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Alu elements, the short interspersed element numbering more than 1 million copies per human genome, can mediate the formation of copy number variants (CNVs) between substrate pairs. These Alu/Alu-mediated rearrangements (AAMRs) can result in pathogenic variants that cause diseases. To investigate the impact of AAMR on gene variation and human health, we first characterized Alus that are involved in mediating CNVs (CNV-Alus) and observed that these Alus tend to be evolutionarily younger. We then computationally generated, with the assistance of a supercomputer, a test data set consisting of 78 million Alu pairs and predicted ∼18% of them are potentially susceptible to AAMR. We further determined the relative risk of AAMR in 12,074 OMIM genes using the count of predicted CNV-Alu pairs and experimentally validated the predictions with 89 samples selected by correlating predicted hotspots with a database of CNVs identified by clinical chromosomal microarrays (CMAs) on the genomes of approximately 54,000 subjects. We fine-mapped 47 duplications, 40 deletions, and two complex rearrangements and examined a total of 52 breakpoint junctions of simple CNVs. Overall, 94% of the candidate breakpoints were at least partially Alu mediated. We successfully predicted all (100%) of Alu pairs that mediated deletions (n = 21) and achieved an 87% positive predictive value overall when including AAMR-generated deletions and duplications. We provided a tool, AluAluCNVpredictor, for assessing AAMR hotspots and their role in human disease. These results demonstrate the utility of our predictive model and provide insights into the genomic features and molecular mechanisms underlying AAMR.

Small supernumerary marker chromosomes (sSMC) are chromosomal fragments difficult to characterize genomically. Here, we detail a proband with schizoaffective disorder and a mother with bipolar disorder with psychotic features who present with a marker chromosome that segregates with disease. We explored the architecture of this marker and investigated its temporal origin. Array comparative genomic hybridization (aCGH) analysis revealed three duplications and three triplications that spanned the short arm of chromosome 9, suggestive of a chromoanasynthesis-like event. Segregation of marker genotypes, phased using sSMC mosaicism in the mother, provided evidence that it was generated during a germline-level event in the proband's maternal grandmother. Whole-genome sequencing (WGS) was performed to resolve the structure and junctions of the chromosomal fragments, revealing further complexities. While structural variations have been previously associated with neuropsychiatric disorders and marker chromosomes, here we detail the precise architecture, human life-cycle genesis, and propose a DNA replicative/repair mechanism underlying formation.

// Hui Zhao 1, 3, * , Ye Hua 2, * , Zhihua Lu 1 , Shen Gu 3 , Laifa Zhu 1 , Yuan Ji 1 , Yudong Qiu 3 , Tu Dai 1 and Huihan Jin 1 1 Department of Hepatopancreatobiliary Surgery, Nanjing Medical University Affiliated Wuxi Second People’s Hospital, Wuxi, Jiangsu, China 2 Department of Neurology, Nanjing Medical University Affiliated Wuxi Second People’s Hospital, Wuxi, Jiangsu, China 3 Department of Hepatopancreatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China * These authors have contributed equally to this work Correspondence to: Huihan Jin, email: 18762806797@163.com Tu Dai, email: 45687061@qq.com Keywords: hepatocellular carcinoma, microvascular invasion, prognosis, preoperative predictors Received: December 15, 2016 Accepted: April 25, 2017 Published: May 22, 2017 ABSTRACT Objectives: The aim of this study was to investigate the prognostic value and preoperative predictors of microvascular invasion (MVI) in solitary hepatocellular carcinoma (HCC) ≤ 5 cm without macrovascular invasion. Methods: A total of 233 consecutive HCC patients underwent curative hepatectomy were included in our study. Independent risk factors influencing the prognosis were identified, and preoperative predictors for MVI were determined. Results: Multivariate regression analysis identified ICG-R15, BCLC staging and MVI as independent risk factors for the overall survival rate. Type of resection and MVI were independent risk factors for the recurrence-free survival rate. Kaplan-Meier analysis showed the overall survival and recurrence-free survival rates in patients with MVI were significantly poorer than those in patients without MVI ( P = 0.002 and P = 0.001). Anatomical resection obviously improved the overall survival and recurrence-free survival rates in patients with MVI compared with non-anatomical resection ( P = 0.017 and P = 0.009). A prediction scoring system for MVI was built up according to the three independent predictors (tumor size > 3.5 cm, AFP > 200 ng/mL and GGT > 53 U/L). The prevalence of MVI in HCC patients with predictive score ≥ 2 was 58.3%, which was obviously higher than patients with predictive score < 2 (20.8%). Conclusions: MVI is associated with a poor prognosis in solitary HCC ≤ 5 cm after hepatectomy. Anatomical resection could improve the prognosis of HCC patients with MVI. The preoperative prediction scoring model has practical value for the prediction of MVI.

