Genetic biomarkers

Noha l. ibrahim

Introduction

•All organisms are subject to mutations as

a result of normal cellular operations or

interactions with the environment, leading

to genetic variation (polymorphism).

•For this variation to be useful, it must be

(1) heritable and (2) discernable.

Introduction

•Types of genetic variation include:

base substitutions, commonly referred to as

single nucleotide polymorphisms (SNPs).

insertions or deletions of nucleotide sequences

(indels) within a locus.

inversion of a segment of DNA within a locus.

rearrangement of DNA segments around a locus

of interest

DNA-based genetic markers•In the past, allozyme and mtDNA markers.

•More recent marker types include:

restriction fragment length polymorphism (RFLP) (1)

randomly amplified polymorphic DNA (RAPD)

amplified fragment length polymorphism (AFLP)

expressed sequence tag (EST) markers

single nucleotide polymorphism (SNP)

microsatellite

Type I (coding) vs. type II (non coding)

markers

•Molecular markers are classified into two

categories:

type I are markers associated with genes

of known function.

type II markers are associated with

anonymous genomic segments

Type I vs. type II markers

•Most RFLP markers are type I markers because

they were identified during analysis of known

genes.

•Allozyme markers are type I markers because the

protein they encode has known function.

•RAPD markers are type II markers because RAPD

bands are amplified from anonymous genomic

regions via the polymerase chain reaction (PCR).

Type I vs. type II markers

• AFLP markers are type II because they are alsoamplified from anonymous genomic regions.

• EST markers are type I markers because theyrepresent transcripts of genes, it is more common inanimals and plants research.

• SNP markers are mostly type II markers unless theyare developed from expressed sequences (eSNP orcSNP) (type l).

• Microsatellite markers are type II markers unlessthey are associated with genes of known function(type l).

polymorphic

information content (PIC)

• The usefulness of molecular markers can be

measured based on their PIC.

• PIC refers to the value of a marker for detecting

polymorphism in a population.

• PIC depends on the number of detectable alleles

and the distribution of their frequencies.

• The greater the number of alleles, the greater the

PIC

Allozyme markers

• Allozymes are allelic variants of proteins producedby a single gene locus, and are of interest asmarkers because polymorphism exists andbecause they represent protein products of genes.

• Amino acid differences in the polypeptide chains ofthe different allelic forms of an enzyme reflectchanges in the underlying DNA sequence.

Allozyme markers• Depending on the nature of the amino acid

changes, the resulting protein products may migrate

at different rates (due to charge and size

differences) when run through a starch gel

subjected to an electrical field.

• Differences in the presence/absence and relative

frequencies of alleles are used to quantify genetic

variation and distinguish among genetic units at the

levels of populations, species, and higher taxonomic

designations.

Allozyme markers• Disadvantages:

heterozygote deficiencies due to null (enzymaticallyinactive) alleles and the amount and quality of tissuesamples required.

some changes in DNA sequence are masked at theprotein level, reducing the level of detectable variation.

some changes in nucleotide sequence do not changethe encoded polypeptide (silent substitutions).

some polypeptide changes do not alter the mobility ofthe protein in an electrophoretic gel (synonymoussubstitutions).

Mitochondrial DNA markers• Sequence divergence accumulates more rapidly in

mitochondrial than in nuclear DNA due to a fastermutation rate result from a lack of repairmechanisms during replication.

• Due to its non-Mendelian mode of inheritance, themtDNA molecule must be considered a singlelocus in genetic investigations.

• Disadvantage: mtDNA data may not reflect those ofthe nuclear genome.

Restriction fragment length

polymorphism (RFLP)• Restriction endonucleases are bacterial enzymes that

recognize specific 4, 5, 6, or 8 bp nucleotide sequencesand cut DNA wherever these sequences areencountered, so that changes in the DNA sequence dueto indels, base substitutions, or rearrangementsinvolving the restriction sites can result in the gain, loss,or relocation of a restriction site.

• Digestion of DNA with restriction enzymes results infragments whose number and size can vary amongindividuals, populations, and species.

Restriction fragment length

polymorphism (RFLP)• Traditionally, fragments were separated using Southern

blot analysis, Most recent analyses replace it with PCR.

• If flanking sequences are known for a locus, thesegment containing the RFLP region is amplified viaPCR.

