Feasibility of gene therapy for late neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis is a progressive childhood neurodegenerative disorder characterized by intracellular accumulation of autofluorescent material resembling lipofuscin in neuronal cells. This report summarizes the new therapies under consideration for late infantile neuronal ceroid lipofuscinosis, with a focus on strategies for in vivo gene therapy for the retinal and central nervous system manifestations of the disease.

Decrease in drug accumulation and in tumour aggressiveness marker expression in a fenretinide-induced resistant ovarian tumour cell line.

We investigated whether the efficacy of fenretinide (HPR) against ovarian tumours may be limited by induction of resistance. The human ovarian carcinoma cell line A2780, which is sensitive to a pharmacologically achievable HPR concentration (IC(50)= 1 microM), became 10-fold more resistant after exposure to increasing HPR concentrations. The cells (A2780/HPR) did not show cross-resistance to the synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and were not sensitive, similarly to the parent line, to all-trans-retinoic acid, 13-cis-retinoic acid or N-(4-methoxyphenyl)retinamide. A2780/HPR cells showed, compared to parental cells, a 3-fold reduction in colony-forming ability in agar. The development of HPR resistance was associated with a marked increase in retinoic acid receptor beta (RARbeta) mRNA and protein levels, which decreased, together with drug resistance, after drug removal. The expression of cell surface molecules associated with tumour progression including HER-2, laminin receptor and beta1 integrin was markedly reduced. The increase in the levels of reactive oxygen species is not involved in HPR-resistance because it was similar in parental and resistant cells. Conversely differences in pharmacokinetics may account for resistance because, in A2780/HPR cells, intracellular peak drug levels were 2 times lower than in A2780 cells and an as yet unidentified polar metabolite was present. These data suggest that acquired resistance to HPR is associated with changes in marker expression, suggestive of a more differentiated status and may be explained, at least in part, by reduced drug accumulation and increased metabolism.

Serial intraoperative magnetic resonance imaging of brain shift.

OBJECTIVE: A major shortcoming of image-guided navigational systems is the use of preoperatively acquired image data, which does not account for intraoperative changes in brain morphology. The occurrence of these surgically induced volumetric deformations ("brain shift") has been well established. Maximal measurements for surface and midline shifts have been reported. There has been no detailed analysis, however, of the changes that occur during surgery. The use of intraoperative magnetic resonance imaging provides a unique opportunity to obtain serial image data and characterize the time course of brain deformations during surgery. METHODS: The vertically open intraoperative magnetic resonance imaging system (SignaSP, 0.5 T; GE Medical Systems, Milwaukee, WI) permits access to the surgical field and allows multiple intraoperative image updates without the need to move the patient. We developed volumetric display software (the 3D Slicer) that allows quantitative analysis of the degree and direction of brain shift. For 25 patients, four or more intraoperative volumetric image acquisitions were extensively evaluated. RESULTS: Serial acquisitions allow comprehensive sequential descriptions of the direction and magnitude of intraoperative deformations. Brain shift occurs at various surgical stages and in different regions. Surface shift occurs throughout surgery and is mainly attributable to gravity. Subsurface shift occurs during resection and involves collapse of the resection cavity and intraparenchymal changes that are difficult to model. CONCLUSION: Brain shift is a continuous dynamic process that evolves differently in distinct brain regions. Therefore, only serial imaging or continuous data acquisition can provide consistently accurate image guidance. Furthermore, only serial intraoperative magnetic resonance imaging provides an accurate basis for the computational analysis of brain deformations, which might lead to an understanding and eventual simulation of brain shift for intraoperative guidance.

Evaluation of two current generation enzyme immunoassays and an improved isolation-based assay for the rapid detection and isolation of rotavirus from stool.

