De Novo Noninversion Variants Implicated in Sporadic Hemophilia A: A Variant Origin and Timing Study.

Sporadic hemophilia A (HA) enables the persistence of HA in the population. F8 gene inversion originates mainly in male germ cells during meiosis. To date, no studies have shown the origin and timing of HA sporadic noninversion variants (NIVs); herein, we assume that HA-sporadic NIVs are generated as a de novo variant. Of the 125 registered families with HA, 22 were eligible for inclusion. We conducted a linkage analysis using F8 gene markers and amplification refractory mutation system-quantitative polymerase chain reaction to confirm the origin of the sporadic NIVs (~0% mutant cells) or the presence of a mosaic variant, which requires further confirmation of the origin in the parent. Nine mothers, four maternal grandmothers, and six maternal grandfathers were confirmed to be the origin of sporadic NIVs, which most likely occurred in the zygote within the first few cell divisions and in single sperm cells, respectively. Three mothers had mosaic variants, which most likely occurred early in postzygotic embryogenesis. All maternal grandparents were free from sporadic NIV. In conclusion, F8 NIVs in sporadic HA were found to be caused primarily by de novo variants. Our studies are essential for understanding the genetic pathogenesis of HA and improving current genetic counseling.

Origin and timing of de novo variants implicated in type 2 von Willebrand disease.

Very few studies have shown the real origin and timing of de novo variants (DNV) implicated in von Willebrand disease (VWD). We investigated four families with type 2 VWD. First, we conducted linkage analysis using single nucleotide variant genotyping to recognize the possible provenance of DNV. Second, we performed amplification refractory mutation system-quantitative polymerase chain reaction to confirm the real origin of variant (~0% mutant cells) or presence of a genetic mosaic variant (0%-50% mutant cells) in three embryonic germ layer-derived tissues and sperm cells. Then, three possible timings of DNV were categorized based on the relative likelihood of occurrence according to the number of cell divisions during embryogenesis. Two each with type 2B VWD (proband 1 p.Arg1308Cys, proband 4 p.Arg1306Trp) and type 2A VWD (proband 2 p.Leu1276Arg, proband 3 p.Ser1506Leu) were identified. Variant origins were identified for families 1, 2 and 3 and confirmed to originate from the mother, father and father, respectively. However, the father of family 4 was confirmed to have isolated germline mosaicism with 2.2% mutant sperm cells. Further investigation confirmed the paternal grandfather to be the origin of variant. Thus, we proposed that DNV originating from the two fathers most likely occurred at the single sperm cell, the one originating from the mother occurred at the zygote during the first few cellular divisions; alternatively, in family 4, the DNV most likely occurred at the early postzygotic development in the father. Our findings are essential for understanding genetic pathogenesis and providing accurate genetic counselling.

Preimplantation genetic diagnosis and screening: Current status and future challenges.

Preimplantation genetic diagnosis (PGD) is a clinically feasible technology to prevent the transmission of monogenic inherited disorders in families afflicted the diseases to the future offsprings. The major technical hurdle is it does not have a general formula for all mutations, thus 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 scarce, whereas timely result is sometimes requested if fresh embryo transfer is desired. On the other hand, preimplantation genetic screening (PGS) screens embryo with aneuploidy and was also known as PGD-A (A denotes aneuploidy) in order to enhance the implantation rates as well as livebirth rates. In contrasts to PGD, PGS is still under ferocious debate, especially recent reports found that euploid babies were born after transferring the aneuploid embryos diagnosed by PGS back to the womb and only very few randomized trials of PGS are available in the literature. We have been doing PGD and/or PGS for more than 10 years as one of the core PGD/PGS laboratories in Taiwan. Here we provide a concise review of PGD/PGS regarding its current status, both domestically and globally, as well as its future challenges.

Application of molecular cytogenetic techniques to characterize the aberrant Y chromosome arising de novo in a male fetus with mosaic 45,X and solve the discrepancy between karyotyping, chromosome microarray, and multiplex ligation dependent probe amplification.

