Usefulness of high resolution comparativ

American Journal of Medical Genetics 113:125– 136 (2002) Usefulness of High-Resolution Comparative Genomic Hybridization (CGH) for Detecting and Characterizing Constitutional Chromosome Abnormalities Gro Oddveig Ness, Helle Lybæk, and Gunnar Houge* Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway Comparative genomic hybridization (CGH) is a technique for detection of chromosomal imbalances in a genomic DNA sample We here report the application of the recently developed method of high-resolution CGH on DNA samples from 66 children having various degrees of delayed psychomotor development with or without clear dysmorphic features and congenital malformations In of 50 patients with apparently normal karyotypes, a deletion or duplication was revealed by CGH Only one of these cases had a subtelomeric rearrangement In one of seven cases with a de novo apparently balanced translocation, deletions were found In all nine cases where the origin of a marker chromosome or additional chromosomal material was difficult to determine, CGH gave a precise identification The following findings were from cases having a deletion or duplication as the sole chromosomal imbalance; dup(2)(p16p21), del(4)(q21q21), del(6) (q14q15), del(6)(p12p12), dup(6)(q24qter), and dup(15)(q11q13) One case had dup(9) (p11pter) combined with a very small subtelomeric deletion on 6q In our hands, CGH is highly useful not only for identifying known chromosomal imbalances, but also for finding elusive deletions or duplications in the large group of children with developmental delay with or without congenital abnormalities In such cases, the diagnostic yield of CGH appears to be higher than what has been reported from subtelomeric FISH screening ß 2002 Wiley-Liss, Inc *Correspondence to: Gunnar Houge, Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, N-5021 Bergen, Norway E-mail: gunnar.houge@haukeland.no Received 10 September 2001; Accepted 14 April 2002 DOI 10.1002/ajmg.10593 ß 2002 Wiley-Liss, Inc KEY WORDS: dysmorphology; cytogenetics; FISH; deletions; duplications INTRODUCTION The complex relationship between genotype and phenotype, the plethora of established and possible syndromes, and the sometimes highly variable phenotypic expression of a given gene or chromosomal defect constrain our ability to find a diagnosis or an explanation for an abnormal child’s condition in the dysmorphology clinic Our ability to accurately predict a mutation in a specific gene or a given chromosomal abnormality based on phenotype investigations is even more limited When lacking phenotypic handles pointing to specific syndromes or a family history that gives indicative clues, the finding of a genetic cause heavily depends on the sensitivity of available screening methods If there is no reason to suspect a metabolic disease, the only screening method for genetic defects in general use is conventional chromosome analysis by G- or R-banding Because our clinical diagnostic skills are likely to remain insufficient also in the future, better molecular screening methods would be extremely useful Comparative genomic hybridization (CGH) is the only DNA-based screening method that can detect chromosomal imbalances (e.g., deletions and duplications) in one experiment All other DNA-based methods (e.g., specific gene tests, or fluorescence in situ hybridization [FISH] tests for chromosomal imbalances such as the microdeletion syndromes) return only the result that is asked for An additional advantage of CGH is the independence of living cells from the tissue to be investigated The usefulness of CGH in tumor cytogenetics for identifying chromosomal gains and losses has been well documented [James, 1999; Gray and Collins, 2000] When searching for constitutional chromosome abnormalities, CGH analysis has so far been less useful than conventional G-banding This is partly because balanced aberrations will not be found, and partly because the sensitivity for detecting imbalances has 126 Ness et al been on the same level as routine G-banding [detection of gains and losses of at least 10 Mb; Kallioniemi et al., 1994; Bentz et al., 1998] Therefore, CGH has been most widely used for identifying or confirming the presence of aberrations that already have been observed by conventional G-banding