Chapter 1 USMLE PDF

Chapter 1: Biochemistry 1 • Introduction to Nucleic Acids Nucleic acids are long chain polymers of nucleotides joined by 3′, 5′-phosphodiester bonds; that is, a phosphate group links the 3′ carbon of a sugar to the 5′ carbon of the next sugar in the chain • Each strand has a distinct 5′ end and...

Chapter 1: Biochemistry 1 • Introduction to Nucleic Acids Nucleic acids are long chain polymers of nucleotides joined by 3′, 5′-phosphodiester bonds; that is, a phosphate group links the 3′ carbon of a sugar to the 5′ carbon of the next sugar in the chain • Each strand has a distinct 5′ end and 3′ end, and thus has polarity • Sequence is always specified as 5′→3′ DNA structure properties • The two strands are antiparallel (opposite in direction). • The two strands are complementary. A always pairs with T (two hydrogen bonds), and G always pairs with C (three hydrogen bonds) Overview: • Nucleic acids (DNA and RNA) are assembled from nucleotides, which consist of three components: • Nitrogenous base (Purines, Pyrimidines) • Five- carbon sugar (pentose) Ribose or Comparison between DNA and RNA Deoxyribose Comparison DNA Found in Nucleus • Phosphate. • Nucleic acids are classified according to pentose Pentose Deoxyribose sugar If the pentose is ribose, the nucleic acid is Nitrogenous base Adenin- Thymin RNA (ribonucleic acid); if the pentose is Guanin- Cytocin Shape Double strand deoxyribose, the nucleic acid is DNA Types (deoxyribonucleic acid). • There are two types of nitrogen-containing bases commonly found in nucleotides: A-purines: • The two purines commonly found in nucleic acids are adenine (A) and guanine (G); both are found in DNA and RNA. • Contain 2 rings in their stricture B-pyrimidines • Cytosine (C) is present in both DNA and RNA. Thymine (T) is usually found only in DNA, whereas uracil (U) is found only in RNA. • Pyrimidines have only one ring. • • • • The bases attached to each other due to hydrogen bonds Guanine binds to cytosine with three hydrogen bonds Adenine binds thymine with two hydrogen bonds Nucleotides are formed when one or more phosphate groups is attached to the 5′ carbon of a nucleoside (Nucleoside di- and triphosphates are high energy compounds because of the hydrolytic energy associated with the acid anhydride bonds RNA Nucleus and Cytoplasm Ribose Adenine-Uracil Guanin- Cytocin Single strand Messenger 8mRNA) Transfer (tRNA) Ribosomal (rRNA) Polymerase enzyme: • Polymerases are enzymes that catalyze the synthesis of DNA or RNA polymers whose 2 • • sequence is complementary to the original template It adds nucleotides to the 3´ end and synthesized from the 5´ ends to the 3´ ends • The parental strand is used as a template for this process DNA polymerases has A 3´→ 5´ proofreading feature. It Formation of replication fork: allows the enzyme to check each nucleotide during • Helicase breaks the hydrogen bonds holding the DNA synthesis and excise mismatched nucleotides base pairs together. This allows the two parental in the 3´ to 5´ direction. strands of DNA to begin unwinding and forms two Helicase enzyme: replication forks • Helicases are enzymes that bind and unwinds the • SSB protein binds the complex to stabilize ssDNA DNA Primer synthesis • Primase synthesizes a short (about 10 nucleotides) RNA primer in the 5′→3′ direction, beginning at the origin on each parental strand. • RNA primers are required because DNA polymerases are unable to initiate synthesis of DNA and can only extend a strand from the 3′ end of a preformed “primer.” Leading strand synthesis • DNA polymerase III begins synthesizing DNA in • There are DNA and RNA helicases. the 5′→3′ direction, beginning at the 3′ end of each • DNA helicases are essential during DNA RNA primer. The newly synthesized strand is replication because they separate double-stranded complementary and antiparallel to the parental DNA into single strands allowing each strand to be strand used as a template. This strand will move copied into the replication fork (Leading strand synthesis). Ligase Lagging strand synthesis • Used to join DNA fragments by catalyzing the • Many DNA fragments are synthesized sequentially formation of phosphodiester bonds between 5' on the DNA template strand in the 5′→3′ direction. phosphate and 3' hydroxyl termini in doubleThese DNA fragments called Okazaki fragments stranded DNA using ATP as a coenzyme • Each Okazaki fragment is initiated by the synthesis of an RNA primer by primase, and then completed by the synthesis of DNA using DNA polymerase III • RNA primers on Okazaki fragments are digested by enzyme RNAase • The gaps are filled by DNA polymerase I in in the 5′→3′ direction • DNA ligase seals the “nicks” between Okazaki Endonuclease fragments, converting them to a continuous strand • Enzyme that breaks down a nucleotide chain into of DNA. two or more shorter chains by cleaving the Termination phosphodiester bonds linking nucleotides • (DNA topoisomerase II) removes positive supercoils ahead of advancing replication forks DNA replication DNA replication • DNA replication: Synthesis of daughter DNAs using parental DNAs as template 3 Summary of DNA replication steps Replication step Origin of replication (ori) One ori site per chromosome Unwinding of DNA double helix Helicase Stabilization of unwound template strands Single-strand DNA-binding protein SSB Primase Synthesis of RNA primers Synthesis of leading, lagging strands (Okazaki fragments) DNA polymerase III Removal of RNA primers DNA polymerase I Joining of Okazaki fragments DNA ligase Removal of positive supercoils ahead of advancing replication forks DNA topoisomerase II Split site involves either insertion or deletion results in one or more introns remaining in mature mRNA Trinucleotide repeat expansion Protein function might be altered Protein product becomes longer than normal Example: Huntington disease Lac Operon Function • The lac operon (Lactose Operon) is required to transport and metabolism of Glucose in Escherichia coli • The lac operon allows the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase • During low glucose levels- Increase adenylate cyclase activity- Increase generation of cAMP from ATP and consequently lead to activation of catabolite activator protein (CAP) and eventually enhance transcription. • In case of high lactose leads to unbinds repressor protein from repressor/operator site and eventually enhance transcription. DNA mutations Overview • One of the common types of mutation is a single base alteration or point mutation • Transition a point mutation in which purinepyrimidine base is replaced with pair with a different purine-pyrimidine base pair • Example: A-T base pair becomes a G-C base pair. • Transversion a point mutation that replaces a purine-pyrimidine base pair with a pyrimidinepurine base pair • Example: A-T base pair becomes a T-A or a C-G base pair. • The severity of damage classified as follow silent << missense < nonsense < frameshift. Common types of mutations in protein structures Three genes associated with Lac operon Type of mutation Silent: New codes for the same amino acid Missense: New codes BUT specifies different amino acid Nonsense: New codon results in stop codon (UAG, UAA, UGA) Frameshift/ in-frame: addition or deletion of bases resulting in misreading of all nucleotides downstream 1. Lac Z codes for B-galactosidase and help breaks down glucose into glucose and galactose 2. Lac y codes for galactose permease which found in E.Coli cytoplasmic membrane which help transport lactose into the cells 3. Lac a code for Galactoside acetyltransferase an enzyme that transfers an acetyl group from acetylCoA to β-galactosides. Effect on protein None Possible decrease in function Example: Sickle cell disease (substitution of glutamic acid with valine). Element Operator (lac O) Promotor (lacP) Repressor Usually nonfunctional Lac I Protein function might be altered Example: Duchenne muscular dystrophy Tay-Sachs disease. 