47th Annual Meeting of the American Society of Human Genetics

October 28 - November 1, 1997 Baltimore, Maryland


774_____COMBINING SPECTRAL KARYOTYPING AND SUBTELOMERIC PROBES TO IMPROVE CYTOGENETIC DIAGNOSIS. Y. Ning1, B. Huang2, J. Meck3, E. Schrock4, T. Reid4. GeneCare/George Washington University, Washington DC1, Genzyme Genetics, Long Beach, CA,2, Georgetown University, Washington DC3; NHGI, Bethesda, MD4.

Spectral karyotyping (SKY) and subtelomeric probes are recently developed molecular cytogenetic tools. Combining these two methods with conventional cytogenetic analysis, we have raised our diagnostic ability to new level. Three cases each with ad different abnormal karyotype were initially detected using G-banding analysis: one had additional material of unknown origin added to 5p, and the other two had a supernumerary marker chromosome. In the case with the 5p+, SKY study revealed that the extra material was from chromosome 10. Further analysis with subtelomeric probes showed that the 5pter probe was absent and the 10pter probe was present on the derivative chromosome 5. Using information from G-banding, SKY, and subtelomeric probes, we refineded the karyotype as 46,XX,der(5)t(5;10)(p15.3;p13). In the first of the marker chromosome cases, subsequent SKY revealed that the marker was derived from chromosome 15. Further analysis with a 15qter probes showed that the probe hybridized to both ends of the marker, suggesting the marker represented an inverted duplication of 15q. The second case with a mosaic supernumerary marker was prenatal diagonsis. SKY led to a rapid identification of the marker as chromosome 14 orgin. Chromosome 14 specific painting probe was subsequently used to confirm the SKY result. Further FISH with alph-satellite, rDNA and 14qter probes indicated that the marker was derived from the proximal part of 14q. These results contributed to the counseling of the family, and the parents chose to continue the pregnancy.

796_____SPECTRAL KARYOTYPING REFINES CYTOGENETIC DIAGNOSTICS OF CONSTITUTIONS CHROMOSOMAL ABNORMALITIES. E. Schrock1, T. Veldman1, H. Padilla-Nash1, Y. Ning1, J. Spurbeck2, S. Jalal2, L.G. Shaffer3, P.I. Papenhausen4, C. Kozma5, M.C. Phelan6,E. Kjeldsen7, S.A. Schonberg8, P.O'Brien9, Les Biesecker1, S. Du Manoir1, T. Ried1. 1National Institute for Human Genome Research (NHGRI/HIH), Bethesda, Maryland; 2Mayo Clinic, Cytogenetic Laboratory, Rochester, MN; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 4Labcorp, Research Triangle Park, NC; 5Georgetown University, Department of Pediatrics, Washington, DC; 6Greenwood Genetic Center, Greenwood, SC; 7Odense Universiteshospital, Department of Clinical Genetics, Odense, Denmark; 8American Medical Laboratories, Chantilly, VA; 9Department of Pathology, Cambridge University, Cambridge, UK.

Karyotype analysis by chromosome banding is the standard method to identify numerical and structural chromosomal aberrations in the pre and postnatal cytogenetic laboratories. However, the chromosomal origins of markers, subtile translocations or complex chromosomal rearrangements are often difficult to identify with certainty. We have developed a novel karyotyping technique, termed spectral karyotyping which is based on the simultaneous hybridization of 24 chromosome specific painting probes labeled with different emission spectra using interferometer based spectral imaging allows for the definitive discernment of all human chromosomes in different colors.

Here we reported the comprehensive karyotype analysis of 16 samples from different cytogenetic laboratories by merging conventional cytogenetic methodology and spectral karyotyping. This approach could become a powerful tool for the cytogeneticist because it results in a considerable improvement of karyotype analysis by identifying chromosomal aberrations not previously by G-banding alone.

