Overview of Chromosome Abnormalities in First Trimester Miscarriages: A Series of 1,011 Consecutive Chorionic Villi Sample Karyotypes.

In order to contribute to the knowledge of type and frequency of chromosome abnormalities in early pregnancy losses, we analyzed the cytogenetic results from a large series of first trimester miscarriages, using a diagnostic approach with a high success rate and no maternal contamination. A total of 1,119 consecutive chorionic villi samples were obtained before evacuation, and karyotypes were prepared after short-term culture (STC). In 603 samples, a long-term culture (LTC) was also performed. The overall and individual frequencies of the different types of chromosome abnormalities were established, including placental mosaicisms, and their relationship with maternal age and gestational weeks was assessed. An abnormal karyotype was detected in 70.3% of the samples. Single autosomal trisomy was the most frequent abnormality (64.6% of the abnormal cases), followed by triploidy (13.1%) and monosomy X (10.4%). Chromosome rearrangements were found in 5.2%, combined abnormalities in 8.9%, and placental mosaicism in 3.5% of the cases with STC and LTC performed. Individual trisomies behaved differently with respect to maternal age and intrauterine survival. Due to the combination of STC and LTC, our study offers reliable information on the incidence and type of chromosome abnormalities and placental mosaicism in miscarriages and contributes to define the cytogenetic implication in their etiology.

MLPA: a prenatal diagnostic tool for the study of congenital heart defects?

Congenital heart defects (CHD) represent the most common birth defects, so they are not a rare finding when performing routine ultrasound examinations during pregnancy. Once chromosome abnormalities have been excluded in a fetus with a CHD, chromosome 22q11.2 deletion is usually investigated by FISH, as it is the most frequent microdeletion syndrome and is generally associated with cardiac malformations. If 22q11.2 microdeletion is ruled out, the etiology of the CHD remains generally unexplained, making familial genetic counseling difficult. To evaluate the usefulness of Multiplex Ligation-dependent Probe Amplification (MLPA) kits designed for the study of 22q11.2 and other genomic regions previously associated with syndromic CHD, we performed MLPA in 55 pregnancies with fetuses presenting CHD, normal karyotype and negative FISH results for 22q11.2 microdeletion, which constitutes the largest prenatal series reported. Definitive MLPA results were obtained in 50 pregnancies, and in this setting such MLPA kits did not detect any imbalance. On the other hand, to compare FISH and MLPA techniques for the study of 22q11.2 microdeletions, we performed MLPA in 4 pregnancies known to have 22q11.2 deletions (by FISH). All four 22q11.2 microdeletions were also detected by MLPA, which corroborates that it is a reliable technique for the diagnosis and characterization of 22q11.2 deletions. Finally, we evaluated the possibility of replacing conventional FISH by MLPA for the prenatal diagnosis of CHD, comparing the diagnostic potential, results delivery times, repetition and failure rates and cost of both techniques, and concluded that FISH should still be the technique of choice for the prenatal diagnosis of fetuses with CHD.

Familial 4.8 MB deletion on 18q23 associated with growth hormone insufficiency and phenotypic variability.

The deletion of the long arm of chromosome 18 causes a contiguous gene deletion syndrome with a highly variable phenotype, usually related to the extent of the deleted region. The most commonly reported clinical features include: decreased growth, microcephaly, facial abnormalities, hypotonia, developmental delay, intellectual disability, congenital aural atresia with hearing impairment and limb anomalies. Here we report on a familial terminal deletion of 18q23 region transmitted from a mother to two daughters, resulting in a remarkable phenotypic variability. The deletion was first detected by conventional cytogenetic analysis in one daughter and subsequently characterized using fluorescence in situ hybridization (FISH) and array-CGH. FISH analysis using subtelomeric 18p and 18q probes confirmed the 18qter deletion in the three patients, and FISH with a whole chromosome painting probe specific for chromosome 18 excluded rearrangements with other chromosomes. Array-CGH analysis allowed us to precisely define the extent of the deletion, which spans 4.8 Mb from 71,236,891 to 76,093,303 genomic positions and includes GALR1 and MBP genes, among others. High-resolution analysis of the deletion, besides a detailed clinical assessment, has provided important data for phenotype-genotype correlation and genetic counseling in this family. Furthermore, this study adds valuable information for phenotype-genotype correlation in 18q- syndrome and might facilitate future search for candidate genes involved in each phenotypic trait.

