Abnormal Variants Research Articles

Evaluation of Haemoglobin F and Haemoglobin A2 among Sickle Cell Patients in Steady State at Selected Hospital in Ogun State, Nigeria

Background: Sickle cell anemia (SCA) is a genetic disorder characterized by abnormal hemoglobin variants, including hemoglobin F (Hb F) and hemoglobin A2 (Hb A2). Evaluating the levels of these hemoglobin variants in steady-state sickle cell patients can provide insights into disease prognosis and management. This study aimed to assess Hb F and Hb A2 levels among sickle cell patients in steady state at a selected hospital in Ogun State, Nigeria. Objective: To evaluate the levels of hemoglobin F (Hb F) and hemoglobin A2 (Hb A2) in sickle cell anemia patients and determine their associations with demographic factors such as age and gender. Materials and methods: A descriptive cross-sectional study was conducted at the Sickle Cell Center, Abeokuta, Ogun State, Nigeria, from September to October 2020. A total of 60 sickle cell patients in steady state were recruited using convenient sampling. Blood samples were collected, and Hb F and Hb A2 levels were measured using high-performance liquid chromatography (HPLC). Data were analyzed using SPSS version 26.0 with independent t-test and bivariate correlation. Results: The mean levels of Hb F and Hb A2 varied across age groups and genders. Hb F levels were highest in patients over 35 years (10.4 ± 12.16%) and lowest in those aged 26-30 years (3.7 ± 1.3%). The majority of participants had Hb F levels between 2-10%, with a significant association between Hb F levels and age (p = 0.038). Hb A2 levels were consistently above 3.1% in 92.6% of the study population, with no significant association with age or gender. Conclusion: There is a significant association between age and Hb F levels among steady-state sickle cell patients, while Hb A2 levels showed no significant demographic correlations. The findings suggest that Hb F may serve as a potential marker for clinical evaluation in SCA patients.

P53 ABNORMAL ENDOMETRIAL CANCERS – ANALYSIS OF PD-1 CO-EXPRESSION BY CD4 HELPER AND CD8 CYTOTOXIC T-CELL SUBTYPES IN THE TUMOR MICROENVIRONMENT

Abstract Introduction/Objective Endometrial cancer is classified into four molecular subtypes of which the p53 abnormal variant is biologically aggressive. Programmed cell death protein 1 (PD-1) is a cell surface receptor and a vital inhibitor of the immune response. Programmed death ligand 1 (PD-L1) is a trans-membrane co-inhibitory factor of the immune response. PD-1 / PDL-1 interaction inhibits the activation and survival of T cells and decreases cytokine secretion. Some cancer cells express PD-L1, which then bind to PD-1 expressed on tumor specific T cells, and escape destruction by the T cells. The CD4/CD8 T cell immune response in the p53 abnormal variant has not been adequately evaluated with respect to the PD-1/PD-L1 pathway. Understanding this immune microenvironment is important to assess the potential use of anti-PD-1 targeted tumor therapy. Methods/Case Report Twenty cases of p53 abnormal endometrial cancers were obtained. All cases were PDL-1 +ve by immunohistochemistry. Two dual stains were performed - PD-1/CD4 and PD-1/CD8 antibodies. Six high-power fields (40x objective) with the greatest concentration of lymphocytes were counted. The total number of CD4 or CD8-only cells was recorded for each of the two stained slides for each case. The total number of PD- 1/CD4 and PD-1/CD8 positive cells were counted and recorded. The results were tabulated, and a paired t-test statistical analysis was performed to compare the total number of CD4 and CD8 cells and the percentage of PD-1/CD4 cells with the percentage of PD-1/CD8 positive cells. Results (if a Case Study enter NA) A statistically significant increase in the number of CD8 (Mean = 383) compared to CD4 cells (Mean = 317) was seen in the tumor microenvironment (p-value=0.0023). Evaluating PD-1 expression, the converse was true, and the percentage of PD-1/CD4 cells (Mean = 13.035%) was increased compared to the percentage of PD-1/CD8 cells (Mean = 9.205%), achieving statistical significance (p-value=0.0116). Conclusion The statistically significant increase in CD8 cells in the tumor and its stroma suggests that a robust cytotoxic response is being mounted to this high-risk molecular variant. The concurrent increase in CD4/PD-1 positive lymphocytes provides an appropriate microenvironment for the use of anti-PD-1 immunotherapy, since blockade of PD-1 on CD4 helper T cells may augment the tumor ablative cytotoxic properties of the CD8 positive lymphocytes. This includes reinvigoration of exhausted CD8+ T cells. Anti-PD-1 may also prevent the inhibition of PD-1 positive CD8 cells.

