Oligonucleotide Array-based Comparative Genomic Hybridization Research Articles

Prenatal Diagnosis Using Chromosomal Microarray Analysis in High-Risk Pregnancies.

Background: To assess the value of chromosomal microarray analysis (CMA) during the prenatal diagnosis of high-risk pregnancies. Methods: Between January 2016 and November 2021, we included 178 chorionic villi and 859 amniocentesis samples from consecutive cases at a multiple tertiary hospital. Each of these high-risk singleton pregnancies had at least one of the following indications: (1) advanced maternal age (AMA; ≥35 years; 546, 52.7%); (2) fetal structural abnormality on ultrasound (197, 19.0%); (3) high-risk first- or second-trimester Down syndrome screen (189, 18.2%), including increased nuchal translucency (≥3.5 mm; 90, 8.7%); or (4) previous pregnancy, child, or family history (105, 10.1%) affected by chromosomal abnormality or genetic disorder. Both G-banding karyotype analysis and CMA were performed. DNA was extracted directly and examined with oligonucleotide array-based comparative genomic hybridization. Results: Aneuploidies were detected by both G-banding karyotyping and CMA in 42/1037 (4.05%) cases. Among the 979 cases with normal karyotypes, 110 (10.6%) cases had copy number variants (CNVs) in CMA, including 30 (2.9%) cases with reported pathogenic and likely pathogenic CNVs ≥ 400 kb, 37 (3.6%) with nonreported VOUS, benign, or likely benign CNVs ≥ 400 kb, and 43 (4.1%) with nonreported CNVs < 400 kb. Of the 58 (5.6%) cases with aneuploidy rearrangements, 42 (4.1%) were diagnosed by both G-banding karyotyping and CMA; four inversions, six balanced translocations, and six low mosaic rates were not detected with CMA. Conclusions: CMA is an effective first step for the prenatal diagnosis of high-risk pregnancies with fetal structural anomalies found in ultrasonography or upon positive findings.

Novel Phenotype of 6p25 Deletion Syndrome Presenting Juvenile Parkinsonism and Brain Calcification.

Chromosome 6p25 deletion syndrome is a rare neurocristopathy with variable clinical features. The objective of the current study was to describe a novel phenotype for autosomal-dominant chromosome 6p25 deletion syndrome. The presentation included bilateral basal ganglia and subcortical calcifications and juvenile parkinsonism, resembling primary familial brain calcification. Phenotypic characterization, exome sequencing, and oligonucleotide array were carried out in the index family. The index patient and her mother had a history of developmental delay, mild facial dysmorphism, Axenfield eye anomalies, slight intellectual disability, and subsequently developed levodopa-responsive parkinsonism in early adulthood. Brain-computed tomography showed bilateral basal ganglia and subcortical calcifications. Magnetic resonance imaging revealed diffuse white matter lesions. A 99mTc TRODAT single-photon emission computed tomography scan revealed bilateral dopaminergic denervation. Whole-exome sequencing and oligonucleotide array-based comparative genomic hybridization revealed a 2.27-Mb chromosome 6pter-p24 deletion, which cosegregated within the family. Our findings extended the current phenotypic spectrum of chromosome 6p25 deletion syndrome. © 2020 International Parkinson and Movement Disorder Society.

A Novel Acquired t(2;4)(q36.1;q24) with a Concurrent Submicroscopic del(4)(q23q24) in An Adult with Polycythemia Vera

