• Each of the 46 chromosomes of humans is made up of single molecule of double-stranded DNA

• if stretched out, the DNA from a single cell would extend approximately 2 meters in lenght

• an elaborate system of coling, which also seem to be involved in the control of gene expression is present in memmalian cells

• basic proteins called histones provide a core around which DNA is wound in a double loop composing approximately 146bp of DNA - „NUCLEOSOME“

• the resulting „beads-on-string“ DNA structure results in a compaction of lenght of about a factor of 7.Further organisation occurs by arrangement of the nucleosomes in solenoid fashion and by higher order structural complexities.

The human genome - the total genetic information (DNA content) in human cells. It comprise two genomes:• a nuclear genome• mitochondrial genome

The human nuclear and mitochondrial genomes

• differ in many aspects of their organisation and expression

Human Genome Project

It is thought that about 5% of all newborn babies come into the world with an inherited disorder. About 0.5% have a clinically relevant chromosome anomaly and about 1% have a monogenic inherited disorder. The remainder of disorders are multifactorial or due to external factors. Among those who die before their 65th year, inherited disorders are still the fifth most frequent cause of death. Most deaths result from inherited heart disorders followed by anomalies of the central nervous system (brain,spinal cord) as well as urogenital anomalies (urinary and reproductive organs) and gastrointestinal anomalies (digestive organs).

Inherited disorders

Major types of Genetic desease

genetically determined diseases are classified into four major categories: chromosomal diseases* are the result of the addition or deletion of entire chromosomes or part of chromosomes* most major chromosome disorders are characterised by growth retardation, mental retardation and variety of somatic

abnormalities* clinically significant chromosome abnormalities occur in nearly 1% of liveborn babies and account for about 1% of

pediatric hospital admissions and 2,5% of childhood death* typical examples of major chromosomal disease is Down syndrom (trisomy 21), Edwards sy(trisomy 18), Patau sy (trisomy13)

monogenic diseases (single gene defects)* are due to single mutant genes with a large effect on the patient´s health* inherited in simple Mendelian fashion* some 6000 distinct disorders are now known ( sicle cell anemia, familial hypercholesterolemia, cystic fibrosis, NFI,

DMD ...)* account for approximately 5-10% of pediatric hospital admissions and childhood mortality

polygenic diseases* result from the interaction of multiplex genes, each of which may have a relatively minor effect* account for 25-50% of pediatric hospital admissions, approximately 25-35% of childhood mortality* examples: diabetes mellitus, hypertension, schizophrenia and variety of common congenital defects such as cleft lip,

cleft palate and most congenital heart diseases (LQT sy)

somatic cell genetic defects* arrise only in specific somatic cells* paradigm for somatic cell genetic diseases in cancer - development of malignancy is the consequence of mutations in

genes that control cellular growth

In monogenic diseases, only a single gene is altered (mutant) with the consequence that the pattern for a specific protein is flawed, which in turn leads to the manifestation (development) of a disease. Sickle cell disease is an example of this. Monogenic diseases are often rare and severe illnesses. At present, over 6,000 genes are known whose mutations lead to various monogenic disorders. Currently, a molecular genetics analysis can be made on 1,000 of these diseases.

With polygenic diseases it is the interaction of several gene alterations (mutations) which leads to the development of an illness. • the mutation of a single gene has a much less serious effect than with a monogenic disease. • another important difference to monogenic diseases is that polygenic diseases are very common in the population. Apart from the mutation of several genes, one or more environmental factors usually contribute to the manifestation of these diseases (multifactorial diseases). Onset, severity and course of these illnesses are also significantly influenced by environmental factors (e.g. nutrition, exercise).For this group of illnesses, the contribution of the gene can be thought of as a “predisposition”. At present, there are only very few illnesses for which the contributory gene has been identified: e.g. in the case of thrombotic diseases (APC [Activated Protein C] resistance).

Genes and diseases The boundaries between monogenic, polygenic and multifactorial diseases can be expressed by the following statement: genes can always be represented as more or less strongly predisposing factors for the development of an illness.

