“Study to Gather Gene Data for All Families”
I work on Diamond-Blackfan anemia (DBA) at the Dana-Farber Cancer Institute, Department of Pediatric Oncology in Boston, MA, in Dr. Colin Sieff's lab. The funding from the DBA Foundation for my grant proposal "Phenotype-genotype correlation in Diamond-Blackfan anemia" has enabled me to screen DNA for ribosomal protein S19 (RPS19) gene mutations from all DBA families from the USA and from outside the country, who want to participate in the study. It also gives me an opportunity to search for relationships between RPS19 mutations and clinical symptoms in DBA. I work with research technician, Ela Latawiec, who pursues blood samples, and with Drs. Donna Neuberg and Shuli Li, the statisticians, who statistically calculate the relationship between RPS19 genotype and DBA phenotype. This project is conducted in collaboration with the Diamon-Blackfan anemia registry run by Drs. Jeffrey Lipton, Adrianna Vlachos, and Clinical Research Coordinator Eva Atsidaftos, who evaluate clinical status and collect blood samples from the patients and their family members.

The first DBA gene, RPS19 identified by Dr. Dahl's group is mutated in approximately 25% of patients, however its role in DBA remains unknown. The clinical heterogeneity of DBA is still among the most challenging and poorly understood aspects of the disease. Although severe anemia is a prominent feature of DBA, mild anemia or only subtle indications of the red cells abnormality such as increased MCV and/or elevated erythrocyte adenosine deaminase activity are found in some patients. In previous reports the clear correlation between phenotype and a specific mutation was not found. However, a recent study in the UK showed the possibility of phenotype-genotype correlations with respect to hematological severity of anemia and physical anomalies. To further address the question whether there are correlations between genotype such specific RPS19 mutation or lack of these mutations and the clinical symptoms, we undertook more detailed investigation based on larger group of DBA patients.

Our objective is to sequence DNA from at least 400 unrelated DBA patients for mutations in RPS19 gene. We expect to find RPS19 mutations in approximately 100 patients and plan to screen DNA from family members of these patients. We will then investigate the relationship between RPS19 genotype and the clinical symptoms, such as severity of anemia, response to steroid treatment, different physical anomalies, and presence of the malignancy in a family. These relationships may be useful in genetic counseling allowing the prediction of the clinical course of the disease in families with certain mutations. The potential phenotype-genotype correlations may further our understanding of the role of RPS19 protein in erythopoiesis and development and the functional significance of specific RPS19 mutations. Other comparisons will be clinical symptoms in two groups of patients with and without the RPS19 mutations. We will investigate whether there is a statistical difference between these two groups of patients with respect to hematological severity of the disease, physical anomalies, cancer, and evolution of the disease over time.

Finally, we will create DNA and EBV immortalized lymphoblastoid cell line repositories for further studies on DBA.
We are actively seeking patients and their families to participate in this study. Participation will involve review of clinical history and donation of approximately 3-5 ml (2 teaspoons) of blood. Anyone who is interested in participating or in learning more about the study, please contact Dr. Gazda at any time by email (hanna_gazda@dfci.harvard.edu) or by phone (617-632-3258).
Hanna Gazda, M.D.
Dana-Farber Cancer Institute, Pediatric Oncology, Rm M615
44 Binney Street, Boston, MA 02115
ph. 617-632-3258
fax: 617-632-6845
hanna_gazda@dfci.harvard.edu

Dr. Stefan Karlsson, a researcher specializing in genetics, has been investigating the possibility of developing gene therapy for patients with DBA who have mutations in the Ribosomal protein S19 gene. In order to continue and accelerate this research, Kr. Karlsson needs more hematopoietic stem cells. Accordingly, he has asked the DBAF to help him identify patients with a deficiency in Ribosomal protein S19 who are willing to undergo peripheral blood stem cell harvest. The peripheral blood stem cell harvest is a relatively routine medical procedure.