4 Function Binding site for repressor Binding site for RNA polymerase Binds to DNA at the operator and blocks binding of RNA polymerase at the promotor Controls production of the repressor protein Summary of different scenarios of glucose and lactose abundance Regulation of gene expression Overview: • Regulation of gene expression is an essential feature in maintaining the functional integrity of a cell. • It involves several mechanisms that are used by the cells to increase or decrease the production of specific gene products (protein or RNA) • It also regulates the rate of transcription by the help of activator and repressor proteins • The regulatory proteins bind to either enhancer or silencer elements associated with eukaryotic gene regions • Gene regulation in prokaryotes is one of the methods of conservation of cell resources by turning OFF and ON of genes transcribing. • Regulations of prokaryotic genes are done in units called as Operons. • The operon is a unit of gene expression and regulation which typically includes structural genes for enzymes involved in a specific biosynthetic pathway whose expression is coordinately controlled. • Operator sequence: DNA sequence that regulates transcription of the structural genes and regulatory gene whose products recognize the control elements, for example a repressor which binds to and regulates an operator sequence. Several steps in gene expression process may be modulated 1. Transcription 2. RNA splicing 3. Translation 4. Post-translation modification Regulation of gene expression divided into 1. Positive regulation 2. Negative regulation • When the expression of genetic information is quantitatively increased by the presence of a specific regulatory element, regulation is said to be positive • when the expression of genetic information is decreased by the presence of a specific regulatory element, regulation is said to be negative. • The element or molecule mediating negative regulation is said to be a negative regulator, a silencer or repressor; that mediating positive regulation is a positive regulator, an enhancer or activator. Lac operon • The lac operon (Lactose Operon) is required to transport and metabolism of Glucose in Escherichia coli • The lac operon allows the effective digestion of lactose when glucose is not available through the Promotor: activity of beta-galactosidase • DNA region that initiates transcription of a • During low glucose levels- Increase adenylate particular gene. cyclase activity- Increase generation of cAMP • Promoters are located near the transcription start from ATP and consequently lead to activation of sites of genes, on the same strand and upstream on catabolite activator protein (CAP) and eventually the DNA (towards the 3' region of the anti-sense enhance transcription. strand). • In case of high lactose leads to unbinds repressor protein from repressor/operator site and eventually enhance transcription. Three genes associated with Lac operon 1. Lac Z codes for B-galactosidase and help breaks down glucose into glucose and galactose 5 2. Lac y codes for galactose permease which found in mRNA splicing E.Coli cytoplasmic membrane which help transport lactose into the cells 3. Lac a code for Galactoside acetyltransferase an enzyme that transfers an acetyl group from acetylCoA to β-galactosides. Lac Repressor • Encoded by lacI gene, which is active as a • tetramer of identical subunits. mRNA splicing: Protein involved in the process by • In the absence of lactose, the repressor occupies which nonsense sequences or intervening the operator-binding site. Both the lac repressor sequences (introns) are removed from pre-mRNA and the RNA polymerase can bind simultaneously to generate a functional mRNA (messenger RNA) to the lac promoter and operator sites. that contains only exons. Induction • Pre-mRNA is processed into a messenger RNA • In the presence of lactose an inducer molecule like (mRNA) Allolactose, which is an isomer of lactose, binds to • A 5' cap is added to the beginning of the RNA a specific site on the lac repressor causing a transcript, and a 3' poly-A tail is added to the end. conformational change which leads to dissociation • During splicing, introns are removed accomplished of repressor from the operator. by spliceosomes (also known as an snRNP), which • β galactosidase molecule in E. coli converts are complexes of snRNA and protein. Meanwhile, lactose into allolactose. exons are joined together to assemble the coding mRNA splicing region of the mature mRNA. Overview • The mature mRNA molecule is transported to the • Genetic information is transferred from genes to cytoplasm, where it is translated to form a protein. the proteins via RNA • In some cases, alternative splicing might occur • Most genes have their protein-coding information leading to the production of different mRNA interrupted by non-coding sequences called molecules “introns”. The coding sequences are then called “exons” • Proteins have a non-coding sequence called introns and a coding sequences called exons • Introns are removed by a process called RNA splicing • • • • 6 Spliceosome: Help remove the introns from premRNA molecules Spliceosome composed of 5 small ribonucleoproteins snRNPs The RNA components of snRNPs interact with the intron and are involved in the catalysis Spliceosome acts by formation of complex E, A, B, C Introns VS Exons Comparison between Introns and Exons Introns Exons Non-coding region Coding region for protein Sequence changed over the time Conserved sequence Found in eukaryotes Found in both prokaryotes and eukaryotes Considered as the bases located between two exons Considered as the bases which encode an amino acid sequence of a protein Stay in the nucleus by splicing out from mRNA Exit the nucleus to the cytoplasm after mRNA production RNA types Ribosomal RNA (rRNA) Transfer RNA (tRNA) mRNA Messenger RNA snRNA small nuclear RNA ● Most abundant RNA ● With ribosomal proteins, makes up the ribosomes, the organelles that translate the mRNA. ● Brings amino acids to ribosomes during translation. ● Carries the information specifying the amino acid sequence of a protein to the ribosome ● The only translated form of RNA ● Involved in splicing of mRNA (Removal of introns) MicroRNAs • MicroRNAs constitute a recently discovered class of non-coding RNAs that play key roles in the regulation of gene expression. • microRNAs play an integral role in numerous biological processes, including the immune response, cell-cycle control, metabolism, viral