Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

Loss-of-function variants in UBAP1L cause autosomal recessive retinal degeneration.

PURPOSE: Inherited retinal diseases (IRDs) are a group of monogenic conditions that can lead to progressive blindness. Their missing heritability is still considerable, due in part to the presence of disease genes that await molecular identification. The purpose of this work was to identify novel genetic associations with IRDs. METHODS: Patients underwent a comprehensive ophthalmological evaluation using standard-of-care tests, such as detailed retinal imaging (macular optical coherence tomography and short-wavelength fundus autofluorescence) and electrophysiological testing. Exome and genome sequencing, as well as computer-assisted data analysis were used for genotyping and detection of DNA variants. A minigene-driven splicing assay was performed to validate the deleterious effects of 1 of such variants. RESULTS: We identified 8 unrelated families from Hungary, the United States, Israel, and The Netherlands with members presenting with a form of autosomal recessive and nonsyndromic retinal degeneration, predominantly described as rod-cone dystrophy but also including cases of cone/cone-rod dystrophy. Age of disease onset was very variable, with some patients experiencing first symptoms during their fourth decade of life or later. Myopia greater than 5 diopters was present in 5 of 7 cases with available refractive data, and retinal detachment was reported in 2 cases. All ascertained patients carried biallelic loss-of-function variants in UBAP1L (HGNC: 40028), a gene with unknown function and with homologies to UBAP1, encoding a protein involved in ubiquitin metabolism. One of these pathogenic variants, the intronic NM_001163692.2:c.910-7G>A substitution, was identified in 5 unrelated families. Minigene-driven splicing assays in HEK293T cells confirmed that this DNA change is responsible for the creation of a new acceptor splice site, resulting in aberrant splicing. CONCLUSION: We identified UBAP1L as a novel IRD gene. Although its function is currently unknown, UBAP1L is almost exclusively expressed in photoreceptors and the retinal pigment epithelium, hence possibly explaining the link between pathogenic variants in this gene and an ocular phenotype.

TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell-derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition-like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.

Acute Increase in Ocular Microcirculation Blood Flow Upon Cholesterol Removal. The Eyes Are the Window of the Heart.

BACKGROUND: Lipoprotein apheresis acutely increases coronary microvascular blood flow. However, measurement techniques are time-consuming, costly, and invasive. The ocular vasculature may be an appropriate surrogate and an easily accessible window to investigate the microcirculation. Recent advances in ocular imaging techniques enable quick, noninvasive quantification of ocular microcirculation blood flow. The insights from these techniques represent a significant opportunity to study the short-term changes in optic disk blood flow after lipoprotein apheresis for inherited hypercholesterolemia. METHODS: This study was performed at the Italian Reference Center for Inherited Dyslipidemias in Tuscany. The study sample was comprised of 22 patients with inherited hypercholesterolemia who were previously studied for coronary microcirculation. Laser speckle flowgraphy (LSFG) was used to measure optic disk blood flow before and after lipoprotein apheresis. The main outcomes measures were average tissue blood flow (referred to as mean tissue) and arteriolar/venular average blood flow (referred to as mean vessel). Eyes were divided into 2 groups based on pre-lipoprotein apheresis optic disk blood flow values. P < .05 was considered statistically significant. RESULTS: After each lipoprotein apheresis treatment resulting in the reduction of plasma lipids, there was a concurrent increase in all optic disk microcirculatory parameters. The increase was statistically significant in eyes with lower pre-apheresis optic disk blood flow values (mean tissue +7.0%, P < .005; mean vessel +7.2%, P < .05). CONCLUSIONS: A single lipoprotein apheresis session resulted in a statistically significant short-term increase in optic disk blood flow. These findings together with previous coronary microcirculation data suggest a similar ocular and coronary blood flow response to lipoprotein apheresis. Ocular microcirculation may represent a versatile biomarker for evaluating systemic microcirculatory health, including coronary microcirculation. Hence, it is plausible that plasma lipoprotein levels may influence optic disk blood flow.

