NON-SYNDROMIC HEARING LOSS AND HIGH- THROUGHPUT STRATEGIES TO DECIPHER ITS GENETIC HETEROGENEITY

Hearing loss (HL) is the most common sensory disorder, affecting all age groups, ethnicities, and gen-ders. According to World Health Organization (WHO) estimates in 2005, 278 million people worldwide have moderate to profound HL in both ears. Results of the 2002 National Health Interview Survey indicate that nearly 31 million of all non-institutionalized adults (aged 18 and over) in the United States have trouble hearing. Epidemiological studies have estimated that approximately 50%of profound HL can be attributed to genetic causes. With over 60 genes implicated in nonsyndromic hearing loss, it is also an extremely het-erogeneous trait. Recent progress in identifying genes responsible for hearing loss enables otolaryngologists and other clinicians to apply molecular diagnosis by genetic testing. The advent of the $1000 genome has the potential to revolutionize the identification of genes and their mutations underlying genetic disorders. This is especially true for extremely heterogeneous Mendelian conditions such as deafness, where the muta-tion, and indeed the gene, may be private. The recent technological advances in target-enrichment methods and next generation sequencing offer a unique opportunity to break through the barriers of limitations im-posed by gene arrays. These approaches now allow for the complete analysis of all known deafness-causing genes and will result in a new wave of discoveries of the remaining genes for Mendelian disorders. This re-view focuses on describing genotype-phenotype correlations of the most frequent genes including GJB2, which is responsible for more than half of cases, followed by other common genes and on discussing the im-pact of genomic advances for comprehensive genetic testing and gene discovery in hereditary hearing loss.

Age-specific heterogeneity of genetic susceptibility to cardiovascular disease might have opposite outcomes depending on the presence of prediabetes

Examining age-specific heterogeneity of susceptibility to cardiovascular disease is also essential in individuals without prediabetes to determine its relative size and direction compared to those with prediabetes.Of particular interest,age-specific heterogeneity in genetic susceptibility may exhibit opposite directions depending on the presence or absence of prediabetes.

Comprehensive functional annotation of susceptibility variants identifies genetic heterogeneity between lung adenocarcinoma and squamous cell carcinoma

Although genome-wide association studies have identified more than eighty genetic variants associated with non-small cell lung cancer(NSCLC)risk,biological mechanisms of these variants remain largely unknown.By integrating a large-scale genotype data of 15581 lung adenocarcinoma(AD)cases,8350 squamous cell carcinoma(SqCC)cases,and 27355 controls,as well as multiple transcriptome and epigenomic databases,we conducted histology-specific meta-analyses and functional annotations of both reported and novel susceptibility variants.We identified 3064 credible risk variants for NSCLC,which were overrepresented in enhancer-like and promoter-like histone modification peaks as well as DNase I hypersensitive sites.Transcription factor enrichment analysis revealed that USF1 was AD-specific while CREB1 was SqCC-specific.Functional annotation and genebased analysis implicated 894 target genes,including 274 specifics for AD and 123 for SqCC,which were overrepresented in somatic driver genes(ER=1.95,P=0.005).Pathway enrichment analysis and Gene-Set Enrichment Analysis revealed that AD genes were primarily involved in immune-related pathways,while SqCC genes were homologous recombination deficiency related.Our results illustrate the molecular basis of both wellstudied and new susceptibility loci of NSCLC,providing not only novel insights into the genetic heterogeneity between AD and SqCC but also a set of plausible gene targets for post-GWAS functional experiments.

Genetic heterogeneity of noncompaction

To the Editor： With interest we read the article by Wu et all on a 53-year male with agenesia of the right lung and left-ventricular hypertrabeculation / noncompaction （LVHT）. The paper raises the following concerns.

