SF33 Medical Genetics

Developmental & Pharmacogenetics.

SF33 Medical Genetics - Developmental.

Developmental Genetics and Pharmacogenetics.
Required Reading: Chapter 17, Genetics in Medicine, 6th Ed., Thompson and Thompson, 2001. Learning Objectives. After this lecture the student should be able to; 1. Define the basic concepts of Pattern formation. 2. Describe how transcription factors, signaling molecules and Growth factor receptors influence human development. 3. Give an example of a birth defect caused by disruption of pattern formation. 4. List the 5 data types used in the field of Pharmacogenetics. 5. Give an example of a genetic condition that shows variability in genotype and drug sensitivity. Developmental Genetics Birth defects are the most common cause of infant death in the USA. There are approximately 100,000 children born in the USA each year with a birth defect. Birth defects are defined as an isolated abnormality or they can be one of the more than 2000 genetic syndromes. Currently the etiology of birth defects is unknown; however it is estimated that a large portion of the caused by mutations in genes that control normal development. Basic concepts Most of what is know about development has be learned from model systems such as D. melanogaster (fruit fly), X laevis (African clawed frog), D rerio (zebrafish), C. elegans (nematode), G. gallus (chicken), M. musculus (mouse), P. hamadryas (baboon). In each of these organisms the fertilization of an egg begins the process of development. This single cell will form each of the different cell types, tissues and organs in the adult organism. The specific body plan arranges the patterns in the body. Many of these instructions for normal development are encoded from by the genome of the organism. The questions of development How do cells that are identical genetically form complex adult organisms composed of many different cell types and tissues? What controls the fate of a single cell, making a cell differentiate into a brain or liver cell? How do cells organize into discrete tissues? How is the body plan of the organism determined? These questions of development have been investigated for at least a century. In the past few years due to the recent technical advances much as been discovered that is helping us understand the causes of human malformations and genetic syndromes.

Brief overview of Major embryonic development The major processes that are involved in the development of the embryo include axis Page 1 of 8

SF33 Medical Genetics

Developmental & Pharmacogenetics. specification, pattern formation, and organogenesis. This involves the arrangement of cells in specific patterns spatially to form tissues and organs. Axis specific organization involves the definition of the major axes of the body: ventral/dorsal and anterior/posterior. Polarity is the direction in any limb or organ and an important part of this process. Induction is the process by which cells influence the organization of surrounding cells. Genetic Mediators of Development The genes required for normal development encode many different products. These include signal transduction molecules such as growth factors and their receptors, DNA transcription factors, components of the extracellular matrix, enzymes, transport systems and other proteins. These controllers of development are expressed in different temporal and spatial patterns to allow the normal development to occur. Table 17-1 lists a number of genes that have been found to be mutated in certain genetic syndromes that affect development. Partial list of Table 17-1 Examples of Human Developmental Abnormalities Condition Clinical Findings Genetics and Pathogenesis Reduced or absent iris, lens or corneal Autosomal semidominant loss Aniridia abnormalities of function PAX6 gene Deafness, white forelock, pale and or Autosomal semidominant loss Waardenburg Syndrome asymmetrical eye pigmentation of function PAX3 gene Interophalangeal webbing and extra digits Semidominant HOXD13 gene Synpolydactyly in hands and feet Holoprosencephaly Defective morphogenesis and bilateral Loss of function in SHH gene cleaveage of the forebrain and midface Mental retardation, broad thumbs and toes, Heterzygosity loss of function Rubenstein-Taybi syndrome downslanting palpebral fissures, hypoplastic maxilla, prominent nose, in the autosome CREB binding protein CBP gene congenital heart disease Growth and mental retardation, upper limb Dominant mutation, sporatic Cornelia de Lange syndrome deficiencies, synophrys, depressed nasal mutation of unknown gene bridge, thin upper lip Paracrine Signalling Molecules The interaction between adjacent cells in a developmental field is usually mediated bye proteins that can diffuse across small distances to induce a response. This is typically called a paracrine factor. To date 4 major paracrine signaling molecules families have been described; 1. fibroblast growth factors family (FGFs) (19) 2. Hedgehog family (First discovered in the Fly) 3. Wingless (Wnt) family (also discovered in the fly) 4. Transforming Growth Factor B family (TGF- ) Each of these factors binds to one or more signaling molecules to generation a response and mutations in genes encoding this molecules lead to abnormal communication between these cells. FGFs and FGFRs Clinical significance FGFR are receptors that detect the presence of FGF. There are 19 different FGFs that

