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© 2014 Habbal et al.Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alka

We characterized the t(7;22)(q32;q11.2) chromosomal translocation in an obese female with coarse features, short stature, developmental delay and a hypoplastic fifth digit. While these clinical features suggest Coffin-Siris Syndrome (CSS), we excluded a CS

Mutations in five canonical Ras pathway genes (NF1, NRAS, KRAS, PTPN11 and CBL) are detected in nearly 90% of patients with juvenile myelomonocytic leukemia (JMML), a frequently fatal malignant neoplasm of early childhood. In this report, we describe seven patients diagnosed with SH2B3-mutated JMML, including five patients who were found to have initiating, loss-of-function mutations in the gene. SH2B3 encodes the adaptor protein LNK, a negative regulator of normal hematopoiesis upstream of the Ras pathway. These mutations were identified to be germline, somatic or a combination of both. Loss of function of LNK, which has been observed in other myeloid malignancies, results in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of the JAK/STAT signaling pathway. In vitro studies of induced pluripotent stem cell-derived JMML-like hematopoietic progenitor cells also demonstrated sensitivity of SH2B3-mutated hematopoietic progenitor cells to JAK inhibition. Lastly, we describe two patients with JMML and SH2B3 mutations who were treated with the JAK1/2 inhibitor ruxolitinib. This report expands the spectrum of initiating mutations in JMML and raises the possibility of targeting the JAK/STAT pathway in patients with SH2B3 mutations.

Background: MGMT (O 6 -methylguanine-DNA methyltransferase) promoter methylation is a commonly assessed prognostic marker in glioblastoma (GBM). Epigenetic silencing of the MGMT gene by promoter methylation is associated with greater overall and progression free survival with alkylating agent regimens. To date, there is marked heterogeneity in how MGMT promoter methylation is tested and which CpG sites are interrogated., Methods: To further elucidate which MGMT promoter CpG sites are of greatest interest, we performed comprehensive searches in PubMed, Web of Science, and Embase and reviewed 2,925 article abstracts. We followed the GRADE scoring system to assess risk of bias and the quality of the studies we included., Results: We included articles on adult glioblastoma that examined significant sites or regions within MGMT promoter for the outcomes: overall survival, progression free survival, and/or MGMT expression. We excluded systemic reviews and articles on lower grade glioma. fifteen articles met inclusion criteria with variable overlap in laboratory and statistical methods employed, as well as CpG sites interrogated. Pyrosequencing or BeadChip arrays were the most popular methods utilized, and CpG sites between CpG's 70-90 were most frequently investigated. Overall, there was moderate concordance between the CpG sites that the studies reported to be highly predictive of prognosis. Combinations or means of sites between CpG's 73-89 were associated with improved OS and PFS. Six studies identified CpG sites associated with prognosis that were closer to the transcription start site: CpG's 8, 19, 22, 25, 27, 32,38, and CpG sites 21-37, as well as low methylation level of the enhancer regions., Conclusion: The following systematic review details a comprehensive investigation of the current literature and highlights several potential key CpG sites that demonstrate significant association with OS, PFS, and MGMT expression. However, the relationship between extent of MGMT promoter methylation and survival may be non-linear and could be influenced by potential CpG hotspots, the extent of methylation at each CpG site, and MGMT enhancer methylation status. There were several limitations within the studies such as smaller sample sizes, variance between methylation testing methods, and differences in the various statistical methods to test for association to outcome. Further studies of high impact CpG sites in MGMT methylation is warranted., (© 2024. The Author(s).)

Background: Despite the intensity of hematopoietic stem cell transplantation (HCT), relapse remains the most common cause of death in juvenile myelomonocytic leukemia (JMML). In contrast to other leukemias where therapy is used to reduce leukemic burden prior to transplant, many patients with JMML proceed directly to HCT with active disease. The objective of this study was to elucidate whether pre-HCT therapy has an effect on the molecular burden of disease and how this affects outcome post-HCT., Procedure: Twenty-one patients with JMML who received pre-HCT therapy and were transplanted at UCSF were analyzed in this study. The mutant allele frequency of the driver mutation was assessed before and after pre-HCT therapy, using custom amplicon next-generation sequencing., Results: Of the 21 patients, seven patients (33%) responded to therapy with a significant reduction in their mutant allele frequency and were classified as molecular responders. Six of these patients received moderate-intensity chemotherapy, one patient received only azacitidine. The 5-year progression-free survival after HCT of molecular responders was 100% versus 61% for nonresponders (P = .12). Survival of molecular nonresponders was not improved by use of high-intensity conditioning, but patients were salvaged if they experienced severe graft versus host disease. There were no baseline clinical characteristics that were associated with response to pre-HCT therapy., Conclusions: Despite the myelodysplastic nature of JMML, patients treated with pre-HCT therapy can achieve molecular remissions. These patients experienced a trend toward improved outcomes post-HCT. Importantly, molecular testing can be helpful to distinguish between responders and nonresponders and should become an integral part of clinical care., (© 2019 Wiley Periodicals, Inc.)

