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A Rare Find

March 2011

“The doctor will see you now.”

A young mother and her four-year-old daughter rose to their feet. It was two days before Thanksgiving 1949, and the mother worried that they might not make it home in time for turkey dinner with the family. But she had to make the trek to the Cleveland Clinic. Dr. Earl Weldon Netherton, the clinic’s chief of dermatology and an expert on rare skin diseases, was her final hope.

The 56-year-old Netherton, clad in a generic white lab coat, greeted the two and asked what brought them to town. The mother answered that something was profoundly wrong with her daughter’s skin. She had been born with a rash from head to toe that made her skin look as though it had been scalded in hot water. Luckily the rash went away, but it flared up now and again on her forehead, neck, and back. The doctors back home said they’d never seen anything like it.

Netherton listened, asked questions, and jotted down her answers. “Frequently restless, irritable, and difficult to feed because of her preference for liquid foods,” he jotted down, his mind cancelling out one diagnosis and moving on to the next possibility. “No familial history of hay fever, asthma, or eczema.

The mother then pointed out several large patches of thin, downy hair on her daughter’s head. Netherton took a closer look. Strange. The strands were short and brittle, and the affected area of the scalp appeared bright red and flaky.

Before the mother and daughter left, Netherton collected hair samples from the child’s head, eyebrows, and eyelashes. He later mounted the strands onto slides, focused the microscope, and couldn’t believe his eyes.

Hair shafts 

Photomicrograph showing nodose swelling of hair shaft that resembles joints of bamboo. From October 1958 Archives of Dermatology: "A Unique Case of Trichorrhexis Nodosa—"Bamboo Hairs," by Earl W. Netherton, M.D.

Each strand was jointed like a bamboo fishing rod. Netherton had never seen anything like it in his four-plus decades as a clinician. He contacted several colleagues and asked if they had ever encountered a condition that involved a severe skin rash and bamboo-like hair? No luck. Like a criminal investigation, Netherton’s case had gone cold.

In September 1956, Netherton re-opened the case when he examined the now 11-year-old girl for a second time. The symptoms remained unchanged, and he recalled a colleague’s comment seven years ago, “I have come to the conclusion that you have in all probability a new type of hair atrophy, a possible variant of trichorrhexis nodosa,” a fracture of the hair shaft.

Now a white-haired professor emeritus at the Cleveland Clinic and more compelled to record his career’s most interesting cases for posterity, Netherton gathered his notes on the little girl and penned his final journal article. It was titled "A Unique Case of Trichorrhexis Nodosa – “Bamboo Hairs.” In October 1958, the A.M.A Archives of Dermatology published the paper.

The condition became known in the medical literature as Netherton syndrome.

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Nearly 50 years later, Katiuchia Uzzun Sales, D.D.S., Ph.D. started her post doc in the NIDCR’s Proteases and Tissue Remodeling Section. She arrived with a Ph.D. in oral pathology from her native Brazil, a strong desire to transition into a research career, and a hope that she’d get to work on an interesting research project.

Thomas Bugge, Ph.D., her lab chief, soon floated a possible project. She could take first crack at crossbreeding two mouse knockout models, meaning each model has a different but distinct gene inactivated. He said one of the gene knockouts produced mice that had a terrible crimson-red skin rash similar to that seen in human babies born with the rare Netherton syndrome.

Sales wrote down the word Netherton. She’d never heard of it.

Sales later typed the name into an internet search engine . . . a rare autosomal recessive disorder . . . characterized by the triad of (a) a severe congenital skin rash that predisposes patients to infection, sepsis, and dehydration, (b) bamboo hair, and (c) markedly increased blood levels of eosinophils, a type of immune cell . . . incidence is undetermined, but the condition is rare.

Thomas Bugge, Ph.D. and Katiuchia Uzzun Sales, D.D.S., Ph.D. 

Dr. Thomas Bugge pictured with Dr. Katiuchia Uzzun Sales.

Soon thereafter, Bugge laid out the project. Bugge said he suspected that if the previously mentioned mouse models were crossbred, Sales eventually could produce double-knockout pups (inheriting two knockout genes from each model) and eliminate the terrible Netherton-like skin rash. If his hunch was correct, Bugge said it would lead to additional experiments to sort out the molecular biology underlying the results.