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Developmental & Pharmacogenetics. participate in cell migration, growth and differentiation. FGFR are widely expressed in developing bones. Dominant mutations of these genes can cause skeletal dysplasias. Achondroplasia is characterized by short stature and autosomal inheritance. FGFR3 is activated by a mutation in the transmembrane domain and gives rise to this genetic condition. Different FGFR3 mutations cause different syndromes. See Table 10-3. Craniosynostosis Syndromes Due to Mutation in Fribroblast Growth Factor Receptors Gene Syndrome Characteristics FGFR1 Pfeiffer Broad first digits, hypertelorism Fusion of digits, mid-face hypoplasia FGFR2 Apert Broad First digits, mid-face hypoplasia Pfeiffer mid-face hypoplasia, Ocular proptosis Crouzon mid-face hypoplasia, corrugated skin Beare-Stevenson mid-face hypoplasia, Foot anomalies Jackson-Weiss mid-face hypoplasia, Ocular proptosis FGFR3 Crouzon Non-syndromic Digital defects, hearing loss Craniosynostosis

Hedgehog family First identified in D melanogaster, vertebrates were shown to have a number of homologues of this gene (Sonic hedgehog, Shh). Participates in axis formation in neural plate and patterning of the limbs. A receptor of Shh is called patched. Shh binding to patched causes suppresses transcription of the TGF- and Wnt genes causing cell growth inhibition. Mutation is the human homologue PATCHED (PTC) causes Gorlin syndrome, a birth defect causing rib defects, cysts of the jaw and basal cell carcinoma. Germ cells that have a mutation in PTC give rise to a birth defect and a somatic mutation cause cancer. Wnt family First identified in D. melanogaster with homologues in vertebrates (integrated). These are glycoproteins that establish the polarity of the limb for the dorso/vental axis. They participate in brain, muscle, gonads and kidney development. TGF-b family This is a large group of genes that produce proteins that form homo and heterodimers. These proteins act in bone formation pathway as well other morphogenetic pathways. Mutations in CDMP1 (cartilage-derived morphogenetic protein 1) cause various skeletal abnormalities. Nonsense mutation causes brachydactyly (short digits). Patients with a 22p duplication have brachydactyly as well as shortening of the long bones of the limbs (AR acromesomelic dysplasia). A homozygous missense mutation causes Grebe chondrodysplasia (severe shortening of the long bones and the digits) (dominant negative mutation type) DNA transcription Factors Transcription factors regulate the expression of genes in specific ways. The can turn off or turn on specific genes. Most transcription factors regulate the expression of many different genes, which in turn can regulation other genes giving rise to a cascade effect. There are many different families of transcription factors families. Some are involved in regulating genes important for

