Inclusion of genotype with fundus phenotype improves accuracy of predicting choroidal neovascularization and geographic atrophy.

PURPOSE: The accuracy of predicting conversion from early-stage age-related macular degeneration (AMD) to the advanced stages of choroidal neovascularization (CNV) or geographic atrophy (GA) was evaluated to determine whether inclusion of clinically relevant genetic markers improved accuracy beyond prediction using phenotypic risk factors alone. DESIGN: Cohort study. PARTICIPANTS: White, non-Hispanic subjects participating in the Age-Related Eye Disease Study (AREDS) sponsored by the National Eye Institute consented to provide a genetic specimen. Of 2415 DNA specimens available, 940 were from disease-free subjects and 1475 were from subjects with early or intermediate AMD. METHODS: DNA specimens from study subjects were genotyped for 14 single nucleotide polymorphisms (SNPs) in genes shown previously to associate with CNV: ARMS2, CFH, C3, C2, FB, CFHR4, CFHR5, and F13B. Clinical demographics and established disease associations, including age, sex, smoking status, body mass index (BMI), AREDS treatment category, and educational level, were evaluated. Four multivariate logistic models (phenotype; genotype; phenotype + genotype; and phenotype + genotype + demographic + environmental factors) were tested using 2 end points (CNV, GA). Models were fitted using Cox proportional hazards regression to use time-to-disease onset data. MAIN OUTCOME MEASURES: Brier score (measure of accuracy) was used to identify the model with the lowest prediction error in the training set. The most accurate model was subjected to independent statistical validation, and final model performance was described using area under the receiver operator curve (AUC) or C-statistic. RESULTS: The CNV prediction models that combined genotype with phenotype with or without age and smoking revealed superior performance (C-statistic = 0.96) compared with the phenotype model based on the simplified severity scale and the presence of CNV in the nonstudy eye (C-statistic = 0.89; P<0.01). For GA, the model that combined genotype with phenotype demonstrated the highest performance (AUC = 0.94). Smoking status and ARMS2 genotype had less of an impact on the prediction of GA compared with CNV. CONCLUSIONS: Inclusion of genotype assessment improves CNV prediction beyond that achievable with phenotype alone and may improve patient management. Separate assessments should be used to predict progression to CNV and GA because genetic markers and smoking status do not equally predict both end points. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Multiplexed analysis of circulating cell-free fetal nucleic acids for noninvasive prenatal diagnostic RHD testing.

OBJECTIVE: The objective of the study was the evaluation of a novel multiplex assay to detect fetal Rh blood group D-antigen gene (RHD) loci in maternal plasma from RhD-negative, pregnant women. STUDY DESIGN: An RHD genotyping assay was designed to detect exons 4, 5, 7, and 10 and RHDΨ (pseudogene) of the RHD gene along with a Y chromosome-specific assay and a generic polymerase chain reaction amplification control. Plasma samples from 150 RhD-negative pregnant women were assayed for fetal RHD genotype using the MassARRAY system. RESULTS: The fetal RHD status of 148 of 150 samples (98.7%) was correctly classified; 86 (57.3%) and 62 (41.3%) were positive and negative, respectively. CONCLUSION: This study demonstrates that noninvasive prenatal diagnostics with a single-reaction multiplexed assay is a viable path toward routine characterization of fetal RHD genotypes using circulating cell-free fetal DNA in maternal plasma on the MassARRAY system and is perhaps preferable to serologic testing as currently used clinically.

Circulating Cell-Free DNA to Determine the Fetal RHD Status in All Three Trimesters of Pregnancy.

OBJECTIVE: To estimate the accuracy of a new assay to determine the fetal RHD status using circulating cell-free DNA. METHODS: This was a prospective, observational study. Maternal blood samples were collected in each trimester of pregnancy in 520 nonalloimmunized RhD-negative patients. Plasma samples were analyzed for circulating cell-free DNA using the SensiGENE RHD test, which used primers for exons 4 and 7 as previously described and incorporated a new primer design for exon 5 of the RHD gene. Neonatal serology for RhD typing using cord blood at birth was undertaken and results were stored in a separate clinical database. After unblinding the data, results of the DNA analysis were compared with the neonatal serology. RESULTS: Inconclusive results secondary to the presence of the RHD pseudogene or an RHD variant were noted in 5.6%, 5.7%, and 6.1% of the first-, second-, and third-trimester samples, respectively. The incidence of false-positive rates for RhD (an RhD-negative fetus with an RHD-positive result) was 1.54% (95% confidence interval [CI] 0.42-5.44%), 1.53% (CI 0.42-5.40%), and 0.82% (CI 0.04-4.50%), respectively. There was only one false-negative diagnosis (an RhD-positive fetus with an RHD-negative result), which occurred in the first trimester (0.32%; 95% CI 0.08-1.78%). Genotyping for mismatches across repeated samples revealed that this error was related to mislabeling of samples from two patients collected on the same day at one of the collection sites. Overall test results were in agreement across all three trimesters (P>.99). CONCLUSION: Circulating cell-free DNA can accurately predict the fetal RhD status in all three trimesters of pregnancy.