BACKGROUND: Rapid and accurate rotavirus testing is important in decisions involving patient care and management. Quality assurance testing needs to be periodically performed, especially among widely used assays having a direct impact on patient care. OBJECTIVES: To evaluate the current generation Kallestad Pathfinder Direct antigen Detection system (PTH), and the widely used Rotaclone(R) Rotavirus EIA Diagnostic Kit (RTC), in comparison with an improved cell culture amplification-antigen detection (CCA-Ag) isolation-based assay. STUDY DESIGN: Two hundred stool specimens (specimen stored at > or =-75 degrees C), which had been previously tested by PTH, were tested by RTC and CCA-Ag. Discordant specimens were retested by PTH, blocking assay, polyacrylamide gel electrophoresis (PAGE), and/or electron microscopy (EM). RESULTS: Among 200 stool specimens, 197 were in accord by PTH, RTC and CCA-Ag. The sensitivity, specificity, positive and negative predictive values for RTC, PTH and CCA-Ag were, 100, 99, 99, 100, 100, 99, 99, 100; and 98, 100, 100, 98%, respectively. Among five initially discordant specimens, two required a period of 10 days to affect isolation. A non-cultivatable (CCA-Ag negative) but true positive specimen, was identified as rotavirus group A serotype G2 by RT-PCR. Four true positive but discordant specimens were blocking assay negative using one or both EIA kits. CONCLUSIONS: PTH and RTC are excellent rotavirus detection system. However, PTH is more expensive (ca. $3.50 vs. $2.00 per test), mandates a slightly longer turn-around time (ca. 1 vs. 1.5 h), and necessitates slightly more hands-on manipulative/preparative steps. Blocking assay was not a reliable confirmatory test for the resolution of specimen discordancy. A combination of CCA-Ag, PAGE, EM, and/or perhaps RT-PCR, is recommended as an appropriate test panel for the resolution of discordant results during assay evaluation. The newly modified and simplified 48-h rotavirus isolation-based assay may serve as a base line methodology in laboratory evalaution studies, as a laboratory support methodology during drug/vaccine efficacy trials, or for the testing of sources (e.g., biopsy/autopsy tissues) not approved for assay by commercial rotavirus kits.

Rheolytic thrombectomy of the occluded internal carotid artery in the setting of acute ischemic stroke.

BACKGROUND AND PURPOSE: Acute thromboembolic stroke complicated by ipsilateral carotid occlusion may present both mechanical and inflow-related barriers to effective intracranial thrombolysis. We sought to review our experience with a novel method of mechanical thrombectomy, in such cases, using the Possis AngioJet system, a rheolytic thrombectomy device. METHODS: A review of our interventional neuroradiology database revealed three patients in whom an occluded cervical internal carotid artery was encountered during endovascular treatment for acute stroke and in whom thrombectomy was attempted, using the 5F Possis AngioJet thrombectomy catheter. The medical records and radiographic studies of these patients were reviewed. RESULTS: Three patients were identified (ages, 52--84 years). Two patients had isolated occlusion of the internal carotid artery; in one patient, thrombus extended down into the common carotid artery. Treatment was initiated within 190 to 360 minutes of stroke onset. Thrombectomy of the carotid artery was deemed necessary because of poor collateral flow to the affected hemisphere (chronic contralateral internal carotid artery occlusion [one patient] and thrombus extending to the carotid "T" [one patient]) or inability to pass a microcatheter through the occluded vessel (one patient). Adjunctive therapy included pharmacologic thrombolysis with tissue plasminogen activator (all patients), carotid angioplasty and stenting (two patients), and middle cerebral artery angioplasty (one patient). Patency of the carotid artery was reestablished in two patients, with some residual thrombus burden. In the third patient, the device was able to create a channel through the column of thrombus, allowing intracranial access. CONCLUSION: Rheolytic thrombectomy shows potential for rapid, large-burden thrombus removal in cases of internal carotid artery thrombosis, allowing expedient access to the intracranial circulation for additional thrombolytic therapy.

Silent thromboembolic events associated with the treatment of unruptured cerebral aneurysms by use of Guglielmi detachable coils: prospective study applying diffusion-weighted imaging.

