Karyotype Results Research Articles

Multiplex Quantitative Fluorescent Polymerase Chain Reaction for Detection of Aneuploidies

Quantitative fluorescent polymerase chain reaction (QF-PCR) is a well-established method in Western countries where commercial kits are being used for rapid prenatal diagnosis of aneuploidies. The limitations of these kits are that these are basically designed for the Caucasian population and are very expensive. Thus there is an urgent need to investigate the informative markers in the Asian population and to develop indigenous informative markers for detection of aneuploidies. This is a cost-effective alternate to the commercial kits and can be easily used in developing countries. We have tested three STR markers (D21S1435, D21S11, and D21S1411) in confirmed cases of Down syndrome and their parents. Origin of nondisjunction was also determined by means of QF-PCR. All the Down syndrome cases were confirmed for trisomy 21 by STR analysis. The results matched 100% with the karyotyping results. Nondisjunction was maternal in 87.5% cases and paternal in 12.5% cases. Nondisjunction during meiosis I was responsible for 87.5% cases of maternal nondisjunction and 100% cases of paternal nondisjunction. The combination of three STR markers is highly informative and can be used for diagnosis of trisomy 21 in India.

Resultados obstétricos en cribado combinado de las aneuploidías en el primer trimestre superior al corte con resultado de amniocentesis con cariotipo fetal normal

IntroductionTo evaluate whether there are adverse pregnancy outcomes in pregnant women with a risk index above the cut-off point in first-trimester screening for fetal chromosomal abnormalities and an amniocentesis result of normal fetal karyotype in a sample of pregnant women attending our clinic at the beginning of pregnancy. Subjects and methodsWe performed a case-control study. A series of patients who underwent first-trimester combined screening as part of antenatal care between January 2009 and January 2010 were selected. ResultsOf the maternal complications registered during the pregnancy, gestational diabetes was more frequent among cases. ConclusionsThe incidence of the remaining complications analyzed (intrauterine restricted growth, preeclampsia, oligoamnios) was similar in the two groups. In most of the pregnant women, delivery occurred after 37 weeks through the vaginal route.

Detection of Numerical Aneuploidy of Chromosomes X, Y, 13, 18 and 21 in 100 Blood and Fetals Samples by QF-PCR Method

Objective: Determination of parents to avoid giving birth to a child with any anomalies has increased the demand for prenatal diagnosis. Two most important criteria of any prenatal diagnosis procedure are accuracy, and speed. They have to minimize burden and anxiety for families. One of the main tests requested during pregnancies is testing for chromosomal abnormalities. This study was carried out with the aim of investigating the use of a rapid diagnosis test for detecting chromosomal numerical aneuploidy in blood and fetal samples, and also to compare the outcomes with cytogenetic method. Materials and methods: In this study, 100 samples from high risk pregnancies or affected individuals, comprising 12 chorionic villi (CV), 43 amniotic fluids (AF) and 45 blood samples were analyzed by QF-PCR. The samples were amplified using the specific microsatellite markers (STRs) for chromosomes X, Y, 13, 18 and 21. The sample analysis was performed based on the peak type of the PCR products, and the results were compared with the cytogenetic findings. Results: In total 26 samples were normal and 74 were diagnosed as aneuploids. Eight sex chromosome aberrations (three 45, X; three 47, XXY; one 47, XXX and one 46, XY (female phenotype), 65 numerical aberrations of X, Y, 13, 18 and 21 chromosomes and one triploidy were recognized. The QF-PCR data were compared with the karyotype results and showed complete concordance. Conclusion: This study showed that QF-PCR method is definitely superior, due to its advantages and few drawbacks in diagnosis of numerical chromosomal aberrations. Low costs and high speed of analysis as well as its automaticity are among the most important advantages of this method. Considering more than 99.4% accuracy of the QF-PCR method (compared with cytogenetics) and the time required to do cytogenetic analysis, QF-PCR is the method of choice for aneuploidy testing.

Medical versus surgical treatment for early pregnancy loss in infertile patients: Which approach facilitates more rapid return to subsequent treatment cycle?