Imposed positive defocus changes choroidal blood flow in young human subjects.

PURPOSE: It has previously been found that imposing positive defocus changes axial length and choroidal thickness after only 30 min. In the present study, we investigated whether these changes may result from an altered choroidal blood flow. METHODS: Eighteen young adult subjects watched a movie from a large screen (65 in.) in a dark room at 2 m distance. A 15-min wash-out period was followed by 30 min of watching the movie with a monocular positive defocus (+ 2.5D). Changes in axial length and ocular blood flow were measured before and after the defocus, by using low-coherent interferometer (LS 900, Haag-Streit, Switzerland) and a laser speckle flowgraphy (LSFG) RetFlow unit (Nidek Co., LTD, Japan), respectively. Three regions were analyzed: (1) the macular area, where choroidal blood flow can be measured, (2) the optic nerve head (ONH), and (3) retinal vessel segments. RESULTS: Changes in choroidal blood flow were significantly and negatively correlated with changes in axial length that followed positive defocus in exposed eyes (R = - 0.67, p < 0.01). The absolute values of changes in choroidal blood flow in the defocused eyes were significantly larger than in the fellow control eyes (2.35 ± 2.16 AU vs. 1.37 ± 1.44 AU, respectively, p < 0.05). ONH and retinal blood flow were not associated with the induced changes in axial length. CONCLUSIONS: Positive defocus selectively alters choroidal, but not retinal or ONH blood flow in young human subjects after short-term visual exposure. The results suggest that blood flow modulation is involved in the mechanism of choroidal responses to optical defocus.

Cataract progression following lens-sparing pars plana vitrectomy for rhegmatogenous retinal detachment.

Lens-sparing pars plana vitrectomy (PPV) is often followed by cataract development. However, there have been few prospective studies evaluating the timing of cataract progression and potential associated factors. This was an observational study conducted at the Ophthalmology Unit of the University Hospital of Parma (Parma, Italy). Patients presenting with rhegmatogenous retinal detachment (RRD), who underwent PPV with preservation of the lens, were examined according to a scheduled follow-up (3, 6 and 12 months after PPV) and then preoperatively when cataract extraction surgery (CES) was indicated, or at the end of the study follow-up period (May 2021). The primary outcome was the interval between PPV and CES indication (based on predefined refractive criteria). A total of 36 eyes of 36 patients (mean age: 52 ± 10 years) were included in the study. Nineteen eyes (53%) were indicated for CES a median of 14.5 months (IQR: 12.0-24.8) after PPV. The nuclear and posterior subcapsular forms of cataract progressed significantly starting at 6 months after PPV. Older age at the time of PPV, silicone oil tamponade and RRD without macular involvement were significantly and independently associated with an earlier indication for CES. Patient age and the use of silicone oil tamponade must be taken into consideration when evaluating the risk of cataract development after PPV.

Tilt and decentration of posterior and anterior iris-claw intraocular lenses: a pilot study using anterior segment optical coherence tomography.