Background and aim The superiority of anatomical resection (AR) in patients with hepatocellular carcinoma compared with non-anatomical resection (NAR) remains controversial. We aimed to investigate the prognostic outcomes of AR and NAR for solitary hepatocellular carcinoma (HCC) patients without macroscopic vascular invasion, using a propensity score matching (PSM) analysis. Methods A total of 305 consecutive HCC patients without macroscopic vascular invasion who underwent curative hepatectomy were included in our study. PSM was performed in order to eliminate possible selection bias. Results By PSM, the patients were divided into propensity-matched anatomical resection (PS-AR) (n = 114) and propensity-matched non-anatomical resection (PS-NAR) (n = 114) groups. The 1-year, 3-year, and 5-year overall survival rates were 90.4%, 77.7%, and 65.7% in PS-AR and 88.6%, 70.7%, and 52.2% in PS-NAR (P = 0.053), respectively. The 1-year, 3-year, and 5-year recurrence-free survival (RFS) rates were 84.1%, 64.9%, and 45.1% in PS-AR and 75.4%, 48.1%, and 31.0% in PS-NAR (P = 0.005), respectively. Multivariate analysis showed that ICG-R15 (P = 0.022); the Barcelona clinic liver cancer staging (P = 0.044) and microvascular invasion (MVI; P = 0.005) were independent risk factors for the overall survival rate, while type of resection (P = 0.027), surgical margin (P = 0.039), and MVI (P = 0.024) were independent risk factors for the RFS rate. Patients who underwent NAR were prone to early recurrence and marginal recurrence. Subgroup analysis indicated that the RFS rate was significantly better in PS-AR than that in PS-NAR (surgical margin ≥ 1 cm) (P = 0.025). Better RFS rate was observed in PS-AR with MVI compared with PS-NAR (P = 0.016). Conclusions Anatomical resection contributed to improve the RFS rate in solitary HCC patients without macroscopic vascular invasion using PSM analysis, especially in patients with MVI.

Gene data on infiltrative hepatocellular carcinoma (iHCC) are still unknown. This study aims to identify the gene expression signature of iHCC compared with single nodular (SN)-type HCC according to the gross classification. The whole-exome sequencing was performed in six matched HCC tumor/normal pairs (three infiltrative type and three single nodular type) from six patients who received curative hepatectomy. Subsequent validation using Sanger sequencing and real-time PCR was performed in 30 HCC tumor samples (15 infiltrative type and 15 single nodular type). Following whole-exome sequencing, Sanger sequencing, and bioinformatics analysis, it revealed significant difference of iHCC from SN-type HCC in gene patterns. Particularly, a typical growth factor receptor tyrosine kinase FGFR3 was predominantly mutated in iHCC. One nonsynonymous variant c.G285T (p.Q95H) and five additional mutations (c.G938A:p.G313D, c.G1291A:p.A431T, c.C1355G:p.T452R, c.C1377T:p.L459L, and c.A1445T:p.E482V) were investigated by whole-exome and Sanger sequencing, respectively. Immunohistochemical studies confirmed the specific expression of FGFR3 in iHCC samples. Our studies indicated that FGFR3 may be a candidate oncogene in tumor progression and a promising therapeutic target in iHCC patients who had early recurrence.

