Hb Waikato [α127(H10)Lys→Gln; HBA1: c.382A>C]: A Novel High Oxygen Affinity Variant.

We report the identification of a novel, high oxygen affinity hemoglobin (Hb) variant [α127(H10)Lys→Gln; HBA1: c.382A>C]. The variant was detected in an adolescent male (proband) of Syrian descent by cation exchange high performance liquid chromatography (HPLC), during Hb A1c analysis. A complete blood count (CBC) showed elevated red blood cells (RBCs) (6.08 × 1012/L), Hb (16.1 g/dL) and packed cell volume (PCV) (0.48 L/L). Capillary electrophoresis (CE) revealed the variant was more negatively charged and represented 18.2% of total Hb. Isopropanol stability was normal. Cyanosis in the subject prompted investigation of oxygen affinity, with a reduced p50 of 20.8 mm Hg and a left shifted oxygen dissociation curve demonstrating increased oxygen affinity. We propose the novel variant be named Hb Waikato, which reflects the Hospital Laboratory where the variant was discovered and region where the proband was born and herein describe characterization.

Hb Tacoma: G>T or G>C, and Does It Matter?

Hb Tacoma [ß30(B12)Arg→Ser] is a missense variant that is caused by either an AGG>AGT or AGG>AGC substitution at codon 30 of the HBB gene. Currently, the latter is classified as a rare cause of ß0-thalassemia (ß0-thal). We propose that HBB: c.93G>C has been incorrectly assigned as ß0-thal and discuss whether HBB: c.93G>T or HBB: c.93G>C should be classified as ß+-thal instead, or as ß-globin variants without thalassemic effect. We present several subjects who are heterozygous for Hb Tacoma, one with HBB: c.93G>T and two with HBB: c.93G>C, to support our conclusions.

Small mammal responses to long-term large-scale woodland creation: the influence of local and landscape-level attributes.

Habitat loss and fragmentation greatly affect biological diversity. Actions to counteract their negative effects include increasing the quality, amount and connectivity of seminatural habitats at the landscape scale. However, much of the scientific evidence underpinning landscape restoration comes from studies of habitat loss and fragmentation, and it is unclear whether the ecological principles derived from habitat removal investigations are applicable to habitat creation. In addition, the relative importance of local- (e.g., improving habitat quality) vs. landscape-level (e.g., increasing habitat connectivity) actions to restore species is largely unknown, partly because studying species responses over sufficiently large spatial and temporal scales is challenging. We studied small mammal responses to large-scale woodland creation spanning 150 yr, and assessed the influence of local- and landscape-level characteristics on three small mammal species of varying woodland affinity. Woodland specialists, generalists, and grassland specialists were present in woodlands across a range of ages from 10 to 160 yr, demonstrating that these species can quickly colonize newly created woodlands. However, we found evidence that woodlands become gradually better over time for some species. The responses of individual species corresponded to their habitat specificity. A grassland specialist (Microtus agrestis) was influenced only by landscape attributes; a woodland generalist (Apodemus sylvaticus) and specialist (Myodes glareolus) were primarily influenced by local habitat attributes, and partially by landscape characteristics. At the local scale, high structural heterogeneity, large amounts of deadwood, and a relatively open understory positively influenced woodland species (both generalists and specialists); livestock grazing had strong negative effects on woodland species abundance. Actions to enhance habitat quality at the patch scale focusing on these attributes would benefit these species. Woodland creation in agricultural landscapes is also likely to benefit larger mammals and birds of prey feeding on small mammals and increase ecosystem processes such as seed dispersal.

Novel α0-Thalassemia Deletion Identified in an Indian Infant with Hb H Disease.

