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Few cases of de novo unbalanced X;autosome translocations associated with a normal or mild dysmorphic phenotype have been described. We report a 3-year-old dizygotic female twin with prenatally ascertained increased nuchal translucency. Prenatal chromosome studies revealed nearly complete trisomy 15 due to a de novo unbalanced translocation t(X;15)(q22;q11.2) confirmed postnatally. A mild phenotype was observed with normal birth measurements, minor facial dysmorphic features (hypertelorism, short broad nose, and a relatively long philtrum), and moderate developmental delay at the age of 3 years in comparison to her male fraternal twin. Replication timing utilizing BrdU and acridine-orange staining showed that the der(X) chromosome was late-replicating with variable spreading of inactivation into the translocated 15q segment. The der(X) was determined to be of paternal origin by analyses of polymorphic markers and CGG-repeat at FMR1. Methylation analysis at the SNRPN locus and analysis of microsatellites on 15q revealed paternal isodisomy with double dosage for all markers and the unmethylated SNRPN gene. The Xq breakpoint was mapped within two overlapping BAC clones RP11-575K24 and RP13-483F6 at Xq22.3 and the 15q breakpoint to 15q11.2, within overlapping clones RP11-509A17 and RP11-382A4 that are all significantly enriched for LINE-1 elements (36.6%, 43.0%, 26.6%, 22.0%, respectively). We speculate that the attenuated phenotype may be due to inactivation spreading into 15q, potentially facilitated by the enrichment of LINE-1 elements at the breakpoints. In silico analysis of breakpoint regions revealed the presence of highly identical low-copy repeats (LCRs) at both breakpoints, potentially involved in generating the translocation.

Short stature refers to the height of an individual which is below expected. The causes are heterogenous and influenced by several genetic and environmental factors. Chromosomal abnormalities are a major cause of diseases and cytogenetic mapping is one of the powerful tools for the identification of novel disease genes. Here we report a three generation family with a heterozygous pericentric inversion of 46, XX, inv(3) (p24.1q26.1) associated with Short stature. Positional cloning strategy was used to physically map the breakpoint regions by Fluorescence in situ hybridization (FISH). Fine mapping was performed with Bacterial Artificial Chromosome (BAC) clones spanning the breakpoint regions. In order to further characterize the breakpoint regions extensive molecular mapping was carried out with the breakpoint spanning BACs which narrowed down the breakpoint region to 2.9 kb and 5.3 kb regions on p and q arm respectively. Although these breakpoints did not disrupt any validated genes, we had identified a novel putative gene in the vicinity of 3q26.1 breakpoint region by in silico analysis. Trying to find the presence of any transcripts of this putative gene we analyzed human total RNA by RT-PCR and identified transcripts containing three new exons confirming the existence of a so far unknown gene close to the 3q breakpoint.

The Alagille syndrome (AGS), a congenital disorder affecting liver, heart, skeleton and eye in association with a typical face, is an autosomal dominant disease with nearly complete penetrance and variable expression. AGS is caused by mutations in the developmentally important JAG1 gene. In our mutation screening, where 61 mutations in JAG1 were detected, we identified five cases where mosaicism is present. Our results point to a significant frequency of mosaicism for JAG1 mutations in AGS of more than 8.2%. Because mosaicism may be associated with a very mild phenotype, the appropriate diagnosis of AGS and consequently the determination of the recurrence risk can be complicated.

Rett syndrome (RS) is a disease of neurological development. First reported 30 years ago in 1966, its biological and genetic basis remains obscure. RS is commonly thought of as an X linked dominant disorder lethal to hemizygous males. The few familial cases would arise through mosaicism or because of occasional females failing to manifest the disorder through skewed X inactivation in relevant cell types. We have one family where the mother and daughter are affected with RS, and which can be explained according to this hypothesis. If the alternative proposal of Thomas (1996) is correct, that the lack of males affected by such disorders is the result of a high male to female ratio of germline mutations rather than of gestational lethality, then the RS gene should be located on the grandpaternal chromosome. Genomic screening with markers covering the whole X chromosome has been performed. Studies using multiple informative markers indicate that the RS locus is likely to be located close to one of the X chromosome telomeres. Further investigations in eight additional families suggest the most likely region for the RS gene to be is the distal part of Xq (Xq28).

The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here we describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.

Prenatal cytogenetic analysis at 11 weeks of gestation revealed an abnormal karyotype 47,XX,+mar in all metaphases obtained from a chorionic villi sample after 24 h culture. Karyotyping of amniotic fluid cells in the second trimester showed mosaicism 47,XX,+i(12p)/46,XX with 10% aneuploid cells. The pregnancy was terminated at 20 weeks of gestation on the patient's request. The aborted fetus showed typical manifestations of the Pallister-Killian mosaic aneuploidy syndrome. The identity of the supernumerary isochromosome 12p was proven by LDH isozyme electrophoresis using cultured fibroblasts and by nonradioactive in situ hybridization using a biotinylated set of chromosome 12-specific DNA probes.

