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: Oclusion 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) 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+lG^T 0-1% 621+1G-VT 0-8% 1898+lG^A 0-1% R117H 0-7% 2184AA 0-1% 3849+10 kbC- 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 Me lie Phe Gly Val Ser I I I SOS S0& 511 In AF508 CFTR; Nucleotide Amino Acid AAT ATC ATC GGT GTT TCC Asn lie Me Gly Val Ser 60S 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-helixTM domains A C7 NBF1 N-terminus out u membrane NBF2 R-domain C-terminus NBF = nucleotide binding fold R = regulatory domain c - c c ^ ~~~~~ Carbohydrate 1» Nucleotide binding domain mm c c c o o c Chloride x c c c Phosphate"^ Cytoplasm Site of common phenylalanine NucleoMe deletion bjndjng 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-M. 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 Sickle Cell Anemia autosomal recessive inheritance Sickle Cell Anemia • mutation in the Hemoglobin Beta Gene which can be found in the chromosome 11 -11p15.5 • haemoglobin A is replaced with what's known as haemoglobin S • 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 globin 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 /—►Deaths- Brain affected t Heart failure t Liver damage A Anemia t Hemolysis Signs and symptoms of sickle cell anemia • Anemia - sickle cells die in 10 to 20 days, leaving a shortage of red blood cells • Episodes of pain- develops when sickle-shaped red blood cells block blood flow through tiny blood vessels, this can lead to bone and joint damage • Painful swelling of hands and feet • Frequent infections • Delayed growth • Vision problems Hemophilia A *-> 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 • Bruising and bleeding • Minor bleeds: early joint and muscle bleeds, bleedig in the mouth and gums, hematuria • Major bleeds: central nervous system, severe injury, neck/throat, eye, gastrointestinal, late joints and muscles,... • Chronic joint deformities from recurrent bleeding 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 Germinal mosaicism Intrinsic Pathway surface HK PK {* Nxila -*i XI Extrinsic Pathway XI XIa VIII-t* Villa- HK IX U IXa |Ca2+ " PL \ VII VIIa+TP^ Xa' \ i vascular in ju^y V i-Va Ca3+ PL Prothrombin ^Thrombin —7^ Fibrinogen ^Fibrinogen monomer XIII r Aprox 40% of severe hemophilia A is caused by a major inversion in the gene - the breakpoint is situated within intron 22 ^etomert int22h3 5' Iffl22h2 ~ -ui I : telomere I-//—I Hill I ■ 111 I 111! 22 23 centromeie 26 -*■ int22hl | Intra-chromosomal recombination mill i ■ iiiii11 in ■—//—# I Inversion of exons 1-22 22 centi omere -*■ null ■ I Mill iiiiizw/—i m m- int22h3 <-Z-— lnt22h2A y A „Royal Disease" □ □ LEOPOLD UHUmCE MFOH90 «ONZM0 O NORMAL 0 CARPJER 0 P08SBLE CARWER Q NORMAL ] HEMOPHILIAC [7] POSSIBLE HEMOPHILIAC ©©© SPANISH ROYAL FAMLY 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 X X" girl (unaffected) girl (carrier) 3 x*1 W 25% boy (unaffected) J boy (with defect) y 5y 25* #Adam r • Dystrofin gene: locus Xp21 • 2,4 MB (1% of X chromosome) • 79 exons • over 200 types of mutations • the most frequent mutation: -Deletion of 1 and 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, walk on toes • curvature of spine • IQ's usually below 75 points. • Increase in bone fractures due to the decrease in bone density • Chest muscles weaken, making • breathing difficult • 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. r Molecular genetic testing of DMD gene • 1. Detection of exon deletions in hot spot regions of DMD gene (multiplex PCR) • 2. MLPA • 3. Sequencing of coding regions of DMD gene • 4. Indirect DNA diagnostics MLPA (Multiplex Ligation-dependent Probe Amplification) • multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences • it only requires a thermocycler and capillary electrophoresis equipment • low cost and technically uncomplicated method • over 300 probe sets • five major steps: • - DNA denaturation and hybridisation of ML PA probes • - ligation reaction • - PCR reaction • - separation of amplification products by electrophoresis • - data analysis 1. Denaturation and Hybridization PCR primer sequence X PCR primer sequence Y Hybridization sequence (left) Hybridization sequence (right) 2. Ligation 3. PCR with universal primers X and Y exponential amplification of hgated probes only 4. Fragment analysis i ft 1 . . Homozygj) LAJU u us dclct 5 J on ASPA cxon 1-6 3 2 6 — »0 Specificity of MLPA probes is very high. MRC-Holland b.v, -MLPA- 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 DMDorBMD Incontinentia pigmenti gonosomal dominant inheritance • affects the skin, hair, teeth, nails, and central nervous system • also known as Bloch-Siemens syndrome, Bloch-Sulzberger syndrome and nevus pigmentosus systematicus • skin abnormalities that begin in childhood. Other symptoms include hair loss, dental abnormalities, eye abnormalities lined or pitted fingernails and toenails. Associated problems can include delayed development, intellectual disability, seizures, and other neurological problems • IP is lethal in most, but not all, males • mutations in a IKK-gamma gene (IKBKG) also called NEMO (NF-kB essential modulator) 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 n= 11 6 • 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. Uriplci Rcpriil Disorders Table 12.7 