A-149 Comparison of Mass Spectrometry to Sickle Solubility for Confirmation of Low Percentages of Hemoglobin S

Abstract Background Hemoglobin S (HbS) causes sickling disorders. In the laboratory, confirmation of HbS in patient samples with varying percentages is essential. Assessment is routinely performed using capillary electrophoresis (CE) and/or high-performance liquid chromatography. Sickle solubility (SS) exploits decreased solubility of HbS under reducing conditions and is a confirmatory test. SS is unreliable with low HbS, elevated HbF, severe anemia, erythrocytosis, hyperglobulinemia, extreme leukocytosis, hyperlipidemia, or increased serum proteins. Infant samples commonly have low percentages of HbS, high HbF, and limited sample volume. Intact globin chain Mass Spectrometry (MS) is used to identify abnormal hemoglobin variants by detecting the mass change (normal α/β/γ mass =15127/15867/15996 amu), respectively). MS has potential advantages in sensitivity and efficiency over SS and requires lower input sample volume. Herein we compare the utility of MS versus SS for confirmation of HbS. Methods Twenty-three EDTA-anticoagulated blood samples containing low percentages of HbS were identified. SS was performed on 30μL washed packed cells (requires 200μL whole blood) per standard methods. Intact Quadrupole Time-of-Flight MS (Accurate-Mass Q-TOF LC/MS 6520, Agilent Technologies, California) was performed using 10μL whole blood sample in acetonitrile solution reagent and results were compared. HbS and HbF levels were assayed by capillary electrophoresis (Sebia, France) per standard methods. Results Select results are summarized in Table 1. SS detected HbS percentages to 4.8% when HbF was low but not 6% HbS when Hb F is high. MS showed no HbF interference and identified HbS peaks (at 15837 amu) down to 3.1%. HbS percentages <3.1 were undetected by both methods. Conclusions Overall, MS was superior to SS for confirming HbS, particularly in samples with low variant percentage and/or elevated HbF. MS is also optimal when sample volume is minimal, such as for infants. Percentages of HbS <3% require methods such as IEF or DNA sequencing for confirmation.

Personalized management of aspirin-exacerbated respiratory disease: From clinical presentation to therapeutic strategies

Aspirin-exacerbated respiratory disease (AERD), defined as the triad of asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and development of upper- and/or lower-respiratory symptoms after exposure to aspirin or other cyclooxygenase-1 inhibitors, has a prevalence of up to 30% for adults with asthma and with CRSwNP. The pathogenesis is likely due to a combination of abnormal genetic variants and environmental exposures, which results in types 1, 2, and/or 3 inflammation and dysregulation of arachidonic acid metabolism. Patients with AERD often fail to respond to conservative medical treatment, including all topical corticosteroids, antileukotrienes, and saline solution irrigation; furthermore, short courses of SCS generally provide only temporary improvement. Advanced therapeutic options include endoscopic sinus surgery; aspirin treatment after aspirin desensitization; and biologics, e.g., dupilumab, omalizumab, and mepolizumab. A successful and long-lasting clinical response often requires combination or sequential treatments with more than one of these therapeutic approaches. After a review of the efficacy, safety, and logistical considerations of each of these therapeutic options, an algorithmic approach for patient management is presented.

