Clinical utility of molecular and flow cytometric markers in chronic lymphocytic leukaemia.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease. While immunoglobulin variable region heavy chain (IgVH) mutational status remains the 'gold standard' in molecular prognostication, a range of additional markers is increasingly being used in clinical trials. As awareness of trial data increases, requests to determine these prognostic markers for new CLL patients are becoming more prevalent in Australia. AIM: To explore the clinical utility of currently available prognostic markers for CLL in an Australian cohort. METHODS: IgVH mutational status and gene usage was determined and compared with other reported immunophenotypic markers, cytogenetics and clinical outcome as defined by treatment-free survival (TFS), lymphocyte doubling time and clinical stage in a cohort of 65 CLL patients. RESULTS: An unmutated IgVH gene, high expression of CD38, ZAP-70, CD25, CD49d, CD54 or low expression of CD49c was associated with shorter TFS indicating an adverse clinical prognosis in our cohort. High expression of each of CD38, ZAP-70, CD49d and CD54 was significantly associated with an unmutated IgVH gene; however, associations were not absolute. IgVH and CD25 expression retained their significance in multivariate analysis. Concordant CD25(high) /IgVH unmutated CLL patients had the shortest median TFS interval (40 months) in our cohort. CONCLUSIONS: Molecular and immunophenotypic markers remain useful as adjuncts to clinical prognostication; however, as single parameters they are unable to dictate the timing of therapeutic intervention. The combined use of CD25 and IgVH mutational status may be clinically relevant to CLL prognostication while also providing insight into the biological pathways involved in disease progression.

Odontoblast dysfunction in osteogenesis imperfecta: an LM, SEM, and ultrastructural study.

The inherited dentin defect dentinogenesis imperfecta (DI), while clinically obvious in osteogenesis imperfecta (OI) Types IB and IC, II, III, and IVB, is now thought to be present in all children with OI, in a continuum from minimal to severe dentin pathology. This collaborative study further clarifies the structural and ultrastructural dentin changes in the teeth of OI children with clinically obvious DI, and attempts to explain these in terms of odontoblast dysfunction. Collaborative studies were carried out in Melbourne, Australia, and Strasbourg, France, using light and polarized-light microscopy, scanning and transmission electron microscopy (SEM, TEM), selected-area diffraction (SAD), and x-ray spectroscopy (EDX). These showed structurally normal enamel (but containing long and broad lamellae) and a normally scalloped dentino-enamel junction (DEJ), but severe pathologic changes in the dentin. An initial narrow band of normal-appearing dentin tubules (including the mantle layer) ceased abruptly and was replaced by a wavelike laminar zone parallel to the DEJ with occluded tubules. Multiple parallel channels of 5-10 microns diameter were present at right angles to the DEJ indenting this zone, some terminating in retro-curved "processes." The abnormal dentin containing these channels almost completely occluded the pulp chamber. The structural and ultrastructural changes seen can be explained on the basis of the collagen defect in OI resulting in odontoblast dysfunction, which produces a distinct phenotype and one that is different from that in bone.

Molecular analysis for genetic counselling in amelogenesis imperfecta.

OBJECTIVE: To use molecular genetics to establish the mode of inheritance in a family with amelogenesis imperfecta. MATERIALS AND METHODS: The polymerase chain reaction was used to amplify exons of the amelogenin gene on the short arm of the X chromosome. RESULTS: A single base deletion mutation in exon 6 of the amelogenin gene was identified. This mutation was a single base deletion of a cytosine residue - 431delC - in codon 96 of exon 6, introducing a stop codon 30 codons downstream of the mutation in codon 126 of the exon. CONCLUSION: The firm establishment of an X-linked mode of inheritance affects the genetic counselling for this family.

SHH mutation is associated with solitary median maxillary central incisor: a study of 13 patients and review of the literature.

