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We aimed to determine whether SNP-microarray genomic testing of saliva had a greater diagnostic yield than blood for pathogenic copy number variants (CNVs). We selected patients who underwent CMA testing of both blood and saliva from 23,289 blood and 21,857 saliva samples. Our cohort comprised 370 individuals who had testing of both, 224 with syndromic intellectual disability (ID) and 146 with isolated ID. Mosaic pathogenic CNVs or aneuploidy were detected in saliva but not in blood in 20/370 (4.4%). All 20 individuals had syndromic ID, accounting for 9.1% of the syndromic ID sub-cohort. Pathogenic CNVs were large in size (median of 46 Mb), and terminal in nature, with median mosaicism of 27.5% (not exceeding 40%). By contrast, non-mosaic pathogenic CNVs were 100% concordant between blood and saliva, considerably smaller in size (median of 0.65 Mb), and predominantly interstitial in location. Given that salivary microarray testing has increased diagnostic utility over blood in individuals with syndromic ID, we recommend it as a first-tier testing in this group.

We aimed to determine whether SNP-microarray genomic testing of saliva had a greater diagnostic yield than blood for pathogenic copy number variants (CNVs). We selected patients who underwent CMA testing of both blood and saliva from 23,289 blood and 21,857 saliva samples. Our cohort comprised 370 individuals who had testing of both, 224 with syndromic intellectual disability (ID) and 146 with isolated ID. Mosaic pathogenic CNVs or aneuploidy were detected in saliva but not in blood in 20/370 (4.4%). All 20 individuals had syndromic ID, accounting for 9.1% of the syndromic ID sub-cohort. Pathogenic CNVs were large in size (median of 46 Mb), and terminal in nature, with median mosaicism of 27.5% (not exceeding 40%). By contrast, non-mosaic pathogenic CNVs were 100% concordant between blood and saliva, considerably smaller in size (median of 0.65 Mb), and predominantly interstitial in location. Given that salivary microarray testing has increased diagnostic utility over blood in individuals with syndromic ID, we recommend it as a first-tier testing in this group.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, and implementation of risk-adapted therapy has been instrumental in the dramatic improvements in clinical outcomes. A key to risk-adapted therapies includes the identification of genomic features of individual tumors, including chromosome number (for hyper- and hypodiploidy) and gene fusions, notably ETV6-RUNX1, TCF3-PBX1, and BCR-ABL1 in B-cell ALL (B-ALL). RNA-sequencing (RNA-seq) of large ALL cohorts has expanded the number of recurrent gene fusions recognized as drivers in ALL, and identification of these new entities will contribute to refining ALL risk stratification. We used RNA-seq on 126 ALL patients from our clinical service to test the utility of including RNA-seq in standard-of-care diagnostic pipelines to detect gene rearrangements and IKZF1 deletions. RNA-seq identified 86% of rearrangements detected by standard-of-care diagnostics. KMT2A (MLL) rearrangements, although usually identified, were the most commonly missed by RNA-seq as a result of low expression. RNA-seq identified rearrangements that were not detected by standard-of-care testing in 9 patients. These were found in patients who were not classifiable using standard molecular assessment. We developed an approach to detect the most common IKZF1 deletion from RNA-seq data and validated this using an RQ-PCR assay. We applied an expression classifier to identify Philadelphia chromosome–like B-ALL patients. T-ALL proved a rich source of novel gene fusions, which have clinical implications or provide insights into disease biology. Our experience shows that RNA-seq can be implemented within an individual clinical service to enhance the current molecular diagnostic risk classification of ALL.

We report two patients with leukaemia driven by the rare CNTRL-FGFR1 fusion oncogene. This fusion arises from a t(8;9)(p12;q33) translocation, and is a rare driver of biphenotypic leukaemia in children. We used RNA sequencing to report novel features of expressed CNTRL-FGFR1, including CNTRL-FGFR1 fusion alternative splicing. From this knowledge, we designed and tested a Droplet Digital PCR assay that detects CNTRL-FGFR1 expression to approximately one cell in 100 000 using fusion breakpoint-specific primers and probes. We also utilised cell-line models to show that effective tyrosine kinase inhibitors, which may be included in treatment regimens for this disease, are only those that block FGFR1 phosphorylation.

