Ancestry resolution of South Brazilians by forensic 165 ancestry-informative SNPs panel.

Forensic DNA phenotyping (FDP) includes biogeographic ancestry (BGA) inference and externally visible characteristics (EVCs) prediction directly from an evidential DNA sample as alternatives to provide valuable intelligence when conventional DNA profiling fails to achieve identification. In this context, the application of Massively Parallel Sequencing (MPS) methodologies, which enables simultaneous typing of multiple samples and hundreds of forensic markers, has been gradually implemented in forensic genetic casework. The Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) is a forensic multiplex assay consisting of 165 autosomal SNPs designed to provide biogeographic ancestry information. In this work, a sample of 250 individuals from Rio Grande do Sul (RS) State, southern Brazil, apportioned into four main population groups (African-, European-, Amerindian-, and Admixed-derived Gauchos), was evaluated with this panel, to assess the feasibility of this approach in a highly heterogeneous population. Forensic descriptive parameters estimated for each population group revealed that this panel has enough polymorphic and informative SNPs to be used as a supplementary instrument in forensic individual identification and kinship testing regardless of ethnicity. No statistically significant deviation from Hardy-Weinberg equilibrium was observed after Bonferroni correction. However, seven loci pairs displayed linkage disequilibrium in pairwise LD testing (p < 3.70 × 10-6). Interpopulation comparisons by FST analysis, MDS plot, and STRUCTURE analysis among the four RS population groups apart and along with 89 reference worldwide populations demonstrated that Admixed- and African-derived Gauchos present the highest levels of admixture and population stratification, whereas European- and Amerindian-derived exhibit a more homogeneous genetic conformation.

Contribution of Clinical and Genetic Approaches for Diagnosing 209 Index Cases With 46,XY Differences of Sex Development.

CONTEXT: Massively parallel sequencing (MPS) technologies have emerged as a first-tier approach for diagnosing several pediatric genetic syndromes. However, MPS has not been systematically integrated into the diagnostic workflow along with clinical/biochemical data for diagnosing 46,XY differences of sex development (DSD). OBJECTIVE: To analyze the contribution of phenotypic classification either alone or in association with genetic evaluations, mainly MPS, for diagnosing a large cohort of 46,XY DSD patients. DESIGN/PATIENTS: 209 nonsyndromic 46,XY DSD index cases from a Brazilian DSD center were included. Patients were initially classified into 3 subgroups according to clinical and biochemical data: gonadal dysgenesis (GD), disorders of androgen secretion/action, and DSD of unknown etiology. Molecular genetic studies were performed by Sanger sequencing and/or MPS. RESULTS: Clinical/biochemical classification into either GD or disorders of hormone secretion/action was obtained in 68.4% of the index cases. Among these, a molecular diagnosis was obtained in 36% and 96.5%, respectively. For the remainder 31.6% classified as DSD of clinically unknown etiology, a molecular diagnosis was achieved in 31.8%. Overall, the molecular diagnosis was achieved in 59.3% of the cohort. The combination of clinical/biochemical and molecular approaches diagnosed 78.9% of the patients. Clinical/biochemical classification matched with the genetic diagnosis in all except 1 case. DHX37 and NR5A1 variants were the most frequent genetic causes among patients with GD and DSD of clinical unknown etiology, respectively. CONCLUSIONS: The combination of clinical/biochemical with genetic approaches significantly improved the diagnosis of 46,XY DSD. MPS potentially decreases the complexity of the diagnostic workup as a first-line approach for diagnosing 46,XY DSD.

Analysis and comparison of the STR genotypes called with HipSTR, STRait Razor and toaSTR by using next generation sequencing data in a Brazilian population sample.

