Multidisciplinary analysis of pediatric T-ALL: 9q34 gene fusions.

T-cell acute lymphoblastic leukemia (T-ALL) is not as frequently reported as the B-cell counterpart (B-ALL), only occurring in about 15% of pediatric cases with a typically heterogeneous etiology. Approximately 8% of childhood T-ALL cases have rearrangements involving the ABL1 tyrosine kinase gene at 9q34.12; although a t(9;22), resulting in a fusion of ABL1 with the BCR gene at 22q11.23 is a common occurrence in B-ALL, it is not a typical finding in T-ALL. A subset of 10 of 40 documented cases of T-ALL analyzed over a 5-year period is presented, each having gene rearrangements within band 9q34 that resulted in fusions other than BCR/ABL1. These cases included fusions involving ABL1, SET (9q34.11), NUP214 (9q34.13), SPTAN1 (9p34.11), and TNRC6B (22q13.1). Among the 10 cases are: six SET/NUP214 fusions, two ABL1/NUP214 fusions (one of which was associated with episomal amplification) and novel SPTAN1/ABL1 and TNRC6B/ABL1 fusions. The evaluations of these clones were each significantly aided by FISH analysis, which directed subsequent microarray and anchored multiplex PCR testing for fusion confirmations.

Diagnostic cytogenetic testing following positive noninvasive prenatal screening results: a clinical laboratory practice resource of the American College of Medical Genetics and Genomics (ACMG).

Disclaimer: ACMG Clinical Laboratory Practice Resources are developed primarily as an educational tool for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these practice resources is voluntary and does not necessarily assure a successful medical outcome. This Clinical Laboratory Practice Resource should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with this Clinical Laboratory Practice Resource. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Noninvasive prenatal screening (NIPS) using cell-free DNA has been rapidly adopted into prenatal care. Since NIPS is a screening test, diagnostic testing is recommended to confirm all cases of screen-positive NIPS results. For cytogenetics laboratories performing confirmatory testing on prenatal diagnostic samples, a standardized testing algorithm is needed to ensure that the appropriate testing takes place. This algorithm includes diagnostic testing by either chorionic villi sampling or amniocentesis samples and encompasses chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray.

Camptodactyly and the 22q11.2 deletion syndrome.

We describe a 5-day-old male with minor facial anomalies, a congenital laryngeal web, severe laryngomalacia, and prominent fixed flexion of the proximal interphalangeal joints of digits 2 through 5 bilaterally. A whole genome SNP microarray analysis identified a 2.55 Mb interstitial deletion of 22q11.21, typical of that seen in the DiGeorge and Velocardiofacial syndromes. A review of the literature identifies 10 other cases with camptodactyly. Camptodactyly appears to be an associated but rarely reported anomaly in patients with the 22q11.2 microdeletion syndrome. © 2016 Wiley Periodicals, Inc.

Status of the anaplastic lymphoma kinase (ALK) gene in inflammatory breast carcinoma.

BACKGROUND: Although preliminary reports suggest that ALK gene amplification may occur in inflammatory breast cancer (IBC), data are limited. We performed a comprehensive investigation of the status of ALK gene in IBC. METHODS: We used core biopsy (CB) samples from 30 IBC patients for immunohistochemistry (IHC), 25 of these samples for fluorescence in situ hybridization (FISH) of ALK gene rearrangement, 8 for chromosome 2 aneusomy, and 20 microdissected frozen CBs for array comparative genomic hybridization (CGH) and mRNA analysis. RESULTS: All 30 samples were negative for ALK protein expression by IHC. FISH analysis showed no EML4-ALK gene rearrangement in any samples, although 16 of the 25 samples (64%) contained 3 to 4 extra copies of the ALK gene, and chromosome 2 aneusomy was found in 7 of 8 samples that had extra copies of the ALK gene. Only 3 of the 20 samples showed evidence of mild ALK gene amplification by array CGH. mRNA analysis revealed that mRNA expression of ALK was not significantly higher in these samples compared with samples that showed no evidence of ALK gene amplification in CGH analysis, nor was mRNA expression of ALK significantly different in tumor compared with 5 normal breast samples (P > 0.05, t test). CONCLUSION: Our comprehensive evaluation suggests that ALK gene rearrangement did not occur in the IBC patients studied. The significance of our finding of mildly increased copy numbers of the ALK gene resulting from chromosome 2 aneusomy rather than mild amplification of the ALK gene requires further investigation.

