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J C Harper

Publications and source records attributed to J C Harper.

At least 37 records · Page 2Linked to original sources
PubMed2000

FISH analysis on day 5 post-insemination of human arrested and blastocyst stage embryos.

Preimplantation genetic diagnosis (PGD) is usually performed on cleavage stage embryos on day 3 post-insemination. Fluorescent in situ hybridization (FISH) has revealed four groups of chromosome patterns in embryos at this stage: uniformly normal, uniformly abnormal, mosaic and chaotic. Recently, some in vitro fertilization (IVF) clinics have started to perform blastocyst stage transfer. In blastocysts, conventional karyotyping has shown that all four groups of chromosome patterns are observed. In the present study, embryos were cultured to day 5 and were subject to a two-round multicolour FISH procedure for chromosome analysis to ensure almost every nucleus was examined. Probes for chromosomes X, Y and 18 were used in the first round and those for chromosomes 13 and 21 in the second round. Twenty arrested embryos (274 cells) and 19 blastocyst stage embryos (1272 cells) were analysed. Four arrested embryos and two blastocysts were uniformly diploid. The remaining 33 embryos were mosaic, including 17 blastocysts. Most of the blastocysts had a high proportion of diploid cells while in the arrested embryos, this proportion varied widely. For PGD, this high prevalence of mosaicism persisting to the blastocyst stage may pose problems similar to mosaicism in cleavage stage embryos.

Adult↗
PubMed2000

A successful strategy for preimplantation diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

Preimplantation genetic diagnosis (PGD) involves the screening of biopsied cells from in vitro fertilization (IVF) generated embryos. This procedure allows the selective transfer of unaffected embryos and thus may be preferable to prenatal diagnosis for couples at high risk of transmitting genetic defects to their offspring. In this way, termination of pregnancy is avoided. We describe here the development and first clinical application of PGD for medium-chain acyl-CoA dehydrogenase deficiency (MCAD). MCAD is a common inherited metabolic disorder affecting fatty acid beta oxidation. The condition is autosomal recessive with an incidence of 1/6000-1/15 000 live births in the UK. It presents usually within the first two years of life with fasting-associated hypoketotic hypoglycaemia which may lead to coma and death. The strategy developed was based on multiplex fluorescent PCR, and adapted for single cell detection. Mutation analysis was carried out using single-strand conformation polymorphism (SSCP) with fluorescent detection. The embryos generated through IVF arrested on day two post-insemination and consequently none were available for transfer; all available blastomeres were used to confirm the accuracy and reliability of the diagnostic assays.

Acyl-CoA Dehydrogenase↗
PubMed2000

Examination of trisomy 13, 18 and 21 foetal tissues at different gestational ages using FISH.

In man high levels of aneuploidy are seen in spontaneous abortions. Very few autosomal trisomies survive to birth, the three most common being those for chromosome 13, 18 and 21 giving rise to the syndromes named Patau, Edwards and Down respectively. Since the majority of these spontaneously abort, what makes the survivors different from the aborters? Could it be that they have tissue specific mosaicism with the additional normal cell line supporting survival? In this study fluorescence in situ hybridisation was used as a convenient way to detect trisomy in interphase cells. To study the level of mosaicism across gestation, different tissues from 21 trisomic foetuses were analysed using probes for chromosome 13, 18, 21, X and Y. Two trisomy 18 foetuses exhibited mosaicism. Two others, one trisomy 13 and one trisomy 18 had mosaic placentas. There was no clear association between the limited mosaicism seen and severity of the phenotype. We conclude that at least for this sample set, tissue-specific mosaicism was not likely to be responsible for potential survival to birth.

Adult↗
PubMed2000

Pre-implantation genetic diagnosis.

Pre-implantation genetic diagnosis (PGD) was developed in the UK over 10 years ago. There are now more than 40 centres worldwide carrying out PGD and 150 babies have been born after genetic testing on day 3 of development, at the cleavage stage. This review covers the current status of PGD, the technology used and the types of genetically determined diseases for which testing has been developed.

