Plasminogen Activator Inhibitor-1 4G/5G Promoter Polymorphism in Indian Patients with Deep Vein Thrombosis.

A single guanosine deletion/insertion (4G/5G) polymorphism in the promoter region of plasminogen activator inhibitor-1 (PAI-1) gene encoding PAI-1 protein has been investigated in deep vein thrombosis (DVT) patients. The association between PAI-1 4G/5G polymorphism and increased risk of DVT has been reported in some studies, while others have reported a lack of association. The present study aimed to investigate if the PAI-1 4G/5G polymorphism is associated with an increased risk of DVT in the Indian population and to assess its association with thrombophilic risk factors. Fifty-two adult patients with a history of chronic or recurrent DVT and 52 healthy adult controls were genotyped for PAI-1 4G/5G polymorphism. Plasma levels of PAI-1 and other thrombophilic risk factors were also measured. PAI-1 4G/5G polymorphism was not significantly associated with an increased risk of DVT. Protein C deficiency was significantly associated with the 4G/4G genotype. Patients with the 4G/4G genotype had significantly reduced PAI-1 levels as compared to the controls. PAI-1 4G/5G polymorphism did not significantly contribute to an increased risk of DVT in the Indian population. However, in the presence of thrombophilic risk factor abnormalities, the risk of DVT is increased in individuals with the 4G/4G genotype in the Indian cohort. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01660-3.

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.

The frequency and clinical outcome of mono-hit and multi-hit TP53 aberrations in newly diagnosed multiple myeloma.

We investigated the frequency and outcome of mono-hit and multi-hit TP53 aberrations [biallelic or ≥1 TP53 mutations (TP53mut) or TP53mut with variant allele frequency (VAF) ≥55%] in an Indian cohort of newly diagnosed multiple myeloma (NDMM) patients. We employed fluorescence insitu hybridisation (FISH; n=457) and targeted next-generation sequencing (NGS; n=244) on plasma cell-enriched samples. We also studied the impact of TP53mut in cases with and without TP53 deletions (TP53del). In our cohort with a median age of 60 years, TP53del and TP53mut were seen in 12.9% (n=59/457; 14-95% cells) and 10.2% (n=25/244; 30 variants; VAF 3.4-98.2%; median 38.2%) respectively. Mono-hit and multi-hit-TP53 aberrations were observed in 10.2% and 7.8%, respectively. Compared to TP53-wild-type (TP53wt), mono-hit and multi-hit TP53 aberrations were associated with significantly poorer progression-free survival (PFS) (22.6 vs 12.1 vs 9.5 months; p=0.004) and overall survival (OS) [not reached (NR) vs 13.1 vs 15.6 months respectively; p=0.024]. However, multi-hit TP53 did not significantly differ in OS/PFS compared to mono-hit cases. Compared to TP53wt, PFS and OS were significantly poorer in patients with TP53mut only (9.5 vs 22.6 months and 12.1 months vs NR, respectively; p=0.020/0.004). TP53mut retained its significance even in the presence of any Revised International Staging System (HR 2.1; 95% CI 1.1-3.8; p=0.015) for OS. The detection of additional cases with TP53 aberrations, as well as poor survival associated with the presence of mutation alone, supports TP53mut testing in NDMM at least in patients without TP53del and other high-risk cytogenetic abnormalities.

Unearthing the genotype-inhibitor phenotype association in severe haemophilia A: A north Indian cohort study.

INTRODUCTION: Various risk factors for inhibitor development in haemophilia A (HA) have been described but Indian data remains scanty. AIM: We aimed to evaluate the genetic changes in Indian HA-patients that are associated with the development of inhibitors. METHODS: All HA-patients with inhibitors who availed coagulation-laboratory services from January-2015 till December-2021 and had their samples preserved for DNA extraction were included in this study. An equal number of severity-matched HA patients without inhibitors were also included as controls. Intron 22 and intron 1 inversions in Factor VIII gene were identified using inverse-shifting-PCR. Inversion-negative patients were further assessed by targeted NGS, MLPA. RESULTS: Thirty HA-patients with inhibitors were identified. All had severe-HA. Thirty severe-HA-patients without inhibitors were also included as controls. Intron 22 inversion (63.3%) and large deletions (15%) were the commonest variants identified. There was no difference in genetic variants in patients with low and high titre inhibitors. A3, A2 and C2 were the most common domains involved in inversion-negative patients with inhibitors. However, there was no significant difference in domain involvement among inversion-negative patients with and without inhibitors. Seven novel-variants were identified, including three large deletions, one large duplication and two nonsense variants in inhibitor-positive patients, and one frameshift variant in inhibitor-negative patient. After adjusting for clinical risk-factors, large deletions were independently associated with the presence of inhibitors [aOR:6.1 (1.41-56.3)]. CONCLUSION: Intron 22 inversions are the commonest variant in Indian patients with severe-HA. Large deletions predispose to inhibitor development independent of clinical risk factors.

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.