Sound good?

Sales nodded, and thus began what would become a three and-a-half year scientific journey. As a former practicing dentist with limited hands-on lab experience, Sales learned to conduct complex experiments in mouse genetics, biochemistry, molecular imaging, histology, and pathology.

But through her dedication and the help of her lab mates and mentors, Sales completed the project and published her first paper last August in the high-impact journal Nature Genetics as the lead author.

The paper could very well lay the biochemical foundation for the development of more targeted treatments to prevent the often debilitating skin loss in children born with the syndrome.

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In 1958, when Dr. Netherton published his seminal paper, he reported that the little girl likely had been born with a tongue-twisting condition called congenital ichthyosiform erythroderma, or CIE.

As always, Netherton’s clinical reasoning was sound. The affected skin appeared abnormally dry, flaky, and inflamed in a manner consistent with CIE. But with only the refractive power of a microscope to guide him, Netherton had to suffice with appearance. The molecules underlying the condition remained like small distant planets that fell hundreds of orders of magnitude outside of his investigative view.

By the late 1990s, however, these molecules started to come into focus when researchers found that the syndrome was caused by mutations in a gene called SPINK5.

The SPINK5 gene encodes a protein called LEKTI, a serine protease inhibitor. Think of the serine proteases as enzymes that work like box cutters, cleaving specific proteins and setting in motion biochemical cascades that result in a specific cellular response. LEKTI, as an inhibitor, attaches to its target serine proteases like a safety mechanism that blocks the blade – and thus the signal – when it isn’t required.

When scientists produced the full-length LEKTI that was encoded by the SPINK5 gene, they found staring back at them the protein equivalent of a giant octopus. It consists of 1,064 amino acids (typically, a protein has 200-300 amino acids) that assembled into 15 inhibitory domains, with each of these distinct regions capable of inhibiting specific serine proteases.

An international team of researchers later determined that LEKTI fragments rapidly bind and inactivate a group of serine proteases called kallikreins. This immediately made things very interesting. Kallikreins play a prominent role in the turnover of dead cells that naturally flake off of the thin, outermost layer of our skin called the stratum corneum. This finding and subsequent work set up the following scenario:

  • Just below the stratum corneum sits the granular layer of our skin. There, pro-kallikreins are synthesized, snipped into smaller pieces, and activated as serine proteases.
  • LEKTI latched onto the activated kallikreins in the granular layer to prevent them from prematurely degrading corneodesmosomes, the protein rivets that hold together skin cells in the stratum corneum.
  • In the absence of LEKTI, the activated kallikreins are free to degrade corneodesmosomes in the granular layer. This leads to a “runaway” kallikrein cascade that results in the premature loss of the stratum corneum and the terrible rash seen in people with Netherton syndrome.

An obvious next question was: What activates the kallikreins? The answer might provide a good target to prevent the abnormal biochemical cascade that results in the skin rash.

And that takes us back to Bugge’s hunch.

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Thomas Bugge studies serine proteases tethered on the surface of cells, with a special focus on their roles in oral tissues. Although Netherton syndrome isn’t a condition that an oral biology laboratory would investigate, Bugge knew of the earlier study that reported the runaway kallikrein cascade in Netherton syndrome. He thought of a cell-surface serine protease called matriptase. Bugge figured matriptase just might activate the epidermal kallikreins and cause the skin rash.

Why’s that? First, matriptase has the unusual ability to activate itself and then activate other serine proteases, serving as the start switch that triggers distinct cascades.

Secondly, he knew that matriptase, LEKTI, and the kallikreins are all produced in exactly the same layer of normal skin.

Thirdly, and even more biologically intriguing, Bugge and his coworkers already had created a matriptase knockout mouse, meaning it lacked the matriptase start switch. He had found that the mice lacked the natural ability to shed their stratum corneum and oral lining – or, exactly the opposite manifestation, or phenotype, of Netherton syndrome.

“That led us to speculate that no matriptase production meant no kallikrein activation,” said Bugge. “If the kallikreins don’t have access to matriptase, they don’t get activated. In other words, there is no proteolytic activity generated in the matriptase knockouts to shed the stratum corneum.”