Aneuploidy↗
PubMed2000

Preimplantation genetic diagnosis.

Embryo biopsy for preimplantation genetic diagnosis can be performed on the oocyte/zygote, cleavage stage embryo, or blastocyst, but the majority of centres perform cleavage stage biopsy. Single-cell diagnosis is undertaken by the polymerase chain reaction or fluorescent in-situ hybridization. Technical difficulties have arisen with preimplantation genetic diagnosis, such as allele dropout and chromosomal mosaicism. However, it is hoped that these difficulties can be overcome in the future with the advent of new techniques.

Chromosome Aberrations↗
PubMed1999

Allelotype of gastric adenocarcinoma.

Gastric adenocarcinoma is a leading cause of cancer mortality world-wide. Yet, the underlying molecular events important in the development of this cancer are largely undefined. Thus, we performed a comprehensive survey for allelic loss on our panel of xenografted human gastric carcinomas. Contaminating normal stromal cells of primary cancers often limit mutational analyses. Xenografted samples of our gastric carcinomas provided optimally enriched tumors for neoplasia that clearly and sensitively demonstrated genetic alterations. Additionally, total absence of allelic signals in these xenografted samples confirmed true loss of alleles rather than just allelic imbalance. Analysis of at least two highly polymorphic microsatellite markers per nonacrocentric chromosomal arm in our xenografted human gastric carcinomas demonstrated significant loss of heterozygosity well above background levels at 3p, 4p, 5q, 8p, 9p, 13q, 17p, and 18q. Several of these loci represent novel findings of significant loss in gastric cancers. On chromosome 17p, p53 is known to be inactivated either by mutation or deletion in a majority of gastric carcinomas. The critical target(s) of inactivation in gastric cancers at these other loci remain to be characterized.

Adenocarcinoma↗
PubMed1999

Recent advances and future developments in PGD.

PGD has now been practised for a decade. The basic techniques currently used involve embryo biopsy, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH). Recent advances in molecular diagnostic techniques have included the use of fluorescent PCR, multiplex PCR and whole genome amplification. For cytogenetic analysis, many centres are now using five or more chromosome probes to examine for chromosome abnormalities, sexing and aneuploidy. Future improvements in molecular diagnosis include the use of quantitative PCR, DNA fingerprinting and microarray technology. Developments in methods to analyse chromosomes from a single cell have included interphase chromosome conversion, which has already been clinically applied, and the use of comparative genomic hybridization, which is still being developed. These methods will hopefully enable more accurate and a greater number of diseases to be diagnosed at the single cell level.

Chromosome Aberrations↗
PubMed1999

Successful preimplantation genetic diagnosis for sex Link Lesch--Nyhan Syndrome using specific diagnosis.

Lesch-Nyhan syndrome (LN) is a severe X-linked recessive disorder caused by a deficiency of the enzyme hypoxanthine phosphoribosyl transferase (HRT). Clinical features displayed by affected boys are particularly severe and disturbing and include hyperuricaemia, Characteristic neurological features including self-mutilation, choreothetosis, spasticity and mental retardation. A couple with a boy diagnosed with LN and a history of pregnancy termination was referred to the Hammersmith Hospital. Their affected son was born in 1982 after an uncomplicated pregnancy and vaginal delivery. Eight subsequent pregnancies had been unsuccessful. There were five therapeutic terminations and three spontaneous abortions, one at least directly caused by the sampling procedure during amniocentesis. From 1989 to 1991 two unsuccessful preimplantation genetic diagnosis (PGD) cycles by sexing were performed by DNA amplification. The mutation was characterized and a nested PCR protocol was designed which allowed the efficient amplification of the affected loci followed by the detection of the mutant allele by restriction digestion. Three PGD cycles were performed using this specific diagnostic test before a successful pregnancy was achieved resulting in the birth of a healthy unaffected baby girl.