We report the identification of a large deletion of the α-globin gene cluster, which removed both HBA2 and HBA1 and included the region from HBZ to HBQ1 on chromosome 16 (16p13.3). The α0-thalassemia (α0-thal) deletion was discovered in an Indian family residing in New Zealand. The proband was a 3-month-old female, who presented with a Hb H disease of unknown molecular origin. Routine hematology showed marked hypochromic microcytic anemia, with numerous Hb H inclusion bodies. In the absence of iron deficiency, there was a strong clinical suspicion of α-thal. On initial screening using a multiplex gap polymerase chain reaction (gap-PCR), only the common rightward deletion (-α3.7) was detected. Investigation of the proband's mother and father revealed the mother was heterozygous for the -α3.7 deletion, while none of the seven most common pathogenic α-thal deletions were detected in the father. Multiplex ligation-dependent probe amplification (MLPA) was employed to detect the presence of a novel α0-thal deletion in both the proband and her father. For the proband, the α0-thal deletion in combination with the -α3.7 deletion, eliminated three copies of HBA consistent with a clinical diagnosis of Hb H disease.

Conjugation of urate-derived electrophiles to proteins during normal metabolism and inflammation.

Urate is often viewed as an antioxidant. Here, we present an alternative perspective by showing that, when oxidized, urate propagates oxidative stress. Oxidation converts urate to the urate radical and the electrophilic products dehydrourate, 5-hydroxyisourate, and urate hydroperoxide, which eventually break down to allantoin. We investigated whether urate-derived electrophiles are intercepted by nucleophilic amino acid residues to form stable adducts on proteins. When urate was oxidized in the presence of various peptides and proteins, two adducts derived from urate (Mr 167 Da) were detected and had mass additions of 140 and 166 Da, occurring mainly on lysine residues and N-terminal amines. The adduct with a 140-Da mass addition was detected more frequently and was stable. Dehydrourate (Mr 166 Da) also formed transient adducts with cysteine residues. Urate-derived adducts were detected on human serum albumin in plasma of healthy donors. Basal adduct levels increased when neutrophils were added to plasma and stimulated, and relied on the NADPH oxidase, myeloperoxidase, hydrogen peroxide, and superoxide. Adducts of oxidized urate on serum albumin were elevated in plasma and synovial fluid from individuals with gout and rheumatoid arthritis. We propose that rather than acting as an antioxidant, urate's conversion to electrophiles contributes to oxidative stress. The addition of urate-derived electrophiles to nucleophilic amino acid residues, a process we call oxidative uratylation, will leave a footprint on proteins that could alter their function when critical sites are modified.

Acetabular Revision Using Trabecular Metal Augments for Paprosky Type 3 Defects.

BACKGROUND: Trabecular Metal (TM) augments are one option when reconstructing bone loss during acetabular side revision surgery. METHODS: We studied 38 consecutive patients with Paprosky type 3 defects that were revised using a TM shell and one or more augments over a 6-year period. There were 29 Paprosky type 3A defects and 9 Paprosky type 3B defects. The mean age of the patients at the time of surgery was 68.2 years (range 48-84). The mean length of follow-up was 36 months (range 18-74). RESULTS: The mean preoperative short form 12 health survey improved from 27.7 before operation to 30.1 at the time of final follow-up (P = .001). The mean Western Ontario and McMaster Universities Osteoarthritis Index score improved from 53 preoperatively to a mean of 78.8 at final follow-up (P < .0001). There was evidence of radiographic loosening in 7 of the cup-augment constructs. One patient developed a deep infection requiring re-revision. Two patients required revision for aseptic loosening. CONCLUSION: The use of TM in complex acetabular reconstruction is associated with good outcome in the short to medium term.

Hb Amsterdam-A1 [α32(B13)Met→Ile; HBA1: c.99G>A]: A Hyperunstable Variant Due to a New Mutation on the α1 Gene.

