Traditional Karyotyping Research Articles

An economic analysis of prenatal cytogenetic technologies for sonographically detected fetal anomalies

When congenital anomalies are diagnosed on prenatal ultrasound, the current standard of care is to perform G-banded karyotyping on cultured amniotic cells. Chromosomal microarray (CMA) can detect smaller genomic deletions and duplications than traditional karyotype analysis. CMA is the first-tier test in the postnatal evaluation of children with multiple congenital anomalies. Recent studies have demonstrated the utility of CMA in the prenatal setting and have advocated for widespread implementation of this technology as the preferred test in prenatal diagnosis. However, CMA remains significantly more expensive than karyotype. In this study, we performed an economic analysis of cytogenetic technologies in the prenatal diagnosis of sonographically detected fetal anomalies comparing four strategies: (i) karyotype alone, (ii) CMA alone, (iii) karyotype and CMA, and (iv) karyotype followed by CMA if the karyotype was normal. In a theoretical cohort of 1,000 patients, CMA alone and karyotype followed by CMA if the karyotype was normal identified a similar number of chromosomal abnormalities. In this model, CMA alone was the most cost-effective strategy, although karyotype alone and CMA following a normal karyotype are both acceptable alternatives. This study supports the clinical utility of CMA in the prenatal diagnosis of sonographically detected fetal anomalies.

Potential diagnostic consequences of applying non‐invasive prenatal testing: population‐based study from a country with existing first‐trimester screening

Targeted non-invasive prenatal testing (NIPT) tests for trisomies 21, 18 and 13 and sex chromosome aneuploidies and could be an alternative to traditional karyotyping. The aim of this study was to determine the risk of missing other abnormal karyotypes of probable phenotypic significance by NIPT. This was a retrospective population-based analysis of all singleton pregnancies booked for combined first-trimester screening (cFTS) in Denmark over a 4-year period. Data concerning maternal demographics, cFTS and prenatal or postnatal karyotypes were collected from the Danish Fetal Medicine database. Karyotypes were classified according to whether the chromosomal anomaly would have been detected by NIPT and whether it was likely to affect phenotype. cFTS was completed in 193638 pregnancies. 10205 (5.3%) had cytogenetic or molecular analysis performed. Of these, 1122 (11.0%) had an abnormal karyotype, of which 262 (23.4%) would have been missed by NIPT, but would probably have been clinically significant. The prevalence of such 'atypical abnormal karyotypes' was increased in women above 45 years of age, in pregnancies with increased nuchal translucency (NT) thickness (≥ 3.5 mm), with abnormal levels of free β-human chorionic gonadotropin (<0.2 or ≥ 5.0 multiples of the median (MoM)) or pregnancy-associated plasma protein-A<0.2 MoM. One or more of these factors was present in 3% of women, and the prevalence of atypical abnormal karyotypes in this high-risk cohort was 1.6%. A significant proportion of karyotypic abnormalities will be missed by targeted NIPT. Women of advanced maternal age, or with increased fetal NT or abnormal biochemistry, have a higher risk of having a fetus affected by an atypical abnormal karyotype and need to be counseled accordingly when considering NIPT.

Non-invasive prenatal diagnosis of the most common aneuploidies with cell-free fetal DNA in maternal serum – preliminary results

The aim of the study was to present initial results of non-invasive prenatal diagnosis of common aneuploidies of chromosomes 21, 18 and 13 based on cell-free fetal DNA in maternal serum in high-risk patients, and to compare the results with routine karyotyping. Before the invasive procedure, 10 ml of peripheral blood from 10 patients was collected to isolate cell-free fetal DNA and to perform a non-invasive fetal trisomy test (NIFTY provided by Beijing Genomics Institute, BGI, Shenzen, China). Three out of 10 samples showed an abnormal karyotype in traditional karyotyping. There were 9 conclusive NIFTY results. NIFTY detected 1 out of 2 trisomies 18. The quantity of cell-free fetal DNA in maternal plasma in the second probe with trisomy 18 was unsatisfactory fora conclusive NIFTY result. In 1 case traditional karyotyping revealed mosaicism impossible to detect with NIFTY

Cytogenetic microarray in prenatal and postnatal diagnosis.

