Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations

PurposeTargeted next-generation sequencing provides a remarkable opportunity to identify variants in known disease genes, particularly in extremely heterogeneous disorders such as nonsyndromic hearing loss. The present study attempts to shed light on the complexity of hearing impairment.MethodsUsing one of two next-generation sequencing panels containing either 80 or 129 deafness genes, we screened 30 individuals with nonsyndromic hearing loss (from 23 unrelated families) and analyzed 9 normal-hearing controls.ResultsOverall, we found an average of 3.7 variants (in 80 genes) with deleterious prediction outcome, including a number of novel variants, in individuals with nonsyndromic hearing loss and 1.4 in controls. By next-generation sequencing alone, 12 of 23 (52%) probands were diagnosed with monogenic forms of nonsyndromic hearing loss; one individual displayed a DNA sequence mutation together with a microdeletion. Two (9%) probands have Usher syndrome. In the undiagnosed individuals (10/23; 43%) we detected a significant enrichment of potentially pathogenic variants as compared to controls.ConclusionNext-generation sequencing combined with microarrays provides the diagnosis for approximately half of the GJB2 mutation–negative individuals. Usher syndrome was found to be more frequent in the study cohort than anticipated. The conditions in a proportion of individuals with nonsyndromic hearing loss, particularly in the undiagnosed group, may have been caused or modified by an accumulation of unfavorable variants across multiple genes.Genet Med16 12, 945–953.     

激素耐药型与激素敏感型肾病综合征差异表达基因分析

分析;定量PCR验证芯片的基因差异表达结果.结果 与正常对照组相比较,与SRNS组和SSNS组相关的差异基因共157条;聚类分析显示SSNS组与对照组基因表达差异较小,SRNS组与前两者差异明显;功能分析发现与SRNS组相关的差异表达基因主要参与细胞核内生物学活动和细胞信号转导.结论 不同临床和病理类型原发性肾病综合征涉及多个不同的差异基因和生物学途径,其中HLA-DRB4和CLNS1A基因可能在不同类型原发性肾病综合征发病机制中发挥重要作用.     

应用下一代半导体靶向测序技术检测Marfan综合征致病突变

目的 应用Ion Torrent PGM半导体测序仪和Ion AmpliSeqTMInherited Disease Panel对3例马凡综合征(Marfan syndrome,MFS)进行致病基因突变检测,明确其致病突变,并评价下一代半导体靶向测序诊断复杂单基因遗传病的效果.方法 在知情同意的基础上采集3例MFS患者及1名正常志愿者外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段.每个样本用特异性序列标签进行标记后,应用Ion One Touch系统进行模板制备、乳化PCR及磁珠颗粒富集;最后用318半导体测序芯片进行高通量测序.用Ion Torrent Suite 3.2软件进行序列比对及SNPs和Indels提取,再用dbSNP 137数据库过滤得到SNPs和indels,剩余的可疑突变经Sanger法测序验证.结果 用一张318芯片得到855.80Mb的总数据量,4个样本的平均测序深度均达到100×以上,对目的区域的覆盖度在98％以上.数据经软件分析及数据库过滤后,在3例MFS患者中分别得到3个FBN1基因可疑突变,并经Sanger法测序验证,一个为已报道FBN1基因错义突变(p.E1811K),另外两个为新发现的突变,包括一个无义突变(p.E2264X),1个插入突变(p.L871FfsX23).结论 在3例MFS患者中都成功检出FBN1基因致病突变,表明半导体靶向测序可对复杂单基因遗传病进行高效、准确的基因诊断.     

Novel mutations and their genotype-phenotype correlations in patients with Noonan syndrome,using next-generation sequencing

Purpose

Noonan Syndrome (NS) is an autosomal dominant disorder with many variable and heterogeneous conditions. The genetic basis for 20–30% of cases is still unknown. This study evaluates Iranian Noonan patients both clinically and genetically for the first time.

Advances in Alport syndrome diagnosis using next-generation sequencing

Alport syndrome (ATS) is a hereditary nephropathy often associated with sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in both autosomal recessive and autosomal dominant ATS. The conventional mutation screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and has relatively high costs because of the absence of hot spots and to the high number of exons per gene: 51 (COL4A5), 48 (COL4A4) and 52 (COL4A3). Several months are usually necessary to complete the diagnosis, especially in cases with less informative pedigrees. To overcome these limitations, we designed a next-generation sequencing (NGS) protocol enabling simultaneous detection of all possible variants in the three genes. We used a method coupling selective amplification to the 454 Roche DNA sequencing platform (Genome Sequencer junior). The application of this technology allowed us to identify the second mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4) and to reconsider the diagnosis of ATS in a third patient. This study, therefore, illustrates the successful application of NGS to mutation screening of Mendelian disorders with locus heterogeneity.     

Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas.     

难治性肾病综合征患儿载脂蛋白E基因多态性的研究

目的：研究难治性肾病综合征（steroid-resistant idiopathic nephrotic syndrome,SRINS)患儿载脂蛋白E基因多态性,为临床上正确选择合适的脂质代谢紊乱病例进行降脂治疗提供依据。方法：用酶法测定了60例SRINS患儿及80例健康儿童血脂、脂蛋白、载脂蛋白3种物质共7个脂质代谢指标,用PCR－SSCP法检测载脂蛋白E（apoE)基因型,并行肾穿刺活检术检查肾病综合征患儿病理类型。结果：SRINS患儿存在明显脂质紊乱,与健康儿童比较差异有显著性（P＜0．01）,随诊半年后仍有绝大多数SRINS患儿存在明显脂质代谢紊乱。难治性肾病综合征apoε2等位基因显著多于健康儿童（P＜0．05）。结论：SRINS患儿脂质代谢紊乱持续的时间较长,这类患儿,尤其携带ε2等位基因者,更易发生进行性肾脏损害,动脉粥样硬化及冠心病。应考虑给这类患儿使用降脂药物。     

Posterior reversible encephalopathy syndrome in a child with steroid-resistant nephrotic syndrome: a case report and review of literature

Posterior reversible encephalopathy syndrome (PRES) is a rare and serious syndrome of central nervous system that can develop in both adults and children. It is characterized by acute onset of headache, confusion, seizures or focal neurological deficits along with radiological findings of white matter abnormalities in the parietal and occipital lobes. In the past ten years, this syndrome has been described mainly in adults, rare in children. Here, we report a case of PRES presenting in a 12-year-old girl with steroid-resistant nephrotic syndrome. Her neurological symptom was rapidly recovered after control of hypertension without discontinuation of cyclosporine A.     

Foothold of NPHS2 mutations in primary nephrotic syndrome

Glomerular podocytes are highly specialized cells with a complex cytoarchitecture. Their most prominent features are interdigitated foot processes with filtration slits in between. These are bridged by the slit diaphragm, which plays a major role in establishing the selective permeability of the glomerular filtration barrier. We searched Medline and Pubmed using the combination of keywords "NPHS2", "podocin", "steroid-resistant nephrotic syndrome," and "genetics" to identify studies describing an association between NPHS2 gene and renal disease. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells. Mutations affecting several podocyte proteins lead to rearrangement of the cytoskeleton, disruption of the filtration barrier, and subsequent renal disease. The fact that the dynamic regulation of the podocyte cytoskeleton is vital to kidney function has led to podocytes emerging as an excellent model system for studying actin cytoskeleton dynamics in a physiological context. Injury to podocytes leads to proteinuria, a hallmark of most glomerular diseases. Recent studies have led to a considerable increase in our understanding of podocyte biology including composition and arrangement of the cytoskeleton involved in the control of ultrafiltration. Moreover, disturbances of podocyte architecture resulting in the retraction of foot processes and proteinuria appear to be a common theme in the progression of an acquired glomerular disease. In hereditary nephrotic syndromes identified over the last few years, all mutated gene products were localized in podocytes. This review integrates our recent physiological and molecular understanding of the role of podocytes during the maintenance and failure of the glomerular filtration barrier.     

Electron microscopic findings suggestive of focal and segmental glomerulosclerosis in patients with steroid-resistant nephrotic syndrome

Distinction between minimal change disease and unsampled Focal Segmental Glomerulosclerosis is a challenging concept in kidney biopsy of patients with nephrotic syndrome with minimal histopathological findings. This study was performed to compare electron microscopic findings in patients with steroid-resistant nephrotic syndrome with minimal histopathological abnormalities and cases with Focal Segmental Glomerulosclerosis. This Cohort study was conducted in Cancer Institute, Imam Khomeini Hospital Complex, Tehran, Iran. Twenty patients with steroid-resistant nephrotic syndrome and minimal changes on the light microscopic study were selected as case group. Similarly, 20 patients with Focal Segmental Glomerulosclerosis were selected as the control group. Ultrastructural findings were re-evaluated and scored qualitatively (0–3+). In patients with minimal changes on light microscopic evaluation, clinical course of the disease was followed after 5 years. Mean ages of the patients (8 women and 12 men) in case and control groups were 12.9 and 15.9 years, respectively (p > 0.05). There was no significant difference in number of examined glomeruli and sampling from cortico–medullary junction area between the groups. The mean percentage of sclerotic glomeruli in control group was 15.4%. Tubular atrophy and interstitial fibrosis were more frequent in control patients. Podocyte proliferation, GBM duplication (involving more than 10% of capillary walls), and moderate to severe multifocal expansion of mesangial matrix were significantly more obvious in FSGS patient samples (p < 0.05). No statistically significant difference was found in severity of cytoplasmic vacuolization, GBM wrinkling and splitting between the groups. Most of (80%) the patients with minimal changes improved during the 5-year follow-up. Generally, we concluded that Podocyte proliferation, GBM remodeling, and moderate to severe mesangial matrix expansion are the most reliable findings on electron microscopic examination in favor of FSGS.     

