Genetic mutations and cellular modifications may provide clues to the associations between slight genetic abnormalities and often fatal diseases

Modern research is delving deep into the human genome to find the answers to medical mysteries and, hopefully, cures for the most deadly diseases. According to four new studies presented during the 46th Annual Meeting of the American Society of Hematology, genetic mutations and cellular modifications may provide clues to the associations between slight genetic abnormalities and often fatal diseases.

"We are hopeful that this type of research will help clearly identify genetic risk factors and markers for some of the most complex diseases we are fighting," said Janis Abkowitz, M.D., professor in the Division of Hematology, Department of Medicine, at the University of Washington in Seattle and head of the Section of Hematology at the University of Washington Medical Center. "More importantly, finding these links could lead to the development of therapies to treat the diseases early, and eventually prevent them entirely."

Mutations in TERT, the Gene Encoding Telomerase Reverse Transcriptase, in "Acquired" Aplastic Anemia Inhibit Enzymatic Function by a Dominant Negative Mechanism of Action

Approximately one-third of patients with aplastic anemia (AA, which occurs when the bone marrow fails to produce blood cells) have short telomeres (the natural end of a chromosome), leading to longer duration of disease and poorer response to therapy. While some of these cases have a specific mutation of the TERC gene (telomerase RNA template subunit), this study by researchers at the National Heart, Lung, and Blood Institute examines another area, the TERT gene (telomerase reverse transcriptase subunit), to analyze the correlation. Telomeres help maintain the life of the cell, as they keep the ends of the various chromosomes in the cell from accidentally attaching to each other.

In examining the gene in 122 patients with AA and 282 controls, the researchers found four new nonsynonymous mutations not present in the controls. They also identified three polymorphisms, two nonsynonymous SNPs (single nucleotide polymorphisms, DNA sequence variations that occur when a single nucleotide in the genome sequence is altered) and one deletion of a single amino acid.

To determine the functional consequences, the team analyzed the telomere lengths. All patients with the TERT mutation had noticeably shorter telomeres as compared to the controls, as opposed to other AA patients with polymorphisms, who exhibited normal telomere lengths. In one patient, the presence of TERT in other family members correlated to shorter telomeres. In all patients with mutations, testing showed little or no telomerase activity compared to the control population.

The researchers reproduced these mutations to analyze the results and found that all mutant TERT cell lysates, or residuals, were severely deficient in enzymes. In addition, combining individual TERT genes and their mutations severely reduced telomerase activity.

"These results indicate that mutations produce nonfunctional proteins, which are responsible for the lack of telomerase activity," said Rodrigo Calado, M.D., of the National Heart, Lung, and Blood Institute of the National Institutes of Health, and lead author of the study. "The mutations affecting the enzyme-binding area of TERC and the actual transcriptase of TERT are, in fact, genetic risk factors for the development of aplastic anemia."

G-CSF Receptor Mutations Found in Patients with Severe Congenital Neutropenia Confer a Strong Competitive Advantage at the Hematopoietic Stem Cell Level that is Dependent on Increased Systemic Levels of G-CSF

Severe congenital neutropenia (SCN) is a rare syndrome that presents shortly after birth with a severe decrease in the number of neutrophils (a type of white blood cell that kills bacteria) in the blood. Children with this syndrome are prone to frequent infections and have a markedly increased risk of developing myelodysplasia (MDS) or acute myeloid leukemia (AML). Treatment with G-CSF increases the number of neutrophils in most patients, is effective in preventing infections, and prolongs survival. However, the risk of developing MDS/AML remains. Mutations of the receptor for G-CSF, the G-CSF receptor (G-CSFR), are found in most patients with SCN who develop AML or MDS, but are rare in other forms of AML. These observations raise several questions: Do G-CSFR mutations contribute to the development of AML/MDS in children with SCN? Why are G-CSFR mutations so common in SCN-related AML and yet rare in other leukemias?

To answer these questions, a team of researchers at the Washington University School of Medicine developed a mouse model in which roughly half of the white blood cells contained a G-CSFR mutation. They showed that the proportion of cells containing the G-CSFR mutation was stable over a period of months, demonstrating that these cells had no growth or survival advantage. In children with SCN, the level of G-CSF is usually elevated, either through increased production in the body or secondary to treatment with G-CSF. To simulate these conditions in their mouse model, the researchers then treated mice for 21 days with G-CSF. At the end of treatment, the percentage of G- CSFR mutant cells in the blood and bone marrow drastically increased to 97.6 percent from 45.7 percent before treatment. This shift also extended to stem cells, where the percentage of cells carrying the G-CSFR mutation increased from 53.3 percent to 97.8 percent. Secondary transplants confirmed that this brief exposure to G-CSF was sufficient to preferentially expand the G-CSFR mutant stem cells.