Due to high resolution cytogenetic microarray (CMA) has replaced traditional karyotyping for evaluation of individuals with intellectual disability, autism and congenital malformations. The diagnostic yield of CMA is 10 to 12 % and is more than any other investigation for evaluation of developmental disabilities. Due to its high resolution CMA is also being used as a prenatal test for chromosomal anomalies. The diagnostic yield is about 3% whatever may be the indication. The yield is higher in cases with fetal anomalies. The main concern with CMA is its ability detection of copy number variations of unknown significance. This is a major problem in prenatal diagnosis and is a challenge for the counselor and dilemma for the family in concern. With accumulation of more and more data of pathological and polymorphic variations in genome the variations of unknown significance will decrease. CMA is also useful in delineating abnormalities picked up by karyotyping. Some cases with double segment rearrangement may point towards familial chromosomal rearrangement and hence, CMA is indicated in familial cases with developmental disabilities and birth defects. Cost and availability of clinical cytogeneticists for appropriate interpretation of CMA results are important concerns for wider application of the technique.

Modified cIg-FISH protocol for multiple myeloma in routine cytogenetic laboratory practice

The International Myeloma Working Group recommends that fluorescence in situ hybridization (FISH) be performed on specifically identified plasma cells (PC). This is because chromosomal abnormalities are not frequently detected by traditional karyotyping due to the low proliferative rate of PC in multiple myeloma (MM). Conventional FISH enhances the sensitivity but lacks the specificity, as it does not distinguish PC from other hematopoetic cells. To fulfill this recommendation, PC need to be selected either by flow cytometry or immunomagnetic bead-based PC sorting or by concomitant labeling of the cytoplasmic immunoglobulin light chain, which allows for unambiguous identification. These techniques require expertise, time, and funding and are not easily incorporated into the routine workflow of the cytogenetic laboratory. We have modified and refined the technique using fixed cell pellets to achieve nicely separated and easily identifiable PC. With immunostaining and subsequent FISH (i.e., cytoplasmic immunoglobulin FISH, cIg-FISH), this technique can be easily incorporated into every cytogenetic laboratory. Twenty samples from patients with MM were subjected to routine FISH, cIg-FISH, and chromosomal karyotyping and the results were compared. Three FISH probes, which enabled detection of the t(4;14), t(14;16) and deletion of TP53, were used to validate this modified technique successfully.

Limited utility of fluorescence in situ hybridization for common abnormalities of myelodysplastic syndrome at first presentation and follow-up of myeloid neoplasms

Fluorescence in situ hybridization for abnormalities common to myelodysplastic syndrome (MDS FISH) is often used with traditional karyotype in the diagnosis and monitoring of myeloid neoplasms. However, its value in these roles has been questioned. To evaluate its utility, we compared MDS FISH results with karyotype in 544 bone marrow specimens obtained for diagnosis (180 cases) or follow-up (364 cases) of myeloid neoplasia. We found excellent concordance between FISH and karyotype, such that FISH is rarely abnormal (1.7% at diagnosis and 3.0% at follow-up) in cases with normal karyotype. Even in the rare discordant cases, the abnormal FISH has little or no clinical value. Thus, we propose that this test should be limited to cases with inadequate karyotype only. Such guidelines could result in significant cost savings with no impact on patient diagnosis.

Key Informants’ Perspectives of Implementing Chromosomal Microarrays Into Clinical Practice in Australia

High-resolution genomic tests have the potential to revolutionize healthcare by vastly improving mutation detection. The use of chromosomal microarray (CMA) represents one of the earliest examples of these new genomic tests being introduced and disseminated in the clinic. While CMA has clear advantages over traditional karyotyping in terms of mutation detection, little research has investigated the process by which CMA was implemented in clinical settings. Fifteen key informants, six clinicians, and nine laboratory scientists from four Australian states were interviewed about their experiences during and in the time since CMA was adopted for clinical use. Participants discussed challenges such as result interpretation and communication. Strengths were also highlighted, including the collaborative approaches of some centers. Clinical experiences and opinions can inform larger studies with a range of stakeholders, including patients. The historical perspectives from this retrospective study can be helpful in guiding the implementation of future genomic technologies such as whole exome/genome sequencing.

