Preimplantation Genetic Testing of Spinocerebellar Ataxia Type 3/Machado-Joseph Disease-Robust Tools for Direct and Indirect Detection of the (CAG) Repeat Expansion.

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a neurodegenerative disorder caused by the CAG repeat expansion. Preimplantation genetic testing for monogenic disorders (PGT-M) of SCA3/MJD should include reliable repeat expansion detection coupled with high-risk allele determination using informative linked markers. One couple underwent SCA3/MJD PGT-M combining (CAG) triplet-primed PCR (TP-PCR) with customized linkage-based risk allele genotyping on whole-genome-amplified trophectoderm cells.

Robust preimplantation genetic testing of the common F8 Inv22 pathogenic variant of severe hemophilia A using a highly polymorphic multi-marker panel encompassing the paracentric inversion.

Background: Hemophilia A (HEMA) is an X-linked bleeding disorder caused by reduced/absent coagulation factor VIII expression, as a result of pathogenic variants in the F8 gene. Preimplantation prevention of HEMA should ideally include direct pathogenic F8 variant detection, complemented by linkage analysis of flanking markers to identify the high-risk F8 allele. Linkage analysis is particularly indispensable when the pathogenic variant cannot be detected directly or identified.

Third-Generation Single-Molecule Sequencing for Preimplantation Genetic Testing of Aneuploidy and Segmental Imbalances.

Background: Current strategies for preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) rely mainly on next-generation sequencing (NGS) and microarray platforms, which are robust but require expensive instrumentation. We explored the suitability of third-generation single-molecule sequencing as a PGT-A/SR screening platform for both aneuploidy and segmental imbalance.

Methods: Single-cell and multicell replicates from aneuploid or segmentally unbalanced cell lines (n = 208) were SurePlex-amplified, randomized, and subjected to (a) Nanopore-based single-molecule sequencing (Oxford Nanopore Technologies) and (b) NGS using a leading commercial PGT-A solution (Illumina VeriSeq PGS).

NAD Degrading Enzymes, Evidence for Roles During Infection.

Declines in cellular nicotinamide adenine dinucleotide (NAD) contribute to metabolic dysfunction, increase susceptibility to disease, and occur as a result of pathogenic infection. The enzymatic cleavage of NAD transfers ADP-ribose (ADPr) to substrate proteins generating mono-ADP-ribose (MAR), poly-ADP-ribose (PAR) or O-acetyl-ADP-ribose (OAADPr). These important post-translational modifications have roles in both immune response activation and the advancement of infection.

Carnosine protects stimulus-secretion coupling through prevention of protein carbonyl adduction events in cells under metabolic stress.

Type 2 diabetes is characterised by failure to control glucose homeostasis, with numerous diabetic complications attributable to the resulting exposure of cells and tissues to chronic elevated concentrations of glucose and fatty acids. This, in part, results from formation of advanced glycation and advanced lipidation end-products that are able to modify protein, lipid, or DNA structure, and disrupt normal cellular function. Herein we used mass spectrometry to identify proteins modified by two such adduction events in serum of individuals with obesity, type 2 diabetes, and gestational diabetes, along with similar analyses of human and mouse skeletal muscle cells and mouse pancreatic islets exposed to glucolipotoxic stress.

Identification of Novel Microsatellite Markers Flanking the and Duplicated Region and Inclusion Into a Single-Tube Tridecaplex Panel for Haplotype-Based Preimplantation Genetic Testing of Spinal Muscular Atrophy.

Preimplantation genetic testing for the monogenic disorder (PGT-M) spinal muscular atrophy (SMA) is significantly improved by supplementation of deletion detection with marker-based linkage analysis. To expand the availability of informative markers for PGT-M of SMA, we identified novel non-duplicated and highly polymorphic microsatellite markers closely flanking the and duplicated region. Six of the novel markers within 0.

Robust Preimplantation Genetic Testing of Huntington Disease by Combined Triplet-Primed PCR Analysis of the HTT CAG Repeat and Multi-Microsatellite Haplotyping.

Huntington disease (HD) is a lethal neurodegenerative disorder caused by expansion of a CAG repeat within the huntingtin (HTT) gene. Disease prevention can be facilitated by preimplantation genetic testing for this monogenic disorder (PGT-M). We developed a strategy for HD PGT-M, involving whole genome amplification (WGA) followed by combined triplet-primed PCR (TP-PCR) for HTT CAG repeat expansion detection and multi-microsatellite marker genotyping for disease haplotype phasing.