Cryptic NUP214-ABL1 fusion with complex karyotype, episomes and intra-tumor genetic heterogeneity in a T-cell lymphoblastic lymphoma

T-lymphoblastic lymphoma(T-LBL)is a rare and aggressive form of non-Hodgkin’s lymphoma and little is known about their molecular background.However,complex karyotypes were already related to this group of malignancy and associated with poor outcome.Here,we describe a 17-year-old female being diagnosed with T-LBL and a normal karyotype after standard G-banding with trypsin-Giemsa(GTG)-banding.However,further analyses including high-resolution molecular approaches,array-comparative genomic hybridization(aCGH),multiplex ligation-dependent probe amplification,fluorescence in situ hybridization and multicolor chromosome banding revealed a cryptic complex karyotype,NUP214-ABL1 gene fusion,episomes and intra-tumor genetic heterogeneity.In addition,homozygous loss of CDKN2A,as well as amplification of oncogene TLX1(HOX11)were detected.Actually,NUP214-ABL1 fusion gene replicated autonomously in this case as episomes.Overall,highly amplification of NUP214-ABL1 fusion gene defines possibly a new subgroup of T-LBL patients which accordingly could benefit from treatment with tyrosine kinase inhibitors.As episomes are missed in standard karyotyping aCGH should be performed routinely in T-LBL to possibly detect more of such cases.

New insights into the role of intra-tumor genetic heterogeneity in carcinogenesis: identification of complex single gene variance within tumors

Aim:Present cancer hypotheses are almost all based on the concept that accumulation of specific driver gene mutations cause carcinogenesis.The discovery of intra-tumor genetic heterogeneity(ITGH),has resulted in this hypothesis being modified by assuming that most of these ITGH mutations are in passenger genes.In addition,accumulating ITGH data on driver gene mutations have revealed considerable genotype/phenotype disconnects.This study proposes to investigate this disconnect by examining the nature and degree of ITGH in breast tumors.Methods:ITGH was examined in tumors using next generation sequencing of up to 68,000 reads and analysis tools that allowed for identification of distinct minority variants within single genes,i.e.,complex single gene variance(CSGV).Results:CSGV was identified in the androgen receptor genes in all breast tumors examined.Conclusion:Evidence of CSGV suggests that a selection-as opposed to a mutation-centric hypothesis could better explain carcinogenesis.Our hypothesis proposes that tumors develop by the selection of preexisting de novo mutations rather than just the accumulation of de novo mutations.Thus,the role of selection pressures,such as changes in tissue microenvironments will likely be critical to our understanding of tumor resistance as well as the development of more effective treatment protocols.

Phenotypic and genetic variation between two populations of the Chinese yellow pond turtle, Mauremys mutica (Cantor, 1842)

Mauremys mutica(Cantor,1842)is an endangered species in China.Main phenotypic variations inbody color,body weight,body shape,clutch size,egg size,and hatchling size were revealed betweenthe southern and northern populations.Both populations have the phenomenon of'larger male'sexualsize dimorphism(SSD),especially in the southern population.Furthermore,genetic variations betweenthe two populations were analyzed by RAPD band patterns of 30 random individuals in each population.The average genetic distance was 0.299±0.108 among the samples of two populations.The average ge-netic distance between southern and northern populations was 0.305±0.046.Cluster analysis indicatedthat all the individuals from the southern and northern populations were clustered among themselves toform two distinct clades.A total of 20 population-specific RAPD fragments were scored from 16 primers,and could be used as RAPD markers for distinguishing the southern and the northern population.Basedon the nucleotide sequences of two RAPD markers,two pairs of SCAR primers(SC1-S and SC2-S)weredesigned,which could be used as SCAR markers for the southern population.According to the significantphenotypic and genetic variations,we suggested that the northern population and southern populationmight be considered as two separate taxa,the'northern taxon'and the'southern taxon',and the con-servation should be respectively conducted on the two taxa.

Heterogeneous genetic architecture by gender for precision medicine of cardiovascular disease

It is well-known that gender differences exist in the onset, progression, and prognosis of cardiovascular diseases （CVDs）, and that risk factors such as high blood pressure and lipid profiles vary between men and women, Cur- rently, sex differences are stressed as important variables to take into account when examining the etiology of CVD. Genome-wide association studies of CVD have employed the sex as a covariate in their analytical models, but generally disregarded potential genetic heterogeneity （GHS） attributable to sex.

