Next Generation Sequencing of Red Blood Cell Antigens in Transfusion Medicine: Systematic Review and Meta-Analysis.

Molecular analysis of blood groups is important in transfusion medicine, allowing the prediction of red blood cell (RBC) antigens. Many blood banks use single nucleotide variant (SNV) based methods for blood group analysis. While this is a well-established approach, it is limited to the polymorphisms included in genotyping panels. Thus, variants that alter antigenic expression may be ignored, resulting in incorrect prediction of phenotypes. The popularization of next-generation sequencing (NGS) has led to its application in transfusion medicine, including for RBC antigens determination. The present review/meta-analysis aimed to evaluate the applicability of the NGS for the prediction of RBC antigens. A systematic review was conducted following a comprehensive literature search in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines. Studies were selected based on predefined criteria and evaluated using Strengthening the Reporting of Observational studies in Epidemiology guidelines. The characteristics and results of the studies were extracted and meta-analysis was performed to verify the agreement between results from standard molecular methods and NGS. Kell (rs8176058), Duffy (rs2814778, rs12078), or Kidd (rs1085396) alleles were selected as a model for comparisons. Additionally, results are presented for other blood group systems. Of the 864 eligible studies identified, 10 met the inclusion criteria and were selected for meta-analysis. The pooled concordance proportion for NGS compared to other methods ranged from 0.982 to 0.994. The sequencing depth coverage was identified as crucial parameters for the reliability of the results. Some studies reported difficulty in analyzing more complex systems, such as Rh and MNS, requiring the adoption of specific strategies. NGS is a technology capable of predicting blood group phenotypes and has many strengths such as the possibility of simultaneously analyzing hundred individuals and gene regions, and the ability to provide comprehensive genetic analysis, which is useful in the description of new alleles and a better understanding of the genetic basis of blood groups. The implementation of NGS in the routine of blood banks depends on several factors such as cost reduction, the availability of widely validated panels, the establishment of clear quality parameters and access to bioinformatics analysis tools that are easy to access and operate.

In deep bioinformatic characterization of a novel fumarate hydratase variant FH c.199T > G; (p.Tyr67Asp) in hereditary leiomyomatosis and renal cell carcinoma.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare, autosomal dominant tumor predisposition syndrome characterized by variable development of multiple skin and uterus leiomyomas and aggressive forms of renal cell carcinoma (RCC). Mutations in fumarate hydratase (FH), one of the proteins in homologous recombination repair, precede the development of HLRCC with high penetrance. Considering the risk of early metastasis of RCC, FH has been included in mutation screening panels. The identification of a pathogenic FH variant guides the screening for tumors in the carriers. However, variants of uncertain significance (VUS) are frequent findings, limiting the clinical value of the mutation screening. Here, we describe the associated phenotype and an in-depth, multi-step Bioinformatic evaluation of the germline FH c.199T > G (p.Tyr67 > Asp) variant segregated in an HLRCC family. Evidence for FH c.199T > G; (p.Tyr67Asp) pathogenicity includes the variant segregation with the disease in three affected family members, its absence in populational databases, and the deep evolutionary conservation of the Tyr67 residue. At the protein level, this residue substitution causes the loss of molecular bonds and ionic interactions, affecting molecular dynamics and protein stability. Considering ACMG/AMP criteria, we propose the reclassification of the FH c.199T > G; (p.Tyr67Asp) variant to "likely pathogenic". In addition, the in-depth, in silico approach used here allowed us to understand how and why FH c.199T > G; (p.Tyr67Asp) could cause HLRCC. This could help in clinical management decisions concerning the monitoring of unaffected family members having this variant.

Phenotypic variation in milk fatty acid composition and its association with stearoyl-CoA desaturase 1 (SCD1) gene polymorphisms in Gir cows.

Individual variation in milk fatty acid (FA) composition has been partially attributed to stearoyl-CoA desaturase 1 (SCD1) gene polymorphisms in taurine breeds, but much less is known for Zebu breeds. This study investigated the phenotypic variation in milk FA composition, and the influence of SCD1 variants on this trait and on milk fat desaturase indices (DI) in Gir cows. The functional impact of SCD1 variants was predicted using bioinformatics tools. Milk and blood samples were collected from 312 cows distributed in 10 herds from five states of Brazil. SCD1 variants were identified through target sequencing, and milk FA composition was determined by gas chromatography. Phenotypic variation in milk FA composition fell within the range reported for taurine breeds, with SCD18 index showing the lowest variation among the DI. Fourteen SCD1 variants were identified, six of which not previously described. Regarding the A293V polymorphism, all cows were homozygous for the C allele (coding for alanine), whereas all genotypes were detected for the second SNP affecting the 293 codon (G > A), with compelling evidence for functional effects. Significant associations (based on raw p-values) were found between this SNP and C12:0, cis-9, trans-11 CLA and short-chain FA, and between another SNP (rs523411937) and C15:0 and odd-chain linear FA. A new SNP on Chr26:21277069 was associated with trans-11 C18:1, cis-9, trans-11 CLA, C18:3 n-3 and n-3 FA. These findings indicate that SCD1 polymorphisms also contributes to the phenotypic variation in milk FA composition of Gir cows, with potential use in their breeding programmes.

Working memory and arithmetic impairments in children with FMR1 premutation and gray zone alleles.