Patients and physicians confront a challenging dynamic when an early pregnancy loss (EPL) occurs after fertility treatment (FT). Our study focused on the time to resumption of FT in patients managed medically (Cytotec) compared to in those managed surgically with dilatation and curettage (DC p = 0.01). More D&C patients received karyotype results (68% vs 5%). A significant difference was not found in resumption time to the next fertility treatment between the medically and surgical treatment. More medically managed patients RPOC requiring D&C. Although a D&C is more likely to provide karyotype results, medical management is a viable alternative.

1: A multicenter, prospective, masked comparison of chromosomal microarray with standard karyotyping for routine and high risk prenatal diagnosis

1: A multicenter, prospective, masked comparison of chromosomal microarray with standard karyotyping for routine and high risk prenatal diagnosis

Chorionic villus sampling for abnormal screening compared to historical indications: prevalence of abnormal karyotypes

Objective: To determine the prevalence of abnormal karyotype results in women undergoing chorionic villus sampling (CVS) for abnormal first trimester screening compared to CVS for historical indications (advanced maternal age (AMA) or prior aneuploidy). Methods: Retrospective cohort of all patients undergoing CVS at Oregon Health & Science University from January 2006 to June 2010. Patients were separated based on CVS indication: (1) positive ultrasound (U/S) or serum screening; or (2) AMA or prior aneuploidy with normal or no screening. Prevalence of abnormal karyotype results were compared between groups. Results: Fetal karyotyping was successful in 500 of 506 CVS procedures performed. 203 CVS were performed for positive screening with 69 abnormal karyotypes (34.0%). 264 CVS were performed for historical indications with 11 abnormal karyotypes (4.2%). This difference was statistically significant (χ2 71.9, p < 0.001; OR 11.8 [95% CI 5.8, 24.6]). There were two age-related aneuplodies in AMA women without positive screening. 42 out of 44 AMA women diagnosed with aneuploidy (95.5%) had abnormal U/S and/or serum screening (35 U/S, 4 serum, 3 U/S and serum). Conclusions: Combined ultrasound and serum screening should be recommended to all women, including AMA women, prior to undergoing invasive testing to improve risk-based counseling and minimize morbidity.

Higher Rates of AML Transformation and Poor Risk Cytogenetics in Patients with Low Average Albumin Levels Confers Poor Prognosis in Myelodysplastic Syndromes,