BACKGROUND AND PURPOSE: Aneurysm embolization using Guglielmi detachable coils (GDC) is gaining increasing acceptance as a viable alternative to surgery in the treatment of cerebral aneurysms. Although recent reports describe a significant rate of symptomatic thromboembolic complications with GDC use, many of the neurologic deficits are transient. We sought to determine the incidence of silent thromboembolic events with the use of diffusion-weighted imaging and to correlate radiologic findings with the results of neurologic examinations. METHODS: Diffusion-weighted MR imaging was performed within 48 hours in 14 consecutive elective GDC aneurysm treatments. Embolizations were performed under systemic heparinization; all flush solutions were heparinized, and both guiding catheters and microcatheters were placed for continuous heparinized infusions. Neurologic examination, including the National Institutes of Health Stroke Scale determination, was performed by a stroke neurologist before the coiling procedures were performed, immediately after the procedures were performed, and before discharge. MR imaging examinations were reviewed by a stroke neurologist and an interventional neuroradiologist, with determination and characterization of diffusion-weighted imaging abnormalities. RESULTS: Small areas of restricted diffusion, presumed to represent procedure-related embolic infarctions, were noted on the images of eight of 14 patients. All except one of the areas were located ipsilateral to the side of the catheterization. Six patients had evidence of multiple infarcts. Most lesions were small (<2 mm); one patient with coil stretch and herniation into the parent vessel had numerous infarcts with a dominant posterior frontal infarct. Pre- and posttreatment National Institutes of Health Stroke Scale scores were unchanged for 13 of 14 patients. Overall, the rate of asymptomatic emboli was 61% (eight of 13 treatments) in uncomplicated treatments. Strokes occurred independently of the number of coils used; the mean number of coils used for patients with strokes was 7.6 (range, two to 13) and for patients without evidence of infarcts was 10.2 (range, one to 30). This was not a significant difference (P > .5). CONCLUSION: Silent thromboembolic events related to the use of the GDC system are a common occurrence, despite meticulous technique and systemic anticoagulation. Although clinical sequelae are rare, the high rate of occurrence suggests that alterations in the technique, such as the addition of antiplatelet agents, should be considered.

Intraoperative diffusion imaging on a 0.5 Tesla interventional scanner.

Intraoperative line scan diffusion imaging (LSDI) on a 0.5 Tesla interventional MRI was performed during neurosurgery in three patients. Diffusion trace images were obtained in acute ischemic cases. Scan time per slice was 46 seconds and 94 seconds, respectively, for diffusion tensor images. Diagnosis of acutely developed vascular occlusion was confirmed with follow-up scans. White matter tracts were displayed with the principal eigenvectors and provided guidance for the tumor surgery. In all cases, the diagnostic utility of LSDI was established. J. Magn. Reson. Imaging 2001;13:115-119.

Intra-operative MR guidance during trans-sphenoidal pituitary resection: preliminary results.

The use of intra-operative MR image guidance has the potential to improve the precision, extent, and safety of trans-sphenoidal pituitary resections. The trans-sphenoidal approach to pituitary surgery has been performed for some time (1--3). Until now these surgeries have relied on direct visualization without the aid of image guidance. An open-bore configuration 0.5T SIGNA SP MR system (GE Medical Systems, Milwaukee, Wisconsin) has been used to provide image guidance for seventeen trans-sphenoidal pituitary adenoma resections (4). The intra-operative MRI system allowed the radiologist to successfully direct the surgeon toward the sella turcica while avoiding the cavernous sinus, optic chiasm and other critical structures. Imaging performed during the surgery monitored the extent of resection and allowed for removal of tumor beyond the surgeon's view in seven cases. Dynamic MR imaging was used to distinguish residual tumor from normal gland and postoperative changes, permitting more precise tumor localization. A heme-sensitive long TE gradient echo sequence was used to find the presence of hemorrhagic debris. All patients tolerated the procedure well without significant complications. J. Magn. Reson. Imaging 2001;13:136-141.

Intraoperative MR systems. Midfield approaches.

Intraoperative MR imaging provides an unrestricted view of intracranial structures and lesions that has revolutionized the way that neurosurgery is performed in the authors' institution. Intraoperative imaging allows the practitioner to update and adjust the approach to intracranial lesions continuously. With this system, important anatomic and vascular structures can be successfully avoided; boundaries of low-grade tumors can be accurately defined, and foci of possible higher grade within these lesions can be identified; foci of high-grade astrocytomas can be differentiated from radiated brain; hyperacute hemorrhage or infarction during and after procedures can be determined; and the possible communication of cystic collections with CSF can be ascertained. These advantages provide a level of comfort to the surgeon and a presumptive margin of safety to the patient that is unattainable during conventional surgical approaches, and given the choice, the authors' neurosurgeons would prefer to operate in the interventional magnet. Preliminary reports concerning the efficacy and usefulness of MR-guided navigational tools for the performance of neurosurgery are encouraging, as noted earlier, Wirtz et al have shown that the more extensive removal of glioblastomas afforded by intraoperative MR leads to significantly prolonged patient survival compared with conventional surgery. Further outcomes analysis must be performed, however, to determine whether these new techniques significantly decrease overall long-term morbidity or increase survival in those patients who have low-grade astrocytomas.

Cartilage and bone regeneration using gene-enhanced tissue engineering.