ObjectiveTo determine how single nucleotide variants (SNVs) and copy number variants (CNVs) contribute to molecular diagnosis in familial Parkinson disease (PD), we integrated exome sequencing (ES) and genome-wide array-based comparative genomic hybridization (aCGH) and further probed CNV structure to reveal mutational mechanisms.MethodsWe performed ES on 110 subjects with PD and a positive family history; 99 subjects were also evaluated using genome-wide aCGH. We interrogated ES and aCGH data for pathogenic SNVs and CNVs at Mendelian PD gene loci. We confirmed SNVs via Sanger sequencing and further characterized CNVs with custom-designed high-density aCGH, droplet digital PCR, and breakpoint sequencing.ResultsUsing ES, we discovered individuals with known pathogenic SNVs in GBA (p.Glu365Lys, p.Thr408Met, p.Asn409Ser, and p.Leu483Pro) and LRRK2 (p.Arg1441Gly and p.Gly2019Ser). Two subjects were each double heterozygotes for variants in GBA and LRRK2. Based on aCGH, we additionally discovered cases with an SNCA duplication and heterozygous intragenic GBA deletion. Five additional subjects harbored both SNVs (p.Asn52Metfs*29, p.Thr240Met, p.Pro437Leu, and p.Trp453*) and likely disrupting CNVs at the PRKN locus, consistent with compound heterozygosity. In nearly all cases, breakpoint sequencing revealed microhomology, a mutational signature consistent with CNV formation due to DNA replication errors.ConclusionsIntegrated ES and aCGH yielded a genetic diagnosis in 19.3% of our familial PD cohort. Our analyses highlight potential mechanisms for SNCA and PRKN CNV formation, uncover multilocus pathogenic variation, and identify novel SNVs and CNVs for further investigation as potential PD risk alleles.

Background We investigated the features of the genomic rearrangements in a cohort of 50 male individuals with proteolipid protein 1 (PLP1) copy number gain events who were ascertained with Pelizaeus-Merzbacher disease (PMD; MIM: 312080). We then compared our new data to previous structural variant mutagenesis studies involving the Xq22 region of the human genome. The aggregate data from 159 sequenced join-points (discontinuous sequences in the reference genome that are joined during the rearrangement process) were studied. Analysis of these data from 150 individuals enabled the spectrum and relative distribution of the underlying genomic mutational signatures to be delineated. Methods Genomic rearrangements in PMD individuals with PLP1 copy number gain events were investigated by high-density customized array or clinical chromosomal microarray analysis and breakpoint junction sequence analysis. Results High-density customized array showed that the majority of cases (33/50; ~ 66%) present with single duplications, although complex genomic rearrangements (CGRs) are also frequent (17/50; ~ 34%). Breakpoint mapping to nucleotide resolution revealed further previously unknown structural and sequence complexities, even in single duplications. Meta-analysis of all studied rearrangements that occur at the PLP1 locus showed that single duplications were found in ~ 54% of individuals and that, among all CGR cases, triplication flanked by duplications is the most frequent CGR array CGH pattern observed. Importantly, in ~ 32% of join-points, there is evidence for a mutational signature of microhomeology (highly similar yet imperfect sequence matches). Conclusions These data reveal a high frequency of CGRs at the PLP1 locus and support the assertion that replication-based mechanisms are prominent contributors to the formation of CGRs at Xq22. We propose that microhomeology can facilitate template switching, by stabilizing strand annealing of the primer using W-C base complementarity, and is a mutational signature for replicative repair.

Hereditary spastic paraplegia (HSP) is a group of disorders with predominant symptoms of lower-extremity weakness and spasticity. Despite the delineation of numerous genetic causes of HSP, a significant portion of individuals with HSP remain molecularly undiagnosed. Through exome sequencing, we identified five unrelated families with childhood-onset nonsyndromic HSP, all presenting with progressive spastic gait, leg clonus, and toe walking starting from 7 to 8 years old. A recurrent two-base pair deletion (c.426_427delGA, p.K143Sfs*15) in the UBAP1 gene was found in four families, and a similar variant (c.475_476delTT, p.F159*) was detected in a fifth family. The variant was confirmed to be de novo in two families and inherited from an affected parent in two other families. RNA studies performed in lymphocytes from one patient with the de novo c.426_427delGA variant demonstrated escape of nonsense-mediated decay of the UBAP1 mutant transcript, suggesting the generation of a truncated protein. Both variants identified in this study are predicted to result in truncated proteins losing the capacity of binding to ubiquitinated proteins, hence appearing to exhibit a dominant-negative effect on the normal function of the endosome-specific endosomal sorting complexes required for the transport-I complex.