Duplications of the distal long arm of the X chromosome are rare and carrier females are usually phenotypically normal. We report on a 14-year-old short statured (height and weight

Alagille syndrome (AGS) is an autosomal dominant disorder with developmental abnormalities of the liver, heart, eyes, vertebrae, and face. Mutations in the JAG1 (Jagged 1) gene, coding a ligand in the evolutionarily conserved Notch signaling pathway, are responsible for AGS. Here we present sixteen different JAG1 gene mutations, among them twelve novel, not described previously. Seven frameshift: c. 172_178del7 (p.Ala58fs), c.509delT (p.Leu170fs), c.1197delG (p.Val399fs), c.1485_1486delCT (p.Pro495fs), c.1809_1810insTGGG (p.Lys604fs), c.2122_2125delCAGT (p.Gln708fs), c.2753delT (p.Ile918fs); five nonsense: c.383GA (p.Trp128X), c.496CT (p.Glu166X), c.841CT (p.Gln281X), c.1207CT (p.Gln403X), c.1603CT (p.Gln535X); two splice site: c.388-1GC, c.3048+1_3048+2insG and two missense mutations: c.359TA (p.Ile120Asn), c.560GA (p.Cys187Tyr) were found. Forty percent of the changes were identified in exons 2 and 4, the remaining mutations are distributed along the entire coding sequence of the gene. Seventy-five percent of the mutations lead to creation of premature termination codons. Family studies revealed that the specific mutations were inherited in 3 out of 11 investigated cases. No correlation between genotype and phenotype was observed.

The genetic background of rheumatoid arthritis (RA) is only partly understood, and several genes seem to be involved. The matrix metalloproteinases MMP1 (interstitial collagenase) and MMP3 (stromelysin 1) are thought to be important in destructive joint changes seen in RA. In the present study, functional relevant promoter polymorphisms of MMP1 and MMP3 were genotyped in 308 patients and in 110 controls, to test whether the polymorphisms contribute to the severity of the disease measured by radiographic progression of joint destruction. For comparison, the shared epitope of HLA DR4 and DR1 (SE) was determined by polymerase chain reaction. There was no association of MMP polymorphisms with susceptibility to RA. However, a strong linkage disequilibrium was observed between the 1G/2G (MMP1) and the 5A/6A (MMP3) polymorphisms (P << 10-6; linkage disequilibrium index D' = 0.46). In factorial regression, the degree of radiographic joint destruction correlated significantly with the 1G-5A haplotype (P = 0.0001) and the interaction term 'estimated number of 1G-5A haplotypes × duration of disease' (P = 0.0007). This association was phasic, indicating that possession of the 1G-5A haplotype has a protective effect over a period of about 15 years of RA, but might be associated with a more pronounced radiographic progression later on. Similar results were also found with the 1G allele of MMP1 alone (P = 0.015) and with the interaction term 'estimated number of 1G alleles × duration of disease' (P = 0.014). The correlation of SE with the Ratingen score was comparable (0.044). The regression model of MMP haplotypes explained 35% of the variance of the radiographic score, whereas the SE explained 29%. The 1G-5A haplotype across the closely linked MMP1 and MMP3 gene loci is a newly described genetic factor strongly associated with the progression of joint damage in RA. Our findings suggest that there are haplotypes in a MMP cluster region that modify the joint destruction in RA in a phasic manner.

A thorough study of the heterochromatin organisation in the pericentromeric region and the proximal long (q) and short (p) arms of human chromosome 9 (HSA 9) revealed homology between 9p12 and 9q13-21.1, two regions that are usually not distinguishable by molecular cytogenetic techniques. Furthermore, the chromosomal regions 9p12 and 9q13-21.1 showed some level of homology with the short arms of the human acrocentric chromosomes. We studied five normal controls and 51 clinical cases: 48 with chromosome 9 heteromorphisms, one with an exceptionally large inversion and two with an additional derivative chromosome 9. Using fluorescence in situ hybridisation (FISH) with three differentially labelled chromosome 9-specific probes we were able to distinguish 12 heteromorphic patterns in addition to the most frequent pattern (defined as normal). In addition, we studied one inversion 9 case with the recently described multicolour banding (MCB) technique. Our results, and previously published findings, suggest several hotspots for recombination in the pericentromeric heterochromatin of HSA 9. They also demonstrate that constitutional inversions affecting the pericentromeric region of chromosome 9 carry breakpoints located preferentially in 9p12 or 9q13-21.1 and less frequently in 9q12.

Although clinical features in Kabuki syndrome (KS; Niikawa-Kuroki syndrome) have been well defined, the underlying genetic mechanism still remains unclear. We report a 9-year-old girl with typical KS-like facial appearance, skeletal and dermatoglyphic abnormalities, severe mental retardation, and growth deficiency. In 60 of 100 GTG-banded metaphases from peripheral blood lymphocytes, a ring chromosome smaller than a G group chromosome was found, which, according to reverse painting, consisted of Xq11.1q13. The proband's karyotype was described as mos45,X/46,X,+r(X). Several loci were analyzed with fluorescence in situ hybridization (FISH) and microsatellite markers revealing that one r(X) breakpoint mapped proximal to DXS422 (Xp11.21) and the second mapped distal to XIST gene, between loci DXS128E and DXS441 (Xq13.2). Uniparental disomy for X and r(X) was excluded and the paternal origin of r(X) was identified. XIST expression was demonstrated by nested reverse transcription polymerase chain reaction (RT-PCR) using primers spanning exons 5, 6i, and 6 in RNA prepared from lymphocytes. The observation of XIST expression is in contrast to two other cases in which the XIST gene was either not present on r(X) or not expressed. To our knowledge, this is the first case of Kabuki-like syndrome manifestations with r(X) and XIST expression.