To review the clinicopathological features and highlight the problems in the diagnosis and management of low grade fibromyxoid sarcomas (LGFMS).Three cases of LGFMS were studied with histology and immunohistochemistry, and cytogenetics in one. The features and problems were compared with those in the literature.Two LGFMS had typical fibrotic and myxoid patterns showing abrupt transition from one to the other. Cellularity was low to moderate. Nuclei were medium sized and regular. In one of these the correct diagnosis was not made in the original needle biopsy resulting in inappropriate management. In the third tumour only myxoid areas were seen and the diagnosis was supported by cytogenetics showing a complex previously unreported translocation, t(7;18;16). One tumour recurred, one metastasised, and one has possible metastasis on imaging of the lungs.LGFMS is a tumour with low grade histological features but a high risk of local recurrence and a significant risk of metastasis which can be very late. There should be a high index of suspicion for this rare tumour and a low threshold for sending tissue for cytogenetics and/or molecular genetics. Special precautions should be exercised in the interpretation of small biopsies of a spindle cell lesion with bland cytological features in children. If the diagnosis is unclear there must be a detailed follow up plan with a person responsible for monitoring the plan.

Apparent reduction in the size of the CGG repeat is reported from seven fragile-X mothers to nine offspring in seven extended families. The overall frequency of the reduction amongst 121 fragile-X mother-fragile-X child transmissions was 7.4%, compared with an 18% reduction in fragile-X father-fragile-X daughter transmissions, but ascertainment bias may account for some underestimation of the frequency of reductions, especially between mothers and their offspring. In one mother-son transmission, a premutation present in the son (with a full mutation present in the mother) was unmethylated. This may be the result of either demethylation with reduction, or undetected mosaicism in the mother. There was no obvious increase in the level of intellectual performance in female offspring who received a reduced fragment from their mothers. Several mechanisms leading to apparent reduction, such as germline mosaicism, deletion or replication slippage, should be considered.

Chimerism analysis has an important role in the management of allogeneic hematopoietic stem cell transplantation. It informs response to disease relapse, graft rejection, and graft-versus-host disease. We have developed a method for chimerism analysis using ubiquitous copy number variation (CNV), which has the benefit of a "negative background" against which multiple independent informative markers are quantified using digital droplet polymerase chain reaction. A panel of up to 38 CNV markers with homozygous deletion frequencies of approximately 0.4-0.6 were used. Sensitivity, precision, reproducibility, and informativity were assessed. CNV chimerism results were compared against established fluorescence in situ hybridization, single nucleotide polymorphism, and short tandem repeat-based methods with excellent correlation. Using 30 ng of input DNA per well, the limit of detection was 0.05% chimerism and the limit of quantification was 0.5% chimerism. High informativity was seen with a median of four informative markers detectable per individual in 39 recipients and 43 donor genomes studied. The strength of this approach was exemplified in a multiple donor case involving four genomes (three related). The precision, sensitivity, and informativity of this approach recommend it for use in clinical practice.

Chorionic villus sampling (CVS) was performed on a 38-year-old woman at 10 weeks' gestation for advanced maternal age. Two long-term cultures showed true mosaicism of cells with a normal karyotype and cells with trisomy 15. Follow-up amniocentesis showed only cells with a normal karyotype. Methylation analysis of amniocyte DNA using the probe PW71B showed a result consistent with a diagnosis of Prader-Willi syndrome. This result was confirmed using dinucleotide microsatellite polymorphism analysis, which showed that the fetus had maternal uniparental heterodisomy for chromosome 15. This report describes the use of methylation analysis for prenatal diagnosis of uniparental disomy 15 but also indicates that there is some doubt regarding the methylation status of all amniocyte samples, as one of nine controls showed hypomethylation. Fetal skin was found to show low-level mosaicism for trisomy 15, indicating a prolonged persistence of mosaic trisomy 15, which raises questions regarding the management of pregnancies found to be mosaic for trisomy 15.

Based on the high incidence of gonadoblastoma in females with XY gonadal dysgenesis or 45,X/46,XY mosaicism, the existence of a susceptibility locus on the Y chromosome (GBY) has been postulated. We attempted to map GBY by making use of a recently developed dense map of Y-chromosomal sequence-tagged sites (STSs). In two female patients with gonadoblastoma, small marker chromosomes contained portions of the Y chromosome, and a single region of overlap could be defined extending from probe pDP97 in interval 4B, which contains the centromere, to marker sY182 in interval 5E of the proximal long arm. This interval is contained in a YAC contig that comprises approximately 4 Mb of DNA. Our findings confirm the previous localization of GBY and greatly refine it. The localization of GBY overlaps with the region to which a putative growth determinant, GCY, was recently assigned.

Cytogenetic analysis was conducted on tumor biopsy material from two pediatric, small, round, blue-cell tumors whose histology failed to give a clearcut diagnosis. The first case showed a complex composite karyotype within which there were two normal chromosomes 11 and one abnormal chromosome 22 present. The composite karyotype in the second case was similarly complex but this time included an abnormal chromosome 11 but no corresponding abnormal chromosome 22. Analysis of tumor mRNA from both cases using a Reverse Transcriptase PCR test with primers derived from a Ewing's sarcoma t(11;22)(q24;q12) breakpoint sequence showed both to have abnormal, chimeric transcribed messengers, each of different lengths. Further analysis of case 2 using chromosome painting and centromeric probing confirmed the abnormal chromosome 11 to be a der(11)t(11;22)(q24;q12) and also revealed two additional minor clones containing a der(22), which may be the karyotypic locations of the t(11;22) fusion sequences. Taken into consideration with clinical and histologic information, the results of these investigations indicated that both were neuroectodermal tumors (Ewing sarcomas of the chest wall/Askin tumors). The comparative values of both cytogenetic and molecular analysis in the diagnosis of neuroectodermal tumors and the detection of covert chromosome rearrangements are discussed.

