Massive Parallel DNA Sequencing Research Articles

Detailed Clinical Features of PTPRQ-Associated Hearing Loss Identified in a Large Japanese Hearing Loss Cohort.

The PTPRQ gene has been identified as one of the genes responsible for non-syndromic sensorineural hearing loss (SNHL), and assigned as DFNA73 and DFNB84. To date, about 30 causative PTPRQ variants have been reported to cause SNHL. However, the detailed clinical features of PTPRQ-associated hearing loss (HL) remain unclear. In this study, 15,684 patients with SNHL were enrolled and genetic analysis was performed using massively parallel DNA sequencing (MPS) for 63 target deafness genes. We identified 17 possibly disease-causing PTPRQ variants in 13 Japanese patients, with 15 of the 17 variants regarded as novel. The majority of variants identified in this study were loss of function. Patients with PTPRQ-associated HL mostly showed congenital or childhood onset. Their hearing levels at high frequency deteriorated earlier than that at low frequency. The severity of HL progressed from moderate to severe or profound HL. Five patients with profound or severe HL received cochlear implantation, and the postoperative sound field threshold levels and discrimination scores were favorable. These findings will contribute to a greater understanding of the clinical features of PTPRQ-associated HL and may be relevant in clinical practice.

Massive Parallel DNA Sequencing of Patients with Inherited Cardiomyopathies in Cyprus and Suggestion of Digenic or Oligogenic Inheritance.

Inherited cardiomyopathies represent a highly heterogeneous group of cardiac diseases. DNA variants in genes expressed in cardiomyocytes cause a diverse spectrum of cardiomyopathies, ultimately leading to heart failure, arrythmias, and sudden cardiac death. We applied massive parallel DNA sequencing using a 72-gene panel for studying inherited cardiomyopathies. We report on variants in 25 families, where pathogenicity was predicted by different computational approaches, databases, and an in-house filtering analysis. All variants were validated using Sanger sequencing. Familial segregation was tested when possible. We identified 41 different variants in 26 genes. Analytically, we identified fifteen variants previously reported in the Human Gene Mutation Database: twelve mentioned as disease-causing mutations (DM) and three as probable disease-causing mutations (DM?). Additionally, we identified 26 novel variants. We classified the forty-one variants as follows: twenty-eight (68.3%) as variants of uncertain significance, eight (19.5%) as likely pathogenic, and five (12.2%) as pathogenic. We genetically characterized families with a cardiac phenotype. The genetic heterogeneity and the multiplicity of candidate variants are making a definite molecular diagnosis challenging, especially when there is a suspicion of incomplete penetrance or digenic-oligogenic inheritance. This is the first systematic study of inherited cardiac conditions in Cyprus, enabling us to develop a genetic baseline and precision cardiology.

Molecular and phenotypic characteristics influencing the degree of cytoreduction in high-grade serous ovarian carcinomas.

High-grade serous ovarian carcinoma (HGSOC) is the deadliest ovarian cancer subtype, and survival relates to initial cytoreductive surgical treatment. The existing tools for surgical outcome prediction remain inadequate for anticipating the outcomes of the complex relationship between tumour biology, clinical phenotypes, co-morbidity and surgical skills. In this genotype-phenotype association study, we combine phenotypic markers with targeted DNA sequencing to discover novel biomarkers to guide the surgical management of primary HGSOC. Primary tumour tissue samples (n = 97) and matched blood from a phenotypically well-characterised treatment-naïve HGSOC patient cohort were analysed by targeted massive parallel DNA sequencing (next generation sequencing [NGS]) of a panel of 360 cancer-related genes. Association analyses were performed on phenotypic traits related to complete cytoreductive surgery, while logistic regression analysis was applied for the predictive model. The positive influence of complete cytoreductive surgery (R0) on overall survival was confirmed (p = 0.003). Before surgery, low volumes of ascitic fluid, lower CA125 levels, higher platelet counts and relatively lower clinical stage at diagnosis were all indicators, alone and combined, for complete cytoreduction (R0). Mutations in either the chromatin remodelling SWI_SNF (p = 0.036) pathway or the histone H3K4 methylation pathway (p = 0.034) correlated with R0. The R0 group also demonstrated higher tumour mutational burden levels (p = 0.028). A predictive model was developed by combining two phenotypes and the mutational status of five genes and one genetic pathway, enabling the prediction of surgical outcomes in 87.6% of the cases in this cohort. Inclusion of molecular biomarkers adds value to the pre-operative stratification of HGSOC patients. A potential preoperative risk stratification model combining phenotypic traits and single-gene mutational status is suggested, but the set-up needs to be validated in larger cohorts.

