Low-level mosaic trisomy 13 at amniocentesis in a pregnancy associated with a positive NIPT result suspicious of trisomy 13, a CVS result of mosaic trisomy 13, cytogenetic discrepancy in various tissues and a favorable fetal outcome.

OBJECTIVE: We present low-level mosaic trisomy 13 at amniocentesis in a pregnancy associated with a positive non-invasive prenatal testing (NIPT) result suspicious of trisomy 13, a chorionic villus sampling (CVS) result of mosaic trisomy 13, cytogenetic discrepancy in various tissues and a favorable fetal outcome. CASE REPORT: A 29-year-old, gravida 2, para 1, woman underwent amniocentesis at 20 weeks of gestation because of a positive NIPT result (Z-score = 20.9, positive ≥3) suspicious of trisomy 13 at 11 weeks of gestation and a CVS result of mosaic trisomy 13 at 14 weeks of gestation. At 14 weeks of gestation, CVS revealed the multiplex ligation-dependent probe amplification (MLPA) result of rea X,Y (P095) × 1, 13 (P095) × 3, 18,21 (P095) × 2/X,Y (P095) × 1, 13,18,21 (P095) × 2 and a karyotype of 48,XY,+13,+mar [9]/47,XY,+mar[16]. She was referred to the hospital for genetic counseling at 15 weeks of gestation, and cytogenetic analysis of parental blood revealed 47,XY,+mar in the father and 46, XX in the mother. Fluorescence in situ hybridization (FISH) analysis on the paternal blood showed that the extra dicentric marker was derived from chromosome 15 without the locus SNRPN (15q11.2), and the result was 47,XY,+mar.ish dic(15) (D15Z1++, SNRPN-, PML-)[20]. Amniocentesis at 20 weeks of gestation revealed a karyotype of 47,XY,+mar pat (20/20). Simultaneous interphase FISH analysis on uncultured amniocytes revealed 32% (32/100 cells) mosaicism for trisomy 13. Quantitative fluorescence polymerase chain reaction (QF-PCR) analysis using the DNA extracted from the parental bloods and uncultured amniocytes excluded uniparental disomy (UPD) 13. Prenatal ultrasound findings were normal. The woman was advised to continue the pregnancy, and a phenotypically normal 2708-g male baby was delivered at 38 weeks of gestation, The cord blood, umbilical cord and placenta had the karyotypes of 47,XY,+mar pat and did not have UPD 13. When follow-up at age two months, the neonate was phenotypically normal. FISH analysis on buccal mucosal cells detected 5.3% (5/95 cells) mosaicism for trisomy 13, compared with 0% in the normal control. CONCLUSION: Low-level mosaic trisomy 13 at amniocentesis can be associated with a positive NIPT result suspicious of trisomy 13, a CVS result of mosaic trisomy 13, cytogenetic discrepancy in various tissues and a favorable fetal outcome.

Whole Exome Sequencing with Comprehensive Gene Set Analysis Identified a Biparental-Origin Homozygous c.509G>A Mutation in PPIB Gene Clustered in Two Taiwanese Families Exhibiting Fetal Skeletal Dysplasia during Prenatal Ultrasound.

Skeletal dysplasia (SD) is a complex group of bone and cartilage disorders often detectable by fetal ultrasound, but the definitive diagnosis remains challenging because the phenotypes are highly variable and often overlap among different disorders. The molecular mechanisms underlying this condition are also diverse. Hundreds of genes are involved in the pathogenesis of SD, but most of them are yet to be elucidated, rendering genotyping almost infeasible except those most common such as fibroblast growth factor receptor 3 (FGFR3), collagen type I alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), diastrophic dysplasia sulfate transporter (DTDST), and SRY-box 9 (SOX9). Here, we report the use of trio-based whole exome sequencing (trio-WES) with comprehensive gene set analysis in two Taiwanese non-consanguineous families with fetal SD at autopsy. A biparental-origin homozygous c.509G>A(p.G170D) mutation in peptidylprolyl isomerase B (PPIB) gene was identified. The results support a diagnosis of a rare form of autosomal recessive SD, osteogenesis imperfecta type IX (OI IX), and confirm that the use of a trio-WES study is helpful to uncover a genetic explanation for observed fetal anomalies (e.g., SD), especially in cases suggesting autosomal recessive inheritance. Moreover, the finding of an identical PPIB mutation in two non-consanguineous families highlights the possibility of the founder effect, which deserves future investigations in the Taiwanese population.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 21q11.2-q21.1 and a literature review.