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Developmental & Pharmacogenetics. development. Examples of homeobox containing genes are HOX, PAX, EMX and MSX families. In addition there are HMG box genes such as the SOX and T-box families. A number of SOX genes act in different developmental pathways. SOX9 is expressed in the genital ridges of both sexes but in males its regulated and in females its down regulated. It regulates chondrogenesis and the expression of Col2A1, a collagen gene. Defects in SOX9 cause skeletal defects as well as sex reversal that produces XY females. Transcription factors regulate the transcription of specific genes and the same transcription factor can be used in different developmental pathways. Therefore disorders caused by mutations in gene encoding transcription factors are said to be Pleiotropic. Extracellular Matrix proteins These are secreted proteins that serve as scaffolding for all tissues and organs. These include collagens, Fibrillins, proteoglycans, and large glycoproteins such as fibronectin, laminin, and tenascin. Mutations in fibrillin-1 and elastin cause Marfan syndrome and supravalvular aortic stenosis, respectively. For EMPs for stick to the cells they interact with cell surface receptors such as integrins and glycosyltransferases. Pattern formation The ordered spatial arrangement of differentiated cells to create tissues and organs is called pattern formation. This is laid down in the embryo. This occurs by; 1. definition of the cells of the region 2. establishment of signaling centers 3. differentiation of cells within a region in response to additional cues. Example, cells in the vertebrate limb will produce many different cells types including cartilage (chrondrocytes) and bone (osteocytes). These cells must also be arranged in a temporal spatial pattern that will create the bones in the limb. Additional information is needed to determine whether an ulna or a humerus is being formed. Questions remaining to be answered; 1. How do particular structures develop in specific places? 2. How do cells acquire information about their relative positions Pattern formation occurs by cells communicating with each other using many different signaling pathways. Research has shown that these pathways are used repeatedly and are integrated with one another to control specific cell fates (the eventual location and function of the cell). The Sonic Hedgehog (Shh) protein in used in vertebrate neural tubes, somites and limbs as well as distinguishing left from right. Point mutations in Shh can cause abnormal midline brain development giving rise to holoprosencephaly (see Figure 10-2). Note; not all affected individuals have holoprosencephaly, this is likely due to the requirement of Shh to be attached to cholesterol. Things that affect cholesterol metabolism in the embryo are likely to enhance the mutation effect. (environmental effects) Basics in Human Embryonic development Gastrulation Cell movements and cell differentiation characterize this stage of development. The three layers of ecotoderm, endoderm and mesoderm are formed. See Figure 17.3

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Developmental & Pharmacogenetics. Neurulation and ectoderm This event produces the neural tube by cellular migration. It also produces the cells of the skin and the neural crest. In humans the neural tube produces the brain and spinal cord and closes at 5 separate sites. Common defects include anencephaly, occiptipal encephalocele and lumbar spina bifida. Mesoderm and endoderm This tissue produces a number of structures including the notochord, the skeletal structures the skeletal muscles and the connective tissue of the skin. Intermediate mesoderm produces the kidneys and the genitourinary system. The lateral plate mesoderm produces the heart and connective tissue viscera and the muscles of the eye and head arise from the head mesenchyme. The endoderm produces the digestive and respiratory tracts. Interactions with the neural crest cells produce the endodermal lined structures such as the middle ear, thymus, parathyroid and thyroids. This process involves budding and branching and involves fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs). Mutations in FGFR3 give rise to shortened long bones, poorly developed vertebral column, a small thoracic cage and a relatively large skull. Axis specification All vertebrates have a notochord and 3 axis, Dorso/ventral, anterior/ posterior, and left/right. Specification of these axes is important for the orientation of the body plan. The Left/Right Axis The left right axis begins with symmetry and progresses to a situation with asymmetric expression of Sonic hedgehog (Shh). This results in left sided expression of the TGB family member called nodal, which is responsible of the rightward looping of the heart tube. The ZIC3 gene can cause laterality defects in humans. Organ laterality is also important. Two transcription factors dHAND and eHAND have been showed to be important the left/right heart axis The Anterior/Posterior Axis The HOX genes are homologues of genes discovered in the fruit fly (homeotic genes such as antennapedia and bithorax). There are 39 Hox genes each on a different chromosome. Typically there are 13 genes in a cluster. The Hox genes are expressed from Anterior to posterior in a temporal collinearity. See Figure 17-7 and 17-8. Disruption of and HOX gene cause body limb and organ patterning disruptions. The Dorso/Ventral Axis This axis is defined signaling molecules and their antagonists that can dorsalize ventral cells. Organ and appendage Formation Many of the proteins used in the early formation of the embryo are used for the formation of organs and appendages. Most of the genes that are known to cause birth defects have prominent roles in this part of development. Craniofacial Development The majority of the craniofacial structures develop from the neural crest cells. The fate of each group of neural crest cells is specified by homeo-box genes. Some of the genes specifying craniofacial development have been identified by the analysis of craniosynostosis syndromes. Limb Development The development of the limb is well understood. See Fig 17-15. Limb bud growth occurs