BackgroundParkinson’s disease (PD) is a genetically heterogeneous condition; both single nucleotide variants (SNVs) and copy number variants (CNVs) are important genetic risk factors. We examined the utility of combining exome sequencing and genome-wide array-based comparative genomic hybridization (aCGH) for identification of PD genetic risk factors.MethodsWe performed exome sequencing on 110 subjects with PD and a positive family history; 99 subjects were also evaluated using genome-wide aCGH. We interrogated exome sequencing and array comparative genomic hybridization data for pathogenic SNVs and CNVs at Mendelian PD gene loci. SNVs were confirmed via Sanger sequencing. CNVs were confirmed with custom-designed high-density aCGH, droplet digital PCR, and breakpoint sequencing.ResultsUsing exome sequencing, we discovered individuals with known pathogenic single nucleotide variants in GBA (p.E365K, p.T408M, p.N409S, p.L483P) and LRRK2 (p.R1441G and p.G2019S). Two subjects were each double heterozygotes for variants in GBA and LRRK2. Based on aCGH, we additionally discovered cases with an SNCA duplication and heterozygous intragenic GBA deletion. Five additional subjects harbored both SNVs (p.N52fs, p.T240M, p.P437L, p.W453*) and likely disrupting CNVs at the PARK2 locus, consistent with compound heterozygosity. In nearly all cases, breakpoint sequencing revealed microhomology, a mutational signature consistent with CNV formation due to DNA replication errors.ConclusionsIntegrated exome sequencing and aCGH yielded a genetic diagnosis in 19.3% of our familial PD cohort. Our analyses highlight potential mechanisms for SNCA and PARK2 CNV formation, uncover multilocus pathogenic variation, and identify novel SNVs and CNVs for further investigation as potential PD risk alleles.