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 ataxia 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 143IOO CAG 10-34 40-121 Personality changes, uncontrollable movements, dementia Jacobsen 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 1 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) Fragile X Syndrome • occurs in about 1 in 4,000 males and 1 in 8,000 females • expansion of the CGG repeat sequence in the FMR1 gene on chromosome Xq27 • FMR1 gene codes for the FMR protein (FMRP), which is expressed in many cell types, especially in neurons. Fragile X syndrome FMR1 gene Xq27 X X Female carrier FMRl gene Xq27 X Y Affected male http://www.gen etics4medics.com/fragile-K-svn drome, htm I • Expansion size: • Normal: 5-44 repeats • Intermediate: 45-54 repeats. May expand to a premutation allele during intergenerational transmission. • Premutation: 55-200 repeats. May expand to a full mutation allele, especially during maternal transmission. • Full mutation: >200 repeats • Premutation alleles in a female may (not always!) expand to full mutation in the subsequent generation. r Males with Full mutation • Development: developmental delay - mainly speech. • Behaviour: Hyperactivity, autistic features, temper tantrums in childhood. Usually shy as adults. • Intellectual difficulties: moderate to severe • Physical features: Prominent forehead, long face, prominent jaw, large ears, joint hypermobility. Large testes (post-pubertal feature) • Premutation carriers Fragile X associated tremor/ataxia syndrome (FXTAS): Occurs in a proportion of premutation carriers (both males & females). Characterised by late onset cerebellar signs (ataxia & intentional tremor) and other neurological features including dementia and cognitive decline. Primary ovarian insufficiency: Occurs in about 20% of females who are premutation carriers. Characterised by menopause before the age of 40 years. r Huntington disease o named for George Huntinton, described this disease in 1872 o begin between 30 and 50 years of age, but can start at any o About 8% of cases start before the age of 20 years Autosomal dominant mutation in either of an individual's two o expansion of CAG triplet repeats in the gene coding for the Huntingtin protein results in an abnormal protein, which gradually damages cells in the brain o 4 to 15 in 100,000 people of European descent, affects men and women equally age Classification of the trinucleotide repeat, and resulting disease status, depends on the number of CAG repeats Repeat count Classification Disease status Risk to offspring <26 Normal Will not be affected None 27-35 Intermediate Will not be affected Elevated but «50% 36-39 Reduced Penetrance May or may not be affected 50% 40+ Full Penetrance Will be affected 50% Symptoms • Cognitive: amnesia, delusion, lack of concentration, memory loss, mental confusion, slowness in activity and thought, or difficulty thinking and understanding • Muscular: abnormality walking, increased muscle activity, involuntary movements, problems with coordination, loss of muscle, or muscle spasms • Behavioral: compulsive behavior, fidgeting, irritability, or lack of restraint • Psychological: delirium, depression, hallucination, or paranoia • Mood: anxiety, apathy, or mood swings • Also common: tremor, weight loss, or impaired voice Myotonie dystrophy • Affects 1 in 8000 people • Autosomal Dominant Disorder with anticipation • Myotonia: hyperexcitability of muscle membrane -» inability of quick muscle relaxation • Progressive muscular weakness and wasting, most prominent in cranial and distal muscles • Cataracts, frontal balding, testicular atrophy • Cardiac abnormalities, mental retardation • 1). "Mild DM" (adult onset): People often lead active lives and may even be unaware that they have the disorder. • 2). "Classical DM" (adult onset): People commonly have muscle weakness and wasting, myotonia, hand and wrist weakness and/or foot drop. • 3). "Congenital Myotonic Dystrophy" (CMD): A very severe form of DM1, often fatal in young children Myotonic dystrophy 1 and 2 • In DM1, the affected gene is called DMPK (myotonic dystrophy protein kinase) which codes for a myosin kinase expressed in skeletal muscle. The gene is located on the long arm of chromosome 19 • DM2 is similarly caused by a defect of the ZNF9 gene on chromosome 21. DMPK gene • gene DMPK (myotonic dystrophy protein kinase gene) -19q13.3 • CTG repeat expansion in a gene on chr. 19 in 3' UTR (3' untranslated region) • This condition occurs when the CTG segment is abnormally repeated from 50 to 5,000 times. The mutated DMPK gene produces an altered version of messenger RNAthat interacts with certain proteins to form clumps within the cell. The abnormal clumps interfere with the production of many other proteins. • People with the classic features of type 1 myotonic dystrophy, including muscle weakness and wasting beginning in adulthood, usually have 100 to 1,000 CTG repeats. • People born with the more severe congenital form of type 1 myotonic dystrophy tend to have a larger number of CTG repeats, often more than 2,000. CNBP gene • "CCHC-type zinc finger, nucleic acid binding protein." • CNBP gene provides instructions for making a protein called CCHC-type zinc finger, nucleic acid binding protein • tetranucleotide repeat CCTG • This condition occurs when the CCTG segment is abnormally repeated 75 to more than 11,000 times.