BackgroundMaintenance of cholesterol homeostasis is crucial for brain development. Brain cholesterol relies on de novo synthesis and is cleared primarily by conversion to 24S-hydroxycholesterol (24S-HC) with brain-specific cholesterol 24-hydroxylase (CYP46A1). We aimed to investigate the impact of hypoxia-ischemia (HI) on brain cholesterol metabolism in the neonatal mice.MethodsPostnatal day 9 C57BL/6 pups were subjected to HI using the Vannucci model. CYP46A1 expression was assessed with western blotting and its cellular localization was determined using immunofluorescence staining. The amount of brain cholesterol, 24S-HC in the cortex and in the serum, was measured with enzyme-linked immunosorbent assay (ELISA).ResultsThere was a transient cholesterol loss at 6 h after HI. CYP46A1 was significantly upregulated at 6 and 24 h following HI with a concomitant increase of 24S-HC in the ipsilateral cortex and in the serum. The serum levels of 24S-HC correlated with those in the brain, as well as with necrotic and apoptotic cell death evaluated by the expression of spectrin breakdown products and cleaved caspase-3 at 6 and 24 h after HI.ConclusionEnhanced cholesterol turnover by activation of CYP46A1 represents disrupted brain cholesterol homeostasis early after neonatal HI. 24S-HC might be a novel blood biomarker for severity of hypoxic-ischemic encephalopathy with potential clinical application.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution.

The association between leptin and reproduction originated with the leptin-mediated correction of sterility in ob/ob mice and initiation of reproductive function in normal female mice. The uncovering of a central leptin pathway regulating food intake prompted the dissection of neuroendocrine mechanisms involving leptin in the metabolic control of reproduction. The absence of leptin receptors on GnRH neurons incited a search for intermediary neurons situated between leptin-responsive and GnRH neurons. This review addresses the most significant findings that have furthered our understanding of recent progress in this new field. The role of leptin in puberty was impacted by the discovery of neurons that co-express kisspeptin, neurokinin B, and dynorphin and these could act as leptin intermediates. Furthermore, the identification of first-order leptin-responsive neurons in the premammilary ventral nucleus and other brain regions opens new avenues to explore their relationship to GnRH neurons. Central to these advances is the unveiling that agouti-related protein/neuropeptide Y neurons project onto GnRH and kisspeptin neurons, allowing for a crosstalk between food intake and reproduction. Finally, while puberty is a state of leptin sensitivity, mid-gestation represents a state of leptin resistance aimed at building energy stores to sustain pregnancy and lactation. The mechanisms underlying leptin resistance in pregnancy have lagged; however, the establishment of this natural state is significant. Reproduction and energy balance are tightly controlled and backed up by redundant mechanisms that are critical for the survival of our species. It will be the goal of the following decade to shed new light on these complex and essential pathways., (© 2014 Society for Endocrinology.)

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule. A key enzymatic reaction, which occurs at the top of the Bloch and Kandutsch-Russell pathways, is the demethylation of lanosterol and dihydrolanosterol (DHL). This reaction is catalyzed by lanosterol 14α demethylase (CYP51) and requires three oxygen molecules. Thus, it is the first step in the distal pathway to be susceptible to oxygen deprivation. Having previously identified that the forkhead transcription factor 4 (FoxO4) represses CYP51 expression, we aimed to characterize its role at the CYP51 promoter. Hypoxia-treated 3T3L1 cells showed decreased cholesterol biosynthesis, accumulation of lanosterol/DHL, and stimulation of FoxO4 expression and its cytoplasmic translocation to the nucleus. Transfection assays with a CYP51 promoter reporter gene revealed that FoxO4 and sterol regulatory element binding protein (SREBP)2 exert a stimulatory effect, whereas FoxO4 and the hypoxia inducible factor (HIF)2α repress CYP51 promoter activity. Electromobility shift, chromatin immunoprecipitation, pull-down, and coimmunoprecipitation assays show that FoxO4 interacts with SREBP2 and HIF2α to modulate CYP51 promoter activity. We also show an inverse correlation between FoxO4 and CYP51 in adipose tissue of ob/ob mice and mouse fetal cortical neurons exposed to hypoxia. Overall, these studies demonstrate a role for FoxO4 in the regulation of CYP51 expression.

We characterized the t(7;22)(q32;q11.2) chromosomal translocation in an obese female with coarse features, short stature, developmental delay and a hypoplastic fifth digit. While these clinical features suggest Coffin-Siris Syndrome (CSS), we excluded a CSS diagnosis by exome sequencing based on the absence of deleterious mutations in six chromatin-remodeling genes recently shown to cause CSS. Thus, molecular characterization of her translocation could delineate genes that underlie other syndromes resembling CSS. Comparative genomic hybridization microarrays revealed on chromosome 7 the duplication of a 434,682 bp region that included the tail end of an uncharacterized gene termed C7orf58 (also called CPED1) and spanned the entire WNT16 and FAM3C genes. Because the translocation breakpoint on chromosome 22 did not disrupt any apparent gene, her disorder was deemed to result from the rearrangement on chromosome 7. Mapping of yeast and bacterial artificial chromosome clones by fluorescent in situ hybridization on chromosome spreads from this patient showed that the duplicated region and all three genes within it were located on both derivative chromosomes 7 and 22. Furthermore, DNA sequencing of exons and splice junctional regions from C7orf58, WNT16 and FAM3C revealed the presence of potential splice site and promoter mutations, thereby augmenting the detrimental effect of the duplicated genes. Hence, dysregulation and/or disruptions of C7orf58, WNT16 and FAM3C underlie the phenotype of this patient, serve as candidate genes for other individuals with similar clinical features and could provide insights into the physiological role of the novel gene C7orf58.

