American Journal of Medical Genetics 103:320±325 (2001)

Molecularly De®ned Interstitial Tandem Duplication6p Case With Mild Manifestations

David Ng,1 Philip Mowrey,2 Jiannis Ragoussis,4 Ghazala Mirza,4 Edward Coll,5 Marc P. Di Fazio,3

Clesson Turner,5 and Sondra W. Levin1,5*1Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland2Department of Cytogenetics, LabCorp, Research Triangle Park, North Carolina3Department of Neurology, Walter Reed Army Medical Center, Washington, DC4Division of Medical Molecular Genetics, GKT Medical School, King's College, London, England5Department of Pediatrics, Walter Reed Army Medical Center, Washington, DC

An interstitial tandem duplication of 6p21.1-p22.2 was found in a girl at 11 months of agewhen she was evaluated for developmentaldelay. Previous cases reported with partial6p duplication usually have involved term-inal duplications, with breakpoints rangingfrom 6p11 to 6p25. Our patient exhibits amilder phenotype compared to the pre-viously reported cases in the literature.Features that she has in common withthe other cases include craniofacial anoma-lies, such as broad nasal bridge and bulboustip, thin lips, incomplete development of thescapha helix bilaterally, mild spastic para-paresis of the lower extremities, gross motordelay, and mild cognitive delays.ß 2001 Wiley-Liss, Inc.

KEY WORDS: pure 6p duplication; FISH;developmental delay

INTRODUCTION

A recent review of duplications of chromosome 6p[Villa et al., 2000] is notable for demonstrating thatmost reported cases of trisomy 6p are, in fact, due tochromosomal translocations. Consequently, such tris-omy 6p cases are also partially monosomic or trisomicfor other regions as well. Indeed, only one reported case[Villa et al., 2000] shows an isolated interstitial tandem

duplication of (6)(p12-p21.3), while other 6p trisomiesinvolve a larger region of 6p up to and including 6pter.Herein we report a patient with de novo 6p trisomywhose duplication of 6p21.1-p22.2 is unique. We alsodescribe specialized chromosome 6 probes that havebeen used to re®ne further the breakpoints of thisnewly described duplication, such that the karyotypeas originally described can now be designated as46,XX, dup(6)(p21.1-p22.2).ish dup(6)(wcp6�, D6S299/D6S1691�, D6S276��, D6S1549��, D6S1604�).

Clinical Report

The patient was born at 37 weeks gestation bynormal spontaneous vaginal delivery to a then G2P124-year-old mother and a 28-year-old father. Her birthweight was 2858 g (50th±75th centile); length, 47.5 cm(50th±75th centile); and head circumference (OFC),34 cm (75th centile). There were no reported prenatal orperinatal complications. Newborn screening for T4,PKU, and galactosemia were reported to be normal.Early in infancy she had problems feeding, necessitat-ing several switches in formula. Frequent episodes ofotitis media occurred resulting in PE tube placementat 9 months of age. By 11 months of age, her parentswere concerned with her delay in attainment of grossmotor milestones. Chromosomal analysis at thistime revealed 46,XX, dir dup(6)(p21.1-p22.2). Parentalchromosomes were normal. Growth has proceeded atthe 10th centile for height and weight and the 50thcentile for head circumference. Speech commenced at18 months of age. A formal developmental assessmentat 7 4/12 years of age was notable for mild to borderlinede®cit of language skills and moderate dif®culties with®ne and gross motor skills. The patient has beenenrolled in a prekindergarten early intervention pro-gram. Formal psychological assessment (Wechsler Pre-School and Primary Scale of Intelligence-Revised) atschool reveals her current functioning at the EducableMentally Disabled (EMD) category both intellectuallyand academically. A lack of attention, hyperactivity,and need for redirection has been observed at home andat school and veri®ed by testing using the Behavior

The opinions or assertions contained herein are the privateviews of the authors and are not to be construed as of®cial or asre¯ecting the views of the Department of the Army or theDepartment of Defense.

*Correspondence to: Sondra W. Levin, M.D., Chief, GeneticsSection, Department of Pediatrics, Building 41, Walter ReedArmy Medical Center, Washington, DC 20307-5001.E-mail: sondra.levin@na.amedd.army.mil

Received 17 January 2001; Accepted 14 June 2001

DOI 10.1002/ajmg.1577

ß 2001 Wiley-Liss, Inc.