Erdem, Haktan Bagis/0000-0002-4391-1387; Albayrak, Hatice Mutlu/0000-0001-5624-3878; isikay, sedat/0000-0003-0103-9612; Grochowski, Christopher/0000-0002-3884-7720; Gezdirici, Alper/0000-0002-2432-9279; KM, Girisha/0000-0002-0139-8239; Gu, Shen/0000-0003-3107-1218; YESIL, GOZDE/0000-0003-1964-6306; Gezdirici, Alper/0000-0002-2432-9279; Tuysuz, Beyhan/0000-0002-9620-5021; Fatih, Jawid/0000-0002-3927-2711; YUKSEL, Zafer/0000-0002-2085-5773; Balta, Burhan/0000-0003-2672-2493; Posey, Jennifer/0000-0003-4814-6765; Bayhan, Ilhan/0000-0001-8308-1309; Punetha, Jaya/0000-0002-6774-4464; YILDIRIM, Timur/0000-0003-0291-7632 WOS:000473723000011 PubMed ID: 31230720 Arthrogryposis is a clinical finding that is present either as a feature of a neuromuscular condition or as part of a systemic disease in over 400 Mendelian conditions. The underlying molecular etiology remains largely unknown because of genetic and phenotypic heterogeneity. We applied exome sequencing (ES) in a cohort of 89 families with the clinical sign of arthrogryposis. Additional molecular techniques including array comparative genomic hybridization (aCGH) and Droplet Digital PCR (ddPCR) were performed on individuals who were found to have pathogenic copy number variants (CNVs) and mosaicism, respectively. A molecular diagnosis was established in 65.2% (58/89) of families. Eleven out of 58 families (19.0%) showed evidence for potential involvement of pathogenic variation at more than one locus, probably driven by absence of heterozygosity (AOH) burden due to identity-by-descent (IBD). RYR3, MYOM2, ERGIC1, SPTBN4, and ABCA7 represent genes, identified in two or more families, for which mutations are probably causative for arthrogryposis. We also provide evidence for the involvement of CNVs in the etiology of arthrogryposis and for the idea that both mono-allelic and bi-allelic variants in the same gene cause either similar or distinct syndromes. We were able to identify the molecular etiology in nine out of 20 families who underwent reanalysis. In summary, our data from family-based ES further delineate the molecular etiology of arthrogryposis, yielded several candidate disease-associated genes, and provide evidence for mutational burden in a biological pathway or network. Our study also highlights the importance of reanalysis of individuals with unsolved diagnoses in conjunction with sequencing extended family members. National Human Genome Research Institute (NHGRI)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Human Genome Research Institute (NHGRI) [UM1 HG006542]; National Heart, Lung, and Blood Institute (NHLBI)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Heart Lung & Blood Institute (NHLBI) [UM1 HG006542]; NHGRIUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Human Genome Research Institute (NHGRI) [K08 HG008986]; National Institutes of Health - Brain Disorders and Development Training Grant [T32 NS043124-17]; Clinical Research Training Scholarship in Neuromuscular Disease; Tubitak project, TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [217S675]; Indian Council of Medical Research, New Delhi, IndiaIndian Council of Medical Research (ICMR) [5/13/58/2015/NCD-III]; [R35 NS105078]; [512848]; NATIONAL HUMAN GENOME RESEARCH INSTITUTEUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Human Genome Research Institute (NHGRI) [K08HG008986, UM1HG006542, K08HG008986, UM1HG006542, UM1HG006542, UM1HG006542, K08HG008986, UM1HG006542] Funding Source: NIH RePORTER; NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKEUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Neurological Disorders & Stroke (NINDS) [R35NS105078, T32NS043124, T32NS043124, T32NS043124, R35NS105078, T32NS043124, T32NS043124, R35NS105078, T32NS043124, T32NS043124, T32NS043124, R35NS105078] Funding Source: NIH RePORTER This work was supported in part by R35 NS105078 and MDA#512848 to J.R.L. and a jointly funded National Human Genome Research Institute (NHGRI) and National Heart, Lung, and Blood Institute (NHLBI) grant to the Baylor-Hopkins Center for Mendelian Genomics (UM1 HG006542). J.E.P. is supported by NHGRI K08 HG008986. D.P. is supported by the National Institutes of Health - Brain Disorders and Development Training Grant (T32 NS043124-17) and a Clinical Research Training Scholarship in Neuromuscular Disease partnered by the American Brain Foundation (ABF) and Muscle Study Group (MSG). This study is partly funded by Tubitak project number 217S675, Turkey to N.E. and B.T.. This study is partly funded by Indian Council of Medical Research, New Delhi, India with File no.: No. 5/13/58/2015/NCD-III to A.S.

Background Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES. Methods In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array. Results The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities. Conclusions Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations. Electronic supplementary material The online version of this article (10.1186/s13073-019-0639-5) contains supplementary material, which is available to authorized users.

Aberrant left-right patterning in the developing human embryo can lead to a broad spectrum of congenital malformations. The causes of most laterality defects are not known, with variants in established genes accounting for

Objective This study aims to establish the incidence and implications of confined placental mosaicism (CPM) in the context of prenatal chromosomal microarray analysis (CMA). Methods We retrospectively reviewed prenatal array data on 1382 consecutive chorionic villus sampling (CVS) specimens spanning the past 6 years, focusing on those for which whole CVS biopsy (both cytotrophoblast and mesenchymal cells) was used for CMA and cultured cells (primarily mesenchyme) was also analyzed or amniotic fluid (AF)/newborn blood was used for confirmation, to determine the frequency of mosaic abnormal findings that were the result of CPM. Results Out of a total of 1382 consecutive CVS cases, we identified 42 (42/1382 = 3.0%) cases with abnormal array findings suggestive of mosaicism. Among them, 10 cases were unequivocally interpreted as CPM based on a normal AF/newborn blood confirmatory result. In addition, another 10 cases were interpreted as provisional CPM based on normal results on cultured cells. Notably, 40% (8/20) of the cases revealed complex findings, including multiple mosaic aneuploidies, mosaic submicroscopic copy number variation (CNV), and mosaic aneuploidy plus mosaic CNV. Conclusion Abnormal CMA results from CVS specimens should be interpreted with caution when mosaicism is evident or suspected. Furthermore, confirmatory testing on amniotic fluid, which contains cells derived from the fetus, is recommended in these cases.