Prenatal cytogenetic diagnosis in Spain: analysis and evaluation of the results obtained from amniotic fluid samples during the last decade.

Chromosome abnormalities are one of the main causes of congenital defects, and establishing their frequency according to the different clinical indications for invasive procedure during pregnancy is especially important for genetic counselling. We analyzed the results of 29,883 amniotic fluid samples referred to our laboratory for cytogenetic studies from 1998 to 2009, which constitutes the largest series of cytogenetic analysis performed on amniotic fluid samples in Spain. The number of samples received tended to increase from 1998 to 2005, but after 2005 it decreased substantially. Cytogenetic results were obtained in 99.5% of the samples, and the detected incidence of chromosome abnormalities was 2.9%. Of these, 48.1% consisted of classical autosomal aneuploidies, trisomy 21 being the most frequent one. The main clinical indications for amniocentesis were positive prenatal screening and advanced maternal age, but referral reasons with highest positive predictive values were, excluding parental chromosome rearrangement, increased nuchal translucency (9.2%) and ultrasound abnormalities (6.6%). In conclusion, performing the karyotype on amniotic fluid samples is a good method for the detection of chromosome abnormalities during pregnancy. The number of cytogenetic studies on amniotic fluid has now decreased, however, due to the implementation of first trimester prenatal screening for the detection of Down syndrome, which allows karyotyping on chorionic villus samples. Our results also show that both ultrasound abnormalities and increased nuchal translucency are excellent clinical indicators for fetal chromosome abnormality.

Subtelomeric MLPA: is it really useful in prenatal diagnosis?

OBJECTIVE: To evaluate the usefulness of subtelomeric multiplex ligation-dependent probe amplification (MLPA) in both the detection of subtelomeric rearrangements in fetuses with ultrasound abnormalities and normal karyotype, and the characterization of cytogenetically detectable rearrangements. METHOD: We studied by subtelomeric MLPA 229 pregnancies with ultrasound findings and normal karyotype (Group 1) and five pregnancies with a cytogenetically visible but not microscopically characterizable rearrangement (Group 2). The detected imbalances were confirmed by fluorescence in situ hybridization (FISH) and parents were also studied. RESULTS: In Group 1, two clinically relevant subtelomeric imbalances (14qter deletion and 20pter deletion) and one subtelomeric imbalance of uncertain significance (X/Ypter duplication) were diagnosed, showing a detection rate of cryptic subtelomeric imbalances in these pregnancies of 1.3%. However, only 14qter deletion seems to be clearly associated with the observed prenatal findings. In Group 2, MLPA contributed to the precise description of the chromosome abnormalities. CONCLUSION: The low detection rate of subtelomeric imbalances and the poor genotype-phenotype correlations in pregnancies with ultrasound abnormalities and normal karyotype suggest that subtelomeric MLPA is not a crucial tool in the prenatal diagnosis of these cases. However, our work provides evidence that MLPA is very useful for the characterization of unbalanced karyotypes. Copyright © 2010 John Wiley & Sons, Ltd.

Assessment of QF-PCR as the first approach in prenatal diagnosis.

Quantitative fluorescent PCR (QF-PCR) has been used by many laboratories for prenatal diagnosis of the most common aneuploidies. QF-PCR is rapid, cost-effective, and suitable for automation and can detect most abnormalities diagnosed by conventional karyotyping. Whether QF-PCR should be used alone in most of the samples and in which karyotyping should also be offered is currently a topic of debate. We evaluated and compared the results obtained from 7679 prenatal samples in which conventional karyotype and QF-PCR had been performed, including 1243 chorionic villi and 6436 amniotic fluid samples. Concordant QF-PCR and karyotype results were obtained in 98.75% of the samples. An abnormal karyotype associated with adverse clinical outcome undetected by QF-PCR was found in 0.05% of samples. Therefore, QF-PCR can be used alone in a large number of samples studied in a prenatal laboratory, thereby reducing both the workload in cytogenetic laboratories and parental anxiety when awaiting results.

