Increased nuchal translucency, hydrops fetalis or hygroma colli. A new test strategy for early fetal aneuploidy detection.

OBJECTIVES: Nuchal translucency measurement of 3 mm or more (> or = 95th centile for gestation age), hydrops fetalis or hygroma colli between the 11th and 14th weeks of gestation is associated with a higher risk of fetal Down syndrome and other aneuploidies. So far, chromosome preparation of chorionic villi samplings (CVS) after short-term (or direct) culture is the only valid, reliable and rapid method of choice for the early detection of chromosomal aberrations. However, because of the placental mosaicisms detected after short-term culture, CVS has to be confirmed by a second method. Moreover, short-term villi preparation does not always provide a sufficient quantity and quality of metaphases to enable cytogenetic analysis. Unfortunately, a predicative cytogenetic result will be available only after long-term cultivation (usually after 1-2 weeks). An alternative rapid method, inexpensive and suitable for diagnosing autosomal trisomies, is the quantitative fluorescence polymerase reaction (QF-PCR) using different polymorphic small tandem repeats (STRs) on CVS-DNA. Therefore, it was the aim of the study to evaluate whether a new CVS test strategy could be employed in early pregnancies at high risk after the rapid detection of fetal chromosomal abnormalities by QF-PCR for chromosomes 13, 18 or 21 and sexing in conjunction with short-term chromosome analysis. MATERIALS: Nineteen CVS were chosen for QF-PCR detection of trisomy 21, 18 or 13 after an increased nuchal translucency measurement (> or = 95th centile for gestation age), a hydrops fetalis or a hygroma colli. The amelogenin locus of chromosomes X and Y (AMXY) were used for sexing. The QF-PCR results were compared with routine karyotyping after short- and/or long-term cultivation of CVS cells. RESULTS: An informative result was demonstrated in all analysed specimens. Nine CVS were diagnosed as a QF-PCR trisomy either for chromosome 21, 18 and 13. The pathological samples also included 4 cases of mosaicism where the normal cell line was not identified by QF-PCR. In 1 additional case with a normal QF-PCR result, short-term CVS chromosome analysis showed a mosaic trisomy 13, whereas longterm CVS culture revealed a normal karyotype. The malformed aborted fetus showed no clinical signs of trisomy 13, confirming the normal results obtained by QF-PCR and long-term CVS chromosome analysis. One pregnancy with a Turner syndrome was not identified by molecular analysis. CONCLUSIONS: This study showed that all early pregnancies with a clinically relevant autosomal trisomy could be detected prenatally in routine practice by QF-PCR. The combined use of both rapid methods - QF-PCR and short-term chromosome analysis - optimise the results by minimising the possibility of false-positive or false-negative findings. We believe that after verification of a pathological result obtained by two independent methods (QF-PCR and short-term CVS chromosome analysis), long-term villi cultivation is no longer necessary. However, in all cases with discrepancies, especially in samples with mosaic findings at short-term CVS cultivation, further studies are still necessary.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by QF-PCR in 662 selected pregnancies at risk.

A quantitative fluorescent-polymerase chain reaction (QF-PCR) test system with different short tandem repeat (STR) markers of the X chromosome (SBMA, DXS8377 and DXS1283E) together with the amelogenin locus (AMXY) was developed for the rapid detection of sex chromosome aneuploidies on uncultured amniotic fluids. The samples (n = 662) were also tested with STRs specific for chromosomes 13, 18 or 21, with two STRs used for each chromosome. In uninformative cases, an additional STR marker was applied. The QF-PCR data were compared with the results of conventional cytogenetics. One dark red stained specimen showed an artificial PCR pattern, probably due to maternal contamination. Six sex chromosome aberrations (four 45,X, one 47,XXY, one mosaic 47,XXY/46,XX) were identified as aneuploid by STRs specific for chromosome X and AMXY. One pregnancy with a mosaic 45, X/46,XX karyotype was not detected by the assay. In all, 12 cases with a numerical aberration involving either chromosome 18 or 21 or with a triploidy were correctly diagnosed by QF-PCR. No information was obtained in one fetal sample with a trisomy 18 due to an uncertain result for two of the three applied STRs specific for chromosome 18 and an uninformative third STR marker. Two samples with an unbalanced Robertsonian translocation could be identified by QF-PCR as trisomic for chromosomes 13 and 21 respectively. The results show an excellent agreement between QF-PCR and cytogenetics with regard to sex chromosome and autosomal aneuploidy detection in prenatal diagnosis.

Presence of the AZF region in a female with an idic(Y)(q11).

