Accurate quantitation of JAK2 V617F allele burden by array-based digital PCR.

INTRODUCTION: The JAK2 V617F mutation is highly prevalent in patients with myeloproliferative neoplasms (MPN). Several studies have shown that allele burden correlates with hematologic characteristics and clinical end-points in patients with MPN. Allele-specific real-time quantitative polymerase chain reaction (RQ-PCR) is probably the most commonly used technique for detection and quantitation of the JAK2 V617F mutation. Alternatively, digital PCR is an emerging technology for absolute DNA quantitation, which is based on dilution and high-grade partitioning of a sample. METHODS: We compared array-based digital PCR using the QuantStudio(™) 3D Digital PCR System platform with a RQ-PCR assay, CE-registered for in vitro diagnostic use, regarding JAK2 V617F allele quantitation. This study included 30 samples positive for the JAK2 V617F mutation and additionally 13 samples without the mutation. RESULTS: JAK2 V617F allele burden of samples ranged between well below 1% and more than 90%. Linear regression analysis showed a high correlation between the results obtained from the two techniques (r(2) = 0.9983). Thirteen samples tested negatively for the JAK2 V617F mutation with RQ-PCR were also found negative with digital PCR. CONCLUSION: We conclude that array-based digital PCR is an appropriate method for the quantitation of JAK2 V617F allele burden demonstrating highest correlation with allele-specific RQ-PCR.

Early and late mismatch negativity elicited by words and speech-like stimuli in children.

In auditory perception the brain's attentional and preattentional mechanisms select certain stimuli for preferential processing and filter out irrelevant input. This study investigated nonattentive auditory processing in children. Event-related potentials (ERPs) provide a means to study neural correlates related to language and speech-sound processing. Mismatch negativity (MMN) is an ERP wave that indicates attention-independent perceptual change detection. In this study cortical ERPs were elicited by complex tones, naturally spoken words, and pseudowords, with each stimulus type containing equal acoustical elements. Tones elicited a bifurcated mismatch negativity (MMN), with early MMN (peaking at 150-200 ms) being more dominant. On the other hand, words elicited a strong late MMN, peaking at about 400-450 ms after stimulus onset. The MMN wave form was significantly weaker for pseudowords than for words. The late MMN wave, especially for word differences, was found to reflect summating MMN generators and memory trace formation on gestalt bases. Results suggest that the auditory processing, even nonattended, is highly associated with the cognitive meaning of the stimuli.

Mismatch negativity and late discriminative negativity in investigating speech perception and learning in children and infants.

For decades, behavioral methods, such as the head-turning or sucking paradigms, have been the primary tools to investigate speech perception and learning of a language in infancy. Recently, however, new methods provided by event-related potentials have emerged. These are called mismatch negativity (MMN) and late discriminative negativity (LDN). MMN, the brain's automatic change-detection response in audition, has been intensively used in adults in both basic and clinical studies for longer than 20 years. LDN, on the other hand, was only recently discovered. There seem to be many differences between these two responses. MMN is developmentally quite stable and can be obtained even from preterm infants. LDN, however, can be obtained most reliably from young children, and its amplitude decreases as a function of age. New data suggest that both of these responses have a special role in language processing, although both of them can also be elicited by nonspeech stimuli.

Automatic detection of frequency changes depends on auditory stimulus intensity.

OBJECTIVE: A cortical cognitive auditory evoked potential, mismatch negativity (MMN), reflects automatic discrimination and echoic memory functions of the auditory system. For this study, we examined whether this potential is dependent on the stimulus intensity. DESIGN: The MMN potentials were recorded from 10 subjects with normal hearing using a sine tone of 1000 Hz as the standard stimulus and a sine tone of 1141 Hz as the deviant stimulus, with probabilities of 90% and 10%, respectively. The intensities were 40, 50, 60, 70, and 80 dB HL for both standard and deviant stimuli in separate blocks. RESULTS: Stimulus intensity had a statistically significant effect on the mean amplitude, rise time parameter, and onset latency of the MMN. CONCLUSION: Automatic auditory discrimination seems to be dependent on the sound pressure level of the stimuli.