replication, stem cell differentiation and human development. • microRNA expression or function is significantly altered in numerous diseases including cancer and fibrosis, as well as CNS, metabolic and inflammatory disorders tRNA structure and Function Overview ● ● ● tRNA structure ● 70 to 80 nucleotides in length, that serves as the physical link between the mRNA and the amino acid sequence of proteins. ● Cloverleaf secondary structure Transcription is the first step in the expression of tRNA composed of genetic information starting from the base ● 5-terminal phosphate group sequence of double-stranded DNA molecules to ● Amino acid acceptor arm at the 3’ form single-stranded molecule of RNA ● Site for amino acid attachment DNA molecule is considered the template strand ● D arm composed of stem and loop with and through which RNA is synthesized in the 5-3 dihydrouracil direction using RNA polymerase ● D arm serve as recognition site for aminoacylHowever, RNA polymerase moves in 3-5 direction tRNA synthetase that activate the amino acid through DNA template therefore, RNA product will be antiparallel and complementary to the templateRNA types 7 ● TΨ C arm composed of stem and loop where Ψ is pseudouridine a modified uridine ● ● ● Anticodon loop Composed of stem and loop Recognize mRNA codon and binds to it Anticodon complementary and antiparallel to codon in mRNA Variable arm ● Located between anticodon loop and T Ψ C loop ● Helps stability of tRNA tRNA function ● During translation, the codons for each amino acid Protein Synthesis Overview in the is transcribed from DNA to mRNA. • Protein synthesis occurs by peptide bond ● tRNAs serve as adapter molecules that couple the formation between successive amino acids whose codons in mRNA with the amino acids, they each order is specified by a gene and thus by an mRNA. specify, thus aligning them in the appropriate • Protein synthesis is carried out through 2 important sequence before peptide bond formation molecules ribosomes and tRNA ● In the cytoplasm, tRNA joins its cognate amino • Each end at tRNA contain a sequence of three acid in a process called amino acid activation nucleotides (Anticodon) that could bind to specific ● Aminoacyl tRNA synthetase activate amino acids, mRNA codon then transfer the activated amino acid to the 3’ end • During translation, the amino acids are attached to of RNA the 3′ ends of their respective tRNAs. ● Next, the amino acid binds to tRNA with high • The aminoacyl–tRNAs are situated in the P and A energy bond that is sufficient enough to make a sites of the ribosome peptide bond linking the amino acid into a protein. • Ribosome is the backbone where polypeptides are ● Each tRNA has an anticodon sequence that allows built it to pair with the codon for its cognate amino acid • Each ribosome has two subunits, a large one (50S) in the mRNA. Because base pairing is involved, and a small one (30S) the orientation of this interaction will be complementary and antiparallel. • The ribosome has three active sites: The A site, the ● Note: Aminoacyl tRNA synthetases have selfP site, and the E site. checking functions to prevent incorrectly paired 1. The A site (Acceptor site), binds to the aminoacyl aminoacyl tRNAs from forming. tRNA 2. The P site is where the peptidyl tRNA is formed in the ribosome. 3. The E site function as threshold which holds empty tRNA as it exits Protein Synthesis • Occur in three steps initiation, elongation and termination • GTP is required as a source of energy • Special protein factors are required for initiation (IF), elongation (EF), and termination (release factors) 8 Initiation • The small ribosomal subunit binds to the mRNA • The charged initiator tRNA becomes bound to the AUG start codon on the message through base Cell Cycle • Overview pairing with its anticodon The large subunit binds to the small subunit, forming the completed initiation complex Elongation • Occurred through three steps cycle, each cycle consumes 4 energy bounds • The mRNA is read one codon at a time, and the amino acid matching each codon is added to a C growing protein chain. • During elongation, the ribosome moves in the 5′ to Overview: 3′ direction along the mRNA, synthesizing the Role of cell division: protein from amino to carboxyl terminus • Reproduction • Growth and development 1. A charged tRNA binds in the A site. The particular • Renewal and repair aminoacyl–tRNA is determined by the mRNA codon aligned with the A site. Cell Cycle phases: 2. Peptidyl transferase catalyzes peptide bond • The M phase (mitosis) is the time in which the cell formation, transfers growing polypeptide to amino divides to form two daughter cells acid in A site (The bond linking the growing • Mitosis consists of 5 phases: peptide to the tRNA in the P site is broken 1. Prophase 3. The ribosome moves exactly three nucleotides 2. Prometaphase (one codon) towards 3’ end of mRNA. This moves 3. Metaphase the growing peptidyl–tRNA into the P site and 4. Anaphase aligns the next codon to be translated with the 5. Telophase empty A site. • Cytokinesis: Division of the cytoplasm • Interphase is the term used to describe the time between two cell divisions or mitoses • During Interphase, gene expression take place Interphase is divided into G1 phase (gap 1) • Period of cellular growth preceding DNA synthesis. • Cell is metabolically active S phase (DNA synthesis) • period of time during which DNA replication occurs G2 phase (gap 2) Termination • Continuation of cell growth after DNA synthesis • The stage in which the release factors recognize but preceding mitosis stop codon (UAG, UAA, or UGA) that eventually G0 stop translation • Exit from cell cycle – Non dividing cell such as • Completed polypeptide is released from its tRNA muscle and nerve cells, are said to be in a special moving out of the ribosome state called G0 Factors that control the cell cycle • 9 • • Control of the cell cycle is done by checkpoints between the various phases Checkpoints ensure that cells will not enter the next phase of the cycle until the molecular events in the previous cell cycle phase are concluded. Cell Types Permanent Stable Definition Cannot reproduce themselves after birth (Remain in G0) Don´t replicate normal but maintain the capacity of proliferation Continuous proliferation through life (Never go to G0) Affected by chemotherapy Examples Neurons, skeletal and cardiac muscle, RBCs. Hepatocytes, lymphocytes, PCT, periosteal cells. Cyclin dependent kinases: Labile Bone marrow, gut • They are also involved in regulating transcription, epithelium, skin, hair mRNA processing, and the differentiation of nerve follicles, cells germ cells. • Constitutive and inactive Cyclin-CDK complexes: Cell organelles • Activates other proteins in the presence of cyclins Endoplasmic reticulum such as Maturation promoting factor (MPF) • Endoplasmic reticulum composed of: • When the cyclins and CDKs that are expressed in a • Rough ER (RER) is involved in some protein specific phase are bonded and activated, they production, protein folding, quality control and phosphorylate the specific serine and threonine despatch. residues of a target protein. • It is called ‘rough’ because it is studded with ribosomes Cyclins • Smooth E