Using mice, lab-grown cells and clues from a related disorder, Johns Hopkins researchers have greatly increased understanding of the causes of systemic sclerosis, showing that a critical culprit is a defect in the way certain cells communicate with their structural scaffolding. They say the new insights point the way toward potentially developing drugs for the disease, which affects approximately 100,000 people in the United States.
‘Until now we’ve had little insight and no effective treatment strategies for systemic sclerosis, and many patients die within a year of diagnosis,’ says Hal Dietz, M.D., the Victor A. McKusick Professor of Genetics and Medicine in the Institute of Genetic Medicine, director of the Smilow Center for Marfan Syndrome Research at Johns Hopkins and Howard Hughes Medical Investigator. ‘Our group created mouse models that allowed us to learn about the sequence of events that leads to the disease’s symptoms, and we hope drugs can be developed that target one or more of these events.’
Patients with systemic sclerosis, also known as systemic scleroderma, experience a sudden hardening, or fibrosis, of the skin. For some patients, this hardening occurs only in limited areas, but for others, it quickly spreads across the body and to organs such as the heart, intestines and kidneys. It is this fibrosis of the internal organs that is often fatal.
Systemic sclerosis rarely runs in families, Dietz says, making the gene for the disease, if it exists, very difficult to find. Without a known genetic mutation, researchers had not been able to create a genetically altered mouse with which to study the condition. But Dietz’s group was struck by the similarities between systemic sclerosis and a less severe, much rarer condition called stiff skin syndrome (SSS), which does run in families, and they suspected that learning more about SSS would also shed light on systemic sclerosis.
In a previous experiment, they pinpointed the genetic mutation responsible for SSS in a gene for a protein called fibrillin-1, which plays a role in other connective tissue disorders. In certain types of tissues, including skin, fibrillin-1 helps make up the scaffolding for cells. The specific changes in fibrillin-1 seen in SSS patients were predicted to impair the ability of cells to make contact with fibrillin-1 through bridging molecules called integrins.
In the current study, M.D./Ph.D. student Elizabeth Gerber created a line of mice with a genetic variant similar to that found in SSS patients. To test the group’s hypothesis, Gerber also created a line of mice with a variant the team knew would prevent fibrillin-1 from interacting with integrin. As the team expected, both groups of mice developed patches of stiff skin, along with elevated levels of proteins and cells involved in the immune response—much like humans with SSS or systemic sclerosis. ‘It seemed we were right that the SSS mutation causes the condition by blocking fibrillin’s interaction with integrin,’ Dietz says. ‘Something else we found was that both types of mice had high levels of integrin in their skin, which made us think their cells were trying to compensate for the lack of fibrillin-integrin interaction by making more and more integrin.’
This still left open the question of what was ultimately causing fibrosis, however: Was it the integrin levels or the immune response? Dietz’s group delved deeper into the question by creating mice that had both the SSS mutation and artificially low levels of integrin, and found that the mice never developed fibrosis or an abnormal immune response. ‘They looked normal,’ Dietz says.
The team next tried waiting until mice with the SSS mutation had developed fibrosis, then treating them with a compound known to block a key molecule with known connections to both fibrosis and the immune response. This reversed the mice’s skin fibrosis and immunologic abnormalities. The team also tested the compounds on lab-grown human skin cells with systemic sclerosis, with the same results. This raises the possibility that systemic sclerosis patients could eventually be treated with similar compounds in humans, Dietz says. A number of the compounds that proved effective in SSS mice and systemic sclerosis cells are currently being explored by drug companies for the treatment of other conditions, prominently including cancer.
The results raised another big question for the team: Which of the several types of skin cells were responsible for the runaway immune response and fibrosis? They traced the activity to so-called plasmacytoid dendritic cells, or pDCs, a cell type known to either tamp down or ramp up immune response, depending on the circumstances.
‘Dietz’s work gives scleroderma patients hope that we have gained fundamental insights into the process of fibrosis in scleroderma. In particular, I am confident that within a relatively short time, novel therapies can be tested in patients, and I am optimistic that such treatments will have a profound effect,’ says Luke Evnin, Ph.D., chairman of the board of directors of the Scleroderma Research Foundation and a scleroderma patient. John Hopkins Medicine

Huskies football defensive lineman Caleb Eidsvik takes up a lot of room as he sits on an examining table in the Huskies trainer’s room at Griffiths Field, patiently waiting for pharmacology student Hungbo Qudus to draw a small sample of his blood.

