Scientists at the Gladstone Institute of Cardiovascular Disease (GICD) and the University of California San Francisco (UCSF) have identified a genetic factor critical to the formation of chambers in the developing heart. The discovery of the role of a microRNA called miR-138, could offer strategies for the treatment of congenital heart defects.The heart is one of the first and most important organs to develop. In fact, embryos cannot survive long with a functioning heart. In vertebrates (animals with backbones), special cells form a heart tube; that tube loops back on itself to form the atrium and ventricle and the canal and valve that separates them. This requires a complicated sequence of genes turning on and off. MicroRNAs are very small RNAs of 20 to 25 nucleotides that regulate numerous gene functions. Approximately 650 human miRNAs are known, but only a few have yet been studied to determine what they actually do in a cell.

Researchers, led by Sarah Morton, an MD/PhD student at UCSF and GICD Director Deepak Srivastava MD, examined zebrafish, which are an ideal model system for understanding genetic functions. Zebrafish are small, reproduce fast, and are essentially transparent so that that events of heart formation can be studied while they are still alive. Yet many of their systems are quite similar to those of humans. For example, miR-138 is exactly the same in zebrafish and humans.

“What’s interesting is that a single microRNA is responsible for setting up the distinct patterning of a developing heart into separate chambers,” said Dr. Srivastava, senior author of the study. “Since many congenital heart defects involve abnormalities in the formation of the chambers, this is important information in finding ways of treating or avoiding those defects.”

The GICD scientists reported in today’s issue of the Proceedings of the National Academy of Sciences USA, that miR-138 is present in the zebrafish heart at specific times and in specific places in the developing heart. Furthermore, they showed that it is required to insure that the cardiac chambers develop properly. When the scientists used genetic engineering techniques to eliminate miR-138, cardiac function was disrupted, and the ventricles did not develop correctly, with the muscle precursor cells failing to mature properly.

“The miR-138 function was required during a discrete developmental window that occurred 24-34 hours after fertilization,” said Sarah Morton. The team also showed that the miRNA controlled development by regulating numerous factors that function jointly to define the chambers, including a key enzyme that makes retinoic acid.

Clubfoot, one of the most common birth defects, has long been thought to have a genetic component.  Now, researchers at Washington University School of Medicine in St. Louis report they have found the first gene linked to clubfoot in humans.

Their research will be published in the Nov. 7 issue of the American Journal of Human Genetics.

By studying a multi-generation family with clubfoot, the scientists traced the condition to a mutation in a gene critical for early development of lower limbs called PITX1.  While other genes are also likely to be linked to clubfoot, the new finding is a first step toward improved genetic counseling and the development of novel therapies.

“To our knowledge this report is the first evidence for PITX1 mutation in human disease,” said Christina Gurnett, M.D., Ph.D., assistant professor of neurology, of pediatrics and of orthopedic surgery at the School of Medicine.  “Once we identified the mutation, we proved that all of the individuals in this family with lower extremity malformations also have the mutation.  Having large families to work with is very helpful in genetic research.”

Gurnett and her colleagues analyzed the DNA of 35 extended family members of an infant male patient of Matthew Dobbs, M.D., associate professor of orthopedic surgery at the School of Medicine and a clubfoot specialist at St. Louis Children’s Hospital and St. Louis Shriners Hospital.  The patient, the most severely affected in the family, had clubfoot in both feet, duplicated first toes and was missing the tibia in the right leg.

Gurnett and Dobbs visited the family members in their community to examine their lower limbs and to take DNA samples.  They found that 13 family members were affected: Five additional family members had clubfoot, which was more severe in the right foot in three of them.  Five others had lower limb abnormalities including flatfoot, an underdeveloped patella and hip dysplasia.

Through the genome-wide study, Gurnett and her colleagues found a region on chromosome 5 that was common to all family members affected.  From there, they identified a mutation in a gene critical for early development of lower limbs called PITX1.  The PITX1 mutation was found in all affected family members and in three carriers who showed no clinical symptoms.