BACKGROUND: Information on the centration and tilt of iris-claw intraocular lenses (IC-IOLs) is limited. In this study, we tested the capacity of an anterior segment optical coherence tomography (AS-OCT) instrument to measure decentration and tilt of anterior and posterior IC-IOLs through an integrated software. METHODS: The present observational, cross-sectional study was conducted at University Eye Clinic of Parma (Parma, Italy). The CASIA2 swept-source AS-OCT (Tomey Corp.) was used to measure the tilt and decentration of posterior and anterior IC-IOLs in patients implanted at least 6 months in advance. After failure with full-automation, semi-manual IOL tracing was applied. In-the-bag (IB) contralateral IOLs, when present, were measured automatically. The Bland-Altman method was used to evaluate the agreement between repeated measurements (2 images for each study eye). The amount and direction of tilt and decentration were recorded and plotted into polar charts for evaluation. RESULTS: A total of 21 patients were included: 14 with posterior and 7 with anterior IC-IOL fixation. In 17 eyes (81%), the AS-OCT provided a repeatable measurement of tilt and decentration. All contralateral eyes with IB IOL were automatically measured. The median decentration was 0.67 mm, 0.24 mm, and 0.24 mm in posterior IC-IOLs, anterior IC-IOLs, and IB IOLs group, respectively. The median tilt was 5.0°, 5.6°, and 5.6° for posterior IC-IOLs, anterior IC-IOLs, and IB IOLs, respectively. Tilt direction was mainly temporal, while decentration was inferior-temporal with posterior IC-IOLs and scattered with anterior IC-IOLs and IB IOLs. CONCLUSIONS: The semi-manual tracing function of the CASIA2 AS-OCT provides repeatable and affordable measurements of the decentration and tilt of IC-IOLs in both the anterior and posterior chamber. Data from the former group were similar to the IB group.

Rare occult macular dystrophy with a pathogenic variant in the RP1L1 gene in a patient of Swiss descent.

Purpose: We report a first case of bilateral occult macular dystrophy (OMD) with a c.133C>T (p.Arg45Trp) pathogenic variant in the retinitis pigmentosa 1-like 1 (RP1L1) gene in a patient of Caucasian Swiss decent. Observations: A 34-year-old man presented with decreased visual acuity known since childhood. Fundus examination of both eyes revealed no pathology other than mildly increased granularity of the foveal retinal pigment epithelium. The full-field electroretinogram (ffERG) presented with normal findings while the multifocal electroretinogram (mfERG) showed severely reduced amplitudes of the foveal response. Optical coherence tomography (OCT) showed foveal outer retinal atrophy. Fundus autofluorescence (FAF) imaging demonstrated near-normal findings with minimal mottling at the posterior pole. The genetic analysis revealed a heterozygous pathogenic variant (c.133C>T, p.Arg45Trp) in the RP1L1 gene. Conclusion and importance: Our present case suggests that OMD shows a wide range of clinical presentations with a variety of ophthalmological findings, age of disease onset, visual acuity, and genetic diversity.

Full-field sensitivity threshold and the relation to the oxygen metabolic retinal function in retinitis pigmentosa.

PURPOSE: The aim of our study was to evaluate retinal function with white light dark-adapted full-field sensitivity threshold (FST) and find possible correlations with metabolic function measured with retinal oximetry (RO) in patients with retinitis pigmentosa (RP). METHODS: In this prospective observational study (BASEC 2020-00,122), FST and RO measurements were performed on 66 RP eyes (33 subjects, 12♀ 21♂) aged between 18 and 80 years (mean 43.2 years); all eyes were graded for disease severity. Main outcome parameters were white FST thresholds using the Diagnosys Espion system with the ColorDomeTM LED full-field stimulator (Diagnosys LLC, Lowell, MA) as well as the main RO parameters: the mean arterial (A-SO2; %), venular (V-SO2; %) oxygen saturation, their difference (A-V SO2; %), and the corresponding mean diameters of the peripapillary retinal arterioles (D-A; µm) and venules (D-V; µm) recorded with the oxygen saturation tool of the Retinal Vessel Analyser (RVA; IMEDOS Systems UG, Jena, Germany). In addition, semi-automated kinetic perimetry (V4e, III4e, I4e, III3e isopters, Octopus 900®, Haag-Streit AG Bern, Switzerland) was performed and included in the linear mixed-effects models analysis calculated with SPSS®. RESULTS: Neither the oxygen saturation parameters (p > 0.21) nor the D-A and D-V (p > 0.13) showed significant correlations with the FST. However, when compared systematically with the visual field (VF) areas of the different isopters, RO parameters V-SO2 (p = 0.024) and A-V SO2 (p < 0.02) showed significant correlations. Furthermore, both V-SO2 and A-V SO2 showed gradual changes with more pronounced impairment in oxygen metabolic function in advanced stages of RP when analyzed in subgroups of disease severity grades. CONCLUSION: In contrast to standardized VF parameters, white dark-adapted FST appears not to correlate with retinal oxygen metabolic function measured with RO in patients with RP, suggesting that the two examinations may capture unrelated aspects of the retinal pathological process. However, RO showed a significant association with standardized VF testing parameters and may, therefore, offer an alternative outcome measure for interventional trials.

