GENETIC DISEASE

1. M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar Classes of Biomolecules Affected in Disease

2. Classes of Biomolecules Affected in Disease • All classes of biomolecules found in cells are affected in structure, function, or amount in one or another disease • Can be affected in a primary manner (e.g., defect in DNA) or secondary manner (e.g., structures, functions, or amounts of other biomolecules)

3. Rate of Biochemical Alterations • Biochemical alterations that cause disease may occur rapidly or slowly • Cyanide (inhibits cytochrome oxidase) kills within a few minutes • Massive loss of water and electrolytes (e.g., cholera) can threaten life within hours • May take years for buildup of biomolecule to affect organ function (e.g., mild cases of Niemann-Pick disease may slowly accumulate sphingomyelin in liver and spleen)

4. Deficiency or Excess of Biomolecules • Diseases can be caused by deficiency or excess of certain biomolecules • deficiency of vitamin D results in rickets, excess results in potentially serious hypercalcemia • Nutritional deficiencies • primary cause - poor diet • secondary causes - inadequate absorption, increased requirement, inadequate utilization, increased excretion

5. Organelle Involvement • Almost every cell organelle has been involved in the genesis of various diseases

6. Different Mechanisms, Similar Effect • Different biochemical mechanisms can produce similar pathologic, clinical, and laboratory findings • The major pathological processes can be produced by a number of different stimuli • e.g., fibrosis of the liver (cirrhosis) can result from chronic intake of EtOH, excess of copper (Wilson’s disease), excess of iron (primary hemochromatosis), deficiency of a1-antitrypsin, etc. • different biochemical lesions producing similar end point when local concentration of a compound exceeds its solubility point (excessive formation or decreased removal)  precipitation to form a calculus • e.g., calcium oxalate, magnesium ammonium phosphate, uric acid, and cystine may all form renal stone, but accumulate for different biochemical reasons

7. Genetic Diseases • Many disease are determined genetically • Three major classes: (1) chromosomal disorders, (2) monogenicdisorders (classic Mendelian), and (3) multifactorial disorders (product of multiple genetic and environmental factors)

8. Genetic Diseases • Polygenic denotes disorder caused by multiple genetic factors independently of environmental influences • Somatic disorders - mutations occur in somatic cells (as in many types of cancer) • Mitochondrial disorders - due to mutations in mitochondrial genome

9. Chromosomal Disorders • Excess or loss of chromosomes, deletion of part of a chromosome, or translocation • e.g., Trisomy 21 (Down syndrome) • Recognized by analysis of karyotype (chromosomal pattern) of individual (if alterations are large enough to be visualized) • Translocations important in activating oncogenes • e.g., Philadelphia chromosome - bcr/abl)

10. Monogenic Disorders • Involve single mutant genes • Classification: (1) autosomal dominant - clinically evident if one chromosome affected (heterozygote) • e.g., Familial hypercholesterolemia (2) autosomal recessive - both chromosomes must be affected (homozygous) • e.g., Sickle cell anemia (3) X-linked - mutation present on X chromosome • females may be either heterozygous or homozygous for affected gene • males affected if they inherit mutant gene • e.g., Duchenne muscular dystrophy

11. Multifactorial Disorders • Interplay of number of genes and environmental factors • pattern of inheritance does not conform to classic Mendelian genetic principles • due to complex genetics, harder to identify affected genes; thus, less is known about this category of disease • e.g., Essential hypertension

12. Inborn Error of Metabolism • A mutation in a structural gene may affect the structure of the encoded protein • If an enzyme is affected, an inborn error of metabolism may result • A genetic disorder in which a specific enzyme is affected, producing a metabolic block, that may have pathological consequences

13. Increased X,Y E  *E S  P Increased S  Decreased P Normal Block Inborn Error of Metabolism • A block can have three results: (1) decreased formation of the product (P) (2) accumulation of the substrate S behind the block (3) increased formation of metabolites (X, Y) of the substrate S, resulting from its accumulation • Any one of these three results may have pathological effects

14. Inborn Error of Metabolism Increased phenylpyruvic acid • Phenylketonuria - mutant enzyme is usually phenylalanine hydroxylase • synthesize less tyrosine (often fair skinned), have  plasma levels of Phe, excrete phenylpyruvate and metabolites • If structural gene for noncatalytic protein affected by mutation can have serious pathologic consequences (e.g.,hemoglobin S)  *E Increased phenylalanine  Decreased tyrosine Block