Abstract 3832Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders characterized by reduced hematopoiesis and progression to leukemia. Albumin is a prognostic biomarker in many malignant and non-malignant conditions, including multiple myeloma, chronic inflammation and aging. Recently, one study (Yepez, M, ASH 2010, abst #4001) showed that hypoalbuminemia was an independent prognostic factor in MDS. However, serum albumin levels can vary with time and be influenced by nutrition, age and presence of other comorbidities which are prevalent in MDS. To have an accurate assessment, we took the average serum albumin level of MDS patients over a period of 6 months. We hypothesize low average albumin will confer worse outcomes We conducted a retrospective analysis of 237 MDS patients seen in Cleveland Clinic between 1999 and 2009 (RARS= 30, RCMD=25, RAEB1/2=61, 5q- syndrome= 4, CMML1/2=39, MDS/MPN-U=32, RARS-T=30, RCUD=26). A total of 194 patients were included since average albumin was able to be calculated. The median age is 66 (20–92) years. Median follow-up time is 15 months (0–81 months). Clinical information including age, SNP-A karyotyping results, hemoglobin and serum albumin at time of diagnosis, average serum albumin level over 6 months, absolute neutrophil count, platelet counts, bone marrow blasts, and metaphase cytogenetic results were obtained.Patients were divided based on IPSS into lower and higher risk disease. We analyzed a total of 123 patients in the lower risk and 71 patients in the higher risk group. Further, we subdivided patients into lower albumin level (<3.5) or higher (3.5 or greater) based on the average albumin over 6 months. The median overall survival (OS) of the cohort is 15 months. Categorical variables were analyzed using Fisher’s exact test. The Cox-proportional hazards model was used to assess univariate and multivariate analyses for OS and progression free survival (PFS); factors assessed included age (≥60 vs. <60 years), disease grouping (MDS and MDS/MPN vs sAML), BM blasts (≥5 vs. <5 %), Hgb level (≥10 vs. <10 g/dL), metaphase cytogenetics (MC) risk groups using IPSS criteria (good, intermediate, poor), presence or absence of new SNP lesions and average albumin level. Each variable was retained in the multivariate model regardless of its statistical significance. Only variables with p<.05 in multivariate analysis were considered significant. Similar to the study by Yepez M et al, we also found that serum albumin level at the time of original diagnosis is prognostic in MDS and related disorders. Patients with low albumin levels at diagnosis (<3.5) have a worse OS compared to those with high albumin levels (6 vs 16 mos, p=.005). However, this was possible with a dichotomized albumin level. More importantly, we also found that the lower average serum albumin level [OS (8 vs 30 mos, p=.0001) and PFS (5 vs 20 mos, p=<.0001)] over 6 months can also be prognostic in MDS but not in MDS/MPN and sAML. Patients with low average albumin have poorer survival compared to patients with high albumin (OS: 6 vs 23 months, p=<.0001; PFS: 5 vs 15 months, p= <.0001). The same statistically significant findings were observed in patients stratified to lower (OS: 9 vs 37 mos, p=.001; PFS: 5 vs 24 mos, p=<.0001) or higher (5 vs 13 mos, p=.03) risk MDS by IPSS. Multivariate analysis showed that low average albumin level is an independent predictor of poor outcomes in MDS (OS: HR=2.12 CI:1.53–2.99, p=<.0001; PFS: HR=1.91 CI:1.31–2.76, p=.0009). Similarly, previously validated poor prognostic factors in MDS remained significant (Age ≥60 [HR:1.87, p=.0003], sAML disease grouping [HR:2.08, p=.0001], Hgb <10 g/dl [HR:1.42, p=.009], BM blasts [HR:1.44, p=.03], Poor risk cytogenetics [HR:2.22, p=.004], and presence of new SNP lesions [HR:1.59, p=.0008]. To identify factors that may result in worse outcomes in low average albumin patients, we assessed treatment response differences between patients which were not significant between low and high albumin levels. However AML transformation [(42/92 (45%) vs 111/150 (74%), p.002] and cytogenetics [Poor vs Good/Intermediate (21/43 (49%) vs 55/185 (30%), p=.02)] correlated with lower average albumin levels. In conclusion, low average serum albumin levels are an independent predictor of worse outcomes in MDS. Moreover, these data suggest that low average serum albumin is associated with poor risk karyotype and with progression to AML. Disclosures:No relevant conflicts of interest to declare.

Introducing array comparative genomic hybridization into routine prenatal diagnosis practice: a prospective study on over 1000 consecutive clinical cases

To assess the feasibility of offering array-based comparative genomic hybridization testing for prenatal diagnosis as a first-line test, a prospective study was performed, comparing the results achieved from array comparative genomic hybridization (aCGH) with those obtained from conventional karyotype. Women undergoing amniocentesis or chorionic villus sampling were offered aCGH analysis. A total of 1037 prenatal samples were processed in parallel using both aCGH and G-banding for standard karyotyping. Specimen types included amniotic fluid (89.0%), chorionic villus sampling (9.5%) and cultured amniocytes (1.5%). Chromosomal abnormalities were identified in 34 (3.3%) samples; in 9 out of 34 cases (26.5%) aCGH detected pathogenic copy number variations that would not have been found if only a standard karyotype had been performed. aCGH was also able to detect chromosomal mosaicism at as low as a 10% level. There was complete concordance between the conventional karyotyping and aCGH results, except for 2 cases that were only correctly diagnosed by aCGH. This study demonstrates that aCGH represents an improved diagnostic tool for prenatal detection of chromosomal abnormalities. Although larger studies are needed, our results provide further evidence on the feasibility of introducing aCGH as a first-line diagnostic test in routine prenatal diagnosis practice.

