A novel mutation L260P of the steroidogenic acute regulatory protein gene in three unrelated patients of Swiss ancestry with congenital lipoid adrenal hyperplasia.

CONTEXT Lipoid congenital adrenal hyperplasia (CAH) is the most severe form of CAH leading to impaired production of all adrenal and gonadal steroids. Mutations in the gene encoding steroidogenic acute regulatory protein (StAR) cause lipoid CAH. OBJECTIVE We investigated three unrelated patients of Swiss ancestry who all carried novel mutations in the StAR gene. All three subjects were phenotypic females with absent Müllerian derivatives, 46,XY karyotype, and presented with adrenal failure. METHODS AND RESULTS StAR gene analysis showed that one patient was homozygous and the other two were heterozygous for the novel missense mutation L260P. Of the heterozygote patients, one carried the novel missense mutation L157P and one had a novel frameshift mutation (629-630delCT) on the second allele. The functional ability of all three StAR mutations to promote pregnenolone production was severely attenuated in COS-1 cells transfected with the cholesterol side-chain cleavage system and mutant vs. wild-type StAR expression vectors. CONCLUSIONS These cases highlight the importance of StAR-dependent steroidogenesis during fetal development and early infancy; expand the geographic distribution of this condition; and finally establish a new, prevalent StAR mutation (L260P) for the Swiss population.

Smooth Muscle Hyperplasia due to ACTA2/MYH11 Mutations: Identification of Novel Pathology and Pathways Leading to Aneurysms and Diverse Vascular Occlusive Diseases

Missense mutations in smooth muscle cell (SMC) specific ACTA2 (á-actin) and MYH11 (â-myosin heavy chain) cause diffuse and diverse vascular diseases, including thoracic aortic aneurysms and dissections (TAAD) and early onset coronary artery disease and stroke. The mechanism by which these mutations lead to dilatation of some arteries but occlusion of others is unknown. We hypothesized that the mutations act through two distinct mechanisms to cause varied vascular diseases: a loss of function, leading to decreased SMC contraction and aneurysms, and a gain of function, leading to increased SMC proliferation and occlusive disease. To test this hypothesis, ACTA2 mutant SMCs and myofibroblasts were assessed and found to not form á-actin filaments whereas control cells did, suggesting a dominant negative effect of ACTA2 mutations on filament formation. A loss of á-actin filaments would be predicted to cause decreased SMC contractility. Histological examination of vascular tissues from patients revealed SMC hyperplasia leading to arterial stenosis and occlusion, supporting a gain of function associated with the mutant gene. Furthermore, ACTA2 mutant SMCs and myofibroblasts proliferated more rapidly in static culture than control cells (p<0.05). We also determined that Acta2-/- mice have ascending aortic aneurysms. Histological examination revealed aortic medial SMC hyperplasia, but minimal features of medial degeneration. Acta2-/- SMCs proliferated more rapidly in culture than wildtype (p<0.05), and microarray analysis of Acta2-/- SMCs revealed increased expression of Actg2, 15 collagen genes, and multiple focal adhesion genes. Acta2-/- SMCs showed altered localization of vinculin and zyxin and increased phosphorylated focal adhesion kinase (FAK) in focal adhesions. A specific FAK inhibitor decreased Acta2-/- SMC proliferation to levels equal to wildtype SMCs (p<0.05), suggesting that FAK activation leads to the increased proliferation. We have described a unique pathology associated with ACTA2 and MYH11 mutations, as well as an aneurysm phenotype in Acta2-/- mice. Additionally, we identified a novel pathogenic pathway for vascular occlusive disease due to loss of SMC contractile filaments, alterations in focal adhesions, and activation of FAK signaling in SMCs with ACTA2 mutations.

A study of the cost and effect of newborn screening for Congenital Adrenal Hyperplasia in Texas

Congenital Adrenal Hyperplasia (CAH), due to 21-Hydroxylase deficiency, has an estimated incidence of 1:15,000 births and can result in death, salt-wasting crisis or impaired growth. It has been proposed that early diagnosis and treatment of infants detected from newborn screening for CAH will decrease the incidence of mortality and morbidity in the affected population. The Texas Department of Health (TDH) began mandatory screening for CAH in June, 1989 and Texas is one of fourteen states to provide neonatal screening for the disorder.^ The purpose of this study was to describe the cost and effect of screening for CAH in Texas during 1994 and to compare cases first detected by screen and first detected clinically between January 1, 1990 and December 31, 1994. This study used a longitudinal descriptive research design. The data was secondary and previously collected by the Texas Department of Health. Along with the descriptive study, an economic analysis was done. The cost of the program was defined, measured and valued for four phases of screening: specimen collection, specimen testing, follow-up and diagnostic evaluation.^ There were 103 infants with Classical CAH diagnosed during the study and 71 of the cases had the more serious Salt-Wasting form of the disease. Of the infants diagnosed with Classical CAH, 60% of the cases were first detected by screen and 40% were first detected because of clinical findings before the screening results were returned. The base case cost of adding newborn screening to an existing program (excluding the cost of specimen collection) was $357,989 for 100,000 infants. The cost per case of Classical CAH diagnosed, based on the number of infants first detected by screen in 1994, was \$126,892. There were 42 infants diagnosed with the more benign Nonclassical form of the disease. When these cases were included in the total, the cost per infant to diagnose Congenital Adrenal/Hyperplasia was $87,848. ^

