Genomic profiling of late-onset basal cell carcinomas from two brothers with nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant genetic disorder. It is commonly caused by mutations in PTCH1 and chiefly characterized by multiple basal cell carcinomas (BCCs) developing prior to the age of 30 years. In rare cases, NBCCS presents with a late onset of BCC development. OBJECTIVE: To investigate BCC tumorigenesis in two brothers, who showed characteristic features of NBCCS but developed their first BCCs only after the age of 40 years. Two other siblings did not show signs of NBCCS. RESULTS: We obtained blood samples from four siblings and nine BCCs from the two brothers with NBCCS. Whole exome sequencing and RNA sequencing revealed loss of heterozygosity (LOH) of PTCH1 in eight out of nine tumours that consistently involved the same haplotype on chromosome 9. This haplotype contained a germinal splice site mutation in PTCH1 (NM_001083605:exon9:c.763-6C>A). Analysis of germline DNA confirmed segregation of this mutation with the disease. All BCCs harboured additional somatic loss-of-function (LoF) mutations in the remaining PTCH1 allele which are not typically seen in other cases of NBCCS. This suggests a hypomorphic nature of the germinal PTCH1 mutation in this family. Furthermore, all BCCs had a similar tumour mutational burden compared to BCCs of unrelated NBCCS patients while harbouring a higher number of damaging PTCH1 mutations. CONCLUSIONS: Our data suggest that a sequence of three genetic hits leads to the late development of BCCs in two brothers with NBCCS: a hypomorphic germline mutation, followed by somatic LOH and additional mutations that complete PTCH1 inactivation. These genetic events are in line with the late occurrence of the first BCC and with the higher number of damaging PTCH1 mutations compared to usual cases of NBCCS.

A Genetic Risk Score for the Estimation of Weight Loss After Bariatric Surgery.

BACKGROUND: Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) are the most frequent bariatric surgery procedures worldwide. In this prospective study, we examined the association of a genetic risk score (GRS) with loss of excess weight after bariatric surgery. METHODS: A total of forty-seven morbidly obese Greek patients who underwent SG (81%) or RYGB were recruited, followed up for 2 years and genotyped. Weight loss after surgery was reported as the percentage of excess weight that was lost (%EWL) at 12 and 24 months after surgery. A GRS was constructed based on previously BMI- and WHR-related single nucleotide polymorphisms (SNPs) that were found significantly correlated with weight loss after bariatric surgery in our population. The level of post-surgery %EWL after 12 and 24 months was estimated through two multiple linear regression models that considered the effects of relevant genetic risk variants. RESULTS: The first proposed model suggested that the predictor variables of GRS, age, and BMI had a significant effect on %EWL12m. GRS was significantly associated with %EWL12m, indicating a 4.618% decrease of %EWL12m per score unit. The second model indicated a positive correlation between %EWL24m and %EWL12m, suggesting that while post-surgery weight loss increased during the first 12 months, an increase was expected in the next 12 months as well. GRS was also significantly associated with %EWL24m, indicating approximately 3% decrease of %EWL24m per score unit. CONCLUSION: GRS can be used in the future together with other preoperative parameters in order to predict the outcome of bariatric surgery.

Chromatin three-dimensional interactions mediate genetic effects on gene expression.

Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.

MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach.

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.

Chromosomal contacts connect loci associated with autism, BMI and head circumference phenotypes.

Copy number variants (CNVs) are major contributors to genomic imbalance disorders. Phenotyping of 137 unrelated deletion and reciprocal duplication carriers of the distal 16p11.2 220 kb BP2-BP3 interval showed that these rearrangements are associated with autism spectrum disorders and mirror phenotypes of obesity/underweight and macrocephaly/microcephaly. Such phenotypes were previously associated with rearrangements of the non-overlapping proximal 16p11.2 600 kb BP4-BP5 interval. These two CNV-prone regions at 16p11.2 are reciprocally engaged in complex chromatin looping, as successfully confirmed by 4C-seq, fluorescence in situ hybridization and Hi-C, as well as coordinated expression and regulation of encompassed genes. We observed that genes differentially expressed in 16p11.2 BP4-BP5 CNV carriers are concomitantly modified in their chromatin interactions, suggesting that disruption of chromatin interplays could participate in the observed phenotypes. We also identified cis- and trans-acting chromatin contacts to other genomic regions previously associated with analogous phenotypes. For example, we uncovered that individuals with reciprocal rearrangements of the trans-contacted 2p15 locus similarly display mirror phenotypes on head circumference and weight. Our results indicate that chromosomal contacts' maps could uncover functionally and clinically related genes.