Short tandem repeats (STRs) are particularly difficult to genotype with rapid evolving next-generation sequencing (NGS) technology. Long amplicons containing repetitive sequences result in alignment and genotyping errors. Stutters arising from polymerase slippage often result in reads with additional or missing repeat copies. Many tools are available for analysis of STR markers from NGS data. This study has evaluated the concordance of the HipSTR, STRait Razor, and toaSTR tools for STR genotype calling; NGS data obtained from a highly genetically diverse Brazilian population sample have been used. We found that toaSTR can retrieve a larger number of genotypes (93.8%), whereas HipSTR (84.9%) and STRait Razor present much lower genotype calling (75.3%). Accuracy levels for genotype calling are very similar (identical genotypes ~95% and correct alleles ~ 97.5%) across the three methods. All the markers presenting the same genotype through the methods are in Hardy-Weinberg equilibrium. We found that combined match probability and combined exclusion power are 2.90 × 10-28 and 0.99999999982, respectively. Although toaSTR has varying locus-specific differences and better overall performance of toaSTR, the three programs are reliable genotyping tools. Notwithstanding, additional effort is necessary to improve the genotype calling accuracy of next-generation sequencing datasets.

Frequency distribution of HCV resistance-associated variants in infected patients treated with direct-acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) is a global public health problem. Second-generation direct-acting antivirals targeting non-structural regions on the viral genome are the cornerstone for treatment of chronic infection. However, resistance-associated variants (RAVs) have been reported to be associated with therapeutic failure. The aim of this study was to assess the frequency of variants, including RAVs, in the NS3, NS5A and NS5B regions at baseline in Brazilian patients with chronic hepatitis C with HCV genotypes 1a, 1b and 3a. METHODS: Serum samples from 13 patients were used to obtain viral RNA. Massively parallel sequencing was performed using genotype-specific amplicons and a panel of Ampliseq technology for all genotypes. RESULTS: Several non-synonymous substitutions were detected at baseline for 11 responders and pre-/post-treatment for two non-responders. HCV genotype 3a was found to have significantly more non-synonymous substitutions than HCV genotype 1 in the NS3 and NS5A regions. Analyses were conducted using quantitative and qualitative inter- and intrapatient comparisons. Variants that confer resistance to the treatment used by the patients were found in both responders and non-responders. CONCLUSIONS: A wide frequency distribution of RAVs was found at baseline, and this did not interfere with the achievement of a sustained response. Evaluation of the presence of RAVs requires additional study in order to determine clinical relevance.

Targeted massively parallel sequencing for congenital generalized lipodystrophy.

OBJECTIVE: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS). METHODS: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed. RESULTS: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in AGPAT2 and three in BSCL2. Three large homozygous deletions in AGPAT2 were identified by copy-number variant analysis. CONCLUSION: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.

Evaluation of Promega PowerSeq™ Auto/Y systems prototype on an admixed sample of Rio de Janeiro, Brazil: Population data, sensitivity, stutter and mixture studies.

Forensic DNA typing typically relies on the length-based (LB) separation of PCR products containing short tandem repeat loci (STRs). Massively parallel sequencing (MPS) elucidates an additional level of STR motif and flanking region variation. Also, MPS enables simultaneous analysis of different marker-types - autosomal STRs, SNPs for lineage and identification purposes, reducing both the amount of sample used and the turn-around-time of analysis. Therefore, MPS methodologies are being considered as an additional tool in forensic genetic casework. The PowerSeq™ Auto/Y System (Promega Corp), a multiplex forensic kit for MPS, enables analysis of the 22 autosomal STR markers (plus Amelogenin) from the PowerPlex® Fusion 6C kit and 23 Y-STR markers from the PowerPlex® Y23 kit. Population data were generated from 140 individuals from an admixed sample from Rio de Janeiro, Brazil. All samples were processed according to the manufacturers' recommended protocols. Raw data (FastQ) were generated for each indexed sample and analyzed using STRait Razor v2s and PowerSeqv2.config file. The subsequent population data showed the largest increase in expected heterozygosity (23%), from LB to sequence-based (SB) analyses at the D5S818 locus. Unreported allele was found at the D21S11 locus. The random match probability across all loci decreased from 5.9 × 10-28 to 7.6 × 10-33. Sensitivity studies using 1, 0.25, 0.062 and 0.016 ng of DNA input were analyzed in triplicate. Full Y-STR profiles were detected in all samples, and no autosomal allele drop-out was observed with 62 pg of input DNA. For mixture studies, 1 ng of genomic DNA from a male and female sample at 1:1, 1:4, 1:9, 1:19 and 1:49 proportions were analyzed in triplicate. Clearly resolvable alleles (i.e., no stacking or shared alleles) were obtained at a 1:19 male to female contributor ratio. The minus one stutter (-1) increased with the longest uninterrupted stretch (LUS) allele size reads and according to simple or compound/complex repeats. The haplotype-specific stutter rates add more information for mixed samples interpretation. These data support the use of the PowerSeqTM Auto/Y systems prototype kit (22 autosomal STR loci, 23 Y-STR loci and Amelogenin) for forensic genetics applications.

