Supernumerary marker chromosomes (SMCs) in Turner syndrome are mostly derived from the Y chromosome

DNA and FISH (fluorescence in situ hybridization) analysis were carried out in 12 patients with stigmata of Turner syndrome to determine whether the Supernumerary M arker C hromosome (SMC) found cytogenetically in each of these patients was derived from the Y chromosome. The presence of a Y chromosome in these patients may predispose them to develop gonadoblastoma. PCR-Southern blot analysis, followed by FISH, was used to detect the presence of Y chromosome material. The S ex determining R egion Y (SRY), T estis S pecific P rotein Y -encoded (TSPY) and Y -chromosome R NA R ecognition M otif (YRRM) genes, which map at Yp11.31, Yp11.1–11.2 and Yp11.2/Yq11.21–11.23, respectively, were selected as markers, because they span the whole Y chromosome, and more importantly, they are considered to be involved in the development of gonadoblastoma. It was shown that in 12 patients, all of whom had an SMC, the SMC of 11 was derived from the Y chromosome. Furthermore, the presence of the SRY, TSPY and YRRM gene sequences was determined and FISH analysis confirmed the Y origin of the SMCs. The methodology described in this report is a rapid, reliable and sensitive approach which may be easily applied to determine the Y origin of an SMC carried in Turner syndrome. The identification of an SMC is important for the clinical management and prognostic counseling of these patients with Turner syndrome.     

Detection and incidence of cryptic Y chromosome sequences in Turner syndrome patients

The presence of Y chromosome sequences in Turner syndrome (TS) patients may predispose them to gonadoblastoma formation with an estimated risk of 15–25%. The aim of this study was to determine the presence and the incidence of cryptic Y chromosome material in the genome of TS patients. The methodology involved a combination of polymerase chain reaction (PCR) and nested PCR followed by Southern blot analysis of three genes—the sex determining region Y (SRY), testis specific protein Y encoded (TSPY) and RNA binding motif protein (RBM) (previously designated as YRRM) and nine additional STSs spanning all seven intervals of the Y chromosome. The methodology has a high sensitivity as it detects one 46, XY cell among 10⁵ 46, XX cells. Reliability was ensured by taking several precautions to avoid false positive results. We report the results of screening 50 TS patients and the identification of cryptic Y chromosome material in 12 (24%) of them. Karyotypes were divided in four groups: 5 (23.8%) patients out of the 21 TS patients which have the 45, X karyotype (group A) also have cryptic Y sequences; none (0%) of the 7 patients who have karyotypes with anomalies on one of the X chromosomes have Y mosaicism (group B); 1 (6.3%) of the 16 patients with a mosaic karyotype have Y material (group C); and 6 (100%) out of 6 patients with a supernumerary marker chromosome (SMC) have Y chromosome sequences (group D). Nine of the 12 patients positive for cryptic Y material were recalled for a repeat study. Following new DNA extraction, molecular analysis was repeated and, in conjunction with fluorescent in situ hybridization (FISH) analysis using the Y centromeric specific probe Yc-2, confirmed the initial positive DNA findings. This study used a reliable and sensitive methodology to identify the presence of Y chromosome material in TS patients thus providing not only a better estimate of a patient's risk in developing either gonadoblastoma or another form of gonadal tumor but also the overall incidence of cryptic Y mosaicism.     

Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination

The epithelial Na+ channel (ENaC) is comprised of three subunits, alpha, beta and gamma, and plays an essential role in Na+ and fluid absorption in the kidney, colon and lung. We had identified proline-rich sequences at the C termini of alpha beta gamma ENaC, which include the sequence PPxY, the PY motif. This sequence in beta or gamma ENaC is deleted or mutated in Liddle's syndrome, a hereditary form of arterial hypertension. Our previous work demonstrated that these PY motifs bind to the WW domains of Nedd4, a ubiquitin protein ligase containing a C2 domain, three or four WW domains and a ubiquitin protein ligase Hect domain. Accordingly, we have recently demonstrated that Nedd4 regulates ENaC function by controlling the number of channels at the cell surface, that this regulation is impaired in ENaC bearing Liddle's syndrome mutations, and that ENaC stability and function are regulated by ubiquitination. The C2 domain is responsible for localizing Nedd4 to the plasma membrane in a Ca(2+)-dependent manner, and in polarized epithelial MDCK cells this localization is primarily apical. In accordance, electrophysiological characterization of ENaC expressed in MDCK cells revealed inhibition of channel activity by elevated intracellular Ca2+ levels. Thus, in response to Ca2+, Nedd4 may be mobilized to the apical membrane via its C2 domain, where it binds ENaC via Nedd4-WW:ENaC-PY motifs' interactions, leading to ubiquitination of the channel by the Nedd4-Hect domain and subsequent channel endocytosis and lysosomal degradation. This process may be at least partially impaired in Liddle's syndrome due to reduced Nedd4 binding, leading to increased retention of ENaC at the cell surface.     

The strength of combined cytogenetic and mate-pair sequencing techniques illustrated by a germline chromothripsis rearrangement involving FOXP2

Next-generation mate-pair sequencing (MPS) has revealed that many constitutional complex chromosomal rearrangements (CCRs) are associated with local shattering of chromosomal regions (chromothripsis). Although MPS promises to identify the molecular basis of the abnormal phenotypes associated with many CCRs, none of the reported mate-pair sequenced complex rearrangements have been simultaneously studied with state-of-the art molecular cytogenetic techniques. Here, we studied chromothripsis-associated CCR involving chromosomes 2, 5 and 7, associated with global developmental and psychomotor delay and severe speech disorder. We identified three truncated genes: CDH12, DGKB and FOXP2, confirming the role of FOXP2 in severe speech disorder, and suggestive roles of CDH12 and/or DGKB for the global developmental and psychomotor delay. Our study confirmes the power of MPS for detecting breakpoints and truncated genes at near nucleotide resolution in chromothripsis. However, only by combining MPS data with conventional G-banding and extensive fluorescence in situ hybridizations could we delineate the precise structure of the derivative chromosomes.     

HNH proteins are a widespread component of phage DNA packaging machines

The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.Tailed bacteriophages and herpes viruses package their large double-stranded DNA genomes into a preformed protein shell, known as the “prohead,” using terminase enzymes. In both types of viruses, the genome is synthesized as concatemers composed of multiple copies of the genome joined end to end. This concatemeric DNA is packaged into the prohead and cleaved into genome-length units by terminase in an ATP-dependent reaction. Phage terminases are composed of two proteins: the large subunit harbors an endonuclease domain and an ATPase that powers the DNA packaging reaction, and the small subunit mediates specific DNA-binding required for recognition of packaging sites in the phage genome. A variety of elegant structural and biophysical studies have recently provided insight into the molecular mechanisms of terminase function (1, 2). However, the factors that affect the action of terminase enzymes in vivo have been less well characterized.Terminase enzymes perform several functions. They specifically recognize and bind the viral genome, interact with the prohead, then drive the DNA into the head through the narrow entry channel formed by the portal protein that is positioned at a single vertex of the head. During this process terminases also cleave the viral DNA, either nonspecifically upon head filling or at a specific site known as “cos.” The efficient packaging of a phage genome in vivo may require phage-encoded cofactors in addition to the terminase enzyme. For example, Escherichia coli phage λ gpFI facilitates interaction of the terminase–DNA complex with proheads (3–6). A wide variety of phages appear to encode proteins with a function similar to λ gpFI (7). Additionally, the activity of Bacillus subtilis phage phi29 terminase requires a phage-encoded RNA molecule bound to its portal protein (8), and in vivo packaging of the E. coli phage T4 genome can only be completed with the participation of the phage-encoded endonuclease, gp49 (9). The general prevalence and importance of terminase cofactors is difficult to evaluate because few studies have addressed this issue.We recently reported that phage genomes often encode proteins possessing an HNH motif near their terminase genes (10). The HNH motif is ∼35 aa long, and is characterized by the presence of two highly conserved His residues and one Asn residue. These HNH motifs, as defined by the large (∼7,400-member) HNH Pfam (11) protein sequence family (PF01844), are often found in proteins that possess endonuclease activity, such as site-specific homing endonucleases (12, 13), colicins (14, 15), S pyocins (16), and restriction enzymes (17–19). HNH motif-containing proteins comprised of primarily an HNH motif as found in E. coli colicins, usually possess nonspecific endonuclease activity. Conversely, HNH motif-containing proteins may contain DNA-recognition domains in addition to the HNH motif and thus possess high sequence specificity, as found in the homing endonucleases.The frequent juxtaposition of HNH and phage terminase genes (10, 20) suggests a unique role for HNH proteins in the endonuclease and/or packaging activities of the terminases. To address this issue, we investigated the function of E. coli phage HK97 gp74, a 119-residue protein containing an HNH motif. The gene encoding gp74 is located at the extreme 3′ end of the mature linear HK97 genome, adjacent to the cos site. In both the lysogen and replicative form of the HK97 genome gene 74 is immediately adjacent to genes 1 and 2, which encode the small and large subunits of terminase (TerS and TerL), respectively. Whereas gp74 was previously found to possess endonuclease activity (10), its role in the HK97 replication cycle remained uncharacterized. In this study we used functional and bioinformatic analyses to investigate its function.     