Alagille syndrome (AGS) or arteriohepatic dysplasia is a rare but well-defined clinical entity that is usually inherited as an autosomal dominant trait. A limited number of patients carry a deletion in chromosome 20p, with 20p11.23-p12.2 as the area of minimal overlap. Recently, a family has been identified in which a balanced translocation with a breakpoint in 20p12 co-segregates with the AGS phenotype. Here, we report a three-generation family with AGS and in which the affected members have a normal karyotype. Linkage analysis was performed with markers from the 20p candidate region. A lod score of Z = 2.96 was obtained with D20S27 at no recombination. Combining D20S27 and D20S61 to a single highly informative locus resulted in a maximum lod score of Z = +3.56 at theta = 0.0. Haplotype analysis positioned AGS between D20S59 and D20S65, markers that define an interval of about 40 cM. Allelic loss was not observed for the tested markers and no abnormalities in the PAX1 candidate gene were detected. These findings demonstrate that the locus on chromosome 20p could be responsible for AGS in cytogenetically normal patients and argues for a general role of this locus in the aetiology of AGS.

Employing the flow-sorted chromosome 20-specific DNA library LL20NS01, we isolated seven novel unique poly- and monomorphic DNA markers specific to human chromosome 20. Initially, 201 phage clones were analyzed regarding insert size and repetitivity. By testing 14 single- and low-copy number clones for their ability to detect RFLPs, three polymorphisms were revealed by two probes, pFMS22-1.4 [D20S22] and pFMS76 [D20S23]. Seven of twenty probes (35%) were assigned to chromosome 20 using a somatic cell hybrid DNA panel. Five of them were regionally mapped by in situ hybridization. Three DNA markers, pFMS51 [D20S29], pFMS76 [D20S23], and pFMS106 [D20S30], were assigned to 20p11.2-p12, and two markers, pFMS22-1.4 [D20S22] and pFMS135 [D20S31], to 20q12-q13.3. Our new chromosome 20-specific DNA markers should be useful for the molecular characterization of this rather underpopulated human chromosome.

SummaryNMRI/Han mice ovulate significant numbers of diploid oocytes after gonadotrophin stimulation, most of them arrested at metaphase I. In contrast, females from other mouse strains ovulate only oocytes having completed meiosis I. We investigated the heritability of the trait Dipl I by analysing F1hybrid females from crosses between the sensitive NMRI/Han stock and two mouse strains (C3H/HeHan and BALB/c) considered as non-sensitive, and females from backcrosses to NMRI/Han males (only crosses with C3H/HeHan). The results show (1) that the trait Dipl I is inheritable; (2) that an X-chromosomal (location proximally from the X–Y pairing region) or autosomal recessive mode of inheritance is excluded; and (3) that the expression of the trait, measured as frequency of diploidy, is modulated by a maternally transmitted factor.

High-resolution chromosome analysis of a 19-year-old female proband with syndromic intrahepatic ductular hypoplasia (Alagille syndrome, AWS) revealed an interstitial deletion of chromosome 20p with breakpoints provisionally located in or close to p11.22 and p12.2. Southern blots from digests of DNA of the proband and her chromosomally normal parents were hybridized with the human DNA probes pR12.21, HuPrPcDNA2, and pDS6-SgI, which have been mapped to the region 20 (p12-pter), and rehybridized with the F IX probe for calibration. Comparing the hybridization signals of the normally sized DNA fragments of the family, we found no evidence for loss of any of the three tested distal chromosome 20p loci in our proband. Furthermore, in situ hybridization with HuPrPcDNA2 revealed a specific accumulation of grains at or around the faint distal G band suspected to represent all or most of band p12.3 of the proband's deleted 20p and at p12 of the normal chromosome 20. Thus the AWS of our proband is associated with an interstitial deletion that preserved the three tested distal loci on 20p. Since nine further reported cases of 20p deletion are clinically similar, we propose AWS as a further "contiguous gene syndrome" and assign it to an approximately 8-Mb-large chromosome 20p segment (provisionally, p11.23-p12.1).

