The Second Allele: A Key to Understanding the Timing of Sporadic and Hereditary Colorectal Tumorigenesis.

Our understanding of the molecular basis of colorectal neoplasia is derived from Mendelian genetics, with tumor suppressor genes contributing more to the deregulation of growth than oncogenes. In patients with hereditary syndromes, expression of one allele of a key tumor suppressor gene is absent at birth. The loss of the expression of the second allele precipitates tumorigenesis. However, there are multiple ways in which the expression of the second allele of a tumor suppressor gene is lost. Here, we review these ways and their possible effect on phenotype.

Re-evaluating the pathogenicity of the c.783+2T>C BAP1 germline variant.

BAP1 germline pathogenic sequence variants (PSVs) underlie a unique tumor predisposition syndrome (BAP1-TPDS) associated with an increased lifetime risk for developing primarily pleural and peritoneal mesothelioma and uveal and cutaneous melanoma. Overwhelmingly, BAP1 PSVs are unique, family-specific inactivating variants. We identified seven families, six of Jewish Iraqi origin, harboring an identical BAP1 splice variant (c.783+2T>C), currently assigned a "likely pathogenic" status. Given a nonclassical BAP1-TPDS tumor type clustering and low penetrance in these families, the pathogenicity of this variant was re-evaluated by a combined approach including literature analysis, revised bioinformatics analysis, allelic loss, effect on the transcript, and tumor protein expression patterns. None of the three available tumors showed an allelic loss, there was no discernable effect on alternative splicing based on reverse-transcription polymerase chain reaction, and there was no decrease or loss of somatic protein expression in 2/3 analyzed tumors. This led to assigning a Benign Strong (BS) criteria, BS4, supporting BS3 criteria, and weakening the Pathogenic Supporting (PP) criteria PP5. Combined, these data suggest that this sequence variant should be reclassified as a variant of unknown significance by American College of Medical Genetics (ACMG) criteria.

Detection and Analysis of 12 Suspected Amelogenin Allelic Loss Cases.

OBJECTIVES: To observe and analyse the Amelogenin allelic loss in parent-child identification cases, and to explore the type and mechanism of Amelogenin allelic loss as well as its influence on gender identification and solutions. METHODS: After the detection by SiFaSTR™ 23plex DNA identification system, samples had the characteristics of the peak area of Amelogenin X was the same as the one of adjacent heterozygote or lower than one half of adjacent homozygote in females while Amelogenin X loss was observed in males were selected. X chromosome STR （X-STR） typing and Amelogenin X sequencing were performed. The samples with Amelogenin Y loss in males were confirmed by the detection of Y chromosome STR typing and sex-determining region of Y （SRY）. The type and rate of Amelogenin allelic loss were confirmed and calculated, and the mechanism and influence of this variation were also analysed. RESULTS: Amelogenin X allelic loss was observed in one male sample, the mutation in primer-binding region was confirmed by sequencing. The suspected Amelogenin X allelic loss was observed in four female samples, but the mutation in primer-binding region was confirmed by sequencing in only one sample. Amelogenin Y allelic loss was observed in seven male samples, SRY positive cases was detected in five of them, and two were SRY negative. Y-STR type was detected in four cases of the five SRY positive cases, which was not detected in the two SRY negative cases. The rate of Amelogenin allelic loss was about 0.029%. CONCLUSIONS: Amelogenin X allelic loss does not affect the gender identification, but Amelogenin Y allelic loss may cause wrong gender identification. Thus, Y-STR or SRY should be detected for gender confirmation. When Y-STR genotypes are not detected in a "male" whose SRY detection is also negative, then the chromosome karyotype analysis and sex differentiation related genes test should be taken to further confirm the gender.

Bi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia.

