Array-based comparative genomic hybridization identifies a high frequency of copy number variations in patients with syndromic overgrowth

Overgrowth syndromes are a heterogeneous group of conditions including endocrine hormone disorders, several genetic syndromes and other disorders with unknown etiopathogenesis. Among genetic causes, chromosomal deletions and duplications such as dup(4)(p16.3), dup(15)(q26qter), del(9)(q22.32q22.33), del(22)(q13) and del(5)(q35) have been identified in patients with overgrowth. Most of them, however, remain undetectable using banding karyotype analysis. In this study, we report on the analysis using a 1-Mb resolution array-based comparative genomic hybridization (CGH) of 93 patients with either a recognizable overgrowth condition (ie, Sotos syndrome or Weaver syndrome) or an unclassified overgrowth syndrome. Five clinically relevant imbalances (three duplications and two deletions) were identified and the pathogenicity of two additional anomalies (one duplication and one deletion) is discussed. Altered segments ranged in size from 0.32 to 18.2 Mb, and no recurrent abnormality was identified. These results show that array-CGH provides a high diagnostic yield in patients with overgrowth syndromes and point to novel chromosomal regions associated with these conditions. Although chromosomal deletions are usually associated with growth retardation, we found that the majority of the imbalances detected in our patients are duplications. Besides their importance for diagnosis and genetic counseling, our results may allow to delineate new contiguous gene syndromes associated with overgrowth, pointing to new genes, the deregulation of which may be responsible for growth defect.     

Is Dandy-Walker malformation associated with "distal 13q deletion syndrome"? Findings in a fetus supporting previous observations

We report on a fetus with a large deletion of the distal part of the long arm of chromosome 13, (del(13)(q14 --> qter)) congenital anomalies of the urinary system, lungs and extremities, and Dandy-Walker malformation (DWM). Although DWM has been associated with many chromosomal abnormalities and genetic syndromes, its relation to the distal 13q has been demonstrated recently. In 2002, McCormack et al., described two patients with deletions of the long arm of chromosome 13 who had multiple congenital abnormalities along with holoprosencephaly (HPE) and DWM. The phenotypic features and autopsy findings of a fetus with "distal 13q deletion syndrome" at 22 weeks gestation are discussed and comparison with the previous two cases is made. The findings support the previous hypothesis suggesting that haploinsufficiency at a locus within 13q22-33 due to microdeletions may be responsible for isolated DWM in some of the patients. Detailed examination of 13q (13q22-33) by means of conventional and molecular cytogenetic methods is necessary in cases with DWM.     

Usefulness of high-resolution comparative genomic hybridization (CGH) for detecting and characterizing constitutional chromosome abnormalities

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 5 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.     

Heterogeneity of NSD1 alterations in 116 patients with Sotos syndrome

Sotos syndrome is an overgrowth syndrome characterized by distinctive facial features, learning difficulties, and macrocephaly with frequent pre- and postnatal overgrowth with advanced bone age. Here, we report on our experience in the molecular diagnostic of Sotos syndrome on 116 patients. Using direct sequencing and a quantitative multiplex PCR of short fluorescent fragments (QMPSF)-based assay allowing accurate detection of both total and partial NSD1 deletions, we identified NSD1 abnormalities in 104 patients corresponding to 102 Sotos families (90%). NSD1 point mutations were detected in 80% of the index cases, large deletions removing the NSD1 gene entirely in 14%, and intragenic NSD1 rearrangements in 6%. Among the 69 detected distinct point mutations, 48 were novel. The QMPSF assay detected an exonic duplication and a mosaic partial deletion. QMPSF mapping of the 15 large deletions revealed the heterogeneity of the deletions, which vary in size from 1 to 4.5 Mb. Clinical features of NSD1-positive Sotos patients revealed that the phenotype in patients with nontruncating mutations was less severe that in patients with truncating mutations. This study confirms the heterogeneity of NSD1 alterations in Sotos syndrome and therefore the need to complete sequencing analysis by screening for partial deletions and duplications to ensure an accurate molecular diagnosis of this syndrome.     