R (SER) is associated with the • Control the progression of cells through the cell production and metabolism of fats and steroid cycle by activating cyclin-dependent kinase (CDK) hormones. It is ‘smooth’ because it is not studded enzymes with ribosomes and is associated with smooth • There are two main groups of cyclins: slippery fats. • G1/s cyclins • Smooth ER also plays a large part in detoxifying a • G2/M cyclins number of organic chemicals converting them to safer water-soluble products Tumor Suppressors Peroxisome Peroxisome Function: • Tumor suppressor genes encode proteins that regulate and suppress cell proliferation by • Known as microbody inhibiting progression of the cell through the cell • Involved in catabolism of, branched fatty acids, cycle. methanol and aminoacids • DNA repair may not occur properly when certain • Beta oxidation of very long chain fatty acids tumor suppressor genes have been inactivated • Reduction of reactive oxygen species (H2O2) through mutation or deletion • Production of Plasmalogens (for brain and lung • The p53 gene encodes a protein that prevents a cell development) with damaged DNA from entering the S phase. • Synthesis of cholesterol, bile acids • p21 represents a major target of P53 activity and thus is associated with linking DNA damage to Diseases associated with Peroxisome dysfunction cell cycle arrest Zellweger Syndrome • Inactivation or deletion associated with Li • Autosomal recessive disorder Fraumeni syndrome and many solid tumors. • Occur due to genetic mutation in any gene • The retinoblastoma protein (RB): prevent involved in peroxisomes biogenesis such as PEX1, excessive cell growth by inhibiting cell cycle PEX2 progression until a cell is ready to divide. • Characterized by: • When the cell is ready to divide, Rb is phosphorylated to pRb inactivating the protein 1. Accumulation of very long chain fatty acid and which allows cell cycle progression branched fatty acids • Growth factors (eg, insulin, PDGF, EPO, EGF) 2. Impaired neuronal migration and brain bind tyrosine kinase receptors to transition the cell development from G1 to S phase 3. Deficiency in myelin production (Hypomyelination) 4. Deficiency in Plasmalogens (important for brain and lung function) 10 Clinical presentations: • Hepatomegaly • Vision problems • Abnormal muscle tone • Decrease brain development • Infants usually die within their first year. Refsum disease • Autosomal recessive disorders due to accumulation of phytanic acid in cells and tissue due to impaired alpha-oxidation of branched fatty acid Characterized by: • Cataracts • Shortening of 4th toe • Ataxia • Epiphyseal dysplasia. • Scaly skin • Treatment: Diet, Plasmapheresis. Adrenoleukodystrophy • X-linked recessive disorder of β-oxidation results in results in the accumulation of very long chain fatty acid in adrenal gland • Lead to destruction of myelin sheath of the nerves resulting in seizures and hyperactivity Signs and symptoms: Adrenomyeloneuropathy (AMN) phenotype symptoms: • Walking and balance problems • Pain, numbness, or tingling in the legs • Urinary problems or incontinence and bowel urgency or incontinence • Sexual dysfunction, or the inability to obtain or maintain an erection. Proteasome • Protein complexes which degrade or damaged proteins by proteolysis (ubiquitin-proteasome system UPS) • Deregulation of the UPS has been implicated in the pathogenesis of many neurodegenerative disorders like Alzheimer's disease, Parkinson's diseases, Huntington disease, Prion-like lethal disorders, in the pathogenesis of several genetic diseases including cystic fibrosis, Angelman's syndrome and Liddle syndrome and in many cancers. Cell trafficking Cell trafficking components: • The Golgi apparatus is involved in the sorting and trafficking of proteins produced within a cell. • Proteins translated within the rough endoplasmic reticulum are transferred to the Golgi. From there they are modified and packaged into vesicles for distribution. • Mannose-6-phosphate (M6P) is a key targeting signal for acid hydrolase precursor proteins that are destined for transport to lysosomes. • The M6P-tagged lysosomal enzymes are shipped to the late endosomes via vesicular transport • Endosomes provide an environment for material to be sorted before it reaches the degradative lysosome • Molecules are also transported to endosomes from the trans- Golgi network and either continue to lysosomes or recycle back to the Golgi • Lysosomes: Organelles whose major function is to digest materials that the cell has ingested by endocytosis. • Lysosomes contain multiple enzymes that, collectively, digest carbohydrates (glycosylases), lipids (lipases), and proteins (proteases). Signal recognition particles (SRP) • Abundant, cytosolic ribonucleoprotein that traffics proteins from the ribosome to the endoplasmic reticulum • Disfunction associated with SRP leads to accumulation of proteins in the cytosol Vesicular trafficking proteins COPI: • Specific coat protein complex that initiates the budding process on the cis-Golgi membrane. • Function as coating vesicles transporting proteins from the cis end of the Golgi complex back to the rough endoplasmic reticulum (ER), where they were originally synthesized. • This type of transport is termed as retrograde transport COPII: • Transport proteins from Endoplasmic reticulum to the Golgi-complex • This type of transport is termed as anterograde transport Clathrin • key molecules in the process of clathrin-mediated endocytosis, which is used for the specific uptake 11 of large extracellular molecules such as proteins, membrane-localized receptors, and ion channels. Microtubules • Function: Involved in movement and cell division • Examples: Cilia, flagella, mitotic spindle, axonal trafficking and centrioles I –cell disease: • When cargo arrive in the Golgi apparatus, specific mannose residues located in their N-linked Intermediate filaments oligosaccharide chains are phosphorylated by N• Elements that connect cells and tissue together acetylglucosamine- 1 phosphotransferase, forming • Examples: Vimentin, desmin, cytokeratin, lamins, a critical mannose-6-phosphate in the glial fibrillary acidic protein (GFAP), oligosaccharide chain neurofilaments. • Genetic defects affecting this phosphorylation produce I-cell disease in which lysosomal enzymes are released into the extracellular space, and inclusion bodies accumulate in the cell, compromising its function. Clinical Presentations: • Coarse facial features, gingival hyperplasia, macroglossia • Craniofacial abnormalities, joint immobility, clubfoot, claw-hand, scoliosis • Psychomotor retardation, growth retardation • Cardiorespiratory failure, death in first decade • Bone fracture and deformities • Mitral valve defect • Secretion of active lysosomal enzymes into blood and extracellular fluid • • • • • Cilia Structure Cytoskeleton Cilia Cilia contain 9 peripheral pairs of microtubules and 2 central microtubules. Composed of Basal body, intermediate junction, Microvillus and Occluding junction Basal body (base of cilium below cell membrane) consists of 9 microtubule triplets B with no central microtubules. The microtubules convey motility to cilia through the ATPase dynein They also form the