Study of Human Diseases - Assignment Example

PART I (50% total) (a) Describe the further examinations that will identify whether or not the boy will suffer from the disease. Make it clear what the difference is between normal and diseased using the various options (10%) The enlarged calves and the way the boy arises from the floor, taken together with the existence of a family case, suggest the occurrence of DMD. The diagnosis is initially based on genetic analysis by PCR. The target gene sequence and the etiology of the disease are known, so the molecular tools to do this type of investigation are available. In physiological condition the dystrophin gene is expected to be expressed, while in the diseased individual dystrophin gene is not expressed. Alternatively, a shorted dystrophin gene may be expressed but still different from normal due to an altered molecular weight. Different probes against different parts of the gene, or alternatively gene sequencing, can be exploited to identify the missing parts. Given the almost asymptomatic patients status, likely due to the young age, no histological features are expected in muscle biopsies. Therefore, a molecular, i.e. immuno-histochemical, analysis will be necessary. Indeed, immunofluorescence (IF) analysis for dystrophin can confirm the genotyping. In physiological conditions, laminin is localized around all muscle fibers and it appears as circles/polygonal shapes in muscle cross-sections, while it is absent in virtually all muscle fibers in diseased individuals (with the notable exception of possible revertant fibers). Given the invasive nature of this procedure, the IF analysis, which requires more tissue to be collected, will be performed as a second option and only in the presence of positive genetic tests. On examining the genomic DNA it was found that exon 52 was absent. (b) Will splicing of exon 51 to 53 produce a functional shortened dystrophin? Explain and justify your decision by using an illustration and text (20%). The splicing of exon 51 to 53 does not produce a shortened dystrophin, since the two exons have different codon boundary. The result of the exon 52 deletion, is thus disruption of the genetic code and the premature stop of protein translation. On the contrary, the splicing of exon 51 to 54 would give rise to a shortened but functional form of dystrophin (see diagram below). In the case presented above, the absence of dystrophin expression and the development of DMD is the diagnosis. Scheme of exon boundary extremities in the dystophin region of interest: After genetic counselling the parents choose to seek help from a specialist in gene therapy. (c) If you were the gene therapy specialist what kind of therapy would you suggest for the boy. Justify your choice. (20%) I would suggest an exon skipping approach with antisense oligonucleotides (AON) aimed to skip exon 53. The loss of the latter in addition to the congenital loss of exon 52 will likely allow to rescue the expression of an almost normal dystrophin, which lacks only two of the repeated motifs that constitute the central body of the protein. Exon skipping has recently been proven an efficient therapeutic approach in large animals (dogs) affected by muscular dystrophy (Yokoda, 2011). PART II (50% total) 1) The picture above shows a family with an inherited disorder. All affected individuals are tall and thin, with long fingers and toes. a) What would a genetic counselor be able to tell an affected individual about the mode of inheritance and the serious complications associated with the disorder (10%) The phenotype of the people in the picture is compatible with the diagnosis of the Marfan. In fact, people with Marfan syndrome tend to be unusually tall, with long, thin fingers. It is inherited as a dominant trait, thus people who have inherited one affected gene from either parent will have Marfan syndrome. This may explain the high penetrance of the disease into a group of individuals, likely members of the same family in the pcture. b) Explain the molecular basis of the condition (15%) Marfan syndrome is a genetic disorder of the connective tissue (Cañadas, 2010). The targeted gene is called FBN1 and it encodes a connective protein called fibrillin-1. The latter is a component of the extracellular matrix (thus providing structural support to connective tissue). Since connective tissue is present is most organs of the body, the mutation exerts pleiotropic effects, giving rise to a complex phenotype – hence the status of syndrome. The severity of the disease is also very variable. In physiological conditions fibrillin-1 protein binds to another protein, the transforming growth factor beta (TGF-β). TGF-β is a growth factor which regulates fibroblasts as well as osteoblasts proliferation and metabolism. The fibroblasts are the cells of the connective tissue, while osteoblasts are cells of the bone tissue. In fact TGF-β is implicated in bone growth and healing. An alteration of fibrillin binding to TGF-β is likely to affect TGF-β release and activity, in turn affecting growth of long bones and ultimately explaining the phenotype reported here. 2) The picture above shows an individual with a connective disorder. a) What could you tell this patient about the probable cause(s) of this disorder? Please include an explanation of the molecular basis and potential complications that are associated with the different forms of the condition (15%) The hallmark shown here is an elastic skin phenotype. The latter characterizes a group of inherited connective tissue disorders called Ehlers–Danlos syndrome (EDS). The latin name of the disease is very descriptive: Cutis hyperelastica, hyper-elastic skin. This group of disorders is caused by a defect in the synthesis of collagen (Malfait, 2010). The latter is a protein in connective tissue, encoded by many different genes. However, a common feature of collagen is being a fibrillar protein resistant to traction. Absent or defective collagen expression, therefore results in exacerbated elasticity. The skin has a thick connective tissue (rich in collagen) underlying the epithelium; thus, the phenotype of this disease is particularly evident for the skin. b) What laboratory investigations can be used to identify the different forms of the condition, and what are the options for treatment? (10%) Clinical observation and family history may help in diagnosing EDS. Both DNA (i.e. PCR analysis) and immuno-histochemical studies on skin biopsies can be used to highlight the type of collagen molecule which is affected in a given case. There is no cure for Ehlers Danlos Syndrome. Recently, myostatin depletion has been proposed as a therapeutical approach against EDS (Prontera, 2010). Medical intervention is limited to symptomatic therapy and to prevention; physically stressful activities should be avoided since there is a high risk for joint integrity. In some cases surgical intervention is required to repair joint defects or injuries. Resources Cañadas, Victoria; Vilacosta, Isidre; Bruna, Isidoro; Fuster, Valentin. “Marfan syndrome. Part 1: pathophysiology and diagnosis.” Nat Rev Cardiol. 7(2010):256-65. Malfait, Fransiska; Wenstrup, Richard J.; De Paepe, Anne. “Clinical and genetic aspects of Ehlers-Danlos syndrome, classic type.” Genet Med; 12(2010):597-605. Print. Prontera, Paolo; Belcastro, Vincenzo; Calabresi, Paolo; Donti, Emilio. “Myostatin depletion: a therapy for Ehlers-Danlos syndrome?” Ann Neurol. 67(2010):147-8. Print. Yokota, Toshifumi; Hoffman, Eric; Takeda, Shinichi. “Antisense oligo-mediated multiple exon skipping in a dog model of duchenne muscular dystrophy.” Methods Mol Biol;709 (2011):299-312. Print. Read More