Fanconi Anemia Genetics - PGD . . .

Mutation Analysis

Introduction

Knowledge of the exact nature of the mutations carried by your family has many uses. First, researchers are starting to correlate mutations to the severity of the disease (correlating genotype to phenotype, to be technical). Knowledge of the mutations affecting your child can therefore be useful in planning treatment. Knowledge of the exact mutations is also essential for preimplantation genetic diagnosis, access to gene therapy trials, carrier detection, and prenatal diagnosis.

Remember that it is unlikely that you and your partner have exactly the same mutation--you most likely each have a different kind of pathogenic mutation. If you are carriers of mutated FANCC genes, it is somewhat more likely that you could have the exact same kind of mutation--but this needs to be verified. The analysis will require blood samples from both parents and the affected child. The analysis will identify mutations in the FA genes of the child, then trace that mutation back to which parent contributed it.  

For use in PGD, this information on the mutation is used to setup a genetic analysis system for the biopsies done to your embryos (see section on system setup on the main PGD page).

FANCC Mutation Analysis

If you and your partner are carriers of pathogenic mutations in the FANCC gene, you will have an easy time having the mutation analysis done for your family. There are only 10 known mutations to the FANCC gene and it is quite easy to test for all 10. You will find many labs willing to do this analysis.

FANCA & Other Mutation Analysis

If you and your partner are carriers of pathogenic mutations of the FANCA gene--and you probably are--you will encounter some discouragement along the way to getting your mutation analysis done.  When you enquire to research labs, you may receive responses like the following:

". . . identification of the precise mutation(s) carried by your family is a much more complex matter, and is generally a procedure which would take many months. The identification of mutations in the FA subtype A gene (the most common subtype) is very difficult, since the gene is so large. Also, once a putative mutation is identified, it must be confirmed by linkage analysis or by functional tests. So in short, even when we find a putative mutation in an FA gene in your family, we cannot be absolutely sure that this mutation is the cause of the disease in your family."

or you may read:

". . . Once the complementation group has been identified, the responsible mutation can be determined by DNA sequencing in research laboratories. Direct sequence analysis is not clinically available given the number of possible affected genes, the large number of possible mutations in each gene, and the large size of many of the FA genes."

In short: it's too hard, it's too big, we can't be sure it's a pathogenic mutation

I think these statements are too discouraging. Technology developed for the human genome project has made genetic sequencing fast and cheap. Machines used to sequence DNA, called capillary electrophoresis-based sequencers, such as the ABI 3700 (from Applied Biosystems, Inc) can sequence 500-600 bases every 2-3 hours. You can have the Fanconi genes in your family sequenced to look for mutations.

How big is FANCA compared to FANCC (which is considered easy to do mutation analysis on)?  Here is a chart summarizing the technical information on all the Fanconi genes:

So how big is FANCA?  It's about 3 times the size of FANCC. FANCD2 is about the same size as FANCA. The others are similar in size to FANCC, except BRCA2, which is much larger. You may have done the math on the sequencing time of FANCA and are saying to yourself, "4365 bases divided by 250 bases per hour, that should take 18 hours to complete!"  That doesn't sound like a long time, does it? Of course, you have to do it six times (twice for mother, father, and child). The truth of the matter is that it does take longer, because of the sample preparation and PCR amplifying that needs to be done. Sequencing of FANCA takes 10-12 weeks to complete by a diligent lab. It's not hard. It's not too big.

So do not be discouraged in your quest to have a mutation analysis done for you family!

Can you be assured of success in finding the pathogenic mutations? Unfortunately, no--but do not be discouraged. The mutation analysis is still worth pursuing. You can be assured that you will know a lot more about your Fanconi genes at the end of the analysis than you do now. 

Why might the analysis not find the pathogenic mutations? First, the technique of sequencing is not useful when the mutation is a large deletion (other techniques, however, can look for deletions). Second, your family may have a "private" pathogenic mutation--a mutation that has never been associated before with Fanconi Anemia.

However, there is a database of known pathogenic mutations--the Fanconi Anemia Mutation Database at Rockefeller University. Your mutations could be already known in the database. The database also tracks polymorphisms--which are mutations which do not cause disease. You might find the pathogenic mutation contributed by, say, the father, and not discover the one contributed by the mother. You may, however, be able to track inheritance of the gene contributed by the mother via a polymorphism on the gene contributed by the mother. This would be adequate for performing PGD.

Recall the uses of the information from a mutation analysis: predicting the severity of the disease, PGD, carrier detection, access to gene therapy trials, and prenatal diagnosis. Not having knowledge of both pathogenic mutations would inhibit your ability to predict the severity of the disease. I can't comment on access to gene therapy trials, as I don't know the requirements. But you'll likely be able to do PGD, carrier detection, and prenatal diagnosis. You can also use another analysis technique--linkage analysis--for PGD (linkage analysis, in fact, is used for the HLA genes if you are also testing for a tissue match).

See the section at the bottom for further discussion of how the mutation analysis can use polymorphisms to track inheritance.