Prenatal diagnosis of trisomy 2 mosaicism: a case report

We report a case of trisomy 2 mosaicism detected upon amniocentesis in a woman with advanced maternal age. A mos 47,XY,+2(4)/46,XY(21) karyotype was revealed using standard GTG banding. There were no pathological sonographic findings and the fetal size was normal for gestational age at 16th week. The use of serial high-resolution ultrasound examination of the fetus to detect major abnormalities was offered as an option to the parents who, however, decided for termination of the pregnancy. Fetal autopsy did not reveal any malformations. Trisomy 2 mosaicism is associated with variable phenotypic abnormalities without a specific pattern, intrauterine growth restriction, fetal demise or stillbirth. The rarity of trisomy-2 mosaicism in prenatal diagnosis, as well as the increased risk of an abnormal outcome makes the diagnostic approach and genetic counseling difficult.     

Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters

ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K⁺ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.

ATP binding cassette (ABC) proteins are a large family of membrane proteins found in all kingdoms of life (1, 2). ABC proteins have a core structure composed of two transmembrane (TM) domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2) (Fig. 1A and SI Appendix, Fig. S1A) (3–5). Through ATP binding and hydrolysis at the NBDs, ABC proteins actively transport solutes across cell membranes, regulate activities of other proteins, or function as channels (1, 2). Thus, ABC proteins are involved in many biological processes, including lipid homeostasis, cellular metal trafficking, and antigen peptide transport. Mutations in human ABC proteins cause diseases such as Tangier disease, adenoleukodystrophy, cystic fibrosis, Dubin–Johnson syndrome, and pseudoxanthoma elasticum (PXE) (1, 2). Furthermore, the export of a wide range of cancer chemotherapeutics, antibiotics, and anti-fungal drugs by ABC transporters confers multidrug resistance to tumor cells, bacteria, and fungal pathogens, respectively (1, 2, 6, 7).Open in a separate windowFig. 1.Ycf1p structure. (A) Ycf1p domain arrangement. TMD, transmembrane domain; L0, L0 linker; NBD, nucleotide binding domain; and R, regulatory (R) region. The Pep4p proteolytic digestion site within the luminal loop 6 of TMD1 is denoted by a pink “*.” Phosphorylation sites in the L0 linker (S251) and R region (S908 and T911) are depicted with a “P” circled in red. (B) Cryo-EM density of Ycf1p with domains colored as in A. (C) Example of the atomic model for individual TM helices in TMD0 and the R region fit into the corresponding map densities. (D) Schematic ribbon diagram of Ycf1p colored as in A and B and with the proteolytic digestion site denoted by a pink “*.”Human ABC proteins are divided into seven subfamilies (A to G) based in part on the sequence of their NBDs and TMDs in the core ABC structure (1, 2). The C-subfamily is the most diverse and includes the cystic fibrosis transmembrane conductance regulator (CFTR), the sulphonylurea receptors that form regulatory subunits in ATP-sensitive K⁺ (K_ATP) channels, and the multidrug resistance proteins (MRPs). In addition to the ABC core, ABCC proteins contain an N-terminal extension that is either composed of an additional TM domain (TMD0) and L0 linker (Fig. 1A, orange and tan, respectively, and SI Appendix, Fig. S1A) or just an L0 tail (5, 8). A TMD0, but not L0 