Applying whole-genome sequencing in relation to phenotype and outcomes in siblings with cystic fibrosis.

Variations in disease onset and/or severity have often been observed in siblings with cystic fibrosis (CF), despite the same CFTR genotype and environment. We postulated that genomic variation (modifier and/or pharmacogenomic variants) might explain these clinical discordances. From a cohort of patients included in the Wisconsin randomized clinical trial (RCT) of newborn screening (NBS) for CF, we identified two brothers who showed discordant lung disease courses as children, with one milder and the other more severe than average, and a third, eldest brother, who also has severe lung disease. Leukocytes were harvested as the source of DNA, and whole-genome sequencing (WGS) was performed. Variants were identified and analyzed using in-house-developed informatics tools. Lung disease onset and severity were quantitatively different between brothers during childhood. The youngest, less severely affected brother is homozygous for HFE p.H63D. He also has a very rare PLG p.D238N variant that may influence host-pathogen interaction during chronic lung infection. Other variants of interest were found differentially between the siblings. Pharmacogenomics findings were consistent with the middle, most severely affected brother having poor outcomes to common CF treatments. We conclude that genomic variation between siblings with CF is expected. Variable lung disease severity may be associated with differences acting as genetic modifiers and/or pharmacogenomic factors, but large cohort studies are needed to assess this hypothesis.

VarSight: prioritizing clinically reported variants with binary classification algorithms.

BACKGROUND: When applying genomic medicine to a rare disease patient, the primary goal is to identify one or more genomic variants that may explain the patient's phenotypes. Typically, this is done through annotation, filtering, and then prioritization of variants for manual curation. However, prioritization of variants in rare disease patients remains a challenging task due to the high degree of variability in phenotype presentation and molecular source of disease. Thus, methods that can identify and/or prioritize variants to be clinically reported in the presence of such variability are of critical importance. METHODS: We tested the application of classification algorithms that ingest variant annotations along with phenotype information for predicting whether a variant will ultimately be clinically reported and returned to a patient. To test the classifiers, we performed a retrospective study on variants that were clinically reported to 237 patients in the Undiagnosed Diseases Network. RESULTS: We treated the classifiers as variant prioritization systems and compared them to four variant prioritization algorithms and two single-measure controls. We showed that the trained classifiers outperformed all other tested methods with the best classifiers ranking 72% of all reported variants and 94% of reported pathogenic variants in the top 20. CONCLUSIONS: We demonstrated how freely available binary classification algorithms can be used to prioritize variants even in the presence of real-world variability. Furthermore, these classifiers outperformed all other tested methods, suggesting that they may be well suited for working with real rare disease patient datasets.

Mortalin (GRP75/HSPA9) Promotes Survival and Proliferation of Thyroid Carcinoma Cells.

We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.

Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD.

Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14-3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.

A Novel Association between the 27-bp Deletion and 538G>A Mutation in the ABCC11 Gene.

A single nucleotide polymorphism in the ABCC11 gene, 538G>A (rs17822931), is known to determine human ear wax type. The G/G and G/A genotypes correspond to the wet type, while the A/A genotype corresponds to the dry type. Another earwax determinant, a 27-bp deletion (Δ27) downstream from the rs17822931 site, is a rare variant that leads to the dry phenotype. In a previous report, we found an individual with the G allele who unexpectedly showed the dry type of earwax, leading to the identification of Δ27. We also demonstrated that the Δ27 allele was present in individuals of Japanese, Thai, native North American, Andean, and Bolivian ancestry but absent in those of European and African ancestry. Here, we assessed the Δ27 allele frequency among Japanese and Ukrainian individuals and identified a novel association between the Δ27 and 538G>A mutations. The Δ27 allele frequency was 0.002 (3/1,520; one individual is heterozygous, and another is homozygous) among Japanese individuals and 0 (0/794) among Ukrainians. We also found a previously unreported homozygous genotype for both the Δ27 and A alleles. Our findings suggest that the Δ27 deletion may have occurred in an ABCC11 gene with the 538G>A mutation.

Kinome sequencing reveals RET G691S polymorphism in human neuroendocrine lung cancer cell lines.

