Genetic alterations in childhood medulloblastoma analyzed by comparative genomic hybridization

Despite intensive therapy, the survival of children with medulloblastoma remains disappointing. Moreover, children who survive are affected by serious long-term sequelae of treatment that impair their quality of life. In search of chromosomal aberrations indicative of sites involved in oncogenic transformation and in an attempt to find reliable prognostic markers, the authors analyzed 15 medulloblastomas by comparative genomic hybridization. All neoplasms showed chromosomal abnormalities. The most frequent losses were 17p (7/15 tumors), 8p and 11p (6/15), 10p, 1lq, 16q, and 20q (5/15), and 20p (4/15). Gains were recurrently found at 7q (10/15 tumors), 17q and 18q (9/15 tumors), 7p and 13q (7/15), 18p (6/15), and 1q, 4q, 6q. and 9p (5/15 tumors). Four tumors showed loss of 17p together with gain of 17q, suggesting an isochromosome 17q. High-level amplifications were seen at 1p34, 5p15, 13q34, and 18p11 (one tumor each), and at 2p15 in two tumors, one of which was proven to be N-Myc amplification. The overall pattern of alterations found in this study confirms the findings of other studies and adds two novel regions with chromosomal gains, at 13q and 18q. Previous reports on the relation between 17q gain and survival could not be confirmed, whereas amplification of N-myc or L-myc seems to indicate poor clinical outcome.     

Genetic alterations in pediatric medulloblastomas detected by comparative genomic hybridization.

Children with medulloblastomas show diverse clinical courses even when receiving similar treatments. In this study, comparative genomic hybridization, which allows the detection of losses and gains in DNA copy number along the entire genome, was used to investigate the genetic alterations in 6 cases of medulloblastoma with adequate follow-up periods and similar treatments, and in a medulloblastoma cell line. In the cell line, the number of aberrations was the highest of all samples examined. In 6 clinical samples, frequently altered regions (more than 3 cases) observed in all medulloblastomas were gains of 7q and 17q (4 cases each), and of 2p, 2q and 7p (3 cases each). High-grade amplification was observed at the loci 8q, 17q and 21q, each in a single case. The case with the most favorable outcome 9 years after surgery had the smallest number of chromosomal changes among the cases examined. Our results may indicate that further acquisition of genetic alterations detected by comparative genomic hybridization are associated with unfavorable prognosis in patients with medulloblastomas.     

Genetic analysis of childhood endodermal sinus tumors by comparative genomic hybridization

Childhood endodermal sinus tumors (CEST) are a distinct category of germ cell tumors that involve the testis and extragonadal sites of young children. Recurrent deletions of 1p and 6q have been reported by classic cytogenetic analysis of a small number of cases. Comparative genomic hybridization, a technique that screens the entire genome for genetic abnormalities, is applied to additionally define the genetic changes present in CESTs. Sixteen frozen CESTs (10 testicular, 6 extragonadal) obtained from Pediatric Oncology Group-affiliated institutions or from the Cooperative Human Tissue Network were analyzed. The most common changes were gain of 20q (10 tumors), 1q (6 tumors), 11q and 22 (4 tumors each), and loss of 6q (8 tumors with common deleted region of 6q24-qter), 16q (4 tumors), and 1p (4 tumors). Localized regions of gain were identified at 8q24 (2 tumors both showing c-myc amplification by fluorescence in situ hybridization). Gain of 12p, characteristic of adolescent germ cell tumor, was present in one testicular tumor. Comparative genomic hybridization was useful in defining genetic differences between adult and childhood tumors, in determining the common regions deleted on chromosome 6, and in identifying other involved loci to be correlated with clinical parameters in future studies.     

