From 1997 to 1999, 22 sets of CFS discordant twins from the University of Washington CFS Twin Registry were chosen for a 7-day in-person evaluation based on registry information and telephone screening establishing the presence or absence of symptoms consistent with the Centers for Disease Control (CDC) diagnostic criteria of CFS (1
). Twins were required to (1
) be at least 18 years of age; (2
) be reared together; (3
) be discordant for CFS (one twin met the CDC CFS criteria, the other did not); (4
) be negative for HIV; (5
) abstain from alcohol and caffeine and, based on their personal physicians’ advice, discontinue all medications at least 2 weeks prior to the evaluation; and (6
) travel to Seattle together.
To determine if a twin met CDC CFS criteria, we used responses to the CFS symptom checklist, diagnoses generated by the Diagnostic Interview Schedule (Version III-A) (32
), and information from review of the subject’s medical records. To meet criteria, debilitating fatigue must have been present for at least 6 months with endorsement of at least 4 of 8 CFS symptoms. Exclusionary medical and psychiatric conditions must have been absent. The same inclusion and exclusion criteria (e.g., body mass index, specific psychiatric disorders) and review processes were applied to the fatigued and non-fatigued twins. Medical records covering the last five years were reviewed by a physician knowledgeable about CFS (DB) for exclusionary medical conditions. A psychologist and infectious disease specialist also independently reviewed the twins’ medical charts to verify health status and approve twins for participation. Prior to the scheduled visit, we confirmed that the ill twin still met CFS criteria and that the control twin was devoid of CFS.
Between 2000 and 2003, the twins were contacted about participating in a follow up study. Of the 22 original pairs of twins, 14 agreed to participate in a second week-long evaluation. Written informed consent was obtained from all twins in accordance with regulations of the University of Washington Institutional Review Board. A waiver of consent was obtained from the University of Michigan Institutional Review Board to conduct the statistical analysis of the data at UM.
Depression was assessed using the Diagnostic Interview Schedule, a structured interview based on Diagnostic and Statistical Manual III (32
). Monozygosity was initially determined using previously validated self-report methods (33
), then confirmed with analysis of restriction fragment length polymorphisms. DNA samples were extracted and digested with the restriction endonuclease Hae
III. The restriction fragments were separated by molecular size in agarose gel, Southern blotted onto nylon membrane, and hybridized with a variable number of tandem repeat probes. With 6 probes, the probability of monozygosity can be ascertained with 99.9% certainty (35
Each pair of twins spent 3 consecutive nights and 1 day in the University of Washington Sleep Research Laboratory in temperature controlled, sound attenuated rooms. All sleep recording equipment was located in a central control room separate from the individual sleeping rooms. Twins were instructed to follow a set sleep schedule for 1 week prior to coming to the laboratory based on an average of their nightly sleep schedule ascertained from a 2-week sleep diary. This schedule was adjusted for twins who traveled to Seattle from Eastern, Central and Mountain time zones.
Throughout the study, the Sleep Research Laboratory investigators and technicians were blind to the illness status of the twins. During the first night, the twins adapted to the laboratory; baseline sleep data from the second night are reported here. The third night was an experimental manipulation night reported elsewhere (37
). The twins completed a 10-item post-sleep questionnaire each morning before getting out of bed.
Clinical Characteristics. Body mass index was computed from measured weight and height. Both history of and current major depression were assessed using the National Institute of Mental Health Diagnostic Interview Schedule. Depression was assessed using the Diagnostic Interview Schedule (Version III-A), (35
), a structured interview based on Diagnostic and Statistical Manual III. Menopause status was ascertained by asking “To your knowledge, have you reached menopause?” The tenderpoint examination was performed according to the published diagnostic recommendations (36
). Participants were considered to have fibromyalgia if they had ≥ 11 tender points and met criteria for widespread pain. Widespread pain was defined by the presence of upper and lower segment, right- and left-sided, and axial pain (36
). The age of onset and duration of CFS were computed based on self-reported dates.