The influence of different chromosomal aberrations on molecular cytogenetic parameters in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia of adults in Western countries. The most frequent recurring chromosomal aberrations identified in B-CLL patients are trisomy 12 and deletions of 13q, 17p, and 11q. Cases with deletions of 11q and 17p have a poor prognosis, whereas cases with deletions in 13q have a favorable prognosis. It was previously shown that CLL patients with trisomy 12 and del(13)(q14) have a higher rate of asynchronous replication of normal structural genes when compared to those with normal karyotypes. We studied the replication pattern of the structural locus 21q22 and the imprinted gene SNRPN and its telomere (15qter) and the random aneuploidy of chromosomes 9 and 18 in CLL patients with trisomy 12 and deletions of 11q and 17p, and compared the results to those of CLL patients without these aberrations and to healthy controls. Random aneuploidy rate was higher in the group of patients with trisomy 12 as compared to all other groups. The replication pattern with higher asynchronous pattern was found in both aberration groups compared to the CLL patients without the aberrations and to the control group with involvement of 21q22 and 15qter, whereas the highest synchronous group was found in the 2 aberrations CLL patient groups compared to the other groups with the imprinted locus SNRPN. The existence and significance of chromosomal aberrations in CLL have a deleterious effect on the processes of cell cycle and gene replication and may have biological and prognostic implications.     

Deletion of chromosome 11 (p11p13) in a patient with Beckwith-Wiedemann syndrome

Four cases of duplication of a segment of 11p have been reported in patients with Beckwith-Wiedemann syndrome (Waziri et al. 1983, Turleau et al. 1984). We describe a patient with Beckwith-Wiedemann syndrome who has a deletion of chromosome 11(p11p13) and suggest involvement of this chromosomal region in both the duplicated and deleted states such as occurs in Prader-Willi syndrome.     

Ossifying fibromyxoid tumor of soft parts. Cytogenetic findings

Ossifying fibromyxoid tumor (OFMT) of soft parts is a recently described, rare but morphologically distinctive soft tissue tumor. The histogenesis of this lesion remains uncertain, although several immunohistochemical and ultrastructural features suggest that it is an unusual neural tumor, possibly of Schwann cell origin. We report here a case of a malignant variant of OFMT that occurred in the foot of a 52-year-old man. The karyotype of a pulmonary metastasis exhibited the following complex numeric and structural aberrations:72 approximately 74,XXY,-5,+6,+del(8)(p21),del(9)(p22),+10,der(11)t(3;11)(p21;p15),del(12) (q13),der(13)t(5;13)(q13;q34),+18,+19,+20,-22 [cp10]. A kidney metastasis exhibited the following karyotypic abnormalities: 46,XY,add(3)(p11),+der(3)t(3;?;11)(3qter-->3p11::?::11q13-->11qter), -5,del(8)(p21),add(9)(q22),del(9)(p22),der(11)t(3;11)(p21;p15),del(12)(q13),+der(13)t(5;13) (q13;q34),-22. To our knowledge, this is the first reported case of OFMT in which clonal chromosomal aberrations have been shown.     

Developmental Biology: Frontiers for Clinical Genetics: Overgrowth syndromes and genomie imprinting: from mouse to man

Beckwith-Wiedemann syndrome (BWS), a human overgrowth syndrome with a complex genetic basis, is caused by alterations to chromosome 11p15, a region subject to genomie imprinting. These alterations include translocations, duplications, single gene mutations of p57^KIP2, and increased expression of insulin-like growth factor 2 (IGF2). A phenotypically related X-linked overgrowth syndrome, Simpson-Golabi-Behmel syndrome (SGBS), is caused by alterations in glypican-3 (GPC3), a molecule that may interact with the gene products identified to be important in generating the BWS phenotype, that is, IGF2 and p57^KIP2. The crucial defect in these overgrowth syndromes is likely to be an imbalance in contributions of growth-promoting genes versus growth-inhibitory genes in critical tissues at specific developmental stages. Murine models have been used to study the effects of targeted deletions of the genes p57^KIP2 and GPC3, as well as overexpression of IGF2. At this time, there are still many issues which remain to be explored before we can fully understand the molecular basis of BWS and SGBS.     