core of the flagella, the motile tail of sperm cells Diseases associated with Cilia dysfunction • • • Kartagener syndrome • Due to an absence of dynein that is required for flagellar motility • Characterized by: • Immotile spermatozoa that lead to male infertility • Dysfunction fallopian tube cilia which increase ectopic pregnancy • Chronic respiratory chronic respiratory infections because of similar defects in cilia of respiratory epithelium • Conductive hearing loss and situs inversus Cytoskeleton is a cellular structure that helps cells maintain their shape and internal organization It also provides mechanical support that enables cells to carry out essential functions like celldivision, anchorage and movement. Cytoskeleton composed of: 1. Microfilaments 2. Intermediate filaments 3. Microtubules Microfilaments • Function: Reinforcing cytoskeletal elements, muscle contraction and cytokinesis • Examples: Fibers of actin subunits, Microvilli 12 Microtubules • Composed of helical array of polymerized heterodimers of α- and β-tubulin • Microtubules form the mitotic spindle during mitosis and meiosis. • Provide intracellular transport of vesicles and molecules specially through the axons • Transport requires specific ATPase motor molecules Dynein drives retrograde transport and kinesin • drives anterograde transport • Microtubules are found in true cilia and flagella, and utilize dynein to convey motility to these structures • Drugs that acts on microtubules: 1. 2. 3. 4. 5. Mebendazole (anthelminthic) Griseofulvin (antifungal) Colchicine (antigout) Vincristine/Vinblastine (anticancer) Paclitaxel (anticancer) • Because the hydroxylase enzymes that perform these reactions require vitamin C as a cofactor, a long-term deficiency in this vitamin results in impaired collagen synthesis and scurvy Collagen and Elastin synthesis and structure Overview • Collagen is the most abundant protein in the C-Glycosylation human body, found in the bones, muscles, skin, • Selected hydroxylysines are glycosylated. and tendons. • Three pro-α chains assemble to form a triple • Collagen forms a scaffold to provide strength and helical structure (procollagen) via hydrogen and structure. disulfide bond • Collagen is an example of a protein that undergoes • At this point, procollagen can now be transferred several important co- and posttranslational to the Golgi. modifications • Modification of oligosaccharide continues in the • Composed of a repeating tripeptide Gly-X-Y-GlyGolgi. X-Yetc. D-Exocytosis • X and Y are proline and lysine • Procollagen is secreted from the cell. Collagen Synthesis E-proteolytic processing • Hydroxyproline is an amino acid unique to • The propeptides are cleaved from the ends of collagen. procollagen by proteases to form collagen • The hydroxyproline is produced by hydroxylation molecules (also called tropocollagen). of prolyl residues at the Y positions in procollagen chains as they pass through the RER. Cross-Linking A-Synthesis • Collagen molecules assemble into fibrils. Cross• Preprocollagen-α chains containing a hydrophobic linking involves lysyl oxidase, an enzyme that signal sequence are synthesized by ribosomes requires O2 and copper. attached to the RER. • Fibrils aggregate and cross-link to form collagen B-Hydroxylation fibers. • Selected prolines and lysines are hydroxylated by prolyl and lysyl hydroxylases. Disorders of collagen biosynthesis Ehlers-Danlos (ED) Syndromes • Autosomal dominant or recessive disease caused by mutations in the gene for type-3 procollagen • Example of Locus heterogeneity 13 • Represent a collection of defects in the normal synthesis and processing of collagen Clinical presentations • Hyperextensible, fragile skin • Hypermobile joints • Dislocations • Varicose veins • Ecchymoses, • Berry and aortic aneurysms • Classical type associated with joint instability (Mutation in type V collagen) Such as COL5A1, COL5A2 • Vascular type III associated with arterial, intestinal, or uterine rupture; and easy bruising Menkes disease • Menkes disease Deficient cross-linking secondary to functional copper deficiency • Impaired Menkes protein (ATP7A) Clinical presentations • Depigmented hair (Brittle and kinky hair) • Growth retardation • Osteoporosis • Anemia • Arterial tortuosity Osteogenesis imperfecta • Mutations in genes encoding type 1 collagen affect Scurvy • Scurvy is caused by a prolonged dietary deficiency the coding of one of the two genes (COL1A1 and of vitamin C COL1A2), accounting for approximately 80% • Risk factors for vitamin C deficiency: cases. A. Babies who are fed only cow's milk or plant-based • The mutations result in the production of a mixture beverages during the first year of life of normal and mutant collagen chains. B. Alcoholic individuals • Substitution of a larger amino acid (eg, cysteine or C. Elderly individuals who eat a tea-and-toast diet alanine) for glycine results in abnormal helix D. Economically disadvantaged persons, who tend to formation not purchase foods high in vitamin C (eg, green • Arise from autosomal dominant mutations vegetables, citrus fruits) Clinical presentations E. Cigarette smokers • Blue sclerae due to the translucent connective F. Pregnant and lactating women and those with tissue over choroidal veins thyrotoxicosis: These individuals require an • Triangular facies increased intake of vitamin C because of increased • Macrocephaly utilization • Hearing loss (abnormal ossicles) G. People with anorexia nervosa or anorexia from • Defective dentition (due to lack of dentin) other diseases such as AIDS or cancer • Barrel chest H. People with type 1 diabetes have increased vitamin • Scoliosis C requirements, as do those on hemodialysis and • Limb deformities peritoneal dialysis • Fractures Elastin Synthesis • Joint laxity • Elastin is the main protein of elastic fibers and • Growth retardation confers the property of elastic recoil to the tissues such as arteries, lung, elastic cartilage • The major protein component of the blood vessels is elastin. Therefore, the loss of elastin may cause 14 • • • • • • • • atherosclerosis. The loss of elastin in lungs causes ` emphysema. Elastin is mainly produced in the fetus. It is no longer produced after puberty. Elastin is an insoluble protein polymer Synthesized from a precursor, tropoelastin Rich in non-hydroxylated proline, glycine, and lysine residues α1-antitrypsin plays an important role in elastin degradation It inhibits the activity of trypsin synthesized by the pancreas Inhibit neutrophil elastase Elastase is a powerful protease that is released into the extracellular space by neutrophils Collagen- Osteogenesis imperfecta & Ehler-Danlos Syndrome Types of Collagen Type Type I Type II Marfan disorders • Spectrum of disorders caused by a heritable genetic defect of connective tissue • The defect itself has been isolated to the FBN1 gene on chromosome 15, which codes for the connective tissue protein fibrillin. • Abnormalities in this protein cause a myriad of distinct clinical problems, of which the musculoskeletal, cardiac, and ocular system problems predominate Clinical presentations • Craniofacial characteristics • Thumb and wrist signs • Pectus carinatum • Severe hindfoot valgus • Subluxation of lenses, typically