微阵列比较基因组杂交技术检测不明原因智力低下/发育迟缓患儿的基因组拷贝数变异

目的应用高分辨微阵列比较基因组杂交技术(Array-CGH),对中国人群不明原因的智力低下/发育迟缓(MR/DD)患儿进行全基因组拷贝数变异(CNVs)筛查,获得在这些不明原因MR/DD患儿中CNVs的检出率,并分析其中的罕见CNVs与MR/DD的相关性,以此评估Array-CGH对不明原因MR/DD可能的遗传病因诊断作用。方法根据特定筛选条件收集在首都儿科研究所临床诊断为不明原因MR/DD患儿,用Oligo244KDNA芯片筛查全基因组CNVs。针对所发现的CNVs,首先将其与国际基因组CNVs多态性数据库(databaseofgenomicvariants)进行比对,剔除常见多态性CNVs,将获得的罕见CNVs应用美国波士顿儿童医院遗传诊断实验室的临床分子诊断平台,结合基因组异常拷贝数数据库(DECIPHER)进行核查并与既往相关文献比对,以发现罕见CNVs在不明原因MR/DD患儿中的检出率。结果2004年7月至2008年7月共收集111例不明原因MR/DD患儿,平均年龄为6岁,男女比例为1.775。28例患儿发现36个罕见CNVs,CNVs平均长度为1326kb(29～8760kb),这些CNVs均无法被常规染色体G带检查所识别。通过评估,19例患儿携带可能与MR/DD相关的CNVs,另1例患儿的CNVs临床意义不明确,Array-CGH在不明原因MR/DD患儿中发现携带与疾病相关的罕见CNVs的诊断率为17.1%(19/111例)。22/36个(66.1%)罕见CNVs曾被美国波士顿儿童医院Array-CGH数据库、DECIPHER数据库、既往MR/DD微阵列研究文献所报道。1例患儿在15q11.2-13.1存在2098kb的基因组缺失,覆盖Prader-Willi综合征/Angelman综合征关键区的多个候选基因,包括SNRPN、NECDIN、SnRNAs和UBE3A,结合该患儿面部表型、临床检查以及Array-CGH结果,诊断为非典型性Prader-Willi综合征。结论基因组CNVs相关的微缺失/重复是中国人群中不明原因MR/DD患儿的原因之一,高分辨Array-CGH技术可在不明原因MR/DD患儿中发现更多的遗传病因,帮助和提高不明原因MR/DD的分子诊断水平。     

Genetic analysis of childhood germ cell tumors with comparative genomic hybridization

BACKGROUND: Germ Cell Tumors (GCTs) in children and adolescents constitute a clinically and histologically heterogeneous group of tumors. Compared to GCTs in adults, the numbers of GCTs in children analyzed with cytogenetic and molecular genetic techniques is limited. However, the data available to date reveal a pattern of cytogenetic aberrations different from that in adults. Comparative genomic hybridization (CGH) is a valuable technique for the genetic profiling of tumors that allows screening for chromosomal imbalances consistent with amplification of oncogenes and loss of putative tumor suppressor genes. As CGH does not require tissue culture, it also allows analysing archival tissue samples. PATIENTS: This study focuses exclusively on GCTs in children younger than ten years of age and summarizes the genetic data of 51 tumors. Eighteen teratomas and 33 malignant GCTs were included. Primary sites were the testis (n=10), coccyx (n=13), mediastinum (n=20), ovary (n=5), CNS (n=2), and the face (n=1). METHODS: The experimental procedure includes differential enzymatic fluorescence labeling of tumor and control DNA followed by comparative hybridization to normal male chromosomes, karyotyping and computerized analysis of the fluorescence profiles. RESULTS: With the exception of one testicular and two ovarian tumors, malignant GCTs in children do not show chromosomal gain of 12p, which is characteristic of GCTs in adult patients. Irrespective of the primary site, childhood GCTs show chromosomal imbalances of chromosome 1 (loss of distal 1p, gain of 1q), deletion of 4q and 6q as well as gain of 20q at a high frequency. CONCLUSIONS: These studies will help guiding further investigations elucidating the role of putative tumor suppressor genes at e.g. 1p36 and 6q. In addition, further studies incorporated in prospective therapeutic protocols are necessary to evaluate the prognostic relevance of specific genetic aberrations.     

Clinical utility of an array comparative genomic hybridization analysis for Williams syndrome

To reveal the relation between intellectual disability and the deleted intervals in Williams syndrome, we performed an array comparative genomic hybridization analysis and standardized developmental testing for 11 patients diagnosed as having Williams syndrome based on fluorescent in situ hybridization testing. One patient had a large 4.2‐Mb deletion spanning distally beyond the common 1.5‐Mb intervals observed in 10/11 patients. We formulated a linear equation describing the developmental age of the 10 patients with the common deletion; the developmental age of the patient with the 4.2‐Mb deletion was significantly below the expectation (developmental age = 0.51 × chronological age). The large deletion may account for the severe intellectual disability; therefore, the use of array comparative genomic hybridization may provide practical information regarding individuals with Williams syndrome.     

