scholarly journals Haplotyping by linked-read sequencing (HLRS) of the genetic disease carriers for preimplantation genetic testing without a proband or relatives

2020 ◽  
Author(s):  
Qing Li ◽  
Yan Mao ◽  
Shaoying Li ◽  
Hongzi Du ◽  
Wenzhi He ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Genetic Disease ◽  
De Novo ◽  
Monogenic Disease ◽  
Nucleotide Polymorphisms ◽  
De Novo Mutations ◽  
Preimplantation Genetic Testing ◽  
Linkage Analyses ◽  
Alpha Thalassemia ◽  
Polar Bodies

Abstract Background: In order to mitigate the risk of allele dropout (ADO) and ensure the accuracy of preimplantation genetic testing for monogenic disease (PGT-M), it is necessary to construct parental haplotypes.. Typically, haplotype resolution is obtained by genotyping multiple polymorphic markers in both parents and a proband or a relative. Sometimes, single sperm typing, or tests on the polar bodies may also be useful. Nevertheless, this process is time-consuming. At present, there was no simple linkage analysis strategy for patients without affected relatives.Method: To solve this problem, we established a haplotyping by linked-read sequencing (HLRS) method without the requirement for additional relatives. First, the haplotype of the genetic disease carriers in the family was constructed by linked-read sequencing, and then the informative single nucleotide polymorphisms (SNPs) in upstream and downstream mutation region were selected to construct the embryo haplotype and to determine whether the embryo was carrying the mutation. Two families were selected to validate this method; one with alpha thalassemia and the other with NDP gene disorder.Results: The haplotyping by linked-read sequencing (HLRS) method was successfully applied to construct parental haplotypes without recruiting additional family members; the method was also validated for PGT-M. The mutation carriers in these families were sequenced by linked-read sequencing, and their haplotypes were successfully phased. Adjacent SNPs of the mutation gene were identified. The informative SNPs were chosen for linkage analyses to identify the carrier embryos. For the alpha thalassemia family, a normal blastocyst was transferred to the uterus and the accuracy of PGT-M was confirmed by amniocentesis at 16 weeks of gestation. Conclusions: Our results suggest that HLRS can be applied for PGT-M of monogenic disorders or de novo mutations where the mutations haplotype cannot be determined due to absence of affected relatives. Keywords: Preimplantation Genetic Testing for monogenic disease, Linked-read sequencing, Linkage analyses, Haplotype

scholarly journals Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dylan G. Chitwood ◽  
Qinghua Wang ◽  
Kathryn Elliott ◽  
Aiyana Bullock ◽  
Dwon Jordana ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Microsatellite Loci ◽  
Cho Cells ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Chinese Hamster ◽  
Functional Genes ◽  
Nucleotide Polymorphisms ◽  
De Novo Mutations

Abstract Background As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter. Most of these gains in productivity can be attributed to increasing cell densities within bioreactors. As such, strategies have been developed to minimize accumulation of metabolic wastes, such as lactate and ammonia. Unfortunately, neither cell growth nor biopharmaceutical production can occur without some waste metabolite accumulation. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a suboptimal culture environment, little is known about the genotoxic properties of these compounds that may lead to global genome instability. In this study, we examined the effects of high and moderate extracellular ammonia on the physiology and genomic integrity of Chinese hamster ovary (CHO) cells. Results Through whole genome sequencing, we discovered 2394 variant sites within functional genes comprised of both single nucleotide polymorphisms and insertion/deletion mutations as a result of ammonia stress with high or moderate impact on functional genes. Furthermore, several of these de novo mutations were found in genes whose functions are to maintain genome stability, such as Tp53, Tnfsf11, Brca1, as well as Nfkb1. Furthermore, we characterized microsatellite content of the cultures using the CriGri-PICR Chinese hamster genome assembly and discovered an abundance of microsatellite loci that are not replicated faithfully in the ammonia-stressed cultures. Unfaithful replication of these loci is a signature of microsatellite instability. With rigorous filtering, we found 124 candidate microsatellite loci that may be suitable for further investigation to determine whether these loci may be reliable biomarkers to predict genome instability in CHO cultures. Conclusion This study advances our knowledge with regards to the effects of ammonia accumulation on CHO cell culture performance by identifying ammonia-sensitive genes linked to genome stability and lays the foundation for the development of a new diagnostic tool for assessing genome stability.