Infertility affects around 7% of men worldwide. Idiopathic non-obstructive azoospermia (NOA) is defined as the absence of spermatozoa in the ejaculate due to failed spermatogenesis. There is a high probability that NOA is caused by rare genetic defects. In this study, whole-exome sequencing (WES) was applied to two Estonian brothers diagnosed with NOA and Sertoli cell-only syndrome (SCOS). Compound heterozygous loss-of-function (LoF) variants in FANCM (Fanconi anemia complementation group M) were detected as the most likely cause for their condition. A rare maternally inherited frameshift variant p.Gln498Thrfs∗7 (rs761250416) and a previously undescribed splicing variant (c.4387-10A>G) derived from the father introduce a premature STOP codon leading to a truncated protein. FANCM exhibits enhanced testicular expression. In control subjects, immunohistochemical staining localized FANCM to the Sertoli and spermatogenic cells of seminiferous tubules with increasing intensity through germ cell development. This is consistent with its role in maintaining genomic stability in meiosis and mitosis. In the individual with SCOS carrying bi-allelic FANCM LoF variants, none or only faint expression was detected in the Sertoli cells. As further evidence, we detected two additional NOA-affected case subjects with independent FANCM homozygous nonsense variants, one from Estonia (p.Gln1701∗; rs147021911) and another from Portugal (p.Arg1931∗; rs144567652). The study convincingly demonstrates that bi-allelic recessive LoF variants in FANCM cause azoospermia. FANCM pathogenic variants have also been linked with doubled risk of familial breast and ovarian cancer, providing an example mechanism for the association between infertility and cancer risk, supported by published data on Fancm mutant mouse models.

NDRG2 gene copy number is not altered in colorectal carcinoma.

AIM: To investigate if the down-regulation of N-myc Downstream Regulated Gene 2 (NDRG2) expression in colorectal carcinoma (CRC) is due to loss of the NDRG2 allele(s). METHODS: The following were investigated in the human colorectal cancer cell lines DLD-1, LoVo and SW-480: NDRG2 mRNA expression levels using quantitative reverse transcription-polymerase chain reaction (qRT-PCR); interaction of the MYC gene-regulatory protein with the NDRG2 promoter using chromatin immunoprecipitation; and NDRG2 promoter methylation using bisulfite sequencing. Furthermore, we performed qPCR to analyse the copy numbers of NDRG2 and MYC genes in the above three cell lines, 8 normal colorectal tissue samples and 40 CRC tissue samples. RESULTS: As expected, NDRG2 mRNA levels were low in the three colorectal cancer cell lines, compared to normal colon. Endogenous MYC protein interacted with the NDRG2 core promoter in all three cell lines. In addition, the NDRG2 promoter was heavily methylated in these cell lines, suggesting an epigenetic regulatory mechanism. Unaltered gene copy numbers of NDRG2 were observed in the three cell lines. In the colorectal tissues, one normal and three CRC samples showed partial or complete loss of one NDRG2 allele. In contrast, the MYC gene was amplified in one cell line and in more than 40% of the CRC cases. CONCLUSION: Our study suggests that the reduction in NDRG2 expression observed in CRC is due to transcriptional repression by MYC and promoter methylation, and is not due to allelic loss.

Anastomotic recurrence of colon cancer: Genetic analysis challenges the widely held theories of cancerous cells' intraluminal implantation and metachronous carcinogenesis.

BACKGROUND AND OBJECTIVES: Anastomotic recurrence (AR), whose etiopathogenesis is attributed to intraluminal implantation of cancerous cells or metachronous carcinogenesis, is a major issue for patients undergoing colon cancer (CC) resection. The objective of the study is to throw some light on AR etiopathogenesis and to identify risk factors of AR in selecting patients to undergo early endoscopy. METHODS: An analysis of clinical and histopathological parameters, including MSI and LOH of seven sites (Myc-L, BAT26, BAT40, D5S346, D18S452, D18S64, D16S402) was performed in primary CC and AR of 18 patients. They were then compared to 36 controls not developing AR. RESULTS: A genetic instability was present in 16/18 patients, with distinct genetic patterns between primaries and ARs. LOH at 5q21 and/or 18p11.23 were found in both primary and AR in >50% of cases, but this rate was no different from control population. CEA resulted as associated with AR (P = 0.03), whereas N status presented a borderline result (P = 0.08). CONCLUSIONS: Our findings challenge present theories about AR development. No "genetic marker" has been found. CEA and, to a lesser extent, N status, appear associated with AR. Rectal washout is seemingly meaningless. Iterative resection should be recommended since a long survival may be expected. J. Surg. Oncol. 2016;114:228-236. © 2016 Wiley Periodicals, Inc.