Flow cytometric DNA index, G-band karyotyping, and comparative genomic hybridization in detection of high hyperdiploidy in childhood acute lymphoblastic leukemia

High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic hybridization (HR-CGH) in detecting high hyperdiploid leukemic clones. Twenty-six girls and 34 boys with acute lymphoblastic leukemia diagnosed in 1998 to 1999 were analyzed by FCM, GBK, and HR-CGH. The correlations between DNA indices obtained by FCM, GBK, and HR-CGH were significant (rs=0.61 to 0.77; P<0.001 for all comparisons). However, in 4 of 18 patients, high hyperdiploidy was overlooked by GBK or HR-CGH, and even when FCM was applied, 2 of 18 patients with high hyperdiploidy by GBK and/or HR-CGH were classified as nonhigh hyperdiploid. If high hyperdiploid subclones were included, FCM could detect all high hyperdiploid patients found by either GBK or HR-CGH, but would then in addition classify 15% to 20% of the remaining patients as high hyperdiploid. Thus, both GBK and HR-CGH overlook patients with high hyperdiploidy, and FCM only detects all high hyperdiploid patients if small high hyperdiploid clones are included. In addition, FCM detects patients with high hyperdiploid subclones, not detected by either GBK or HR-CGH, and the challenge remains to determine the prognosis of patients with such high hyperdiploid subclones.     

微阵列技术检测Wolf-Hirschhorn综合征1例报告

患儿女,8月龄,因精神运动发育迟缓7个月就诊。患儿系第1胎第1产,胎龄37+3周,因孕母产检时发现“胎儿多发畸形,宫内发育迟缓,臀位妊娠”行剖宫产出生,出生体重为1.9?kg。因“小于胎龄儿”在我院新生儿科住院治疗,行宫内致畸四项、遗传代谢病氨基酸和酰基肉碱谱及尿液有机酸综合分析等代谢相关检查、染色体检查均未见明显异常。心脏彩超提示先天性心脏病：Ⅱ型房间隔缺损、动脉导管未闭等。泌尿系B超提示左肾积水。头颅MRI检查提示：考虑左侧中央旁小叶少许渗液;右侧脑室较左侧脑室明显变窄,考虑发育变异。患儿现8月龄,竖头不稳,尚不能坐,不能认人。以“精神运动发育迟缓：足月小于胎龄儿;先天性心脏病：房间隔缺损、动脉导管未闭;左肾积水”收入我科。其父母非近亲结婚,父亲生育年龄为28岁,母亲为30岁,均体健,均未从事有毒有害工作。家族中无类似病史,无家族遗传病。     

Distal chromosome 17 gains in neuroblastomas detected by comparative genomic hybridization (CGH) are associated with a poor clinical outcome

PROCEDURE: To establish the significance of chromosome 17 aberrations in the biology of neuroblastomas, the fresh-frozen material of 53 primary neuroblastomas (average patient age: 20.8 months; stage 1 or 2: n = 10; stage 3: n = 10; stage 4: n = 10; stage 4s: n = 23) was studied by means of comparative genomic hybridization (CGH). Follow-up data were available for 52 of 53 cases studied (average follow-up period: 26.4 months). Except for one, all cases had previously been analyzed for MYCN status (semiquantitative Southern blot analysis). Studies of LOH 1p36 (VNTR-PCR) had been performed on 28 of 53 cases. RESULTS: Chromosome 17 gains were detected in 46 of 53 (86.8%) cases. Whole chromosome gains were mostly restricted to localized tumors (stage 1 or 2: 9 of 10 cases; stage 4s:19 of 23; stage 3: 2 of 10; stage 4:0 of 10 cases), whereas distal 17 gains were significantly associated with clinically advanced tumor stages and patients aged over 1 year at diagnosis. Univariate analyses revealed a statistically significant correlation of distal 17q gains with overall survival (P< 0.01, MYCN amplification: P< 0.01; 1p deletion: P< 0.01) and an elevated recurrency rate (17q: P= 0.02, MYCN amplification: P = 0.05; 1p deletion P= 0.3). There was a strong coincidence of distal 17q gains and 1p deletion or MYCN amplification (P < 0.01). CONCLUSION: Our data indicate that distal chromosome 17q gains are of major prognostic relevance for neuroblastoma patients. However, studies on a larger series of tumors have to be performed to assess whether or not these alterations are independent prognostic markers of a poor clinical outcome.     

Incidence of bacteremias and invasive mycoses in children with high risk neuroblastoma