Rapid Whole-Genome Sequencing in Acutely Ill Children: A Single Pediatric Center Experience

AbstractThe aim of this study was to describe the turnaround time, diagnostic yield, and clinical impact of rapid whole-genomic sequencing (rWGS). We conducted a prospective observational study in acutely ill children (0–21 years) with an undiagnosed, potentially genetic abnormality in a children's hospital. A phenotype-prioritized analysis approach for rWGS was utilized. The turnaround times, diagnostic yield, number of genes detected, inheritance pattern, zygosity, and the clinical impact of positive or negative tests were analyzed. Out of a total of 109 children, 92 abnormal (pathogenic or likely pathogenic) gene variants were detected in 60 (55%) patients. There were 45 neonates, 35 infants, and 29 children. The admission location was 49.5, 34.9, and 15.6% in the pediatric intensive care unit (PICU), neonatal intensive care unit (NICU), and cardiac intensive care unit (CICU), respectively. The median (interquartile range [IQR]) times for the return of preliminary and final results were 3 (2–5) and 10 (6–14) days, respectively. With ultra-rapid processing, the median time to final results was shorter (5 [3–7] vs. 12 [7.75–15] days). Neurologic issues were the most common underlying admission diagnoses. The diagnostic yield for a causative gene was 47.7%. The diagnostic yield was not different based on age group or location of admission but higher in metabolic issues (78.6 vs. 43.2%; odds ratio [OR]: 4.8; 95% confidence interval [CI]: 1.3–18.4). There was a change in clinical management in 39.4%. In acutely ill children with undiagnosed conditions and with clinical suspicion of a genetic disorder, rWGS detected gene variants in 55% with a diagnostic yield of 47.7% and resulted in a change in the management in 39.4%. The diagnostic yield in patients with metabolic conditions was the highest.

Generation of an induced pluripotent stem cell line, JHUi005-A, from a Marfan Syndrome patient harboring a pathogenic c.3338-2A>C intronic splicing variant

Marfan Syndrome, a connective tissue disorder caused by Fibrillin-1 (FBN1) gene mutations, induces disease in the ocular, musculoskeletal, and cardiovascular systems and increases aortic vulnerability to rupture associated with high mortality rates. We describe an induced pluripotent stem cell line (HFD1) generated from patient-derived human dermal fibroblasts harboring a heterozygous c.3338-2A>C intronic splice acceptor site variant preceding Exon 28 of FBN1. The clonal line, which produces abnormal FBN1 splice variants, has a normal karyotype, expresses appropriate stemness markers, and maintains trilineage differentiation potential. This line represents a valuable resource for studying how abnormal splicing variants contribute to Marfan Syndrome.

Saturation genome editing-based functional evaluation and clinical classification of BRCA2 single nucleotide variants.

10511 Background: Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n > 4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing (SGE)-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot encoded by exons 15 to 26 for pathogenic missense variants. Methods: SGE of exons 15 to 26 of BRCA2(MANE transcript ENST00000380152.8) was performed in the haploid human HAP1 cell line. Each exon along with 10bp flanking intronic sequence was studied as an independent target region. The most efficient sgRNA for each target region was selected and cloned into a Cas9 expressing plasmid. Site-saturation mutagenesis (SSM) libraries for each of the 14 target regions were designed to target every nucleotide and were generated by site directed mutagenesis using NNN-tailed PCR primers. HAP1 cells were transfected and harvested at Day5 and 14. Cell survival in response to each variant was assessed based on deep sequence analysis of the surviving endogenously targeted HAP1 cells at Day 5 and Day 14. Results: A total of 7013 SNVs were characterized as functionally abnormal (n = 955), intermediate/uncertain, or functionally normal (n = 5224) based on 95% precision for ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. Conclusions: The production of functional maps for 97% of all SNVs in the BRCA2 DBD allowed for separation of nucleotide/protein-level functional aberrations. When coupled with genetic and clinical sources of evidence this study led to clinical classification of 5500 individual variants. These data will prove useful in the future, through integration with other datasets, for characterization and clinical classification of all variants in this region in individuals from all racial and ethnic backgrounds and for all BRCA2 associated forms of cancer.