What can we learn on rodent fearfulness/anxiety from the genetically heterogeneous NIH-HS rat stock?

The “National Institutes of Health” genetically heterogeneous (NIH-HS) rat stock was created in the 1980s through an eight-way cross of as much as possible separate inbred rat strains (i.e. the MR/N, WN/N, WKY/N, M520/N, F344/N, ACI/N, BN/SsN and BUF/N strains) which were readily available at that time. Hansen and Spuhler [1] developed a more naturalistic, genetically heterogeneous rat stock with the aim of optimizing the distribution of genotypic frequencies and recombination and under the hypothesis that the NIH-HS stock could yield a broad-range distribution of responses (broader than commonly used laboratory rat strains) to experimental conditions, and thus serve as a base population for selection studies. Along the last decade, in a series of studies we have phenotypically characterized the NIH-HS rat stock (a colony exists at our laboratory since 2004) for their anxiety/fearfulness profiles (using a battery of both unconditioned and conditioned tests/tasks), as well as regarding their stress-induced hormonal responses, coping style under inescapable stress and spatial learning ability. We have also compared the phenotypic profiles of NIH-HS rats with those of the low anxious RHA-I and the high anxious RLA-I rat strains. The NIH-HS rat stock is, as a population, a rather anxious type of rat, with predominantly reactive/passive coping style in unlearned and learned anxiety/fear tests, and elevated stress hormone responses (as well as enhanced “depressive” symptoms in the forced swimming test). Genetic studies currently under way have thus far revealed that the genetically heterogeneous NIH-HS rat stock constitutes a unique tool for fine mapping of QTL (for multiple behavioural and biological complex traits) to megabase resolution levels, thus enabling candidate gene identification. We give some examples of this in the present paper, while also highlighting that microarray gene expression studies reveal that HPA-axis- and prolactin-related genes (among others) in the amygdala appear to be related with (or associated to) the coping style and anxiety/fearfulness responses of NIH-HS rats.

Role of discs large homolog 5

In 2004, an association of genetic variation in the discs large homolog 5 （DLG5＇） gene with inflammatory bowel disease （IBD） was described in two large European study samples. The initial report of DLG5 as a novel IBD susceptibility gene sparked a multitude of studies investigating its effect on CD and IBD, respectively, leading to controversial findings and ongoing discussions concerning the validity of the initial association finding and its role in the aetiology of Crohn disease. This review aims to summarize the current state of knowledge and to place the reported findings in the context of current concepts of complex diseases. This includes aspects of statistical power, phenotype differences and genetic heterogeneity between different populations as well as gene-gene and gene-environment interactions.

Single-cell profiling of the copy-number heterogeneity in colorectal cancer

Background:With functionally heterogeneous cells,tumors comprise a complex ecosystem to promote tumor adaptability and evolution under strong selective pressure from the given microenvironment.Diversifying tumor cells or intra-tumor heterogeneity is essential for tumor growth,invasion,and immune evasion.However,no reliable method to classify tumor cell subtypes is yet available.In this study,we introduced the single-cell sequencing combined with copy number characteristics to identify the types of tumor cells in microsatellite stable(MSS)colorectal cancer(CRC).Methods:To characterize the somatic copy number alteration(SCNA)of MSS CRC in a single cell profile,we analyzed 26 tissue samples from 19 Korean patients(GSE132465,the Samsung Medical Center[SMC]dataset)and then verified our findings with 15 tissue samples from five Belgian patients(GSE144735,the Katholieke Universiteit Leuven 3[KUL3]dataset).The Cancer Genome Atlas(TCGA)cohort,GSE39582 cohort,and National Cancer Center(NCC)cohort(24 MSS CRC patients were enrolled in this study between March 2017 and October 2017)were used to validate the clinical features of prognostic signatures.Results:We employed single cell RNA-sequencing data to identify three types of tumor cells in MSS CRC by their SCNA characteristics.Among these three types of tumor cells,C1 and C3 had a higher SCNA burden;C1 had significant chromosome 13 and 20 amplification,whereas C3 was the polar opposite of C1,which exhibited deletion in chromosome 13 and 20.The three types of tumor cells exhibited various functions in the tumor microenvironment and harbored different mutations.C1 and C2 were linked to the immune response and hypoxia,respectively,while C3 was critical for cell adhesion activity and tumor angiogenesis.Additionally,one gene(OLFM4)was identified as epithelium-specific biomarker of better prognosis of CRC(TCGA cohort:P=0.0110;GSE39582 cohort:P=0.0098;NCC cohort:P=0.0360).Conclusions:On the basis of copy number characteristics,we illustrated tumor heterogeneity in MSS CRC and identified three types of tumor cells with distinct roles in tumor microenvironment.By understanding heterogeneity in the intricate tumor microenvironment,we gained an insight into the mechanisms of tumor evolution,which may support the development of therapeutic strategies.

