An important unresolved question in sensory neuroscience is whether, and if so with what time course, tactile perception is enhanced by visual deprivation. In three experiments involving 158 normally sighted human participants, we assessed whether tactile spatial acuity improves with short-term visual deprivation over periods ranging from under 10 to over 110 minutes. We used an automated, precisely controlled two-interval forced-choice grating orientation task to assess each participant's ability to discern the orientation of square-wave gratings pressed against the stationary index finger pad of the dominant hand. A two-down one-up staircase (Experiment 1) or a Bayesian adaptive procedure (Experiments 2 and 3) was used to determine the groove width of the grating whose orientation each participant could reliably discriminate. The experiments consistently showed that tactile grating orientation discrimination does not improve with short-term visual deprivation. In fact, we found that tactile performance degraded slightly but significantly upon a brief period of visual deprivation (Experiment 1) and did not improve over periods of up to 110 minutes of deprivation (Experiments 2 and 3). The results additionally showed that grating orientation discrimination tends to improve upon repeated testing, and confirmed that women significantly outperform men on the grating orientation task. We conclude that, contrary to two recent reports but consistent with an earlier literature, passive tactile spatial acuity is not enhanced by short-term visual deprivation. Our findings have important theoretical and practical implications. On the theoretical side, the findings set limits on the time course over which neural mechanisms such as crossmodal plasticity may operate to drive sensory changes; on the practical side, the findings suggest that researchers who compare tactile acuity of blind and sighted participants should not blindfold the sighted participants. Does visual deprivation cause tactile acuity enhancement? This question, important to neuroscientific understanding of tactile perception and of the interaction between the senses, has been investigated for decades.Early studies reported that tactile perception improved upon prolonged simultaneous deprivation of multiple sensory modalities. Doane and colleagues These findings were soon followed by reports that prolonged visual deprivation alone sufficed to improve tactile perception. Zubek et al. For a period of several decades following these intriguing early studies, interest in the field seems to have faded. With the advent of functional imaging, interest resurged as many studies revealed that tactile stimuli activate occipital cortical areas in blind participants  It was soon discovered that the occipital cortex of visually deprived sighted participants becomes hyperexcitable Because tactile responsiveness of occipital cortex occurred within 90 minutes of blindfolding according to one study In contrast to the early literature, two modern studies reported significant effects of short-term visual deprivation on tactile acuity. Comparing a “non-deprived” control group to a visually deprived experimental group, Facchini and Aglioti Nevertheless, particular technical aspects of these modern studies may have led the investigators to mistaken conclusions. For instance, Facchini and Aglioti Here, we report the results of a study designed to resolve the controversy surrounding the effects of short-term visual deprivation on passive tactile spatial acuity. Ours is the first study of short-term visual deprivation to use a precision-controlled automated tactile grating orientation task In three experiments involving 158 participants, we assessed whether tactile spatial acuity improves with short-term visual deprivation. We tested 48 participants in Experiment 1, 44 participants in Experiment 2, and 66 participants in Experiment 3. We used the GOT, a rigorous test of tactile spatial acuity To investigate whether tactile spatial acuity improves upon brief periods  of visual deprivation, we used a 2x2 counterbalanced repeated-measures design, testing 48 sighted participants under all four combinations of ambient lighting (light or pitch-dark) and eyelid state (eyes opened or eyes closed) . After tTo examine the effects of ambient lighting and eyelid state, we performed a 2 (ambient lighting) x 2 (eyelid state) x 2 (iteration) x 2 (sex) ANOVA. This analysis revealed significant main effects of ambient lighting (p = 0.010) and of sex (p = 0.029). Participants' tactile acuity worsened significantly with visual deprivation, and women significantly outperformed men. On average, thresholds in the dark were 0.09 mm higher than in the light  , and menAlthough the effect of eyelid state was not significant (p = 0.077), participants tended to perform better with eyes opened than closed. The effect of iteration was not significant (p = 0.396), but participants tended to perform better in iteration 2 than iteration 1, suggestive of a practice effect .We next examined whether the elevation of tactile threshold in the dark depended upon the dark/light testing order. For each participant we computed a difference score: threshold of first iteration 1 test in the dark – threshold of first iteration 1 test in the light. For instance, for a participant tested in the order LC, DC, DO, LO , we wondered whether a longer period of visual deprivation would improve participants' tactile spatial acuity and, if so, whether the improvement would occur abruptly or gradually. Accordingly, in Experiment 2 we lengthened the visual deprivation period to 70 minutes.Participants were assigned to one of four groups. In the non-deprived group, participants were tested in the light 10 times. In the three visually deprived groups, participants were tested in the light twice before and three times after a period of 90 minutes in the pitch-dark. The sequence of events in the dark differed by group . We condTo analyze the data from each group, we performed a one-way repeated-measures ANOVA across testing blocks. We observed no significant change in GOT performance within any group : non-depTo examine the effect of sex, we averaged the threshold of each participant across all test blocks and performed an independent-samples t test to compare the mean thresholds for women and men. This analysis revealed that women significantly outperformed men (p = 0.015). On average, men's thresholds were 0.35 mm higher than women's .Having observed no improvement in tactile spatial acuity in Experiments 1 and 2, we wondered whether a somewhat longer period of deprivation might result in acuity enhancement. In addition, we wondered whether participants might have lost alertness during the visual deprivation period in Experiment 2, perhaps resulting in a worsening of performance that masked a true benefit of visual deprivation. Accordingly, we further lengthened the visual deprivation period to 110 minutes, and to safeguard participant alertness we recruited participants in sets of three and encouraged conversation during the visual deprivation period. In keeping with Facchini and Aglioti We tested 66 participants. Five participants could not perform the task beyond chance level, and were therefore excluded from data analysis. Each set of three participants was assigned to one of two groups: a non-deprived group (n = 29) and a visually deprived group (n = 32). Participants in both groups were tested three times: before a 110-minute conversation period, immediately following the conversation period, and 120 minutes following the second test. Whereas participants in the non-deprived group were always in the light, those in the visually deprived group were in the pitch-dark during the conversation period and the second test .To examine whether 110 minutes of visual deprivation improves GOT performance, we performed a 3 (test block) x 2  x 2 (sex) ANOVA. This analysis revealed a significant main effect of sex (p = 0.008), indicating that women outperformed men. There was no significant main effect of test block or of group, nor was there a significant test block x group interaction. Thus, visual deprivation did not affect tactile spatial acuity.One-way repeated-measures ANOVAs performed separately for each group confirmed that across the three test blocks there was no significant change in the performance of participants in the visually deprived group (p = 0.435)  or the nTo quantify the difference between thresholds of men and women, we averaged each participant's thresholds across the three tests (without regard to group). On average, men's thresholds were 0.28 mm higher than women's .Contrary to previous reports The experiments reported here provide clear and consistent evidence that short-term visual deprivation does not enhance passive tactile spatial acuity.Using a counterbalanced repeated-measures design, we found in Experiment 1 that tactile spatial acuity actually worsened to a small but significant degree upon short-term visual deprivation. Participants performed significantly worse in the dark than in the light, and tended  to perform worse with their eyes closed than opened. Of the four conditions under which they were tested, participants performed best on average in the condition with the greatest visual stimulation (eyes opened in the light). These results may be attributable to a loss of alertness experienced during visual deprivation, or to some other cause.Tracking tactile spatial acuity over 70 minutes of visual deprivation, we found in Experiment 2 that this period of deprivation did not result in tactile perceptual improvement. This was true irrespective of whether during visual deprivation the participant received no tactile stimulation, unattended tactile stimulation, or repeated GOT testing. Similarly, in Experiment 3, participants performed equivalently before and after 110 minutes of visual deprivation. Thus, our results consistently show that tactile spatial acuity does not improve during short-term visual deprivation.In contrast to Experiment 1, in Experiments 2 and 3 we did not observe a significant worsening of GOT performance upon visual deprivation. We propose that this apparent discrepancy is explained by two factors: 1) The fact that all visually-deprived participants in Experiments 2 and 3 were first tested in the light, whereas half the participants in Experiment 1 were first tested in the dark, and 2) The fact that all three experiments revealed a trend  for thresholds to decrease slightly with repeated testing, consistent with previous reports  was large and positive only among participants who were initially tested in the dark (0.16 mm). The corresponding difference score for participants who were initially tested in the light (−0.03 mm) indicates that those participants did not on average worsen when subsequently tested in the dark. This trend repeated in iteration 2 (0.20 mm vs. −0.02 mm). A parsimonious explanation for this order effect is that it is due to a trend for practice to improve acuity (lower thresholds) from one testing block to the next, together with a trend for visual deprivation to worsen acuity (raise thresholds).This explanation reconciles the apparent discrepancy between Experiment 1, which revealed a slight but significant worsening of acuity under conditions of visual deprivation, and Experiments 2 and 3, which did not. Experiment 1 used a counterbalanced design so that the average difference observed between conditions was robust against practice effects, whereas Experiments 2 and 3 always tested participants in the light prior to testing them in the dark.improve tactile spatial acuity. If our explanation above is correct, all three experiments indeed lend support to the conclusion that tactile spatial acuity tends to worsen under short-term visual deprivation.Most importantly, we note that Experiments 1, 2 and 3 all clearly support the conclusion that short-term visual deprivation does not Our results stand in stark contrast to those of Facchini and Aglioti How might the results of Facchini and Aglioti If the discrepancy between studies did not arise from statistical fluctuation, another possibility is that it arose from unintended variability in stimulus-delivery parameters. The GOT provides a rigorous measure of tactile spatial acuity by assessing participants' ability