Inheritance rules for genetic diseases

The following inheritance processes are known for monogenic diseases. In order to understand these rules of inheritance it is important to emphasize that :• every gene in the cell nucleus is expressed twice – in the same way as all chromosomes are expressed twice (one set from the maternal and one set from the paternal parental set).• What is decisive for the onset of a genetic disease is whether a mutated gene can prevail in its pathological action against its “healthy” counterpart (gene homologue) or whether the other gene also has to be mutated. These inheritance patterns were already observed in the 19 century by Gregor Mendel in his famous cross-breeding experiments with peas, which form the basis for today's comprehensive family tree analyses.

Autosomal dominant inheritance process

In this inheritance pattern only one of the two homologous genes is mutated and although another normal gene is present (heterozygosity), the illness still appears (dominant gene effect). If, therefore, one of the parents carries this gene, there is a 50% probability that it will be transmitted to each child. Both men and women can be affected by this.This inheritance pattern accounts for over 60% of monogenic diseases,representing by far the most common inheritance process.

This inheritance pattern is followed by many diseases which exhibitchanges in the proteins forming the basis for connective and supportingtissue (e.g. Marfan’s syndrome, achondroplasia, hypertrophiccardiomyopathy). Obviously a mutated protein in just half the amount willhave a pathological effect on the human organism in such cases.

Autosomal recessive inheritance

In this inheritance pattern, both homologous genes must be mutated (homozygosity) in order to produce an illness in the affected person. • The individual must therefore have inherited a corresponding gene mutation from both the father and the mother.• Individuals, who only receive one version of the mutated gene are called carriers. • Both sexes can be affected. If, for example, both parents are carriers, there is a 25% chance that the child will receive both mutated genes and so develop theillness.

Many metabolic diseases fall into this category (e.g. cystic fibrosis, phenylketonuria, adrenogenital syndrome, haemochromatosis). In the heterozygote state (i.e. only one gene copy is mutant) it seems that the human organism can often compensate for this state and make do with half of the unchanged protein. Only when both gene copies are mutated does the amount of faulty enzymes have a pathological effect which can then no longer be compensated.

X chromosome inheritance (sex-linked inheritance)

Women have two X chromosomes. If they have a recessively acting mutated gene on one X chromosome, they are carriers for the corresponding illness. Men have only one X chromosome, since the other sex chromosome is a Y chromosome. If they have the mutated gene on the X chromosome, they will develop the illness as a rule.If a woman is a carrier for the illness inherited by the X chromosome, there is a 50% chance that she will pass on this illness to her son. Her daughters have a 50% chance of becoming a carrier for this illness.

Inheritance process in polygenic diseases

Of course, in these cases too the individual genes take part in the abovementioned inheritance processes. However, there are always severalgenes involved in causing polygenic diseases with different types ofinteraction possible. Sometimes the effect of several genes must beadded together and at other times a certain threshold has first to becrossed before an illness becomes manifest. Often individual genes havea complex relationship with each other (control and regulation genes ina gene network). In addition, one or more environmental factors maycontribute to the manifestation of the corresponding illness. Given thelarge number of causative factors and their interrelationships, it is easy tounderstand that there are also a large number of variations in the genesisof multifactorial diseases. These are expressed in different ages of onsetof the illness, different courses of the illness and different degrees ofseverity.

In general, there is a fluid transition from the healthy state to the pathological state in polygenic diseases if there is no threshold effect.This breadth in the variation of mutations leading to the same illnessmakes it impossible to make a prediction of symptoms and prognosis withcurrent scientific knowledge.

Identification of inherited diseases) Phenotype analysis

The simplest and for centuries the most common type of genetic analysisis the analysis of the so-called phenotype. The analysis involves the endproduct of the genes which are translated into proteins by transcriptionand translation. These proteins determine the way an individual’s bodylooks.

