Purpose: Despite advances in harvesting and culturing techniques, analysis of the impact of these improvements on the observed frequency of chromosomal abnormalities in spontaneous abortions (SAB) has not been determined. We sought to evaluate the effect of these refinements on the success rate of our cultures and on the resulting frequency of detected chromosomal abnormalities. Methods: Between 1990 and 2002, 2301 specimens obtained from the products of conception (POC) of SABs were submitted to our laboratory for cytogenetic analysis.Due to refinements in specimen processing and culture techniques introduced at the end of 1997, our data were analyzed for two periods: Period A from 1990 through 1997 with 907 eligible specimens and Period B from 1998 through 2002 with 1273 eligible specimens. Results: Modifications in physician communication and sample processing contributed to significant improvements in the culture success rate and in the ratio of male-to-female cases with normal karyotypes. Additionally, increased detection of trisomic, triploid, and multiple aneuploid cases in Period B resulted in a significant increase in the percentage of cases with abnormal karyotypes (42.8% in Period A vs. 65.8% in Period B). Monosomy X accounted for Ͻ 10% of all abnormalities in Period B. Eighty five multiple aneuploid karyotypes, including 57 double trisomies, comprised 7.7% of our 1099 abnormal cases. These karyotypes were detected predominantly in POCs from the older women in our study. This collection of multiple aneuploidies is the largest published to date and includes abnormalities not reported in prior studies. We also present a table empirically derived from the data in Period B that indicates the likelihood of a specific abnormal karyotype based on maternal age. The table can be utilized by health care providers, who counsel patients after a spontaneous miscarriage. Conclusion: Improvements in laboratory technique have led to reduced contamination and growth failure of POCs, irrespective of maternal age. This in turn has led to a more balanced male-to-female ratio and to the detection of an increased number of abnormal cases. Genet Med 2005:7(4):251-263. Key Words: cytogenetics, spontaneous abortion, aneuploidy, karyotype, chromosome abnormalityA correlation between chromosomal abnormalities and spontaneous abortions (SABs) has been observed since the 1960s. 1 This correlation was strengthened in the 1970s when Boue et al. 2 published one of the earliest large cytogenetic studies. In the study, almost 1500 samples of fetal tissue were karyotyped and an abnormality rate of over 60% was found. Subsequent large studies using harvested products of conception (POC) failed to match this abnormality rate, with rates of 32%-54% reported. [3][4][5][6][7][8][9] Furthermore, a large number of specimens in these studies failed to grow successfully in culture and thus could not be evaluated for abnormalities. The data culled from these studies have served as the basis for the estimated abnormality rates in the general popu...

Molecular analysis of a patient affected by the autosomal recessive skeletal dysplasia, pycnodysostosis (cathepsin K deficiency; MIM 265800), revealed homozygosity for a novel missense mutation (A277V). Since the A277V mutation was carried by the patient's father but not by his mother, who had two normal cathepsin K alleles, paternal uniparental disomy was suspected. Karyotyping of the patient and of both parents was normal, and high-resolution cytogenetic analyses of chromosome 1, to which cathepsin K is mapped, revealed no abnormalities. Evaluation of polymorphic DNA markers spanning chromosome 1 demonstrated that the patient had inherited two paternal chromosome 1 homologues, whereas alleles for markers from other chromosomes were inherited in a Mendelian fashion. The patient was homoallelic for informative markers mapping near the chromosome 1 centromere, but he was heteroallelic for markers near both telomeres, establishing that the paternal uniparental disomy with partial isodisomy was caused by a meiosis II nondisjunction event. Phenotypically, the patient had normal birth height and weight, had normal psychomotor development at age 7 years, and had only the usual features of pycnodysostosis. This patient represents the first case of paternal uniparental disomy of chromosome 1 and provides conclusive evidence that paternally derived genes on human chromosome 1 are not imprinted.