OBJECTIVE: We present prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 21q11.2-q21.1, and we review the literature of an sSMC(21) with a duplication of 21q11.2-q21.1. CASE REPORT: A 40-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XX,+mar [18]/46,XX [4]. The parental karyotypes were normal. Prenatal ultrasound findings were unremarkable. aCGH analysis of cultured amniocytes revealed a 2.855-Mb duplication of 21q11.2-q21.1 encompassing the genes of LIPI, ABCC13 and NRIP1. Metaphase fluorescence in situ hybridization analysis on cultured amniocytes revealed a result of 47,XX,+mar .ish der(13/21) (D13/21Z1+) [10]. Spectral karyotyping analysis determined the origin of chromosome 21 in the sSMC. A female fetus was delivered with no phenotypic features of Down syndrome and no structural abnormalities. We discuss the genotype-phenotype correlation of LIPI, ABCC13 and NRIP1, and review the literature of an sSMC(21) associated with dup(21)(q11.2q21.1). CONCLUSION: aCGH is useful for identification of the nature and genetic component of a prenatally detected sSMC.

Preimplantation genetic diagnosis of hemophilia A.

Preimplantation genetic diagnosis (PGD) is a powerful tool to tackle the transmission of monogenic inherited disorders in families carrying the diseases from generation to generation. It currently remains a challenging task, despite PGD having been developed over 25 years ago. The major difficulty is it does not have an easy and general formula for all mutations. Different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scanty, whereas timely laboratory diagnosis is mandatory if fresh embryo transfer is desired occasionally. Indicators for outcome assessment of a successful PGD program include the successful diagnosis rate on blastomeres (Day 3 cleavage-stage embryo biopsy) or trophectoderm cells (Day 5/6 blastocyst biopsy), the implantation rate per embryo transferred, and the livebirth rate per oocyte retrieval cycle. Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8). The mutation spectrum of the F8 is complex, according to our previous report, including large segmental intra-gene inversions, large segmental deletions spanning a few exons, point mutations, and total deletion caused by chromosomal structural rearrangements. In this review, the molecular methodologies used to tackle different mutants of the F8 in the PGD of HA are to be explained, and the experiences of successful use of amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) and linkage analysis for PGD of HA in our laboratory are also provided.

Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: validation and verification.

BACKGROUND: Aneuploidy is an important etiology of implantation failure and quantitative real-time polymerase chain reaction (qPCR) seems a promising preimplantation genetic screening (PGS) technology to detect aneuploidies. This verification study aimed at verifying the impact on reproductive outcomes in in vitro fertilization (IVF) cycles using fresh embryo transfer (FET) in which the embryos were selected by blastocyst biopsy with qPCR-based PGS in our settings. RESULTS: A total of 13 infertile couples with more than once failed in vitro fertilization were enrolled during July to October of 2014. PGS was conducted by qPCR with selectively amplified markers to detect common aneuploidies (chromosomes 13, 18, 21, X, and Y). The design of the qPCR molecular markers adopted the locked nucleic acid (LNA) strategy. The blastocyst biopsy was performed on Day 5/6 and the PGS was done on the same day, which enabled FET. A total of 72 blastocysts were biopsied. Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %. The aneuploidy rate was 38.9 % (28/72). 28 embryos were transferred. The clinical pregnancy rate was 61.5 % (8/13) per cycle. Early first trimester abortion was encountered in 1 and the ongoing pregnancy rate was 53.8 % (7/13) per cycle. CONCLUSION: This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting. Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.

Validating a rapid, real-time, PCR-based direct mutation detection assay for preimplantation genetic diagnosis.

Although co-amplification of polymorphic microsatellite markers is the current gold standard for preimplantation genetic diagnosis (PGD) of single-gene disorders (SGD), this approach can be hampered by the lack of availability of informative markers. We recently (2011) devised a novel in-house assay for PGD of aromatic L-amino acid decarboxylase deficiency, based on an amplification refractory mutation system and quantitative PCR (ARMS-qPCR). The objective of the present study was to verify ARMS-qPCR in a cohort of 20 PGD cycles with a diverse group of SGDs (15 couples at risk for 10 SGDs). Day-3 cleavage-stage embryos were subjected to biopsy and genotyping, followed by fresh embryo transfer (FET). The diagnostic rate was 82.9%; unaffected live births were achieved in 9 of 20 FET cycles (45%), with only one false negative (among 54 transferred embryos). Overall, the ARMS-qPCR had frequent allele-dropout (ADO), rendering it inappropriate as the sole diagnostic method (despite a favorable live-birth rate). Regardless, it has the potential to complement the current gold-standard methodology, especially when trophectoderm biopsy becomes a preferred option and genotyping needs to be timely enough to enable FET.

