Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

Maternal and Newborn Blood Aluminum Levels and Neurodevelopment of Infants: Is there a Need for Concern?

Aluminum is a neurotoxic element that enters the human body due to its widespread usage in daily life. It has the potential to affect the neurological development of the fetus and infant adversely. This study aimed to evaluate the relationship between maternal and umbilical cord serum aluminum level and infant neurodevelopment. Over a period of March 2018 to September 2019, we conducted a prospective cohort study; 173 Mother-new-born pairs were enrolled. Aluminum levels were measured using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The correlation with Bayley Scales of Infant Development (BSID) -3rd edition score and maternal and cord serum aluminum were assessed via linear regression model. The mean concentration of maternal and cord serum aluminum was 2.58 ± 1.14 µg/dL and 1.44 ± 0.62 µg/dL, respectively. There was a significant correlation in aluminum level between maternal and umbilical cord serum (Pearson's r = 0.591, p < 0.000). There is no significant correlation between maternal and serum aluminum level, and BSID-3rd edition (cognitive, motor, language, and social-emotion) score at the average age of 6.5 months. In conclusion, maternal and cord serum aluminum levels were significantly correlated but did not correlate with infant neurodevelopment. Thus, low serum aluminum concentration and their association with child neurodevelopment deserve further investigation longitudinally in a large cohort. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-021-01002-y.

Monitoring tools for early identification of children with developmental delay in India: an update.

Developmental screening is challenging in both developed countries and developing countries. An estimated 10% of children experience developmental delay leading to disability in India. Unfortunately, the Indian literature is limited regarding early identification and intervention of developmental delays in youngsters, given multiple barriers in implementing screening. Quick screening tools can easily be integrated into the routine work-up of infants and young children in the primary health-care system and the literature has shown the efficiency of early identification and intervention for children born with high-risk profiles. Therefore, validated screening tools may improve the early identification of developmental delay in young children, further enhancing the clinical outcome of children with developmental delays. India introduced the first development screening tool in 1991, the Baroda Development Screening Test (BDST), followed by Developmental Assessment Scale for Indian Infants (DASII), Trivandrum Development Screening Chart (TDSC). Indian researchers have adapted few development screening tools from standardized tools available from Western countries. This review highlights the different screening tools adapted in India, psychometric properties and adapted items to the gold standard, critical appraisal of most commonly used screening tools and description on strengths and limitations. The goal is to help clinicians understand the rationale behind each tool and the different domains evaluated as per the child's needs.

Critical appraisal of droplet digital polymerase chain reaction application for noninvasive prenatal testing.

Maternal-fetal medicine (FM) is currently a highly demanding branch and is gaining importance as increasing number of genetic disorders rise in incidence. Prenatal testing helps to detect such abnormalities that could affect the health status of the developing fetus like birth defects or genetic disorders. Considering the rising trend of genetic disorders, there is a need for a highly sensitive way of noninvasive prenatal testing (NIPT) that may reduce the incidence of unnecessary invasive procedures and iatrogenic fetal loss. The concept of NIPT for screening of genetic disorders is continuously evolving over the last two decades and multiple techniques have come up to utilize this in the field of FM. The crucial factor which decides the accuracy of NIPS is cell free fetal DNA (cffDNA) that is present in extremely low fraction (10%-15%) in the maternal plasma. Among the available methods, the next generation sequencing (NGS) is considered as the gold standard. However, the higher cost diminishes its utility in low-resource settings. Droplet digital Polymerase chain reaction (ddPCR), a type of digital PCR is a novel technique that is frugal, equally sensitive, less labor intensive, less time-consuming and plain algorithm dependent method for detecting cffDNA fraction. Considering these impressive attributes of ddPCR, we decided to critically review the existing literature on ddPCR for NIPT whilst highlighting the clinical utility, challenges and its advantages over NGS.

Association of newborn blood lead concentration with neurodevelopment outcome in early infancy.

BACKGROUND: In utero exposure to toxic metal substances can cause severe neurodevelopmental deficits in developing fetus and infant. METHODS: We evaluated the association of newborn umbilical cord blood lead concentration with early neurodevelopmental performance (cognitive, receptive language, expressive language, fine motor, gross motor and social-emotional development). The Bayley Scale of Infants Developments-III (BSID-III) was used to perform neurodevelopment outcomes at an average age of 6.5 months. In this prospective study, total of 167 mother-child pairs were enrolled from Western Rajasthan, India. Association between risk factors of lead contamination and newborn umbilical cord blood lead levels was observed. Multivariate regression was performed to see the association of cord blood lead level with infant neurodevelopment outcome. RESULTS: The obtained newborn umbilical cord blood lead concentration 5.0-10.5 µg/dL was negatively associated with the sub-scale score of gross motor development (ß-coefficient with 95 % CI; -0.29 (-5.0-0.11), p = 0.04). However, no associations were found with the score of cognitive, language, gross motor, and social-emotional development. The umbilical cord blood lead concentration <5.0 µg/dL was also not associated with the BSID-III scores. The mother's regular intake of calcium supplements during the antenatal period was significantly associated with a lower umbilical cord blood lead level (p-value 0.031). CONCLUSION: The data suggest that newborn umbilical cord blood lead concentration 0.5-10.5 µg/dL has a negative association with early gross motor development during infancy.