Description of the smallest critical region for Dandy-Walker malformation in chromosome 13 in a girl with a cryptic deletion related to t(6;13)(q23;q32).

We report on a girl with mild dysmorphic facial features, Dandy-Walker malformation (DWM), iris coloboma, profound hearing loss, and hyperlaxity of skin and joints, whose karyotype is 46,XX,t(6;13)(q23;q32)dn and who has a cryptic imbalance at the 13q32 translocation breakpoint assessed by array-CGH. Our patient has many clinical manifestations in common with those of the previously reported cases of 13q32 deletions, which suggests that at least part of the abnormal phenotype is probably due to the imbalance. The recurrent finding of DWM among patients with 13q deletions has led to the suggestions that candidate gene/s for its development are on chromosome 13. We describe the smallest 13q deletion associated to DWM, which allows further narrowing of the previously established critical region for this brain malformation to 13q32.2-32.3. Among the few genes of the deleted region, ZIC2 and ZIC5 seem the most plausible candidates, which is in keeping with some previous reports. This work also illustrates the usefulness of array-CGH platforms in the identification of cryptic imbalances in carriers of apparently balanced rearrangements with abnormal phenotypes.

Non-mosaic trisomy 20 of paternal origin in chorionic villus and amniotic fluid also detected in fetal blood and other tissues.

Trisomy 20 mosaicism is a common abnormality found in prenatal diagnosis. Its clinical significance remains unclear since approximately 90-93% of cases result in normal phenotype. Only 5 cases of non-mosaic trisomy 20 in amniotic fluid culture surviving beyond the first trimester have been reported. Moreover, trisomic cells are generally not detectable in blood and have only been reported in three cases. We present a case of non-mosaic trisomy 20 found in chorionic villi sample and amniotic fluid culture in a fetus with minor abnormalities not detected by ultrasound examination. Pathological examination of the fetus only revealed right pulmonary isomerism and camptodactily, and no major malformations were disclosed. Trisomic lineage was also detected in fetal blood, kidney, skin and brain tissue cultures. Molecular analysis revealed that the extra chromosome 20 was originated in paternal meiosis. To our knowledge, we report the first prenatal case of non-mosaic trisomy 20 of paternal origin that has been confirmed in several fetal tissues, including blood, in a fetus with minor malformations not detected prenatally.

Prenatal diagnosis of two different unbalanced forms of an inherited (Y;12) translocation.

The identification of an unexpected structural chromosome rearrangement at prenatal diagnosis can be problematic and raises unique genetic counseling issues. We describe two consecutive prenatal cases within a family with an inherited unbalanced (Y;12) translocation and discuss the genotype-phenotype correlation. The first fetus presented with 12qter monosomy and pseudoautosomal region 2 trisomy, while the second fetus had the alternative unbalanced state. Although the first fetus had a structural heart defect, such small imbalances might not give sonographic findings, making their prenatal diagnosis difficult. However, congenital abnormalities are expected in both unbalanced forms of the translocation, including mental retardation, which could be explained by the gene dosage variation of P2RX2. To our knowledge, these are the first published cases reporting this subtype of (Y;12) translocation, in both balanced and unbalanced states.

Reproductive consequences of genome-wide paternal uniparental disomy mosaicism: description of two cases with different mechanisms of origin and pregnancy outcomes.

