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written by Anne Vascik

The NIGMS Respository at Coriell Medical Institute is the biobank where Cure CMD has decided to start a CMD cell repository.  Coriell is a nonprofit organization that maintains a repository for researchers to utilize in developing treatments and cures.  Samples can be sent from all over the country and turned into cell lines and induce pluripotent stem cells (iPSc).  IP stem cells are not embryonic stem cells. They are cells that start as skin cells or white blood cells that are coaxed into becoming a new type of cell that can now divide and become many different kinds of cell types, such as brain cells (neurons), heart cells (cardiac cells), pancreas cells and muscle cells.  Having an iPSc allows researchers to study a cell type such as a brain cell (astrocyte or neuron) from tissue that is otherwise not accessible.

When a sample arrives at Coriell it is first processed.  All samples have their information entered into the data base, and are also given a Coriell bar code and code number.  This ensures that the samples remain anonymous.  From the in take room samples go to different places based on whether they are blood or skin (fibroblast) samples.  Blood samples go to the lymphoblast lab, skin cells go to the fibroblast lab to generate cell lines. Cell lines are clusters of cells that are replicated to create a larger supply of the cells so that they can be divided into separate tubes, frozen separately and then sent out to different scientists.  Creating a cell line is like taking a town and making a city of cells.

Blood samples are taken to the lymphoblast lab.  This lab is where the cells are divided out in preparation to become cell lines.  These labs are cleaned daily and work is done under clean benches to reduce the chance of contamination.  After a sample arrives its records are brought up and any observations about the sample are recorded.  Only the white blood cells are needed for making cell lines or iPSc and so all the other components of the blood need to be filtered out.  Filtering is done using a layer of sugar, which separates out the red blood cells, plasma, anti-coagulant, plasma, and white blood cells into layers.  A technician is then able to suck the white blood cells out of the tube and clean them.  The cells are placed in labeled bottles with media and a virus.  The media contains all of the nutrients that a cell needs to grow.  The virus speeds up the cell replication,  with out the virus a cell would not dived very quickly.  The bottles are then placed in an incubator set a 37C, the temperature of the human body.  The samples are repeatedly checked and more media is added based off of how the bottles appear.  Each time a a change is made it is recorded.  Once a bottle is full of cells, it is split off into two bottles to ensure that if something goes wrong with one bottle there is a backup.  The end goal is growing a lot of wbc cells to create a multiple vials (called aliquots) of cells.
 
Biopsy samples are sent to the fibroblast lab.  Many of the techniques such as incubation and bottle contents are identical.  The skin samples are separated and dissected in a petri dish prior to incubation, because of their size.  To break things down further the samples are also treated with and enzyme.  With fibroblasts the bottles sit on their sides so that the cells grow on to attacked to the sides. 

Once the growth is done both blood and fibroblast samples are ready for cryo-preservation.  Samples are prepped by placing them in labeled glass ampules (aliquots).  One initial sample can produce many tubes if things go well.  The tubes are then sealed and placed in a dye bath to test for leaks.  In order to preserve the samples they are frozen at -316F in liquid nitrogen.  The liquid nitrogen helps to maintain a constant temperature.  This location of the samples are then recorded.

This is the final step of prepping cells to become cell lines.  Cells are now considered an official part of the biobank and ready to be used by researchers.

To generate iPScells, cell lines are either coaxed to becoming iPS cells using a series of viruses or chemical compounds.

To learn more about the CMD BioBank and how you can donate to this important resource, go to http://www.cmdir.org/index.php?option=com_content&view=article&id=139&Itemid=245&lang=en

Link to update on PTC 124/Ataluren, http://www.parentprojectmd.org/site/DocServer/Final_ICNMD_Data_Summary_2010-07-29.pdf?docID=9941

Ataluren, formerly known as PTC124, is a drug that works on nonsense mutations. There are several different types of mutations a person can have who has CMD, including missense mutation, nonsense mutation, deletion, duplication and frame shift. See below for a definition of these terms.
 
A nonsense mutation is a stop sign that doesn't allow the copying machinery of the DNA to complete copying into RNA, the step before making the protein.  The goal with PTC 124 and compounds like PTC 124 is to sit on the stop sign and allow the copying machinery to copy the full length of DNA into RNA to make a functional if not normal protein.
 