A 137-kb deletion within the Potocki-Shaffer syndrome interval on chromosome 11p11.2 associated with developmental delay and hypotonia.

Potocki-Shaffer syndrome (PSS) is a rare disorder caused by haploinsufficiency of genes located on the proximal short arm of chromosome 11 (11p11.2p12). Classic features include biparietal foramina, multiple exostoses, profound hypotonia, dysmorphic features, and developmental delay/intellectual disability. Fewer than 40 individuals with PSS have been reported, with variable clinical presentations due in part to disparity in deletion sizes. We report on a boy who presented for initial evaluation at age 13 months because of a history of developmental delay, hypotonia, subtle dysmorphic features, and neurobehavioral abnormalities. SNP microarray analysis identified a 137 kb deletion at 11p11.2, which maps within the classically defined PSS interval. This deletion results in haploinsufficiency for all or portions of six OMIM genes: SLC35C1, CRY2, MAPK8IP1, PEX16, GYLTL1B, and PHF21A. Recently, translocations interrupting PHF21A have been associated with intellectual disability and craniofacial anomalies similar to those seen in PSS. The identification of this small deletion in a child with developmental delay and hypotonia provides further evidence for the genetic basis of developmental disability and identifies a critical region sufficient to cause hypotonia in this syndrome. Additionally, this case illustrates the utility of high resolution genomic approaches in correlating clinical phenotypes with specific genes in contiguous gene deletion syndromes.

Subtelomeric deletion of chromosome 10p15.3: clinical findings and molecular cytogenetic characterization.

We describe 19 unrelated individuals with submicroscopic deletions involving 10p15.3 characterized by chromosomal microarray (CMA). Interestingly, to our knowledge, only two individuals with isolated, submicroscopic 10p15.3 deletion have been reported to date; however, only limited clinical information is available for these probands and the deleted region has not been molecularly mapped. Comprehensive clinical history was obtained for 12 of the 19 individuals described in this study. Common features among these 12 individuals include: cognitive/behavioral/developmental differences (11/11), speech delay/language disorder (10/10), motor delay (10/10), craniofacial dysmorphism (9/12), hypotonia (7/11), brain anomalies (4/6) and seizures (3/7). Parental studies were performed for nine of the 19 individuals; the 10p15.3 deletion was de novo in seven of the probands, not maternally inherited in one proband and inherited from an apparently affected mother in one proband. Molecular mapping of the 19 individuals reported in this study has identified two genes, ZMYND11 (OMIM 608668) and DIP2C (OMIM 611380; UCSC Genome Browser), mapping within 10p15.3 which are most commonly deleted. Although no single gene has been identified which is deleted in all 19 individuals studied, the deleted region in all but one individual includes ZMYND11 and the deleted region in all but one other individual includes DIP2C. There is not a clearly identifiable phenotypic difference between these two individuals and the size of the deleted region does not generally predict clinical features. Little is currently known about these genes complicating a direct genotype/phenotype correlation at this time. These data however, suggest that ZMYND11 and/or DIP2C haploinsufficiency contributes to the clinical features associated with 10p15 deletions in probands described in this study.

Tetrasomy 15q26: a distinct syndrome or Shprintzen-Goldberg syndrome phenocopy?