"These results show that the expression of mutant G-CSFR results in a strong growth advantage at the stem cell level, but only in the presence of an increased concentration of G-CSF," said David Grenda, M.D., of the Washington University School of Medicine and lead author of the study. "This may explain the nearly unique association of these G-CSFR mutations with SCN, and provides further evidence that these mutations may contribute to the development of leukemia, as most leukemias are thought to arise in stem cells."

Mesenchymal Stem Cell Engraftment in Bone Following In Utero Transplantation in a Patient with Severe Osteogenesis Imperfecta

An important factor for successful allogeneic (between humans) stem cell transplants has been obtaining similar markers, or traits, between the donor and recipient cells. The purpose of this study by a team of researchers at the Karolinska University Hospital in Sweden was to assess the ability of fetal stem cells (MSCs) to engraft (grow together with the original cells) and reproduce after being transplanted, even without matched cell similarities.

The subject of the study was a human female fetus that had been diagnosed with severe osteogenesis imperfecta, a brittle bone disorder, with multiple intrauterine fractures. In the 32nd week of gestation, the team transplanted male fetus MSCs not matched to the female fetus's own cells. At nine months of age, bone tests showed regularly arranged and configured bone framework, and tests showed positive protein cells and marrow consistency for bone density and growth. The researchers found no evidence of the patient's cells reacting against the male donor MSCs in in vitro co-cultures.

Supplemental bisphosphonate therapy (treatment to increase bone density) was started at four months, and at two years of age, psychomotor development was normal, as was growth.

"These exciting results suggest that these fetal stem cells can be transplanted and successfully incorporated into bone in a human fetus even when the cells are not necessarily compatible to the recipient," said Katarina LeBlanc, M.D., Ph.D., of the Karolinska University Hospital, and lead author of the study. "We hope to further our research to determine the potential for early therapy in utero in other categories."

Developmental Stage-Selective Effect of Somatically Mutated GATA-1 in Down Syndrome AML M7-a Potential Basis for Transient Myeloproliferative Disorder

Doctors have identified acquired mutations of the gene GATA-1 in nearly all newborns with Down Syndrome (DS) who develop a transient myeloproliferative disorder (termed TMD), an abnormal hematologic condition seen shortly after birth or in the neonatal period that is characterized by leukocytosis, an abnormal increase in white blood cell count, and thrombocytopenia, a low platelet count in the blood. The mutation has also been found in most DS neonates diagnosed with leukemia. The GATA-1 gene encodes a protein in the GATA family of transcription factors, which plays an important role in red blood cell and megakaryocytic development.

There are two hypotheses for the temporal nature of TMD and restriction of DS-AMKL (acute megakaryocytic leukemia, a rare subtype of acute myeloid leukemia resulting from the overproduction of cells responsible for platelet development in the blood) to infants and young children. The first suggests that the GATA-1 mutation in DS may occur only during a specific development window. The other hypothesis suggests that the mutation may occur at any time, but only specific target cells present during development are sensitive to its effects. This study by researchers at Harvard Medical School attempts to distinguish between the two hypotheses.

In their study, the team created mice from embryonic stem cells engineered to harbor the specific mutant form of GATA-1 observed in DS patients. Adult mice produced normal platelet and red blood cell counts, and, at the fetal stage, the mutant embryos were temporarily anemic, but later returned to normal. The fetal livers showed increased numbers of CD41+ platelet precursors, particularly early on. Of importance in distinguishing the models, the investigators found that yolk sac and fetal liver cells showed a large number of overactive megakaryocytic cell colonies, which were greatly reduced after mid-stage and absent after birth. In vitro, embryonic stem cells with the mutant GATA-1 also gave rise to the abnormal megakaryocytic colonies, as did wild-type cells forced to express the mutant protein.

The biological effects of mutant GATA-1 appear to be restricted to progenitor cells that are present only at embryonic and early fetal liver stages of development. The investigators suggest that this finding accounts for the transient nature and restriction to DS-AMKL. The results support the theory of exaggerated expansion of fetal progenitors (precursor cells) under the action of mutant GATA-1. This is predicted to lead to TMD, which then evolves into full DS-AMKL following additional, yet unknown, mutations.

"Our study found that the mutation of the GATA-1 gene seems to separate growth and development from differentiation," said Stuart Orkin, M.D., from Harvard Medical School, and lead author of the study. "The restricted effect of the mutation on a specific cell type may apply as well to other forms of infant leukemias or other malignancies."