Hallazgos incidentales en medicina genómica / Incidental Findings in Genomic Medicine

Las técnicas de Biología Molecular de última generación, como es la secuenciación masiva en paralelo o NGS (Next Generation Sequencing), permite obtener gran cantidad de información genómica, la cual muchas veces va más allá de la detección de una variante patogénica en un gen que explique la patología (hallazgo primario). Es así como surgió desde hace años la discusión internacional respecto a la decisión a tomar frente a los hallazgos secundarios accionables, es decir, aquellos hallazgos de variantes clasificadas como patogénicas o probablemente patogénicas que no están relacionadas con el fenotipo del paciente, pero que tiene alguna medida preventiva o tratamiento posible y, por lo tanto, podría ser de utilidad para la salud del paciente. Luego de revisar la bibliografía internacional y debatir entre los expertos del Hospital de Pediatría Garrahan, se logró establecer una política institucional y reforzar el hecho de que se trata de una disciplina multidisciplinaria. Así, fue posible definir que solo se atenderá las cuestiones relacionadas con la edad pediátrica, dejando para un tratamiento posterior aquellas variantes detectadas en genes que sean accionables en edad adulta. En el Hospital Garrahan, ha sido posible definir claramente cómo proceder frente a los hallazgos secundarios, al adaptar el consentimiento informado a esta necesidad, definiendo cuándo serán informados, y sabiendo que serán buscados intencionalmente en los genes clínicamente accionables enlistados en la última publicación del American College of Medical Genetics and Genomics, siempre y cuando el paciente/padre/tutor lo consienta (AU)

The latest generation of molecular biology techniques, including massive parallel sequencing or NGS (Next Generation Sequencing), allows us to obtain a whealth of genomic information, which often goes beyond the detection of a pathogenic variant in a gene that explains the pathology (primary finding). As a result, an international discussion has arisen over the years regarding the decision-making concerning actionable secondary findings, it means, those findings of variants classified as pathogenic or probably pathogenic that are not related to the patient's phenotype, but which have some possible preventive measure or treatment and, therefore, could be useful for the patient's health. After reviewing the international literature and discussing among the experts of the Hospital de Pediatría Garrahan, an institutional policy was established and the concept that this is a multidisciplinary discipline was reinforced. Consequently, it has been defined that only issues related to children will be addressed, reserving those variants detected in genes that are actionable in adulthood for later treatment. At Garrahan Hospital, we were able to clearly define how to proceed with secondary findings by adapting the informed consent to this need, defining when they will be reported, and knowing that they will be intentionally searched for in the clinically actionable genes listed in the latest publication of the American College of Medical Genetics and Genomics, as long as the patient/parent/guardian consents (AU)

Assessment of left ventricular performance during laparoscopy.

OBJECTIVES: Cardiovascular changes during laparoscopic surgery have been described in several studies. Pneumoperitoneum effects on cardiac performance instead have not been much investigated and are less known. The carbon dioxide insufflation necessary in order to perform laparoscopic procedures represents a higher force against which the myocardial fibers must shorten during ventricular contraction. Hypothesis of this study is that the intra-abdominal pressure at 12 mmHg could acutely affect the left ventricular wall stress and work. Aim of the study was to evaluate the impact of relationship pneumoperitoneum on the echocardiographic measures of left ventricular contractile function. PATIENTS AND METHODS: We studied 20 healthy, ASA I women, undergoing laparoscopic hysterectomy. Transthoracic echocardiography was performed preoperatively, after induction of anaesthesia, 15' after pneumoperitoneum, 15' after Trendelenburg positioning and 15' after the end of surgery. Left ventricular end-systolic wall stress, stroke work, left ventricular ejection time and mean velocity of fiber shortening were registered. RESULTS: Carbon dioxide insufflation caused a consistent increase in left ventricular end-systolic wall stress and left ventricular ejection time, while the mean velocity of fiber shortening decreased. The Trendelenburg position did not produce any other signficant effects. These changes were transient with a return to preinsufflation values at the last measurement. Pneumoperitoneum adversely affects left ventricular performance during laparoscopy, leading to an increase of left ventricular end-systolic wall stress and reducing the mean velocity of circumferential fiber shortening. These cardiac consequences appear to be reversible since all the echocardiographic parameters normalized at the end of surgery. CONCLUSIONS: Our results suggest that there is an adaptation of the cardiac systolic contractile status to the pressure overload during laparoscopy.

