In January, 2001, the two mutations that cause FD were identified. These mutations cause part of the gene to skip, resulting in a shortened form of IKAP protein. Understanding how the mutation causes the skipping is necessary for figuring out how to get cells to ignore the skip; figuring out how to successfully turn off the mutation will bring about the cure to FD. Such was the goal of Dr. Gil Ast, FD Hope-funded researcher at Tel Aviv University in Israel, who in April, 2004 published two articles describing in part how the FD mutation causes its effect.

Genes are the basic unit of heredity. Each cell in our bodies contains two copies of eachofourgenes, one inherited from each parent. Genes are made up of DNA, chains as beads on a string. They act like templates for making proteins, the molecules which carry out the work in cells. A gene has areas that are used to make proteins (exons) and other areas (introns) that help guide the process of protein production. Most genes are made up of many exons, separated by introns.

The steps necessary to produce a protein from a gene are very complex but consist of two main steps, known as transcription and translation. The process of transcription and translation is known as gene expression. When a gene undergoes transcription, the information stored in its DNA is transferred to a molecule called RNA (ribonucleic acid) in the cell nucleus. RNA and DNA are both chains of nucleotide bases, although they have different chemical properties. The messenger RNA (mRNA) carries the information for making a protein out of the cell nucleus, where the DNA lives, into the main part of the cell where translation occurs. During the process of creating mRNA, the introns of a gene are spliced out and the exons are pasted together.

The mRNA combines with a ribosome complex, which reads the chain of mRNA bases. Proteins are made up of hundreds or thousands of amino acids, which are strung together in chains. The ribosome and a second type of RNA called transfer RNA build the protein one amino acid at a time by substituting an amino acid for each sequence of three nucleotide bases (called a codon). The protein is finished when the ribosome finds a "stop" codon (three base pairs that do not code for an amino acid).

The exact function of the IKAP protein is not clearly understood, but it is believed to play a role in transcription and it may also have other functions in the cell, such as regulating the cell's response to stress. The "major" or more common mutation in FD is found in an intron segment of IKBKAP called a donor splice site (5' ss) that has direct control over the splicing (or cutting) out of introns and pasting together of exons. Because of the mutation, exon 20 is skipped in the formation of IKAP mRNA. The result is a shorter and dysfunctional IKAP protein.

The splicing is carried out by a series of proteins that make up a splicesome complex, labeled Ul, U2,...U6. These proteins help cut and then paste two exons together, thus peeling away the unneeded intervening intron portion.

In FD, some cells are able to ignore the mutation and produce normal protein, while other cell types (particularly the autonomic and sensory nervous systems) end up producing more mutated proteins and only a small proportion of normal proteins. Determining what controls the differences in gene expression is essential to controlling the mutation's effect.

In the first of his two papers, Dr. Ast identified some of the factors found in the IKAP gene which may cause the mutation to have its effect. One such factor, which seems to influence the function of the IKAP gene expression in FD, is known as an Alu sequence. Alu elements are sections of DNA that are found only in primates (man, apes) and are scattered among introns. Only a small percentage of introns contain Alu elements and Alu elements are found in different stages of becoming "exonized" (i.e.. transforming from an Alu element into an exon).

Because it is nearly an exon, the Alu element in intron 20 of the FD gene may affect the binding of the spliceosome complex to the region of the FD mutation, which may result in the skipping of exon 20. This finding has significant impact for the study of how genes produce proteins, particularly in FD. If the nearly exonized Alu element of intron 20 does indeed influence the effect of the FD mutation on the 1KAP gene, then Dr. Ast's research suggests that only primate neurons would be able to fully express FD in the same manner as humans.

In his second publication, Dr. Ast examines how differences in various positions of the nucleotide bases affect how an exon is spliced. The FD mutation is found in the 6th position (~6) of intron 20's 5" splice site. The mutation in this position. in combination with the nucleotide found in the last position (-1) of exon 20, negatively affects the binding with the splicesome protein called Ul. "We found that base-pairing of this position {+6 } to U1,. . . partially restored the normal splicing of exon 20. . . .." (Carmel, et al, 2004, p. 834-5)

The FD mutation doesn't allow the U1 to bind well and the occurrence of an adenosine (A) nucleotide in the last position of exon 20 contributes to this inefficient binding. "T-to-C mutation at position +6 on mRNA splicing of exon 20 implies that it weakens the 5'ss:Ul snRNA interaction. We then examined whether this weakness could be related to the presence of adenosine at position -1, which mispairs with Ul snRNA." (Ibid, p. 835). The researchers tested this hypothesis by creating a U1 splicesome protein with a mutation ot match the one found in the FD gene, allowing them to bind together well. This designer splicesome protein "restored the splicing of exon 20 in 24% of the splicing events (which is likely the ratio between endogenous and exogenous Ul)." (Ibid., p. 835)

They further evaluated the influence of the nucleotide base at position -1 by changing the nucleotide found in that position of the FD mutation (Adenosine or A) to a more common form (Guanine or G) and found that "..., a mutation of A to G at position -1 of exon 20 restored normal splicing of the mRNA derived from the FD mini-gene. ... it shows that increasing the base-pairing of Ul snRNA to the exonic portion of the 5'ss can compensate for loss of function due to a mispair with the intronic portion, which is in agreement with the exonic-intronic linkage previously shown." (Ibid., p. 835). In other words, despite the mutation found in intron 20 of IKAP. other factors can be manipulated to increase the binding of the Ul splicesome protein, which is needed for normal gene transcription.