On routine chromosome analysis a moderately retarded 18-year-old man was found to have an unusual short arm on one chromosome 14. With GTL-banding this chromosome showed an enlarged short arm with no evident secondary constriction. Negative CBG-banding of the short arm suggested the possibility of a translocation involving euchromatin. Interpretation of the abnormality as an unbalanced translocation relied on chromosome analysis using GTL-, CBG-, and Ag-NOR-banding of the proband's phenotypically normal mother, who was found to be carrying a balanced translocation involving chromosomes 8 and 14. In situ hybridization of sequences known to map to the short arm of chromosome 14 confirmed the interpretation and established that the breakpoint was within p11. The patient, whose karyotype is 46,XY, -14, +der(14)t(8;14)(q24.1;p11), is trisomic for the terminal end of the long arm of chromosome 8. The patient's clinical features are described and compared with those reported in patients trisomic for this region. This study demonstrates the importance of using a number of different banding techniques in conjunction with in situ hybridization for the investigation of morphologically unusual acrocentric short arm variants seen at routine diagnosis.

Over 30% of female carriers of the fragile X [fra(X)] syndrome are clinically affected. A nonrandom X chromosome inactivation in these cases could be a plausible explanation. A review of previous studies addressing this question showed inconclusive results; thus, we analysed the X inactivation pattern in fibroblasts of 4 unrelated, mentally retarded fra(X) carriers with a high expression of the fragile site Xq27.3. Using Southern analysis with a highly polymorphic probe M27 beta that recognizes methylation differences between the active and inactive X chromosome we found a 50/50 inactivation pattern in 2 cases and skewed patterns in the other 2. As biased patterns were also observed in control females we conclude that at present no evidence exists for a nonrandom X chromosome inactivation in the fra(X) syndrome in females.

Loss of genetic material from chromosome arm 8p occurs frequently in human breast carcinomas, consistent with this region of the genome harboring one or more tumor suppressor genes (TSGs). We used the complementary techniques of microsatellite-based LOH, high-density FISH, and conventional CGH on 6 breast cancer cell lines (MCF7, SKBR3, T47D, MDA MB453, BT549, and BT474) to investigate the molecular cytogenetic changes occurring on chromosome 8 during tumorigenesis, with particular emphasis on 6 potential TSGs on 8p. We identified multiple alterations of chromosome 8, including partial or complete deletion of 8p or 8q, duplication of 8q, and isochromosome 8q. The detailed FISH analysis showed several complex rearrangements of 8p with differing breakpoints of varying proximity to the genes of interest. High rates of LOH were observed at markers adjacent to or within PCM1, DUSP4/MKP2, NKX3A, and DLC1, supporting their status as candidate TSGs. Due to the complex ploidy status of these cell lines, relative loss of 8p material detected by CGH did not always correlate with microsatellite-based LOH results. These results extend our understanding of the mechanisms accompanying the dysregulation of candidate tumor suppressor loci on chromosome arm 8p, and identify appropriate cellular systems for further investigation of their biological properties.

Mosaicism for two forms of ring 13 was found in a child with embryonal rhabdomyo-sarcoma of the bladder, minor anomalies, and developmental delay. Her chromosome constitution was 46, XX, r(13)(p11q34)/46, XX, r del(13)(p11q14). Both cell lines were present in lymphocytes and fibroblasts. The cell line with the smaller ring chromosome predominated in both tissues. The child's manifestations reflect the presence of both cell lines.

A 12 year old girl referred for chromosome analysis because of short stature was found to have karyotype mos 45,X/46,X,+mar. The marker chromosome was observed in 58% of her blood lymphocytes. It was a small, pale staining, spherical fragment with GTL banding and showed faint differentiation along its length with CBG banding. DNA analysis using Y specific probes showed the absence of the testicular determining region and the presence of some short arm and centromeric Y chromosomal material. In situ hybridisation confirmed that the Y chromosomal material was associated with the marker chromosome. At laparotomy the patient was found to have streak gonads. Gonadectomy was subsequently performed and histological examination showed dysgenetic gonads with a dysgerminoma arising from a gonadoblastoma in the left gonad. This case shows that even very small Y derived marker chromosomes with pericentric material can predispose the phenotypic female to gonadal neoplasia.