Hyperplasia to neoplasia sequence of duodenal and pancreatic neuroendocrine diseases and pseudohyperplasia of the PP-cells in the pancreas.

Hyperplastic changes of the neuroendocrine cell system may have the potential to evolve into neoplastic diseases. This is particularly the case in the setting of genetically determined and hereditary neuroendocrine tumor syndromes such as MEN1. The review discusses the MEN1-associated hyperplasia-neoplasia sequence in the development of gastrinomas in the duodenum and glucagon-producing tumors in the pancreas. It also presents other newly described diseases (e.g., glucagon cell adenomatosis and insulinomatosis) in which the tumors are (or most likely) also preceded by islet cell hyperplasia. Finally, the pseudohyperplasia of PP-rich islets in the pancreatic head is defined as a physiologic condition clearly differing from other hyperplastic-neoplastic neuroendocrine diseases.

An Intergenic Regulatory Region Mediates Drosophila Myc -Induced Apoptosis and Blocks Tissue Hyperplasia

Induction of cell-autonomous apoptosis following oncogene-induced overproliferation is a major tumor-suppressive mechanism in vertebrates. However, the detailed mechanism mediating this process remains enigmatic. In this study, we demonstrate that dMyc-induced cell-autonomous apoptosis in the fruit fly Drosophila melanogaster relies on an intergenic sequence termed the IRER (irradiation-responsive enhancer region). The IRER mediates the expression of surrounding proapoptotic genes, and we use an in vivo reporter of the IRER chromatin state to gather evidence that epigenetic control of DNA accessibility within the IRER is an important determinant of the strength of this response to excess dMyc. In a previous work, we showed that the IRER also mediates P53-dependent induction of proapoptotic genes following DNA damage, and the chromatin conformation within IRER is regulated by polycomb group-mediated histone modifications. dMyc-induced apoptosis and the P53-mediated DNA damage response thus overlap in a requirement for the IRER. The epigenetic mechanisms controlling IRER accessibility appear to set thresholds for the P53- and dMyc-induced expression of apoptotic genes in vivo and may have a profound impact on cellular sensitivity to oncogene-induced stress.

The role of endogenous IFN-beta in cutaneous melanoma: Consequences of epidermal hyperplasia

The purpose of this study was to characterize epidermal hyperplasia overlying malignant melanoma, to determine the mitogenic factor responsible for the induction of this hyperplasia and to investigate its biological consequence. Whether increased keratinocyte proliferation overlying melanoma is due to production of growth factors by the tumor cells or to other mechanisms is unknown. Epidermal hyperplasia overlying human melanoma was found overlying thick (>4.0mm), but not thin (<1.0mm) tumors. Immunostaining of the sections for growth factors related to angiogenesis revealed that epidermal hyperplasia was associated with loss of IFN-β production by the keratinocytes directly overlying the tumors. Since previous studies from our laboratory have demonstrated that exogenous administration of IFN-β negatively regulates angiogenesis, we hypothesize that tumors are able to produce growth factors which stimulate the proliferation of cells in the surrounding tissues. This hyperplasia leads to a decrease in the endogenous negative regulator of angiogenesis, IFN-β. ^ The human melanoma cell line, DM-4 and several of its clones were studied to identify the mitogenic factor for keratinocytes. The expression of TGF-α directly correlated with epidermal hyperplasia in the DM-4 clones. A375SM, a human melanoma cell line that produces high levels of TGF-α, was transfected with a plasmid encoding full-length antisense TGF-α. The parental and transfected cells were implanted intradermally into nude mice. The extent of epidermal hyperplasia directly correlated with expression of TGF-α and decreased production of IFN-β, hence, increased angiogenesis. ^ In the next set of experiments, we determined the role of IFN-β on angiogenesis, tumor growth and metastasis of skin tumors. Transgenic mice containing a functional mutation in the receptor for IFN α/β were obtained. A375SM melanoma cells were implanted both s.c. and i.v. into IFN α/βR −/− mice. Tumors in the IFN α/β R −/− mice exhibited increased angiogenesis and metastasis. IFN α/βR −/− mice were exposed to chronic UV irradiation. Autochthonous tumors developed earlier in the transgenic mice than the wild-type mice. ^ Collectively, the data show that TGF-α produced by tumor cells induces proliferation of keratinocytes, leading to epidermal hyperplasia overlying malignant melanoma associated with loss of IFN-β and enhanced angiogenesis, tumorigenicity and metastasis. ^