Assessment System for Children. There has beenneither aggressive behavior nor regression of mile-stones noted to date.

The patient has remained in good health. She hasrequired surgery to replace a ¯exor tendon of the righthand and underwent a tonsillectomy to alleviate loudsnoring and apnea. She has recurrent chronic lumbarspine pain of uncertain etiology. A head and lumbarspine magnetic resonance imaging (MRI) has beenunrevealing. She continues to have dif®culty with trip-ping secondary to tight heel cords. No surgical inter-vention for the tight heel cords has been recommendedat this time. Screening audiogram and ophthalmologicexam has been normal. Renal ultrasound reveals nostructural abnormality.

Symptoms of hyperactivity and attention de®cit haveincreased over the past 2 years despite attempts attreatment with adderall and ritalin. Extremely poorconcentration remains the biggest issue at school. Shecannot write letters or numbers nor can she read thealphabet consistently. During school she receivesphysical therapy for ambulation, occupational therapyto help relax her chronic clenched ®sts, speech therapyfor hyper-nasal speech, and extra tutoring for readinginstruction. She is currently in a second-grade class forchildren with special education needs.

Physical exam at age 7 4/12 years is notable for pro-minent forehead, slightly up-slanted palpebral ®ssures,tubular nose with bulbous tip, microstomia, thin lips,normally set ears with incomplete development ofthe scapha helix bilaterally, high narrow palate withnormal uvula, micrognathia, crowded carious teethwith an overbite, slight pectus excavatum, 2.3 cmelliptical cafeÂ-au-lait spot on the right lower quadrantof the abdomen, normal palmar creases, tapered®ngers, surgical scar over the dorsal surface of theright second metacarpophalangeal joint, bilateral ®fth®nger clinodactyly, bilateral tight heel cords with higharches of both feet, and long second toes bilaterally. SeeFigures 1±4. Cranial nerves are intact. Muscle toneand bulk are normal in the upper extremities. Thedistal portions of the lower extremities have increasedtone. Patient has a normal walk and tends to toe runand trips on stooping to recover objects. The remainderof the exam is unremarkable except for a heart murmurdetermined to be functional based on a recent normalechocardiogram exam.

METHODOLOGY

Initial cytogenetic analysis was performed at 1 yearof age and repeated at 5.5 years of age. Peripheral bloodlymphocytes were studied after phytohemagglutinin(PHA) stimulation and giemsa-trypsin-wright (GTW)banding.

Metaphase chromosome preparations were readiedfor ¯uorescence in situ hybridization (FISH) analysisusing a ®xed suspension remaining from the cytoge-netics preparation. Whole-chromosome paint for chro-mosome 6 (Coatasome, Oncor, Inc., Gaithersburg, MD)painted the entire length of the normal chromosome 6and abnormal chromosome 6.

DNA was extracted and puri®ed as recommended bythe HGMP Resource Centre. All clones were labeledwith biotin-dATP or digoxigenin-11-dUTP by nicktranslation (Bio-Nick Labeling System or Nick Trans-lation System, respectively (BRL Life Technologies,Carlsbad, CA). In situ hybridization was performed aspreviously described [Davies et al., 1996, 1999].

In total, 16 P1-derived arti®cial chromosome (PAC),1 bacterial arti®cial chromosome (BAC), and 2 cosmidclones were used (Table I) that had been previouslyFISH-mapped to de®ned locations on 6p or containedwell-established genetic markers. The PACs dJ180E22,dJ261G23, dJ75E20, dJ202121, dJ207H1, dJ288E7,dJ97D16, dJ103C14, dJ126C21, dJ8G21, dJ217E5, anddJ59B16 were selected from the Sanger Centre data-base due to their reported cytogenetic location and/orde®ned genetic marker content (see Table I). The clonesdJ50J22, dJ178D20, and dJ99J17 and the BACclone b121L5 are taken from Tripodis et al. [2000].PAC clone dJ45P21 was reported by Lauer et al. [1997].The cosmids TN82 and E913, mapping within the MHCclass III region, were kindly donated by Dr. R.D.Campbell (UK's HGMP Resource Centre, Hinxton,Cambridge, UK).

