Medical BiochemistryMedical Biochemistry

Biochemical and Genetic Basis of Disease

Lecture 77

Biochemical and Genetic Basis of Disease

Lecture 77

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)

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)

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

Organelle Involvement

• Almost every cell organelle has been involved in the genesis of various diseases

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 1-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

Genetic Diseases

• Many disease are determined genetically– Three major classes: (1) chromosomal disorders, (2)

monogenic disorders (classic Mendelian), and (3) multifactorial disorders (product of multiple genetic and environmental factors)

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

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)

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

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

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

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

S P Increased S Decreased PE

Normal Block

Increased X,Y

*E

Inborn Error of Metabolism

• 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)

Increased phenylalanine Decreased tyrosineBlock

Increased phenylpyruvic acid

*E

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)

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

Methods to clone disease genes

• Functional approach– gene identified on basis of biochemical defect

– e.g., found that phenotypic defect in HbS was GluVal, evident that mutation in gene encoding -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

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

Identifying defect in disease gene

• Once disease gene identified, still can be arduous task identifying actual genetic defect

Mutations in CFTR geneStructure of CFTR gene and

deduced protein

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

Molecular Medicine

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

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

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

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)

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 be

actively growing forintegration into genome

• usually performed ex vivo

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

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

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