Spontaneous abortions were studied by analyzing chromosomes directly from chorionic villi. The frequency and the type of anomalies detected among 140 abortuses are in good agreement with those observed by others using conventional tissue cultures. Abnormal karyotypes were found in 48.6% of the cases. Trisomy predominated (66.2%), followed by polyploidy (22.1%), monosomy X (7.4%), and structural anomalies (4.4%). Among the trisomies, the most prevalent were of chromosome 22 (22.2%), 16 (22.2%), and 13 (9.5%). The relative frequencies of trisomies, monosomy X, and the different chromosomes involved in trisomies seem to differ between our study and those in which tissue cultures were analyzed. Our low frequency of 45,XO karyotypes and the shift to trisomies of chromosomes whose involvement increases steeply with maternal age are considered due to the approximately 3 year higher mean maternal age in our sample. The sex ratio (male to female) in chromosomally abnormal abortuses was 1.28, which is nearly identical to the 1.2 found in earlier studies. Surprisingly, in chromosomally normal abortions males were significantly outnumbered by females (sex ratio 0.76). Since maternal cell contamination cannot have influenced the sex ratio in our study, we consider it worthwhile to investigate whether failures associated with X inactivation are responsible for pregnancy wastage of some euploid female conceptuses. Knowledge of the karyotypes may serve as a prerequisite for the investigation of non-chromosomal genetic causes of pregnancy wastage.

Testing the hypothesis of amplified developmental instability in autosomal trisomies as proposed by Shapiro we predicted and found an increased frequency of symphalangies in the toes of patients with Down syndrome. In our X-ray study of the feet of 71 adults with trisomy 21 we also detected a greater than normal number of sesamoid bones. A corollary to Shapiro's hypothesis is a dependence on ethnic origin of the frequency of symptoms in Down syndrome. Compared to data from Europe, toe symphalangies are more prevalent in Japan. We predict this anomaly to occur even more often in Japanese patients with trisomy 21.

In order to study the mechanisms of nondisjunction at meiosis I in oocytes gonadotropin-stimulated Djungarian hamsters were treated at two stages [4.5 and 6 h post human chorionic gonadotropin (HCG)] during the preovulatory period with 1000 mg/kg Carbendazim (MBC). The compound, known to bind fast but reversibly to mammalian tubulin, was chosen to investigate whether the stage at which spindle function is inhibited affects the pattern of nondisjunction. Ovulated oocytes were cytologically prepared and scored for hyperhaploidy, diploidy and presegregation. Application at an early spindle phase, 4.5 h post HCG, to females stimulated with a low gonadotropin dose [3 IU pregnant mares serum (PMS); 2 IU HCG] caused a high frequency of nondisjunction (40.6%) with a more or less nonspecific pattern of malsegregated bivalents. Treatment at a late stage of spindle function (6 h post HCG) resulted in a less frequent (22.5%) but highly preferential malsegregation of those A-D group bivalents thought earlier to be late segregators. On the other hand, oocytes from females primed with a high (10 IU PMS and HCG) gonadotropin dose, a treatment assumed to delay meiosis by approximately 1.5 h, responded to MBC treatment at the late stage (6 h) with a nonspecific pattern and a high frequency (71.2%) of nondisjunction. The latter result is comparable to that in which MBC was given at the early stage (4.5 h) and after a low gonadotropin dose. The high nondisjunction response additionally indicates that spindles in hypergonadotropic stimulated oocytes are more susceptible and/or that the concentration of the inhibitor is higher in such oocytes. Only few oocytes with presegregation (3.1%; 0.0%; 1.7%) and few diploid oocytes (3.3%; 1.5%; 3.2%) with complete inhibition of meiosis I were observed. We conclude, that in Djungarian hamsters (1) the segregation of bivalents at meiosis I is asynchronous with the large A-D bivalents segregating last, (2) the phase in which spindle function is inhibited determines the pattern of nondisjunction, and (3) the resumption of meiosis I - from dictyotene to metaphase II - does not follow a rigidly timed programme but depends on the conditions of follicular maturation.

Females from the NMRI/Han mouse strain ovulate a high number of diploid oocytes (about 12%) after gonadotrophin-stimulated ovulation. These oocytes can be fertilized and develop into triploid embryos subsequently. The exposure of such gonadotrophin-primed females to X-ray doses of 0.05, 0.10, 0.20 or 0.40 Gy during the preovulatory period (2 h after the HCG dose) significantly decreased the percentage of diploid oocytes. After the highest dose used, i.e. 0.80 Gy, however, the incidence was on the level from unirradiated females, again. We suggest that the observed negative hump-shaped dose response of diploidy is not caused by secondary modifications induced by irradiation, such as a selective killing of diploid oocytes before ovulation, or a (compensatory) super-ovulation of only normal oocytes, but rather is caused by a direct radiobiological interference of low doses in protecting from gonadotrophin-induced aneuploidy.

Chorionic villi sampling at week 9–11 of gestation is increasingly applied as a new method in prenatal diagnosis of genetic disorders. Cytogenetical, biochemical or recombinant DNA methods have been used successfully in more than 6000 pregnancies at risk. Different obstetric techniques for sampling chorionic villi using a catheter or a biopsy forceps, both under ultrasound guidance only, are practised so far. At the present stage of experience main problems of this method seem to be the risk of foetal loss, diagnostic errors caused by maternal cell contamination, as well as the difficulty in the interpretation of chromosomal mosaicism.