upward and temporally • Hypermobile joints; long, tapering fingers and toes (arachnodactyly) • Cystic medial necrosis of aorta 15 Type III Type IV • • • Example Most common-bone, skin, tendon, dentin, fascia, cornea, late wound repair Cartilage (including hyaline), vitreous body, Nucleus pulposus Reticulin, Skin, blood vessels, uterus, fetal tissue, granulation tissue. (early wound healing) Associated with Ehlers-Danlos Syndrome Basement membrane, basal lamina, lens Hearing loss, vision loss, renal disease Function Provide tensile strength to connective tissue Provide tensile strength to connective tissue Provide structural networks of spleen, Liver, Lymph nodes, smooth muscle and adipose tissue Associated with Alport syndrome and Goodpasture syndrome Goodpasture syndrome (GPS), also known as antiglomerular basement membrane disease, is a rare autoimmune disease in which antibodies attack the basement membrane in lungs and kidneys, leading to bleeding from the lungs and kidney failure. Alport syndrome is a genetic condition characterized by kidney disease, hearing loss, and eye abnormalities. Characterized by progressive loss of kidney function. Almost all affected individuals have blood in their urine (hematuria), which indicates abnormal functioning of the kidneys. Ehlers-Danlos (ED) Syndromes • Inhibit neutrophil elastase • Autosomal dominant or recessive disease caused • Elastase is a powerful protease that is released by mutations in the gene for type-3 procollagen into the extracellular space by neutrophils • Example of Locus heterogeneity Polymerase chain reaction (PCR) • Represent a collection of defects in the normal Principle: synthesis and processing of collagen Clinical presentations • Hyperextensible, fragile skin • Hypermobile joints • Dislocations • Varicose veins • Ecchymoses, • Berry and aortic aneurysms • Classical type associated with joint instability • A common laboratory technique used to make (Mutation in type V collagen) Such as COL5A1, many copies (millions or billions!) within a few COL5A2 hours of a selected region of a chromosome • Vascular type III associated with arterial, • The main goal of PCR is to make enough DNA intestinal, or uterine rupture; and easy bruising that can be used for many application Osteogenesis imperfecta • only a small amount of DNA is available e.g. drop • Mutations in genes encoding type 1 collagen affect of blood, Semen strains, Single hair, vaginal swabs the coding of one of the two genes (COL1A1 and etc. COL1A2), accounting for approximately 80% • The region of a chromosome to be amplified by a cases. PCR is referred to as the target sequence and may • The mutations result in the production of a mixture be an area containing a suspected mutation, a short of normal and mutant collagen chains. tandem repeat (STR, or microsatellite sequence), • Substitution of a larger amino acid (eg, cysteine or or really any area of interest. alanine) for glycine results in abnormal helix • This technique involves repeating different heating formation and cooling cycles over and over again, as many as • Arise from autosomal dominant mutations 35 or more times. • Each complete cycle doubles the amount of DNA Clinical presentations present, so in just 20 cycles there are over one • Blue sclerae due to the translucent connective million (220) copies of that specific segment of tissue over choroidal veins DNA. • Triangular facies • Macrocephaly PCR components: • Hearing loss (abnormal ossicles) • Defective dentition (due to lack of dentin) • Barrel chest • Scoliosis • Limb deformities • Fractures • Joint laxity • Growth retardation • For PCR there are five chemical components needed, including: • Elastin is mainly produced in the fetus. It is no • DNA template: Contain the fragments needed to longer produced after puberty. be amplified • Elastin is an insoluble protein polymer MgCl2: Essential cofactor for Taq DNA • • Synthesized from a precursor, tropoelastin polymerase • Rich in non-hydroxylated proline, glycine, and • Taq DNA polymerase: Enzyme that helps catalyze lysine residues the polymerization of the deoxynucleotides into a • α1-antitrypsin plays an important role in elastin DNA strand degradation Primers: A primer is a strand of nucleic acid that • • It inhibits the activity of trypsin synthesized by the serves as a starting point for DNA replication. Two pancreas 16 primers are needed to amplify the target DNA fragment • DNTPs Nucleotides: the building blocks from which the DNA polymerase synthesizes a new DNA strand • Reaction buffer: providing a suitable chemical environment for optimum activity and stability of the DNA polymerase The reaction is commonly carried out in a volume of 10–200 μL in small reaction tubes (0.2–0.5 mL volumes) in a thermal cycler. Detailed steps of PCR 1. Add the sample containing DNA to be amplified. 2. Add excess amounts of primers complementary to both 3′ flanks of the target sequence. This selects the region to be amplified. 3. Add a heat-stable DNA polymerase (Taq DNA polymerase) and deoxyribonucleotides (dNTPs) for DNA synthesis. 4. Heat the sample to melt the DNA (convert dsDNA to ssDNA). 5. Cool the sample to re-anneal the DNA. Because the ratio of primers to complementary strands is extremely high, primers bind at the 3′ flanking regions 6. Heat the sample to increase the activity of the Taq DNA polymerase. 7. Primer elongation occurs, and new complementary strands are synthesized Elongation • Elongation Taq polymerase binds to the template DNA and starts adding nucleotides that are complementary to the first strand. • This happens at 72°C as it is the optimum temperature for Taq Polymerase. PCR applications: • Comparing DNA samples in forensic cases • Paternity testing • Direct mutation testing • Diagnosing bacterial and viral infections • HIV testing in situations where antibody tests are uninformative (importantly, infants whose mothers are HIV positive) Crisper Cas9 Principle: • Genome editing is a group of technologies that give scientists the ability to change an organism's DNA. • Different genome editing techniques include: • ZFNs (Zinc Finger Nucleases) • TALENs (Transcription Activator Like Effector Nucleases) • CRISPR • Crisper Cas9 Is an RNA- guided DNA endonuclease enzyme associated with the CRISPR. • It catalyzes site-specific cleavage of double stranded DNA. • CRISPR are sections of genetic code containing short repetitions of base sequences followed by spacer DNA segments. • It was identified in a prokaryotic defense system. • It is associated with the CRISPR adaptive immunity system in Streptococcus pyogenes, among other bacteria. • Considered as a tool for genome engineering. By replacing the endogenous RNA with the sequence Major PCR steps of a target gene the endonuclease component (Cas) Initiation can be induced to cleave almost any DNA sequence. • In this step the samples are heated to 94-96 C with the main purpose to activate taq DNA polymerase Mechanism of CRISPR/Cas9-mediated gene disruption. Denaturation • DNA strands are separated by heating to 95°C. • The Hydrogen bonds between the two strands breaks down and the two strands separates. Annealing • The process of allowing two sequences of DNA to form hydrogen bonds. • The annealing of the target sequences and primers is done by cooling the DNA to 55°C. • Time taken to anneal is 45 seconds. 