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Developmental & Pharmacogenetics. from the apical ectodermal ridge, which will have a dorso/ventral determination. The proximal/distal growth is controlled by the fibroblast growth factors (FGF2, FGF4 & FGF8). Sonic hedgehog is also used to signal in the Zone of polarizing activity. (in the nervous system D/V and in embryo L/R). The HOX gene provides signals for growth and differentiation. A HOXD13 mutation causes Synpolydactyly in humans. Organ formation The formation of organs involves the reciprocal intereactions between epithilium and mesenchyme. This interaction is mediated by secreted signaling molecules that bind to receptors, conduct signals through various interconnected pathways and stimulate or repress DNA (gene) transcription.

Pharmacogenetics. "One Size Doesn't Fit All"

It is well known that everyone does not respond to medication in the same way. A person's environment, diet, and general state of health can all influence this. In addition, genes play a critical role. Pharmacogenetics is the study of how genes affect the way people respond to medication. This includes antidepressants, chemotherapy drugs, drugs for asthma and heart disease, as well as others. The ultimate goal of pharmacogenetics research is to help tailor medicines to people's unique genetic make-ups. This will make medicines safer and more effective for everyone. The PharmGKB website (http://www.pharmgkb.org/) is a comprehensive website of medical conditions that have been found to be applicable to pharmacogenetics classification and characterization. Glossary of Pharmacogenetic terms. Pharmacodynamics is the study of the relationships between the concentration of a drug at its site(s) of action and the magnitude of the biological or physiological effect that is achieved. Pharmacokinetics is the study of the bodily absorption, distribution, metabolism and excretion of drugs. Drug - A chemical or biological substance used in the diagnosis, treatment, or prevention of a disease or phenotype, or as a component of a medication. Molecular Assay - A molecular assay is an experiment in which the characteristics of a molecule (or ensemble of molecules) are measured. Cellular Assay - A cellular assay is an experiment in which the response or characteristics of a cell (or population of cells) is measured. Genotype - Genotype is the internally coded, heritable information carried by the organism. Variation in genotype represents differences in sequence within a species, such as SNPs, the location or the number of repeats, deletions, or critical splice sites. Phenotype - Phenotypes are the observable properties of an organism produced by the interaction of the genotype with the environment. For pharmacogenetics, the "environment" is often defined as exposure to a drug, although it may include other variables as well. There are numerous kinds of data important to the field of Pharmacogenetics. The PharmGKB website has annotated their entries with their associated genes and/or drugs. The pharmacogenetic knowledge base is classified into five general categories. These categories range from observations of genetic variation to assessment of variation in clinical outcomes in response to drugs. The categories are: Clinical Outcome Page 6 of 8

