The copy number variant involving part of the alpha7 nicotinic receptor gene contains a polymorphic inversion.

The alpha7 nicotinic acetylcholine receptor gene (CHRNA7) is located at 15q13-q14 in a region that is strongly linked to the P50 sensory gating deficit, an endophenotype of schizophrenia and bipolar disorder. Part of the gene is a copy number variant, due to a duplication of exons 5-10 and 3' sequence in CHRFAM7A, which is present in many but not all humans. Maps of this region show that the two genes are in opposite orientation in the individual mainly represented in the public access human DNA sequence database (Build 36), suggesting that an inversion had occurred since the duplication. We have used fluorescent in situ hybridization to investigate this putative inversion. Analysis of interphase chromosomes in 12 individuals confirms the occurrence of an inversion and indicates that CHRFAM7A exists in both orientations with similar frequency. We showed that the 2 bp deletion polymorphism in exon 6 of CHRFAM7A is in strong linkage disequilibrium with the inversion polymorphism (r(2)=0.82, CI 0.53-1.00, P=0.00003), which can therefore be used as a surrogate marker. Previous associations of endophenotypes of schizophrenia with the 2 bp deletion might therefore be due to the orientation of the duplicon containing CHRFAM7A.

Array comparative genomic hybridization (array CGH) for detection of genomic copy number variants.

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient's sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Meiotic outcomes in reciprocal translocation carriers ascertained in 3-day human embryos.

Chromosomes involved in reciprocal translocations form quadrivalents at meiosis. These quadrivalents segregate, with or without recombination, to give 32 different meiotic outcomes, only two of which are normal or balanced. This paper presents data collected from 25 cycles of preimplantation genetic diagnosis for 18 couples carrying 15 different reciprocal translocations. Embryos were tested using fluorescence in situ hybridisation with probes for the translocated and centric segments. Overall, 47.7% (71 out of 149) of embryos tested showed signal patterns consistent with alternate segregation, 24.8% adjacent-1 segregation, 10.1% adjacent-2 segregation, 15.4% 3 : 1 segregation and 2% 4 : 0 segregation. For most translocations, alternate segregation was apparently the most frequent mode. Alternate and adjacent-1 frequencies were similar in male and female carriers; however, 5.7% of embryos from female translocation carriers showed adjacent-2 segregation and 20.0% showed 3 : 1 segregation, whilst the corresponding figures for male carriers were 20.5 and 4.5%. Overall, 2.8% of embryos were mosaic and 2.3% of embryos showed chaotic constitutions for the chromosomes tested. The pregnancy success rate for these 25 cycles was 38.8% per embryo transfer and also 38.8% per couple.

Delineation of an estimated 6.7 MB candidate interval for an anophthalmia gene at 3q26.33-q28 and description of the syndrome associated with visible chromosome deletions of this region.

Anophthalmia or microphthalmia occur in approximately one in 10 children who have severe visual impairment. These eye malformations are often of unknown aetiology, but can be inherited in autosomal dominant, recessive or X-linked forms, and can also occur in association with specific chromosome abnormalities. Four children are described in the medical literature with microphthalmia or anophthalmia in association with chromosome rearrangements involving distal 3q, suggesting the presence of a micro/anophthalmia gene in this region. We have identified two further patients with micro/anophthalmia and chromosome rearrangements involving 3q26-->3q27 and identified a 6.7 MB common deleted region. Patient 1 had multiple abnormalities including bilateral anophthalmia, abnormalities of the first and second cranial nerves and partial absence of the corpus callosum. His karyotype was 46,XY,del(3)(q26.33q28). Patient 2 had right anophthalmia and left extreme microphthalmia. Her karyotype was 46,XX,del(3)(q26.33q28)t(3;7)(q28;q21.1). Both patients had intrauterine growth retardation (IUGR) and strikingly similar dysmorphic facies consisting of bossed forehead, downward-slanting palpebral fissures, grooved bridge of the nose, prominent low-set ears, small down-turned mouth and small mandible. We identified BAC clones mapping to distal 3q from the ENSEMBL and NCBI Entrez databases. These BAC clones were used as fluorescence in situ hybridisation (FISH) probes to identify the minimum deleted region common to both patients. This interval, between clones RPC11-134F2 and RPC11-132N15, was estimated to be 6.7 MB. We conclude that there is an anophthalmia locus within this interval. Candidate genes mapping to this region include Chordin and DVL3, a homologue of the Drosophila Dishevelled gene.