Low-frequency Mosaicism of Trisomy 14, Missed by Array CGH

before and after birth, mental retardation, developmental delay, cardiac anomalies, minor anomalies of genitalia in boys, abnormal skin pigmentation and dysmorphic features. Array comparative genomic hybridization (CGH) analysis has recently become a widely used method for detecting DNA copy number changes, in place of traditional karyotype analysis. In particular, an international consensus statement has now recommended array CGH as a first-line test in patients with unexplained developmental delay, intellectual disability and dysmorphisms. However, the ability of array CGH is limited in case of low-level mosaicism. Here, we report the detailed clinical and cytogenetic findings of the first case in Korea to show lowfrequency mosaicism of trisomy 14, diagnosed by array CGH analysis supplemented with karyotyping. Low-frequency Mosaicism of Trisomy 14, Missed by Array CGH

Re: Use of prenatal chromosomal microarray: prospective cohort study and systematic review and meta-analysis. S. C. Hillman, D. J. McMullan, G. Hall, F. S. Togneri, N. James, E. J. Maher, C. H. Meller, D. Williams, R. J. Wapner, E. R. Maher and M. D. K

Linked Comment: Ultrasound Obstet Gynecol 2013:41:610–620 With their prospective cohort study on chromosomal microarray analysis (CMA) performed in high-risk pregnancies, Hillman and colleagues contribute another study to this field and also present their data in the context of an up-to-date systematic review and meta-analysis of the literature. They provide overall detection rates of abnormalities by CMA compared with conventional karyotyping for various indications, as well as of variants of unknown clinical significance (VOUS), and report how these have changed over time. This is a timely publication, as it draws together the significant number of important and large prospective clinical trials on CMA for prenatal testing that have been published recently. The combined data from these studies represent a cohort of over 18 000 prenatal samples. Taken together, their findings clearly indicate that, compared with conventional karyotyping, the use of CMA in prenatal diagnosis improves substantially the detection rate of pathogenic chromosomal abnormalities. The findings provide evidence for the feasibility of introducing CMA into routine prenatal diagnosis practice, indicating that it is acceptable to apply CMA as a first-line diagnostic test, at least concurrently with conventional karyotyping. Statements issued by the American College of Obstetrics and Gynecology (ACOG), Canadian College of Medical Geneticists (CCMG) and Italian Society of Human Genetics (SIGU) have recommended that CMA should be offered as an adjunctive tool to selected groups of high-risk pregnancies (e.g. those with abnormal ultrasound findings and normal conventional karyotyping results), using the technique as a second-line test only, after standard karyotyping. The main reasoning behind this relates to the fact that CMA performed for indications other than abnormal ultrasound findings would likely be associated with a low positive predictive value, since the vast majority of fetuses tested would be clinically unaffected. The higher detection rate by CMA compared with conventional karyotyping reported by Hillman et al. is not confined to cases with abnormal ultrasound findings; the results of their meta-analysis demonstrate the improved diagnostic ability of CMA to detect clinically relevant abnormalities and the utility of bringing CMA into routine prenatal practice as a primary diagnostic tool for a number of other indications. Furthermore, the findings clearly indicate that offering CMA only as a second-line test in high-risk pregnancies may substantially limit prenatal diagnostic potential, since a significant proportion of copy number variants (CNVs) that can cause serious disability are not detected by traditional karyotyping. There is still understandable concern regarding the use of CMA on prenatal samples, related mainly to the potential detection by CMA of mild or unpredictable phenotypes and/or VOUS. In the meta-analysis of Hillman et al., VOUS are reported to occur in ∼1.4% of prenatal samples. However, it is well described that the incidence of VOUS detected is related mainly to the specific array platform used and its resolution. In fact, the ideal microarray platform and resolution for use in the prenatal setting remain to be determined. High-resolution oligonucleotide or single-nucleotide polymorphism (SNP) microarray platforms offer the theoretical opportunity to find additional sub-microscopic chromosomal abnormalities, compared to bacterial artificial chromosome (BAC) arrays at lower resolution, and are the default array choice for postnatal cytogenetics. The potential benefits of using high-resolution microarrays for prenatal diagnosis are greater, but the probability of finding VOUS may also be greater. In contrast, BAC arrays usually detect substantially fewer VOUS than do high-resolution microarrays, but their potential in detecting small clinically relevant CNVs may also be lower. In conclusion, the work of Hillman et al. is an important addition to the growing body of evidence on the diagnostic superiority of CMA over conventional karyotyping, with the ultimate goal of improved prenatal diagnosis and a lower risk of giving birth to a chromosomally abnormal neonate. F. Fiorentino GENOMA - Molecular Genetics Laboratory, Via di Castel Giubileo, 11, 00138 Rome, Italy (e-mail: fiorentino@laboratoriogenoma.it)

