Y SINH

DNA diagnostics in human disease
Introduction 1
Three talks to cover the subject of diagnosis of human disease through the application of DNA technology.

1. DNA Diagnostics in Human disease
What diseases?
Why?

2. Mutation Detection Applied to Diagnostic Service
What kinds of techniques ?
What kinds of mutations?

3. Models of Service Provision
UK diagnostic model
Population screening
Introduction 2
DNA diagnostics: a new discipline
Diagnostic labs have existed for about 10 years
About 40 labs in UK offer diagnostic services covering major single gene disorders
Classification of Genetic disease
1. Single Gene disorders
In 1966 about 1500 single gene disorders known. By 1994 over 6000 identified. There are between 50000-100000 structural genes so more disorders likely to be identified.

2. Chromosome abnormalities
Studied cytogenetically. Deletions, rearrangements, translocations which can be identified microscopically. Also, variations in chromosome number.

3.Multifactorial disorders
Involve other genes or factors such as environment
Why provide Genetics diagnostic services?
Cumulative impact of genetic disease on community. Increased public awareness.
Molecular genetics using recombinant DNA technology, PCR, sequencing now available. Large effort in research made diagnostics economic.
Impact of genetic disease 1
Impact of genetic disease: Childhood

1. Chromosome abnormality present in at least 50% of all spontaneous miscarriage

2. 2-3% of all neonates have one major genetic abnormality often caused by genetic factors. 2% of all neonates have a chromosome abnormality or single gene disorder.

3. Genetic disorders account for 50% of all childhood blindness, 50% deafness, and 50% all severe mental retardation. Genetic disorders and congenital malformations together account for 30% of all childhood hospital admissions and 40-50% of all childhood deaths.
Impact of Genetic disease 2
Adult life
Approximately 1% all malignancy due to genetic factors. 10% common cancers have strong genetic component for example: breast, colon, ovary.


By 25 years age, 5% of population will have a disorder in which genetic factors play important role.
Examples of major single gene disorders in UK and Northern Europe
Disorder Type Chromosome

CF AR 7
Fragile X XL X
Huntington AD 4
Myotonic dyst. AD 19
Haemophilia XL X
Duchenne MD XL X Haemochromatosis AR 6
Incidence of common Mendelian disorders in UK
Disorder Incidence/1000 births

APKD 0.8
HD 0.5
NF 0.4
Myotonic dystrophy 0.2
Cystic fibrosis 0.4
Thalassaemia 0.05
Fragile X 0.5
DMD 0.3
Haemophilia A 0.1

Familial combined hyperlipidaemia 5.0
Familial hypercholesterolaemia 2.0

Sample process through laboratory
Blood sample received
DNA extracted
Sample for analysis or storage?
Storage
DNA bank
Analysis
Urgent Non urgent
Immediate
analysis
Batch
collected
What is urgent?
It is important to remember that all of this
technology is applied for the benefit of the patient. What is urgent reflects the interest of the patient. Examples:

1. Pre-natal diagnosis

2. Exclusion of a genetic disease in pregnancy

3. Pre-operative mutation testing in Myotonic dystrophy

4. Peace of mind
Most DNA laboratories attempt to produce an answer in 5-10 working days.
DNA preparation and storage
DNA can be prepared from blood or tissue. The most common source for diagnostic purposes is blood which must be in anticoagulant. EDTA is best anticoag. The Aberdeen laboratory extracts about 1200 samples /year. Process takes about 4hours.

10ml blood sample gives about 50-100g of DNA. Many DNA samples are stored as archive material especially important if genes not yet identified for a particular disease. Aberdeen archive contains about 11,000 samples, some from people who are now dead.
Gene tracking 1
In diagnostic laboratories 2 forms of gene tracking are employed, DIRECT and INDIRECT.

If the gene for a disorder has been isolated and the mutation or mutations identified, a simple DIRECT test may be devised for the mutation(s) which can be applied to an individual patient. Such tests will be described in more detail in the next lecture.

Where the presence of many mutations makes a simple test difficult to devise (such as in Polyposis coli) a combination of direct and indirect methods may be required to identify carriers or those affected by a particular condition.
Gene tracking 2
Indirect tracking

Before the genes and mutations for disorders such as Myotonic dystrophy, CF and HD discovered, diagnostic procedures were of linkage type, ie, following or tracking linked marker as indicator of gene inheritance. RFLP’s were commonly used.

RFLP problem Low informativeness in many cases.

If an indirect (tracking) method required today, marker of choice would be highly polymorphic CA repeat. Repeat elements can be very important in certain diagnostic situations.
Dinucleotide Repeats
Dinucleotide repeat elements of the form:

...CACACACACACACACACA.....

are found throughout genome. Possibly 50,000 exist with variable units of 10-60 copies. Referred to as microsatellites. The high degree of variability means that chances of finding homozygote are low so informativeness is high.

Microsatellites are very useful in FAP, PW/AS, CF (rare mutation forms).
Dinucleotide Repeat Analysis
Repeat elements are small in size so PCR used to amplify with radiolabel and the products resolved on acrylamide gels.

Detected by autoradiography.

Products differing in size by one base pair can easily be separated this way
Tracking a microsatellite
....
7
14
21
26
Portion of FAP gene
Dinuceotide repeats in intron
26
21
14
7
?
26/7
21/14
14/7
21/7
References
Best single textbook:
Strachan T & Read A Human Molecular Genetics 2nd ed 1999

Websites
MRCPath. www.ich.ucl.ac.uk/cmgs/cmgshelp.htm
Lecture slides