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Developmental & Pharmacogenetics. Genetic variations in the response to drugs can cause measurable differences in clinical endpoints such as rates of cure, morbidity, side effects, and death. Pharmacodynamics and Drug Response Genetic variation in drug targets can cause measurable differences in the response of an organism to a drug. Data in this category document that the biological or physiological response to a drug varies, and that this variation can be associated with the variation of one or more genes. This variation is often measured at the whole-organism level. Pharmacokinetics Genetic variation in processes involved in the absorption, distribution, metabolism, or elimination of a drug can result in changes in drug availability. This category is concerned with demonstrating that genetic polymorphisms lead to variations in the levels or concentrations of drugs or their metabolites at the site of action. Molecular and Cellular Functional Assays Genetic variation can alter results of molecular and cellular functional assays, and this may correlate with variations in the organism's drug response. This category demonstrates associations between genetic variation and laboratory assays of function at the molecular or cellular level. The assays may test the molecular properties of drug targets or drug metabolizing enzymes, or may test the cellular properties of cells involved in the response to a drug (such as whole-cell gene expression). Genotype Genotype is collection of alleles for each gene carried by the organism. Variation in genotype is fundamental to pharmacogenetics and is measured as sequence variation in individual genes--the type and location of the variation, and the frequency of the variation in the populations of interest. This genetic variation is independent of individual drugs, but forms the basis for variation in response to drugs. There are currently over 100 conditions being studied to understand how physicians can better treat these individuals with medications. A few examples are listed below. Breast Cancer Therapy;Individuals with a specific genetic variation in an enzyme called CYP2D6 do not respond well to tamoxifen, a common breast cancer drug. Blood Thinner Dosing; differences in a gene called VKORC1 influence how much warfarin is optimal for each person. This has lead to a faster and more precise dosing. Glucose 6-phosphate dehydrogenase (G6PD) deficiency Glucose 6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder, is the most common enzymatic disorder of red blood cells in humans, affecting 200 to 400 million people. The clinical expression of G6PD variants encompasses a spectrum of hemolytic syndromes. Affected patients are usually asymptomatic, but some patients have episodic anemia while a few have chronic hemolysis. With the most prevalent G6PD variants (G6PD A- and G6PD Mediterranean), severe hemolysis is induced by the sudden destruction of older, more deficient erythrocytes after exposure to drugs having a high redox potential (including the antimalarial drug primaquine and certain sulfa drugs) or to fava beans, selected infections, or metabolic abnormalities.

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Developmental & Pharmacogenetics. Malignant Hyperthermia (MH) Malignant Hyperthermia (MH) is a lifethreatening, acute pharmacogenetic disorder, developping during or after a general anaesthesia. Both a genetic predisposition, and one or more triggering agents are necessary to evoke MH. Triggering agents include all volatile anaesthetics (Chloroform, Ether, Halothane, Enflurane, Isoflurane, Sevoflurane, Deflurane) and depolarizing muscle relaxants (Suxamethonium). The classical MH crisis shows a hypermetabolic state, caused primarly by the muscles of the sceletal system. Besides this classical form of MH exist abortive forms with unspecific signs like tachycardia, arrhythmia and a raise in temperature. Modern monitoring, better knowledge of MH by the anaesthetists and the therapy using dantrolene reduces the incidence of the classic MH crisis. Nevertheless, MH is a dangerous disease, and anyone who is involved with anaesthesia and anaesthetics should have up to date knowledge about diagnosis and therapy of MH. Some MH families showed a defect on chromosome 19, on the ryanodinereceptor gene. Ryanodine, an alkaloid, binds selectivly to the ryanodine receptor, a calcium channel in the sarcoplasmatic reticulum. Other families with MH predisposition showed no ryanodinereceptor defect. Porphyria Porphyria is a disorder in which the body produces too much of the chemical porphyrin. Porphyrin is used to make heme, the part of blood that carries oxygen. Heme also gives blood its color. Any circulating porphyrin the body doesn't use is excreted in urine and stool. When the body produces and excretes too much porphyrin, as happens with porphyria, not enough heme remains to keep a person healthy. Porphyria affects either the nervous system or the skin. When porphyria affects the nervous system, it can cause chest pain, abdominal pain, muscle cramps, weakness, hallucinations, seizures, purple-red-colored urine, or mental disorders like depression, anxiety, and paranoia. When porphyria affects the skin, blisters, itching, swelling, and sensitivity to the sun can result. Porphyria is an inherited condition. Attacks of the disease can be triggered by drugs (barbiturates, tranquilizers, birth control pills, sedatives), chemicals, certain foods, and exposure to the sun.

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