Dobbs, senior author of the study, said the finding is an exciting step in developing a better understanding of the genetic basis of clubfoot, which affects about 1 in 1,000 new births.

“Clubfoot is a complex disorder meaning that more than one gene as well as environmental factors will be discovered to play a role in its etiology,” Dobbs said.  “Identifying the genes for clubfoot will allow for improved genetic counseling and may potentially lead to new and improved treatment and preventive strategies for this disorder.”

Dobbs treats children with clubfoot and other orthopedic abnormalities using the Ponseti method, a treatment that involves weekly casting and the manipulation of clubfoot soon after birth.  In 2007, Dobbs developed a new dynamic brace called the Dobbs brace for clubfoot that allows active movement, preservation of muscle strength in the foot and ankle and fewer restrictions on the child than the traditional brace.

About 80 percent of clubfoot cases are idiopathic, meaning the cause is unknown and the patient has no other birth defects.  A familial link plays a role in about 25 percent of cases.  The condition occurs in males twice as often as in females and occurs more often in the right foot.  About half of the cases affect both feet, including the bones, muscles, tendons and blood vessels.  If untreated, those affected walk on the outside of their feet, which can lead to long-term pain and disability.

Gurnett said some clinical characteristics of the family members with the PITX1 mutation suggest that the genetic defect may be linked to idiopathic clubfoot.  First, the majority of the affected family members had clubfoot, but no other abnormalities.  Second, there were five females who carried the gene but did not have clubfoot, which supports the lower incidence of clubfoot in females.  Third, clubfoot affects the right foot more frequently, a hallmark of mutations in PITX1.

Previous studies had shown a relation between PITX1 and the development of hindlimbs in other vertebrates.  In mice, a loss of PITX1 leads to shorter femur length and fewer digits on the right foot than on the left.  An alteration of the gene in a developing chick wing changes it so that it looks more like a leg.  In vertebrates such as the manatee and stickleback fish, an alteration has resulted in evolutionary changes in the development of the pelvis.

“It’s our job to prove that this is going to be important for many kids with clubfoot,” Gurnett said.  “Until now, we didn’t know whether clubfoot was a muscle, nerve, spinal cord or brain problem.  Now, we have an idea that clubfoot may result from mutations of genes that are involved in early limb development.”

Gurnett said she and her colleagues will take the finding back to the lab to look for other factors involved in the pathway or how environmental effects may influence the gene.  She and Dobbs, who have been studying the genetics of clubfoot for a decade, plan to investigate the frequency of PITX1 gene mutations in other families with clubfoot.

Breakthroughs in cytogenetic technologies, which focus on subtle alterations in genes and chromosomes, are enabling a new level of detail and accuracy in the diagnosis of complex and unexplained developmental problems in children.

The availability of this new information can help clinicians shift to a “genotype first” model of diagnosis, according to David H. Ledbetter, PhD, Woodruff professor of human genetics at Emory University and director of the Division of Medical Genetics.

Ledbetter’s editorial on “Cytogenetic Technology–Genotype and Phenotype,” is published online this week by the New England Journal of Medicine.  It accompanies an article by Heather Mefford and colleagues about using new cytogenetic technologies to identify microdeletions and microduplications in a specific region of chomosome 1q21.1 in patients with unexplained mental retardation, autism or congenital anomalies

Cytogenetic arrays that reveal DNA microdeletions and additions, including single-copy changes of a few hundred base pairs, beadchips that detect single-nucleotide polymorphisms (SNPs) and tests called comparative genomic hybridization have led to an exciting renaissance of genetics-based syndrome delineation, says Ledbetter in his editorial.

“In the early 1960s we began discovering the relationship between chromosome imbalance and diseases and syndromes, such as Down syndrome,” says Ledbetter.  “This was based on identifying multiple patients with the same cytogenetic abnormality and similar clinical symptoms.  Ever since then, technology breakthroughs have allowed us to identify new syndromes and ever more subtle genetic differences.”

