Variability in retinoblastoma genome stability is driven by age and not heritability

Retinoblastoma (RB) is a childhood intraocular cancer initiated by biallelic inactivation of the RB tumor suppressor gene (RB1^?/?). RB can be hereditary (germline RB1 pathogenic allele is present) or non‐hereditary. Somatic copy number alterations (SCNAs) contribute to subsequent tumorigenesis. Previous studies of only enucleated RB eyes have reported associations between heritability status and the prevalence of SCNAs. Herein, we use an aqueous humor (AH) liquid biopsy to investigate RB genomic profiles in the context of germline RB1 status, age, and International Intraocular Retinoblastoma Classification (IIRC) clinical grouping for both enucleated and salvaged eyes. Between 2014 and 2019, AH was sampled from a total of 54 eyes of 50 patients. Germline RB1 status was determined from clinical blood testing, and cell‐free DNA from AH was analyzed for SCNAs. Of the 50 patients, 23 (46.0%; 27 eyes) had hereditary RB, and 27 (54.0%, 27 eyes) had non‐hereditary RB. Median age at diagnosis was comparable between hereditary (13 ± 10 months) and non‐hereditary (13 ± 8 months) eyes (P = 0.818). There was no significant difference in the prevalence or number of SCNAs based on (1) hereditary status (P > 0.56) or (2) IIRC grouping (P > 0.47). There was, however, a significant correlation between patient age at diagnosis, and (1) number of total SCNAs (r[52] = 0.672, P < 0.00001) and (2) number of highly‐recurrent RB SCNAs (r[52] = 0.616, P < 0.00001). This evidence does not support the theory that specific molecular or genomic subtypes exist between hereditary and non‐hereditary RB; rather, the prevalence of genomic alterations in RB eyes is strongly related to patient age at diagnosis.     

Limitations of exome sequencing in detecting rare and undiagnosed diseases

While exome sequencing (ES) is commonly the final diagnostic step in clinical genetics, it may miss diagnoses. To clarify the limitations of ES, we investigated the diagnostic yield of genetic tests beyond ES in our Undiagnosed Diseases Network (UDN) participants. We reviewed the yield of additional genetic testing including genome sequencing (GS), copy number variant (CNV), noncoding variant (NCV), repeat expansion (RE), or methylation testing in UDN cases with nondiagnostic ES results. Overall, 36/54 (67%) of total diagnoses were based on clinical findings and coding variants found by ES and 3/54 (6%) were based on clinical findings only. The remaining 15/54 (28%) required testing beyond ES. Of these, 7/15 (47%) had NCV, 6/15 (40%) CNV, and 2/15 (13%) had a RE or a DNA methylation disorder. Thus 18/54 (33%) of diagnoses were not solved exclusively by ES. Several methods were needed to detect and/or confirm the functional effects of the variants missed by ES, and in some cases by GS. These results indicate that tests to detect elusive variants should be considered after nondiagnostic preliminary steps. Further studies are needed to determine the cost‐effectiveness of tests beyond ES that provide diagnoses and insights to possible treatment.     

中长型结肠镜检查回肠末段的临床价值

目的：探讨运用中长型结肠镜检查回肠末段的临床价值。方法自2001年1月~2013年12月用富士能EC-25WI5型中长型结肠镜单人操作法检查回肠末段。结果中长型结肠镜检查回肠末段8320例,检出回肠末段疾病192例,病变检出率2.3%。结论结肠镜检查延伸到回肠末段检查,杜绝了回肠末段疾病的漏诊,并可显著提高回肠末段疾病的检出率,具有很高的临床意义。     

EGFR protein expression and gene amplification in squamous intraepithelial lesions and squamous cell carcinomas of the cervix

The purpose of this study was to evaluate the protein expression and gene amplification of epithelial growth factor receptor (EGFR) in intraepithelial neoplasias and squamous cell carcinoma of the cervix and to determine the value of EGFR in carcinogenesis, progression, and prognosis of cervical cancer. EGFR protein expression and gene amplification involved gene copy number in 75 cases of cervical various lesions were evaluated using immunohistochemistry and by fluorescence in situ hybridization (FISH) techniques. Expression of EGFR was observed in 76.00% of the high-grade CIN and 79.17% of the invasive carcinomas. In contrast, there were low levels of EGFR expression in chronic cervicitis (1/10) and low-grade CIN (7/16). There were statistically significant differences among them (P<0.05). Gene amplification was detected in 20.51% high-grade CIN and invasive carcinoma, but there only 4.35% EGFR gene amplification was observed in chronic cervicitis and low grade CIN. Among the 42 patients with negative or low levels of EGFR expression, 26 patients (61.90%) were found to have diploidy and 11 patients (26.20%) to have balanced triploidy. However, among the 20 patients with an intermediate and high levels of EGFR protein expression, 13 (65.00%) were found to have balanced polyploidy or gene amplification. All cases of EGFR gene amplification involved intermediate and high levels of protein expression. EGFR may be involved in the carcinogenesis of the cervix and may be an early event during the carcinogenesis. Overexpression of EGFR protein may result from gene amplification and increases in gene copy number.     