Most individuals with cat eye syndrome (CES) have a supernumerary bisatellited chromosome which, on the basis of cytogenetic evidence, has been reported to originate from either chromosome 13 or 22. To resolve this question, a single-copy DNA probe, D22S9, was isolated and localized to 22q11 by in situ hybridization to metaphase chromosomes. The number of copies of this sequence was determined in CES patients by means of Southern blots and densitometry analysis of autoradiographs. In patients with the supernumerary chromosome, four copies were found, whereas in one patient with a duplication of part of chromosome 22, there were three copies. Therefore, the syndrome results from the presence of either three or four copies of DNA sequences from 22q11; there is no evidence that sequences from other chromosomes are involved. This work demonstrates how DNA sequence dosage analysis can be used to study genetic disorders that are not readily amenable to standard cytogenetic analysis.

Purpose: Comparative genomic hybridization (CGH) is a powerful DNA-based cytogenetic technique that allows the entire genome to be scanned for chromosomal imbalances without requiring the sample material to be mitotically active. During the past 2 years we received many requests from various medical centers around the country to use CGH to resolve the identity of aberrant chromosomal material. Methods I M U C T I O NApproximately 3% to 4% of liveborn babies have a major congenital defect.' These defects include unbalanced chromosome abnormalities which occur in approximately 1 in 250 newborm2 Between 15% and 20% of all pregnancies end in a spontaneous abortion, and of these approximately 50% are associated with chromosome abn~rmalities.~ A precise diagnosis in a newborn or prenatal sample with a chromosome abnormality is critical for appropriate genetic counseling as well as for the clinical management of an infant. It also provides the parents with a realistic prognosis. There are many instances when cytogenetic analysis is unsuccessful in producing a result because the specimen cannot be cultured. There are also many occasions when extrachromosomal material remains unidentifiable even after numerous standard cytogenetic staining methods have been attempted. Molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH), have brought a new depth to clinical cytogenetics by facilitating the identification of chromosomal material of unknown origin. This approach often requires using multiple whole chromosome paints (WCP) until the source chromosome is identified.s5 Alternative approaches include reverse FISH, using probes derived from the microdissected chromosome region of interestM; and multicolor FISH p r~b i n g .~. 'O However, these techniques require specialized equipment in addition to the regular cytogenetic/FISH image analysis equipment that now is commonly found in many comprehensive cytogenetic laboratories. Although multicolor FISH can determine the origin of the unknown material, it does not identify the specific location or breakpoints on the chromosome from which the extra material originated.Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic technique that allows for the identification of chromosomal gains or losses by scanning the entire genome in a single step. It has the distinct advantage of providing a genome-wide search without any prior &formation about the chromosomal aberration in question. It is accomplished by in situ hybridization of differentially labeled total genornic specimen DNA and normal reference DNA to normal human metaphase chromosome ~preads.'l-~~ Hybridization of the specimen and reference DNA can be distinguished by their Merent fluorescent colors. The relative amounts of specimen and reference DNA hybridized at a particular chromosome position are contingent on the relative excess of those sequences in the two DNA samples and can be quantified by calculation of the ratio of their different fluorescent c~l o r s .~~-l~...

Neocentromeres are fully functional centromeres found on rearranged or marker chromosomes that have separated from endogenous centromeres. Neocentromeres often result in partial tri- or tetrasomy because their formation confers mitotic stability to acentric chromosome fragments that would normally be lost. We describe the prenatal identification and characterization of a de novo supernumerary marker chromosome (SMC) containing a neocentromere in a 20-wk fetus by the combined use of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). GTG-banding of fetal metaphases revealed a 47,XY,+mar karyotype in 100% of cultured amniocytes; parental karyotypes were both normal. Although sequential tricolor FISH using chromosome-specific painting probes identified a chromosome 10 origin of the marker, a complete panel of chromosome-specific centromeric satellite DNA probes failed to hybridize to any portion of the marker. The presence of a neocentromere on the marker chromosome was confirmed by the absence of hybridization of an all-human-centromere alpha-satellite DNA probe, which hybridizes to all normal centromeres, and the presence of centromere protein (CENP)-C, which is associated specifically with active kinetochores. Based on CGH analysis and FISH with a chromosome 10p subtelomeric probe, the marker was found to be an inversion duplication of the distal portion of chromosome 10p. Thus, the proband’s karyotype was 47,XY,+inv dup(10)(pter→p14∼15::p14∼15→neo→pter), which is the first report of partial tetrasomy 10p resulting from an analphoid marker chromosome with a neocentromere. This study illustrates the use of several molecular strategies in distinguishing centric alphoid markers from neocentric analphoid markers.