Attached is the letter sent by Drs. Karlsson and Richter asking the DBAF for help with its study:
Lund University
Institute of Laboratory Medicine, Lund
Department of Molecular Medicine and Gene Therapy
Stefan Karlsson, MD, PhD
Professor of Molecular Medicine
Director, Gene Therapy Center
July 14, 2004

Re: Stem Cells and Gene Therapy for DBA

Dear Members of the DBA Foundation:
I am writing to you to solicit your help. During the past 4-5 years we have been investigating the possibility of developing gene therapy for patients with DBA who have mutations in the Ribosomal protein S19 gene. The work has progressed well and we have published tow recent papers which present evidence for that gene therapy of RPS19 deficient DBA may indeed be feasible (Hamaguchi et al, Blood, 100:2724-273 , 2002 and Hamaguchi et al, Mol Ther 7:613-622, 2003). The progress of the project has gone well scientifically, but we have difficulties obtaining enough hematopoietic stem cells (stem cells that can generate all blood cells) to do the required experiments. Before we can apply to the authorities (FDA and equivalent organizations in countries outside the US) to perform clinical trials to develop gene therapy for DBA, we have to do many additional experiments. So far, we have performed experiments in culture that demonstrate feasibility to develop gene therapy for RPS 19 deficient DBA but experiments in animal models remain to be done before clinical trials begin. For these animal experiments, we need hematopoietic (blood) stem cells. In the published experiments, we have used stem cells from the bone marrow of patients which have agreed to donate small bone marrow samples for these studies. Unfortunately, we can only obtain 2 million CD34+ cells (contain progenitor and stem cells) from each patient and this is only enough for one gene therapy experiment. Since we now need to transplant gene-modified stem cells into appropriate animal models, more stem cells are required. It is possible to harvest a lot of stem cells from patients by mobilizing hematopoietic stem cells from the bone marrow using growth factors. The stem cells can thereafter be harvested from the blood stream in a Blood Bank (Dept. of Transfusion Medicine) by having the patient's blood flow through a leukapheresis machine that separates the stem cells from other blood cells using principles that are analogous to the ones that are used in a cream separator. The advantage with this method is that many stem cells can be harvested, approximately 100 times the number that can be harvested by bone marrow aspirations under local anesthesia. Dr. Cynthia Dunbar at the National Institutes of Health in Bethesda has already harvested peripheral blood stem cells from two DBA patients successfully and without complications.
We are now writing to ask the DBA Foundation to help us to identify DBA patients with a deficiency in Ribosomal Protein 519 who is willing to undergo peripheral blood stem cell harvest to help in the development of gene therapy for DBA. We are looking for patients that are more than 12 kg in weight. The peripheral blood stem cell harvest can be performed at the NIH in Bethesda MD or in Lund, Sweden. Ninety percent of the stem cells harvested at NIH by Dr. Dunbar will be stored frozen and can be used at a later stage to treat bone marrow failure or leukemia t a later stage, if required. Ten percent of the cells will be used for gene therapy research. This is a protocol that was established a few years ago to give patients an opportunity to keep stored blood stem cells. The protocol in Lund will be solely to develop gene therapy and all the cells harvested will be used for gene therapy research aimed towards developing a cure for RPS 19 deficient DBA.
We sincerely hope that you are willing to help us to identify patients that want to help us to cure DBA.

Best wishes,

Stefan Karlsson MD, PhD Johan Richter MD, PhD
Professor of Molecular Medicine Senior Physician

Harvest of peripheral stem cells from patients with Diamond-Blackfan Anemia and Mutation in the RPS19 Gene.Description of Procedure.
The blood forming stem cells in the body normally reside in the bone marrow but they can, under certain circumstances, be mobilized to the bloodstream temporarily. One way to mobilize stem cells from the marrow to the peripheral blood is through treatment with a drug called granulocyte-colony stimulating factor (G-CSF). G-CSF is a bone marrow stimulating hormone produced in the human body under normal circumstances. By giving this substance as daily subcutaneous injections for 4-5 days at somewhat higher doses than normally produced, blood forming stem cells can be made to move from the marrow out into the blood stream.
From the blood stream, these cells can be harvested through a procedure called leukapheresis. During this procedure blood is led through a leukapheresis machine which can separate the portion of blood cells containing stem cells from red blood cells, platelets and plasma which can be returned to the individual in question. To gain access to the bloodstream fro this procedure needles have to be inserted into veins in both arms or into the large vein in the groin. The leukapheresis procedure takes 3-4 hours and can normally be performed on an outpatient basis. Mobilization and harvest of peripheral stem cells has been a routine medical procedure for more than 10 years and it is performed on thousands of patients and healthy stem cell donors all over the world every year.
The side effects from the above procedure are rare and mild. Treatment with G-DSF can lead to mild to moderate bone pain for a few days. Treatment with an ordinary pain killer can alleviate this problem. Sometimes G-CSF treatment can lead to a transient, harmless elevation of liver enzymes which return to normal when the medication is stopped. The procedure involves only a minimal blood loss and has not been shown to affect the future blood forming capacity of the individual.
From a practical standpoint, participation in our study involving harvest of peripheral stem cells from patients with Diamond-Blackfan Anemia and mutation in RPS19 will first include a medical check-up including a physical exam, electrocardiogram and blood tests. If all of these are satisfactory the person can be included in the study. G-CSF can be administered in the home of the patient as a subcutaneous injection for 4-5 days. The leukapheresis can then normally be performed as an out-patient procedure.
The part of the stem cells harvested under this program that will be used for gene therapy research cannot be returned to the individual in question even if the gene transfer experiments performed are successful. However, we hope that our research will help pave the way for future clinical gene therapy of Diamond-Blackfan Anemia.
Lund, Sweden