Labs for Mutation Analysis

Talk to your PGD provider first. They may be able to recommend a lab, or have a lab they prefer to work with. However, if your PGD provider recommends a lab, be sure to talk to that lab to understand what kind of committment you'll get for getting your analysis done in a timely manner. Make sure your case is pursued diligently and is not simply a research project (pursued on "research time").

The labs listed below are all outside of the United States. Shipping blood is not a problem between countries. I recommend you use Federal Express for your blood shipments. They have shipping instructions for non-infectious blood on their web page--or email me for simpler instructions. 

How much does the analysis cost?  For FANCA, expect to pay $3000-$4000 for the analysis--less (say, $1000) if the pathogenic mutations are found early in the analysis. Many other labs than the ones listed below can test for FANCC.

Gene Analysis Service, GmbH
Goerzallee 253
D 14167 Berlin
Germany
Email: info@gene-analysis-service.de

The principle scientist here is Dr. Alfred C. Looman. You can correspond with him regarding your case via email. He will answer you promptly and is very patient in answering all of your questions. Mutation screening for FANCA takes 10-12 weeks (less if the mutations are found early in the analysis).

Free University Medical Center
Amsterdam, The Netherlands
Dr. Gerard Pals
Email: g.pals@vumc.nl

This lab offers mutation analysis for the seven known FA genes. Turnaround time is 1-3 months.  Patients for whom no mutations can be found are referred to the research division at no further cost. This lab is affiliated with Dr. Hans Joenje, one of the pioneering researchers in FA. They also use a technique that can identify large deletions.

Analysis Report

The outcome of your mutation analysis work will be an analysis report that should cover the methods used in the analysis, a summary of the results, a table summarizing the mutations/polymorphisms found and their location in the gene, and then may also include detailed information on the sequencing data for each mutation/polymorphism.

Notes on the Analysis and Polymorphisms

The goal of the mutation analysis is to determine which pathogenic mutations the child carries, and then determine which parent contributed which mutation. Note that a "mutation" is any change to the reference genetic sequence--it doesn't mean that it's harmful. That's why I'm always writing "pathogenic mutation"--a mutation that causes disease. Those mutations that do not cause disease are called polymorphisms--they are just benign differences in the genetic sequence of the individual compared to the reference sequence. Where does the reference sequence come from?  Either from the researcher who first discovered the gene, or from the Human Genome Project.

If you look at the Mendelian cross tables, this seems simpler than it is. From the cross tables, the father is Aa, the mother is Aa, and the child is aa. So obviously the child inherited an "a" for each parent. This is good enough for education and population statistics, but it's not specific enough for tracking a mutation through your family.

The reality is that there are 4 unknown genes involved.  For simplicity, let's say the father's two genes are denoted AB.  The mother is CD. The child can be four possibilities: AC, AD, BC, BD. At the beginning of the analysis, you have no idea which two genes the child has inherited--the child is "??" at that point. To simplify the explanations below, let me refer to the child's genes as EF--the analysis will determine if "E" is really A or B and if "F" is really C or D.

So the analysis starts by sequencing both chromosomes containing the gene of the child. A mutation to the reference genetic sequence is discovered in one of the child's genes, let's say it's E. You then sequence the same section of the gene in the parents--all four genes, A, B, C, D. If the mutation is only found in one of the parents genes, you have discovered the identity of E. Let's say the mutation was only found in A--this means that "E" is really A. This sounds easy, but it gets harder.

Let's say you continue sequencing the child's genes. You find a mutation in F. You find the same mutation in E (which we now know is really A)--the child is homozygous for the mutation. This mutation is not informative--you can't use it to differentiate between E and F (since it is the same in both).

So you continue to sequence the child's genes. You find a mutation in F that is not present in E (child is heterozygous for it)..  You then sequence the parents genes. The same mutation is found in A and C. This is also not informative--you can't determine which parent contributed it, since both parents have the same mutation.

You continue on once again. You find a mutation the child is heterozygous for. You sequence the parents genes in the same section--the mutation is only found in C. Victory! The identity of "F" is really C. The child is AC.

Note that in the above discussion, I never referred to the mutations as "pathogenic". The entire procedure above can be done--and usually is done--with polymorphisms. So you determine inheritance of genes via polymorphic mutations. So you don't need to know the mutation causing disease (the pathogenic mutation)--you can track inheritance via polymorphisms. This will let you do PGD, carrier detection, and prenatal diagnosis. It's desirable to know the pathogenic mutations--it would increase the accuracy of PGD and give you information on the severity of the disease. Remember that the child is unlikely to be homozygous for a pathogenic mutation--it is likely the mutation in each copy of the gene is different, so the child is heterozygous. You may discover the pathogenic mutation in only one gene and will have to use polymorphisms to trace the inheritance of the other.

How are pathogenic mutations identified?  By comparing the mutation sequences to a database of know pathogenic mutations. These pathogenic mutations were discovered by researchers during past research--from studying how the mutation affects the cells.

Note to genetics professionals: I know the exact procedure to identify the genes inherited by the child from the parents is more complicated than my presentation above.  I hope, though, the above is adequate to give a flavor of the analysis that has to be done to trace genes back to parents.

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Last updated: 07 Feb 2004