9p deletion and distal 9q duplication due to a paternal pericentric inversion 9(p22q32)

A female infant with 46,XX,rec(9), dup q,inv(9)(p22q32)pat is presented. She had a duplication from 9q32 to qter and a deletion from 9p22 to 9pter. Phenotypical abnormalities observed corresponded with features noted in cases with distal dup (9q), while pathognomonic features of del(9p) syndrome were not observed.     

Cryptic subtelomeric translocations in the 22q13 deletion syndrome

Cryptic subtelomeric rearrangements are suspected to underlie a substantial portion of terminal chromosomal deletions. We have previously described two children, one with an unbalanced subtelomeric rearrangement resulting in deletion of 22q13→qter and duplication of 1qter, and a second with an apparently simple 22q13→qter deletion. We have examined two additional patients with deletions of 22q13→qter. In one of the new patients presented here, clinical findings were suggestive of the 22q13 deletion syndrome and FISH for 22qter was requested. Chromosome studies suggested an abnormality involving the telomere of one 22q (46,XX,?add(22)(q13.3)). FISH using Oncor D22S39 and Vysis ARSA probes confirmed a terminal deletion. A multi-telomere FISH assay showed a signal from 19qter on the deleted chromosome 22. Results were confirmed with 19qtel and 22qtel specific probes. The patient is therefore trisomic for 19qter and monosomic for 22qter. The patient''s mother was found to have a translocation (19;22)(q13.42;q13.31). We also re-examined chromosomes from two patients previously diagnosed with 22q deletions who were not known to have a rearrangement using the multi-telomere assay. One of these patients was found to have a derivative chromosome 22 (der(22)t(6;22)(p25;q13)). No evidence of rearrangement was detected in the other patient. Thus we have found the 22q13 deletion to be associated with a translocation in three of four patients. This report illustrates the usefulness of examining patients with hypotonia, severe language delay, and mild facial dysmorphism for this syndrome and suggests that most of these deletions may be unbalanced subtelomeric rearrangements.     

9p Deletion and distal 9q duplication due to a paternal pericentric inversion 9(p22q32)

A female infant with 46,XX,rec(9), dup q,inv(9)(p22q32)pat is presented. She had a duplication from 9q32 to qter and a deletion from 9q22 to 9pter. Phenotypical abnormalities observed corresponded with features noted in cases with distal dup (9q), while pathognomonic features of del(9p) syndrome were not observed.     

Evaluation of NSD2 and NSD3 in overgrowth syndromes

Sotos syndrome is an overgrowth condition predominantly caused by truncating mutations, missense mutations restricted to functional domains, or deletions of NSD1. NSD1 is a member of a protein family that includes NSD2 and NSD3, both of which show 70-75% sequence identity with NSD1. This strong sequence similarity suggests that abrogation of NSD2 or NSD3 function may cause non-NSD1 Sotos cases or other overgrowth phenotypes. To evaluate this hypothesis, we mutationally screened NSD2 and NSD3 in 78 overgrowth syndrome cases in which NSD1 mutations and deletions had been excluded. Additionally, we used microsatellite markers within the vicinity of the genes to look for whole gene deletions. No truncating mutations or gene deletions were identified in either gene. We identified two conservative missense NSD2 alterations in two non-Sotos overgrowth cases but neither was within a functional domain. We identified three synonymous and two intronic variants in NSD2 and two synonymous base substitutions in NSD3. Our results suggest that despite strong sequence similarity between NSD1, NSD2 and NSD3, the latter genes are unlikely to be making a substantial contribution to overgrowth phenotypes and thus may operate in distinct functional pathways from NSD1.     

