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Genetic Linkage

Kids With 2 Upper Jaws -- And My Fruit Flies

3D CT scan of child with ACS. Lower jaw is small and malformed (left); same aged child with normal jaw (middle); lower jaw of child with ACS inverted over upper jaw of normal skull (right). (Credit: Image courtesy of Seattle Children’s).
Body-Altering Mutations – In Children and Flies

I became a science writer, circa 1980, because I didn’t think flies with legs growing out of their heads – my PhD research – had much to do with human health. So when I spied “A Human Homeotic Transformation” way down on the Table of Contents in the May issue of the American Journal of Human Genetics, I was as riveted as a normal person would be getting a copy of People with a celebrity on the cover.

Homeotic Mutations
A homeotic mutation mixes up body parts, so that a fly grows a leg on its head, antennae on its mouth, or sports a double set of wings. Designation of body parts begins in the early embryo, when cells look alike but are already fated, thanks to gradients of “morphogen” proteins that program a particular region to elaborate particular structures. Mix up the messages, and a leg becomes an antenna – or, as in the AJHG article, a child develops two upper jaws, instead of an upper and a lower.

I once knew the homeotic mutants of Drosophila melanogaster intimately, as I archaically mapped their genes. Shortly after I left Thom Kaufman’s lab at Indiana University (where I penned a fruit fly romance novella, in addition to my thesis), post-doc Matt Scott and fellow grad student Amy Weiner took the science a quantum leap forward, discovering the homeobox. This 180-base-sequence encodes a protein domain that binds other proteins that turn on sets of other genes – crafting an embryo, section by section.

Soon, homeoboxes turned up in all manner of genomes, affecting the positions of petals, legs, and larval segments, the genes mysteriously arrayed on their chromosomes in the precise order in which they’re deployed in development. Homeotic mutants even starred in an episode of The X-Files.

Homeotic mutations cause a few human diseases. In lymphomas, white blood cells detour onto the wrong lineage, and in DiGeorge syndrome, the missing thymus and parathyroids and abnormal ears, nose, mouth, and throat echo the abnormalities in Antennapedia, the legs-on-the-head flies I worked with. Extra and fused fingers and various bony alterations also stem from homeotic mutations.

Alas, no human homeotic seemed as compelling to me as a double-winged fly -- until I saw photos of the tiny faces of the children with upper lower jaws.

Two Upper Jaws
Discovery of the homeotic mutations that turn a lower jaw (mandible) into an upper jaw (maxilla) began with an astute pediatrician. Michael L. Cunningham, MD, PhD, director of Seattle Children’s Craniofacial Center who also has training in anatomy and embryology, was examining the jaw of a little girl with what would become known as auriculocondylar syndrome or ACS.

The condition, originally described in 1978 and also called “Question Mark Ears” syndrome, can twist the ears into the shape of said punctuation marks, and disrupts development of the temporomandibular joint and mandible. The head and mouth are so small that children must undergo surgeries to be able to breathe and eat normally. ACS is a rare disease: fewer than 1 in 50,000 newborns have it.

Dr. Cunningham noted, in examining the girl in 1998, that the lower jawbone had unusual bony areas that fused with her cheekbones. “Seeing her mandible doing that gave us the idea that her lower jaw was patterned like an upper jaw. And the fact that her mother was also affected made me think that we had found a novel condition,” he said.

Over the years, when Dr. Cunningham’s team cared for the little girl, he noted fleshy tissue forming inside her mouth on both sides of her mandible that looked like halves of a duplicated soft palate with a uvula on each side – which is exactly what they were, just in the wrong place. “It was obvious that her lower jaw was patterned like a maxilla and zygoma (cheekbone),” he recalled.

Whole Exome Sequencing
The search to find a causative mutation began, as such searches often do, with an animal model – the Dlx5/Dlx6 mouse. Mutations in this Hox gene cause a small, malformed jaw in mice, “split-hand/foot malformation” in humans, and legs and antennae popping up where they don’t belong, or missing where they do belong, in flies.

But when Cunningham’s group and collaborators sequenced Dlx5/Dlx6 as well as a downstream gene called endothelin, in the patient and in a few others, the genes had no mutations. Something else was causing the oddly duplicated/deficient jaw of ACS.

The next step: whole exome sequencing, thanks to collaboration with Mark J. Rieder, PhD, from the department of genome sciences at the University of Washington and co-workers from France, Australia, San Francisco and Tucson. They compared the protein-encoding parts of the genomes in child-parent trios from five families, consulting a few additional pedigrees that other investigators provided.

The results were remarkable, in a few ways.

First, the researchers discovered “two distinct genetic causes of a single human malformation syndrome…in the same pathway….in one experiment,” said Cunningham, referring to genes called PLCB4 and GNAI3. Both affect the endothelin signaling pathway, but through different routes: PLCB4 mutations inactivate stimulation, whereas GNAI3 mutations boost an inhibitory signal. Clues came from zebrafish with similar jaws and a PLCB4 mutation. The GNAI3 mutation, however, had no known animal counterpart. (The researchers do not yet know exactly how the mutations cause ACS.)

The second unexpected result was that all of the mutations in both genes affect amino acids that are identical in all vertebrates, flies, and even fungus, indicating that the genes are essential to multicellular life.

Third, the mutations are not in Hox genes, but in their controls.

The Bigger Picture
Discovery of two genes behind ACS will surely help in diagnosis of this syndrome and related ones. But the implications are broader, in four ways.

#1 EVOLUTION When mutation derails development similarly in such different species as a human and a fly, descent from a common ancestor is a much more logical explanation than repeated identical genetic changes or being plopped down by a Creator.

#2 WHOLE EXOME SEQUENCING Obsolescence looms. “Exome sequencing is so powerful that the mutations will soon no longer be what we search for. Mutations will be easy to find, even boring. It’s the biology that will be tricky to figure out: protein function, regulation of expression, epigenetics, and developmental biology…..this is were we will be spending more and more of our time,” said Cunningham.

#3 MY CAREER CHOICE I realized, with the elegant work on the double-jaw in which a maxilla is seen from two perspectives, that the homeotic mutations are a metaphor for my career – using my knowledge of genetics to communicate research results, rather than investigating molecules and mechanisms.

#4 MODEL ORGANISMS Discovering the mutations behind ACS illuminates the value of research on model organisms. I’ll be writing news releases for the upcoming 2012 Model Organisms to Human Biology - Cancer Genetics Meeting in Washington, D.C. June 17-20. I will blog from the world of worms, zebrafish, frogs and mice – and of course, the noble fruit fly, whose homeotic mutations I once deemed unimportant to human health.

This blog first appeared on the Scientific American blog on May 9.

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