Full Karyotype Research Articles

Amniocentesis and chorionic villus sampling: Green-top Guideline No. 8 July 2021: Green-top Guideline No. 8.

Amniocentesis and chorionic villus sampling: Green-top Guideline No. 8 July 2021: Green-top Guideline No. 8.

Generation of an induced pluripotent stem cell line (FDCHi006-A) from a 7-year-old girl with central precocious puberty

Central precocious puberty (CPP) refers to a pediatric endocrine disease in which boys have secondary sexual characteristics before the age of 9 and girls before the age of 8. In this study, we generated an induced pluripotent stem cell line FDCHi006-A from the Peripheral blood cell of a typical central precocious patient who was only 7 years old. This iPSC line showed full pluripotency, normal karyotype and differentiation capacity without integrating vectors. It provides a useful model to study the underlying molecular mechanisms of CPP.

Outcome of Acute Myeloid Leukemia Patients with Abnormal 12p after Allogeneic Hematopoietic Cell Transplantation at Fred Hutchinson Cancer Research Center

BackgroundClonal abnormalities of the short arm of chromosome 12 (Abn12p) are seen in 3~5% of patients with acute myeloid leukemia (AML). Anecdotal reports associate these aberrations with adverse disease prognosis; however, no large studies have been published to formally document its clinical impact on prognosis, especially in patients undergoing allogeneic hematopoietic cell transplantation (HCT). This study aimed to test the hypothesis that AML patients with Abn12p have an inferior survival outcome after HCT. Here we present data from 110 patients with Abn12p, to contrast with 442 comparable AML patients but without Abn12p, all who have received HCT at Fred Hutch.Patients and MethodsWe performed a retrospective comparative cohort analysis based on available data from the Gateway database at Fred Hutch. Inclusion criteria for the testing cohort was AML patients diagnosed according to the current WHO criteria with any chromosome 12p abnormality by karyotype analysis who underwent a HSCT at Fred Hutch. A comparison cohort of AML patients without 12p abnormality was identified based on the following matching criteria: patient age difference within 5 years, transplant time difference within 2 years, conditioning intensity, and donor type. Four matching patients were identified for each Abn12p patient. Overall survival (OS), cumulative incidence of relapse (CIR) and non-relapse-mortality (NRM) after HCT were analysed for the whole cohort and for patients receiving HCT in first complete remission (CR1).ResultsData from 552 patients with full karyotype information were analysed. The median age was 50 years with a range from 2 years to 80 years. Twenty-seven patients were younger than 18 years. Patients transplanted in CR1 accounted for 32% (=177/552), slightly lower among Abn12p patients than among the non-Abn12p patients (24% vs. 34%, P=0.045). 450 patients (82%) were treated with standard myeloablative conditioning (MAC) regimens while the remaining patients received reduced intensity conditioning (RIC). Donors were matched related in 38% (including 4% haploidentical), matched unrelated donors in 36%, and partially matched or mismatched unrelated donors in 14% of the patients. Thirty-nine patients received unrelated cord blood. With a median follow-up of 12 months (range from 0 to 370 months), the 5-year OS probabilities were 12% for the Abn12p patients and 41% for the non-Abn12p patients (Figure 1. Hazard ratio 2.33, P<0.0001, and 2.28 after adjustment for pre-transplant disease status). Patients transplanted in CR1 showed better outcome even among those with Abn12p. The 5-year survival probabilities were 25% for the Abn12p patients among those transplanted in CR1 and 6% for the other Abn12p patients (Figure 2, P=0.005). The primary cause of treatment failure was relapse. Cumulative incidence of relapse at three years was 29% and 56% for patients with and without Abn12p, respectively (P<0.0001). NRM was not significantly different between the two groups. Different types of 12p abnormalities - translocation, deletion, additional material of undefined origin, monosomy, and inversion - showed similar clinical associations with mortality and relapse. Co-variation analysis of cytogenetic abnormalities showed that complex and monosomal karyotypes were present in 82% and 60% of Abn12p patients, respectively, compared with 19% and 7% of non-Abn12p patients, respectively (both P-values < 0.0001). Abnormal chromosome 17p abnormalities were seen in 37% of Abn12p patients compared with 5% of non-Abn12p patients (P<0.0001). ELN classification of the Abn12p patients showed 85% unfavorable cytogenetics, 14% intermediate, and 2% favorable compared with 24%, 72%, and 4%, respectively, among non-Abn12p patients (P<0.0001).ConclusionIn conclusion, AML patients with Abn12p have a poor outcome after HCT. Abn12p tends to be present as part of a complex karyotype and hence considered unfavorable per ELN classification. Abn12p also overlaps with monosomal karyotype, but less so with 17p abnormalities. HCT in first complete remission still remains the treatment of choice for these patients. Clinical trial protocols should be considered for patients with Abn12p whenever possible. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