Navigating highly homologous genes in a molecular diagnostic setting: a resource for clinical next-generation sequencing

PurposeNext-generation sequencing (NGS) is now routinely used to interrogate large sets of genes in a diagnostic setting. Regions of high sequence homology continue to be a major challenge for short-read technologies and can lead to false-positive and false-negative diagnostic errors. At the scale of whole-exome sequencing (WES), laboratories may be limited in their knowledge of genes and regions that pose technical hurdles due to high homology. We have created an exome-wide resource that catalogs highly homologous regions that is tailored toward diagnostic applications.MethodsThis resource was developed using a mappability-based approach tailored to current Sanger and NGS protocols.ResultsGene-level and exon-level lists delineate regions that are difficult or impossible to analyze via standard NGS. These regions are ranked by degree of affectedness, annotated for medical relevance, and classified by the type of homology (within-gene, different functional gene, known pseudogene, uncharacterized noncoding region). Additionally, we provide a list of exons that cannot be analyzed by short-amplicon Sanger sequencing.ConclusionThis resource can help guide clinical test design, supplemental assay implementation, and results interpretation in the context of high homology.Genet Med 18 12, 1282–1289.     

Autozygome-guided exome sequencing in retinal dystrophy patients reveals pathogenetic mutations and novel candidate disease genes

Retinal dystrophy (RD) is a heterogeneous group of hereditary diseases caused by loss of photoreceptor function and contributes significantly to the etiology of blindness globally but especially in the industrialized world. The extreme locus and allelic heterogeneity of these disorders poses a major diagnostic challenge and often impedes the ability to provide a molecular diagnosis that can inform counseling and gene-specific treatment strategies. In a large cohort of nearly 150 RD families, we used genomic approaches in the form of autozygome-guided mutation analysis and exome sequencing to identify the likely causative genetic lesion in the majority of cases. Additionally, our study revealed six novel candidate disease genes (C21orf2, EMC1, KIAA1549, GPR125, ACBD5, and DTHD1), two of which (ACBD5 and DTHD1) were observed in the context of syndromic forms of RD that are described for the first time.Deprivation of visual perception is a major form of morbidity worldwide with a wide array of causes that cover the entire spectrum from primarily environmental to primarily genetic. Representing the Mendelian end of the spectrum, retinal dystrophy (RD) is a vast group of blinding diseases that are characterized by loss of photoreceptor function, usually due to mono- or biallelic mutations in an expansive list of genes (Wright et al. 2010). Collectively, RD is a major cause of blindness, particularly in industrialized countries where infectious causes are less common and where treatable blinding diseases such as cataract and glaucoma receive adequate management.Clinically, RD can take various forms, retinitis pigmentosa (RP) being the most common (Buch et al. 2004). RP patients typically present with a predominantly rod dysfunction, which manifests as night blindness, progressively worsening peripheral vision, and typical fundus appearance (Ho 2003; Hamel 2006). In cone dystrophies, it is the cone photoreceptors that are primarily involved, causing a substantial decrease in visual acuity and photophobia (Hamel 2007). In both classes, the other photoreceptor subtype is inevitably affected as the disease progresses, hence the terms rod-cone and cone-rod dystrophy, although the mechanism for this sympathetic cell loss is poorly understood. When severe RD is congenital or early-infantile in onset, it is usually referred to as Leber congenital amaurosis (LCA). Interestingly, the clinical boundaries between these subclasses are blurred by the increasing appreciation of the marked phenotypic variability that is associated with mutations in a large number of RD genes (Daiger et al. 2007).The remarkable genetic heterogeneity (179 genes as of January 2012; https://sph.uth.tmc.edu/Retnet/sum-dis.htm) and the poor predictive value of the clinical assessment to the specific genetic etiology (at least in nonsyndromic cases) make it extremely challenging to offer a molecular diagnosis to these patients (Koenekoop et al. 2007). Thus, of all Mendelian disorders, this is one disease category where most patients remain unaware of their underlying causative mutation even though such information is critical for informed genetic counseling that aims at prevention and expansion of available reproductive options. This is compounded by estimates that, even if all known RD were to be sequenced in a given patient, the yield is probably 50% (Farrar et al. 2002; Hartong et al. 2006; den Hollander et al. 2008). An additional value in securing a molecular diagnosis lies in the recent progress in gene therapy, which has prompted many RD patients to seek to determine their mutation status in order to know whether they are eligible for these gene-specific treatment protocols (Maguire et al. 2008). In addition, certain classes of mutations have been found to be amenable to treatment in other diseases, e.g., nonsense mutations, which offers hope that RD patients with such mutations could similarly benefit from such innovative strategies, but this will require prior knowledge of the underlying genetic defect (Kerem et al. 2008).Research in the genetics of RD has greatly improved our understanding of the molecular machinery that enables the retina to play a critical role in the perception of visual stimuli (Inglehearn 1998). While some of the genes were predicted to cause RD based on established physiological roles of the protein they encode, e.g., phototransduction genes, it came as a surprise that almost one in four RD genes plays a role in the photoreceptor cilium (Adams et al. 2007; Wright et al. 2010). Moreover, many genes were completely unsuspected, e.g., pre-mRNA splicing genes, and the function of some remains unknown (Vithana et al. 2001; Faustino and Cooper 2003; Wright et al. 2010). Indeed, the increasing pace of discovery of RD genes over the past few years has widened the gap between our knowledge of the genetic architecture of RD and its functional context.In this study, we aimed to investigate the utility of genomic approaches in the study of RD genetics. Specifically, we implemented autozygome analysis (Woods et al. 2006; Alkuraya 2010) and exome sequencing in a large cohort of simplex and multiplex patients with different clinical RD subtypes. In addition to providing the most comprehensive analysis to date on the actual contribution of known RD genes to the overall mutation pool, our study reveals six novel RD genes, including two involved in novel syndromic forms of RD, and suggests a framework for mutation identification in these patients.     

Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration

Mutations of the novel renal glomerular genes NPHS1 and NPHS2 encoding nephrin and podocin cause two types of severe nephrotic syndrome presenting in early life, Finnish type congenital nephrotic syndrome (CNF) and a form of autosomal recessive familial focal segmental glomerulosclerosis (SRN1), respectively. To investigate the mechanisms by which mutations might cause glomerular protein leak, we analysed NPHS1/NPHS2 genotype/phenotype relationships in 41 non-Finnish CNF patients, four patients with congenital (onset 0 to 3 months) focal segmental glomerulosclerosis and five patients with possible SRN1 (onset 6 months to 2 years). We clarify the range of NPHS1 mutations in CNF, detecting mutation 'hot-spots' within the NPHS1 coding sequence. In addition, we describe a novel discordant CNF phenotype characterized by variable clinical severity, apparently influenced by gender. Moreover, we provide evidence that CNF may be genetically heterogeneous by detection of NPHS2 mutations in some CNF patients in whom NPHS1 mutations were not found. We confirm an overlap in the NPHS1/NPHS2 mutation spectrum with the characterization of a unique di-genic inheritance of NPHS1 and NPHS2 mutations, which results in a 'tri-allelic' hit and appears to modify the phenotype from CNF to one of congenital focal segmental glomerulosclerosis (FSGS). This may result from an epistatic gene interaction, and provides a rare example of multiple allelic hits being able to modify an autosomal recessive disease phenotype in humans. Our findings provide the first evidence for a functional inter-relationship between NPHS1 and NPHS2 in human nephrotic disease, thus underscoring their critical role in the regulation of glomerular filtration.     

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome

X-linked Alport syndrome (XLAS) is a progressive, hereditary nephropathy. Although men with XLAS usually develop end-stage renal disease before 30 years of age, some men show a milder phenotype and develop end-stage renal disease later in life. However, the molecular mechanisms associated with this milder phenotype have not been fully identified. We genetically diagnosed 186 patients with suspected XLAS between January 2006 and August 2014. Genetic examination involved: (1) extraction and analysis of genomic DNA using PCR and direct sequencing using Sanger''s method and (2) next-generation sequencing to detect variant allele frequencies. We identified somatic mosaic variants in the type VI collagen, α5 gene (COL4A5) in four patients. Interestingly, two of these four patients with variant frequencies in kidney biopsies or urinary sediment cells of ≥50% showed hematuria and moderate proteinuria, whereas the other two with variant frequencies of <50% were asymptomatic or only had hematuria. De novo variants can occur even in asymptomatic male cases of XLAS resulting in mosaicism, with important implications for genetic counseling. This is the first study to show a tendency between the variant allele frequency and disease severity in male XLAS patients with somatic mosaic variants in COL4A5. Although this is a very rare status of somatic mosaicism, further analysis is needed to show this correlation in a larger population.