dna genetic testing, mar04, prof ron trent

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Professor Ron Trent DPhil (Oxon), FRACP, FRCPA.

Director, department of molecular & clinical

genetics, Royal Prince Alfred Hospital,

and professor of molecular genetics,

University of Sydney.

DNA genetic t esting

IntroductionDNA genetic testing has been used in patho logy practice for more than 20 years. D espite this, the general

practitioner has not had much direct exposure to the DNA genetic test, which has usually been ordered by

specialists in clinical genetics. This has come about because:

(1) A relat ively small number o f labora tories, usually in the public health system, have been involved;

(2) No Medicare reimbursement was availab le until 1998 when the first item number w as issued fo r

haemochromatosis (HFE) DNA testing. Toda y, only six D NA tests associated with venous thrombosis are

funded through Medicare: haemochromatosis (HFE); fragile X mental retardation (FMR1); and four

enzyme deficiencies (Facto r V Leiden, antithrombin III, protein C and protein S) , despite the fact that

many hundreds of different DNA tests are undertaken;

(3) On the w hole, DNA genetic tests are mostly directed to relat ively rare Mendelian genetic disorders to

which GPs have little exposure; and(4) The necessary req uirement for genetic counselling needs expertise, a s well as resources, particularly

time, to deal with the patient and family. It should be noted that genes (ie, DNA) are shared between

family members, so that any DNA test result for a genetic disorder will automatically have implications for

other fa mily members.

For a more detailed version of this article (including referen ces and further samp le case), please go to the college web site at

www.rcpa.edu.auClick on Publications and Forms then Common Sense Pathology. Or visit www.australiando ctor.com .au

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Utility of DNA genetic testingTwo unique properties of D NA set genetic DNA tests apart from other pathology tests:

(1) Any tissue can be used, including blood, tissue scrapings and hair follicles, since DNA is the same in all tis-

sues in the body. This also allows DNA to be used for first trimester prenatal diagnosis, as well as testing for

various genetic diseases; and

(2) DNA is the same from conception to death. This allow s DN A testing to predict the onset o f a genetic dis-

order years before signs or symptoms a re present.

A DNA test can be used for many purposes, from confirming a clinical diagnosis or predicting a patients

response to treatment, t o predicting the likelihood of developing a disease in the future. Five broad classes of

DNA tests can be identified (see Table 1). Some DNA tests are straightforward , eg, diagnostic, w hile others

are more complex, eg, screening and predictive DNA tests. The complexity arises because of the predictive

Table 1: Five classes of DNA tests

*For simplicity, the terms predictive and presymptomatic DNA testing will be considered as the same.

Examples

A case of venous thrombosis in a youngpatient with no other predisposing factorshould be investigated for an underlyingcause, including the Factor V LeidenDNA mutation. Finding this mutationprovides a possible cause for the venousthrombosis.

In a fetus at risk of a genetic disorder,chorionic villus sampling or amniotic fluidsampling provides a source of fetal DNA.In some circumstances, pre-implantationgenetic diagnosis allows a diagnosis tobe made in the developing pre-embryoin association with IVF.

Various screening options are available:i. Pregnancy screening; ii. Newbornscreening; and iii. Community screening.

i. The DNA predictive test for Hunting-tons disease is used to identify whetheran at-risk individual has inherited the

mutant or normal gene from an affectedparent; and ii. Individuals with familialbreast cancer can be tested for BRCA1and BRCA2 mutations to guide them infuture treatment decisions, and identifyrisks in family members.

Individuals exposed to anaestheticagents can develop life-threateningcomplications such as malignanthyperthermia.

Explanation

Confirms that the patient has asuspected disease. The DNAtest in this circumstance iscomparable to most otherpathology tests, although apositive result has implications forfamily members.

Detects a genetic disorder in thefetus or embryo.

Investigates asymptomaticindividuals or populations todetermine carriers or those with agenetic predisposition.

Predicts the development of agenetic disorder in advance of anysigns or symptoms.