A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells.

AIM: To determine the impact of a functional human islet clock on insulin secretion and gene transcription. METHODS: Efficient circadian clock disruption was achieved in human pancreatic islet cells by small interfering RNA-mediated knockdown of CLOCK. Human islet secretory function was assessed in the presence or absence of a functional circadian clock by stimulated insulin secretion assays, and by continuous around-the-clock monitoring of basal insulin secretion. Large-scale transcription analysis was accomplished by RNA sequencing, followed by quantitative RT-PCR analysis of selected targets. RESULTS: Circadian clock disruption resulted in a significant decrease in both acute and chronic glucose-stimulated insulin secretion. Moreover, basal insulin secretion by human islet cells synchronized in vitro exhibited a circadian pattern, which was perturbed upon clock disruption. RNA sequencing analysis suggested alterations in 352 transcript levels upon circadian clock disruption. Among them, key regulators of the insulin secretion pathway (GNAQ, ATP1A1, ATP5G2, KCNJ11) and transcripts required for granule maturation and release (VAMP3, STX6, SLC30A8) were affected. CONCLUSIONS: Using our newly developed experimental approach for efficient clock disruption in human pancreatic islet cells, we show for the first time that a functional ß-cell clock is required for proper basal and stimulated insulin secretion. Moreover, clock disruption has a profound impact on the human islet transcriptome, in particular, on the genes involved in insulin secretion.

The impact of oxaliplatin-based chemotherapy for colorectal cancer on the autonomous nervous system.

BACKGROUND: The oxaliplatin (ΟΧΑ)-based regimens FOLFOX and XELOX can cause peripheral neuropathy. It is unknown if ΟΧΑ, alone or in combination regimens, affects the Autonomous Nervous System (ANS). Accordingly, we evaluated the impact of ΟΧΑ-based chemotherapy on the ANS. METHODS: We enrolled 36 patients with colorectal cancer, treated with adjuvant mFOLFOX6 or XELOX chemotherapy, and 22 healthy volunteers. For the assessment of ANS function, participants completed a questionnaire and underwent neurophysiological examination at three time points (baseline, 3-4 months and 6-8 months after the first chemotherapy cycle). ANS testing included assessment of the adrenergic cardiovascular function (orthostatic hypotension-OH), parasympathetic heart innervation (ratio 30/15) and Sympathetic Skin Response (SSR). RESULTS: The values of the 30/15 ratio were significantly reduced at the two time point assessments compared to baseline (Wilcoxon signed ranks test, both P < 0.001), while patients had more often diastolic OH at the 6-8 month evaluation compared to baseline (P = 0.039). In contrast, SSR was not affected. The incidence of positive responses in the questionnaire assessing the subjective impact of symptoms attributable to ANS dysfunction was higher at the two time points compared to baseline (P = 0.036 and P = 0.020). CONCLUSIONS: Oxaliplatin-based chemotherapy is associated with significant effects on the adrenergic cardiovascular reaction and the parasympathetic heart innervation, whereas SSR remains untouched.

Predictors of laterality of motor block during epidural analgesia in a mixed surgical population.