Embryo Transfer↗
PubMed1999

Preimplantation genetic diagnosis for couples at high risk of Down syndrome pregnancy owing to parental translocation or mosaicism.

The population risk for trisomy 21 is 1 in 700 births but some couples are at a much higher risk owing to parental translocation or mosaicism. We report on the first attempt to carry out preimplantation genetic diagnosis for two such couples using cleavage stage embryo biopsy and dual colour FISH analysis. Each couple underwent two treatment cycles. Couple 1 (suspected gonadal mosaicism for trisomy 21) had two embryos normal for chromosome 21 transferred, but no pregnancy resulted; 64% (7/11) unfertilised oocytes/embryos showed chromosome 21 aneuploidy. Couple 2 (46,XX,t(6;21)(q13;q22.3)) had a single embryo transferred resulting in a biochemical pregnancy; 91% (10/11) oocytes/embryos showed chromosome 21 imbalance, most resulting from 3:1 segregation of this translocation at gametogenesis. The opportunity to test embryos before implantation enables the outcome of female meiosis to be studied for the first time and the recurrence risk for a Down syndrome pregnancy to be assessed.

Adult↗
PubMed1998

Consistent genetic alterations in xenografts of proximal stomach and gastro-esophageal junction adenocarcinomas.

The genetic alterations underlying the development of gastric and gastro-esophageal carcinoma remain largely undefined. DNA copy number changes were determined by comparative genomic hybridization in eight xenografts of proximal gastric and gastro-esophageal junction adenocarcinomas of the intestinal type. All tumors exhibited DNA copy number changes, with a total of 139 changes detected (range, 11-24 per tumor; mean = 17), indicating numerous and widespread alterations within these cancers. Gains (65%) in DNA copy number were more frequent than losses (35%). Our most striking finding was gain (all eight cases) or high-level amplification (four cases) in 20q, with a minimal common overlapping region at 20q13. Other frequent gains were observed at 6p, 7q, and 17q (six cases each) and at 1q, 2q, and 8q (five cases each). Frequent losses were observed at 4q and 5q (six cases each) and at 9p (five cases). No differences in DNA copy number changes were seen in tumors arising from the gastro-esophageal junction compared to those of the proximal stomach. The presence of common and consistent DNA copy number changes in these tumors implicate a number of chromosomal regions that may harbor important genes that are involved in tumorigenesis of the proximal stomach and gastro-esophageal junction.

Adenocarcinoma↗
PubMed1998

Infertile couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions.

Preimplantation genetic diagnosis (PGD) may provide a feasible option for some Robertsonian translocation carriers who experience severe difficulty in achieving a normal pregnancy. We report on five PGD cycles for two such couples, 45,XY,der(13;14)(q10:q10) and 45,XX,der(13;21)(q10;q10), carried out by biopsy of two cells from day 3 post-insemination embryos generated by in vitro fertilisation. Locus-specific YAC probes for chromosomes 13, 14 and 21 were used to detect the chromosomes involved in the translocation using multicolour FISH. Three embryos transfers were carried out (two single embryo transfers and one double transfer) but no clinical pregnancies were established. In two cycles no embryos were transferred as all those biopsied were chromosomally abnormal. Combined results from both couples show 13% (6/45) of embryos analysed were normal for the translocation chromosomes and 87% (39/45) were chromosomally abnormal; these were categorised as 36% aneuploid or aneuploid mosaic and 51% chaotic where the chromosome constitution varied randomly from cell to cell. This suggests two factors may be acting to reduce fertility in these couples; the aneuploid segregation of the parental Robertsonian translocation and also a post-zygotic factor leading to uncontrolled chromosome distribution in early cleavage stages in an exceptionally high proportion of embryos.

Blastomeres↗
PubMed1997

Inactivation of Smad4 in gastric carcinomas.