Patients with hyperunstable α chain variants usually present with a thalassemic, rather than hemolytic, phenotype. Electrophoretic, ion exchange and reverse phase separations usually fail to detect the variant and when DNA sequencing identifies a 'silent' substitution it is usually presumed to be hyperunstable. We report the identification of such a variant, α32(B13)Met→Ile; HBA1: c.99G>A, arising from a new mutation on the α1 gene. The hemoglobin (Hb) was unequivocally detected by the isopropanol stability test and confirmed as hyperunstable by mass spectrometry (MS) of the precipitate and lysate, which showed proportions of 55% and 2.5% of α chains, respectively. The instability appears to be driven by perturbation of globin-heme, and possibly α1ß1 subunit, interactions.

Hb Ashburton [ß12(A9)Thr → Pro; HBB: c.37A > C], a novel, mildly unstable variant and the first substitution identified at codon 12.

We report the identification of a novel, slightly unstable hemoglobin (Hb) variant [ß12(A9)Thr → Pro; HBB: c.37A > C] that came to our attention during Hb A1C ion exchange chromatography where it migrated as a trailing shoulder on the Hb A0 peak. On electrospray ionization mass spectrometry (ESI MS), this electrophoretically silent variant was detected as an unresolved ß component with a 2 Da decrease in average ß chain mass. Reversed phase high performance liquid chromatography (HPLC) confirmed the presence of a more hydrophilic ß chain with a mass 4 Da less than normal ß(A) and showed it represented 40.0% of the total ß-globin. Tryptic mapping revealed the [M + 1H] ion of peptide ßT-2 had shifted from 932.5 to 928.5 m/z, suggesting a point mutation of Thr → Pro at position ß12(A9). This substitution was confirmed by fragmentation analysis of the [M + 2H] ion (464.8 m/z) of the new ßT-2 peptide and it represents a novel mutation which we have named Hb Ashburton.

A novel mutation in the FGB: c.1105C>T turns the codon for amino acid Bß Q339 into a stop codon causing hypofibrinogenemia.

Routine coagulation tests on a 14year-old male with frequent epistaxis showed a prolonged thrombin time together with diminished functional (162mg/dl) and gravimetric (122mg/dl) fibrinogen concentrations. His father showed similar aberrant results and sequencing of the three fibrinogen genes revealed a novel heterozygous nonsense mutation in the FGB gene c.1105C>T, which converts the codon for residue Bß 339Q to stop, causing deletion of Bß chain residues 339-461. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC (reverse-phase high-pressure liquid chromatography) of purified fibrinogen showed only normal Aα, Bß, and γ chains, indicating that molecules with the truncated 37,990Da ß chain were not secreted into plasma. Functional analysis showed impaired fibrin polymerization, fibrin porosity, and elasticity compared to controls. By laser scanning confocal microscopy the patient's fibers were slightly thinner than normal. Electrospray ionization mass spectrometry (ESI MS) presented normal sialylation of the oligosaccharide chains, and liver function tests showed no evidence of liver dysfunction that might explain the functional abnormalities.

Hb Papanui [α99(G8)Lys→Arg; HBA2: c.299A>G]: a novel silent substitution interfering in Hb A1c determination.

We report a novel hemoglobin (Hb) mutation [Hb Papanui [α99(G8)Lys→Arg; HBA2: c.299A>G] that was identified in an individual with an inappropriately low Hb A1c value of 2.8% when using an ion exchange method. Although occurring at a well conserved site, the conservative substitution was not associated with any adverse clinical features.

Novel α2 gene deletion (c.349_359 del GAGTTCACCCC) identified in association with the -α3.7 deletion.

We report a novel α2 gene mutation identified in compound heterozygosity with the common -α(3.7) deletion. The mutation (c.349_359 del GAGTTCACCCC), causes a frameshift after codon α115 with the predicted extension of the αchain from 141 to 166 residues. The new polypeptide would be expected to have a mass of 17,463 Da, but no such product was detected in hemolysates, confirming the mutation's thalassemic phenotype. Mechanistically, the deletion is probably caused by replication slippage during DNA synthesis as the sequence is bracketed by a CC repeat. The lack of protein expression is probably caused by loss of critical binding sites to the chaperone, α Hb stabilizing protein (AHSP).