Helplessness-like escape deficits of NIH-HS rats predict passive behavior in the forced swimming test:Relevance for the concurrent validity of rat models of depression

The genetically heterogeneous NIH-HS rat stock has been characterized by its response to anxiety- and fear-inducing situations, thus leading to the conclusion that they are a rather anxious and passive coping type of rats. Taking advantage of these profiles, and knowing that they show very poor performance in the two-way active (shuttle box) escape/avoidance task, we have tested NIH-HS rats (n = 80) in the forced swimming test (FST) as well as we have studied escape response deficits (i.e. response failures) of the same animals in the two-way shuttle box task. They were also tested for anxiety in the elevated zero-maze. The goal of such a study was that of investigating whether there are associations or relationships among helplessness-like or passive coping responses between both models of depression, i.e. the FST and the helplessness-like escape deficits in the shuttle box task. The results for the first time show associations among responses from both depression models and that selecting rats for displaying extreme (active or passive) responses in one of the models predict in a coherent manner (according to the hypothesis) their behaviour in the other model. These findings are discussed in the context of the concurrent validity of both models of depression as well as concerning the possible relevance of NIH-HS rats as a tool for future studies on this field.

Relationships of open-field behaviour with anxiety in the elevated zero-maze test:Focus on freezing and grooming

The National Institutes of Health Genetically Heterogeneous Rat Stock (NIH-HS) is a unique tool for genetic studies of complex traits due to its high genetic heterogeneity and to its high level of genetic recombinants accumulated along many outbreeding generations. In the present study, 90 NIH-HS male rats were tested for anxiety/fearfulness in the elevated zero-maze and in the open-field test in order to investigate the associations among defensive responses from both tests and, in particular, those among open- field self-grooming and freezing. These associations were evaluated by means of a correlational-factorial approach and an analysis of differences between sub- groups displaying extreme scores in representative variables. The final factor analysis revealed a first factor with high loadings of all variables from the zero-maze (“Maze timidity/conflict” factor), and a second (independent) factor dominated by open-field crossings (-0.74), rearings (-0.62) and freezing (0.65), with lower loadings of open-field grooming (-0.39) and stretched attend postures, as well as of entries and time (loadings of -0.32 to -0.25) in the open sections of the zero-maze (“Open Behavior inhibition/ desinhibition” factor), suggesting that open-field self-grooming is a response associated to activity, in the present study, rather than to inhibition. Furthermore, the finding that grooming in the OF loaded negatively in a second factor supports a relationship between grooming and dearousal. Present results, thus, are in accordance with the usefulness of these tests for the purposes they are commonly employed and add new evidence supporting their concurrent validity, as indicated by the relationships observed among measures from both tests.

