Jan Blancato

The Syk tyrosine kinase suppresses malignant growth of human breast cancercells

Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.

Absence of donor-derived keratinocyte stem cells in skin tissues cultured from patients after mobilized peripheral blood hematopoietic stem cell transplantation

OBJECTIVE Recent studies suggest that primitive bone marrow-derived cells contribute to regeneration of many tissues, including muscle, endothelium, myocardium, neural tissues, liver, and skin. Conversely, primitive cells resident in muscle and other tissues have been reported to reconstitute hematopoiesis. We investigated the contribution of cells with a primitive hematopoietic phenotype to human epidermal skin formation in recipients of allogeneic mobilized peripheral blood hematopoietic stem cell (HSC) transplantation. PATIENTS AND METHODS Our study population included female patients who had received granulocyte colony-stimulating factor mobilized peripheral blood HSC transplants from male donors for a variety of benign and malignant hematologic disorders at least 6 months before study entry, with a history of skin graft-vs-host disease. Epidermal skin cells (keratinocytes) obtained from punch biopsies of the skin were cultured under conditions specific for growth and expansion of homogenous populations of keratinocytes from keratinocyte stem cells. After multiple passages, DNA was extracted from cultured cells and evaluated by two different polymerase chain reaction (PCR) method for detection of Y chromosome specific sequences. RESULTS Neither sensitive PCR-based technique revealed the presence of male donor-derived keratinocyte stem cells in keratinocytes cultured from skin biopsies of female allogeneic transplantation recipients. CONCLUSIONS We could not confirm the contribution of donor mobilized peripheral blood hematopoietic stem cells to keratinocyte stem cell populations after HSC transplantation. These results cannot explain the presence of donor-derived cells with keratinocyte phenotypic markers in tissue sections of HSC transplant recipients.

Respiratory insufficiency in desminopathy patients caused by introduction of proline residues in desmin c-terminal alpha-helical segment

Mutations in desmin gene have been identified in patients with cardiac and skeletal myopathy characterized by intracytoplasmic accumulation of desmin‐reactive deposits and electron‐dense granular aggregates. We characterized two new desminopathy families with unusual features of adult‐onset, slowly progressive, diffuse skeletal myopathy and respiratory insufficiency. Progressive reduction of respiratory muscle strength became clinically detectable between the 3rd and the 8th years of illness and led to recurrent chest infections and death in one of the patients. Novel mutations, A357P and L370P, predicted to introduce proline residue into a highly conserved α‐helical region of desmin, were identified. Proline is known to disrupt the α‐helix. In addition, the A357P mutation distorts a unique stutter sequence that is considered to be critically important for proper filament assembly. Functional assessment in two cell‐lines, one of which does and the other of which does not constitutively produce type III intermediate filaments, demonstrated the inability of mutant desmin carrying either the A357P or the L370P mutation to polymerize and form an intracellular filamentous network. The results of this study indicate that respiratory insufficiency is an intrinsic feature of disease associated with specific desmin mutations; in some patients, respiratory weakness may present as a dominant clinical manifestation and a major cause of disability and death. Muscle Nerve 27: 669–675, 2003

Clinical and molecular delineation of the Greig cephalopolysyndactyly contiguous gene deletion syndrome and its distinction from acrocallosal syndrome

Greig cephalopolysyndactyly syndrome (GCPS) is caused by haploinsufficiency of GLI3 on 7p13. Features of GCPS include polydactyly, macrocephaly, and hypertelorism, and may be associated with cognitive deficits and abnormalities of the corpus callosum. GLI3 mutations in GCPS patients include point, frameshift, translocation, and gross deletion mutations. FISH and STRP analyses were applied to 34 patients with characteristics of GCPS. Deletions were identified in 11 patients and the extent of their deletion was determined. Nine patients with deletions had mental retardation (MR) or developmental delay (DD) and were classified as severe GCPS. These severe GCPS patients have manifestations that overlap with the acrocallosal syndrome (ACLS). The deletion breakpoints were analyzed in six patients whose deletions ranged in size from 151 kb to 10.6 Mb. Junction fragments were found to be distinct with no common sequences flanking the breakpoints. We conclude that patients with GCPS caused by large deletions that include GLI3 are likely to have cognitive deficits, and we hypothesize that this severe GCPS phenotype is caused by deletion of contiguous genes.

