Nuclei size in relation to nuclear status and aneuploidy rate for 13 chromosomes in donated four cells embryos.

PURPOSE: The aim was to elucidate if the nuclear size and number are indicative of aberrant chromosome content in human blastomeres and embryos. METHODS: The number of nuclei and the nucleus and blastomere size were measured by a computer controlled system for multilevel analysis. Then the nuclei were enumerated for 13 chromosomes by a combination of PNA and DNA probes. RESULTS: In the mononucleated embryos there was no difference in the mean size of chromosomally normal and abnormal nuclei but a significant difference in the mean nuclei size of nuclei that had gained chromosomes compared to nuclei that had lost chromosomes. The nuclei from multinucleated blastomeres had a significant smaller mean size and the frequency of chromosomally aberrant blastomeres was significantly higher. CONCLUSION: The mean nuclear size is not a marker for the chromosome content in mononucleated embryos. However, it seems that the nuclei size can be related to multinucleation and maybe to the chromosome content.

Genetic analysis of males from intracytoplasmic sperm injection couples.

A total of 392 men referred for intracytoplasmic sperm injection (ICSI) participated in genetic analysis. The control group consisted of 100 normal fertile males. Chromosome and DNA analyses were performed to investigate the frequency of Y-chromosome microdeletions and CFTR mutations (the controls underwent DNA analysis only). An abnormal karyotype was found in 4.6% of all males, but the frequency among men with azoospermia was higher, at 11.7%. Y-chromosome microdeletions were found only among men with azoospermia (6.5%) and men with extreme oligospermia (2%). Compound heterozygosity for CFTR mutations was found in men with azoospermia (3.9%) and congenital bilateral absence of vas deferens (CBAVD) only. We conclude that all couples referred for ICSI should be offered chromosome analysis. DNA analysis for Y-chromosome microdeletions should be reserved for men with azoospermia or extreme oligospermia (<1 x 106 spermatozoa). Analysis for CFTR mutations should be limited to those with obstructive azoospermia or those with a family history of cystic fibrosis.

Fertility and pregnancy outcome in Danish women with Turner syndrome.

The aim of the investigation was to study fertility in Danish women diagnosed with Turner syndrome (TS), and to describe their offspring. In total, 410 women in the fertile age were registered in the Danish Cytogenetic Central Register with TS between January 1973 and December 1993. Their karyotype were as follows: 49% with 45,X, 23% with mosaicism and a structural abnormality of the second X, 19% with 45,X/46,XX mosaicism, and 9% with 46,XX and a structural abnormality of the second X. Thirty-three women, one with 45,X, 27 with mosaicism and 5 with 46,XX and a structural abnormality of the second X, gave birth to 64 children. Two women had become pregnant after in vitro fertilization, including a woman with 45,X after an egg donation. Thus, 31 women(7.6%) had achieved at least one spontaneous pregnancy, but 48% of the fertile women registered with 45,X/46,XX had 45,X in less than 10% of the analysed cells. Twenty-five of the 64 children had a chromosome analyzed. Six of the 25 examined children, including three siblings, had chromosomal aberrations. No case of Down's syndrome was present, and only two children had malformations. Fertility in women registered with TS is higher than earlier reported. However, only women with 45,X/46,XX mosaicism or 46,XX and structural abnormality of the second X, gave birth to live children after spontaneous pregnancies.

Birth of a healthy girl after ICSI with ejaculated spermatozoa from a man with non-mosaic Klinefelter's syndrome.

Klinefelter's syndrome is a major contributor to male infertility. Recent reports of births after ICSI with especially testicular spermatozoa from infertile men with this syndrome are promising. The birth of a healthy girl after ICSI treatment with ejaculated spermatozoa from a man with non-mosaic Klinefelter's syndrome is reported. The non-mosaic karyotype was confirmed by chromosome analysis of both peripheral blood leukocytes and fibroblasts from a skin biopsy. In conclusion, in a very few cases, men with apparently non-mosaic Klinefelter's syndrome have ejaculated spermatozoa that can result in a birth of a healthy child following ICSI.