Preimplantation Genetics

2021 ◽  
pp. 98-109
Author(s):  
Valerie Gutmann Koch
Keyword(s):  
Genetic Testing ◽  
Informed Consent ◽  
Genetic Disease ◽  
Chromosomal Abnormalities ◽  
Preimplantation Genetic Testing ◽  
Economic Implications ◽  
Genetic Traits ◽  
Ethical Implications ◽  
Wrongful Life ◽  
Wrongful Birth

This chapter highlights the uses and ethical implications of preimplantation genetic testing and addresses the topic of liability as it applies to use of this technology to screen and select embryos for chromosomal abnormalities and genetic traits prior to implantation. When errors or wrongs occur, there may be significant medical, psychological, and economic implications for those individuals who sought preimplantation testing to avoid a genetic disease or to improve the chance of achieving pregnancy. Informed consent, wrongful birth, and wrongful life claims may be available to those who are harmed due to these errors.

68. PREIMPLANTATION GENETIC TESTING OF MONOGENIC DISEASE: EXPERIENCE IN RUSSIA

2019 ◽  
Vol 39 ◽  
pp. e68-e69
Author(s):  
S. Zhikrivetskaya Olegovna ◽  
Y. Volkova Leonidovna ◽  
E. Musatova Valerievna ◽  
Y. Sofronova Vladislavovna ◽  
N. Shirokova Anatolievna ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Monogenic Disease ◽  
Preimplantation Genetic Testing ◽  
Disease Experience

scholarly journals Preimplantation genetic testing for a family with usher syndrome through targeted sequencing and haplotype analysis

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Haining Luo ◽  
Chao Chen ◽  
Yun Yang ◽  
Yinfeng Zhang ◽  
Yuan Yuan ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Rare Variant ◽  
Genomic Dna ◽  
Haplotype Analysis ◽  
Multiple Displacement Amplification ◽  
Usher Syndrome ◽  
Nucleotide Polymorphisms ◽  
Preimplantation Genetic Testing ◽  
Targeted Capture ◽  
Capture Sequencing

Abstract Background Preimplantation genetic testing for monogenic defects (PGT-M) has been available in clinical practice. This study aimed to validate the applicability of targeted capture sequencing in developing personalized PGT-M assay. Methods One couple at risk of transmitting Usher Syndrome to their offspring was recruited to this study. Customized capture probe targeted at USH2A gene and 350 kb flanking region were designed for PGT-M. Eleven blastocysts were biopsied and amplified by using multiple displacement amplification (MDA) and capture sequencing. A hidden Markov model (HMM) assisted haplotype analysis was performed to deduce embryo’s genotype by using single nucleotide polymorphisms (SNPs) identified in each sample. The embryo without paternal rare variant was implanted and validated by conventional prenatal or postnatal diagnostic means. Results Four embryos were diagnosed as free of father’s rare variant, two were transferred and one achieved a successful pregnancy. The fetal genotype was confirmed by Sanger sequencing of fetal genomic DNA obtained by amniocentesis. The PGT-M and prenatal diagnosis results were further confirmed by the molecular diagnosis of the baby’s genomic DNA sample. The auditory test showed that the hearing was normal. Conclusions Targeted capture sequencing is an effective and convenient strategy to develop customized PGT-M assay.

scholarly journals The human gene damage index as a gene-level approach to prioritizing exome variants

2015 ◽  
Vol 112 (44) ◽  
pp. 13615-13620 ◽  
Author(s):  
Yuval Itan ◽  
Lei Shang ◽  
Bertrand Boisson ◽  
Etienne Patin ◽  
Alexandre Bolze ◽  
...  
Keyword(s):  
General Population ◽  
De Novo ◽  
Damage Index ◽  
Monogenic Disease ◽  
Sequence Length ◽  
De Novo Mutations ◽  
Protein Coding ◽  
A Genome ◽  
Gene Level ◽  
Gene Damage

The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing). The GDI server, data, and software are freely available to noncommercial users from lab.rockefeller.edu/casanova/GDI.