Generation of an induced pluripotent stem cell line from a patient with global development delay carrying DYRK1A mutation (c.1730T>A) and a gene correction isogenic iPSC line

Mental retardation autosomal dominant 7 (MRD7), or DYRK1A Related Intellectual Disability Syndrome (OMIM 614104) is a developmental syndrome with microcephaly, intellectual disability, language delay and epileptic seizures. Haploinsufficiency of DYRK1A is the cause of MRD7. Here, we generated an induced pluripotent stem cell (iPSC) line with the mutation (DYRK1Ac.1730T>A) from the Peripheral blood mononuclear cell (PBMC) of a MRD7 patient along with an isogenic gene-corrected control iPSC line by CRISPR/Cas9 genome editing. Both iPSC lines showed full pluripotency, normal karyotype and differentiation capacity without integrating vectors. These DYRK1A mutant and isogenic gene-corrected iPSC control line provides a useful model to study the underlying molecular mechanisms of MRD7.

Rapid fluorescence in situ hybridisation optimises induction therapy for acute myeloid leukaemia.

Rapid fluorescence in situ hybridisation optimises induction therapy for acute myeloid leukaemia.

Prenatal Detection of Chromosome Aneuploidy by Quantitative Fluorescence PCR.

Autosomal chromosome aneuploid pregnancies that survive to term, namely, trisomies 13, 18, and 21, account for 89% of chromosome abnormalities with a severe phenotype identified in prenatal samples. They are traditionally detected by full karyotype analysis of cultured cells. The average reporting time for a prenatal karyotype analysis is approximately 14days, and in recent years, there has been increasing demand for more rapid prenatal results with respect to the common chromosome aneuploidies, to relieve maternal anxiety and facilitate options in pregnancy. The rapid tests that have been developed negate the requirement for cultured cells, instead directly testing cells from the amniotic fluid or chorionic villus sample, with the aim of generating results within 48h of sample receipt. Interphase fluorescence in situ hybridization is the method of choice in some genetic laboratories, usually because the expertise and equipment are readily available. However, a quantitative fluorescence (QF)-PCR-based approach is now widely used and reported as a clinical diagnostic service in many studies. It may be used as a stand-alone test or as an adjunct test to full karyotype or array CGH analysis, which scan for other chromosome abnormalities not detected by the QF-PCR assay.

Hypodiploidy in Childhood Acute Myeloid Leukemia: A Retrospective Cohort Study within the International Berlin-Frankfurt-Münster Study Group