In a child with some features of Turner's syndrome, gonosomal mosaicism with an isodicentric nonfluorescent (idic)Y chromosome was detected (mos 45,X/47,X,idic(Y)(q11),idic(Y)(11)/46,X,idic(Y)(q11)). Histopathological examination showed streak gonads with some evidence of ovarian stroma and no sign of gonadoblastoma. Polymerase chain reaction (PCR) analysis in blood lymphocytes and gonadal tissues using primers of seven loci along the Y chromosome, including the sex determined region (SRY), azoospermia factor region (AZF) and the deleted in azoospermia (DAZ) gene was positive for all loci tested, confirming the isodicentric character of the Y chromosome and indicating the presence of the AZF region. It is remarkable that the existence of spermatogenesis controlling genes does not play an important role in gonadal development and differentiation in a phenotypic female with some Turner stigmata. The data presented here are briefly discussed with previously-described patients.

Isochromosome Xq in Klinefelter syndrome: report of 7 new cases.

In this collaborative study we report on 2 prenatally and 5 postnatally diagnosed cases with a 47,X,i(Xq),Y chromosomal constitution. Excepting tall stature, the 5 adult patients showed all typical manifestations of Klinefelter syndrome. Taken together with previously reported cases, these data suggest that Klinefelter syndrome with isochromosome Xq has a favorable prognosis with normal mental development, and with normal-to-short stature. The prevalence of this Klinefelter variant is calculated to be between 0.3-0.9% in males with X chromosome polysomies.

Ullrich-Turner syndrome is not caused by haploinsufficiency of RPS4X.

Ullrich-Turner syndrome (UTS) is frequently associated with monosomy X but may also occur with structural aberrations of the X and the Y chromosomes. It has been hypothesized that the ribosomal protein genes RPS4X and RPS4Y play a critical role in the prevention of UTS. Individual patients with a 46,X,i(Xq) karyotype cannot be differentiated phenotypically from 45,X UTS patients and carry three gene copies of RPS4X. Since haploinsufficiency of one or several gene(s) is thought to cause the UTS phenotype, direct assessment of RPS4X expression levels in these patients should establish whether RPS4X is involved in UTS. We have investigated fibroblasts of four 46,X,i(Xq) UTS patients with typical symptoms and a non-mosaic chromosome complement, and have found significantly increased RPS4X mRNA levels in all patients. Based on our results, we conclude that haploinsufficiency of RPS4X is not the cause of UTS.

Assignment of an autosomal sex reversal locus (SRA1) and campomelic dysplasia (CMPD1) to 17q24.3-q25.1.

We have mapped the autosomal sex reversal locus, SRA1, associated with campomelic dysplasia (CMPD1) to 17q24.3-q25.1 by three independent apparently balanced de novo reciprocal translocations. Chromosome painting indicates that the translocated segment of 17q involves about 15% of chromosome 17 in all three translocations, corresponding to a breakpoint at the interphase between 17q24-q25. All three 17q breakpoints were localized distal to the growth hormone locus (GH), and proximal to thymidine kinase (TK1). Due to the distal location of the breakpoints, previously mentioned candidate genes, HOX2 and COL1A1, can be excluded as being involved in CMPD1/SRA1. The mouse mutant tail-short (Ts) which maps to the homologous syntenic region on mouse chromosome 11, displays some of the features of CMPD1.

Early genetic amniocentesis--4 years' experience.

Four hundred and thirty early amniocenteses (EAC) from 10 to 14 weeks' gestation were compared with 300 routine amniocenteses (RAC) from 15 weeks' gestation (control A) and 733 routine amniocenteses from 16 to 18 weeks' gestation (control B) with regard to success rates, various growth parameters, and cytogenetic results. Using both in situ and trypsinization techniques, the success rate was 99.8 per cent for EAC versus 100 per cent for RAC. The average turn-around time for establishing a diagnosis was 8.4 days in EAC versus 8.3 days in 15 weeks' specimens (n.s.) and 7.7 days in 16 to 18 weeks' specimens (p < 0.0001) for the last 200 samples. The banding quality of early specimens compared favourably with that of controls (both 500-550 bphs) and was much better than that in long-term cultured chorionic villus sampling (CVS) (350-400 bphs). For level I and level II mosaicism, no statistically significant differences were noted between EAC and control group A. Comparing EAC with control group B, a significant increase in the number of numerical and structural single cell aberrations was observed (p < 0.025 and p << 0.001, respectively), whereas for multiple cell aberrations only the increase in numerical aberrations was statistically significant (p << 0.001) (chi 2-test). Clinical problems arising from the detection of mosaicism were solved in all cases by investigating parallel cultures. It is concluded that early amniocentesis is a reliable procedure which permits prenatal diagnosis of numerical and structural chromosome aberrations to a high standard.