Testicular Juvenile Granulosa Cell Tumors Demonstrate Recurrent Loss of Chromosome 10 and Absence of Molecular Alterations Described in Ovarian Counterparts

Testicular juvenile granulosa cell tumors (JGCTs) are a rare type of sex cord–stromal tumor, accounting for <5% of all neoplasms of the prepubertal testis. Previous reports have demonstrated sex chromosome anomalies in a small subset of cases, but the molecular alterations associated with JGCTs remain largely undescribed. We evaluated 18 JGCTs using massive parallel DNA and RNA sequencing panels. The median patient age was <1 month (range, newborn to 5 months). The patients presented with scrotal or intra-abdominal masses/enlargement, and all underwent radical orchiectomy (17 unilateral and 1 bilateral). The median tumor size was 1.8 cm (range, 1.3-10.5 cm). Histologically, the tumors were purely cystic/follicular or mixed (ie, solid and cystic/follicular). All cases were predominantly epithelioid, with 2 exhibiting prominent spindle cell components. Nuclear atypia was mild or absent, and the median number of mitoses was 0.4/mm2 (range, 0-10/mm2). Tumors frequently expressed SF-1 (11/12 cases, 92%), inhibin (6/7 cases, 86%), calretinin (3/4 cases, 75%), and keratins (2/4 cases, 50%). Single-nucleotide variant analysis demonstrated the absence of recurrent mutations. RNA sequencing did not detect gene fusions in 3 cases that were sequenced successfully. Recurrent monosomy 10 was identified in 8 of 14 cases (57%) with interpretable copy number variant data, and multiple whole-chromosome gains were present in the 2 cases with significant spindle cell components. This study demonstrated that testicular JGCTs harbor recurrent loss of chromosome 10 and lack the GNAS and AKT1 variants described in their ovarian counterparts.

Methylation classifiers: Brain tumors, sarcomas, and what's next.

Tumor classification has evolved over the last decades with technical progress contributing much to our current concepts. Among diagnostic hallmarks, novelties were immunostaining, Fluorescence in situ hybridization, Sanger sequencing followed by massive parallel DNA sequencing, and recently, epigenetic analyses have entered the stage. Although each of these techniques was revolutionary and, in some way, also disruptive in certain diagnostic fields, it took years to decades for broad implementation into standard pathological-diagnostic algorithms. In contrast, DNA methylation profiling has been accepted in short time as a game changer with lasting impact on brain tumor classification and with potential for classification of other tumor types. This review provides a brief introduction in DNA methylation-based tumor classification. We present why DNA methylation signatures are attractive diagnostic biomarkers, discuss present achievements and future aims and explain the integration of methylation-based classifiers in diagnostic procedure. Finally, we provide an outlook on the challenges and opportunities associated with DNA methylation-based tumor profiling.