Newborn screening in Colombia: The experience of a private program in Bogotá

Introduction. The first neonatal screening program in Colombia – PREGEN – was set up in the medical private sector of Bogotá in 1988. We report the results from recent years that, given the scarcity of similar information in our country, may help estimate the frequency of the evaluated neonatal disorders and which ones should be included in the neonatal screening programs in our country. Objective. To describe the results of PREGEN´s newborn screening program between 2006 and 2019. Materials and methods. We analyzed databases and other informative documents preserved in PREGEN from the 2006-2019 period. Results. One in every 164 newborns screened in our program had an abnormal hemoglobin variant, and one in every 194 carried some hemoglobin S variant. Glucose-6- phosphate dehydrogenase deficiency and congenital hypothyroidism are next as the more common disorders. Conclusions. Abnormal hemoglobin causes the most frequent monogenic disorder in the world. Glucose-6-phosphate dehydrogenase deficiency is the most common enzymopathy affecting nearly 400 million individuals worldwide. Since both disorders are more common in people of African descent and confer some resistance to malaria, we believe that screening for both disorders may be more relevant in the areas with African ancestry in our country.

Hemoglobin Variants in Patients Attending Aden Diagnostic Center by High Performance Liquid Chromatography

Introduction: High performance liquid chromatography (HPLC) is emerging as the method of choice for initial screening and diagnosis of hemoglobinopathies. The use of alkaline and acid gel electrophoresis may result in incorrect diagnosis of hemoglobinopathies. The aim of the study is to investigate the hemoglobin pattern using the HPLC and to correlate the hematological profile with the types of hemoglobin. Methods: A cross-sectional study was conducted in Aden Diagnostic Center from July– December 2022. Over a six-month period, 250 samples of patients aged between six months to thirty years, were evaluated by HPLC for detection of hemoglobinopathies using the Lifotronic Hemoglobin Analyzer H9: β-thalassemia Analysis Mode. Red blood cell and red cell indices were determined using automated hematology analyzer. Results: A total of 152 samples (60.8%) showed different abnormal hemoglobin variants. Sixty-four (25.6%) were diagnosed to have sickle cell anemia, 46 (18.4%) as sickle cell trait, two (0.8) as beta – heterozygous thalassemia based on high level of HbA2 (>3.9%), four (1.6%) as beta – homozygous thalassemia (HbF 25–91%), 18 (7.2%) as compound heterozygous state of sickle - β+ thalassemia, 17 (6.8%) as beta thalassemia trait, and one (0.4%) sample had HbD variant on HPLC was diagnosed as HbD trait. Conclusion: H9-HPLC is suitable for the routine investigation of hemoglobinopatheis because it is very powerful tool in the evaluation of Hb variants, rapid assay time and accurate quantification.

Establishment of a human induced pluripotent stem cell line, KMUGMCi010-A, from a patient with X-linked Ohdo syndrome bearing missense mutation in the MED12 gene

X-linkded Ohdo syndrome is characterized mainly by intellectual disability, delays in reaching development, feeding difficulties, thyroid dysfunction, and dysmorphic appearance with blepharophimosis, immobile mask-like face and bulbous nose. The X-linked Ohdo syndrome is caused by loss of function mutation in MED12 gene on X chromosome. The peripheral blood mononuclear cells from a patient carrying missense mutation of the MED12 gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. The missense mutation in MED12 gene causes the abnormal protein variant. The established human induced pluripotent cell line will enable proper in vitro disease modelling of X-linked Ohdo syndrome.

Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.

Quality and Diagnosis on the Lateral View of Pediatric Upper Gastro-Intestinal Series.