Background: Polycythemia vera (PV) is a clonal myeloid stem cell disease characterized by a growth-factor independent erythroid proliferation with an inherent tendency to transform into overt acute myeloid malignancy. Approximately 95% of the PV patients harbor the JAK2V617F mutation while less than 35% of the patients harbor cytogenetic abnormalities at the time of diagnosis. Methods and Results: Here we present a JAK2V617F positive PV patient where G-banding revealed an apparently balanced t(2;4)(q35;q21), which was confirmed by 24-color karyotyping. Oligonucleotide array-based Comparative Genomic Hybridization (aCGH) analysis revealed an interstitial 5.4 Mb large deletion at 4q23q24. Locus-specific fluorescent in situ hybridization (FISH) analyses confirmed the mono-allelic 4q deletion and that it was located on der(4)t(2;4). Additional locus-specific bacterial artificial chromosome (BAC) probes and mBanding refined the breakpoint on chromosome 2. With these methods the karyotype was revised to 46,XX,t(2;4)(q36.1;q24)[18]/46,XX[7]. Conclusions: This is the first report on a PV patient associated with an acquired novel t(2;4)(q36.1;q24) and a concurrent submicroscopic deletion del(4)(q23q24). The study also underscores the benefit of combined usage of FISH and oligo-based aCGH analysis in characterizing chromosomal abnormalities. The present findings provide additional clues to unravel important molecular pathways in PV to obtain the full spectrum of acquired chromosomal and genomic aberrations, which eventually may improve treatment options.

Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies

Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development.

Oligonucleotide Array-based Comparative Genomic Hybridization Approach in Hematologic Malignancies With Normal/Failed Conventional Cytogenetics and Fluorescent In Situ Hybridization.

Oligonucleotide array-based comparative genomic hybridization (oaCGH) was used to investigate 60 cases of hematologic malignancies, mainly acute myeloid leukemias and myelodysplastic syndromes, in order to evaluate its sensitivity and specificity and to search for genomic alterations undetected by previous investigation with conventional cytogenetics (CC) and fluorescent in situ hybridization (FISH). On the basis of CC and FISH results, we subdivided the series into group A (36 cases with a normal karyotype after CC and/or FISH testing) and group B (24 cases with anomalies detected by CC and/or FISH). oaCGH did not show alterations in 21 cases of the group A (58.3%); in the remaining 15 cases (41.7%), it detected 19 new abnormalities (14 amplifications and 5 deletions). In the group B, oaCGH confirmed 32/55 aneuploidies detected by FISH (58.1%). The sensitivity increased at 27/33 confirmed aneuploidies (81.8%) by placing as a cutoff a mosaic of 50%. Moreover, in the cases of this group oaCGH revealed 36 new alterations (15 amplifications and 21 deletions). From these results it is possible to assess a strong overlap between results obtained by FISH and oaCGH. However, oaCGH is a reliable alternative where CC and FISH are not feasible and is able to identify new alterations unexplored by FISH.

Chromosome 17/17q gain and unaltered profiles in high resolution array-CGH are prognostically informative in neuroblastoma.

The prognostic relevance of chromosome 17 gain in neuroblastoma is still discussed. This investigation specifies the frequency, type, size, and transcriptional relevance in a large patient cohort. Primary tumor material of 202 patients was analyzed using high-resolution oligonucleotide array-based comparative genomic hybridization (aCGH) and correlated with clinical and survival data. A subset (n = 145) was correlated for differentially expressed genes (DEG) by microarray analysis. Chromosome 17 aCGH analysis showed numerical gain in 94/202 patients (47%), partial gain in 93/202 patients (46%), and no gain in 15/202 patients (7%). The frequency of partial gain was higher in stage 4 neuroblastoma (stage 1 15%; stage 2 12%; stage 3 16%; stage 4S 7%; and stage 4 50%). Overall survival (OS) was superior in patients with numerical gain compared with patients with partial gain or no gain (5-y-OS: 0.95 ± 0.02 vs. 0.63 ± 0.05 vs. 0.60 ± 0.13; P < 0.001). Gene expression analysis demonstrated 95/130 DEGs between tumors with numerical or partial chromosome/no gain. Only one DEG (CCKBR) was detected comparing tumors with partial gain and those with no gain. In patients with partial gain, the distribution of breakpoints did not correlate with stage and 11q status, but with MYCN amplification and 1p status. The "best" breakpoints in cases with partial 17q gain were at 42.5 Mb for event-free and 26.6 Mb for OS. Numerical gain of chromosome 17 is associated with a better prognosis than partial and no gain. The group of tumors with partial gain was similar to the group without gain with respect to stage distribution, outcome, and gene expression profile.