Validation of amplicon-based next generation sequencing panel for second-tier test in newborn screening for inborn errors of metabolism

Abstract Objectives Next generation sequencing (NGS) technology has allowed cost-effective massive parallel DNA sequencing. To evaluate the utility of NGS for newborn screening (NBS) of inborn errors of metabolism (IEM), a custom panel was designed to target 87 disease-related genes. The pilot study was primarily proposed for second-tier testing under the NBSIEM program in Hong Kong. Methods The validation of the panel was performed with two reference genomes and an external quality assurance (EQA) sample. Sequencing libraries were synthesized with amplicon-based approach. The libraries were pooled, spiked-in with 2% PhiX DNA as technical control, for 16-plex sequencing runs. Sequenced reads were analyzed using a commercially available pipeline. Results The average target region coverage was 208× and the fraction of region with target depth ≥20× was 95.7%, with a sensitivity of 91.2%. There were 85 out of 87 genes with acceptable coverage, and EQA result was satisfactory. The turnaround time from DNA extraction to completion of variant calling and quality control (QC) procedures was 2.5 days. Conclusions The NGS approach with the amplicon-based panel has been validated for analytical performance and is suitable for second-tier NBSIEM test.

Application of Massive Parallel DNA Sequencing to Diagnose Drug-Resistant Epilepsy in Children

Application of Massive Parallel DNA Sequencing to Diagnose Drug-Resistant Epilepsy in Children

Microbiome analysis, the immune response and transplantation in the era of next generation sequencing

The human gastrointestinal tract, skin and mucosal surfaces are inhabited by a complex system of bacteria, viruses, fungi, archaea, protists, and eukaryotic parasites with predominance of bacteria and bacterial viruses (bacteriophages). Collectively these microbes form the microbiota of the microecosystem of humans. Recent advancement in technologies for nucleic acid isolation from various environmental samples, feces and body secretions and advancements in shotgun throughput massive parallel DNA and RNA sequencing along with 16S ribosomal gene sequencing have unraveled the identity of otherwise unknown microbial entities constituting the human microecosystem. The improved transcriptome analysis, technological developments in biochemical analytical methods and availability of complex bioinformatics tools have allowed us to begin to understand the metabolome of the microbiome and the biochemical pathways and potential signal transduction pathways in human cells in response to microbial infections and their products. Also, developments in human whole genome sequencing, targeted gene sequencing of histocompatibility genes and other immune response associated genes by Next Generation Sequencing (NGS) have allowed us to have a better conceptualization of immune responses, and alloimmune responses. These modern technologies have enabled us to dive into the intricate relationship between commensal symbiotic and pathogenic microbiome and immune system. For the most part, the commensal symbiotic microbiota helps to maintain normal immune homeostasis besides providing healthy nutrients, facilitating digestion, and protecting the skin, mucosal and intestinal barriers. However, changes in diets, administration of therapeutic agents like antibiotics, chemotherapeutic agents, immunosuppressants etc. along with certain host factors including human histocompatibility antigens may alter the microbial ecosystem balance by causing changes in microbial constituents, hierarchy of microbial species and even dysbiosis. Such alterations may cause immune dysregulation, breach of barrier protection and lead to immunopathogenesis rather than immune homeostasis. The effects of human microbiome on immunity, health and disease are currently under intense research with cutting edge technologies in molecular biology, biochemistry, and bioinformatics along with tremendous ability to characterize immune response at single cell level. This review will discuss the contemporary status on human microbiome immune system interactions and their potential effects on health, immune homeostasis and allograft transplantation.

Sequencing Cell-free Fetal DNA in Pregnant Women With GCK-MODY.

Individuals with monogenic diabetes due to inactivating glucokinase (GCK) variants typically do not require treatment, except potentially during pregnancy. In pregnancy, fetal GCK genotype determines whether treatment is indicated, but noninvasive methods are not clinically available. This work aims to develop a method to determine fetal GCK genotype noninvasively using maternal cell-free fetal DNA. This was a proof-of-concept study involving 3 pregnant women with a causal GCK variant that used information from 1) massive parallel sequencing of maternal plasma cell-free DNA, 2) direct haplotype sequences of maternal genomic DNA, and 3) the paternal genotypes to estimate relative haplotype dosage of the pathogenic variant-linked haplotype. Statistical testing of variant inheritance was performed using a sequential probability ratio test (SPRT). In each of the 3 cases, plasma cell-free DNA was extracted once between gestational weeks 24 and 36. The fetal fraction of cell-free DNA ranged from 21.8% to 23.0%. Paternal homozygous alleles that were identical to the maternal GCK variant-linked allele were not overrepresented in the cell-free DNA. Paternal homozygous alleles that were identical to the maternal wild-type-linked allele were significantly overrepresented. Based on the SPRT, we predicted that all 3 cases did not inherit the GCK variant. Postnatal infant genotyping confirmed our prediction in each case. We have successfully implemented a noninvasive method to predict fetal GCK genotype using cell-free DNA in 3 pregnant women carrying an inactivating GCK variant. This method could guide tailoring of hyperglycemia treatment in pregnancies of women with GCK monogenic diabetes.