Longitudinal Study of Optic Disk Perfusion and Retinal Structure in Leber's Hereditary Optic Neuropathy.

Purpose: The purpose of this study was to evaluate optic disk perfusion and neural retinal structure in patients with subacute Leber's hereditary optic neuropathy (LHON) and LHON carriers, as compared with healthy controls. Methods: This study included 8 patients with LHON in the subacute stage, 10 asymptomatic carriers of a LHON-associated mitochondrial DNA mutation, and 40 controls. All subjects underwent measurement of the retinal nerve fiber layer (RNFL) thickness, the ganglion cell-inner plexiform layer (GCIPL) thickness using optical coherence tomography and optic disk microvascular perfusion (Mean Tissue [MT]) using laser speckle flowgraphy (LSFG). Patients were re-examined after a median interval of 3 months from the baseline visit. Results: LHON carriers had higher values of RNFL thickness, GCIPL thickness, and disk area than controls (P < 0.05), whereas MT was not different between the two groups (P = 0.936). Median MT and RNFL thickness were 32% and 15% higher in the early subacute stage of the disease than in controls (P < 0.001 and P = 0.001). MT declined below the values of controls during the late subacute stage (P = 0.024), whereas RNFL thickness declined later during the dynamic stage (P < 0.001). GCIPL thickness was lower in patients with LHON than in controls independently of the stage of the disease (P < 0.001). Conclusions: The high blood flow at the optic disk during the early subacute stage may be the consequence of vasodilation due to nitric oxide release as compensation to mitochondrial impairment. Optic disk perfusion as measured by LSFG is a promising biomarker for LHON diagnosis and monitoring as well as an objective outcome measure for assessing response to therapies.

Measuring optic nerve head perfusion to monitor glaucoma: a study on structure-function relationships using laser speckle flowgraphy.

PURPOSE: We aimed to describe the global and localized correlations among visual field (VF) sensitivity, optic nerve head (ONH) perfusion measured by laser speckle flowgraphy (LSFG) and neural structure measured by optical coherence tomography (OCT) in open-angle glaucoma (OAG) and to compare the floor effect for LSFG and OCT. METHODS: Cross-sectional, multicenter study including one eye each from fifty OAG patients (mean age 69.3 years; average VF mean deviation, MD, -8.5 dB, range -25.17 to 0.85 dB) and fifty-one controls. Patients underwent SITA standard 24-2 automated perimetry and measurement of ONH perfusion, peripapillary retinal nerve fibre layer thickness (RNFLT) and macular ganglion cell-inner plexiform layer thickness (GCIPLT). We tested the presence of a significant change (breakpoint) in the correlation slope with VF sensitivity to assess floor effect. RESULTS: The correlation between the LSFG parameter Mean All (MA) of the global disc area and MD (r = 0.56, p < 0.001) did not show a breakpoint, in contrast to the correlations between MD and OCT global parameters, which showed breakpoints at -8.53 and -4.05 dB for RNFLT and GCIPLT, respectively. Global and localized correlations with VF sensitivity were stronger for LSFG compared to OCT. In particular, LSFG outperformed OCT in the correlation with the central VF sector (r = 0.50, p < 0.001 and r = 0.06, p = 0.67 for MA and RNFLT, respectively). CONCLUSION: The global and sectoral correlations with VF sensitivity and the favourable floor effect compared to OCT indicate LSFG as a promising tool to monitor progression particularly in late-stage glaucoma. Further longitudinal studies are warranted.