analysis [Erdel et al., 1997; Boceno et al., 1998; Levy et al., 1998; Breen et al., 1999; Weimer et al., 2000; Rigola et al., 2001] Recently, a method of analyzing CGH data based on dynamic standard reference intervals instead of fixed intervals was reported [Kirchhoff et al., 1998], resulting in a two-to-threefold improvement of resolution, i.e., approaching Mb In their hands, such high-resolution CGH analysis proved useful for detecting aberrations in patients with an apparently balanced karyotype [Kirchhoff et al., 2000, 2001] In the present work, we have explored the usefulness of this novel CGH analysis software in our routine diagnostic service In addition to being the most rational and precise way to identify chromosomal material of unknown nature, the novel CGH analysis software has sufficient sensitivity to detect deletions and duplications missed by previous good-quality G-banding Some of our presented cases, with a small deletion or duplication as the only finding, might add to our understanding of genotype/phenotype correlation In conclusion, we find that CGH is a highly useful tool also in the routine cytogenetic laboratory, providing an explanation for an aberrant phenotype in approximately 10% of previously undiagnosed cases MATERIALS AND METHODS Patient Samples Patient samples were received for routine cytogenetic analysis at Center for Medical Genetics and Molecular Medicine at Haukeland University Hospital After conventional karyotyping, some samples were selected for CGH analysis, either because the patient phenotype was highly suggestive of a chromosome abnormality (even though the karyogram appeared to be normal), or because observed aberrations were difficult to classify or investigate thoroughly Most cases with aberrations detected by CGH have later been referred to us for phenotypic evaluation of the child and genetic counseling of the parents Brief information about the patients’ phenotypes is given in Table I Cytogenetic Analysis Metaphases from peripheral blood lymphocytes were prepared according to standard procedures, using phytohemagglutinin for stimulation of the lymphocytes, and methotrexate for synchronization of the cell cycle Metaphase chromosomes of good quality were karyotyped by conventional G-banding, giving a band level of approximately 500 CGH Slides with normal lymphocyte metaphase chromosomes for CGH analysis were postfixed in 1% formaldehyde for at 48C, dehydrated in an ethanol series (70%, 85%, and 100%) and stored at À208C before hybridization CGH was performed essentially as described by Kallioniemi et al [1994] Normal male or female DNA was used as reference DNA after labeling with Texas Red-5-dUTP (NEN Life Science Products, Zaventem, Belgium) using nick translation Patient DNA was labeled with fluorescein isothiocyanate (FITC)-12dUTP (NEN Life Science Products) Genomic DNA was digested to fragment lengths of 0.3–2 kb Labeled patient DNA (800 ng) and reference DNA (800 ng) together with excess unlabeled Cot-1 DNA (Life Technologies Ltd., Gibco BRL, Paisley, UK) were hybridized to normal lymphocyte metaphase chromosomes Slides were counterstained with 4,6-diamidine-2-phenylindole in an anti-fade solution (Vector Laboratories, Burlingame, CA) Each sample was hybridized twice, with both sex-matched and mismatched reference DNA Digital Image Analysis The hybridizations were analyzed using the CytoVision System with the Version 2.7 High Resolution CGH analysis software (Applied Imaging, Newcastle, UK) Fifteen to 20 metaphases were collected using a Nikon Eclipse E800 epifluorescence microscope mounted with a CCD camera interfaced to a CytoVision Station The green (patient DNA) to red (reference DNA) fluorescence ratio along the length of the chromosomes was calculated The CGH profiles were compared to a dynamic standard reference interval based on an average of normal cases, as described by Kirchhoff et al [1998] The dynamic standard reference intervals are wide at regions known to produce unreliable CGH profiles The intervals were scaled automatically to fit the test case The mean ratio profile of each case with 99.5% confidence intervals was compared to the average ratio profile of the normal cases with similar confidence intervals To increase sensitivity in cases of mosaicism, 95% confidence intervals were initially used