[Recurrent hemolysis and iron overload of unclear origin]. / Rezidivierende Hämolysen und Eisenüberladung unklarer Genese.

The case of a 33-year-old male with recurrent icterus and hemolysis since childhood that was long mistaken for Gilbert disease is presented. Subsequently, the patient also developed splenomegaly and gallstones together with iron overload. Genetic testing revealed the diagnosis of hereditary xerocytosis, which is an erythrocyte membrane disorder causing recurrent hemolysis. Xerocytosis is often challenging to diagnose and the frequency of the condition might be underestimated as there are often no typical findings in the microscopic differential blood count, and Eosin-5-maleimide dye (EMA) test, which is used to diagnose other erythrocyte membrane disorders, is normal. In cases of splenomegaly, iron overload and recurrent hemolysis, or in the case of a clinical diagnosis of Gilbert disease together with one of the above-mentioned symptoms, further investigations and possibly also genetic testing should be considered.

Insertases scramble lipids: Molecular simulations of MTCH2.

Scramblases play a pivotal role in facilitating bidirectional lipid transport across cell membranes, thereby influencing lipid metabolism, membrane homeostasis, and cellular signaling. MTCH2, a mitochondrial outer membrane protein insertase, has a membrane-spanning hydrophilic groove resembling those that form the lipid transit pathway in known scramblases. Employing both coarse-grained and atomistic molecular dynamics simulations, we show that MTCH2 significantly reduces the free energy barrier for lipid movement along the groove and therefore can indeed function as a scramblase. Notably, the scrambling rate of MTCH2 in silico is similar to that of voltage-dependent anion channel (VDAC), a recently discovered scramblase of the outer mitochondrial membrane, suggesting a potential complementary physiological role for these mitochondrial proteins. Finally, our findings suggest that other insertases which possess a hydrophilic path across the membrane like MTCH2, can also function as scramblases.

Cobblestone-like brain malformation with a new bi-allelic ADGRG1 (GPR-56) mutation: Fetal imaging-pathology correlation.

BACKGROUND AND PURPOSE: Autosomal recessive cobblestone-like cortical malformation of the brain, with no eye or muscle involvement, has been reported in patients with biallelic mutations in ADGRG1 (formerly GPR56) and in other brain surface defects (eg, variants in COL3A1). We reported the intra-uterine brain MRI (iu-MRI), post-mortem MRI (pm-MRI), and neuropathology findings of a new ADGRG1 mutation in a fetus at early gestation. Imaging findings were compared with those of the sibling harboring the same mutation, to provide insights about the evolving morphology of such malformation. METHODS: A 21-week fetus underwent iu-MRI for a suspected cortical anomaly on ultrasound. After the MRI results, the termination of the pregnancy was carried out. A pm-MRI scan and autopsy were performed. A neuropathology-imaging correlation was achieved. The 5-year old sibling affected by developmental impairment also underwent a brain MRI. Both subjects underwent a genetic investigation. RESULTS: Two patterns of abnormality of the cerebral surface were identified on both fetal MRI: one at the vertex resembling a cobblestone-cortex due to neuronal overmigration into the subarchnoid space and the other in the occipital areas resembling polymicrogyria. These details closely matched the neuropathology findings. MRI findings of the sibling consisted of typical ADGRG1/GPR56-related brain findings showing a polymicrogyric-like cortex, also reported as bilateral frontal-parietal polymicrogyria. A flattened pons and small cerebellar vermis were present in both cases. Genetic testing demonstrated a novel homozygous variant c.1484T>C in the c gene in both cases. CONCLUSION: Our findings provide further evidence of the overlap of ADGRG1/GPR56-related brain dysgenesis with cobblestone-like cortical malformation of the brain.