Correction to: Genetic Spectrum in F13A1 Detected by Next-Generation Sequencing Among North Indian Patients with FXIII Deficiency.

[This corrects the article DOI: 10.1007/s12288-022-01579-1.].

Preclinical safety assessment of photoluminescent metal quantum clusters stabilized with autologous serum proteins for host specific theranostics.

Host derived serum proteome stabilised red-emitting gold quantum clusters (or Au-QC-NanoSera or QCNS) of size range ~2 nm have been synthesised in a first reported study. The host serum was taken from bovine, murine and human origins to establish the proof of concept. In-vitro biocompatibility with normal murine L929 fibroblast cells and radiosensitisation ability against PLC/PRF/5 hepatoma cells was established. A concentration dependant radiosensitisation effect of QCNS at differential γ-radiation doses was observed with almost 90% killing of cancer cells at a radiation dose of 5Gy. Acute and subacute safety, and non-immunogenicity of autologously derived QCNS was established in in-bred C57BL/6 mice. The biodistribution analysis revealed that the QCNS were effectively cleared from the body over a course of 28 days and were found to pose no major threat to the proper functioning and morphology of the mice.

Genetic Spectrum in F13A1 Detected by Next-Generation Sequencing Among North Indian Patients with FXIII Deficiency.

Purpose: The study aimed to explore the molecular defects underlying FXIII deficiency. Materials and Methods: Sixteen unrelated cases were enrolled based on the indication of the urea clot solubility test and Factor XIII-A antigen levels. Cases were further subjected to targeted next-generation sequencing (custom gene panel: F7, F8, VWF, F9, F13A1, F13B). The pathogenic/likely pathogenic variants were validated by Sanger sequencing in the patients and family members. Results: Mean age of referral to our center was 27.2 years (8 week-67 years). Consanguinity was found in only one of the 16 cases and 9 cases presented in infancy. The most common symptoms were skin bleeds (69%) and umbilical cord bleed (50%). The clot solubility test was positive in 12, inconclusive in 1, and normal in 3. Mean FXIII-A levels were 15.7 IU/dL (range 0.6 to 49.5 IU/dL). Pathogenic/likely pathogenic variants in F13A1 were found in 11 (69%). Nine cases (82%) were homozygous, and two were compound heterozygous. Total eleven variants were found of which four were missense (c.1226G>A; c.998C>T; c.631G>C; c.2134A>C); three deletion (c.521delG; c.742delA; c.1405_1408delCAAA); two nonsense (c.1112G>A; c.1127G>A) and two splice site (c.1909-1G>C; c.2045G>A). No probably pathogenic variant was found in the F13B. Conclusion: Inherited FXIII deficiency with bleeding is associated with genetic defects in predominantly the F13A1 gene. A variety of variants were seen in this cohort. A nonsense variant c.1127G>A found in three of our cases seems to be recurrent. This data will contribute to designing functional studies and antenatal testing in affected families. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01579-1.

Molecular spectrum of inherited FVII deficiency in North India revealed a recurrent variant with a founder effect.

INTRODUCTION: Inherited Factor VII (FVII) deficiency is commonest among the rare bleeding disorders. A small number of patients present in infancy with severe bleeding, and many may remain asymptomatic but detected before surgery/invasive procedures. Genetic testing may be helpful in predictive testing/prenatal diagnosis in severe cases. AIM: Characterisation of clinical and genotypic spectrum of patients with inherited FVII deficiency. METHODS: Retro-prospectively, 35 cases with prolonged prothrombin time and FVII activity (FVII:C) <50 IU/dl were subjected to targeted resequencing. After in-silico analysis, variant/s were validated by Sanger sequencing in index cases and family members. Haplotype analysis was done for F7 polymorphisms. RESULTS: Severe FVII deficiency was found in 50% of patients (FVII:C ≤1 IU/dl), and 42.9% were asymptomatic. Clinical severity assessment revealed 17% severe, 17% moderate and 22.9% patients with mild bleeds. FVII levels ranged from .3 to 38 IU/dl. Molecular analysis revealed variants in 30/35 cases, of which 17 were homozygous, 10 were compound heterozygous and 3 were heterozygous. Twelve genetic variants were identified, one promoter variant c.-30A>C; seven missense (c.215C>G, c.244T>C, c.253G>C, c.904G>A, c.961C>T, c.1109G>T, c.1211G>A), two deletions (c.21delG, c.868_870delATC), and one each of nonsense c.634C>T and splice-site variant c.316+1G>A. Recurrent variants c.1109G>T and c.215C>G were found in 17 and 8 cases, 12 of the former cases were homozygous. They had the same haplotype, indicating the founder effect in North Indians. CONCLUSION: This is the largest cohort of FVII genotyping from India, confirming heterogeneity in terms of clinical manifestations, FVII activity and zygosity of the variants with a limited genotypic phenotypic correlation.

Prenatal diagnosis for hemophilia A (intron 22 inversion) reveals a rare association with Klinefelter syndrome with diagnostic difficulties in molecular interpretation.