Prediction of eye and hair pigmentation phenotypes using the HIrisPlex system in a Brazilian admixed population sample.

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.

Global expression of noncoding RNome reveals dysregulation of small RNAs in patients with HTLV-1-associated adult T-cell leukemia: a pilot study.

BACKGROUND: Adult T cell lymphoma/leukemia (ATLL) is a peripheral T-cell neoplasm caused by human T-cell lymphotropic virus-1 (HTLV-1). Small RNAs (sRNAs), including microRNAs (miRNAs), play a pivotal role in the initiation and development of hematological malignancies and may represent potential therapeutic target molecules. However, little is known about how these molecules impact the pathogenesis of ATLL. In this study, we aimed to identify sRNA expression signatures associated with ATLL and to investigate their potential implication in the pathophysiology of the disease. METHODS: Small-RNAseq analysis was performed in peripheral blood mononuclear cells from HTLV-1- associated ATLL (n = 10) in comparison to asymptomatic carriers (n = 8) and healthy controls (n = 5). Sequencing was carried out using the Illumina MiSeq platform, and the deregulation of selected miRNAs was validated by real-time PCR. Pathway analyses of most deregulated miRNA were performed and their global profiling was combined with transcriptome data in ATLL. RESULTS: The sequencing identified specific sRNAs signatures associated with ATLL patients that target pathways relevant in ATLL, such as the transforming growth factor-(ßTGF-ß), Wnt, p53, apoptosis, and mitogen-activated protein kinase (MAPK) signaling cascades. Network analysis revealed several miRNAs regulating highly connected genes within the ATLL transcriptome. miR-451-3p was the most downregulated miRNA in active patients. CONCLUSIONS: Our findings shed light on the expression of specific sRNAs in HTLV-1 associated ATLL, which may represent promising candidates as biomarkers that help monitor the disease activity.

Global expression of noncoding RNome reveals dysregulation of small RNAs in patients with HTLV-1–associated adult T-cell leukemia: a pilot study

Background: Adult T cell lymphoma/leukemia (ATLL) is a peripheral T-cell neoplasm caused by human T-cell lymphotropic virus-1 (HTLV-1). Small RNAs (sRNAs), including microRNAs (miRNAs), play a pivotal role in the initiation and development of hematological malignancies and may represent potential therapeutic target molecules. However, little is known about how these molecules impact the pathogenesis of ATLL. In this study, we aimed to identify sRNA expression signatures associated with ATLL and to investigate their potential implication in the pathophysiology of the disease. Methods: Small-RNAseq analysis was performed in peripheral blood mononuclear cells from HTLV-1- associated ATLL (n = 10) in comparison to asymptomatic carriers (n = 8) and healthy controls (n = 5). Sequencing was carried out using the Illumina MiSeq platform, and the deregulation of selected miRNAs was validated by real-time PCR. Pathway analyses of most deregulated miRNA were performed and their global profiling was combined with transcriptome data in ATLL. Results: The sequencing identified specific sRNAs signatures associated with ATLL patients that target pathways relevant in ATLL, such as the transforming growth factor-(βTGF-β), Wnt, p53, apoptosis, and mitogen-activated protein kinase (MAPK) signaling cascades. Network analysis revealed several miRNAs regulating highly connected genes within the ATLL transcriptome. miR-451-3p was the most downregulated miRNA in active patients. Conclusions: Our findings shed light on the expression of specific sRNAs in HTLV-1 associated ATLL, which may represent promising candidates as biomarkers that help monitor the disease activity.