Background: Despite major improvements in survival, relapse is still a frequent and severe event in pediatric acute myeloid leukemia (AML). Cytogenetic abnormalities represent important predictors of outcome. Improved risk stratification encompassing the identification of novel cytogenetic subsets may allow refinements in treatment strategies and increase survival rates.Hypodiploidy, defined as a modal number (MN) below 46 chromosomes, is associated with an adverse outcome in pediatric acute lymphoblastic leukemia. In childhood AML, the predominant cause of hypodiploidy is loss of a sex chromosome accompanying t(8;21). The most commonly lost autosome is chromosome 7, associated with a poor prognosis. However, other chromosome losses in pediatric AML have not been independently explored.We aimed to investigate the occurrence, genetic characteristics, and prognostic impact of hypodiploidy in childhood AML.Methods: This study was conducted as a retrospective cohort study within the I-BFM-AML framework. Children 0-18 years of age diagnosed with de novo AML between January 2000 to December 2015 and a hypodiploid karyotype were eligible for inclusion. Patients with constitutional hypodiploidy, monosomy 7, composite karyotypes, and t(8;21) with loss of a sex chromosome were excluded.Cytogenetic review was performed according to International System for Human Cytogenetic Nomenclature. Hypodiploidy was considered clonal when occurring in at least three metaphases. The clone with the lowest MN defined the ploidy level. The log-rank test was applied for estimating difference in survival. Considering allografting in first complete remission (CR1) as a time-dependent event, the effect of stem cell transplantation (SCT) on overall survival (OS) was estimated by the Mantel-Byar method. Multivariate analyses included sex, complex karyotype (≥3 non-recurrent aberrations), FLT3-ITD mutation status, and white blood cell count as covariates. As all patients with FLT3-ITD mutations had same MN (MN=45) leading to a skewed imputation model, multiple imputation was not performed, and multivariate analyses included complete cases only (n=54).Results: Hypodiploidy was detected in 81/6,409 patients with full karyotyping from 14 collaborative study groups, yielding a frequency of 1.2% in childhood AML. The cohort displayed a balanced sex distribution (male/female: 42/39) and a median age of 6 (range: 0-17). MNs 45 (n=66), 44 (n=10) and 43 (n=5) were observed. No patient had MN below 43. Eight patients (9.8%) had chromosome loss as the only aberration. Forty-eight patients (59%) had a complex karyotype. Core-binding factor abnormalities were observed in three patients (3.7%). Four patients harbored FLT3-ITD mutations (9.3%, 54 tested).Median follow-time for patients alive was 4.5 years (range: 0.2-15.3). Five-year event-free survival (EFS) and OS were 34% (CI 95%: 23% - 45%) and 52% (CI 95%: 40% - 63%), respectively. Cumulative incidence of relapse was 50% with a median time-to-relapse of 0.8 years. Twelve out of 38 relapsed children (32%) were alive at the end of follow-up. Children with MN≤44 (n=15) had lower EFS (21%, CI 95%: 4% - 46%) and OS (33%, CI 95%: 10% - 59%) than children with MN=45 (n=66, EFS: 37%, CI 95%: 24% - 49%, OS: 56%, CI 95%: 42% - 68%). Crude hazard ratios (HR) for MN≤44 vs MN=45 were 1.8 (p=0.08) for EFS and 1.9 (p=0.1) for OS. After adjustment HRs increased to 4.9 (p=0.001) and 6.1 (p=0.003), respectively. Loss of chromosome Y (n=12) displayed a non-significant superior survival (EFS: 58%, OS: 75%). Poor survival rates were especially observed in monosomy 9 (n=11, EFS: 14%, OS 15%), 10 (n=5, EFS: 20%, OS: 20%), and 16 (n=5, EFS: 25%, OS: 25%), although not reaching statistical significance.Allogenic SCT did not offer any advantage in survival. For children receiving SCT in CR1 (n=18), crude HR for OS was 1.4, p=0.39. Multivariate analysis showed HR for OS at 1.5, p=0.42. Exclusion of children not reaching CR1 (n=7) did not significantly change HRs.This large international study reveals that hypodiploidy is rare in pediatric AML. Children with loss of chromosome 9, 10, or 16 all displayed unfavorable survival rates, whereas loss of chromosome Y did not have influence on prognosis. SCT in CR1 did not improve survival. Our results suggest that modal number has an impact on outcome and may be important for risk stratification. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Origin and Composition of Cell-Free DNA in Spent Medium From Human Embryo Culture During Preimplantation Development

Study question What is the origin and composition of cell-free DNA in human embryo spent culture media? Summary answer Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. What is known already In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. Study design, size, duration This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. Participants/materials, setting, methods Trophectoderm biopsies and culture media samples (20 μl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. Main results and the role of chance We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. Limitations, reasons for caution This study was limited by the sample size and the number of cells analyzed by FISH. Wider implications of the findings This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. Study funding/ competing interest This work was funded by Igenomix S.L. There are no competing interests.

Full Karyotype Interphase Cell Analysis.

Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphases simultaneously, especially in single cells. Here, we present the selection of human probe DNA and a technique using multistep fluorescence in situ hybridization (FISH) employing four sets of six labeled FISH probes able to delineate all 24 human chromosomes in interphase cells. This full karyotype analysis approach will provide additional diagnostic potential for single cell analysis. The use of spectral imaging (SIm) has enabled the use of up to eight different fluorochrome labels simultaneously. Thus, scoring can be easily assessed by visual inspection, because SIm permits computer-assigned and distinguishable pseudo-colors to each probe during image processing. This enables full karyotype analysis by FISH of single-cell interphase nuclei.

Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

What is the origin and composition of cell-free DNA in human embryo spent culture media? Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. Trophectoderm biopsies and culture media samples (20 μl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. This study was limited by the sample size and the number of cells analyzed by FISH. This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. This work was funded by Igenomix S.L. There are no competing interests.

126: Amniocentesis: QFPCR vs karyotype

To review the number of amniocentesis carried out over a 5 year period in a tertiary maternity unit and compare results between Quantitative Fluorescence-Polymerase Chain Reaction (QFPCR) and full karyotyping obtained from amniotic fluid. All patients who underwent amniocentesis between the years 2012-2016 were included in the study. For each case, QFPCR and Karyotype results were collected. Data was analysed using IBM SPSS version 20. 528 amniocentesis were performed between the years 2012 and 2016. There was a downward trend observed in the numbers performed over that period, following the introduction of non-invasive prenatal testing (NIPT) in June 2013. Results were analysed using both QFPCR and karyotype. In relation to QFPCR, 32.2% (n=170) returned an abnormal result. The most common mutations were: Trisomy 21 (11.7%, n=62), Trisomy 18 (10.6%, n=56) and Trisomy 13 (5.3%, n=28). Other mutations identified were 45X0 (2.5%, n=13) and 69XXY (1.1%, n=6). Karyotype analysis returned an abnormal result in 36.2% (n=191) of cases, with no discrepancy between karyotype and QFPCR for the most common aneuploidies (Trisomy 21, 18 and 13). However, 3.8% (n=21) of normal QFPCR results were subsequently found to be abnormal when karyotype results become available. DiGeorge Syndrome (n=7) was the most common clinically significant missed mutation. This study shows the number of amniocentesis performed in our unit each year is steadily declining since the introduction of NIPT. This is in keeping with worldwide trends. QFPCR offers a more rapid and cost effective alternative to full karyotype. In our population, a policy of QFPCR alone would detect 96.2% of abnormalities. While 100% of the most common chromosomal abnormalities were reliably detected by QFPCR, 3.8% of clinically significant mutations were missed. Therefore, whilst it would be more economical to offer QFPCR alone, without karyotype a significant number of clinically relevant mutations would be missed with this approach.

An adaptive detection method for fetal chromosomal aneuploidy using cell-free DNA from 447 Korean women.

BackgroundNoninvasive prenatal testing (NIPT) using massively parallel sequencing of cell-free DNA (cfDNA) is increasingly being used to predict fetal chromosomal abnormalities. However, concerns over erroneous predictions which occur while performing NIPT still exist in pregnant women at high risk for fetal aneuploidy. We performed the largest-scale clinical NIPT study in Korea to date to assess the risk of false negatives and false positives using next-generation sequencing.MethodsA total of 447 pregnant women at high risk for fetal aneuploidy were enrolled at 12 hospitals in Korea. They underwent definitive diagnoses by full karyotyping by blind analysis and received aneuploidy screening at 11–22 weeks of gestation. Three steps were employed for cfDNA analyses. First, cfDNA was sequenced. Second, the effect of GC bias was corrected using normalization of samples as well as LOESS and linear regressions. Finally, statistical analysis was performed after selecting a set of reference samples optimally adapted to a test sample from the whole reference samples. We evaluated our approach by performing cfDNA testing to assess the risk of trisomies 13, 18, and 21 using the sets of extracted reference samples.ResultsThe adaptive selection algorithm presented here was used to choose a more optimized reference sample, which was evaluated by the coefficient of variation (CV), demonstrated a lower CV and higher sensitivity than standard approaches. Our adaptive approach also showed that fetal aneuploidies could be detected correctly by clearly splitting the z scores obtained for positive and negative samples.ConclusionsWe show that our adaptive reference selection algorithm for optimizing trisomy detection showed improved reliability and will further support practitioners in reducing both false negative and positive results.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-016-0222-5) contains supplementary material, which is available to authorized users.