Assessment of the frequency of genetic alterations (LOH/MSI) in patients with intraepithelial cervical lesions with HPV infection: a pilot study.

In the present study, we analyzed (1) the type of HPV infection and (2) the frequency of loss of heterozygosity and microsatellite imbalance (LOH/MSI) in normal cytology and cervical intraepithelial neoplasia (CIN1-3). The cytological material included: low-grade squamous intraepithelial lesions (CIN1, n = 11), high-grade lesions (CIN2 and CIN3, n = 13), and cytologically normal cells from non-neoplastic cervical samples (n = 8). HPV genotyping was done using RealLine HPV 16/18 kit. We used 20 microsatellite markers from: 1p31.2, 3p14.3, 3p21.3, 3p22.2, 3p24.2, 3p25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1. LOH/MSI was correlated with clinicopathological parameters. The presence of HPV DNA was revealed in 78.13 % samples, including normal cytology. LOH/MSI was the most frequent for: 3p25.3 (39 %), 3p22.2 (20.83 %), 3p24.2 (20 %), and 3p14.3 (16.67 %). It was demonstrated that D3S1234 (FHIT; 3p14.3), D3S1611 (MLH1; 3p22.2), D3S1583 (RARB; 3p24.2), D3S1317 and D3S3611 (VHL; 3p25.3) could differentiate patients with CIN2/CIN3 versus CIN1, showing significantly higher frequency in CIN2/CIN3. LOH/MSI frequency for other than 3p markers was lower, 10-22.2 %. The simultaneous occurrence of LOH/MSI for several markers (OFAL) was higher in CIN2/CIN3. Significant differences in OFAL were found between samples with versus without HPV infection. In HPV-positive patients, significant differences in OFAL were found between normal cytology, CIN1 and CIN2/CIN3. HPV infection influences the increase in LOH/MSI frequency, especially in tumor suppressor gene loci. Several studied microsatellite markers seem to be useful for CIN grading. Hopefully, the obtained results, if confirmed on larger patient cohort, would allow creating a panel of markers supporting clinical diagnosis in patients with HPV infection.

Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas.

Malignant pleural mesothelioma (MPM), a rare malignancy with a poor prognosis, is mainly caused by exposure to asbestos or other organic fibers, but the underlying genetic mechanism is not fully understood. Genetic alterations and causes for multiple primary cancer development including MPM are unknown. We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. We found that the MPM had R282W, a key TP53 mutation, and genome-wide allelic loss or loss of heterozygosity, a distinct genomic alteration not previously described in MPM. We identified frequent inactivating SETDB1 mutations in this patient and in 68 additional MPM patients (mutation frequency: 10%, 7/69) by targeted deep sequencing. Our observations suggest the possibility of a new genetic mechanism in the development of either MPM or multiple primary cancers. The frequent SETDB1 inactivating mutations suggest there could be new diagnostic or therapeutic options for MPM.

Analysis of 41 cancer cell lines reveals excessive allelic loss and novel mutations in the SIRT1 gene.