Atypical chronic myelogenous leukemia in a patient with trisomy 8 mosaicism syndrome.

A 17-year-old woman was admitted for bone marrow transplantation with the diagnosis of atypical Philadelphia-negative chronic myelogenous leukemia (aCML), cytogenetically characterized by trisomy 8 as the sole chromosome aberration. A striking feature was a congenital opacity of the right cornea. Chromosomal analysis of skin fibroblasts were performed and revealed a mosaic for trisomy 8. Commonly, a distinct clinical picture leads to the diagnosis of trisomy 8 mosaicism syndrome (T8ms), but an extreme phenotypic variability has been observed. To our knowledge the development of an aCML in a patient with T8ms has not been reported. A review of the literature revealed that an association to other hematological disorders had been described in two cases. The question of whether our patient's aCML was a random event or not is discussed. The patient is now 24 months post transplant and shows no evidence of disease. Her Karnofsky score is 100%. We conclude that it might be worthwhile to look for an associated constitutional trisomy 8 mosaicism in all patients with trisomy 8 leukemia.

Expression of RPS4X in fibroblasts from patients with structural aberrations of the X chromosome.

A series of fibroblasts from patients with numerical or structural aberrations of the X chromosome were scored for the amount of mRNA of ribosomal protein S4 (RPS4X). Haplo-insufficiency of this gene has been reported previously to be a possible cause of Turner syndrome. Our results show that the transcription rate of RPS4X correlates with the number of gene copies. This confirms earlier findings indicating that this gene escapes X inactivation. In addition, we demonstrate that this applies to structurally aberrant X chromosomes. Our results show that RPS4X does not give rise to a type of haplo-insufficiency in these cases, because it escapes inactivation, even on structurally aberrant X chromosomes from patients with Turner syndrome. We therefore assume that RPS4X is not the most prominent candidate gene for Turner syndrome.

Mosaicism in 45,X Turner syndrome: does survival in early pregnancy depend on the presence of two sex chromosomes?

Cytogenetic and molecular genetic findings in 91 patients with Turner syndrome are reported. In 87 patients, chromosome studies were carried out both in lymphocyte and fibroblast cultures. Mosaicism was demonstrated in 58 of these patients (66.7%), whereas only 18 (20.7%) were apparent non-mosaic 45,X, and 11 patients (12.6%) showed non-mosaic structural aberrations of the X chromosome. Among the mosaic cases 16 (18.4% of all patients) displayed a second cell line containing small marker chromosomes. The association of Y-specific chromosomal material with the presence of marker chromosomes was demonstrated in 6 out of 7 mixoploid fibroblast cell lines by polymerase chain reaction amplification and by Southern-blot analysis. The observation of ring formation and morphological variability in vivo and in vitro, and the continuous reduction in the percentage of cells containing marker chromosomes in longterm cultivation experiments indicated an increased instability of marker chromosomes. The findings suggest that in vivo selection of structurally altered sex chromosomes exists. Thus, the observation of apparent non-mosaic 45,X chromosomal complements in liveborn individuals with Turner syndrome does not contradict the hypothesis that some degree of mosaicism is necessary for survival in early pregnancy.

Chromosomal mosaicism in prenatal diagnosis: a problem still unsolved.

The problems in differentiating chromosomal mosaicism from pseudomosaicism after amniocentesis and CVS are demonstrated in 6 cases. Two cases of true mosaicism (45, X/46, XX and 46, XY/47, XXY) were of clinical relevance. In both cases the aberrant cell line was less expressed in amniotic fluid cells than in fetal blood and cultivated fibroblasts. Two cases of pseudomosaicism (chromosome 2 and chromosome 10 trisomy) originated either from preexisting mutants or from in vitro mutations whereas a case of true chromosome 20 mosaicism indicated the possibility of a mosaic confined to a single fetal tissue. The problems of interpreting mosaicism after CVS is illustrated in a 45, X/46, XY case, in which the abnormal cell line was detectable only in extrembryonic tissue.

[Early amniocentesis for cytogenetic diagnosis]. / Frühzeitige Amniozentese zur zytogenetischen Diagnostik.