to discern the orientation of grating stimuli pressed orthogonally against a body part Two additional methodological considerations may explain the discrepancy between these studies and ours. First, Leon-Sarmiento et al. Second, Facchini and Aglioti In light of the results of Experiment 1, we caution against the blindfolding of sighted participants in tactile psychophysics studies, as this procedure may inadvertently worsen participants' tactile acuity. For instance, although it is becoming increasingly clear that the tactile acuity of blind participants is better than that of sighted participants Another practical consequence of this study is that investigators of tactile spatial acuity should be aware of the tendency for women to outperform men, and design and analyze their studies accordingly. In all three experiments reported here, we found that women significantly outperformed men on the GOT. This result is consistent with previous reports In contrast to short-term visual deprivation, several studies have reported that prolonged visual deprivation does drive tactile acuity enhancement What neural mechanism might underlie visual deprivation-induced tactile acuity enhancement?In the absence of vision, the visual cortex becomes responsive to tactile inputs  Crossmodal plasticity coupled with extensive daily reliance on the sense of touch may also underlie tactile acuity enhancement in blindness In three experiments, we show consistently that short-term visual deprivation for periods up to 110 minutes does not enhance passive tactile spatial acuity. We note that in contrast to short-term visual deprivation, prolonged visual deprivation does reportedly drive tactile acuity enhancement. Investigations that couple perceptual testing with neural imaging will help to elucidate the mechanism by which prolonged visual deprivation enhances tactile acuity.We conducted three experiments involving 158 participants. None of the participants tested in one experiment were tested in any other. None of the participants had previous experience with the grating orientation task. Experiment 1 was conducted at Duquesne University  and Experiments 2 and 3 at McMaster University .Experiment 1 was approved by the Duquesne University Institutional Review Board; Experiments 2 and 3 were approved by the McMaster University Research Ethics Board. All participants provided written consent and received monetary compensation and/or course credit for their participation.Forty-eight normally sighted participants  took part in Experiment 1. Inclusion criteria ensured that participants did not have (by self report) dyslexia, diabetes, nervous system disorders, or injuries or calluses on the index finger of the dominant hand (the finger was inspected in the laboratory to verify its condition). Dyslexia was an exclusion criterion because it has been shown to adversely affect tactile spatial perception We assessed each participant's ability to discern the orientation of grating stimuli applied to the distal index finger pad of the dominant hand. The stimuli were a set of custom-made square-wave gratings, with groove widths ranging from 0.25 mm to 3.1 mm (in increments of 0.15 mm). We used the Tactile Automated Passive-finger Stimulator (TAPS) to mechanically deliver the grating stimuli; see In each two-interval forced-choice (2-IFC) trial, the participant's tested finger pad was contacted twice by the grating stimuli, once with the gratings aligned parallel to the long axis of the finger , and once with the gratings aligned transverse to the long axis of the finger ; the presentation order was chosen randomly . An interstimulus interval of 2s separated the presentation of the two orientations. The participant indicated, by pressing one of two buttons with the non-tested hand, whether the horizontally aligned gratings contacted the tested finger in the first or second interval . ParticiWe used a two-down one-up adaptive staircase procedure We tested every participant twice under all four combinations of ambient lighting (light or pitch-dark) and eyelid state (eyes opened or eyes closed). The order of conditions was counterbalanced across participants, such that each of the 24 men was tested on one of the 24  possible combinations of these four conditions, and similarly for each of the 24 women. After completing the four conditions, the participant was tested again on the same conditions and in the same testing order.Participants took on average 8 minutes to complete a testing block. The mean elapsed time between the end of one block and the start of the next was 2 minutes for blocks within the same iteration. The mean elapsed time between iterations (end of block 4 of iteration 1 to start of block 1 of iteration 2) was 3 minutes. The experiment duration averaged approximately 80 minutes.During the light conditions, the participants were tested under fluorescent overhead room lighting typical of a well-lit indoor environment. During the dark conditions, the participants were tested in the pitch-dark. Room darkness was such that no visual input was perceptible, even of large nearby objects . The light intensity was less than 0.01 lux, the lower detection limit of our light meter (Mannix DLM2000). To achieve visual deprivation, we chose here (and in Experiments 2 and 3) to use a pitch-dark room rather than blindfolding the participants. A simple cloth blindfold does not screen out all light, and also rubs and tickles against the eyes and face, causing a tactile distraction. Opaque goggles (such as painted swim goggles) can screen out all light, but require tight fits to the eye sockets, and are consequently both distracting and uncomfortable. We wished to test participants without light, and without inducing discomfort or distraction.An experimenter remained in the testing room at all times to ensure the participants' compliance with the eyelid state (eyes opened or closed) instructions appropriate to the condition. During the light conditions, the experimenter simply viewed the participant's eyes with unaided vision. During the dark conditions, the experimenter periodically verified that the participant's eyes were opened or closed as per condition with the aid of an infrared night vision monocular (Bushnell). Because the infrared beam cast by the night vision device bled somewhat into the visible red, we secured an opaque occluder with a pinhole cutout over the beam source to reduce the size of the beam to the bare minimum needed to obtain a view of the participant.Forty-four normally sighted right hand-dominant students from McMaster University  participated in Experiment 2. Hand dominance was confirmed by questionnaire . Participants were not blindfolded, nor were they instructed to close their eyes during the test blocks. Previous studies have shown that tactile acuity improves when the participant views the tested hand As in Experiment 1, participants were tested in the light (fluorescent overhead room lighting) and the pitch-dark (<0.01 lux). Unlike in Experiment 1, the investigator did not remain in the testing room with the participants. Therefore, participants in the dark were required to put on light-occluding goggles for a brief period (approximately 2–3 minutes) as the experimenter entered the room to initialize the equipment before each stimulation block. Except for these very brief periods, the participants were not blindfolded.Participants were assigned to one of four groups in pseudorandom order. Participants in the non-deprived group completed 10 test blocks in the light and were never visually deprived. Participants in the other three  groups completed two test blocks before (in the light) – to obtain baseline tactile acuity – and three test blocks after (in the light) experiencing a period of visual deprivation. The sequence of events during the visual deprivation period (in the pitch-dark) differed by visual deprivation group .To investigate whether short-term visual deprivation alone improves tactile spatial acuity, as reported To investigate whether and how tactile acuity changes over time with visual deprivation, we administered five test blocks during the visual deprivation period to participants in the repeatedly tested group.To investigate whether unattended grating stimulation in the dark would improve tactile acuity, we administered four passive stimulation blocks followed by one test block during the visual deprivation period to participants in the passively stimulated group. These participants were instructed to ignore the grating stimuli contacting the finger during a passive stimulation block; during the passive stimulation, they listened to music of their choice. As in the test blocks, in each trial of a passive stimulation block the participant's tested finger was contacted with a grating twice, once oriented vertically and once horizontally (order chosen randomly). However, unlike during testing, the participant did not make any response . The sequence of grating groove widths contacting the participant's finger in a passive stimulation block was the same sequence the participant experienced during the first or second test block, chosen randomly .Participants took on average 7 minutes to complete a test or passive stimulation block; including set-up time by the experimenter, each block lasted approximately 9 minutes. Successive blocks were separated by 8-minute break periods during which participants were free to listen to music of their choice. For participants in the repeatedly tested and passively stimulated groups, the average elapsed time between the start of the initial block in the dark and the start of the final block in the dark was 68 minutes; participants in the unstimulated group sat in the dark for exactly 70 minutes before being tested. Participants in all three visually deprived groups remained in pitch-darkness during the break following the final testing block in the dark; these participants sat in a pitch-dark room for approximately 90 minutes .Sixty-six normally sighted right hand-dominant students from McMaster University  participated in Experiment 3. The same qualification criteria and handedness questionnaire used in Experiment 2 were used here.The psychophysical procedures were identical to those used in Experiment 2.To ensure participant alertness, we recruited participants in sets of three and encouraged conversation during the visual deprivation period. Each set of three was assigned to one of two groups in alternating order: non-deprived and visually deprived.Every participant was tested three times. The first test block served as a measure of the participant's baseline tactile acuity. This was followed by a 110-minute conversation period during which participants talked with one another or with an experimenter. The conversation period was followed by a second test block, after which the participant left the laboratory to take a 120-minute break. Following the break, the participant returned to the laboratory to complete a final test block. Participants took on average 8 minutes to complete a test block.Non-deprived participants were always in the light. Visually deprived participants were in the pitch-dark during the conversation period and while completing the second test block. The visually deprived participants were in the pitch-dark for an average duration of 120 minutes.The test blocks were administered in a testing room, and the conversation period took place in a separate conversation room. Participants in each set of three were tested sequentially . It was We performed analyses of variance (ANOVA) with SPSS v19  for Macintosh, with an alpha-level of 0.05. The dependent measure used in the statistical analysis of Experiment 1 was the participant's 70.71% correct threshold, obtained using a two-down one-up staircase procedure Figure S1Sequence of events in Experiment 3. (A–C) The participants were tested sequentially in the testing room, and then seated sequentially in the conversation room. (D) Each participant spent a total of 110-minutes in the conversation room. (E–G) The participants were then tested sequentially a second time. (H) All three participants left the laboratory for a 120-minute break and returned sequentially to be tested a final time (not shown). The image is not drawn to spatial or temporal scale.(TIF)Click here for additional data file.


