The correlation between CpG methylation and gene expression is driven by sequence variants.

Gene promoter and enhancer sequences are bound by transcription factors and are depleted of methylated CpG sites (cytosines preceding guanines in DNA). The absence of methylated CpGs in these sequences typically correlates with increased gene expression, indicating a regulatory role for methylation. We used nanopore sequencing to determine haplotype-specific methylation rates of 15.3 million CpG units in 7,179 whole-blood genomes. We identified 189,178 methylation depleted sequences where three or more proximal CpGs were unmethylated on at least one haplotype. A total of 77,789 methylation depleted sequences (~41%) associated with 80,503 cis-acting sequence variants, which we termed allele-specific methylation quantitative trait loci (ASM-QTLs). RNA sequencing of 896 samples from the same blood draws used to perform nanopore sequencing showed that the ASM-QTL, that is, DNA sequence variability, drives most of the correlation found between gene expression and CpG methylation. ASM-QTLs were enriched 40.2-fold (95% confidence interval 32.2, 49.9) among sequence variants associating with hematological traits, demonstrating that ASM-QTLs are important functional units in the noncoding genome.

A PRPH splice-donor variant associates with reduced sural nerve amplitude and risk of peripheral neuropathy.

Nerve conduction (NC) studies generate measures of peripheral nerve function that can reveal underlying pathology due to axonal loss, demyelination or both. We perform a genome-wide association study of sural NC amplitude and velocity in 7045 Icelanders and find a low-frequency splice-donor variant in PRPH (c.996+1G>A; MAF = 1.32%) associating with decreased NC amplitude but not velocity. PRPH encodes peripherin, an intermediate filament (IF) protein involved in cytoskeletal development and maintenance of neurons. Through RNA and protein studies, we show that the variant leads to loss-of-function (LoF), as when over-expressed in a cell line devoid of other IFs, it does not allow formation of the normal filamentous structure of peripherin, yielding instead punctate protein inclusions. Recall of carriers for neurological assessment confirms that from an early age, homozygotes have significantly lower sural NC amplitude than non-carriers and are at risk of a mild, early-onset, sensory-negative, axonal polyneuropathy.

Retinal oximetry with a scanning laser ophthalmoscope.

PURPOSE: The purpose of the study was to assess if a scanning laser ophthalmoscope (SLO), Optomap 200Tx, could be used for measurements of hemoglobin oxygen saturation in retinal blood vessels. METHODS: Optomap 200Tx uses two lasers for image acquisition, 532 and 633 nm. Retinal images of healthy individuals and patients with retinal vein occlusion were analyzed with modified Oxymap Analyzer software, which tracks retinal vessels and calculates relative hemoglobin oxygen saturation. RESULTS: Oxygen saturation in healthy individuals was measured as 92% ± 13% for arterioles and 57% ± 12% for venules (mean ± SD, n = 11, P = 0.0001). Standard deviation for repeated measurements of the same eye was 3.5% for arterioles and 4.4% for venules. In patients with confirmed venular hypoxia, central retinal vein occlusion (CRVO) or hemivein occlusion, the average venular oxygen saturation was measured as 23% ± 3% in the affected eyes and 59% ± 3% in the fellow eyes (n = 4, P = 0.0009). CONCLUSIONS: Technically, it is possible to derive information on retinal oxygen saturation from an SLO with a 2-wavelength oximetry algorithm. The system produced both sensitive and repeatable results. The remaining challenges include decreasing variability between vessels of the same eye and variability between individuals. Given the advantages that SLO imaging has over conventional fundus camera optics in retinal oximetry, further development of SLO oximetry may provide the optimal approach to retinal oximetry.

Reliability of vessel diameter measurements with a retinal oximeter.

BACKGROUND: The purpose of this study was to test the reliability of vessel diameter measurements with a newly developed retinal oximeter. METHODS: Twelve healthy individuals participated in the study. Retinal images were taken with the Oxymap Retinal Oximeter. Diameters of retinal vessels were measured automatically with the Oxymap Analyzer software. Repeated measurements on the same vessel segments were compared. The automatic measurements were also compared with semi-automatic measurements with a plug-in for the ImageJ software. RESULTS: Variance coefficient (standard deviation/mean) from the repeatability test was 2.8 and 4.0% for first- and second-degree venules and 3.5 and 5.4% for first- and second-degree arterioles, respectively. ImageJ measured larger mean diameters than Oxymap Analyzer in all cases. Means of differences were 5.1 ± 2.2, 2.9 ± 1.3, and 2.7 ± 1.6 pixels for first-, second-, and third-degree venules and 3.1 ± 1.2, 2.7 ± 0.9, and 2.9 ± 1.4 pixels for first-, second-, and third-degree arterioles. CONCLUSIONS: Vessel diameter measurements with the oximeter are repeatable and comparison with an established method demonstrates a relatively stable offset where the standard deviation of the difference is rather small. Different definitions of vessel borders may be the cause of this difference.