Progress in the functional studies of human olfactory receptors has been largely hampered by the lack of a reliable experimental model system. Although transgenic approaches in mice could characterize the function of individual olfactory receptors, the presence of over 300 functional genes in the human genome becomes a daunting task. Thus, the characterization of individuals with a genetic susceptibility to altered olfaction coupled with the absence of particular olfactory receptor genes will allow phenotype/genotype correlations and vindicate the function of specific olfactory receptors with their cognate ligands. We characterized a 118 kb β-globin deletion and found that its 3' end breakpoint extends to the neighboring olfactory receptor region downstream of the β-globin gene cluster. This deletion encompasses six contiguous olfactory receptor genes (OR51V1, OR52Z1, OR51A1P, OR52A1, OR52A5, and OR52A4) all of which are expressed in the brain. Topology analysis of the encoded proteins from these olfactory receptor genes revealed that OR52Z1, OR52A1, OR52A5, and OR52A4 are predicted to be functional receptors as they display integral characteristics of G-proteins coupled receptors. Individuals homozygous for the 118 kb β-globin deletion are afflicted with β-thalassemia due to a homozygous deletion of the β-globin gene and have no alleles for the above mentioned olfactory receptors genes. This is the first example of a homozygous deletion of olfactory receptor genes in human. Although altered olfaction remains to be ascertained in these individuals, such a study can be carried out in β-thalassemia patients from Malaysia, Indonesia and the Philippines where this mutation is common. Furthermore, OR52A1 contains a γ-globin enhancer, which was previously shown to confer continuous expression of the fetal γ-globin genes. Thus, the hypothesis that β-thalassemia individuals, who are homozygous for the 118 kb deletion, may also have an exacerbation of their anemia due to the deletion of two copies of the γ-globin enhancer element is worthy of consideration.

The Forkhead transcription factors FoxO1, FoxO3a, and FoxO4 play a prominent role in regulating cell survival and cell cycle. Whereas FOXO1 was shown to mediate insulin sensitivity and adipocyte differentiation, the role of the transcription factor FoxO4 in metabolism remains ill defined. To uncover the effects of FoxO4, we generated a cellular model of stable FoxO4 overexpression and subjected it to microarray-based gene expression profiling. While pathway analysis revealed a disruption of cholesterol biosynthesis gene expression, biochemical studies revealed an inhibition of cholesterol biosynthesis, which was coupled with decreased mRNA levels of lanosterol 14alpha demethylase (CYP51). FoxO4-mediated repression of CYP51 led to the accumulation of 24,25 dihydrolano-sterol (DHL), which independently and unlike lanosterol inhibited cholesterol biosynthesis. Furthermore, FoxO4-overexpressing cells accumulated lipid droplets and triacylglycerols and had an increase in basal glucose uptake. Recapitulation of these effects was obtained following treatment with CYP51 inhibitors, which also induce DHL buildup. Moreover, DHL but not lanosterol strongly stimulated liver X receptor alpha (LXRalpha) activity, suggesting that DHL and LXRalpha mediate the downstream effects initiated by FoxO4. Together, these studies suggest that FoxO4 acts on CYP51 to regulate the late steps of cholesterol biosynthesis.

Leptin treatment ameliorates lipoatrophic diabetes in animal models and humans. Transgenic mice overexpressing leptin (LepTg) are lipoatrophic but not diabetic and thus represent a model for elucidating mechanisms of leptin-mediated glucose homeostasis. In this communication, we show that LepTg mice overexpress the forkhead transcription factor foxo4 in their remnant adipose tissue. To further characterize the role of foxo4 in adipose tissue, we generated transgenic mice overexpressing a constitutive active form of foxo4 (A3foxo4) under the control of the aP2 promoter/enhancer. aP2-A3foxo4 mice are not lipoatrophic but are able to clear glucose rapidly similar to LepTg mice. In addition, both LepTg and A3foxo4 mice show in their adipocytes increased AMP-activated protein kinase (AMPK) phosphorylation, suggesting a link between AMPK, glucose clearance, foxo4 and the leptin axis. These studies shed new light on mechanisms by which leptin treatment improves glucose disposal.

Adipose tissue is unique in that it can undergo significant hypertrophy and atrophy, resulting in wide ranges of obesities and lipodystrophies. At the base of this elasticity is the lipid-filled adipocyte, which can either overfill by storing large amounts of triglycerides or shrink to a tiny cell by depleting its lipids and as such is remarkable in sustaining insults. As a major energy reservoir, the adipocyte may hold considerable calories necessary for survival and reproduction, two functions that are essential for the survival of the species. This review will summarize some of the recent studies that have advanced our understanding of the central and peripheral mechanisms that are initiated by adipocyte-secreted factors such as leptin, adiponectin, resistin, and retinol-binding protein 4. The intersection of obesity and lipodystrophy results in insulin resistance, which may be unlocked by elucidating the roles of these factors in pathways that control insulin sensitivity and glucose uptake.