Utility of chromosomal microarray in five cases with cytogenetic abnormalities detected by traditional karyotype

Utility of chromosomal microarray in five cases with cytogenetic abnormalities detected by traditional karyotype

Effectiveness of multiplex ligation dependent probe amplification (MLPA) in prenatal diagnosis of common aneuploidies

To assess the effectiveness of the MLPA method (multiplex ligation dependent probe amplification) in prenatal diagnosis of common aneuploidies and compare its concordance with traditional karyotyping. From October 2008 until July 2012 we performed 195 MLPA (MRC-Holland) tests with the P095 probe mix on DNA extracted from chorionic villi or amniotic fluid from pregnant women with elevated risk for abnormal fetal karyotype and 5 tests on miscarriage DNA samples. Cell culture and traditional karyotyping were performed in parallel. Traditional karyotyping was successfully performed in 192 cases (98.5%; 192/195). In 52 cases the fetal karyotype was abnormal (26.8%). The most common findings included aneuploidies of the following chromosomes: 13, 18, 21, X, Y (86.5%, 45/52). There were 179 conclusive and 1 inconclusive MLPA result (92.3%; 180/195). The absolute specificity and sensitivity of the MLPA test were 100%. In 9 cases traditional karyotyping revealed aberrations impossible to detect with the MLPA P095 kit. The MLPA reaction was successfully performed on all miscarriage DNA samples. MLPA is an effective method for detecting common aneuploidies. Its effectiveness for miscarriage DNA samples remains to be elucidated in further studies.

CIg-FISH On Patients with Multiple Myeloma – A Modified and Simple Technique Easily Incorporated Into Routine Clinical Service

AbstractAbstract 4792Chromosomal abnormalities associated with unfavorable prognosis in multiple myeloma patients include involvement of the TP53, FGRFR3 and MAF genes. It is important to accurately detect these chromosomal abnormalities to aid clinicians in the decision of therapeutic plans. Chromosomal abnormalities are not detected by traditional karyotyping due to low proliferative rate of myeloma cells. Conventional Fluorescence In-situ Hybridization (FISH) enhances the sensitivity but lacks the specificity as it does not distinguish plasma cells (PC) from the other hematopoetic cells. This can be overcome by identification of PCs by cytoplasmic immunoglobulin staining followed by FISH (cIg-FISH).Currently, cIg-FISH is done by two methods – lysing red cells in the bone marrow aspirates and spinning the pellet onto charged slides using the Cytospin machine (Ahmann et al Cancer Genet Cytogenet 1998). The second technique uses cultured and fixed cells stored in Carnoy’s fixative (Filkova et al 2006 http://www.myeloma.cz/res/file/archiv/2007-cytogen-sbornik-workshop.pdf). This second technique is more easily incorporated into the routine cytogenetic protocols used for chromosomal analysis. However, clumping and the small size of plasma cells seemed to be a major setback with the protocol. We have made minor modifications to this technique, with a different approach to fixing and dropping the cells on to the slides to give nicely separated plasma cells. These when subjected to immunostaining with kappa or/and lambda antibodies, followed by FISH, result in easily identifiable plasma cells with good bright signals under the fluorescence microscope. Twenty samples from patients with multiple myeloma were subjected to routine FISH, cIg-FISH, chromosomal karyotyping along with flow cytometry and the results were compared. Three FISH probes from Vysis for t(4;14), t(14;16) and deletion of TP53 were used. To test the utility of the technique for stored cell pellets, 4 fixed pellets stored from 2005–2008 were also tested with good results.Table 1Comparative analysis of 20 patients with multiple myeloma by three different methodsNo% Plasma cellsConventional KaryotypingConventional FISHConventional cIg FISH147NormalNormalNormal255Monosomy 14Monosomy 14Monosomy 14371der(1;16)Monosomy 16Monosomy 1646NormalNormalNormal52NormalNormalNormal672der(12;16)Monosomy 16Monosomy 16713NormalNormalTrisomy 14852NormalNormalNormal98Monosomy 17t(4;14),del17pt(4;14),monosomy 16, del17p1025NormalNormalNormal111NormalNormalNormal1231NormalTrisomy 4Trisomy 413Not doneNot doneNormalNormal145NormalTrisomy 17Monosomy 14, Trisomy 171519.5Monosomy 14t(4;14), monosomy 16t(4;14), monosomy 161634NormalNormalNormal1750NormalNormalNormal181.8NormalNormalNormal1928NormalNormalNormal2047NormalNormalNormalThe percent of plasma cells seen in bone marrow aspirates ranged from 1.8–71%. Of 20 samples, 3 samples showed a positive cIg-FISH but a normal conventional FISH result. All samples showed a uniformly higher percent of abnormality with the cIg-FISH protocol due to selection of plasma cells as expected. [Display omitted] In conclusion, this technique using fixed cells will be a major asset to all cytogenetic laboratories as 1.It saves sample as the entire quantity of bone marrow aspirate can be used to set up cultures for chromosomal karyotyping and part of the fixed pellet can be used for cIg-FISH.2.There is no necessity of making Cytospin slides - saving time and effort.3.Stored pellets, especially if cells are well stored and preserved in fixative can be used for retrospective analysis.The technique can be easily incorporated as a routine targeted FISH test for MM samples, which is not practiced in most clinical cytogenetics laboratories at present. Disclosures:No relevant conflicts of interest to declare.