We present a rare male fetus with karyotype of mosaic 45,X that comprises two types of aberrant Y chromosomes arising de novo (Yq12 deletion and isodicentric Yq11.22). Both types of the aberrant Y chromosomes lack the AZFc region which are expected to result in oligospermia but unaffected male external genitalia. Genetic analyses by karyotyping, chromosome microarray (CMA), and multiplex ligation-dependent probe amplification (MLPA) for the fetus revealed conflicting results. Additional molecular cytogenetics tools including fluorescence in situ hybridization (FISH) and multicolor banding (mBAND) were performed, which help resolving the discrepancy and delineated the composition of the aberrant Y chromosomes. This report highlighted the importance of incorporating multiple genetic technologies for accurate characterization of complex chromosomal rearrangements, which aid in the prenatal diagnosis and genetic counseling.

Low-molecular-weight-heparin can benefit women with recurrent pregnancy loss and sole protein S deficiency: a historical control cohort study from Taiwan.

BACKGROUND: Heritable thrombophilias are assumed important etiologies for recurrent pregnancy loss. Unlike in the Caucasian populations, protein S and protein C deficiencies, instead of Factor V Lieden and Prothrombin mutations, are relatively common in the Han Chinese population. In this study we aimed to investigate the therapeutic effect of low molecular weight heparin upon women with recurrent pregnancy loss and documented protein S deficiency. METHODS: During 2011-2016, 68 women with recurrent pregnancy loss (RPL) and protein S deficiency (both the free antigen and function of protein S were reduced) were initially enrolled. All the women must have experienced at least three recurrent miscarriages. After excluding those carrying balanced translocation, medical condition such as diabetes mellitus, chronic hypertension, and autoimmune disorders (including systemic lupus erythematosus and anti-phospholipid syndrome), coexisting thrombophilias other than persistent protein S deficiency (including transient low protein S level, protein C deficiency, and antithrombin III), only 51 women with RPL and sole protein S deficiency were enrolled. Initially they were prescribed low dose Aspirin (ASA: 100 mg/day) and unfortunately there were still 39 women ended up again with early pregnancy loss (12 livebirths were achieved though). Low-molecular-weight-heparin (LMWH) was given for the 39 women in a dose of 1 mg/Kg every 12 h from the day when the next clinical pregnancy was confirmed to the timing at least 24 h before delivery. The perinatal outcomes were assessed. RESULTS: Of 50 treatment subjects performed for the 39 women (i.e. 11 women enrolled twice for two pregnancies), 46 singletons and one twin achieved livebirths. The successful live-birth rate in the whole series was 94 % (47/50). Nineteen livebirths delivered vaginally whereas 28 delivered by cesarean section. The cesarean delivery rate is thus 59.57 %. Emergent deliveries occurred in 3 but no postpartum hemorrhage had been noted. CONCLUSIONS: Our pilot study in Taiwan, an East Asian population, indicated anti-coagulation therapy is of benefit to women with recurrent pregnancy loss who had documented sole protein S deficiency. TRIAL REGISTRATION: ISRCTN64574169. Retrospectively registered 29 Jun 2016.

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.

De novo mutation and somatic mosaicism of gene mutation in type 2A, 2B and 2M VWD.

BACKGROUND: Von Willebrand disease (VWD) is not uncommon in Taiwan. In type 2 or type 3 VWD hemorrhagic symptoms are severer and laboratory data relatively more distinctive. De novo mutation and somatic mosaicism of type 2 VWD gene were rarely reported. Therefore clinical, laboratory and genetic studies of only type 2A, 2B and 2M VWD will be presented and issues of de novo mutation and somatic mosaicism will be explored. METHODS: Fifty-four patients belonging to 23 unrelated families from all around the country in whom type 2 VWD exclusive of type 2N has been diagnosed not only by clinical and routine laboratory studies but also by genetic confirmation during 1990-2015 were investigated. A novel technique named amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) was used to confirm the presence of somatic mosaicism. Informed consent was obtained for study. RESULTS: De novo mutation was identified in 4 families among 15 families (26.7 %) in whom family members including parents were available for examination. All their parents were free from bleeding symptoms and had no similar mutation as their respective affected daughter. An interesting example of somatic mosaicism of VWF gene mutation was found in a large family with type 2A VWD. The father carrying a mutated VWF gene, p.Arg1597Trp, transmitted this mutation to his 3 daughters, 1 son, 3 granddaughters and 2 grandsons. However, the father had normal laboratory findings and experienced no abnormal bleeding, while his offspring who inherited the mutation showed abnormal laboratory findings compatible with type 2A VWD and had history of abnormal bleedings. ARMS-qPCR revealed that the father had only 25.5 % mutant in his blood cells and 31.1 % mutant in his oral mucosal cells, while all his offspring had about 49 % mutant in their blood cells. CONCLUSION: De novo mutation of type 2 VWD gene was identified in 4 out of 15 families (26.7 %) examined. Since only one child was affected in each family, germline mosaicism was not likely. A somatic mosaicism of type 2A VWD gene was documented in a big family by a newly in-house developed technique ARMS-qPCR.

