Application Factors Associated With Clinical Performance During Pediatric Internship.

OBJECTIVE: Our goal was to identify aspects of residency applications predictive of subsequent performance during pediatric internship. METHODS: We conducted a retrospective cohort study of graduates of US medical schools who began pediatric internship in a large pediatric residency program in the summers of 2013 to 2017. The primary outcome was the weighted average of subjects' Accreditation Council for Graduate Medical Education pediatric Milestone scores at the end of pediatric internship. To determine factors independently associated with performance, we conducted multivariate linear mixed-effects models controlling for match year and Milestone grading committee as random effects and the following application factors as fixed effects: letter of recommendation strength, clerkship grades, medical school reputation, master's or PhD degrees, gender, US Medical Licensing Examination Step 1 score, Alpha Omega Alpha membership, private medical school, and interview score. RESULTS: Our study population included 195 interns. In multivariate analyses, the aspects of applications significantly associated with composite Milestone scores at the end of internship were letter of recommendation strength (estimate 0.09, 95% confidence intervals [CI]: 0.04, 0.15), numbers of clerkship honors (est. 0.05, 95% CI: 0.01-0.09), medical school ranking (est. 0.04, 95% CI: 0.08-0.01), having a master's degree (est. 0.19, 95% CI: 0.03-0.36), and not having a PhD (est. 0.14, 95% CI: 0.02-0.26). Overall, the final model explained 18% of the variance in milestone scoring. CONCLUSIONS: Letter of recommendation strength, clerkship grades, medical school ranking, and having obtained a Master's degree were significantly associated with higher clinical performance during pediatric internship.

Exome sequencing results in successful diagnosis and treatment of a severe congenital anemia.

Whole-exome sequencing is increasingly used for diagnosis and identification of appropriate therapies in patients. Here, we present the case of a 3-yr-old male with a lifelong and severe transfusion-dependent anemia of unclear etiology, despite an extensive clinical workup. Given the difficulty of making the diagnosis and the potential side effects from performing interventions in patients with a congenital anemia of unknown etiology, we opted to perform whole-exome sequencing on the patient and his parents. This resulted in the identification of homozygous loss-of-function mutations in the EPB41 gene, encoding erythrocyte protein band 4.1, which therefore causes a rare and severe form of hereditary elliptocytosis in the patient. Based on prior clinical experience in similar patients, a surgical splenectomy was performed that resulted in subsequent transfusion independence in the patient. This case illustrates how whole-exome sequencing can lead to accurate diagnoses (and exclusion of diagnoses where interventions, such as splenectomy, would be contraindicated), thereby resulting in appropriate and successful therapeutic intervention-a major goal of precision medicine.

Anatomy of the red cell membrane skeleton: unanswered questions.

The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S.

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.

The carboxyterminal EF domain of erythroid alpha-spectrin is necessary for optimal spectrin-actin binding.

Spectrin and protein 4.1R crosslink F-actin, forming the membrane skeleton. Actin and 4.1R bind to one end of ß-spectrin. The adjacent end of α-spectrin, called the EF domain, is calmodulin-like, with calcium-dependent and calcium-independent EF hands. The severely anemic sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF domain, implying that the domain is critical for skeletal integrity. To test this, we constructed a minispectrin heterodimer from the actin-binding domain, the EF domain, and 4 adjacent spectrin repeats in each chain. The minispectrin bound to F-actin in the presence of native human protein 4.1R. Formation of the spectrin-actin-4.1R complex was markedly attenuated when the minispectrin contained the shortened sph(1J) α-spectrin. The α-spectrin deletion did not interfere with spectrin heterodimer assembly or 4.1R binding but abolished the binary interaction between spectrin and F-actin. The data show that the α-spectrin EF domain greatly amplifies the function of the ß-spectrin actin-binding domain (ABD) in forming the spectrin-actin-4.1R complex. A model, based on the structure of α-actinin, suggests that the EF domain modulates the function of the ABD and that the C-terminal EF hands (EF(34)) may bind to the linker that connects the ABD to the first spectrin repeat.

Targeted deletion of betaIII spectrin impairs synaptogenesis and generates ataxic and seizure phenotypes.

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain betaIII spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in betaIII spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact betaIII spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3(-/-) animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluRdelta, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of alphaII spectrin. It appears that betaIII spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y.

