The role of molecular microsatellite identity testing to detect sampling errors in prenatal diagnosis.

OBJECTIVE: The objective was to determine the risk of sampling error in amniocentesis and chorionic villus sampling (CVS) in singleton and multiple pregnancies. Data from this and other published studies were used to discuss current practice guidelines for molecular identity testing. METHOD: Clinical and laboratory records of all patients undergoing molecular-based identity testing in our clinical laboratory from July 2002 until March 2008 were reviewed. DNA microsatellite testing was performed to determine zygosity in multiple pregnancies and maternal cell contamination (MCC) in both singleton and multiple pregnancies. RESULTS: MCC was detected in 6/148 (4%) CVS and 1/87 (1%) amniotic fluids from singleton pregnancies. In two of the CVS, only maternal cells were found. In 2/24 (8%) twin pregnancies, the same fetus was tested twice. In a total of 285 pregnancies (235 singleton, 24 twin, 26 with >or= 3 fetuses), without molecular identity testing, four women would have received erroneous results. CONCLUSION: Current guidelines recommend molecular identity testing for MCC in conjunction with molecular diagnostic testing, but not for cytogenetic testing. No published guidelines were found for zygosity testing in multiple pregnancies. We suggest that identity testing be considered for all prenatal testing of multiple pregnancies, especially if CVS is performed.

Extracardiac lesions and chromosomal abnormalities associated with major fetal heart defects: comparison of intrauterine, postnatal and postmortem diagnoses.

OBJECTIVES: The clinical outcome of prenatally diagnosed congenital heart defects (CHD) continues to be affected significantly by associated extracardiac and chromosomal abnormalities. We sought to: determine the frequency and type of major extracardiac abnormalities (with impact on quality of life) and chromosomal abnormalities associated with fetal CHD; and compare the extracardiac abnormalities detected prenatally to the postnatal and autopsy findings in affected fetuses, to find the incidence of extracardiac abnormalities missed on prenatal ultrasound. METHODS: We reviewed the computerized database of the Division of Cardiology of the Hospital for Sick Children in Toronto to identify all cases of major CHD detected prenatally from 1990 to 2002. Medical records, fetal echocardiograms and ultrasound, cytogenetic and autopsy reports were reviewed. The types of CHD detected were grouped into categories and the frequencies of major extracardiac and chromosomal abnormalities in these categories were noted. Prenatal ultrasound findings were compared with those at autopsy or postnatal examination. RESULTS: Of 491 fetuses with major structural CHD, complete data were obtained for 382. Of these, there were 141 (36.9%) with major extracardiac abnormalities at autopsy or postnatal exam, of which 46 had chromosomal abnormalities and 95 did not. In the absence of chromosomal abnormalities, the organ systems most affected were urogenital (12.2%) and gastrointestinal (11.6%). CHDs with the highest incidence of extracardiac abnormalities (>25%) included: heterotaxy, single left ventricle and tricuspid atresia, hypoplastic left heart syndrome and tetralogy of Fallot. Ninety-four of 334 (28.1%) fetuses tested had chromosomal abnormalities. The most common chromosomal abnormalities were trisomies 21 (43.6%), 18 (19.1%) and 13 (9.6%), monosomy X (7.4%) and 22q11.2 deletion (7.4%). Of 289 extracardiac abnormalities from the complete series, 134 (46.4%) were not identified prenatally. Of the missed extracardiac abnormalities, 65 were considered not detectable at prenatal ultrasound, so 23.9% (69/289) of detectable extracardiac abnormalities were missed prenatally. CONCLUSIONS: Major extracardiac and chromosomal abnormalities are common in fetuses with major fetal CHD. Many important associated extracardiac abnormalities may be missed prenatally, which should be taken into consideration in the prenatal counseling for fetal CHD.

Isodicentric Yp: prenatal diagnosis and outcome in 12 cases.