Joint cartilage injury remains a major problem in orthopaedics with more than 500,000 cartilage repair procedures performed yearly in the United States at a cost of hundreds of millions of dollars. No consistently reliable means to regenerate joint cartilage currently exists. The technologies of gene therapy and tissue engineering were combined using a retroviral vector to stably introduce the human bone morphogenic protein-7 complementary deoxyribonucleic acid into periosteal-derived rabbit mesenchymal stem cells. Bone morphogenic protein-7 secreting gene modified cells subsequently were expanded in monolayer culture, seeded onto polyglycolic acid grafts, implanted into a rabbit knee osteochondral defect model, and evaluated for bone and cartilage repair after 4, 8, and 12 weeks. The grafts containing bone morphogenic protein-7 gene modified cells consistently showed complete or near complete bone and articular cartilage regeneration at 8 and 12 weeks whereas the grafts from the control groups had poor repair as judged by macroscopic, histologic, and immunohistologic criteria. This is the first report of articular cartilage regeneration using a combined gene therapy and tissue engineering approach.

Presence of N-cadherin transcripts in mature spermatozoa.

The essential mechanism involved in sperm-oolemma fusion has yet to be elucidated. Recognition and binding is initiated by specific cell surface receptor engagement between gametes. Fusion between hamster oolemma and spermatozoa is prevented in the presence of trypsin in Ca(2+)-free media, as is oocyte activation, implicating a cadherin-like adhesion. Cadherins are a family of Ca(2+)-dependent adhesion molecules that bind homotypically with their target, are morphoregulatory and function eptopically to affect tissue form and function. Cadherins and cadherin-associated molecules have been identified in testes and germinal cells, as well as ejaculated spermatozoa. Moreover, cadherins are also present in oocytes and may suggest a cadherin-mediated adhesion in sperm-oocyte interaction. We have detected antigenic epitopes recognized by N-cadherin monoclonal antibodies diffusely distributed over the entire sperm head. In addition, Western blot analysis confirmed the presence of an antibody reactive peptide in spermatozoa, testis and ovary protein extracts at the expected molecular weight for authentic N-cadherin. Total RNA was isolated from mature motile spermatozoa, as well as ovary and testis tissue, and served as template for reverse transcription-polymerase chain reaction (RT-PCR) with N-cadherin specific primers. Alignment of sequences from PCR products of testis, ovary and spermatozoa with published N-cadherin sequence was identical except for occasional base changes. We intend to develop methods to analyse this transcript from small numbers of spermatozoa from a variety of donors to determine if defects in cadherin distribution or structure may predict reduced male fertility.

L-type voltage-dependent calcium channel alpha-1C subunit mRNA is present in ejaculated human spermatozoa.

The current study adds to the growing body of evidence that RNA is present in mature ejaculated human spermatozoa. We report that a sodium dodecyl sulphate (SDS)/citric acid extraction method is superior to guanidinium isothiocyanate in terms of reproducibility of RNA recovery from motile sperm populations from individual ejaculates. Using the SDS/citric acid method, RNA was recovered from both fresh and frozen-thawed motile spermatozoa. Sperm RNA were used as templates in reverse transcription-polymerase chain reaction (RT-PCR), in an attempt to identify partial RNA transcripts of a highly conserved region within the alpha-1C (pore-forming) subunit of L-type voltage-dependent calcium channels from 11 individual donors. Control reactions employed primers derived from the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence. In nine of the 11 specimens, gene-specific PCR products were obtained with both the GAPDH and alpha-1C primer pairs. DNA sequencing analysis confirmed that the respective spliced transcripts were amplified. The two cases in which no amplification was obtained were attributed to reduced RNA yield. These data are consistent with results from in-situ RT-PCR of rat testis sections indicating that the testis-specific calcium channel of that species was expressed uniformly in all stages of the germinal epithelium, including mature spermatozoa.

Image-guided therapy and intraoperative MRI in neurosurgery.

Computer-assisted 3D planning, navigation and the possibilities offered by intra-operative imaging updates have made a large impact on neurological surgery. Three-dimensional rendering of complex medical image information, as well as co-registration of multimodal sources has reached a highly sophisticated level. When introduced into surgical navigation however, this pre-operative data is unable to account for intra-operative changes, ('brain-shift'). To update structural information during surgery, an open-configured, intra-operative MRI (Signa SP, 0.5 T) was realised at our institution in 1995. The design, advantages, limitations and current applications of this system are discussed, with emphasis on the integration of imaging into procedures. We also introduce our integrated platform for intra-operative visualisation and navigation, the 3D Slicer.