The binding of leptin to hypothalamic neurons elicits inhibition of orexigenic NPY/AgRP neurons and stimulation of anorexigenic POMC/CART neurons. Projections of serotonergic neurons onto POMC neurons suggest that leptin and serotonin converge onto POMC neurons to regulate body weight. We probed the interaction of these pathways by generating transgenic mice overexpressing leptin (LepTg) without 5HT2c receptors. On a chow diet, the lean phenotype of LepTg mice was unaffected by the absence of 5HT2c receptors, whereas on a high fat diet, LepTg/5HT2c receptors knockout mice showed an exacerbation of diet-induced obesity. POMC mRNA levels were low in LepTg, 5HT2c receptors knockout and LepTg/5HT2c receptors knockout mice, demonstrating that perturbations of the 5HT2c receptor and leptin pathways, either alone or in combination, negatively impact on POMC expression. Thus, on a chow diet, leptin action is independent of 5HT2c receptors whereas on a high fat diet 5HT2c receptors are required for the attenuation of obesity.

The molecular basis in the majority of fragile X patients results from expansion of the CGG repeats in the FMR1 gene causing its transcriptional silencing and deficiency of its encoded protein FMRP. In this communication, we report on a male patient who lacks the characteristic physical features of fragile X and carries a fully methylated mutation, a premutation, a non-methylated full mutation, and a microdeletion encompassing the entire CGG repeat region and 42 bp of upstream flanking sequence. Southern blot analysis revealed that the methylated full mutation accounted for only 10% of his genotype while the premutation/non-methylated full mutation and the microdeletion constituted 37% and 53%, respectively. Immunofluorescent staining of FMRP demonstrated the presence of 22% FMRP in his peripheral blood leukocytes and quantitative RT-PCR revealed a 3.6-fold elevation of FMR1 mRNA levels. Developmental assessments indicated that while he has a learning disability, he does not have mental retardation. Because previous reports had noted that 28% FMRP expression is associated with a characteristic fragile X phenotype, we propose that in our patient the association of 22% FMRP levels with normal physical features and a high-functioning status may have resulted from increased FMRP stability by a mechanism that takes into account the CGG microdeletion and elevated mRNA levels., ((c) 2006 Wiley-Liss, Inc.)

Our understanding of the effects of leptin have stemmed mainly from animal studies, which continue to leave important clues of its roles in physiology, metabolism, neuroscience, and cell signaling. Since its discovery, leptin has been linked to various pathways, either directly at its primary site of action in the hypothalamus, or indirectly via downstream effector pathways such as in adipocytes and muscle. Leptin's importance is exemplified by the lack of redundant backup mechanisms, since leptin-deficient mice are obese, diabetic, and sterile. Investigations uncovering the pleiotropic actions of leptin were unfolded mainly from rodent models. Thus, this chapter focuses on these studies and, more specifically, on those findings recently brought forward by transgenic mice overexpressing leptin. The vast amount of biology that has been ascribed to leptin encompasses effects on food intake, insulin sensitivity, adiposity, thermogenesis, reproduction, immunity, and bone regulation. Mechanisms underlying leptin's action revolve essentially around neural pathways but also encompass to a lesser extent peripheral mechanisms. The roles of leptin along these axes are reviewed, with particular emphasis on pathways and phenotypes generated by transgenic hyperleptinemia. An evolutionary significance of hyperleptinemia in association with development of leptin resistance is suggested as a protective armament against some of the detrimental effects caused by hyperleptinemia in transgenic mice overexpressing leptin.

Transgenic mice overexpressing leptin (LepTg) exhibit substantial reductions in adipose mass. Since the binding of leptin to its receptor activates the sympathetic nervous system, we reasoned that the lean state of the LepTg mice could be caused by chronic lipolysis. Instead, the LepTg mice exhibited a low basal lipolysis state and their lean phenotype was not dependent on the presence of beta3-adrenergic receptors. In their white adipose tissue, protein levels of protein kinase A, hormone-sensitive lipase, and ADRP were not impaired. However, compared to normal mice, perilipin, perilipin mRNA, and cAMP-stimulated PKA activity were significantly attenuated. Overall, we demonstrate that the lean phenotype of the LepTg mice does not result in a chronically elevated lipolytic state, but instead in a low basal lipolysis state characterized by a decrease in perilipin and PKA activity in white fat.