Value of Oligonucleotide-Based Array Comparative Genomic Hybridization for Diagnosis of Acute Promyelocytic Leukemia in a Patient Negative for t(15;17)(q24.1;q21.2)/Promyelocytic Leukemia-Retinoic Acid Receptor, Alpha by Conventional Cytogenetics and Fluorescence In Situ Hybridization

Acute promyelocytic leukemia (APL) is characterized by promyelocytic leukemia (PML)-retinoic acid receptor, alpha (RARA) fusion gene as a result of t(15;17)(q24.1;q21.2).1 Current advances in APL therapy have dramatically improved patient outcome.2,3 However, patients with APL are at risk of potentially lethal coagulopathy if appropriate therapy is delayed.4 The t(15;17)(q24.1;q21.2) is detected using conventional cytogenetic analysis in approximately 90% of APL cases.5,6 A small subset of APL cases (4%) lack the classical t(15;17)(q24.1;q21.2) and have other complex translocations.1,2 Rare APL cases have submicroscopic insertions of PML or RARA, in which reverse transcription (RT) polymerase chain reaction (PCR) detects PML-RARA transcripts and yet conventional cytogenetics and fluorescence in situ hybridization (FISH) results are negative.1,5,7–9

Prenatal diagnosis: array comparative genomic hybridization in fetuses with abnormal sonographic findings

To evaluate the clinical value of a high-resolution whole-genome array method for examination of genomic imbalances in prenatal samples (46 amniotic fluid, 17 chorionic villus, and 26 products of conception) from fetuses with abnormal ultrasound, in a clinical setting where more than 90% of pregnant women receive first-trimester combined screening and a second-trimester anomaly scan. Cross-sectional study. Fetal medicine units (national healthcare system) in Central and North Denmark Regions from March 2009 to April 2012. Eighty-nine samples obtained at 11.5-35.0 (mean 19.3) gestational weeks, either during ongoing pregnancy or after termination. DNA was extracted directly from amniotic fluid cells and chorionic villus samples, or from cultured cells, and examined with 80-kb resolution oligonucleotide array-based comparative genomic hybridization (aCGH). Clinically significant copy number variations identified by aCGH. We detected clinically significant copy number variations in 11 fetuses (12%, confidence interval 6.0-19%) with structural malformations. Three fetuses (3.4%) had uncertain clinical significant variations and incidental findings. aCGH is a valuable diagnostic tool when fetal malformations are detected. More affected fetuses may be diagnosed at an earlier gestational age providing better possibilities for postnatal treatment and allowing for women to decide earlier on termination of pregnancy. When a normal result has reduced the risk of significant chromosomal aberration, aCGH may facilitate parental decision-making on whether to continue the pregnancy.

Evolution of Molecular Diagnosis of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the commonest of the muscular dystrophies. The DMD gene (DMD) is the biggest human gene and the most common molecular defect in the DMD gene, accounting for approximately 65 % of cases of DMD, is the deletion of one or more exons. The most basic method still in regular use involves multiplex PCR of the exons, known to be most commonly deleted. The multiplex is relatively simple. Quantitative analysis of all exons of the gene and multiplex ligation-dependent probe amplification have brought about an improvement in mutation detection rate, as they will detect all exon scale deletions as well as duplications, widely used to detect exonic and intronic mutations. As a sensitive and discriminative tool, MLPA can be used for prenatal testing. A more recent development in quantitative analysis is the use of oligonucleotide-based array comparative genomic hybridization.

Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements

Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.