Dopamine infusion and fluid administration improve renal function during laparoscopic surgery.

BACKGROUND: Previous studies have documented the negative influence of pneumoperitoneum on cardiac and renal function during laparoscopy. In this respect, regard there is a lack of data regarding the use of an appropriate management of fluid therapy to decrease these side effects. The aim of this study was to investigate the cardiac and renal responses to the administration of different amounts of crystalloid solution, alone or in association with dopamine during laparoscopy. METHODS: Sixty ASA I patients undergoing laparoscopic surgery for endometriosis were randomly assigned to three groups. Group A was given saline solution at 5 mL/kg/h; group B received saline solution at 5 mL/kg/h and dopamine 3 mg/kg/min, and group C received saline solution at 10 mL/kg/h. Patients received A, B or C perioperatively. Renal function was evaluated by assessing total intraoperative diuresis and estimated glomerular filtration rate. Vasopressin plasmatic levels before and after surgery were measured. Transthoracic echocardiography was performed to estimate left ventricular filling pressure by using Tissue Doppler Imaging and registering the E/Ea ratio every hour after pneumoperitoneum. RESULTS: Total intraoperative diuresis was impaired in group A and significantly increased in group C (P<0.001). In group A estimated glomerular filtration rate significantly decreased after the end of surgery (P<0.003) and vasopressin significantly increased (P<0.001). The E/Ea ratio was significantly increased in group C (P<0.001). The infusion of saline solutions with the addition of dopamine slightly affected this parameter. CONCLUSION: A low rate of saline infusion could lead to an impaired renal function during laparoscopic surgery. This could be prevented by increasing the amount of saline solutions infused per hour or by adding a dopamine infusion.

Short-term neurodevelopmental outcome of babies operated on for low-risk esophageal atresia: a pilot study.

Data on the neurodevelopmental outcome of esophageal atresia (EA) survivors are scarce, controversial, and based on small samples. This is an observational prospective longitudinal study on a selected cohort of low-risk EA survivors. We considered a low-risk EA survivor a patient with the following characteristics: gestational age >32 weeks, no long gap, no genetic or chromosomic anomaly associated with neurodevelopmental delay, and no further major surgical congenital anomalies. Infants were evaluated with scales derived from the Bayley Scales of Infant and Toddler Development - 3rd Edition at 6 and 12 months, with a score of 100 considered normal for each scale. Analysis of variance was used to assess differences of cognitive and motor development. Linear regression was used to assess the impact of the following clinical and sociodemographic variables: gender, birthweight, gestational age, length of hospital stay, number of surgeries and number of esophageal dilatations during first hospitalization, days of mechanical ventilation, weight at follow up, number of surgeries and esophageal dilatations at follow up, parental age, educational level, and socioeconomic status. Thirty children form the object of the study. The mean (standard deviation [SD]) cognitive scale's score was 93.7 (7.5) and 98.2 (9.6) at 6 and 12 months, respectively (P < 0.05). The mean (SD) motor scale's score was 97.6 (9.3) and 98.0 (12.1) at 6 and 12 months, respectively (P = n.s.). Children with a body weight <5° percentile at 12 months showed a mean (SD) cognitive score significantly lower when compared with those with a body weight >5° percentile: 88.8 (6.3) and 100.5 (8.9), respectively. At 12 months, children with unemployed mothers had a mean (SD) motor score significantly lower when compared with those in the other socioeconomic classes: 87.7 (9.8) and 100.6 (12.4), respectively. In conclusion, parents of babies operated on for low-risk EA can be reassured about neurodevelopmental outcome at least up to 1 year of age. When offering a multidisciplinary follow-up program, underweight patients should deserve particular attention to promote their quality of life and support their global development.

Desflurane versus sevoflurane: a comparison on stress response.