Autonomic dysfunction is a well-known feature in neurodegenerative dementias, especially common in α-synucleinopathies like dementia with Lewy bodies and Parkinson's disease with dementia. The most common symptoms are orthostatic hypotension, incontinence and constipation, but its relevance in clinical practice is poorly understood. There are no earlier studies addressing the influence of autonomic dysfunction on clinical course and survival. The aim of this study was to investigate the frequency of the three most common features of autonomic dysfunction and analyze how it affects survival.Thirty patients with dementia with Lewy bodies and Parkinson's disease with dementia were included in this prospective, longitudinal follow-up study. Presence of incontinence and constipation was recorded at baseline. Blood pressure was measured at baseline, after 3 months and after 6 months according to standardized procedures, with 5 measurements during 10 minutes after rising. Orthostatic hypotension was defined using consensus definitions and persistent orthostatic hypotension was defined as 5 or more measurements with orthostatic hypotension. Difference in survival was analyzed 36 months after baseline.2 = 4.47, p = 0.034). Patients with constipation and/or urinary incontinence, in addition to persistent orthostatic hypotension, had a poorer prognosis compared to those with isolated persistent orthostatic hypotension or no orthostatic hypotension .There was a high frequency of persistent orthostatic blood pressure (50%), constipation (30%) and incontinence (30%). Patients with persistent orthostatic hypotension had a significantly shorter survival compared to those with no or non-persistent orthostatic hypotension (Log rank xAccording to our findings, the identification of autonomic dysfunction seems to be of great importance in clinical practice, not only to avoid falls and other complications, but also as a possible predictor of survival. Dementia with Lewy bodies (DLB) and dementia associated with Parkinson's disease (PDD) are neurodegenerative disorders with similar clinical and neuropathological features. Together they account for approximately 15–20% of all clinically diagnosed dementia cases The clinical course of DLB, PDD and all other types of neurodegenerative dementia shows a high degree of inter individual variability. There are studies reporting differences between diagnoses, where DLB seems to be a more aggressive disorder than AD Autonomic dysfunction is a well-known feature in all α-synucleinopathies and in the revised diagnostic criteria for DLB it is a supportive feature. Three of the supportive features; repeated falls, syncope and transient loss of consciousness, can also be partly attributable to the presence of autonomic dysfunction. The principal autonomic symptoms are urinary incontinence, constipation and orthostatic hypotension. Autonomic dysfunction occurs to a lesser extent in AD, vascular dementia and in frontotemporal dementia Many studies have been performed on autonomic dysfunction in PD and MSA, but there is a lack of well-designed prospective studies. MSA is the α-synucleinopathy with the most pronounced autonomic dysfunction. Tada et al have shown that in MSA, an early development of autonomic dysfunction predict a poorer prognosis The objective in this study is therefore to investigate the frequency of symptoms related to autonomic dysfunction  in a DLB/PDD population and find out whether its presence or severity is correlated to a shorter survival in these patients.th edition (DSM IV); APA, 1994) or met the revised consensus criteria for DLB This longitudinal prospective study is a continuation of a double-blind 24-week randomized placebo-controlled trial (RCT) conducted in 2005–2008 In the original study, patients were assigned to placebo or Memantine treatment (20 mg daily) and summoned for visits at baseline, 12 and at 24 weeks. This study comprises a 4-week washout period followed by open-label treatment and ordinary yearly clinical visits within a structured follow up program at our clinic. Randomization was kept strictly double-blinded during wash out, but not during the open label treatment. Discontinuation of the double blind medication was performed by the end of the RCT without sequentially decreasing the doses. The open label medication doses were increased during a titration period of 4 weeks until reaching 20 mg daily.In this study, outcome was recorded 36 months from baseline. Survival was the primary outcome measure.At baseline, at 12 and 24 week visits, patients completed an orthostatic test. The procedure was performed according to a standardized scheme. A validated digital spyngomanometer (OMRON M5-1) over the brachial artery was used To illustrate the extent of orthostatic hypotension, each patient's systolic and diastolic blood pressure values were analyzed individually. Each measurement point was dichotomized as orthostatic or not orthostatic. The sum of all orthostatic values  in each patient was calculated. Not everybody was able to remain in standing position for 10 minutes. At baseline all patients completed a thorough clinical examination including their medical history and concomitant medication. Cardiovascular medication was defined as drugs included in ATC group C, i.e. antihypertensives, diuretics, beta blockers, calcium channel blockers, renin-angiotensin II receptor blockers and treatment of hyperlipidemia. Information about continence was acquired through the disability assessment for dementia (DAD) Statistical analyses were performed using PASW Statistics 18. All binary variables were compared using Chi-Square Tests. Due to the small number of subjects in this study, non-parametric statistics (Mann-Whitney U-test) were used to detect significant differences in groups for normally and non-normally distributed continuous variables. Since orthostatic hypotension was the most common autonomic disturbance and was assessed with the most systematic measurement, we explored the effect of persistent orthostatic hypotension on survival separately. To assess a possible dose-effect, we also studied the effect of the additional presence of urinary incontinence and/or constipation. Kaplan–Meyer curves were performed to compare survival in two groups. The p values were two sided exclusively and level of significance defined as less than 0.05.The RCT was conducted in accordance with the Declaration of Helsinki as revised in 2000 and approved by the regional ethics committee at Lund University, as was the open-label extension study. Renewed written informed consent was obtained from patients and their caregivers before entering the open-label extension.The frequency of symptoms related to autonomic dysfunction in this population is described in Comparisons were made between the groups with and without persistent orthostatic hypotension . The medSeven (23%) of the 30 patients died during the follow up, 5 from the DLB group and 2 from the PDD group.2 = 4.47, p = 0.034). There were no significant differences in survival between patients with and without constipation or with and without urinary incontinence. (Data not shown).The Kaplan Meyer curve in 2 = 6.370, p = 0.041). Patients in the third group had the shortest survival and the second group had the next shortest  a multivariate analysis to adjust for possible confounders, was not feasible. In our population with DLB/PDD patients, the frequency of coexisting heart disease and the use of antihypertensive and antiparkinson medication were equal in both groups. There was no significant difference in the use of antidepressants between groups, but it tended to be more common in the subgroup with persistent orthostatic hypotension. It is possible that the more severe orthostatic hypotension is at least partly influenced by concomitant medication.The pathophysiological mechanism of the association between autonomous dysfunction and mortality is not clear. Autonomic dysfunction is thought to be related to the α-synuclein pathology, since several neuropathological studies describe the presence of lewy bodies in strategic locations like locus coeruleus, sympathetic ganglia and parasympathetic plexus Studies comparing survival in AD and DLB are inconclusive. Some studies report a similar rate of cognitive decline, but a shorter survival The extent and pattern of autonomic dysfunction in dementia with Lewy bodies are poorly documented, even though many authors mention it as a well-known clinical feature. To our knowledge, this is the first study to investigate its influence on prognosis and survival in DLB even though there are a few studies proposing that repeated hypotensive episodes may exaggerate cognitive decline on the basis of cerebral hypoperfusion and microvascular lesions In summary we have found a high frequency of symptoms related to autonomic dysfunction in patients with DLB/PDD and importantly, patients with persistent orthostatic hypotension had a shorter survival. This needs to be verified in larger studies. Orthostatic hypotension seems to be the most important feature in autonomic dysfunction and we strongly recommend orthostatic blood pressure measurement on a routine basis in all patients with DLB/PDD.