877_____IS MATERNAL SERUM UNCONJUGATED ESTRIOL LOW IN TRISOMY 18? TW Hallahan1, DA Krantz1, PD Buchanan2, JW Larsen, Jr.3, JN Macri1. 1NTD Laboratories, Inc., Huntington Station, NY; 1GeneCare Medical Genetics Center, Chapel Hill, NC; 1George Washington Univ. Med. Center, Washington, DC.

Unconjugated estriol was analyzed retrospectively in maternal serum samples from 15 pregnancies affected with Trisomy 18 and 60 mateched control samples using a radio immunoassay kit (Diagnostic Systems Laboratories). Controls were matched for gestational age and ethnicity. All samples were collected between 14 and 20 weeks gestation. None of the cases or controls were previously evaluated for unconjugated estriol. Free Beta hCG and Alpha-fetoprotein (AFP) values were available from perspective second trimester maternal serum screening.

The median MoM in the Trisomy 18 cases from free Beta, AFP and unconjugated estriol were 0.14, 0.63, and 0.76 MoM respectively. The number of cases of Trisomy 18 below 0.5 MoM was 13/15 (87%) for free Beta, 5/15 (33%) for unconjugated estriol. Using a patient specific term risk cut-off of 1/200, the combination of free Beta and AFP detected 11/15 (74%) Trisomy 18 cases with only 1 false positive result. Adding unconjugated estriol to the free Beta/AFP protocol did not change the detection efficiency and false positive rate.

These results confirm that low free-beta hCG and AFP levels observed in the second trimester are predictive of Trisomy 18. Based on this study unconjugated estriol levels in cases of Trisomy 18 are not as low as those of free Beta and AFP. Consequently, addition of unconjugated estriol to free Beta and AFP does not appear to be useful when screening for Trisomy 18.

892_____PRENATAL DIAGNOSIS OF A MOSIAC MARKER CHROMOSOME 14 BY SPECTRAL KARYOTYPING. C.H. Laundon1, Y. Ning2, T. Ried2, P. Buchanan1. 1GeneCare Medical Genetics Center, Chapel Hill, NC; 2NHGRI, Bethesda, MD.

Small supernumerary marker chromosomes occur with a frequency of 0.6-1.5 per 1000 in prenatal cytogenetic analysis. The chromosomal origin of the marker chromosomes is largely unknown due to the limited detection of sensitivity of stand chromosomal banding methods. Fluorescence in situ hybridization (FISH) with whole chromosome painting probes has enhanced our ability to identify the origin of marker chromosomes, but the process is limited to one or a few chromosomes at a time. The development of a novel technology, termed spectral karyotyping (SKY), has allowed simultaneous hybridization of 24 chromosomes in 24 colors. We report here what to our knowledge is the first use of SKY in the prospective prenatal diagnosis of a mosaicism for a small marker chromosome in amniotic fluid cells. Cells from a second trimester amniotic fluid specimen were cultured in situ because of increased maternal age. The amniotic fluid alpha fetoprotein (AFAFP) was within normative range. Chromosome analysis of GTG banded preparations detected a small marker chromosome of unknown origin in 7 out or 21 colonies from 3 separate cover slip cultures. Analysis of parental blood chromosomes showed no evidence of the marker. SKY analysis revealed the marker was derived from chromosome 14. The result was confirmed by FISH with a chromosome specific painting probe. Further analysis with alpha-satellite, rDNA and 14q. Genetic counseling was provided to the parents and an increased risk of birth defects and/or mental retardation of as high as 15-20% due to the presence of a de novo marker chromosome was cited. Four patients with small marker 14 chromosome have been reported to be phenotypically normal at ages ranging from 6-21 years (1). The parents have chosen to continue the pregnancy with a expected delivery date in October. Gravholt, C.H., Frederick U. (1995) Am. J. Hum. Gen. 56:106-111.

2206_____PRENATAL DETECTION OF A DELETION IN THE 15(Q11.1Q13) PRADER WILLI SYNDROME/ANGELMAN SYNDROME REGION. P.Buchanan1, C. H. Laundon1, Y. Ning2, T. Donlon3. 1GeneCare Medical Genetics Center, Chapel Hill, NC; 2NHGRI, Bethesda, MD; 3Kapiolani Medical Center, Honolulu, HI.