OBJECTIVES: 1. To present the prenatal cytogenetic findings and postnatal outcome of 12 cases with an isodicentric chromosome composed of the short arm of the Y chromosome.2. To review the literature and provide recommendations for cytogenetic analysis and counseling. METHODS: Prenatal and postnatal cytogenetic data and clinical findings of isodicentric Yp ascertained in six institutions were gathered and reviewed. RESULTS: Nine of the twelve cases were referred for advanced maternal age (AMA), one of which was a twin pregnancy with one twin having an increased nuchal translucency measurement. The remaining cases were referred owing to a family history of hemophilia and an abnormal maternal serum screen, respectively. Nine of these pregnancies resulted in the birth of a normal-appearing male infant with subsequent normal growth and psychomotor development. Follow-up ranged from birth to 7 years. In two cases, the pregnancy was terminated and the fetuses showed male external genitalia. In the case ascertained because of an increased nuchal translucency measurement, the prenatal diagnosis of 45,X was made. At birth, there were ambiguous genitalia, and postnatal cytogenetic studies found an isodicentric Yp. In 11 of the 12 cases, mosaicism was present. CONCLUSION: Our cases show that the prenatal finding of an isodicentric Yp, with or without 45,X mosaicism, is compatible with normal male phenotype in most cases, particularly in the absence of other anomalies. To ensure accuracy in cytogenetic reporting and prenatal counseling, the identification of a structurally abnormal or small Y chromosome, either alone or in the presence of 45,X colonies, should be followed immediately by confirmatory molecular cytogenetic investigations as well as by ultrasound determination of the phenotypic sex of the fetus.

Functional disomy of Xp: prenatal findings and postnatal outcome.

We report on trisomy of the short arm of the X chromosome (Xp11.2 --> pter) due to a de novo unbalanced X;13 translocation diagnosed prenatally in a female fetus. Amniocentesis was performed at 20-weeks' gestation following ultrasound finding of a Dandy-Walker malformation. The trisomy of Xp11.2 --> pter was confirmed with fluorescence in situ hybridization (FISH), using an X chromosome painting probe and telomeric FISH probes specific for the short arm of chromosome X. The karyotype was defined as 46,XX,der(13)t(X;13)(p11.2;p11.2). Molecular analysis suggested that the extra Xp material was of paternal origin. FISH analysis with an XIST probe showed that the derivative chromosome 13 did not include the XIST locus at the X-inactivation center (XIC). A complex phenotype was seen at birth including macrosomia, facial dysmorphism with preauricular tag, congenital heart defects, and structural brain malformations. Because the derivative chromosome was not subject to X inactivation, functional disomy of Xp11.2 --> pter most likely accounts for the abnormal phenotype in this patient.

Prenatal ultrasound findings of lissencephaly associated with Miller-Dieker syndrome and comparison with pre- and postnatal magnetic resonance imaging.

OBJECTIVE: To report on the prenatal ultrasound findings in fetuses with lissencephaly associated with Miller-Dieker syndrome (MDS) and to compare these findings with those of magnetic resonance imaging (MRI). METHODS: Cases of MDS confirmed by postnatal chromosome microdeletion analysis were identified through review of patient records. Prenatal ultrasound scans were reviewed retrospectively by two radiologists. For cerebral cortical development, the Sylvian, parieto-occipital and calcarine fissures, and the cingulate sulcus and sulci over the cerebral convexity were evaluated. If one or more of these fissures or sulci were not visualized at the expected gestational age or their appearance was abnormal for gestational age, cortical development was considered delayed. Prenatal and postnatal MRI examinations were reviewed by a pediatric neuroradiologist. RESULTS: There were seven cases of MDS. In three cases, the prenatal diagnosis of agyria/lissencephaly was prospectively suspected by ultrasound at 23, 26 and 30 weeks, and subsequently confirmed by prenatal MRI. When we retrospectively reviewed the prenatal ultrasound scans of all fetuses, all had delayed cortical development identified on ultrasound performed after 23 weeks' gestation. In all cases the Sylvian fissure was abnormal on both ultrasound and MRI. In one fetus, a normal cortical appearance for gestational age was seen at the initial 20-week ultrasound examination, but delayed cortical development was identified at a 24-week scan. Mild ventriculomegaly was seen in six fetuses and dysgenesis of the corpus callosum in one. Extracranial abnormalities were detected in five fetuses. Delayed cortical development was seen in two fetuses with mild ventriculomegaly, but no other fetal anomalies. CONCLUSIONS: In fetuses with MDS, delayed cortical development can be suspected on ultrasound as early as 23 weeks' gestation. This finding warrants further investigations including MRI and FISH analysis for chromosome 17p13.3 deletion.

Role of amniotic fluid interphase fluorescence in situ hybridization (FISH) analysis in patient management.