Identification of a Novel Copy Number Variation of EYA4 Causing Autosomal Dominant Non-syndromic Hearing Loss.

Eyes absent 4 (EYA4) is the causative gene of autosomal dominant non-syndromic hereditary hearing loss, DFNA10. We aimed to identify a copy number variation of EYA4 in a non-syndromic sensory neural hearing loss pedigree. A Japanese family showing late-onset and progressive hearing loss was evaluated. A pattern of autosomal dominant inheritance of hearing loss was recognized in the pedigree. No cardiac disease was observed in any of the individuals. Targeted exon sequencing was performed using massively parallel DNA sequencing (MPS) analysis. Scanning of the array comparative genomic hybridization (aCGH) was completed and the copy number variation (CNV) data from the aCGH analysis was confirmed by matching all CNV calls with MPS analysis. Breakpoint detection was performed by whole-genome sequencing and direct sequencing. Sequencing results were examined, and co-segregation analysis of hearing loss was completed. We identified a novel hemizygous indel that showed CNV in the EYA4 gene from the position 133,457,057 to 133,469,892 on chromosome 6 (build GRCh38/hg38) predicted as p.(Val124_Pro323del), and that was segregated with post-lingual and progressive autosomal dominant sensorineural hearing loss by aCGH analysis. Based on the theory of genotype-phenotype correlation with EYA4 mutations in terms of hearing loss and comorbid dilated cardiomyopathy, the region of amino acids 124 to 343 is hypothesized not to be the pathogenic region causing dilated cardiomyopathy. Additionally, the theory of genotype-phenotype correlation about the prevalence of dilated cardiomyopathy is thought to be rejected because of no correlation of deleted amino acid region with the prevalence of dilated cardiomyopathy. These results will help expand the research on both the coordination of cochlear transcriptional regulation and normal cardiac gene regulation via EYA4 transcripts and provide information on the genotype-phenotype correlations of DFNA10 hearing loss.

Massive parallel DNA sequencing for the diagnosis of drug-resistant epilepsy in children

Massive parallel DNA sequencing for the diagnosis of drug-resistant epilepsy in children

Ultra-Deep Massive Parallel Sequencing of Plasma Cell-Free DNA Enables Large-Scale Profiling of Driver Mutations in Vietnamese Patients With Advanced Non-Small Cell Lung Cancer.

Population-specific profiling of mutations in cancer genes is of critical importance for the understanding of cancer biology in general as well as the establishment of optimal diagnostics and treatment guidelines for that particular population. Although genetic analysis of tumor tissue is often used to detect mutations in cancer genes, the invasiveness and limited accessibility hinders its application in large-scale population studies. Here, we used ultra-deep massive parallel sequencing of plasma cell free DNA (cfDNA) to identify the mutation profiles of 265 Vietnamese patients with advanced non-small cell lung cancer (NSCLC). Compared to a cohort of advanced NSCLC patients characterized by sequencing of tissue samples, cfDNA genomic testing, despite lower mutation detection rates, was able to detect major mutations in tested driver genes that reflected similar mutation composition and distribution pattern, as well as major associations between mutation prevalence and clinical features. In conclusion, ultra-deep sequencing of plasma cfDNA represents an alternative approach for population-wide genetic profiling of cancer genes where recruitment of patients is limited to the accessibility of tumor tissue site.

Exploring the Molecular Aetiology of Preeclampsia by Massive Parallel Sequencing of DNA.