Germline copy-number variants (CNVs) involving quadruplications are rare and the mechanisms generating them are largely unknown. Previously, we reported a 20-week gestation fetus with split-hand malformation; clinical microarray detected two maternally inherited triplications separated by a copy-number neutral region at 17p13.3, involving BHLHA9 and part of YWHAE. Here, we describe an 18-month-old male sibling of the previously described fetus with split-hand malformation. Custom high-density microarray and digital droplet PCR revealed the copy-number gains were actually quadruplications in the mother, the fetus, and her later born son. This quadruplication-normal-quadruplication pattern was shown to be expanded from the triplication-normal-triplication CNV at the same loci in the maternal grandmother. We mapped two breakpoint junctions and demonstrated that both are mediated by Alu repetitive elements and identical in these four individuals. We propose a three-step process combining Alu-mediated replicative-repair-based mechanism(s) and intergenerational, intrachromosomal nonallelic homologous recombination to generate the quadruplications in this family.

Background Juvenile‐onset cataracts are known among the Hutterites of North America. Despite being identified over 30 years ago, this autosomal recessive condition has not been mapped, and the disease gene is unknown. Methods We performed whole exome sequencing of three Hutterite‐type cataract trios and follow‐up genotyping and mapping in four extended kindreds. Results Trio exomes enabled genome‐wide autozygosity mapping, which localized the disease gene to a 9.5‐Mb region on chromosome 6p. This region contained two candidate variants, LEMD2 c.T38G and MUC21 c.665delC. Extended pedigrees recruited for variant genotyping revealed multiple additional relatives with juvenile‐onset cataract, as well as six deceased relatives with both cataracts and sudden cardiac death. The candidate variants were genotyped in 84 family members, including 17 with cataracts; only the variant in LEMD2 cosegregated with cataracts (LOD = 9.62). SNP‐based fine mapping within the 9.5 Mb linked region supported this finding by refining the cataract locus to a 0.5‐ to 2.9‐Mb subregion (6p21.32‐p21.31) containing LEMD2 but not MUC21. LEMD2 is expressed in mouse and human lenses and encodes a LEM domain‐containing protein; the c.T38G missense mutation is predicted to mutate a highly conserved residue within this domain (p.Leu13Arg). Conclusion We performed a genetic and genomic study of Hutterite‐type cataract and found evidence for an association of this phenotype with sudden cardiac death. Using combined genetic and genomic approaches, we mapped cataracts to a small portion of chromosome 6 and propose that they result from a homozygous missense mutation in LEMD2.

The genomic duplication associated with Potocki-Lupski syndrome (PTLS) maps in close proximity to the duplication associated with Charcot-Marie-Tooth disease type 1A (CMT1A). PTLS is characterized by hypotonia, failure to thrive, reduced body weight, intellectual disability, and autistic features. CMT1A is a common autosomal dominant distal symmetric peripheral polyneuropathy. The key dosage-sensitive genes RAI1 and PMP22 are respectively associated with PTLS and CMT1A. Recurrent duplications accounting for the majority of subjects with these conditions are mediated by nonallelic homologous recombination between distinct low-copy repeat (LCR) substrates. The LCRs flanking a contiguous genomic interval encompassing both RAI1 and PMP22 do not share extensive homology; thus, duplications encompassing both loci are rare and potentially generated by a different mutational mechanism. We characterized genomic rearrangements that simultaneously duplicate PMP22 and RAI1, including nine potential complex genomic rearrangements, in 23 subjects by high-resolution array comparative genomic hybridization and breakpoint junction sequencing. Insertions and microhomologies were found at the breakpoint junctions, suggesting potential replicative mechanisms for rearrangement formation. At the breakpoint junctions of these nonrecurrent rearrangements, enrichment of repetitive DNA sequences was observed, indicating that they might predispose to genomic instability and rearrangement. Clinical evaluation revealed blended PTLS and CMT1A phenotypes with a potential earlier onset of neuropathy. Moreover, additional clinical findings might be observed due to the extra duplicated material included in the rearrangements. Our genomic analysis suggests replicative mechanisms as a predominant mechanism underlying PMP22-RAI1 contiguous gene duplications and provides further evidence supporting the role of complex genomic architecture in genomic instability.

Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.