Neuroendocrine (NE) lung tumors comprise 20-25% of all invasive lung malignancies. Currently, no effective treatments are available to cure these tumors, and it is necessary to identify a molecular alteration(s) that characterizes NE lung tumor cells. We aimed to identify a kinase mutation(s) associated with NE lung tumor by screening 517 kinase-encoding genes in human lung cancer cell lines. Our next-generation sequencing analysis of six NE lung tumor cell lines (four small cell lung cancer lines and two non-small cell lung cancer lines) and three non-NE lung tumor lines revealed various kinase mutations, including a nonsynonymous mutation in the proto-oncogene RET (c.2071G>A; p.G691S). Further evaluation of the RET polymorphism in total 15 lung cancer cell lines by capillary sequencing suggested that the frequency of the minor allele (A-allele) in NE lung tumor lines was significantly higher than its frequency in a reference population (p = 0.0001). However, no significant difference between non-NE lung tumor lines and a reference group was detected (p = 1.0). Nevertheless, neither RET expression levels were correlated with the levels of neuron-specific enolase (NSE), a key NE marker, nor vandetanib and cabozantinib, small molecule compounds that inhibit RET, affected NSE levels in lung cancer cells. Our data suggest a potential association of G691S RET polymorphism with NE lung tumor, proposing the necessity of more thorough evaluation of this possibility. The dataset of kinase mutation profiles in this report may help choosing cell line models for study of lung cancer.

The Role of STAT3 in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy and its global incidence rates are rapidly increasing. Although the mortality of thyroid cancer is relatively low, its rate of recurrence or persistence is relatively high, contributing to incurability and morbidity of the disease. Thyroid cancer is mainly treated by surgery and radioiodine remnant ablation, which is effective only for non-metastasized primary tumors. Therefore, better understanding of the molecular targets available in this tumor is necessary. Similarly to many other tumor types, oncogenic molecular alterations in thyroid epithelium include aberrant signal transduction of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase/AKT (also known as protein kinase B), NF-кB, and WNT/ß-catenin pathways. However, the role of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT3) pathway, a well-known mediator of tumorigenesis in different tumor types, is relatively less understood in thyroid cancer. Intriguingly, recent studies have demonstrated that, in thyroid cancer, the JAK/STAT3 pathway may function in the context of tumor suppression rather than promoting tumorigenesis. In this review, we provide an update of STAT3 function in thyroid cancer and discuss some of the evidences that support this hypothesis.

Microarray comparative genomic hybridization analysis of 59 patients with schizophrenia.

Schizophrenia is a common psychiatric disorder with a strong genetic contribution. Disease-associated chromosomal abnormalities in this condition may provide important clues, such as DISC1. In this study, 59 schizophrenia patients were analyzed by microarray comparative genomic hybridization (CGH) using custom bacterial artificial chromosome (BAC) microarray (4,219 BACs with 0.7-Mb resolution). Chromosomal abnormalities were found in six patients (10%): 46,XY,der(13)t(12;13)(p12.1; p11).ish del(5)(p11p12); 46,XY, ish del(17)(p12p12); 46,XX.ish dup(11)(p13p13); and 46,X,idic(Y)(q11.2); and in two cases, mos 45,X/46XX. Autosomal abnormalities in three cases are likely to be pathogenic, and sex chromosome abnormalities in three follow previous findings. It is noteworthy that 10% of patients with schizophrenia have (sub)microscopic chromosomal abnormalities, indicating that genome-wide copy number survey should be considered in genetic studies of schizophrenia.

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid.

Cell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.

A SNP in the ABCC11 gene is the determinant of human earwax type.

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G --> A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G --> A SNP is the first example of DNA polymorphism determining a visible genetic trait.

Molecular characterization of del(8)(p23.1p23.1) in a case of congenital diaphragmatic hernia.

A 36-week-old fetus was referred to the medical center because of his cystic mass and fluid in left thoracic cavity, and was delivered by cesarean section to manage neonatal problems at 37 weeks of gestation. Emergent surgical repair of the left diaphragmatic hernia was performed, but severe hypoxia persisted, and he expired on the following day. Chromosome analysis of cultured amniotic fluid cells indicated 46,XY,del(8)(p23.1p23.1). This is the fourth case of 8p23.1 deletion associated with diaphragmatic hernia. Microarray comparative genomic hybridization analysis using DNA of cultured amniotic fluid cells showed that six clones were deleted, which were mapped to the region between two low copy repeats (LCRs) at 8p23.1 previously described. Microsatellite analysis revealed that the deletion was of paternal origin, and his parents did not carry 8p23.1 polymorphic inversion. These data strongly suggested that the 8p23.1 interstitial deletion should have arisen through a different mechanism from that of inv dup del(8p) whose structural abnormality is always of maternal origin and accompanies heterozygous 8p23.1 polymorphic inversion in mother.