DNA and You

Mutations in RAD51C: A New Genetic Cause for a Fanconi Anemia-Like Disorder

Today in an advance online publication at Nature Genetics, Christopher Mathew, Helmut Hananberg, Detlev Schindler, and colleagues from an international collaboration report the identification of a new cause for a Fanconi anemia-like disorder (that ultimately may be recognized as a new genetic cause for Fanconi anemia).

Fanconi Anemia

Fanconi anemia is a rare condition that includes various physical abnormalities, bone marrow failure (low counts of blood cells because the bone marrow is not churning them out like it should), and an increased risk for certain types of cancer.  The physical abnormalities are present in about two-thirds to three-quarters of affected individuals and can include the following:

• Short stature
• Malformations of the thumb, forearm, and other parts of the skeleton
• Malformations of the kidneys and/or urinary tract
• Male genital malformations
• Gastrointestinal tract malformations
• Malformations of the heart
• Hearing loss
• Central nervous system abnormalities
• Abnormalities of skin pigmentation (color)
• Other abnormalities and malformations

Although there is a wide variety of both major and minor malformations in Fanconi anemia (FA), the fact that they are not present in everyone with FA means that their absence cannot be used to rule out the disorder.

Most individuals with FA develop bone marrow failure leading to decreased counts of blood cells (platelets, white blood cells and/or red blood cells before the age of 10).  However, the blood count problems can also develop later in life. 

Unfortunately, there is a very high risk of several types of cancer in Fanconi anemia.  By the fifth decade of life, approximately one in ten to one in three people with FA will have developed a blood cancer (particularly acute myeloid leukemia), and about three in ten people will develop a solid tumor (non-blood cancer), especially those of the head and neck and GI tract.

Diagnosis of Fanconi Anemia

When doctors suspect FA, they order a test that looks for certain chromosome abnormalities after cells from the patient are treated in the lab with something called a "DNA interstrand cross-linking agent".  The genetics of Fanconi Anemia are complicated by the fact that FA can be caused by mutations in a number of different genes.  In fact, mutations in at least 13 different genes have been known to cause FA.

Fanconi Anemia Genes

• FANCA
• FANCB
• FANCC
• BRCA2 (FANCD1)
• FANCD2
• FANCE
• FANCF
• FANCG
• FANCI
• BRIP1 (FANCJ)
• FANCL
• FANCM
• PALB2 (FANCN)

For all but one of these examples, inheritance of FA occurs in an "autosomal recessive" manner.  This means that an affected child must inherit a mutated copy of the gene from each parent.  Thus, the parents are FA carriers, but don't have FA themselves (although being a FA carrier can have significant implications for cancer risk if the FA gene is BRCA2, PALB2, or BRIP1!!).  The lone example of non-autosomal recessive inheritance in FA is X-linked inheritance in FA due to FANCB.

Study of a Family with a Distinct Fanconi Anemia-Like Disorder Not Explained by the Previously Known FA Genes

Christopher Mathew and colleagues studied a family in which three children had multiple severe congenital anomalies that were consistent with FA.  The parents of the affected children were first cousins and were of Pakistani origin.  Two of the affected children were shown to have elevated chromosome abnormalities when their cells were treated in the lab with a DNA interstrand cross-linking agent.  Given the congenital anomalies in the children and the results of the lab tests, the children were diagnosed with a Fanconi anemia-like syndrome (the authors elected to call it this rather than Fanconi anemia because none of the children had yet developed any abnormal blood cell counts).

The researchers then did several different types of tests on cells from the patients to determine whether their FA-like disorder was due to a problem with one of the 13 known FA genes.  The tests suggested that a different gene was behind the disorder in this family. 

Because the parents were cousins, the researchers chose to use a strategy called "autozygosity mapping" to identify the region of the genome responsible for the FA-like disorder.  This allowed them to focus on a region of chromosome 17 where they ultimately were able to determine that mutations in the RAD51C gene were responsible. 

Their assertion that RAD51C was the culprit was supported both by evidence that the amino acid changed by the mutation in this family is evolutionarily conserved.  In this family, the mutation leads to the substitution of the amino acid histidine for arginine at a key area of the protein.  Across species, this amino acid is an arginine in RAD51C, RAD51, RAD51B, or the related protein XRCC3, whether the protein is in a human, a chicken, a zebrafish, a sea urchin or thale cress (which I just had to Google to determine it is Arabidopsis thaliana, a small flowering plant that is often studied in biology labs). 