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.

Proposal for Practical Approach in Prenatal Diagnosis of Beckwith-Wiedemann Syndrome and Review of the Literature.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a phenotypically and genetically heterogeneous disorder associated with epigenetic/genetic aberrations on chromosome 11p15.4p15.5. There is no consensus criterion for prenatal diagnosis of BWS. METHODS: Three BWS patients with their clinical histories, prenatal ultrasonographic features, and results of molecular diagnosis were presented. Likewise, by incorporating the findings of our cases and literature review, the phenotypic spectrum and genotype-phenotype correlations of fetal BWS were summarized, and a practical approach in prenatal diagnosis of BWS was proposed. RESULTS: A total of 166 BWS cases with prenatal features were included for analysis. Common fetal features include abdominal wall defects (42.8%), polyhydramnios (33.1%), and macrosomia (32.5%). Molecular pathologies include methylation changes in imprinting control region 1 and 2 (ICR1 and ICR2), paternal uniparental disomy of chromosome 11p15.5, copy number change involving 11p15, etc. Some genotype-phenotype correlations were observed. However, the broad phenotypic spectrum but limited features manifested by affected fetuses rendering ultrasonographic diagnosis not easy. CONCLUSIONS: Molecular tests are used for prenatal diagnosis of BWS suspected by ultrasonography. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is recommended as the first-line molecular tool because it simultaneously detects ICR1/ICR2 methylation statuses and copy numbers that solve the majority of clinical cases in the prenatal scenario.

Benefits and risks of low molecular weight heparin use on reproductive outcomes: A retrospective cohort study.

OBJECTIVE: Low molecular weight heparin (LMWH) has been given to reproductive-age women with various indications. This study aims to assess the benefits and risks of such use. MATERIALS AND METHODS: We retrospectively reviewed data (n = 204) between Jan 2016 and May 2019. Logistic regression analysis was conducted to evaluate the correlation between indications and reproductive outcomes. RESULTS: LMWH use had higher odds of live birth in women less than 30 years of age (OR: 4.98; 95% CI = 1.13-21.98; p = 0.034) and with protein S deficiency (OR: 3.90; 95% CI = 1.77-8.59; p = 0.001). For the subgroup of recurrent pregnant loss, LMWH use was only advantageous to women with protein S deficiency (OR: 2.45; 95%:1.01-5.97; p = 0.048). Risks such as preterm delivery, small-for-gestational-age, placental abruption, antepartum/postpartum hemorrhage were not significantly increased among subgroups. Women treated with LMWH and who had successful live births (n = 171) had a slightly increased risk of postpartum hemorrhage compared to controls (n = 8058) during this period in our institution (2.9% vs 1.2%, p < 0.001). CONCLUSION: LMWH administration produces a higher chance of live-birth to women younger than 30 years of age or with protein S deficiency. However, risk of postpartum hemorrhage is increased.

Polyhydramnios as a sole ultrasonographic finding for detecting fetal hemolytic anemia caused by anti-c alloimmunization.

OBJECTIVE: The prenatal course of a rare case with fetal anemia caused by maternal anti-c alloimmunization was reported. CASE REPORT: A 39-year-old female with anti-c and anti-E antibodies against red cells had previously experienced a stillbirth. At her present pregnancy, titers of maternal antibodies and fetal middle cerebral artery peak systolic velocity (MCA-PSV) were frequently monitored to investigate the severity of fetal hemolytic anemia. Rather than manifesting as an increase in MCA-PSV, the anemic fetus was delivered at 32 weeks and one day of gestation with a sole presentation: polyhydramnios. Neonatal hospitalization course were compatible with hemolytic anemia. The baby was discharged at 48 days of age. CONCLUSION: This case illustrated the complexities of dealing with maternal red cell alloimmunization during pregnancy and the limitations of noninvasive diagnostic modalities for detecting fetal anemia, and highlighted that obstetricians should refer all available clinical parameters in order to offer appropriate perinatal care.

Skewed X-Chromosome Inactivation and Parental Gonadal Mosaicism Are Implicated in X-Linked Recessive Female Hemophilia Patients.