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-like Cytotoxic Nanostructures.

Recent reports have revealed the intrinsic propensity of single aromatic metabolites to undergo self-assembly and form nanostructures of amyloid nature. Hence, identifying whether aspartame, a universally consumed artificial sweetener, is inherently aggregation prone becomes an important area of investigation. Although the reports on aspartame-linked side effects describe a multitude of metabolic disorders, the mechanistic understanding of such destructive effects is largely mysterious. Since aromaticity, an aggregation-promoting factor, is intrinsic to aspartame's chemistry, it is important to know whether aspartame can undergo self-association and if such a property can predispose any cytotoxicity to biological systems. Our study finds that aspartame molecules, under mimicked physiological conditions, undergo a spontaneous self-assembly process yielding regular ß-sheet-like cytotoxic nanofibrils of amyloid nature. The resultant aspartame fibrils were found to trigger amyloid cross-seeding and become a toxic aggregation trap for globular proteins, Aß peptides, and aromatic metabolites that convert native structures to ß-sheet-like fibrils. Aspartame fibrils were also found to induce hemolysis, causing DNA damage resulting in both apoptosis and necrosis-mediated cell death. Specific spatial arrangement between aspartame molecules is predicted to form a regular amyloid-like architecture with a sticky exterior that is capable of promoting viable H-bonds, electrostatic interactions, and hydrophobic contacts with biomolecules, leading to the onset of protein aggregation and cell death. Results reveal that the aspartame molecule is inherently amyloidogenic, and the self-assembly of aspartame becomes a toxic trap for proteins and cells, exposing the bitter side of such a ubiquitously used artificial sweetener.

Strategically Designed Antifibrotic Gold Nanoparticles to Prevent Collagen Fibril Formation.

Because uncontrolled accumulation of collagen fibrils has been implicated in a series of pathologies, inhibition of collagen fibril formation has become one of the necessary strategies to target such collagen-linked complications. The presence of hydroxyproline (Hyp) at the Y position in (Gly-X-Y)n sequence pattern of collagen is known to facilitate crucial hydrophobic and hydration-linked interactions that promote collagen fibril formation. Here, to target such Hyp-mediated interactions, we have synthesized uniform, thermostable, and hemocompatible Hyp coated gold nanoparticles (AuNPsHYP) and have examined their inhibition effect on the fibril formation of type I collagen. We found that collagen fibril formation is strongly suppressed in the presence of AuNPsHYP and no such suppression effect was observed in the presence of free Hyp and control Gly-coated nanoparticles at similar concentrations. Both isothermal titration calorimetric studies and bioinformatics analysis reveal possible interaction between Hyp and (Gly-Pro-Hyp) stretches of collagen triple-helical model peptides. Further, gold nanoparticles coated with proline (AuNPsPRO) and tryptophan (AuNPsTRP) also suppressed collagen fibril formation, suggesting their ability to interfere with aromatic-proline as well as hydrophobic interactions between collagen molecules. The Hyp molecules, when surface functionalized, are predicted to interfere with the Hyp-mediated forces that drive collagen self-assembly, and such inhibition effect may help in targeting collagen linked pathologies.

Eugenol prevents amyloid formation of proteins and inhibits amyloid-induced hemolysis.

Eugenol has attracted considerable attention because of its potential for many pharmaceutical applications including anti-inflammatory, anti-tumorigenic and anti-oxidant properties. Here, we have investigated the effect of eugenol on amyloid formation of selected globular proteins. We find that both spontaneous and seed-induced aggregation processes of insulin and serum albumin (BSA) are significantly suppressed in the presence of eugenol. Isothermal titration calorimetric data predict a single binding site for eugenol-insulin complex confirming the affinity of eugenol for native soluble insulin species. We also find that eugenol suppresses amyloid-induced hemolysis. Our findings reveal the inherent ability of eugenol to stabilize native proteins and to delay the conversion of protein species of native conformation into ß-sheet assembled mature fibrils, which seems to be crucial for its inhibitory effect.

Capsaicin-Coated Silver Nanoparticles Inhibit Amyloid Fibril Formation of Serum Albumin.

We have synthesized capsaicin-coated silver nanoparticles (AgNPs(Cap)) and have tested their anti-amyloid activity, considering serum albumin (BSA) as a model protein. We found that amyloid formation of BSA was strongly suppressed in the presence of AgNPs(Cap). However, isolated capsaicin and uncapped control nanoparticles did not show such an inhibition effect. Bioinformatics analysis reveals CH-π and H-bonding interactions between capsaicin and BSA in the formation of the protein-ligand complex. These results suggest the significance of surface functionalization of nanoparticles with capsaicin, which probably allows capsaicin to effectively interact with the key residues of the amyloidogenic core of BSA.