Newborn screening for lipid disorders.

PURPOSE OF REVIEW: Newborn screening is one of the most successful public health programs of the last century and offers unparalleled access to universal screening for a variety of metabolic and other disorders. Interest in development of newborn screening for lipid disorders has intensified in recent years. Screening newborns for lipid disorders has important implications for the health of the newborn as well as their relatives, and in the case of more common lipid disorders like familial hypercholesterolemia, could have important public health implications. RECENT FINDINGS: Recent studies have demonstrated feasibility of measuring biomarkers for heterozygous familial hypercholesterolemia from newborn screening dried blood spot specimens. Another lipid disorder, cerebrotendinous xanthomatosis, is currently amenable to newborn screening utilizing currently available assays. New research in next-generation sequencing as a primary screen in newborns will also identify both common and rare lipid disorders in newborns. SUMMARY: Historically, newborn screening for lipid disorders was not done for many reasons, but new research has developed testing methods that may successfully identify common and rare lipid disorders. This will impact the health of the newborn but could also impact family members and public health.

Familial Hypercholesterolemia Biomarker Distribution in Dried Blood Spots.

OBJECTIVE: To evaluate distribution profiles of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and apolipoprotein B (apoB) as candidate markers of familial hypercholesterolemia in newborns, taking into consideration potential confounding factors, such as gestational age, birth weight, sex, and race. STUDY DESIGN: TC, LDL-C, and apoB were measured from 10 000 residual deidentified newborn dried blood spot cards. Concentrations for each biomarker were reported as multiples of the median, with emphasis on describing the 99th percentile values based on birth weight, gestational age, sex, and race. Seasonal variation of biomarkers was also explored. RESULTS: LDL-C and apoB had distribution curves with tails showing extreme elevation, whereas the distribution of TC was less elevated and had the smallest range. Neonates born at early gestational age and low birth weight had significantly greater 99th percentile of multiples of the median values for apoB but not TC or LDL-C. Differences in biomarker concentration based on sex and race were minimal. All biomarkers showed greatest concentrations in the winter as compared with summer months. CONCLUSIONS: LDL-C and apoB had distribution curves supporting candidacy for neonatal familial hypercholesterolemia screening. Future studies are needed to correlate newborn screening results with molecular testing to validate these 2 biomarkers, along with measured cholesterol levels later in childhood.

C. elegans srGAP is an α-catenin M domain-binding protein that strengthens cadherin-dependent adhesion during morphogenesis.

The cadherin-catenin complex (CCC) is central to embryonic development and tissue repair, yet how CCC binding partners function alongside core CCC components remains poorly understood. Here, we establish a previously unappreciated role for an evolutionarily conserved protein, the slit-robo GTPase-activating protein SRGP-1/srGAP, in cadherin-dependent morphogenetic processes in the Caenorhabditis elegans embryo. SRGP-1 binds to the M domain of the core CCC component, HMP-1/α-catenin, via its C terminus. The SRGP-1 C terminus is sufficient to target it to adherens junctions, but only during later embryonic morphogenesis, when junctional tension is known to increase. Surprisingly, mutations that disrupt stabilizing salt bridges in the M domain block this recruitment. Loss of SRGP-1 leads to an increase in mobility and decrease of junctional HMP-1. In sensitized genetic backgrounds with weakened adherens junctions, loss of SRGP-1 leads to late embryonic failure. Rescue of these phenotypes requires the C terminus of SRGP-1 but also other domains of the protein. Taken together, these data establish a role for an srGAP in stabilizing and organizing the CCC during epithelial morphogenesis by binding to a partially closed conformation of α-catenin at junctions.

A "Null" Pattern of p16 Immunostaining in Endometrial Serous Carcinoma: An Under-recognized and Important Aberrant Staining Pattern.

The ability to distinguish endometrial serous carcinoma (SC) from high-grade endometrioid adenocarcinoma is of great importance given their differences in prognosis and management. In practice, this distinction typically relies upon the use of a focused immunohistochemical panel including p53, p16, and mismatch repair proteins. The expression of p16 is characteristically strong and diffuse in SCs, and weak and/or patchy in many high-grade endometrioid adenocarcinomas. Here, we report a subset of SCs that are entirely negative for p16 immunostaining, a pattern we refer to as "p16 null." This pattern was identified in 2 of 63 cases of SC diagnosed at our institution-1 with histologically classic features and 1 with ambiguous high-grade histologic features. These tumors otherwise showed a SC signature by immunohistochemical and demonstrated an SC pattern of genetic mutations. No mutation in the gene for p16, cyclin-dependent kinase inhibitor 2A (CDKN2A), was identified in either case. However, molecular correlates for the absent p16 expression were present, including homozygous deletion of CDKN2A in one case and hemizygous deletion of CDKN2A with promotor hypermethylation of the remaining allele in the other case. To our knowledge, this constitutes the first report conclusively demonstrating the existence of a small subset of SCs that are completely negative by p16 immunohistochemistry, and the molecular lesions responsible for this pattern. In the context of an otherwise clinically and histologically classic example of SC, we endorse this "null" p16 staining pattern as an alternative aberrant staining pattern that should not deter one from committing to this diagnosis.