Karyological studies of two hot chilli pepper cultivars from two different geographical regions of India: Bhut jolokia, Capsicum chinense Jacq. and Bullet Lanka, Capsicum annuum L.

The present paper deals with the detailed karyological studies of two hot chilli cultivars of India; one is world’s third hottest chilli Bhut jolokia (BJ) [Capsicum chinense Jacq. cultivar] collected from the hilly area of North-East India, and another is Bullet Lanka (BL) [Capsicum annuum L. cultivar] from plains of West Bengal. Comparative study showed that there was no difference in the chromosome count (2n = 24), but significant differences in chromosome characteristics were found between these two species. All 24 chromosomes of both species are metacentric with full symmetric karyotype. Both species have mono-modal chromosome with longest to shortest 5.75–4.60 µm in BJ and 8.91–6.02 µm in BL. The total chromosome length (TCL) of the haploid complements of BJ and BL are 62.56 and 87.36 μm, respectively. The total form percentage (TF %) of BJ and BL are 47.34 and 47.68 %, respectively. A pair of satellite (SAT) chromosomes with prominent secondary constriction is present in BL, but BJ does not contain any SAT chromosome. Thus, from the karyotype features, both BJ and BL exhibit close relationships which point out their probable origin from a common ancestor. Overall, the TF % and the TCL of BL was greater than BJ; indicating BL is evolutionarily slightly more advanced than BJ.

P14 Significance and associations of aberrant right subclavian artery in the fetal cardiology setting

Background The aberrant right subclavian artery (ARSA) is the most common branching abnormality of the aortic arch, with an estimated prevalence of 0.5–2% of the population. This anomaly is defined anatomically by the unusual course of the right subclavian artery (RSA), which arises as a fourth vessel from the descending aorta, arriving at the right arm via a path looping behind the trachea and the oesophagus. ARSA is usually asymptomatic in children and poses no implications from the cardiovascular point of view. Pathological and clinical studies have shown the prevalence of ARSA to be significantly increased in individuals with congenital heart defects (CHD) and particularly so, in those with trisomy 21. Several papers have also suggested a strong association of ARSA with other chromosomal and genetic syndromes including 22q11.2.deletion. Objectives The aim of this study was to describe the prevalence of chromosomal and non-chromosomal syndromes and CHD in a series of 360 consecutive fetuses diagnosed with ARSA in our fetal cardiology clinic. Material and methods Retrospective analysis of fetuses diagnosed with ARSA during an eight years period (2006–2014). Diagnosis of ARSA was made in transverse three-vessels/arch view with colour flow mapping (Figure 1). Invasive prenatal diagnosis was discussed according to the risk for aneuploidies as adjusted for the presence of ARSA and/or associated cardiac and extra-cardiac abnormalities. Prenatal data were combined with outcome obtained from medical records, pathological examinations and telephone interviews with patients. Results ARSA was an isolated cardiac finding in 78% (281/360) of cases and associated with a major CHD such as tetralogy of Fallot and hypoplastic left heart syndrome in 22% (79/360). A full karyotype was performed in 60% (217/360) of pregnancies, including 83% of those with ARSA and CHD (66/79) and 54% (151/281) of those where ARSA was an isolated cardiac finding. An abnormal karyotype including trisomy 21 and 22q11.2 deletion was found in 64% (42/66) of cases where ARSA was associated with complex CHD. Trisomy 21 was detected in 25% (37/151) of fetuses with ARSA as an isolated cardiac finding and other aneuploidies in further 7% (11/151) of cases. In our series, there were no cases with 22q11.2 deletion identified where ARSA was an isolated cardiac finding and the majority (76%; 213/281) were livebirths with uneventful postnatal outcome. Conclusions Our results are consistent with published literature, which suggests that finding of an ARSA should prompt referral to a specialist fetal cardiologist to rule out underlying cardiac defects. A detailed fetal anomaly scan should be performed in order to examine for features of chromosomal abnormalities and invasive testing should be discussed accordingly. If the fetal karyotype is normal and there are no significant cardiac/extracardiac defects, an isolated ARSA is a benign finding with a good postnatal outcome. There is insufficient data from our study to report on association of ARSA with 22q11.2deletion.

