Objective To evaluate the utility of trophectoderm biopsy for preimplantation genetic diagnosis (PGD) of mitochondrial (mt) DNA mutation load. Design A PGD case and analysis of blastocyst mosaicism. ...Setting Academic center for reproductive medicine. Patient(s) A 30-year-old carrier of 35% 3243A>G mtDNA mutation load with a daughter affected by mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Intervention(s) Blastocyst biopsy for PGD of mutation load and gender. Main Outcome Measure(s) Variation in mutation load within and among embryos, and newborn mutation load after PGD-based selection. Result(s) Oocytes and embryos were found to possess a variety of 3243A>G mutation loads from 9% to 90% in oocytes and 7% to 91% in embryos, demonstrating that PGD would be a relevant procedure. Highly consistent results were obtained within multiple biopsies of both cleavage- and blastocyst-stage embryos. Importantly, mutation loads observed in trophectoderm were predictive of the inner cell mass ( r2 = 0.97). Transfer of a male embryo, predicted to possess 12% mutation load by analysis of a trophectoderm biopsy, resulted in the delivery of a boy with tissue-specific mutation loads ranging from undetectable to 15%. Conclusion(s) This study represents the first successful clinical application of PGD to reduce the transgenerational risk of transmitting an mtDNA disorder and supports the applicability of blastocyst trophectoderm PGD for carriers of mtDNA mutations attempting reproduction.
Revertant mosaicism is a naturally occurring phenomenon involving spontaneous correction of a pathogenic gene mutation in a somatic cell. It has been observed in several genetic diseases, including ...epidermolysis bullosa (EB), a group of inherited skin disorders characterized by blistering and scarring. Induced pluripotent stem cells (iPSCs), generated from fibroblasts or keratinocytes, have been proposed as a treatment for EB. However, this requires genome editing to correct the mutations, and, in gene therapy, efficiency of targeted gene correction and deleterious genomic modifications are still limitations of translation. We demonstrate the generation of iPSCs from revertant keratinocytes of a junctional EB patient with compound heterozygous COL17A1 mutations. These revertant iPSCs were then differentiated into naturally genetically corrected keratinocytes that expressed type XVII collagen (Col17). Gene expression profiling showed a strong correlation between gene expression in revertant iPSC-derived keratinocytes and the original revertant keratinocytes, indicating the successful differentiation of iPSCs into the keratinocyte lineage. Revertant-iPSC keratinocytes were then used to create in vitro three-dimensional skin equivalents and reconstitute human skin in vivo in mice, both of which expressed Col17 in the basal layer. Therefore, revertant keratinocytes may be a viable source of spontaneously gene-corrected cells for developing iPSC-based therapeutic approaches in EB.
Non-invasive prenatal testing (NIPT) for aneuploidy using cell-free DNA in maternal plasma has been widely adopted. Recently, NIPT coverage has expanded to detect subchromosomal abnormalities ...including the 22q11.2 deletion. Validation of a SNP-based NIPT for detection of 22q11.2 deletions demonstrating a high sensitivity (97.8%) and specificity (99.75%) has been reported. We sought to further demonstrate the performance of a revised version of the test in a larger set of pregnancy plasma samples.
Blood samples from pregnant women (10 with 22q11.2-deletion‒affected fetuses and 390 negative controls) were successfully analyzed using a revised SNP-based NIPT for the 22q11.2 deletion. The sensitivity and specificity of the assay were measured.
Sensitivity of the assay was 90% (9/10), and specificity of the assay was 99.74% (389/390), with a corresponding false positive-rate of 0.26%.
The data presented in this study add to the growing body of evidence demonstrating the ability of the SNP-based NIPT to detect 22q11.2 deletions with high sensitivity and specificity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, ...all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Diagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the ...opportunity to make definitive molecular diagnoses using a single assay capable of detecting variants, characterizing methylation patterns, resolving complex rearrangements, and assigning findings to long-range haplotypes. Here, we demonstrate the clinical utility of Nanopore long-read sequencing by validating a confirmatory test for copy number variants (CNVs) in neurodevelopmental disorders and illustrate the broader applications of this platform to assess genomic features with significant clinical implications.
We used adaptive sampling on the Oxford Nanopore platform to sequence 25 genomic DNA samples and 5 blood samples collected from patients with known or false-positive copy number changes originally detected using short-read sequencing. Across the 30 samples (a total of 50 with replicates), we assayed 35 known unique CNVs (a total of 55 with replicates) and one false-positive CNV, ranging in size from 40 kb to 155 Mb, and assessed the presence or absence of suspected CNVs using normalized read depth.
Across 50 samples (including replicates) sequenced on individual MinION flow cells, we achieved an average on-target mean depth of 9.5X and an average on-target read length of 4805 bp. Using a custom read depth-based analysis, we successfully confirmed the presence of all 55 known CNVs (including replicates) and the absence of one false-positive CNV. Using the same CNV-targeted data, we compared genotypes of single nucleotide variant loci to verify that no sample mix-ups occurred between assays. For one case, we also used methylation detection and phasing to investigate the parental origin of a 15q11.2-q13 duplication with implications for clinical prognosis.