Positive findings were those where the confidence intervals of the patient profile and normal averaged profile did not overlap FISH Whole chromosome painting probes were from Vysis Ltd (Downers Grove, IL) or Oncor (Appligene Oncor, Illeirch, France), whereas the telomer probes were from Cytocell Ltd (Oxfordshire, U.K.) BAC probes were selected from a human genomic library at the Julie R Korenberg Lab at Cedars-Sinai Medical Center, and were delivered by Research Genetics (Paisley, U.K.) Metaphase slides were postfixed in 1% formaldehyde and dehydrated in an ethanol series (70%, 85%, and 100%) before denaturation followed by hybridization FISH with whole chromosome painting probes or telomer probes were performed according to the manufacturer’s protocol The BAC clone was labeled with biotin-16-dUTP (Roche Diagnostics GmbH, Mannheim, Germany) by standard nick translation The probe was resuspended in 10 mL hybridization buffer (50% formamide, 2xSSC, 10% dextran sulfate) After CGH in Clinical Dysmorphology 127 TABLE I Clinical Features of Cases With Genomic Imbalances Found by CGH Analysis Case no Sex/YoB Genomic imbalance Available information on patient phenotype F/1995 del 10p15 dup 20q13.3 Short stature, mental retardation, cri de chat–like cry, dysmorphic features (hypertelorism, epicanthus, hypognatia) F/1988 del 6q14-6q15 F/1990 del 4q21.1-4q21.3 M/1990 M/1992 dup 15q11-15q13 del 6p12 M/1987 mosaic dup 4p12-4p13 F/1984 F/2001 mosaic dup 8p11-8p12 dup 2p16-2p21 Short stature, severe mental retardation with autistic-like features, hypotonia, atrial septal defect, dysmorphic features (frontal bossing, protruding jaw, hypoplastic midface, deep-seated eyes, small mouth) Short stature, severe mental retardation, epilepsy (infantile spasms), atrial septal defect, dysmorphic features (frontal bossing, synophrys, mongoloid slant, short philtrum, depressed angles of the mouth, small hands and feet) Mental retardation with autistic-like features Attention deficit disorder (without hyperactivity), subnormal auditive perception with delayed language development, motorically clumssy Attention deficit and hyperactivity disorder, poor social adaptation, rigid personality, delayed development of language and motoric skills, bilateral cryptorchism, congenital hip dysplasia, unequal lower extremity length (3 cm difference), scoliosis Short stature, mental retardation, motorically clumsy M/1987 10 M/1998 del 3p25-3pter dup 3q26-3qter dup 6q24-6qter 11 M/2000 dup 9p11-9pter 12 M/2000 del 5p14-5pter dup 1q42-1qter 13 M/2000 14 F/1996 del 11q24-11qter dup 4q26-4qter del 10q21 del 11q22 15 F/1987 mosaic del 21q22.2-21qter Delayed development of motoric functions, no obvious mental retardation (at age months), dysmorphic features (anteverted nostrils, frontal bossing, small hands) Moderate mental retardation, minimal dysmorphic features (slight synophrys, anteverted nostrils), normal stature, chronic obstipation Short stature, neonatal hypotonia, moderate mental retardation, neonatal transient hyperglycemia, prominent joint contractures, left-sided club foot, left-sided inguinal hernia, many dysmorphic features (telecanthus with prominent eyes, small mandible, low-set ears, webbed and short neck) Neonatal hypotonia with feeding problems, atrial septal defect, mild dysmorphic features (transverse palmar crease, small mandible, five-finger clinodactyly, broad-based bulbous nose, antimongoloid eye slant, low-set cupped ears), still no obvious mental retardation (age months) Multiple congenital abnormalities, severe respiratory problems requiring assisted ventilation, dysmorphic features (cleft lip/palate, preauricular tags, increased intermammary distance), early neonatal death Hypoplastic left ventricle, dysmorphic features, bilateral cryptorchism, curved penis, early neonatal death Short stature, mental retardation with moderately delayed psychomotor and language development, bilateral club feet, congenital hip dislocations, some dysmorphic features (mongoloid eye slant, deep set eye, posteriorly rotated low-set ears) Mild mental retardation with normal motor but late language development, bilateral congenital hip dysplasia, club feet and inguinal hernia, epilepsy (onset 12 years), dysmorphic features (short philtrum, short and broad neck, very curly hair) YoB, year of birth preannealing with