Asthma is a heterogeneous outcome and how the condition should be measured to best capture clinically relevant disease in epidemiologic studies remains unclear. We compared three methods of measuring asthma in the Danish National Birth Cohort (n>50.000). When the children were 7 years old, the prevalence of asthma was estimated from a self-administered questionnaire using parental report of doctor diagnoses, ICD-10 diagnoses from a population-based hospitalization registry, and data on anti-asthmatic medication from a population-based prescription registry. We assessed the agreement between the methods using kappa statistics. Highest prevalence of asthma was found using the prescription registry (32.2%) followed by the self-report (12.0%) and the hospitalization registry (6.6%). We found a substantial non-overlap between the methods (kappa = 0.21–0.38). When all three methods were combined the asthma prevalence was 3.6%. In conclusion, self-reported asthma, ICD-10 diagnoses from a hospitalization registry and data on anti-asthmatic medication use from a prescription registry lead to different prevalences of asthma in the same cohort of children. The non-overlap between the methods may be due to different abilities of the methods to identify cases with different phenotypes, in which case they should be treated as separate outcomes in future aetiological studies. Recent decades have shown a concerning increase in the prevalence of asthma in both developed and developing countries Asthma is a complex and poorly defined syndrome characterized by several phenotypes, possibly with different aetiologies This study aimed to determine the prevalence of asthma in a population of children using three classification methods, including self-report, population-based hospitalization data, and population-based prescription data in a large prospective birth cohort, and to determine the agreement between the methods.The study is based on a follow-up of the Danish National Birth Cohort that enrolled more than 100,000 pregnancies from 1996–2003 and has been described in detail elsewhere Mothers provided written informed consent on behalf of their children. The Regional Scientific Ethics Committee for the municipalities of Copenhagen and Frederiksberg approved all study protocols, and all procedures were in accordance with the Declaration of Helsinki.“Has a doctor ever said that your child had asthma?”. Current self-reported asthma was defined as a child with a self-reported doctor diagnosis of asthma plus a positive answer to the question “Has your child had any wheezing symptoms within the past 12 months?”.Self-reported asthma was assessed from the follow-up questionnaire when the children were 7 years old. We defined a self-reported asthma case as a child with a positive answer to the question We used the unique Danish Personal Identification Number (CPR number) to extract information about asthma from two population-based registers: the Danish National Patient Register (DNPR) and the Register of Medicinal Product Statistics (RMPS).Information about hospital contacts was extracted from the DNPR, which includes mandatory information about all hospital admissions in Denmark collected since 1977, and since 1995, also emergency room and outpatient contacts. Diagnoses of asthma were based on the International Classification of Diagnosis system 10 (ICD-10). We defined a hospitalization case of asthma as a child who had an asthma diagnosis  registered in the DNPR from birth to 7 years of age. Only the first registered asthma case was used.Information about asthma medication use was extracted from the RMPS, which includes mandatory information about all prescriptions redeemed at Danish pharmacies since 1995. Medication use was based on the Anatomical Therapeutic Chemical Classification System (ATC). We extracted information using the following ATC codes: R03A, R03B, R03C, and R03D. We used a previously validated definition of asthma based on prescription data We calculated the 7-year prevalences of asthma by classification method and for the methods in combination. The prevalence according to the registries was calculated for the complete study population; that is 93,616 children, whereas the self-reported prevalence was calculated for those who had filled out the questions on asthma.Asthma cases from the three classification methods were compared in two-by-two tables and the agreement among cases and non-cases for each method calculated. We looked at all possible combinations of the classification methods and therefore alternatively used each of the three methods as the comparison measure or ‘gold standard’. Cohen’s kappa coefficient was calculated to describe the overall agreement between the classification methods.In supplementary analyses we modified the case definitions of asthma in order to ascertain possible phenotypes. Current self-reported asthma was compared to cases ascertained by the registries, excluding the first 3 years of age from the register data. We expected that the modified definitions would to a larger extent reflect current asthma and exclude transient or viral wheeze diagnosed as asthma in early childhood which later resolved.All analyses were carried out in SAS statistical software .The 7-year prevalence of asthma for each classification method is shown in . The overall agreement between cases ascertained by the two registries was lower (k = 0.21).The overall agreement between self-reported asthma cases compared with cases ascertained by the hospitalization registry (k = 0.38) and the prescription registry (k = 0.37) was similar Among cases identified in the hospitalization registry and the prescription registry, the proportion of children who also had a self-reported doctor diagnosis of asthma was 65% and 33%, respectively Table 3.To our knowledge, this is the largest population-based study to simultaneously employ and compare three different asthma classification methods that are commonly used separately in epidemiological studies. Case ascertainment by means of self-report, a hospitalization registry, and a prescription registry yielded variable estimates of asthma prevalence during the first 7 years of age ranging from 6.6%–32.7%. Moreover, our study revealed a substantial non-overlap between cases identified by the three methods resulting in a prevalence of 3.6% if all three methods were combined to define asthma. Measures of agreement were low (kappa = 0.21–0.38).Prevalence measures of asthma are difficult to compare across settings because of the lack of a “gold standard”. Another Danish study that employed ICD-10 codes of asthma in the DNPR found a prevalence of 5.0% of asthma in a cohort of children followed from birth to 12 years of age Overall, studies using self-report of asthma have yielded a range of different prevalences, illustrated for example by the ISAAC study, which reported 12-months prevalences of asthma ranging from 1.6–36.8% using the same standardized questionnaire in different populations The overall agreement of the asthma classification methods in our study was low, with kappa-values below 0.40 for all comparisons. This is in contrast to the results reported in the aforementioned study by Yang et al. that compared self-reported asthma with asthma diagnosis based on health claims data and found an overall agreement of kappa = 0.60 A recent study by Nwaru et al. compared self-reported measures of asthma from a Finnish version of the ISAAC questionnaire with purchase of at least one anti-asthmatic medication during the preceding 12 months and found that the self-reported measures had a 98% agreement both with regard to classifying cases and non-cases of asthma et al. to have a specificity of 0.86 and a sensitivity of 0.63 compared with discharge information and a questionnaire completed by general practitioners in those without a discharge diagnosis The variable prevalence estimates across the three methods that we employed, and the substantial non-overlap between the cases they identified, are noteworthy. The correct interpretation of this is hampered by the fact that we have not assessed the true prevalence of asthma in our population which would require a clinical assessment of each individual. It is reasonable to assume that the three methods we employed may have different error sources and thus variable degrees of misclassification for a given biological phenotype. However, for the case definition using the prescription register data, we used a validated definition of asthma that has previously been shown in a study by Moth While different error sources may explain some of the variance in the prevalences of asthma among the assessment methods, it is also possible that the three methods identify asthma cases with biologically distinct phenotypes. For example, the hospitalization registry may capture more severe phenotypes than the prescription registry or maternal self-report. The overrepresentation of more severe cases when using this classification method may however be appropriate for studies of aetiological associations if hospitalized cases have a different phenotypical aetiology compared to the cases identified using the other methods. In contrast, the prescription registry may identify a heterogeneous mix of cases, ranging from suspected asthma cases that may have been prescribed anti-asthmatic medication to clarify a diagnosis to more severe cases that continuously require anti-asthmatic treatment. A study showed that 13.9% of all children in Denmark aged 0–15 years had redeemed prescriptions of at least one anti-asthmatic drug in one year but not all of these children had received an asthma diagnosis leading to an overestimation of the asthma prevalence Wheezing symptoms in young children aged 0–3 years are common and many are prescribed anti-asthmatic mediations The extent to which the non-overlap between the methods may be due to misclassification or, to different phenotypes being identified by each method, has implications for how to use these methods in studies where each individual has been assessed by more than one method simultaneously. Assuming that the methods identify a single phenotype, methods can be combined. In aetiologic studies of asthma where the aim is to estimate a relative risk for a given factor high specificity may be desirable even at the expense of lower sensitivity; this could be obtained by requiring that two or more methods agree on the asthma case ascertainment. For example, the self-reported measures could be used in combination with the prescription register definition to define an asthma case which in our study would lead to an asthma prevalence of 10.2% On the other hand, assuming that the methods identify different phenotypes we may need to do separate analyses, one for each case definition.In conclusion, our large-scale prospective study comparing three different methods for asthma ascertainment revealed a substantial non-overlap between cases identified by the three methods. Reflective of either different biases or different phenotypical expressions of asthma, these classifications need to be carefully considered when deciding on asthma outcomes in future aetiological studies.


The rationale behind active treatment and ablation in asymptomatic WPW is to prevent sudden cardiac death (SCD). SCD in asymptomatic WPW syndrome has been noted to vary from 0.15 to 0.39% over 3-10 years follow up .SCD happens in WPW in relation to rapid pre-excited atrial fibrillation (AF) with fast ventricular response. It happens in 50% of SCD as the first manifestation. Hence, though the incidence of SCD is low in WPW, the same is avoidable in suitably selected cases. It can presently be taken care of, by radiofrequency (RF) ablation of the bypass tract without significant morbidity.What then are the criteria to select patients of Asymptomatic WPW who may be predisposed to SCD? The markers identified for SCD include: 1. Anterograde effective refractory period of accessory pathway (APERP) 240- 250ms; (on isoproternol APERP <200ms?) 2. Shortest Pre-excited RR interval (SPERRI) less than 250ms during or induced AF. 3. Multiple accessory pathways 4. Ebsteins anomaly 5. Familial WPW syndrome. The Non invasive parameters of a presumed long refractory period of bypass tract (>250ms) include: 1. Intermittent pre-excitation. 2. Disappearance of pre-excitation during exercise and 3. Disappearance of pre-excitation with procainamide. The predictive value of the non invasive parameters is low and when combined with the low risk of RF ablation, most electrophysiologists rely on invasive testing to risk stratify asymptomatic patients. In a European survey 44% of EP centers perform invasive approach as the first line approach [Regardless of how the EP protocol is set, one of the specific parameter taken is anterograde ERP <240ms at baseline to decide RF ablation. Additional parameters include minimum RR interval in AF: SPERRI <250ms, and AF induction. Considering all boundary parameters, the best binary discriminators are APERP <240ms at baseline and SPERRI <250ms, which we follow at our centre. In perhaps the best prospective follow up study in children age 8-12 yrs, with a follow up of 57 months, Pappone et al [The "PACES /HRS Consensus statement on the management of the Asymptomatic Young Patient with a WPW Electrocardiographic pattern" published this week  has noteTherefore the most important binary parameter to the invasive electrophysiologist is SPERRI <250ms and APERP <240ms at baseline (without isoproternol) to decide RF Ablation.The important question raised in this issue by Oliver C et al in this issue of the journal  is the sThe changes of baseline APERP temporally in the basal state, as noted by the authors,  are variThe response to APERP on isoproternol has been studied by multiple authors; though acceptance of a standard value has not been universally agreed upon. A value of APERP <200ms on isoproternol has been noted as a cut off value, but the dose of the drug has ranged from 1-20μg per minute. The authors  have notI would like to conclude by noting that APERP estimations are temporally variable. The variations are more pronounced during isoproternol infusion. As a matter of fact all EP parameters ranging from venricular ERP to VT Inducibility are temporally variable; what is important is to know the significance of a baseline parameter in relation to the natural history of the disease. On the background of the natural history studies by previous authors, the variations in the basal state are acceptable and a value of APERP <240ms can serve as a binary cut off value to indicate and select a potentially high risk group that can undergo RF ablation to reduce SCD. EP studies of APERP on isoproternol show significant variations temporally and cannot be relied upon as a sole predictor of future risk, independently; however it can contribute to the basal study. . Based on the Consensus Statement and Recommendations SPERRI <250 ms can serve as a parameter to select patients for catheter ablation  and is s