Chromosomal Copy Number Alterations Are Associated with Persistent Lymph Node Metastasis After Chemoradiation in Locally Advanced Rectal Cancer

Lymph node metastasis is an important indicator of oncologic outcome for patients with rectal cancer. Identifying predictive biomarkers of lymph node metastasis could therefore be clinically useful. The aim of this study was to assess whether chromosomal copy number alterations can assist in predicting persistent lymph node metastasis in patients with locally advanced rectal cancer treated with preoperative chemoradiation therapy. This is a nonrandomized, prospective phase II study. This study took place in a multi-institutional setting. Ninety-five patients with stage II (cT3-4, cN0) or stage III (any cT, cN1-2) rectal cancer were included. Patients were treated with preoperative chemoradiation therapy followed by total mesorectal excision. Pretreatment biopsy tumor DNA and surgical margin control DNA were extracted and analyzed by oligonucleotide array-based comparative genomic hybridization. Chromosomal copy number alterations were correlated with persistent lymph node metastasis. Finally, a model for predicting persistent lymph node metastasis was built. The primary outcomes assessed were whether chromosomal copy number alterations are associated with persistent lymph node metastasis in patients with rectal cancer and the accuracy of oligonucleotide array-based comparative genomic hybridization for predicting lymph node metastasis. Twenty-five of 95 (26%) patients had lymph node metastasis after chemoradiation. Losses of 28 chromosomal regions, most notably in chromosome 4, were significantly associated with lymph node metastasis. Our predictive model contained 65 probes and predicted persistent lymph node metastasis with 68% sensitivity, 93% specificity, and positive and negative predictive values of 77% and 89%. The use of this model accurately predicted lymph node status (positive or negative) after chemoradiation therapy in 82 of 95 patients (86%). The patient cohort was not completely homogeneous, which may have influenced their clinical outcome. In addition, although we performed rigorous, statistically sound internal validation, external validation will be important to further corroborate our findings. Copy number alterations can help identify patients with rectal cancer who are at risk of lymph node metastasis after chemoradiation.

Familial Pelizaeus–Merzbacher disease caused by a 320.6‐kb Xq22.2 duplication and the pathological findings of a male fetus

Pelizaeus-Merzbacher disease (PMD) is a recessive, X-linked leukoencephalopathy attributed to impaired myelination during central nervous system development, caused by defects in the proteolipid protein 1 (PLP1) gene. PMD presents clinical variability, ranging from the severe connatal form to the classic form. We report the clinical and molecular findings of two affected males, three carrier females, and an aborted male fetus with familial PMD. The two male probands presented with severe PMD phenotype and intellectual disability. High-resolution oligonucleotide-based array comparative genomic hybridization (aCGH) identified an Xq22.2 duplication of 320.6 kb (102641391-102961998, hg18), including the PLP1 gene and surrounding chromosomal region. Postmortem examination of the aborted fetus at 25 weeks' gestation showed focal subcortical white matter degeneration, focal gliosis, and cerebellar atrophy. Genotype-phenotype correlation is provided. In the connatal form of PMD, leukodystrophy and cerebellar atrophy can occur antenatally and be established at 25 weeks' gestation. The observation of degenerative brain lesions occurring before the onset of subcortical myelination suggests that the PLP1 gene has a more complex role in human brain development, exceeding its structural function in myelin formation.

Targeted array CGH as a valuable molecular diagnostic approach: Experience in the diagnosis of mitochondrial and metabolic disorders

Oligonucleotide array-based comparative genomic hybridization (aCGH) targeted to coding exons of genes of interest has been proven to be a valuable diagnostic tool to complement with Sanger sequencing for the detection of large deletions/duplications. We have developed a custom designed oligonucleotide aCGH platform for this purpose. This array platform provides tiled coverage of the entire mitochondrial genome and high-density coverage of a set of nuclear genes involving mitochondrial and metabolic disorders and can be used to evaluate large deletions in targeted genes. A total of 1280 DNA samples from patients suspected of having mitochondrial or metabolic disorders were evaluated using this targeted aCGH. We detected 40 (3%) pathogenic large deletions in unrelated individuals, including 6 in genes responsible for mitochondrial DNA (mtDNA) depletion syndromes, 23 in urea cycle genes, 11 in metabolic and related genes. Deletion breakpoints have been confirmed in 31 cases by PCR and sequencing. The possible deletion mechanism has been discussed. These results illustrate the successful utilization of targeted aCGH to detect large deletions in nuclear and mitochondrial genomes. This technology is particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing. For female carriers of X-linked disorders, if sequencing analysis does not detect point mutations, targeted aCGH should be considered for the detection of large heterozygous deletions.