Comprehensive Genetic Analysis of 128 Candidate Genes in a Cohort With Idiopathic, Severe, or Familial Osteoporosis.

CONTEXT: The genetic bases of osteoporosis (OP), a disorder with high heritability, are poorly understood at an individual level. Cases of idiopathic or familial OP have long puzzled clinicians as to whether an actionable genetic cause could be identified. OBJECTIVE: We performed a genetic analysis of 28 cases of idiopathic, severe, or familial osteoporosis using targeted massively parallel sequencing. DESIGN: Targeted sequencing of 128 candidate genes was performed using Illumina NextSeq. Variants of interest were confirmed by Sanger sequencing or SNP array. PATIENTS AND SETTING: Thirty-seven patients in an academic tertiary hospital participated (54% male; median age, 44 years; 86% with fractures), corresponding to 28 sporadic or familial cases. MAIN OUTCOME MEASURE: The identification of rare stop-gain, indel, splice site, copy-number, or nonsynonymous variants altering protein function. RESULTS: Altogether, we identified 28 variants of interest, but only 3 were classified as pathogenic or likely pathogenic variants: COL1A2 p.(Arg708Gln), WNT1 p.(Gly169Asp), and IDUA p.(His82Gln). An association of variants in different genes was found in 21% of cases, including a young woman with severe OP bearing WNT1, PLS3, and NOTCH2 variants. Among genes of uncertain significance analyzed, a potential additional line of evidence has arisen for GWAS candidates GPR68 and NBR1, warranting further studies. CONCLUSIONS: While we hope that continuing efforts to identify genetic predisposition to OP will lead to improved and personalized care in the future, the likelihood of identifying actionable pathogenic variants in intriguing cases of idiopathic or familial osteoporosis is seemingly low.

Circulating microRNAs associated with liver fibrosis in chronic hepatitis C patients.

A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.

Targeted massively parallel sequencing for congenital generalized lipodystrophy

ABSTRACT Objective: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS). Subjects and methods: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed. Results: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in AGPAT2 and three in BSCL2. Three large homozygous deletions in AGPAT2 were identified by copy-number variant analysis. Conclusions: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.

Small RNA profiles of HTLV 1 asymptomatic carriers with monoclonal and polyclonal rearrangement of the T cell antigen receptor γ chain using massively parallel sequencing: A pilot study

In the present pilot study, massively parallel sequencing (MPS) technology was used to investigate cellular small RNA (sRNA) levels in the peripheral blood mononuclear cells (PBMCs) of human T­lymphotropic virus type I (HTLV­I) infected asymptomatic carriers with monoclonal (ASM) and polyclonal (ASP) T cell receptor (TCR) γ gene. Blood samples from 15 HTLV­I asymptomatic carriers (seven ASM and eight ASP) were tested for the clonal TCR­Î³ gene and submitted for sRNA library construction together with blood samples of five healthy controls (HCs) using Illumina sequencing platform. The sRNA­sequencing reads were aligned, annotated and profiled using various bioinformatics tools. Based on these results, possible markers were validated in the study samples by performing reverse transcription­quantitative (RT­q)PCR analysis. A total of 76 known sRNAs and 52 putative novel sRNAs were identified. Among them, 44 known and 34 potential novel sRNAs were differentially expressed in the ASM and ASP libraries compared with HCs. In addition, 10 known sRNAs were exclusively dysregulated in the ASM group and one (transfer RNA 65) was significantly upregulated in the ASP group. Homo sapiens (hsa) microRNA (miRNA/mir)­23a­3p, ­28­5p, hsa­let­7e­5p and hsa­mir­28­3p and ­361­5p were the most abundantly upregulated mature miRNAs and hsa­mir­363­3p, ­532­5p, ­106a­5p, ­25­3p and ­30e­5p were significantly downregulated miRNAs (P<0.05) with a >2­fold difference between the ASM and ASP groups compared with HCs. Based on these results, hsa­mir­23a­3p and ­363­3p were selected for additional validation. However, the quantification of these two miRNAs using RT­qPCR did not provide any significant differences. While the present study failed to identify predictive sRNA markers to distinguish between ASM and ASP, the MPS results revealed differential sRNA expression profiles in the PBMCs of HTLV­1 asymptomatic carriers (ASM and ASP) compared with HCs

Small RNA profiles of HTLV-1 asymptomatic carriers with monoclonal and polyclonal rearrangement of the T-cell antigen receptor γ-chain using massively parallel sequencing: A pilot study.

