This corrects the article DOI:10.3325/cmj.2024.65.180.

Hereditary protein S deficiency, a risk factor for venous thromboembolism, presents diagnostic challenges due to the PROS1 gene's homologous pseudogene, PROSP. The pseudogene's homology risks co-amplification and misinterpretation of sequencing data, leading to false results and compromising clinical management. We report a case of a 28-year-old male who presented dural venous sinus thrombosis and low free protein S. After a novel PROS1 variant was identified via next-generation sequencing, a "pseudogene-aware" Sanger sequencing assay was developed to validate the finding. This assay utilized primers designed in silico method and high-temperature PCR to exploit nucleotide mismatches between PROS1 and PROSP, ensuring targeted amplification. The assay successfully validated a novel heterozygous missense variant, PROS1:c.1484T>C. This variant co-segregated with low protein S levels in the family and was classified as likely pathogenic. This pseudogene-aware strategy enabled the accurate identification of a novel variant causing severe thrombophilia, highlighting the importance of such assays for reliable molecular diagnosis and patient management.

INTRODUCTION:Iron deficiency (ID) is the most common micronutrient deficiency globally, often underdiagnosed due to nonspecific symptoms and limitations of standard laboratory tests. Reticulocyte-derived indices from modern hematology analyzers offer enhanced early detection of iron-restricted erythropoiesis. METHODS:We studied 103 self-reported asymptomatic healthy adults who underwent complete blood counts and reticulocyte analysis on XN-2000 analyzers (Sysmex Corp., Kobe, Japan). Detailed biochemical iron profile and vitamin B12/folate testing were done. Diagnostic performance of hematologic parameters was assessed and combinatorial logistic regression models and discriminant indices were developed. RESULTS:Participants' median age was 28 years (range 22-79); 69.9% (n = 72) were women. 54.4% (n = 56) had ID and 21.4% (n = 22) were anemic. Latent ID was detected in 34.9% (36/103). B12 and folate deficiencies were present in 41% and 40%, respectively - extensively overlapping with ID. Key individual indices for ID included RET-He, RET-Y, and RET-RBC-Y, each with area under the ROC curve (AUC) >85%. A comprehensive 21-variable logistic regression model yielded AUC 93.7% (95% C.I. 88.9-98.5), sensitivity 89.1%, and specificity 70.2%. A pared-down 3-variable model achieved AUC 90.1% (95% C.I. 84.1-96.1), sensitivity 87.5%, and specificity 70.9%. Of the two heuristic composite indices tested, one - [RDW-CV × PLT × 105] / [RET-He × RET-RBC-Y × IRF-Y × RET-Y] - showed AUC 89.8% (95% C.I. 83.7-95.9), sensitivity 87%, and specificity 72.3%. CONCLUSION:In a cohort with high rates of nutritional deficiency, cost-effective equations combining conventional and advanced reticulocyte indices demonstrated strong diagnostic utility for screening ID, with potential for broader application in resource-limited settings.

Background: Micronutrient deficiencies remain prevalent among preschool-aged children (PSC) in India. Quintuply-fortified salt (QFS) is one of many potential interventions to improve micronutrient intake and status at the population level. Objectives: To determine the effect of QFS compared with iodized salt (IS) for 12 mo on the micronutrient status of PSC. Methods: This was a substudy of a double-blinded, household-randomized, controlled, community-based trial involving nonpregnant females of reproductive age whose households were randomly assigned to receive: 1) QFS with zinc, vitamin B-12, folic acid, iodine, and iron as encapsulated ferrous fumarate (eFF-QFS); 2) QFS with the same micronutrients, but iron as encapsulated ferric pyrophosphate plus ethylenediaminetetraacetic acid (eFePP-QFS); or 3) IS. The micronutrient status of 470 PSC (aged 12-59 mo) residing in these households was assessed at enrollment, 6 mo, and 12 mo. Continuous outcomes were analyzed with linear regression and reported as means or geometric mean ratios, and binary outcomes were analyzed with logistic regression and reported as odds ratios. Results: At baseline, the prevalence of anemia, iron deficiency anemia, hypozincemia, vitamin B-12 insufficiency, and folate deficiency was 35%, 30%, 14%, 17%, and 5.5%, respectively. Effects of QFS at 6 and 12 mo were greatest for vitamin B-12 and folate. At 12 mo, the eFePP-QFS group had higher serum vitamin B-12 [geometric mean ratio (GMR) = 1.16, 95% confidence interval (CI) = 1.03, 1.30], serum folate (GMR = 1.29, 95% CI = 1.09, 1.53), and red blood cell folate (GMR = 1.21, 95% CI = 1.05, 1.39) concentrations compared with the IS group. Effects were similar among the 2 QFS groups. There were no significant differences in serum zinc, ferritin, hemoglobin, or urinary iodine between groups at 6 and 12 mo. Conclusions: Preschool children consuming QFS for 12 mo demonstrated greater improvements in vitamin B-12 and folate status compared with children consuming IS. QFS may be a useful vehicle to address micronutrient deficiencies, especially vitamin B-12 and folate, in this population.