Solitary median maxillary central incisor (SMMCI) or single central incisor is a rare dental anomaly. It has been reported in holoprosencephaly (HPE) cases with severe facial anomalies or as a microform in autosomal dominant HPE (ADHPE). In our review of the literature, we note that SMMCI may also occur as an isolated finding or in association with other systemic abnormalities. These anomalies include short stature, pituitary insufficiency, microcephaly, choanal atresia, midnasal stenosis, and congenital nasal pyriform aperture stenosis. SMMCI can also be a feature of recognized syndromes or associations or a finding in patients with specific chromosomal abnormalities. We performed a molecular study on a cohort of 13 SMMCI patients who did not have HPE. We studied two genes, Sonic Hedgehog (SHH) and SIX3, in which mutations have been reported in patients showing SMMCI as part of the HPE spectrum. A new missense mutation in SHH (I111F), segregating in one SMMCI family, was identified. Our results suggest that this mutation may be specific for the SMMCI phenotype since it has not been found in the HPE population or in normal controls. Published 2001 Wiley-Liss, Inc.

Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein-1 gene expression by insulin. The role of winged helix/forkhead proteins.

Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Previous mutational analyses of the PEPCK and IGFBP-1 IREs revealed mutations which do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provided compelling evidence that HNF3 proteins, per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein.

Insulin regulates expression of metabolic genes through divergent signaling pathways.

The regulation of metabolic gene expression is a major mechanism by which insulin modulates glucose homeostasis. Defective transcription factors or signal transduction molecules that are required for insulin regulated gene expression could contribute to insulin resistance. The phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase II (HKII) genes are involved in metabolic processes that represent opposing facets of glucose homeostasis, namely gluconeogenesis and glucose utilization. The regulation of the PEPCK and HKII genes by insulin has been studied in great detail at the level of both transcription and signal transduction. Recent work on the insulin signaling pathways that lead to down-regulation of PEPCK gene expression and upregulation of HKII gene expression has shown that they both require activation of phosphatidylinositol 3-kinase (PI3K) for the transmission of the insulin signal. However, the pathways diverge after PI3K and lead to activation of different downstream effectors. In this paper we review the results of studies on the transcriptional regulation of these genes by insulin and the signal transduction pathways that mediate these responses.

GAPO syndrome: a new case of this rare syndrome and a review of the relative importance of different phenotypic features in diagnosis.

The case of GAPO syndrome reported here is the 24th recorded case, 23 cases having been published previously. The 29-year-old male under discussion presents all the typical features of the syndrome, having short stature, dysmorphic craniofacial features, total alopecia and pseudoanodontia. Orally, the erupted primary dentition was extremely worn and on radiographic examination, the second mandibular molars were found to be unerupted, together with the entire permanent dentition. Cephalometry revealed the absence of facial pneumatisation, a deficient cranial base with diminished upper face height and maxillary and mandibular hypoplasia with a prognathic skeletal pattern. Histological examination of an extracted primary incisor and its surrounding root bone revealed extensive ankylosis. This paper describes in detail the clinical findings and reviews, and discusses previously published cases in relation to the present one. As with prior cases, parental consanguinity was present in the pedigree.

The interaction of maintenance interferon with cytolytic cells in patients with multiple myeloma who responded to cytotoxic chemotherapy.

STUDY OBJECTIVE: To determine the long-term effects of maintenance interferon on CD56+ and CD3+ cell activity. DESIGN: Prospective phase II trial. SETTING: Tertiary medical center and level 2 Veterans Administration hospital. PATIENTS: Seven patients (age 45-74 yrs) with multiple myeloma who had reached the plateau phase from cytotoxic chemotherapy, and seven age- and sex-matched controls. INTERVENTIONS: All patients were given interferon-alpha 2b 3 x 10(6) U/m2 3 times/week. MEASUREMENTS AND MAIN RESULTS: The CD56+, CD3+, and CD16+ counts were determined by flow cytometry in both peripheral blood and bone marrow. Natural killer (NK) cell functional activity was determined by a 51chromium release assay. Monocyte cell numbers were determined from the white blood cell count with differential. Interleukin-6 (IL-6) concentrations were determined by a commercially available enzyme-linked immunosorbent assay. During the 24-week study, the peripheral blood CD3+ and monocyte counts in patients with myeloma remained constant (p > or = 0.39) but their absolute CD56+ counts decreased significantly (p = 0.05). In peripheral blood, CD56+, CD16-, CD3- was the predominant phenotype in patients. The predominant phenotype in bone marrow was CD56+, CD16-, CD3+ at baseline but changed to CD56+, CD16-, CD3- by week 24. The cytolytic activity of NK cells significantly increased in bone marrow (p = 0.05) whereas it remained stable in the peripheral blood (p = 0.55), but only half that of the controls. Concentrations of IL-6 did not increase significantly during the study. CONCLUSION: In peripheral blood, NK cell activity remained stable in patients but was significantly lower than that in controls, probably secondary to the predominance of the CD56+, CD16-, CD3- phenotype in the patients. In contrast, NK cell activity increased significantly in bone marrow despite the predominance of the CD56+, CD16-, CD3- phenotype by week 24.