The adhesion modulation domain of Caenorhabditis elegans α-catenin regulates actin binding during morphogenesis.

Maintaining tissue integrity during epidermal morphogenesis depends on α-catenin, which connects the cadherin complex to F-actin. We show that the adhesion modulation domain (AMD) of Caenorhabditis elegans HMP-1/α-catenin regulates its F-actin-binding activity and organization of junctional-proximal actin in vivo. Deleting the AMD increases F-actin binding in vitro and leads to excess actin recruitment to adherens junctions in vivo. Reducing actin binding through a compensatory mutation in the C-terminus leads to improved function. Based on the effects of phosphomimetic and nonphosphorylatable mutations, phosphorylation of S509, within the AMD, may regulate F-actin binding. Taken together, these data establish a novel role for the AMD in regulating the actin-binding ability of an α-catenin and its proper function during epithelial morphogenesis.

Cell-cell adhesion in metazoans relies on evolutionarily conserved features of the α-catenin·ß-catenin-binding interface.

Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and ß-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/ß-catenin is higher than that of α-catenin for ß-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalian α-catenin·ß-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40-100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans Our data provide novel insights into how cadherin-dependent cell-cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.

Structural and functional characterization of Caenorhabditis elegans α-catenin reveals constitutive binding to ß-catenin and F-actin.

Intercellular epithelial junctions formed by classical cadherins, ß-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the ß-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds ß-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

Phosphoregulation of the C. elegans cadherin-catenin complex.

Adherens junctions play key roles in mediating cell-cell contacts during tissue development. In Caenorhabditis elegans embryos, the cadherin-catenin complex (CCC), composed of the classical cadherin HMR-1 and members of three catenin families, HMP-1, HMP-2 and JAC-1, is necessary for normal blastomere adhesion, gastrulation, ventral enclosure of the epidermis and embryo elongation. Disruption of CCC assembly or function results in embryonic lethality. Previous work suggests that components of the CCC are subject to phosphorylation. However, the identity of phosphorylated residues in CCC components and their contributions to CCC stability and function in a living organism remain speculative. Using mass spectrometry, we systematically identify phosphorylated residues in the essential CCC subunits HMR-1, HMP-1 and HMP-2 in vivo. We demonstrate that HMR-1/cadherin phosphorylation occurs on three sites within its ß-catenin binding domain that each contributes to CCC assembly on lipid bilayers. In contrast, phosphorylation of HMP-2/ß-catenin inhibits its association with HMR-1/cadherin in vitro, suggesting a role in CCC disassembly. Although HMP-1/α-catenin is also phosphorylated in vivo, phosphomimetic mutations do not affect its ability to associate with other CCC components or interact with actin in vitro. Collectively, our findings support a model in which distinct phosphorylation events contribute to rapid CCC assembly and disassembly, both of which are essential for morphogenetic rearrangements during development.

Genome-wide analysis of microRNA and mRNA expression signatures in hydroxycamptothecin-resistant gastric cancer cells.

AIM: To investigate the involvement of microRNAs (miRNAs) in intrinsic drug resistance to hydroxycamptothecin (HCPT) of six gastric cancer cell lines (BGC-823, SGC-7901, MGC-803, HGC-27, NCI-N87, and AGS). METHODS: A sulforhodamine B (SRB) assay was used to analyze the sensitivity to HCPT of six gastric cancer cell lines. The miRNA and mRNA expression signatures in HCPT-resistant cell lines were then identified using DNA microarrays. Gene ontology and pathway analysis was conducted using GenMAPP2. A combined analysis was used to explore the relationship between the miRNAs and mRNAs. RESULTS: The sensitivity to HCPT was significantly different among the six cell lines. In the HCPT-resistant gastric cancer cells, the levels of 25 miRNAs were deregulated, including miR-196a, miR-200 family, miR-338, miR-126, miR-31, miR-98, let-7g, and miR-7. Their target genes were related to cancer development, progression and chemosensitivity. Moreover, 307 genes were differentially expressed in HCPT-resistant cell lines, including apoptosis-related genes (BAX, TIAL1), cell division-related genes (MCM2), cell adhesion- or migration-related genes (TIMP2, VSNL1) and checkpoint genes (RAD1). The combined analysis revealed 78 relation pairs between the miRNAs and mRNAs. CONCLUSION: Hierarchical clustering showed that the miRNA and mRNA signatures in our results were informative for discriminating cell lines with different sensitivities to HCPT. However, there was slightly lower correlation between the expression patterns of the miRNA and those of the predicted target transcripts.