BACKGROUND: Hemophilia A (HA) and B (HB) are X-linked recessive disorders that mainly affect males born from a mother carrier. Females are rarely affected but a number of mechanisms have been suggested in symptomatic females, such as skewed X-chromosome inactivation (XCI), chromosomal rearrangements, and hermaphrodites. Different methodologies are required to elucidate the underlying causes of such diseases in female patients. METHODS: Three families with female hemophilia patients, including two HA and one HB, were enrolled for genetic analyses. Cytogenetics, molecular examinations on F8 and F9 genes, XCI assay, and linkage analysis were performed. RESULTS: All three female patients are demonstrated to be heterozygous for an F8, or F9 mutation: one patient is inherited from her unaffected mother and the other two are sporadic cases. All three patients exhibit skewed XCI. The inherited patient is found to be unmethylated in the maternal X chromosome, which increases the potential for the expression of the mutant allele. The two sporadic cases are hypomethylated or unmethylated in the paternal X chromosome, suggesting that paternal gonadal mosaicism may exist in these families. CONCLUSIONS: In addition to screening for coagulation function, different genetic analyses are mandatory to explore the nature of mechanisms responsible for the X-linked recessive disorders in female patients as shown in this study. Our results confirm that skewed XCI is responsible for hemophilia in heterozygous female patients. Likewise, our results implicate that parental gonadal mosaicism, followed by skewed XCI, contributes to hemophilia in "sporadic" female patients.

Molecular cytogenetic characterization of a de novo small supernumerary marker chromosome derived from chromosome 15 in a pregnancy with incidental detection of a maternal Robertsonian translocation of 45,XX,der(13;14) (q10;q10).

OBJECTIVE: We present molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) derived from chromosome 15 in a pregnancy with incidental detection of a maternal Robertsonian translocation of 45,XX,der(13; 14) (q10; q10). CASE REPORT: A 37-year-old, primigravid woman underwent amniocentesis at 16 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XY,+mar. Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes showed the result of no genomic imbalance or arr (1-22) × 2, (X,Y) × 1. Cytogenetic analysis of the parents showed a karyotype of 45,XX,der(13; 14) (q10; q10) in the mother and a karyotype of 46,XY in the father. Prenatal ultrasound was unremarkable. At 38 weeks of gestation, a 2790-g phenotypically normal male baby was delivered. The cord blood had a karyotype of 47,XY,+mar. Metaphase fluorescence in situ hybridization (FISH) analysis showed the result of +mar.ish dic(15) (D15Z1++, SNRPN-, PML-) (18/20). The extra chromosome was derived from chromosome 15. CONCLUSION: Metaphase FISH analysis is useful for the identification of the origin of an sSMC in the presence of no genomic imbalance at aCGH analysis. Prenatal diagnosis of a de novo sSMC may be associated with a Robertsonian translocation in the parents, and parental cytogenetic analysis is necessary under such a circumstance.

Prenatal diagnosis and molecular cytogenetic characterization of a familial small supernumerary marker chromosome derived from the acrocentric chromosome 14/22.

OBJECTIVE: We present prenatal diagnosis and molecular cytogenetic characterization of a familial small supernumerary marker chromosome (sSMC) derived from the acrocentric chromosome 14/22. CASE REPORT: A 40-year-old, gravida 2, para 1, woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XY,+mar. Prenatal ultrasound was unremarkable. Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed no genomic imbalance. Cytogenetic analysis of the parental bloods revealed a karyotype of 47,XY,inv (9) (p12q13),+mar in the father and a karyotype of 46, XX in the mother. The sSMC was investigated by fluorescence in situ hybridization (FISH) analysis on cultured amniocytes using the CEP 13/21 α-satellite specific gene probe labeled with fluorescein isothiocyanate (FITC) fluorophore and the CEP 14/21 α-satellite specific gene probe labeled with Texas Red fluorophore (Cytocell Inc.). The result showed that the sSMC was derived from the chromosome 14/22, or+mar.ish dic (14/22) (D13Z1/D21Z1-, D14Z1/D22Z1+)[20]. A healthy male baby was delivered at term with no phenotypic abnormality. Quantitative fluorescent polymerase chain reaction (QF-PCR) analysis on parental bloods and the child's peripheral blood was used to exclude uniparental disomy (UPD) (14) and UPD(22). CONCLUSION: FISH analysis is useful for the determination of an sSMC derived from an acrocentric chromosome under the circumstance of no genomic imbalance at amniocentesis. QF-PCR analysis is useful for excluding the possible associated UPD.

Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon.

BACKGROUND: The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses. RESULTS: We found that microsatellite sequences were highly abundant in the P. monodon genome, comprising 8.3% of the total length. The density and the average length of microsatellites were evidently higher in comparison to those of other taxa. AT-rich microsatellite motifs, especially poly (AT) and poly (AAT), were the most abundant. High abundance of microsatellite sequences were also found in the transcribed regions. Furthermore, via self-BlastN analysis we identified 103 novel repetitive element families which were categorized into four groups, i.e., 33 WSSV-like repeats, 14 retrotransposons, 5 gene-like repeats, and 51 unannotated repeats. Overall, various types of repeats comprise 51.18% of the P. monodon genome in length. Approximately 7.4% of the FESs contained protein-coding sequences, and the Inhibitor of Apoptosis Protein (IAP) gene and the Innexin 3 gene homologues appear to be present in high abundance in the P. monodon genome. CONCLUSIONS: The redundancy of various repeat types in the P. monodon genome illustrates its highly repetitive nature. In particular, long and dense microsatellite sequences as well as abundant WSSV-like sequences highlight the uniqueness of genome organization of penaeid shrimp from those of other taxa. These results provide substantial improvement to our current knowledge not only for shrimp but also for marine crustaceans of large genome size.

Difficulties of Prenatal Genetic Counseling for a Subsequent Child in a Family With Multiple Genetic Variations.

Many parents with a disabled child caused by a genetic condition appreciate the option of prenatal genetic diagnosis to understand the chance of recurrence in a future pregnancy. Genome-wide tests, such as chromosomal microarray analysis and whole-exome sequencing, have been increasingly used for prenatal diagnosis, but prenatal counseling can be challenging due to the complexity of genomic data. This situation is further complicated by incidental findings of additional genetic variations in subsequent pregnancies. Here, we report the prenatal identification of a baby with a MECP2 missense variant and 15q11.2 microduplication in a family that has had a child with developmental and epileptic encephalopathy caused by a de novo KCNQ2 variant. An extended segregation analysis including extended relatives, in addition to the parents, was carried out to provide further information for genetic counseling. This case illustrates the challenges of prenatal counseling and highlights the need to understand the clinical and ethical implications of genome-wide tests.

Low-level mosaicism for trisomy 16 at amniocentesis in a pregnancy associated with intrauterine growth restriction and a favorable outcome.

OBJECTIVE: We present low-level mosaicism for trisomy 16 at amniocentesis in a pregnancy associated with intrauterine growth restriction (IUGR) and a favorable outcome. CASE REPORT: A 31-year-old woman underwent amniocentesis at 24 weeks of gestation because of IUGR. Amniocentesis revealed a karyotype of 47,XX,+16 [3]/46,XX [22]. Simultaneous array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed gene dosage increase in chromosome 16 consistent with 28% mosaicism for trisomy 16. Uniparental disomy (UPD) 7 and UPD 11 were excluded. She underwent repeat amniocentesis at 27 weeks of gestation. Repeat amniocentesis revealed a karyotype of 47,XX,+16 [1]/46,XX [24]. Simultaneous aCGH analysis on the DNA extracted from uncultured amniocytes revealed 25%-35% (log2 ratio = 0.17-0.25) mosaicism for trisomy 16. Interphase fluorescence in situ hybridization (FISH) analysis detected trisomy 16 signals in 28/100 (28%) uncultured amniocytes. Polymorphic DNA marker analysis excluded UPD 16. Level II ultrasound revealed no fetal abnormalities except symmetric IUGR. The pregnancy was continued to 37 weeks of gestation, and a 2306-g phenotypically normal baby was delivered. The cord blood had a karyotype of 46, XX in 50/50 lymphocytes. The umbilical cord had a karyotype of 47,XX,+16 [14]/46,XX [36]. Interphase FISH analysis on buccal mucosal cells and urinary cells at age three days revealed trisomy 16 signals in 3.8% (4/106) buccal mucosal cells and 6.5% (7/107) urinary cells, compared with 1% in the normal control. Polymorphic DNA marker analysis on placenta confirmed trisomy 16 in the placenta and a maternal origin of the extra chromosome 16. CONCLUSION: Cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes may present in mosaic trisomy 16 at amniocentesis. Low-level mosaicism for trisomy 16 at amniocentesis without maternal UPD 16 can be associated with a favorable outcome despite the presence of IUGR.

