Molecular and Biochemical Basis

of

genetic Disorder

Prof. Dr. Müjgan CENGİZ

Principles of molecular disease

* Molecular reason of a genetic

disease is a mutation. This

mutation either inherited or

acquired.

*The biochemical genetic is study of

phenotype at the level of proteins,

biochemistry and metabolism.

A genetic disease occurs when an

alteration in the DNA of an essential

gene changes the amount or function

or both, of the gene product -mRNA

and protein.

Single Gene disorders almost always

results from mutations that alter the

function of a protein.

The pathologies because of the

defaults of a protein expression can

be divided to 2.

1-Mutations at the control genes.

2-Mutations at the structural genes.

The mutations at the control genes

causes single nucleotide change. The

amount of the polypeptide and

function does not effected.

Only the velocity of protein synthesis

changes, phenotype is normal.

Mutations taking place at the structural genes causes a genetic disease .

The amounts and functions of gene products decreases.

To uderstand the pathogenesis of a genetic disease we have to learn the knowledge of primary abnormalities the results of the alteration of the gene function.

Disease causing mutations have been identified in more than 1990 of the 3310 single gene disease.

There are four effects of disease causing mutations on protein function.

1- Loss of function of a protein.

2-Gain a function.

3-Acquision of a novel property by mutant protein.

4- Expression of a gene at the wrong time or place.

Examples of these effects:

1-α thalasemias are commonly examples for

deletions of α globin genes.

2-Gain function mutations can alter the

biochemical phenotype by increasing the

function of a protein.

This effect because of

a-İncrease in the level of protein’s

expression(Trisomy 21).

b- İncrease in the ability of each protein to

perform one or more normal function.

Achondroplasia (fibroblast growth factor

mutation active in the absence of the ligand).

3-Acquising of a novel property:In a

few disease the change in amino acid

sequence does not change function.

Ex.Sickle cell disease(β chain:Glu6Val)

no effect on on transport of oxygen but

this hemoglobin chains aggregate when

deoxigenated, it forms polymeric fibres.

4-Expression of a gene at the wrong

time: the regulatory regions of the

gene can alter and cause an

inappropriate expression of the gene

in normal time or place. ex oncogene

normally promotes cell proliferation.

But if is not normally expressed

causes cancer.

Some mutations in hemoglobin regulatory

elements lead to the continued expression in the adult life γ globulin gene.

This gama globulin is normally expressed

at high levels only in fetal not postnatal

life.

This mutation led to phenotype called persistance of fetal hemoglobin.

The molecular and Biochemical causes of

genetic diseaes:

Mutation protein coding

genes(DNA)→mutation→Defective

protein production.

These are examples of single gene

disorders. Some examples of proteins

associated with monogenic diseases.

Some examples of Classes of Proteins

Associated with Monogenic Diseases

1-Transport and storage

İnterorgan Hemoglobin

(thalassemias)(AR)

İntracellular transport (copper

transport prot. menkes syndr. (AR)

Epitel membr. Cystic fibrosis (CFRT

AR)

2-Enzyme defects

Amino acids - PKU(phenyl alanine hydroxilase AR)

Complex lipid-Tay sachs(Hexosaminidase A AR)

Purines-immundeficiency( Adenosine deaminidase AR)

Carbohydrates Galactose 1 phosphate uridyl transferase

3-Structure of cells and organs

Duschene muscular distrophy(dystrophin XR)

4-Control of growth and differantiation-Tumor suppressors,RB gene products (AR), oncogenes(AD).

5-Intracellular metabolism and comunication

Growth gormon(dwarfizm,AR),insulin(AD)

Familial hypercholesterolemia(LDL receptor).

1-Transport and Storage

THALASSEMIAS

Hemoglobin is the oxygen carrier in

vertebrate red blood cells.

The molecule contains four subunits

2 α , 2 βglobin chains

Each subunit composed of polypeptide

chain globin and prostetic group heme.

Heme is a iron containing pigment that

combines with oxygen to give the

molecule its O transport ability.

Thalassemia is drived from greek

word of sea –Thalassa,mia-

mediterenian origin.

Hemoglobin A is normal adult

hemoglobin

2 α 141 aa chain folded and fitted

2 β 146 aa

Four chain folded and fitted

The chains resemble each other.

β thalassemias

Decreasd β globin production causes

hypochromic, microcytic anemia.

Imbalance in globin synthesis leads to precipitation of excess α chains,

leads damage of red cel membrane.

β chain is important in post natal

period. Onset of β thalassemia

apperent untill a few monts after birth (β

globin replaces γ ).

Synthesis of Hb A reduces.

Beta thalassemias usually single base

pair mutations.

They are so many different type of

mutations.

1- 2β thalassemia-allel thalassemia

major(severe anemia)

2-So little production of βglobin No Hb A

present. β° thalassemia.

3- Some Hb A is detectable. β+

thalassemia

4-Carriers of one β+ thalassemia are

clinically thalassemia minor.

Thalassemia minor patients have

hypochromic,microcytic red blood cells

.

They may have a slight anemia that

can miss diagnosed.

MOLECULAR BASIS OF β THALASSEMIA

Simple β thalassemias results many different types of molecular abnormalities mostly point mutations in the β globin gene.