We describe clinical features and laboratory findings in a physically and mentally retarded male with under-developed testes, a seemingly monocentric isochromosome of Yq but the presence of a Yp-specific DNA sequence at a single dose of unknown genomic localisation, and the presence of H-Y antigen at normal male titer. Our data contribute to the fine mapping of the human Y chromosome by correlating phenotypic features with results from karyotypic, immunologic, and molecular hybridisation analyses.

A significant number of diploid oocytes is ovulated from adult NMRI/Han mice treated with high doses of gonadotrophins. This inhibition of the first meiotic division is very likely caused by an altered communication between the germ cell and the surrounding somatic cells leading to a failure of the endocrine control of meiosis. The present study examined the role of mitochondria in follicular development, oocyte maturation and chromosomal segregation during first meiotic division in NMRI/Han mice. To affect mitochondrial function during the late phase of follicular maturation, chloramphenicol, a potent inhibitor of mitochondrial peptidyl transferase, was used. Adult mice were treated with chloramphenicol (CAM; 18.8 or 37.5 mg/kg b.w.) at different times after the pregnant mare serum injection. The results revealed that CAM inhibited the characteristic increase of ovarian weight, reduced the number of oocytes ovulated per female, lowered the progesterone concentration in the postovulatory ovary and increased the incidence of ovulated diploid oocytes. It was concluded that an irregular mitochondrial function may affect normal follicular development and oocyte maturation, and potentially interferes with the order chromosome segregation during the first meiotic division.

Clinical and cytogenetic examinations were performed on eight unrelated infants with duplication of part of the long arm of chromosome 3. A review of published cases shows a clinical syndrome characterized by statomotoric retardation, shortened life span, and a multiple congenital anomalies (MCA) syndrome of abnormal head configuration, hypertrichosis, hypertelorism, ocular anomalies, anteverted nostrils, long philtrum, maxillary prognathia, down-turned corners of the mouth, highly arched or cleft plate, micrognathia, malformed auricles, short, webbed neck, clinodactyly, simian crease, talipes, and congenital heart disease. The dup(3q) syndrome is a clinically easily recognizable entity.

ANEUPLOIDY can result either from non-disjunction, giving trisomy and monosomy, or from chromosome damage leading to loss of chromosomes (monosomy) only. It is known that chromosome loss can be induced after irradiating post-meiotic male germ cells1, but apart from findings on radiation-induced non-disjunction in oocytes from mice2–5, there is no evidence that paternal exposure to X rays induces chromosomal mal-segregation during male meiosis to produce monosomic and trisomic offspring. Szemere and Chandley6 did observe significantly more mal-segregated X and Y chromosomes in spermatocytes II after 100 and 200 rad had been given during preleptotene and pachytene, but failed to recover aneuploid fetuses in progeny from irradiated males. They suggested that only a few aneuploid spermatocytes would be transmitted to the next generation. We have now shown that such induced aneuploidy is indeed transmitted and can be detected in a monosomic and trisomic condition in F1 fetuses on day 9.5 of gestation.

H-Y antigen was determined in seven XO-, nine XO/XX patients, in one patient with i(Xq), and in one patient with a mosaic XO/XYqh-. It turned out that all patients are H-Y antigen positive, confirming the results of earlier investigations of H-Y antigen in patients with Turner's syndrome. The results in XO/XX mosaics clearly demonstrate that the XO-cell is H-Y antigen positive and support the view of a regulatory gene for H-Y antigen gene expression which is located on the X chromosome.

Adult female mice of the "sensitive" NMRI/Han strain ovulate diploid oocytes after gonadotropin treatment. Other mouse strains are "non-sensitive" with respect to the ovulation of such diploid oocytes. In this study we combined the impaired ovarian situation in the XO karyotype with the trait "diploidy", which is determined genetically, by mating Ta/O (Ta = Tabby) females of C3H X 101 background to males of the NMRI/Han strain. The adult female F1 hybrids were stimulated to ovulation by gonadotropins and identified by their karyotype (XX or XO). The cytogenetic analysis of ovulated oocytes revealed a low level of diploidy in the XX littermates (1.0%), but a very high level in females with the XO karyotype (24.6%). All of the XO females ovulated at least one diploid oocyte. We suggest that it is the XO status which drastically impairs meiosis I in our "gonadotropin-sensitive" F1 females due to (1) alterations of the developmental program within the oocyte, (2) a disturbed communication between oocyte and follicle, (3) a preferential maturation and ovulation of "follicles at risk", or (4) an exceptional recruitment of many such follicles, by, e.g., a premature responsiveness to gonadotropins in our XO females. An interdependence of several such mechanisms is possible.

Two carriers of a balanced translocation, one paternal t(9q-;13p+) and one maternal t(3q-;15p+), were studied cytogenetically with selective silver staining to localize more precisely the break points on the short arms of the two acrocentric chromosomes involved. The distal ends of both chromosomes 3 and 9 reacted positively to the selective silver staining. This observation allows the conclusion that the NOR (nucleolar organizer region), or parts of it, is in fact active in organizing nucleoli and by implication produces rRNA after the translocation onto chromosomes 3 and 9.