Global sequence diversity of BRCA2: analysis of 71 breast cancer families and 95 control individuals of worldwide populations.

The aim of this study was to evaluate the prevalence of simple sequence variation in the BRCA2 gene. To this end, 71 breast and breast-ovarian cancer (HBC/HBOC) families along with 95 control individuals from a wide range of ethnicities were analyzed by means of denaturing high-performance liquid chromatography (DHPLC) and direct sequence analysis. In the coding (10 257 bp) and non-coding (2799 bp) sequences of BRCA2, 82 sequence variants were identified. Three different, apparently disease-associated BRCA2 mutations were found in six HBC/HBOC families (8%): two splice site mutations in introns 5 and 21, and one frameshift mutation in exon 11. In the coding region, 53 simple sequence variants were found: 35 missense mutations, one 2 bp deletion (CT) resulting in a stop at codon 3364, one nonsense mutation with a stop at codon 3326, one deletion of a complete codon (AAA) resulting in the loss of leucine, and 15 silent mutations. In the non-coding region, 26 polymorphisms were detected. Of the 79 sequence variants that were not obviously disease-associated, eight were detected only in HBC/HBOC families. The remaining 71 variants were identified in both HBC/HBOC families and control individuals. Sixty three sequence variants (80%) were specific for a continent. Forty two percent (33 out of 79) of the sequence variants were detected exclusively in Africa, though only 13% of the 332 chromosomes screened were of African origin. Our data indicate that, in BRCA2, simple sequence variation is frequent [in the coding region 1 in 194 bp (straight theta = 2.2 x 10(-4)), and in the non-coding region 1 in 108 bp (straight theta = 4.4 x 10(-4)), respectively].

Event-related alpha synchronization/desynchronization in a memory-search task in adolescent survivors of childhood cancer.

OBJECTIVES: Event-related desynchronization (ERD) and synchronization (ERS) of the 8-10 and 10-12 Hz frequency bands of the background EEG were studied in 19 adolescent survivors of childhood cancer (11 leukemias, 8 solid tumors) and in 10 healthy control subjects performing an auditory memory task. METHODS: The stimuli were auditory Finnish words presented as a Sternberg-type memory-scanning paradigm. Each trial started with the presentation of a 4 word set for memorization whereafter a probe word was presented to be identified by the subject as belonging or not belonging to the memorized set. RESULTS: Encoding of the memory set elicited ERS and retrieval ERD at both frequency bands. However, in the survivors of leukemia, ERS was turned to ERD during encoding at the lower alpha frequency band. ERD was lasting longer at the lower frequency band than at the higher frequency band, in each study group. At both frequency bands, the maximum of ERD was achieved later in the cancer survivors than in the control group. CONCLUSION: The previously reported type of ERD/ERS during an auditory memory task was reproducible also in the survivors of childhood cancer, at different alpha frequency bands. However, the temporal deviance in ERD/ERS magnitudes, in the cancer survivors, was interpreted to indicate that both survivor groups had prolonged information processing time and/or they used ineffective cognitive strategies. This finding was more pronounced in the group of leukemia survivors, at the lower alpha frequency band, suggesting that the main problem of this patient group might be in the field of attention.

Event-related desynchronization and synchronization during an auditory lexical matching task.

OBJECTIVES: Event-related desynchronization (ERD) and synchronization (ERS) of the 8-10 and 10-12 Hz frequency bands of the background EEG were studied in 10 subjects performing an auditory lexical matching task. METHODS: The stimuli were words and pseudowords presented sequentially in pairs. The subject was prompted to answer whether the two stimuli shared the same lexical status (words or pseudowords). RESULTS: Regardless of lexicality, the presentation of the first stimulus elicited a significant late frontal ERD in both alpha frequency bands. When preceded by a pseudoword, the presentation of the second stimulus elicited a significant ERS at 200-400 ms and a significant, long-lasting and topographically-widespread ERD at 600-2200 ms in both frequency bands. When preceded by a word, the second stimulus did not elicit ERS in the initial time window, but a late ERD which was similar to the one observed in the previous condition. The complexity of ERD/ERS changes in the present task was revealed by significant interactions that time had with frequency band, stimulus type, stimulus order and lexicality of the preceding stimulus. CONCLUSIONS: The results suggest that ERD/ERS does not reflect primary auditory stimulus processing. Rather, the ERD/ERS observed in this experiment most probably reflected task difficulty and differences between lexical-semantic and phonological memory functions.