Allelic loss of chromosome 18q has been noted in intestinal type gastric adenocarcinomas. Smad4 is a gene located at 18q that was recently cloned in humans and found to be significantly altered in pancreatic cancers. We sought to determine whether Smad4 genetic alterations played a significant role in gastric tumorigenesis by studying 35 gastric adenocarcinomas of all histopathological types and pathological stages. Microdissected specimens were used for mutational analysis of Smad4 at the nucleotide level, including the entire coding region and intron/exon boundaries. Allelic imbalance was also analyzed at the Smad4 locus using two nearby microsatellite markers. One case of apparent biallelic inactivation of Smad4 was found in our study of 35 gastric carcinomas. A nonsense point mutation at codon 334 was demonstrated, which, similar to other Smad4 mutations, is predicted to truncate the conserved COOH-terminal domain of this protein. This Smad4 C to T transition mutation was proven to be somatically acquired. Allelic loss was also noted on chromosome 18q at a marker near Smad4 in this mutated gastric cancer, apparently producing complete inactivation of Smad4 in this tumor. Significant 18q allelic loss (56% of 34 informative cases) was noted in our gastric carcinomas using microsatellite markers near the Smad4 locus, regardless of histological subtype or pathological stage. Additionally, three cases of microsatellite instability were observed. Thus, Smad4 inactivation was noted in our gastric carcinomas; however, this event was rare. The frequent loss of chromosomal arm 18q observed in gastric cancers suggests the presence of other tumor suppressor genes in this region that are involved in gastric tumorigenesis. Further studies are needed to identify these other targets of inactivation during gastric cancer development.

Adenocarcinoma↗
PubMed1997

Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients.

We have used multicolour fluorescent in situ hybridisation (FISH) with DNA probes for chromosomes X, Y and l to analyse spare untransferred cleavage-stage embryos after preimplantation diagnosis to avoid X-linked disease. In total, 93 morphologically normal embryos were available from seven patients (six of proven fertility) who had undergone fourteen in vitro fertilisation (IVF) cycles. The chromosome patterns observed were classified into four groups; normal, abnormal (non-mosaic), mosaic and chaotic (uncontrolled division). Approximately half of the embryos were normal for the chromosomes tested. Two embryos only were aneuploid (non-mosaic) throughout but, after excluding those showing chaotic division, 30% were considered to be chromosomal mosaics. Of these, a minority had arisen because of mitotic non-disjunction or chromosome loss or gain, whereas the majority were ploidy mosaics, with haploidy being the most common. The occurrence of chaotically dividing embryos was strongly patient-related, i.e. some patients had 'chaotic' embryos in repeated cycles, whereas other patients were completely free of this type of anomaly. 'Chaotic' embryos are unlikely to progress beyond implantation. These findings have important implications both for routine IVF and preimplantation genetic diagnosis.

Adult↗
PubMed1996

Pregnancies resulting from embryos biopsied for preimplantation diagnosis of genetic disease: biochemical and ultrasonic studies in the first trimester of pregnancy.

PURPOSE: Our purpose was to investigate early biochemical and ultrasonic measurements of pregnancies resulting from embryos biopsied for preimplantation diagnosis of inherited disease. RESULTS: Singleton pregnancies following biopsy had lower initial hCG levels [10 or 12 days after oocyte recovery (OR)], which rose steeply to match the controls by 16 days after OR. Twin biopsied pregnancies showed hCG levels lower than those of twin control pregnancies, which rose in parallel with the controls but remained lower for a longer period than the singletons. Progesterone levels showed a wide variation. Ultrasound measurements showed that overall the mean sac diameter and crown-rump length at 28 and 42 days after egg collection were similar in biopsied and control pregnancies. CONCLUSIONS: Pregnancies resulting from biopsied embryos behave similarly to control IVF pregnancies. However, the reduction in cell mass following embryo biopsy occasionally results in reduced levels of circulating serum hCG and smaller ultrasound measurements in early pregnancy.

Biopsy↗
PubMed1996

Preimplantation diagnosis of inherited disease by embryo biopsy: an update of the world figures.

