMD. RESULTS: We found a population frequency of 0.217 for R102G and 0.211 for P314L in the Rotterdam Study. Both alleles significantly increased the risk of early AMD and all subtypes of late AMD, and this risk seemed to be independent of CFH Y402H, LOC387715 A69S, and smoking. Detailed analysis showed that the haplotype carrying both alleles had the highest frequency difference between cases and controls (P=0.006). We estimated a total population-attributable risk of 14.6%. A meta-analysis of all currently available data yielded a pooled odds ratio (OR) of 1.61 (95% confidence interval [CI], 1.46-1.78) for the R102G allele, and an OR of 1.50 (95% CI, 1.31-1.71) for the P314L allele. CONCLUSIONS: Our study showed a significant association between variants in the C3 gene and AMD and further highlights the crucial role of the complement pathway in the etiology of AMD.

Comprehensive analysis of the candidate genes CCL2, CCR2, and TLR4 in age-related macular degeneration.

PURPOSE: To determine whether variants in the candidate genes TLR4, CCL2, and CCR2 are associated with age-related macular degeneration (AMD). METHODS: This study was performed in two independent Caucasian populations that included 357 cases and 173 controls from the Netherlands and 368 cases and 368 controls from the United States. Exon 4 of the TLR4 gene and the promoter, all exons, and flanking intronic regions of the CCL2 and CCR2 genes were analyzed in the Dutch study and common variants were validated in the U.S. study. Quantitative (q)PCR reactions were performed to evaluate expression of these genes in laser-dissected retinal pigment epithelium from 13 donor AMD and 13 control eyes. RESULTS: Analysis of single nucleotide polymorphisms (SNPs) in the TLR4 gene did not show a significant association between D299G or T399I and AMD, nor did haplotypes containing these variants. Univariate analyses of the SNPs in CCL2 and CCR2 did not demonstrate an association with AMD. For CCR2, haplotype frequencies were not significantly different between cases and controls. For CCL2, one haplotype containing the minor allele of C35C was significantly associated with AMD (P = 0.03), but this did not sustain after adjustment for multiple testing (q = 0.30). Expression analysis did not demonstrate altered RNA expression of CCL2 and CCR2 in the retinal pigment epithelium from AMD eyes (for CCL2 P = 0.62; for CCR2 P = 0.97). CONCLUSIONS: No evidence was found of an association between TLR4, CCR2, and CCL2 and AMD, which implies that the common genetic variation in these genes does not play a significant role in the etiology of AMD.

High-throughput identification of potential minor histocompatibility antigens by MHC tetramer-based screening: feasibility and limitations.

T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1(IMA) antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent. Collectively these results demonstrate the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack biological relevance.

Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury.

Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.