Disease associated balanced chromosome rearrangements: a resource for large scale genotype-phenotype delineation in man.

Disease associated balanced chromosomal rearrangements (DBCRs), which truncate, delete, or otherwise inactivate specific genes, have been instrumental for positional cloning of many disease genes. A network of cytogenetic laboratories, Mendelian Cytogenetics Network (MCN), has been established to facilitate the identification and mapping of DBCRs. To get an estimate of the potential of this approach, we surveyed all cytogenetic archives in Denmark and southern Sweden, with a population of approximately 6.6 million. The nine laboratories have performed 71 739 postnatal cytogenetic tests. Excluding Robertsonian translocations and chromosome 9 inversions, we identified 216 DBCRs ( approximately 0.3%), including a minimum estimate of 114 de novo reciprocal translocations (0.16%) and eight de novo inversions (0.01%). Altogether, this is six times more frequent than in the general population, suggesting a causal relationship with the traits involved in most of these cases. Of the identified cases, only 25 (12%) have been published, including 12 cases with known syndromes and 13 cases with unspecified mental retardation/congenital malformations. The remaining DBCRs were associated with a plethora of traits including mental retardation, dysmorphic features, major congenital malformations, autism, and male and female infertility. Several of the unpublished DBCRs defined candidate breakpoints for nail-patella, Prader-Willi, and Schmidt syndromes, ataxia, and ulna aplasia. The implication of the survey is apparent when compared with MCN; altogether, the 292 participating laboratories have performed >2.5 million postnatal analyses, with an estimated approximately 7500 DBCRs stored in their archives, of which more than half might be causative mutations. In addition, an estimated 450-500 novel cases should be detected each year. Our data illustrate that DBCRs and MCN are resources for large scale establishment of phenotype-genotype relationships in man.

Quantitative evaluation of fluorescence in situ hybridization (FISH) signals in uncultured coelomic cells.

Uncultured coelomic cells were hybridized with alpha satellite DNA probes representing chromosomes X, Y, 18, and 13/21 in order to evaluate the distribution of hybridization signals obtained by fluorescence in situ-hybridization (FISH) analysis of this cell type. Cells from 26 samples were hybridized with the X probe and the Y probe was hybridized with cells from 25 of the samples. Cells from 16 and 11 samples were hybridized with an 18 alpha satellite DNA probe and 13/21 alpha satellite probe, respectively. The evaluation demonstrated that FISH with X, Y, 18, and 13/21 alpha satellite DNA probes on uncultured coelomic cells is a reliable technique since the distribution of hybridization signals is comparable to that seen in uncultured amniotic fluid cells.

Early prenatal diagnosis: standard cytogenetic analysis of coelomic cells obtained by coelocentesis.

Coelomic fluid samples (n = 30) were obtained from normal pregnancies between 6.0 and 10.0 weeks and attempts to culture the coelomic cells were made. With one culture system, nine of ten samples were cultured and successful cytogenetic analysis was performed. There have been no previous reports of traditional cytogenetic analysis of coelomic cells and this might be the breakthrough for coelocentesis as a future method for early prenatal diagnosis.

Pseudodicentric chromosome 18 diagnosed by chromosome painting and primed in situ labelling (PRINS).

We report on a newborn white male infant with marked dysmorphic features and various congenital malformations. The initial clinical evaluation showed Crouzon-like features as well as some features of trisomy 18 syndrome and trisomy 13 syndrome. The results from conventional cytogenetic analysis showed a structurally abnormal chromosome replacing one normal chromosome 18, but only by applying molecular cytogenetic methods could the architecture of this abnormal chromosome be characterised clearly. The primed in situ labelling (PRINS) technique, using a newly synthesised alpha 18 oligonucleotide, showed the dicentric pattern and direct chromosome painting established the origin to be from chromosome 18. The combination of conventional cytogenetics and molecular cytogenetics showed the karyotype in the proband to be 45,XY,-14,-18,-21,+t(14;21),+psu dic(18) (qter-->cen-->p11.3: :p11.3-->psu cen-->qter). This was supported by molecular analysis using chromosome 18 specific DNA markers, which showed the paternal origin of the abnormal chromosome.