The liver plays a vital role in glucose homeostasis, the synthesis of bile acids and the detoxification of foreign substances. Liver culture systems are widely used to test adverse effects of drugs and environmental toxicants. The two most prevalent liver culture systems are hepatocyte monolayers (HMs) and collagen sandwiches (CS). Despite their wide use, comprehensive transcriptional programs and interaction networks in these culture systems have not been systematically investigated. We integrated an existing temporal transcriptional dataset for HM and CS cultures of rat hepatocytes with a functional interaction network of rat genes. We aimed to exploit the functional interactions to identify statistically significant linkages between perturbed biological processes. To this end, we developed a novel approach to compute Contextual Biological Process Linkage Networks (CBPLNs). CBPLNs revealed numerous meaningful connections between different biological processes and gene sets, which we were successful in interpreting within the context of liver metabolism. Multiple phenomena captured by CBPLNs at the process level such as regulation, downstream effects, and feedback loops have well described counterparts at the gene and protein level. CBPLNs reveal high-level linkages between pathways and processes, making the identification of important biological trends more tractable than through interactions between individual genes and molecules alone. Our approach may provide a new route to explore, analyze, and understand cellular responses to internal and external cues within the context of the intricate networks of molecular interactions that control cellular behavior. The liver is one of the important organs in our bodies, playing a vital role in glucose homeostasis, the synthesis of bile acids for the metabolism of cholesterol, and the secretion of proteins to aid clotting This analysis did not consider the fact that a cell's response to its environment is governed by an intricate network of molecular interactions. These interactions dynamically change in response to a myriad of cues. Therefore, discovering the set of molecular interactions that are active in a given cellular context is a fundamental question in computational systems biology expression -values,a functional or physical interaction network connecting genes and proteins, anda dataset of functional annotations for genes and proteins.et al.et al.We extend the method developed by Dotan-Cohen et al. to incorporate gene expression measurements to determine which inter-process links are significantly perturbed between the measured conditions. Informally, we compute a score for a link from process et al. The second hypothesis tests the dependence of the score on the specific interactions in the network, i.e., it asks if we would observe the score from link -value, to distinguish it from the expression In this work, we extend the ideas of Dotan-Cohen We used the Affymetrix Rat Genome 230 2.0 GeneChip to measure genomewide transcriptional profiles in rat hepatocytes grown in monolayers and in collagen sandwiches functional association, which the authors define as “the specific and meaningful interaction between two proteins that jointly contribute to the same functional process.” Apart from incorporating experimental interaction data, STRING uses multiple methods to predict possible functional linkages including interolog-based interaction transfer, similar transcriptional response across a variety of conditions (co-expression), text-mining, and gene families that share above-random similarities in their evolutionary histories. STRING includes a scheme to score each predicted interaction in the range 150–1000 against a common reference of functional partnership based on the KEGG database Existing databases of protein interactions contain very few experimentally detected Protein-Protein Interactions (PPIs) for rat: seven different widely-used sources In our earlier work et al.We considered only those links with a link Several salient trends emerged. First, in 0.1) see . Third, Finally, the overlap between the intersection of the results from both hypothesis tests and the BPLN was small in days 1 and 2 and more substantial in days 3 and 8 , althougAlthough both hypothesis tests find very similar sets of process pairs to be significantly linked at the 0.01 level, especially in later days, we found that the actual link ther see . Based other see , noting ther see . When weet al.The 251 genes in the HSIAO_LIVER_SPECIFIC_GENES gene set are expressed selectively in the liver, as determined by Hsiao 5′-GTTAATNATTAAC-3′. The promoter regions of genes in the MSigDB set V$HNF1_Q6 match this binding site for HNF1 Hepatic nuclear factor 1 (HNF1), also known as albumin proximal factor, is a transcription factor required for the expression of several liver-specific genes including albumin CBPLNs assist us in elaborating upon these earlier observations. We studied the link between V$HNF1_Q6 and HSIAO_LIVER_SPECIFIC_GENES in the day 8 CBPLN by examining the functional interactions in the STRING database connecting genes in V$HNF1_Q6 to genes in HSIAO_LIVER_SPECIFIC_GENES. HNF1 gene Hepatocyte nuclear factor 4Plasminogen (Plg) is a secreted protein that is proteolysed to plasmin and angiostatin. Plasmin dissolves fibrin in blood clots while angiostatin inhibits angiogenesis. In Cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1) encodes a member of the cytochrome P450 superfamily of enzymes. Cytochrome P450s proteins are monooxygenases, which carry out the liver's prominent role in xenobiotic metabolism and synthesis of cholesterol, steroids and other lipids. CYP2E1 is an important member of this family, implicated in the metabolism of exogenous compounds such as benzene, carbon tetrachloride, ethylene glycol, and substances found in cigarette smoke as well as endogenous compounds including ethanol, acetone, and acetal Two of the most important functions that hepatocytes in the liver carry out are lipid homeostasis and bile acid synthesis. These two functions are intrinsically linked. As illustrated schematically in tabolism .Before we examine some of these links in more detail, we stress that the links in CBPLNs  must be interpreted with caution. Both HSA03320_PPAR_SIGNALING_PATHWAY and HSA00120_BILE_ACID_BIOSYNTHESIS are up-regulated in CS cultures in contrast to HMs . Bile acWe also note modest changes in the interconnections between the gene sets in Two other noticeable changes over the time series have immediate biological interpretations. First, the link from NUCLEAR_RECEPTORS to HSA03320_PPAR_SIGNALING_PATHWAY appears at day 2, which we interpret as a regulatory relationship reflected in the underlying functional interaction network and the corresponding up-regulation of the two gene sets. Second, the link from HSA00071_FATTY_ACID_METABOLISM to HSA03320_PPAR_SIGNALING_PATHWAY also appears at day 2, which we interpret in light of feedback in the fatty acid metabolic pathway. In the rest of this section, we discuss the linkages between these three gene sets, anchoring our discussing on the underlying functional interaction networks on day 8  and 10. In in vitroIn vitro studies have shown that Nr5a2 both promotes and represses the expression of CYP7A1 Lrh-1 (Nr5a2) performed selectively in cells that developed into mouse hepatocytes demonstrated that the absence of Nr5a2 had little effect on expression of CYP7A1All these nuclear receptors exhibit increasing perturbation over time, and interact with CYP7A1, a cytochrome P450 enzyme that is a member of the PPAR signaling pathway. Note that CYP7A1 itself shows no significant perturbation until day 8. We discuss the support in the literature for a subset of the interactions with CYP7A1. HNF4CYP7A1 until day 8. However, in vitro studies indicate that CYP7A1 protein exhibits low turnover Liver X receptors regulate cholesterol and lipid homeostasis in multiple tissues via two isoforms: LXRLrh-1 (Nr5a2) in mice CYP7A1, the knockout caused a significant drop in expression of CYP8B1, demonstrating a very strong regulatory relationship between Nr5a2 and CYP8B1CYB8B1 expression exists CYP8B1 also increases over time, although it lags the expression of its regulatory receptors Hnf4a and Nr5a2.Another set of contributions to the linkage between these two gene sets come from interaction of the nuclear receptors Hnf4a and Nr5a2/Lrh1 with sterol 12CYP27A1 concomitant to but lagging that of Lrh-1 (Nr5a2). The knockout of Lrh-1 led to significantly decreased expression of CYP27A1Nr5a2 is also predicted to interact with 27-hydroxylase (CYP27A1), a mitochondrial cytochrome P450 enzyme that is responsible for a step in the conversion of cholesterol to approximately 25% of the bile acids in mouse Next, we focus on the role played by PPARIn  as well . RXR hasScd1 transcription through an upstream response element Scd1Stearoyl-Coenzyme A desaturase 1  with PPARThe genes in NUCLEAR_RECEPTORS are responsible for initiating cellular responses to a wide variety of conditions and for starting appropriate signal cascades. The nuclear receptors in HSA03320_PPAR_SIGNALING_PATHWAY are the specific subset responsible for initiating the signaling cascade leading to the breakdown of long chain fatty acids Keeping the examples of the previous sections in mind, we now discuss how links in CBPLNs might be interpreted.Gene set Multiple gene sets may link to a gene set Links that exist in both directions between on day 2  but bidion day 2 .A link from process A gene set et al.We have presented an approach that represents cellular responses at the granularity of biological processes and connections among them. Our approach extends the work of Dotan-Cohen response network, which may be defined as the sub-network of interactions that are perturbed in a particular condition. A wide variety of methods have been developed for computing such response networks Our approach is a complement to a suite of methodologies that integrate physical, signaling, regulatory, and functional networks with measurements of molecular profiles such as transcriptional, proteomic, or metabolic data to compute the Generalizing our approach to the entire spectrum of MSigDB gene sets or to the set of all biological processes in the Gene Ontology raises several interesting challenges. First, gene sets can have considerable overlap, leading to redundant links. Second, scaling this approach up to thousands of gene sets may result in tens to hundreds of thousands of links that are deemed to be statistically significant. This deluge of links will be hard to interpret. Third, it will be challenging to computationally scale our permutation-based sampling to the large number of process pairs we will have to test. We are currently investigating these issues.In this work, we computed CBPLNs for two conventional hepatocyte culture systems. Three dimensional liver mimics We applied Linear Models for Microarray Data (LIMMA) et al. to identify linkages between biological processes et al. compute what they term a Biological Process Linkage Network (BPLN). Informally, given two biological processes, they defined the first process as being linked to the second process if genes annotated by the first process interact with a significant number of genes annotated by the second process. By definition, such links are directed. The resulting output of the algorithm by Dotan-Cohen et al. is, for each ordered pair of processes, the probability that the first process is linked to the second.We first present the approach developed by Dotan-Cohen linked to process universeFormally, let gene gene In other words,With this background, we extend the formulation of BPLN to take transcriptional measurements and interaction weights into account. For each interaction The function contextual linkage scoreWe define the In this formulation, some pairs of processes may have a high contextual linkage score even if all genes were perturbed by the same amount. To account for this possibility, we compute a normalized score et al. it is unclear how to compute its statistical significance analytically. Therefore, we use two different approaches in order to assess the significance of the observed score Since the contextual linkage score is a weighted generalization of the statistic measured by Dotan-Cohen et al.The first approach is an empirical version of the test performed by Dotan-Cohen Two different processes may annotate some genes in common. To preserve this property even in the random selections of the set The second approach accounts for the role played by the interactions between the genes in To construct We use the method of Benjamini and Hochberg File S1et al.File S1 is in tab-separated values format. It contains results of comparisons on the number of links identified to be significant under the two hypothesis tests, as well as under the original BPLN algorithm by Dotan-Cohen, (TSV)Click here for additional data file.File S2et al.File S2 is in tab-separated values format. It contains results of comparisons between the two different hypothesis tests, as well as the original BPLN algorithm by Dotan-Cohen, (TSV)Click here for additional data file.File S3-value ) by least one of the hypothesis tests (based on gene set randomization or on network randomization) with normalization. Each plot corresponds to a single day. Each point on a plot corresponds to one pair of processes, with the -coordinate being the -value from gene set randomization and -coordinate representing the -value from network randomization. In each plot, both axes are on a logarithmic scale.File S3 contains scatter plots of link (TIFF)Click here for additional data file.