Pathway-based Analysis of the Hidden Genetic Heterogeneities in Cancers

Many cancers apparently showing similar phenotypes are actually distinct at the molecular level,leading to very different responses to the same treatment.It has been recently demonstrated that pathway-based approaches are robust and reliable for genetic analysis of cancers.Nevertheless,it remains unclear whether such function-based approaches are useful in deciphering molecular heterogeneities in cancers.Therefore,we aimed to test this possibility in the present study.First,we used a NCI60 dataset to validate the ability of pathways to correctly partition samples.Next,we applied the proposed method to identify the hidden subtypes in diffuse large B-cell lymphoma （DLBCL）.Finally,the clinical significance of the identified subtypes was verified using survival analysis.For the NCI60 dataset,we achieved highly accurate partitions that best fit the clinical cancer phenotypes.Subsequently,for a DLBCL dataset,we identified three hidden subtypes that showed very different 10-year overall survival rates （90％,46％ and 20％） and were highly significantly （P =0.008） correlated with the clinical survival rate.This study demonstrated that the pathwaybased approach is promising for unveiling genetic heterogeneities in complex human diseases.

Intratumoral heterogeneity of HER2 gene amplification in occult breast cancer

Occult breast cancer （OBC） is an uncommon type of breast cancer, with palpable masses in the axillary area as the initial symptom. We report a case of OBC with HER2 genetic heterogeneity and discuss the impact of an unusual HER2 amplification pattern.A 56-year-old female was discovered a palpable mass in the right axillary area. She underwent a lymph node excisional biopsy at another hospital approximately one month before referral to our hospital service for further evaluation. The biopsy showed lymph tissue with tumor cell infiltrating in the form of sheets or nests. The tumor cells were larging in size, obviously atypical, and showed prominent nucleoli and intracellular keratosis. Mammography, chest X-ray, chest computed tomography （CT）, abdominal ultrasonography （US）, and positron emission tomography- CT （PET-CT）, which were also performed at the initial hospital, showed no abnormal findings.

Intra-tumor heterogeneity in head and neck cancer and its clinical implications

The presence of heritable differences among cancer cells within a tumor, called intra-tumor genetic heterogeneity, has long been suspected of playing a role in poor responses to therapy. Research over the past decade has documented the existence of such heterogene-ity within tumors of individual patients and documented its potential clinical significance. The research methods for identifying this heterogeneity were not, however, readily adaptable to widespread clinical application. After a brief review of this background, we describe the devel-opment of a measure of intra-tumor genetic heterogeneity, based on whole-exome sequencing of individual tumor samples, that could be applied to biopsy specimens in a clinical setting. This measure has now been used in head and neck squamous cell carcinoma (HNSCC) to docu-ment, for the first time, a relation of high intra-tumor genetic heterogeneity to shorter overall survival in a large, multi-institutional study. The implications of heterogeneity for research and clinical care thus now need to be addressed. Copyright a 2016 Chinese Medical Association. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5

Objective There is a paucity of published works that systematically evaluate gene anomalies or clinical features of patients with renal cysts and diabetes syndrome （RCAD）/maturity onset diabetes of the young type 5 （MODY5）. The purpose of this review was to systematically assess the detection rate, genetic and phenotypic implications of heterozygous autosomal dominant TCF2 anomalies. Data sources MEDLINE database was searched to select articles recorded in English from 1997 to 2008. The focus was monoallelic germline TCF2 gene mutations/deletions. Biallelic inactivation, polymorphisms, DNA modification （hypomethylation and hypermethylation）, loci associated with cancer risk, and somatic TCF2 anomalies were all excluded. Study selection After searching the literature, 50 articles were selected. Results The detection rate of TCF2anomalies was 9.7% and varied considerably among MODY （1.4%）, renal structure anomalies （RSA） （21.4%） and RSA with MODY （41.2%） subgroups. Mutations were strikingly located within the DNA binding domain and varied among exons of the DNA binding domain： exons 2 and 4 were the hottest spots, while mutations were sporadically distributed in exon 3. The consistent phenotypes were RSA （89.6%） and diabetes mellitus （DM） （45.0%）. However, the concurrence of RSA and DM was relatively low （27.5%）, which hinders the optimal performance of genetic testing and obtainment of timely diagnosis. Other organ involvements were complementary and necessary for the early identification of patients with TCF2 anomalies. Analysis of phenotypes of TCF2 point mutations showed significant differences in the detection rates of RSA, impaired renal function （IRF） and DM according to mutation type but not mutation location. Conclusion These valuable features of TCF2 anomalies that previously did not receive sufficient attention should not be neglected.