Although the exact rate of nonsense mutations in the CMDs in unknown, estimates are that for every subtype there is a 10-15 % rate of nonsense mutations. Having a drug that causes read through is only half of the issue. Some nonsense mutations are in regions of the DNA that cannot tolerate any abnormal change and these mutations may not be easily "fixed" by a read through drug. More work needs to be done on CMD mutations to characterize with nonsense mutations might be amenable to read through, resulting in a abnormal but still functional protein to be made that could "convert" the disease to a milder course.
 
A clinical trial of Ataluren in Duchenne muscular dystrophy just finished. In that trial, giving the drug did not show a difference in the boy's ability to improve distance walked during the 6 minute walk test (timed test).  However, a recent analysis shows that boys who received a lower dose of the drug did have a significant improvement in their ability to walk in the time test. What does this mean?  It means that at a lower dose, Ataluren may show benefit in ambulatory boys with Duchenne.  To read more, click here to read an update prepared by PTC Therapeutics for Parent Project Muscular Dystrophy.
 
What does this mean for CMD?  Nonsense mutation read through may be a viable strategy for those with CMD who have a nonsense mutation and whose nonsense mutation is not in a "critically sensitive" area related to the protein's function.  What are the steps needed to show that nonsense read through could work in the CMDs? 1. testing drugs that read through in cell lines from people with CMD  2. testing drugs that read through in CMD animal models 3. identifying the rate of nonsense mutations in the CMDs 4. identifying which nonsense mutations are amenable to being "fixed" by read through.


Mutation

A change in the genetic code, either brought about spontaneously, though chemicals or radiation or inherited. Point mutations refer to changes in a single nucleotide, such as the insertion of an adenine for a guanine. Point mutations can either be silent (meaning they do not cause a change in the triplet/codon resulting in no net change in amino acid insertion), nonsense (a nucleotide change that results in a stop codon, thus a shortened or truncated often nonfunctional protein) and a missense mutation (a nucleotide change that results in a substitution for a different amino acid, leading to a structurally misshapen but possibly still somewhat functional protein). Other mutations result from insertions or deletions during replication or transcription.

Nonsense mutation (premature stop codon)

Nonsense mutation: a nonsense mutation refers to a point mutation that changes a genetic sequence into a 3 letter code for a stop sign. If a stop sign arises in the middle of a gene because of a mutation, then when the gene encodes first mRNA and then a protein, a truncated or shortened protein will be made. Most of these shortened proteins are not functional and are destroyed by the cell.
Deletion Mutation

DNA is the genetic language and is read from left to right in 3 letter words. Let us say a normal piece of DNA says “THE MAN HAS CAT AND HAT” . DNA language does not have spaces so in DNA language, above would read: THEMANHASCATANDHAT.  In a deletion mutation, one or more letters are removed.  For example, if the E in THE was deleted, you would have THMANHASCATANDHAT. When the copying machinery would read it out by 3, it would no longer make sense THM ANH ASC ATA NDH AT.
Frameshift mutation

DNA is the genetic language and is read from left to right in 3 letter words. Let us say a normal piece of DNA says “THE MAN HAS CAT AND HAT” . DNA language does not have spaces so in DNA language, above would read: THEMANHASCATANDHAT. A frameshift mutation occurs when you either have a deletion, (one or more letters are removed) or an insertion (one or more letters are inserted). In the case of a deletion, if the E in THE was deleted, you would have THMANHASCATANDHAT. When the copying machinery would read it out by 3, it would no longer make sense THM ANH ASC ATA NDH AT. Similarly with an insertion, if two letters BX and were inserted after MAN, reading the genetic language in DNA would not make sense: THE MAN BXH ASC ATA NDH AT.
Inframe mutation

A mutation that does not cause a shift in the triplet reading frame; such mutations can, however, lead to an abnormal protein product
Insertion mutation

DNA is the genetic language and is read from left to right in 3 letter words. Let us say a normal piece of DNA says “THE MAN HAS CAT AND HAT” . DNA language does not have spaces so in DNA language, above would read: THEMANHASCATANDHAT. In an insertion, one or more letters are inserted. If two letters BX and were inserted after MAN, reading the genetic language in DNA would not make sense: THE MAN BXH ASC ATA NDH AT.
Introns/exons