Cot-1 and salmon sperm DNA, the probes were hybridized overnight at 378C in a HYBrite machine (Vysis) Hybridization was detected by FITCanti DIG (Appligene Oncor) Chromosomes were counterstained with 4,6-diamidine-2-phenylindole in an antifade solution (Vector Laboratories) RESULTS The clinical features of patients, whose exact karyotypes were determined by CGH analysis, are listed in Table I The initial karyotypes after G-banding, CGH results and final karyotypes are shown in Table II In all cases the CGH findings were confirmed, either by FISH (Cases 1, 4, 6–14) or careful retrospective analysis of G-banded chromosomes (Cases 2, 3, 5, 15; see Figs and 4–6, and Table II) Cases with Normal Karyotypes After Routine Chromosome Analysis Cases 1–5 had negative (normal) results on routine chromosome analyses, but were selected for CGH because of clinical features suggesting a chromosomal aberration (Case 1–3), our special interest in autism (Case 4), or for further investigation of what we considered to be a variant chromosome 15 satellite (Case 5) In Case 1, we found an unbalanced terminal 10;20translocation (Fig 1) We still not know if one of the parents carries a translocation [approximately 50% do; see Knight et al., 1999] The translocation was impossible to see on G-banded chromosomes (analyzed in 1995 and 1997), even when knowing what to look for (Fig 1) Whole chromosome paint probes for chromosome 10 and 128 Ness et al TABLE II Initial Karyotype, CGH Results, Confirmatory Studies, and Final Adjusted Karyotype Karyotype after initial G-banding Case no 46,XX CGH result: rev isha CGH result confirmation 46,XX 46,XX 46,XY 46,XY 47,XY,ỵmar[19]/46,XY[11] dim(10p15) enh(20q13.3) dim(6)(q14q15) dim(4q21)d enh(15q12)d dim(6p12)d enh(4)(p12p13)d tel10p tel20px3 G-banding G-banding SNRPNỵỵ G-banding wcp4ỵ 47,XX,ỵmar[18]/46,XX[12] enh(8)(p11p12)d cen8ỵ 46,XX,dup(2)(p?) 46,XY,add(3)(p25) 10 46,XY,add(6)(q25) enh(2)(p16p21) enh(3)(q26qter) dim(3)(p25pter) enh(6)(q24qter) 11 46,XY,add(6)(q25) enh(9)(p11pter) 12 46,XY,der(5)t(5;?)(p14;?) 13 46,XY,der(11)t(11;?)(q24;?) 14 46,XX,t(10;11)(q21;q22) 15 46,XX,r(21)[17]/46,XX[13] enh(1)(q42qter) dim(5)(p14pter) enh(4)(q26qter) dim(11)(q24qter)d dim(10q21)d dim(11q22)d dim(21q22)d G-banding wcp2ỵ wcp3ỵ tel3qx3 wcp6ỵ G-banding tel9px3 tel6qwcp5tel1qx3 wcp4ỵ wcp11BAC11q22.3G-banding Karyotype after CGH analysis and follow-up studiesb,c 46, XX.ish der(10)t(10;20)(p14;q13) (tel10p-,tel20px3) 46,XX,del(6)(q14q15) de novo 46,XX,del(4)(q21.1q21.3) 46,XY.ish dup(15)(q11q13)(SNRPNỵỵ) 46,XY,del(6)(p12p12) de novo 47,XY,ỵder(4)del(4)(p13)del(4)(q10)[19]/ 46,XY[11] 47,XX,ỵder(8)del(8)(p12)del(8)(q10)[18]/ 46,XX[12] 46,XX,dup(2)(p21p16) 46,XY,der(3)t(3;3)(p25;q26.2)del(3)(p25) dup(3)(q26.2qter) 46,XY,dup(6)(q27q24.3) 46,XY,der(6)t(6;9)(q27;p12) 46,XX,der(5)t(1;5)(q42;p14) 46,XY,der(11)t(4;11)(q26;q24) 46,XX,t(10;11)(q21;q22)del(10)(q21q21) del(11)(q22q22) 46,XX,r(21)(p11?q22.1)([17]/46,XX[13] a rev ish, reverse in situ hybridization; dim, diminished (signal intensity), enh, enhanced Nomenclature follows the 1995 revision of the International System for human Cytogenetic Nomenclature For simplicity, the ish karyotype is only included if an aberration is impossible to detect, also in retrospect, on high-quality G-banded chromosomes d Not observed using fixed threshold 0.8–1.2 b c 20 used retrospectively were also unable to detect any abnormality (data not shown) We have found no reports on the phenotype of small terminal 10p deletions, but it appears that the phenotype is quite unlike the DiGeorge syndrome-like phenotype of more proximal 10p deletions [Daw et al., 1996] Likewise, we have not found any published cases with similar terminal duplications of 20q In Case 2, it was fairly easy to confirm the aberration on G-banded chromosomes when they were examined retrospectively for the deletion (Fig 1) The aberration had previously been missed twice by routine analysis of G-banded chromosomes This region of chromosome has a banding pattern that easily obscures the presence of abnormalities Only 12 children with proximal interstitial 6q deletions have so far been described [Roland et al., 1993; Kumar et al., 1997; Passarge, 2000] The phenotype of our case (Table I, Fig 2), a severely retarded 13-year-old girl, was only slightly reminiscent of the previously