BRCA1-related breast cancer in Austrian breast and ovarian cancer families: specific BRCA1 mutations and pathological characteristics.

We identified 17 BRCA1 mutations in 86 Austrian breast and ovarian cancer families (20%) that were screened for mutations by denaturing high-performance liquid chromatography (DHPLC) and the protein truncation test (PTT). Eleven distinct mutations were detected, 4 of them (962del4, 2795del4, 3135del4 and L3376stop) not previously reported in families of non-Austrian origin. In addition, 6 rare missense mutations (allele frequency < 1%) with unknown biological effects were identified. Four mutations occurred more than once in the Austrian population: 2795del4 (3 times), Cys61Gly (3 times) 5382insC (2 times) and Q1806stop (2 times). Haplotype analysis of the 4 recurrent mutations suggested a common ancestor for each of these. Thirty-four breast cancer cases from 17 families with BRCA1 mutations were further analyzed. We observed a low median age of onset (39.5 years). Sixty-eight percent of all BRCA1 breast cancer cases had negative axillary lymph nodes. This group showed a significant prevalence of a negative estrogen and progesterone receptor status and stage I tumors compared with an age-related, node-negative control group. The prevalence of grade III tumors was marginally significant. Survival analysis either with a control group matched for age (within 5 years), grade, histologic subtype and estrogen receptor status, or with an age-related, node-negative comparison group, showed no statistical difference.

Relative alpha desynchronization and synchronization during speech perception.

Brain processes elicited by speech were studied in 10 right-handed subjects by means of examining the desynchronization and synchronization of the 8-10 Hz and 10-12 Hz EEG alpha frequency bands. The subjects listened to an auditorily presented 5 min text passage. The text was presented both forward and backward. Listening to the text forward elicited alpha desynchronization in both of the frequency bands studied, whereas listening to the same text presented backward elicited synchronization in the 10-12 Hz frequency band only. Listening to the text forward elicited greater desynchronization than listening to the text backward. In the 10-12 Hz frequency band, listening to the text forward elicited desynchronization whereas listening to the same text backward elicited synchronization. This dissociation was not observed in the 8-10 Hz frequency band. The results suggest that the lower and upper alpha bands differ such that the 10-12 Hz frequency band exhibits reactivity to the presence of linguistic content while the 8-10 Hz band shows an unspecific response.

Perceptual magnet effect in the light of behavioral and psychophysiological data.

Finnish speaking adults categorized synthetic vowels, varying in the frequency of the second formant (F2), as either /y/ or /i/. Two subject groups emerged: "good" and "poor" categorizers. In a /i/ rating experiment, only the good categorizers could consistently label their best /i/ (the prototype, P), being low in the F2 continuum. Poor categorizers rated /i/'s with high F2 values as Ps. In a same/different (AX) discrimination experiment, using the individual Ps and nonprototypes (NPs), it was more difficult for good categorizers to detect small F2 deviations from the P than from an NP (the "perceptual magnet effect"). For poor categorizers, the opposite effect was found. The same stimuli were used to record the mismatch negativity (MMN), an ERP component reflecting preattentive detection of deviations from a standard sound. For the good categorizers the MMNs were lower for Ps than for NPs; for the poor categorizers the MMNs for Ps and NPs did not differ significantly. The results show that individual listeners behaved differently in categorization and goodness rating but in the same way in attentive (AX) discrimination, being the poorest at about the same F2 location. The perceptual magnet effect was indicated in the good categorizers both by behavioral and psychophysiological (MMN) discrimination data.

Neurophysiological findings in the northern epilepsy syndrome.