Solitary median maxillary central incisor, short stature, choanal atresia/midnasal stenosis (SMMCI) syndrome.

This article describes a series of 21 consecutive cases, each involving a solitary median maxillary central incisor; the patients were seen in the Department of Dentistry or the Victorian Clinical Genetics Unit, Murdoch Institute, at the Royal Children's Hospital, Melbourne, from 1966 to 1997. The spectrum of anomalies and associated features present in these cases--solitary median maxillary central incisor, choanal atresia, and holoprosencephaly--is described, and the literature related to the features, including genetic studies in these conditions, is reviewed. We relate our findings in these cases to current knowledge of developmental embryology. It is hoped that the findings, together with our interpretation of them, will help to clarify understanding of solitary median maxillary central incisor syndrome. This syndrome was previously considered a simple midline defect of the dental lamina, but it is now recognized as a possible predictor of holoprosencephalies of varying degrees in the proband, in members of the proband's family, and in the family's descendants.

Dental health of children with cerebral palsy following sialodochoplasty.

Drooling occurs commonly in children with cerebral palsy (CP). Surgical procedures, known as slalodochoplasties, are often performed for the control of drooling. These include major salivary gland excision, parasympathetic nerve section, duct ligation, and duct re-routing. Alterations in saliva amount, flow, and consistency occur following sialodochoplasty, and the resultant effect on dental homeostasis requires further investigation. This controlled study investigated 19 children with CP following sialodochoplasty (surgery group) and 75 children with CP treated nonsurgically (control group) who attended our hospital. Dental caries experience-including dmft, DMFT, and partial DMFS scores of mandibular incisors and canines only-plaque index, and enamel developmental defects index were recorded. Saliva buffering capacity and bacterial counts were assessed. The surgical group (median DMFT = 5.00) had significantly more dental caries when compared with the control group (median DMFT = 0.00), Wilcoxon Signed-rank Test, P < 0.0001. This study has shown that children with CP following sialodochoplasty have increased risk of dental caries when compared with those treated nonsurgically for drooling. Although no caries predictors were identified, alterations to the caries-protective role of saliva are considered the likely cause. Children who undergo this procedure should receive intensive pre- and postsurgical preventive dental therapy.

The role of the TATA box in the hormonal regulation of phosphoenolpyruvate carboxykinase gene expression.

In this study, we analyzed the role of the TATA box in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression by dexamethasone (DEX), retinoic acid (RA), glucagon (via cAMP) and insulin (INS). The PEPCK TATA box (TATTTAAA) was absolutely required for both basal promoter activity and hormone-mediated transactivation. However, the relative induction of PEPCK gene expression by DEX, RA and cAMP, and its repression by INS, remained unaltered despite the substitution of the PEPCK TATA box with TATA elements from the herpes simplex virus-thymidine kinase gene, gene 33 or a consensus TATA box sequence, TATAAA. The results indicate that the TATA box serves a permissive, but not defining, function in the response of the PEPCK gene to hormones, and that this function can be equally facilitated by heterologous TATA box elements.

Retinoid receptors cause distortion of the retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter.

