Prenatal Detection of De Novo Duplication of theShort Arm of Chromosome 18 Confirmed byFluorescence In Situ Hybridization (FISH)

Shibo Li,* Cathy M. Tuck-Muller, Jose E. Martınez, Ewellonda R. Rowley, Harold Chen, andWladimir WerteleckiDepartment of Medical Genetics, University of South Alabama, Mobile, Alabama

We present a patient with developmentaldelay, minor anomalies, and duplication 18pconfirmed by fluorescence in situ hybridiza-tion with whole chromosome 18 paintingprobe (Oncor p5218). Our observation con-firms the findings of other investigatorsthat duplication 18p is not associated withmajor malformations. Am. J. Med. Genet. 80:487–490, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: chromosome 18; duplication18p; fluorescence in situ hy-bridization (FISH); prenataldiagnosis

INTRODUCTION

Duplication of the short arm of chromosome 18 israrely reported [Moog et al., 1994]. Most patients re-ported either had an apparently normal phenotype orminor anomalies. Mental development ranged fromnormal to marked retardation. Most reported patientswere ascertained indirectly through family studies be-cause of monosomy or tetrasomy 18p in another rela-tive, most often due to an unbalanced familial translo-cation. Pure trisomy 18p has been reported in onlyseven previous patients [Moog et al., 1994; Takeda etal., 1989; Taylor et al., 1975; Wolff et al., 1991]. How-ever, underreporting of this abnormality is probablebecause the clinical phenotype is usually mild. We pre-sent a patient with duplication 18p initially detected byroutine cytogenetic analysis of amniocytes and con-firmed by fluorescence in situ hybridization (FISH) ofperipheral blood lymphocytes. Consistent with otherreports of pure 18p trisomy, the patient had only minoranomalies.

CLINICAL REPORT

A 36-year-old woman was referred for amniocentesisat 18 weeks of gestation because of advanced maternalage. Nine pregnancies from a previous partner had re-sulted in one healthy daughter, two miscarriages, andsix elective terminations. The pregnancy was compli-cated by maternal alcohol abuse. Genetic counselingwas provided, and the patient decided to continue thepregnancy and delivered a white male infant at 40weeks gestation by normal vaginal delivery. Birthweight was 3,200 g (50th centile), length 50.5 cm (50thcentile), and head circumference (OFC) 34 cm (25thcentile). Family history was noncontributory. Therewere no neonatal complications.

At age 8 months the developmental level was at 4months in every domain and the infant had hypotonia(Fig. 1), deep-set eyes, bilateral epicanthic folds, shortupturned nose, a faint hemangioma in the right malararea (1 × 1.5 cm), micrognathia, overturned helix bilat-erally, and redundant nuchal skin. A single transversecrease of the left palm was also noted. Growth param-eters were OFC 41.8 cm (<5th centile), length 71.5 cm(50th centile), and weight 7.7 kg (5th centile).

CYTOGENETIC ANALYSIS

The initial chromosome analysis was performed pre-natally and the results were confirmed by studies ofhigh-resolution methotrexate-synchronized peripheralblood lymphocytes [Yunis et al., 1978]. Karyotypicanalysis of the patient’s parents was performed on lym-phocytes. Chromosomes were analyzed by trypsin-Giemsa banding (GTG-banding) [Seabright, 1971] andby CBG banding [Sumner, 1972].

Analysis of the patient’s chromosomes from amnio-cytes and peripheral blood demonstrated an abnormalchromosome 18 with extra material in the short arm(Fig. 2). CBG banding indicated only one centromere inthe abnormal chromosome, and that the extra materialwas not heterochromatic (data not shown). The originof the additional chromosome material could not be de-termined with certainty by GTG-banding analysis. Noother chromosomes appeared to be involved in the re-arrangement. Parental chromosomes were normal.

Correspondence to: Shibo Li, M.D., Department of Medical Ge-netics, University of South Alabama, CCCB 214, 307 UniversityBoulevard, Mobile, AL 36688-0002. E-mail: sli@usamail.usouthal.edu

Received 30 April 1998; Accepted 17 August 1998

American Journal of Medical Genetics 80:487–490 (1998)

© 1998 Wiley-Liss, Inc.

FISH ANALYSIS

To determine the origin of extra chromosomal mate-rial, FISH analysis was performed on peripheral bloodlymphocytes using a biotinylated painting probe spe-cific for chromosome 18. The probe (COATA-SOME™18, p5218) was purchased from Oncor (Gai-thersburg, MD) and used according to the manufactur-er’s instructions with minor modifications. The probewas hybridized overnight and detected by FITC-labeled avidin with a single round of amplification of

antiavidin antibody. The slides were examined using aZeiss Axioskop microscope equipped with epifluores-cence.

