Molecular Karyotyping Research Articles

The influence of single nucleotide polymorphism microarray-based molecular karyotype abnormalities on preimplantation embryonic development potential

The influence of single nucleotide polymorphism microarray-based molecular karyotype abnormalities on preimplantation embryonic development potential

Informatics-based molecular karyotyping of products of conception (POC): focus on molar pregnancies

Report incidence of paternal triploidy and full paternal uniparental disomy (UPD) identified in >6,500 POC samples analyzed via single nucleotide polymorphism (SNP) microarray and a bioinformatics technique (Parental Support™). A high correlation between dispermia or duplication of the male genome exists with complete or partial molar pregnancy (CMP or PMP). Both are common causes of miscarriage (affecting 1/1000 pregnancies in Caucasian women) and convey risk of gonadotrophoblastic disease/neoplasia (GTD/GTN).

Combined De-Novo Mutation and Non-Random X-Chromosome Inactivation Causing Wiskott-Aldrich Syndrome in a Female with Thrombocytopenia

Disorders linked to mutations in the X chromosomes typically affect males. The aim of the study is to decipher the mechanism of disease expression in a female patient with a heterozygous mutation on the X-chromosome. Clinical data was extracted from the Canadian Inherited Marrow Failure Registry. Genomic ribonucleic acid (DNA) and complementary DNA (cDNA) underwent Sanger sequencing. Protein analysis was performed by flow cytometry. X-inactivation patterns were analyzed by evaluating the DNA methylation status and cDNA clonal expression of several genes on the X-chromosome. SNP array was used for molecular karyotyping of the X-chromosome. A female with thrombocytopenia, eczema and mild T-lymphocyte abnormalities with extensive negative diagnostic testing, was suspected to have Wiskott-Aldrich syndrome (WAS)/X-linked thrombocytopenia. Although the girl had a mutation (c.397G > A, p.E133K) in only one allele, she was found to have an extremely skewed X-inactivation pattern and no expression of the WAS protein. Family studies using DNA methylation analysis and cDNA clonal expression of several genes on the X-chromosome demonstrated that the patient developed de-novo non-random inactivation of the X-chromosome that does not carry the mutation. Genome-wide high-density molecular karyotyping excluded deletions and amplifications as a cause for the non-random inactivation of one X-chromosome. Our study emphasizes the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of a family history.

The phenotypic spectrum of duplication 5q35.2–q35.3 encompassing NSD1: Is it really a reversed sotos syndrome?

Loss-of-function mutations of NSD1 and 5q35 microdeletions encompassing NSD1 are a major cause of Sotos syndrome (Sos), which is characterized by overgrowth, macrocephaly, characteristic facies, and variable intellectual disability (ID). Microduplications of 5q35.2-q35.3 including NSD1 have been reported in only five patients so far and described clinically as a reversed Sos resulting from a hypothetical gene dosage effect of NSD1. Here, we report on nine patients from five families with interstitial duplication 5q35 including NSD1 detected by molecular karyotyping. The clinical features of all 14 individuals are reviewed. Patients with microduplications including NSD1 appear to have a consistent phenotype consisting of short stature, microcephaly, learning disability or mild to moderate ID, and distinctive facial features comprising periorbital fullness, short palpebral fissures, a long nose with broad or long nasal tip, a smooth philtrum and a thin upper lip vermilion. Behavioral problems, ocular and minor hand anomalies may be associated. Based on our findings, we discuss the possible etiology and conclude that it is possible, but so far unproven, that a gene dosage effect of NSD1 may be the major cause.