Value of spectral domain-optical coherence tomography parameters on evaluating visual acuity improvement after internal limiting membrane peeling of macular hole / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To analyze the application value of spectral domain-optical coherence tomography(SD-OCT)parameters on evaluating visual acuity improvement after internal limiting membrane peeling of macular hole.METHODS: The retrospective analysis was performed on the clinical data of 82 patients(82 eyes)with idiopathic macular hole(IMH)who underwent vitrectomy + internal limiting membrane peeling + long-acting gas tamponade in the hospital between May 2019 and February 2021. The correlation between IMH closure and SD-OCT parameters at 3mo after surgery was analyzed, and the risk factors for poor postoperative visual acuity improvement were evaluated.RESULTS: Spearman rank correlation coefficient analysis showed that IMH closure at 3mo after operation was positively correlated with preoperative external limiting membrane(ELM)defect diameter(rs=0.308, P<0.05), and it was negatively correlated with preoperative macular hole index(MHI; rs=-0.266, P<0.05). Logistic regression analysis revealed that preoperative MHI≥0.5 was a protective factor affecting poor postoperative visual acuity improvement(OR=0.691, P<0.05).CONCLUSION: SD-OCT can predict the surgical efficacy by detecting the preoperative MHI and ELM defect diameter, and it is beneficial to judging the improvement of visual function.

Intraoperative Cell-Saver Caused More Autologous Salvage Hemolysis in a Hereditary Spherocytosis Patient Than in a Normal Erythrocyte Patient.

Hereditary spherocytosis is a common red blood cell disease caused by an inherited red blood cell membrane defect, leading to a spherical shape and propensity for hemolysis. There is a lack of reports on intraoperative autologous blood transfusion for hereditary spherocytosis patients. We hereby report our recent experience with using the Cell Saver® system for intraoperative red blood cell salvage on a hereditary spherocytosis patient. There was a drastic increase in salvaged blood free-hemoglobin compared with the preoperative sample (82.6 mg/dl vs. 6.2 mg/dl) which indicated severe hemolysis. Although our patient recovered smoothly with a normal liver and renal function test and reported no adverse reaction during follow-up, it is noteworthy that severe hemolysis could happen during the cell salvage process for patients with hemolytic anemia, as there are similar reports on sickle cell anemia, beta-thalassemia intermedia, and paroxysmal nocturnal hemoglobinuria. Therefore, more clinical attention and thorough research should be drawn into this perspective, namely, hemolysis during the red blood cell salvage process for patients with hemolytic anemia.

Whole-exome sequencing uncovered genetic diagnosis of severe inherited haemolytic anaemia: Correlation with clinical phenotypes.

Next-generation sequencing has shed light on the diagnosis of previously unsolved cases of inherited haemolytic anaemia (IHA). We employed whole-exome sequencing to explore the molecular diagnostic spectrum of 21 unrelated Thai paediatric patients with non-thalassemic IHA, presenting hydrops fetalis and/or becoming transfusion-dependent for 1 year or more or throughout their lifespan. Anaemia was detected prenatally, within the first month and the fifth year of life in three, 12 and six patients respectively. Molecular diagnosis obtained from all patients revealed SPTB as the most frequently mutated gene (four reported, three novel), found in 31 of 42 studied alleles. The other two mutated genes identified were ANK1 (three novel) and KLF1 (two reported). Four recurring mutations within exon 29/30 (NM_001024858.2) accounted for the vast majority (90%) of mutated SPTB alleles, biallelic inheritance of which resulted in the most severe phenotypes: hydrops fetalis and life-long transfusion dependency. Dominant ANK1 (n = 3) and SPTB (n = 2) mutations and biallelic class 2 KLF1 mutations (n = 1) led to a shorter period of transfusion dependency. Our study demonstrated that mutated SPTB causing red-cell membranopathy is likely the most common cause of severe non-thalassemic IHA among Thai patients. This urges carrier screening in the population to prevent subsequent, severely affected births.

Dissociated optic nerve fiber layer-like appearance indicating an internal limiting membrane defect associated with an epiretinal membrane: two case reports.

BACKGROUND: We report for the first time a way to predict the 2-dimensional extension of an internal limiting membrane (ILM) defect by detecting the area with dissociated optic nerve fiber layer (DONFL)-like spots in the preoperative optical coherence tomography (OCT) en-face images. CASE PRESENTATIONS: Case 1 was a 67-year-old man with metamorphopsia and decreased vision in his right eye. His best-corrected visual acuity (BCVA) was 20/100, with a pterygium, a moderate nuclear cataract, and an epiretinal membrane (ERM). Case 2 was a 73-year-old man with metamorphopsia and decreased vision in his left eye. His BCVA was 20/25, with a moderate nuclear cataract and an ERM. Both patients underwent simultaneous cataract surgery and pars plana vitrectomy with ERM and ILM peeling. Brilliant Blue G staining, performed before ERM and ILM peeling, revealed an unstained area. A careful evaluation of the area showed that it was not covered by either the ERM or ILM. A postoperative evaluation of the preoperative OCT images obtained from these cases showed DONFL-like low-brightness spots in the ILM defect area on the OCT en-face images. CONCLUSIONS: OCT en-face images may indicate the area of the ILM defect. To avoid iatrogenic damage to the retinal nerve fiber layer by touching/pinching it with forceps, detecting areas with DONFL-like spots in the preoperative OCT en-face images may be useful to predict an ILM defect.