Investigation of the molecular mechanisms of endometrial hyperplasia

While there is considerable information on the molecular aberrations associated with the development of endometrial cancer, very little is known of changes in gene expression associated with its antecedent premalignant condition, endometrial hyperplasia. In order to address this, we have compared the level of expression of components of the IGF-I signaling pathway in human endometrial hyperplasia to their level of expression in both the normal pre-menopausal endometrium and endometrial carcinoma. We have also characterized the molecular characteristics of endometrial hyperplasia as it occurs in a murine model of hormone-dependent tumorigenesis of the female reproductive tract. ^ There was a significant and selective increase in the expression of the IGF-I Receptor (IGF-IR) in both human hyperplasia and carcinoma as compared to the normal endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in hyperplasia and carcinoma there is activation of the downstream component Akt. The expression of the PTEN tumor suppressor is decreased in a subset of subjects with hyperplasia and in all of the carcinomas. The simultaneous loss of PTEN expression and increased IGF-IR activation in the hyperplastic endometrium was associated with an increased incidence of endometrial carcinoma elsewhere within the uterus. In the rodent hyperplasia, there was a significant increase in the expression and activation of Akt that appears to be attributable to a marked increase in the expression of IGF-II. ^ Our studies have demonstrated the pathologic proliferation of both the human and rodent endometrium is linked to a marked activation of the Akt pathway. However the cause of this dysregulation is different in the human disease and the animal model. In rodents, hyperplasia is linked to increased expression of one of the ligands of the IGF-IR, IGF-II. In humans the IGF-I receptor itself is upregulated and activated. Additional activation of the Akt pathway via the suppression of PTEN activity, results in conditions that are associated with the marked increase in the probability of developing endometrial cancer. Our data suggests that increased activity of the IGF-I pathway plays the key role in the hyperproliferative state characteristic of endometrial hyperplasia and cancer.^

Retrospective assesment of the levonorgestrel intrauterine device to treat complex atypical hyperplasia and early endometrioid endometrial cancer

Objective: The primary objective of this project was to describe the efficacy of the Levonorgestrel Intrauterine Device (LIUD) for treatment of Complex Endometrial Cancer (CAH) and Grade 1 Endometrial Cancer (G1EEC) in terms of rate of Complete Response (CR) and Partial Response (PR) after 6 months of therapy. Finally, we assessed if any clinical or pathologic features were associated with response to the LIUD. ^ Methods: This study was a retrospective case series designed to report the response rate of patients with CAH or G1EEC treated with LIUD therapy. In addition, this study has a laboratory component to assess molecular predictors of response to LIUD therapy. Retrospective data already collected from patients diagnosed with CAH or EEC grade 1 and treated with LIUD therapy at MD Anderson Cancer Center (MDACC) were used for this study. Patients from all ethnic and race groups were included. A Complete Response (CR) was defined in patients diagnosed with CAH if pathologic report at 6 months demonstrated either no evidence of hyperplasia or no atypia in the setting of simple or complex hyperplasia. Partial Response (PR) was recorded if disease downgraded to only CAH from G1EEC. No Response (NR) was recorded if pathologic report demonstrates no change (Stable Disease, SD) or progression to cancer (Progressive Disease, PD). We calculated the proportion of patients with complete response to LIUD therapy with 95% confidence interval. We compared the response rates (CR/PR vs NR) by obesity status (Obese if BMI > 40 kg/m2 vs non-obese if BMI <= 40 kg/m2) as well as other clinical and pathologic factors, such as age, uterine size (median size), and presence of exogenous progesterone effect. ^ Results: There were 39 patients diagnosed with either CAH or G1EEC treated with the LIUD. Of 39 patients, 12 did not have pathological results of biopsy at 6months time period. Of 27 evaluable patients, 17 were diagnosed with CAH and 10 with G1EEC. Overall response rate (RR) was 78% (95% CI = 62-94%) at 6 months, 18 patients had CR (4 in G1EEC; 14 in CAH), 3 patients had PR (3 in G1EEC), 3 had SD (1 in CAH; 2 in G1EEC), 3 had PD (2 in CAH; 1 in G1EEC). After histology stratification, RR at 6 months was 82.35% (14/17; 95%CI = 67.4-97.3%) in CAH and 70% (7/10; 95% CI = 41-98.4%) in G1EEC. ^ There was no difference in response (R) and no response (NR) based on BMI (p=0.56). He observed a trend showing association between age with response (p=0.1). There was no association between uterine size and response to therapy (p=0.17). We recorded strong association between exogenous progesterone effect and response. ^ Conclusion: LIUD therapy for the treatment of CAH and G1EEC may be effective and safe. Presence of exogenous progesterone effect may predict the response to LIUD therapy at earlier time points. There is need of further studies with larger sample size to explore the relationship of response with other clinical and pathologic factors^

