Cystic fibrosis (CF) -inherited autosomal recessive disorder incidence of 1 in 3 000 live births carrier frequency of 1 in 25 CF affects roughly 70 000 worldwide Hallmarks of CF Very salty-tasting skin Appetite, but poor growth & weight gain Coughing, wheezing & shortness of breath Lung infections, e.g. pneumonia/bronchitis Clinical Aspects Cystic fibrosis affects the entire body • Lungs and sinuses • Gl, liver and pancreas • Endocrine system • Reproductive system Organs Affected by Cystic Fibrosis AIRWEAYS: Clogging and infection of bronchial passages impede breathing. The infection progressively destroy the lungs. LIVER: Plugging of small bile ducts impedes digestion and discrupts liver function in perhaps 5% of patients PANCREAS: delusion of ducts prevents the pancreas from delivering critical digestive enzymes to the bowel in 65% of patients. Diabetes can result as well. SMALL INTESTINE: Obstruction of the gut by thick stool necessitates surgerry in about 10% of newborns REPRODUCTIVE TRACT: Absence of fine ducts, such as the vas deferans, renders 95% of males infertile. Occasionally, women are made infertile by a dense plug of mucus that blocks sperm from entering the uterus. SKIN: Malfunctioning of sweat glands causes perspiration to contain excessive salt (NaCI) The Sweat Test • Measures the concentration of chloride and sodium that is excreted in sweat. • Two reliable positive results on two separate days is diagnostic for CF. • Clinical presentation, family history and patient age must be considered to interpret the results. CFTR gene (cystic fibrosis transmembrane conductance regulator) Location: 7q31.2 Over 1,000 mutations in CFTR have been found AF508 accounts for just 70% of CF cases Panel 1: Frequencies of CFTR mutations* CFTR Allele CFTR Allele mutation frequency (%) mutation frequency AF508 69-4% 2789+5G^A 0-3% Unknown 15-7% R1162X 0-3% G542X 2-3% G85E 0-3% G551D 2-2% R560T 0-2% AI507 1-6% R334W 0-2% W1282X 1-4% 3659AC 0-2% N1303K 1-2% A455E 0-1% R553X 0-9% 711+1G-»T 0-1% 621+lG^T 0-8% 1898+lG^A 0-1% R117H 0-7% 2184AA 0-1% 3849+10 kbC^T 0-7% S549N 0-1% 1717-IG-»A 0-5% 1078AT 0-03% R347P 0-3% *n=17 853. The AF508 Mutation A 3 base pair deletion called AF508 is the most common mutation causing cystic fibrosis The mutation results in the deletion of a single amino acid (Phe) at position 508. In Normal CFTR: Nucleotide AAT ATC ATC TTT GGT GTT TCC Amino Acid Asn lie He Phe Gly Val Ser I I I SOS BOB 511 In AF508 CFTR: Nucleotide Amino Acid AAT ATC ATC GGT GTT TCC Asn lie lie Gly Val Ser SOS Benefits of AF508 The AF508 mutation most likely occurred over 50,000 years ago in Northern Europe. Individuals with two copies of AF508 get cystic fibrosis and often cannot reproduce. Having one copy of AF508 reduces water loss during cholera, greatly increasing the chance of survival. The Function of CFTR CFTR encodes a 170 kDa, membrane-based protein with an active transport function CFTR Clchannel 12 a-helix TM domains NBF N-terminus L7 out membrane R-domain NBF2^L C-terminus NBF = nucleotide binding fold R = regulatory domain coo ^ Carbohydrate Pore Nucleotide binding domain 0 0 * phenylalanine NuCieotide V Chloride x c Phosphate Cytoplasm deletion binding domain Regulatory domain From Mutation to Disease The mutant form of CFTR Mucus clogs the airways prevents chloride transport, and disrupts the function of causing mucus build-up the pancreas & intestines. 5 Classes of CFTR Mutations CF Mutations can be classified by the effect they have on the CFTR protein. Panel 2: Functional classification of CFTR alleles Class Functional effect of Allele mutation 1 Defective protein G542X, R553X, W1282X, production R1162X, 621-1G->T, 1717-1G-»A, 1078AT, 3659AC II Defective protein AF508, AI507, N1303K, processing S549N III Defective protein G551D, R560T regulation IV Defective protein R117H, R334W, G85E, conductance R347P V Reduced amounts of 3849+10KbC->T, functioning CFTR protein 2789+5G^A, A455E Unknown 711+1G->T, 2184DA, 1898+lG^A 5 Classes of CFTR Mutations I II III IV v Defective Defective Defective Defective Reduced Production Processing Regulation Conductance Amounts Probability of producing a child with CF IF: Both parents have CF THEN: 100% chance child will have CF. IF: One parent has CF, the other is *not* a carrier THEN: 0% chance child will have CF (barring the very unlikely event of spontaneous mutation); 100% chance child will be a carrier. IF: One parent has CF, the other is a carrier THEN: 50% chance that child will have CF; 50% chance that child will be a carrier. IF: Both parents are carriers THEN: 25% chance that child will have CF; 50% chance that child will be a carrier; 25% chance that child will not have CF or be a carrier. Sickle Cell Anemia autosomal recessive inheritance Sickle Cell Anemia Normal «II Spherical ceil Sichle cell mutation in the Hemoglobin Beta Gene which can be found in the chromosome 11 abnormally shapes red blood cells. substitution of the second nucleotide base of codon 6, adenin (A) to thymine (T) changes the codon GAG for glutamic acid to the codon GTG for valine Mutation in codon 6 of ß qlobin GAG ► GTG (Glu) (Val) Hemoglobin A Hemoglobin S Normal solubility Less soluble, cristalizes 1 Erythrocytes I Normal Sickle cell — Sickle cells Unlike normal erythrocytes, sickle cells are unable to pass through small arteries and capillaries. These become clogged and cause local oxygen deficiency in the tisues, followed by infection. Defective erythrocytes are destroyed (hemolysis). The result is chronic anemia and its numerous sequelae such as heart failure, liver damage and infection Sickle cefl Learning deficit t Frequently ill t Infections t Oxygen deficit t Small arteries and capillaries plugged Brain affected t Heart failure t Liver damage A Anemia t Hemolysis Hemophilia A s-> X linked recessive hereditary disorder incidence about 1 in 5 000 males Hemophilia A • 2 types of hemophilia: A and B • Hemophilia A: X linked recessive hereditary disorder • Hemophilia A results from the deficiency of blood coagulation factor VIII, which function as a cofactor in the activation of factor X to factor Xa during the intermediate phase of the coagulation cascade Genetics Dj9 "■ l±l SrO O thrC 1 2 3 4 5 | e III 1 2 3 4 5 6 7 B Transmitted by females, suffered by males The female carrier transmits the disorder to half their sons and the carrier state to half her dtrs The affected male does not transmit the disease to his sons but all his dtrs are all carriers (transmission of defected X) Genetics Factor VIII gene - Xq28, one of the largest genes -186kb, 26 exons. Its large size predisposes it to mutations In Hemophilia A there is no uniform abnormality. There are deletions, insertions, and mutations Aprox 40% of severe hemophilia A is caused by a major inversion in the gene- the breakpoint is situated within intron 22 Intrinsic Pathway surface HK PK Xli \XIIa IX La IXa Extrinsic Pathway \ VII Um-^VIHa-9-fpL X \a'^ VIIa+TP r""\ t vascular injury V-fVa- PL Prothrombin ^Thrombin ,,XIII Fibrinogen ^Fibrinogen monomer r IV Prince Albert von Sachsen-Coburg-Gotha o Alice Queen Victoria Ludwig v. Hessen ODO Heinrich von Preussen Irene Alexandra Frederick / / / D Leopold Duke of Albany a Nikolaus II Mi ö Alice Beatrice 6ÍÍ im / # / A. X-Chromosomal inheritance of hemophilia A ó? ^ & ď # # # ď Prince Heinrich von Battenberg Duchenne Muscular Dystrophy /-> X - recesive Occuring in 1 in 3000 males Duchenne Muscular Dystrophy Occuring in 1 in 3000 males X - recesive Duchenne Muscular Dystrophy Females carry the DMD gene on the X chromosome. • Females are carriers and have a 50% chance of transmitting the disease in each pregnancy. • Sons who inherit the mutation will have the disease. • Daughters that inherit the mutation will be carriers. The DMD gene is located on the Xp 21 band of the X chromosome glrUunaffected) XX 25% boy (unaffected) X Y 1 25% girl (carrier) boy (with defect) » t *Abam • Dystrofin gene: locus Xp21 • 2,4 MB (1% of X chromosome) • 79 exons • The most frequent mutation: -Deletion of land more exons (65%) -Frameshift mutations - 1/3 patients has de novo mutation Clinical Features - Phenotype of DMD • Delays in early childhood stages involving muscle use • Learning difficulties in 5% of patients. • Speech problems in 3% of patients. • Leg and calf pain. • IQ's usually below 75 points. • Increase in bone fractures due to