The following points apply to phenotype analyses (here specifically: biochemical investigations):• Genes are directly responsible for the production of hormones, enzymes and other proteins. To this extent, the analysis of these substances and metabolic products can also be classified as a genetictest in a wider sense.• Indication: Especially in the case of metabolic diseases in newborn babies.• Investigation procedure: Diagnostic measurement of altered or missing proteins using blood or urine analysis. This provides indirect evidence of a mutation of the gene responsible for this.• Examples: Phenylketonuria, alpha1-antitrypsin deficiency

There is clinical evidence of a connection between severe alpha1-antitrypsin deficiency and liver cirrhosis as well as lung emphysema. There are several different courses of the illness. In the early childhood form, in which liver cirrhosis and lung emphysema occur in infancy, the children usually die from the complications before their twelfth year. Another form primarily affects adults, in which emphysema with a rapidly progressive course occurs before their 40th year. Environmentalfactors (nicotine, dust) play an important part in promoting the manifestation of this illness .

B) Chromosome analysis (cytogenetic investigations)

• This includes microscope examinations to investigate chromosomealterations in terms of number (duplication or loss of individual chromosomes= numeric chromosome aberration) and in terms of structure(wrong composition, chromosome breaking = structural chromosomeaberration). There is no detailed investigation of individual genes insuch cases.• Indication: Anomalies in children (malformations, retarded development)in the context of prenatal diagnosis, tendency to miscarriages, infertility.

• A relatively new technique is molecular cytogenetics, which uses a combination of cytogenetic and molecular genetics methods.Fluorescence-labelled DNA sequences are often used as diagnostic “probes”, hence the designation “FISH” (Fluorescence in situ hybridization)

C) Molecular genetics testing (DNA analysis, genome analysis DNA tests)

• This provides evidence of a gene mutation responsible for producingthe illness. Here it is determined whether the sequence of the DNA bases (nucleotide sequence) has changed within the affected

DNA/RNA diagnosis of genetic diseases

Not all mutation test use DNA . Testing RNA by RT-PCR has advantages when screening genes with many exons ( NF1 gene, DMD gene...) or seeking splicing mutations.Very important in molecular genetic testing is using a protein-based functional assay, which may classify the products into two simple groups: functional and nonfunctional – essential question in most diagnostics

Limitations of DNA analysis It has long been known from research into identical twins that monogenic and also polygenic diseases sometimes do not occur in both twins, even though the genetic information is the same in identical twins. So it is not surprising either that in monogenic diseases in which only a single gene is altered, the phenotypic consequences are often difficult to predict even in the case of a positive test result. This is due to several factors:• Penetrance: not every pathogenic mutation leads to the manifestation of a disease in the lifetime of a person. For example, the gene mutation for neurofibromatosis is manifested in almost all affected individuals. In inherited ovarian cancer this proportion is considerably reduced. This can be described as “reduced penetrance”.• Expressivity on the other hand describes quantitative differences in the manifestation of the disease/symptoms. Sometimes, the two concepts are difficult to separate, when, for example, a disease is so weakly manifested that it can no longer be diagnosed. The expressivity can fluctuate strongly especially in dominant monogenic diseases (e.g. Marfan’s syndrome, neurofibromatosis).• The age at which the disease manifests itself can vary strongly. An example of this is Huntington’s chorea. Differences in the onset of diseases are sometimes explained by so-called dynamic mutations. In passing on to the next generation, the disease-inducing mutation can lead to an earlier onset of the illness (anticipation) involving the extension of a mutated sequence of bases.• In many cases, genetic information is manifested in a different way when it is inherited from the mother than when it is inherited from the father. Here one speaks of imprinting.

DNA/RNA - based diagnosis can be made in two different ways:

Direct testing – DNA from a consultand is tested to see whether or not it carriesa given pathogenic mutation

Indirect testing (gene tracking)- linked markers are used in family studies todiscover whether or not the consultand inherited the disease-carrying chromosomefrom a parent

Direct testing

to see wheter the DNA of tested person has a gene normal or mutant

Detection of mutation in relevant gene always confirms theclinical diagnosis

we must know- which gene to examine- the relevant „normal“ (wild type) sequence

1. mutation screening methods - screen a sample for any deviation from the standard sequence

The mutation screening is possible for diseases where a good proportion ofpatients carry independent mutations.