Gene-enhanced tissue engineering: applications for wound healing using cultured dermal fibroblasts transduced retrovirally with the PDGF-B gene.

The treatment of difficult wounds remains a considerable clinical challenge. The goal of this study was to determine whether genetic augmentation of dermal cells on resorbable matrices can stimulate the healing process, leading to increased tissue repair in a rat full-thickness excisional wound repair model. The human platelet-derived growth factor B (PDGF-B) gene was the initial gene chosen to test this hypothesis. The human PDGF-B gene was obtained from human umbilical vein endothelial cells (HUVEC) by reverse transcriptase-polymerase chain reaction, cloned into retroviral vectors under control of either the cytomegalovirus promoter or the rat beta-actin promoter, and introduced into primary rat dermal cells. In vitro results demonstrate that rat dermal cells are transduced and selected readily using retroviral vectors, and engineered to secrete PDGF-B at a steady-state level of approximately 2 ng per milliliter culture per 1 million cells per 24 hours. Seeding of the gene-modified cells onto polyglycolic acid (PGA) scaffold matrices and introduction into the rat model resulted in substantially increased fibroblast hypercellularity over control wounds at both 7 and 14 days posttreatment. Our results demonstrate that gene augmentation of rat dermal fibroblasts with the PDGF-B gene introduced into this animal model via PGA matrices modulates wound healing and suggests that experimentation with additional genes for use separately or in combination with PDGF-B for additional, improved wound healing is warranted.

Human serum-resistant retroviral vector particles from galactosyl (alpha1-3) galactosyl containing nonprimate cell lines.

Retroviral vector particles (RVP) which are resistant to inactivation by human serum will be needed for many in vivo gene therapy applications. Murine-based producer cell lines generate RVP which are inactivated by human serum, reportedly due to the presence of the galactosyl (alpha1-3) galactosyl carbohydrate moiety (alphaGal) on these and other nonprimate producer cells and RVP. Consequently, human cells (which lack the alphaGal moiety) have been developed as producer cell lines for generation of human serum-resistant RVP. In this study, we report that contrary to earlier reports, the presence of the alphaGal moiety on producer cells and RVP does not necessarily correlate with cell killing or RVP inactivation by human serum. We show that the alphaGal-positive ferret brain cell line, Mpf, is an excellent basal cell line for generation of RVP which have titers and serum resistance levels equal to or greater than RVP produced in human cell lines such as HT1080. Therefore, packaging cell lines need not be limited to those of human or primate origin for production of human serum-resistant RVP.

Role of retinoic acid receptor overexpression in sensitivity to fenretinide and tumorigenicity of human ovarian carcinoma cells.

The role of retinoic acid receptor (RAR) expression in sensitivity to N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide) as well as on the tumorigenicity of human ovarian carcinoma cells was examined. Two human ovarian cancer cell lines, A2780 and IGROV-1, with a 10-fold difference in sensitivity to 4HPR were chosen to study RAR involvement in the response to 4HPR. To determine which RAR was effective, RARalpha, beta and gamma were individually overexpressed in A2780 cells, which are the most sensitive to 4HPR. Sensitivity to 4HPR was increased in RARbeta-overexpressing clones, whereas it was slightly decreased in RARalpha transfectants (which had diminished RARbeta expression) and was unchanged in clones transfected with RARgamma. IGROV-1 cells, which are RARbeta negative, were transfected with RARbeta. Surprisingly, none of the obtained IGROV-1 RARbeta transfectants expressed RARbeta protein, in spite of RARbeta mRNA transcription. All clones were similar to the parental IGROV-1 cells in their sensitivity to 4HPR. Treatment with a pharmacologically achievable concentration of 4HPR (1 microM) led to a rapid 2-fold increase in RARbeta mRNA levels in A2780 cells, but it did not induce RARbeta expression in IGROV-1 cells. Analysis of the tumorigenicity of A2780-transfected clones revealed that overexpression of RARalpha was associated with a significant reduction in tumor takes (50% and 67%, respectively, vs. 96% for the parent line) and with a reduced growth rate. Oncogenicity was clearly decreased in only 1 of the 2 RARbeta-overexpressing clones (33% takes) and was unchanged in the 2 clones with increased RARgamma expression. Our results demonstrate that basal expression and 4HPR inducibility of RARbeta play a role in mediating 4HPR response in ovarian cancer cells. The findings of reduced oncogenicity of clones overexpressing RARalpha and of one clone overexpressing RARbeta indicate that RARalpha and RARbeta might have a tumor-suppressive effect in ovarian tumors.