A case of a prenatal diagnosis at the second trimester is presented showing a normal karyotype in 12 metaphases from chorionic villi. In all cultured amniotic cells, however, and also in all fetal fibroblasts analyzed after abortion a structural anomaly (46,XY;del 4(pter----p15.2) was detected. Prenatal diagnosis was performed because of intrauterine growth retardation, cleft lip and esophagus atresia by ultrasound. The fetal stigmata are compatible with the Wolf Hirschhorn syndrome. We conclude that amniocentesis may be indicated notwithstanding a normal CV-diagnosis in those rare pregnancies with a characteristically abnormal ultrasound.

Cytogenetic studies were performed on 136 couples with a history of two or more abortions referred to us after gynaecological causes of the abortions had been excluded. Fifteen (11%) of the couples were found to have a chromosome anomaly, and when the couples were subdivided according to number of abortions, surprisingly 6 (10%) of the 59 couples with a history of only two abortions had a chromosome anomaly. An increased frequency of mosaicism for X-chromosome aneuploidy (2.2%) in the women from the 136 couples was also found. A review of the literature shows that translocations of some chromosomes (e.g. nos. 1, 7 or 22) preferentially lead to fetal wastage, while those involving, for example, chromosome nos. 5, 9, 14 or 21 are more likely to result in the birth of a handicapped child. Couples with a history of two abortions should be investigated cytogenetically. Other causes of miscarriages must, however, be excluded first.

Ten new cases of accessory bisatellited marker chromosomes examined in different laboratories are reported. As a basis for genetic counselling in the context of prenatal diagnosis a cytogenetic categorization of such marker chromosomes is proposed and an estimation of the genetic risk associated with each category is carried out. The results are as follows: 1. There is no increased risk for offspring with abnormal phenotype born to a healthy carrier of an accessory bisatellited marker chromosome with either a single or two closely adjacent C-bands (Category AI or AII). 2. The unbiased sample of cases with de novo accessory bisatellited marker chromosomes of categories AI and AII is too small to allow a satisfactory estimation of the actual risk that, in case of such a prenatal finding, the foetus may not show a normal phenotype as a consequence of the marker chromosome. There is, however, evidence that this risk may be lower than 10%. 3. Accessory bisatellited marker chromosomes showing a discrete pattern of G- or R-bands situated between two distant C-bands (Category AIII) usually indicate a chromosomal imbalance giving rise to an abnormal phenotype. Mosaic carriers of such dicentric marker chromosomes may, however, present a normal phenotype.

Oocytes from parental mice strains NMRI/Han, C57/bl and Balb/c and from F1 hybrid lines were analysed for aneuploidy due to non-disjunction after gonadotropin-stimulated ovulation. No hyperploid oocytes were present in five of the strains studied. F1 hybrids from crosses of NMRI/HanxC57/bl did ovulate, however, a significantly increased number of hyperploid oocytes, although females from their parental strains show a rather low incidence of non-disjunction. The evidence for a genetic basis for non-disjunction is assessed and possible causative factors are discussed.

NMRI mouse and Djungarian hamster females ovulate diploid and/or hyperploid oocytes with increased frequencies after gonadotrophin stimulation, suggesting that somatic cells are involved in the failures of endocrine control resulting in aneuploidy. To study the inheritance of gonadotrophin-induced aneuploidy as well as the fate of sensitive oocytes in a resistant somatic environment and vice versa, we analysed the frequency of diploid oocytes in NMRI/Han, C57BL/6J and their F1 hybrids (C57BL/6J X NMRI/Han), (NMRI/Han X C57BL/6J) as well as in NMRI/Han in equilibrium C57BL/6J chimeric females after gonadotrophin injections. Ovulated oocytes were analysed in all females for the appearance of diploidy, characterized as premature arrest of development at metaphase I. Our data suggest that the trait of induced diploidy is genetically determined and can be transmitted either maternally or paternally. A maternal effect modulated the expression of that trait. Several mechanisms acting on the feed-back control ovary-hypothalamus/pituitary, within the ovary or even within a chimeric follicle, may be responsible that 'sensitive' oocytes ovulated from chimeras are all normal haploid. These data suggest that not only oocyte maturation but also chromosome disjunction during meiosis I is controlled by somatic cells.

The effect of maternal ageing on the meiotic rate, on chiasma and univalent frequency as well as on heteroploidy in secondary oocytes from Djungarian hamsters was examined. The frequency of hyperhaploid oocytes increased from 0.6% in young (8-14 weeks) to 2.8% in middle-aged (26-46 weeks) and reached 3.6% in the oldest females (49-75 weeks). On the basis of malsegregated bivalents per oocyte, nondisjunction occurred most often in the middle-aged group (5.42 X 10(-2) bivalents per oocyte). Hereby, the large meta- and submetacentric A-D chromosomes were preferentially involved. Furthermore, the pattern of nondisjunction was not different from that expected on the basis of chromosome length or induced by colchicine. The large A-D chromosomes did not show any alteration in chiasma or univalent frequency. Terminalized chiasmata were only detected in the E group and univalents increased slightly, but not significantly in the small chromosomes (G group). At higher ages, both chromosome groups were not preferentially involved in nondisjunction. Presegregation slightly increased with age and affected more or less all bivalents, whereas the incidence of diploidy significantly decreased. With respect to the rate of meiosis in oocytes from aged females, the resumption was delayed at metaphase I. Our data suggest that failures in the control of oocyte proliferation are involved in nondisjunction rather than the "production-line." Furthermore, a model is proposed to explain nondisjunction of specific bivalents at certain maternal ages.