Mosaic ring chromosome 21, monosomy 21, and isodicentric ring chromosome 21: Prenatal diagnosis, molecular cytogenetic characterization, and association with 2-Mb deletion of 21q21.1–q21.2 and 5-Mb deletion of 21q22.3

ObjectiveTo present the perinatal findings and molecular cytogenetic characterization of prenatally detected mosaic r(21). Materials, Methods, and ResultsA 29-year-old primigravid woman underwent amniocentesis at 22 weeks’ gestation because of hyperechogenic cardiac foci and intrauterine growth restriction. Amniocentesis revealed a karyotype of 46,XY,r(21)[15]/45,XY,–21[5]. The parental karyotypes were normal. The woman requested repeat amniocentesis. Oligonucleotide-based array comparative genomic hybridization was applied to the uncultured amniocytes, rapidly detecting a 2.09-Mb deletion of 21q21.1–q21.2 (21,495,262–23,580,815bp) and a 5.03-Mb deletion of 21q22.3–q22.3 (41,887,412–46,914,715bp). Cytogenetic analysis revealed a karyotype of 46,XY,r(21)[8]/45,XY,–21[3]/46,XY,idic r(21)[1]. The pregnancy was terminated, and a malformed fetus was delivered with clinodactyly, short big toes, separation between the first and second toes, prominent nasal bridge, downward slanting palpebral fissures, protuberant occiput, prominent forehead, broad anteverted nasal tip, long philtrum, thin upper lip, small mouth, and micrognathia. The placenta had a karyotype of 46,XY,r(21)[83]/45,XY,–21[11]/46,XY,idic r(21)[6], and the cord blood lymphocytes had a karyotype of 46,XY,r(21)[88]/45,XY,–21[9]/46,XY,idic r(21)[3]. Polymorphic DNA marker analysis determined a maternal origin for the deletion. ConclusionAn extra interstitial 21q deletion can be associated with mosaic r(21) in addition to a terminal 21q deletion. aCGH is useful in determining the breakpoints and associated subtle structural abnormalities in cases of prenatally detected ring chromosome in order to facilitate genetic counseling.

Chromosomal 16p microdeletion in Rubinstein-Taybi syndrome detected by oligonucleotide-based array comparative genomic hybridization: a case report

IntroductionChromosomal aberrations of chromosome 16 are uncommon and submicroscopic deletions have rarely been reported. At present, a cytogenetic or molecular abnormality can only be detected in 55% of Rubinstein-Taybi syndrome patients, leaving the diagnosis in 45% of patients to rest on clinical features only. Interestingly, this microdeletion of 16 p13.3 was found in a young child with an unexplained syndromic condition due to an indistinct etiological diagnosis. To the best of our knowledge, no evidence of a microdeletion of 16 p13.3 with contiguous gene deletion, comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes, has been described in typical Rubinstein-Taybi syndrome.Case presentationWe present the case of a three-year-old Malaysian Chinese girl with a de novo microdeletion on the short arm of chromosome 16, identified by oligonucleotide array-based comparative genomic hybridization. Our patient showed mild to moderate global developmental delay, facial dysmorphism, bilateral broad thumbs and great toes, a moderate size atrial septal defect, hypotonia and feeding difficulties. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XX karyotype. Further investigation by high resolution array-based comparative genomic hybridization revealed a 120 kb microdeletion on chromosomal band 16 p13.3.ConclusionA mutation or abnormality in the cyclic adenosine monophosphate-response element-binding protein has previously been determined as a cause of Rubinstein-Taybi syndrome. However, microdeletion of 16 p13.3 comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes is a rare scenario in the pathogenesis of Rubinstein-Taybi syndrome. Additionally, due to insufficient coverage of the human genome by conventional techniques, clinically significant genomic imbalances may be undetected in unexplained syndromic conditions of young children. This case report demonstrates the ability of array-based comparative genomic hybridization to offer a genome-wide analysis at high resolution and provide information directly linked to the physical and genetic maps of the human genome. This will contribute to more accurate genetic counseling and provide further insight into the syndrome.

