Pradeep.ii

I D E N T I F Y I N G H U M A N D I S E A S E S G E N E S

Presented BY:Pradeep Jaswani

M.S.c MHG III semesterJiwaji University Gwalior

(M.P)9713435733,882303344

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ContentsIntroductionPrinciples & strategies in identifying disease genePosition independent strategies for identifying disease gene Identifying disease gene though knowing the protein product Identifying disease gene though an animal model Identification of a diseases gene using position independent

DNA sequence knowledgePositional cloning Single stand conformation polymorphism analysis Hetero duplex analysisCandidate gene approach

INTRODUCTION“Identifying genetic determinants of human

phenotypes”.

The approach described are equally applicable to identifying determinants of diseases or of normal variations such as red hair or red-green color blindness.

• There are many different ways of arriving at the final identification, but all path converge on a candidate gene.

• One way or another, a candidate gene is identified; the researcher then test the hypothesis that these gene is disease gene by screening it for mutation

PRINCIPLES & STRATEGIES IN IDENTIFYING DISEASE GENE

• Candidate genes may be identified without reference to their chromosomal location but more commonly, first a candidate chromosomal region is pinpointed, and then candidate genes are identified from within that region.

• Positional information reduces the list of possible candidates from all 30000 or so on human genes to may be 10-30genes in a candidate region.

• Over and over again, when a disease gene is finally identified, it remains a complete mystery why mutation should cause that particular disease

• For e.g. why should loss of function of the FMR1 protein, involved in transporting RNA from nucleus to cytoplasm cause, cause mental retardation (Fragile-X syndrome)

POSITION INDEPENDENT STRATEGIES FOR IDENTIFYING DISEASE GENE

Historically, the first diseases genes were identified by position independent methods, simply because no relevant mapping information existed an no techniques were available to generate it.

Under those circumstances the candidate must be suggested by the knowledge of the gene product: β-globin for sickle cell disease, phenylalanine hydroxylase for PKU.

B3

Model organism

Database searching Clinical Input Laboratory work

A3

A1

B3

B2

B1

B4

C1

C2

C5

D1

D2

D3

D4

D5

E1

E2

E3

E5

Mapped candidate homolog?

Cloned candidate homolog?

Candidate chromosomal region?

Check database for genes

Possible candidate gene

Has it been fully characterized

Collect families for mapping

Chromosomal deletions or translocations

Collect unrelated patients

Genetic mapping: genome search

Clone the chromosomal breakpointsIdentify

new human genes

Work out full sequence & structure

Look for mutations

Successfully located?

Think again about mode of inheritance, heterogeneity

Think again about candidate gene & go to A3 B2 D3

Pathogenic mutations found?

success

N

N

N

N

N

Y

Y

Y

Y

Y

Modern proteomic technique allow even very tiny quantities of protein to be identified or partially sequenced by mass spectrophotometery.

As only one of the nucleotide in the mixture will corresponding to the authentic sequence, it is important to keep the number of different oligonucleotides low so as to increase the chance of identifying correct target.

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IDENTIFYING DISEASE GENE THOUGH KNOWING THE PROTEIN PRODUCT

The number of possible permutations should be reduced by ligating the target cDNA to a vector and using one vector-specific primer and one degenerate protein specific-primer

Host cells containing clones with the desired gene should produce the protein or at least parts of the protein, and could be indentified using colony filters from the library with an appropriate antibody.

A more rapid alternative is to use partially degenerate oligonucleotides as PCR primers

IDENTIFYING DISEASE GENE THOUGH AN ANIMAL MODEL

• Many human diseases genes have been identified with the help of animal models-but nearly always this has been after checking positional information's

A mouse mutant and a phenotypically similar human diseases are mapped to chromosomal location that are corresponds.

If the mouse gene is cloned its human homolog became a natural candidate.

• Alternatively a diseases gene may be identified in the mouse and then the human homolog isolated; this can be mapped by fluorescence in situ Hybridization, and becomes a candidate gene for any relevant diseases mapping to that location.

• This is how the MIFT gene was identified as a cause of type2 waardenburg syndrome (Hughes et al 1994).

IDENTIFICATION OF A DISEASES GENE USING POSITION INDEPENDENT DNA SEQUENCE KNOWLEDGE

• Positional independent candidates are also generated by expression array experiment in which mRNA samples from patient and controls are compared to produce a list of genes whose expression is alter in the disease.

• An interesting application of positional independent DNA sequence knowledge is attempt to clone genes containing novel trinucleotide repeats.

• The repeat expansion detection method of schalling et al 1993 permits detection of expanded repeats in unfractionated genomic DNA of affected patient and method have been developed for cloning expanded repeats detected (koob et al 1998).

Positional cloningA diseases is identified knowing nothing except its

appropriate chromosomal location.The first successful application was identification

of the gene for X-linked chronic granulomatous disease (Royer- Pokora et al 1985)

The successful conclusion of these work in 1986 marked the start of triumphant new era for human molecular genetics.

One after another, the genes underlying important disorder such as cystic fibrosis, Huntington's diseases.

Define the candidate region

Obtain clones of all the DNA of the region

Identify all the genes in the region

Prioritize them for mutation screening

Test candidate genes for mutation in affected people

Fig: Logic of positional cloning

A NUMBER OF POSITIONAL CLONING METHODS ARE USED AS FOLLOWS:-

Single strand conformation polymorphism (SSCP)

Denaturing gradient gel electrophoresis (DGGE)

Heteroduplex analysisChemical mismatch cleavage protein

truncation test (PTT)

SINGLE STRAND CONFORMATION POLYMORPHISM (SSCP)

Reference: http://www.wikilectures.eu/index.php/DNA_Diagnostic_Direct_Methods

CANDIDATE GENE APPROACH• A functional/candidate gene cloning project

starts with either the known protein that is responsible for an inherited disorder or a protein that is considered a likely candidate based on the symptoms and biochemistry of the disease.

• The amino acid sequence of the protein is used to deduce the possible cloning sequence of the corresponding gene.

Mutation phenotype studies

Known (or candidate) protein

Deduce nucleic acid sequence

Examine human genome database

Localize chromosome region

Retrieve bacterial artificial chromosome (BAC) clones

Characterize gene structure

Develop mutation detection assays

Devise DNA diagnostic tests

Identify exons

Fig: candidate gene approach

REFERENCES HUMAN MOLECULAR GENETICS “Tom strachan &

Read” AN INTRODUCTION TO HUMAN MOLECULAR

GENETICS “Jack J.Pasternek”http://www.wikilectures.eu/index.php/DNA_Di

agnostic_Direct_Methods