In the present pilot study, massively parallel sequencing (MPS) technology was used to investigate cellular small RNA (sRNA) levels in the peripheral blood mononuclear cells (PBMCs) of human T-lymphotropic virus type I (HTLV-I) infected asymptomatic carriers with monoclonal (ASM) and polyclonal (ASP) T cell receptor (TCR) γ gene. Blood samples from 15 HTLV-I asymptomatic carriers (seven ASM and eight ASP) were tested for the clonal TCR-γ gene and submitted for sRNA library construction together with blood samples of five healthy controls (HCs) using Illumina sequencing platform. The sRNA-sequencing reads were aligned, annotated and profiled using various bioinformatics tools. Based on these results, possible markers were validated in the study samples by performing reverse transcription-quantitative (RT-q)PCR analysis. A total of 76 known sRNAs and 52 putative novel sRNAs were identified. Among them, 44 known and 34 potential novel sRNAs were differentially expressed in the ASM and ASP libraries compared with HCs. In addition, 10 known sRNAs were exclusively dysregulated in the ASM group and one (transfer RNA 65) was significantly upregulated in the ASP group. Homo sapiens (hsa) microRNA (miRNA/mir)-23a-3p, -28-5p, hsa-let-7e-5p and hsa-mir-28-3p and -361-5p were the most abundantly upregulated mature miRNAs and hsa-mir-363-3p, -532-5p, -106a-5p, -25-3p and -30e-5p were significantly downregulated miRNAs (P<0.05) with a >2-fold difference between the ASM and ASP groups compared with HCs. Based on these results, hsa-mir-23a-3p and -363-3p were selected for additional validation. However, the quantification of these two miRNAs using RT-qPCR did not provide any significant differences. While the present study failed to identify predictive sRNA markers to distinguish between ASM and ASP, the MPS results revealed differential sRNA expression profiles in the PBMCs of HTLV-1 asymptomatic carriers (ASM and ASP) compared with HCs.

The molecular landscape of osteogenesis imperfecta in a Brazilian tertiary service cohort.

We have sought the molecular diagnosis of OI in 38 Brazilian cases through targeted sequencing of 15 candidate genes. While 71% had type 1 collagen-related OI, defects in FKBP10, PLOD2 and SERPINF1, and a potential digenic P3H1/WNT1 interaction were prominent causes of OI in this underrepresented population. INTRODUCTION: Defects in type 1 collagen reportedly account for 85-90% of osteogenesis imperfecta (OI) cases, but most available molecular data has derived from Sanger sequencing-based approaches in developed countries. Massively parallel sequencing (MPS) allows for systematic and comprehensive analysis of OI genes simultaneously. Our objective was to obtain the molecular diagnosis of OI in a single Brazilian tertiary center cohort. METHODS: Forty-nine individuals (84% adults) with a clinical diagnosis of OI, corresponding to 30 sporadic and 8 familial cases, were studied. Sixty-three percent had moderate to severe OI, and consanguinity was common (26%). Coding regions and 25-bp boundaries of 15 OI genes (COL1A1, COL1A2, IFITM5 [plus 5'UTR], SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, PLOD2, BMP1, SP7, TMEM38B, WNT1, CREB3L1) were analyzed by targeted MPS and variants of interest were confirmed by Sanger sequencing or SNP array. RESULTS: A molecular diagnosis was obtained in 97% of cases. COL1A1/COL1A2 variants were identified in 71%, whereas 26% had variants in other genes, predominantly FKBP10, PLOD2, and SERPINF1. A potential digenic interaction involving P3H1 and WNT1 was identified in one case. Phenotypic variability with collagen defects could not be explained by evident modifying variants. Four consanguineous cases were associated to heterozygous COL1A1/COL1A2 variants, and two nonconsanguineous cases had compound PLOD2 heterozygosity. CONCLUSIONS: Novel disease-causing variants were identified in 29%, and a higher proportion of non-collagen defects was seen. Obtaining a precise diagnosis of OI in underrepresented populations allows expanding our understanding of its molecular landscape, potentially leading to improved personalized care in the future.