Genes have both noncoding (introns) and coding (exons) regions. The noncoding regions contain genetic information that gets cut out when the genetic information goes from DNA to RNA and is not integral to protein manufacture. Point mutations in the introns usually have no effect, unless they cause a shift in the reading frame through an insertion or deletion. Exons contain the actual genetic information that will be used to manufacture proteins. Point mutations, insertions, duplications and deletions all are types of mutations that change the genetic code and can lead to disease
Missense mutation

A missense mutation is a  genetic change involving the substitution of one base in the DNA for another which results in the substitution of one amino acid in a polypeptide for another. A missense mutation is a “readable” genetic message although its “sense” (its meaning) is changed. This is in contrast to a nonsense mutation which has no meaning except to halt the reading of the genetic message.
Variant of Unknown Significance

An alteration in the normal sequence of a gene, the significance of which is unclear until further study of the genotype and corresponding phenotype in a sufficiently large population; complete gene sequencing often identifies numerous (sometimes hundreds) allelic variants for a given gene

As you may know, Cure CMD now offers a travel bug.  What is it?  Before we answer, that, you should know what geocaching is.  In short, it's a treasure hunt with a gps device. 

For example, someone might hide a tupperware container, filled with say... a logbook, pencil and a matchbox car and an army man, then post the coordinates on geocaching.com.  A cache is any container used to hold anything you'd like that is hidden to be found using only a gps device.  Other people will go out "geocaching" and search for it.  Once found, they will sign and date the log book.  Maybe take the army man and leave trading card.  They can also log the item as "found" on the geocaching website, and leave a note for everyone to see online.

What's a travel bug?  In short, it's a dog tag with a unique number.  It's meant to have a travel destination(s), or goal(s).  It should be placed in a cache, and folks will move it to another cache in intended direction.

Whoopee right?  It's actually a lot of fun, especially for the family, especially with a kid in a wheelchair.  We even take the dog.  Around our house, there are many "caches" hidden in local parks.

I had heard about it, but never tried.  I started looking into when Cure CMD announced the travel bug.  We ordered one so I thought we should learn a little more.  You can't use a TomTom or a car gps.  Many caches are located off of the street, and car gps devices are not meant to navigate to such a destination.

Funny story, after weeks of doing this, trying with our TomTom, then buying and returning a handheld gps tracker, I learned you can do this with a smartphone.  I've been resisting one, but finally broke down and got a droid.  On Saturday, we were out geocaching when Anne R. sent me an email and asked me to post about geocaching in the forum.  Hehe

In the past my husband has benefited from medical massages.  Has anyone had luck with health insurance covering it?  We were spending over $300 a month or more.
Charlene

Wow!! Thanks for the update.  I am so glad that we got a good wide spectrum of donors.
We must remember that we are the fuel to the research.

Finding treatments requires scientific resources, including blood and skin cells from people with CMD and their unaffected older siblings and parents. Help build a research resource for scientists studying CMD.

At the Family conference alone, we collected 31 samples (15 skin biopsies & 16 blood samples) which will significantly help in research, to eventually find treatments. See list below for details of what samples were collected. If you are planning a blood draw or a skin biopsy, consider coordinating with the CMD BioBank. A biopsy collection kit can be shipped to you 1 week before your scheduled appointment. After the biopsy's have been collected it is ok that they are shipped at room temperature because the air bill we include is to return ship them overnight delivery. As you know-it is important that the biopsies are shipped to be received at Coriell on a weekday, so that they can be processed upon arrival.

For more information, contact Tara Schmidlen lead genetic counselor at the CMD BioBank, tschmidl@coriell.org


Read more about the how Cure CMD partners with Coriell Institute to Launch CMD BioBank

http://curecmd.org/forum/index.php?topic=313.msg1134#msg1134


31 samples (15 skin biopsies & 16 blood samples)
Diagnosis Donor Status Sample Type
merosin deficient CMD affected biopsy
merosin deficient CMD carrier biopsy
merosin deficient CMD carrier biopsy
Ullrich CMD affected biopsy
Ullrich CMD carrier biopsy
Ullrich CMD carrier blood
Ullrich CMD carrier blood
Ryanodine Receptor 1 Congenital Myopathy RYR1 carrier blood
Ryanodine Receptor 1 Congenital Myopathy RYR1 carrier blood
 