A new direction for prenatal chromosome microarray testing: software-targeting for detection of clinically significant chromosome imbalance without equivocal findings.

Purpose. To design and validate a prenatal chromosomal microarray testing strategy that moves away from size-based detection thresholds, towards a more clinically relevant analysis, providing higher resolution than G-banded chromosomes but avoiding the detection of copy number variants (CNVs) of unclear prognosis that cause parental anxiety. Methods. All prenatal samples fulfilling our criteria for karyotype analysis (n = 342) were tested by chromosomal microarray and only CNVs of established deletion/duplication syndrome regions and any other CNV >3 Mb were detected and reported. A retrospective full-resolution analysis of 249 of these samples was carried out to ascertain the performance of this testing strategy. Results. Using our prenatal analysis, 23/342 (6.7%) samples were found to be abnormal. Of the remaining samples, 249 were anonymized and reanalyzed at full-resolution; a further 46 CNVs were detected in 44 of these cases (17.7%). None of these additional CNVs were of clear clinical significance. Conclusion. This prenatal chromosomal microarray strategy detected all CNVs of clear prognostic value and did not miss any CNVs of clear clinical significance. This strategy avoided both the problems associated with interpreting CNVs of uncertain prognosis and the parental anxiety that are a result of such findings.

Benefits and drawbacks of preimplantation genetic diagnosis (PGD) for reciprocal translocations: lessons from a prospective cohort study.

Preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridisation probes was carried out for 59 couples carrying reciprocal translocations. Before treatment, 85% of pregnancies had resulted in spontaneous miscarriage and five couples had achieved a healthy live-birth delivery. Following treatment, 33% of pregnancies failed and 21 of 59 couples had a healthy live-born child. The accuracy of diagnosis was 92% (8% false abnormal and 0% false normal results). The overall incidence of 2:2 alternate segregation products was 44%; however, products consistent with 2:2 adjacent segregation were ~twice as likely from male heterozygotes, and those with 3:1 disjunction were three times more likely from female heterozygotes. Our results indicate that up to three stimulation cycles per couple would give an ~50% chance of a successful live birth, with the risk of miscarriage reduced to the level found in the general population. In our study, 87% of all normal/balanced embryos available were identified as being suitable for transfer. We conclude that PGD provides benefit for couples with high-risk translocations by reducing the risk of miscarriage and avoiding a pregnancy with an unbalanced form of the translocation; however, for fertile carriers of translocations with a low risk of conceiving a chromosomally unbalanced offspring, natural conception may be a more viable option.

Preimplantation genetic diagnosis for a carrier of a Y;autosome translocation resulting in a healthy male offspring.

OBJECTIVE: To demonstrate the feasibility of establishing a successful pregnancy for a carrier of a balanced Y;autosome translocation. DESIGN: Four locus-specific fluorescence in situ hybridization (FISH) probes, informative for the translocation, were identified and tested on peripheral lymphocyte metaphase chromosomes and interphase preparations from the translocation carrier and his partner. SETTING: National health service genetics center, cytogenetics laboratory, and assisted conception unit. PATIENT(S): An infertile man, presenting with a balanced Y;13 translocation, and his reproductive partner. INTERVENTION(S): After ovarian stimulation, 15 eggs were collected, nine were injected, and three were suitable for blastomere biopsy on day 3; a single blastomere was taken from each embryo and tested with four locus-specific FISH probes. MAIN OUTCOME MEASURE(S): Birth of a healthy child. RESULT(S): One embryo showed a triploid signal pattern and one had fragmented nuclei; neither was suitable for transfer. One embryo showed a balanced male signal pattern and was transferred. A singleton pregnancy was established, resulting in the birth of a healthy male child. CONCLUSION(S): This first report of successful preimplantation genetic diagnosis treatment for infertile males with y:autosome translocations demonstrates that this treatment option can result in successful pregnancies and healthy offspring.

Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance.