Fig. 1. Side pro®le of proband showing prominent forehead, incompletedevelopment of the scapha helix, and micrognathia.

Molecularly De®ned 6p Duplication 321

RESULTS

In each cell evaluated, one chromosome 6 wasobserved to have extra material in the middle of theshort arm region (Fig. 5). The extra material appearedto result from a partial duplication of the chromosomematerial between bands 21.1 and 22.2 of the short arm.The karyotype was determined to be 46,XX, dirdup(6)(p21.1-p22.2).ish dir dup(6)(wcp6�). Parentalchromosomes were normal.

In order to de®ne the duplicated region by FISH, weused bacterial clones mapping to the short arm ofchromosome 6. Nineteen clones mapping to the 6p21.1-6p22.2 interval were used to further de®ne the break-points in the duplicated segment. The order of signalsfrom the duplicated region con®rmed that this is atandem duplication (Table I). Probes that gave only onesignal de®ned the breakpoints of the duplication. Thesewere dJ180E22 and dJ261G23, marking the proximalpart of 6p that is not duplicated, and dJ59B16 for theunduplicated part of 6p22.1-p22.2. At the telomericend, we de®ned the breakpoint as being in the regionbetween the markers D6S276 (dJ217E5/dJ8G21) and

D6S299/D6S1691 (dJ59B16), which is positioned imme-diately telomeric of D6S276. The distance betweendJ59B16 and dJ217E5/dJ8G21 is less than 500 kb.These two markers are present within the 1.3 MbpYAC clone y874b3 [Lauer et al., 1997], indicatingtheir close proximity. At the centromeric end, thebreakpoint is located between the clone dJ75E20,positioned at genetic marker D6S1549 in 6p21.1, andclone dJ261G23, containing D6S1604, mapped in6p12.3. The distance between the two genetic markersis 3 cM [Dib et al., 1996], and the breakpoint can bepositioned in this interval. Based on these results,the expanded karyotype designation is 46,XX, dup(6)(p21.1p22.2).ish dup(6)(wcp6�, D6S299/D6S1691�,D6S276��, D6S1549��, D6S1604�).

Overall the FISH results show that the entire majorhistocompatability complex (MHC), including theextended MHC, is duplicated. There are 100 genesduplicated in the MHC region alone. The extent ofthe duplication can be approximated as containing theentire 4 Mbp of the extended MHC (telomeric side),the 4 Mbp of the MHC itself, and the 2.5 Mbp distancebetween MHC and dJ50J22 [Tripodis et al., 2000], inaddition to the approximately 20 Mbp from the 6p12.3-p21.2 region (calculated from the genetic distance ofDib et al. [1996]).

Fig. 2. Front pro®le of proband showing prominent forehead, slightlyup-slanted palpebral ®ssures, tubular nose with bulbous tip, microstomia,and thin lips.

Fig. 3. Proband's feet with bilateral elongated second toes.

Fig. 4. Proband's hands with bilateral ®fth ®nger clinodactyly.

322 Ng et al.

DISCUSSION

Isolated interstitial tandem duplication of 6p is a rareoccurrence with only one other case noted so far in theliterature by Villa et al. [2000]. In addition, Phelan et al.[1986] described a case with terminal duplication from6p21-pter. There are two cases reported with distal 6pduplication resulting from an inverted insertion of 6pmaterial into an acrocentric chromosome short arm. Ineach case the child received the derivative acrocentricchromosome and therefore had partial trisomy 6p[Chiyo et al., 1975; Morton et al., 1980]. In general,these larger segmental duplications seem to cause amore severe phenotype with associated prenatal and/orpostnatal growth retardation, in utero demise, increa-sed number of facial anomalies, seizures, and severemental retardation.

Duplication of the MHC region has been previouslyreported by Pearson et al. [1979] and Morton et al.[1986]. Human leukocyte antigen surface expressionstudies on lymphocytes ®nd that the duplicated genesare co-expressed. Neither case seemed to have alteredimmune function as a result of gene dosage imbalance.Thus, it appears that duplication of approximately 100genes in this segment is clinically silent.