Currents in Contemporary Bioethics

Increasingly, genomic analysis is being utilized to diagnose children with developmental delay or dysmorphic facial features suggestive of a congenital disorder. Genetic testing has rapidly evolved, and the genome-wide tests that we use today are significantly different from the more targeted single-gene tests of the last decade. Chromosomal microarray analysis (CMA) is now a first line test for children with multiple birth defects, children with intellectual impairment (including autism), and children with an unusual constellation of symptoms that do not fit with a known disease. There are three types of CMA that are currently clinically available. CMA by oligonucleotide array-based comparative genomic hybridization (aCGH) compares the hybridization signal from the patient's DNA to that of a reference DNA sample for each oligonucleotide on the array. Depending on the specific array, this can range from tens of thousands to hundreds of thousands of oligonucleotides.

Detection of ≥1 Mb microdeletions and microduplications in a single cell using custom oligonucleotide arrays

High resolution detection of genomic copy number abnormalities in a single cell is relevant to preimplantation genetic diagnosis and potentially to noninvasive prenatal diagnosis. Our objective is to develop a reliable array comparative genomic hybridization (CGH) platform to detect genomic imbalances as small as ~1Mb ina single cell. We empirically optimized the conditions for oligonucleotide-based array CGH using single cells from multiple lymphoblastoid cell lines with known copy number abnormalities. To improve resolution, we designed custom arrays with high density probes covering clinically relevant genomic regions. The detection of megabase-sized copy number variations (CNVs) in a single cell was influenced by the number of probes clustered in the interrogated region. Using our custom array, we reproducibly detected multiple chromosome abnormalities including trisomy 21, a 1.2Mb Williams syndrome deletion, and a 1.3Mb CMT1A duplication. Replicate analyses yielded consistent results. Aneuploidy and genomic imbalances with CNVs as small as 1.2Mb in a single cell are detectable by array CGH using arrays with high-density coverage in the targeted regions. This approach has the potential to be applied for preimplantation genetic diagnosis to detect aneuploidy and common microdeletion/duplication syndromes and for noninvasive prenatal diagnosis if single fetal cells can be isolated.

Chromosomal copy number alterations are associated with tumor response to chemoradiation in locally advanced rectal cancer

Rectal cancer response to chemoradiation (CRT) varies from no response to a pathologic complete response (pCR). Identifying predictive biomarkers of response would therefore be useful. We assessed whether chromosomal copy number alterations (CNAs) can assist in predicting pCR. Pretreatment tumor biopsies and paired normal surgical tissues from the proximal resection margin were collected from 95 rectal cancer patients treated with preoperative CRT and total mesorectal excision in a prospective Phase II study. Tumor and control DNA were extracted, and oligonucleotide array-based comparative genomic hybridization (aCGH) was used to identify CNAs, which were correlated with pCR. Ingenuity pathway analysis (IPA) was then used to identify functionally relevant genes in aberrant regions. Finally, a predictive model for pCR was built using support vector machine (SVM), and leave-one-out cross validation assessed the accuracy of aCGH. Chromosomal regions most commonly affected by gains were 20q11.21-q13.33, 13q11.32-23, 7p22.3-p22.2, and 8q23.3-q24.3, and losses were present at 18q11.32-q23, 17p13.3-q11.1, 10q23.1, and 4q32.1-q32.3. The 25 (26%) patients who achieved a pCR had significantly fewer high copy gains overall than non-pCR patients (P = 0.01). Loss of chromosomal region 15q11.1-q26.3 was significantly associated with non-pCR (P < 0.00002; Q-bound < 0.0391), while loss of 12p13.31 was significantly associated with pCR (P < 0.0003; Q-bound < 0.097). IPA identified eight genes in the imbalanced chromosomal regions that associated with tumor response. SVM identified 58 probes that predict pCR with 76% sensitivity, 97% specificity, and positive and negative predictive values of 91% and 92%. Our data indicate that chromosomal CNAs can help identify rectal cancer patients more likely to develop a pCR to CRT.