We report a case of an X-autosome translocation t(X;4)(q13;p16) found in both sexes in three generations. The anomaly was diagnosed in a couple referred for cytogenetic investigation as a result of three spontaneous abortions. With the exception of the miscarriages there are no particularities in the gynecologic data of the woman or in the pedigree. In all 50 lymphocytes and in 66 of 68 fibroblasts investigated the normal X chromosome was the late replicating one.

Chromosomal mosaicism confined to the placenta is a serious problem in first-trimester fetal diagnosis. We report a case of mosaicism of trisomy 7. The aneuploid cell line could not be confirmed in fetal tissue.

The differential staining methods for chromosomes have led to the demonstration of more chromosomal polymorphisms. Not rarely, these polymorphisms allow in autosomal trisomies the detection of parental origin of the supernumerary chromosome. In addition, the malsegregation may be ascribed to 1st or 2nd meiotic division in informative families. This approach of analyzing possible causes of trisomies is subject to a considerable bias. Trisomic phenotypes are twice as frequent for 2nd meiotic errors than for 1st meiotic errors. Also, rare chromosome variants seldom occur in matings where malsegregation in 1st meiotic division can be detected. In the present paper this bias is analyzed mathematically on the family as well as on the population level. From this mathematical analysis and from the data in the literature we conclude that Down's syndrome as a whole is caused about 5–10 times more often by a malsegregation in 1st meiotic than by an error in 2nd meiotic division. Mainly from experimental studies in rodents, causes for errors in 1st and 2nd meiotic division are becoming apparent. They are summarized in the context of the results of the present paper.

To evaluate the significance of placental histology, a collaborative histological and cytogenetic study on the products of 123 spontaneous abortions of 6-19 week pregnancies was performed. From each sample, 2-3 g was dissected randomly and analysed histologically with no prior knowledge of the karyotype. Chromosomes were prepared from the remaining chorionic villi after an overnight incubation in culture medium; they were analysed by Q- or G-banding. The frequency and type of chromosome anomalies detected are comparable to those seen in other studies. Altogether, 49.6% were chromosomally abnormal, trisomies predominating (49.2%), followed by polyploidy (27.9%) and monosomy X (14.5%). The histological classification failed for technical reasons in 11 cases (8.9%). To classify the remaining cases, 18 different histological criteria were considered independently by two pathologists. A definite diagnosis was not possible for 27 abortions (22.0%), and 50 were classified as being chromosomally abnormal. This proved to be correct in 36 (72.0%), but incorrect in as many as 14 (28.0%). No evidence for an abnormal karyotype was found histologically in samples from 35 abortuses. Nevertheless, 9 of them (25.7%) had an abnormal karyotype. The predictive value of chorionic villus histology seems to be inadequate, as only 62 of 112 samples were correctly classified by histology (55.4%). The value differs little from the a priori probability of an abnormal or normal karyotype in abortuses of corresponding gestational ages.

Cytogenetic studies of early human abortuses revealed not only information of clinical importance to the respective couple, but also provided basic data, e.g., on the frequency of chromosome anomalies in various age and ethnic groups (for review see Sankaranarayanan 1982; Bou6 et al. 1975; Hassold and Chiu 1985; Hassold and Jacobs 1984), on their recurrence risk (Hassold 1980, 1985), and on the effect of preconceptional exposure to various agents (Yamamoto et al. 1982). In connection with the analysis of chromosome--and recently also DNA-polymorphisms (Hassold et al. 1985), information on the origin of the supernumerary chromosome as well as on the stage of meiotic failure in about 400 aneuploid abortuses was obtained. In the past, however, analysis of such early abortuses required long-term cell cultures and since these are rather laborious and costly, not many laboratories were involved. With the recently developed method of direct preparation of metaphases from chorionic villi (Simoni et al. 1983), chromosomes from early abortuses may now be analyzed more quickly and with the aid of various banding techniques. To date we have avalysed 34 spontaneous abortuses by direct preparation or overnight-culture of chorionic villi and obtained metaphases sufficient in number and quality in 28 cases. In four of these six abortuses the failure was most likely due to the long time interval between abortion and setting up villi culture (two cases) or due to the late gestational age (one case at the 17th week). In one case no villi could be obtained. Fifty percent (14 cases) of the remaining 28 abortuses were chromosomally abnormal: one 45,XO, nine trisomies (3, 13 (three cases), 16 (two cases), 22 (three cases)), one triploidy, one 2n/4n mosaic, and two structural chromosome anomalies (one 13/14 Robertsonian translocation, one unbalanced der(5)pat due to a paternal 5/22 reciprocal translocation). This approach of chromosome analysis from direct preparations of chorionic villi appears to be very useful in collecting information on various aspects of aneuploidy in man. Early abortions can be studied now on a large scale and, impor