After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations  in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE). Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form.Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in  zinc finger protein 644 isoform 1 (ZNF644) were identified as potentially responsible for the phenotype of high myopia. The main feature of high myopia is axial elongation of the eye globe. Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, a mutant ZNF644 protein may impact the normal eye development and therefore may underlie the axial elongation of the eye globe in high myopia patients. Further study of the biological function of ZNF644 will provide insight into the pathogenesis of myopia.People with myopia see near objects more clearly than objects far away. Myopia is the most common ocular disorder worldwide, with a high prevalence in Asian (40%–70%) and Caucasian (20%–30%) populations. Although the etiologies of myopia have not yet been established, previous studies have indicated the involvement of genetic and environmental factors . Genetic factors play a critical role in the development of myopia, especially high myopia. In this study, we use exome sequencing, a powerful tool for a disease gene identification, to identify a gene involved in high myopia in a monogenic form among Han Chinese. Mutations in  Myopia is the most common ocular disorder worldwide, with a prevalence of 20–30% in North American, European and Australian populations In high myopia, affected patients' eyes have a spherical equivalent of less than or equal to −6.00 diopter sphere (DS) and an axial length longer than or equal to 26.0 mm. In some cases, high myopia may also show retinal pathological changes with progressive choroidal degeneration in the posterior pole and other complications, potentially resulting in severe vision loss. In such cases, high myopia is referred to as pathological or degenerative myopia, which is one of the leading causes of blindness in the world The exact pathogenesis of myopia remains unclear. There are indications that environmental factors  Myopia can be inherited as a complex trait or in a monogenic form. For the complex form, myopia appears to be the result of an interaction of multiple genes and environmental factors. Recently, several loci have been identified by genome-wide association study (GWAS) as being responsible for complex myopia Here we describe a Han Chinese family (951) from Chengdu, China, that has monogenic high myopia with a dominant inheritance model. The clinical features of the nineteen of 20 living family members who participated in this study are shown in For this analysis, we selected two affected family members (V:1 and III:2) , Table 1We used exome sequencing to identify potential variants responsible for high myopia in this family. We generated an average of 2.4 Gb of sequence with 30× average coverage for each individual as single-end, 80-bp reads, and about 97% (∼32.98 Mb in length) of the targeted bases were covered sufficiently to pass our thresholds for calling SNPs and short insertions or deletions (indels) . The basfttp://www.1000genome.org). This left a total of 393 variants that were shared between these two patients. Of these, 332 genetic variants  were predicted to potentially have a functional impact on the gene   . We obtaZNF644 exons in an additional 300 unrelated , sporadic high myopia patients. The 300 patients had refractive errors ranging from −6.0 to −29.0 DS for both eyes and an axial eye globe length from 26 to 33 mm for both eyes  were absent in all the 600 controls. Among these five mutations identified from the sporadic cases, three were in exon 3  and two were in the 3′ untranslated region (UTR) (+12 C>G and +592 G>A) (ZNF644 and one was located in the 3′UTR (+1015 C>G). The P-value for the 17 potentially functional variants in 301 patients with high myopia (One mutation identified in one member of the high myopia family 951 plus the 6 mutations identified in the 16 unrelated patients from the 300 sporadic cases) compared with these variants being seen in 600 controls (3 in 600 controls) was 2.28×10−6 by Fisher's exact test. This data suggests that there are multiple rare variants in ZNF644 associated with high myopia.We further carried out direct PCR sequencing of the oth eyes . Some ofoth eyes , Table 5592 G>A) , Table 5ZNF644 gene is expressed in the eye, we examined ZNF644 expression in different human tissues using reverse transcript polymerase chain reaction (RT-PCR). The ZNF644 gene was expressed in the human retina and RPE as well as in the liver and placenta (To make sure that the placenta .http://www.hgmd.cf.ac.uk/ac/index.php). Therefore, through sequencing and comparing the coding region of affected and unaffected individuals within a family and filtering the benign changes using a public database, such as 1000 Genomes Project and dbSNP databases, the mutation in the coding region can be identified even within small families and without knowing the pathway of a disease and marked locus heterogeneity. Currently, the cost of the exome sequencing method is even less than that of the positional cloning strategy. So, this method will not only speed up disease gene identification but will enable us to systematically tackle previously intractable monogenic disorders. In fact, exome sequencing approaches have been successfully used to identify disease genes for Mendelian disorders in recent studies For the past several decades, standard methods for identifying genes underlying disease in a monogenic form have primarily been through selecting candidate genes for testing or by using positional cloning. The candidate gene approach requires less work and costs less because only the candidate gene needs to be sequenced, but this method requires prior knowledge of the pathogenesis of a disease for gene selection. This fundamentally impedes the disease gene identification speed because the pathogeneses of many diseases have not yet been unmasked. Without pathogenesis information of a disease, the traditional positional cloning strategy can be used first to map the disease gene in the chromosome and then to identify the disease-causing gene within a specific interval. Thus the pathogenesis of a disease can be explored based on the identified disease gene. However, the positional cloning method requires marked locus heterogeneity and the availability of a large family. Focusing on the exome can be especially fruitful in disease gene identification given that previous studies have indicated that approximately 85% of causal mutations for human diseases are located within the coding region and canonical splice acceptor and donor sites  only the S672G mutation identified in the two affected patients showed complete co-segregation with the disease phenotype in the family studied; 2) our analysis of the ZNF644 gene in 300 unrelated, sporadic high myopia patients identified an additional three missense mutations and two mutations in the 3′ UTR which may affect mRNA stability or microRNA interaction; 3) none of these identified mutations were present in the 30 genomes of Han Chinese Beijing in the 1000 Genomes Project database, the Han Chinese Beijing SNPs in the dbSNP131 database, or 600 normal ethnicity-matched controls; and 4) Comparative analyses of ZNF644 in other species showed that I587 is conserved and S672, R680, C699 are highly conserved among primates, placental animals, and other vertebrate species . Based on protein structure, ZNF644 is predicted to be a transcription factor (http://www.genecards.org/cgi-bin/carddisp.pl?gene=ZNF644), and given that it has potentially deleterious mutations in patients with high myopia, it may play a role in gene expression regulation in the retina and retinal pigment epithelium (RPE). One important issue in the genetic study of high myopia is the age of disease onset. We would have an informative censoring problem if family members of 951 did not show the disease phenotype because their age was too young. However, the disease phenotype studied in family 951 is very special. The disease onset was at 3–4 years old for all affected patients in the family 951 with high myopia; all affected patients developed high myopia by the age of seven. The youngest unaffected member in the family (V:2) is 9 years old now; he does not show any signs of myopia at all. In addition, all affected patients in the family had severe high myopia, which allowed us identify the affected patients easily. Therefore, there is very little chance that an unaffected family member does not show the trait by virtue of being too young.Using this technique, we successfully identified a gene for high myopia in an affected family. Several lines of evidence provided support for the mutations in PRPC8 is ubiquitously expressed in human tissues ZNF644 gene having activity in the eye. Note that, given that less than 4% of the sporadic high myopia cases had mutations in ZNF644 (we identified 5 different mutations in 11 patients out of 300 cases), the ZNF644 gene is unlikely to play a major role in sporadic high myopia.Although it clearly has a ubiquitous level of expression, this is common for other genes involved in eye diseases  with high myopia that had 30 (20 living) family members , Table 1http://www.ncbi.nlm.nih.gov/projects/CCDS/). The exon-enriched DNA libraries were then subjected to a second library construction in preparation for Illumina GA sequencing and were sequenced using the Illumina Genome Analyzer II platform, following the manufacturer's instructions  The exome sequencing approach was used to identify the disease-causing genetic variant for the high myopia family in the study (951). Genomic DNA was extracted from peripheral white blood cells, using Gentra Systems PUREGENE DNA purification kit . Fifteen µg of genomic DNA from each of the two selected individuals , version hg18 (build36). Alignment of the sequences from the two affected individuals was performed using SOAPaligner after we removed the duplicated reads fttp://www.1000genome.org), and against the Han Chinese Beijing SNPs in the dbSNP131. Sanger sequencing was then used to validate the identified potential disease-causing variants. SIFT (http://sift.jcvi.org) was used to predict whether an amino acid substitution affects protein function.The human reference genome, together with its gene annotation, was downloaded from the UCSC database (ZNF644 were designed using primer 3 (http://frodo.wi.mit.edu/primer3/) -product sequencing using Bigdye terminator v3.1 cycle sequencing kits  and analyzed on an ABI 3130XL Genetic Analyzer. We used Sanger sequencing to determine whether any of the remaining variants co-segregated with the disease phenotype in family 951 and used MLINK of the LINKAGE program to calculate a two-point LOD score for the detected variants to assess the locus position of the predicted disease gene in family 951 rimer3/) , and allZNF644 gene in the human liver, retina, RPE, and placenta. The human liver, retina, and RPE were donated by a deceased 55-year-old Han Chinese male and the human placenta was donated by a 29-year-old Han Chinese female. Total RNA from the human liver, placenta, retina, and RPE was extracted by trizol , and reverse transcription was performed using a reverse transcription kit . The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control Click here for additional data file.Table S2Primers for gene expression by RT-PCR.(DOC)Click here for additional data file.