• If the length polymorphism is caused by a relativelylarge (> approx. 100 bp depending on the size of theundigested PCR product) deletion or insertion, gelelectrophoresis of the PCR products should reveal thesize difference.

Restriction fragment length

polymorphism (RFLP)• By using a ‘universal’ primers on a target DNA, PCR

products can be digested with restriction enzymes andvisualized by simple staining with ethidium bromide due tothe increased amount of DNA produced by the PCRmethod.

• Advantage: they are codominant markers, because thesize difference is often large, scoring is relatively easy.

• Disadvantage: the relatively low level of polymorphism. Inaddition, either sequence information (for PCR analysis)or probes (for Southern blot analysis) are required,making it difficult and time-consuming

Random amplified polymorphic

DNA (RAPD)

• RAPD procedures were using PCR to randomlyamplify anonymous segments of nuclear DNA withan identical pair of primers 8 – 10 bp in length.

• Because the primers are short and relatively lowannealing temperatures (often 36– 40 C) are used,the likelihood of amplifying multiple products isgreat, with each product representing a differentlocus.

Random amplified polymorphic

DNA (RAPD)• The potential power is relatively high for detection of

polymorphism; typically, 5 –20 bands can beproduced using a given primer pair, and multiple setsof random primers can be used to scan the entiregenome for differential RAPD bands.

• Because each band is considered a bi-allelic locus(presence or absence of an amplified product), PICvalues for RAPDs fall below those for microsatellitesand SNPs, and RAPDs may not be as informative asAFLPs because fewer loci are generatedsimultaneously.

Amplified fragment length

polymorphism (AFLP)

• AFLP is a PCR-based, multi-locus fingerprintingtechnique that combines the strengths andovercomes the weaknesses of the RFLP and RAPDmethods.

• Like RFLPs, the molecular basis of AFLPpolymorphisms includes indels between restrictionsites and base substitutions at restriction sites; likeRAPDs, it also includes base substitutions at PCRprimer binding sites.

Amplified fragment length

polymorphism (AFLP)• The unique feature of the technique is the addition

of adaptors of known sequence to DNA fragments

generated by digestion of whole genomic DNA.

• This allows for the subsequent PCR amplification of

a subset of the total fragments for ease of

separation by gel electrophoresis.

• It is the same as RFLP, but instead of analyzing

one locus at a time, it allows for the analysis of

many loci simultaneously.

Single nucleotide polymorphism

(SNP)• It describes polymorphisms caused by point

mutations that give rise to different allelescontaining alternative bases at a given nucleotideposition within a locus. lt is used for DNAsequencing.

• SNP markers are inherited as co-dominantmarkers.

• Its PIC is not as high as multi-allele microsatellites.

• Random shotgun sequencing, ampliconsequencing using PCR, and comparative ESTanalysis are among the most popular sequencingmethods for SNP discovery.

Expressed sequence tags (ESTs)• ESTs are single-pass sequences generated from

random sequencing of cDNA clones used for geneprofiling and genomic mapping.

• It offers a rapid and valuable first look at genesexpressed in specific tissue types, under specificphysiological conditions, or during specificdevelopmental stages.

• ESTs are useful for the development of cDNAmicroarrays that allow analysis of differentiallyexpressed genes.

What is microsatellites & SSRs? • Microsatellites or simple sequence repeats (SSRs),

represent codominant molecular genetic markers, i.e., bothallele in an individual are present in the analysis.

• Microsatellites are stretches of DNA consisting of tandemlyrepeated short units of 1–6 base pairs (bp) in length. SSRstypically span between twenty and a few hundred bases

• Due to their high level of polymorphism, relatively small size,multiallelic nature, codominant inheritance and rapiddetection protocols, easily amplified with the PCR using twounique oligonucleotide primers that flank the microsatelliteand hence define the microsatellite locus, these markers arewidely used in a variety of fundamental and applied fields oflife and medical sciences.

Microsatellites &SSRs • Application in biology and medicine including:

forensics, molecular epidemiology, parasitology,population and conservation genetics, geneticmapping and genetic dissection of complex traits.

• Microsatellites are considered selectively neutralmarkers, found anywhere in the genome, both inprotein-encoding (9-15%) and noncoding DNA.

• SSRs contribute to DNA structure, chromatinorganization, regulation of DNA recombination,transcription and translation, gene expression andcell cycle dynamics.

