American Journal of Medical Genetics 132A:206–208 (2005)

Clinical ReportDown Syndrome With Pure Partial Trisomy 21q22 due to aPaternal Insertion (4;21) Uncovered by UnculturedAmniotic Fluid Interphase FISHJ. Lee,1 J.R. Stanley,2 S.A. Vaz,2 J.J. Mulvihill,1 P. Wilson,1 D. Hopcus-Niccum,1 and Shibo Li1*1Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma2Prenatal Assessment Center, Department of Obstetrics/Gynecology, University of Oklahoma Health Sciences Center,Oklahoma City, Oklahoma

Objective: To emphasize the usefulness and relia-bility of fluorescence in situ hybridization (FISH)on uncultured amniotic fluid cells in the prenataldiagnosis of common chromosomal aneuploidies.Methods: FISH analyses utilizing centromeric,locus-specific or whole chromosome paint DNAprobes specific for chromosomes X, Y, 13, 18, 21,and 4 were performed on uncultured amnioticfluid cells or the peripheral blood specimenfrom the father. Routine chromosome analysiswas carried out as well. Results: A prenatalcase with partial trisomy 21 due to a paternalcryptic insertion (4;21) was ascertained by arapid overnight FISH on uncultured amnioticfluid cells. The fetus was delivered at term andhad classical features of Down syndrome. Conclu-sion: Our results stress the importance of FISH onuncultured amniotic fluid cells to supplementroutine cytogenetics, especially in cases withabnormal ultrasound findings.� 2004 Wiley-Liss, Inc.

KEY WORDS: prenatal diagnostics; Down syn-drome; partial trisomy 21; inter-phase FISH

INTRODUCTION

FISH analysis to screen for aneuploidies of chromosomes X,Y, 13, 18, and 21 on uncultured fetal cells utilizing eithercommercial or ‘‘home brew’’ DNA probes is becoming astandard test in some cytogenetic laboratories despite the rareoccurrence of false-positive or false-negative cases [Eiben et al.,1999; Tepperberg et al., 2001; George et al., 2003]. Withoutdoubt FISH is a useful adjunct technique because it quicklyand reliably detects the common fetal chromosomal aneuploi-dies; the results are available in fewer than 24 hr instead of the7–14 days of standard techniques [Li et al., 2002]. Here wepresent a prenatal case with partial trisomy 21q22 due to anunbalanced form of a cryptic insertion (4;21)(q21;q22.1q22.3)inherited from the father, who is a carrier. Routine karyotyp-ing might have missed the abnormal chromosome. The fetuswas delivered at term and had classical features of Downsyndrome.

CASE REPORT

A 32-year-old Caucasian patient (gravida 4 para 0121) wasreferred to the Prenatal Assessment Center at the Universityof Oklahoma Health Sciences Center because of a positivescreen for Down syndrome, a greater than 1:10 risk by mater-nal serum quad screen. The patient had a targeted ultrasoundperformed and her due date was changed by 15 days. The quadscreen, recalculated using the new information, decreased therisk estimate to 1:17. The ultrasound examination also showeda moderately thick nuchal fold of 0.58 cm (normal range¼0.6 cm). Both the patient and her husband were counseledregarding the implication of the findings. The patient wasoffered the option of an amniocentesis with FISH testing, butthis testing was declined at the first visit. The patient returned3 weeks later and elected amniocentesis. The nuchal foldcontinued to remain borderline thick at 0.56 cm at 18 weeksand 4 days. The femurs measured 16 weeks and 6 days (2.3 cm),which is short for gestational age. The couple was informedabout the abnormal cytogenetic findings and elected tocontinue the pregnancy. The fetus was delivered at term andthe newborn baby had classical features of Down syndrome,including brachycephaly, upward slanting palpebral fissures,and a depressed nasal bridge. At the age of 5-month, the babygirl was evaluated by a clinical geneticist at our medical center.Height was 56.4 cm, weight was 5.1 kg, and occipitofrontalcircumference was 40.1 cm. On the Down syndrome curvesthese measurements were at 40th, 15th, and 50th centiles,respectively. Head and skull anomalies included a flattenedocciput, a 6� 6 cm anterior frontanelle, a closed posteriorfontanelle, and redundant skin on the neck. The eyes had eccen-tric pupils, upslanted palpebral fissures, lateral displacement of

Fig. 1. Photograph of the baby girl with typical facial features of Downsyndrome at the age of 5-month.

*Correspondence to: Shibo Li, Department of Pediatrics,OUHSC, BSEB 224, Oklahoma City, OK 73104.E-mail: Shibo-li@ouhsc.edu

Received 1 March 2004; Accepted 16 September 2004

DOI 10.1002/ajmg.a.30449

� 2004 Wiley-Liss, Inc.

the inner canthus, and no epicanthal folds. Palpebral fissurelength was at �2 standard deviations. The nose was short andupturned. The philtrum was normally formed as was the upperlip. The palate was highly arched, and the bifid uvula and bulky.The tongue was normal and did not protrude. Chest configura-tion was normal except for inverted, hypoplastic, and low spaced

nipples. Lung sounds were clear. There was no heart murmur.Echocardiogram showed VSD. There was bilaterally brachycli-nodactyly of the fifth digits with close flexion phalangealcreases, and a single transverse palmar crease on the righthand and a Sydney line on the left hand. She was slightlyhypotonic. There were dimples over both elbows (Fig. 1).

Fig. 2. A: A representative nucleus from the uncultured amniotic fluidcells showing three clear hybridization signals of LSI 21 (orange) and twosignals of LSI 13 (green). B: Probes LSI 21 (orange) and LSI 13 (green) wereused to hybridize to a metaphase of amniotic fluid cell cultures. C: Aninsertion of chromosome 21 to chromosome 4q was confirmed by hybridiza-tion of a metaphase cell of the amniotic fluid culture using a combination ofchromosome 4 paint probe (orange) and chromosome 21 paint probe (green).

