Comprehensive Genomic Analysis Identifies a Diverse Landscape of Sideroblastic and Nonsideroblastic Iron-Related Anemias with Novel and Pathogenic Variants in an Iron-Deficient Endemic Setting.

Inherited iron metabolism defects are possibly missed or underdiagnosed in iron-deficient endemic settings because of a lack of awareness or a methodical screening approach. Hence, we systematically evaluated anemia cases (2019 to 2021) based on clinical phenotype, normal screening tests (high-performance liquid chromatography, α gene sequencing, erythrocyte sedimentation rate, C-reactive protein, and tissue transglutaminase), and abnormal iron profile by targeted next-generation sequencing (26-gene panel) supplemented with whole-exome sequencing, multiplex ligation probe amplification/mitochondrial DNA sequencing, and chromosomal microarray. Novel variants in ALAS2, STEAP3, and HSPA9 genes were functionally validated. A total of 290 anemia cases were screened, and 41 (14%) enrolled for genomic testing as per inclusion criteria. Comprehensive genomic testing revealed pathogenic variants in 23 of 41 cases (56%). Congenital sideroblastic anemia was the most common diagnosis (14/23; 61%), with pathogenic variations in ALAS2 (n = 6), SLC25A38 (n = 3), HSPA9 (n = 2) and HSCB, SLC19A2, and mitochondrial DNA deletion (n = 1 each). Nonsideroblastic iron defects included STEAP3-related microcytic anemia (2/23; 8.7%) and hypotransferrenemia (1/23; 4.3%). A total of 6 of 22 cases (27%) revealed a non-iron metabolism gene defect on whole-exome sequencing. Eleven novel variants (including variants of uncertain significance) were noted in 13 cases. Genotype-phenotype correlation revealed a significant association of frameshift/nonsense/splice variants with lower presentation age (0.8 months versus 9 years; P < 0.01) compared with missense variants. The systematic evaluation helped uncover an inherited iron defect in 41% (17/41) of cases, suggesting the need for active screening and awareness for these rare diseases in an iron-deficient endemic population.

Women in Selected Communities of Punjab, India Have a High Prevalence of Iron, Zinc, Vitamin B12, and Folate Deficiencies: Implications for a Multiply-Fortified Salt Intervention.

Dietary intake and biomarkers of micronutrient status of 100 non-pregnant women of reproductive age (NPWRA) were assessed to determine optimal levels of iron, zinc, vitamin B12, and folic acid to include in multiply-fortified salt (MFS) that will be evaluated in an upcoming trial. Weighed food records were obtained from participants to measure intake of micronutrients and discretionary salt, and to assess adequacy using Indian Nutrient Reference Values (NRVs). Statistical modeling was used to determine optimal fortification levels to reduce inadequate micronutrient intake while limiting intake above the upper limit. Fasting blood samples were obtained to assess iron, zinc, vitamin B12, and folate status. In usual diets, inadequate intake of iron (46%), zinc (95%), vitamin B12 (83%), and folate (36%) was high. Mean intake of discretionary salt was 4.7 g/day. Prevalence estimates of anemia (37%), iron deficiency (67%), zinc deficiency (34%), vitamin B12 insufficiency (37%), and folate insufficiency (70%) were also high. Simulating the addition of optimized MFS to usual diets resulted in percentage point (pp) reductions in inadequate intake by 29 pp for iron, 76 pp for zinc, 81 pp for vitamin B12, and 36 pp for folate. MFS holds potential to reduce the burden of micronutrient deficiencies in this setting.

Impact of HFE-2 and HAMP Gene Variations on Iron Overload in Pediatric Patients with Non-Transfusion Dependent Thalassemia: A Pilot Study.

Patients with non-transfusion dependent thalassemia (NTDT) develop variable degrees of iron overload. Possible genes which may be implicated in causing iron overload are hepcidin (HAMP) and hemojuvelin (HFE). There is variable data assessing the role of c.-582Y A > G HAMP gene and H63D hotspot in HFE-1 gene in causing iron overload, while role of HFE-2 gene is undetermined. Twenty-five patients with NTDT (≥ 10 years) were assessed for iron overload. Genetic analysis for ß-globin, α-globin, HAMP, HFE-2 and C282Y and H63D hotspots in HFE-1 genes was performed. T2*MRI demonstrated elevated LIC in 48% patients. No mutations were detected in HAMP gene or HFE-1 hotspots. Four single nucleotide variations (SNV) were detected in HFE-2 gene in 4 (20%) patients, including a novel SNV, p.Gln315Arg in 2 patients in heterozygous state. This is a likely pathogenic mutation; however, in heterozygous state, it did not lead to iron overload. HAMP and HFE-2 gene variations were infrequently seen in this pilot study, with no significant impact on iron overload. Presence of SNV p.Gln315Argin HFE-2 gene needs to be evaluated in larger sample sizes in our population to determine the incidence in homozygous state and its association with iron overload. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12288-021-01442-9.