DNA test involving drugmetabolism pathways to predict apatients response to treatmentand so individualise drug dose.

Class of DNA test

Diagnostic

Prenatal

Screening

Predictive (pre-symptomatic*)

Therapeutic(pharma-cogenetic)


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Case study 1A 50-year-old man of Celtic ethnic origin comes to see you because he is tired. You suspect

haemochroma tosis and order a serum ferritin, w hich is elevated a t 2000 g/L. This is confirmed on

repeated testing. You next order a H FE DNA test, which shows the presence of homozygous C282Y.

Question 1: What are the implications of the HFE DNA test result for the patient?The clinical suspicion has now been confirmed. Treatment should start. Liver biopsy is indicated only if it is

necessary to assess the patient for liver damage. Specialist referral is indicated because life-long treatment will

be needed. There remains a risk for complications, such a s cirrhosis and hepatocellular carcinoma.

Table 2: Clinical significance of HFE mutations

Phenotype (ie, clinical or laboratory features)

No clinical significance to the patient

This is the usual mutation found in patients with clinicalhaemochromatosis. If present in a patient with features ofhaemochromatosis, it confirms the diagnosis.

This is another combination associated with the develop-ment of clinical haemochromatosis, although the severityin this case is considerably less than what is seen with

homozygous C282Y

There is some doubt about the significance of these muta-tions in clinical haemochromatosis. It is best to seek expertadvice about the implications of these test results forindividual cases.

Genotype (ie, DNA mutation status)

Heterozygosity for any of the threemutations

Homozygous C282Y*

Compound heterozygote for C282Yand H63D*

Homozygous H63D and the S65C*mutation alone, or in combination withother HFE mutations

*Abbreviations: C282Y cysteine is replaced by tyrosine at amino acid 282. Histidine is replaced by aspartic acidat position 63 and serine is replaced by cysteine at position 65.

Table 3: Indications for ordering HFE DNA testing

Medicare prerequisites

The patient has an elevated transferrin saturation orelevated serum ferritin on testing of repeatedspecimens

The patient has first-degree relative with:i. Clinical haemochromatosis; ii. Homozygosityfor C282Y, or a compound heterozygote for HFEmutations

Clinical or laboratoryscenario

Patient has clinical orlaboratory features ofhaemochromatosis

Testing first-degree familymembers who do nothave clinical or laboratoryfeatures of haemochro-matosis

Class of test

Diagnostic

Predictive


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Question 2: What are the implications of the HFE DNA test for a patients family?

There are several options here. Relatives could be screened with ferritin levels, or alternatively gene

H FE testing. In this particular fa mily (Figure 1), the patient w ith D NA proven clinical haemochromato-

sis who came to see you is II-1 (). He has three brothers and one sister. After the family has agreed to

predictive DNA testing, it is evident that two brothers (II-3 and II-4) have genetic haemochromatosis.

These two should be followed carefully with regular estimations of serum ferritin. A third brother (II-6)

is a compound heterozygote, and so at lesser risk, but should also be followed. One sister (II-7) has

refused testing and so her genetic status is unknown, although she is at less risk of clinical haemochro-

matosis because of her sex. Incidental findings in this family include, firstly, II-5, the wife of II-4, also

having genetic ha emochroma to sis. She needs her ferritin levels checked. All children of II-4 w ill have

genetic haemochromatosis. They should be warned about this, especially the male children. A second

finding is that I-2, the living father of II-1, is heterozygous for C282Y and therefore the deceased

mother must have been a double heterozygote for C282Y a nd H 63D. This can be concluded even w ith-out a D NA test (II-6 could have inherited the H63D mutation only from her) and she must a lso have

been heterozygous for C 282Y to have sons who a re homozygous for this muta tion.