Microsatellites &SSRs• The majority of microsatellites (30–67%) found are

dinucleotides, mostly represented by poly (A/T)

tracts, which are the most frequent classes of

SSRs, where (tri-, tetra-, penta-and

hexanucluotides) are about 1.5-fold less common in

genomic DNA.

• In the human genome, one microsatellite was found

every 6 kb and one CA repeat (the most common

type of tandem repeat) occurred every 30 kb of

DNA.

Microsatellites &SSRs

• Di- and tetranucleotide motifs are mostly clustered

in noncoding regions. In vertebrates, they are

distributed 42- and 30-fold less frequently in exons

than in intronic sequences and intergenic regions,

respectively.

• Long dimeric motifs are highly unstable within

expressed sequences, while in noncoding regions

most dinucleotide repeats can have surprisingly

long stretches, probably due to the high tolerance of

noncoding DNA to mutations.

Microsatellites &SSRs• In contrast, triplets are found in both coding and

non-coding genomic regions with a high frequency.

• In humans, the expansion of trinucleotides,encoding polyproline (CCG)n, polyarginine(CGG)n, polyalanine [(GCC)n and (GCG)n] andpolyglutamine (CAG)n tracts within exons has beendescribed.

• Such expansions can lead to variousneurodegenerative and neuromuscular disorders,including myotonic distrophy, fragile X syndrome,Huntington's disease and spinocerebellar ataxia.

Function of microsatellites

1. DNA & chromosome structure• Microsatellites are involved in forming a wide variety of

unusual DNA structures with simple and complex loop-folding patterns.

• Telomeric and centromeric chromosome regions havebeen shown to be rich in long arrays of a variety ofmono-, di-, tri-, tetra-and hexanucleotide motifs.

• The (TTAGGG)n hexamer sequence is recognized byribonucleoprotein polymerase, a telomerase, whichsynthesizes telomere repeats onto the chromosomeends to overcome the loss of sequences during DNAreplication, whereas other proteins prevent nucleolyticdegradation and confer stability of chromosomes.

Function of microsatellites

2. DNA recombination• Dinucleotide motifs are preferential sites for

recombination events due to their high affinity forrecombination enzymes.

• Some SSR sequences, such as GT, CA, CT, GAand others, may influence recombination throughtheir effects on DNA structure.

• SSRs were shown to be associated with theassignment of some Rh phenotypes, and to beinvolved in the molecular evolution of the human Rhgene family and its orthologs in other eukaryotesvia replication slippage and recombination (geneconversion) mechanisms.

Function of microsatellites

3. DNA replication• Human genes encoding important cell fidelity and

growth factors, such as the B-cellleukemia/lymphoma 2 (BCL2)-associated X protein,insulin-like growth factor 2 receptor (IGF2R), breastcancer early onset protein 2 (BRCA2) andtransforming growth factor beta 2 (TGF-β2), containshort repeated sequences.

• Frame-shift mutations, resulting in both insertionsand deletions of repeat units within thesesequences that affect these genes and couldtherefore initiate tumorigenes and can affectenzymes controlling mutation rate and cell cycles.

Function of microsatellites

4. Gene expression• SSRs located in promoter regions can influence drastic

or quantitative variations in gene expression andchange the level of promoter activity. The human insulinminisatellite is highly polymorphic, and some of itsalleles were shown to regulate the expression of theinsulin gene.

• Intronic SSRs also can affect gene transcription affectmRNA stability, representing binding sites fortranslation factors. For example, such an effect wasmeasured for the tetrameric microsatellite located inintron 1 of the human tyrosine hydroxylase gene andthe (CA)n dinucleotide repeat in the first intron of thehuman epidermal growth factor receptor gene.

Development of type I (coding) and type II

(non-coding) microsatellite markers

• Type I markers are more difficult to develop. While non-gene sequences are free to mutate, causing higher levelsof polymorphism, sequences within protein-coding regionsgenerally show lower levels of polymorphism because offunctional selection pressure.

• The most effective and rapid way for producing type Imicrosatellites is the sequencing of clones from cDNAlibraries. Both 5′- and 3′-ends of a cDNA clone can besequenced to produce expressed sequence tags (ESTs).

• An EST represents a short, usually 200–600 bp-longnucleotide sequence, which represents a uniquelyexpressed region of the genome.

Development of type I (coding) and type II

(non-coding) microsattellite markers

• EST sequences are archived in a special branch of theGenBank nucleotide database (dbEST). In Nov. 2005, theEST database contained more than 31.3 million sequenceentries from around 500 species.