We present an assay that efficiently targets genomic regions to confirm clinically relevant CNVs with a concordance rate of 100%. Furthermore, we demonstrate how integration of genotype, methylation, and phasing data from the Nanopore sequencing platform can potentially simplify and shorten the diagnostic odyssey.
Hereditary hypertrichoses are a group of hair overgrowth syndromes that are extremely rare in humans. We have previously demonstrated that a position effect on TRPS1 is associated with hypertrichosis ...in humans and mice. To gain insight into the functional role of Trps1, we analyzed the late morphogenesis vibrissae phenotype of Trps1(Δgt) mutant mice, which is characterized by follicle degeneration after peg downgrowth has been initiated. We found that Trps1 directly represses expression of the hair follicle stem cell regulator Sox9 to control proliferation of the follicle epithelium. Furthermore, we identified a copy number variation upstream of SOX9 in a family with hypertrichosis that significantly decreases expression of the gene in the hair follicle, providing new insights into the long-range regulation of SOX9. Our findings uncover a novel transcriptional hierarchy that regulates epithelial proliferation in the developing hair follicle and contributes to the pathology of hypertrichosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective To develop, validate, and apply a single nucleotide polymorphism (SNP) microarray–based method for simultaneous preimplantation genetic diagnosis (PGD) of unbalanced inheritance of ...rearranged chromosomes and 24-chromosome aneuploidy screening. Design Prospective clinical research study. Setting Academic reproductive medicine center. Patient(s) Eighteen couples carrying a balanced reciprocal or Robertsonian chromosomal rearrangement. Intervention(s) PGD on blastocyst trophectoderm biopsy specimens. Main Outcome Measure(s) Aneuploidy, implantation, pregnancy, and delivery rates after SNP microarray–based aneuploidy and translocation screening. Result(s) Single nucleotide polymorphism microarray was capable of detecting translocation-associated imbalances as small as 9.0 megabases. In the 12 transfers performed, sustained implantation occurred for 9 (45%) of 20 balanced-normal and euploid embryos replaced. The clinical pregnancy rate in patients receiving a transfer was 75% with six singleton deliveries and three ongoing singleton pregnancies thus far. Significantly fewer embryos were eligible for transfer with the incorporation of simultaneous 24-chromosome aneuploidy screening. Arrested embryos were also significantly more likely to possess unbalanced chromosomes when compared with developmentally competent blastocysts. Conclusion(s) This SNP microarray–based method provides the first opportunity to improve outcomes through comprehensive identification of euploid embryos from translocation carrier couples.
Objective:
The Glut1 deficiency syndrome (Glut1 DS) phenotype has expanded dramatically since first described in 1991. Hypoglycorrhachia and decreased erythrocyte 3‐OMG uptake are confirmatory ...laboratory biomarkers. The objective is to expand previous observations regarding the diagnostic value of the uptake assay.
Methods:
One hundred and nine suspected cases of Glut‐1 DS were studied. All cases had a consistent clinical picture and hypoglycorrhachia. The uptake assay was decreased in 74 cases (group 1) and normal in 35 cases (group 2). We identified disease‐causing mutations in 70 group 1 patients (95%) and one group 2 patient (3%).
Results:
The cut‐off for an abnormally low uptake value was increased from 60% to 74% with a corresponding sensitivity of 99% and specificity of 100%. The correlation between the uptake values for the time‐curve and the kinetic concentration curve were strongly positive (R2 = 0.85). Significant group differences were found in CSF glucose and lactate values, tone abnormalities, and degree of microcephaly. Group 2 patients were less affected in all domains. We also noted a significant correlation between the mean erythrocyte 3‐OMG uptake and clinical severity (R2 = 0.94).
Interpretation:
These findings validate the erythrocyte glucose uptake assay as a confirmatory functional test for Glut1 DS and as a surrogate marker for GLUT1 haploinsufficiency. ANN NEUROL 2011;70:996–1005
In the nearly 60 years since prenatal diagnosis for genetic disease was first offered, the field of prenatal diagnosis has progressed far past rudimentary uterine puncture to provide fetal material ...to assess gender and interpret risk. Concurrent with the improvements in invasive fetal sampling came technological advances in cytogenetics and molecular biology that widened both the scope of genetic disorders that could be diagnosed and also the resolution at which the human genome could be interrogated. Nowadays, routine blood work available to all pregnant women can determine the risk for common chromosome abnormalities; chorionic villus sampling (CVS) and amniocentesis can be used to diagnose nearly all conditions with a known genetic cause; and the genome and/or exome of a fetus with multiple anomalies can be sequenced in an attempt to determine the underlying etiology. This chapter will discuss some of the major advances in prenatal sampling and prenatal diagnostic laboratory techniques that have occurred over the past six decades.
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