Cytogenetic,spectral karyotyping,fluorescence in situ hybridization,and comparative genomic hybridization characterization of two new secondary leukemia cell lines with 5q deletions,and MYC and MLL amplification

Cytogenetic studies of patients with therapy-induced acute myeloid leukemia (t-AML) have demonstrated whole chromosome loss or q-arm deletion of chromosomes 5 and/or 7 in a majority of cases. We have established two cell lines, SAML-1 and SAML-2, from two patients who developed t-AML after radiation and chemotherapy for Hodgkin disease. In both cases, the leukemia cells contained 5q deletions. SAML-1 has 58 chromosomes and numerous abnormalities, including der(1)(1qter-->1p22::5q31-->5qter), der(5)(5pter-->5q22::1p22-->1pter), +8, der(13)i(13)(q10)del(13)(q11q14.1), and t(10;11). Fluorescence in situ hybridization (FISH) with unique sequence probes for the 5q31 region showed loss of IL4, IL5, IRF1, and IL3, and translocation of IL9, DS5S89, EGR1, and CSFIR to 1p. SAML-2 has 45 chromosomes, del(5)(q11.2q31) with a t(12;13)ins(12;5), leading to the proximity of IRF1 and RB1, and complex translocations of chromosomes 8 and 11, resulting in amplification of MYC and MLL. Comparative genomic hybridization and spectral karyotyping were consistent with the G-banding karyotype and FISH analyses. Because a potential tumor suppressor(s) in the 5q31 region has yet to be identified, these cell lines should prove useful in the study of the mechanisms leading to the development of t-AML.     

Cytogenetic studies of primary cultures of esophageal squamous cell carcinoma

Cytogenetic analysis was performed on primary cultures of 21 squamous cell carcinomas of the esophagus (SCCE). Seven cases exhibited mosaic clonal chromosome abnormalities distributed as follows: two contained tetraploid cell populations, one with t(3;7)(p21;q11); two showed loss of the Y chromosome, one with double minutes; single cases demonstrated der(11)t(4;11)(q?27;q23); add(1)(p35) and del(4)(p12); and del(7)(p13), del(7)(q22q34), and der(11)t(7;11)(p?15;p?13). The remaining 14 cases had apparently normal karyotypes, possibly derived from stromal elements. These results demonstrate numerical abnormalities and the multiple occurrence of rearrangements involving chromosomes 7 and 11 in SCCE.     

Monosomy 6q: report on four new cases

We report on four patients with partial monosomy of the long arm of chromosome 6: two children presenting with an interstitial deletion del(6)(q14q16), the two others presenting with a terminal deletion del(6)(q25qter). These patients are compared with previous reports in the literature: 16 cases of terminal deletion and 17 cases of interstitial deletion. The deletions most often occur de novo. Mental retardation is always described. Dysmorphic facial features range between minor and major. There may be associated visceral abnormalities. After comparing the size and the localisation of the deletions with clinical data, we are now able to suggest a clinical localisation on chromosome 6.     

Williams-Beuren syndrome: phenotypic variability and deletions of chromosomes 7, 11, and 22 in a series of 52 patients.

Fluorescence in situ hybridisation (FISH) and conventional chromosome analysis were performed on a series of 52 patients with classical Williams-Beuren syndrome (WBS), suspected WBS, or supravalvular aortic stenosis (SVAS). In the classical WBS group, 22/23 (96%) had a submicroscopic deletion of the elastin locus on chromosome 7, but the remaining patient had a unique interstitial deletion of chromosome 11 (del(11)(q13.5q14.2)). In the suspected WBS group 2/22 (9%) patients had elastin deletions but a third patient had a complex karyotype including a ring chromosome 22 with a deletion of the long arm (r(22)(p11-->q13)). In the SVAS group, 1/7 (14%) had an elastin gene deletion, despite having normal development and minimal signs of WBS. Overall, some patients with submicroscopic elastin deletions have fewer features of Williams-Beuren syndrome than those with other cytogenetic abnormalities. These results, therefore, emphasise the importance of a combined conventional and molecular cytogenetic approach to diagnosis and suggest that the degree to which submicroscopic deletions of chromosome 7 extend beyond the elastin locus may explain some of the phenotypic variability found in Williams-Beuren syndrome.     