Number of somatic mutations in the mitochondrial D-loop region indicates poor prognosis in breast cancer, independent of TP53 mutation.

The objective of this study was to investigate whether somatic mutations in the mitochondrial DNA (mtDNA) D-loop region correlate with known prognostic factors, namely, age, tumor size, lymph node status, metastasis, tumor-node-metastasis stage, lymphovascular invasion, and status of the progesterone receptor, estrogen receptor, ERBB2 (alias HER2/neu), and TP53 proteins (as determined by immunohistochemistry) and to investigate their relationship, if any, to TP53 mutations in human breast cancer. Thirty breast tumors without BRCA mutation, along with adjacent nontumorous tissues, were genotyped for the mtDNA D-loop region and for the promoter as well as the coding region of the TP53 gene. Clinicopathological parameters were recorded and assessed. In all, 17 somatic mtDNA D-loop mutations were identified, in 13 of 30 tumor samples (43%); two mutations were novel: 544C>T and 16510A>C. Four TP53 mutations were found in six tumor samples (20%), and two (c.437G>A and c.706T>C) were novel. Only progesterone receptor status correlated with the number of somatic mtDNA D-loop mutations (likelihood chi-square test; P < 0.05). Somatic mutations in the mtDNA D-loop and in TP53 were independent of each other (Fisher's exact test; P > 0.05). These results suggest that the number of somatic mtDNA D-loop mutations may be an indicator of poor prognosis through a mechanism independent of TP53.

Differential expression of NUDT9 at different phases of the menstrual cycle and in different components of normal and neoplastic human endometrium.

OBJECTIVE: The human endometrium manifests different and distinct morphologies and physiologies during the different phases of the menstrual cycle. We aimed to determine which candidate genes demonstrate differential expression patterns in the endometrium during different phases of the menstrual cycle. MATERIALS AND METHODS: Using differential display reverse transcription polymerase chain reaction to compare day 5 and day 18 human glandular endometrium obtained by laser capture microdissection, we identified a specific gene, NUDT9 (nucleoside diphosphate-linked moiety X motif 9). NUDT9 is known to function as a highly specific adenosine diphosphate ribose pyrophosphatase and has been mapped to chromosome 4q22.1. It gives rise to two alternatively spliced messenger RNAs, NUDT9alpha and NUDT9beta, encoding a member of the Nudix hydrolase family. In this study, we purified NUDT9 protein and produced an antibody, which we then used for immunohistochemical studies. RESULTS: Using this anti-NUDT9 antibody, we successfully demonstrated that NUDT9 protein was differentially expressed in endometrial glandular cells at different phases of the menstrual cycle. NUDT9 was also found to be expressed more prominently in the epithelial glandular component than in the stromal component of human endometrial carcinomas. CONCLUSION: We suggest that NUDT9 may be involved in the regulation of the menstrual cycle and may be related to the proliferation of glandular cells in the human endometrium.

Phenotype and genotype of two Taiwanese cystic fibrosis siblings and a survey of delta F508 in East Asians.

BACKGROUND: Cystic fibrosis (CF) is considered to be a rare disease in Asians. We report two cases of CF in a 5-year-old girl and her newborn brother. They are of mixed parentage: a Taiwanese mother and an Australian father. METHODS: A comprehensive mutational analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was completed. Literature was reviewed for delta F508 in East Asians. RESULTS: Two mutation sites were identified in the siblings. The carrier status of their parents and elder brother were also confirmed: heterozygous delta F508 mutation from the father; 13 TG repeats in the IVS8-5T from the mother. An update of delta F508 mutation reported in East Asian patients from various ethnicities is included; most of them were of mixed parentage. CONCLUSION: These two cases are the first report of cystic fibrosis associated with a delta F508 mutation in a Taiwanese patient attributable to a mutation most commonly seen in Caucasians. We found that the delta F508 mutation is of western origin. Asian patients are seldom found with this mutation unless they are of mixed parentage. Our findings provide further evidence that different ethnicities have their own set of CFTR mutations.