Distinctive phenotype in 9 patients with deletion of chromosome 1q24‐q25

Reports of individuals with deletions of 1q24→q25 share common features of prenatal onset growth deficiency, microcephaly, small hands and feet, dysmorphic face and severe cognitive deficits. We report nine individuals with 1q24q25 deletions, who show distinctive features of a clinically recognizable 1q24q25 microdeletion syndrome: prenatal-onset microcephaly and proportionate growth deficiency, severe cognitive disability, small hands and feet with distinctive brachydactyly, single transverse palmar flexion creases, fifth finger clinodactyly and distinctive facial features: upper eyelid fullness, small ears, short nose with bulbous nasal tip, tented upper lip, and micrognathia. Radiographs demonstrate disharmonic osseous maturation with markedly delayed bone age. Occasional features include cleft lip and/or palate, cryptorchidism, brain and spinal cord defects, and seizures. Using oligonucleotide-based array comparative genomic hybridization, we defined the critical deletion region as 1.9 Mb at 1q24.3q25.1 (chr1: 170,135,865-172,099,327, hg18 coordinates), containing 13 genes and including CENPL, which encodes centromeric protein L, a protein essential for proper kinetochore function and mitotic progression. The growth deficiency in this syndrome is similar to what is seen in other types of primordial short stature with microcephaly, such as Majewski osteodysplastic primordial dwarfism, type II (MOPD2) and Seckel syndrome, which result from loss-of-function mutations in genes coding for centrosomal proteins. DNM3 is also in the deleted region and expressed in the brain, where it participates in the Shank-Homer complex and increases synaptic strength. Therefore, DNM3 is a candidate for the cognitive disability, and CENPL is a candidate for growth deficiency in this 1q24q25 microdeletion syndrome.

Array Comparative Genomic Hybridization Analysis Identifies Recurrent Gain of Chromosome 2p25.3 Involving the ACP1 and MYCN Genes in Chronic Lymphocytic Leukemia

Chromosomal aberrations are independent prognostic markers in chronic lymphocytic leukemia (CLL). Recent studies using genomic arrays have shown recurrent gains of the short arm of chromosome 2 (2p) in a subset of CLL. We evaluated 178 CLL cases for 2p gains using custom-designed oligonucleotide array-based comparative genomic hybridization (aCGH). A high frequency of 2p gains was observed in 53 of 178 (30%) cases, which ranged from a small 29-kb region to large segments involving the entire short arm. Besides several common chromosomal aberrations associated with 2p gain, we demonstrated a novel observation that gain of the telomeric region 2p25.3 harboring the ACP1 gene is common in CLL (25%, 44 of 178 cases). The ACP1 gene has been previously shown to regulate T-cell receptor signaling through ZAP-70, and both genes are unfavorable clinical markers for CLL. Quantitative polymerase chain reaction (qPCR) confirmed the presence of 3-6 copies of ACP1 in 35 of 40 (88%) of these cases. Interestingly, none of the aCGH diploid CLL cases showed gain of ACP1. Assessment of 73 healthy individuals by qPCR revealed ACP1 copy number gain in only two cases (2.7%). Gain of 2p25.3 was associated with ZAP-70 expression (P < .002) and unmutated immunoglobulin heavy chain variable (IGHV) gene mutation (P < .0001). A high frequency of MYCN co-amplication with ACP1 was observed (14 of 40 cases, 35%). The frequent 2p25.3 gain involving the ACP1 and MYCN genes may help define the critical region of 2p that contributes to pathogenesis of CLL together with other chromosomal abnormalities.