Cartilaginous bending spring tympanoplasty: a temporal bone study and first clinical results.

OBJECTIVE: Anchoring grafts for tympanic membrane (TM) reconstruction in anterior and subtotal TM defects is essential to prevent medialisation and can be facilitated by cartilaginous bending spring tympanoplasty (CBST). The purpose of this study was to analyse the impact of spring cartilages on middle ear transfer functions and patient hearing levels. METHODS: In six fresh-frozen human temporal bones a cartilage graft (measuring 6 × 2 mm with a thickness of 0.1-0.2 mm) was formed into a 'U'-shaped bending spring, to be placed between the medial tympanic wall and the tympanic underlay grafts. The stapes velocity for excitation by exponential sweeps from 400 to 10,000 Hz was measured with a laser Doppler vibrometer. The resulting middle ear transfer functions were compared with the reconstructed middle ear. For clinical evaluation, 23 ears in 21 patients with chronic otitis media and an intact ossicular chain were operated using CBST. At each follow-up visit, the patients underwent pure-tone audiometry and the Freiburg monosyllabic speech test at a presentation level of 65 dB SPL for the word recognition score (WRS). RESULTS: The measured stapes velocities at one-third octave midband frequencies averaged 3.56 × 10-2 ± 9.46 × 10-3 (mm/s/Pa) compared to 3.06 × 10-2 ± 6.86 × 10-3 (mm/s/Pa) with the bending and underlay cartilage in place (p = 0.319; r = 0.32). The bending spring tympanoplasty reduced the transfer function by 1.41 ± 0.98 dB on average. In the clinical part of the study, the graft success rate was 96% (22 out of 23 patients) after a mean follow-up of 5.8 ± 2.4 months (min. 3.5 months, max. 12.0 months). The air-bone gap improved significantly by 6.2 dB (± 6.6 dB; p < 0.001; r = 0.69), as well as the WRS from 61.8 ± 33.3% preoperatively to 80.0 ± 20.9% postoperatively (p = 0.031; r = 0.35). CONCLUSION: Experimental data as well as initial clinical results suggest that CBST is an effective method for reconstructing anterior or subtotal defects of the tympanic membrane with satisfactory audiologic results and graft success rates comparable to previously described methods. It can, therefore, be added to the arsenal of tympanoplasty techniques for anterior and subtotal TM perforations.

Targeted next-generation sequencing identified novel mutations associated with hereditary anemias in Brazil.

Hereditary anemias are a group of heterogeneous disorders including hemolytic anemias and hyporegenerative anemias, as congenital dyserythropoietic anemia (CDA). Causative mutations occur in a wide range of genes leading to deficiencies in red cell production, structure, or function. The genetic screening of the main genes is important for timely diagnosis, since routine laboratory tests fail in a percentage of the cases, appropriate treatment decisions, and genetic counseling purposes. A conventional gene-by-gene sequencing approach is expensive and highly time-consuming, due to the genetic complexity of these diseases. To overcome this problem, we customized a targeted sequencing panel covering 35 genes previously associated to red cell disorders. We analyzed 36 patients, and potentially pathogenic variants were identified in 26 cases (72%). Twenty variants were novel. Remarkably, mutations in the SPTB gene (ß-spectrin) were found in 34.6% of the patients with hereditary spherocytosis (HS), suggesting that SPTB is a major HS gene in the Southeast of Brazil. We also identified two cases with dominant HS presenting null mutations in trans with α-LELY in SPTA1 gene. This is the first comprehensive genetic analysis for hereditary anemias in the Brazilian population, contributing to a better understanding of the genetic basis and phenotypic consequences of these rare conditions in our population.

[Compensating the Descemet's membrane defect (a clinical case study)]. / Kompensatsiia defekta destsemetovoi membrany (klinicheskoe nabliudenie).