Hypercholesterolemia in children with Smith-Magenis syndrome: del (17) (p11.2p11.2).

Purpose: Smith-Magenis syndrome (SMS), a probable contiguous gene syndrome due to an interstitial deletion of chromosome 17 band p11.2, is associated with a distinct and complex phenotype, including physical, developmental, and neurobehavioral features. The majority of SMS patients are deleted for a common ∼ 4 Mb interval that includes the gene SREBF1, a transmembrane transcription factor that regulates the low density lipoprotein (LDL) receptor and plays a crucial role in cholesterol homeostasis. A systematic study of fasting lipid profiles of patients with SMS was conducted to determine the frequency of cholesterol abnormalities.Methods: Fasting lipid profiles were examined in 49 children (27F/22M) between the ages of 0.6 years to 17.6 years (mean, 6.9 years) with a cytogenetically confirmed diagnosis of SMS. Observed values for serum total cholesterol (TC), triglycerides (TG), LDL cholesterol, and high density lipoprotein cholesterol were compared with published norms. The body mass index (BMI) was used as a measure of nutritional status.Results: Mean TC was significantly higher than published NHANES III pediatric norms (P < 0.0008). Overall 28 of 49 (57%) SMS subjects had lipid values greater than the 95th percentile for age and gender for at least one or more of the following: TC, TG, and/or LDL. Only 16 SMS subjects (32%) were within normal limits for all three of these variables. BMI values showed minimal positive correlation to SMS lipid values; however, no consistent effect was found. Thus BMI values alone do not explain the marked trend in increased TC, TG, and/or LDL observed in the SMS group. Based on the American Academy of Pediatrics recommended lipid levels for children and adolescents, only one third of SMS subjects fall within normal range for TC and LDL; an additional one third each measure “borderline” or “high” for these values.Conclusion: Hypercholesterolemia is common in SMS and may serve as a useful early clinical biochemical marker of the syndrome.

Detection of chromosomal aberrations by a whole-genome microsatellite screen.

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include developmental and growth retardation. Current microscopic techniques are useful for the detection of such aberrations but have a limit of resolution that is above the threshold for phenotypic effect. We hypothesized that a genomewide microsatellite screen could detect chromosomal aberrations that were not detected by standard cytogenetic techniques in a portion of these individuals. To test this hypothesis, we performed a genomewide microsatellite screen of patients, by use of a currently available genetic-marker panel that was originally designed for meiotic mapping of Mendelian traits. We genotyped approximately 400 markers on 17 pairs of parents and their children who had normal karyotypes. By using this approach, we detected and confirmed two cases of segmental aneusomy among 11 children with multiple congenital anomalies. These data demonstrate that a genomewide microsatellite scan can be used to detect chromosomal aberrations that are not detected by microscopic techniques.

Diagnostic FISH probes for del(17)(p11.2p11.2) associated with Smith–Magenis syndrome should contain the RAI1 gene

Smith–Magenis syndrome (SMS) is a mental retardation syndrome with distinctive behavioral characteristics, dysmorphic features, and congenital anomalies usually associated with an interstitial deletion of chromosome 17p11.2. While high quality G‐banding will identify most SMS patients, fluorescent in situ hybridization (FISH) is the recommended test for confirmation of an SMS diagnosis. Recently, haploinsufficiency of the RAI1 gene due to deletion or mutation was determined to be the likely cause of SMS. All diagnostic FISH probes available commercially contain the FLII gene and are approximately 580 kb centromeric to RAI1. We present two patients with SMS who have interstitial deletions at 17p11.2 but are not deleted for currently available commercial FISH probes that include FLII; both patients have deletions that are demonstrated with probes containing the RAI1 gene. We recommend that for diagnostic accuracy, all future FISH tests for SMS be performed with probes containing the RAI1 gene, as some atypical deletions in the region critical to the SMS phenotype will otherwise be missed.