Polysomnography. EEG electrodes were positioned at 2 frontal (F7, F8), 2 central (C3, C4), and 2 occipital (O1, O2) locations (International 10–20 system of measurement) and were referenced to the contralateral mastoids. Chin electromyogram electrodes and electrodes for right and left electro-oculogram also were attached. To monitor the twins for sleep-disordered breathing, airflow was measured using a nasal pressure cannula placed in the nose (Pro-Tech Services, Inc. Mukilteo, WA). Chest and abdominal respiratory effort was measured by Piezo Respiratory Effort bands placed around the chest and abdomen (Pro-Tech Services, Inc. Mukilteo, WA). Oxygen saturation was measured from the left or right index finger by a pulse oximeter (EMBLA, Broomfield, CO). Snoring was assessed by a small microphone sensor (Pro-Tech Services, Inc. Mukilteo, WA) placed on the throat, just lateral to the trachea. Electromyogram electrodes were placed on the anterior tibialis of each leg to monitor the occurrence of periodic leg movements during sleep. Two electrodes were placed on the chest to measure the electrocardiogram, according to the modified Lead II configuration.
Electrophysiological signals were recorded and digitized by the EMBLA somnologica data acquisition recording system (A-Ay-101, EMBLA, Broomfield, CO) and displayed and stored on a desktop computer. The sampling rates were set as follows: EEG, electromyogram, periodic leg movements, and electrocardiogram data = 200 Hz; electro-oculogram signal and snoring sensor = 100 Hz; nasal airflow and respiratory effort = 20 Hz, and oximeter = 1 Hz. All digitized data were acquired and stored unfiltered. Prior to each recording session, a standard 50 microvolt, 10 Hz calibration signal was recorded for 5 minutes. Data were displayed in 30 second epochs, on a continuous basis during recording.
Sleep Stage Scoring
All channels of recorded data were displayed on a high-resolution 21-inch color monitor for visual sleep stage scoring. Filter settings for display were set at 0.3 Hz to 40 Hz. Sleep and wake stages were scored in 30 second epochs according to standard criteria (38
). Key sleep architectural variables reported here include sleep latency to Stage 1 and sleep latency to Stage 2, time in bed (lights out to final arising), total sleep period (time in minutes from the first epoch of Stage 2 until final awakening), sleep efficiency (total sleep time/time in bed), sleep latency (time from lights out to first epoch of Stage 2 sleep), REM latency (time from sleep latency to first epoch of REM), time spent awake, and the percentage of NREM and REM sleep stages, expressed as a percentage of the sleep period time.
Power Spectral Analysis
On-board power spectral software from the EMBLA Somnologica data acquisition system was used to evaluate power in each of delta (0.5–3.9 Hz), theta (4.0–7.9 Hz), alpha (8.0–11.9 Hz), sigma (12.0–15.9 Hz), and beta (16.0–31.9 Hz) bands. The algorithm used a 512 point fast Fourier Transform with Hamming windows (−53 dB stop band, filter degree 1068, transition bandwidth 0.622 Hz), in 6 second blocks. The resultant power values, expressed in μV2, were then averaged in consecutive 30 second epochs in each frequency band to correspond to visual stage scoring to prepare for averaging data by sleep stage. In addition, relative power measures we also computed for each frequency band, expressed as a proportion of total power per epoch of sleep. Both raw EEG and power spectral data were inspected epoch by epoch for evidence of movement artifact. Epochs with high amplitude artifact were excluded from all EEG analyses. Only data from C3 electrodes are reported here. All-night power spectral data were plotted and inspected visually for evidence of alpha and delta power that were in phase across the night in each subject.
Data were coded for CFS status, sleep stage (REM, Stage 1, 2 and combined Stage 3 and 4) and frequency band (delta through beta), which were used as repeated measures. MANOVAs evaluated potential statistical differences. Univariate analyses, contrasting twin pairs within each sleep stage, were only computed if a significant overall MANOVA effect was obtained. In addition, within-subject Pearson’s correlation coefficients were computed to evaluate the statistical relationship between all-night alpha and delta power by stage and across the whole night independent of sleep stage. These correlations were coded for twin pair and a within-subject ANOVA evaluated potential differences. Differences in demographic or clinical characteristics were compared between the CFS ill and non-CFS twins with t-tests of Chi-square statistics.