Background:Young children in India often face multiple micronutrient deficiencies, yet interventions such as micronutrient powders have raised concerns about potential adverse effects on the gut microbiome. Large-scale food fortification is an effective strategy to improve micronutrient intake; however, its impact on the gut microbiome of children remains unclear. Objectives:To determine whether intake of quintuply-fortified salt (QFS) for 12 mo adversely affects gut microbiome composition in children aged 1-5 y. Methods:In a double-blind, randomized, controlled trial in Punjab, India, children received: 1) QFS with iron as encapsulated ferrous fumarate [eFF], zinc, vitamin B12, folic acid, and iodine (eFF-QFS); 2) QFS with the same micronutrients, but iron as encapsulated ferric pyrophosphate [eFePP] plus ethylenediaminetetraacetic acid (eFePP-QFS); or 3) standard iodized salt for 12 mo. Stool samples were collected from 125 children (eFF-QFS, n = 43; eFePP-QFS, n = 45; iodized salt, n= 37) at baseline and 12 mo and analyzed via 16S rRNA gene sequencing. Changes in alpha diversity (Shannon, abundance-based estimator index) between groups were assessed with linear mixed models, beta diversity (Bray-Curtis dissimilarity) with linear regression and permutational multivariate analysis of variance, and relative abundance of Enterobacteriaceae, Lactobacillus, Bifidobacterium, Bacteroides, Prevotella, or Escherichia-Shigella with zero-inflated negative binomial mixed models. Results:Average discretionary salt utilization was estimated to be 3.5 g/child equivalent/d across groups. Abundance-based estimator index was higher in the iodized salt arm compared with eFePP-QFS, but similar to eFF-QFS. Permutational multivariate analysis of variance revealed no overall group differences; however, pairwise Bray-Curtis distances from baseline were modestly greater in eFF-QFS compared with the other groups. No significant changes in relative abundance were identified. Conclusions:After 12 mo, QFS resulted no major changes in abundance of key taxa and minimal, inconsistent shifts in certain diversity metrics and relative to the iodized salt control, suggesting no adverse effects on microbiome composition among young children in this setting. Additional studies in settings with improved iron status are needed.This trial was registered at clinicaltrials.gov as NCT05166980 and at Clinical Trials Registry-India as CTRI/2022/02/040333.

MYH9-related disorders (MYH9-RD) are rare autosomal dominant disorder caused by pathogenic variants in MYH9 gene. They are characterized by macrothrombocytopenia, variably accompanied by neutrophilic inclusions, nephropathy, hearing loss, or cataracts. Due to mild symptoms and lack of awareness of the extrahematological manifestations, many cases may remain undiagnosed or misdiagnosed as immune thrombocytopenia (ITP). We report the first Indian family with three affected members with a heterozygous missense variant MYH9:c.5521G > A (p.Glu1841Lys) on targeted next generation sequencing. The index case, a 10-month-old male, was incidentally detected thrombocytopenia and giant platelets but no bleeding manifestations. Peripheral smear revealed inclusion bodies in neutrophils. His father and paternal grandfather also had macrothrombocytopenia with inclusion bodies. The father had undergone a nephrectomy for hydronephrosis, while the grandfather exhibited late-onset proteinuria and coronary artery disease. Literature review revealed this variant in 34 studies with 71 families and 133 affected individuals worldwide, who had macrothrombocytopenia and inclusion bodies but variable bleeding and extrahematological features. We describe the first Indian family with MYH9:c.5521G > A (p.Glu1841Lys) hotspot variant. The family had the characteristic macrothrombocytopenia and neutrophil inclusions. MYH9-RD should be considered in patients with macrothrombocytopenia, even without bleeding. Early molecular diagnosis is crucial for appropriate management and to avoid misclassification as ITP.