Genome-wide screening of DNA copy number alterations in cervical carcinoma patients with CGH+SNP microarrays and HPV-FISH

Alterations in the genome that lead to changes in DNA sequence copy number are characteristic features of solid tumors. We used CGH+SNP microarray and HPV-FISH techniques for detailed screening of copy number alterations (CNAs) in a cohort of 26 patients with cervical carcinoma (CC). This approach identified CNAs in 96.2% (25/26) of tumors. Array-CGH discovered CNAs in 73.1% (19/26) of samples, HPV-FISH experiments revealed CNAs in additional 23.1% (6/26) of samples. Common gains of genetic sequences were observed in 3q (50.0%), 1q (42.4%), 19q (23.1%), while losses were frequently found in 11q (30.8%), 4q (23.1%) and 13q (19.2%). Chromosomal regions involved in loss of heterozygosity were observed in 15.4% of samples in 8q21, 11q23, 14q21 and 18q12.2. Incidence of gain 3q was associated with HPV 16 and HPV 18 positive samples and simultaneous presence of gain 1q (P = 0.033). We did not found a correlation between incidence of CNAs identified by array-CGH and HPV strain infection and incidence of lymph node metastases. Subsequently, HPV-FISH was used for validation of array-CGH results in 23 patients for incidence of hTERC (3q26) and MYC (8q24) amplification. Using HPV-FISH, we found chromosomal lesions of hTERC in 87.0% and MYC in 65.2% of specimens. Our findings confirmed the important role of HPV infection and specific genomic alterations in the development of invasive cervical cancer. This study also indicates that CGH+SNP microarrays allow detecting genome-wide CNAs and copy-neutral loss of heterozygosity more precisely, however, it may be less sensitive than FISH in samples with low level clonal CNAs.     

产前诊断样本中母体细胞污染对染色体微阵列技术检测的影响

目的评估产前诊断样本中母体细胞污染(MCC)对染色体微阵列技术(CMA)检测拷贝数变异(CNV)的影响。方法前瞻性研究。挑选2016年12月至2018年8月于杭州市妇产科医院产前诊断中心行羊水产前诊断且经CMA检测出拷贝数变异的DNA标本5份,加入正常女性基因组DNA以模拟不同比例母体细胞污染。采用Agilent微阵列染色体芯片180K CGH(Agilent 180K CGH)对模拟标本进行检测,检测结果通过Agilent CytoGenomics软件进行分析。结果当MCC>38.4%(95%可信区间:36.6%~40.2%)时重复CNV无法检出,MCC>41.3%(95%可信区间:39.9%~42.7%)时缺失CNV无法检出,且随MCC比例增高,CNV检出率逐步降低;大片段的CNV较小片段CNV对MCC耐受程度高;相同大小CNV,缺失型CNV的检出能力较重复型略微升高。在男性标本中,当MCC>10%时,可通过微阵列检测到X/Y染色体发生位移。结论MCC高于一定比例时可以对CMA检测CNV产生影响。基于Agilent 180K CGH对MCC模拟物的检测结果及CNV检测特异性原则,鉴于保守原则,本中心Agilent 180K CGH的MCC阈值设定为30%。     

SNP array mapping of chromosome 20p deletions: Genotypes,phenotypes, and copy number variation

The use of array technology to define chromosome deletions and duplications is bringing us closer to establishing a genotype/phenotype map of genomic copy number alterations. We studied 21 patients and five relatives with deletions of the short arm of chromosome 20 using the Illumina HumanHap550 SNP array to: 1) more accurately determine the deletion sizes; 2) identify and compare breakpoints; 3) establish genotype/phenotype correlations; and 4) investigate the use of the HumanHap550 platform for analysis of chromosome deletions. Deletions ranged from 95 kb to 14.62 Mb, and all of the breakpoints were unique. Eleven patients had deletions between 95 kb and 4 Mb and these individuals had normal development, with no anomalies outside of those associated with Alagille syndrome (AGS). The proximal and distal boundaries of these 11 deletions constitute a 5.4‐Mb region, and we propose that haploinsufficiency for only 1 of the 12 genes in this region causes phenotypic abnormalities. This defines the JAG1‐associated critical region, in which deletions do not confer findings other than those associated with AGS. The other 10 patients had deletions between 3.28 Mb and 14.62 Mb, which extended outside the critical region, and, notably, all of these patients had developmental delay. This group had other findings such as autism, scoliosis, and bifid uvula. We identified 47 additional polymorphic genome‐wide copy number variants (>20 SNPs), with 0 to 5 variants called per patient. Deletions of the short arm of chromosome 20 are associated with relatively mild and limited clinical anomalies. The use of SNP arrays provides accurate high‐resolution definition of genomic abnormalities. Hum Mutat 0,1–8, 2008. © 2008 Wiley‐Liss, Inc.