BACKGROUND: Neurohumoral, immunologic and metabolic alteration characterize surgical procedures in relation with the intensity of injury, the total operating time and the anesthetic technique. We, therefore, compared the effects of desflurane versus sevoflurane anesthesia on intra and postoperative release of the stress hormones and inflammatory cytokines. METHODS: Fifty Caucasian women undergoing laparoscopic surgery for benign ovarian cysts were randomized to receive inhaled anesthesia with desflurane (DES group; N.=25) or sevoflurane (SEVO group; N.=25), with fentanyl bolus and remifentanil infusion. Plasma levels of noradrenaline, adrenaline, ACTH and cortisol were measured preoperatively (T1), 30 minutes after the beginning of surgery (T2), and 30 minutes, 2 and 4 hours after the end of surgery (T3, T4, T5 respectively). Interleukin 6 (IL-6), glucose and C-reactive protein (CRP) were measured at T1, T2,T3, T4, T5 and 12 hours after the end of surgery (T6). RESULTS: An increase of catecholamines during (T2) and immediately after surgery (T3) was observed in both groups. However, adrenaline and noradrenaline levels were significantly higher in the DES group compared to the SEVO group. Despite a drop of cortisol concentration was observed in both groups, only in the DES group there was a significant difference intraoperatively as compared to the baseline levels and to the SEVO group. While, the consequent increase of ACTH was significantly higher in the SEVO group at T2-T4. The preoperatory levels were restored at T5. Glucose, IL-6, CRP levels and postoperative pain did not show significant differences in timing within the same group and comparing DES vs SEVO group. CONCLUSION: In the present study we demonstrated that desflurane and sevoflurane produced a different stress response in the setting of laparoscopic surgery. The greater release of catecholamines during desflurane anesthesia could have adverse effects in patients with pre-existing cardiovascular disease. In low stress surgery desflurane, as compared to sevoflurane, was associated with a better control of intraoperative cortisol and ACTH response (T2). Moreover, the ACTH secretion resulted attenuated also postoperatively (T3-T4). Both gases did not influence the plasmatic levels of Il-6, CRP and glucose.

Influence of pneumoperitoneum on left ventricular filling pressures and NT-proBNP levels.

BACKGROUND: We recently demonstrated that pneumoperitoneum affects diastolic echocardiographic findings in healthy women scheduled for gynaecologic laparoscopy. No reports have been conducted in order to assess the echocardiographic consequences in hypertensive subjects during laparoscopic procedures. AIM: The aim of this study was to evaluate Left Ventricular filling pressures in hypertensive women with and without diastolic dysfunction, combining the tissue Doppler imaging technique and the plasmatic levels of amino terminal proBNP. MATERIALS AND METHODS: Doppler recordings of mitral inflow, tissue Doppler imaging of mitral annulus and N-terminal-proBNP plasmatic levels were obtained in 40 hypertensive women with or without diastolic dysfunction. Measurements were executed in awake patients (T0), after the induction of anesthesia (T1), 10 and 20 minutes after the creation of the pneumoperitoneum (T2 and T3, respectively) and at the end of the surgery (T4). Furthermore, we collected the last blood sample after 12 hours (T5). RESULTS: The E/Ea ratio for the evaluation of left ventricular filling pressures were higher in the diastolic dysfunction group than in the non diastolic dysfunction and significantly increased after pneumoperitoneum. Pneumoperitoneum increased the plasmatic levels of natriuretic peptide in both groups. At the end of the procedure we did not observe any further significant alteration. CONCLUSIONS: Pneumoperitoneum produces a consistent increase of ventricular filling pressures in a population of hypertensive patients with and without diastolic dysfunction. Moreover, there is a significant but transient rise in NT-proBNP after gas insufflation in both groups, most accentuated in the diastolic dysfunction group.

Efficacy of scheduled time ketorolac administration compared to continuous infusion for post-operative pain after abdominal surgery.