Acute myeloid leukemia (AML) with the RUNX1::RUNX1T1 fusion is typically associated with a favorable prognosis. However, when it occurs alongside systemic mastocytosis (SM), the outcome is usually adverse. This report describes a case involving a 41-year-old male diagnosed with AML harboring the RUNX1::RUNX1T1 fusion, who was initially misdiagnosed due to the lack of a thorough bone marrow examination. Although molecular testing confirmed the RUNX1::RUNX1T1 fusion, the associated mast cell component was overlooked, as the initial evaluation focused on peripheral blood. Follow-up bone marrow aspiration revealed an increased population of spindle-shaped mast cells, leading to a revised diagnosis of systemic mastocytosis with associated hematological neoplasm (SM-AHN) upon detection of a C-KIT D816Y mutation. This case emphasizes the necessity for comprehensive diagnostic evaluations, including bone marrow analysis and molecular testing for KIT mutations, to accurately identify concurrent neoplasms. While AML with RUNX1::RUNX1T1 fusion generally has a favorable prognosis, the presence of systemic mastocytosis and KIT mutations complicate the clinical landscape, requiring careful monitoring and potential modification of therapeutic strategies.

Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of inherited red cell disorders characterized by ineffective erythropoiesis, and distinctive bone marrow dyserythropoiesis. Diagnostic challenges arise from genetic heterogeneity and overlapping marrow morphology with other inherited hemolytic anemia and bone marrow failure syndromes. Over the period of 15-year, we analyzed 83 individuals from 76 families with suspected CDA to delineate the genetic basis and assess clinical implications of molecular diagnosis. Cases were enrolled following exclusion of common acquired and inherited causes of anemia and hemolysis. Bone marrow in all patients showed significant dyserythropoiesis. Genetic evaluation began with Sanger screening for the SEC23B:p.Tyr462Cys founder variant, followed by next-generation sequencing (NGS) in negative cases. Pathogenic/likely pathogenic variants were confirmed by Sanger sequencing and family studies (where available). The SEC23B:p.Tyr462Cys variant was identified in homozygous form in 35 individuals from 31 families and in heterozygous form in four families using Sanger sequencing. NGS in remaining cases revealed compound heterozygous SEC23B variants in five families and a single heterozygous variant in two families. In six families, only one SEC23B variant was detected, suggesting potential structural variants or intronic splice sites. NGS identified CDA subtypes with genotype phenotype concordance: KLF1-related CDA IV with elevated HbF, RACGAP1-associated CDA IIIb with giant multinucleated erythroblasts, and two cases with GATA1 variants presenting as X-linked thrombocytopenia with dyserythropoiesis. Phenotype-Genotype Discordance: Importantly, 17 cases were reclassified based on genetic findings, including PKLR-related pyruvate kinase deficiency (7 cases), hereditary pyropoikilocytosis (SPTA1, SPTB, 4 cases), PIEZO1-related xerocytosis (2 cases), and single cases of MTRR (Homocystinuria-megaloblastic anemia, cbl E type), HbH disease (HBA2), Diamond-Blackfan anemia (RPS19), and SAMD9L-associated anemia. These reclassified cases had significant management implications. A patient with MTRR defect responded to a change in therapy. Splenectomy in a PKLR-deficient patient led to transfusion independence in all except one where tanfusion requirement was reduced. Iron overload was noted in several CDA II transfusion-independent, requiring chelation. Five families underwent successful prenatal diagnosis. One patient with KLF1-related CDA IV underwent bone marrow transplantation with favorable outcome. This study represents the largest series of genetically characterized CDA from South Asia. It demonstrates the utility of tiered molecular testing, starting with cost-effective Sanger screening for common variant SEC23B:p.Tyr462Cys, and extending to NGS for comprehensive diagnosis. Accurate molecular characterization enabled precise therapy, avoided inappropriate interventions (e.g., splenectomy in stomatocytosis), informed genetic counseling, and facilitated prenatal and curative interventions. Our findings underscore the diverse genetic landscape of suspected CDAs in the Indian population and highlight the pivotal role of integrative diagnostics in optimizing patient care.