published proximal interstitial 6q deletion syndrome [Kumar et al., 1997] Our patient lacked the dolichocephaly (she is in fact brachycephalic) and long philtrum (her philtrum is short) often found in this syndrome More in line with previously described stigmata was neonatal hypotonia, a cardiac abnormality (atrial septal defect), large ears, short nose with a broad nasal tip, and a thin upper lip (Fig 2) [Kumar et al., 1997] In Case 3, the aberration had been missed twice by routine G-banding Even after informing the cytogenetic technicians that a deletion was present, it was still very hard to detect on high-quality G-banded chromosomes (Fig 1) This case has been through many rounds of evaluation by well-known experts on dysmorphology Brachmann-de Lange syndrome was initially suggested because of the presence of synophrys, increased body hair, and severe mental retardation, but later was discarded The patient phenotype was slightly reminiscent of the previously described deletion 4q21/4q22syndrome with frontal bossing, hypotonia, and short Fig CGH and follow-up studies for cases where no aberration was initially found on the G-banded karyotype (Cases 1–5, see Table I) Case 1: CGH indicating a deletion of terminal 10p and duplication of terminal 20q FISH analysis with subtelomeric probes confirmed the suspected presence of an unbalanced 10;20 translocation (two normal chromosome 20, one normal chromosome 10 and one der(10) with terminal 20q replacing terminal 10p) Case 2: CGH showing a deletion on 6q, confirmed by G-banding analysis Case 3: CGH found a 4q21 deletion, confirmed by G-banding analysis Case 4: CGH indicated a duplication of proximal 15q to be present This could not be seen on G-banded chromosomes, but FISH with the SNRPN probe confirmed the duplication Case 5: The observed deletion of 6p12 in the CGH profiles could retrospectively also be seen on long G-banded chromosomes General comment to Figures and 4–6: In each case, the CGH profiles are on the left side, a picture of G-banded chromosomes in the middle/right (with a red arrow pointing to the aberration if visible), and FISH pictures (if any) on the right All the CGH profiles, except in Cases and (99%), are shown with confidence intervals of 99.5% The CGH profiles include ideograms of the involved chromosomes indicating the gains (green bar) or losses (red bar) detected by CGH On the CGH plots the average (pink lines), the 99.5% confidence intervals (yellow lines) and the 99.5% standard reference intervals (black lines) can be seen An aberration is recorded when the intervals are nonoverlapping n indicates the number of chromosomes analyzed The FISH-probes used are denoted wcp for whole chromosome paints, tel for telomeric probes, and cen for centromeric probe The name of the probe is written with the same color as the probes FISH signal [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] CGH in Clinical Dysmorphology hands and feet (between the 10th and 25th centile), but she did not have the macrocephaly reported to be common, and her facial appearance was quite different (Fig 3) [Nowaczyk et al., 1997] Her head circumference was between the 50th and 75th centile, her length a few centimeters below the 2.5 centile 129 Case was screened for a proximal duplication on chromosome 15 because several reports had linked autistic-like features to maternal duplications or triplications of the Prader-Willi/Angelman critical region [e.g., Cook et al., 1997], sometimes as a part of the socalled inv dup(15)-syndrome [Battaglia et al., 1997] 130 Ness et al Cases with Marker Chromosome Mosaicisms In Case 6, a small marker chromosome was observed in 60% of lymphocyte metaphases By using a confidence interval of 99% (and not 99.5%) to increase sensitivity, the marker could be identified by CGH as a chromosome derivative, containing 4p12-4p13 DNA (Fig 4) A painting probe as well as a centromer probe for chromosome confirmed this finding (Fig 4, Table II) In Case too, it was necessary to compare profiles with less stringent confidence intervals (99% instead of 99.5%) in order to identify the marker, which also in this case was present in 60% of the lymphocyte metaphases The marker was a der(8), containing 8p11-8p12 derived DNA The finding was confirmed by a FISH centromer probe (Fig 4) Fig Case 2, girl born 