At six-foot three and 260 pounds, Eidsvik exudes strength and good health. And that’s the problem, according to researcher Changiz Taghibiglou, since Eidsvik is suspected of having a concussion.

‘There’s no easy way to conclusively diagnose concussion now. You need an MRI or a CT scan,’ he said. ‘Whether it’s car accidents, falls or sports injuries, we actually don’t have any simple tests.’

Taghibiglou is an assistant professor in the College of Medicine’s Department of Pharmacology. If he gets his way, testing for concussion will be so simple that a test kit will be a standard item in every medical bag, to be used by trainers and coaches at football fields and hockey arenas, and even by first responders and EMTs.

Diagnosis of concussion is critical. While short term symptoms such as vomiting, confusion and headache may be easy to spot, Taghibiglou explained that long-term effects can be more subtle and easier to brush off. This can be extremely dangerous: if the person suffers a second concussion before fully recovering from the first, they are at high risk of developing permanent brain damage, psychiatric problems or even dying. There are also risks of long term effects, including Parkinson’s and Alzheimer diseases, and post-traumatic stress disorder.

At the heart of Taghibiglou’s concussion test is a molecule that exists on the surface of brain cells. Through research carried out with scientists at the Canadian Department of National Defence, a link was found between the molecule and brain trauma. This research is ongoing and represents one of the agency’s many inquiries into the effects of battlefield blasts on soldiers.

‘Physical injuries are easy to spot but with a concussion a person can appear fine,’ Taghibiglou said. ‘In the worst case, there are no outward signs of injury so they are sent back out, re-injured, and suffer significant neurological issues later.’

Taghibiglou explains that head trauma – whether from an accidental blow to the head, a hard slam on the gridiron or a forceful check against the boards – can knock certain brain cell molecules loose. Once free, they circulate in the blood where they can be detected by a simple blood test (a patent for the test has been applied for through the U of S Industry Liaison Office).

Working with Huskie Athletics, Taghibiglou, Qudus and graduate student Nathan Pham are gathering blood samples from athletes pre- and post-injury. Taghibiglou praised Director of Huskie Athletics Basil Hughton and Huskies Head Therapist Rhonda Shishkin for arranging access, particularly during peak season.

‘We’re collecting from the football team and are also looking for concussion in other teams such as soccer and hockey,’ he said.

Since the test is so new, the research team also needs about 300 male and female volunteers to donate small blood samples to establish the normal level of the concussion-associated molecules in the blood.

‘There are no values in the reference books, simply because no one has gathered the data yet. Our ultimate goal is a simple diagnostic test, much like the blood sugar tests used by diabetics.’ The test would be particularly valuable for rural and remote communities that lack the medical equipment typically used for trauma diagnosis.

‘Small health clinics don’t have an MRI. It may help rural doctors refer their patients to larger centres and know what’s going on.’ University of Saskatchewan

A Chinese research team composed of Shenzhen Second People’s Hospital, BGI and other institutes reports their latest study on bladder cancer genomics. The discoveries were made using whole-genome and exome sequencing technologies and provide evidence that genetic alterations affecting the sister chromatid cohesion and segregation (SCCS) process may be involved in bladder tumorigenesis and open a new way for the treatment of bladder cancer.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer diagnosed, accounting for 90% of all bladder malignancies in North America, South America, Europe, and Asia. It’s reported that there were an estimated 386,300 new bladder cancer cases and 150,200 deaths in 2008 alone. And the number was up to 170,000 deaths in 2010. Until now, there has been no complete genomic data available for developing new therapeutic approaches to combat bladder cancer.

To have a deeper understanding of the genetic basis underlying TCC, Chinese scientists conducted exome sequencing on the tumour and matched peripheral blood samples from 99 TCC patients, and identified 1,023 somatic substitutions and 67 indels respectively. They performed whole genome sequencing (WGS) to detect copy number alterations (CNAs) and obtained 4-fold mean haploid coverage for each sample.
After evaluating the genetic alterations or variants, researchers found frequent alterations in two genes, STAG2 and ESPL1, which are associated with the sister chromatid cohesion and segregation (SCCS) process. Among them, STAG2 was particularly notable as to harbour a greater number of non-synonymous mutations and a higher ratio of non-synonymous to synonymous mutations. Their study indicated that chromosomal instability and aneuploidy had an influence on bladder cancer, and provided evidence that bladder cancer became the first type of cancer with major genetic lesions in SCCS genes.