Individuals with abnormalities in the proximal long arm region (q11.1q13) of chromosome 15 usually have either Prader Willi Syndrome (PWS) or Angelman Syndrome (AS) depending upon the parental orgin. When the genetic material in this region is altered from a paternal 15 chromosome, PWS can occur. When the same region is altered from a maternal 15 chromosome, AS can occur. PWS is characterized by hypotonia and failure to thrive in early infancy, short stature,hypogonadism, hypopigmentation, mental retardation, small hands and feet and obesity due to hyperphagia beginining in early childhood. Characteristics of AS include mental retardation of a greater severity, poor to absent speech, paroxysms of laughter, ataxia, microcephaly and seizure disorders. These differences in phenotype between PWS and AS depending on which on parental chromosome 15 is altered is due to genomic imprinting. Approximately 70% of PWS and AS are due to deletions in the 15(q11.1q13) deletion [del(15)] in amniotic fluids cells from an amniocentesis performed at 18wks gestations for increased maternal age. GTG banding at the 550+ band level showed a suspected deletion in one of the 15 chromosomes. FISH using two probes for the detection of the PWS/AS (SNRPN and D15S 10-Vysis) confirmed the deletion of either the SNRPN or D15S10 probes. QM variant analysis of the parental and fetal chromosomes shows a unique polymorphism which suggests maternal origin of the fetal del (15) chromosome and the fetus would most likely be affected with AS. Methylation and microsatellite studies are being performed to determine the parental origin of the del (15).
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18th Annual Meeting of the Society of Perinatal Obstetricians

February 2 - 7, 1998 Miami, Florida


450_____FIRST TRIMESTER DOWN SYNDROME SCREENING. D. Krantzx1, T. Hallanhan1, F. Orlandi2, C. Rossi2, J. Larsen3, P. Buchanan4,J. Macri1. 1NTD Laboratories, Inc., Huntington Station, NY, 2Centro Di Diagnosi Prenatale, Palermo, Italy, 3George Washington Univ. Med. Center, Washington, D.C., 4GeneCare Medical Genetics Center, Chapel Hill, NC
OBJECTIVE: To assess detection capability of freeBeta hCG, PAPP-A and nuchal translucency (NT) in first trimester Down syndrome screening.
STUDY DESIGN: 81 DS and 3947 unaffected controls were evaluated biochemically. NT was available in a limited number of cases (7 DS and 2151 controls) between 10-13 weeks. Cases were from retrospective and ongoing prospective studies. Geometric means, standard deviations (LOGe) and correlations for DS and unaffected pregnancies were determined. Detection efficiency was modeled based on these parameters for biochemistry and observed likelihood ratios for NT.
RESULTS:
Down Syndrome Cases
Free-Beta
PAPP-A
GANMeanSDMeanSDr
10-11531.73.46570.46.6141-.193
12-13282.07.48680.66.47940.134
10-13811.85.40770.53.5919-.055
Unaffected Cases
10-1339471.0.51291.0.49860.206
DS Detection at 5% FP Rate
Marker(s) +Age10-11 wk12-13 wk10-13 wks
Free-Beta43%47%45%
PAPP-A56%36%46%
Free-Beta + PAPP-A69%64%68%

DS detection efficiency was 72% using NT+Age and using NT+FreeBeta+PAPP-A+Age.
CONCLUSION: Biochemical markers alone detect 65-70% of DS cases in first trimester. The combination of biochemistry with NT measurement appears capable of detecting close to 90% of DS cases.