Testing for unknown mutations in laboratory suffer two limitations:

- methods are quite laborious and expensive for use in diagnostic service, whichneeds to produce answers quickly

- detect differences between the patient´s sequence and published normalsequence ( not distinguish between pathogenic and nonpathogenic changes.)

- mutation: (synonym: sequence alteration) Any alteration in a gene from itsnatural state; may be disease causing or a benign, normal variant

Principles of PCR method

- rapid in vitro method for amplifying defined target DNA sequences

- selective amplification of specific target DNA sequence withinheterogeneous collection of DNA ( total genomic DNA or complex cDNA)required:

sequence information from the target sequence for construction twooligonucleotide primer sequences ( 15 – 30 nucleotides long ) amplimers

denatured genomic DNA heat stable DNA polymerase DNA precursors ( four deoxynucleotide triphosphates dATP, dCTP,

dGTP and dTTP)

PCR is a chain reaction because newly synthesized DNA strands will actas templates for further DNA synthesis in subsequent cycles. After about 30cycles of DNA synthesis PCR product will be about 105 copies of specifictarget sequence (an amount is easily visualised as a discrete band of specificsize when submitted to agarose or polyacrylamide gel electrophoresis).

PCR involves sequential cycles composed of three steps:

- Denaturation ( typically at about 93 – 95o C )

- Reannealling (at temperatures usually from about 50 o –70oC, depending on Tm of the expected duplex

- DNA synthesis – typically at about 70 –75o

Senzitivity of PCR allows us to use a wide range ofsamples: blood samples monthwashes or buccal scrapes chorionic villus biopsy samples amniocentesis speciments ome or two cells (removed from eight-cell stageembgryos) hair, semen archived pathological specimensGuthrie cards (spot of dried blood)

sequence analysis: (synonym: sequencing) Process by which the nucleotidesequence is determined for a segment of DNA

nucleotide: A molecule consisting of a nitrogenous base (adenine, guanine,thymine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), aphosphate group, and a sugar (deoxyribose in DNA; ribose in RNA). DNA andRNA are polymers of many nucleotides.

Some Clinical ImplicationsAn entire gene may be sequenced or, more commonly, only select regions of the gene most likely to containmutations (e.g., the exons and intron-exon boundaries) are sequenced.

Sequencing a segment of DNA identifies most variations from the wild-type. (In contrast, mutationanalysis identifies only specific targeted mutations within a given segment of DNA.)

Types of sequence alterations that may be detected1

Pathogenic sequence alteration reported in the literature Sequence alteration predicted to be pathogenic but not reported in the literature Unknown sequence alteration of unpredictable clinical significance Sequence alteration predicted to be benign but not reported in the literature Benign sequence alteration reported in the literature

Possibilities if a sequence alteration is not detected Patient does not have a mutation in the tested gene (e.g., a sequence alteration exists in another gene

at another locus) Patient has a sequence alteration that cannot be detected by sequence analysis (e.g., a large deletion) Patient has a sequence alteration in a region of the gene (e.g., an intron or regulatory region) not

covered by the laboratory's test1Adapted from the ACMG Recommendations for Standards for Interpretation of Sequence Variations (2000).

Some Clinical Implications Mutation analysis is generally not the first

molecular genetic test to be used for mutationidentification for diseases known to be associatedwith a high proportion of rare (i.e., family-specific)mutations.

If an affected individual does not have a mutationdetectable by mutation analysis, sequence analysisor mutation scanning may be necessary formutation identification

mutation scanning: (synonym: mutation screening) A process by which a segmentof DNA is screened via one of a variety of methods to identify variant gene region(s).Variant regions are further analyzed (by sequence analysis or mutation analysis) toidentify the sequence alteration.

Some Clinical Implications Mutation scanning is used when mutations are distributed throughout a gene,

when most families have different mutations, and when sequence analysiswould be excessively time-consuming due to the size of a given gene.

