The-Hung Bui

Molecular cytogenetic and rapid aneuploidy detection methods in prenatal diagnosis

Cytogenetic analysis is an important component of prenatal diagnosis. The ability to rapidly detect aneuploidy and identify small structural abnormalities of fetal chromosomes has been greatly enhanced by the use of molecular cytogenetic technologies. In this review, we will present some of the molecular cytogenetic techniques available to the clinical cytogenetics laboratory. These include fluorescence in situ hybridization (FISH), quantitative fluorescence PCR (QF‐PCR), multiplex ligation‐dependent probe amplification (MLPA) and microarray‐based comparative genomic hybridization (array CGH). The benefits and limitations of each technology will be discussed in the context of prenatal diagnosis.

Current controversies in prenatal diagnosis 3: is conventional chromosome analysis necessary in the post‐array CGH era?

Microscopic chromosome analysis of cultured cells has been regarded as the standard method for prenatal cytogenetic diagnosis since its first application to prenatal testing in 1966 (Steele and Breg, 1966) and has become routine since the first use of chromosome banding (karyotyping) in the early 1970s. Karyotyping has proved to be highly reliable for the diagnosis of numerical chromosome abnormalities (aneuploidies) and large structural rearrangements [>5–10 million base (Mb) pairs] in fetal cells obtained invasively by either amniocentesis in the second trimester of pregnancy or chorionic villus sampling in the first trimester. The supremacy of karyotyping in prenatal cytogenetic diagnosis has been challenged by the introduction of molecular cytogenetic methods including interphase fluorescence in situ hybridization (FISH), quantitative fluorescent PCR and more recently, multiplex ligation-dependent probe amplification (Boormans et al., 2010) for the rapid detection of aneuploidies for chromosomes 13, 18, 21 and the sex chromosomes (Shaffer and Bui, 2007). In addition to the common aneuploidies, many submicroscopic chromosomal rearrangements that lead to copy-number gains or losses have been shown to cause distinctive and recognizable clinical phenotypes. The sensitivity of detection of copy-number alterations has increased significantly with the advent of microarraybased comparative genomic hybridization (aCGH) with its commercially available multiple platforms. Together with improved assemblies and annotation of genome sequence data, these methods allow for the rapid identification of new syndromes that are associated with submicroscopic genomic changes in children with idiopathic intellectual disabilities, autism, developmental

Rapid methods for targeted prenatal diagnosis of common chromosome aneuploidies

Improvements in non-invasive screening methods for trisomy 21 (Down syndrome) and other aneuploidies during the first and second trimester of pregnancy have radically changed the indications for prenatal diagnosis over the last decade. Consequently, there was a need for rapid tests for the detection of common chromosome aneuploidies resulting in the development of molecular methods for the rapid, targeted detection of (an)euploidies of the chromosomes 13, 18, 21 and the sex chromosomes. The analysis of large series of prenatal samples has shown that such tests can detect the great majority of chromosome abnormalities in prenatal diagnosis. This resulted in lively discussions on whether conventional karyotyping should remain the standard method for the majority of prenatal cases or can be replaced by rapid tests only. This review gives an overview of different aspects of the three most common tests for rapid, targeted prenatal detection of (an)euploidies, i.e. interphase fluorescence in-situ hybridisation (iFISH), quantitative fluorescent polymerase chain reaction (QF-PCR) and multiplex ligation-dependent probe amplification (MLPA).

Current controversies in prenatal diagnosis 2: should incidental findings arising from prenatal testing always be reported to patients?

The Karolinska Institute, Center for Molecular Medicine, Clinical Genetics Unit and Center for Fetal Medicine, Karolinska University Hospital, Stockholm, Sweden Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Departments of Obstetrics and Gynecology and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA *Correspondence to: The-Hung Bui. E-mail: the.bui@karolinska.se All authors contributed equally.

Characterization of double ring chromosome 4 mosaicism associated with bilateral hip dislocation, cortical dysgenesis, and epilepsy†‡

We present the clinical and molecular findings in a Turkish child with a de novo mosaic ring derived from chromosome 4 with multiple cell‐lines; the karyotype was 46,XY,r(4)[83]/45,XY, −4[6]/47,XY,r(4),+r(4)[5]/48,XY,r(4),+r(4),+dic r(4)[1]/46,XY[5]. The patient is a 20‐month‐old male who was the first pregnancy of nonconsanguineous parents. The baby was delivered at term with a birth weight of 1,700 g (<3rd centile) and a length of 46 cm. The baby had feeding difficulties and vomiting problems. He started walking at age 2 years and delayed language was observed. Facial appearance was normal, but the ears were large with abnormal structure. The hands showed bilateral clinodactyly of the 5th fingers. He had mild mental retardation, and epilepsy. Analysis of chromosomes showed 46,XY,r(4)(::p16.3 → qter::)[67]/46,XY,r(4;4)(::p16.3 → qter::p16.3 → qter::)[2]/46,XY[3] by multicolor banding (MCB) technique. Array CGH delineated the size of the terminal deletion as 900 kb in 4p16.3. The Wolf–Hirschhorn critical region was preserved even though our patient had mild mental and motor retardation. While the mosaicism of the ring 4 could affect the phenotype, the deleted 900 kb distal deletion and clinical features of the patient may provide further insight into characteristic phenotype of the 4p− related syndromes.

Screening of fetal stem cells for infection and cytogenetic abnormalities.

Fetal stem cell transplantation may rely on material from therapeutic abortions. It is essential that the stem cell transplant does not transmit any microorganisms that may affect the fetus and that genetically abnormal cells are avoided. To evaluate such contamination, human fetal stem cells collected February 1992 - December 1993 were analyzed for bacterial and fungal growth, and the placentas were karyotyped. Four samples of 70 were positive for different pathogens. Serological screening of 43 women during this period resulted in five seroconversions and revealed one carrier of anti-HCV. Karyotyping revealed two abnormal findings out of 72 samples. Thus, the concept of using material from therapeutic abortions is safe.