The current proliferation of new genetic information has led researchers to discover that many small genetic variations are common and mostly benign in the human population.  This means the relationship between DNA variations and disease must be analyzed even more carefully in order to find accurate connections.  In order to prove that a genetic difference is directly related to a particular syndrome, notes Ledbetter, researchers must show that the difference is never found in normal control individuals or at least is found with significantly less frequency.

Also, researchers have found that a particular genetic variation may have only a mild effect in a parent but a much more severe effect in a child who inherits the same variant.  And a group of children may have a variety of different problems resulting from the same gene variation.  Whole-genome cytogenetic arrays are becoming much more common, however, which is bringing genetic testing to the level of everyday medicine.

“So many variations of developmental disorders and syndromes have been discovered that genetic testing has become essential for making a specific clinical diagnosis,” says Ledbetter.  “Although more information has made the job of a diagnostician even more challenging, it also is leading to more accurate diagnoses and should lead to much more effective treatments.”

Prenatal biochemical screening tests are widely used to look for chromosomal abnormalities in the fetus which can lead to serious handicap, or even death during gestation or in the first few days after birth. But these tests are only able to detect fewer than half of the total chromosomal abnormalities in the fetus, a scientist will tell the annual conference of the European Society of Human Genetics tomorrow (Monday 2 June) Dr. Francesca R. Grati, of the TOMA Laboratory, Busto Arsizio, Italy, says that these findings mean that women should be better informed on the limitations of such diagnostic tests.The researchers studied 115,576 prenatal diagnoses carried out during the last fourteen years. 84,847 were amniocenteses, usually carried out around the 16th week of pregnancy, and 30,729 chorionic villus samplings, which can be undertaken from 12 weeks into the pregnancy. Both these tests carry an increased risk of miscarriage, so the decision on whether or not to undertake them can be difficult to weigh up. “Since our sample included a large number of women aged less than 35 who underwent invasive prenatal diagnosis without any pathological indication to do so, we felt that the results could be useful in helping to inform pre-test counselling of such women”, says Dr. Grati. “Up until now, the information we had came from smaller studies which only looked at the performance of these tests in detecting a limited number of chromosomal abnormalities.”

After analysing the results of the chromosomal abnormalities from their own dataset, the researchers combined them with the official detection rates for these abnormalities published by SURUSS and FASTER consortia. These are multi-centre research groups involved in the investigation of screening and diagnostic tests performed in pregnancy, whose results are being used to optimise prenatal care for pregnant patients. They found that current screening procedures were only able to detect half the total chromosomal abnormalities in women both younger and older than 35.

The TOMA laboratory is particularly suited to carry out this kind of research, says Dr. Grati, because it was among the first in the world to deal with prenatal diagnosis, and has a vast number of prenatal diagnostic samples at its disposal.

Current tests do not detect all fetal chromosomal abnormalities, but only trisomies 21 (Down syndrome), 18 (Edward’s syndrome), and 13 (Patau syndrome), monosomy X (Turner syndrome), and triploids (conceptuses with 69 chromosomes instead of 46). “These are common vital chromosomal abnormalities, but there are many others which are not picked up by these tests”, says Dr. Grati. “And the tests do not even detect 100% of the common abnormalities.”

At conception, 23 chromosomes from each parent combine to create a fetus with 46 chromosomes in all its cells. Trisomy occurs when the fetus has one additional chromosome (47 instead 46). The extra genetic material from the additional chromosome causes a range of problems of varying severity.

In Down syndrome, for example, where the fetus has three copies of chromosome 21, babies are usually born with impaired cognitive ability and physical growth, cardiac defects and a characteristic facial appearance. Unlike many other such abnormalities, however, babies born with Down syndrome are able to lead relatively normal lives and their life expectancy is around 50 years.