Mutation scanning may cover the entire gene or select regions.The sequence alteration identified in a segment of DNA may be a benign variant(polymorphism), a disease-causing mutation, or an alteration of undeterminedsignificance.

Indirect testing

- historically first type of DNA diagnostic method- most of the mendelian diseases went through a phase of gene tracking and moved

on to direct test once the genes were cloned (DMD, CF, HD, MD...)- with some diseases, even though the gene has been cloned, mutations are hard to

find mutations are scattered widely over a large gene the existence of homologous pseudogenes the lack of mutational hot spots

- never confirm clinical diagnosis!

linkage analysis: (synonym: indirect DNA analysis) Testing DNA sequencepolymorphisms (normal variants) that are near or within a gene of interest totrack within a family the inheritance of a disease-causing mutation in a givengene

Genetic markers used for undirect testing

two-allelic markers – RFLP analysis (restriction fragment lenghtpolymorphism), first generation of DNA markerstheir informativeness as markers is limited, because restriction sitepolymorphism has only two alleles: the restriction site is present orabsent.RFLP analysis can still be useful if strategically placed near or withina gene of intertest

multi-allelic markers – VNTR (variable number of tandem repeats orminisatellite markers) – hypervariable tandem repeatpolymorphism on DNAClassical minisatellites are difficult to handle by standard PCRprotocols because large alleles may fail to amplify.Now, minisatellites are little used for gene tracking.

highly informative multiallelic markers – the standard tools for linkage analysisare now microsatellites (short tandem repeats /STR/, usually di-,tri- or tetranucleotides) within introns sequences of the target genes.The bulk of these are (CA)n repeats

The three steps of linkage analysis are:

1. Establish haplotypes: Multiple DNA markers lying on eitherside of (flanking) or within (intragenic) a gene-region ofinterest are tested to determine the set of markers (haplotypes)of each family member.

2. Establish phase: The haplotypes are compared betweenfamily members whose genetic status is known (e.g., affected,unaffected) in order to establish the haplotype associated withthe disease-causing allele.

3. Determine genetic status: Once the disease-associatedhaplotype is established, it is possible to determine the geneticstatus of at-risk family members.

The main principles of application of indirect DNA analysis:

Indirect DNA analysis

gene CFTR - intron 8 - polymorphic site (CA)n

chr.7

from motherfrom father

GTATCACACACATTCGG

allele A1:------ GTATCACATTCGG----

the lenght of this allele is 130 bp

allele A2:-----GTATCACACACATTCGG---

the lenght of this allele is 134 bp

chr.7 chr.7

chr.7chr.7

mutation in CFTRgene

dF508 / non non / ?

dF508 / ? non / non

A1 / A3 A1 / A2

A1 / A2 A1 / A3 informative

A1 / A3 A1 / A1

A1 / A1 A1 / A3 informative

dF508 / non non / non

dF508 / non non / non dF508 / G542X

A1 / A3 A2 / A5

A1 / A2 A3 / A5 A1 / A4

Linkage analysis is often used when direct DNA analysis is notpossible because the gene of interest is unknown or a mutation withinthat gene cannot be detected in a specific family.

In most instances, the haplotype itself has no significance; it hasmeaning only in the context of a family study. Only in the rare instanceof linkage disequilibrium is a specific haplotype highly likely to beassociated with a specific disease-causing mutation.

The accuracy of linkage analysis is dependent on: The accuracy of the clinical diagnosis in affected family member(s). The distance between the disease-causing mutation and the markers.

Linkage analysis may yield false positive or false negative results ifrecombination of markers between maternally and paternally-inheritedchromosomes occurs during gamete formation. The risk ofrecombination is proportional to the distance between the disease-causing mutation and the markers. The risk of recombination is lowestif intragenic markers are used.

The informativeness of genetic markers in the patient's family. If the DNAsequence for a given variant differs on the maternally-inherited andpaternally-inherited chromosomes, that marker is informative. If the DNAsequence for a given variant does not differ on the two chromosomes, thatmarker is not informative.

Some Clinical Implication