The Rate of Sex Chromosome Aneuploidies in Prenatal Diagnosis and Subsequent Decisions in Western Turkey

Sex chromosome abnormalities (SCAs) are the most common genetic disorder with a frequency of 1/400 or 1/500 live births. In this study we aimed to evaluate the initial indications, frequencies, and pregnancy termination rates of pregnancies with SCAs referred to Ege University Medical Faculty, Department of Medical Genetics. Prenatal diagnosis was performed in 7505 cases in the period of January 1998 through December 2009. In this study, their initial indications and fetal karyotype results were evaluated retrospectively. A total of 60 pregnancies (0.80%) with SCA were evaluated. Turner syndrome was the most commonly diagnosed SCA in prenatal diagnosis (60%). The most common referral reason for pregnancies with Turner syndrome was cystic hygroma on ultrasonography. Of 14 pregnancies having a prenatal diagnosis with SCA (Turner syndrome: 7, Klinefelter syndrome: 5, Mosaic Turner syndrome: 2), 12 with SCA (85.7%) were terminated. The ratio of SCA in the prenatally diagnosed cases was similar to those reported in the literature. Although the ratio of terminated pregnancies with Turner syndrome was similar to those reported from European countries, all the pregnancies with Klinefelter syndrome have chosen termination, which showed a regional difference in Turkey. It is important to consider the decisions of the families during the genetic counseling sessions of the couples having SCAs.

Women with chromosomally normal male fetuses are at increased risk of being referred for invasive testing following first‐trimester risk assessment

This study investigated the gender distribution in karyotype results from chorionic villus samples and amniocenteses performed due to an increased risk for Down syndrome based on first-trimester combined risk assessment. All prenatal karyotypes performed from 2006-2008 due to a risk assessment above 1:300 were retrieved from the Danish Central Cytogenetic Register. The distribution of gender for all newborns was obtained from Statistics Denmark. The χ(2) test and odds ratios were used for intergroup comparison. We retrieved 5 157 karyotype results (54.9% male and 45.1% female karyotypes), of which 4 662 were normal. During the same period, 100 112 boys and 94 732 girls were born. Women carrying male fetuses were significantly more often referred for invasive testing than women carrying female fetuses (2.8 vs. 2.5%, p < 0.0001). However, the proportion of chromosomally normal male fetuses was significantly greater than that for female fetuses (92.0 vs. 88.4%, p < 0.0001). A significantly higher proportion of women pregnant with male fetuses compared with female fetuses had an invasive diagnostic test performed prenatally following a risk assessment for Down syndrome.

De novo exceptional complex chromosomal rearrangement in a healthy fertile male: case report and review of the literature

To report a de novo exceptional complex chromosomal rearrangement (CCR) with four breakpoints in the male partner of a couple with recurrent abortions. Case report and review of the literature. Genetics laboratory in a private hospital. A couple referred for recurrent abortions. Cytogenetic and sperm fluorescence in situ hybridization (FISH) techniques. Karyotype and FISH sperm results. The couple was phenotypically normal, with no family history of miscarriage or infertility. Female karyotype was normal. Male karyotype followed by FISH analysis showed a de novo CCR with four breakpoints: t(5,13,16)(q11.1, q14.3, q12.2), ins(16;13)(q12.2;q?q14.2). ish t(5;13;16)(wcp5+,wcp13+), ins(16;13)(wcp13+). Exceptional de novo CCR male carriers with recurrent abortions are extremely rare. Patients with CCRs have limited options to achieve a normal pregnancy. Careful consideration and assessment should be provided upon counseling of couples with CCRs.

Female overweight is not associated with a higher embryo euploidy rate in first trimester miscarriages karyotyped by hysteroembryoscopy

Overweight women (body mass index ≥ 25 kg/m(2)) present an embryo euploidy rate in first trimester miscarriages similar to normoweight controls after a selective biopsy and karyotyping of embryo and/or chorion samples taken by hysteroembryoscopy.

Adjusted risk for trisomy 21 after the anomaly scan. How accurate is the Fetal Medicine Foundation algorithm?