17 1. 2. 3. 4. • A single guide RNA (sgRNA), consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein Next, sgRNA will bind to a recombinant form of Cas9 protein that has DNA endonuclease activity. The resulting complex will cause target-specific double-stranded DNA cleavage. The cleavage site will be repaired by the nonhomologous end joining (NHEJ) DNA repair pathway, an error-prone process that may result in insertions/deletions (INDELs) that may disrupt gene function. • • This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labelled probe hybridization. Basically, the DNA fragments are separated on the basis of size and charge during electrophoresis. Separated DNA fragments after transferring on nylon membrane, the desired DNA is detected using specific DNA probe that is complementary to the desired DNA. A hybridization probe is a short (100-500bp), single stranded DNA. The probes are labeled with a marker so that they can be detected after hybridization. Applications of CRISPR/Cas9 Blotting procedures Blotting: • Blotting is a technique used to separate DNA, RNA and proteins. Blotting is generally done by letting a mixture of • DNA, RNA or protein flow through a slab of gel. • This gel allows small molecules to move faster than bigger ones. Southern blot: • Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. Northern blot • Northern blotting is used to detect RNA. • Cells can be broken open to release their RNA. • Northern blotting may reveal how a disease is working at the level of RNA production. • Probe remains DNA Western blot • Western blotting is a common technique for separating proteins by size using electrophoresis • After separation, proteins are transferred to an appropriate blotting matrix such as PVDF membrane • Western blotting uses specific antibodies to identify proteins that have been separated based on size • Visualization is done using secondary antibodies and detection reagents. • Western blot is a confirmatory test in a presumed HIV infection Western blot procedure 18 • Up to thousands of particles per second can be analyzed as they pass through the liquid stream. Composition of Flow Cytometry: • Flow cell • Measuring system • Detector • Amplification system • Computer for analysis of the signals Southwestern blot • Used to investigate DNA-Protein interaction • Used for proteins that regulate transcription • It provides information regarding the molecular weight of unknown protein factor. • This method combines the features of Southern and Western blotting techniques, Separation of proteins is done first by electrophoresis, followed by transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence Basic principle of flow cytometry • When a cell suspension is run through the cytometer • Light scattered from the cells or particles is detected as they go through the laser beam. • A detector in front of the light beam measures forward scatter (FS) and several detectors to the side measure side scatter (SS). • Fluorescence detectors measure the fluorescence emitted from positively stained cells or particles. Flow Cytometry Overview Different types of light used in a flow cytometry • Flow cytometry measures various properties of experiment. single particles flowing in a single file in a stream of fluid Forward scattered light Side scattered light • Light scattering at different angles can distinguish differences in size and internal complexity, • Detected by a • Detected by a whereas light emitted from fluorescently labeled sensor in the light sensor that is antibodies can identify a wide array of cell surface path orthogonal to the and cytoplasmic antigens original light • 19 Used to detect the size of the object in the light path. • path. used to make a determination • Larger objects will produce more forward scattered light than smaller objects, and larger cells will have a stronger forward scatter signal • regarding the granularity and complexity of the cell in the light path. Highly granular cells with a large amount of internal complexity, like neutrophils, will produce more side-scattered light, and a higher sidescatter signal than cells with a low-granularity and complexity. • DNA microarrays is known as DNA chips, gene chips, DNA arrays, gene arrays and biochips Principle (Indirect assessment of mRNAs) • mRNA carries the genetic information from the cell nucleus to the cytoplasm for protein synthesis. • These mRNAs synthesize the corresponding protein by translation. • So, indirectly by assessing the various mRNAs, we can assess the genetic information or the gene expression. • This helps in the understanding of various processes behind every altered genetic expression. Thus, mRNA acts as a surrogate marker. Since mRNA is degraded easily, it is necessary to convert it into a more stable cDNA form. Labeling of cDNA is done by fluorochrome dyes Cy3 (green) and Cy5 (red). Applications of flow cytometry • Basic research: Cell counting and Cell sorting • Protein engineering: Identify cell surface protein variants • Medicine: used in transplantation, oncology, hematology and genetics • Marine biology • Diagnosis of health disorders such as blood cancers • Biomarker detection Microarray (Microchips) Outline: • Overview • Principle • Component of DNA microarray • Application of DNA microarray Overview: • DNA microarrays used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. • Used to compare differences in gene expression between two mRNA samples. For example, up or down-regulation of certain genes could highlight the implication of certain genes in diseases and might help identify possible therapeutic targets • • 20 The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology, the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization. • The property of complementary nucleic acid sequences is to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. 1. 2. 3. 4. 5. First, mRNA is extracted from the ‘normal’ sample, and a fluorescent labeled cDNA probe is produced, representing all of the genes expressed in the reference sample. A second cDNA probe is generated using a different-colored fluorescent label and mRNA extracted from the ‘affected’ cells (tumor cells or cells exposed to drugs). The two fluorescent probe samples are mixed and applied to a single microarray chip, where they react with the arrayed cDNA molecules. Each well of the microarray is scanned for the fluorescence intensity of each probe, the intensity of which is proportional to the expression level of that gene in the sample. The ratio of the two fluorescent intensities provides a highly accurate and quantitative measurement of the relative gene expression level in the two cell samples. • The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used for serological analysis • Used to measure antibodies, antigens, proteins and glycoproteins in biological samples. • Efficient technique allows measuring several samples at once using 96 well plate Principle • • Components of DNA microarray • DNA Chip • Target Sample (Fluorescently labelled) • Enzymes • • ELISA relies on antigen-antibody interaction, where an antigen