Osteonecrosis of the femoral head (ONFH) is a very common complication after femoral neck fracture. The purpose of this study was to assess the femoral head vascularity after femoral neck fracture using single photon emission computerized tomography and computerized tomography (SPECT/CT), and to evaluate its value in predicting ONFH. Between January 2008 and March 2011, 120 patients diagnosed with femoral neck fracture underwent SPECT-CT before the internal fixation. The fracture was classified according to the Garden classification. The ratios of the radionuclide uptake of the fractured femoral head to that of the contralateral femoral head (F/N) were calculated to assess the femoral head vascularity. After a minimum of two years’ follow-up, magnetic resonance imaging (MRI) was used as the “gold standard” for the diagnosis of possible ONFH. A total of 114 patients completed the study. The SPECT/CT examination showed that the F/N ratios of Garden I, II, III and IV were 2.6, 1.8, 0.8, and 0.6, respectively. At the time of the most recent follow-up, osteonecrosis developed in two of the 27 patients who had a Garden Stage-II fracture, in eight of the 34 patients who had a Garden Stage-III fracture, and nine of the 27 patients who had a Garden Stage-VI fracture. With a cutoff of 0.55 from the receiver operating characteristic (ROC) curve, F/N ratio showed a sensitivity of 97 %, a specificity of 79 %, a positive predictive value of 95 %, and a negative predictive value of 19 %. SPECT-CT proved to be reliable and valid for predicting ONFH after femoral neck fracture.

In this study, we determined the effects of transforming growth factor-beta (TGF-β) and Wnt/β-catenin signaling on myofibroblast differentiation of NIH/3T3 fibroblasts in vitro and evaluated the therapeutic efficacy of NSC668036 in bleomycin-induced pulmonary fibrosis murine model. In vitro study, NSC668036, a small organic inhibitor of the PDZ domain in Dvl, suppressed β-catenin-driven gene transcription and abolished TGF-β1-induced migration, expression of collagen I and α-smooth muscle actin (α-SMA) in fibroblasts. In vivo study, we found that NSC668036 significantly suppressed accumulation of collagen I, α-SMA, and TGF-β1 but increased the expression of CK19, Occludin and E-cadherin that can inhibit pulmonary fibrogenesis. Because fibrotic lung exhibit aberrant activation of Wnt/β-catenin signaling, these data collectively suggest that inhibition of Wnt/β-catenin signaling at the Dvl level may be an effective approach to the treatment of fibrotic lung diseases.

ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.

To compare the effect difference for stroke between meridian-collateral diagnosis and therapy byTotally 148 patients were assigned into an observation group(72 cases) and a control group(76 cases) by random number table,with 10 cases dropping out in the observation group. In the observation group,meridians were examined and differentiated and then the treating meridians and acupoints were defined. Corresponding acupuncture was used according to them. In the control group,acupuncture was applied at acupoints by internal differentiation and experience. Treatment was given once a day and five times a week,with total 20 times. The motion function of limbs and coloboma degree of nerve function were assessed by Fugl-Meyer score and National Institutes of Health Stroke Score(NIHSS) before and after treatment as well as at three-month follow-up.After treatment,the Fugl-Meyer scores increased and the NIHSS scores decreased in the two groups compared with those before treatment(allMeridian-collateral diagnosis and therapy by

MicroRNAs (miRNAs) are known to be crucial players in governing the differentiation of human induced pluripotent stem cells (hiPSCs). Despite their utter importance, identifying key lineage specifiers among the myriads of expressed miRNAs remains challenging. We believe that the current practice in mining miRNA specifiers via delineating dynamic fold-changes only is inadequate. Our study, therefore, provides evidence to pronounce “lineage specificity” as another important attribute to qualify for these lineage specifiers. Adopted hiPSCs were differentiated into representative lineages (hepatic, nephric and neuronal) over all three germ layers whilst the depicted miRNA expression changes compiled into an integrated atlas. We demonstrated inter-lineage analysis shall aid in the identification of key miRNAs with lineage-specificity, while these shortlisted candidates were collectively known as “lineage-specific miRNAs”. Subsequently, we followed through the fold-changes along differentiation via computational analysis to identify miR-192 and miR-372-3p, respectively, as representative candidate key miRNAs for the hepatic and nephric lineages. Indeed, functional characterization validated that miR-192 and miR-372-3p regulate lineage differentiation via modulation of the expressions of lineage-specific genes. In summary, our presented miRNA atlas is a resourceful ore for the mining of key miRNAs responsible for lineage specification.

