Seeing Cell to Cell Differences to Explain Symptoms of Rare Genetic Disorders

Every cell in the body has two genomes, one from the mother and one from the father. Until now, researchers have lacked the tools to examine — in a single cell –the exact readout from each genome to make RNA. Scientists at the University of Pennsylvania Perelman School of Medicine and the Children’s Hospital of Philadelphia (CHOP), have been able to examine a rare disease in which the maternal and paternal genomes are expressed differently throughout the body, sometimes even in the same organ. They found that at the single-cell level, gene expression was highly variable and quite different than expected, which is now shedding light on the molecular causes of rare diseases and perhaps the complex nature of tumors.

“This is a great example of cross-school collaboration,” stated co-senior study author Marisa Bartolomei, Ph.D., professor of cell and developmental biology in the Perelman School of Medicine. “With this new technique, we can now see which cells express which genome.”

Most individuals inherit two copies, one maternal and one paternal. However, in the case of a phenomenon called imprinting, offspring inherit only one working copy of a gene, and depending on its parental origin, expression of the gene is controlled by an added methyl group during egg or sperm formation to tighten DNA physically so it cannot be read to make RNA and ultimately protein.

For a minuscule number of genes, imprinting is a typical phenomenon and required for normal development. However, in human imprinting disorders, imprinted genes are either abnormally turned on for both the maternal and paternal genomes, or abnormally turned off from both genomes. Abnormal expression has been linked to genetic mutations and mistakes in the placement of certain methyl groups (a function of epigenetics).

Variability from cell to cell in which genes are expressed in the same organ may help explain why many diseases show a “mosaic” pattern, with different parts of the body exhibiting varying degrees of disease.

Yet, defects in imprinting can lead to inappropriate expression of the normally silenced version of the gene, but it remains unclear whether every cell in a given organ expresses the abnormal version. “If this were the case, it would have profound implications for human imprinting disorders,” Dr. Kalish noted.

The investigators were interested in two rare imprinting disorders—Russell-Silver syndrome, an undergrowth disorder occurring in approximately 1 out of 50,000 to out of 100,000 births, with symptoms of small stature and limb, body, or facial asymmetry, and Beckwith-Wiedemann syndrome, an overgrowth disorder usually present at birth, characterized by an increased risk of childhood cancer and such congenital features as a large tongue, large birth weight, and limb, body, organ, and facial asymmetry.

The team focused their attention on Russell-Silver syndrome, which involves the undermethylation of two genes, H19, and IGF2. H19 encodes a noncoding RNA that limits growth whereas insulin-like growth factor 2 (IGF2) is a growth factor.

These syndromes display a range of symptoms, which happens because some cells in a given organ express maternal genes while others express paternal genes. “But the mom genes are the ones that limit growth of cells,” explained co-lead study author Jennifer Kalish, M.D., Ph.D., pediatric geneticist at CHOP. “This means that the mosaic pattern is really a mix of normal cells and abnormal cells—with a disrupted balance between mom’s do-not-grow signals and dad’s grow signals, in the same tissue. This is what accounts for an enlarged or too-small organ in the same person.”

Using mice, the team compared cell genomes in normal mice versus Russell-Silver syndrome mice. The researchers found that in Russell-Silver mice both maternal and paternal H19 was expressed.

The scientists went on to develop a molecular probe to detect a single-nucleotide change in the RNA expressed in mom’s versus dad’s genomes—which in essence measured gene-variant specific expression in individual cells. The team found that found that some cells had RNA from both the maternal and paternal version (abnormal, as seen in Russell-Silver patients) whereas other cells had only the maternal RNA (normal).

“We showed that mutant mouse embryo fibroblast cells are comprised of two subpopulations: those expressing both maternal and paternal H19 versions [abnormal] and those expressing only the maternal copy [normal],” Dr. Bartolomei noted. “Only in the latter normal cell population is Igf2 expression detected.”

The findings from this study were published recently in Genes & Development in an article entitled “Visualizing Allele-Specific Expression in Single Cells Reveals Epigenetic Mosaicism in an H19 Loss-of-Imprinting Mutant.”

“This is the first time that epigenetic mosaicism has been demonstrated at a single cell level,” Dr. Bartolomei remarked. “What this means is that epigenetics—the balance of tightening or loosening of DNA to control which genes are expressed when—is the driver of mosaicism at the cell population level. Our results establish that imprinting disorders can display striking single-cell heterogeneity and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.”

“The epigenetic mosaicism shown in this work explains the spectrum of clinical features we see in our patients—it all makes sense,” Dr. Kalish concluded. “Now we know what is going on.”