1988, at age 12 Cases with Additional Chromosome Material of Unknown Origin CGH analysis detected a duplication of band 15q12 (Fig 1) This duplication was not evident on goodquality G-banded chromosomes, although a few chromosome pairs appeared suspicious FISH with a probe for the SNRPN locus showed that this region indeed was duplicated (Fig 1) Case was selected for CGH in order to analyze a large and strange-looking satellite on 15p, which later turned out to be an unusual normal variant that the boy had inherited from his mother However, we discovered an unexpected deletion of band 6p12.1 (Fig 1) The patient’s phenotype was quite mild (some learning difficulties, ADHD-like behavior, motorically clumsy) The presence of a small deletion could be confirmed by careful analysis of good quality metaphases None of his parents had a deletion of the same band Further analyses using BAC and YAC probes are in progress in order to define this deletion better To our knowledge, a similar case has not previously been published Fig In Case 8, it was obvious that additional material was present on 2p FISH paint suggested a duplication, but it was not possible to determine which part of chromosome was duplicated The clinical features at age months appeared to be mild (not investigated by us), and of little help in defining the exact duplication because of the lack of dysmorphic handles pointing to a specific region CGH identified the duplicated segment as 2p16-2p21, and the banding pattern was most consistent with an inverse duplication (Fig 5, Table II) Approximately 20 patients with internal duplications in 2p have so far been described [Aviram-Goldring et al., 2000], but none of these patients were reported to have a duplication similar to our patient’s The difficulty in defining the exact origin of a suspected duplication is also well illustrated by Case In this case, the cytogenetic laboratory initially (in 1995) interpreted the G-banded karyotype as an inverse terminal duplication of 3p (Fig 5) Only recently the family was referred for genetic counseling, and CGH was done to confirm the presence of a distal partial trisomy 3p Case 3, girl born 1990, at age 10 CGH in Clinical Dysmorphology Fig CGH and supplementary FISH studies for cases with marker chromosomes (Cases and 7, see Table I) Case 6: CGH identified the 60% mosaic marker as a der(4) with the 4p12-p13 region, confirmed by cen4 FISH Case 7: Here the 60% mosaic marker was from 8p11-p12, and a cen8 FISH confirmed chromosome origin Mar, marker chromosome, also called ESAC or SMC For further explanations, see legend to Figure [Color figure can be viewed in the online issue, which is available at www.interscience wiley.com.] Such patients can have psychomotor retardation without dysmorphic features [Smeets et al., 2001], well in line with our patient’s phenotype (Table I) However, instead of a 3p duplication, CGH revealed a duplication of terminal 3q combined with a small deletion of terminal 3p (Fig 5, Table II) The CGH analysis was confirmed by subtelomer 3q FISH (Fig 5) It thus turned out that the patient had a duplication of the proposed critical region of the dup(3q)-syndrome [Aqua et al., 1995], which superficially resembles Brachmann-de Lange syndrome His phenotype was consistent with a mild variant of the dup(3q)-syndrome: moderate mental retardation, slight synophrys, and anteverted nares, but no hirsutism or malformations of internal organs [Rizzu et al., 1997] Case 10 is one of the few examples where the patient’s features are highly suggestive of a specific chromosome abnormality (Table I) The transient neonatal hyperglycemia combined with dysmorphic features indicated in itself of a paternal duplication of terminal 6q [Cave et al., 2000], and the boy’s joint contractures were typical for patients with such duplications [Schinzel, 1983] As a newborn, he appeared severely mentally retarded, and he was expected to live a short life He was resuscitated once, at age months With time, however, he improved, and he now seems to have a moderate mental retardation Routine chromosome analysis showed extra material on the terminal part of chromosome 6q, identified as a terminal duplication of 6q25-qter by CGH This 131 finding was confirmed by FISH paint chromosome (Fig 5) Case 11 appeared to have a G-banded karyotype identical