Aberrant right subclavian artery in fetuses with Down syndrome: a systematic review and meta-analysis.

The primary objective was to estimate the prevalence of aberrant right subclavian artery (ARSA) in fetuses with Down syndrome. Secondary objectives were to assess the prevalence of ARSA in euploid fetuses, the feasibility of ultrasound evaluation of the right subclavian artery (RSA) in the first and second trimesters of pregnancy, the performance of ARSA in screening for trisomy 21 and its association with other abnormalities. Web-based databases (PubMed, EMBASE and MEDLINE) were searched up to July 2014. The STROBE, PRISMA and QUIPS instruments were used to assess all included studies and for reporting of methodology, results and conclusions. Original studies that reported prenatal ultrasound evaluation of ARSA, assessment of its prevalence in Down-syndrome and euploid fetuses, feasibility of ultrasound evaluation of the RSA in the first and second trimesters of pregnancy and correlation of ARSA with other abnormalities were included, excluding duplications and case reports. Collected data were summarized to estimate prevalence and feasibility. A meta-analysis was performed pooling the study-specific positive and negative likelihood ratios (LR+ and LR-), detection rates and false-positive rates for trisomy 21. Prevalence of ARSA in Down-syndrome fetuses was 23.6% (95% CI, 19.4-27.9%), whereas in euploid fetuses it was 1.02% (95% CI, 0.86-1.10%). Ultrasound evaluation of the RSA course and origin in the first and second trimesters of pregnancy was feasible in 85% and 98% of cases (first and second trimester, respectively) and it was directly related to sonographic experience and fetal crown-rump length and inversely related to maternal body mass index. In more than 20% of fetuses with ARSA there was an association with other abnormalities but ARSA seemed to be an independent marker of trisomy 21. The meta-analysis showed that ARSA is a significant risk factor for Down syndrome (pooled LR+ = 26.93, 95% CI, 19.36-37.47, P for effect < 0.001, P for Q = 0.3, I(2) = 17.3%), whereas normal RSA is a significant protective marker (pooled LR- = 0.71, 95% CI, 0.51-0.99, P for effect = 0.043, P for Q = 0.9, I(2) = 0%). ARSA appears to be a clinically useful prenatal ultrasound marker of Down syndrome. Additional testing when ARSA is diagnosed should involve evaluation of all risk factors by applying a mathematical model. There is insufficient evidence to recommend fetal karyotyping in cases with isolated ARSA. If the background risk is higher or additional markers are present, full fetal karyotyping is advisable, including analysis for 22q11 microdeletion.

Estimation of Detection Rates of Aneuploidy in High-Risk Pregnancy Using an Approach Based on Nuchal Translucency and Non-Invasive Prenatal Testing: A Cohort Study

Objectives: The aim was to investigate aneuploidy detection using an approach based on nuchal translucency (NT) and non-invasive prenatal testing (NIPT). Methods: This was a cohort study including 5,306 high-risk pregnancies with NT measurements and chorionic villus samples (CVS) tested for full karyotype. Results: The fetal karyotype was normal in 4,172 (78.6%) cases and abnormal in 1,134 (21.4%), including 1,009 with a likely clinically significant adverse outcome. Universal CVS with full karyotyping would lead to the diagnosis of all clinically significant abnormalities. A policy of relying solely on NIPT would have led to the diagnosis of 88.9% of clinically significant abnormalities. A strategy whereby NIPT is the main method, with CVS reserved for cases with NT ≥3.0 mm, would require CVS in 21.7% of cases, identify 94.8% of significant abnormalities and avoid miscarriage in 41 pregnancies compared to CVS for all. Conclusions: A policy of NIPT for increased-risk cases and CVS with full karyotype if the NT was ≥3.0 mm reduced the risk of miscarriage yet still identified 95% of clinically significant aneuploidy.