PURPOSE: The aim of this study was to characterize the clinical phenotype of patients with tetrasomy of the distal 15q chromosome in the form of a neocentric marker chromosome and to evaluate whether the phenotype represents a new clinical syndrome or is a phenocopy of Shprintzen-Goldberg syndrome. METHODS: We carried out comprehensive clinical evaluation of four patients who were identified with a supernumerary marker chromosome. The marker chromosome was characterized by G-banding, fluorescence in situ hybridization, single nucleotide polymorphism oligonucleotide microarray analysis, and immunofluorescence with antibodies to centromere protein C. RESULTS: The marker chromosomes were categorized as being neocentric with all showing tetrasomy for regions distal to 15q25 and the common region of overlap being 15q26→qter. CONCLUSION: Tetrasomy of 15q26 likely results in a distinct syndrome as the patients with tetrasomy 15q26 share a strikingly more consistent phenotype than do the patients with Shprintzen-Goldberg syndrome, who show remarkable clinical variation.

Clinical and molecular characterization of chromosome 7p22.1 microduplication detected by array CGH.

A 28-month-old Peruvian male presented with speech delay and unusual facial features including prominent forehead, anteverted nares, ocular hypertelorism, and low-set and posteriorly rotated ears with a unilateral preauricular pit. The patient had poor speech with no other developmental delays. Height and weight were normal, although closure of the anterior fontanel and bone age were delayed. Head circumference approximated the 95th centile for age. Following normal routine chromosome analysis and subtelomeric FISH, whole genome microarray revealed a novel interstitial duplication at 7p22.1, approximately 1.7 Mb in size, and containing 13 OMIM annotated genes. FISH studies on the propositus and his parents confirmed that the duplication had occurred de novo. This finding represents the smallest interstitial 7p duplication reported to date, and does not include genes previously implicated as candidates for a 7p duplication syndrome. Common phenotypic features of 7p duplication include distinctive facies with hypertelorism,large anterior fontanel, and intellectual disability. Based on the findings in our patient, and those in previously reported cases of 7p duplication, we propose that genes within this duplicated interval may have a role in skeletal maturation,craniofacial development, and speech acquisition.

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres.

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.

Section E9 of the American College of Medical Genetics technical standards and guidelines: fluorescence in situ hybridization.

This updated Section E9 has been incorporated into and supersedes the previous Section E9 in Section E: Clinical Cytogenetics of the 2008 Edition (Revised 02/2007) American College of Medical Genetics Standards and Guidelines for Clinical Genetics Laboratories. This section deals specifically with the standards and guidelines applicable to fluorescence in situ hybridization analysis.

College of American Pathologists/American College of Medical Genetics proficiency testing for constitutional cytogenomic microarray analysis.

PURPOSE: To evaluate the feasibility of administering a newly established proficiency test offered through the College of American Pathologists and the American College of Medical Genetics for genomic copy number assessment by microarray analysis, and to determine the reproducibility and concordance among laboratory results from this test. METHODS: Surveys were designed through the Cytogenetic Resource Committee of the two colleges to assess the ability of testing laboratories to process DNA samples provided and interpret results. Supplemental questions were asked with each Survey to determine laboratory practice trends. RESULTS: Twelve DNA specimens, representing 2 pilot and 10 Survey challenges, were distributed to as many as 74 different laboratories, yielding 493 individual responses. The mean consensus for matching result interpretations was 95.7%. Responses to supplemental questions indicate that the number of laboratories offering this testing is increasing, methods for analysis and evaluation are becoming standardized, and array platforms used are increasing in probe density. CONCLUSION: The College of American Pathologists/American College of Medical Genetics proficiency testing program for copy number assessment by cytogenomic microarray is a successful and efficient mechanism for assessing interlaboratory reproducibility. This will provide laboratories the opportunity to evaluate their performance and assure overall accuracy of patient results. The high level of concordance in laboratory responses across all testing platforms by multiple facilities highlights the robustness of this technology.

UPD detection using homozygosity profiling with a SNP genotyping microarray.