We retrospectively reviewed 309 amniotic fluid interphase fluorescence in situ hybridization (FISH) analyses performed from October 1995 to June 1999 to assess the role of interphase FISH in the management of patients at increased risk for fetal aneuploidies. Gestational age and indications for amniocentesis, clinical interventions after FISH results, as well as interventions after final culture reports were analyzed. There were 244 (79%) normal, 50 (16%) abnormal and 15 (5%) inconclusive FISH results. There were no false-positive or false-negative results, but there were nine (3%) clinically significant chromosomal abnormalities not detectable by FISH. Of the 50 women with abnormal FISH results, 26 (52%) elected to terminate the pregnancy prior to the availability of the standard chromosome analysis. In two of the fetuses with trisomy 21 no abnormalities were reported by ultrasound examination. Our experience indicates that interphase FISH results played an important role in decision making, especially for pregnancies close to 24 weeks' gestation. Standard karyotype analysis is still required for detection of chromosome abnormalities not detectable by interphase FISH techniques and for clarification of unusual or inconclusive FISH results.

Inverted duplication of the distal short arm of chromosome 3 associated with lobar holoprosencephaly and lumbosacral meningomyelocele.

A fetus with lobar holoprosencephaly and lumbosacral meningomyelocele associated with duplication of the short arm of chromosome 3 is reported. The anomalies were detected on fetal ultrasound at 20 weeks' gestation and the autopsy findings correlated well with the prenatal findings. The fetal karyotype was 46,XY,der(3)del(3)(p26) dup(3)(p26p21.3). The association of holoprosencephaly with duplication 3p is well known, but to the best of our knowledge this is the first reported association of meningomyelocele with 3p duplication. These findings suggest that a gene or genes with a crucial role in central nervous system development are located on the short arm of chromosome 3.

De novo 46,XX,t(6;7)(q27;q11;23) associated with severe cardiovascular manifestations characteristic of supravalvular aortic stenosis and Williams syndrome.

Supravalvular aortic stenosis may present as an isolated finding or as part of Williams syndrome. Williams syndrome is a contiguous gene syndrome associated with neurodevelopmental and multisystemic manifestations caused by hemizygous deletion at 7q11.23. We report on the prenatal and histopathological findings in a patient with a chromosome translocation involving the Williams syndrome critical region. The initial abnormality on fetal ultrasound was hydrops fetalis detected at 30 weeks and echocardiography showed narrowing of the aorta and the pulmonary arteries. The baby died shortly after delivery and an autopsy revealed diffuse tubular thickening with luminal narrowing of the aorta, aortic branches, and the pulmonary arteries. Histopathology showed dysplasia of the media with reduced elastic content and "cartwheel" arrangement of collagen, elastic, and muscle fascicles. The karyotype was 46,XX,t(6;7)(q27;q11.23). Three signals were detected using the Oncor fluorescent in situ hybridization probe for elastin-Williams syndrome (WSCR) suggesting that the break in chromosome 7 is within the elastin-Williams gene. This patient is of special interest because of the prenatal presentation and the chromosomal translocation involving the elastin-Williams syndrome locus.

Risk of false-positive prenatal diagnosis using interphase FISH testing: hybridization of alpha-satellite X probe to chromosome 19.

FISH analysis of uncultured interphase amniotic fluid cells from a male fetus revealed two signals using an alpha-satellite X-chromosome DNA probe. One of the signals was much smaller than the other. It was subsequently shown that the normal sized signal was located on the X chromosome and the smaller signal was located at the centromere of chromosome 19. This hybridization pattern was confirmed in the newborn infant and in his phenotypically normal father. The use of alpha-satellite DNA probes on interphase cells could result in false-positive errors due to rare variants such as the X-chromosome alpha-satellite found on chromosome 19 in our patient.

Cytogenetic aspects of the Canadian early and mid-trimester amniotic fluid trial (CEMAT).

Cytogenetic results from a large multicentre randomized controlled study of 2108 amniotic fluids obtained at 11+0-12+6 weeks (EA) and 1999 fluids at 15+0-16+6 weeks (MA) were compared. There was no statistically significant difference in the rate of chromosome abnormalities (EA =1.9 per cent; MA=1.7 per cent) or level III mosaicism (EA=0.2 per cent; MA= 0.2 per cent) between the groups. Level I and Level II mosaicism occurred more frequently in MA. Maternal cell contamination was not significantly different between the groups, but maternal cells only were analysed from one bloody EA fluid. The number of repeat amniocenteses because of cytogenetic problems was 2.2 per cent in the EA group compared with only 0.3 per cent in the MA group. On average, culture of EA fluids required one day more than MA fluids. Although both culture success (97.7 per cent) and accuracy (99.8 per cent) were high for patients randomized to the EA group, routine amniocentesis prior to 13 weeks' gestation is not recommended for clinical reasons including an increased risk of fetal loss and talipes equinovarus.