The researchers were also able to prove that RAD51C was the culprit here by reinserting a functioning normal RAD51C gene into cells with the mutations; this led to correction of cellular abnormalities observed in the cells from the patients in this family.

RAD51C

As RAD51C, like previously known FA genes, was already known (from studies of its function in the lab) to protect against hypersensitivity to DNA interstrand cross-linking agents (the key cellular abnormality seen in humans with FA), it is perhaps not so surprising that it turns out to cause the syndrome found in the family that Mathew and colleagues studied.  However, this appears to be the first time that mutations in this gene have been shown to cause a human disorder (this study led to another published online in Nature Genetics today demonstrating that heterozygous mutations in RAD51C are associated with Hereditary Breast and Ovarian Cancer - we've covered that paper now at Cancer and Your Genes).  Although it might have been expected that mutations in this gene would be a more common cause of FA-like disorders or FA, it has been shown that loss of the gene in mice is associated with embryonic lethality (i.e., is not compatible with life).  This could explain why inherited mutations in this gene have not been seen more commonly in human disease.

Key References

Vaz F, Hanenberg H, Schuster B, et al.  Mutation of the RAD51C gene in a Fanconi anemia-like disorder.  Nature Genetics 2010; published online 18 April 2010; doi:10.1038/ng.570

Auerbach AD, Buchwald M, Joenje H.  Fanconi anemia.  In Scriver CR, Beaudet AL, Sly WS, et al., Eds., The Metabolic & Molecular Bases of Inherited Disease, 8th Ed.  New York, McGraw-Hill, 2001, pp 753-68.

Taniguchi T.  Fanconi anemia.  In GeneReviews (online resource), Last Revision: March 27, 2008.  Accessed April 18, 2010.

New Blog: Cancer and Your Genes

I've started another blog, "Cancer and Your Genes," which can be found here.  It will be focused on the intersection of our genomes and cancer risk, with particular emphasis on educating the lay public about genetic risks for cancer.

Mountains Out of Molehills? Or the Real Deal?

Despite some significant challenges in bringing effective personalized medicine approaches to the clinic, cancer treatment is one area in which personalized medicine and molecularly-targeted therapies have begun to come to fruition already.  In addition to Gleevec, which effectively targets the BCR-ABL fusion protein that is characteristic of chronic myelogenous leukemia (CML) in humans, Herceptin is important in the treatment of the subset of breast cancer patients whose cancers have substantial expression of the "Her2/Neu" protein.

However, these molecularly targeted therapies that focus on specific proteins important to the causation and maintenance of a subset of cancer cases are only available for a small percentage of all cancers.  To move closer to truly targeted therapy (which hopefully would have less severe side effects than current approaches with relatively non-specific cytotoxic drugs and radiation), we must know more about the relatively complicated changes in the DNA of the cancer cell.

Although it frequently seems like the more we learn about cancer the more complex the problem becomes, a paper published in the online edition of Science today makes a significant contribution to our knowledge of what goes awry in the development of breast and colon cancers.  (Although the full paper is available only by subscription, there is a press release with more information about the findings at the HHMI website) 

In this work, groups led by Giovanni Parmigiani, Kenneth Kinzler, Victor Velculescu, and Bert Vogelstein utilized a high-throughput DNA sequencing approach to look at the coding sequences (the DNA sequence coding for protein sequence) of more than 18,000 genes in each of 11 different breast cancer cases and 11 colon cancer cases.  Utilizing a careful approach to determine that mutations occurred somatically in the development of the cancer (as opposed to being polymorphisms present in all of the DNA of the individual at birth), the study basically showed that an impressively high percentage of the genes had a non-silent mutation (i.e., one that altered the protein coding sequence) in at least 1 cancer case: 9.4% of the total number of genes analyzed.  Putative cancer-related genes were evaluated in another set of cancers.  There were a total of 280 genes (equally distributed between breast and colon cancers) in all that were validated by the presence of mutations in both the initial cancer set and the validation tumor set.  Further analyses were performed to assess the plausibility that these genes were mutated more often in the cancers than would be predicted by chance. 