But in science, as in life, talk is cheap. To prove his hunch, Bugge refocused on the SPINK5 knockouts, the mouse model with the Netherton-like skin rash. What if Sales also knocked out the matriptase gene? If his hunch was correct, the double knockouts (lacking both matriptase and LEKTI) would have no skin rash.

For Sales, it was the perfect first project. “I did some work in mouse genetics while earning my Ph.D. in oral biology, and I loved it,” she said. “Just seeing the consequences of knocking out a gene in vivo was a real eye opener for me. Until then, genetics had been an abstract concept to me. I couldn’t wait to get started.”

But as Sales added, “Of course, I had no idea at the time how complex and challenging these crossbreeding experiments would be.”

Sales crossbred the two knockouts and eventually produced pups that had one working copy of SPINK5 and one working copy of matriptase. She then crossbred them and waited to see if the next litter contained a subset of mice with no working copies of either gene and, most importantly, no peeling skin.

“It took about six months before I got the final version of the mouse,” she said of this make-or-break experiment. “I could see some of the pups had skin that was flaking and red like Netherton syndrome, while others in the litter looked normal. I snipped a small piece of the tail from one of the normal-looking pups to get some DNA, did the genotyping, and said, ‘Oh, my goodness.’ The normal-looking pup was a double knockout for SPINK5 and matriptase.”

Sales repeated the crossbreeding experiment. Same results. But her task was anything but over. Now Sales had to show biochemically that matriptase was indeed the activator of the epidermal kallikreins.

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The American sportscaster Warner Wolf built a career on a signature phrase, “Let’s go to the videotape,” meaning let’s see what really happened in last night’s game. Sales now did the research equivalent. She performed in situ zymography, a gel-based technique to show, like a freeze-frame photograph, the actual enzymatic activity in the skin.

“I’d never performed in situ zymography,” she said. “But I talked with Thomas [Bugge] about it, read the protocol very carefully, and worked my way step by step through it. It was a great learning experience.”

Sales compared the serine protease activity in the skin of the double knockout with the original SPINK5 knockout. If the activity was nonexistent in the former and prominent in the latter, she would have her first level of evidence to explain the different phenotypes.

Sure enough, that’s exactly what the in situ zymography showed. Further supporting this point, the proteolytic profile in the double knockout matched that seen in normal mice.

Then, taking a page from pathology, she carefully stained the various skin samples and compared their appearance under the microscope. She saw the corneodesmonsomes, the rivets that hold skin cells in place in the stratum corneum, were indeed intact in the double knockouts. The implication being, the runaway kallikrein cascade wasn’t in play to prematurely shed the stratum corneum.

That left just one possibility on the table. What if, unbeknownst to Sales, LEKTI was a direct inhibitor of matriptase, and matriptase was the culprit that degraded corneodesmosomes in Netherton syndrome?

To get the answer, Sales had to make a recombinant copy of LEKTI - all 15 inhibitory domains long. It was like asking an apprentice baker to whip up a 15-layer chocolate torte.

“It was a great deal of work, and things didn’t always go smoothly,” said Sales. “But, in the end, I did it. It was fantastic to see the finished product, the first protein that I’d ever made. ”

Sales evaluated meticulously the ability of all of the inhibitory domains to inhibit matriptase and epidermal kallikreins. She confirmed that the LEKTI domains do inhibit kallikreins but don’t affect matriptase.

“I see two really exciting aspects coming out of this work,” said Bugge. “One, it shows exactly how matriptase regulates the normal and abnormal shedding of the corneum stratum. Two, it suggests that a matriptase-inhibiting cream one day could be helpful for babies born with Netherton syndrome and also possibly for other severe skin rashes whose molecular origins are still not understood.”

“This is a classic case of why it’s so important to study rare conditions,” he continued. “I know it’s a cliché, but these conditions are nature’s own experiments. Understanding them can tell us profound things about human health and disease, and that’s certainly the case here.”

After completing the project, Sales moved on to her next learning experience. When last seen, she was finishing a project on proteases in oral cancer.

“When I came to NIDCR, I had fallen in love with science,” said Sales. “The love affair continues, and I’ve learned a great deal about being a bench scientist during my post doc. I feel prepared to continue on with my career.”

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This page last updated: July 31, 2014