Transgenic mice overexpressing leptin backcrossed to the C57BL/6J genetic background (LepTg) have a lean phenotype, characterized by a 95% reduction in adipose mass; reduced plasma levels of glucose, triglycerides, insulin, and IGF-1; and a 75% decrease in adipocyte size. High-fat diet treatment for 20 wk revealed that, compared with normal mice, the LepTg mice had an increased susceptibility to diet-induced obesity, as demonstrated by their rate of weight gain, higher accumulation of sc white adipose tissue mass, hypertrophy of adipocytes, and normalization of their reduced metabolic parameters. The stromal vascular fraction of white adipose tissue from the LepTg mice was highly cellular and contained cells capable of rapid lipid accumulation in primary cultures. The precipitous diet-induced obesity of the LepTg mice was accompanied with 10-fold and 1.6-fold elevations in insulin and IGF-1, respectively, suggesting that the trophic action of insulin and IGF-1 on the preadipocytes and small adipocytes may have caused them to rapidly differentiate and accumulate triacylglycerol stores. Other contributing factors may involve a shift in insulin sensitivity triggered by hyperleptinemia and a decrease in energy expenditure. These studies demonstrate that a chronic response to hyperleptinemia as in the LepTg mice is a predisposing factor to diet-induced obesity and suggest that individuals who are particularly lean because of increased leptin secretion may develop rapid obesity under conditions of a high-fat diet.

A deficiency of leptin synthesis in mice results in a complex phenotype characterized by morbid obesity, diabetes, sterility, and defective thermogenesis. To determine whether the genetic background could alter the pleiotropic effects of leptin deficiency, we backcrossed the ob mutation for 10 generations from the C57BL/6J to the BALB/cJ genetic background. Compared with C57BL/6J ob/ob mice, BALB/cJ ob/ob mice showed at 27 wk of age a 35-40% reduction in body weight attributed to a 60% decrease in white adipose tissue mass. Food intake was not significantly different between the two obese strains, suggesting distinct utilization of energy intake. In the fed state, BALB/cJ ob/ob mice had elevated insulin and triglycerides levels, demonstrating a worsening effect on diabetes. At the reproductive level and in contrast to sterile C57BL/6J ob/ob mice, male and female BALB/cJ ob/ob mice were capable of reproducing after a mating period of 16 and 32 wk, respectively. At thermoneutrality, the body temperature of BALB/cJ ob/ob mice was 2.9 C higher than that of C57BL/6J ob/ob mice, whereas exposure of both groups to 4 C demonstrated a prolonged cold tolerance of BALB/cJ ob/ob mice. These studies show that the abnormalities caused by leptin deficiency can be genetically dissected and separated from each other, suggesting discrete pathways controlled by leptin modifier genes.

Sensitivity to leptin is associated with a normal regulation of the adipose mass, whereas decreased leptin sensitivity results in elevated adipose tissue stores. To address whether the effects of chronic hyperleptinemia are sustained with age, we generated transgenic mice that overexpress leptin under the control of the fat specific aP2 promoter/enhancer. At 6-9 weeks of age, transgenic mice overexpressed 5-fold more human leptin than endogenous mouse levels and had consistently low body weights, with reduced brown and white fat depots characterized by adipocytes either devoid of or containing minute lipid droplets. However, at 33-37 weeks, despite continuous secretion of human leptin, the transgenic mice showed a rebound effect characterized by an increase in body weight, accumulation of adipose mass, and lipid-filled adipocytes. Thus, this mouse model exhibits a two-stage phenotype, with respect to fat accumulation. In addition, plasma glucose, triglycerides, and cholesterol levels were markedly depressed in young, but not older, transgenic mice. A detrimental consequence of early hyperleptinemia was a failure of the transgenic mice to acclimatize to the cold, as a result of depleted fat stores within their brown adipocytes. Cold exposure was tolerated after a 2-week high-fat diet or at an older age when fat depots had naturally accumulated. Treatment of the older transgenic mice with large doses of leptin stimulated weight loss, demonstrating that the leptin pathway still responds to pharmacological levels of leptin. Overall, these studies show that moderate hyperleptinemia in normal mice results in a sensitivity of the adipose mass to leptin at a younger (but not older) age. The mechanisms that lead to the accumulation of fat at an older age remain largely unknown, and this hyperleptinemic mouse model will allow the uncovering of at least some of these mechanisms.

Leptin, an adipocyte-derived hormone, informs neuroendocrine pathways about the status of energy stores in adipose tissue. The integration of this peripheral signal in hypothalamic networks results in activation of peripheral metabolic pathways that control energy build-up and expenditure. Firing of the reproductive cascade of hormones at puberty and its regulation in adults is tightly associated with energy metabolism and is thus regulated by leptin. This article provides an update of past and present theories that link nutrition and reproduction in light of new research.

