The first comprehensive study of gene activity in the X chromosome of women reveals an unexpected level of variation among individual females. This extensive variation means there is not ONE human genome, but TWO – Male and Female.

Men routinely complain that they just can’t figure women out, and now there is scientific evidence to support their feelings. Genetic research supported by the National Institutes of Health reveals the puzzling and unpredictable influence of women’s second X chromosome, and turns a long-held assumption into a falsehood.

Chromosomes are the set of genetic instructions that guide the creation of an organism. Every human embryo begins with two X chromosomes, but in order to be a male, one of the X chromosomes turns into a Y chromosome. The Y chromosome is solely responsible for all the characteristics that make men male, including male sexual organs and the ability to produce sperm. Conversely, women retain two copies of the X chromosome.

Many genes on the Y chromosome have been lost over evolutionary time, leaving the chromosome with fewer than 100 functional genes. In contrast, the X chromosome – present in at least one fully functional copy in both sexes – encodes more than 1,000 genes. Since the X chromosome carries a larger instruction manual than the Y chromosome, biology’s solution to such excessive genetic coding is to switch off genes, a modification referred to as X-inactivation, on the females’ second X chromosome.

More than 45 years ago researchers discovered that the genes on one copy of the female’s X chromosome undergo inactivation. It has been previously taken for granted that the inactivation process resulted in a complete silencing of the genes on the second X chromosome in order to leave both sexes with the same activity level, or amount, of the genes encoded on the X chromosomes. Scientists had also implicitly assumed that the X chromosomes in all women were identical.

However, the second X chromosome is not fully inactivated as previously believed. “Our study shows that the inactive X in women is not as silent as we thought,” said Laura Carrel, Ph.D., assistant professor of biochemistry and molecular biology, Penn State College of Medicine, Penn State Milton S. Hershey Medical Center. “The effects of these genes from the inactive X chromosome could explain some of the differences between men and women that aren’t attributable to sex hormones.”

Depending on the gene, having two active copies can matter very little or very much. When genes on the second X chromosome that escape inactivation are expressed, this can create a stronger overall concentration of particular genes. Carrel and her co-author, Huntington F. Willard of the Institute for Genome Sciences & Policy, Duke University, employed two testing models in separate labs to better verify and validate the results.

In the first lab, the researchers isolated which genes were escaping inactivation and where they were located on the second X chromosome. Using primary skin cells, the researchers compared gene expression between the primary X chromosome and the secondary X chromosome for 94 genes spanning the X chromosome in 40 human samples. They also measured the activity level of each of the 471 genes to determine whether the second copy was turned on or off. Only 65 percent of the genes were inactive in all samples. Twenty percent were inactivated in some samples but not in others, and 15 percent escaped inactivation in all samples. Additionally, many of those from the second X chromosome were only partially expressed. Furthermore, the proportion of genes that remain active differs dramatically among regions of the X chromosome. Also, in some women but not others, an additional 10 percent of the X-linked genes demonstrate variable patterns of inactivation and different levels of activity in the second X chromosome.

In the other lab, other cell lines were used to compare inactive X chromosomes and the genes expressed from the inactive X chromosome were recorded. Of the 624 genes on the chromosome that were tested, 16 percent of the genes on the second X chromosome escaped inactivation, which confirmed the first model’s lab results.

“We now know that 25 percent of the X chromosome – 200 to 300 genes – can be uniquely expressed in one sex relative to the other,” Willard said. “In essence; therefore, there is not one human genome, but two – male and female.” This data strongly suggests that the female genome differs from the male genome in at least three ways. First, previous studies had shown that the Y chromosome gives males several genes that are absent in females. Second, since some genes on the second X chromosome are expressed means that about 15 percent of genes are expressed at higher levels in females than they are in males. The incomplete nature of X-inactivation means that at least 15 percent of X-linked genes, and their protein products, are present not only in differing levels as compared to males, but also at more variable levels in females compared to males. Third, this study shows that an additional 10 percent of genes on the second X chromosome are expressed in variable ways between females, whereas men only have a single copy of these genes. “Such characteristic genomic differences should be recognized as a potential factor to explain sex-specific traits both in complex disease, as well as normal gender difference,” Willard said.

A surprising revelation from this research is that there is more variability among females than scientists thought. “We looked at the X chromosomes of 40 women and every one of them had a unique pattern of gene expression,” Willard said. “All of that variation is completely unique to women. The X chromosomes of males are all the same in this regard.”

Something else they found is that most of the still active genes were grouped together. “This tells us that neighborhoods matter,” Carrel said. “Genes on the X chromosome evolved in five sequential segments or layers. The older segments have fewer genes that escape inactivation than those that developed later in the chromosome’s evolutionary path. This suggests that, as the human species continues to evolve, more and more of the genes that are escaping inactivation may lose their ability to do so.” In other words, as evolution eliminates the need for certain genes, they will increasingly continue to be inactivated in women’s second X chromosome, whereas other genes will continue to defy inactivation.

“Although we’ve shown sex-specific differences, the clinical implications remain unexplored,” Carrel said. “We can; however, conclude that these differences should be recognized as potential factors for explaining normal differences between the sexes, but also gender differences in how certain diseases are manifested, progress and respond to treatment. Further studies will be required to establish such a role for these genes.”

Earlier work in the late 1980s by Willard and others shattered some first assumptions by showing evidence that some portions of the genes on the second X chromosome in women remained active. The new work of this 2005 study titled, “X-inactivation profile reveals extensive variability in X-linked gene expression in females,” extends these earlier findings to the full set of the X-linked genes and also further reveals that individual women exhibit extensive differences among them with respect to X inactivation. This study was first published in the March 17, 2005 issue of the journal Nature. In the same issue, more than 250 researchers, including Willard and Carrel, reported the complete DNA sequence of the human X chromosome.