A comprehensive molecular study on Coffin–Siris and Nicolaides–Baraitser syndromes identifies a broad molecular and clinical spectrum converging on altered chromatin remodeling

Chromatin remodeling complexes are known to modify chemical marks on histones or to induce conformational changes in the chromatin in order to regulate transcription. De novo dominant mutations in different members of the SWI/SNF chromatin remodeling complex have recently been described in individuals with Coffin-Siris (CSS) and Nicolaides-Baraitser (NCBRS) syndromes. Using a combination of whole-exome sequencing, NGS-based sequencing of 23 SWI/SNF complex genes, and molecular karyotyping in 46 previously undescribed individuals with CSS and NCBRS, we identified a de novo 1-bp deletion (c.677delG, p.Gly226Glufs*53) and a de novo missense mutation (c.914G>T, p.Cys305Phe) in PHF6 in two individuals diagnosed with CSS. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex implicating dysfunction of a second chromatin remodeling complex in the pathogenesis of CSS-like phenotypes. Altogether, we identified mutations in 60% of the studied individuals (28/46), located in the genes ARID1A, ARID1B, SMARCB1, SMARCE1, SMARCA2, and PHF6. We show that mutations in ARID1B are the main cause of CSS, accounting for 76% of identified mutations. ARID1B and SMARCB1 mutations were also found in individuals with the initial diagnosis of NCBRS. These individuals apparently belong to a small subset who display an intermediate CSS/NCBRS phenotype. Our proposed genotype-phenotype correlations are important for molecular screening strategies.

Dual-Color Fluorescence In Situ Hybridization Reveals an Association of Chromosome 8q22 but Not 8p21 Imbalance with High Grade Invasive Breast Carcinoma

We previously reported molecular karyotype analysis of invasive breast tumour core needle biopsies by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) (Walker et al, Genes Chromosomes Cancer, 2008 May;47(5):405-17). That study identified frequently recurring gains and losses involving chromosome bands 8q22 and 8p21, respectively. Moreover, these data highlighted an association between 8q22 gain and typically aggressive grade 3 tumors. Here we validate and extend our previous investigations through FISH analysis of tumor touch imprints prepared from excised breast tumor specimens. Compared to post-surgical tumor excisions, core needle biopsies are known to be histologically less precise when predicting tumor grade. Therefore investigating these chromosomal aberrations in tumor samples that offer more reliable pathological assessment is likely to give a better overall indication of association. A series of 60 breast tumors were screened for genomic copy number changes at 8q22 and 8p21 by dual-color FISH. Results confirm previous findings that 8p loss (39%) and 8q gain (74%) occur frequently in invasive breast cancer. Both absolute quantification of 8q22 gain across the sample cohort, and a separate relative assessment by 8q22:8p21 copy number ratio, showed that the incidence of 8q22 gain significantly increased with grade (p = 0.004, absolute and p = 0.02, relative). In contrast, no association was found between 8p21 loss and tumor grade. These findings support the notion that 8q22 is a region of interest for invasive breast cancer pathogenesis, potentially harboring one or more genes that, when amplified, precipitate the molecular events that define high tumor grade.

Monozygotic twins discordant for 18q21.2qter deletion detected by array CGH in amniotic fluid

Discordant chromosomal anomalies in monozygotic twins may be caused by various timing issues of erroneous mitosis and twinning events. Here, we report a prenatal diagnosis of heterokaryotypic monozygotic twins discordant for phenotype. In a 28-year-old woman, ultrasound examination performed at 26 weeks of gestation, detected intrauterine growth restriction and unilateral cleft lip and palate in twin B, whereas twin A had normal fluid, growth and anatomy. Molecular karyotyping in twin B identified a 18q21.2qter deletion, further confirmed by FISH analysis on amniocytes. Interestingly, in twin A, cytogenetic studies (FISH analysis and karyotype) on amniocytes were normal. Genotyping with microsatellite markers confirmed the monozygosity of the twins. At 32 weeks of gestation, selective termination of twin B was performed by umbilical cord coagulation and fetal blood samples were taken from the umbilical cord in both twins. FISH analyses detected mosaicism in both twins with 75% of cells being normal and 25% harboring the 18qter deletion. After genetic counseling, the parents elected to terminate the second twin at 36 weeks of gestation. In postmortem studies, FISH analyses revealed mosaicism on several tissues in both twins. Taking into account this observation, we discuss the difficulties of genetic counseling and management concerning heterokaryotypic monozygotic twins.

Further delineation of genotype–phenotype correlation in homozygous 2p21 deletion syndromes: First description of patients without cystinuria

Homozygous contiguous gene deletion syndromes are rare. On 2p21, however, several overlapping homozygous gene deletion syndromes have been described, all presenting with cystinuria but otherwise distinct phenotypes. Hypotonia-cystinuria syndrome (HCS, OMIM606407) is characterized by infantile hypotonia, poor feeding, and growth hormone deficiency. Affected individuals carry homozygous deletions including the cystinuria gene SLC3A1 and the adjacent PREPL gene. Larger homozygous deletions in this region encompassing the PPM1B, SLC3A1, PREPL, and C2orf34 (CAMKMT) genes result in a more severe phenotype, the 2p21 deletion syndrome. A phenotype intermediate to HCS and the 2p21 deletion syndrome is termed atypical HCS and is caused by deletion of SLC3A1, PREPL, and C2orf34 (CAMKMT). Using high resolution SNP array molecular karyotyping we identified two siblings with a homozygous deletion of 83 kb partially encompassing the genes PREPL and C2orf34 (CAMKMT), but not the SLC3A1 gene. The affected siblings display a recognizable phenotype which is similar to atypical HCS with regard to growth failure and neuro-muscular features, but is characterized by lack of cystinuria. The patients also exhibit features which have not been reported to date such as cleft palate and genital abnormalities. In conclusion, we report the first patients with a homozygous 2p21 deletion syndrome without cystinuria and further delineate the complex genotype-phenotype correlations of homozygous microdeletion syndromes of this region.

Tetraploidy in the era of molecular karyotyping – What we need to remember

Tetraploidy is a condition in which there are four complete sets of chromosomes in a single cell. In humans, this would be 92 pairs of chromosomes per cell. A great majority of pregnancies with a tetraploid fetus end in miscarriage, or if the pregnancy goes to full term, the infant dies shortly after birth. Longer surveillance is rarely described. The only method for confirming tetraploidy is karyotyping, in many cases using classical G-banding methods. In this paper we would like to present another living individual with tetraploidy and to revise the syndrome in the light of its diagnostics in the era of molecular karyotyping, with array Comparative Genomic Hybridization (aCGH, arrayCGH).

FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage

Does comprehensive chromosome screening (CCS) of cells sampled from the blastocyst trophectoderm (TE) accurately predict the chromosome complement of the inner cell mass (ICM)? Comprehensive chromosome screening of a TE sample is unlikely to be confounded by mosaicism and has the potential for high diagnostic accuracy. The effectiveness of chromosome aneuploidy screening is limited by the technologies available and chromosome mosaicism in the embryo. Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select diploid embryos for transfer. The study was performed between January 2011 and August 2011. In the first part, a new ICM isolation method was developed and tested on 20 good morphology blastocysts. In the main phase of the study, fluorescence in situ hybridization (FISH) was used to reanalyse the ICMs and TEs separated from 70 embryos obtained from 26 patients undergoing blastocyst stage array comparative genome hybridization (aCGH) PGS cycles. The isolated ICM and TE fractions were characterized by immunostaining for KRT18. Then, non-transferrable cryopreserved embryos were selected for the FISH reanalysis based on previous genetic diagnosis obtained by TE aCGH analysis. Blastocysts either diploid for chromosome copy number (20) or diagnosed as single- (40) or double aneuploid (10) were included after preparing the embryo into one ICM and three equal-sized TE sections. Accuracy of the aCGH was measured based on FISH reanalysis. Chromosomal segregations resulting in diploid/aneuploid mosaicism were classified as 'low-', 'medium-' and 'high-' grade and categorized with respect to their distribution (1TE, 2TE, 3TE, ICM or ALL embryo). Linear regression model was used to test the relationship between the distributions and the proportion of aneuploid cells across the four embryo sections. Fisher's exact test was used to test for random allocation of aneuploid cells between TE and ICM. All ICM biopsy procedures displayed ICM cells in the recovered fraction with a mean number of ICM cells of 26.2 and a mean TE cell contamination rate of 2%. By FISH reanalysis of previously aCGH-screened blastocysts, a total of 66 aneuploidies were scored, 52 (78.8%) observed in all cells and 14 (21.2%) mosaic. Overall, mosaic chromosomal errors were observed only in 11 out of 70 blastocysts (15.7%) but only 2 cases were classified as mosaic diploid/aneuploid (2.9%). Sensitivity and specificity of aCGH on TE clinical biopsies were 98.0 and 100% per embryo and 95.2 and 99.8% per chromosome, respectively. Linear regression analysis performed on the 11 mosaic diploid/aneuploid chromosomal segregations showed a significant positive correlation between the distribution and the proportion of aneuploid cells across the four-blastocyst sections (P < 0.01). In addition, regression analysis revealed that both the grade and the distribution of mosaic abnormal cells were significantly correlated with the likelihood of being diagnosed by aCGH performed on clinical TE biopsies (P = 0.019 and P < 0.01, respectively). Fisher's exact test for the 66 aneuploidies recorded showed no preferential allocation of abnormal cells between ICM and TE (P = 0.33). The study is limited to non-transferable embryos, reanalyzed for only nine chromosomes and excludes segmental imbalance and uniparental disomy. The prevalence of aneuploidy in the study group is likely to be higher than in the general population of clinical PGD embryos. This study showed high accuracy of diagnosis achievable during blastocyst stage PGS cycles coupled with 24-chromosomes molecular karyotyping analysis. The new ICM isolation strategy developed may open new possibilities for basic research in embryology and for clinical grade derivation of human embryonic stem cells. No specific funding was sought or obtained for this study.

Array-based comparative genomic hybridization (ARRAY-CGH) in analysis of chromosomalaberrations and CNV in blighted ovum pregnancies

Application of modern molecular cytogenetic technologies in research and clinical practice provides unprecedented possibilities for high resolution molecular karyotyping in different areas of clinical cytogenetics. In this study the oligonucleotide-based array-CGH data about unbalanced chromosomal aberrations and copy number variations (CNV) in blighted ovum pregnancies are presented. Analysis of genes content of involved chromosomal regions was done. Scopes, perspectives and limitations of aCGH application into analysis of early pregnancy losses are discussed.

CNV Analysis in Monozygotic Twin Pairs Discordant for Urorectal Malformations

Early post-twinning mutational events can account for discordant phenotypes in monozygotic (MZ) twin pairs. Such mutational events may comprise genomic alterations of different sizes, ranging from single nucleotides to large copy-number variations (CNVs). Anorectal malformations (ARM) and the bladder exstrophy-epispadias complex (BEEC) represent the most severe end of the urorectal malformation spectrum. Recently, CNV studies in patients with sporadic ARM and the BEEC have identified de novo events that occur in specific chromosomal regions. We hypothesized that early arising, post-twinning CNVs might contribute to discordance in MZ twin pairs with ARM or the BEEC; knowledge of such CNVs might help to identify additional chromosomal regions involved in the development of these malformations. We investigated four discordant MZ twin pairs (three ARM and one BEEC) using molecular karyotyping arrays comprising 1,140,419 markers with a median marker spacing of 1.5 kb. Filtering the coding regions for possible disease-causing post-twinning de novo CNVs present only in the affected twin, but not in the unaffected twin or the parents, identified a total of 136 CNVs. These 136 CNVs were then filtered against publicly available databases and finally re-evaluated visually. No potentially causative CNV remained after applying these filter criteria. Our results suggest that post-twinning CNV events that affect coding regions of the genome did not contribute to the discordant phenotypes in MZ twin pairs that we investigated. Possible causes for the discordant phenotypes include changes in regulatory elements or smaller genetic changes within coding regions which may be detectable by whole-exome sequencing.

Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

Molecular Karyotyping: A Revolutionary Concept in Prenatal Diagnosis

The ‘standard of care’ for chromosome testing has recently changed. For the last 30 years it has involved chromosome testing using microscope analysis. New technology using ‘DNA chips’, i.e. molecular karyotyping or microarray has now become available and is routinely offered to pregnant women undergoing invasive testing for an ultrasound detected fetal abnormality. Women who choose to take advantage of this technology undergo extensive counselling in a multidisciplinary setting regarding the possible implications of an abnormal result. They are advised that there are some molecular changes which are known to be harmless and will not be reported. They are also counselled that some molecular changes will be reported but their clinical significance is uncertain. This article reviews the current evidence-based role of molecular karyotyping in routine clinical practice in the setting of ultrasound detected fetal abnormality.

Molecular karyotyping of an isolated partial trisomy 11q patient with additional findings

Isolated partial duplication of the long arm of chromosome 11 is very rare. The main features are dysmorphic facial features, pre/postnatal growth retardation, speech delay, mental retardation, hypotonia, microcephaly, and cardiac, vertebral, limb and genital anomalies. In this case, we report a patient with partial trisomy of 11q13.5→qter due to a de novo rearrangement consisting of the whole X chromosome and part of chromosome 11; 46,X,der(X)(Xqter→Xp22.33::11q13.5→11qter). Additional findings were a separated clavicle, lacrimal duct stenosis and prenatally detected renal hypoplasia. SNP array results revealed a duplication between 11q13.5 and 11qter, measuring 58Mb, from nucleotide 76,601,607 to 134,926,021. As a result, molecular karyotyping could be performed in such cases in order to establish a definite phenotype–genotype correlation using conventional or molecular cytogenetics techniques.

Single-nucleotide polymorphism-array improves detection rate of genomic alterations in core-binding factor leukemia

Acute myeloid leukemia (AML) is a group of clonal diseases, resulting from two classes of mutation. Investigation for additional abnormalities associated with a well-recognized subtype, core-binding factor AML (CBF-AML) can provide further understanding and discrimination to this special group of leukemia. In order to better define genetic alterations in CBF-AML and identify possible cooperating lesions, a single-nucleotide polymorphism-array (SNP-array) analysis was performed, combined to KIT mutation screening, in a set of cases. Validation of SNP-array results was done by array comparative genomic hybridization and FISH. Fifteen cases were analyzed. Three cases had microscopic lesions better delineated by arrays. One case had +22 not identified by arrays. Submicroscopic abnormalities were mostly non-recurrent between samples. Of relevance, four regions were more frequently affected: 4q28, 9p11, 16q22.1, and 16q23. One case had an uncovered unbalanced inv(16) due to submicroscopic deletion of 5´MYH11 and 3´CBFB. Telomeric and large copy number neutral loss of heterozygosity (CNN-LOH) regions (>25 Mb), likely representing uniparental disomy, were detected in four out of fifteen cases. Only three cases had mutation on KIT gene, enhancing the role of abnormalities by SNP-array as presumptive cooperating alterations. Molecular karyotyping can add valuable information to metaphase karyotype analysis, emerging as an important tool to uncover and characterize microscopic, submicroscopic genomic alterations, and CNN-LOH events in the search for cooperating lesions.

Ethical considerations when offering noninvasive prenatal testing

Technology in the prenatal setting is advancing at an exceptional rate, and these advancements will likely result in major changes to current pregnancy screening and testing paradigms. In Australia, prenatal testing is increasingly becoming a routine part of antenatal care and pregnant women are offered an assortment of screening and diagnostic tests, which give them information about their fetus, and can identify potential anomalies before it is born. In Victoria (Australia), each year approximately 4% of babies will be born with a birth defect or fetal abnormality many of which are diagnosed in uterus. Greater than 97% of women in Victoria have at least one or more ultrasounds during pregnancy and greater than 80% of pregnant women take up screening for Down syndrome, with the majority having First Trimester Combined Screening (FTCS). FTCS combines maternal serum analytes — pregnancy associated plasma protein-A (PAAP-A) and beta human chorionic gonadotrophin (free β-hCG) — with results from the nuchal translucency ultrasound to give a risk figure for Down syndrome and Trisomy 18. Approximately 5% of women will receive a false positive result from FTCS, meaning they screen positive for Down syndrome or Trisomy 18 and do not have an affected pregnancy. Women who are considered to be “increased risk” either because of a screening result or ultrasound finding are offered invasive diagnostic testing whereby a fetal sample is obtained via chorionic villus sampling (CVS) or amniocentesis and sent for conventional or molecular karyotyping (microarray). These tests carry a miscarriage risk of up to 1% above the background rate of miscarriage. There has been increasing demand for a safe and reliable alternative to invasive diagnostic testing and very recently, noninvasive prenatal testing (NIPT) has become commercially available to women in America (2012), Australia and many other countries, including Brazil (2013). NIPT is an advanced screening test, which relies on the fact that small fragments of cell-free fetal DNA and RNA circulate in maternal serum. In the first and second trimesters of pregnancy, approximately 6–10% of total cell free DNA (cfDNA) circulating in maternal serum is thought to be fetal in origin; this fetal fraction rises to 10–20% in the third trimester. Using massively parallel sequencing technology, scientists can sequence cfDNA fragments in maternal plasma and detect specific chromosome aneuploidies such as trisomies 21, 13 and 18, much earlier in pregnancy than has previously been possible,

CNVs Provide the Clue for a Cryptic t(5;11) in Acute Myeloid Leukemia

s from the Cancer Cytogenomics Microarray Consortium/CAGdb meeting, Chicago, IL, August 5e7, 2013 Massive Parallel Sequencing of Leukemia Samples Using a Custom Designed Cancer Gene Mutation Panel Fengqi Chang, Liu Liu, Erica Fang, Guangcheng Zhang, Yanchun Li, Marilyn M. Li Baylor College of Medicine, Houston, TX, USA Leukemia is the result of accumulation of somatic mutations including point mutations, copy number variations, and chromosomal abnormalities. Since multiple mutations can be present in a given leukemia genome, a technology that can detect many mutations in different genes simultaneously is desirable. Next generation sequencing (NGS) technologies allow deep sequencing of numerous mutations and multiple samples at the same time. Among NGS technologies, targeted sequencing is appealing in clinical oncology due to its shorter turnaround time, lower costs, and simpler data analysis compared to whole genome or whole exome sequencing. We introduce here a new panel specific for leukemia using multiplex PCR followed by ion semiconductor deep sequencing technologies. The leukemia panel covers 48 genes and can detect over 3,200 leukemia-associated mutations, many of which are clinically actionable mutations. Twenty-five samples were used to validate the panel on different sample types, including blood, bone marrow, FFPE and cell free DNA. The uniformity of coverage was about 87.75% for all cases. The average depth of coverage for targeted region was more than 3000X and the mean 100X coverage of targeted mutations was 97.07%. The mutation panel detected mutations in NPM1, TP53, ABL1, JAK3, IDH1, IDH2, KRAS etc in these samples with as little as 20 ng DNA and the positive rate was about 56%. The most commonly mutated genes were: TP53 (7 cases) followed by CEBPA (3 cases), NPM1 (3 cases). Overall, the leukemia panel showed improved coverage uniformity, reading length and depth compared to certain commercially available kits. The turnaround time for the test can be as short as 3 to 5 days, which fits the needs of personalized oncology. Furthermore, samples can be multiplexed using barcoding, which leads to tremendous increase of sequencing efficiency and decrease of cost while maintaining the high quality of sequencing results. CNVs Provide the Clue for a Cryptic t(5;11) in Acute Myeloid Leukemia Nicole L. Chia , Howard R. Slater , Julia M. Potter a,b NU Medical School, Australian National University, Canberra ACT 0200, Australia; ACT Pathology, The Canberra Hospital, Canberra ACT, 2606 Australia; Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Vic 3052, Australia Molecular karyotyping has been introduced into diagnostic pathology services for first-tier investigation of constitutional anomalies. The clinical utility of this mode of diagnostic investigation in neoplasms is currently being explored. Evidence based interpretation of copy number change requires comparison with control cohorts. Cytogenetic investigation of neoplasms frequently requires identification of apparently balanced structural rearrangements that result in critical gene fusion. High resolution microarray investigations have shown that not all balanced rearrangements are in fact balanced at the sequence level. We describe a 25 year old male with acute myelomonocytic leukemia. A del(5)(q35) was identified as the sole abnormality by conventional cytogenetic analysis. SNP microarray analysis confirmed an acquired 6.0 Mb interstitial deletion at 5q35.1q35.3 and an adjacent 350kb deletion. In addition, two acquired adjacent deletions (2.1 Mb and 850kb) were observed at chromosome 11p15.5p15.4 and a 675kb gain at 10q11.22. Subsequent subtelomere FISH identified a cryptic t(5;11)(q35;p15) with putative NUP98/NSD1 fusion. Review of the “Aussie Normal” control cohort yielded no evidence of CNVs on chromosome 5 and 11 at the putative breakpoints. However, common CNVs were observed within the deleted regions for each chromosome. These are located within repetitive DNA (LINES and SINES). A low frequency CNV gain (220kb) in the Aussie Normal cohort, harboring the ANXA8L2 gene at 10q11.2 coincided with the CNV reported in the patient here. This CNV is located within a region of segmental duplication and may represent a rearrangement due to NAHR mechanisms. This case demonstrates the potential value for the application of molecular karyotyping of neoplasms. Comparison with control cohorts provides evidence upon which pathogenicity, predisposition, and putative mechanisms can be investigated.

Implications of a Chr7q21.11 Microdeletion and the Role of the PCLO Gene in Developmental Delay = الأثار المترتبة على فقدان الخبن الصبغي الجزيئي و دور جين PCLO في صعوبات التعلم

We report here a 4-year-old boy with global developmental delay who was referred for karyotyping and fragile X studies. A small interstitial deletion on chromosome 7 at band 7q21 was detected in all cells examined. Subsequent molecular karyotype analysis gave the more detailed result of a 6.3 Mb heterozygous deletion involving the interstitial chromosome region 7q21.11. In this relatively gene-poor region, the presynaptic cytomatrix protein, Piccolo (PCLO) gene appears to be the most likely candidate for copy number loss leading to a clinical phenotype. G-banded chromosome analysis of the parents showed this deletion was inherited from the father. Molecular karyotype analysis of the father's genome confirmed that it was the same deletion as that seen in the son; however, the father did not share the severity of his son's phenotype. This cytogenetically-visible deletion may represent another example of a chromosomal rearrangement conferring a variable phenotype on different family members.

Clinical Outcomes and Counselling Issues Regarding Partial Trisomy of Terminal Xp in a Child with Developmental Delay

Female carriers of balanced translocations involving an X chromosome and an autosome offer genetic counselling challenges. This is in view of the number of possible meiotic outcomes, but also due to the impact of X chromosome-localised genes that are no longer subject to gene silencing through the X chromosome inactivation centre. We present a case where delineation of the extent of X chromosome-localised genes on the derivative autosome using molecular karyotyping offers critical information in the context of genetic counselling.