Array-based comparative genomic hybridization is more informative than conventional karyotyping and fluorescence in situ hybridization in the analysis of first-trimester spontaneous abortion

BackgroundArray-based comparative genomic hybridization (aCGH) is a new technique for detecting submicroscopic deletions and duplications, and can overcome many of the limitations associated with classic cytogenetic analysis. However, its clinical use in spontaneous abortion needs comprehensive evaluation. We used aCGH to investigate chromosomal imbalances in 100 spontaneous abortions and compared the results with G-banding karyotyping and fluorescence in situ hybridization (FISH). Inconsistent results were verified by quantitative fluorescence PCR.ResultsAbnormalities were detected in 61 cases. aCGH achieved the highest detection rate (93.4%, 57/61) compared with traditional karyotyping (77%, 47/61) and FISH analysis (68.9%, 42/61). aCGH identified all chromosome abnormalities reported by traditional karyotyping and interphase FISH analysis, with the exception of four triploids. It also detected three additional aneuploidy cases in 37 specimens with ‘normal’ karyotypes, one mosaicism and 10 abnormalities in 14 specimens that failed to grow in vitro.ConclusionsaCGH analysis circumvents many limitations in traditional karyotyping or FISH. The accuracy and efficiency of aCGH in spontaneous abortions highlights its clinical usefulness for the future. As aborted tissues have the potential to be contaminated with maternal cells, the threshold value of detection in aCGH should be lowered to avoid false negatives.

Rapid Diagnosis of Aneuploidy by High-Resolution Melting Analysis of Segmental Duplications

Several molecular methods, such as quantitative fluorescence PCR and multiplex ligation-dependent probe amplification, currently serve as important adjuncts to traditional karyotyping for the diagnosis of aneuploidy; however, the performance or throughput limitations of these methods hinder their use for routine prenatal diagnosis and population-based postnatal screening. We developed a novel approach, called "high-resolution melting analysis of segmental duplications," to detect common aneuploidies. In this method, similar sequences located on different chromosomes are amplified simultaneously with a single primer set; the PCR products are then analyzed by high-resolution melting. Aneuploidy-associated dosage abnormalities produce different ratios of similar amplicons, which produce melting curves that are detectably different from those of samples from unaffected individuals. We applied this method to DNA samples isolated from individuals with trisomy 21 (n = 48), trisomy 18 (n = 10), trisomy 13 (n = 3), 45,X (n = 8), and 47,XXY (n = 14), and from unaffected controls (n = 48). As judged by the karyotyping results, our method attained 100% diagnostic sensitivity and 99.6% diagnostic specificity. Moreover, our method was able to detect a change in chromosome dosage as low as 1.05-fold. This novel method clearly differentiates samples of patients with common aneuploidies from those of unaffected controls, while markedly simplifying the assays and reducing time and costs. The assay has sufficient throughput to meet the demands of large-scale testing, such as population-based postnatal screening, and is thus suitable for routine use.