APOE and reflects an apparent early disturbance in lipid metabolism. The rate of biological aging in AD patients, which cannot be explained by BioAge, is associated instead with NdStress, which includes genes related to protein folding and metabolism. Inflame, comprised of inflammatory cytokines and microglial genes, is broadly activated and appears early in the disease process. In contrast, the disease-specific biomarker Alz was selectively present only in the affected areas of the AD brain, appears later in pathogenesis, and is enriched in genes associated with the signaling and cell adhesion changes during the epithelial to mesenchymal (EMT) transition. Together these biomarkers provide detailed description of the aging process and its contribution to Alzheimer's disease progression.Alzheimer's disease (AD) is a complex neurodegenerative disorder that diverges from the process of normal brain aging by unknown mechanisms. We analyzed the global structure of age- and disease-dependent gene expression patterns in three regions from more than 600 brains. Gene expression variation could be almost completely explained by four transcriptional biomarkers that we named BioAge , Alz (Alzheimer), Inflame (inflammation), and NdStress (neurodegenerative stress). BioAge captures the first principal component of variation and includes genes statistically associated with neuronal loss, glial activation, and lipid metabolism. Normally BioAge increases with chronological age, but in AD it is prematurely expressed as if some of the subjects were 140 years old. A component of BioAge, Lipa, contains the AD risk factor  A number of genetic risk factors for AD have been proposed An important goal of AD research is to identify interventions that maintain brain function, potentially by inhibiting the formation or improving the clearance of neurotoxic aggregates, or by promoting resistance to, or recovery from, damage. A number of biological processes have been associated with AD, including cholesterol metabolism, inflammation, and response to misfolded proteins such as increased expression of heat shock proteins http://www.brainbank.mclean.org/PDF files/Consent.pdf). All tissue and research were conducted according to the Harvard Brain Tissue Resource Center Guidelines . The study was approved by McLean Hospital Institutional Review Board.The dataset comprises gene expression data from brain tissues that were posthumously collected from more than 600 individuals with AD diagnosis, HD diagnosis, or with normal non-demented brains. All brains were obtained from individuals for whom both the donor and the next of kin had completed the Harvard Brain Tissue Resource Center Informed Consent   http://national_databank.mclean.harvard.edu/brainbank/Main). Any researcher wishing to obtain the de-identified dataset can do so by contacting the National Brain Databank at McLean Hospital, Harvard Medical School.We refer to each batch of tissue samples using the abbreviated brain region and the phase of the experiment . This microarray dataset is MIAME compliant. The raw and final processed data for each hybridization are available to any researcher upon request. The essential sample annotation including experimental factors and their values  is available and summarized in z-scores more than 3.We used the log10-ratio of the individual microarray intensities to the average intensities of all samples from the same brain region profiled in the same phase as the primary measure of gene expression. Quality control of gene expression data was performed by principal component analysis using MATLAB R2007a . Outlier samples (less than 2%) were removed from the data set based on extreme standardized values of the first, second, or third principal components, with absolute p, assuming normal distribution for Fisher z-transformed values, atanh ρ The first principal component (PC1) was used to assess the major pattern of gene expression variability in the dataset. Genes that were highly correlated with PC1 were used to build a surrogate biomarker. Throughout this work we used Pearson correlation coefficients, ρ, and assessed their significance, Gene expression changes associated with aging and disease were characterized by metagenes combining sets of genes with significant association with a disease trait and a very strong Pearson correlation with each other. We utilized a procedure of exploring covariance structure of the gene expression data similar to metagene identification The biological nature of the metagenes was assessed using the gene set overlap analysis with known biological processes described in GeneGo or Ingenuity. Significance of the overlaps between the lists was assessed using Bonferroni-corrected p-values biomarker to refer to a metagene together with associated score that quantifies it in each brain tissue sample. The biomarker score for each sample was calculated as the mean expression levels of the comprising gene probes or as the arithmetic difference between the means in the positive and negative arms of the metagene when both arms were specified.I/I0 is normalized intensity of the metagene probes. To produce a robust score, all samples have to be normalized to the same reference. The reference intensity I0 for each gene corresponded to the average intensity in the cohort. Importantly, averaging genes that correlate with each other produced a measure that is more accurate than individual genes. For all metagenes identified in this work, the biomarker score represented a quantitative measure of a particular disease aspect in each brain sample. To evaluate the performance of the biomarker score as a classifier between diseased and normal samples, we used the area under the curve for the receiver operating characteristic (AUROC) Throughout this work we adopted the term To validate biomarkers identified in this work, we tested their coherence and predictive power in the context of independent gene expression dataset, GSE1572 An additional set of public gene expression data used to validate the coherence and predictive power of the biomarkers was obtained from hippocampus samples from elderly control and AD subjects, GSE1297 p<1E–6) by more than 28% (see p = 9E–13) and concluded that this gene expression pattern captures normal aging processes in prefrontal cortex. Interestingly, this correlation did not exist in AD patients . Analysis of differential gene expression in prefrontal cortex samples between non-demented individuals and AD patients revealed massive changes, with more than 18,000 transcripts significantly regulated  (p = 4E–11), validating that aging is a major reproducible source of variance in gene expression in PFC. Similar prediction of chronological age using gene expression was recently proposed As an independent test of the power of BioAge to predict normal chronological age, we applied this biomarker to an independent cohort of prefrontal cortex samples from non-demented individuals (GSE1572). This gene expression data were used to qualitatively describe aging in an earlier study  = 8E–7) . In addip = 4E–4). p = 1E–5), capturing a major source of gene expression variance and an overall brain condition.We performed another validation of BioAge as a predictive biomarker of the brain condition in elderly subjects both with and without AD using publicly available gene expression data from hippocampus samples (GSE1297). These data were used in prior research to qualitatively describe AD progression based on gene expression correlation with the disease severity, from control group through severe AD GFAP , shown to be strongly correlated with BioAge in The massive gene expression changes associated with aging that we detected involved a constellation of biological processes. A gene set annotation analysis revealed that the genes downregulated with increasing BioAge showed significant enrichment for neuronal and synaptic processes, possibly reflecting neuronal depletion or loss of plasticity . The uprGFAP . In agreAPOE, PPARA, γ-protocadherins, and other genes involved in lipid metabolism, amino acid metabolism and cell adhesion. Other notable Lipa genes included HES1, TGFB2, NTRK2, and WIF1. This metagene was much more coherent in normal samples than in AD samples. The corresponding Lipa biomarker indicated an average 3-fold upregulation of these genes early in the aging process. We also noticed that the genes upregulated with age in normal samples could be further dissected using a metagene discovery approach see . We focup<0.005, absolute fold change >25%, FDR<0.1). Higher BioAge of AD patients explained more than 50% of the differential expression between normal and AD cohorts. In the range of BioAge scores in which AD and normal individuals overlap, there was a significant residual differential expression, composed of several distinct subpatterns that explain a large fraction of the normal-to-AD variance. We focused on 88 AD and 43 normal brain samples with matched moderate levels of BioAge between −0.1 and 0.3. We identified 625 genes that are differentially expressed between the two cohorts . At the same time, NdStress and chronological age correlated negatively . This biomarker score explained 22% of variance in differentially expressed genes and demonstrated AUROC of 0.75 in separating AD and normal samples. See The first and the largest group of about 2000 genes, further referred to as NdStress, was associated with various metabolic disruptions. This metagene contained some genes that were upregulated and others that were downregulated in AD samples. In normal brain samples with BioAge<0, the expression of these genes was maintained in a relatively stable narrow range with relatively low coherence. In AD samples, however, the expression of these genes varied dramatically and was highly correlated . AlthougCTSK, MFAP2/4, FN1, VIM, WNT6 and TWIST1 (p<1E–7) and chronological age , see also The second metagene, further referred to as Alz, consisted of about 200 genes upregulated in AD . This med TWIST1 , Fig. S4IL1B, IL10, IL16, IL18, and HLA genes, as well as markers of macrophages, such as VSIG4, SLC11A1, and apoptosis, such as CASP1/4, TNFRSF1B (p75 death receptor) (p = 1E–10) and chronological age  in AD samples. When used as a classifier, the Inflame score was capable of discriminating AD and normal brain with AUROC of 0.69. These genes maintained their mutual correlation in both normal and AD samples but reached significantly higher levels in AD  , Fig. S4ls in AD .p = 0.004). We also observed a low correlation between NdStress and Alz  and moderate correlation between Alz and Inflame  in AD samples.p = 0.04). We tested performance of these biomarkers in the public gene expression data from hippocampus samples from elder non-demented and AD subjects (GSE1297). We found that at least half of genes in each of the biomarkers preserved their coherence. In addition, we found that Alz was strongly elevated in 5 out of 7 severe cases of AD and correlated with MMSE  and cerebellum (CR). We confirmed that BioAge and the disease-specific