Purpose: Copy-number analysis to detect disease-causing losses and gains across the genome is recommended for the evaluation of individuals with neurodevelopmental disorders and/or multiple congenital anomalies, as well as for fetuses with ultrasound abnormalities. In the decade that this analysis has been in widespread clinical use, tremendous strides have been made in understanding the effects of copy-number variants (CNVs) in both affected individuals and the general population. However, continued broad implementation of array and next-generation sequencing-based technologies will expand the types of CNVs encountered in the clinical setting, as well as our understanding of their impact on human health.
human cytogenetics rooney pdf 14
Local myogenesis, neoangiogenesis and homing of progenitor cells from the bone marrow appear to contribute to repair of the infarcted myocardium. Implantation into heart tissues of autologous skeletal myoblasts has been associated with improved contractile function in animal models and in humans with acute myocardial ischemia. Since heart infarction is most prevalent in individuals of over 40 years of age, we tested whether culture methods available in our laboratory were adequate to obtain sufficient numbers of differentiated skeletal myoblasts from muscle biopsy specimens obtained from patients aged 41 to 91.
When over 3 108 viable cells were obtained by in vitro culture (i.e., over day-35 post-plating), an aliquot of each culture was trypsinized and plated in two different chamber slides. Cells were grown in PM, treated with colchicine for 30 min, subjected to hypotonic treatment, fixed in methanol:acetic acid (3:1), and processed according to standard methods [23]. Fifty metaphases were analyzed by QFQ-banding with an automated cytogenetics system (Genikon; Nikon) following the rules of the International System for Human Cytogenetic Nomenclature [24].
Morphologic aspects of primary human skeletal muscle cultures are shown in Figure 2. The morphology of an adherent SC is shown in Figure 2A (day-5 post-plating). Figures 2B and 2C show cell monolayers cultured in PM for 7 and 10 days, respectively. Experience with muscle cultures from adult donors shows that changing the environmental conditions of cultured myoblasts from PM to DM is followed by cell fusion within a few days. Fusion was only used to confirm the presence of myoblasts. Confluent myoblasts in the process of fusing to form myotubes are shown in Figure 2D.
Morphologic aspects of primary human skeletal muscle cultured in vitro. Adherent satellite cell 5 days post-plating in proliferation medium (A; 40). Semiconfluent monolayers of myoblasts cultured in proliferation medium for 7 (B; 20) and 10 days (C; 10). Confluent myoblasts cultured in differentiation medium are in the process of fusing to form myotubes (D; 10).
Cytogenetic analysis and the search for human viruses were performed in muscle cell cultures grown in PM for 35 to 40 days. Normal diploid karyotypes were obtained from muscle cultures of all investigated patients. Figure 4 shows a representative metaphase and the normal male karyotype of patient #5.
At the time when cultures were subjected to cytogenetic analysis, samples were also processed for detecting human viral pathogens. After DNA and RNA extraction, PCR and RT-PCR methods were used for detecting the genome of CMV, EBV, HBV, parvovirus B19, HIV, and HCV. All cultures gave negative results with virus-specific primers. Amplification for beta-globin (PCR), GAPDH (RT-PCR), and virus-positive controls consistently gave the expected results (data not shown). At the time of muscle biopsy, RT-PCR had shown that patient #4 was HCV-positive both in blood (53,000 genome equivalents/ml) and in the muscle biopsy specimen. Interestingly, HCV genome was not detected in cultured muscle cells obtained from her biopsy at passage numbers 3, 6, and 9, indicating that HCV failed to replicate in muscle cell cultures.
In humans, phase I clinical studies begin to demonstrate the clinical benefits of autologous myoblast transplantation [22, 12, 34, 14, 15, 11] and, to a minor extent, of autologous mesenchymal/hematopoietic cells [16, 35, 36].
In order to make clinical applications possible on a larger scale, conditions for the reproducible and safe in vitro expansion of human skeletal muscle need to be set. To this end, five points are of particular relevance: 1) processing of bioptic tissue as to obtain appropriate muscle stem cells; 2) use of culture media free of non-human components; 3) methods to evaluate the differentiation of cultured cells along myogenic lineages; 4) methods to evidence genetic alterations of cells to be implanted; 5) methods to assure the absence of pathogenic microorganisms (viruses causing persistent infections and TSE agents). In the present study, several of these conditions have been satisfied. Bioptic specimens have been processed with a widely available bacterial protease with pleasing results. Preliminary selection of muscle stem cells by fluorescence-activated sorting and/or antibodies has not been attempted in this study, but seems promising [37]. The medium employed has been supplemented with carefully selected human recombinant growth factors. Addition of FBS and fetuin remained however indispensable. Recently, media supplemented with autologous human serum have been proposed and appear to be associated with superior clinical results [22]. In particular, no malignant arrhythmias were reported among 20 patients and the post-infarctual LV ejection fraction was significantly improved. Myogenic differentiation of human myoblasts obtained with the proposed technique has been comparable to what reported by others. Using desmin as a cytoplastic marker, about 50% of cells cultured from adult donors were differentiated along myogenic lineages. This result is comparable to what previously reported using either the desmin marker [9] or the CD56 surface marker [15]. Detailed characterization showed that expanded muscle cell cultures maintained the ability to express proteins proper of skeletal muscle (myosin type I and II, actin, actinin, spectrin and dystrophin). Since the regulation of myosin heavy chain gene expression is strongly regulated by transcriptional events and by physical exercise [38], and since in old subjects muscle fibers co-expressing myosin type I and myosin type IIA are more frequent than in young subjects [39], immunostaining of in vitro cultured cells should not be expected to strictly reproduce what observed in tissue sections.
Of particular interest for clinical applications is that cytogenetic alterations were not detected in cultured cells that had undergone at least 10 population doublings. This was particularly reassuring since the investigated samples were derived from adult/old donors and chromosomal alterations are known to occur at an increased frequency in tissues of adult/old peoples [40]. In future studies, the new CGH technology could offer superior sensitivity for detecting minor cytogenetic changes [41]. Finally, molecular methods for common human viruses are widely available and need to be employed in human clinical trials to validate myoblast cultures prior to implant. Of interest, is the chance observation that HCV failed to replicate in one muscle culture derived from an HCV-infected donor. This result is in agreement with experimental observations showing that HCV has no effects on liver myofibroblasts [42].
The results indicate that in about one month it is possible to produce in vitro approximately one billion of adequately differentiated skeletal muscle cells from human donors, independently of age. Clinical experience indicates that approximately 300 millions of autologous skeletal muscle cells are sufficient for the cellular therapy of infarcted heart [15, 14]. Thus, an early intervention may be possible by processing a muscle biopsy of about 2 grams. The proposed tissue culture methods may also represent a basis on which to envisage applications in the urologic [18, 19] and orthopedic fields [20, 21].
Know the genetic basis of the main diseases with a base or genetic component. Relate the genetic dysfunction with the pathological phenotype. Perform the genetic interpretation of the diagnosis, prognosis, prevention and therapy of the most frequent genetic pathologies in the human population. Understand the distribution of genetic-based diseases in a population taking into account their origin. Analyze genetically the probands-family relationship that facilitates the offert of a genetic counseling. 2ff7e9595c
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