The use of arrays to detect copy-number variations in clinical practice

Since their introduction, array-based detection techniques have flooded diagnostic practice worldwide. Technical innovation and commercialization of array platforms have brought down their costs and put them within reach of many diagnostic laboratories. Array diagnostics are, therefore, rapidly replacing traditional karyotyping techniques for a range of indications. Such innovations give rise to many questions related to indications for testing, test outcome interpretation, quality control, and the ethics of using arrays for particular applications. There is a growing need for guidelines on these topics. To address the key issues in this field, an international workshop was organized in Amsterdam in 2011 under the auspices of the Genetic Services Quality Committee of the European Society of Human Genetics. The aim was to explore whether consensus could be achieved on the major quality issues and to produce recommendations for the use of arrays to detect copy-number variations (CNVs) in a diagnostic setting. The outcome of the workshop has been summarized in five articles that formulate recommendations and guidelines to cover the different aspects of CNV detection using diagnostic arrays in genetics. In general, it can be concluded that genome-wide array analysis is well established in postnatal cytogenetics. Most commercially available platforms and data analysis software packages are of high quality and relatively easy to use. The article by Vermeesch et al. (2012) aims to guide laboratories in assuring the quality of their array experiments, by suggesting standards for the minimal resolution of the platform used, and for the interpretation and reporting of the results. Unfortunately, the interpretation of CNVs is not always straightforward since the distinction between pathogenic and benign variants can be difficult as, for example, CNVs with a highly variable phenotype and incomplete penetrance are common. Web-based databases, especially when used in combination, can be helpful in interpreting CNVs. A general clinical interpretation strategy for CNVs is presented by de Leeuw et al. (2012). Because of the genome-wide nature of the use of arrays, clinically relevant CNVs will be detected that are not related to the patient's known phenotype. This clearly raises ethical questions, which may be especially complex if the tested individuals are children, investigated prenatally or postnatally. Dondorp et al. (2012) discuss these ethical issues, trying to find a solution to possible “information overload” in counseling, and exploring the balance between the “right to know” and the “right not to know” in different situations. Two particular clinical applications of array diagnostics are discussed in more detail. Vetro et al. (2012) explore the use of arrays in prenatal diagnosis and provide recommendations for the various steps of the process, starting with pretest counseling. In their opinion, array diagnostics have a place in the case of abnormal ultrasound findings as long as local guidelines have been established, whereas the value of offering array diagnostics to all pregnant women is highly debatable. Changing focus from constitutional to acquired chromosomal abnormalities, Simons et al. (2012) review the clinical value of array-based genome-wide screens in leukemia. The genetic complexity of cancer cells requires the detection of small genomic changes in a mixed cell population, as well as the ability to detect regions of homozygosity. The authors discuss these technical challenges and, again, stress the need for international data sharing. We are proud to present this special collection of articles that report on a set of recommendations and suggested guidelines for CNV array diagnostics. They are meant to encourage further discussion, development, and implementation of local, national, and preferably, international guidelines. The development of such guidelines will not only be important for the use of array diagnostics as part of good clinical and laboratory practice. There should be important spin-off as well. In the rapidly changing landscape of genetic diagnostics, other genome-wide techniques are being introduced as well, including whole-exome sequencing and whole-genome sequencing. Those who endeavor to apply these new technologies to standard clinical use will likely face many challenges similar to those faced in the routine application of array diagnostics. We thank the workshop organizers, workshop leaders, and speakers (in alphabetical order): Trijnie Dijkhuizen, Ros Hastings, Jayne Hehir-Kwa, Klaas Kok, Nicole de Leeuw, David Ledbetter, Conny van Ravenswaaij, Joris Vermeesch, Guido de Wert, and Orsetta Zuffardi. We also thank all authors and workshop participants for their enthusiastic participation and professional input.