Background Innovative fortification solutions are needed to address micronutrient deficiencies, which remain highly prevalent among adult females in India. Objectives The objective of this trial was to evaluate the effects of quintuply-fortified salt (QFS) compared with iodized salt on the micronutrient status of nonpregnant females of reproductive age (NPFRA) in Punjab, India. Methods We conducted a double-blind, randomized, controlled, community-based trial. A total of 998 NPFRA were randomly assigned to receive: 1) QFS with iron as encapsulated ferrous fumarate, zinc, vitamin B12, folic acid, and iodine (eFF-QFS); 2) QFS with the same micronutrients, but iron as encapsulated ferric pyrophosphate plus ethylenediaminetetraacetic acid (eFePP-QFS); or 3) iodized salt. Biomarkers of micronutrient status were assessed at enrollment, 6 mo and 12 mo. Results At enrollment, the prevalence of anemia, iron deficiency, hypozincemia, vitamin B12 insufficiency, and folate insufficiency among trial participants was 47.9%, 59.7%, 35.5%, 61.5%, and 69.7%, respectively. Mean household salt disappearance, measured at monthly home visits, was 6.0 g/adult female equivalent/day [95% confidence interval (CI): 5.9, 6.1] and did not vary across groups or time. At 6 mo, the odds of vitamin B12 insufficiency, folate insufficiency, and hypozincemia were, respectively, 80% [odds ratio (OR): 0.20; 95% CI: 0.13, 0.31], 86% (OR: 0.14; 95% CI: 0.09, 0.21), and 38% (OR: 0.62; 95% CI: 0.41, 0.93) lower in the eFF-QFS compared with the iodized salt group. Effects on vitamin B12 and folate status were sustained at 12 mo, and were comparable in the eFePP-QFS compared with the iodized salt group. There was a small, marginally significant, reduction in iron deficiency in the eFF-QFS compared with the iodized salt group at 6 (OR: 0.64; 95% CI: 0.42, 0.98; P = 0.08) and 12 mo (OR: 0.58; 95% CI: 0.35, 0.95; P = 0.06), but not in the eFePP-QFS compared with the iodized salt group. There were no groupwise differences in anemia at either time point. Conclusions Multiple micronutrient salt fortification may be an effective strategy to improve micronutrient status, especially vitamin B12 and folate, among NPFRA at high risk of deficiency. Trial registration number This study was registered at clinicaltrials.gov, with NCT05166980 and at Clinical Trials Registry-India with CTRI/2022/02/040333.

INTRODUCTION:Recent World Health Organization (WHO) and International Consensus Classifications have introduced numerous molecular entities in B-lineage acute lymphoblastic leukemia (B-ALL), necessitating comprehensive genomic characterization by detecting gene fusions, expression, mutations, and exon deletions. While whole-genome plus transcriptome sequencing is the ideal strategy, it remains cost-prohibitive for routine use. This study reports a cost-effective and reasonably efficient alternate approach integrating a customized targeted hybrid capture RNA sequencing (RNAseq) into the routine workup. METHODOLOGY:A total of 95 consecutive adolescent/adult B-ALL cases negative for common chimeric gene fusions (CGF) (BCR::ABL1, KMT2A::AFF1, TCF3::PBX1, and ETV6::RUNX1) were analyzed using a customized 69-gene targeted RNAseq panel. In total, three fusion detection pipelines, the Trinity Cancer Transcriptome Analysis Toolkit (CTAT) Mutations pipeline, and the Toblerone alignment tool were employed, and the results were compared with fluorescence in situ hybridization (FISH)/multiplex ligation-dependent probe amplification (MLPA) testing. RESULTS:RNAseq identified fusions in 43% of cases (including BCR::ABL1-like: 15.8% and IGH::DUX4: 10.5%), demonstrating superior detection of cryptic intrachromosomal rearrangements. Somatic variants were detected in 30% of cases (including rat sarcoma (RAS) pathway and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) variants in 18% and 5.3% respectively), and IKZF1 deletions were detected in 25% (77% concordance with MLPA). The integration of targeted RNAseq and comprehensive bioinformatic analysis with flow-cytometry-based ploidy analysis and FISH-based IGH rearrangements helped categorize 79% of common CGF-negative B-ALL. The BCR::ABL1/BCR::ABL1-like group showed a higher frequency of pathogenic IKZF1 deletions (50% versus 21.7%; p = 0.011), measurable residual disease (92% versus 51%; p = 0.009), and poorer overall survival (8.6 versus 22.8 months; p = 0.07). DISCUSSION AND CONCLUSIONS:Effective utilization of RNAseq data by comprehensive bioinformatic analysis to test fusions, mutations, and deletions, supported by only minimal supplementary FISH testing, provides a practical, cost-effective solution for the molecular characterization of B-ALL in real-world scenarios until a single alternative and cost-effective test is available.

