De Novo Mutations and Autism

Rates of autism have risen to extraordinary levels over the last several decades. Whereas 50 years ago an autism diagnosis was made for 1 in 360 children, today, autism spectrum disorder affects 1 in every 66 boys. Some of this increase can be attributed to changes in the timing and nature of diagnoses, but even accounting for these factors the rates have risen dramatically and continue to rise. Autism is a difficult disease to study because it is not likely to have a single functional cause. The disease onset is in children and it affects a core human characteristic: it impairs social communication. In trying to identify causal factors that lead to autism, researchers have been focused on environmental agents that interact with heritable traits as possible causes for the disease. The results of these studies have not yet shown a single pathway or mechanism. Researchers are also trying to define distinct mechanisms for different sub-groups with the goal of accounting for the heterogeneous aspects of the disease.

A new study used modern genetic techniques to examine unique properties of autistic children’s DNA that might be associated with the disorder. The authors searched for point mutations that occur spontaneously or what are called “de novo mutations.” These are mutations in the child that are not present in the parents. Researchers at the University of Washington selectively sequenced the exomes (protein coding regions) of 20 families with autistic children by what they called trio-based exome sequencing (TBES). In contrast to relatively coarse micro-array studies, TBES can look with high resolution at individual point mutations in patients.

The study reports that the de novo mutation rate in autistic children does not differ from the rate in controls. However, the de novo mutations in the autistic children were located in crucial places in the genome, and that such de novo mutations do not occur at similar places in controls. The study estimates that the new technique may identify up to 40-50 percent of the genetic causes of the disease.

In several children they found mutations they believed were causative to their diagnosis. The four genes implicated were FOXP1, GRIN2B, SCN1A, and LAMC3. LAMC3 was not previously implicated in autism, but the gene does have some association with the limbic system and frontal cortex; these are brain regions that researchers believe are abnormal in some autistic brains. Looking at the specific mutations that arise spontaneously in autism is an innovative approach to studying autism. As whole genome sequencing becomes more accessible and less expensive, more data can be collected to find all of the genes implicated in autism.

O’Roak et al, “Exome Sequencing In Sporadic Autism Spectrum Disorders Identifies Severe de Novo Mutations”. Nature Genetics. Online May 15, 2011.

Gray L. “Sporadic Mutations Idenitified in Children with Autsim Spectrum Disorders.” University of Washington Press Release. May 16th, 2011.

New Cystic Fibrosis Gene Modifier Loci Discovered

Cystic Fibrosis (CF) is one of the most common hereditary diseases afflicting today's global population. CF causes body-wide defects including exocrine pancreatic inefficiency, male infertility, poor digestion, and excess mucus buildup in sinuses. However, 90% of deaths caused by CF are due to the excess mucosal blockages in pulmonary air channels. Past research showed that the recessive allele mutation of the cystic fibrosis transmembrane conductance regulator protein (CFTR) is the sole culprit of causing CF when inherited from a carrier at the parental generation. Researchers concluded at the time that the recessive allele inheritance of CFTR was the sole reason of acquiring CF, yet the severity of the symptoms a patient suffers with CF can vary. This led researchers to believe that the diverse severity of CF symptoms observed from different patients attributed to secondary factors besides just the CFTR recessive mutant.

This month, researchers from the University of North Carolina, Chapel Hill have discovered two loci that could be the so called secondary factors. Using an extensive genome-wide association study (GWAS), researchers took samples from three research study groups, totaling over 3,000 patients suffering from CF, and analyzed them by their severity of their phenotype symptoms. After narrowing down to using subjects with the common homozygous CFTR variant, p.Phe508del, and eliminating any outlier cases, the study found that chromosome loci 11p13 and 20q13 heavily influenced the severity of CF symptoms expressed in various patients from the study groups. Furthermore, both genes were found to be inhibitory factors involved in apoptotic suppression pathways. Their roles as inhibitors could explain the decrease of neutrophilic-triggering apoptosis, in which inflamed neutrophils within pulmonary air channels would fail to be eradicated, and thus impair pulmonary function.

This study was not the first attempt from CF researchers in finding genetic modifiers. While three previous small-scale association studies on other loci modifiers. There modifiers could not be replicated in a genome-wide scale by both this study and a previous one conducted in 2009. Nevertheless, the discovery of two possible modifiers from this GWAS study has shed new light and potential opportunities in creating better or possibly more individualized therapies to CF sufferers. For instance, a new therapy treatment could be created to suppress the activation of these modifiers as a method to reduce the severity of the symptoms CF patients exhibit or bring a new perspective road map for scientists to now tackle the disease by also integrating these modifiers in the equation. Therefore, this discovery of CF-modifying genes could completely change the methods we could use against this devastating disease.

Witt, Heiko. Nature Genetics 43, 508–509 (2011).

Wright, Fred A. et al. Nature Genetics 43, 539–549 (2011).

Epigenetics during Pregnancy Can Influence Obesity

Obesity has become prevalent all across the world, yet what is to blame for the condition, genetics or the environment? Recently, a third factor has been linked to obesity: epigenetics. Studies have found that the connection between skinny mothers and obese children is partly due to epigenetics in the womb that causes the child to have an increase in appetite. The mothers must have felt a little relieved after finding this out!