BACKGROUND: Ketorolac tromethanime is a non steroidal anti inflammatory drug and its efficacy on acute pain control after abdominal surgery has been well documented. It has a rapid onset and it can be given both for intra operative and for post operative pain management. AIM: In this study we aimed to evaluate if there were any differences in relieving post operative pain when Ketorolac was administered with continuous infusion or if it was given at prearranged times. PATIENTS AND METHODS: 80 ASA I patients, scheduled for major gynecological surgery, were randomly assigned to 2 groups: group A patients were connected after surgical incision with a 24h analgesic infusor (2 ml/h) containing morphine (0.02 mg/kg/h) and Ketorolac (90 mg). Group B patients were connected after surgical incision with a 24h analgesic infusor (2 ml/h) containing morphine (0.02 mg/kg/h) at first and Ketorolac was then given in bolus after surgical incision and then every 8 hours for the first 24 hours. Post-operative pain scores were assessed using the Visual Analogue Scale (VAS) every 8 hours for 24 h. For a VAS value greater than 6, patients received Tramadol 100 mg. RESULTS: Post-operative pain scores showed a better pain relief for patients in the group B. Furthermore, the requirements of rescue analgesic were less in the group B [Tramadol was used for only 8 patients] than in the group A [Tramadol was used for 31 patients]. No adverse effects were registered in both groups. CONCLUSIONS: For post-operative pain Ketorolac administration at prearranged times, every 8 hours, offers greater benefits in respect to its continuous infusion.

Terracotta polychrome sculptures examined before and after their conservation work: contributions from non-invasive in situ analytical techniques.

The potential of non-invasive in situ analytical techniques such as portable Raman, portable X-ray fluorescence, portable optical microscope and fibre optics reflectance spectroscopy has been shown studying painted layers of Renaissance terracotta polychrome sculptures belonging to the statuary of Santo Sepolcro Church in Milan. The results obtained allowed pointing out the contribution of these techniques to the compositional diagnostic, providing complete information, in some cases, better than micro-destructive techniques, on the kind of pigments used on the external painted layers. Moreover, a comparison with the results obtained before the last conservation work (2009) with micro-destructive techniques allowed ascertaining the removal of the external painted layers during the conservation operations.

Spectroscopy enhances the information content of optical mammography.

Near-infrared (NIR) diffuse optical spectroscopy and imaging may enhance existing technologies for breast cancer screening, diagnosis, and treatment. NIR techniques are based on quantitative measurements of functional contrast between healthy and diseased tissue. In this study we measured the spectral dependence of tissue absorption (mu(a)) and reduced scattering (mu'(s)) in the breasts of 30 healthy women and one woman with a fibroadenoma using a seven-wavelength frequency-domain photon migration probe. Subjects included pre- and postmenopausal women between the ages of 18 and 64. Multi-spectral measurements were used along with a four-component fit to determine the concentrations of de-oxy and oxy-hemoglobin, water and lipids in breast. The scattering spectral shape was also quantified. Our measurements demonstrate that the measured concentrations of NIR analytes correlate well with known breast physiology. Although the tissue scattering at a single wavelength was found to have little value as a functional parameter, the dependence of the scattering on wavelength provided key insights into breast composition and physiology. Lipids and scattering spectra in the breast were found to increase and decrease, respectively, with increasing body mass index. Simple calculations are also provided to demonstrate potential penalties from ignoring the contributions of water and lipids in breast measurements. Finally, water is shown to be a possible indicator for detecting a fibroadenoma, whereas the hemoglobin saturation was found to be a poor indicator. Multi-spectral measurements, compared to measurements restricted to one or two wavelengths, provide additional information that may be useful in managing breast disease.

Sources of absorption and scattering contrast for near-infrared optical mammography.

RATIONALE AND OBJECTIVES: Near-infrared (NIR) diffuse optical spectroscopy and imaging may enhance existing technologies for breast cancer screening, diagnosis, and treatment. NIR techniques are based on sensitive, quantitative measurements of functional contrast between healthy and diseased tissue. In this study, the authors quantified the origins of this contrast in healthy breasts. MATERIALS AND METHODS: A seven-wavelength frequency-domain photon migration probe was used to perform noninvasive NIR measurements in the breasts of 28 healthy women, both pre- and postmenopausal, aged 18-64 years. A diffusive model of light transport quantified oxygenated and deoxygenated hemoglobin, water, and lipid by their absorption signatures. Changes in the measured light-scattering spectra were quantified by means of a "scatter power" parameter. RESULTS: Substantial quantitative differences were observed in both absorption and scattering spectra of breast as a function of subject age. These physiologic changes were consistent with long-term hormone-dependent transformations that occur in breast. Instrument response was not adversely affected by subject age or menopausal status. CONCLUSION: These measurements provide new insight into endogenous optical absorption and scattering contrast mechanisms and have important implications for the development of optical mammography. NIR spectroscopy yields quantitative functional information that cannot be obtained with other noninvasive radiologic techniques.