BACKGROUND: Several studies have demonstrated that array comparative genomic hybridisation (CGH) for genome-wide imbalance provides a substantial increase in diagnostic yield for patients traditionally referred for karyotyping by G-banded chromosome analysis. The purpose of this study was to demonstrate the feasibility of and strategies for, the use of array CGH in place of karyotyping for genome imbalance, and to report on the results of the implementation of this approach. RESULTS: Following a validation period, an oligoarray platform was chosen. In order to minimise costs and increase efficiency, a patient/patient hybridisation strategy was used, and analysis criteria were set to optimise detection of pathogenic imbalance. A customised database application with direct links to a number of online resources was developed to allow efficient management and tracking of patient samples and facilitate interpretation of results. Following introduction into our routine diagnostic service for patients with suspected genome imbalance, array CGH as a follow-on test for patients with normal karyotypes (n = 1245) and as a first-line test (n = 1169) gave imbalance detection rates of 26% and 22% respectively (excluding common, benign variants). At least 89% of the abnormalities detected by first line testing would not have been detected by standard karyotype analysis. The average reporting time for first-line tests was 25 days from receipt of sample. CONCLUSIONS: Array CGH can be used in a diagnostic service setting in place of G-banded chromosome analysis, providing a more comprehensive and objective test for patients with suspected genome imbalance. The increase in consumable costs can be minimised by employing appropriate hybridisation strategies; the use of robotics and a customised database application to process multiple samples reduces staffing costs and streamlines analysis, interpretation and reporting of results. Array CGH provides a substantially higher diagnostic yield than G-banded chromosome analysis, thereby alleviating the burden of further clinical investigations.

Submicroscopic chromosome imbalance in patients with developmental delay and/or dysmorphism referred specifically for Fragile X testing and karyotype analysis.

BACKGROUND: Microdeletion syndromes are generally identified because they usually give rise to specific phenotypic features; many of these deletions are mediated by duplicons or LCRs. The phenotypes associated with subtelomeric deletions are also becoming recognised. However, reciprocal duplication events at these loci are less easily recognised and identified, as they may give rise to milder phenotypic features, and the individuals carrying them may not therefore be referred for appropriate testing. 403 patients with developmental delay and/or dysmorphism, referred to our Genetics Centre for karyotyping and Fragile X expansion testing, were assessed for chromosome imbalance by Multiplex Ligation-dependent Probe Amplification (MLPA). Two MLPA kits were used, one containing probes for the subtelomere regions, and one containing probes for common microdeletion loci. 321 patients were tested with both kits, 75 with the subtelomere kit alone, and 7 with the microdeletion kit alone. RESULTS: 32 patients had abnormal results; the overall abnormality detection rate was 2.5% for karyotype analysis and 7.2% for MLPA testing; 5.5% of subtelomere tests and 2.1% of microdeletion tests gave abnormal results. Of the abnormal MLPA results, 5 were in cases with cytogenetically visible abnormalities; of the remaining, submicroscopic, changes, 3 results were established as de novo and 8 were inherited; parental samples were not available for the remaining cases. None of the patients was found to have a Fragile X expansion. CONCLUSION: Karyotype analysis in combination with MLPA assays for subtelomeres and microdeletion loci may be recommended for this patient group.

Maternal cell contamination of prenatal samples assessed by QF-PCR genotyping.

OBJECTIVES: To establish the genotype of cultured cells from a cohort of amniotic fluid and chorionic villus samples, and compare this genotype with that obtained from uncultured material from the same sample, in order to assess the frequency and significance of maternal cell contamination of prenatal samples. METHODS: Quantitative fluorescence-polymerase chain reaction (QF-PCR) was carried out by amplification of microsatellite markers using fluorescence-labelled primers, followed by quantitative analysis of the allele peaks on a genetic analyser. A multiplex of 12 primer pairs for four loci on each of chromosomes 13, 18 and 21 was used. RESULTS: A total of 307 prenatal samples were tested. Of the 254 amniotic fluid samples, 39.8% had some degree of bloodstaining, ranging from 5% bloodstaining in the cell pellet to heavily bloodstained fluid. Uncultured samples were tested by QF-PCR analysis and the cultured cells were tested by both QF-PCR and karyotype analysis. Of the samples, 90.2% had the same single genotype on direct and cultured material. Two samples (0.65%) were mosaic for an aneuploidy cell line. A second genotype, interpreted as maternal cell contamination, was identified in direct and/or cultured preparations in 9.1% of samples, 17.8% of which were not bloodstained. Seven amniotic fluid samples (2.8%) showed maternal cell contamination in cultured material. CONCLUSIONS: For heavily bloodstained amniotic fluid samples, a maternal blood specimen may help interpret the results of rapid trisomy testing, followed by confirmation of the fetal origin of cultured cells. QF-PCR analysis has established a higher incidence of maternal cell contamination of cultured amniocytes than previous reports; the presence of MCC (maternal cell contamination) in cultured cells from samples with no bloodstaining underlines the need for karyotype analysis of more than one XX culture.