MicroRNAs in glioblastoma multiforme pathogenesis and therapeutics.

Glioblastoma multiforme (GBM) is the most common and lethal cancer of the adult brain, remaining incurable with a median survival time of only 15 months. In an effort to identify new targets for GBM diagnostics and therapeutics, recent studies have focused on molecular phenotyping of GBM subtypes. This has resulted in mounting interest in microRNAs (miRNAs) due to their regulatory capacities in both normal development and in pathological conditions such as cancer. miRNAs have a wide range of targets, allowing them to modulate many pathways critical to cancer progression, including proliferation, cell death, metastasis, angiogenesis, and drug resistance. This review explores our current understanding of miRNAs that are differentially modulated and pathologically involved in GBM as well as the current state of miRNA‐based therapeutics. As the role of miRNAs in GBM becomes more well understood and novel delivery methods are developed and optimized, miRNA‐based therapies could provide a critical step forward in cancer treatment.

Review of Disrupted Sleep Patterns in Smith-Magenis Syndrome and Normal Melatonin Secretion in a Patient with an Atypical Interstitial 17p11.2 Deletion

Smith–Magenis syndrome (SMS) is a disorder characterized by multiple congenital anomalies and behavior problems, including abnormal sleep patterns. It is most commonly due to a 3.5 Mb interstitial deletion of chromosome 17 band p11.2. Secretion of melatonin, a hormone produced by the pineal gland, is the bodys signal for nighttime darkness. Published reports of 24‐hr melatonin secretion patterns in two independent SMS cohorts (US and France) document an inverted endogenous melatonin pattern in virtually all cases (96%), suggesting that this finding is pathognomic for the syndrome. We report on a woman with SMS due to an atypical large proximal deletion (∼6Mb; cen<‐>TNFRSFproteinB) of chromosome band (17)(p11.2p11.2) who presents with typical sleep disturbances but a normal pattern of melatonin secretion. We further describe a melatonin light suppression test in this patient. This is the second reported patient with a normal endogenous melatonin rhythm in SMS associated with an atypical large deletion. These two patients are significant because they suggest that the sleep disturbances in SMS cannot be solely attributed to the abnormal diurnal melatonin secretion versus the normal nocturnal pattern.

Molecular Analysis of the Retinoic Acid Induced 1 Gene (RAI1) in Patients with Suspected Smith-Magenis Syndrome without the 17p11.2 Deletion

Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder characterized by multiple congenital anomalies. The syndrome is primarily ascribed to a ∼3.7 Mb de novo deletion on chromosome 17p11.2. Haploinsufficiency of multiple genes likely underlies the complex clinical phenotype. RAI1 (Retinoic Acid Induced 1) is recognized as a major gene involved in the SMS phenotype. Extensive genetic and clinical analyses of 36 patients with SMS-like features, but without the 17p11.2 microdeletion, yielded 10 patients with RAI1 variants, including 4 with de novo deleterious mutations, and 6 with novel missense variants, 5 of which were familial. Haplotype analysis showed two major RAI1 haplotypes in our primarily Caucasian cohort; the novel RAI1 variants did not occur in a preferred haplotype. RNA analysis revealed that RAI1 mRNA expression was significantly decreased in cells of patients with the common 17p11.2 deletion, as well as in those with de novo RAI1 variants. Expression levels varied in patients with familial RAI1 variants and in non-17p11.2 deleted patients without identified RAI1 defects. No correlation between SNP haplotype and RAI1 expression was found. Two clinical features, ocular abnormalities and polyembolokoilomania (object insertion), were significantly correlated with decreased RAI1 expression. While not significantly correlated, the presence of hearing loss, seizures, hoarse voice, childhood onset of obesity and specific behavioral aspects and the absence of immunologic abnormalities and cardiovascular or renal structural anomalies, appeared to be specific for the de novo RAI1 subgroup. Recognition of the combination of these features will assist in referral for RAI1 analysis of patients with SMS-like features without detectable microdeletion of 17p11.2. Moreover, RAI1 expression emerged as a genetic target for development of therapeutic interventions for SMS.