Perturbation Monte Carlo methods to solve inverse photon migration problems in heterogeneous tissues.

We introduce a novel and efficient method to provide solutions to inverse photon migration problems in heterogeneous turbid media. The method extracts derivative information from a single Monte Carlo simulation to permit the rapid determination of rates of change in the detected photon signal with respect to perturbations in background tissue optical properties. We then feed this derivative information to a nonlinear optimization algorithm to determine the optical properties of the tissue heterogeneity under examination. We demonstrate the use of this approach to solve rapidly a two-region inverse problem of photon migration in the transport regime, for which diffusion-approximation-based approaches are not applicable.

Quantifying the absorption and reduced scattering coefficients of tissuelike turbid media over a broad spectral range with noncontact Fourier-transform hyperspectral imaging.

Absorption (mu(a)) and reduced scattering (mu(s)') spectra of turbid media were quantified with a noncontact imaging approach based on a Fourier-transform interferometric imaging system (FTIIS). The FTIIS was used to collect hyperspectral images of the steady-state diffuse reflectance from turbid media. Spatially resolved reflectance data from Monte Carlo simulations were fitted to the recorded hyperspectral images to quantify mu(a) and mu(s)' spectra in the 550-850-nm region. A simple and effective calibration approach was introduced to account for the instrument response. With reflectance data that were close to and far from the source (0.5-6.5 mm), mu(a) and mu(s)' of homogeneous, semi-infinite turbid phantoms with optical property ranges comparable with those of tissues were determined with an accuracy of +/-7% and +/-3%, respectively. Prediction accuracy for mu(a) and mu(s)' degraded to +/-12% and +/-4%, respectively, when only reflectance data close to the source (0.5-2.5 mm) were used. Results indicate that reflectance data close to and far from the source are necessary for optimal quantification of mu(a) and mu(s)'. The spectral properties of mu(a) and mu(s)' values were used to determine the concentrations of absorbers and scatterers, respectively. Absorber and scatterer concentrations of two-chromophore turbid media were determined with an accuracy of +/-5% and +/-3%, respectively.

Broadband absorption spectroscopy in turbid media by combined frequency-domain and steady-state methods.

A technique for measuring broadband near-infrared absorption spectra of turbid media that uses a combination of frequency-domain (FD) and steady-state (SS) reflectance methods is presented. Most of the wavelength coverage is provided by a white-light SS measurement, whereas the FD data are acquired at a few selected wavelengths. Coefficients of absorption (mu(a)) and reduced scattering (mu(s)') derived from the FD data are used to calibrate the intensity of the SS measurements and to estimate mu(s)' at all wavelengths in the spectral window of interest. After these steps are performed, one can determine mu(a) by comparing the SS reflectance values with the predictions of diffusion theory, wavelength by wavelength. Absorption spectra of a turbid phantom and of human breast tissue in vivo, derived with the combined SSFD technique, agree well with expected reference values. All measurements can be performed at a single source-detector separation distance, reducing the variations in sampling volume that exist in multidistance methods. The technique uses relatively inexpensive light sources and detectors and is easily implemented on an existing multiwavelength FD system.

Surgical crown lengthening of the maxillary anterior dentition: aesthetic considerations.

Recent patient interest in aesthetic treatment has resulted in the development of advanced surgical and restorative procedures that are capable of conservatively addressing such objectives. While numerous materials and techniques have been developed accordingly, each must respect specific biological criteria in order to be successful. This article demonstrates the implementation of surgical periodontal plastic and restorative procedures that facilitate the full-mouth rehabilitation of a patient who presented with excessive wear of the natural dentition. The procedure described is specific for the maxillary anterior.

Digital holography for quantitative phase-contrast imaging.