R-CHOP resistance in diffuse large B-cell lymphoma:biological and molecular mechanisms

Although the first-line rituximab plus cyclophosphamide,doxorubicin,vincristine,and prednisone regimen(R-CHOP)substantially improved outcomes for patients with diffuse large B-cell lymphoma(DLBCL),40%of the patients suffered from relapsed/refractory disease and had poor survival outcomes.The detailed mechanism underlying R-CHOP resistance has not been well defined.For this review,we conducted a thorough search for literature and clinical trials involving DLBCL resistance.We discussed DLBCL biology,epigenetics,and aberrant signaling of the B-cell receptor(BCR),phosphatidylinositol 3-kinase(PI3K)/Akt,nuclear factor kappa light chain enhancer of activated B-cells(NF-κB),and the Janus kinase(JAK)/signal transducer and activator of transcription 3(STAT3)pathways as defining mechanisms of DLBCL heterogeneity and R-CHOP resistance.The cell of origin,double-or triple-hit lymphoma and double-protein-expression,clonal evolution,tumor microenvironment,and multi-drug resistance help to contextualize DLBCL resistance in an(epi)genetically and biologically comparative manner.With better understanding of the biological and molecular landscape of DLBCL,a more detailed classification system and tailored treatments will ideally become available to further improve the prognosis of DLBCL patients.

Identification of Seven Novel Mutations in the Acid Alpha-glucosidase Gene in Five Chinese Patients with Late-onset Pompe Disease

Background： Pompe disease is a rare lysosomal glycogen storage disorder linked to the acid alpha-glucosidase gene （GAA）. A wide clinical and genetic variability exists between patients from different ethnic populations, and the genotype-phenotype correlations are still not well understood. The aim of this study was to report the clinicopathological and genetic characteristics of five Chinese patients with late-onset Pompe disease （LOPD） who carried novel GAA gene mutations. Methods： Clinical and pathological data of patients diagnosed with glycogen storage disease at our institution from April 1986 to August 2017 were collected, and next-generation sequencing of frozen muscle specimens was conducted. Results： Of the five patients included in the study, the median disease onset age was 13 years, with a median 5 years delay in diagnosis. The patients mainly manifested as progressive weakness in the proximal and axial muscles, while one patient developed respiratory insufficiency that required artificial ventilation. In muscle biopsies, vacuoles with variable sizes and shapes appeared inside muscle fibers, and they stained positive for both periodic acid-Schiff and acid phosphatase staining. Ten GAA gene mutations, including seven novel ones （c.796C〉A, c. 1057C〉T, c. 1201C〉A, c. 1780C〉T, c. 1799G〉C, c.2051C〉A, c.2235dupG）, were identified by genetic tests. Conclusions： The seven novel GAA gene mutations revealed in this study broaden the genetic spectrum of LOPD and highlight the genetic heterogeneity in Chinese LOPD patients.

Expression of human WFDC2 protein from patients with serous carcinomas by the two-tier system

The human WFDC2 protein, also known as human epididymal secretory protein 4 （HE4）, is a new markerof tumor progressionJ that is used for the differential diagnosis of ovarian cystadenocarcinomas. At present, the pathology of ovarian carcinomas is defined by the “dualistic model”2 and “heterogeneity of ovarian carcinomas”3 theories. Previously, it was considered that ovarian carcinomas originate from the ovaries themselves. The ＂dualistic model＂ assumes an external origin for ovarian carcinomas, in which it is considered that serous ovarian carcinomas originate from secondary implantation of oviduct mucosa epithelium. Epithelial ovarian carcinomas are not a single disease, but a set of ＂heterogeneous diseases＂.3