Ullrich-Collagen VI carrier blood
Ullrich-Collagen VI carrier biopsy
Bethlem affected biopsy
merosin deficient CMD affected biopsy
merosin deficient CMD carrier biopsy
merosin deficient CMD carrier biopsy
walker-warburg carrier blood
merosin deficient CMD carrier blood
merosin deficient CMD carrier blood
Collagen VI deficiency (Ullrich or Bethlem?) carrier blood
Dystroglycanopathy POMT1 carrier blood
Lamin A/C related CMD CMD carrier blood
Ryanodine Receptor 1 Congenital Myopathy RYR1 carrier blood
merosin deficient CMD carrier blood
 
Ullrich affected biopsy
Ullrich carrier biopsy
Dystoglycanopathy POMT1 carrier blood
walker-warburg carrier biopsy
Ullrich carrier blood
Unknown Dystoglycanopathy-Merosin + carrier biopsy
Unknown Dystoglycanopathy-Merosin + carrier biopsy
Bethlem  affected blood

Dear CMD Community!

Cure CMD was thrilled by the turnout and support shown at this year's Family Conference.  Increased vendor support, extended family participation and a roster of 16 presenters with representation of CMD families from across the US showed how our CMD community is growing!

The best part of the conference is watching families connect and reach out to support each other- whether relaxing at the Friday night welcome dinner, catching up with people met at last year's conference, welcoming newly diagnosed families and avoiding getting hit with a hockey puck.

We are family.

Our family includes the scientists, allied health professionals, extended relatives (grandparents, aunts and uncles), doctors, advocacy, including the MDA and NIH who support and help our community thrive.

Cure CMD would like to thank its volunteer staff who made the 2010 conference possible: Susan Sklaroff- Van Hook, Diane Smith-Hoban, Lucinda Rosso, Susan Lee Miller, Meera Gandhi, Mike Newton, Terri Clausen, Mari Schindele, Simon Cantos, Linda Gowarty, Shawn Rudman,Tracy McCausland and Wayne Ogata.
 
We would also like to thank both Diane's Smith-Hoban's family for providing critical support in making sure we were well fed and CHOP staff: Dr. Xilma Ortiz, Livija Medne, Allan Glanzman, Meganne Leach and Alan Tuttle for their annual contribution to the conference and unflagging support of the CMD community.
 
We anticipate additional practical information breakout sessions for next year's conference, including one for newly diagnosed families, children with cognitive involvement/behavioral issues, cardiac issues and exercise (ambulatory and nonambulatory). We will also extend the caregiver and Dad's sessions to an hour and half.  Cure CMD appreciates feedback and will launch a brief survey to gather information to be used to plan next year's conference (info@curecmd.org)
 
2010 CMD Family Conference power point presentations will be posted here on the forum.
 
Cure CMD will also release a 2010 CMD Family Conference video for a suggested donation on merchandise page in upcoming months.

Conference pictures to be posted on Cure CMD forum. If you have pictures you would like to share, please email: info@curecmd.org

Luke Hoban is leading an exciting project to develop 4 targeted videos for CMD breathing issues:
1. General Advice from CMD BiPap Users
2. Luke's story: A Journey with BiPap
3. Lucinda Rosso: Parent Perspectives on Starting BiPap in Your Young Child
4. Dr. Hank Mayer explains Bipap and Cough Assist.

We are indebted to Sarah Dillon for her continuing work in editing videos and John Rosso for posting videos.

To watch an inspiring Cure CMD/SAM scrapbook video: 2008-2010,  http://www.youtube.com/watch?v=MH8fzaYSQ4E

We look forward to seeing you at an upcoming Cure CMD event and at next year's conference, August 2011!