biomarkers were also expressed coherently and differentially between normal and AD samples. We then performed direct correlation analysis between the biomarker scores in these regions. BioAge demonstrated relatively high correlations of 0.81 between VC1 and PFC1, with residual differences possibly reflecting different levels of aging between the brain regions. The Lipa biomarker also demonstrated high correlation of 0.80 between these regions. We determined that correlation between Inflame scores in PFC1 and VC1 was equal to 0.83. The highest correlation of 0.93 between PFC1 and VC1 was observed in the NdStress biomarker. These results are also shown in p<1E–6). In general, these biomarkers reached similar average levels in AD and HD samples in all profiled brain regions. In PFC2, however, the average BioAge reached in HD samples was significantly lower than that of AD samples (p = 1E–17). These biomarkers, therefore, seemed to capture general systemic neurodegenerative processes rather than being specific to AD. The most striking difference between AD and HD samples was reflected in the Alz biomarker, which again was specific to the presence of AD was not significantly elevated in any brain region in HD samples . In addition, “astrocytic” M15 positively correlates with BioAge (ρ = 0.83) and “neuronal” M16 negatively correlates with it (ρ = −0.93). We also found that none of the major brain transcriptome modules strongly correlated with either the neurodegenerative NdStress or AD-specific Alz biomarkers. This confirms that these expression patterns are novel patterns that can only be detected in brains affected by disease.This genome-wide gene expression profiling study of a large cohort of AD and normal aging brains revealed large groups of genes that vary as a function of age and disease status. When the hundreds of gene expression values contained in each of these sets are converted into a single quantitative trait, new molecular biomarkers of biological aging and disease progression emerge. The transcriptional profiles of AD brains were profoundly different from those in non-demented individuals, with thousands of genes differing in their levels of expression between the two cohorts. To reduce the complexity of the observed changes, we focused on key gene expression patterns that explained the most variability across the cohorts. We demonstrated that the most significant pattern in terms of variance explained both within and between AD and non-demented cohorts was BioAge, a biomarker of the level of biological aging in the brain. BioAge captured the extent of gradual molecular changes in the normal aging brain by averaging the gene expression changes associated with a multitude of synchronous physiological events. BioAge can be accurately and reliably assigned to each brain tissue sample in the dataset and used to describe the molecular state of the brain in the same way as we use other clinical and physiological measurements.Genes upregulated with BioAge point to activation of cell cycle regulation pathways, lipid metabolism and axon guidance pathways . MisexprAPOE, HES1, and TGFB2  contained genes associated with cell adhesion, migration, and morphogenesis. This metagene prominently featured genes characteristic of epithelial-to-mesenchymal transition (EMT), such as Finally, BioAge and Inflame are consistent with published analysis of the healthy brain transcriptome and are associated with neuronal, astrocytic, and microglial modules Our analysis of gene expression changes in the brains of Alzheimer's patients confirms that AD is both similar and distinct from the process of normal aging. Although each brain was captured only in a particular (postmortem) state and was not studied longitudinally, we can assemble these data as a function of time to propose a few generalized aging trajectories. BioAge and chronological age showed a significant association in non-demented individuals and no association in AD patients, who had consistently high BioAge scores regardless of their chronological age. We attribute this observation to a difference in the strength of the aging drivers, distribution of the aging rates, and different causes of death in the two cohorts. In non-demented individuals, the drivers of aging were weak; the rates of aging were relatively slow and consistent across the population; and, in the absence of unnatural causes, death was likely related to aging issues other than the health of the brain. Since non-demented individuals likely died from causes largely unrelated to neurodegeneration, each individual death is conceptually a random event along the generalized brain aging trajectory. In AD patients, the drivers of aging were stronger and variable across the cohort; and death was generally related to the health of the brain that became incompatible with life regardless of the chronological age. The extrapolated BioAge of normal patients would not reach the highest AD levels until the age of 140 years. Thus, AD can be viewed as an aberrant aging of the brain, which retains the gene expression hallmarks of normal aging combined with additional patterns associated with pathological drivers of the disease and response of the brain tissue to disease-related processes.For AD patients, we are missing early stages of the aging trajectory and observe only late stages with terminally high BioAge. Unlike the normal cohort that can be represented by a single trajectory, the AD cohort covers a family of trajectories with different rates of biological aging. Patients with a fast rate of biological aging would succumb to disease at younger ages and generally would have higher levels of BioAge relative to their chronological age in the early phases of disease. However, since we do not have longitudinal specimens from subjects before they develop the disease, a second biomarker is required to explain disease progression rates after BioAge is maximal. The expression profile of NdStress fits the properties expected of this progression rate biomarker: it is highest in chronologically young AD patients, and it significantly correlates with (+) BioAge and (−) chronological age. Alz, on the other hand, is the highest in chronologically older patients and does not correlate with BioAge. Thus, patients with high NdStress likely have more accelerated aging trajectories than patients with high Alz. The older chronological age of Alz onset may suggest that the acceleration of BioAge due to Alz does not occur until the level of BioAge of the brain reaches a certain threshold. The quantitative assessment of the brain biological age in terms of BioAge and the rate of its disease-related acceleration in terms of NdStress are two critical hypotheses proposed in this work.APOE and other lipid metabolic genes together with Notch and TGFβ signaling, signifying the transition from N0 to N1. The following upregulation of the Inflame biomarker is associated with transition from N1 to N2. The brains in these states were diagnosed as normal because the subjects did not yet exhibit any cognitive impairment associated with AD. The next transition, from N2 to A1, is associated with massive disruptions in metabolic pathways and marked acceleration of aging follows. Some brains, however, avoid transitioning to A1 and continue to age into N3. Another transition to AD state A2 can happen later, since we observe brains with high scores for both NdStress and Alz, which may be associated with a different path to AD. Alternatively, A2 is possibly localized to a brain region not covered in the dataset. This transition may, therefore, appear later than A1 in a particular brain region and happen much earlier in some other brain region.Another way to look at the aging trajectory is to model it as a set of molecular transitions that lead to changes in BioAge. Examination of biomarker scores for BioAge-low brains in The proposed model is most consistent with an age-based hypothesis of Alzheimer's disease that postulates three fundamental steps: initial injury aggravated by age, chronic neuroinflammation, and transition of most brain cells to a new state Figure S1Differential gene expression and variance in PFC1. (A) Cumulative p-value distribution in the t-test between AD and normal samples. The blue line shows the number of sequences that can be detected for a given p-value cutoff. For example, at p<1E–6, about 18,000 genes can be detected. The green line shows the level of false positives due to multiple testing. (B) Pareto diagram of variance explained by the first 10 principal components. The first principal component dominates the distribution explaining 33% of the data variance. (C) Comparison of correlations between PC1 and individual genes in normal and AD samples. Related to (PDF)Click here for additional data file.Figure S2Validation of BioAge and Alz biomarkers in GSE1297. Panel (A) demonstrates the relationships between projected BioAge score and the disease severity as MMSE. The points are colored according to the assigned severity level. The box plots represent the distribution of the biomarker scores in the hippocampus samples from non-demented control subjects and subjects with AD of different severity. Panel (B) shows the same analysis for the Alz biomarker.(PDF)Click here for additional data file.Figure S3Regulation of selected sell cycle regulation genes with BioAge. The heat map shows hierarchical clustering of selected 17 genes involved in cell cycle regulation and DNA repair. The samples (rows) are sorted according to the values of the first principal component of the complete dataset and labeled according to diagnosis . Related to (PDF)Click here for additional data file.Figure S4Expression of selected genes and their relationships with biomarkers. The heat map shows hierarchical clustering of 17 selected genes and 5 biomarkers developed in this work. The samples (rows) are sorted according to the values of the first principal component of the complete dataset and labeled according to diagnosis . Only samples with BioAge<0.4 are shown. Related to (PDF)Click here for additional data file.Figure S5Correlation between biomarker scores between PFC1 and CR1 of the same individuals. Each plot shows relationships between the biomarker values in PFC1 and CR1. Samples from non-demented and AD subjects are shown in black and red respectively. Related to (PDF)Click here for additional data file.Figure S6Validation of mutual relationships between key biomarkers in PFC2 cohort, which contained non-demented (black), AD (red), and HD (green) samples. Compare with (PDF)Click here for additional data file.Table S1Demographic, clinical, and experimental composition of the HBTRC gene expression dataset.(PDF)Click here for additional data file.Table S2Gene sets that comprise gene expression biomarkers of aging and AD progression.(XLS)Click here for additional data file.Table S3Functional annotations of the biomarker gene sets based on pathway enrichment. The abridged summary of this analysis is provided in .(XLS)Click here for additional data file.