Hemophilia A is an X-linked recessive disorder caused by genetic abnormalities in the F8 . Klinefelter syndrome is sex chromosome aneuploidy caused by nondisjunction during meiosis in the germ cells or mitotic cell divisions in the early embryonic cells. We here report an intriguing case of a prenatal diagnosis where a rare association of hemophilia A and Klinefelter syndrome was found in a fetus. This case highlights the diagnostic difficulty where the inverse-PCR for intron 22 inversion defect leading to hemophilia A did not amplify. Indirect molecular testing was done using multiallelic extragenic variable number tandem repeat (VNTR) DXS52 (St14) and polymorphic markers. The interpretation was further complicated by the presence of Klinefelter syndrome. This case highlights the challenges faced when such rare combinations are found during prenatal diagnosis.

High Drug Loading Nanoparticles Stabilized with Autologous Serum Proteins Passively Inhibits Tumor Growth.

We report drug nanocrystals stabilized with host-specific serum proteins with high loading (∼63% w/w). The human serum derived curcumin nanoparticles (Cur-NanoSera) showed superior in vitro anticancer efficiency compared to a free drug with substantial hemocompatibility. The preadsorbed protein coating impeded further protein corona formation, even with repeated serum exposures. Acute and subacute toxicity evaluations post single and dual injections of C57BL/6 mice indicated that Cur-NanoSera showed no prominent inflammatory response or organ damage in the in-bred mice. Passive accumulation of Cur-NanoSera in tumor tissue significantly suppressed its growth in a syngeneic breast tumor model in addition to controlling tumor burden associated splenomegaly.

Quintuply-fortified salt for the improvement of micronutrient status among women of reproductive age and preschool-aged children in Punjab, India: protocol for a randomized, controlled, community-based trial.

BACKGROUND: Multiple micronutrient (MN) deficiencies remain highly prevalent among women of reproductive age (WRA) and preschool-aged children (PSC) in many areas within India. Salt is an attractive vehicle for MN fortification in this context, as it is universally consumed in fairly consistent amounts and coverage of iodized salt (IS) is 94%. The overall objective of this trial is to evaluate the nutritional impact of quintuply-fortified salt with iron in the form of encapsulated ferrous fumarate, zinc, vitamin B12, folic acid, and iodine (eFF-Q5S) vs. quintuply-fortified salt with iron in the form of ferric pyrophosphate plus EDTA, zinc, vitamin B12, folic acid, and iodine (FePP-Q5S) vs. IS for the improvement of MN status among non-pregnant WRA and PSC. METHODS: The study is a community-based, randomized, controlled trial that will be conducted in Punjab, India. 780 non-pregnant WRA 18-49 years old and 468 PSC 12-59 months old will be enrolled and assigned to one of three intervention groups. Salt will be provided to participants monthly for 12 months. Primary outcomes include changes in mean concentration of biomarkers of iron, zinc, vitamin B12, folate and iodine. Secondary outcomes include changes in the composition of the gut microbiome, and discretionary salt intake of PSC. DISCUSSION: If proven efficacious, multiply-fortified salt (MFS) has the potential to drastically reduce the burden of MN deficiencies in India, and around the world. Although effectiveness research will be needed to examine the impact of MFS under programmatic conditions, salt fortification will piggy-back on existing platforms to produce IS and doubly-fortified salt (DFS), making it possible to scale-up the intervention quickly. TRIAL REGISTRATION: Clinicaltrials.gov: NCT05166980; date of registration: December 22, 2021. Clinical Trials Registry-India: CTRI/2022/040332 and CTRI/2022/02/040333; date of registration: February 15, 2022.

Correlating clinical and laboratory diagnostic modalities in the diagnosis of epidermolysis bullosa in a resource-poor setting.

BACKGROUND: Mutational analysis and immunofluorescence antigen mapping (IFM) are recommended as the laboratory tools of choice for diagnosing EB. In the past, transmission electron microscopy (TEM) was considered the gold standard, and more recently, clinical diagnostic matrix (CDM) has shown good concordance with next-generation sequencing (NGS). METHODS: In this prospective diagnostic study, a skin biopsy was taken for TEM and IFM in consecutive patients with EB (aged >6 months) diagnosed clinically with CDM. Wherever possible, mutational analysis was done using targeted NGS. RESULTS: Of the 80 patients diagnosed with CDM, skin biopsy specimens of 42 patients were assessed using TEM, and of 59 patients using IFM. NGS was done in 39 patients. Taking NGS as the gold standard for diagnosing EB (n = 39 patients), the concordance with CDM, TEM, and IFM were estimated at 84.6% (33/39), 78.5% (11/14), and 76% (19/25) respectively. CDM showed a substantial agreement with NGS (k = 0.69, p < 0.001). CONCLUSIONS: In comparison to NGS, the highest concordance was seen with CDM followed by TEM and IFM in diagnosing major subtypes of EB.