Small duplications of chromosomal segments are animportant evolutionary mechanism for the develop-ment of extra copies of genes that later may acquirealtered functional activity due to decreased selectionpressure on the duplicated genes. Once a duplication isestablished, additional unequal crossing over betweenthe homologous regions can lead to further duplication,excision, or dispersion of the gene cluster. As a result,structurally related genes and pseudogenes may beformed. This has led to the development of genefamilies such as the hemoglobin and haptoglobingene cluster with now evolutionary divergent func-tions. Interstitial tandem duplication as seen in thiscase occurs as a de novo phenomenon arising frommismatched pairing of homologs, or between thechromatids of one homolog followed by recombinationor unequal sister chromatid exchange during meioticrecombination. A direct duplication results when thelinear orientation of the chromosome is maintained.Direct duplications have also been observed in themosaic state, suggesting that it may arise duringpostzygotic mitosis, in addition to meiotic recombina-tion. In some known microdeletion disorders, such asSmith-Magenis syndrome [Chen et al., 1997], a hot spotfor chromosomal recombination and unequal crossingover occurs as a result of ¯anking repeat gene clusters.Unequal crossing over results in either a deletion orduplication of the intervening segment.

Most cases of 6p duplication include aneuploidies ofother chromosomes involved with the reciprocal trans-location. Thus, features such as congenital heartdefects and renal anomalies may be due to theseadditional aneuploidies, as suggested by Villa et al.[2000]. A comparison of our case with that of Villa et al.[2000] (Table II) reveals that a smaller tandemduplication of 6p21.1-p22.2, compared to 6p12-p21.3,does modify and decrease the severity of the facialdysmorphism and mental/psychomotor retardation.Indeed, the child described by Villa et al. [2000] diedat age 16 months. The smaller gene dosage imbalancedocumented for our patient apparently obviates thedevelopment of gross malformation of the brain, genitalanomalies, and seizures. Features that our patient hasin common with that of Villa et al. [2000] includemental/psychomotor retardation, early feeding dif®cul-ties, prominent forehead, abnormal folding of the ears,broad nasal bridge with a bulbous nasal tip, smallmouth with thin lips, high arched palate, and micro-gnathia. However, our patient has much mildercognitive delay and normal somatic growth, comparedto the case of Villa et al. [2000], thereby improving ourpatient's overall prognosis. Without precise delineationof the breakpoints and con®rmation of the linear genecontent in the duplicated segment in the case reported

TABLE I. List of Nineteen Probes With Cytogenetic LocationUsed to De®ne the Breakpoints and Duplicated Segment

ProbeRegion of

hybridization Results

dJ180E22 6p12.3 Not duplicateddJ261G23 6p12.3 (D6S1604) Not duplicateddJ75E20 6p21.1 (D6S1549) DuplicateddJ202121 6p21.2 DuplicateddJ207H1 6p21.1-21.3 DuplicateddJ288E7 6p21.1-21.2 DuplicateddJ50J22 6p21.2 DuplicateddJ178D20 6p21.2 Duplicatedb121L5 6p21.2 DuplicateddJ99J17 6p21.2-21.3 DuplicatedTN82 6p21.3 DuplicatedE913 6p21.3 DuplicateddJ45P21 6p21.3 DuplicateddJ97D16 6p21.3 (D6S105) DuplicateddJ103C14 6p21.3-22.1 DuplicateddJ126C21 6p22.1 (W 3878) DuplicateddJ8G21 6p22.2 (D6S276) DuplicateddJ217E5 6p22.2 (D6S276) DuplicateddJ59B16 6p22.2-22.3

(D6S299/1691)Not duplicated

Fig. 5. Ideogram and cytogenetic photograph of normal chromosome 6on left and ideogram of chromosome 6 with tandem duplication of 6p21.1-p22.2 alongside a cytogenetic photograph of chromosome 6 with tandemduplication of 6p21.1-p22.2 on right.

Molecularly De®ned 6p Duplication 323

by Villa et al. [2000], it is dif®cult to ascertain likelycandidate genes that may contribute to the phenotypicdifferences that have been documented between thetwo cases. Delineation of the breakpoints with probesre®nes the actual boundary in physical distance sothat genes within and bordering that segment can bestudied for evidence of disruption or gene dosageeffects. Once the interval in physical distance isde®ned, evaluation of the nucleotide sequence in thevicinity may help identify repeat gene clusters thatincrease the propensity for unequal crossing over,leading to duplication or deletion in that chromosomalregion. With ascertainment of the proximal and distalbreakpoints using molecular probes, additional docu-mented cases of pure 6p duplication may help de®negenotype-phenotype correlation and possibly aid in

the prognostication of the clinical severity of futurepatients with 6p duplication.