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome.

Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single-tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta-thalassemia.

Beta (β)-thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β-globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage-based PGD for this disorder.

Single-tube nonaplex microsatellite PCR panel for preimplantation genetic diagnosis of Hb Bart's hydrops fetalis syndrome.

Objective: To develop a single-tube multi-marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non-deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity.

Methods: We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha-globin gene cluster, and optimized a single-tube assay combining detection of α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application.

Evidence of differential selection for the -α(3.7) and -α(4.2) single-α-globin gene deletions within the same population.

Since the 1950s, a strong correlation between high carrier rates for β-thalassemia mutations and selective survival advantage in tropical and subtropical 'malarial belt' regions has been established. Due to the relatively more complex genetics of α-thalassemia, a similar relationship was demonstrated for α-globin gene mutations only from the 1980s, with both single- and double-α-globin gene deletions prevalent in the malarial belt. Mechanistically, the single-α-globin gene deletions arise from non-allelic recombination between the homologous α1 (HBA1) and α2 (HBA2) globin genes.

Preimplantation genetic diagnosis of chromosome translocations by analysis of polymorphic short tandem repeats.

Introduction: We aimed to develop and implement a short tandem repeat (STR) polymerase chain reaction alternative to fluorescence in situ hybridisation (FISH) for the preimplantation genetic diagnosis (PGD) of chromosomal translocations.

Methods: Selected informative STRs located on translocated arms of relevant chromosomes were used to discriminate between normal and unbalanced chromosome states in each embryo.

Results: PGD cycles were performed on five couples where one spouse carried a balanced translocation.

Molecular strategies for pre-implantation genetic diagnosis of single gene and chromosomal disorders.

Pre-implantation genetic diagnosis is used to analyse pre-implantation stage embryos or oocytes for genetic defects, generally for severe Mendelian disorders and chromosome abnormalities. New but controversial indications for pre-implantation genetic diagnosis include identifying human leukocyte antigen compatible embryos suitable as donor, sex selection and adult-onset disorders, particularly cancer. Pre-implantation genetic screening is a variant of pre-implantation genetic diagnosis to improve outcomes of in-vitro fertilisation.

Simplified PGD of common determinants of haemoglobin Bart's hydrops fetalis syndrome using multiplex-microsatellite PCR.

The high incidence of double-gene deletions in α-thalassaemia increases the risk of having pregnancies with homozygous α(0)-thalassaemia, the cause of the lethal haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. However, the current gap-PCR based PGD protocol for deletional α-thalassaemia requires specific primer design for each specific deletion.

Successful preimplantation genetic diagnosis of Hb Bart's hydrops fetalis in Singapore after fresh and frozen embryo replacement cycles.

Introduction: We report the fi rst successful preimplantation genetic diagnosis (PGD) for Hb Bart's hydrops fetalis in Singapore, involving both fresh and frozen embryo replacement cycles.

Clinical Picture: Two couples who were carriers of the Southeast Asian type double gene deletion (--(SEA) deletion carriers) requested for PGD. Couple A had 2 previous affected pregnancies, while couple B have a child of unknown genotypic status.

First successful preimplantation genetic diagnosis in Singapore--avoidance of beta-thalassaemia major.

Introduction: We report on the first successful preimplantation genetic diagnosis (PGD) in Singapore.

Clinical Picture: A couple who are beta-thalassaemia carriers and have an affected daughter requested for PGD.

Treatment: Two cycles of PGD were performed on the couple.

Rapid carrier screening for beta-thalassemia by single-step allele-specific PCR and detection.

Objectives: This study aimed to evaluate a rapid molecular carrier screening strategy for beta-thalassemia.

Design And Methods: Allele-specific PCR was combined with amplicon detection by dissociation curve analysis of SYBR Green I fluorescence in a single step.

Results: The presence of a particular mutation results in the amplification of a mutation-specific product and the dissociation temperature of each amplicon was highly reproducible.

Clinical report: a case of Williams Syndrome and Klinefelter Syndrome.

Introduction: Williams syndrome (WS) is a rare but well recognised neurodevelopmental disease affecting the connective tissue and the central nervous system. Many patients are identified through the presence of dysmorphic features and associated cardiac abnormalities. Klinefelter syndrome (KS) is associated with gynaecomastia, small testes, azoospermia and elevated gonadotropin levels.