Genetic Analysis of Consanguineous Pakistani Families with Congenital Stationary Night Blindness.

INTRODUCTION: Congenital stationary night blindness (CSNB) is a rare, largely nonprogressive, inherited retinal disorder that can be clinically classified on the basis of fundus and electroretinogram abnormalities. METHODS: We analyzed four large consanguineous families from the Southern Punjab region of Pakistan including multiple individuals affected with CSNB. Exome sequencing was performed in probands of all four families; Sanger sequencing was performed in additional members to test co-segregation of the variants identified. RESULTS: We identified two novel and likely pathogenic variants in two pedigrees, namely, NM_002905.4:c.668A>C (p.Gln223Pro) in RDH5 and NM_022567.2:c.908del (p.Gly303ValfsTer45) in NYX. In the two other families, the variants NM_002905.4:c.319G>C (p.Gly107Arg) in RDH5 and NM_000541.5:c.874C>T (p.Arg292Ter) in SAG were identified. These latter mutations have been reported previously, but not in the Pakistani population. CONCLUSIONS: Our findings expand the mutational spectrum of CSNB, in particular within the population of Southern Punjab.

Short-term changes in retinal and choroidal relative flow volume after anti-VEGF treatment for neovascular age-related macular degeneration.

The effects of anti-vascular endothelial growth factor (anti-VEGF) agents on the native ocular vasculature are poorly understood. This pilot study aimed to assess short-term changes in retinal and choroidal perfusion after anti-VEGF treatment for neovascular exudative age-related macular degeneration (nAMD) using the relative flow volume (RFV) parameter derived from laser speckle flowgraphy. Ten treatment-naïve nAMD patients underwent measurements of mean, maximum, minimum, and differential RFV within a retinal arteriolar segment and a choroidal vessel segment outside the neovascular area. Measurement of retinal RFV (rRFV), choroidal RFV (cRFV), and subfoveal choroidal thickness (SCT) was repeated 9 and 35 days after a single anti-VEGF injection. The treatment caused a statistically significant decrease in the mean rRFV, mean cRFV, and SCT during the follow-up (p < 0.05). At the intermediate visit, the mean cRFV and SCT were - 17.6% and - 6.4% compared to baseline, respectively. However, at the final measurement, the mean cRFV was not different from the baseline value, which indicated waning of the anti-VEGF effect. In conclusion, a single anti-VEGF injection in treatment-naïve nAMD resulted in a decrease in retinal arteriolar and choroidal perfusion, according to the RFV parameter, which is a promising tool to simultaneously assess retinal and choroidal perfusion changes in response to anti-VEGF therapy.

Parsplana vitrectomy alone versus parsplana vitrectomy combined with phacoemulsification for the treatment of rhegmatogenous retinal detachment: a randomized study.

BACKGROUND: To compare parsplana vitrectomy (PPV) with and without phacoemulsification to treat rhegmatogenous retinal detachment (RRD). METHODS: Subjects aged 48-65 years with RRD in a phakic eye due to superior retinal tears with an overall extension of retinal breaks < 90° underwent to PPV alone (group A); or PPV plus phacoemulsification (phacovitrectomy, PCV, group B). Post-operative follow-up visits occurred at 1 week, 1 month (m1), 3 months (m3), and 6 months (m6) after surgery. The main outcome was the rate of retinal reattachment. Secondary outcomes included best-corrected visual acuity (BCVA), intraocular pressure (IOP), central macular thickness (CMT), and cataract progression (in the lens-sparing [PPV-alone] group). RESULTS: In this initial phase of the study a total of 59 patients (mean age: 55 years, 59 eyes) were enrolled: 29 eyes in group A and 30 eyes in group B. Both groups had similar gas tamponade. During the follow-up there were three cases of RRD recurrence in group A and one in group B. The relative risk of recurrence in group A was 3.22 times higher but the difference was not significant (p = 0.3). The two groups were also similar in terms of BCVA and IOP variation. At m3, CMT was significantly higher in group B (p = 0.014). In group A, cataract progression was significant at m6 (p = 0.003). CONCLUSIONS: In a cohort of RRD patients selected according to their preoperative clinical characteristics, PPV was comparable to PCV in terms of the rate of retinal reattachment after 6 months. TRIAL REGISTRATION: ISRCTN15940019 . Date registered: 15/01/2021 (retrospectively registered).