Functional retinoic acid response elements (RAREs) have been described wherein the direct repeats are separated by 1, 2 or 5 bp (termed DR1, DR2 and DR5 respectively). We have previously shown that retinoic acid receptor/retinoid X receptor (RAR/RXR) binds a DR1 RARE within the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter and is the trans-acting complex that mediates the retinoic acid (RA) response. However, the mechanism of trans-activation is unknown. The consequences of RAR/RXR binding to the PEPCK RARE were examined using a circular permutation analysis as a first step to explore the possible role of DNA conformational changes in the RA response. The RAR/RXR heterodimer produced a distortion angle of 78 degrees. The DNA distortion was shown to be at the centre of the PEPCK RARE; RA did not affect the severity of the distortion angle or the location of the distortion centre. Monomers and homodimers of RAR also distorted the DNA, but to a lesser extent than did RAR/RXR. The results of a phasing analysis demonstrated that RAR/RXR heterodimers did not induce a static DNA bend, in either the presence or the absence of RA. A cyclization kinetics assay was employed to show that RAR/RXR binding affected DNA ring closure in a phase-sensitive, RA-insensitive, manner. Taken together, these observations support the idea that RAR/RXR heterodimers distort the structure of the PEPCK RARE, at least in part, by altering DNA flexibility. The conformational change in the PEPCK RARE upon RAR/RXR binding has implications for how RAR/RXR heterodimers recognize various RARE structures.

Gingival fibromatosis and Klippel-Trénaunay-Weber syndrome. Case report.

A case of a young male with the Klippel-Trénaunay-Weber syndrome is described. Typical features of hemihypertrophy, hemangiomata, macrodactyly, and macrocephaly were present. The most striking oral feature was generalized severe gingival hypertrophy confirmed histologically, ultrastructurally, and by collagen analysis. In the absence of other known systemic causes of gingival enlargement, a diagnosis of familial gingival fibromatosis in association with Klippel-Trénaunay-Weber-syndrome is concluded. The combination of gingival fibromatosis and Klippel-Trénaunay-Weber syndrome has not been reported to our knowledge, it is uncertain whether this occurrence is significant or coincidental.

Amelogenesis imperfecta and nephrocalcinosis syndrome. Case studies of clinical features and ultrastructure of tooth enamel in two siblings.

This article describes the enamel ultrastructure and clinical features in two siblings with the little known syndrome of Amelogenesis imperfecta and nephrocalcinosis. Nephrocalcinosis was diagnosed by x-ray examination of the abdomen, intravenous pyelography, ultrasonography, and computed tomography scan. Amelogenesis imperfecta was diagnosed from clinical and histologic examinations. The affected enamel was hypoplastic (approximately 0.2 mm thick), positively birefringent, generally aprismatic, porous, and consisted of loosely packed, randomly orientated, thin (approximately 10 nm wide), ribbonlike crystals. The enamel surface was rough, extensively cracked, and covered with ovoid or globular protrusions. Observations showed that in this case hypoplasia, hypocalcification, or hypomaturation defects were present in the same tooth, indicating that both secretory and maturation phases may have been affected. The study suggested the possibility of an abnormality in interstitial matrix, which could lead to dystrophic calcification in the kidney and abnormal tooth enamel formation. It also suggested the possibility of involvement of two separate but closely linked genes.

Mucopolysaccharidosis: MPS VI and associated delayed tooth eruption.

Eight cases of mucopolysaccharidosis Type VI (Maroteaux-Lamy syndrome) are reviewed and two cases are presented in detail. Developmental dental anomalies including unerupted and impacted permanent teeth and associated hyperplastic tooth follicles are seen frequently in MPS patients. The surgical implications and management are discussed. All patients reviewed had significant cardiovalvular disease. It is essential for the primary medical provider to establish early basic dental care and evaluation for delayed eruption of primary and permanent dentition. This will probably minimize the possibility of infective endocarditis and allow for early treatment of impacted teeth.

Adjuvant therapy of node-negative breast cancer.