Mosaicism for a small marker chromosome resulting from a familial Robertsonian translocation (21;22).

A mosaic marker chromosome found in amniotic fluid was shown to have originated from the proximal part of the long arm of chromosome 22. This marker is unusual because it is the result of a deletion of a maternally inherited Robertsonian 21;22 translocation. It is suggested that the deletion and marker formation probably occurred post zygotically in the fetus. This rare case illustrates the difficulty in estimating risk of fetal abnormalities associated with de novo marker chromosomes. In this example, although the 'extra' marker chromosome contains euchromatin, the karyotype may still be 'balanced'.

Prenatally diagnosed neural tube defects: ultrasound, chromosome, and autopsy or postnatal findings in 212 cases.

From January 1990 until December 1996, 212 cases of neural tube defect (NTD) were seen through the Prenatal Diagnosis Program of the University of Toronto. Of the 212 cases, 200 were karyotyped successfully and of these, 13 (6.5%) had chromosome abnormalities. When classified according to the site of the NTD, 2.3% (2/88) of anencephalics, 7.1% (1/14) of encephaloceles, and 10.2% (10/98) of meningomyeloceles had abnormal karyotypes. The absence of associated ultrasound abnormalities was not necessarily predictive of a chromosomally normal fetus; 4/167 (2.4%) of fetuses with isolated NTDs had chromosome abnormalities. Conversely, 24/33 (72%) of fetuses with additional findings on ultrasound had normal chromosomes. The diagnosis of a chromosome abnormality associated with NTD has important implications for recurrence risk and prenatal diagnosis, not only for the parents but potentially for other relatives. Based on our finding that 6.5% of prenatally detected NTDs are associated with chromosome abnormalities, we recommend karyotyping of all fetuses and/or newborns with NTD.

The early amniocentesis study: a randomized clinical trial of early amniocentesis and midtrimester amniocentesis. II. Evaluation of procedure details and neonatal congenital anomalies.

The present study provides detailed neonatal and congenital malformation follow-up from 695 women enrolled in a prospective randomized multicenter study comparing the safety and accuracy of early (11-12 weeks of gestation) and midtrimester amniocentesis (15-16 weeks of gestation). No differences were found for total pregnancy loss (difference 0.4%; CI -3.6 to 4.4%), obstetrical or neonatal outcome. The incidence of congenital anomalies was 2.4 and 2.6% for the early amniocentesis and midtrimester amniocentesis groups, respectively. Respiratory problems were present in 2.1 and 1.6%, respectively. Musculoskeletal problems were present in 0.9 and 2.4%, respectively. It must be emphasized that before early amniocentesis can be considered as an alternative to midtrimester amniocentesis or chorionic villus sampling, careful evaluation of any fetal effects must be considered and further large randomized trials are necessary.

The role of fetal karyotyping from unconventional sources.

OBJECTIVE: Our purpose was to determine whether fetal specimens, including pleural, ascitic, pericardial, facial, and cystic hygroma fluid or urine, are suitable sources for accurate chromosomal analysis. STUDY DESIGN: Thirty-nine samples of fetal fluid (pleural, n = 11; ascitic, n = 5; pericardial, n = 1; lung cyst, n = 1, facial cyst, n = 1; cystic hygroma, n = 7; and urine, n = 13) were cultured and analyzed with standard cytogenetic techniques for lymphocytes or amniotic fluid. These samples were obtained as part of the routine obstetric investigation and management. Conventional backup samples were also obtained. RESULTS: Karyotyping was successful in 34 of 39 samples. Cells were harvested from all pleural samples, three ascitic samples, and one hygroma fluid sample in 2 to 4 days from 11 urine samples, one ascitic sample, and the remaining six hygroma samples in 7 to 11 days. Five cultures were unsuccessful. Samples with high lymphocyte counts yielded results as quickly as fetal blood. CONCLUSION: The use of "alternative" samples of fetal fluids for karyotyping may be considered when amniotic fluid or fetal blood is difficult to obtain. In selected cases this approach avoids the unnecessary risk of additional invasive procedures solely to obtain a karyotype.