The probe hybridized along the entire length of theabnormal chromosome, indicating that the extra mate-rial derives from chromosome 18 (Fig. 3). However, thisprobe does not give a homogenous signal for the entirelength of chromosome 18; the short arm fluorescesmore brightly than the long arm. The extra material inthe patient’s abnormal chromosome 18 was found tofluoresce brightly, giving indirect evidence that the ex-tra material derives from 18p. FISH analysis using ana-satellite probe hybridizing to chromosome 18 (D18Z1from Oncor used according to the manufacturer’s in-structions) confirmed the presence of a single centro-mere in the abnormal chromosome (data not shown).The karyotype was interpreted as 46,XY,add(18)(p11.3).ish dup(18)(p11.2p11.32)(wcp18+,D18Z1+)[ISCN, 1995].

DISCUSSION

Severe phenotypic abnormalities and considerablemental retardation are observed in most patients withautosomal duplications. However, this does not seem tobe the case with duplication of the short arm of chro-mosome 18, as demonstrated by reviews of previouslyreported cases [Moog et al., 1994; Takeda et al., 1989;Taylor et al., 1975; Wolff et al., 1991]. Approximately20 patients with dup(18p) have been reported, two-thirds resulting from unbalanced segregation of recip-rocal translocations and the remainder from duplica-tion of 18p. They presented with minor anomalies andlack of mental retardation. Facial anomalies, smallfemoral heads, and mental retardation, which are someof the phenotypic abnormalities found in these pa-tients, may have been due to the duplication or deletionof part of the long arm of chromosome 18 or, in cases of

Fig. 2. GTG-banded partial karyotype and idiogram showing the du-plicated chromosome 18 of the propositus. Arrow indicates the abnormalchromosome.

Fig. 1. Patient at 8 months. Note hypotonic face, deep-set eyes, epican-thic folds, short upturned nose (a); micrognathia and overturned helix (b).

Fig. 3. FISH analysis with COATASOME 18 total chromosome DNAprobe used to paint metaphase chromosomes from the patient. Note non-homogeneous painting of the chromosome 18 homologues with brighterfluorescence on the short arms than the long arms of both chromosomes.Arrowhead indicates normal chromosome 18; arrow indicates duplicatedchromosome 18.

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unbalanced reciprocal translocations, of another chro-mosome [Gardner et al., 1978; Johansson et al., 1988;Meinecke and Koske-Westphal, 1981; Sujansky andSmith, 1981].

Pure trisomy 18p has been reported previously inonly seven patients. Clinical findings range from nor-mal to various minor anomalies and mild mental re-tardation with no clear syndromatic phenotype. Twopatients, who were reportedly ‘‘normal’’ in appearanceand intelligence, were ascertained because of offspringwith 18p syndrome and/or tetrasomy 18p [Takeda etal., 1989; Taylor et al., 1975]. Wolff et al. [1991] re-ported on mother-to-child transmission of pure trisomy18p; both mother and child were physically and intel-lectually apparently normal. However, Moog et al.[1994] reported on a child with direct tandem duplica-tion 18p inherited from a mother who was mosaic forthe same chromosome abnormality. The child had milddevelopmental delay, moderate mental retardation,and slight craniofacial anomalies including deep-seteyes and small mouth. The mother was mildly men-tally retarded and microcephalic but was otherwisenormal. Moog et al. [1994] reported a 3-year-old girlwith a de novo inverted duplication of 18p, with a de-letion of 18p11.32, whose developmental level was nearlynormal and who had no physical anomalies apart fromhigh-arched palate and bilateral epicanthus.

Reevaluation of the G-banded chromosome 18showed that this duplication is likely due to a tandemduplication based on banding characteristics of chro-mosome 18p. FISH analysis using unique DNA mark-ers on chromosome 18p could confirm G-banded chro-mosome results. Unfortunately, parents were unwill-ing to cooperate in providing a further specimen fromthe propositus.

Thirteen genes have been mapped to the short arm ofchromosome 18 (Fig. 4). One of them, melanocortin 2receptor, (MC2R) (adrenocorticotropic hormone recep-tor) was found to be associated with familial glucocor-ticoid deficiency, an autosomal recessive disorder. The

remaining genes have not been determined to haveclinical significance. Cytogenetic analysis of duplica-tion 18p cases provides evidence that genes on theshort arm of chromosome 18 are unlikely to have amajor role in the pathogenesis of trisomy 18 syndrome.

Pittalis et al. [1994] reported a rare case of a hetero-chromatic heteromorphism on the short arm of chro-mosome 18 found in the amniocytes. This possibilitywas ruled out with our case by the CBG-banding tech-nique.