Obesity is often associated with an impairment of the hypothalamic-pituitary-gonadal axis. The leptin-deficient ob/ob mouse model is characterized by a morbid obesity with a sterility in males and females that is corrected by continuous leptin treatment. Since ob/ob mice are maintained on the C57BL/6J inbred genetic background, we sought to determine whether their infertility can be corrected without leptin treatment but via the effect of modifier genes brought into the obese-sterile phenotype by a different genetic background. Thus, we generated via an F2 intercross ob/ob mice on a mixed C57BL/6J-BALB/cJ genetic background and assayed them for fertility by mating with wild-type C57BL/6J mice. Whereas genetically heterogeneous F2 obese females remained sterile like male and female C57BL/6J ob/ob mice, 41% of F2 C57BL/6J-BALB/cJ obese males were capable of reproducing despite a morbidly obese state. Therefore, the sterility of the original C57BL/6J ob/ob mouse model was genetically corrected independently of its obese state via the effects of modifier genes. Unlike testosterone levels, triglyceride levels, and testes weight-to-body weight ratios, which were all higher in fertile vs. sterile mice, glucose levels were similar in both groups, indicating that the underlying hyperglycemia of ob/ob mice was not an impediment to the onset of fertility. A genome-wide scan in F2 ob/ob males resulted in the localization of four modifier loci on chromosomes 1, 3, 5, and 14 with respective quantitative traits consisting of number of pregnancies, testes weights normalized to body weights, body weight at 8 weeks of age, and circulating testosterone. We conclude that the inheritance of modifier genes at the identified loci acts to promote fertility of otherwise sterile leptin-deficient obese male mice.

Leptin levels are significantly elevated in pregnant mice, rats and humans suggesting a critical role for leptin during gestation. To address whether leptin plays a putative role in the physiology of pregnancy, we asked whether a mouse pregnancy would be affected by the complete absence of leptin from both the mother and fetuses. Thus, leptin-deficient ob/ob females were first treated with exogenous leptin and then mated to similarly treated ob/ob males. All resulting fetuses have an ob/ob genotype and lack like their mothers any endogenous leptin production. Withdrawal of leptin treatment at 0.5, 6.5, 10.5 and 19.5 days p.c. did not affect any stage of the pregnancy despite a gradual return of the mothers to an obese state. However, some mice had delayed gestation periods of 21-23 days which were associated with prolonged parturition. The pups were normally delivered with no obvious signs of deformities although none survived beyond a day after delivery due to failure of lactation. Monitoring daily food intake of pregnant ob/ob females treated throughout gestation with leptin revealed significantly elevated levels of food intake from day 10 p.c. and onward demonstrating an attenuation of a leptin response during pregnancy and a leptin resistance effect. These studies demonstrate that in the mouse, leptin is not a critical molecule for implantation, gestation, fetal growth and parturition but that the leptin resistance effect at mid-gestation aims to stimulate food intake thus providing sustained energy resources for pregnancy.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is highly conserved within vertebrate species. Its pattern of expression in vivo seems to be tightly regulated both developmentally and in a tissue-specific manner, but shows differences with species. To identify transcriptional regulatory elements in the CFTR promoter region, we have used a combined approach based both on the analysis of the chromatin structure in vivo in rat tissues and on evolutionary clues (i.e. phylogenetic footprinting). In CFTR-expressing tissues, 15 DNase I-hypersensitive sites were identified within a 36 kb region encompassing exon 1. Eleven of them are clustered in a 3.5 kb region that exhibits eleven phylogenetic footprints observed when comparing sequences from eight mammalian species representing four orders (Primates, Artiodactylia, Lagomorpha and Rodentia). Comparison of the two sets of data allows the identification of two types of regulatory elements. Some are conserved between species, such as a non-consensus cAMP response element (CRE) and a PMA-responsive element (TRE) located respectively at positions -0.1 and -1.3 kb relative to ATG. Some are species-specific elements such as a 300 bp purine.pyrimidine (Pu.Py) stretch that is present only in rodents. Analysis of protein/DNA interactions in vitro with rat tissue protein extracts on the conserved elements revealed that the TRE site binds a specific heterodimeric complex composed of Fra-2, Jun D and a protein immunologically related to Jun/CRE-binding protein in the duodenum, whereas the CRE-like site binds ATF-1 ubiquitously. Functional analysis in Caco-2 cells showed that the CRE-like site supports a high basal transcriptional activity but is not able by itself to induce a response to cAMP, whereas the TRE site acts as a weak transactivator stimulated by PMA. Lastly, we found that the rodent-specific Pu.Py stretch confers nuclease S1 hypersensitivity under conditions of acidic pH and supercoiling. This indicates a non-B DNA conformation and thus reinforces the biological significance of non-random Pu.Py strand asymmetry in the regulation of transcription. Thus the tight transcriptional regulation of CFTR expression involves the combination of multiple regulatory elements that act in the chromatin environment in vivo. Some of them are conserved throughout evolution, such as the CRE-like element, which is clearly involved in the basal level of transcription; others are species-specific.

Thirteen patients with sickle cell anemia (SS) were found to have two alpha gene deletions with a presumptive genotype of beta(S)/beta(S); -alpha/-alpha. Hematological data showed that this group of patients had elevated Hb A2 level. In order to determine whether the elevation of Hb A2 is typical of SS with a two alpha gene deletion or is due to undiagnosed S-beta(O)-thalassemia with a two alpha gene deletion we looked for the presence or absence of beta(O)-thalassemia by molecular techniques. The latter included reverse dot-blot hybridization to rule out a beta-thalassemia mutation, digestion with CvnI endonuclease followed by Southern blotting and hybridization with a beta genomic probe, and, in selected patients, determination of the synthetic alpha/beta ratio. One of the 13 patients had S-beta(O)-thalassemia with a G-->A mutation at IVS-II-1 indicating that her genotype was beta(S)/beta(O) thalassemia; -alpha/-alpha. The remaining 12 patients were homozygous for the sickle gene, had relatively elevated Hb levels, increased Hb A2 values, and Hb F levels similar to those in patients with SS and four or three alpha genes. At the clinical level, the 12 patients with SS and a two alpha gene deletion had increased prevalence of avascular necrosis, retinopathy, and splenomegaly, but decreased prevalence of leg ulcers and cerebrovascular accidents. Together, the data indicate that SS with a two alpha gene deletion (beta(S)/beta(S); -alpha/-alpha) is a unique subset of patients with SS characterised by distinct hematological and clinical features.