Figure 1: Haemochromatosis famil y and DNA testingInterestingly, DNA testing of II-5 would not have been funded by M edicare becausethis individual does not satisfy the prerequisites for an HFE DNA test (Table 2). Therisks for genetic haemochromatosis in t he children of II-1 (your patient) cannot beaccurately determined w ithout testing t he mother (II-2), who is not eligible forMedicare benefits f or an HFE DNA test. At present, all that can be said is that allchildren of II-1 are at least obligatory carriers of the C282Y mutation.

??

1

I

II

1 2 3 4 5 6 7

2

C282Y H63D


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Case study 2Another 50-year-old ma n, this time of G reek ethnic backgro und, com es to see you a w eek later,

presenting with tiredness. Again, you suspect haemochromatosis, but on this occasion the ferritin

level is marginally raised, even on repeat testing. You order the HFE DNA test and the result comes

back normal, ie, no mutation found.

What are the implications of this DNA test result for the patient?Interpretation of this DNA result is more difficult because of the ethnic background of the patient. The

C282Y muta tion is more commonly found in those of north-western European b ackground, so a nega-

tive finding in a patient of southern Mediterranean origin does not necessarily exclude genetic

haemochromatosis. As with other DNA genetic tests, a positive result is much more meaningful than a

negat ive one. C linical haemochromato sis is not excluded b y the D NA test a nd the pat ient requires fur-

ther investigation.

Future challengesMethodology: the methodologies of testing are improving continually, making DNA testing more versa-tile, a nd G Ps w ill be faced w ith an increasing number of o ptions for D NA tests.

Role of the GP and specialists in DNA testing: rare diseases, including those for which predictive testingis being sought, will continue to remain restricted to referral by genetics specialists. However, common

diseases or tho se diseases requiring long-term follow -up require the GP t o be involved in the DN A test-

ing in some capacity.

Duty of care:this is an interesting medicolegal dilemma that remains unresolved. Because DNA is sharedw ith other fa mily members, w here does the doctors duty of care end in terms of ad vising or w arning

others about their risks? In the US, one judge has ruled that the doctors duty of care stops with t he

patient and t hat advising the pat ient tha t the fa mily is at risk is sufficient. H ow ever, this judgment w asreversed on appeal.

Continuing education: information abo ut new genes and their effects on health a nd w ellbeing w ill con-tinue to emerge as information ga ined fro m the H uman G enome Project becomes translated into know l-

edge abo ut how genes w ork. This will be a challenge for continuing educat ion and, in the case of DN A-

based genetics, it w ill be necessary t o use Internet a nd comput er-ba sed resources to ga in regular

updates.

Sourcesn www.ncbi.nlm.nih.gov/Omim/searchomim.html This is the web address for Online Mendelian Inheritance

in Man, an invaluable and free Internet resource providing information about all genetic disorders.

n www.hgsa.com.au This is the Internet site for the Human G enetics Society of Australa sia. A list of all DN A

tests conducted in Australian laboratories can be found at this address.n Harrison SA, Bacon BR. Hereditary hemochomatosis: update for 2003. Journal of H epatology2003;

38:S14-S23.n Mullis KB. The unusual origin of the polymerase chain reaction. Scienti fi c American1990; 262:56-65.n The Medical Journal of Australiaseries on the new genetics, ie, DNA-based genetics, provides insights into

various a spects of genetics and clinical pra ctice, including:

Trent RJ A, Williamson R, Sutherland G R. The new genetics and clinical pra ctice. MJA 2003; 178:406-09.

Ma nn L. The general pra ctitioner and the new genetics. M JA 2003; 179:109-11.

McCusker EA. The specialist neurologist and the new genetics. M JA 2003; 179:167-69.

Haan EA. The clinical geneticist and the new genetics. M JA 2003; 178:458-62.

Otlow ski MFA, Williamson R . Ethical a nd legal issues and the new genetics. MJA 2003; 178:582-85.

Barlow -Stewart KK, G aff CL. Working in partnership with support services in the era of the new genetics.

M JA 2003; 178:515-19.

dna genetic testing, mar04, prof ron trent

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