Advancing Horizons in Fetal Medicine

The four issues of 2014 of the Journal of Fetal Medicine have just rolled out. It has been an exciting and tough year for us at the Editorial Board. We struggled through the process of having sufficient articles to publish. But at long last, we feel we have turned the corner, and now there is a steady stream of articles being submitted for publication. The Society of Fetal Medicine, on the other hand, is flourishing, and the membership is ever expanding. This is largely through the untiring and enthusiastic efforts of our Secretary Dr Ashok Khurana. A large number of cities in India (Mumbai, Kochi, Bangalore, Hyderabad, Jodhpur, Ludhiana, Patiala, Faridkot, Agra, Aurangabad, and Kochi have opened branches of the Society of Fetal Medicine, and have organized numerous regional continuing medical educational programs. This is testimony to the popularity of fetal medicine in the country, as well as, to the influence of our Society. Given that there are about 26 million births per year in India, fetal medicine will continue to flourish for years to come. What is amazing is that in spite of this huge number of births, the IVF clinics are flourishing at an even faster pace than fetal medicine and obstetrics. This emphasizes the strong desire of the couples to have a baby in our society. Fortunately, this desire is matched by the fervent wish of the couples to have a normal baby. Couples who have a pregnancy from conception through IVF, making the baby very precious, are willing to undergo tests to ensure normality of the baby. We would like to examine briefly the advances that have occurred in the field of fetal medicine in 2014. No doubt the single most advance, which has shaken the obstetricians and fetal medicine specialists, is the noninvasive prenatal testing of chromosomal disorders.The Journal had its share of articles on this topic. Cuckle emphasized the changing scene brought on by the NIPT technology [1]. Benn provided a good overview of the subject [2], while Verma [3] and Dash and colleagues [4] presented their experience with this technology in India. They reiterated that NIPT is still a screening test, and needs confirmation of a positive result with an invasive test. Secondly, it is not a good option if the nuchal translucency in the first trimester or nuchal fold thickness in the second trimester is increased, or there are ultrasound abnormalities, or there is history of miscarriages or abnormalities in past pregnancies. In all these instances, it would be good to have a full fetal karyotype, and also have fetal DNA for further studies once the five aneuploidies are excluded. Numerous papers on the use of NIPT in high-risk pregnancies have been published in 2014. Recently, papers have appeared demonstrating its utility in the low-risk population as well [5, 6]. Once the cost comes down, it would be routinely used in all pregnancies. Recently, there have been two excellent papers on the experience in detecting microdeletions [7, 8] Those using massively parallel sequencing technology claim they would be able to detect all deletions and duplications in the genome, and this advance in technology is awaitedwith baited breath. We also look forward, with excitement, to the application of this technology for the diagnosis of single-gene disorders.What is heartening to see is the establishment of this technology right in India by one company, to avoid the dispatch of samples abroad. More companies are likely to follow suit. Hopefully, thiswill also lead to lowering of the cost of the test, and the turn-around time for the reports. Lo et al. have recently described how the massively parallel sequencing technology has been used to determine the fetal genome, methylome, and transcriptome [9]. & Ishwar C. Verma jfm200@yahoo.co.in

Clinical Impact of Additional Cytogenetic Aberrations, cKIT- and RAS Mutations and Other Factors in Pediatric t(8;21)-AML