Ovulated mouse oocytes and preimplantation embryos were examined for NOR activity by means of selective silver staining. Evidence of the first staining activity appeared in two cell embryos, which was later followed by an increase in nucleolar activity, whereas the ovulated oocytes and pronuclei showed no such activity whatsoever. The staining of chromosomes was restricted to the nucleolus organizing region. Our results agree with earlier observations that genes for ribosomal RNA (rRNA) are transcribed as early as in the 2-cell stage in mouse embryogenesis. In addition to the nuclear staining we also observed some silver staining within the cytoplasm, at least from 4-cell stages onwards. Cytoplasmic staining was resistant to incubation with cycloheximide and actinomycin D. Nuclear staining was depressed, or even totally blocked, after actinomycin D incubation but was not blocked by cycloheximide. The onset of silver staining depends not on a specific embryonic stage but on the time interval following ovulation. This appears to indicate that the initiation of ribosomal cistrons is regulated by molecules which are activated or synthesized within the oocyte soon after ovulation.

A case of a false-negative first-trimester diagnosis following chorionic villus sampling is reported that ended with the birth of a child with Down syndrome. Chromosome analysis of 30 metaphases from 24 h-cultured chorionic villi obtained in week 12 of gestation showed a normal chromosome constitution. However, the newborn showed manifestations of Down syndrome, and 99 of 100 metaphases analysed from cultured lymphocytes showed 47, XY, + 21. The remaining metaphase was normal (46, XY).

The ovulation of diploid oocytes, abnormally arrested at or during the first meiotic division, is an inheritable trait (DiplI) in mice and modulated by a maternally transmitted factor. By repeated backcrossing, mouse strains with identical nuclear encoded genes and differing only in their mitochondrial genomes can be created. NMB mice represent such a strain having acquired the nuclear genome of C57BL/6J but still possessing mitochondria and therewith mitochondrial DNA (mtDNA) of NMRI/Bom, their female progenitor. The strains NMB and C57BL/6J were used to characterize a new mitochondrial trait, namely the ability to modulate the expression of the inheritable meiosis I error Dipl I in oocytes. We show that an increased rate of ovulated diploid oocytes is associated with the mtDNA type of C57BL/6J. These results corroborate the assumption that mitochondria do play an important role in meiosis of mammalian oocytes and hence seem to be involved also in the orderly segregation of chromosomes.

The influence of age and hormones on chromosome segregation at meiosis I was studied in female mice heterozygous for the T(2;4)1Go translocation. Females of two age groups (18-22 and 40-56 weeks old) were stimulated for ovulation with different doses of gonadotropins (1.5 IU PMS/1.0 IU HCG or 10 IU PMS/10 IU HCG). Analysis of metaphase II oocytes revealed the highest level of hyperhaploidy (1.8%) and presegregation (4.4%) in the young females receiving the low dose. Presegregation preferentially affected the small 4(2) marker chromosome. There was no significant interference of the tetravalent with disjunction of the nontranslocated normal bivalents. Moreover, no remarkable difference in the mode of segregation (adjacent I, II or alternate) was observed. Recombination within the interstitial pairing segments of the chromosomes involved in the translocation allowed us to calculate cross-over frequencies in ovulated oocytes. For both the large 2(4) and the small 4(2) marker chromosomes, this frequency was higher in old than in young T(2;4)1Go/+ females. Our data do not support the production line hypothesis of Henderson and Edwards (1968) which claims that chiasma frequency in oocytes decreases with maternal age.

ALTHOUGH many unfertilised mouse oocytes have been variously stimulated to develop into embryos—parthenogenones—all have died soon after implantation1. The suggested causes of this failure include homozygosity of recessive lethals and an incomplete cortical reaction1. It is not known whether the lack of the paternally-derived genome inhibits their proper development. Using selective silver staining2,3 we have examined the activity of the genes for 18S and 28S ribosomal RNA during preimplantation development of parthenogenones. From studies of the activity of nucleolus organisers on the chromosomes of human–mouse cell hybrids, it has been assumed that silver precipitation was a consequence of gene activity for 18S and 28S rRNA (refs 4–6). Our results indicate that these genes are activated during early embryogenesis and without the need for paternally-derived factors.

Cytogenetic investigations were carried out on 103 women presenting with either primary (n = 88) or secondary (n = 15) amenorrhea. A sex chromosome anomaly was found in 26% and 33% of these patients, respectively. Other studies on women with primary amenorrhea have found a similar or even higher percentage of patients with an abnormal karyotype. It is therefore suggested that all women with absence of menstruation after the age of 16 years should be investigated cytogenetically. The surprisingly high percentage of pathological karyotypes among the secondary amenorrhea group does indicate that sex chromosome anomalies cannot be ruled out in women who have had apparently normal ovarian function for at least some time, and therefore more patients from this group should be selected for chromosome analysis.