Iron Deficiency and Iron Deficiency Anemia in 3-5 months-old, Breastfed Healthy Infants.

OBJECTIVE: To assess the prevalence of iron deficiency (ID) and iron deficiency anemia (IDA) in predominantly breastfed, 3-5-mo-old infants, born at term, with a birth weight ≥ 2.5 kg. METHODS: The cross-sectional study was conducted in the outpatient department of a tertiary care center from January 2013 through December 2014. INCLUSION CRITERIA: Age: 90-180 d, exclusively/predominantly breastfed, birth weight ≥ 2.5 kg and term gestation. EXCLUSION CRITERIA: systemic illness, leucocytosis, leucopenia, thrombocytopenia, peripheral smear abnormality or iron supplementation. Blood sample was collected for complete blood count and ferritin assay. ID was defined as serum ferritin <12 µg/L. IDA was defined as ID plus Hb ≤ 10.5 g/dl. RESULTS: Two hundred ninety six infants were initially recruited; 29 declined consent; 22 had leukocytosis, leucopenia or eosinophilia; 15 had thrombocytopenia; 15 samples were hemolyzed or insufficient. Finally, 215 infants were evaluated. The male-female ratio was 1.8:1. The mean birth weight was 2.9 (0.4) kg. The mean Hb was 10.8 (1.2) g/dl. The median serum ferritin was 44 µg/L (18, 120). The prevalence of ID at 3, 4 and 5 mo of age was 5.4%, 21.4% and 36.4%, while that of IDA was 4.6%, 16.7% and 11.4%, respectively. CONCLUSIONS: The prevalence of ID at 4 and 5 mo of age in predominantly breastfed, term infants was 21.4% and 36.4%, respectively. The study generates evidence for considering iron supplementation for well-babies from 4 mo of age, instead of the currently recommended 6 mo by National Iron plus Initiative in India.

Developmental profile in children with iron deficiency anemia and its changes after therapeutic iron supplementation.

OBJECTIVE: To evaluate the developmental profile of children with iron deficiency anemia (IDA) and the changes following iron supplementation. METHODS: Study was conducted prospectively in a tertiary care teaching institution. Subjects were children aged 6 months to 5-years, with IDA, proven by hematological parameters and iron studies. Complete blood counts and iron studies were performed at the beginning and following 3-months therapy with iron. Simultaneously, development was assessed by Developmental profile II (DPII), which was interpreted using IQ equivalent (IQE) scores and 'fractional months differential' (FMD). RESULTS: Thirty five children fulfilled predetermined inclusion criteria. The mean-age was 22.3+/-13.4 months. Majority (71.4%) had moderate, while 5 (14.3%), each had mild and severe anemia. Significant developmental delay was observed in iron deficient children. Maximum delay was observed in academic and communication domains. 6 (17.2%) failed developmental screening, with IQE scores of <70. Significant improvement in DPII scores was noticed following therapy. Although some gain in IQE scores was noticed in the majority (88.6%), significant improvement (e => 10-point gain) was observed in about half (51.4%). Interpretation of DPII by FMD revealed significant improvement in all the domains as well. CONCLUSION: Children with IDA have suboptimal developmental scores. The delayed development is variably reversible following oral iron therapy. Hb =< 7 g/dl and age >24 months predicts suboptimal outcome. FMD is a useful method of interpreting DPII.

UGT1A1 gene polymorphisms in North Indian neonates presenting with unconjugated hyperbilirubinemia.

Genetic factors are implicated in pathogenesis of neonatal hyperbilirubinemia. In this nested case-control study, we determined 1) frequency of thymine-adenine (TA)n promoter polymorphism and Gly71Arg mutation in uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) gene in neonates > or =35-wk gestation presenting with bilirubin levels > or =18 mg/dL and controls, 2) interaction among (TA)n promoter polymorphism, glucose-6-phosphate dehydrogenase (G6PD) gene mutations, and peak bilirubin. The number of TA repeats was assessed by PCR-single-strand conformation polymorphism (SSCP) analysis and Gly71Arg mutation by PCR-RFLP. Fifty samples of both mutations were verified with DNA sequencing. One hundred twenty-seven neonates were enrolled (77 hyperbilirubinemics, 50 controls). The incidence of (TA)n polymorphism was higher in babies with hyperbilirubinemia [89.6% vs. 50%, OR 8.63 (95% CI, 3.2-24.1)]. Gly71Arg mutation was not found either in hyperbilirubinemics or controls. A novel polymorphism (Ala72Pro) at codon position 72 of exon 1 was detected in all 50 samples (21 hyperbilirubinemics, 29 controls), which were sequenced. Presence of variant (TA)n promoter (adjusted OR, 10.6; 95% CI, 3.3-34.2), G6PD deficiency (adjusted OR, 20.6; 95% CI, 3.6-117.3), and history of jaundice in sibling requiring phototherapy (adjusted OR, 12.6; 95% CI, 1.1-141.6) were independent risk factors for bilirubin levels > or =18 mg/dL.