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing.

Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFKMR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twenty-three STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples.

Small RNA profiles of HTLV-1 asymptomatic carriers with monoclonal and polyclonal rearrangement of the t-cell antigen receptor γ-chain using massively parallel sequencing: a pilot study

In the present pilot study, massively parallel sequencing (MPS) technology was used to investigate cellular small RNA (sRNA) levels in the peripheral blood mononuclear cells (PBMCs) of human T‑lymphotropic virus type I (HTLV‑I) infected asymptomatic carriers with monoclonal (ASM) and polyclonal (ASP) T cell receptor (TCR) γ gene. Blood samples from 15 HTLV‑I asymptomatic carriers (seven ASM and eight ASP) were tested for the clonal TCR‑γ gene and submitted for sRNA library construction together with blood samples of five healthy controls (HCs) using Illumina sequencing platform. The sRNA‑sequencing reads were aligned, annotated and profiled using various bioinformatics tools. Based on these results, possible markers were validated in the study samples by performing reverse transcription‑quantitative (RT‑q)PCR analysis. A total of 76 known sRNAs and 52 putative novel sRNAs were identified. Among them, 44 known and 34 potential novel sRNAs were differentially expressed in the ASM and ASP libraries compared with HCs. In addition, 10 known sRNAs were exclusively dysregulated in the ASM group and one (transfer RNA 65) was significantly upregulated in the ASP group. Homo sapiens (hsa) microRNA (miRNA/mir)‑23a‑3p, ‑28‑5p, hsa‑let‑7e‑5p and hsa‑mir‑28‑3p and ‑361‑5p were the most abundantly upregulated mature miRNAs and hsa‑mir‑363‑3p, ‑532‑5p, ‑106a‑5p, ‑25‑3p and ‑30e‑5p were significantly downregulated miRNAs (P<0.05) with a >2‑fold difference between the ASM and ASP groups compared with HCs. Based on these results, hsa‑mir‑23a‑3p and ‑363‑3p were selected for additional validation. However, the quantification of these two miRNAs using RT‑qPCR did not provide any significant differences. While the present study failed to identify predictive sRNA markers to distinguish between ASM and ASP, the MPS results revealed differential sRNA expression profiles in the PBMCs of HTLV‑1 asymptomatic carriers (ASM and ASP) compared with HCs.

Osteogénesis imperfecta tipo IV originada en una rara variante de cambio de sentido en COL1A2 / Osteogenesis imperfecta type IV originated in a rare variant of change of direction in COL1A2

Resumen La osteogénesis imperfecta es una rara anomalía genética que se carac teriza por una baja masa ósea y susceptibilidad aumentada a fracturas. La mayoría de los casos se asocian a variantes en los genes COL1A1 y COL1A2 que codifican para el colágeno tipo I. Se ha clasificado en cua tro tipos, siendo el tipo IV el menos frecuente. Se presenta un caso de osteogénesis imperfecta tipo IV en una niña de seis meses, quien tenía escleras azules y acortamiento bilateral del fémur y desviación en varo de la tibia. Las radiografías mostraron desproporción craneofacial y hue sos wormianos, fusión atlanto-odontoidea; luxación coxo-femoral bila teral congénita, acortamiento y desviación del fémur bilateral, fractura antigua en fémur derecho y osteopenia generalizada. Se realizó panel molecular que incluyó los genes ALPL, COL1A1, COL1A2 e IFITM5, mos trando en COL1A2 una transición en heterocigosis de guanina a adenina (c.2531G>A), cambio asociado con osteogénesis imperfecta. El objetivo de este reporte es brindar información sobre la presentación clínica, los métodos diagnósticos y las posibilidades terapéuticas de una rara enfer medad genética.