The early amniocentesis study: a randomized clinical trial of early amniocentesis versus midtrimester amniocentesis.

OBJECTIVES: The primary purpose of this pilot study was to determine whether the safety of early amniocentesis (EA; 11 weeks to 12 weeks and 6 days) is similar to midtrimester amniocentesis (MA; 15 weeks to 16 weeks and 6 days). The secondary objectives were to determine the cytogenetic success and accuracy of EA compared with MA. METHODS: This prospective, randomized clinical trial compared continuous ultrasound-guided EA and MA (22-gauge needle) in patients at a late maternal age (> or = 35 years). The procedures were compared for safety, success and accuracy. RESULTS: Among the 683 women randomized and followed to pregnancy completion, there was a total of 27/344 (7.8%) and 25/339 (7.4%) fetal losses (spontaneous and induced abortions) in the EA and MA groups, respectively (difference 0.4%; CI -3.6 to 4.4%). The rate of postprocedure spontaneous fetal loss was 2.4% (8/330) in the EA group and 3.3% (10/299) in the MA group (NS). The procedure success rate at the first attempt was 97.6% in the EA group and 99.7% in the MA group. There were no diagnostic errors, and all but 2 EA cultures were successful (both repeated successfully). The perinatal outcome was similar in both groups. CONCLUSIONS: EA appears to be as safe and accurate as MA. A large multicentered, randomized trial is currently underway to verify these results.

Maternal cell contamination in uncultured amniotic fluid.

The presence of maternal cells in uncultured amniotic fluid may result in error in the interpretation of prenatal tests such as direct DNA analysis and rapid aneuploidy detection by fluorescence in situ hybridization (FISH). Using simultaneous dual colour X and Y FISH, we assessed maternal cell contamination in uncultured amniotic fluids from 500 women carrying male fetuses. The presence of maternal cells was correlated with the amount of blood present in the amniotic fluid as defined by visual examination of the cell pellet after centrifugation. The overall rate of maternal cell contamination in uncultured amniotic fluid as identified using X and Y-specific probes was 21.4 per cent, compared with 0.2 per cent in cultured fluid. Sixteen per cent of slightly bloody and 55 per cent of moderately bloody uncultured fluids had at least 20 per cent maternal cells and were classified as uninformative according to our protocol for rapid aneuploidy detection. Maternal and fetal cells could not be distinguished based on morphological characteristics alone.

Moving satellites and unstable chromosome translocations: clinical and cytogenetic implications.

We describe 2 families in which acrocentric short arm material moved from one chromosome to another. In case 1, a meiotic event resulted in movement of an unusually large paternal 21p to chromosome 13 in the fetus. In case 2, a mitotic event resulted in fetal mosaicism. The short arm material from a paternal chromosome 15 moved to chromosome 14 in some of the fetal cells. Movement of acrocentric short arm material resulted from breakage and exchange in centromeric areas of repetitive DNA. We suggest the mechanism may be similar to that of previously reported "jumping" translocations. Failure to recognize movement of the short arms of acrocentric chromosomes can result in erroneous interpretation of prenatal cytogenetic results and of other cytogenetic assays dependent on acrocentric short arm polymorphisms.

Trisomy 18 and a constitutional maternal translocation (2;18).

Parental chromosomes are usually not analyzed in cases of trisomy 18 because the extra 18 is assumed to have arisen through a meiotic nondisjunctional event. We report on a case of a trisomy 18 and a maternal translocation (2;18)(q34;q12).

Fetal gastroschisis associated with monosomy 22 mosaicism and absent cerebral diastolic flow.

A case of fetal gastroschisis associated with 45,XY,-22/46,XY mosaicism and absent cerebral diastolic flow is described. This is the third case reported of monosomy 22 mosaicism, and the first one to be diagnosed antenatally.

Familial marker chromosome due to 3:1 disjunction of t(9;15) in a grandparent.

An extra small chromosome detected in amniotic fluid was identified as the product of a translocation [46,XX,t(9;15)(p24;q11.2)]. This case is unusual in that individuals with the unbalanced karyotype resulting from a 3:1 disjunction are phenotypically normal.