Multifetal pregnancy reduction in Sweden: Utilization rate and pregnancy outcome (1986‐1992)

All departments of Obstetrics and Gynecology, as well as all private clinics in Sweden offering assisted cwnception, were surveyed by means of a questionnaire to determine the utilization rate and outcome of multifetal pregnancy reduction (MFR) for the period I January 1986 to 30 June 1992. The response rate was 100%. Multifetal reduction was performed in 26 women, giving an average utilization rate of 1/7 multiple births of three or more for the entire period. Of the various techniques used, intracardiac or intrathoracic injection of a potassiuni chloride solution was predominant. The experience of each center with multifetal reductioiis varied between one and six procedures. In this series, the overall complete pregnancy loss was 27% (n = 7). In 73% (n= 19) of women the pregnancy continued to delivery. One fetus died in utero in the second trimester, one child died from a subtentorial hemorrhage perinatall). and one child had a malformation of the right foot and hand. It seems necessary to limit MFR to a few centers in Sweden in order to maintain and increase the experience of the operators involved, and to decrease the fetal loss rate associated with the procedure. However. the ultimate goal is to make these procedures unnecessary when methods of avoiding excessive ovulation are refined and by limiting the number of replaced embryos in IVF‐treatment.

Information related to prenatal genetic counseling : interpretation by adolescents, effects on risk perception and ethical implications

Being raised in the genomic era may not only increase knowledge of available genetic testing but may also have an impact on how genetic information is perceived. However, little is known about how current adolescents react to the language commonly used by health care professionals providing prenatal counseling. In addition, as risk communication is related to numbers and figures, having different educational backgrounds may be associated with variability in risk perceptions. In order to investigate these issues, a previously developed questionnaire studying different ways of being told about hypothetical anomalies in a baby and corresponding risks (Abramsky and Fletcher Prenatal Diagnosis 22(13):1188–1194, 2002) was administered to high-school students in Sweden. A total of 344 questionnaires were completed by students belonging to a natural science or a social science program. The data show that teenage participants found technical jargon and words such as rare and abnormal more worrying than the presented comparison terms. Negative framing effects and perception differences related to numeric risk formats were also present. Additionally, participants’ gender and educational program did not seem to have an effect on risk assessment. In addition to reporting the questionnaire results, we discuss the ethical implications of the data based on the norm of non-directiveness and make some recommendations for practice. In general, genetic counselors should be aware that the language used within clinical services can be influential on this group of upcoming counselees.

Establishing a national program for fetoscopic guided laser occlusion for twin-to-twin transfusion syndrome in Sweden.

Objective. To describe the establishment of the fetoscopic guided laser occlusion (FLOC) technique for treatment of twin‐to‐twin transfusion syndrome (TTTS) and the initial results in a Swedish national center. Design. Retrospective, descriptive study. Setting. Tertiary level university hospital. Population. All referred and treated cases suffering significant TTTS. Methods. The present study includes all cases of FLOC for TTTS at the Center of Fetal Medicine at Karolinska University Hospital, Stockholm, Sweden from October 2001 until December 2009. Patients were referred from all over Sweden and a few from other Nordic countries. The patients were evaluated with ultrasound examination between gestational ages of 18 and 26 weeks. Data from patients were extracted from our electronic medical record system and, in addition, families were contacted and medical records requested from referring hospitals. Main outcome measures. Pregnancies with one or more surviving infants after FLOC treatment categorized according to stage of TTTS. Results. In 75% of pregnancies, one or more infant was born alive. At stage I, both infants survived in one pregnancy and one survived in the second. There was no significant difference between cases at stage II or III, i.e. 73 vs. 78% of pregnancies resulted in one or more surviving infant. At stage IV, 66% of pregnancies ended with one or more surviving infant. Conclusions. Treatment of TTTS is feasible in a rather small country like Sweden, with comparable results to other centers. There are strong arguments for centralization and further improvement of this kind of highly specialized treatment.

Rapid method for targeted prenatal diagnosis of Duchenne muscular dystrophy in Vietnam.

OBJECTIVE Since there is no effective curative treatment for Duchenne muscular dystrophy (DMD), prevention mostly depends on genetic counseling and prenatal diagnosis. About two-thirds of the affected patients have large deletions or duplications, which can be detected by multiplex ligation-dependent amplification (MLPA). The remaining cases include small mutations, which cannot be easily identified by routine techniques. In such cases, linkage analysis may be a useful tool for prenatal diagnosis. Here we compared results obtained from linkage using short tandem repeats (STRs) with those by MLPA and sequencing analysis. MATERIALS AND METHODS Eight Vietnamese pregnant women at risk of having a baby with DMD and requesting prenatal diagnosis were recruited in this study. MLPA and direct sequencing were applied to screen large rearrangements and point mutations in the dystrophin gene in the DMD probands and the fetal samples. STR linkage was also performed to analyze fetal mutation status. RESULTS By MLPA and sequencing analysis, five DMD patients showed deletions of the dystrophin gene, and no deletions of exons were detected in seven amniotic fluid cell samples; one patient harbored the out-of-frame small deletion of exon 43, which was also found in the fetal sample of this family. STR analysis revealed the transmission of a mutant allele inside each family. CONCLUSION Our results suggest that the combination of STR and MLPA could be a rapid, reliable, and affordable detection protocol for determination of the carriers status and prenatal diagnosis of DMD in a developing country such as Vietnam.