To examine the accuracy of the Fetal Medicine Foundation algorithm used to adjust the risk for trisomy 21 after ultrasound findings detected or not at the time of the anomaly scan. This was a retrospective study of all amniocenteses performed in a single centre, in singleton pregnancies, between 1998 and 2008. Maternal demographic characteristics, second-trimester ultrasound findings, indications for amniocentesis and karyotype results were reviewed. The algorithm introduced by the Fetal Medicine Foundation was used to calculate the patient specific risk for trisomy 21 using the age related background risk and anomaly scan findings. Expected trisomy 21 cases based on these risks was compared with the actual karyotype results. Overall, 4,511 cases of singleton pregnancies that underwent second-trimester amniocentesis were reviewed. In 572 cases (12.7%), there were markers of chromosomal abnormality and no previous screening for trisomy 21. The expected number of trisomy 21 cases based on maternal age for this population of 572 cases was 1. The expected number of chromosomal abnormalities after adjusting for ultrasound findings based on the algorithm introduced by the Fetal Medicine Foundation was 6.9 (95% confidence interval 3.4-14.3). After karyotyping, in this population there were 6 cases of trisomy 21, 1 case of Trisomy 18 and 1 case of XXY. The algorithm that adjusts the age related risk of trisomy 21 according to second-trimester anomaly scan findings is very accurate in predicting the modified risk.

**504 Detection of Cytogenetic Abnormalities Associated with Outcome Differences in Acute Myeloid Leukemia Using Array-Based Comparative Genomic Hybridization (aCGH) Analysis

Abstract 1633 Poster Board I-659 Background The presence of abnormal cytogenetic findings at diagnosis is an independent indicator of the outcome in patients with acute myeloid leukemias (AML). Recent availability of high-resolution analysis by aCGH technology has facilitated rapid detection of cytogenetic abnormalities previously undetected by the conventional G-banded karyotyping. Aim: To determine whether specific cytogenetic abnormalities, as detected by the aCGH analysis, are associated with differences in clinical outcomes in a group of patients with AML treated uniformly with the standard chemotherapy regimen of Idarubicin and Cytarabine. Methods A total of 111 patients with newly diagnosed AML (Median age: 55 years; range, 22 to 73 years) were enrolled in the study. All patients were treated with Idarubicin 12 mg/m 2 IV daily x 3 days and Cytarabine 1.5 g/m 2 by continuous IV infusion daily x 4 days (3 days in patients older than 60). The diagnostic bone marrow samples from 48 of these patients were analyzed by aCGH using a 44K CGH array with a spatial resolution of 50-75 kb (Agilent Inc). The aberrations detected by aCGH using CGH Analytics (Agilent Inc, Santa Clara, CA) and the Nexus Copy Number Software (Biodiscovery Inc, El Segundo, CA) were compared to conventional G-banded karyotyping results. Correlation of aCGH-detected aberrations with clinical outcome was performed by Kaplan-Meier analysis and log-rank test. Results Complete remission (CR) was achieved in 34/48 (71%) patients, out of which 16 (47%) patients relapsed in less than 1 year (Median: 18.5 weeks, range: 1-44 weeks) and the remaining 18 (53%) patients sustained CR beyond one year (Median: 89 weeks, range: 58-132 weeks). The comparison of aCGH findings between the patients achieving CR and the resistant patients showed significant association of loss of a 155 kilobase region on 5q33.3 with achievement of CR (p TP53 gene in 3/12 (25%) patients, although these three patients showed genetic instability and poor clinical outcome. Overall, the aCGH findings were in concordance with the conventional karyotyping results. The aCGH analysis was able to detect aberrations in samples containing blast count as low as 5%. In addition, aCGH identified previously undetected aberrations, as small as 5 kb, of currently unknown significance. Conclusion The aCGH analysis indicates that the loss of 5q33.3 is associated with achievement of complete remission and the loss of 17p11.2-q11.1 is associated with poor overall survival in AML patients treated with Idarubicin and Cytarabine. Array CGH analysis provides a useful and rapid diagnostic tool for identifying these high-risk patients. The biologic roles of EBF1 and RNF145 in therapy response warrant further investigation. Disclosures No relevant conflicts of interest to declare.

Genetic identification of African cultured yeasts of the genus Saccharomyces

Based on the results of genetic analysis and molecular karyotyping, a total of 11 strains involved in fermentation processes employed in various regions of Africa were identified as representatives of the species Saccharomyces cerevisiae. These stains exhibited a high degree of chromosomal polymorphism. The obtained fertile genetic lines of the studied African strains may be of considerable interest for evolutionary genetics and breeding of yeasts of the genus Saccharomyces.