Using microarray to diagnose Noonan Syndrome and predict phenotype

Patient NM was referred to the Geneticists following an ophthalmology appointment where dysmorphic features were noted. Patient NM was born at 40 weeks gestation following a normal pregnancy weighing 3220 g (25th centile). His first day check was unremarkable. There is no consanguinity and his female sibling is fit and well. There is a family history of squint. On examination, he was documented as having the following dysmorphic features: low set ears, hypertelorism, down slanting palpable fissures, broad forehead, short neck, broad chest with wide spaced nipples, foetal fingertip pads and undescended testes. On auscultation of his chest, patient NM had a pan-systolic murmur but no other stigmata of cardiac disease. An echo showed a tiny low muscular ventricular septal defect which was of no clinical concern. Developmental examination at 18 months revealed a globally delayed child. Patient NM was unable to walk but had been pulling to stand since 15 months. He did not have any language nor use gestures. He had only recently progressed to textured foods. There was no concern with hearing. He continued to be reviewed by the ophthalmologists for astigmatism, hypermetropia and an alternating divergent squint and he wears a pair of prescription glasses. Genetic testing of patient NM included a microarray which revealed a 3p duplication including the RAF1 gene and a diagnosis of Noonan Syndrome was made. There is a much higher incidence of hypertrophic cardiomyopathy in patients with RAF1 mutations and therefore this investigation not only confirmed the clinical suspicion but will also determine future monitoring. An array is a relatively new high resolution test for looking at all 46 chromosomes simultaneously and is slowing replacing the traditional karyotype. It looks for added chromosomes (duplications) or missing chromosomes (deletions) and hence specific genes that are duplicated or deleted can be identified. As in this case, it can help predict phenotype and may prevent further unnecessary investigations. However, arrays may pick up harmless changes that are common in the general population (copy number variants) which may make interpretation difficult. It is also a more expensive test and takes longer to yield a result.

Re: Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011

Re: Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011

Molecular cytogenetics : an indispensable tool for cancer diagnosis

Cytogenetic aberrations may escape detection or recognition in traditional karyotyping. The past decade has seen an explosion of methodological advances in molecular cytogenetics technology. These cytogenetics techniques add color to the black and white world of conventional banding. Fluorescence in-situ hybridization (FISH) study has emerged as an indispensable tool for both basic and clinical research, as well as diagnostics, in leukemia and cancers. FISH can be used to identify chromosomal abnormalities through fluorescent labeled DNA probes that target specific DNA sequences. Subsequently, FISH-based tests such as multicolor karyotyping, comparative genomic hybridization (CGH) and array CGH have been used in emerging clinical applications as they enable resolution of complex karyotypic aberrations and whole global scanning of genomic imbalances. More recently, crossspecies array CGH analysis has also been employed in cancer gene identification. The clinical impact of FISH is pivotal, especially in the diagnosis, prognosis and treatment decisions for hematological diseases, all of which facilitate the practice of personalized medicine. This review summarizes the methodology and current utilization of these FISH techniques in unraveling chromosomal changes and highlights how the field is moving away from conventional methods towards molecular cytogenetics approaches. In addition, the potential of the more recently developed FISH tests in contributing information to genetic abnormalities is illustrated.

Multiplex ligation-dependent probe amplification (MLPA): a reliable alternative for fetal chromosome analysis?

Objective: To determine whether molecular karyotyping using multiple ligation probe amplification (MLPA) is a reliable alternative for quick and accurate diagnosis of fetal chromosomal abnormalities. Methods: MLPA, using specialised probe sets designed to detect aneuploidy, major chromosomal rearrangements and recognised microdeletion syndromes, was used to analyse chorionic villi or amniocytes left after traditional karyotypingof 476 fetuses for clinical indications. Results: An abnormal result was obtained in 190 cases, including 124 trisomies, 21 sex chromosome anomalies, 14 triploidies, and 31 rearrangements or mosaics. All trisomies were detected by all three techniques, but triploidies were only detected by karyotyping and QF-PCR. In 19 of the 31 cases of rearrangements or mosaicism there was an uncertain or high risk of adverse outcome. Traditional karyotyping detected 13 of the 19 pathogenic rearrangements, MLPA detected 18, and QF-PCR did not detect any. Conclusion: MLPA, using specialized probe sets, detects more chromosomal rearrangements, conferring significant risk of adverse outcome than karyotyping. A combination of qfPCR and MLPA could be a good, rapid alternative to current practice. In the future, used in conjunction with non-invasive prenatal diagnosis based on cell free fetal DNA it might provide a rapid and efficient approach to fetal karyotyping.

Frequent Aneuploidy Among Normal Human Hepatocytes

Murine hepatocytes become polyploid and then undergo ploidy reversal and become aneuploid in a dynamic process called the ploidy conveyor. Although polyploidization occurs in some types of human cells, the degree of aneuploidy in human hepatocytes is not known. We isolated hepatocytes derived from healthy human liver samples and determined chromosome number and identity using traditional karyotyping and fluorescence in situ hybridization. Similar to murine hepatocytes, human hepatocytes are highly aneuploid. Moreover, imaging studies revealed multipolar spindles and chromosome segregation defects in dividing human hepatocytes. Aneuploidy therefore does not necessarily predispose liver cells to transformation but might promote genetic diversity among hepatocytes.