IntroductionWhilst pediatric t(8;21)-positive AML has an overall survival rate of up to 80-90%, the relapse rate at 30-40% remains unacceptably high. Factors that reliably discriminate between patients with t(8;21) who do or do not relapse have been poorly described. The prognostic impact of additional cytogenetic aberrations and of cKIT or RAS mutations in this subgroup remains unclear. In this large retrospective cohort study, supported by the International-BFM study group, we aimed firstly to determine outcome in relation to these aberrations and, secondly to identify any treatments associated with outcome.MethodsData on diagnostics, treatment, response, relapse or other events and follow-up were collected from 19 childhood cancer study groups, who provided data from 916 pediatric AML-patients positive for t(8;21) and/or AML1-ETO fusion gene. Karyotypes were reviewed and classified (CH&BB). Aberrations occurring in at least 10 patients were considered for analyses and involved loss of sex chromosome, deletions of chromosome 9q (del9(q)), abnormalities of chromosome 7q, gain of chromosome 4 (+4) and gain of chromosome 8. A total of 228 samples could be evaluated for cKIT and/or RAS mutations. Significant characteristics in explorative analysis were used in 2 multivariate Cox proportional-hazard regression models for assessing effects of induction and overall treatment elements. Induction was defined as first two courses of chemotherapy. Endpoints were probabilities of achieving complete remission (pCR), of event free survival (pEFS), and of overall survival (pOS) and cumulative incidence of relapse (CIR). Only results of multivariate analyses are shown.ResultsIn final analyses 838 patients with a full karyotype were included. CR was achieved in 92% of the patients. Five-year pOS was 74% (SE 1.5%), five-year pEFS 58% (SE 1.7%) and five-year CIR 26% (SE 1.5%). WBC count >20 x109/L was associated with poorer outcome for all endpoints (p<0.01). CNS involvement at presentation led to higher CIR (p<0.01). Twenty five percent of the patients had cKIT mutation(s). No differences in clinical or leukemic characteristics between mutated and wild-type patients were identified. Seven out of 10 patients with +4 had cKIT mutation(s) (p<0.01). Patients with RAS mutations (13%) had higher WBC counts (p=0.03). No clinical differences between N-RAS (n=17) and K-RAS (n=5) mutated patients were found. No associations between other additional aberrations and cKIT- or RAS mutations were identified. Patients with del(9)(q) (n=104) had lower pCR (p=0.01), without influencing pEFS or pOS. Additional +4 (n=21) was strongly associated with higher CIR (p<0.01) and poorer survival rates (5ypEFS 18% versus 59%, plogrank<0.01 and 5ypOS 33% versus 75%, plogrank<0.01). Presence of other cytogenetic aberrations including complex karyotype or cKIT or RAS mutations did not affect outcome.Anthracyclines >150mg/m2 used in first induction course was associated with higher pCR (p=0.04) and higher pOS (p=0.03). Etoposide >500mg/m2 used in first course was associated with higher pEFS (p=0.01) and pOS (p=0.02). The use of HD-AraC 3g/m2 during induction was associated with higher pCR (p=0.02). When including consolidation therapy, cumulative dosages of cytarabine >30g/m2 and etoposide >1500mg/m2 were associated with lower CIR (p=0.02 and p<0.01 respectively).ConclusionThis study identifies novel negative prognostic factors in pediatric t(8;21)-AML patients - del(9q) associated with an inferior pCR, and +4 significantly associated with an increased relapse rate and inferior survival. This challenges the favorable classification of patients with t(8;21) and +4. The presence of del(9q) may indicate the need for intensified induction therapy in view of the lower pCR. Although cKIT or RAS mutations did not influence outcome in conventionally treated t(8;21)-patients in this study, mutated patients may benefit further from treatment with tyrosine kinase inhibitors.This study suggests benefit from protocols containing high-dose anthracyclines, etoposide and HD-AraC 3g/m2 during induction, and the use of cumulative dosages of cytarabine >30g/m2 and of etoposide >1500mg/m2 in pediatric t(8;21)-positive AML. Optimal dosages and combinations of these agents should be established in prospective randomized trials. DisclosuresNo relevant conflicts of interest to declare.

Clinical application of noninvasive prenatal testing for the detection of trisomies 21, 18, and 13: a hospital experience.

The aim of this study is to report the clinical application of noninvasive prenatal testing (NIPT) to detect chromosomal aneuploidies, especially trisomies 21, 18, and 13 in Chinese singleton pregnancies. Pilot validation between NIPT with full karyotyping was conducted blindly on 306 cases. Subsequently, 7705 pregnancies were offered with NIPT. Follow-up data was obtained in all cases. In the validation stage, a total of five NIPT positive cases were observed for trisomies 21 and 18, and results were confirmed by karyotyping; there were no cases of trisomy 13. Thus, the overall sensitivity and specificity in the validation stage was 100%. In 7705 cases, NIPT results were obtained in 7701 cases; 66 cases were classified as positive, including 48 cases of trisomy 21, 14 cases of trisomy 18, and 4 cases of trisomy 13. Subsequent karyotyping documented two false positive diagnoses for trisomies 21, 18, and 13, respectively. Sensitivity and specificity for detection of trisomies 21 and 18 and 13 were 100% and 99.9%, respectively. Additionally, prenatal chromosomal detection for pregnancies with NIPT has shown a gradual increase since its implementation. Noninvasive prenatal testing allows a more suitable and efficient workflow for our patients' needs, together with invasive procedures allows a higher prenatal detection of chromosomal aneuploidies.