linker, is also found in some ABCB proteins (3, 5). These N-terminal extensions are involved in trafficking, endosomal recycling, protein interactions, and/or regulation of ABC activity (9–18). The existence of disease-causing mutations in TMD0 and the L0 linker of different ABCC proteins (8, 13, 18) indicates that these regions play important roles in protein function when present.High-resolution structural information for TMD0 is available only for the atypical ABCC protein SUR1 (19, 20), which is part of the large hetero-octameric K_ATP channel complex. In contrast, structures of monomeric ABC transporters showed only low-resolution density for TMD0 that was insufficient for building a full atomic model or lacked density for the domain altogether (14, 21–24). The vacuolar ABCC protein yeast cadmium factor 1 (Ycf1p) from Saccharomyces cerevisiae is a close homolog of human MRPs and a model for ABCC proteins that function as monomers. Ycf1p transports glutathione-conjugated heavy metals, such as Cd²⁺, from the cytosol into the vacuole, detoxifying the cell (25, 26). Human MRP1 can rescue Cd²⁺ transport activity in a YCF1 deletion strain (27).Like other ABCC proteins, Ycf1p contains a relatively long and mostly disordered linker that connects NBD1 and TMD2 (25, 28, 29) (Fig. 1A and SI Appendix, Fig. S1A). This linker contains stimulatory phosphorylation sites (25, 28), similar to the phospho-regulatory (R) region in the ABCC protein CFTR (30–32). Ycf1p also contains an inhibitory phosphorylation site in the L0 linker (33). However, how the R region interacts with the ABCC core and how its phosphorylation modulates protein function remain poorly understood for most ABCC proteins. Structural studies of Ycf1p presented here reveal how TMD0 and the R region exert their regulatory functions in MRP-like ABCC proteins.     

Patients' behavior at the time of injury: effect on nurses' perception of pain level and subsequent treatment.

This study examined whether nurses who currently provide direct patient care would perceive and treat pain differently in patients whose pain resulted from activities involving different levels of socially acceptable behavior. Clinical vignettes (differing only with respect to information provided about the patient's behavior at the time of injury) were randomly distributed to all medical/surgical, critical care, and emergency room nurses at this institution. Nurses were asked to record their perception of the patient's pain level and to indicate how much morphine should be given. Nurses (n = 167) did not differ significantly in their mean pain ratings (p =.86) or in the average amount of morphine (p =.80) given to the 2 patients. However, medical/surgical nurses indicated that they would give significantly less morphine to the patient who was presented as engaging in less socially acceptable behavior at the time of injury (p =.03). Overall, significantly more nurses correctly increased the next dose of morphine for the patient who was presented as having injured him or herself while participating in more socially acceptable behavior (p =.003). Thus, although the level of social acceptability of the patient's behavior at the time of injury did not significantly affect the average pain rating or morphine dose that the nurse would have reportedly given, there is evidence that the nurses would have been less aggressive in ensuring adequate pain treatment in the patient exhibiting less socially acceptable behavior.