Rat studies at the University of Auckland in New Zealand showed that undernourished rats during pregnancy produced overweight offspring. In a later study in 2005 they were able to show that the offspring turned out normal when the methyl tags were removed.

As for humans, two studies have been performed in the United Kingdom. The studies analyzed the diets of pregnant women, extracted the DNA from the child’s umbilical cord, then measured the body fat of the child to look for specific epigenetic marks correlated with obesity. The first test analyzed children at nine and the second test analyzed children at six. Both of the tests gave similar results, showing that epigenetics can be seen early in a child’s development. Seventy-eight genes were analyzed for epigenetic marks and only the methylation at RXRα gene was correlated with obesity. The genetic changes in RXRα had no correlation with fat levels, which indicates that epigenetics, not genetics, explains the differences in RXRα expression. The researchers also found a link between a low carbohydrate diet early in pregnancy and methylation levels of RXRα. Methylation of RXRα is thought to contribute 25% of the differences in fat levels. The statistics of the studies show that “as the percentage of the RXRα genes that were methylated went from 40% to 80%, the children’s percentage of body fat went up from 17% to 21%”. Obesity is classified as being greater than 30% BMI, so 4% can make a difference in a person’s preset conditions for obesity.

The RXRα gene is called Retinoid X Receptor-α and codes for a protein involved in the fat cell and fat metabolism pathway. Researchers think that the reason for RXRα gene methylation in mothers with a low carb diet is that somehow the mom programs the child to live in a world with scarce food resources by increasing the child’s appetite and food storage capacities. Since many of us do not live in a food-scarce world, however, these children overeat and become obese compared to children with less RXRα methylation. Fortunately, there are preventative measures in treating RXRα methylation. Mothers can add carbs to their diet to prevent methylation of the RXRα gene, and treatments for altering epigenetic tags are available. One type of treatment would be to administer micronutrients to the child. While these results have not yet been proven to be considered true, the take home message here is that pregnant women should not try to diet but should eat a healthy amount of carbs just to be safe.

Finkel, Elizabeth. "Why Skinny Moms Sometimes Produce Fat Children - ScienceNOW." Science/AAAS | News - Up to the minute news and features from Science.. Science Now, 22 Apr. 2011. Web. 27 May 2011. .

"Obesity and Overweight for Professionals: Defining | DNPAO | CDC." Centers for Disease Control and Prevention. Centers of Disease Control and Prevention, 10 June 2010. Web. 27 May 2011. .

Obesity and Genetics!

Obesity is one of the biggest concerns of the American population. In 2007-2008, 34% of adults over 20 years old had been diagnosed with obesity. One of the mysteries of obesity is locating the genes responsible for it and understanding its inheritance. We all know that obesity is mostly caused by the environment, and we know there is a genetic component involved, but we do not clearly understand it. A paper was published in 2010 that discussed rare variants discovered in early onset obese patients. Since researchers knew obesity was heritable, they wanted to investigate the copy number variation to obesity in Caucasian patients who were diagnosed with early onset obesity. They first found that early onset obesity is associated with rare number variants causing rare developmental disease, such as autism and mental retardation. They discovered a large chromosomal deletion about 500 kilobases on chromosome 16p11.2, and these deletions were occurring very rarely at <1%. The 16p11.2 region of the chromosome is involved in many genes, but there is a gene that has been involved in leptin and insulin signaling called SH2B1. They found a delayed and exaggerated insulin secretion in patients who have a SH2B1 deletion. In Gene-wide association studies, they identified common single nucleotide polymorphisms (SNPs) near the SH2B1 locus which were associated with Body Mass Index (BMI). After gathering all the data, researchers proposed a mechanism to explain why the deletions are a more frequent source of loss of function at SH2B1 loci; they believe this could occur through a segmental duplication of 16p11.2 by non-allelic homologous recombination. This paper sheds light on a topic that has been very difficult to understand for a very long time.

Bochukova et al. “Large, rare chromosomal deletions associated with severe early-onset obesity.” Nature 463, 666-670.

Centers for Disease Control and Prevention. http://www.cdc.gov/nchs/fastats/overwt.htm

And Then There Was Life!

Just over a year ago Craig Venter and his team created the first form of synthetic life. The team's fifteen year lead-up began with the sequencing of various organism's genomes-- most pertinently, the smallest genomes. By looking at small genomes the Venter group began to understand exactly what genes were necessary to sustain life. The group then produced and linked code for each essential gene creating a 1.08 megabase chromosome. The synthesized code was created in a computer, inserted into a bacterial cell and allowed to replicate. The newly formed replicating cell produced an entirely new set of proteins and eventually became an novel species.

So what does this mean for science?

Synthetic life-- to me--opens an almost limitless set of doors. Synthetic life can benefit not only humanity but also the rest of the animal kingdom by producing bacteria geared to help decrease carbon dioxide emissions, produce biofuels and create vaccines. Organisms can be created for particular niches with extreme specificity and efficiency. Synthetic life and medicine may trim off the scraps of organisms we see potential for using but aren't energetically suitable for use.