Overexpression of transforming growth factor β1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor β1 (TGF-β1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-β1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-β1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-β1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-β1 in vascular disease.

Steroid disorders in children: Congenital adrenal hyperplasia and apparent mineralocorticoid excess

Our research team and laboratories have concentrated on two inherited endocrine disorders, congenital adrenal hyperplasia (CAH) and apparent mineralocorticoid excess, in thier investigations of the pathophysiology of adrenal steroid hormone disorders in children. CAH refers to a family of inherited disorders in which defects occur in one of the enzymatic steps required to synthesize cortisol from cholesterol in the adrenal gland. Because of the impaired cortisol secretion, adrenocorticotropic hormone levels rise due to impairment of a negative feedback system, which results in hyperplasia of the adrenal cortex. The majority of cases is due to 21-hydroxylase deficiency (21-OHD). Owing to the blocked enzymatic step, cortisol precursors accumulate in excess and are converted to potent androgens, which are secreted and cause in utero virilization of the affected female fetus genitalia in the classical form of CAH. A mild form of the 21-OHD, termed nonclassical 21-OHD, is the most common autosomal recessive disorder in humans, and occurs in 1/27 Ashkenazic Jews. Mutations in the CYP21 gene have been identified that cause both classical and nonclassical CAH. Apparent mineralocorticoid excess is a potentially fatal genetic disorder causing severe juvenile hypertension, pre- and postnatal growth failure, and low to undetectable levels of potassium, renin, and aldosterone. It is caused by autosomal recessive mutations in the HSD11B2 gene, which result in a deficiency of 11β-hydroxysteroid dehydrogenase type 2. In 1998, we reported a mild form of this disease, which may represent an important cause of low-renin hypertension.

Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia

An essential component of regulated steroidogenesis is the translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroidogenesis. Recent studies showed that a 30-kDa mitochondrial phosphoprotein, designated steroidogenic acute regulatory protein (StAR), is essential for this translocation. To allow us to explore the roles of StAR in a system amenable to experimental manipulation and to develop an animal model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce StAR knockout mice. These StAR knockout mice were indistinguishable initially from wild-type littermates, except that males and females had female external genitalia. After birth, they failed to grow normally and died from adrenocortical insufficiency. Hormone assays confirmed severe defects in adrenal steroids—with loss of negative feedback regulation at hypothalamic–pituitary levels—whereas hormones constituting the gonadal axis did not differ significantly from levels in wild-type littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, with lesser deposits in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement support a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation inducing progressive accumulation of lipids within the steroidogenic cells and ultimately causing their death. These StAR knockout mice provide a useful model system in which to determine the mechanisms of StAR’s essential roles in adrenocortical and gonadal steroidogenesis.

NF-κB activation provides the potential link between inflammation and hyperplasia in the arthritic joint

The transcription factor NF-κB is a pivotal regulator of inflammatory responses. While the activation of NF-κB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-κB in animal models of RA. We demonstrate that in vitro, NF-κB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor α (TNFα) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-κB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-κB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IκBα profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-κB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-κB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-κB.

Analysis of masked mutations in familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal-dominant disease characterized by the development of hundreds of adenomatous polyps of the colorectum. Approximately 80% of FAP patients can be shown to have truncating mutations of the APC gene. To determine the cause of FAP in the other 20% of patients, MAMA (monoallelic mutation analysis) was used to independently examine the status of each of the two APC alleles. Seven of nine patients analyzed were found to have significantly reduced expression from one of their two alleles whereas two patients were found to have full-length expression from both alleles. We conclude that more than 95% of patients with FAP have inactivating mutations in APC and that a combination of MAMA and standard genetic tests will identify APC abnormalities in the vast majority of such patients. That no APC expression from the mutant allele is found in some FAP patients argues strongly against the requirement for dominant negative effects of APC mutations. The results also suggest that there may be at least one additional gene, besides APC, that can give rise to FAP.

The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules. Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium.