Molecular Karyotyping Research Articles

Russell–Silver syndrome

In comparison to Prader-Willi or Angelman syndrome, Russell-Silver syndrome (RSS) is a relatively "young" imprinting disorder. This congenital disease is characterized by intrauterine and postnatal growth retardation, relative macrocephaly, a typical triangular face, asymmetry, and further less constant characteristic features. Genetic and epigenetic disturbances can meanwhile be detected in approximately 50% of patients with typical RSS features. Up to 5% of patients carry a maternal uniparental disomy of chromosome 7 (UPD(7)mat), at least 44% show hypomethylation in the chromosome 11p15 imprinting center 1 (IC). In 1-2% of RSS patients, (sub)microscopic chromosomal aberrations can be observed. The diagnostic workup should therefore include methylation/genomic testing for chromosome 11p15, UPD(7)mat analysis and molecular karyotyping. The recurrence risk is generally low in RSS but it can be strongly increased in cases of familial epimutations or a chromosomal rearrangement. Interestingly, in approximately 7% of cases with chromosome 11p15 hypomethylation, hypomethylation of additional imprinted loci can be detected. Clinically, patients with hypomethylation at multiple loci do not differ from those with isolated 11p15 hypomethylation whereas the UPD(7)mat patients generally show a milder phenotype. Nevertheless, (epi)genotype-phenotype correlations are still evolving. Furthermore, the pathophysiological mechanisms resulting in the RSS phenotype still remain unknown despite the recent progress in deciphering the molecular defects associated with this condition.

Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass

Aneuploidy has been well-documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening in a 24-h protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy and distinguishes certain meiotic from mitotic errors. In this study, our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from 17 couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e. one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of 51 (96.1%) ICM samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that TE karyotype is an excellent predictor of ICM karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations.

Distinct molecular signatures in pediatric infratentorial glioblastomas defined by aCGH

Glioblastomas (GBM) are rare in children, but reportedly have more varied outcome which suggests differences in tumor etiology compared to typical GBM of adults. To investigate this we performed high resolution array comparative genomic hybridization (aCGH) analysis on three pediatric infratentorial GBM, ages 3.5, 7 and 14 years. Two of these tumors occurred in the brainstem and one in the spinal cord. While histologically typical, one brainstem tumor showed mainly pleomorphic astrocytic cells, whereas the other brainstem and spinal tumors showed a GFAP positive small cell component. Whole chromosomal gains (#1 and #2) and loss (#20) were seen only in the pleomorphic brainstem GBM, which also showed a high level of segmental genomic copy number changes. Segmental loss involving chromosome 8 was seen in all three tumors (Chr8;133039446-136869494, Chr8;pter-3581577, and Chr8;pter-30480019 respectively), whereas loss involving chromosome 16 was seen in only 2 cases with small cell components (Chr16;31827239-qter and Chr16;pter-29754532). Segmental gain of chromosome 7 was shared only between the 2 brainstem cases (Chr7;17187166-qter and Chr7;69824947-qter). Chromosome 17 showed segmental gain of 17q in the backdrop of loss of 17p only in case 1. Segmental gain of chromosome 1q was seen only in case 2. The spinal GBM showed a relatively stable karyotype with a unique loss of Chr19;32848902-qter. None of the frequent losses, gains and amplifications known to occur in adult GBM were identified, suggesting that pediatric infratentorial glioblastomas show a molecular karyotype that was more characteristic of pediatric embryonal tumors than adult GBM.

Familial 3q29 microdeletion syndrome providing further evidence of involvement of the 3q29 region in bipolar disorder

The 3q29 microdeletion syndrome is caused by a recurrent 1.6 Mb deletion of the 3q subtelomeric region. Though sometimes visible on routine microscopy, the deletion is detected more reliably using subtelomeric fluorescence in-situ hybridization (FISH) or molecular karyotyping. The clinical features associated with a 3q29 microdeletion are variable and include developmental delay, autistic features, skeletal abnormalities and dysmorphic facial features with a relatively long face, long nose with a high bridge and broad tip, short philtrum and large ears. Orofacial clefting, cardiac defects, ocular anomalies and genitourinary malformations have been reported occasionally. We report a three generation family where four individuals were confirmed to have a 3q29 microdeletion and compare their clinical features to those of previously reported patients. This family shows that the learning difficulties associated with a 3q29 deletion may be relatively mild. The history of a severe depressive disorder commencing in adulthood in the affected grandmother also supports previous studies linking the 3q29 region to bipolar disorder and links with the observation of Digilio et al. (2009) who also reported a history of depression in an adult woman with a similar deletion.

C32 Comprehensive microarray genetic analyses on single cell(s) from polar bodies or embryos to determine 23-chromosome aneuploidy, structural chromosome aberrations and genome-wide scans using single nucleotide polymorphisms (SNPs); to identify what partner provided the extra chromosome in aneuploid embryos; and to determine what embryo implanted following an in vitro fertilization (IVF) cycle

not available at time of publication. C32 Comprehensive microarray genetic analyses on single cell(s) from polar bodies or embryos to determine 23-chromosome aneuploidy, structural chromosome aberrations and genome-wide scans using single nucleotide polymorphisms (SNPs); to identify what partner provided the extra chromosome in aneuploid embryos; and to determine what embryo implanted following an in vitro fertilization (IVF) cycle W.G. Kearns1, R. Pen1, A. Benner1, A. Siegel1, E. Widra2, R. Leach1. 1Shady Grove Center for Preimplantation Genetics, 2Shady Grove Fertility Reproductive Science Center, Rockville, Maryland, USA *E-mail: Kearnsw@LabCorp.com (W.G. Kearns) Objective: To amplify DNA from a polar body, a single embryonic blastomere or trophectoderm cell(s) and to perform complex genetic analyses. Design: Prospective study. Materials and Methods: A modified whole genome amplification (WGA) protocol was performed on 513 single cells (112 WBCs, 367 human blastomeres from 61 embryos and 34 known cell lines). We used invariant DNA genomic loci to ensure the entire genome was amplified and TaqMan PCR to ensure heterozygous allele amplification. The Illumina HumanHap370 microarray was employed to determine chromosome aberrations and genotype data for ~370K SNPs. Two-channel intensity values from the microarray data files were used to create high-resolution copynumber profiles to identify copy number variations (CNVs) for each cell. Data was analyzed with deCODE genetics Disease Miner Professional® and Illumina BeadStudio software. Results: Analyses of blastomeres (n = 367) from 61 day-3 preimplantation embryos and cell lines (n = 34) showed in many cases, a genomic coverage >98%, a heterozygous allele detection rate >90% and a microarray detection rate and genotype call rate >90%. In some cases, call rates exceeded 99%. A 23-chromosome molecular karyotype was obtained from all 367 blastomeres (>8400 individual chromosomes) and all 34 cell lines (>780 individual chromosomes). Structural chromosome imbalances were identified from all 9 cytogenetically abnormal cell lines. Some of these structural chromosome imbalances included: del(8q), add(17p), del(17p), add(4q), add(9p), add(14q), dup(18p), dic(5), del(12p) and del(9p). Genotype information was also obtained for ~370K SNPs for each cell analyzed. These genome wide scans identified disease risks in embryos for prostate cancer, glaucoma, certain cardiovascular conditions and estrogen responsive breast cancer. Our SNP scans also identified subtle DNA aberrations associated with disorders such as Beckwith Wiedemann syndrome, some types of Prader Willi/ Angelman syndrome, DiGeorge syndrome 1/Velocardiofacial syndrome and some forms of autism. Uniparental disomy can also be identified. Our microarrays can also identify single gene mutations for diseases such as cystic fibrosis, Marfan syndrome, Retinoblastoma or Neurofibromatosis Type 1. A high-resolution copy-number profile also identified CNVs throughout the genome. Using parental, embryonic and fetal genotypic data, we can also determine which partner provided the extra chromosome in aneuploid embryos and which embryo implanted. Conclusions: We successfully obtained complex genetic information from single embryonic cells using a modified WGA protocol and microarray analyses. These analyses may be performed on polar bodies, blastomeres or trophectoderm cells. Support: EMD Serono

Abstract 782: Characterization of genetic changes in oligodendroglial tumors including glioblastomas with oligodendroglial component

Abstract In contrast to astrocytic tumors including glioblastomas (GBM), oligodendrogliomas (O) show a better prognosis and increased responsiveness to chemotherapy. Interestingly, a small subgroup of glioblastomas contains areas with histological features of oligodendroglial differentiation. In the current WHO classification (2007) these tumors are now included as a variant of glioblastomas and are called glioblastomas with oligodendroglial component (GBMO). However, definitive diagnostic criteria still do not exist. In this study we used a genome wide approach (chromosomal comparative genomic hybridization (CGH) and molecular karyotyping) in combination with interphase fluoreszence in situ hybridization (FISH) (regions 1p, 1q, 7q, 10q, 17p, 19q, cen18, 21q) to genetically characterize GBMO and “classical” O. We analyzed the oligodendroglial and the astrocytic glioblastoma parts of 13 GBMO separately by chromosomal CGH of microdissected paraffin embedded tumor tissue and interphase FISH on paraffin sections. Furthermore, freshly frozen material of six oligodendrogliomas (4 WHO grade II, 6 WHO-grade III) was examined genome wide by using the standard Agilent 244A chip. We identified four distinct genetic subtypes in GBMO: an “astrocytic” subtype (9/13) characterized by +7/-10; an “oligodendroglial” subtype with −1p/-19q (1/13); an “intermediate” subtype showing +7/-1p (1/13), and an “other” subtype having none of the former aberrations typical for gliomas (2/13). In contrast, most “classical” O (5/6) showed combined 1p/19q deletion, corresponding genetically to the “oligodendroglial” subtype. Four of the O with 1p/19q codeletion showed additional aberrations (e. g. −4, −14, −9, −18). The different histological tumor parts of GBMO revealed common genetic changes in all tumors and showed additional aberrations specific for their oligodendroglial and astrocytic parts. These findings demonstrate the monoclonal origin of the tumor followed by the formation of the astrocytic and oligodendroglial components. We are currently evaluating the array CGH results of six oligodendrogliomas, which will also be presented. The diagnostic determination of these genetic signatures may allow for a better prognostication of the patients. Our results underline the importance of molecular cytogenetic analyses to supplement the histological diagnosis of gliomas. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 782.

Abstract 2143: High-Resolution Molecular Karyotyping of Chronic Myeloid Leukemia Patients in Blast Crisis by 6.0 SNP-Arrays Identifies Focal Copy Number Alterations Affecting the Whole Sequence or Specific Exons of Oncogenes and Tumor Suppressor Genes

Abstract Despite the striking efficacy of targeted therapy for chronic myeloid leukemia (CML), a proportion of patients (pts) still experience progression from the initial chronic phase to an acute phase (blast crisis; BC) characterized by high disease aggressiveness and poor prognosis. BC is known to be associated with accumulation of additional genetic alterations, but these alterations have so far been only partially characterized. We used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution molecular karyotyping of 29 DNA samples from BC (myeloid, n=17 lymphoid, n=11) CML pts. The 6.0 SNP Array technology relies on 1.8 million markers evenly spaced across the genome, with a median inter-marker distance <700 bp. We decided to exploit this unprecedented resolution aiming our analysis to the identification of very small CNAs that may have been missed by previous studies using less sensitive assays. Gains/losses mapping to known regions of copy number variation (CNV) were excluded. Our approach revealed a number of focal gains or losses ranging from 4 to 47Kb, affecting a single gene or, more frequently, only part of a gene. Amplifications were as frequent as monoallelic deletions and involved the promoter region and/or one or more exons. In some cases, a complex pattern of amplification of some exons and monoallelic deletion of others was recognized within the same gene. Alterations involved the following genes: AKT3; CDC73; RB1; JAK2; JAK1;K ERG; ETS1; SMAD; PIK3CA; EPHA3; RUNX1T1; ETV1; AKT2; MDM4; KALRN; FHIT; K-RAS; PTEN; FAF1; SKAP2; PTCH1; GAS2; FGFR2; SOS1; NRG1; MET; PBX4; ETV5; N-RAS, HGF, TEC; PAK2; H-RAS. The precise anatomy of alterations involving each gene will be presented. Deeper characterization of these alterations at the DNA and RNA levels by polymerase chain reaction and sequencing is now ongoing; results will be presented. All the genes identified in our screening were transcription factors, adaptor proteins, receptor and non-receptor kinases involved in cell proliferation and apoptosis - with a known role as oncogenes or tumor suppressors or oncogene/tumor suppressor interactors. Although these results confirm a high degree of heterogeneity in the alterations detectable in BC CML pts, members of the RAS pathway were the most frequently altered genes. Deeper characterization by polymerase chain reaction and sequencing is ongoing and will be presented. In conclusion, the power of 6.0 SNP Array technology allowed us to detect previously unidentified alterations targeting whole or part of key oncogenes or tumor suppressors whose deregulation may play a role in determining the aggressive phenotype of BC CML and which may represent potential therapeutic targets. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2143.

Mutations in MEF2C from the 5q14.3q15 microdeletion syndrome region are a frequent cause of severe mental retardation and diminish MECP2 and CDKL5 expression

The etiology of mental retardation remains elusive in the majority of cases. Microdeletions within chromosomal bands 5q14.3q15 were recently identified as a recurrent cause of severe mental retardation, epilepsy, muscular hypotonia, and variable minor anomalies. By molecular karyotyping we identified two novel 2.4- and 1.5-Mb microdeletions of this region in patients with a similar phenotype. Both deletions contained the MEF2C gene, which is located proximally to the previously defined smallest region of overlap. Nevertheless, due to its known role in neurogenesis, we considered MEF2C as a phenocritical candidate gene for the 5q14.3q15 microdeletion phenotype. We therefore performed mutational analysis in 362 patients with severe mental retardation and found two truncating and two missense de novo mutations in MEF2C, establishing defects in this transcription factor as a novel relatively frequent autosomal dominant cause of severe mental retardation accounting for as much as 1.1% of patients. In these patients we found diminished MECP2 and CDKL5 expression in vivo, and transcriptional reporter assays indicated that MEF2C mutations diminish synergistic transactivation of E-box promoters including that of MECP2 and CDKL5. We therefore conclude that the phenotypic overlap of patients with MEF2C mutations and atypical Rett syndrome is due to the involvement of a common pathway.

Polygenic control for fermentation of β-fructosides in the yeast Saccharomyces cerevisiae: New genes SUC9 and SUC10

Using molecular karyotyping and genetic hybridization analysis, two new polymeric β-fructosidase genes, SUC9 and SUC10, were identified in the yeast Saccharomyces cerevisiae, which are located on chromosome XIV and on the chromosome XVI/XIII doublet, respectively. The genes are responsible for fermentation of sucrose and raffinose. The SUC gene genotypes of strains VKM Y-1831 and DBVPG 1340 are SUC2 SUC9 and suc2 0 SUC10, respectively. suc2 0 is a silent sequence. The scientific and applied significance of SUC genes is discussed.

Microduplications At 22q11.21 are Associated with Classic Bladder Exstrophy

Purpose Classic exstrophy of the bladder (CBE) is part of the exstrophy-epispadias complex (EEC), a spectrum of urogenital anomalies in which part or all of the distal urinary tract fails to close. Familial occurrence has been observed, and previous studies have suggested an underlying multifactorial mode of inheritance. To date, no causative genetic or non-genetic factor has been unequivocally identified in humans. The present study aimed to identify microaberrations characterized by loss or gain of genomic material that contribute to the EEC at a genome-wide level. Material and Methods Molecular karyotyping, utilizing 549,839 single nucleotide polymorphisms (SNPs) with an average spacing of 5.7 kilobases, was performed to screen an initial cohort of 16 EEC patients. Results A de novo microduplication involving chromosomal region 22q11.21 was identified in one CBE patient. Subsequent multiplex ligation-dependent probe amplification (MLPA) analysis was performed in a further 50 EEC cases. One patient with an overlapping 22q11.21 duplication was identified in whom the duplication had been transmitted from the unaffected mother. Conclusions Chromosomal region 22q11 is well known for its susceptibility to genomic rearrangements, and these are associated with various syndromes including the velo-cardio-facial/DiGeorge syndrome (VCFS/DGS), the der(22) syndrome, and the cat-eye syndrome. Duplications in this region result in a wide and variable spectrum of clinical presentations including features of the VCFS/DGS, while some carriers present with a completely normal phenotype.Our findings extent the phenotypic spectrum of the 22q11.2 duplication syndrome, and indicate that this aberration predisposes to CBE with incomplete penetrance.

Severe clinical course of Hirschsprung disease in a Mowat-Wilson syndrome patient

We present a clinical case of a female infant with multiple anomalies and distinctive facial features, with an exceptionally severe clinical course of Hirschsprung disease. The girl was also diagnosed with Mowat-Wilson syndrome, confirmed by molecular analysis as a heterozygous deletion of the ZEB2 gene. Moreover, molecular karyotyping revealed a deletion involving further genes (KYNU, ARHGAP15, and GTDC1).

Terminal 3p deletions in two families—Correlation between molecular karyotype and phenotype

The 3p deletion syndrome is a rare disorder caused by deletions of different sizes in the 3p25-pter region. It is characterized by growth retardation, developmental delay, mental retardation, dysmorphism, microcephaly, and ptosis. The phenotype of individuals with deletions varies from normal to severe. Most cases occur de novo, but a few familial cases have been reported. We describe two families with terminal 3p deletions and extremely variable clinical features. In family A, the mother and daughter were extremely mildly affected whereas the son had more severe clinical features. In family B, the mother was normal and her son was affected, having some symptoms that had not been described in the 3p deletion syndrome before. The deletions were characterized by genome-wide SNP array analysis and were 9 and 1.1 Mb in size. Sequencing analysis of the CHL1, CNTN4, and CRBN genes did not reveal any masked recessive alleles that might explain the more severe phenotypes in the probands. In family A, the 9 Mb deletion can be considered causal for the 3p deletion syndrome in the proband, but the extremely mild phenotype in the other family members remains unexplained. In family B, the 1.1 Mb terminal deletion encompasses only the CHL1 gene, which is insufficient to cause 3p deletion symptoms; thus the clinical features observed in this family may have a different cause. The variable penetrance of 3p deletions creates challenges in genetic counseling, as the phenotype of the offspring cannot be predicted based on chromosomal and/or genome-wide array analytical findings.

Deletion of 7q11.21‐q11.23 and infantile spasms without deletion of MAGI2

We report on the clinical and cytogenetic findings and on the array-based characterization of an interstitial 7q11.21-q11.23 deletion initially recognized by standard karyotyping in a 15-month-old female patient. Beginning at the age of 3 months and 2 weeks the patient had severe infantile spasms. Recently, it was reported that infantile spasms are associated with deletion of the MAGI2 gene on chromosome 7q11.23. Nevertheless, not all patients reported with deletions of MAGI2 developed infantile spasms and at least one reported patient with a deletion 7q11.23 without missing the MAGI2 gene was diagnosed with infantile spasms. Molecular karyotyping of our patient confirmed a large 13 Mb deletion encompassing the 7q11.21-q11.23 region without involvement of MAGI2. Critical review of published data and the results of our patient underline the importance to map precisely the deletion boundaries of further patients to reevaluate the significance of MAGI2 hemizygosity in the pathogenesis of infantile spasms.

Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol

BACKGROUNDPreimplantation genetic screening (PGS) has been used in an attempt to determine embryonic aneuploidy. Techniques that use new molecular methods to determine the karyotype of an embryo are expanding the scope of PGS.METHODSWe introduce a new method for PGS, termed ‘parental support’, which leverages microarray measurements from parental DNA to ‘clean’ single-cell microarray measurements on embryonic cells and explicitly computes confidence in each copy number call. The method distinguishes mitotic and meiotic copy errors and determines parental source of aneuploidy.RESULTSValidation with 459 single cells of known karyotype indicated that per-cell false-positive and false-negative rates are roughly equivalent to the ‘gold standard’ metaphase karyotype. The majority of the cells were run in parallel with a clinical commercial PGS service. Computed confidences were conservative and roughly concordant with accuracy. To examine ploidy in human embryos, the method was then applied to 26 disaggregated, cryopreserved, cleavage-stage embryos for a total of 134 single blastomeres. Only 23.1% of the embryos were euploid, though 46.2% of embryos were mosaic euploid. Mosaicism affected 57.7% of the embryos. Counts of mitotic and meiotic errors were roughly equivalent. Maternal meiotic trisomy predominated over paternal trisomy, and maternal meiotic trisomies were negatively predictive of mosaic euploid embryos.CONCLUSIONSWe have performed a major preclinical validation of a new method for PGS and found that the technology performs approximately as well as a metaphase karyotype. We also directly measured the mechanism of aneuploidy in cleavage-stage human embryos and found high rates and distinct patterns of mitotic and meiotic aneuploidy.

Microduplications at 22q11.21 are associated with non-syndromic classic bladder exstrophy

The exstrophy-epispadias complex (EEC) comprises a spectrum of urogenital anomalies in which part or all of the distal urinary tract fails to close. The present study aimed to identify microaberrations characterized by loss or gain of genomic material that contribute to the EEC at a genome-wide level. Molecular karyotyping, utilizing 549,839 single nucleotide polymorphisms (SNPs) with an average spacing of 5.7 kilobases, was performed to screen an initial cohort of 16 patients with non-syndromic EEC. A de novo microduplication involving chromosomal region 22q11.21 was identified in one patient with classic exstrophy of the bladder (CBE). Subsequent multiplex ligation-dependent probe amplification (MLPA) analysis was performed with an MLPA 22q11 kit in a further 50 non-syndromic EEC cases. We identified one CBE patient with an overlapping 22q11.21 duplication in whom the duplication had been transmitted from the unaffected mother. Chromosomal region 22q11 is well known for its susceptibility to genomic rearrangements, and these are associated with various syndromes including the velo-cardio-facial/DiGeorge syndrome (VCFS/DGS), the der(22) syndrome, and the cat-eye syndrome. Duplications in this region result in a wide and variable spectrum of clinical presentations that include features of the VCFS/DGS, while some carriers present with a completely normal phenotype. Our findings extend the phenotypic spectrum of the 22q11.2 duplication syndrome, and indicate that this aberration predisposes to CBE with incomplete penetrance.

Diagnosis of miscarriages by molecular karyotyping: Benefits and pitfalls

Diagnosis of miscarriages by molecular karyotyping: Benefits and pitfalls

Conflicting results of prenatal FISH with different probes for Down's Syndrome critical regions associated with mosaicism for a de novo del(21)(q22) characterised by molecular karyotyping: Case report

For the rapid detection of common aneuploidies either PCR or Fluorescence in situ hybridisation (FISH) on uncultured amniotic fluid cells are widely used. There are different commercial suppliers providing FISH assays for the detection of trisomies affecting the Down's syndrome critical regions (DSCR) in 21q22. We present a case in which rapid FISH screening with different commercial probes for the DSCR yielded conflicting results. Chromosome analysis revealed a deletion of one chromosome 21 in q22 which explained the findings. Prenatally an additional small supernumerary marker chromosome (sSMC) was discovered as well, which could not be characterised. Postnatal chromosome analysis in lymphocytes of the infant revealed complex mosaicism with four cell lines. By arrayCGH the sSMC was provisionally described as derivative chromosome 21 which was confirmed by targeted FISH experiments.

Recent advances in molecular diagnostics of colorectal cancer by genomic arrays: proposal for a procedural shift in biological sampling and pathological report

Two forms of genetic instability have been described in colorectal cancer: chromosomal instability, characterized by structural and numerical chromosomal abnormalities and associated to aneuploidy; and microsatellite instability, characterized by a deficiency in the mismatch repair system that leads to slippage in microsatellites and is associated to euploidy. Thirteen colorectal cancer sample DNAs were analyzed after colectomy. High-resolution genome-wide DNA copy number and Single Nucleotide Polimorphism genotyping analysis was performed by Affymetrix SNP 6.0 arrays that interrogates 906,600 single nucleotide polymorphisms and 945,826 copy number probes. We implemented this analysis as part of a routine procedure that includes the sampling of fresh tissue from the tumor mass without affecting the subsequent standard histopathological procedure. The novel molecular technology allows the determination of a genome-wide molecular karyotype using only 500 ng of high-quality tumor DNA; it distinguishes the two main types of genomic instability, discriminating between chromosomal instability positive and negative tumors. It also detects loss of heterozygosity (LOH) regions, called copy neutral-LOH. Tumor-associated copy neutral-LOH regions may play a pivotal role in oncogenesis when they determine duplications of either activating or loss of function gene mutation. We observed recurrent gains of chromosomes 2, 7, 8q, 9, 12, 13, 20 and losses of chromosomes 4, 5, 8p, 15, 17p, 18, 22, and Y, in agreement with previous cytogenetic studies. The use of such sampling procedure could stimulate the routine detection of point mutations in specific genes, thus avoiding subsequent sectioning of formalin-fixed and paraffin-embedded samples.

Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34− cell population with intrinsic resistance to imatinib

AbstractWe show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34+ cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34− cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin−CD34− cells in vitro. Moreover, leukemic CD34− cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

Comprehensive genotype–phenotype analysis in 230 patients with tetralogy of Fallot

BackgroundTetralogy of Fallot (ToF), the most frequent cyanotic congenital heart disease, is associated with a wide range of intra- and extracardiac phenotypes. In order to get further insight into genotype–phenotype...