SIRT1 is an evolutionarily conserved protein deacetylase that modulates stress response, cellular metabolism and aging in model organisms. While SIRT1 exerts beneficial effects in protecting against age-related diseases, the role of SIRT1 in cancer has been controversial. SIRT1 promotes cell survival by deacetylating, and thereby negatively regulating the activity of important tumor suppressors such as p53. In this regard, SIRT1 has been considered to be a potential oncogene, and SIRT1 inhibitors have been studied for possible anticancer therapeutic effects. In contrast, it has been shown that SIRT1 deficiency leads to increased genomic instability and tumorigenesis, and that overexpression of SIRT1 attenuates cancer formation in mice, suggesting it may also act as a tumor suppressor. Based on this evidence, SIRT1-activating molecules could act as candidate chemotherapeutic drugs. In order to gain insight into the role of SIRT1 in cancer, we performed a comprehensive resequencing analysis of the SIRT1 gene in 41 tumor cell lines and found an unusually excessive homozygosity, which was confirmed to be allelic loss by microsatellite analysis. Furthermore, we found two novel SIRT1 mutations (D739Y and R65_A72del) in addition to the known, rare non-synonymous variation resulting in I731V. In vitro assays using purified SIRT1 protein showed that these mutations do not alter SIRT1 deacetylase activity or telomerase activity, which was shown to be regulated by SIRT1. We conclude that allelic loss or mutations in the SIRT1 gene occur prevalently during tumorigenesis, supporting the assertion that SIRT1 may serve as a tumor suppressor.

Repeated anastomotic recurrence of colorectal tumors: genetic analysis of two cases.

AIM: To investigate genetics of two cases of colorectal tumor local recurrence and throw some light on the etiopathogenesis of anastomotic recurrence. METHODS: Two cases are presented: a 65-year-old female receiving two colonic resections for primary anastomotic recurrences within 21 mo, and a 57-year-old female undergoing two local excisions of recurrent anastomotic adenomas within 26 mo. A loss of heterozygosity (LOH) study of 25 microsatellite markers and a mutational analysis of genes BRAF, K-RAS and APC were performed in samples of neoplastic and normal colonic mucosa collected over the years. RESULTS: A diffuse genetic instability was present in all samples, including neoplastic and normal colonic mucosa. Two different patterns of genetic alterations (LOH at 5q21 and 18p11.23 in the first case, and LOH at 1p34 and 3p14 in the second) were found to be associated with carcinogenesis over the years. A role for the genes MYC-L (mapping at 1p34) and FIHT (mapping at 3p14.2) is suggested, whereas a role for APC (mapping at 5q21) is not shown. CONCLUSION: The study challenges the most credited intraluminal implantation and metachronous carcinogenesis theories, and suggests a persistent, patient-specific alteration as the trigger of colorectal cancer anastomotic recurrence.

Prevalence and spectrum of microsatellite alterations in nonmuscle invasive bladder cancers.

We aimed to identify interesting deleted chromosomal regions for bladder cancer diagnosis and carcinogenesis, and to evaluate the association between loss of heterozygosity (LOH) and clinico-pathological parameters. Microsatellite analysis was performed on urine sediment and tumor tissue from 43 consecutive patients with superficial transitional cell carcinoma (TCC) and from 42 consecutive controls. Informative cases were scored as LOH or allelic loss (AL) according to the decrease of the allelic-imbalance ratio. The prevalence of LOH and AL was 39.5% and 86%, respectively. Chromosome 9 was the most frequently altered, especially at 9p (35%). The total number of microsatellite alterations per analysis was correlated with age, grade, stade and EAU classification. The locus 17p13.1 was strongly associated with high-stage (p=0.01) and high-grade tumors (p=0.02). Specificity and sensitivity of LOH was 100% and 39.3% for diagnosis of malignant urinary disease. Specificity and sensitivity of AL was 73.8% and 88%, respectively. Allelic losses are a frequent and early event in bladder cancer, especially at 9p. Thanks to its high specificity, LOH may serve as a complementary tool for non invasive diagnosis of bladder cancer. Further study is warranted to evaluate the prognostic value of LOH on recurrence, progression and muscle invasion.