The beta-thalassemia syndromes are a heterogeneous group of genetic disorders characterized by reduced or absent expression of the beta-globin gene. To date, over 300 beta-thalassemia alleles have been characterized in or around the beta-globin region. Thalassemia major is severe anemia necessitating chronic blood transfusions, splenectomy, iron chelation therapy, and bone marrow transplantation. Usually thalassemia major results from homozygosity or compound heterozygosity for severe betaO- and/or beta+-thalassemia mutations. Thalassemia intermedia is a clinical diagnosis that describes a symptomatic but less severe condition than beta-thalassemia major. beta-thalassemia intermedia may arise from several different combinations of alpha- and/or beta-thalassemia mutations. Heterozygous beta-thalassemia is typically characterized by a mild microcytic hypochromic anemia without any significant clinical implications. In this report, we describe a 63-year-old Africian American woman with asymptomatic homozygous beta-thalassemia, who seems to carry 2 copies of the -29 mutation in the promoter region of the beta-globin gene. Her elevated hemoglobin F level of 83% was associated with heterozygosity for the Xmn I polymorphism upstream of the Ggamma-globin gene. Southern blot analysis at the alpha-globin locus did not show any deletion that would account for the mildness of her phenotype. Therefore, homozygosity for the -29 mutation along with the Xmn I polymorphism appears to confer an extremely mild beta-thalassemia phenotype. This observation has important implications in the prenatal diagnosis and genetic counseling of families segregating this type of genetic defect.

Leptin, a hormone secreted from white adipose tissue, has been shown to normalize the body weight of ob/ob but not db/db mice as postulated by Coleman in his classical parabiosis experiments. The major effect of leptin is therefore to decrease food intake, thus resulting in a breakdown of fat stores. Recently, we have suggested that leptin plays a role in reproductive physiology based on the observation that leptin treatment but not food restriction rescues the sterility of ob/ob females. In the present communication, we treated sterile ob/ob males with leptin and asked whether fertility could be induced, thus selecting their reproductive ability as the endpoint of the experiment. Our results show that all food-restricted ob/ob males are unable to impregnate normal C57BL/6J females. However, all leptin-treated ob/ob males fertilized normal females mice that carried out normal pregnancies and deliveries, demonstrating that the reproductive capacity of ob/ob males was corrected only with leptin treatment. Furthermore, reproductive indices such as testicular weight and histology are normalized in leptin-treated animals. Therefore, as in ob/ob females, leptin plays a significant role in the male mouse reproductive pathways.

Numerous studies have revealed an association between nutritional status, adiposity, and reproductive maturity. The role of leptin, a hormone secreted from adipose tissue, in the onset of reproductive function was investigated. Normal prepubertal female mice injected with leptin grew at a slower rate than controls as a result of the hormone's thinning effects, but they reproduced up to 9 days earlier than controls and showed earlier maturation of the reproductive tract. These results suggest that leptin acts as a signal triggering puberty, thus supporting the hypothesis that fat accumulation enhances maturation of the reproductive tract.

The sterility of male and female homozygous ob/ob mice is a recognized feature of the ob mutation (1). Whereas ob/ob males can occasionally reproduce if maintained on a restricted diet, ob/ob females are always sterile (2). Thinning of the ob/ob females to normal weight by diet-restriction failed to correct their sterility. Early sexual development is normal in ob/ob females; however, ovulation never follows and the mice remain prepuberal indefinitely with no occurrence of oestrus cycles. Reproductive hormones are reduced in ob/ob females (3) demonstrating a functional defect from the hypothalamic-pituitary axis (4-6). The ovaries of ob/ob females are capable of producing viable eggs when transplanted into lean female recipients (7). Reconstitution of reproductive functions in the ob/ob female necessitates delivery of hypothalamic extracts to the third ventricle (8) and administration of pituitary extract (9), gonadotropic hormones (10), progesterone (11) and relaxin (12). These previous findings demonstrate that the sterility of ob/ob females is caused by an insufficiency of hormones at the hypothalamic-pituitary level rather than physical hindrance of copulatory activity, pregnancy and parturition caused by excess adipose tissue. We show here that repeated administration of only the recombinant human ob protein, leptin, into homozygous female ob/ob mice can correct their sterility, thus resulting in ovulation, pregnancy and parturition.