Furthermore, researchers detected a recurrent fusion involving two other SCCS-associated genes, FGFR3 and TACC3, by transcriptome sequencing of 42 DNA-sequenced tumors. They suggested that FGFR3/TACC3 is related with bladder tumorigenesis, and the high expression of TACC3 was mediated by transcriptional regulatory elements in the promoter of the fusion partner, FGFR3, not the amplification of TACC3. BGI

Johns Hopkins researchers say that by measuring levels of certain proteins in cerebrospinal fluid (CSF), they can predict when people will develop the cognitive impairment associated with Alzheimer’s disease years before the first symptoms of memory loss appear.
Identifying such biomarkers could provide a long-sought tool to guide earlier use of potential drug treatments to prevent or halt the progression of Alzheimer’s while people are still cognitively normal.
To date, medications designed to stop the brain damage have failed in clinical trials, possibly, many researchers say, because they are given to those who already have symptoms and too much damage to overcome.
‘When we see patients with high blood pressure and high cholesterol, we don’t say we will wait to treat you until you get congestive heart failure. Early treatments keep heart disease patients from getting worse, and it’s possible the same may be true for those with pre-symptomatic Alzheimer’s,’ says Marilyn Albert, Ph.D., a professor of neurology at the Johns Hopkins University School of Medicine. ‘But it has been hard to see Alzheimer’s disease coming, even though we believe it begins developing in the brain a decade or more before the onset of symptoms,’ she adds.
For the new study, the Hopkins team used CSF collected for the Biomarkers for Older Controls at Risk for Dementia (BIOCARD) project between 1995 and 2005, from 265 middle-aged healthy volunteers. Some three-quarters of the group had a close family member with Alzheimer’s disease, a factor putting them at higher than normal risk of developing the disorder. Annually during those years and again beginning in 2009, researchers gave the subjects a battery of neuropsychological tests and a physical exam.
They found that particular baseline ratios of two proteins — phosphorylated tau and beta amyloid found in CSF — were a harbinger of mild cognitive impairment (often a precursor to Alzheimer’s) more than five years before symptom onset. They also found that the rate of change over time in the ratio was also predictive. The more tau and the less beta amyloid found in the spinal fluid, the more likely the development of symptoms. And, Albert says, the more rapidly the ratio of tau to beta amyloid goes up, the more likely the eventual development of symptoms.
Researchers have known that these proteins were in the spinal fluid of patients with advanced disease. ‘But we wondered if we could measure something in the cerebral spinal fluid when people are cognitively normal to give us some idea of when they will develop difficulty,’ Albert says. ‘The answer is yes.’ John Hopkins Medicine

A collaborative team of researchers has used next generation sequencing to identify clinically relevant genetic variants associated with a rare pediatric speech disorder.
Childhood apraxia of speech (CAS) is a rare, severe speech disorder that in some patients also affects cognitive, language, and learning processes.

In this study, Elizabeth Worthey, PhD, assistant professor of paediatrics (genomic paediatrics and bioinformatics) at the Medical College of Wisconsin, working with Dr. Lawrence Shriberg at the Waisman Center, University of Wisconsin – Madison and their colleagues used whole exome sequencing to search for variants associated with CAS.

Prior studies have identified a few genes associated with CAS. In this study, ten pediatric patients were sequenced, and in eight of the cases, clinically significant variants associated with CAS were identified. In some cases patients were found to have apparently deleterious variants in more than one gene. The findings both confirmed previous reports of candidate causal genes and identified novel candidate associations.

‘This study exemplifies the potential productivity of whole exome sequencing for complex neurodevelopmental disorders such as CAS. The current list price to test individual genes is far in excess of the cost of whole exome, and it is also more time effective to perform these tests concurrently rather than looking at one gene at a time,’ said Dr. Worthey. ‘It is likely that a significant proportion of patients with complex phenotypes will be found to have deleterious variants in multiple genes; single gene testing would be unlikely to identify such cases.’ Medical College of Wisconsin