We present a new application of digital holography for phase-contrast imaging and optical metrology. This holographic imaging technique uses a CCD camera for recording of a digital Fresnel off-axis hologram and a numerical method for hologram reconstruction. The method simultaneously provides an amplitude-contrast image and a quantitative phase-contrast image. An application to surface profilometry is presented and shows excellent agreement with contact-stylus probe measurements.

In vivo local determination of tissue optical properties: applications to human brain.

Local and superficial near-infrared (NIR) optical-property characterization of turbid biological tissues can be achieved by measurement of spatially resolved diffuse reflectance at small source-detector separations (<1.4 mm). However, in these conditions the inverse problem, i.e., calculation of localized absorption and the reduced scattering coefficients, is necessarily sensitive to the scattering phase function. This effect can be minimized if a new parameter of the phase function gamma, which depends on the first and the second moments of the phase function, is known. If gamma is unknown, an estimation of this parameter can be obtained by the measurement, but the uncertainty of the absorption coefficient is increased. A spatially resolved reflectance probe employing multiple detector fibers (0.3-1.4 mm from the source) is described. Monte Carlo simulations are used to determine gamma, the reduced scattering and absorption coefficients from reflectance data. Probe performance is assessed by measurements on phantoms, the optical properties of which were measured by other techniques [frequency domain photon migration (FDPM) and spatially resolved transmittance]. Our results show that changes in the absorption coefficient, the reduced scattering coefficient, and gamma can be measured to within +/-0.005 mm(-1), +/-0.05 mm(-1), and +/-0.2, respectively. In vivo measurements performed intraoperatively on a human skull and brain are reported for four NIR wavelengths (674, 811, 849, 956 nm) when the spatially resolved probe and FDPM are used. The spatially resolved probe shows optimum measurement sensitivity in the measurement volume immediately beneath the probe (typically 1 mm(3) in tissues), whereas FDPM typically samples larger regions of tissues. Optical-property values for human skull, white matter, scar tissue, optic nerve, and tumors are reported that show distinct absorption and scattering differences between structures and a dependence on the phase-function parameter gamma.

Role of tissue structure in photon migration through breast tissues.

Photon migration has been investigated experimentally in vitro on human breast tissues, bovine liver, tissue phantoms, and theoretically by Monte Carlo simulations and diffusion theory. The spatial intensity profiles have been measured at the output surface of a sample illuminated by a collimated beam. Experimental results have then been compared with simulations that assume the sample to be homogeneous. Measurements on phantoms, i.e., fat emulsion and microspheres suspension, and on liver are in good agreement with theory. On the other hand, the width of the intensity profiles measured on breast tissues (adipose and fibrous) are systematically larger than those measured on phantoms or calculated by simulations. The structure of these samples, not considered in simulations and not present in phantoms, explains these differences.

3'-5' L-capped-antisense oligodeoxynucleotides targeted at the SV40 T-antigen gene: pharmacological and biological properties.

This paper reports on some pharmacological and biological properties of 22-mer antisense oligodeoxynucleotides which contain an L-deoxyribonucleoside at each terminus. Compared with natural compounds, of which they retain the DNA hybridizing ability and the cell uptake mechanism, the L-22-mers exhibited an increased resistance to phosphodiesterase degradation, an apparent higher intracellular concentration and a longer intracellular half life. Antiviral activity was not prominent.

Simultaneous polymerase chain reaction detection and restriction typing for the diagnosis of human genital papillomavirus infection.

A polymerase chain reaction method has been developed which allows the simultaneous detection of the majority of clinically relevant HPV types. Degenerate HPV-specific primers direct the one-step amplification of a DNA region spanning E1 and E7 genes. This enables an immediate distinction between the two groups of papillomaviruses, characterized by high or low oncogenic potential, simply from the size of amplified DNA. The PCR product can be subjected to a second round of amplification with internal primers, which are specific for 7 high-risk HPV types, HPV-16, -18, -31, -33, -35, -45 and -58. Precise identification of one-step or two-step amplified DNA is done by endonuclease digestion with one or two enzymes. The detection sensitivity, which has been assessed using cloned HPV genomes and HeLa and CaSki cell lines, varies from a few tens to a few hundreds of viral genome equivalents. The accuracy of the method has been confirmed by examining cervical scrapings of 44 patients.