Other than trisomy, the fetus can also have the loss of genetic material (deletions) or chromosomal abnormalities in a non-homogeneous form, where there is a mixture of two cell lines, one normal and the other abnormal. “Some of these disorders are relatively common in the fetus, which may have as much chance of surviving as children who are born with Down syndrome, and it is worrying that current biochemical tests are not always able to detect them” says Dr. Grati. “Our research confirms that it is fundamental for doctors to counsel patients about the limitations of current screening methods, so that they can make an informed decision on whether or not to undergo invasive diagnostic testing.”

With improved resolution, tissue-specific molecular markers and precise timing, University of Oregon biologist James A. Weston and colleagues have possibly overturned a long-standing assumption about the origin of embryonic cells that give rise to connective and skeletal tissues that form the base of the skull and facial structures in back-boned creatures from fish to humans.

Weston and co-authors from the Max Planck Institute of Immunology in Germany and the French National Scientific Research Centre at the Curie Institute document their potentially textbook-changing case in an article appearing online this week (May 19-23) ahead of regular publication in the Proceedings of the National Academy of Sciences.

The cells in question, they argue, do not come from a portion of embryonic neural epithelium called the neural crest, as widely believed, but rather from a distinct thin layer of epidermal epithelial cells next to it. “Our results,” Weston said, “could lead to a better understanding of the etiology of craniofacial defects, as well as the evolution of the head that distinguishes vertebrates from other creatures.”

The neural crest was first identified by classical embryologists in the late 19th and early 20th centuries and has been one of the most studied embryonic tissues. Conventional wisdom says that the neural crest gives rise to skeletal and connective tissue of the head and face, as well as a wide diversity of other stem cells that migrate to many places in the vertebrate embryo, where they spawn the cells that create the peripheral nervous system, and pigment cells in skin and hair (or scales and feathers).

The new study is part of research done over 25 years in Weston’s quest to understand early development of the neural crest and explore alternative explanations for sometimes differing findings involving its assumed cell lineages. Weston noted that mutations in mice that adversely affected development of the peripheral nervous system or pigmentation did not affect craniofacial structures, whereas mutations that caused abnormal development of skeletal and connective tissue of the head and face did not alter neural crest-derived pigment or peripheral nervous system cells.

This paradox, he said, led him to wonder if different genetic programs were required to function in distinct embryonic precursors of these tissues. “In our new paper,” he said, “we finally were able to re-examine some of the underlying assumptions that have led to the conventional wisdom about the source of the embryonic cell lineages that give rise to the skeleton and connective tissue of the head and face.”

In the mouse embryo at eight days gestation, Weston and collaborators used high-resolution imaging and immunostaining techniques to identify and track the dispersal of cells known to jump start connective and skeletal tissue development. They were able to see clearly that these cells came from the non-neural layer of cells rather than from the neural crest. The same distinction also exists in chicken embryos during the first few days of gestation, Weston noted. “Looking at the right time is very important,” he said.

Weston argues that this non-neural epithelium is indeed distinct from the neural crest, because its cells contain characteristically different molecules. He and colleagues dispute suggestions that this non-neural structure is simply a sub-domain of the neural crest. “These cells emerge at a different time in development and disperse in the embryo before neural crest cells begin to migrate,” Weston said.

“New technologies let us see cell types more clearly than ever before,” said Weston, a member of the UO’s Institute of Neuroscience. “We previously had discovered that a molecule that marks cell surfaces in the non-neural epithelium reveals a very sharp boundary between this non-neural epithelium and the neural tissue connected to the neural crest. In this study, we took a closer look.”

They located a population of cells in the non-neural epithelium that express other molecules that “do not appear to originate from the neural crest,” said Weston, who retired in 2001 but continued to teach in the College of Arts and Sciences until 2006. He still collaborates in some research with colleagues at the UO and at various labs around the world.

“I think our results have two important messages,” he said. “First, it is important to identify and validate — rather than ignore — assumptions; and second, because we identified an alternative embryonic cell lineage as the source of the head and facial structures, we can now more effectively analyze and understand the molecular-genetic mechanisms that regulate the normal and abnormal development of these structures.”