must be immobilized on a solid surface and then complexes with an antibody that is linked to an enzyme Detection is observed through evaluation of conjugated enzyme activity upon substrate addition One of the key aspects of ELISA technique is the specificity of antigen-antibody interaction Each ELISA measures a specific antigen, and kits for a variety of antigens are widely available. Applications of ELISA • Diagnosis of HIV infection • Pregnancy tests • Serum antibody concentration • Detection of potential food allergens Cystic fibrosis • • • Fluorescent dyes Probes Scanner Applications of DNA microarray • Gene expression analysis • Diagnosis of certain diseases • Drug discovery Detection of IDs of organisms in food and mycoplasma in cell culture ELISA Overview Overview: • Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasians. • Autosomal recessive disorder • Cystic fibrosis transmembrane conductance regulator (CFTR) is highly expressed in epithelial cells of airways, GIT (pancreas) and sweat glands • CFTR regulate epithelial chloride channel, sodium, potassium and bicarbonate ions Pathophysiology • Cystic fibrosis occurs due to a mutation in CFTR gene which is located on chromosome 7 and most commonly has been damaged by a deletion of the amino acid phenylalanine at position 508 (ΔF508). 21 • • • • • • • • • • • • • • • • • • • Normal CFTR increases the reabsorption of chloride ion and enhance the reabsorption of sodium ions The alteration in chloride transport is associated with production of abnormally thick secretions in glandular tissues (hypertonic salty sweat) In Lung, GIT and Pancreas: Mutated CFTR decreases the secretion of Chloride ion and augments the reabsorption of Sodium ion (with passive Water reabsorption) that lead to hyper concentrated “dehydrated” viscid secretions In cystic fibrosis the apical Cl channels do not open. Failure of water transport results in thickening of the mucous layer covering the epithelia The lung bronchioles and pancreatic ducts are primarily affected, often resulting in progressive destruction of these organs Cystic fibrosis entails 2 mutations: G551D mutation (4-5 %), in which the amino acid glycine (G) in position 551 is replaced with aspartic acid (D). G551D is characterized by a dysfunctional CFTR protein on the cell surface. In the case of G551D, the protein is trafficked to the correct area, the epithelial cell surface, but once there the protein cannot transport chloride through the channel. F508del mutation, the most common CF mutation, is primarily considered to be a processing mutation. The F508del mutation removes a single amino acid from the CFTR protein. Bronchiectasis: is an abnormal permanent airway dilatation due to chronic necrotizing inflammation. Clinical findings include cough, fever, malodorous purulent sputum, and dyspnea. • • • • • • • • • • • • • • Clinical Presentations: • Signs and symptoms entail several organs include • In the lungs, CF may cause: • Recurrent pulmonary infections • chronic bronchitis • Bronchiectasis Chest Wheezing Chronic cough Hemoptysis, Digital clubbing Pansinusitis and nasal polyps Reticulonodular pattern on CXR Opacification of sinuses In the GIT, thick secretions may cause: intestinal obstruction (meconium ileus) (10-15% of newborns with CF) Meconium peritonitis Rectal prolapse In the pancreas, CF may cause Plugging of pancreatic ducts resulting in atrophy and fibrosis Pancreatic insufficiency leading to fat malabsorption Malodorous steatorrhea (frequent, bulky, greasy, large, foul-smelling stools) Deficiency of fat-soluble vitamins (A, D, E and K) In the liver, plugging of the biliary canaliculi may result in biliary cirrhosis, portal hypertension In the male reproductive system, CF may be associated with absence or obstruction of the vas deferens and epididymis, which often leads to male infertility CF is also associated with secondary amenorrhea, dry skin, salty taste to skin and failure to thrive The 3 most common pulmonary infections are S. Aureus (infancy), H. influenzae, and P. aeruginosa (adolescence) Diagnosis • Sweet chloride test (elevated NaCl) • Due to improved therapies, some patients live into their forties, but with this increase in longevity there has been an increase in liver disease. • Prenatal diagnosis: 1. fetus with hyperechogenic fetal bowel 2. Increase immunoreactivity trypsinogen • Radiography: chest x-ray reveals bronchiectasis and pulmonary arterial enlargement Treatment: • Ivacaftor is a "potentiator" of CFTR, meaning it increases the probability that the defective channel will be open and allow chloride ions pass through the channel pore (less thick mucus) 22 • • • Chest physiotherapy Hypertonic saline to enhance mucus clearance Bronchodilators: albuterol, aerosolized dornase alfa (DNase) • Azithromycin • Ibuprofen • N-acetyl cysteine • Pancreatic enzymes • High protein diet • Combination therapy is recommended in case of patients with Phe508 deletion (lumacaftor+ ivacaftor) • N.B Cystic fibrosis (heterozygote resistance to typhoid fever) Modes of inheritance Mitochondrial Inheritance: • • • • • • • • Because affected individuals must receive a disease-causing gene from an affected parent, the disease is typically observed in multiple generations of a pedigree The recurrence risk is thus 50%, and half the children, on average, will be affected with the disease. If both parents are heterozygous, the recurrence risk is 75%. Autosomal dominant disorder: • Familial hypercholesterolemia (LDL receptor deficiency) • Huntington disease • Neurofibromatosis type 1 • Marfan syndrome • Acute intermittent porphyria Mitochondrial DNA is inherited exclusively through females. Diseases are transmitted only from affected females to their offspring Both males and females are affected. All offspring of an affected female are affected. None of the offspring of an affected male is affected. Autosomal Recessive Inheritance Clinical presentations • Leber hereditary optic neuropathy • MELAS: mitochondrial encephalomyopathy • Lactic acidosis • Stroke-like episodes • Myoclonic epilepsy with ragged red muscle fibers • Autosomal Dominant Inheritance • Due to a defect in a single copy of autosomal gene • Because these genes are located on autosomal gene, both males and females are affected 23 • • Occur only when two copies of the mutated gene are inherited. If one gene is defective whereas the other inherited gene is normal, the individual is simply a carrier of the gene, and does not suffer from the disease. Autosomal recessive diseases are typically seen in only one generation of a pedigree • • • Males and females are affected in roughly equal frequencies. With Heterozygous parents, 25 % of children will be affected (homozygous), 50% will be carrier and 25% will be neither affected nor carrier Autosomal Recessive disorders • Sickle cell anemia • Cystic fibrosis • Phenylketonuria (PKU) • Tay-Sachs disease (hexosaminidase A deficiency) X linked dominant • Due to mutations in genes located on the X chromosome • An X-linked dominant disorder arises when a single copy of the gene leads to expression of the abnormal protein. • Most common in females • A characteristic of X-linked disorders is that an affected father does not pass on the