The standard imaging technique for the diagnosis of intestinal malrotation remains the upper gastro-intestinal series (UGIS). The lateral view is promoted as important for making a diagnosis. For this, the lateral view should be of adequate quality, and radiologists must know the normal appearance as well as the appearance of duodenal variants, as misdiagnosis may lead to unnecessary surgery. We aimed to evaluate the quality, findings including the prevalence of the "descending staircase" configuration and its correspondence to a diagnosis of duodenum redundum. This was a retrospective study and was conducted in a large tertiary children's hospital in the United States. A retrospective review of UGI fluoroscopy exams in children aged ≤ 18 years between January and December 2018 was performed by a pediatric radiologist. First, the lateral view images/cine-loops were assessed independently, followed by the anteroposterior (AP) view. The studies which were designated to have an adequate lateral view were evaluated for configuration of the duodenum and recorded as: normal, abnormal, or normal variant. Also, the presence of a descending staircase configuration was correlated with an AP view for a diagnosis of duodenum redundum. A total of 26 children (26%) (males:16; females:10) with age range 0 to 16 years had adequate lateral views during UGI exams for inclusion. Of the 26, 18 (69%) were reported as normal, 7 (27%) were reported as having a descending staircase and 1 (4%) was reported as abnormal. The AP view demonstrated 2 abnormal studies (1 malrotation and 1 non-rotation), 6 duodenum redundum and 18 normal exams. The one abnormal lateral duodenum was confirmed as a non-rotation on AP view; the second patient with an abnormal AP view had a normal appearance on the lateral view. A total of 26% of UGI studies had adequate lateral views for interpretation. Of these, nearly a quarter (23%) demonstrated the descending stair-case sign corresponding to a diagnosis of duodenum redundum on the AP view. If the lateral view had been used alone, there would have been a missed diagnosis in one patient.

Identification of a Novel Variant c.163delG in HBB Gene Resulting in a Beta Null Phenotype in a Proband with Thalassemia Intermedia

A 21-year-old patient presented with a previous medical history of pallor, mild icterus, increased fatigue, low hemoglobin, and abnormal hemoglobin variant analysis with more than 70 transfusions. He was referred for genetic analysis to identify the pathogenic variations in the β-globin gene. Sanger’s sequencing of the proband and his family revealed the presence of a novel frame shift variant HBB:c.163delG in a compound heterozygous state with hemoglobin E (HbE) (HBB:c.79G > A) variant. The father and the sibling of the patient were found to be normal for the HBB gene. Mother was found to be heterozygous for HbE (HBB:c.79G > A) variant. In silico analysis by Mutalyzer predicted that c.163delG variant generated a premature stop codon after seven codons, leading to a truncated protein. FoldX protein stability analysis showed a positive ΔΔG value of 45.27 kcal/mol suggesting a decrease in protein stability. HBB:c.79G > A is a known variant coding for HbE variant, which results in the reduced synthesis of β-globin chain and shows mild thalassemia. Combined effect of HBB:c.163delG and HBB:c.79G > A variants in the proband might have led to the reduced synthesis of β-globin chains resulting in a thalassemia intermedia type of clinical manifestation.

What is the impact of an Abnormal pathogenic variant in COQ8A/CYP17A1 in pediatric patient - A case report

Background: Primary coenzyme Q10 deficiency-4 (CoQ10D4) and isolated 17,20-lyase deficiency (ILD) are both distinct and rare congenital mutations. Mutations in the CoQ8A gene are rare and cause CoQ10D4. Patients with pathogenic mutations in the CoQ8A gene exhibit symptoms such as ataxic gait, dystonia, seizures, exercise intolerance, and cognitive disabilities. Case presentation: The presence of these two mutations in a patient is extremely rare. In this present case report, a 6-year-old identical twin female with CoQ10D4 and ILD began presenting with microcephaly with developmental delay at 4 years of age. She also had regression of milestones (loss of social interaction, grasp, visual interaction, and speech-babbling) without febrile seizures. Conclusion: This case emphasizes the need for further research to elucidate the underlying mechanisms and potential targeted therapies for these rare genetic disorders, emphasizing the importance of a multidisciplinary approach in providing comprehensive care for affected individuals.

Anesthetic Management of a Patient With Hemoglobin M Disease Undergoing Laparoscopic Uterine Myomectomy: A Case Report

Hemoglobin M (Hb M) is a group of abnormal Hb variants that form methemoglobin, which leads to cyanosis. Patients with Hb M appear cyanotic but are usually asymptomatic. Cyanosis with low peripheral oxygen saturation is unresponsive to oxygen therapy despite normal partial pressure of oxygen. As such, close attention should be paid during anesthesia. We report the first case of a Hb M patient undergoing laparoscopic uterine myomectomy under general anesthesia.

Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We perform saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identify 3432 functionally abnormal variants, in three distinct classes. We train a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants can account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X-driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation.