to Case 10 (Fig 5), but the clinical features were completely different (Table 1) CGH identified the material to correspond to almost the whole of 9p, which was confirmed by subtelomer 9p FISH (Fig 5) Because this is a 6;9 translocation, a small deletion of terminal 6q was also suspected, and this was confirmed by 6q subtelomer FISH (Fig 5) The deletion was not detected by the CGH analysis software, indicating that it was smaller than Mb Retrospectively, the patient features were recognized as typical for a dup(9p)-syndrome: downslanting eyes, broad-based nose with globous tip, asymmetric upper lip, cup-shaped ears, and no congenital malformations [Schinzel, 1983] In Cases 12 and 13, CGH was used to classify a suspected unbalanced translocation Both cases had lethal congenital malformations and obvious dysmorphic features (Table I) In Case 12, chromosome had an aberrant banding pattern on the p-arm (Fig 5) CGH showed that almost the entire 5p was deleted and replaced by a duplicated terminal 1q, thus revealing an unbalanced 1;5 translocation The finding was confirmed by FISH subtelomer 1q and FISH paint chromosome (Fig 5) The 5q deletion and terminal 1q duplication are by themselves reported compatible with life [Schinzel, 1983], but their combination is unlikely to survive until birth [Gardner and Sutherland, 1996], as in this case In Case 13, the additional material on chromosome was identified as the terminal half of 4q, revealing an unbalanced 4;11 translocation (Fig 5) Again, each abnormality has previously been reported to be compatible with life [Schinzel, 1983], but the combination is expected to be lethal, even though this case shows that survival until birth is possible Cases with Chromosomal Breaks Possibly Associated with Deletions Case 14 had a de novo apparently balanced 10;11 translocation (Fig 6), but because the patient’s diverse phenotypic features were more compatible with a deletion than a break in one or two critical genes, we used CGH to investigate whether a deletion could be found This was indeed the case, and CGH suggested a deletion in both breakpoints (10q21 and 11q22) Only the deletion corresponding to band 11q22 has so far been verified by FISH, using a BAC probe recognizing 11q22.3 (Fig 6) A few children with deletions of 10q21 and 11q14-11q22 have previously been described [Wakazono et al., 1992; Doheny et al., 1997], but in these cases the deletions were larger The shape of the nose of our patient resembles the small nose with broad nasal root and tendency to anteverted nostrils shown in a patient with del(10) (q21.2q22.1) [Wakazono et al., 1992], but otherwise the phenotype is quite different [Doheny et al., 1997] Case 15 had mosaicism for a ring chromosome 21 (Fig 6), and we wanted to test the usefulness of CGH in finding the extent of a deletion in such a case, with a marker being present in 57% of lymphocyte metaphases CGH picked up the deletion even at high stringency 132 Ness et al Fig CGH and FISH confirmatory studies for cases with an aberration of unknown origin found by G-banding (Cases 8–13, see Table I) Case 8: CGH indicated that an elongated 2p contained a duplication of 2p16-p21, consistent with the wcp2 FISH results Case 9: CGH showed a duplication of terminal 3q and deletion of terminal 3p The extra material on terminal 3p (arrow) is thus a duplicated 3q26-qter region, replacing terminal 3p sequence as in an unbalanced interchromosomal 3q;3p translocation, as also shown by tel3q FISH Case 10: The addition to 6q (arrow) represented an inverse terminal 6q duplication, consistent with wcp6 FISH Case 11: Here, CGH showed the addition to 6q to be 9p11-pter, confirmed by tel9p FISH Tel-6/9 FISH showed this to be an unbalanced 6;9 translocation, with a der(6) having most of 9p replacing the very distal part of 6q The latter deletion was not revealed by CGH Case 12: CGH showed an aberrant 5p (arrow) to result from an unbalanced 1;5 translocation, having a der(5) with a large part of 5p deleted and terminal 1q duplicated, confirmed by wcp5 and tel1 FISH Case 13: CGH identified an aberrant chromosome 11 (arrow) to result from an unbalanced 4;11 translocation, having a der(11) with terminal 11q deleted and half of 4q duplicated, confirmed by wcp4 and wcp11 FISH For further explanations, see legend to Figure [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] CGH in Clinical Dysmorphology 133 Fig CGH and the FISH follow-up studies for cases with chromosomal breaks (Cases 14 and 15, see Table I) Case 14: CGH revealed deletions in both breakpoints (arrows) in an apparently balanced 10;11 translocation (G-banding) FISH with a BAC probe toward 11q22.3 confirmed the deletion on chromosome 11 Case 15: CGH identified a deletion of terminal 21q in a ring 21 chromosome (arrow) For further explanations, see legend to Figure [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] testing (99.5% confidence intervals), indicating that the percentage of metaphases containing a ring chromosome might be biased because of poorer growth of r(21)containing cells Ring chromosome 21 gives a highly variable phenotype (from normal development to severe mental retardation with multiple congenital malformations), dependent on the presence and extent of deletions and sometimes even duplications [Schinzel, 1983; McGinniss et al., 1992] DISCUSSION In our cytogenetic laboratory, CGH has become an important supplement to the routine diagnostic procedures for two main reasons First, CGH is a more informative and often faster way of identifying chromosome pieces of unknown origin (additions, marker chromosomes) than targeted FISH investigations [Erdel et al., 1997; Boceno et al., 1998; Levy et al., 1998; Breen et al., 1999; Rigola et al., 2001] The guesswork concerning which FISH probes to use is eliminated (e.g., Cases 6–7, 11–13), a single experiment (and not consecutive hybridizations) returns an answer, and not only the chromosome but also the chromosomal region, domain, and sometimes even the subdomain is identified (e.g., Cases 6–11) In addition, one gets a clue for predicting if a marker chromosome is likely to cause a phenotypic abnormality or not It is often difficult to determine if a small G-banded marker chromosome contains genecontaining euchromatin or only heterochromatin with structural/repetitive DNA (e.g., Cases and 7) Because this latter type of DNA is excluded from the CGH analysis (blocked by Cot-1 DNA), a positive CGH result indicates that genes, with a potentially dose-dependent influence on normal embryonic and brain development, might be present in the extra chromosomal material The novel CGH software is also sufficiently sensitive to be used for identification of mosaicisms, at least when the chromosomal abnormality (usually a marker chromosome) is present in more than 50% of the metaphases (Cases and 7) Cases 6, 7, and 15 all had marker chromosomes that could be characterized only by the high-resolution CGH analysis and not by applying fixed thresholds on the CGH profiles The second important point is that the high-resolution CGH software with its two- to threefold improved sensitivity [Kirchhoff et al., 1998, 1999], has made CGH a method of choice for finding cryptic chromosomal aberrations [Cases 1–5 and 14–15) [Kirchhoff et al., 2000, 2001] Alternatively, screening for cryptic aberrations can be done with subtelomeric probe kits [Knight et al., 1999], multicolor FISH techniques involving differential chromosome painting (M-FISH) [Uhrig et al., 1999; Jalal et al., 2001], spectral karyotyping [Schrock et al., 1997; Haddad et al., 1998], or multisubtelomer FISH [Brown et al., 2001] Multicolor FISH techniques are useful for deciphering complex chromosomal rearrangements difficult to resolve by G-banding [Schrock et al., 1997; Eils et al., 1998; Haddad et al., 1998; Uhrig et al., 1999; Jalal et al., 2001], and may 134 Ness et al also detect subtle interchromosomal rearrangements [Azofeifa et al., 2000; Bezrookove et al., 2000], but for several reasons (workload, complexity, cost, level of sensitivity, methodological problems) the method is not well suited when screening for cryptic aberrations [Lee et al., 2001] We have not applied M-FISH on our cases, but noticed that whole chromosome painting probes used retrospectively did not reveal the unbalanced translocation in Case Probe sets for subtelomer FISH screening appears to be more useful and has been claimed to find aberrations in 7.4% of a selected group of patients (children having moderate to severe mental retardation, congenital malformations, and dysmorphic features) [Knight et al., 1999] The technique is more sensitive than CGH for small (