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48th Annual Meeting of The American Society of Human Genetics
October 27 - 31, 1998, Denver, Colorado


215_____ Mechanism of origin of chromosome duplications and deletions: Insight from patients with unique reciprocal duplication-deletion mosaicism. A.T. Tharapel1, G.V.N. Velagaleti1, R.C. Michaelis2, C.H. Laundon3, P.R. Martens1, P.D. Buchanan3, K.E. Teague4, S.A. Tharapel1, R.S. Wilroy, Jr1.. 1) Department of Pediatrics, University of Tennessee, Memphis, TN, USA.; 2) Greenwood Genetics Center, Greenwood, SC, USA.; 3) GeneCare Medical Genetics Center, Chapel Hill, NC, USA.; 4) Naval Medical Center, Portsmouth, VA, USA. Segmental aneusomy due to interstitial duplications and deletions for the same gene loci or chromosome regions is known to produce different phenoytypes, and is a significant factor unique mosaicism for reciprocal duplication- and deletion allowed us to further understand the origin of these abnormalities. The first patient with minor anomalies showed mosaicism for duplication-deletion cell lines: 46,XX,inv dup(11)(q23q13)/46,XX,del(11)(q13q23) in multiple tissues. In the second patient, cultured amniotic fluid cells and other tissues showed 46,XY,dup(7)(p11.2p13)/46,XY,del(7)(p11.2p13)/46,XY. Extensive cytogenetic and FISH analyses using a series of cosmid probes (INT2, sCRL1h6, MLL, c11q4b7) and YAC probes (y963-d-9, y966-e-4, y943-b-7, y961-b-12, y956-e-1) allowed us to document the reciprocal nature of the abnormalities. FISH studies on the first patient showed the duplicated region to be inverted and placed the breakpoints around the two folate-sensitive fragile sites FRA11A and FRA11B. The repeated sequences responsible for the fragile sites may have been involved in the patient's duplication-deletion. Our investigation leads us to conclude that in addition to the known mechanisms such as unequal cross-overs between homologs, unequal sister chromatid exchanges, excision of intrachromatid loops and meiotic recombination within a single chromatid - all ostensibly mediated by repeat sequences (STS) - duplication-deletions can simultaneously arise from loop formation and unequal cross-over at the level of single DNA strand.

264_____Detection of multiple chromosomal aberrations by whole genome microsatellite screening. M.J. Rosenberg1, C. Killoran1, Y. Ning2, A. Dutrah1, D. Vaske3, J. Weber3, L.G. Biesecker1, UMCAS research group. 1) National Human Genome Research Institute, NIH, Bethesda, MD; 2) GeneCare, Wilson Genetics laboratory, George Washington University Medical Center, Washington, DC; 3) Marshfield Medical Research Foundation, Marshfield, WI. Chromosomal aberrations are a common cause of multiple anomaly syndromes with developmental and growth retardation. It is clear from cytogenetic and molecular research that there is a wide spectrum of genomic alterations that can cause anomaly syndromes. A whole genome STRP scan using approximately 400 markers was performed on 17 children with multiple malformations and their parents. The resulting genotypes were used to infer non-Mendelian inheritance patterns including monosomy, trisomy and uniparental iso- and heterodisomy. We calculated the informativeness of our markers for detecting deletions and duplications as 75% and 60% respectively. Eleven children with undiagnosed multiple congenital anomaly syndrome were tested and aberrations were found in 3 of them. The microsatellite analysis indicated that one child has an 11-15 Mb interstitial deletion of 4q as well as a 0.6 Mb deletion on 1q, the second child has a 10Mb interstitial deletion of 19q and a third child has a 1-2 Mb duplication on 9p. Additional molecular testing indicates that this duplication is likely to be only 25-100 Kb. A repeat karyotype of lymphoblasts from child one confirmed the deletion of 4q28-q31.3. FISH analysis using a PAC probe containing the abnormal marker on 1q resulted in signals on both copies of chromosome 1 indicating that any deletion is likely to be less that 50Kb. We are currently using cytogenetic techniques to confirm our results for the other children. These results suggest that whole genome scanning for cytogenetic aberrations by molecular techniques may prove a valuable adjunct to microscopic cytogenetics. As marker density of a whole genome scan increases and genotyping cost decrease, this approach has potential utility for clinical diagnostic evaluations and for delineating the spectrum of chromosomal aberrations that is not biased by cytogenetic banding features.