This study was designed to assess the feasibility of amniocentesis and amnion cell culture for prenatal diagnosis in early weeks of gestation (less than 15 weeks). Within a period of 18 months (1/88-6/89) 135 diagnostic amniocenteses were performed between 10 and 14 weeks and amniotic fluid was obtained in all instances. In all cases but one, sufficient cell cultures and chromosomal analyses were achieved. In the follow up, one miscarriage (0.7%) occurred, in two cases transient amniotic fluid leakage ceased spontaneously. All of the aforementioned complications were related to amniocenteses in weeks 11 and 12. The course of the continued pregnancies as well as fetal outcome were without any further complications. Comparing specimens of early and regular (16-18 weeks) amniocenteses in terms of time required for cell culture and karyotyping as well as the quality of chromosomal banding no significant differences were found. In contrast to maternal serum AFP which increased continuously, amniotic AFP levels could be seen to increase to a peak at 13 weeks' gestation but afterwards gradually declined. Based on these first experiences and results of early amniocenteses the specific problems of this method are discussed. In summary, it is concluded, that early amniocentesis between 12 and 14 weeks of gestation is feasible with regard to both the technique of amniotic fluid retrieval and the technique of chromosomal analysis. Early amniocentesis, therefore, could fill up the diagnostic gap between chorionic villi sampling (CVS) between 9 and 11 weeks of gestation and "classical" amniocentesis during weeks 16-18.

Clinical pattern and the genetics of the fetal iodine deficiency disorder (endemic cretinism): results of a field study in Highland Ecuador.

The clinical manifestations of various degrees of mental retardation, spastic diplegia, and deaf mutism are known as the neurologic type of endemic cretinism (EC), occurring in countries with high goiter endemicity. Maternal iodine deficiency has been established as the major cause in EC, whereas a genetic predisposition has not been well-documented. Genetic data on 70 families with EC from Highland Ecuador are reported. A segregation analysis of 49 fully classified families yielded an estimate of P = 0.245 (var [P] = 0.00167). Half-sibs were all unaffected and no significant birth order effect was observed among 101 probands. The data indicate an autosomal recessive predisposition as a major etiological factor. Because the neurologic type of EC represents a defined section of the spectrum of iodine deficiency disorders (IDD), the term fetal iodine deficiency disorder (FIDD) rather than cretinism is suggested. The clinical findings in 70 patients were used to delineate the minimal diagnostic criteria of FIDD.

Clonal derivatives of a human B-lymphoblastoid cell line producing prolactin--a cytogenetic characterization.

The IM-9-P cell line is a variant of the human B-lymphoblastoid cell line IM-9 which ectopically secretes prolactin (hPRL). The heterogeneous line IM-9-P and three sublines of clonal origin, two of them positive and one negative for PRL gene expression, were subjected to cytogenetic analysis and compared with the reference line IM-9 which showed a normal female diploid karyotype. G-banding revealed several rearrangements in the chromosomes. Nine altered chromosomes including one stable marker chromosome were common to all analysed karyotypes of IM-9-P cells and their clones. A second marker chromosome 'mar2' occurred only in the karyotypes of the hPRL producing clones, but not in the non-producing clone. None of the visible alterations involve chromosome 6 which carries the PRL gene in humans.

Dermatoglyphics in congenital adrenal hyperplasia (CAH).

Dermatoglyphic findings were compared in 42 patients (32 females, 10 males) with Congenital Adrenal Hyperplasia (CAH) and 110 normal controls (70 females, 40 males). In CAH males, an excess of whorls (p less than 0.001), an increased total finger ridge count (p less than 0.05), and an increased frequency of patterns in the fourth interdigital area (p less than 0.025) was found. A main line A terminating high in the hypothenar area (p less than 0.05), and a missing c-triradius or an abortive main line C (p less than 0.05) was observed in CAH females. Both sexes displayed an increase in the frequency of small radially directed hypothenar patterns (p less than 0.05) and sydney lines (p less than 0.01).

A case of lipogranulomatosis Farber: some clinical and ultrastructural aspects.

A 20-month-old girl showed typical clinical signs of Farber disease: hoarseness since birth, and periarticular subcutaneous painful nodules. Complete deficiency of acid ceramidase activity was found in cultured skin fibroblasts. An electron microscopic examination of a dermal nodule disclosed pathognomonic tubular inclusions in histiocytes. In epidermal cells zebra-body-like and needle-like lysosomal inclusions were found. Their ultrastructure is different from that of the intrahistiocytic lysosomal inclusions. Probably three clinical types of Farber disease may be distinguished according to the symptomatology and the course of the disease: a severe type, an intermediate type and a relatively mild type. The activity of acid ceramidase does not correlate with prognosis of the disease, while a correlation between first appearance of dermal nodules and clinical course appears likely.