This manuscript aims to review (for the first time) studies describing NGS sequencing of preeclampsia (PE) women's DNA. Describing markers for the early detection of PE is an essential task because, although associated molecular dysfunction begins early on during pregnancy, the disease's clinical signs usually appear late in pregnancy. Although several biochemical biomarkers have been proposed, their use in clinical environments is still limited, thereby encouraging research into PE's genetic origin. Hundreds of genes involved in numerous implantation- and placentation-related biological processes may be coherent candidates for PE aetiology. Next-generation sequencing (NGS) offers new technical possibilities for PE studying, as it enables large genomic regions to be analysed at affordable cost. This technique has facilitated the description of genes contributing to the molecular origin of a significant amount of monogenic and complex diseases. Regarding PE, NGS of DNA has been used in familial and isolated cases, thereby enabling new genes potentially related to the phenotype to be proposed. For a better understanding of NGS, technical aspects, applications and limitations are presented initially. Thereafter, NGS studies of DNA in familial and non-familial cases are described, including pitfalls and positive findings. The information given here should enable scientists and clinicians to analyse and design new studies permitting the identification of novel clinically useful molecular PE markers.

Clinical Characteristics and In Vitro Analysis of MYO6 Variants Causing Late-Onset Progressive Hearing Loss.

MYO6 is known as a genetic cause of autosomal dominant and autosomal recessive inherited hearing loss. In this study, to clarify the frequency and clinical characteristics of hearing loss caused by MYO6 gene mutations, a large-scale genetic analysis of Japanese patients with hearing loss was performed. By means of massively parallel DNA sequencing (MPS) using next-generation sequencing for 8074 Japanese families, we found 27 MYO6 variants in 33 families, 22 of which are novel. In total, 2.40% of autosomal dominant sensorineural hearing loss (ADSNHL) in families in this study (32 out of 1336) was found to be caused by MYO6 mutations. The present study clarified that most cases showed juvenile-onset progressive hearing loss and their hearing deteriorated markedly after 40 years of age. The estimated hearing deterioration was found to be 0.57 dB per year; when restricted to change after 40 years of age, the deterioration speed was accelerated to 1.07 dB per year. To obtain supportive evidence for pathogenicity, variants identified in the patients were introduced to MYO6 cDNA by site-directed mutagenesis and overexpressed in epithelial cells. They were then assessed for their effects on espin1-induced microvilli formation. Cells with wildtype myosin 6 and espin1 co-expressed created long microvilli, while co-expression with mutant constructs resulted in severely shortened microvilli. In conclusion, the present data clearly showed that MYO6 is one of the genes to keep in mind with regard to ADSNHL, and the molecular characteristics of the identified gene variants suggest that a possible pathology seems to result from malformed stereocilia of the cochlear hair cells.

Use of Spiked Normalizers to More Precisely Quantify Tumor Markers and Viral Genomes by Massive Parallel Sequencing of Plasma DNA

A problematic aspect of massive parallel sequencing is that somatic mutations and viral loads are typically quantified as a fraction relative to wild-type human DNA, yet wild-type levels vary with diverse biologic and preanalytic interferences. A novel strategy was devised to quantify target analytes in copies per mL of plasma after normalizing for read counts of spiked DNAs. Five synthetic DNAs (called EndoGenus spikes) were added to plasma before library preparation (modified ArcherDX LiquidPlex 28). By normalizing to the fractional recovery of EndoGenus spike reads, numerical values for each disease marker were reportable in units of copies per mL. To show how well this system operates, replicate assays were performed on 40 mock plasmas having 23 engineered mutations and on 21 natural plasmas. Reads for all five EndoGenus spikes were recovered (means, 313 and 376 copies/mL in mock and natural plasmas, respectively). Normalizing read counts for the proportional recovery of spikes helped control for variables in the multistep protocol, reducing the CV in replicate tests from 34% to 22% for mutations and from 25% to 7% for viral loads. In conclusion, the EndoGenus system is useful for evaluating efficiency of the total test system and for precisely quantifying target molecules. This system may benefit patients being monitored for disease burden while also tracking emerging subclones.