D: The AML1 gene probe was hybridized to a metaphase cell from the fatherand revealed that one of the AML1 gene signals was present on thederivative chromosome 4. E: The 21q subtelomeric probe was present onboth the normal and the derivative chromosomes 21 in the father’s specimen.F: Partial karyotypes from both the fetus (top panel) and the father(bottom panel).

Down Syndrome With Pure Partial Trisomy 21q22 207

FISH AND CYTOGENETIC STUDIES

An overnight FISH analysis was performed on unculturedamniotic fluid cells with the AneuVysion1 assay kit (DownersGrove, Illinois, USA), a combination of centromere probes ofchromosome X, Y, and 18, and locus-specific probes specific forchromosome 13 (LSI 13, 13q14) and 21 (LSI 21, 21q22). A locus-specific probe, AML1, a subtelomeric probe of 21q, and wholechromosomal paint probes specific for chromosomes 4 and 21were also used on the specimens of the long term back upculture collected from amniotic fluid cells as well as the father’speripheral blood sample. All the probes were purchased from acommercial source (Vysis, Downers Grove, IL) and usedaccording to the manufacturer’s protocols with minor mod-ifications. Routine chromosome analysis was performed onchromosomes derived from amniotic fluid cells as well asperipheral blood from both parents. Chromosome preparationswere treated and stained by trypsin-Giemsa banding (GTG-banding) (Seabright, 1973).

RESULTS AND DISCUSSION

An overnight FISH analysis and routine chromosomeanalysis were requested because of the positive biochemicalscreen for Down syndrome as well as abnormal ultrasoundfindings. The initial FISH analysis performed utilizing theAneuVysion1 assay kit on uncultured amniotic fluid cellsrevealed that all 30 cells analyzed had three signals of LSI 21,supporting the conclusion that the fetus has Down syndrome(Fig. 2A). Seven days after the preliminary report was given,routine chromosome analysis of the long-term in situ culturesrevealed the unexpected finding that all cells analyzed had amodal number of 46 chromosomes, including 2 X chromosomesand 10 normal acrocentric chromosomes, including 2 normalchromosomes 21. In other words, neither an extra chromosome21, nor a Robertsonian translocation involving chromosome21 was found. Instead, in some cells with higher resolutionmetaphases, one of the chromosomes 4 at the q21 region had arelatively larger pale area than the other homologue ofchromosome 4, which suggested that this pale material couldcome from chromosome 21q (Fig. 2F, top panel). To confirm thefindings, FISH analysis of the cultured cells utilizing the locusspecific probes LSI 13/LSI 21 (Fig. 2B) and whole paint probesof chromosome 4 (orange) and chromosome 21 (green) (Fig. 2C)showed that the extra chromosomal material was indeedderived from chromosome 21. Chromosomal analysis of bothparents revealed that the father carried an apparentlybalanced insertion (4;21) (Fig. 2F, bottom panel). To furtherdetermine the size and the breakpoint of the inserted chromo-somal 21 segment, FISH analysis utilizing a locus-specificprobe, AML1, and a 21q subtelomeric DNA probe wasperformed on metaphases from the father’s blood. One of theAML1 gene signals was clearly visualized on the derivativechromosome 4 (Fig. 2D). The 21q subtelomeric probe waspresent on both the normal and the derivative chromosomes21 in the father’s specimen (Fig. 2E). These findings allow arough estimate of the minimum size of the inserted chro-mosomal segment 21q. Since the distance between LSI21(D21S259, D21S341, and D21S342) and AML1 is under 6Mb, at least 5 Mb were duplicated in this case (http://gdbwww.dkfz-heidelberg.de). This estimate exceeds by threefolds the reported size of the ‘‘critical region’’ of chromosome 21for phenotype of Down syndrome, which is 1.6 Mb [Shibuyaet al., 2000]. The final karyotype of the fetus was 46,XX,der(4)-ins(4;21)(q21;q22.13q22.2)pat.

Genetic counseling was provided to the couple regarding theimplications of the laboratory findings, and the couplecontinued the pregnancy. The baby, with typical features of

Down syndrome as expected, was delivered without complica-tion at 40 weeks’ gestation. At birth, the newborn baby hadbrachycephaly with a relatively flat occiput, upslanting palpe-bral fissures, and a small nose with low nasal bridge.

Down syndrome due to partial trisomy of chromosome 21 israre. The majority of cases reported had tandem duplications orunbalanced translocations [Korenberg et al., 1990; Delabaret al., 1993; Garcia-Heras and Rao, 1999; Foster-Gibson et al.,2001]. To our knowledge, an insertion of 21q22 causing Downsyndrome has never been reported. One implication of thisfinding is that some patients with typical or even atypical fea-tures of Down syndrome may have an insertion or an unbalanc-ed translocation leading to the duplication of the chromosome 21‘‘critical region’’ for phenotypes of Down syndrome.

This case clearly demonstrates the critical value andreliability of FISH on uncultured amniotic fluid cells in theprenatal diagnosis of common chromosomal aneuploidies.Without knowing of the presence of three signals of 21q thediagnosis could have been missed. Alternatively, if the extrachromosomal material was detected, the origin would havebeen very difficult to determine without resorting to M-FISHwhich is difficult to perform in prenatal cases. Also, the authorsare aware that additional observations of similar cases arerequired to clarify the prognosis for fetuses and children withpartial trisomy 21q.

ACKNOWLEDGMENTS

The authors thank Kelley V. Scott for assistance in thepreparation of the manuscript.

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