• A typical strategy for the development of ESTderivedmicrosatellite markers (data mining) includes preliminaryanalysis of EST sequences from the DNA database toremove poly(A) and poly(T) stretches which are common inESTs developed from the 3′-ends of cDNA clones andcorrespond to the poly(A)-tails in eukaryotic mRNA.

• Sequences are further screened for putative SSRs (allSSR-containing EST sequences). Following theidentification of ESTs, flanking primers should be designedto amplify a microsatellite.

Applications of microsatellites

1. Genetic mapping

2. Individual DNA identification and

parentage assignment

3. Phylogeny, population and conservation

genetics

4. Molecular epidemiology and pathology

5. Quantitative trait loci mapping

6. Marker-assisted selection

1. Genetic mapping

• SSRs remain the markers of choice for the

construction of linkage maps, because they are

highly polymorphic (and highly informative) and

require a small amount of DNA for each test.

• However, type II (noncoding) microsatellites are

very helpful for building a dense linkage map

framework into which type I (coding) markers can

then be incorporated (type I markers directly shows

the location of genes within the linkage map).

1. Genetic mapping• Linkage map is known as recombination maps and define

the order and distance of loci along a chromosome on thebasis of inheritance in families or populations.

• During meiosis, one random copy of each chromosomepair is passed on to the gamete. Only genes located nextto each other are tightly linked.

• Crossingover results from physical exchange ofchromosome segments between two homologouschromosomes of meiosis.

• Recombination results in the exchange of grandparentalalleles of genes further apart on that chromosome

1. Genetic mapping

• Genetic distance is usually measured in

centimorgans (cM), where 1 cM is equivalent to

1% recombination between markers.

• Linkage map length differs between sexes. In

species with the XY sex determination system,

the female map is usually longer than the male

map because of higher recombination rates in

females compared to males.

2. Individual DNA identification and

parentage assignment• Microsatellites represent codominant single-locus DNA

markers. For each SSR, a progeny inherits one allele fromthe father and another allele from the mother.

• Appropriate mathematical tools are available to evaluategenetic relatedness and inheritance in these systems.

• A suitable methodology should be chosen for accurate andcorrect analysis of genotyping data to reconstruct parentageand pedigree structure.

• Due to the small size of SSRs, they are relatively stable indegraded DNA. This is one reason why polymorphic SSRsare widely used in forensic science, as microsatellite lociremain relatively stable in bone remnants and dental tissue,providing the basis for the successful application of ancientDNA.

3. Phylogeny, population and

conservation genetics• By using variability within stretches of tandem

repeats, which evolve significantly more rapidlythan flanking regions.

• Flanking regions of microsatellites have proventheir value in establishing phylogeneticrelationships between species and families,because they evolve much more slowly thannumbers of tandem repeats.

• Phylogeographical applications of micro-satellitesare eminently suitable, where population structureis observed over a large geographical scale.

4. Molecular epidemiology and

pathology• Genomic instability of microsatellites has been extensively

evaluated in the field of carcinogenesis, where chromosomalrearrangements (e.g., translocations, insertions anddeletions of genomic regions) occur.

• Carcinogenic events often happen within a genomic regionharboring a tumour suppressor gene and hence inactivatethe gene.

• Carcinogenic rearrangements are associated with loss ofheterozygosity (LOH) in microsatellites located within theaffected chromosome region.

• Thus, detecting microsatellite LOH in tumour tissuescontributes not only to molecular diagnosis of cancer, butalso points the possible location of a tumour suppressorgene.

5. Quantitative trait loci mapping• A quantitative trait is one that has measurable

phenotypic variation owing to genetic and/orenvironmental influences.

• The variation can be measured numerically (forexample, height, size or blood pressure) andquantified.

• Generally, quantitative traits are complex(multifactorial) and influenced by severalpolymorphic genes and by environmentalconditions.

6. Marker-assisted selection

• Marker-assisted selection is based on the concept that itis possible to infer the presence of a gene from thepresence of a marker tightly linked to that gene.

• So, it is important to have high-density and high-resolutiongenetic maps, which are saturated by markers in thevicinity of a target locus (gene) that will be selected.

• The degree of saturation is the proportion of the genomethat will be covered by markers at the density such thatthe maximum separation between markers is no greaterthan a few centimorgans (usually 1–2 cM), within whichlinkage of markers can be detected.