Protocols for Tn-seq Analyses in the Group A Streptococcus.

Transposon-sequencing (Tn-seq) has revolutionized forward-genetic analyses to study genotype-phenotype associations and interrogate bacterial cell physiology. The Tn-seq approach allows the en masse monitoring of highly complex mutant libraries, leveraging massive parallel DNA sequencing as a means to characterize the composition of these mutant pools on a genome-scale with unprecedented nucleotide-level high resolution. In this chapter, we present step-by-step protocols for Tn-seq analyses in the human pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) using the mariner-based Krmit transposon. We detail how to generate highly complex Krmit mutant libraries in GAS and the en masse production of Krmit insertion tags for Illumina sequencing of the transposon-genome junctions for Tn-seq analyses. Most of the protocols presented here were developed and implemented using the S. pyogenes M1T1 serotype clinical isolate 5448, but they have been successfully applied to multiple GAS serotypes as well as other pathogenic Streptococci.

Clinical results and importance of next-generation sequencing (NGS) in detecting targeted mutations in the treatment of metastatic Lung Cancer: Single center initial results

Objective: In Lung cancer (LC), which is one of the most deadly cancers, longer survival has been achieved with targeted agents. For this reason, it is important to find the patients who are suitable for targeted therapies. Next-generation sequencing (NGS) is a method that allows multiple genetic variants to be detected simultaneously by performing massive parallel DNA sequencing at the same time. We wanted to reveal the clinical effects and benefits of genetic variant analysis with NGS for our patients.&#x0D; Material and Methods: Patients with stage 4 non-squamous and not otherwise specified (NOS) Non-small cell LC who underwent genetic variant analysis with NGS were included in the study, retrospectively.&#x0D; Results: Total of the 51 patients, 41 (80.4%) were male and the median age was 64 (35-85) years. According to TNM, 21 (41.2%) patients were stage 4A, 30 (58.8%) patients were stage 4B and 39 (76.5%) patients had adenocarcinoma and 12 (23.5%) had NOS histology. NGS analyzes were performed in median 14 days (8-43) and determined 24 pathogenic variants in 17 (%25) patients: 9EGFR (%17,6), 6PIKC3A (%11,7), 5KRAS (%9,8), 2PTEN (%3,9), 1BRAF (%1,9), 1MET (%1,6) (7 of them concomitantly). Cytotoxic chemotherapy was recommended in 41, anti-EGFR agents in 8 (afatinib in 4, erlotinib in 4 patients) patients and anti-BRAF+MEK inhibitor agent (dabrafenib+trametinib) in 1 patient.&#x0D; Conclusion: With the NGS, in just two weeks, both target and resistance genetic variants of our patients were detected at the same time and individualized treatments were applied. In this way, both time and cost were saved.

Integrative analysis of genomic and transcriptomic characteristics associated with progression of aggressive thyroid cancer

Anaplastic thyroid cancer (ATC) and advanced differentiated thyroid cancers (DTCs) show fatal outcomes, unlike DTCs. Here, we demonstrate mutational landscape of 27 ATCs and 86 advanced DTCs by massively-parallel DNA sequencing, and transcriptome of 13 ATCs and 12 advanced DTCs were profiled by RNA sequencing. TERT, AKT1, PIK3CA, and EIF1AX were frequently co-mutated with driver genes (BRAFV600E and RAS) in advanced DTCs as well as ATC, but tumor suppressors (e.g., TP53 and CDKN2A) were predominantly altered in ATC. CDKN2A loss was significantly associated with poor disease-specific survival in patients with ATC or advanced DTCs, and up-regulation of CD274 (PD-L1) and PDCD1LG2 (PD-L2). Transcriptome analysis revealed a fourth molecular subtype of thyroid cancer (TC), ATC-like, which hardly reflects the molecular signatures in DTC. Furthermore, the activation of JAK-STAT signaling pathway could be a potential druggable target in RAS-positive ATC. Our findings provide insights for precision medicine in patients with advanced TCs.