July 14, 2004

Johan Richter Stefan Karlsson

Associate Professor Professor

Department of Molecular Medicine and Gene Therapy

Lesson Learned: "I Am My Child's Greatest Advocate."

Our Thanksgiving baby truly gives us a real reminder each year how much we have to be thankful for. Joshua Lorenz was born Thanksgiving weekend 1992. Thankfully he was our second born. I knew something was wrong carrying him. He rarely moved and never kicked. Maybe itÌs a girl, I thought, remembering how I would cry from the pain of carrying our first son who kicked nonstop under my rib cage. It was scary and very different having hardly no movement.

I gained over sixty pounds, and Josh weighed a hefty 8 pounds 10 ounces that afternoon he was born. "Josh's vitals are fine," I was told. Luckily we were blessed with a nurse, Barb Weber, that went beyond the call of duty to make sure things were all right. I remember Barb telling us that she was running "a test." She just thought our baby was a little pale. I was pale, too. She said it was probably nothing to be concerned with because his vitals were fine. Nurse Barb came back and said, "Sorry they made a mistake running the test. TheyÌll have to run it again." I didnÌt think anything of it.

Josh looked fine and healthy. I remember a relative asking "Is everything all right? Your nurse seems to be a little concerned and is checking on Josh a lot." By the time nurse Barb came back the third time it was later, and Greg and I were by ourselves. The mood had definitely changed. The first two tests canÌt be right. WeÌre running a third. When Barb entered the room with the third test results she was holding back tears and apologizing. We owe our sonÌs life to nurse Barb Weber. If she would not have run that simple test early on we would not have been alerted to the distress Josh was under. His hemoglobin was dropping fast. The specialists at our hospital said it was out of their league. It was touch and go. We were not allowed to see our baby. We were told "There is no room for you while we are working on him." Josh was being transferred and an ambulance was enroute with the needed NICU medical staff. When the NICU staff arrived, they told us exactly the way it was Û no chocolate covering. To be told that after giving birth - that your child may die Û I couldnÌt believe it. It was almost midnight and our baby was enroute to the needed hospital and hooked up to all kinds of machines. Greg and I wrestled to let them release me to be able to travel to the new hospital to be with our son. With all the commotion and not knowing how to get to the new hospital, we were in awe that we beat the ambulance that left much earlier than us.

We found out months later that Josh had to be revived while enroute. If Nurse Barb would not have caught it (which the NICU doctors said was a miracle) then Josh would have been wrapped up; when they would have gone to check on him, he would already have passed on. All I could think of was SIDS (Sudden Infant Death Syndrome). They revived Josh, and he got three transfusions over time. He was strong as ever after getting the transfusions Û he even pulled out his breathing tube! He looked so big in those cubicles, 8 pounds 10 ounces next to 1 and 2 pound babies. After one week he came home. Big brother Tyler (20 months old) had mixed emotions. Has Mom abandoned me for my new little brother? Life just started to get over the roller coaster event when Josh started sleeping nonstop-not even to eat. Looking pale, I took him into our regular doctor. He said I was overreacting and that there is nothing wrong with Josh! Yeah I thought, "You didnÌt even run the test I asked you to run!" Luckily, I called the NICU doctor that took care of Josh. He knew something had to be done and spoke with me about what was needed. He stated "I just told your regular doctor the same advice just before you called." I was fortunate to have consulted the NICU doctor. Our regular doctor called shortly after, and he told a different story from what I was told by the specialist. I confronted him that what he said was not what the specialist recommended. He stated that he did not agree with the specialist and that there was no need for what was recommended. I was outraged. Within minutes I had a referral from the NICU specialist and an appointment for Josh to see the new doctor. That decision saved JoshÌs life.