We describe the neurophysiological findings in the northern epilepsy syndrome (NES), an autosomal recessively inherited childhood onset epilepsy with associated mental deterioration. Sixty-five EEGs of 18 patients (10 females and 8 males) from the age of 5 years to 52 years were analyzed. EEG showed relatively slight changes at the outbreak of epilepsy at the mean age of 6.6 years (range 5-10 years). Slowing of the background activity to 6-7 Hz theta activity, abundant diffuse or intermittent theta and delta activity and disappearance of sleep-specific activity characterized the EEGs at puberty. The amount of diffuse delta and theta activity diminished in adulthood. Epileptiform findings were scanty. Spikes and sharp waves occurred in 43% of the recordings with varying localization, form and extent. Intermittent 2-4 Hz sharp and slow wave rhythm was seen in 32% of the recordings. Of the three ictal recordings, one showed a primarily generalized discharge pattern, while two were clearly asymmetric. Clonazepam was the most effective antiepileptic drug, and it also normalized the EEG when started in childhood or at the onset of puberty. Visual evoked potentials were abnormal in 44% and brainstem auditory evoked potentials in 35%. The neurophysiological findings suggest an extensive, probably multifocal degenerative brain process, which reaches its peak at puberty. Although the abnormal features of EEG often decreased in adulthood, the clinical course of NES showed slow progression throughout the lifetime.

Event-related EEG desynchronization and synchronization during an auditory memory task.

Event-related desynchronization (ERD) and synchronization (ERS) of the lower (8-10 Hz) and upper (10-12 Hz) alpha bands of background EEG were studied in 10 subjects during an auditory memory scanning paradigm. Each experimental trial started with the presentation of a visual warning signal, after which an auditory 4-vowel memory set was presented for memorization. Thereafter the probe, a fifth vowel, was presented and identified by the subject as belonging or not belonging to the memorized set. In 50% of the cases, the probe was among the previously presented memory set. The presentation of the memory set elicited a significant ERS in the both alpha frequency bands. In contrast, the presentation of the probe elicited a significant bilateral ERD in both alpha frequency bands studied. The results suggest that the ERD phenomenon is closely associated with higher cortical processes such as memory functions rather than with auditory stimulus processing per se. Event-related desynchronization provides a potentially valuable tool for studying cortical activity during cognitive processing in the auditory stimulus modality.

Practical issues in the clinical application of mismatch negativity.

The mismatch negativity (MMN) recordings provide an objective measure of the preattentive, automatic auditory discrimination function. This article deals with issues central to the recording of the MMN and the interpretation of the results for clinical and electrodiagnostic purposes. The methods of acquiring as pure an MMN response as possible, i.e., one not contaminated by auditory cortical responses reflecting other functions, are discussed first. Second, other technical questions associated with the recording are reported on, e.g., what MMN parameters should be recorded and how, what is the smallest recordable MMN response, and what is the repeatability of the MMN recordings. Then, the effect of various physiological factors on the MMN (age, alertness, gender, topographic distribution of the MMN) is considered. The correlation between auditory discrimination performance and the MMN amplitude, observed in normal population, is dealt with. Finally, there is a short concluding overview on clinical findings of MMN recordings and discussion on their electrodiagnostic applications.

Effect of contralateral white noise masking on the mismatch negativity.

Mismatch negativity (MMN), an auditive event-related potential (ERP) component, evoked by deviant stimuli in a homogeneous stream of standard stimuli was studied in a unilateral stimulation and contralateral white noise masking condition. Eleven subjects (Ss) with normal hearing (aged 20-35 years) were examined using sine tone stimuli (70 dB HL, interstimulus interval 300 ms, duration 40 ms with 5 ms rise and fall times). Three blocks of standard (std)/deviant (dev) series of stimuli were used: std 500/dev 600 Hz, std 2000/dev 1900 Hz, and std 2000/dev 1600 Hz. The first block was repeated for another group of 11 Ss with normal hearing (aged 17-27 years). The MMN was analysed from the difference curves recorded at Fz, Cz and Pz. The stimuli were delivered unilaterally, either with or without 50 dB effective masking level white noise to the contralateral ear. The MMN amplitude attenuated significantly when contralateral masking was used. In addition, there was interaction between noise masking and the stimulated ear. The MMN latencies were not affected by white noise masking.