Expansive mutations in familial mental retardation 1 (FMR1) gene have been associated with different phenotypes. Full mutations are associated with intellectual disability and autism spectrum disorder; premutations are associated with math learning difficulties and working memory impairments. In gray zone, neuropsychological development has not yet been described. Objectives: This study aimed to describe the frequency of FMR1 premutation and gray zone alleles in a school population sample representing a broad spectrum of variation in math achievement and detail school achievement and cognitive performance in the children identified with FMR1 premutation or gray zone alleles. Methods: We described a two-phase study. In the first phase, 2,195 school-age children were screened for math achievement. In the second phase, 378 children with normal intelligence were neuropsychologically assessed and genotyped for FMR1. Of these, 121 children (61 girls) performed below percentile 25 in mathematics (MD group) and 257 children (146 girls) performed above percentile 25 (control group). Results: Four pupils presented expanded alleles, one premutation and three gray zone alleles. The girl with the premutation and one boy with a gray zone allele presented impairments in working memory and arithmetic performance below percentile 6, compatible with the diagnosis of developmental dyscalculia. These children's difficulties were not associated with inaccuracy of nonsymbolic number representations or literacy impairments. Dyscalculia in these children seems to be associated mainly with working memory impairments. Conclusions: FMR1 expansions in the gray zone may contribute to dyscalculia in otherwise healthy and normally intelligent children.

Mutações expansivas no gene FMR1 têm sido associadas a diferentes fenótipos. Mutações completas estão associadas a deficiência intelectual e transtorno do espectro do autismo; pré-mutações, com dificuldades de aprendizagem de matemática e comprometimentos de memória de trabalho. Na zona cinzenta o desenvolvimento neuropsicológico ainda não foi descrito. Objetivos: Descrever a frequência de alelos pré-mutados e zona cinzenta em uma amostra escolar que representa amplo espectro de variação do desempenho em Matemática e detalhar o desempenho escolar e cognitivo em crianças identificadas com alelos pré-mutados ou zona cinzenta. Métodos: Aqui, descrevemos um estudo de duas fases. Na primeira fase, 2.195 crianças em idade escolar foram selecionadas para desempenho em Matemática. Na segunda fase, 378 crianças com inteligência normal foram avaliadas neuropsicologicamente e, em seguida, por genotipagem FMR1. Resultados: Tiveram desempenho abaixo do percentil 25 em Matemática (grupo DM) 121 crianças (61 meninas), e tiveram desempenho acima do percentil 25 (grupo controle) 257 crianças (146 meninas). Quatro alunos apresentaram alelos expandidos, sendo uma pré-mutação e três alelos da zona cinza. A menina com a pré-mutação e um menino com o alelo da zona cinza apresentaram prejuízos na memória de trabalho e desempenho aritmético abaixo do percentil 6, compatíveis com o diagnóstico de discalculia do desenvolvimento. As dificuldades dessas crianças não foram associadas à imprecisão de representações não simbólicas de números ou deficiências de alfabetização. A discalculia nessas crianças parece estar associada principalmente a deficiências da memória de trabalho. Conclusões: Em conclusão, expansões na zona cinzenta do FMR1 podem contribuir para a discalculia em crianças saudáveis com inteligência normal.

Identification and in silico characterization of structural and functional impacts of genetic variants in milk protein genes in the Zebu breeds Guzerat and Gyr.

Whole genome sequencing of bovine breeds has allowed identification of genetic variants in milk protein genes. However, functional repercussion of such variants at a molecular level has seldom been investigated. Here, the results of a multistep Bioinformatic analysis for functional characterization of recently identified genetic variants in Brazilian Gyr and Guzerat breeds is described, including predicted effects on the following: (i) evolutionary conserved nucleotide positions/regions; (ii) protein function, stability, and interactions; (iii) splicing, branching, and miRNA binding sites; (iv) promoters and transcription factor binding sites; and (v) collocation with QTL. Seventy-one genetic variants were identified in the caseins (CSN1S1, CSN2, CSN1S2, and CSN3), LALBA, LGB, and LTF genes. Eleven potentially regulatory variants and two missense mutations were identified. LALBA Ile60Val was predicted to affect protein stability and flexibility, by reducing the number the disulfide bonds established. LTF Thr546Asn is predicted to generate steric clashes, which could mildly affect iron coordination. In addition, LALBA Ile60Val and LTF Thr546Asn affect exonic splicing enhancers and silencers. Consequently, both mutations have the potential of affecting immune response at individual level, not only in the mammary gland. Although laborious, this multistep procedure for classifying variants allowed the identification of potentially functional variants for milk protein genes.

Genetic variants of VWF gene in type 2 von Willebrand disease.

INTRODUCTION: von Willebrand disease (VWD) is the most common inherited bleeding disorder. Few studies have explored the molecular basis of type 2 VWD. AIM: This study aimed to identify variants associated with type 2 VWD. METHODS: We collected clinical and laboratory data, as well as response to desmopressin and bleeding assessment tool (BAT) score in patients diagnosed with type 2 VWD. We sequenced exons 17, 18, 20 and 28 of the VWF gene. RESULTS: We identified 19 different variants in 40 unrelated patients (47.5%). Most of the variants (84.2%) were found in exon 28. A total of 10/19 variants (52.6%) were identified as "likely causative" in 17/40 patients (42.5%), according to the ISTH-SSC and EAHAD VWF gene mutations databases. Nine variants were initially identified as potentially benign. However, through analyses in silico, four of these variants were reclassified as "likely pathogenic" (Ile1380Val, Asn1435Ser, Ser1486Leu and Tyr1584Cys). Response to desmopressin was associated with three variants: Met740Ile, Arg1597Gln and Tyr1584Cys. Major bleeding was associated with variants related to VWD subtypes 2B and 2M. CONCLUSION: In conclusion, we identified 19 variants, of which 14 are "likely pathogenic" and therefore associated with VWD. We suggest a possible association of pathogenic variants with major bleeding, response to desmopressin and BAT score ≥10, although this requires further confirmation.