Glaucoma filtration surgery and retinal oxygen saturation.

PURPOSE: Glaucoma may involve disturbances in retinal oxygenation and blood flow. The purpose of this study was to measure the effect of glaucoma filtration surgery on retinal vessel oxygen saturation. METHODS: A noninvasive spectrophotometric retinal oximeter was used to measure hemoglobin oxygen saturation in retinal arterioles and venules before and after glaucoma filtration surgery. Twenty-five consecutive patients were recruited, and 19 had adequate image quality. Fourteen underwent trabeculectomy and five glaucoma tube surgery. Twelve had primary open-angle glaucoma and seven had exfoliative glaucoma. IOP decreased from 23 +/- 7 to 10 +/- 4 mm Hg (mean +/- SD, P = 0.0001). RESULTS: Oxygen saturation increased in retinal arterioles from 97% +/- 4% to 99% +/- 6% (n = 19; P = 0.046) after surgery and was unchanged in venules (63% +/- 5% before surgery and 64% +/- 6% after, P = 0.76). There were no significant changes in saturation in the fellow eyes (P > 0.60). The arteriovenous difference was 34% before and 36% after surgery (P = 0.35). CONCLUSIONS: Glaucoma filtration surgery had almost no effect on retinal vessel oxygen saturation.

Oxygen saturation in human retinal vessels is higher in dark than in light.

PURPOSE: Animal studies have indicated that retinal oxygen consumption is greater in dark than light. In this study, oxygen saturation is measured in retinal vessels of healthy humans during dark and light. METHODS: The oximeter consists of a fundus camera, a beam splitter, a digital camera and software, which calculates hemoglobin oxygen saturation in the retinal vessels. In the first experiment, 18 healthy individuals underwent oximetry measurements after 30 minutes in the dark, followed by alternating 5-minute periods of white light (80 cd/m(2)) and dark. In the second experiment, 23 volunteers underwent oximetry measurements after 30 minutes in the dark, followed by light at 1, 10, and 100 cd/m(2). Three subjects were excluded from analysis in the first experiment and four in the second experiment because of poor image quality. RESULTS: In the first experiment, the arteriolar saturation decreased from 92% +/- 4% (n = 15; mean +/- SD) after 30 minutes in the dark to 89% +/- 5% after 5 minutes in the light (P = 0.008). Corresponding numbers for venules are 60% +/- 5% in the dark and 55% +/- 10% (P = 0.020) in the light. In the second experiment, the arteriolar saturation was 92% +/- 4% in the dark and 88% +/- 7% in 100 cd/m(2) light (n = 19, P = 0.012). The corresponding values for venules were 59% +/- 9% in the dark and 55% +/- 10% in 100 cd/m(2) light (P = 0.065). CONCLUSIONS: Oxygen saturation in retinal blood vessels is higher in dark than in 80 or 100 cd/m(2) light in human retinal arterioles and venules. The authors propose that this is a consequence of increased oxygen demand in the outer retina in the dark.

Automatic retinal oximetry.

PURPOSE: To measure hemoglobin oxygen saturation (SO(2)) in retinal vessels and to test the reproducibility and sensitivity of an automatic spectrophotometric oximeter. METHODS: Specialized software automatically identifies the retinal blood vessels on fundus images, which are obtained with four different wavelengths of light. The software calculates optical density ratios (ODRs) for each vessel. The reproducibility was evaluated by analyzing five repeated measurements of the same vessels. A linear relationship between SO(2) and ODR was assumed and a linear model derived. After calibration, reproducibility and sensitivity were calculated in terms of SO(2). Systemic hyperoxia (n = 16) was induced in healthy volunteers by changing the O(2) concentration in inhaled air from 21% to 100%. RESULTS: The automatic software enhanced reproducibility, and the mean SD for repeated measurements was 3.7% for arterioles and 5.3% venules, in terms of percentage of SO(2) (five repeats, 10 individuals). The model derived for calibration was SO(2) = 125 - 142 . ODR. The arterial SO(2) measured 96% +/- 9% (mean +/- SD) during normoxia and 101% +/- 8% during hyperoxia (n = 16). The difference between normoxia and hyperoxia was significant (P = 0.0027, paired t-test). Corresponding numbers for venules were 55% +/- 14% and 78% +/- 15% (P < 0.0001). SO(2) is displayed as a pseudocolor map drawn on fundus images. CONCLUSIONS: The retinal oximeter is reliable, easy to use, and sensitive to changes in SO(2) when concentration of O(2) in inhaled air is changed.