The epidemiology of Moebius syndrome in Italy.

BACKGROUND: The epidemiology of Moebius syndrome (MBS) is difficult to assess. In the present study, we investigated the epidemiology of MBS in a well-defined population within a precise geographical area. MATERIALS AND METHODS: Our university hospital is the only national referral center for the diagnosis and treatment of MBS. Participants in this cross-sectional study were patients affected by MBS who had been periodically followed by our medical staff since 1998. Most of the patients were referred to our hospital by the Italian Association of Moebius Syndrome (AISMO). Demographic data necessary for study purposes were made available in the AISMO database, updated to April 2018. Subjects were assigned to geographical macroareas that are conventionally used in surveys and epidemiological investigations by the Italian National Institute of Statistics. The rates and prevalence of MBS cases were calculated on the basis of the last available survey of the Italian population. Each study parameter was then calculated with reference to the whole country and macroarea partition. The sex rate and the corresponding prevalence were calculated with respect to the weighted whole population and to the respective sex population. Chi-square analysis was adopted to investigate possible differences among geographical regions and/or sexes. A p value < 0.05 was considered statistically significant. RESULTS: One hundred and sixty-four out of 212 MBS patients fulfilled our inclusion criteria. All cases occurred in Caucasian patients and were sporadic. The median age at diagnosis was 3.6 years, ranging from 0 to 55 years; this range was significantly reduced to 0-5 years (median age at diagnosis: 2.2 years) in patients included after 2007. The calculated prevalence at birth was 0.06 cases per 10,000 live births, with an overall prevalence of 0.27/100,000, without any sex or geographical predominance. CONCLUSIONS: The prevalence of MBS observed herein, rounded for possible underestimation, was 0.3/100,000 people, without any regional difference in the distribution of cases. Our data confirm the rarity of the disease on a national level.

Assessment of Choroidal Neovascularization Perfusion: A Pilot Study With Laser Speckle Flowgraphy.

Purpose: The purpose of this study was to quantify perfusion in the area of choroidal neovascularization (CNV) using laser speckle flowgraphy (LSFG) before and after intravitreal anti-vascular endothelial growth factor (VEGF) injection. Methods: Retrospective case series. Fifteen eyes of 15 patients with treatment-naïve CNV due to age-related macular degeneration (AMD) and with available LSFG images were included. The main outcome was the mean blur rate (MBR) quantified as a measure of perfusion within the CNV area observed on indocyanine green angiography. Twelve patients had available longitudinal data until one month after the injection, used to evaluate changes in perfusion, central macular thickness (CMT), visual acuity, and ocular perfusion pressure. Reproducibility of LSFG measurements was investigated at each time point from two images taken within five minutes. Results: Intraclass correlation coefficients for LSFG measurements were higher than 0.8 indicating excellent reproducibility. There was a significant decrease in perfusion after one week (-26.4 ± 14.4%; P = 0.027), whereas, after one month, perfusion was no longer significantly different from baseline (P = 0.121). CMT showed a progressive decrease over the follow-up period. Changes in perfusion were strongly correlated with changes in CMT after one week, but not thereafter. Conclusions: This pilot study suggests a method to select a region in the CNV area to quantify perfusion using LSFG. MBR could represent a parameter possibly related to regrowth of the CNV after anti-VEGF treatment. Large-scale studies are needed to assess the usefulness of LSFG in defining re-treatment criteria for neovascular AMD. Translational Relevance: LSFG technology to quantify perfusion could provide useful biomarkers for therapeutic management of CNV.