BACKGROUND: Predictors of laterality of motor block during epidural analgesia are currently unknown, as studies so far have yielded conflicting results. We aimed to evaluate predictors of post-operative asymmetric lower extremity motor blockade in a mixed surgical population. METHODS: This is a retrospective analysis of 578 consecutive patients with post-operative epidural analgesia for a variety of surgical procedures. A priori determined potential predictors of unilateral motor block were age, gender, body mass index, type of surgical procedure, vertebral level of puncture, catheter insertion depth into the epidural space and concentration of local anaesthetic. Logistic regression analysis was employed for evaluating predictors of laterality. RESULTS: Unilateral motor block occurred in 29.2% of the patients. Univariate logistic regression analysis showed that young age, female gender, gynaecologic procedures, a low puncture level, an increased depth of catheter insertion and a high ropivacaine concentration (2 mg/ml vs. less than 2 mg/ml) were significantly associated with increased incidence of laterality. Multivariate analysis revealed that age (OR = 0.73 per decade increase, P = 0.00001), the vertebral level of epidural puncture (OR = 1.37 per lowering vertebral level, P < 0.000001) and the depth of catheter insertion (OR = 1.46 per centimetre deeper, P = 0.001) were independent predictors of unilateral motor block. CONCLUSION: These results suggest that young patients with lumbar epidural analgesia or deep catheter insertion should be frequently monitored for the occurrence of laterality of motor block. Also, these results provide support for a prospective study to determine the optimal catheter insertion depth to decrease the risk of unilateral motor block.

ZNF232: structure and expression analysis of a novel human C(2)H(2) zinc finger gene, member of the SCAN/LeR domain subfamily.

We have identified a novel zinc finger gene, ZNF232, mapped to human chromosome 17p12. The coding region of the gene is organized in three exons corresponding to a 417 amino acid long polypeptide containing a SCAN/LeR domain and five C(2)H(2)-type zinc fingers. ZNF232 is possibly a nuclear protein, as suggested by expression analysis of GFP/ZNF232 chimeric constructs. ZNF232 transcripts were detected in a wide collection of adult human tissues. The gene is possibly subjected to tissue-specific post-transcriptional regulation by means of alternative splicing.

Differential selection after duplication in mammalian developmental genes.

Gene duplication provides the opportunity for subsequent refinement of distinct functions of the duplicated copies. Either through changes in coding sequence or changes in regulatory regions, duplicate copies appear to obtain new or tissue-specific functions. If this divergence were driven by natural selection, we would expect duplicated copies to have differentiated patterns of substitutions. We tested this hypothesis using genes that duplicated before the human/mouse split and whose orthologous relations were clear. The null hypothesis is that the number of amino acid changes between humans and mice was distributed similarly across different paralogs. We used a method modified from Tang and Lewontin to detect heterogeneity in the amino acid substitution pattern between those different paralogs. Our results show that many of the paralogous gene pairs appear to be under differential selection in the human/mouse comparison. The properties that led to diversification appear to have arisen before the split of the human and mouse lineages. Further study of the diverged genes revealed insights regarding the patterns of amino acid substitution that resulted in differences in function and/or expression of these genes. This approach has utility in the study of newly identified members of gene families in genomewide data mining and for contrasting the merits of alternative hypotheses for the evolutionary divergence of function of duplicated genes.

A muscle mass in a patient with polymyalgia rheumatica.

We report the extremely uncommon case of a 77-year-old woman in whom a tumor found to be a melanotic schwannoma, arising from the right rectus abdominis muscle, was detected during investigation for a rheumatic disorder, finally identified as polymyalgia rheumatica (PMR). Tumors of this type most commonly occur in spinal nerve roots, and their clinical behavior is very difficult to predict. As far as we are aware, this is the first reported case affecting the aforementioned site. The challenging issue in this case concerns the possible link between the 2 clinical conditions, in particular the development of PMR as a paraneoplastic syndrome. Although such an association has not been reported, PMR is included among the rheumatic disorders reported to be associated with malignancies and occasionally with benign tumors. However, given that tumor resection did not result in remission of PMR and symptoms improved rapidly with prednisone, it seems likely that the above disorders might simply coexist.

Nontransitivity of sperm precedence in Drosophila.