21 páginas Hereditary neuropathy with liability to pressure palsies (HNPP) and Smith-Magenis syndrome (SMS) are genomic disorders associated with deletion copy number variants involving chromosome 17p12 and 17p11.2, respectively. Nonallelic homologous recombination (NAHR)-mediated recurrent deletions are responsible for the majority of HNPP and SMS cases; the rearrangement products encompass the key dosage-sensitive genes PMP22 and RAI1, respectively, and result in haploinsufficiency for these genes. Less frequently, nonrecurrent genomic rearrangements occur at this locus. Contiguous gene duplications encompassing both PMP22 and RAI1, i.e., PMP22-RAI1 duplications, have been investigated, and replication-based mechanisms rather than NAHR have been proposed for these rearrangements. In the current study, we report molecular and clinical characterizations of six subjects with the reciprocal phenomenon of deletions spanning both genes, i.e., PMP22-RAI1 deletions. Molecular studies utilizing high-resolution array comparative genomic hybridization and breakpoint junction sequencing identified mutational signatures that were suggestive of replication-based mechanisms. Systematic clinical studies revealed features consistent with SMS, including features of intellectual disability, speech and gross motor delays, behavioral problems and ocular abnormalities. Five out of six subjects presented clinical signs and/or objective electrophysiologic studies of peripheral neuropathy. Clinical profiling may improve the clinical management of this unique group of subjects, as the peripheral neuropathy can be more severe or of earlier onset as compared to SMS patients having the common recurrent deletion. Moreover, the current study, in combination with the previous report of PMP22-RAI1 duplications, contributes to the understanding of rare complex phenotypes involving multiple dosage-sensitive genes from a genetic mechanistic standpoint.

Background: Although various inflammation-based indexes in esophageal carcinoma have been documented, but the prognostic value of the albumin-to-globulin ratio(AGR) and its correlation with fibrinogen in resectable ESCC remain unknown. Methods: The levels of pre-treatment serum common acute phase proteins (including CRP, albumin and fribrinogen) were retrospectively analyzed in 447 patients with ESCC who underwent surgical resection at our department. The prognostic value was explored by univariate and multivariate cox hazard analysis. The correlation between AGR and acute phase proteins were also analyzed. Results: Patients with decreased levels of AGR and increased CRP had significantly lower 5-year survival rates than those with higher AGR, not only in the whole ESCC cohort but also in the subgroups stratified according to the disease T, N classifications, and metastasis, whereas the other acute phase proteins were not independent prognostic factors for ESCC. In addition, a lower AGR level was observed more often in patients with a high fibrinogen level than in those with a low fibrinogen level. Spearman's rank correlation analysis revealed that the AGR level presented a negative correlation with the fibrinogen level (r =-0.317, p

WOS: 000393996800025, PubMed: 27577878, Background: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective: We sought to investigate the ability of whole-exome screening methods to detect disease-causing variants in patients with PIDDs. Methods: Patients with PIDDs from 278 families from 22 countries were investigated by using whole-exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. Results: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/ 110) and management was directly altered in nearly a quarter (26/ 110) of families based on molecular findings. Twelve PIDD-causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion: This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes; permitted detection of low-grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes., South-Eastern Norway Health Authority; American Women's club of Oslo; National Human Genome Research InstituteUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Human Genome Research Institute (NHGRI); National Heart, Lung, and BloodUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Heart Lung & Blood Institute (NHLBI) [U54HG006542]; Jeffrey Modell Foundation; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [AI-120989], Funding for the work performed in Oslo was provided by the South-Eastern Norway Health Authority, and A. S.-P. received research scholarship from the American Women's club of Oslo. The BHCMG is supported by the National Human Genome Research Institute and the National Heart, Lung, and Blood (U54HG006542). Funding was also provided by the Jeffrey Modell Foundation and NIH AI-120989 (to J.S.O.).