The article describes a clinical case of Descemet's membrane detachment that occurred during cataract phacoemulsification with implantation of intraocular lens. Significant corneal edema developed in the early post-op period. Considering the ability of endothelium to close its own defects even with large injury area, the patient was followed-up for 2 years. Corneal edema was treated during 4 months and concluded with instillations of osmotic and anti-inflammatory drugs. At 1.5-2 months of therapy, the edema was gradually decreasing, and by the 4th month of the follow-up the cornea was practically transparent. Density of endothelial cells in central cornea, where the Descemet's membrane was absent, comprised 1000 cells/mm2. Further observation revealed cornea staying transparent for 2 years after the surgery. Complete optical recovery of patients with Descemet's membrane defect in this case study is supported by similar clinical cases briefly described in the article, and evidence that structural functional recovery of the corneal endothelial layer is possible even when it has large defects (more than 2.5 mm in diameter).

Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias.

Hereditary haemolytic anaemias are genetically and phenotypically heterogeneous disorders characterized by increased red cell destruction, with consequences ranging from innocuous to severe life-threatening anaemia. Diagnostic laboratories endeavour to assist clinicians reach the exact patient diagnosis by using tests principally based on morphological and biochemical techniques. However, these routine studies may be inconclusive, particularly in newborn infants and when transfusions have recently been administered. Large numbers and size of the potentially involved genes also impose a practical challenge for molecular diagnosis using routine sequencing approaches. To overcome these diagnostic shortcomings, we have utilized next-generation sequencing to provide a high-throughput, highly sensitive assay. We developed a panel interrogating 28 genes encoding cytoskeletal proteins and enzymes with sequencing coverage of the coding regions, splice site junctions, deep intronic and regulatory regions. We then evaluated 19 samples, including infants with unexplained extreme hyperbilirubinaemia and patients with transfusion-dependent haemolytic anaemia. Where possible, inheritance patterns of pathogenic mutations were determined by sequencing of immediate relatives. We conclude that this next-generation sequencing panel could be a cost-effective approach to molecular diagnosis of hereditary haemolytic anaemia, especially when the family history is uninformative or when routine laboratory testing fails to identify the causative haemolytic process.

Hemolytic anemia in pediatrics / 소아과

To understand the hemolytic anemia (HA) in children, the diagnostic approach and management of hereditary and acquired HA are described. The hereditary hemolytic anemia (HHA) can be classified according to the pathogenesis into three types:RBC membrane defects, hemoglobinopathies, and RBC enzymopathies. Clinical characteristics, laboratory findings and molecular defects of these three types are presented briefly. In Korea, HHA due to the RBC membrane defect, hereditary spherocytosis had been reported often but HHA due to hemoglobinopathies and RBC enzymopathies had been thought to be relatively rare. With recent development in the molecular diagnosis, beta thalassemia, mostly heterozygote, G6PD and pyruvate kinase deficiency have been reported with gene characterization. If the patients with microcytic hypochromic anemia show unproportionally low MCV or MCH or refractory to the iron therapy, hemoglobin electrophoresis and gene analysis for thalassemia or other unstable hemoglobinopathies need to be done accordingly. The global movement of the population especially from the region prevalent of hemoglobinopathies or enzymopathies to Korea warrants considering broad spectrum of etiology for the diagnosis of HHA. Aquired HA resulting from extracellular factors such as autoimmune HA from warm antibody, cold agglutinin and paroxysmal cold hemoglobinuria as well as nonimmune HA are described briefly.

Hereditary Hemolytic Anemia

The hereditary hemolytic anemia (HHA) can be classified into three types according to the pathogenesis: RBC membrane defects, hemoglobinopathies, and RBC enzymopathies. Clinical characteristics of these three types of HHA are presented briefly in this paper. In Korea, HHA due to RBC membrane defect such as hereditary spherocytosis had been relatively well recognized, while HHA due to hemoglobinopathies and RBC enzymopathies had been considered rare. However, with the recent development of molecular testing, beta thalassemia, G6PD and pyruvate kinase deficiency have been reported with identification of disease-causing mutations. If a patient with microcytic hypochromic anemia shows unproportionally low MCV or MCH or refractory to iron therapy, hemoglobin electrophoresis and gene study for thalassemia or other unstable hemoglobinopathies are needed. It should be noted that the recent population migration to Korea from the regions where hemoglobinopathies or enzymopathies are prevalent warrants considering a broad spectrum of etiologies for the diagnosis of HHA.