OBJECTIVE: To describe the molecular and cytogenetic characterization of two different prenatal cases of androgenetic/biparental mosaicism and review the different possible mechanisms of origin in each case. DESIGN: Case study and literature review. SETTING: Tertiary medical center (prenatal diagnosis unit). PATIENT(S): A 26-year-old pregnant woman referred for suspected partial mole placenta and a 33-year-old pregnant woman referred for polyhydramnios and fetal malformations. INTERVENTION(S): Ultrasound examination, prenatal invasive procedures, molecular and cytogenetic analysis, physical and pathologic evaluation, and genetic counseling. MAIN OUTCOME MEASURE(S): Cytogenetic analysis, fluorescent in situ hybridization, and quantitative fluorescence polymerase chain reaction (QF-PCR) analysis. RESULT(S): The finding of a normal karyotype together with a triploidy-like QF-PCR profile led to the diagnosis of two cases of androgenetic (genome-wide paternal uniparental disomy)/biparental mosaicism. The first case showed placental mesenchymal dysplasia and a normal fetus, and the second one presented a fetus showing Beckwith-Wiedemann syndrome features and an apparently normal placenta. CONCLUSION(S): These cases highlight the wide range of possible clinical presentations of androgenetic/biparental mosaicism, the variety of mechanisms of their origin, and the importance of the combination of molecular and cytogenetic analysis to achieve an accurate diagnosis and provide reproductive counseling.

Atypical XX male with the SRY gene located at the long arm of chromosome 1 and a 1qter microdeletion.

Male individuals with a 46,XX karyotype have been designated as XX males. In 80% of the cases, the presence of Yp sequences, including the male sex-determining gene, SRY, has been demonstrated by molecular and/or fluorescence in situ hybridization (FISH) analyses. In most cases, Yp sequences are located on the short arm of the X chromosome, resulting from unequal recombination between Yp and Xp during paternal meiosis. Much less frequent in XX males is the localization of the SRY gene to an autosome. Here we report on the genetic investigation of an atypical XX male in which the SRY gene was located at the end of the long arm of chromosome 1. The patient, with a normal male phenotype, was referred for azoospermia. Conventional cytogenetic analysis showed a 46,XX karyotype. Molecular-cytogenetics (FISH) and molecular (PCR and MLPA) studies identified not only Yp-specific sequences located on the distal long arm of chromosome 1 but also the deletion of the subtelomeric 1qter region. A specific phenotype has been reported for a deletion of the 1qter region associated with mental retardation. The molecular investigation of the 1qter region showed that in our patient the microdeletion is more telomeric than in patients reported with mental retardation. To our knowledge, this is the first report of a XX male with the Yp region transferred to the terminal long arm of chromosome 1. This is also the first microdeletion of the subtelomeric 1qter region not associated with mental retardation.

Cytogenetic study of spontaneous abortions using semi-direct analysis of chorionic villi samples detects the broadest spectrum of chromosome abnormalities.

Conventional tissue culturing and karyotyping of spontaneous abortions has limitations such as culture failure, external contamination and selective growth of maternal cells. Molecular cytogenetic techniques such as FISH, QF-PCR, and CGH allow diagnosis on uncultured cells but are also limited as to the spectrum of cytogenetic abnormalities detected. We describe the cytogenetic findings in a series of 116 first trimester arrested pregnancies, obtained through chorionic villi sampling (CVS) and semi-direct analysis that avoids some of the long-culture pitfalls such as maternal contamination, and compare our results with those that would have been obtained theoretically using molecular cytogenetic techniques. Samples were obtained by transcervical CVS from women with a diagnosis of missed abortion, most of them referred for cytogenetic prenatal diagnosis. Cytogenetic analysis was performed using semi-direct technique. A karyotype was obtained in 103 cases. Eighty-two abnormal karyotypes were found (80%), including 12 triploidies, 10 monosomies, 61 trisomies, and 9 structural abnormalities; a double abnormality being present in 10 cases. Between 10% and 50% of our abnormal results would have been missed using the most common molecular cytogenetic techniques. Semi-direct analysis of CVS may still be considered as a comprehensive, reasonably rapid, cost-effective and reliable method for detecting the broadest spectrum of chromosome abnormalities in missed abortions.

A retrospective and theoretical evaluation of rapid methods for detecting chromosome abnormalities and their implications on genetic counseling based on a series of 3868 CVS diagnoses.