Single nucleotide polymorphism (SNP) based chromosome microarrays provide both a high-density whole genome analysis of copy number and genotype. In the past 21 months we have analyzed over 13,000 samples primarily referred for developmental delay using the Affymetrix SNP/CN 6.0 version array platform. In addition to copy number, we have focused on the relative distribution of allele homozygosity (HZ) throughout the genome to confirm a strong association of uniparental disomy (UPD) with regions of isoallelism found in most confirmed cases of UPD. We sought to determine whether a long contiguous stretch of HZ (LCSH) greater than a threshold value found only in a single chromosome would correlate with UPD of that chromosome. Nine confirmed UPD cases were retrospectively analyzed with the array in the study, each showing the anticipated LCSH with the smallest 13.5 Mb in length. This length is well above the average longest run of HZ in a set of control patients and was then set as the prospective threshold for reporting possible UPD correlation. Ninety-two cases qualified at that threshold, 46 of those had molecular UPD testing and 29 were positive. Including retrospective cases, 16 showed complete HZ across the chromosome, consistent with total isoUPD. The average size LCSH in the 19 cases that were not completely HZ was 46.3 Mb with a range of 13.5-127.8 Mb. Three patients showed only segmental UPD. Both the size and location of the LCSH are relevant to correlation with UPD. Further studies will continue to delineate an optimal threshold for LCSH/UPD correlation.

Microdeletion/microduplication of proximal 15q11.2 between BP1 and BP2: a susceptibility region for neurological dysfunction including developmental and language delay.

The proximal long arm of chromosome 15 has segmental duplications located at breakpoints BP1-BP5 that mediate the generation of NAHR-related microdeletions and microduplications. The classical Prader-Willi/Angelman syndrome deletion is flanked by either of the proximal BP1 or BP2 breakpoints and the distal BP3 breakpoint. The larger Type I deletions are flanked by BP1 and BP3 in both Prader-Willi and Angelman syndrome subjects. Those with this deletion are reported to have a more severe phenotype than individuals with either Type II deletions (BP2-BP3) or uniparental disomy 15. The BP1-BP2 region spans approximately 500 kb and contains four evolutionarily conserved genes that are not imprinted. Reports of mutations or disturbed expression of these genes appear to impact behavioral and neurological function in affected individuals. Recently, reports of deletions and duplications flanked by BP1 and BP2 suggest an association with speech and motor delays, behavioral problems, seizures, and autism. We present a large cohort of subjects with copy number alteration of BP1 to BP2 with common phenotypic features. These include autism, developmental delay, motor and language delays, and behavioral problems, which were present in both cytogenetic groups. Parental studies demonstrated phenotypically normal carriers in several instances, and mildly affected carriers in others, complicating phenotypic association and/or causality. Possible explanations for these results include reduced penetrance, altered gene dosage on a particular genetic background, or a susceptibility region as reported for other areas of the genome implicated in autism and behavior disturbances.

Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

Variability in interpreting and reporting copy number changes detected by array-based technology in clinical laboratories.

PURPOSE: : The purpose of this study was to assess the variability in interpretation and reporting of copy number changes that are detected by array-based technology in the clinical laboratory. METHODS: : Thirteen different copy number changes, detected by array comparative genomic hybridization, that have not been associated with an abnormal phenotype in the literature were evaluated by directors from 11 different clinical laboratories to determine how they would interpret and report the findings. RESULTS: : For none of the thirteen copy number changes was there complete agreement in the interpretation of the clinical significance of the deletion or duplication. For some cases, the interpretations ranged from normal to abnormal. CONCLUSION: : There is a need for more specific guidelines for interpreting and reporting copy number changes detected by array-based technology to clearly and more consistently communicate the clinical significance of these findings to ordering providers.

Ovotesticular disorder of sexual development (true hermaphroditism).

OBJECTIVES: To determine the mechanism for the 46,XX/46,XY karyotype observed in a patient with an ovotesticular disorder of sexual development (ie, true hermaphroditism). METHODS: Cytogenetic, molecular cytogenetic, and molecular DNA analyses were performed on the blood, skin, and left and right gonadal tissue from 2 surgical procedures. The results of these studies were used to determine whether the ovotesticular disorder of sexual development resulted from mosaicism or tetragametic chimerism. RESULTS: Cytogenetic and molecular analyses revealed a mixture of 46,XX and 46,XY cells in most tissues. DNA analysis from the gonadal tissues from surgeries 1 and 2 was performed. Highly polymorphic loci from 12 different chromosomes were examined for the presence of > or = 1 paternal or maternal alleles. Three loci were highly informative: D14S544 (14q32.2), DS14S583 (14q21.3), and SE33 (6q14). Each demonstrated the presence of 2 paternal and 2 maternal alleles, indicating that the ovotesticular disorder of sexual development resulted from tetragametic chimerism. CONCLUSIONS: Based on the findings of the cytogenetic, molecular cytogenetic, and DNA analyses of the polymorphic markers from several different loci, it was confirmed that the patient had tetragametic chimerism. This case has assisted in increasing our knowledge of the possible mechanisms causing this rare and complex disorder.