932_____First trimester maternal serum screening for Down syndrome: Intact hCG vs freeBeta hCG. T. Hallahan1, D. Krantz1, E. Khabbaza1, F. Orlandi2, C. Rossi2, P. Curcio2, S. Marci1, J. Larsen3, P. Buchanan4, J. Marci1. 1) NTD Laboratories, Inc., Huntington Station, NY; 2) Centro Di Diagnosi Prenatale, Palermo, Italy; 3) The George Washington University, Washington, DC; 4) GeneCare Medical Genetics Center, Chapel Hill, NC. We analyzed intact hCG, freeBeta hCG and Pregnancy Associated Plasma Protein A (PAPP-A) in 63 cases of Down syndrome (35 liquid serum and 28 dried blood) and 400 unaffected control (200 liquid serum and 200 dried blood) first-trimester maternal serum specimens separately based on 50 unaffected control specimens/week for gestational weeks 10 to 13. The median multiple of the median (MoM) in 63 cases of Down syndrome was significantly higher (p-0.001) for freeBeta (1.9 MoM) than for intact hCG (1.3 MoM). Overall, 52% of DS cases were above the 90%-tile for freeBeta hCG compared to 30% for intact hCG. Combining with maternal age freeBeta hCG and intact hCG could detect 42% and 32% respectively at a fixed 5% false positive rate. Combining freeBeta hCG, PAPP-A and maternal age results in 3.8% false positive rate at a fixed 60% detection efficiency. Substituting intact hCG for freeBeta hCG resulted in a 24% increase in false positive rate (4.7%). In contrast with our results, a recent publication (Haddow et al. NEJM, Vol. 338; 955-961) reported that intact hCG performed better than freeBeta hCG (29% vs. 25% detection efficiency, respectively). However, our re-analysis based on the distribution parameters provided in the Haddow et al study shows that freeBeta actually was the better marker (23% detection for intact hCG vs. 29% for freeBeta hCG) even though the median intact hCG level in DS cases in the Haddow et al study (1.54 MoM) was higher that of a metanalysis of 15 previous studies (1.29 MoM). In conclusion, freeBeta + PAPP-A offers the best combination of first trimester biochemical markers detecting 65% of cases at a 5% false positive rate.

953_____Down syndrome screening with nuchal translucency: Correlation with biochemical and other maternal factors.J. Macri1, D. Krantz1, T. Hallahan1, F. Orlandi2, C. Rossi2, P. Curcio2, S. Macri1, P. Buchanan3, J. Larsen4. 1) NTD Laboratories, Inc., Huntington Station, NY; 2) Centro Di Diagnosi Prenatale, Palermo, Italy; 3) GeneCare Medical Genetics Center, Chapel Hill, NC; 4) The George Washington University, Washington DC. Nuchal translucency measurement during the first trimester of pregnancy is an effective screen for Down syndrome. FreeBeta hCG and PAPP-A are biochemical markers also effective in Down syndrome screening. In an effort to determine whether these biochemical and ultrasound markers could be combined we assessed correlation of nuchal translucency with freeBeta hCG, PAPP-A, maternal age and maternal weight. Data from 3,143 patients between 10 and 13 weeks of pregnancy were analyzed. Nuchal translucency, freeBeta hCG and PAPP-A results were adjusted for gestational age by converting to multiples of the gestational age specific medians (MOMs). The observed correlations with nuchal translucency are listed in the table below. With the exception of maternal age, none of the correlations with NT were statistically significant. However, the correlation of NT with maternal age is low and unlikely to be clinically significant. The data indicated that ultrasound assessment of Down syndrome risk is independent from the biochemistry and that NT values do not need to be adjusted for maternal weight or maternal age.

Factors Correlation (r) Significance (p)
NT vs FreeBeta hCG -0.009 0.6164
NT vs PAPP-A -0.017 0.3478
NT vs Maternal Age 0.041 0.0206
NT vs Maternal Weight 0.021 0.2396
Abstracts Continued
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