15. Genetic Linkage Studies • The more distant two genes are from each other on the same chromosome, the greater the chance of recombination occurring between them • To identify disease-causing genes, perform linkage analysis using RFLP or other marker to study inheritance of the disease (marker)

16. Genetic Linkage Studies • Simple sequence repeats (SSRs), or microsatellites, small tandem repeat units of 2-6 bp are more informative polymorphisms than RFLPs; thus currently used more

17. Methods to clone disease genes • Functional approach • gene identified on basis of biochemical defect • e.g., found that phenotypic defect in HbS was GluVal, evident that mutation in gene encoding b-globin • Candidate gene approach • genes whose function, if lost by mutation, could explain the nature of the disease • e.g., mutations in rhodopsin considered one of the causes of blindness due to retinitis pigmentosa

18. Methods to clone disease genes • Positional cloning • no functional information about gene product, isolated solely by it chromosomal position (information from linkage analysis • e.g., cloning CF gene based on two markers that segregated with affected individuals • Positional candidate approach • chromosomal subregion identified by linkage studies, subregion surveyed to see what candidate genes reside there • with human genome sequenced, becoming method of choice

19. Structure of CFTR gene and deduced protein Mutations in CFTR gene Identifying defect in disease gene • Once disease gene identified, still can be arduous task identifying actual genetic defect

20. Ethical Issues • Once genetic defect identified, no treatment options may be available • Will patients want to know? • Is prenatal screening appropriate? • Will identification of disease gene affect insurability? • e.g., Hungtington’s disease - mutation due to trinucleotide (CAG) repeat expansion (microsatellite instability) • normal individual (10 to 30 repeats) • affected individual (38 to 120) - increasing length of polyglutamine extension appears to correlate with  toxicity

21. Molecular Medicine • Knowledge of human genome will aid in the development of molecular diagnostics, gene therapy, and drug therapy

22. Gene expression in diagnosis • Diffuse large B-cell lymphoma (DLBCL), a disease that includes a clinically and morphologically varied group of tumors that affect the lymph system and blood. Most common subtype of non-Hodgkin’s lymphoma. • Performed gene-expression profiling with microarray containing 18,000 cDNA clones to monitor genes involved in normal and abnormal lymphocyte development • Able to separate DLBCL into two categories with marked differences in overall patient survival. • May provide differential therapeutic approaches to patients

23. Treatment for Genetic Diseases • Treatment strategies (1) correct metabolic consequences of disease by administration of missing product or limiting availability of substrate • e.g., dietary treatment of PKU (2) replace absent enzyme or protein or to increase its activity • e.g., replacement therapy for hemophilia (3) remove excess of stored compound • e.g., removal of iron by periodic bleeding in hemochromatosis (4) correct basic genetic abnormality • e.g., gene therapy

24. Gene Therapy • Only somatic gene therapy is permissible in humans at present • Three theoretical types of gene therapy • replacement - mutant gene removed and replace with a normal gene • correction - mutated area of affected gene would be corrected and remainder left unchanged • augmentation - introduction of foreign genetic material into cell to compensate for defective product of mutant gene (only gene therapy currently available)

25. Gene Therapy • Three major routes of delivery of genes into humans (1) retroviruses • foreign gene integrates at random sites on chromosomes, may interrupt (insertional mutagenesis) the expression of host cell genes • replication-deficient • recipient cells must beactively growing forintegration into genome • usually performed ex vivo

26. Gene Therapy (2) adenoviruses • replication-deficient • does not integrate into host cell genome • disadvantage: expression of transgene gradually declines requiring additional treatments (may develop immune response to vector) • treatment in vivo, vector can be introduced into upper respiratory tract in aerosolized form (3) plasmid-liposome complexes

27. Gene Therapy • Conclusions based on recent gene therapy trials • gene therapy is feasible (i.e., evidence for expression of transgene, and transient improvements in clinical condition in some cases • so far it has proved safe (only inflammatory or immune reactions directed toward vector or some aspect of administration method rather than toward transgene • no genetic disease cured by this method • major problem is efficacy, levels of transgene product expression often low or transient

28. Genetic Medicines • Antisense oligonucleotides • complementary to specific mRNA sequence • block translation or promote nuclease degradation of mRNA, thereby inhibit synthesis of protein products of specific genes • e.g., block HIV-1 replication by targeting gag gene • Double-stranded DNA to form triplex molecule