Influence of weather variables on pain severity in end-stage osteoarthritis.

PURPOSE: Patients often attribute increasing pain in an arthritic joint to changing weather patterns. Studies examining the impact of weather on pain severity have yielded equivocal and sometimes contradictory results. The relationship between subchondral pseudocysts and the role they play in this phenomenon has not been explored. METHODS: Fifty-three patients with end-stage osteoarthritis of the hip completed daily pain severity visual analogue scale (VAS) scores over a one month period. Radiographs were reviewed to determine the presence of pseudocysts. Data pertaining to precipitation, atmospheric pressure and temperature were collected from the nearest weather station. A generalised linear mixed model was used to explore the relationship between weather variables, cysts and pain severity. RESULTS: Pain levels increased as a function of absolute change in atmospheric pressure from one day to the next. Precipitation, temperature and the presence of subchondral pseudocysts were not shown to influence pain severity. CONCLUSIONS: This data supports the belief held by many osteoarthritic patients that changing weather patterns influence their pain severity.

Talonavicular synostosis with lateral ankle instability--A case report and review of the literature.

Talonavicular coalition is a rare autosomal recessive congenital anomaly that is usually asymptomatic and detected incidentally on radiographs. It is associated with symphalangism, clinodactyly, a great toe that is shorter than the second toe, clubfoot, calcaneonavicular coalition, talocalcaneal coalition and a ball-and-socket ankle joint. The authors present a review of the literature and case report of a patient with complete osseous talonavicular coalition, talocalcaneal coalition and lateral ankle instability which was successfully treated with subtalar fusion and lateral ligament reconstruction.

Left ventricular pseudoaneurysm: an inadvertent consequence of COVID-19-a case report.

BACKGROUND: Left ventricular pseudoaneurysm (LVP) is an uncommon but serious mechanical complication of acute myocardial infarction (AMI). The immediate medical complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are well recognized, but its indirect effect on patients and healthcare systems is potentially less perceivable. CASE SUMMARY: In this report, a 72-year-old man who was anxious about attending hospital during the SARS-CoV-2 pandemic was eventually found to have a total right coronary artery occlusion after a delayed emergency department presentation. He ultimately developed severe symptomatic heart failure and cardiac magnetic resonance imaging (CMR) revealed that a large LVP with concomitant severe ischaemic mitral regurgitation had evolved from his infarct. The patient was successfully discharged home after the surgical replacement of his mitral valve and repair of his LVP. DISCUSSION: This case highlights a salient downstream effect of Coronavirus disease 2019 (COVID-19): the delay in presentation, diagnosis, and management of common treatable conditions such as AMI. It also underscores the importance of non-invasive multimodal imaging on the timely identification of the mechanical complications of AMI. In particular, CMR can play a crucial role in the characterization and management of LVP.

Expression of four mutant fibrinogen gammaC domains in Pichia pastoris confirms them as causes of hypofibrinogenaemia.

Mutations in the fibrinogen gene cluster can cause low plasma fibrinogen concentrations, known as hypofibrinogenaemia. It is important to verify whether a detected sequence variant in this cluster is deleterious or benign and this can be accomplished using protein expression systems. In this study, four mutations in the fibrinogen gammaC domain that had previously been described in patients with hypofibrinogenaemia were introduced into a gammaC construct and expressed in a Pichia pastoris yeast system to investigate their effects on protein stability and secretion. These experiments showed that the fibrinogen Middlemore (N230D), Dorfen (A289V), Mannheim II (H307Y), and Muncie (T371I) mutations were not secreted, supporting their causative role in hypofibrinogenaemia. Overexpression of the N230D, A289V and H307Y mutants revealed that the majority of the synthesised protein was retained in the endoplasmic reticulum, with only a minor proportion reaching the trans-Golgi network. Regardless, none of this protein was secreted which confirms that the four mutations investigated are indeed responsible for hypofibrinogenaemia.