OTOF mutation analysis with massively parallel DNA sequencing in 2,265 Japanese sensorineural hearing loss patients.

The OTOF gene (Locus: DFNB9), encoding otoferlin, is reported to be one of the major causes of non-syndromic recessive sensorineural hearing loss, and is also reported to be the most common cause of non-syndromic recessive auditory neuropathy spectrum disorder (ANSD). In the present study, we performed OTOF mutation analysis using massively parallel DNA sequencing (MPS). The purpose of this study was to reveal the frequency and precise genetic and clinical background of OTOF-related hearing loss in a large hearing loss population. A total of 2,265 Japanese sensorineural hearing loss (SNHL) patients compatible with autosomal recessive inheritance (including sporadic cases) from 53 otorhinolaryngology departments nationwide participated in this study. The mutation analysis of 68 genes, including the OTOF gene, reported to cause non-syndromic hearing loss was performed using MPS. Thirty-nine out of the 2,265 patients (1.72%) carried homozygous or compound heterozygous mutations in the OTOF gene. It is assumed that the frequency of hearing loss associated with OTOF mutations is about 1.72% of autosomal recessive or sporadic SNHL cases. Hearing level information was available for 32 of 39 patients with biallelic OTOF mutations; 24 of them (75.0%) showed profound hearing loss, 7 (21.9%) showed severe hearing loss and 1 (3.1%) showed mild hearing loss. The hearing level of patients with biallelic OTOF mutations in this study was mostly severe to profound, which is consistent with the results of past reports. Eleven of the 39 patients with biallelic OTOF mutations had been diagnosed with ANSD. The genetic diagnosis of OTOF mutations has significant benefits in terms of clinical decision-making. Patients with OTOF mutations would be good candidates for cochlear implantation; therefore, the detection of OTOF mutations is quite beneficial for patients, especially for those with ANSD.

Evaluation of a microhaplotypes panel for forensic genetics using massive parallel sequencing technology

Massive parallel DNA sequencing (MPS) makes it possible to explore a new type of genetic marker, known as microhaplotypes or microhaps. These loci were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Microhaplotypes loci, based on 89 loci in ALFRED, were selected and their genetic variations in 100 Italian individuals were evaluated by using MPS, in order to make inference about utility of a set of microhaps in forensic genetics. After MPS, the panel was reduced to 87 microhaps, comprised of 266 different SNPs and spread across 22 human autosomes. Genotype and haplotype frequencies were estimated, as well as the effective number of alleles at each locus (Ae), which relates to the usefulness of the locus in resolution of relationships and deconvolution of DNA mixtures. Overall, the Ae values for the 87 microhaps range from 1.010 to 8.344, with about 80% showing values greater than 2.0. Noteworthy, 32 microhaps display Ae values greater than 3.0 and 18 loci Ae above 4.0.To explore the suitability of microhaplotypes in mixture deconvolution, the probability of detecting a mixture, as a function of Ae, was inferred for different groups of loci. Considering the fourteen loci with Ae between 3.0 and 3.999 the probability of detecting a mixture was at least 0.99973, while considering the ten loci with Ae between 4.0 and 4.999 the probability was at least 0.99998. Moreover, when considering just the six loci with Ae between 5.0 and 5.999 the probability of detecting a mixture was at least 0.99984, while when considering just the two loci with Ae above 6 the probability was 0.97228. Combining these 32 MH loci, the theoretical probability of detecting a mixture was 0.999999999999973. These results make the subset of 32 loci with Ae above three informative for mixture resolution.The individual matching probabilities (PI) of the 87 microhaps ranged from 0.032 to 0.9802. Considering the 32 microhap loci with Ae greater than 3.0, the cumulative PI value was 1.6 × 10−33, while considering the 18 microhap loci with Ae above 4.0, the cumulative PI value was 2.34 × 10-21. Overall the results of this study confirmed the utility of microhaps in forensic genetics.