Pure 18p duplication apparently can present withminimal phenotypic manifestations. The explanationfor seemingly similar chromosome aberrations present-ing with varying phenotypes may be the presence ofdifferent submicroscopic chromosomal rearrange-ments, such as duplications or deletions. Our patient’smanifestations are in accord with previous observa-tions that pure trisomy 18p is not associated with ma-jor malformations. The contribution of maternal alco-hol abuse to the infant’s phenotype is unclear, althoughthe usual signs of fetal alcohol syndrome, with the ex-ception of developmental delay and microcephaly, werenot present. Because of the maternal history of alcoholabuse during pregnancy, our patient would probablynot have been karyotyped if not for the prenatal diag-nosis for advanced maternal age. This case introducesthe issue of whether all patients with minor anomaliesand developmental delay should be karyotyped evenwhen the phenotypic manifestations and the maternalhistory are suggestive for fetal alcohol syndrome. Fur-ther, this study illustrates the importance of FISHanalysis for confirmation of the origin of abnormalchromosomes, information that is critical for diagnosis,prognosis, and counseling issues.

ACKNOWLEDGMENTS

We thank Mae Jean Reeves for secretarial support,Lawrence Fletcher for photographic assistance, andQian Yan for technical assistance.

REFERENCES

Gardner RJM, Rudd NL, Stevens LJ, Worton RG (1978): Autosomal im-balance with a near-normal phenotype: The small effect of trisomy forthe short arm of chromosome 18. In Summitt RL, Bergsma D (eds):‘‘Sex Differentiation and Chromosomal Abnormalities.’’ New York:Alan R. Liss, Inc., for the National Foundation—March of Dimes.BD:OAS XIV (6C):359–363.

ISCN (1995): ‘‘An International System for Human Cytogenetic Nomen-clature.’’ F. Mitelman (ed.). Basel, Switzerland: S. Karger.

Johansson B, Mutens F, Palm L, Engelsson I, Kristoffersson U (1988):Duplication 18p with mild influence on the phenotype. Am J MedGenet 19:871–874.

Meinecke P, Koske-Westphal T (1981): Partielle trisomie 18 (trisomie 18p)als folge einer familiaren balancierten translokation t(14;18). Klin Pae-diatr 193:433–438.

Moog U, Engelen JJM, de Die-Smulders CEM, Albrechts JCM, LoneusWH, Haagen AAM, Raven EJM, Hamers AJH (1994): Partial trisomy ofthe short arm of chromosome 18 due to inversion duplication and directduplication. Clin Genet 46:423–429.

Pittalis MC, Santarni L, Bovicelli L (1994): Prenatal diagnosis of a hetero-chroatic 18p+ heteromorphism. Prenat Diagn 14:72–73.

Fig. 4. Genes mapped on the short arm of chromosome 18. TYMS (thy-midylate synthetase); LAMA1 (laminin, alpha 1); HPE4 (holoprosen-cephaly 4); YES1 (v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1);NDUFV2 (NADH dehydrogenase (ubiquinone) flavoprotein 2 (24kD));PTPN2 (protein tyrosine phosphatase, nonreceptor type 2); ERV1 (endog-enous retroviral sequence 1); EIF4A2 (eukaryotic translation initiation fac-tor 4A, isoform 2); MC2R (melanocortin 2 receptor); MC5R (melanocortin 5receptor); PTPRM (protein tyrosine phosphatase, receptor type, mu poly-peptide); ADCYAP1 (adenylate cyclase activating polypeptide 1 (pitu-itary)); MAFD1 (major affective disorder 1).

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Seabright M (1971): A rapid banding technique for human chromosomes.Lancet, 2:971–972.

Sujansky E, Smith ACM (1981): Recombinant chromosome 18 in two malesibs with first and second brachial arch syndrome. Am J Hum Genet33:92A.

Sumner AT (1972): A simple technique for demonstrating centromeric het-erochromatin. Exp Cell Res 75:304–306.

Takeda K, Okamura T, Hasegawa T (1989): Sibs with tetrasomy 18p bornto a mother with trisomy 18p. J Med Genet 26:195–197.

Taylor KM, Wolfinger HL, Brown MG, Chadwick DL (1989): Origin of asmall metacentric chromosome: Familial and cytogenetic evidence.Clin Genet 8:364–369.

Wolff DJ, Raffel LJ, Ferre MM, Schwartz S (1991): Prenatal ascertainmentof an inherited dup(18p) associated with an apparently normal pheno-type. Am J Med Genet 41:319–321.

Yunis JJ, Sawyer JR, Ball DW (1978): The characterization of high-resolution G-banded chromosomes of man. Chromosoma 67:293–307.

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