Fully automated FISH examination of amniotic fluid cells.

OBJECTIVE: Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. RESULTS: A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. CONCLUSION: These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes.

Automated microscopy of amniotic fluid cells: detection of FISH signals using the FastFISH imaging system.

OBJECTIVE: FISH (fluorescence in situ hybridization) analysis is a valuable adjunct to cytogenetics that provides a rapid screen for common abnormalities. However, FISH is expensive, labor-intensive, and requires a high skill level and subjective signal interpretation. A fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: In this study we blindly compared automated FISH signal acquisition and display against standard FISH analysis. A total of 62 amniocentesis samples were prepared using the AneuVysion multicolor DNA probe kit and probed for chromosomes 13, 18, 21, X, and Y. Two sets of slides were produced from each sample. Fifty cells were scored in each slide. One set was evaluated using standard manual microscopy and the other using the automated image acquisition and display capabilities of the Ikoniscope fastFISH amnio Test System. This system uses epifluorescence optics, along with optimized slide management to process slides automatically. RESULTS: A 100% concordance was observed between the results obtained using manual microscopy and the automated system. There was also 100% concordance between the FISH results and those obtained by conventional karyotyping. CONCLUSION: Our data suggest that the automated system is capable of providing accurate and rapid identification and display of cells and FISH signals.

Oncogene amplification in transforming myelodysplasia.

The MLL gene, located within band 11q23, has been shown to be involved in translocations with a large variety of reciprocal sites in both lymphoid and myeloid leukemia and has also been shown to undergo submicroscopic self-fusion/partial duplication. We report 29 patients with cytogenetic evidence of 11q23 alteration, all of which demonstrate molecular cytogenetic evidence of amplification of the MLL gene by fluorescence in situ hybridization (FISH). In all MLL cases, the patients were clinically classified as having transforming myelodysplasia (RAEB/RAEBT) or AML. An additional patient with AML was found by 24-color and gene-specific FISH to have AML1 oncogene amplification. Four patients had been previously diagnosed with cancer and had received topoisomerase II targeted drug therapy which is known to be associated with fusion transcripts involving the MLL and AML1 genes. MLL amplification appeared in various forms: an atypical banded region that bridges from 11q23 into a dicentric chromosome, expanded regions emanating from band 11q23, chromosome 11 paint-positive rings with "spoke-like" MLL amplification, and expansion at sites other than chromosome 11 (including extra markers) in the absence of one of the 11 homologues. The fluorescence pattern in most cases suggests palindromic duplication with neighboring sequences in the long arm of chromosome 11. As opposed to MYCN amplification in hsrs (homogeneously staining regions) and double minutes in neuroblastoma, amplification of MLL in most cases occurred at the site of the gene. All of our patients rapidly developed refractory AML. The frequency and clinical correlations of MLL gene amplification in leukemia will need careful follow-up, since the frequently cryptic amplification described in these cases may not generally provoke confirmatory FISH studies. The reported MLL cases represented about 1% of the total abnormal MDS/AML cases over 8 years. A common cytogenetic profile of 5 q-, -17/17 p-, -18/18 q-, and a missing or abnormal chromosome 11, may help direct appropriate follow-up studies. The MLL and the AML1 oncogenes appear to be the only oncogenes amplified at the natural site of the gene. Both genes also show a high degree of diversity of pathogenic mechanisms of leukemia evolution, including numerous reciprocal fusion genes in transformation to either AML or ALL and gain of function amplification.