Within a couple of days I went from being told that I was overreacting to being told that if I had waited another ten days Josh would have been dead. JoshÌs original doctor could have cost Josh his life. I learned a very valuable lesson. I am the one responsible for my sonÌs care. And because I have the greatest concern for my child, I am my childÌs greatest advocate. JoshÌs hemoglobin was once again dangerously low. We were told Josh needed a bone marrow tap to find the cause. Anesthesia was not recommended because they thought the anesthesia could cause problems. The bone marrow confirmed what Dr. Shahidi at UW ChildrenÌs Hospital in Madison suspected. I remember Dr. Shahidi telling Greg and I that Josh had Diamond-Blackfan Anemia. Dr. Shahidi explained DBA to us and Josh started on the steroid regimen. Josh was steroid responsive. Then, Valentines week of 1995 Josh was hospitalized with RSV. 1995 was a difficult year. Josh went to the doctor over 70 times which did not include hospitalizations. We were told we may lose Josh again. We frantically hoped that JoshÌs older brother was an HLA match for bone marrow. They were not a match. On a weekly basis I was asked to consider having another baby in hope that the stem cells from the umbilical cord would be a match for Josh. We were under so much stress in 1995 that a baby, we felt, would be too much. Our lives revolved around doctors and Josh.

In June we switched to a stronger steroid- dexamethasone. Then we added cyclosporin to enhance the stronger steroid even more. Friday of Labor Day weekend I could feel Josh slipping away. On September. 6, 1995 Josh got his first blood transfusion since the week he was born in November of 1992. His hemoglobin went from 4.5 to 7.4. We continued with the higher dose medicines in hope that his bone marrow would bounce back. We sought other medical doctors in the DBA field for advice. Fortunately, very late in October, our doctor found an article and got the protocol for an experimental treatment by Dr. Bernini for intravenous methylprednisone treatment. In November we knew the medicines were not working at all. We started to begin weaning him of all medicines and began blood transfusions. In the spring we would begin this new protocol. During the winter of 1995 we let his body heal, his immune system get stronger, his bones heal, and all the side effects from the medicines dissipate. The 1996 year would be about hope and fear. January and February we prepared. Baselines were done, surgeries performed, and a portacathe put in for treatment. And importantly, we talked with parents whose children had completed the original protocol. We learned from them what to expect and to prepare for. We would have not been emotionally prepared if it wasnÌt for all the DBA families that we have a close bond with. And then, March came.

We started the protocol in March. Within six days we saw his retics jump up. On day nine, his retics were 6.2 with a hemoglobin (hgb) of 8.7. On day 13, his retics were 6.3 with a hgb of 9.3. On day 15, his retic count was 4.5 with a hgb of 10.1. Fortunately, Josh had very minimal side effects. Many were not as lucky. We continued on with the protocol. Home health care came out to our home so I could learn daily care for Josh. His daily blood pressure was monitored and daily intravenous medication was mixed with the saline solution bag. It was not uncommon for Josh to get up and start doing his vitals after he got his breakfast. Near the end of breakfast, I hooked up his IV and started the IV pump for his medicine. On May 2, we switched to oral prelone. On May 23, we switched to an even weaker oral steroid. On February 17, 1997, we were unable to access his port for labs. We couldnÌt access it for several weeks so Josh then had his chest portacathe removed as it only served as a threat of infection. By this time Josh was stable. We continued to reduce his steroids much slower. Three times we tried every other day steroids. Thankfully, our doctor insisted that we keep trying. Josh needed his medicine bumped up for the initial every other day until his body got used to it. Once we recognized that Josh was not bottoming out, we started seeing the side effects that we were accustomed to seeing fade away. Josh started to grow, and we started to lower his every other day steroid. For the last two and a half years we have not reduced his steroids. Josh has been weaning himself by gaining weight. His normal hgb is in the 8-9 range. When Josh was on the experimental protocol we lived in a bubble. We were afraid of catching anything because of his immune system being compromised by long term high dose steroid use. We are less concerned about the little sniffles now. There will always be that twitch in the bottom of my stomach reminding me that all it takes is one episode with pneumonia, RSV, or chicken pox to send us back down to that roller coaster.