Objective: The purpose of this study was to determine whether viral deoxyribonucleic acid is detectable in the amniotic fluid of pregnancies at low risk for fetal viral infection., Study Design: Amniotic fluid samples were prospectively collected from 277 patients. Selected primer pairs amplified deoxyribonucleic acid sequences unique to adenovirus, cytomegalovirus, herpes simplex virus, and parvovirus. Amplified deoxyribonucleic acid was detected by gel electrophoresis. Sensitivity of the adenovirus, cytomegalovirus, and herpes virus primers were determined by serial dilution of 10(3) PFU/ml controls., Results: Of the 277 extracted samples, 243 had detectable deoxyribonucleic acid. None of these samples had detectable viral deoxyribonucleic acid by polymerase chain reaction. The sensitivity of the adenovirus primer pairs was 10(-3) PFU/ml, cytomegalovirus 10(-2) PFU/ml, and herpes simplex virus 10(-1) PFU/ml., Conclusion: This study did not detect viral deoxyribonucleic acid in a low-risk population, supporting the clinical significance of detecting viral deoxyribonucleic acid in pregnancies at risk for infection.

To determine whether a relationship exists between DNA methylation and CFTR gene expression, we investigated the methylation status of CpG sites in the mouse and human CFTR promoters. Tissues and previously characterized cell lines that vary with respect to CFTR expression were selected for analysis using the methylation sensitive restriction endonuclease Hha I. We find that CpG sites are not methylated in high and low CFTR-expressing cell lines, whereas in the very low or non-CFTR-expressing cell lines, the CpG sites are partially or completely methylated. However, none of these sites were methylated in any of the tissues examined irrespective of the state of CFTR expression. Therefore, we conclude that the CFTR promoter belongs to the class of CpG-rich promoters in which the associated CpG sites are not methylated in tissues and that an inverse correlation between methylation and CFTR expression can only be found in cell lines.

We devised a set of allele-specific probes to detect simultaneously 31 known cystic fibrosis mutations using PCR and the reverse dot blot detection format. The assay has been implemented in a clinical setting to the screening of over 750 individuals. Of these 102 Caucasians, 20 Hispanics and 1 Indian patient were affected with cystic fibrosis. The mutation detection rate in the 204 Caucasian and 40 Hispanic CF chromosomes was respectively, 88% and 85%. The availability of the probe sequences to CF screening laboratories should allow implementation of this assay in a clinical setting and comparison of its mutation typing rate among different centers.

Uniparental isodisomy resulting from the simultaneous presence of isochromosomes of the p and q arms of a chromosome and absence of a normal homologue is an exceptionally rare event. We have observed a growth-retarded female infant in whom the normal chromosome 7 homologues were replaced by what appeared cytogenetically to be isochromosomes of 7p and 7q. Polymorphic microsatellite loci spanning the length of 7p and 7q were analyzed in the proband and her parents to ascertain the parental origin and extent of heterozygosity of the proband's rearranged chromosomes. These studies demonstrated that the 7p alleles of the proband were derived only from the father, the 7q alleles were derived only from the mother, and there was homozygosity for all chromosome 7 loci analyzed. The mechanisms leading to the formation of the proband's isochromosomes could reflect abnormalities of cell division occurring at meiosis, postfertilization mitosis, or both. We believe that the present case may result from incomplete mitotic interchange in the pericentromeric regions of chromosome 7 homologues, with resolution by sister-chromatid reunion in an early, if not first, zygotic division. Phenotypically, our proband resembled three previously reported cases of maternal isodisomy for chromosome 7, suggesting that lack of paternal genes from 7q may result in a phenotype of short stature and growth retardation.

To gain insights into the regulation of the mouse and rat cystic fibrosis transmembrane conductance regulator (CFTR) genes, we cloned and sequenced their respective upstream promoter regions. DNA sequence analysis from either side of exon 1 revealed a complete divergence from the human DNA sequence except for three DNA motifs which consist of a 34 bp stretch and two intron specific elements. Highlights of both the rat and mouse promoter sequences include an extensive purine rich stretch, a Y-box motif and putative Sp1, AP1 sites. Transfection of mouse promoter deletional constructs into expressing and non-expressing CFTR murine cell lines revealed that the Pu.Py stretch and the Y-box act, respectively, as negative and positive elements of basal transcription that confer no apparent tissue specificity.

We have optimized a battery of reverse dot blot oligonucleotide probes for detecting the most common beta-globin gene mutations in Asians and American Blacks. These probes allow a high degree of coverage of mutant chromosomes in these two populations and are useful in mutation screening and prenatal diagnosis. When coupled with the Mediterranean subset of probes, a 95% worldwide coverage of beta-thalassemia mutations should be possible. The use of these probes in the reverse dot blot system should allow the distribution of premade strips and application to automated screening.

Molecular diagnostics (MDx) is currently a clinical reality that has its roots deep in the study of gene function, structure, and regulation. The multitude of human mutations identified in the various genetic diseases can now be assayed in the clinical molecular diagnostics laboratory. The polymerase chain reaction (PCR) has facilitated the transition from the research to the clinical laboratory, however, many methods which scan and identify known mutations may not be applicable in a clinical environment. A few of these methods are discussed and one technology that is well suited for clinical use is suggested. Well-trained personnel, regulation of MDx laboratories, and automation are a few of the requirements that will carry us into the promising future of molecular diagnosis.