Deletions

-β globin gene deletions (619 base pair deletion)

Defective mRNA synthesis

- RNA splicing defects

- promoter mutations

- poly adenylation signal defects

Nonfunctional mRNA

- Nonsense mutations

- Frameshift mutations

TRANSPORT DEFECTS

CYSTİC FİBROSİS

Autosomal recessive genetic disorders in Caucasian population

İncidence ~ 1/ 2500

CF gene (called CFTR - Cystic fibrosis conductance Regulatory protein)

The protein encoded by the CTFR gene is a regulated Cl¯ channel located in the apical membrane of epithelial cells affected by the disease.

PHENOTYPES

Lungs,

Pancreas (deficiency of pancreatic enzymes)

Sweat glands (eleveted level of Cl. >60meq/lt)

Chronic obstructive lung disease ( because of thick secretion and recurrent infection)

Infertility (in male)

Half of the patients survive to 26 years

CF gene located on chorosome 7q31 spans about 250 kb DNA

It has 27 exons

Encode large integral protein 170 kD.

Gene called CFTR

CTFR Cl¯ channel has 5 domain:

1. MSD1 (membrane spaining domain)

2. MSD2

3. NBD1 ( nucleotid binding domain) (ATP

binding

domain)

4. NBD2

5. Regulatory domain

The pore of Cl channel is formed by the

12 transmembrane segments.

ATP is bound and hydrolyzed by the

NBDs.

Energy released is used for ion

transport.

Regulation of the channel is mediated by

phosphorylation of the R domain.

It has 4 type mutation

-class I : Defect in protein production

Premature stop codons or mutations generates

-class II : Defective protein processing due to misfolding of protein

Deletion at phenylalanine residue at position 508 (∆F 508)

The first ATP binding fold (NBD1)

Accounts ~ 70% of CF allels

GER → Golgi →Plasma membrane

-class III : Mutations of the NBDs

Defective regulation of protein

S1255P (mutation at NBD2)

-class IV : Mutation at regulatory domain

Defective conduction due to alteration of Cl¯ channel (R334W)

Detection and Treatment

Detection of Na and Cl amount

DNA mutation analysis

Prenatal Diagnosis

Population screening

2-ENZYME DEFECTS

A-AMINOACIDOPATHIES

Hyperphenylalainemias

The abnormalities that lead to an increase in the

blood level of phenylalaline, is called PKU.

There is mutations at phenylalaline coding gene

loci.

There is loss of function mutations on the gene

encoding PAH.

PAH gene isolated in 1986, more than 400 different

allels have been recognized.

Phenylalaninehydroxylase gene

mutations

Arg 408 trp- activity<1% -- %31

european

Tyr414Cys- activity30% -- %5 “

Ile 65 Thr -- activity 25% --%5 “

B-LYOSOSOMAL STORAGE DISEASES

Lyososomes are membrane bound organels

containing hydrolitic enzymes.

They involved in the degradation of biologic

macromolecules.

Genetic defects in these hydrolases lead to the

accumulation of their substrates in the lysosome.

They lead cellular dysfunction-cell death .

When brain is affected it causes

neurodegeneration.

There are more than 48 lyososome hydrolase.

Almost all autosomal recessive.

TAY-SACHS DISEASE

Lyososomal storage disease (GM2

gangliosides)

The inability to degrade a sphingolipid GM2

ganglioside.

The biochemical lesion is deficiency of

hexoaminidase A.

The disease has its clinical impact especially on

the brain.

Hex A is the product of 3 gene system.

α subunit Hex A

β subunit Hex B genes

Activator protein

Mutations of the Hex A

Mutation Effect homozygote

phenotype

-4 bp insertion premature Tay-Sachs

(exon 11) stop codon

-Exon 12 splice defective Tay-Sachs

Junction G→C mRNA spilicing

3-DISORDER OF STRUCTURAL PROTEINS

DUCHENE-AND –BECKER MUSCULAR DISTROPHY

Severe untreatable, relatively common X linked disorder.

Related with Dystrophin protein.

Affected boys normal at first year.

Muscle weakness developed age 3-5.

The child confined to wheelchair at age 12 and deads at the age of 20.

Creatine kinase level is elevated 50-100 times upper limit of normal

Patients die of respiratory failure or

cardiac failure, brain is also effected.

Becker (BMP) is also due to mutation

in dystrophin gene

İt produces a phenotype is much

milder.

If patients is still walking at the age of

16 the disease is BMD.

İncidence of DMDis 1 in 3300 live male

birth

Calculated mutation rate is (10)-4

Normal male produces sperm with new

mutation of DMD gene every 10 to 11

seckonds(mens produces 8.107

sperm/day)

Carier females have no clinical

manifestations. 70% have↑creatine

kinase

İn rare instances females have been

reported with DMD.

Size of DMD gene 2300 kb

%1.5 of X chromosome

Gene is a complex gene it have 79

exons 7 tissue spesific promoter.

It encodes 427 kD protein.

Mechanism of mutation in Duchenne

or Becker muscular distrophy

Gene deletion %60 dmd or bmd

Point mut %34 "

Partial duplication %6 "