Normal karyotype in cultured chorionic villus cells, but mosaicism in amniotic and fetal cells.

A case with a normal male karyotype in cultured chorionic villus cells, but 46,XY/45,X/46,X,i(Yq) mosaicism in amniotic and fetal tissue is reported. The fetus was a phenotypic male. Pathological examination revealed discrete features, which might indicate a syndrome, and histological examination showed large, bright cells in the tubules of the testes. Possible explanations for discordance between the karyotype of embryonic and extraembryonic tissue are discussed.

Alpha 1-antitrypsin alleles in patients with pulmonary emphysema, detected by DNA amplification (PCR) and oligonucleotide probes.

Alpha 1-antitrypsin (AAT) deficiency is a serious predisposing factor for the development of pulmonary emphysema. Twelve representative Danish families were studied. AAT typing was performed as a comparative study between the traditional protein typing by isoelectrical focusing and the deoxyribonucleic acid (DNA) technique of enzymatic amplification and subsequent typing with radioactively labelled oligonucleotide probes. On the basis of clinical and radiological signs of pulmonary emphysema, 25 patients were selected. AAT typing was performed by use of the two techniques in combination, in search for new point-mutations among the patients. Results obtained with the two techniques were discordant in one patient, suggesting an unknown variant. The unexpectedly high PiZ frequency of 0.22 found in the study group is discussed.

The C3-F gene in patients with intracranial saccular aneurysms.

The C3-F gene has previously been found associated with atherosclerotic vascular diseases. The occurrence of the C3-F gene was therefore investigated in a group of 110 hospitalized patients with intracranial saccular aneurysms. The C3-F gene occurred equally often in patients and controls. In patients with a ruptured aneurysm, however, the C3-F gene frequency was significantly increased in subjects aged 40-49 years followed by a marked and statistically significant decline with increasing age. The findings suggest that the C3-F gene may be a risk factor for early aneurysm rupture.

HLA antigens and complement types in patients with intracranial saccular aneurysms.

The occurrence of HLA-A, -B, -C antigens and the HLA controlled complement factors (Bf, C2, C4) was investigated in an unselected group of 116 consecutively admitted patients with intracranial saccular aneurysms, and compared to that of healthy controls (blood donors). When multiplying the p-values with the number of comparisons made, none remained significant. However, a rather high etiologic fraction of the BfS gene (0.59) was obtained. Moreover, for HLA -B7 a significant deviation from the Hardy-Weinberg equilibrium with an increase of homozygotes was found. Due to linkage disequilibria this could indicate a strong association between HLA-DR2 and saccular aneurysms. The presence of HLA-DR2 was therefore investigated in a series of 15 aneurysm patients used as cadaver kidney donors and not included among the 116 consecutively admitted patients. In this group the HLA-DR2 antigen frequency was significantly increased (66.7% vs. 29.7%, p less than 0.01). The present study thus demonstrates an association of saccular aneurysm and the major histocompatibility complex and shows the existence of a genetic predisposition to saccular aneurysm.

Familial cranial diabetes insipidus: a report of five families. Genetic, diagnostic and therapeutic aspects.

Five families were studied in which cranial diabetes insipidus occurred. In the pedigrees presented, the disease clearly followed an autosomal dominant mode of inheritance. Linkage analysis was performed in one large family by calculating lod scores for linkage between loci for cranial diabetes insipidus and 18 polymorphic markers and chromosome heteromorphisms. No significant genetic linkage was found and only one of the polymorphic markers gave a positive hint of linkage. A water deprivation test was performed in nine patients from three of the families and in healthy control subjects. The plasma concentration of arginine vasopressin was very low or undetectable in the patients, and unlike the control subjects did not increase significantly during water deprivation. Arginine vasopressin and serum osmolality (Sosm) were significantly positively correlated in the controls, but not in the patients. The results indicated that an arginine vasopressin-level lower than 2 pg/ml strongly suggests a diagnosis of cranial diabetes insipidus if at the same time Sosm is higher than 295 mosmol/kg. Studies with different intranasal dosages of 1-deamino-D-arginine-vasopressin (DDAVP) given once or twice a day showed that 20 micrograms effectively reduced urinary output and that administration once a day could be sufficient.