Karyotype result prior to surgery in patients with suspected ovarian germ cell tumors

Karyotype result prior to surgery in patients with suspected ovarian germ cell tumors

Karyotype result prior to surgery in patients with suspected ovarian germ cell tumors-response

Karyotype result prior to surgery in patients with suspected ovarian germ cell tumors-response

Subfertility phenotype, chromosome polymorphism and conception failures

To evaluate the prevalence of cytogenetic alterations and chromosomic polymorphism in couples with a subfertility phenotype in a Brazilian population. Karyotype analysis through G and C banding of 1,236 individuals who presented the subfertility phenotype, from two different centers (public and private) were included in the study. These patients were classified in two sub-groups: one with two or more gestational consecutive losses or not and the o with, at least, one gestacional loss or absence of conception. Karyotype results were evaluated in different groups and frequencies were calculated. Statistical analyses were carried out through Fisher's exact test and Odds Ratio analysis. Approximately 25% of the cases presented abnormal karyotype results, including numerical and structural alterations and also polymorphic variants. In both centers, the prevalence of polymorphic variants was 8.9 and 3.8%, respectively. There was no significant difference between the prevalence of polymorphic variants and other abnormalities in individuals with or without previous history of reproductive loss. The results of the present study reinforce the need of adequate disclosure of complete cytogenetic information in the karyotype results, with specific attention in relation to the polymorphic variants.

PREDICTIVE VALUE OF FETAL NUCHAL TRANSLUCENCY IN SCREENING OF CHROMOSOMAL ABERRATIONS

In search for specific early ultrasound signs that could indicate an increased risk of hereditary or acquired disorders of the fetus, scientific researches confirm the value of exceptional ultrasound findings of nuchal translucencey (NT). The aim of the study was to determine the predictive value of the diameter of fetal NT in the detection of chromosomopathy. The investigation included 317 pregnant women with monofetal pregnancies, gestational age 11-14 weeks. The control group consisted of pregnant women in whom, after amniocentesis, a regular result of fetal karyotype was obtained. The limits of physiological and pathological findings of the NT value were not determined; instead, we used the diameters of NT that were obtained in pregnant women with pathological score of amniocentesis as potentially pathological values. Mean value of NT in the control group was 1.92±0.39mm, and in the group with pathological findings of fetal karyotype it was 2.49±0.37mm, which is a statistically significant difference (p 0.05).The probability that a patient with negative NT findings be healthy is 0.97. NT sensitivity as a marker for chromosomopathy was 0.97. The rate of false positive findings was 0.027. Specificity of NT as a marker for chromosomopathy was 0.97. The probability that a patient with positive findings NT really be ill was 0.66. Valid findings of NT can be considered safe ultrasonographic markers in the assessment of absence of chromosomopathy. Pathological finding, given the low positive predictive value of NT, must be amended by other prenatal tests before a pregnant woman is advised on prenatal invasive diagnostics. .

Sonogenetics: A Breakthrough in Prenatal Diagnosis

ABSTRACT G-band and rapid FISH/QF-PCR are regarded as the gold standards for prenatal chromosomal diagnosis. Numerous microdeletion/ microduplication syndromes, however, are not detectable by conventional karyotyping. So far, we had a dilemma between fetal developmental/structural abnormalities with strong suspicion of chromosomal abnormalities and normal karyotype results. Fetal DNA chip includes more than 6,450 genetic loci and covers more than 100 common genetic diseases with numeric, structural chromosomal anomalies. In April 2009, we launched prenatal diagnosis by fetal DNA chip of amniotic fluid samples or chorionic villi samples in the selected fetuses with sonographic abnormalities and suspicion of familial genetic disorders. We had seven cases with both abnormal ultrasound findings and pathologic copy number variations by DNA chip. In all cases, normal karyotype was confirmed by G-banding analysis. Fetal DNA chip (array CGH) may become a strong modality to solve some part of this dilemma. Although we have to be prudent to select the patients, deal with DNA chip results and parental counseling, “sonogenetics” is one of the breakthroughs in prenatal diagnosis, and the further accumulation of case studies will be required in this new field.