Sperm competition is an important component of fitness in Drosophila, but we still do not have a clear understanding of the unit of selection that is relevant to sperm competition. Here we demonstrate that sperm competitive ability is not a property of the sperm haplotype, but rather of the diploid male's genotype. Then we test whether the relative sperm competitive ability of males can be ranked on a linear array or whether competitive ability instead depends on particular pairwise contests among males. Sperm precedence of six chromosome-extracted lines was tested against three different visible marker lines (cn bw, bwD, and Cy), and the rank order of the six lines differed markedly among the mutant lines. Population genetic theory has shown that departures from transitivity of sperm precedence may be important to the maintenance of polymorphism for genes that influence sperm competitive ability. The nontransitivity seen in sperm precedence should theoretically increase the opportunity for polymorphism in genes that influence this phenotype.

Non-Mendelian segregation of sex chromosomes in heterospecific Drosophila males.

Interspecific hybrids and backcrossed organisms generally suffer from reduced viability and/or fertility. To identify and genetically map these defects, we introgressed regions of the Drosophila sechellia genome into the D. simulans genome. A female-biased sex ratio was observed in 24 of the 221 recombinant inbred lines, and subsequent tests attributed the skew to failure of Y-bearing sperm to fertilize the eggs. Apparently these introgressed lines fail to suppress a normally silent meiotic drive system. Using molecular markers we mapped two regions of the Drosophila genome that appear to exhibit differences between D. simulans and D. sechellia in their regulation of sex chromosome segregation distortion. The data indicate that the sex ratio phenotype results from an epistatic interaction between at least two factors. We discuss whether this observation is relevant to the meiotic drive theory of hybrid male sterility.

Comparison of genetic variability and parentage in different ploidy classes of the Japanese oyster Crassostrea gigas.

Chemical treatments with cytochalasin B were used to induce triploidy in the progeny of a mass fertilization of 3 male and 7 female Crassostrea gigas parents. Triploids were produced either by retention of the first (meiosis I (MI) triploids) or the second (meiosis II (MII) triploids) polar bodies. These animals, together with their diploid siblings, were divided for two experiments. One set was used to compare physiological performance, and the other set deployed to compare growth in two different natural environments. For both experiments, genetic variability in different ploidy classes was estimated using three microsatellite loci and eight allozyme loci. The microsatellite loci were highly polymorphic, allowing independent confirmation of ploidy status and the unambiguous identification of parentage for each oyster. Significant differences in parentage were found between ploidy classes, despite the fact they originated from the same mass fertilization. This indicates that the assumptions of a common genetic background among random samples of animals taken from the same mass fertilization may not be generally valid. Knowledge of parentage also allowed the more accurate scoring of allozyme loci. As expected, triploids were found to be significantly more polymorphic than diploids. However, MI triploids were not significantly more polymorphic than MII triploids. MII triploid genotypes were used to estimate recombination rates between loci and their centromeres. These rates varied between 0.29 and 0.71, indicating only moderate chiasma interference.

Negative covariance suggests mutation bias in a two-locus microsatellite system in the fish Sparus aurata.

Constraints on microsatellite length appear to vary in a species-specific manner. We know very little about the nature of these constraints and why they should vary among species. While surveying microsatellite variation in the Mediterranean gilthead sea bream, Sparus aurata, we discovered an unusual pattern of covariation between two closely linked microsatellite loci. One- and two-locus haplotypes were scored from PCR amplification products of each locus separately and both loci together. In a sample of 211 fish, there was a strong negative covariance in repeat number between the two loci, which suggests a mechanism that maintains the combined length below a constrained size. In addition, there were two clusters of the same combined haplotype length, one consisting of a long repeat array at one locus and a short array at the other and vice versa. We demonstrate that several models of biased mutation or natural selection, in theory, could generate this pattern of covariance. The common feature of all the models is the idea that tightly linked microsatellites do not evolve in complete independence, and that whatever size dependence there is to the process, it appears to "read" the combined size of the two loci.

Serum cholinesterase levels in patients with cancer.

Serum cholinesterase levels were determined in 180 patients with carcinoma and in 146 normal subjects. Serum cholinesterase activity was significantly lower in patients suffering from cancer than in normal controls, though still within the normal range. The degree of depression of serum cholinesterase activity was influenced by the extent to which the malignancy had spread and by the site of the primary lesion.