OBJECTIVE: To present the controversies regarding adjuvant cytotoxic chemotherapy or hormonal therapy in patients with node-negative breast cancer, and to evaluate the use of prognostic factors in identifying patients with node-negative breast cancer who will benefit from adjuvant therapy. DATA SOURCE: A MEDLINE search was performed to identify pertinent primary literature and review articles. Articles also were identified through Current Contents, textbooks, and bibliographies of selected articles. DATA EXTRACTION: The most recent clinical trials that evaluated cytotoxic chemotherapy or hormonal therapy in patients with node-negative breast cancer were chosen. Recent review articles and clinical trials that analyzed prognostic factors also were evaluated. DATA SYNTHESIS: The treatment of patients with node-negative breast cancer remains controversial. Approximately 60-80% of patients with this diagnosis will be alive 10 years after initial treatment without adjuvant therapy. The use of chemotherapy or hormonal therapy in node-negative disease increased after the 1988 National Cancer Institute Clinical Alert. Since that time, the research in node-negative breast cancer has focused on identifying prognostic factors and evaluating new treatment regimens. It is hoped that prognostic factors will help direct treatment decisions by identifying subgroups of patients who may benefit from adjuvant therapy. Prognostic factors currently being evaluated include tumor size, hormonal receptors, ploidy status, S-phase fraction, and cathepsin D. CONCLUSIONS: Many patients with node-negative breast cancer will be cured by local therapy alone. Even so, up to 58% of node-negative patients may develop recurrent disease. Reduction of breast cancer recurrence in patients with node-negative breast cancer has been documented as a result of adjuvant chemotherapy or tamoxifen. Of utmost priority is the identification of patients with node-negative breast cancer at highest risk for recurrence so that they may receive appropriate adjuvant therapy with curative intent, while sparing patients at lower risk for recurrence the toxic effects and financial burden incurred by unnecessary adjuvant treatment.

The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

Glucocorticoids stimulate hepatic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) gene expression, thereby increasing the rate of gluconeogenesis. The effect of glucocorticoids on PEPCK gene expression is mediated by a set of promoter elements collectively referred to as the glucocorticoid response unit. The response unit spans a 100-bp segment and includes two glucocorticoid receptor binding sites (GR1 and GR2) and two accessory factor binding sites (AF1 and AF2), all of which are required for a maximal glucocorticoid response. The AF1 element also serves as a retinoic acid response element and may be involved in developmental and tissue-specific expression of the gene. In this study we report that COUP-TF and HNF-4, two orphan members of the nuclear receptor superfamily, bind to the AF1 element and function as accessory factors for the glucocorticoid response of the PEPCK gene.

Congenital adrenal hyperplasia and enamel hypoplasia: case report.

A female with congenital adrenal hyperplasia and enamel defects involving the permanent maxillary incisors and all canines and premolars received composite veneer splint overlays under general anesthesia. Possible etiological factors involved in the formation of the enamel defects and overall case management is discussed.

Ultrastructural study of tooth enamel with amelogenesis imperfecta in AI-nephrocalcinosis syndrome.

This paper describes the ultrastructure of the affected enamel and the clinical features in two siblings with the syndrome of nephrocalcinosis and amelogenesis imperfecta. Nephrocalcinosis was diagnosed by intravenous pyelography, and confirmed by ultrasonography and CT scan. Amelogenesis imperfecta AI was diagnosed clinically and histologically. Light microscopy showed that the affected enamel surfaces were rough and the enamel was hypoplastic and mainly positively birefringent. Scanning electron microscopy revealed a rough and extensively cracked enamel surface covered with oval shaped blister-like protrusions. TEM showed porous enamel consisting of loosely packed and randomly oriented thin ribbon-like crystals with little or no prismatic structure. Observations showed that hypoplasia together with hypocalcification and/or hypomaturation defects were present in the same tooth, indicating the possibility of an abnormality in interstitial matrix, leading to dystrophic calcification in the kidney and abnormal tooth enamel formation, or alternatively an involvement of two separate but closely linked genes.

The role of CT, MRI and 3D imaging in the diagnosis of temporomandibular joint and other orofacial disorders in children.

Advances in medical imaging techniques add a new third dimension (3D computer reconstruction of CT, MR and Ultrasound images) to the diagnostic armamentarium of practitioners in orofacial medicine, surgery and Orthodontics. In particular, Computerized Tomography and Magnetic Resonance Imaging with, to a lesser extent, Nuclear Medicine Imaging and Sonography provide more accurate and detailed information on abnormalities and disorders of craniofacial osseous and soft tissues, especially the temporomandibular joint, than was previously available from plain films or tomography. Greater familiarity with these new imaging modalities by dental specialists will result in their more effective use in paediatric patients.