BACKGROUND:Women of reproductive age in India are vulnerable to multiple micronutrient deficiencies. Large-scale food fortification of staple foods and condiments offers a cost-effective approach to improving micronutrient intake. However, the impact of large-scale food fortification on the gut microbiome remains poorly understood. OBJECTIVES:This study aims to determine whether quintuply-fortified salt (QFS) alters the gut microbiome of nonpregnant women of reproductive age after 12 mo. METHODS:A double-blind, randomized, controlled community-based trial was conducted among 998 women (18-49 y) in Punjab, India. Eligible participants were randomly assigned to receive 1) QFS with iron as encapsulated ferrous fumarate (eFF), zinc, vitamin B12, folic acid, and iodine (eFF-QFS); 2) QFS with the same micronutrients, but iron as encapsulated ferric pyrophosphate (eFePP) plus ethylenediaminetetraacetic acid (eFePP-QFS); or 3) standard iodized salt. Stool samples were collected from a subsample of women at baseline and 12 mo and subjected to 16S ribosomal RNA gene sequencing. Outcomes included intervention effects on alpha diversity (Shannon index and abundance-based estimator (ACE) index) assessed via linear mixed regression models, Bray-Curtis dissimilarity (beta diversity) assessed via permutational multivariate analysis of variance (PERMANOVA), and relative abundance of Enterobacteriaceae, Lactobacillus, and Bifidobacterium, Prevotella or Streptococcus modeled using zero-inflated negative binomial mixed regression. RESULTS:Among the 129 women who provided both a baseline and 12-mo stool sample, 86 had sufficient read depth following sequencing (eFF-Q5S, n = 33; eFePP-Q5S, n = 26; iodized salt, n= 27). Neither alpha diversity nor beta diversity differed significantly at baseline or after the 12-mo intervention. There was no intervention effect on relative abundance of individual taxa (q-value > 0.05). CONCLUSIONS:QFS did not appear to alter the gut microbiome of nonpregnant women of reproductive age in Punjab, India.

Background:Accurate and precise estimates of discretionary salt intake are critical for the design of salt fortification programs and salt reduction interventions. Objectives:This study aimed to compare 4 methods of estimating discretionary salt intake among nonpregnant females of reproductive age in Punjab, India. Methods:One-day, observer-recorded, weighed food records (WFRs), household salt disappearance (HHSD) data, duplicate diet composites, and samples of household salt and milk were collected from 100 females and repeated in a subset of 40 to adjust for intraperson variation and estimate usual discretionary salt intake. Diet composites were also replicated from 40 randomly selected WFR but prepared without the addition of discretionary salt. The duplicate diet composites' sodium and iodine contents were analyzed using inductively coupled plasma (ICP)-optical emission spectrometry and ICP-mass spectrometry, respectively. The iodine content of household salt samples was analyzed using the ion-selective electrode method. The association and agreement between the WFR method, the selected reference method, and the HHSD, replicate diet (RD), and iodine methods (IMs) were explored using correlation and Bland-Altman analyses. Results:Mean ± standard deviation (SD) discretionary salt intakes according to the WFR, HHSD, RD, and IM methods were 4.7 ± 1.8 g/d, 5.7 ± 2.6 g/d, 4.1 ± 2.1 g/d, and 7.8 ± 5.3 g/d, respectively. The RD method showed the strongest correlation (ρ = 0.76; P < 0.001) and the smallest mean difference ± SD (-0.68 ± 1.25 g/d), with limits of agreement from -3.18 to 1.82 g/d, compared with the WFR method. However, the HHSD method was also moderately correlated (ρ = 0.48; P < 0.001) and showed good agreement [0.98 ± 2.12 (-3.27, 5.23) g/d] with the WFR despite lower precision. Conclusions:Although intensive to implement, the WFR and RD methods produce precise estimates of discretionary salt intake. Repeated measurements may improve the precision of the HHSD method for large population-based surveys.