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Protein 4.2 binds to the carboxyl-terminal EF-hands of erythroid alpha-spectrin in a calcium- and calmodulin-dependent manner.

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the alpha-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G(3) peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K(d) = 0.30 microM). EF-domains bearing the sph(1J) mutation are inactive. Binding of protein 4.2 to band 3 (K(d) = 0.45 microM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca(2+) (but not Mg(2+)) by three to five times. Calmodulin also binds to the EF-domain (K(d) = 17 microM), and Ca(2+)-calmodulin blocks Ca(2+)-dependent binding of protein 4.2 but not Ca(2+)-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate.

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.

Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency.

Defects in red blood cell (RBC) membrane skeleton components cause hereditary spherocytosis (HS). Clinically, HS varies significantly even among individuals with identical gene defects, illustrating the profound effects of genetic background on disease severity. We exploited a new spontaneous mouse model, wan, which arose on the inbred C3H/HeJ strain, to identify quantitative trait loci (QTL) that modify the HS phenotype. Homozygous wan mice have severe HS due to a complete deficiency of erythroid band 3. A QTL analysis of RBC count, hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed in wan/wan mice from an F2 intercross between C3H/HeJ(+/wan) and CAST/Ei(+/+) F1 hybrids. Hematologic and survival data from C3H, CAST/Ei F2 wan homozygotes support the hypothesis that genetic modifiers significantly influence the band-3 null HS phenotype. Significant QTL were identified for the MCV trait only, suggesting that RBC membrane characteristics are a target for modifier gene action. The most significant quantitative trait locus, Hsm1 (hereditary spherocytosis modifier 1), localizes to mouse Chromosome 12 and is dominant. The peak LOD score was obtained with a marker for Spnb1 encoding erythroid beta-spectrin, an obvious candidate gene.

Hereditary spherocytosis--defects in proteins that connect the membrane skeleton to the lipid bilayer.

The molecular causes of hereditary spherocytosis (HS) have been unraveled in the past decade. No frequent defect is found, and nearly every family has a unique mutation. In dominant HS, nonsense and frameshift mutations of ankyrin, band 3, and beta-spectrin predominate. Recessive HS is most often due to compound heterozygosity of defects in ankyrin, alpha-spectrin, or protein 4.2. Common combinations include a defect in the promoter or 5'-untranslated region of ankyrin paired with a missense mutation, a low expression allele of alpha-spectrin plus a missense mutation, and various mutations in the gene for protein 4.2. In most patients' red cells, no abnormal protein is present. Only rare missense mutations, like ankyrin Walsrode (V463I) or beta-spectrin Kissimmee (W202R), have given any insight into the functional domains of the respective proteins. Although the eminent role of the spleen in the premature hemolysis of red cells in HS is unquestioned, the molecular events that cause splenic conditioning of spherocytes are unclear. Electron micrographs show that small membrane vesicles are shed during the formation of spherocytes. Animal models give further insight into the pathogenetic consequences of membrane protein defects as well as the causes of the variability of disease severity.

Simultaneous (AC)n microsatellite polymorphism analysis and single-stranded conformation polymorphism screening is an efficient strategy for detecting ankyrin-1 mutations in dominant hereditary spherocytosis.

Nonsense/stop mutations in the ankyrin-1 gene (ANK1) are a major cause of dominant HS (dHS) (frequency of 23% in German dHS patients). To date, no common mutation has been found and therefore a simple mutation screening is not feasible. The reduced expression of one cDNA allele in the (AC)n microsatellite polymorphism of the ankyrin-1 gene, as seen in about 20% of Czech patients with dHS, may identify candidates with a possible frameshift/nonsense mutation. In order to verify the efficiency of this screening we screened the ankyrin-1 gene of 22 Czech dHS patients for both the reduced cDNA allele expression in the frequent (AC)n and the common exonic 26/39 polymorphisms, as well as for polymerase chain reaction (PCR) single-stranded conformation polymorphisms in any one of the 42 exons of ANK1. Anomalous PCR products were sequenced. We found seven new ANK1 frameshift/nonsense mutations in nine patients with, but in none of six patients without, a reduced cDNA allele expression (efficiency of 78%). We conclude that screening of dHS patients for such a reduced allele expression in common ANK1 polymorphisms is an efficient procedure for the identification of candidates for frameshift/nonsense mutations in the ankyrin-1 gene.

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.