Although it is not news that cancer is a genetic disease associated with the accumulation of mutations in several key genes (that presumably differ by tumor/tissue type), the high-throughput resequencing approach utilized in this work suggests much more complexity in the mutated cancer genome than was previously recognized.  By plotting a score related to gene mutation frequency for each gene on something resembling a topographical map, the authors point out that while there are certainly still several "mountains" that appear to likely to be very important in cancer development, perhaps the more striking feature is an extremely high number of "hills" (genes mutated at a much lower frequency in a given cancer type, but which still likely play a role in causation or maintenance). 

Although some may suggest that the authors are making mountains out of molehills, I believe that their interpretation is likely correct.  The ability to sequence virtually the entire coding genome of a tumor is very exciting; with the coming drop in sequencing costs, this could be more realistically applied to individual patient tumors in a clinical context.  The hard part will be devising strategies to act on this information in a way that improves clinical outcomes.  However, as the authors suggest, many of the "hills" can be grouped into one of a small number of biochemical pathways.  Future studies will no doubt assess whether drugs targeting these pathways can be applied on a rational basis based on high-throughput sequencing of patient tumor DNA. 

Despite some significant challenges in bringing effective personalized medicine approaches to the clinic, cancer treatment is one area in which personalized medicine and molecularly-targeted therapies have begun to come to fruition already.  In addition to Gleevec, which effectively targets the BCR-ABL fusion protein that is characteristic of chronic myelogenous leukemia (CML) in humans, Herceptin is important in the treatment of the subset of breast cancer patients whose cancers have substantial expression of the "Her2/Neu" protein.

However, these molecularly targeted therapies that focus on specific proteins important to the causation and maintenance of a subset of cancer cases are only available for a small percentage of all cancers.  To move closer to truly targeted therapy (which hopefully would have less severe side effects than current approaches with relatively non-specific cytotoxic drugs and radiation), we must know more about the relatively complicated changes in the DNA of the cancer cell.

Although it frequently seems like the more we learn about cancer the more complex the problem becomes, a paper published in the online edition of Science today makes a significant contribution to our knowledge of what goes awry in the development of breast and colon cancers.  (Although the full paper is available only by subscription, there is a press release with more information about the findings at the HHMI website) 

In this work, groups led by Giovanni Parmigiani, Kenneth Kinzler, Victor Velculescu, and Bert Vogelstein utilized a high-throughput DNA sequencing approach to look at the coding sequences (the DNA sequence coding for protein sequence) of more than 18,000 genes in each of 11 different breast cancer cases and 11 colon cancer cases.  Utilizing a careful approach to determine that mutations occurred somatically in the development of the cancer (as opposed to being polymorphisms present in all of the DNA of the individual at birth), the study basically showed that an impressively high percentage of the genes had a non-silent mutation (i.e., one that altered the protein coding sequence) in at least 1 cancer case: 9.4% of the total number of genes analyzed.  Putative cancer-related genes were evaluated in another set of cancers.  There were a total of 280 genes (equally distributed between breast and colon cancers) in all that were validated by the presence of mutations in both the initial cancer set and the validation tumor set.  Further analyses were performed to assess the plausibility that these genes were mutated more often in the cancers than would be predicted by chance. 

Although it is not news that cancer is a genetic disease associated with the accumulation of mutations in several key genes (that presumably differ by tumor/tissue type), the high-throughput resequencing approach utilized in this work suggests much more complexity in the mutated cancer genome than was previously recognized.  By plotting a score related to gene mutation frequency for each gene on something resembling a topographical map, the authors point out that while there are certainly still several "mountains" that appear to likely to be very important in cancer development, perhaps the more striking feature is an extremely high number of "hills" (genes mutated at a much lower frequency in a given cancer type, but which still likely play a role in causation or maintenance). 

Although some may suggest that the authors are making mountains out of molehills, I believe that their interpretation is likely correct.  The ability to sequence virtually the entire coding genome of a tumor is very exciting; with the coming drop in sequencing costs, this could be more realistically applied to individual patient tumors in a clinical context.  The hard part will be devising strategies to act on this information in a way that improves clinical outcomes.  However, as the authors suggest, many of the "hills" can be grouped into one of a small number of biochemical pathways.  Future studies will no doubt assess whether drugs targeting these pathways can be applied on a rational basis based on high-throughput sequencing of patient tumor DNA.