BACKGROUND: Information on the incidence of infectious complications during for treatment for high risk neuroblastoma (HR-NB) is limited. Bacteremias and invasive mycoses may be considered surrogate markers of the infection burden. PATIENTS AND METHODS: Data on bacteremias and invasive mycoses occurring during 3 consecutive protocols for front line (NB-89; NB-92; NB-97) or salvage therapy (TVD) for HR-NB were reviewed. The cumulative risk of developing a first episode and the rate of infections during the entire length of each protocol were evaluated. RESULTS: Front line protocols were given to 80 patients for a total of 22,070 days at risk; salvage treatment was given to 24 children for 2,909 days at risk. During front line therapy 41 infectious episodes were diagnosed in 29 (36%) patients, for a 45% cumulative risk and an infection rate (IR) of 0.19/100 patient-days-at risk. Salvage therapy determined five infectious episodes in four (17%) patients, with a 39% cumulative risk, and an IR of 0.17. The IR during the phase of high dose chemotherapy with hematopoietic stem cell rescue (megatherapy) included in the three front line protocols decreased over time (1.54 in NB-89; 0.52 in NB-92 and 0.0 in NB 97; P = 0.001), possibly because of the use of less aggressive conditioning regimens, without radiotherapy. CONCLUSIONS: The IRs of protocols for HR-NB did not change over time. The megatherapy-related phases are those at highest risk.     

Comparative genomic hybridization in pediatric acute lymphoblastic leukemia

Comparative genomic hybridization (CGH) was used to clarify the chromosomal status of 15 patients diagnosed with childhood acute lymphoblastic leukemia (ALL). Bone marrow samples from 10 of the 15 patients were selected because no metaphases were obtained for cytogenetic analysis. Three patients with normal trypsin and giemsa banding (GTG) karyotypes were also studied by CGH to determine whether significant abnormalities might have been missed by banding analysis, and samples from an additional 2 patients with hyperdiploidy were also included. Seven of the 10 patients with failed GTG banding analysis were found to be chromosomally abnormal by CGH; 2 out of 3 patients with normal GTG band karyotypes were abnormal, indicating that the metaphases available for karyotyping were not malignant cells, and that CGH analysis of hyperdiploid samples provided more accurate resolution than karyotyping alone. The prognostic value of chromosomal aberrations detected by CGH and the efficiency of the technique suggest a central role for CGH in routine clinical cytogenetics.     

Array-based comparative genome hybridization in clinical genetics

Abnormalities in DNA copy number are frequently found in patients with multiple anomaly syndromes and mental retardation. Array-based comparative genomic hybridization (array-CGH) is a high-resolution, whole-genome technology that improves detection of submicroscopic aberrations underlying these syndromes. Eight patients with mental disability, multiple congenital anomalies, and dysmorphic features were screened for submicroscopic chromosomal imbalances using the GenoSensor Array 300 Chip. Subtelomeric aberrations previously detected by fluorescence in situ hybridization (FISH) analysis were confirmed in two patients, and accurate diagnosis was provided in two previously undiagnosed complex cases. Microdeletions at 15q11.2-q13 in a newborn with hypotonia, cryptorchidism, and hypopigmentation were detected with few discrepancies between the array results and FISH analysis. Contiguous microdeletion of GSCL, HIRA and TBX1 genes at 22q11.2 was identified in a previously undiagnosed boy with an unusual presentation of the VCF/DiGeorge spectrum. In a newborn with aniridia, a borderline false-negative WT1 deletion was observed, most probably because of differences between the size of the genomic deletion and the microarray probe. A false-positive rate of 0.2% was calculated for clone-by-clone analysis, whereas the per patient false-positive rate was 20%. Array-CGH is a powerful tool for the rapid and accurate detection of genetic disorders associated with copy number abnormalities and can significantly improve clinical genetic diagnosis and care.     

Solving the puzzle: case examples of array comparative genomic hybridization as a tool to end the diagnostic odyssey

We review 3 cases where array comparative genomic hybridization made a difference in the medical management of the patient, ended the diagnostic odyssey, predicted prognosis for the patient, and/or provided closure to the family. Comparative genomic hybridization is a useful tool for testing individuals with clinical examinations suggestive of a genetic syndrome but in which a specific syndrome may be difficult to pinpoint. The cost is similar to that of a standard karyotype but there is a higher yield in children and adults with clinical signs of a genetic syndrome.     

比较基因组杂交技术及其在医学遗传学中的应用

比较基因组杂交（comparative genomic hybridization．CGH）是1992年Kallioniemi创立的基于荧光标记和分子、细胞遗传学技术鉴定基因组DNA的获得、丢失和扩增,并且可以把这些遗传变异定位在正常的中期染色体上的一种技术,也称之为中期CGH（metaphase—CGH）。主要特点是在一个实验中,用1张中期染色体涂片（metaphage spreads）,就能在全基因组范围内分析大的DNA拷贝数的不平衡改变,检测和定位DNA序列拷贝数的获得和丢失,即基因剂量的变化而不需要分裂的细胞。在其创立初期主要应用于肿瘤基因组学的研究。近年来,随着分子生物学技术的进步,经典的中期-CGH技术也有了很大的发展,在了解人类基因组的组织结构、基因定位和遗传病的诊断等方面发挥着越来越重要的作用。