REFERENCES

Chen KS, Manian P, Koeuth T, Potocki L, Zhao Q, Chinault AC, Lee CC,Lupski JR. 1997. Homologous recombination of a ¯anking repeat genecluster is a mechanism for a common contiguous gene deletionsyndrome. Nat Genet 17:154±163.

Chiyo H, Kuroki Y, Matsui I, Yanagida K, Nakagome Y. 1975. A 6p trisomydetected in a family with a ``giant satellite.'' Humangenetik 30:63±67.

Davies AF, Olavesen MG, Stephens RJ, Davidson R, Delneste D, VanRegemorter N, Vamos E, Flinter F, Abusaad I, Ragoussis J. 1996.A detailed investigation of two cases exhibiting characteristics of the 6pdeletion syndrome. Hum Genet 98:454±459.

Davies AF, Mirza G, Sekhon G, Turnpenny P, Leroy F, Speleman F, Law C,van Regemorter N, Vamos E, Flinter F, Ragoussis J. 1999. Delineationof two distinct 6p deletion syndromes. Hum Genet 104:64±72.

TABLE II. A Comparison of the Phenotype Between the Case Reported by Villa et al. [2000] andthe Present Case

Duplicated segmentVilla et al. [2000]

p12-p21.3Present casep21.1-p22.2

Sex Male FemaleGestational age (weeks) 36 37Birth weight (g) 2,490 2,858Age at exam 7 months 7 yearsDeath age 16 months Still livingMental retardation/psychomotor delay � �Pre-and/or postnatal growth retardation � ±Microcephaly � ±Abnormal sutures � ±Prominent occiput � ±High/prominent forehead ± �Small/short palpebral ®ssures � ±Long eyelashes � ±Epicanthus � ±Blepharophimosis/blepharoptosis � ±Strabismus ± ±Cataract/corneal clouding � ±Low-set and or abnormal ears � �Broad/high nasal bridge � �Hooked/bulbous nose � �Anteverted nares � ±Choanal atresia � ±Long/smooth philtrum � �Small mouth and/or thin lips � �High arched palate � �Micrognathia � �Short limbs � ±Upper and lower limb deformities ± ±Talipes � ±Syndactyly of ®ngers and/or toes ± ±Abnormalities of ®ngers and/or toes � �Scoliosis ± ±Sacral dimple ± ±Genital anomalies � ±Congenital heart defects ± ±Brain anomalies � ±Renal anomalies ± ±Gastrointestinal anomalies ± ±Inguinal hernia � ±Recurrent respiratory infections � ±Hypertonia � �Seizures � ±

�, present; ±, absent.

324 Ng et al.

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Lauer P, Meyer NC, Prass CE, Starnes SM, Wolff RK, Gnirke A. 1997.Clone-contig and STS maps of the hereditary hemochromatosis regionon human chromosome 6p21.3-p22. Genome Res 7:457±470.

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Morton CC, Brown JA, Kirsch IR, Evans GA, Mohanakumar T, Nance WE.1986. Detection and localization of an extra HLA locus in a

karyotypically normal male by chromosomal in situ hybridization.Clin Genet 29:62±72.

Pearson G, Mann JD, Bensen J, Bull RW. 1979. Inversion duplication ofchromosome 6 with trisomic codominant expression of HLA antigens.Am J Hum Genet 31:29±34.

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Tripodis N, Palmer S, Phillips S, Milne S, Beck S, Ragoussis J.2000. Construction of a high-resolution 2.5-Mb transcript map of thehuman 6p21.2-6p21.3 region immediately centromeric of the majorhistocompatability complex. Genome Res 10:454±472.

Villa A, Gomez EG, Rodriguez L, Rastrollo RH, Tallo MEM, Martinez-FriasM.2000. Interstitial tandemduplication of6p: a casewithpartial trisomy(6)(p12p21.3). Am J Med Genet 90:369±375.

Molecularly De®ned 6p Duplication 325