PURPOSE: Cleavage stage embryo biopsy and preimplantation diagnosis was first reported five years ago. The annual collation of the world figures for centres offering this procedure is important to continually assess the efficiency and success of the biopsy, diagnosis and pregnancies obtained. MATERIALS AND METHODS: Data was collected from 14 centres worldwide. The largest series of diagnosis has been performed for those patients carrying X-linked disease where the embryos have been sexed either by PCR or FISH. PCR has also been used for the specific diagnosis of a number of single gene defects, mainly for cystic fibrosis but also for the diagnosis of Lesch Nyhan syndrome, Fragile X, Duchenne muscular dystrophy, Tay Sachs, haemophilia and RhD blood typing. RESULTS: For PCR sexing, a total of 62 cycles have been performed with 14 pregnancies, 8 deliveries and 11 babies born. For FISH sexing, 70 cycles have resulted in 15 pregnancies, 8 deliveries and 11 babies born. For the specific diagnosis of single genes defects, a total of 65 cycles resulted in 21 pregnancies, 12 deliveries and 12 babies born. Overall, 197 cycles were performed, with 171 embryo transfers (86%), 50 pregnancies (25% per cycle, 29% per transfer), 28 deliveries and 34 babies born. CONCLUSIONS: New methods are being developed to increase the scope of preimplantation diagnoses that can be offered. From next year, the biochemical and miscarriage rates will also be recorded.

Abortion, Spontaneous↗
PubMed1996

Detection of chromosomal abnormalities in human preimplantation embryos using FISH.

PURPOSE: Multicolour FISH has been used for the preimplantation diagnosis of sex for X-linked disorders and to examine the chromosome constitution of early human embryos. MATERIALS AND METHODS: Single blastomeres and whole embryos were spread using HCI and Tween 20. Multicolour FISH was performed using directly-labelled human DNA probes for chromosomes X, Y, and 1 in a two hour FISH procedure. RESULTS: Four groups of chromosome arrangements have been found in human preimplantation embryos (i) normal, all nuclei uniformly diploid, (ii) diploid mosaics, majority of the nuclei diploid, with a small number of nuclei aneuploid (iii) chromosomally abnormal, all nuclei uniformly chromosomally abnormal, e.g. XO, XXY, XXX and (iv) chaotic, all nuclei showing different chromosome complements. CONCLUSIONS: For the preimplantation diagnosis of sex, an XX nucleus has always been representative of a female embryo. However, for the diagnosis of dominant disorders or chromosome abnormalities, two cells should be analysed to reduce the chance of misdiagnosis which may arise from chromosomal mosaicism. Implantation and further embryo development may be possible from mosaic or chromosomally abnormal embryos, but those showing chaotic chromosome arrangements would be unlikely to implant.

Blastocyst↗
PubMed1996

Obstetric outcome of pregnancies resulting from embryos biopsied for pre-implantation diagnosis of inherited disease.

OBJECTIVE: Pre-implantation diagnosis of inherited disease is now a viable option for some couples at risk of transmitting inherited disorders to their children. Since the pregnancy begins knowing that the embryo is not at risk, the need for repeated terminations is eliminated. Up to 25% of the embryo is removed during the procedure, and so it is important to study the resulting pregnancies. Here we report on the obstetric outcome of our first 16 pregnancies resulting from embryo biopsy and preimplantation diagnosis of inherited disease. SETTING: Teaching hospital. SAMPLE: The first 16 pregnancies (12 singletons and 4 twins) following pre-implantation diagnosis. RESULTS: Three singleton pregnancies were lost in the first trimester. Of the remaining pregnancies, two had no prenatal diagnosis, six cases of X-linked disease had the sex confirmed by ultrasound and chorionic villus sampling was performed in the remaining five. All the singleton pregnancies had an uneventful antenatal course and the birthweights and Apgar scores of the babies were normal. The twin pregnancies presented obstetric complications but these were not unusual. CONCLUSIONS: Fifteen healthy infants were born, but for the foreseeable future pre-implantation diagnosis pregnancies should be closely followed up.

Biopsy↗