Automatic discrimination of phonetically relevant and irrelevant vowel parameters as reflected by mismatch negativity.

An auditory event-related brain potential called mismatch negativity (MMN) was measured to study the perception of vowel pitch and formant frequency. In the MMN paradigm, deviant vowels differed from the standards either in F0 or F2 with equal relative steps. Pure tones of corresponding frequencies were used as control stimuli. The results indicate that the changes in F0 or F2 of vowels significantly affected the MMN amplitudes. The only variable significantly affecting the MMN latencies was sex which, however, did not have any effect on the amplitudes of the MMN. As expected, the MMN amplitudes increased with an increase in the acoustical difference between the standards and the deviants in all cases. On the average, the amplitudes were lower for the vowels than for the pure tones of equal loudness. However, in vowels, minor frequency changes in F0 produced higher MMN amplitudes than similar relative changes in F2. It was also noted that even the smallest and phonetically irrelevant change in F2 was detected by the MMN process. In overall, the results demonstrate that the MMN can be measured separately for F0 and F2 of vowels, although the MMN responses show large interindividual differences.

Transient asymmetries of steady-state visual evoked potentials in classic migraine.

Unilateral abnormalities in the regional cerebral blood flow (rCBF) and visual evoked potentials (VEPs) during the visual aura of classic migraine have been reported. On the other hand, there are only few observations suggesting unilateral abnormality of neural function during the headache-free period. An experiment is described in which the symmetries of steady state VEPs (SVEPs) are compared in healthy subjects and classic migraineurs in the headache-free interval. In the patients an increased number of strong short-lasting interhemispheric asymmetries was found. In the serial recordings performed, pathologic recordings were obtained intermittently, usually with complete normalisation in the following session. The incidence of abnormalities was relatively independent of the interval between the recording and attack, except for the next few hours. The findings may be related to instability of occipital cortical function in classic migraine.

Difference of steady-state visual evoked potentials in classic and common migraine.

The present study was designed to find evidence for the neural hypothesis of migraine and to evaluate possible interictal differences in the two varieties of migraine by electrophysiological means. Steady-state visual evoked potentials (SVEPs) in response to sinusoidally modulated light were measured in 20 patients with classic migraine and compared with those of 30 common migraineurs and 49 reference subjects. SVEPs to stimuli at 10-24 Hz were recorded occipitally from a pair of midline electrodes and, in classic migraineurs and controls, additionally from left and right occipital areas. The response was processed by the Fast Fourier Transform and automatically analysed. The fundamental component of the midline response to medium frequency stimuli (16-22 Hz) appeared normal in patients with classic migraine, contrary to an augmented response in common migraineurs (ANOVA between groups, P = 0.006). In classic migraine the 2nd harmonic component was attenuated (P less than 0.01 at 18-20 Hz) and the amount of strong interhemispheric f1 asymmetries was increased in about half of the patients. The groups also diverged significantly in the SVEP dynamics during stimulation. The results support the hypothesis of a primary neural disorder in both types of migraine. Different sites and mechanisms of brain dysfunction in classic and common migraine are suggested. Hypothetical neuroanatomical correlates for the abnormalities are presented.

[Pump-guided continuous subcutaneous opiate infusion for the treatment of the most severe pain]. / Pumpengesteuerte kontinuierliche subkutane Opiat-Infusion zur Behandlung schwerster Schmerzen.

The continuous subcutaneous infusion of opiate, a new approach to the alleviation of severe chronic pain, has been carried out using a pump system normally employed for the infusion of insulin. Relapses of pain can be controlled with bolus doses. This mode of application was compared with conventional therapy in 11 patients. All patients were free of pain during the continuous infusion, but none showed a satisfactory response during conventional treatment. The improved response under continuous opiate infusion was attained with much lower doses and thus with fewer side effects. The procedure is therefore highly effective and well tolerated.