An international reference typing for Ch and Rg determinants on rare human C4 allotypes.

Red cells, serum and plasma samples of 20 individuals, selected for their C4 allotypes, were distributed from Bonn to five laboratories, for investigation of their Chido (Ch) and Rodgers (Rg) determinants. One anti-Ch (M.H.) and one anti-Rg(Prest.) were distributed, but the individual laboratories also used their own reagents and their own typing methods. There was general agreement in interpretation of the majority of samples. Partial inhibition for Ch and Rg was detected. Two samples gave anomalous results; one sample with C4 A1,3 BQO, QO had Ch determinants on the red cells and in plasma (partial inhibition), and another sample with C4 A3,4 B5, QO apparently lacked Ch determinants on the red cells and in plasma. Heterogeneity of anti-Ch and anti-Rg was suggested in testing red cells, perhaps, reflecting a quantitative effect. This heterogeneity was confirmed by inhibition studies. The capacity of some reagents to detect partial inhibition probably reflects qualitative as well as quantitative differences.

Intra HLA-D/DR region recombinant detected by primed lymphocyte typing (PLT).

The chromosome 6 markers, HLA-ABC, D, DR, MT, properdin factor Bf, and complement factors 2 (C2) and 5 (C4), were studied in three families, each of which included two HLA identical siblings, one or both of whom were known to be HLA-B: GLO recombinants. The families were also typed with primed lymphocyte typing (PLT) for HLA-D/DR region associated DP antigens. None of these studies gave evidence that the recombinations had occurred within the HLA region. Mixed leucocyte culture (MLC) tests within the families showed no detectable stimulation between the HLA identical siblings in two of the families, but a very weak stimulation between the HLA identical siblings (H and G) in the third family (GG). No reactive PLT reagents were generated when cells from the HLA identical siblings of the first two families were primed against each other. In contrast, priming between cells of H and G gave rise to reactive reagents. One of these (GHx), reacted with a determinant which segregated within the GG family as if child G was a paternal recombinant between the HLA-D, DR, DP, and C4 loci, on the one hand, and on the other hand one or more loci governing other HLA-D/DR region controlled lymphocyte activating determinants. This reagent was only restimulated by cells from two of 47 unrelated individuals. The other PLT reagent (HGx) did not give a clearcut pattern within the family because it was weakly positive with all family members (most of whom were D/DR2-positive) except the specific responder; in the panel it reacted with a determinant significantly associated with D/DR/DP2. Other PLT reagents could be generated within the family against lymphocyte activating determinants controlled by genes in the two paternal haplotypes telomeric to the assumed recombinational site. These reagents gave stronger reactions than the HGx and GHx reagents and reacted with two determinants in the unrelated panel strongly associated with D/DR/DP2 and D/DR/DP6, respectively. It seems likely that the GG family represents a third example of a recombination between the HLA-DR and SB loci. Our findings further support the assumption that the DR determinants may be immunodominant in lymphocyte activation.

Genetics of complement C4. Two homoduplication haplotypes C4S C4S and C4F C4F in a family.

A family in which two homoduplicated C4 haplotypes (or supergenes) segregate is described. One haplotype C4F*3 C4F*2.2 is composed of two C4F alleles and the other C4S*5.1 C4S*1 of two C4S alleles. The C4F duplication haplotype is a partial inhibitor of the Rodgers antigen, and judged from our family and population material, it seems to be rather frequent and associated with HLAB*35, Bf*F, and HLAD/DR*1. The C4S duplication haplotype is Rg(a-) and is not identified in individuals without another S, Ch(A+) variant.