OBJECTIVES: To report our experience over the past 10 years of chorionic villi sampling (CVS) prenatal diagnosis in a high-risk population for chromosomal anomalies, and to analyze, according to the results, the advantages and disadvantages of using quantitative fluorescence polymerase chain reaction (QF-PCR) in amniotic fluid with respect to a conventional semi-direct cytogenetic CVS method in a retrospective theoretical review. METHODS: We performed 3,868 cytogenetic analyses from CVS using a semi-direct culture method in a selected high-risk population for chromosomal abnormalities and we compare our findings with the theoretical results obtained using QF-PCR on amniotic fluid. RESULTS: The rate of chromosomal anomalies detected with the semi-direct CVS cytogenetic study, excluding confined placental mosaicism (CPM), was 6.8%. 26.3% of all them would be missed by using QF-PCR only and among them, 21.4% of cases would represent a severe adverse obstetric outcome. CONCLUSIONS: We think that semi-direct CVS cytogenetic analysis in comparison with QF-PCR in amniotic fluid is similarly rapid, performed earlier and more complete, allowing the chromosomal diagnosis in the first trimester of gestation. We propose the use of QF-PCR as an additional method to semi-direct CVS analysis in order to avoid false-negative results, as a rapid alternative to long-term culture.

Diagnóstico citogenético posnatal en España: análisis, evolución y evaluación de resultados en una década / Postnatal cytogenetic diagnosis in Spain: analysis, evolution and outcome evaluation over a decade

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Trisomy of 19.4 Mb region of chromosome 22 and subtelomeric 17p identified in a male without clinical affectation.

Supernumerary marker chromosomes (SMCs) have a reported frequency in the prenatal and newborn population ranging from 0.04% to 0.08% and about 37% of diagnosed SMCs are associated with an abnormal phenotype. Around 7.5% of them are derived from chromosome 22. SMCs(22) that result in tri- or tetrasomy of band 22q11.2 are associated with Cat-eye syndrome (CES), a syndrome of variable penetrance and affectation. CES-like phenotype has been also related to 22q11.2 interstitial duplications and der(22) syndrome. The 22q11.2 region, also involved in the velocardiofacial microdeletional syndrome, presents high susceptibility to chromosomal rearrangements due to the presence of low-copy repeats sequences (LCR22). Another region in the genome rich in LCR is 17p and five recurrent disorders have been mapped on the region 17p11-p13. Some chromosomal imbalances affecting the 17p13.3 subtelomeric region have been reported, related to cryptic unbalanced translocations and associated, in most cases, to mental retardation and dysmorphic features. We report on a healthy male carrier of a SMC that was identified as a +der(22)t(17;22)(p13.3;q11.2) consequence of an abnormal 3:1 segregation of the paternal t(17;22) and we have determined the approximate size of the trisomic regions, comparing the obtained results with other reported imbalances involving 22q11.2 and 17pter.

Duplication/deletion mosaicism of the 7q(21.1 --> 31.3) region.

Mosaicism for structural aberrations is a rare event and the coexistence of a cell line with a duplication and another with a deletion of the same chromosome segment is even more infrequent. We report a boy with a 46,XY,del(7q)/46,XY,dup(7q) mosaicism. High-resolution cytogenetic analysis and fluorescent in situ hybridization (FISH) performed at birth showed a trisomy for region 7q21.1 to 7q31.3 in 90% of metaphases analyzed and monosomy for the same region in 10% of metaphases. At the age of 12 months, karyotype on peripheral blood and exfoliated urinary epithelial cells was 46,XY,dup(7)(q21.1q31.3) in all cells analyzed. The patient presented malformations and psychomotor retardation. His phenotype is compared with other previously case reports describing patients with an interstitial duplication of 7(q21 or q22 --> q31.3). Due to the absence of a normal cell line, we propose a post-zygotic origin of the abnormality during the first mitotic division and a progressive loss of the deleted cells during pre- and post-natal development by selective pressure. The patient described here emphasizes the possible existence of an undetectable cell line in patients previously diagnosed of pure partial 7q trisomy or monosomy to explain the great clinical variability between reported patients. We also describe the culture of urinary epithelial cells in order to perform cytogenetic analysis as a useful non-invasive method.