Identification of structural elements of the testis-specific voltage dependent calcium channel that potentially regulate its biophysical properties.

Calcium influx through voltage-dependent calcium channels regulates the physiological acrosome reaction of mammalian spermatozoa. Expression of the mRNA for these voltage-dependent calcium channels and its co-ordinated translation is initiated early in rat male germ line development and continues throughout spermatogenesis. Herein, we report the complete mRNA and deduced amino acid sequence of the alpha1c pore-forming subunit of the rat testis-specific L-type calcium channel. This subunit is transcribed from the alpha1c gene, which is also expressed in brain and cardiac muscle. The cardiac- and testis-specific isoforms of the alpha1c subunit are produced by alternate splicing of the same primary transcript. The testis-specific isoform differs from that of cardiac tissue at its amino terminus and in transmembrane segments IS6, IIIS2 and IVS3, which are also dihydropyridine binding sites. In somatic tissues, segments S2 and S3 regulate channel activation while the amino terminus and segment IS6 contribute to channel inactivation kinetics. The amino terminus and IS6 segment of the testis-specific alpha1c subunit are also expressed respectively, in the brain and in smooth muscle from lung where they alter the electrophysiological characteristics of the subunit to produce relatively slow inactivation kinetics. These findings provide a molecular explanation for the detection by others, by patch clamp analysis, of T-type calcium currents in immature spermatogenic cells and of atypical L-type calcium currents in mature spermatozoa.

Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia.

We investigated the feasibility and diagnostic utility of genotyping 9 CYP21 mutations, linked chromosome 6p markers, and a dimorphic X-Y marker from neonatal screening samples. Blood-impregnated filter papers (Guthrie cards) from 603 randomly chosen New Zealand neonates were genotyped blind to 17-hydroxyprogesterone (17-OHP) levels. Another 50 samples from Swiss and North American infants with correlative hormonal data were also genotyped. DNA was extracted, and gene-specific PCR was performed. CYP21 PCR products were subjected to ligase detection reaction, simultaneously analyzing 9 CYP21 mutations; PCR products of other genes were subjected to direct gel analysis. CYP21 genotyping indicated a heterozygote rate of 2.8% for classic mutations (excluding CYP21 deletions), and 2.0% for nonclassic mutations in New Zealanders. Ten full-term affected neonates showed a wide range of 17-OHP levels (15-1400 nmol/L). Sick or preterm infants or infants screened on the first day of life with high 17-OHP proved genetically unaffected. Genetic linkage disequilibrium was found between two CYP21 mutations and chromosome 6p markers. Guthrie cards can be used to accurately genotype CYP21 and other relevant markers, potentially enhancing the specificity and sensitivity of congenital adrenal hyperplasia screening. CYP21 heterozygote frequency for classic mutations is higher than expected based on genotype compared with that predicted by hormonal newborn screening.

Structural abnormalities in the G-CSF receptor in severe congenital neutropenia.

Structural abnormalities in the cytoplasmic region of the G-CSF receptor (G-CSF-R) or defects in signal transduction pathways triggered by the G-CSF-R or both have been implicated in the development of neutropenia and increased prediposition to leukemia in patients with severe congenital neutropenia (SCN). To assess the structural integrity of the G-CSF-R in SCN patients, the transmembrane and cytoplasmic regions of the G-CSF-R from 5 SCN patients were cloned and sequenced. DNA mutations (point, deletion, frame-shift, and silent) were observed in 3 patients. In 2 of these, the DNA mutations resulted in altered G-CSF-R protein sequences, including additions of novel C-terminal sequences. Three of the 5 mutant receptor clones lacked 115-121 amino acids in the cytoplasmic region, and 2 showed complete loss of the transmembrane and cytoplasmic regions. Neutrophils from 1 patient expressing these mutant receptors showed normal binding of radiolabeled G-CSF. G-CSF-R in 2 other patients with SCN showed no mutations. Our results indicate that structural abnormalities in the G-CSF-R may be present in some SCN patients. They may not affect the binding of G-CSF to the receptor but may contribute to the pathogenesis of SCN through impaired signal transduction pathways of the mutant G-CSF-R.