Hemoglobin Monza: A New Variant of Unstable Hemoglobin

Introduction: Unstable hemoglobins are variants with structural abnormalities; while not hindering protein formation, they lead to protein destabilization and can cause hemolytic crises. Most unstable hemoglobins are caused by point mutations that result in the premature dissociation of heme from the globin chain and usually involve His residues (Weatherall et al. 2001 Nat.Rev.Genetics; Scheps et al. 2018 EJH). Here a new unstable hemoglobin, named Hb-Monza is presented, exhibiting a long duplication in the HBB gene without apparent alterations in α-β subunit interaction or involvement of specific His residues. The variant was identified in a family, with four out of six members involved and is associated with mild chronic hemolysis and acute severe hemolytic crises during febrile infections. Methods: The presence of erythrocyte membrane abnormalities was tested by glycerol lysis, Pink, osmotic resistance and by EMA binding test. G6PD and PK activity levels were tested both during the hemolytic crisis and at resolution. Abnormal variants were screened by HPLC and electrophoresis. Stability of the Hb variant was assessed by the Isopropanol Precipitation Test. DNA was extracted from peripheral blood leukocytes, followed by PCR amplification of HBB, HBA1 and HBA2 genes. Sequencing was performed using Genetic Analyzer 3500. A 3D model of the variants was produced by SWISS-MODEL using the crystal structure of the deoxy human hemoglobin (PDB 1A3N) as template and refined by Maestro Suite. Results: The index patient (pt) was a 13-year-old girl of Chinese origin; she was the firstborn of 4 children (2 sisters and 1 brother) and came to ER with fever and hyperchromic urine. Blood tests revealed severe anemia (Hb 5.2 g/dL) and signs of hemolysis. Direct Coombs test was negative both for warm and cold antibodies. She tested positive for Mycoplasma Pneumoniae, Epstein Barr Virus (EBV) and Parvovirus B19 (IgG and IgM) antibodies. The child's infection cleared during hospitalization, and hemoglobin levels increased without transfusion. Her hemoglobin further rose to 11 g/dL in outpatient follow-up although signs of low-level chronic hemolysis persisted. All affected family members exhibit mild chronic hemolytic anemia, with all, except the mother, experiencing at least one acute severe hemolytic state triggered by infection. HPLC showed the presence of an abnormal fraction of hemoglobin with slow migration speed in alkaline electrophoresis and extremely slow elution in HPLC (4.8 min). The abnormal Hb fraction in the proband, mother, brother and sister was 20.8%, 17.7%, 28.1%, and 25.6% respectively. The father and the youngest sibling did not show this band. Sequencing of HBB demonstrated an in-frame duplication of 69 bases corresponding to 23 amino acids located in exon 2 (c.176_244dup; p.Pro59_Asn81dup); this was present in all pts exhibiting the abnormal fraction at HPLC. A functional validation of Hb Monza utilizing the isopropanol test confirmed the unstable nature of the variant (Figure 1). The 3D-model analysis (Figure 2) showed that the duplication is situated amidst helix E and F of HBB resulting in a disordered conformation protruding from the Hb structure The binding of the heme group remains unaffected and the coordination of the heme group with His residues of both the E and F helix is preserved. The tertiary structure of the Hb beta chain is scarcely affected. Conclusions: This report introduces a novel HBB gene variant, Hb Monza (HBB: c.176_244dup), associated with subclinical chronic hemolysis and acute hemolytic crises during fever-inducing infections. Only a few HBB duplications exceeding 20 bp have been documented, all leading to Beta-Thalassemia clinical features through production of a truncated beta globin chain or failure of HBB to bind the alpha chain (e.g. Frischknecht et al. 2007 Haematologica). However, 3D-modeling of Hb Monza indicates minimal tertiary structure alteration. The inclusion of 23 additional amino acids results in a disordered conformation protruding from the Hb structure possibly facilitates direct bonding between the iron molecule in the heme group and internal amino acid side chains due to increased structural flexibility. This could lead during thermal or metabolic stress, to Hb precipitation. Further data from molecular dynamic experiments will be presented. Dr. Civettini and Dr. Corti contributed equally to this article

An Empirical Comparison of Commonly Used Universal Rating Scales for Dystonia.