Comparison of Genetic Profiling between Primary Tumor and Circulating Tumor Cells Captured by Microfluidics in Epithelial Ovarian Cancer: Tumor Heterogeneity or Allele Dropout?

Epithelial ovarian cancer (EOC) is a leading cause of cancer mortality among women but unfortunately is usually not diagnosed until advanced stage. Early detection of EOC is of paramount importance to improve outcomes. Liquid biopsy of circulating tumor cells (CTCs) is emerging as one of the promising biomarkers for early detection of solid tumors. However, discrepancies in terms of oncogenomics (i.e., different genetic defects detected) between the germline, primary tumor, and liquid biopsy are a serious concern and may adversely affect downstream cancer management. Here, we illustrate the potential and pitfalls of CTCs by presenting two patients of Stage I EOC. We successfully isolated and recovered CTCs by a silicon-based nanostructured microfluidics system, the automated Cell RevealTM. We examined the genomics of CTCs as well as the primary tumor and germline control (peripheral blood mononuclear cells) by whole exome sequencing. Different signatures were then investigated by comparisons of identified mutation loci distinguishing those that may only arise in the primary tumor or CTCs. A novel model is proposed to test if the highly variable allele frequencies, between primary tumor and CTCs results, are due to allele dropout in plural CTCs or tumor heterogeneity. This proof-of-principle study provides a strategy to elucidate the possible cause of genomic discrepancy between the germline, primary tumor, and CTCs, which is helpful for further large-scale use of such technology to be integrated into clinical management protocols.

Prenatal Diagnosis of True Fetal Mosaicism with Small Supernumerary Marker Chromosome Derived from Chromosome 16 by Funipuncture and Molecular Cytogenetics Including Chromosome Microarray.

This study examined the molecular characterization of a prenatal case with true fetal mosaicism of small supernumerary marker chromosome 16 (sSMC(16)). A 41-year-old female underwent amniocentesis at 19 weeks of gestation due to advanced maternal age. Chromosomal analysis for cultured amniocytes revealed a karyotype of 47,XY,+mar[4]/46,XY[16]. Spectral karyotyping and metaphase fluorescence in situ hybridization (FISH) demonstrated that the sSMC was derived from chromosome 16 (47,XY,+mar.ish der(16)(D16Z1+)[13/20]). Confined placental mosaicism was initially suspected because the prenatal ultrasound revealed a normal structure and the pregnancy was uneventful. However, interphase FISH of cord blood performed at 28 weeks of gestation showed 20% mosaicism of trisomy chromosome 16 (nuc ish(D16Z2×3)[40/200]). Chromosome microarray analysis further demonstrated 55% mosaicism of an 8.02 Mb segmental duplication at the subcentromeric region of 16p12.1p11.1 (arr[GRCh37] 16p12.1p11.1(27021975_35045499)×3[0.55]). The results demonstrated a true fetal mosaicism of sSMC(16) involving chromosome16p12.1p11.1 that is associated with chromosome 16p11.2 duplication syndrome (OMIM #614671). After non-directive genetic counseling, the couple opted for late termination of pregnancy. This case illustrated the use of multiple molecular cytogenetic tools to elucidate the origin and structure of sSMC, which is crucial for prenatal counseling, decision making, and clinical management.

Preimplantation Genetic Diagnosis in Hereditary Hearing Impairment.

Sensorineural hearing impairment is a common sensory deficit in children and more than 50% of these cases are caused by genetic etiologies, that is, hereditary hearing impairment (HHI). Recent advances in genomic medicine have revolutionized the diagnostics of, and counseling for, HHI, including preimplantation genetic diagnosis (PGD), thus providing parents-to-be with better reproductive choices. Over the past decade, we have performed PGD using the amplification refractory mutation system quantitative polymerase chain reaction (ARMS-qPCR) technique in 11 couples with a history of HHI, namely eight with GJB2 variants, one with OTOF variants, one with SLC26A4 variants, and one with an MITF variant. We demonstrated that PGD can be successfully applied to HHI of different inheritance modes, namely autosomal dominant or recessive, and phenotypes, namely syndromic or non-syndromic HHI. However, certain ethical concerns warrant scrutiny before PGD can be widely applied to at-risk couples with a history of HHI.