Cure CMD- Anne, Eunice and Pat

Our daughter Kendal was diagnosed with Partial Merosin Deficiency in February of 2003. We spent the first few years of her diagnosis with a lot of concerns and questions of what this would mean for her life. In addition, we felt there was a lack of resources to help alleviate those feelings.  Around Christmas of 2009, I registered with the Cure CMD. In May of this year, Cure CMD contacted us in regards to the Comparative Outcome Measure Study at the National Institute of Health in Bethesda, Maryland. This past June we participated in this study. It was there that we had the opportunity to meet some truly remarkable and wonderful people like Dr. Anne Rutkowski, Eunice Kim, along with several other doctors and families whose children also are affected by CMD. It was such an awesome experience for Kendal to meet other children, for the first time, with her same disease. For me, it provided a network of families who have the same experiences and concerns as I do. Now even though we still have concerns for Kendal's future, we feel we have found a wonderful resource in Cure CMD.
    After leaving there I was inspired by Anne and the rest of the medical staff to try and do something to help Cure CMD. So on August 7th we celebrated Kendal's 7th birthday by having an after hours pool party in our hometown. It was titled "Kendal's Birthday Bash To Splash Out CMD." We invited the students in her class, our church congregation, relatives, and friends. In lieu of gifts we asked for donations to be made to Cure CMD. It was so wonderful to see the outpour of support that our small town extended to Kendal. Words alone cannot express how thankful we are to everyone who came to help us celebrate, and offered their monetary support in helping us find a cure. Our family is left with a feeling of being truly blessed by our community, family, and friends, as well as the Cure CMD organization. Thank you Cure CMD  for all you do to help us find us fight this battle. We look forward to offering you our continued support in the future.
 
Sincerely,
Jay, Leslie, Kendal, Drake, and Bergan Johnsen

Scientists at McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center, NC, have recently created for the first time a viable mouse model of muscular dystrophy with mutations in fukutin-related protein (FKRP). Mutations of FKRP cause diseases ranging from mild limb-girdle muscular dystrophy type (LGMD2I), more severe congenital muscular dystrophy (CMD), to Walker-Warburg syndrome and muscle-eye-brain disease with striking brain and eye defects.   McColl-Lockwood Laboratory has generated a mouse model with one specific mutation in the FKRP gene. This mutation (called P448L mutation with a change of one amino acid in the protein product) has also been seen in CMD and LGMD2I patients. This small change alters the function of the FKRP gene.  

The FKRP mouse muscles and brain have almost completely lost the ability to produce functional sugars on one important muscle cell surface protein, called alpha-dystroglycan (DG). Since the sugars are critical for normal muscle functions, the absence of the sugars is thought to be the direct cause of muscle wasting in these related diseases.  The functional sugars are also considered to be important for neuron development.

Dr Lu’s team found that the FKRP mouse indeed develops a wide range of abnormalities in the central nervous system, particularly in the cerebrum and cerebellum. The brain and eye defects are highly reminiscent of what seen in severely affected CMD and other diseases. In addition, skeletal muscles of the mouse model develop progressive muscular dystrophy.

Despite the severe defects in central nervous system, some of the mice have survived more than 10 months. The mouse model therefore recapitulates the wide clinical manifestation associated with FKRP mutations and mutations in other genes affecting sugar modifications of the alpha-dystroglycan. The mouse represents a valuable model to further elucidate the functions of FKRP and develop therapies for FKRP-related muscular dystrophies. The McColl laboratory is now planning to use the mouse model for the tests of gene therapy and new drugs with the aim to alleviate disease progression related to the loss of functional sugars on diseased muscles.

Why are mouse models important in a quest to find treatments?  Mouse models are a relatively cost effective way of understanding how certain genetic mutations cause disease.   Having a mouse model allows scientists to study the disease, understand the impact of the mutation on multiple organ systems (heart, brain, muscle) and determine if a compound has the potential to improve the disease.

Mice are not humans. Thus, not all drug compounds that work in mice work in humans and vice versa. However, in order to build a body of evidence showing that a drug compound has great potential, it is important to demonstrate that the drug compound impacts survival, function and if possible, biomarkers in a positive way in the mouse. Establishing clinical evidence of drug effect in the mouse, greatly enhances the opportunity for the drug to make it into human clinical trials.

(Please contact Dr Yiumo Chan (yiumo.chan@carolinashealthcare.org) or Dr Qi Lu (qi.lu@carolinashealthcare.org) for learning more of the FKRP mutant models and collaboration. The results are now published online, Human Molecular Genetics, July 30, PMID: 20675713).

Lockwood Laboratory for Muscular Dystrophy Research directed by Dr Qi Lu, MD, PhD focuses translational research on muscular dystrophy, specifically on drug development, gene therapy and antisense therapy for FKRP-related muscular dystrophies and DMD. The laboratory is mainly supported by Carolinas Muscular Dystrophy Research Endowment at the Carolinas HealthCare Foundation, NIH and Department of Defense).