Philosophically perhaps Dr. Venter said what the change in species marks best, "When we look at life forms we see them as fixed entities but this shows, in fact, how dynamic they are. That they change from second to second. And that life is basically the result of an information process. Our genetic code is our software." I'm surprised I haven't heard of a new blockbuster coming out with a transforming lead morphing into whatever new life form fits best.

Of course the Venter group has a series of critics. They receive accusations of Venter playing god to concerns about potential risk of biological warfare. This leads me to ask the question: Where do you see synthetic life going in the next five, ten or 100 years? We discovered genes and genetics in the 1800's and in less than 200 years we have not only mapped out the entire human genome but we have created life. My guess may be as wild as proposing we could transform one organism into and an entirely new species to Gregor Mendel. I believe synthetic life will branch into multicellular organisms. Specialization of cells will allow us vast increases in medicine and treatment of diseases. Specialized genomes of humans, though very far off, may allow us to slow aging and more importantly make less of a footprint on our home--the Earth.

Here's to life only limited by imagination!

Gibson et al."Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome." Nature 329, 52-54.

Are you a Neanderthal?

When you hear Neanderthal, you probably think of a primitive barbarian with a low brow, big forehead, and a huge nose. Research from the past up until recently has hailed the Neanderthal as a brutish species exterminated by early humans which also led to the claim that the interbreeding between humans and Neanderthals was impossible. However, a recent article published 2010 in Science begs to differ. Using bioinformatics scientist compared the Neanderthal genomes to European Americans, East Asians, West Africans, and chimpanzee genomes. The results show that present day non-Africans share genetic ancestry with Neanderthals (1-4%).

This amazing new finding suggests a whole new perspective on the origins of humans. The original theory says there was bottleneck event due to the expansion of a small population out of Africa. This small population of early humans was believed to be biologically distinct from other early hominoids. The idea of early humans interbreeding with Neanderthals has been a controversial dispute. However these new findings suggest that the small population that migrated out of Africa did in fact mate with Neanderthals then spread out to populate the rest of the world.

Many may look at this new information and shrug it off. I think this is an extraordinary finding that challenges not only past scientific research done on Neanderthals but the underlying social beliefs of people. The popular belief is that the Neanderthal is a lesser human species that was hairy, ugly, and had little cognitive ability and thus was driven to extinction. Many believe that Neanderthals were thwarted and driven to extinction by the mighty early Homo sapiens. Although history is written by the victors, apparently the Neanderthals had the last laugh. Genetics show that all non-African Homo sapien lineages are descended from Neanderthals. So think twice before you try insulting someone by calling them a “Neanderthal” because chances are (if your not 100% African) you share 1-4% of your genome with those “barbarians”.

Green, Richard E. “A Draft Sequence of the Neandertal Genome.” Science 328

Green, Richard E. “Anaylsis of one million base pairs of Neanderthal DNA.” Nature 444, 330-336

Genetic Engineering using TAL proteins in Yeast

Using model organisms is a needed intermediate step when developing potential therapeutics. In this study, yeast is used as a model organism to find out how TAL effector nuclease work inside a living cell. TALEs are a family of protein that contains a DNA binding domain which consists of a number of repeats. Each repeat is identical except at the 13^th and 14^th amino acid which is unique and codes for the nucleotide that the specific repeat binds to. Nucleases can be attached to these TALEs which can then act to both knockout genes through non-homologous end joining (NHEJ) or to add a gene sequence through homologous recombination.

To test these proteins in yeast, three gene targets were selected in yeast: URA3, LYS2, and ADE2. The first test looked at how well mutations, either deletions or insertions, could be induced through NHEJ using TALEs with nucleases attached to them. The DNA binding regions were built to recognize and bind to regions within the three genes of interest. The various mutant genotypes for each gene were used to measure the efficacy of each TALE. Most of the data showed a fairly small change in the overall number of cells from .15-5.4% of them showing some sort of mutation from a TALE nuclease. For studying homologous recombination, resistance genes were added that had homologous regions on either side matching with the targeted area in the URA3 gene. The addition of TALE nucleases revealed a fairly high degree of recombination with up to 34% of the cells having the resistance gene.

Even if these proteins could target the right segment of DNA that does not mean that is the only place they can be targeting. To test against this the different TALEs were inserted into haploid cells and grown. If the TALE nucleases are cutting at off target sites, we would expect to see decreased growth due to deleterious mutations. Yeast is a good model for looking at this, as haploid cells would show these mutations more than a diploid would. The data shown in the study show that the TALE infused cells were identical in growth to that of the control. In addition, the sequence of these cells was determined and no insertion / deletions were found other than the ones targeted for in the specific TALE tested.

So what we have here is a protein that can effectively alter DNA within cells in an efficient and specific manner. The implication of this study is that these proteins could one day be used in humans to alter genetic information in vivo. Although gene therapy is still a work in progress, TALE proteins seem to be another potential, and powerful, tool. All this thanks to your favorite model organism, Saccharomyces cerevisiae.

Li, T. et al. Modularly Assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acid Research. (2011). Dpo:1-.093/nar/gkr188.