There are several widely used clinical rating scales for documenting the severity and distribution of various types of dystonia. The goal of this study was to evaluate the performance of the most commonly used scales in a large group of adults with the most common types of isolated dystonia. Global Dystonia Rating Scale (GDRS) and the Burke-Fahn-Marsden Dystonia Rating Scale (BFM) scores were obtained for 3067 participants. Most had focal or segmental dystonia, with smaller numbers of multifocal or generalized dystonia. These scales were also compared for 209 adults with cervical dystonia that had Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) scores and 210 adults with blepharospasm that had Blepharospasm Severity Scale (BSRS) scores. There were strong correlations between the GDRS and BFM total scores (r = 0.79) and moderate correlations for their sub scores (r > 0.5). Scores for both scales showed positive skew, with an overabundance of low scores. BFM sub-scores were not normally distributed, due to artifacts caused by the provoking factor. Relevant sub-scores of the GDRS and BFM also showed moderate correlations with the TWSTRS (r > 0.5) for cervical dystonia and the BSRS (r > 0.5) for blepharospasm. The BFM is more widely used than the GDRS, but these results suggest the GDRS may be preferable for focal and segmental dystonias. The overabundance of very low scores for both scales highlights challenges associated with discriminating very mild dystonia from other abnormal movements or variants of normal behavior.

Multiplexed assay of variant effect reveals residues of functional importance in the BRCA1 coiled-coil and serine cluster domains.

Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.

THU253 The Hemoglobin Wayne Variant And Association With Falsely Elevated HbA1c

Abstract Disclosure: N. Mulpuri: None. A. Bryant: None. D. Shahin: None. K. Soe: None. Background: Hemoglobin Wayne (Hb Wayne) is a rare, silent alpha chain variant caused by a frameshift mutation in the HbA2 gene. Although clinically silent, this mutation falsely elevates HbA1c values. Clinical cases: We are presenting three cases of Hb Wayne patients referred to our diabetes clinic within the last 5 years. All three cases were Caucasian-American males diagnosed with uncontrolled diabetes based on high HbA1c values of ranging from 10.1-10.6 %. One of the patients was initially started on metformin while the other patients’ treatment plan included dietary and lifestyle modifications. Review of the labs showed that renal function, complete blood count and fasting plasma glucose were within the normal range. High HbA1c value was noted by routine screening lab without having any symptoms related to DM or features of insulin resistance. The discrepancy between high HbA1c values and other glycemic index parameters (serum glucose and FSBG values) was noted. Subsequently, further work up was done (repeat HbA1c with different assay and fructosamine) to verify the diagnosis of uncontrolled DM. Fructosamine levels and repeat HbA1c with a different assay (enzymatic assay at Quest lab) were normal. Hemoglobin electrophoresis confirmed presence of an abnormal alpha variant consistent with Hb Wayne. The patients were reassured and educated about this Hb variant, and no further treatment for DM was pursued. Conclusion: Rare hemolgobinopathies may be more common than previously expected as they can be clinically silent and easily overlooked. While HbA1c has been an important clinical tool for diagnosis and management of diabetes, clinicians should look for underlying reasons which can affect HbA1c assay if there is any discrepancy between HbA1c, FSBG and plasma glucose values. The laboratory at our institution uses cation- exchange High Performance Liquid Chromatography method (Bio-Rad D-100 instrument) resulting in falsely high HbA1c values. Some hemoglobinopathies can affect the widely used HbA1c testing method such as CE-HPLC. The impact on HbA1c values will differ depending on hemoglobin variant and specific method and assay used. Given the limitations of HbA1c testing, clinicians should avoid relying heavily on HbA1c values in management and diagnosis of diabetes. It is vital that clinicians use their clinical judgment to correlate HbA1c with other clinical parameters such as continuous glucose monitoring data, glycated albumin,1,5 anhydroglucitrol (1,5 –AG), fingerstick blood glucose, plasma glucose and fructosamine values. Presentation: Thursday, June 15, 2023