Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses

In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), and preimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosome abnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the false-positive and false-negative single-nucleotide variations (SNVs), which are not acceptable in IVF and can be circumvented by linkage analyses, such as short tandem repeats or karyomapping. It is noteworthy that existing methods of detecting SNV/copy number variation (CNV) and linkage analysis often require separate procedures for the same embryo. Here we report an NGS-based PGD/PGS procedure that can simultaneously detect a single-gene disorder and aneuploidy and is capable of linkage analysis in a cost-effective way. This method, called “mutated allele revealed by sequencing with aneuploidy and linkage analyses” (MARSALA), involves multiple annealing and looping-based amplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated with the monogenic diseases are detected by PCR amplification of the MALBAC product. The false-positive and -negative SNVs are avoided by an NGS-based linkage analysis. Two healthy babies, free of the monogenic diseases of their parents, were born after such embryo selection. The monogenic diseases originated from a single base mutation on the autosome and the X-chromosome of the disease-carrying father and mother, respectively.

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Diagnostic Approach to Monogenic Inflammatory Bowel Disease in Clinical Practice: A Ten-Year Multicentric Experience

Inflammatory Bowel Diseases ◽

10.1093/ibd/izz178 ◽

2019 ◽

Vol 26 (5) ◽

pp. 720-727 ◽

Cited By ~ 4

Author(s):

Sara Lega ◽

Alessia Pin ◽

Serena Arrigo ◽

Cristina Cifaldi ◽

Martina Girardelli ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Next Generation Sequencing ◽

Bowel Disease ◽

Early Onset ◽

Genetic Diagnosis ◽

Diagnostic Approach ◽

Next Generation ◽

Monogenic Diseases ◽

Inflammatory Bowel ◽

Generation Sequencing

Abstract Background and aims Multiple monogenic disorders present as very early onset inflammatory bowel disease (VEO-IBD) or as IBD with severe and atypical features. Establishing a genetic diagnosis may change patients’ management and prognosis. In this study, we describe the diagnostic approach to suspected monogenic IBD in a real clinical setting, discussing genetic and phenotypic findings and therapeutic implications of molecular diagnosis. Methods Information of patients with VEO-IBD and early onset IBD with severe/atypical phenotypes (EO-IBD s/a) managed between 2008–2017 who underwent a genetic workup were collected. Results Ninety-three patients were included, and 12 (13%) reached a genetic diagnosis. Candidate sequencing (CS) was performed in 47 patients (50%), and next generation sequencing (NGS) was performed in 84 patients (90%). Candidate sequencing had a good diagnostic performance only when guided by clinical features specific for known monogenic diseases, whereas NGS helped finding new causative genetic variants and would have anticipated one monogenic diagnosis (XIAP) and consequent bone marrow transplant (BMT). Patients with monogenic IBD more frequently were male (92% vs 54%; P = 0.02), had extraintestinal findings (100% vs 34%; P < 0.001), and had disease onset ≤1 month of life (25% vs 1%; P = 0.006). Genetic diagnosis impacted patient management in 11 patients (92%), 7 of whom underwent BMT. Conclusion A genetic diagnosis can be established in a significant proportion of suspected monogenic IBD and has an impact on patients’ management. Candidate sequencing may be deployed when clinical findings orientate toward a specific diagnosis. Next generation sequencing should be preferred in patients with nonspecific phenotypes.

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A novel preimplantation genetic diagnosis strategy for Duchenne Muscular Dystrophy based on targeted Next-generation sequencing and linkage analysis

10.26226/morressier.573c1513d462b80296c98b86 ◽

2016 ◽

Author(s):

Juan Du

Keyword(s):

Next Generation Sequencing ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Linkage Analysis ◽

Preimplantation Genetic Diagnosis ◽

Genetic Diagnosis ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Generation Sequencing

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Mitochondrial disorders in the Arab Middle East population: the impact of next generation sequencing on the genetic diagnosis.

Journal of Biochemical and Clinical Genetics ◽

10.24911/jbcgenetics/183-1548325196 ◽

2019 ◽

pp. 54-64

Author(s):

Ahmad Alahmad ◽

Hebatallah Muhammad ◽

Angela Pyle ◽

Buthaina Albash ◽

Robert McFarland ◽

...

Keyword(s):

Next Generation Sequencing ◽

Middle East ◽

Genetic Diagnosis ◽

Mitochondrial Disorders ◽

Next Generation ◽

The Impact ◽

Generation Sequencing ◽

Arab Middle East

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Oocyte vitrification is a valid strategy to accumulate gametes for Preimplantation Genetic Diagnosis for Aneuploidy using Next Generation Sequencing

10.26226/morressier.573c1513d462b80296c98b52 ◽

2016 ◽

Author(s):

Sandrine Chamayou

Keyword(s):

Next Generation Sequencing ◽

Preimplantation Genetic Diagnosis ◽

Genetic Diagnosis ◽

Next Generation ◽

Oocyte Vitrification ◽

Generation Sequencing

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Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

Foot & Ankle Orthopaedics ◽

10.1177/2473011420s00027 ◽

2020 ◽

Vol 5 (4) ◽

pp. 2473011420S0002

Author(s):

Yoonjung Choi ◽

Irvin Oh

Keyword(s):

Next Generation Sequencing ◽

False Positive ◽

Bacterial Genome ◽

Rrna Gene ◽

Next Generation ◽

Antibiotic Resistant ◽

Streptococcus Species ◽

Resistant Genes ◽

Standard Culture ◽

Generation Sequencing

Category: Other Introduction/Purpose: Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method. Methods: We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed. Results: In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens. Conclusion: NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy. [Table: see text]

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