GPC3 mutation analysis in a spectrum of patients with overgrowth expands the phenotype of Simpson-Golabi-Behmel syndrome

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth syndrome caused by deletions in glypican 3 (GPC3). SGBS is characterized by pre- and postnatal overgrowth, a characteristic facial appearance, and a spectrum of congenital malformations which overlaps that of other overgrowth syndromes. We performed GPC3 deletion screening on 80 male patients with somatic overgrowth in the following categories: SGBS (n = 19), possible SGBS (n = 26), including families in which individuals had previously been diagnosed with other overgrowth syndromes, and Wiedemann-Beckwith syndrome (WBS) (n = 35). Using exon-specific PCR and Southern blot analysis, we identified seven GPC3 deletions. In most cases a clear X-linked family history was not present. In two cases, GPC3 deletions were identified in patients belonging to pedigrees published previously as other overgrowth syndromes: one with a diagnosis of Sotos syndrome and the other Perlman syndrome with nephroblastomatosis. A third patient developed hepatoblastoma, a tumor type not previously described in SGBS. No GPC3 deletions were identified among the WBS patients. Direct sequencing of all GPC3 exons in the remaining 13 SGBS patients without GPC3 deletions did not identify any further mutations, raising the possibility of alternative silencing mechanisms and/or other genes in the pathogenesis of SGBS. Our results validate the clinical specificity of the facial appearance, skeletal/hand anomalies, and supernumerary nipples in patients with GPC3 deletions. Our data also suggest that nephroblastomatosis and hepatoblastoma are included in the phenotypic spectrum of GPC3 deletions and SGBS, underscoring the importance of tumor surveillance in these children.     

Nonrandom rearrangements of 6p in malignant hematological disorders

It is very uncommon to observe nontranslocation abnormalities (NTAs) involving the short arm of chromosome 6 (6p) in malignant hematological disorders (MHDs). By using conventional cytogenetics and fluorescence in situ hybridization (FISH) with chromosome-microdissection probes specific for 6p21 and 6p25, we observed five patients with myeloid malignancies and two patients with lymphoid malignancies to have 6p NTAs. On the basis of our data and those in the literature, it is possible to divide 6p NTAs into the following three groups in MHD: The first group presents with 6p NTAs as a sole or primary change in myeloid malignancies. There are only two cases reported in this group, including one case with del(6)(p23) and the present case with ins(6)(q23p23p25) identified by FISH only. The second group presents with 6p deletions as a sole or primary change in lymphoid malignancies. Three cases have been reported in this group, including one case with del(6)(p21p23), one with del(6)(p21), and the present case 2 with del(6)(p21). The third group has 6p deletions in addition to other known primary changes, present in both myeloid and lymphoid disorders, with 36 cases reported, including five cases from our series. Deletions involving 6p21, 6p22, or 6p23 have been observed in both myeloid and lymphoid disorders. The present data provide cogent information for further molecular characterization of 6p anomalies in MHD.     

Unique karyotypes in two patients with Prader-Willi syndrome

A physical disruption of the Prader-Willi syndrome (PWS) chromosome region is thought to cause PWS. We describe 2 girls with PWS phenotype, who had unique chromosome 15 abnormalities. The first patient showed mosaicism: 45,XX,t(15;15)(qter→p11.1::q11.200→ qter)/46,XX,t(15;15)(qter → p11.1::q11.200→ qter), + mar. The band 15q11.2 apparently remained intact in the t(15;15) chromosome, and the mar chromosome was considered as r(15) (p11.1q11.1). The second patient had a karyo-type of 47,XX,del(15)(q11.200→q11.207), + idic (15)(pter → q11.1::q11.1→pter). The complex breakage and reunion involving the 15q11.2 regions of the father's homologous chromosomes 15 at meiosis appeared to have resulted in the idic(15) and the del(15) chromosomes. These cytogenetic findings suggest that the PWS chromosome region may be localized on the very proximal portion of band 15q11.2.