the decrease in bone density. • Wheelchair bound by 12 years of age. • Cardiomyopathy at 14 to 18 years. • Few patients live beyond 30 years of age. • Reparatory problems and cardiomyopathy leading to congestive heart failure are the usual cause of death. r DMD Gene and Dystrofin - Function • The DMD gene encodes for the protein dystrofin, found in muscle cells and some neurons. • Dystrofhin provides strength to muscle cells by linking the internal cytoskeleton to the surface membrane. • Without this structural support, the cell membrane becomes permeable. As components from outside the cell are allowed to enter the internal pressure of the cell increases until the cell bursts and dies. Allelic Variants Disease Mutation Effect of Mutation Phenotype Duchenne Muscular Dystrophy Very Large Deletions caused by: Stop mutations Splicing mutations Deletions Duplications Severely Functionally Impaired Dystrophin Protein As Discussed In Prior Slides Becker Muscular Dystrophy Deletion or Duplication That Change In-Frame Exons Creates A Protein That Is Partially Functional Same As But Less Sever Then DMD But Onset At Greater Then 7 Years Old DMD Related Dilated Cardiomyopathy Effects The Cardiac Muscle Promoter and The First Exon No Dystrophin Transcriptions Being Carried Out In Cardiac Muscle Tachycardia (Fat Heart Beat) Leads To Congestive Hear Failure Limb-Girdle Muscular Dystrophy In Gene That Encodes Scarcoglycans and Other Proteins of Muscle Cells Decrease In Scarcoglycans Proteins Pelvic and Shoulder Girdle Can Look Like DMD or BMD r TR mutation trinucleotide repeat TREs - trinucleotide repaet expansion TRED trinucleotide repeat expansion diseases expansion New type of mutation, described 1991 r Trinucleotide repeat disorders • caused by an unusual form of mutation called trinucleotide repeat expansion (TNRE) • The term refers to the phenomenon that a sequence of 3 nucleotides can increase from one generation to the next • These diseases include • Huntington disease (HD) • Fragile X syndrome (FRAXA) • Certain regions of the chromosome contain trinucleotide sequences repeated in tandem • In normal individuals, these sequences are transmitted from parent to offspring without mutation • However, in persons with TRNE disorders, the length of a trinucleotide repeat increases above a certain critical size • It also becomes prone to frequent expansion • This phenomenon is shown here with the trinucleotide repeat CAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 77= 11 G • In some cases, the expansion is within the coding sequence of the gene • Typically the trinucleotide expansion is CAG (glutamine) • Therefore, the encoded protein will contain long tracks of glutamine • This causes the proteins to aggregate with each other • This aggregation is correlated with the progression of the disease • In other cases, the expansions are located in noncoding regions of genes • These expansions are hypothesized to cause abnormal changes in RNA structure • Thereby producing disease symptoms Triplet Repeat Disorders Copyright © The McGraw-Hill Companies. Inc. Permission required for reproduction or display. Table 12.7 Triple! Repeat Disorders mRNA Normal Disease Signs and Disorder OMIM Repeat Number of Copies Number of Copies Symptoms (Phenotype) Fragile X syndrome 309550 CGG or CCG 6-50 200-2,000 Mental retardation, large testicles, long face Friedreich alaxi.i 229300 GAA 6-29 200-900 Loss of coordination and certain reflexes. spine curvature, knee and ankle jerks Haw River syndrome 140340 CAG 7-25 49-75 Loss of coordination, uncontrollable movements, dementia Huntington disease 143100 CAG 10-34 40-121 Personality changes, uncontrollable movements, dementia lacobsen syndrome 147791 CGG 11 100-1,000 Poor growth, abnormal face, slow movement Myotonic dystrophy 160900 CTG 5-37 80-1,000 Progressive muscle weakness; heart. type I brain, and hormone abnormalities Myotonic dystrophy 602668 CCTG 100 Progressive muscle weakness; heart, type II brain, and hormone abnormalities Spinal and bulbar 313200 CAG 14-32 40-55 Muscle weakness and wasting in muscular atrophy adulthood Spinocerebellar ataxia 271245 CAG 4-44 40-130 Loss of coordination (5 types)