Abstract Osteogenesis imperfecta is a rare genetic anomaly characterized by low bone mass and increased susceptibility to fractures. The majority of cases are associated with variants in the COL1A1 and COL1A2 genes that code for type I collagen. It has been classified into four types, with type IV being the least frequent. We present a case of osteogenesis imperfecta type IV in a six-month-old girl, who had blue scleras and bilateral shortening of the femur and varus deviation of the tibia. X-rays showed craniofacial disproportion and wormian bones, atlanto-odontoid fusion; bilateral congenital bilateral coxo-femoral dislocation, shortening and deviation of the bilateral femur, old fracture in the right femur and generalized osteopenia. A molecular panel was carried out that included the ALPL, COL1A1, COL1A2 and IFITM5 genes, showing in COL1A2 a transition in guanine to adenine heterozygosis (c.2531G>A), a change associated with osteogenesis imperfecta. The objective of this report is to provide information on the clinical presentation, diagnostic methods and therapeutic possibilities of a rare genetic disease.

Genetic diagnosis of congenital hypopituitarism by a target gene panel: novel pathogenic variants in GLI2, OTX2 and GHRHR.

AIM: Congenital hypopituitarism has an incidence of 1:3500-10,000 births and is defined by the impaired production of pituitary hormones. Early diagnosis has an impact on management and genetic counselling. The clinical and genetic heterogeneity of hypopituitarism poses difficulties to select the order of genes to analyse. The objective of our study is to screen hypopituitarism genes (candidate and previously related genes) simultaneously using a target gene panel in patients with congenital hypopituitarism. METHODS: Screening of 117 subjects with congenital hypopituitarism for pathogenic variants in 26 genes associated with congenital hypopituitarism by massively parallel sequencing using a customized target gene panel. RESULTS: We found three novel pathogenic variants in OTX2 c.295C>T:p.Gln99*, GLI2 c.1681G>T:p.Glu561* and GHRHR c.820_821insC:p.Asp274Alafs*113, and the previously reported variants in GHRHR c.57+1G>A and PROP1 [c.301_302delAG];[c.109+1G>A]. CONCLUSIONS: Our results indicate that a custom-designed panel is an efficient method to screen simultaneously variants of biological and clinical relevance for congenital GH deficiency. A genetic diagnosis was possible in 5 out of 117 (4%) patients of our cohort. We identified three novel pathogenic variants in GHRHR, OTX2 and GLI2 expanding the spectrum of variants associated with congenital hypopituitarism.

Applicability of the SNPforID 52-plex panel for human identification and ancestry evaluation in a Brazilian population sample by next-generation sequencing.

SNP analysis is of paramount importance in forensic genetics. The development of new technologies in next-generation sequencing allowed processing a large number of markers in various samples simultaneously. Although SNPs are less informative than STRs, they present lower mutation rates and perform better when using degraded samples. Some SNP systems were developed for forensic usage, such as the SNPforID 52-plex, from the SNPforID Consortium, containing 52 bi-allelic SNPs for human identification. In this paper we evaluated the informativeness of this system in a Brazilian population sample (n = 340). DNA libraries were prepared using a customized HaloPlex Target Enrichment System kit (Agilent Technologies, Inc.) and sequenced in the MiSeq Personal Sequencer platform (Illumina Inc.). The methodology presented here allowed the analysis of 51 out of 52 SNPforID markers. Allele frequencies and forensic parameters were estimated, revealing high informativeness: the combined match probability and power of exclusion were 6.48 × 10-21 and 0.9997, respectively. Population admixture analysis indicates high European contribution (more than 70%) and low Amerindian contribution (less than 10%) in our population, while individual admixture analyses were consistent with the majority of individuals presenting high European contribution. This study demonstrates that the 52-plex kit is suitable for forensic cases in a Brazilian population, presenting results comparable with those obtained using a 16 STR panel.