De novo mutations in human genetic disease

2012 ◽  
Vol 13 (8) ◽  
pp. 565-575 ◽  
Author(s):  
Joris A. Veltman ◽  
Han G. Brunner
Keyword(s):  
Genetic Disease ◽  
De Novo ◽  
De Novo Mutations ◽  
Human Genetic Disease

scholarly journals Case Report: Identification of a de novo Missense Mutation in the F8 Gene, p.(Phe690Leu)/c.2070C > A, Causing Hemophilia A: A Case Report

2021 ◽  
Vol 11 ◽  
Author(s):  
Haiyan Bai ◽  
Xia Xue ◽  
Li Tian ◽  
Xi Tong Liu ◽  
Qian Li
Keyword(s):  
Case Report ◽  
Missense Mutation ◽  
Sanger Sequencing ◽  
Hemophilia A ◽  
De Novo ◽  
Pathogenic Mutation ◽  
Site Amplification ◽  
Monogenic Disease ◽  
Inherited Disorders ◽  
Preimplantation Genetic Testing

Hemophilia A is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8/FVIII). Preimplantation genetic testing for monogenic disease (PGT-M) is a powerful tool to tackle the transmission of monogenic inherited disorders from generation to generation. In our case, a mutation in F8 had passed through female carriers in a hemophilia A family and resulted in two male patients with hemophilia A. To identify the etiological genetic variants of F8, next-generation sequencing (NGS) was used for chromosome copy number variation detection, Sanger sequencing to verify mutation sites, single nucleotide polymorphism (SNP) for site amplification, and sequencing to validate the genetic linkage. Finally, a novel missense mutation, p. (Phe690Leu)/c.2070C > A, occurring in exon 13 of F8, was screened out as a pathogenic mutation. Following this, an F8 normal euploid blastocyst was transferred. At the 18th week, the pregnant mother underwent amniocentesis, NGS, Sanger sequencing, and SNP typing that further confirmed that the fetus had a healthy genotype. After delivery, a neonatal blood sample was sent for FVIII concentration detection, and the result established that the FVIII protein was rescued to a nearly average level. We first identified a new type of pathogenic mutation in F8, which has not been previously reported, selected a genetically healthy progeny for an affected family, and provided valuable knowledge of the diagnosis and treatment of hemophilia A.

scholarly journals Local ancestry analysis reveals genomic convergence in extremophile fishes

2019 ◽  
Vol 374 (1777) ◽  
pp. 20180240 ◽  
Author(s):  
Anthony P. Brown ◽  
Kerry L. McGowan ◽  
Enrique J. Schwarzkopf ◽  
Ryan Greenway ◽  
Lenin Arias Rodriguez ◽  
...  
Keyword(s):  
Candidate Genes ◽  
De Novo ◽  
Large Population ◽  
Significant Proportion ◽  
Nucleotide Polymorphisms ◽  
De Novo Mutations ◽  
Genetic Constraints ◽  
Standing Variation ◽  
Whole Genome Resequencing ◽  
Genomic Convergence

The molecular basis of convergent phenotypes is often unknown. However, convergence at a genomic level is predicted when there are large population sizes, gene flow among diverging lineages or strong genetic constraints. We used whole-genome resequencing to investigate genomic convergence in fishes ( Poecilia spp.) that have repeatedly colonized hydrogen sulfide (H 2 S)-rich environments in Mexico. We identified genomic similarities in both single nucleotide polymorphisms (SNPs) and structural variants (SVs) among independently derived sulfide spring populations, with approximately 1.2% of the genome being shared among sulfidic ecotypes. We compared these convergent genomic regions to candidate genes for H 2 S adaptation identified from transcriptomic analyses and found that a significant proportion of these candidate genes (8%) were also in regions where sulfidic individuals had similar SNPs, while only 1.7% were in regions where sulfidic individuals had similar SVs. Those candidate genes included genes involved in sulfide detoxification, the electron transport chain (the main toxicity target of H 2 S) and other processes putatively important for adaptation to sulfidic environments. Regional genomic similarity across independent populations exposed to the same source of selection is consistent with selection on standing variation or introgression of adaptive alleles across divergent lineages. However, combined with previous analyses, our data also support that adaptive changes in mitochondrially encoded subunits arose independently via selection on de novo mutations. Pressing questions remain on what conditions ultimately facilitate the independent rise of adaptive alleles at the same loci in separate populations, and thus, the degree to which evolution is repeatable or predictable. This article is part of the theme issue ‘Convergent evolution in the genomics era: new insights and directions'.

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