Novel fibrinogen mutation γ314Thr→Pro (fibrinogen AI duPont) associated with hepatic fibrinogen storage disease and hypofibrinogenaemia.

Mutation in fibrinogen genes may lead to quantitative or qualitative disorders that result in bleeding, thrombosis or hepatic fibrinogen storage disease. Only three mutations in the fibrinogen γ gene have been identified that cause hepatic endoplasmic reticulum storage of mutant fibrinogen. To investigate the possibility of hepatic fibrinogen storage disease in a 4-year-old male with persistently elevated serum aminotransferases and preserved synthetic function except for a prolonged INR. After informed consent, liver and blood samples were obtained. Liver sections were examined by light microscopy, anti-fibrinogen immunolabelling and electron microscopy. Purified fibrinogen was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography; DNA sequencing was performed using a BigDye Terminator (v. 3.1) cycle sequencing kit. Four-year-old male with persistently elevated transaminases with an INR 1.5 but otherwise normal synthetic function. Fibrinogen activity and thrombin clotting time were abnormal at 0.47 g/L and 46 s respectively. Hepatic histological examination revealed portal inflammatory infiltrates with bridging fibrosis. Clumped eosinophilic material was observed in hepatocytes that was immunoreactive to fibrinogen antisera. Ultrastructural examination showed cytoplasmic inclusions arrayed in fingerprint-like patterns. DNA sequence analysis revealed heterozygosity for a novel γ314Thr →Pro mutation (fibrinogen AI duPont) in the fibrinogen γ gene. Protein analyses showed normal patterns of Aα, Bß and γ chains suggesting that the variant γ allele was not expressed in plasma fibrinogen. We describe only the fourth mutation to be identified, γ314Thr→Pro (fibrinogen AI duPont), giving rise to hypofibrinogenaemia and hepatic fibrinogen storage disease.

Imaging of soft tissues adjacent to orthopedic hardware: comparison of 3-T and 1.5-T MRI.

OBJECTIVE: The purpose of this study was to compare metal artifact reduction techniques at 1.5-T and 3-T MRI. MATERIALS AND METHODS: A titanium plate with steel screws was placed in a freshly harvested pig leg. The leg was imaged with 1.5-T and 3-T MRI. A T2-weighted turbo spin-echo sequence was used with echo-train lengths of 8, 16, 32, and 64 and a constant readout bandwidth of 31.2 kHz. The images were compared qualitatively, and the optimal echo-train length was selected. Images were acquired at the optimal echo-train length with four different readout bandwidths. Artifact was measured quantitatively, and image quality was ranked qualitatively. The qualitatively best image acquired at 1.5 T was compared with the qualitatively highest-ranked image acquired at 3 T. RESULTS: At both 1.5 T and 3 T, optimal images of equal quality were produced at echo-train lengths of 8 and 16. At higher readout bandwidths, there was quantitatively less artifact. The qualitatively best images were acquired at a readout bandwidth of 31.2 kHz at 1.5 T and 62.5 kHz at 3 T (Cronbach's alpha=1.00). The optimal image at 3 T was qualitatively superior to that at 1.5 T. CONCLUSION: Optimizing image acquisition parameters in this phantom model resulted in similar quantitative susceptibility artifact at 3 T and 1.5 T and better qualitative images at 3 T than at 1.5 T.

Hb Perpignan [beta136(H14)Gly-->Ser], a silent variant associated with normal hematology.

A second case of Hb Perpignan [beta136(H14)Gly-->Ser] was identified in a Burmese woman living in New Zealand. Although previously detected in France, this is the first formal description of the variant, which is electrophoretically silent and hematologically normal. This presentation as a benign substitution is in keeping with the low level of phylogenetic conservation of the H14 glycine.