Next-Generation Sequencing for Molecular Diagnosis of Cystic Fibrosis in a Brazilian Cohort.

Cystic fibrosis (CF), an autosomal recessive genetic disease, is recognized as one of the most prevalent diseases in Caucasian populations. Epidemiological data show that the incidence of CF varies between countries and ethnic groups in the same region. CF occurs due to pathogenic variants in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), located on chromosome 7q31.2. To date, more than 2,000 variants have been registered in the CFTR database. The study of these variants leads to the diagnosis and the possibility of a specific treatment for each patient through precision medicine. In this study, complete screening of CFTR was performed through next-generation sequencing (NGS) to gain insight into the variants circulating in the population of Rio de Janeiro and to provide patient access to treatment through genotype-specific therapies. Samples from 93 patients with an inconclusive molecular diagnosis were subjected to full-length screening of CFTR using an Illumina NGS HiSeq platform. Among these patients, 46 had two pathogenic variants, whereas 12 had only one CFTR variant. Twenty-four variants were not part of our routine screening. Of these 24 variants, V938Gfs∗37 had not been described in the CF databases previously. This research achieved a molecular diagnosis of the patients with CF and identification of possible molecular candidates for genotype-specific treatments.

A fully automated artificial intelligence method for non-invasive, imaging-based identification of genetic alterations in glioblastomas.

Glioblastoma is the most common malignant brain parenchymal tumor yet remains challenging to treat. The current standard of care-resection and chemoradiation-is limited in part due to the genetic heterogeneity of glioblastoma. Previous studies have identified several tumor genetic biomarkers that are frequently present in glioblastoma and can alter clinical management. Currently, genetic biomarker status is confirmed with tissue sampling, which is costly and only available after tumor resection or biopsy. The purpose of this study was to evaluate a fully automated artificial intelligence approach for predicting the status of several common glioblastoma genetic biomarkers on preoperative MRI. We retrospectively analyzed multisequence preoperative brain MRI from 199 adult patients with glioblastoma who subsequently underwent tumor resection and genetic testing. Radiomics features extracted from fully automated deep learning-based tumor segmentations were used to predict nine common glioblastoma genetic biomarkers with random forest regression. The proposed fully automated method was useful for predicting IDH mutations (sensitivity = 0.93, specificity = 0.88), ATRX mutations (sensitivity = 0.94, specificity = 0.92), chromosome 7/10 aneuploidies (sensitivity = 0.90, specificity = 0.88), and CDKN2 family mutations (sensitivity = 0.76, specificity = 0.86).

A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus.

Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.4 kb fragment from the human genome. Through a streamlined process of substrate-linked directed evolution we generated two separate recombinases that, when expressed together, act as a heterodimer to delete a human genomic sequence from chromosome 7. Our data indicates that designer-recombinases can be generated in a manageable timeframe for precision genome editing. A large-scale bioinformatics analysis suggests that around 13% of all human protein-coding genes could be targetable by dual designer-recombinase induced genomic deletion (dDRiGD). We propose that heterospecific designer-recombinases, which work independently of the host DNA repair machinery, represent an efficient and safe alternative to nuclease-based genome editing technologies.

Multi-allelic exact tests for Hardy-Weinberg equilibrium that account for gender.

Statistical tests for Hardy-Weinberg equilibrium are important elementary tools in genetic data analysis. X-chromosomal variants have long been tested by applying autosomal test procedures to females only, and gender is usually not considered when testing autosomal variants for equilibrium. Recently, we proposed specific X-chromosomal exact test procedures for bi-allelic variants that include the hemizygous males, as well as autosomal tests that consider gender. In this study, we present the extension of the previous work for variants with multiple alleles. A full enumeration algorithm is used for the exact calculations of tri-allelic variants. For variants with many alternate alleles, we use a permutation test. Some empirical examples with data from the 1,000 genomes project are discussed.

Gain-of-function SAMD9L mutations cause a syndrome of cytopenia, immunodeficiency, MDS, and neurological symptoms.

Several monogenic causes of familial myelodysplastic syndrome (MDS) have recently been identified. We studied 2 families with cytopenia, predisposition to MDS with chromosome 7 aberrations, immunodeficiency, and progressive cerebellar dysfunction. Genetic studies uncovered heterozygous missense mutations in SAMD9L, a tumor suppressor gene located on chromosome arm 7q. Consistent with a gain-of-function effect, ectopic expression of the 2 identified SAMD9L mutants decreased cell proliferation relative to wild-type protein. Of the 10 individuals identified who were heterozygous for either SAMD9L mutation, 3 developed MDS upon loss of the mutated SAMD9L allele following intracellular infections associated with myeloid, B-, and natural killer (NK)-cell deficiency. Five other individuals, 3 with spontaneously resolved cytopenic episodes in infancy, harbored hematopoietic revertant mosaicism by uniparental disomy of 7q, with loss of the mutated allele or additional in cisSAMD9L truncating mutations. Examination of 1 individual indicated that somatic reversions were postnatally selected. Somatic mutations were tracked to CD34+ hematopoietic progenitor cell populations, being further enriched in B and NK cells. Stimulation of these cell types with interferon (IFN)-α or IFN-γ induced SAMD9L expression. Clinically, revertant mosaicism was associated with milder disease, yet neurological manifestations persisted in 3 individuals. Two carriers also harbored a rare, in trans germ line SAMD9L missense loss-of-function variant, potentially counteracting the SAMD9L mutation. Our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with -7/del(7q), whereas hematopoietic revertant mosaicism commonly ameliorated clinical manifestations. The findings suggest a role for SAMD9L in regulating IFN-driven, demand-adapted hematopoiesis.

A case of prenatal detection of a de novo unbalanced complex chromosomal rearrangement involving four chromosomes.

Complex chromosomal rearrangements are rarely observed prenatally. Genetic counceling of CCR carriers is complicated, especially in cases of de novo origin of the rearrangement. Here we present a new case of a de novo CCR involving four chromosomes observed in amniotic fluid cells of the fetus at 17 weeks of gestation. The rearrangement was characterized as an apparently balanced four-way translocation t(1;11;7;13)(~p21;~q13.5;~q32;~q22)dn by conventional cytogenetic studies. However, array-based comparative genomic hybridization revealed 5 submicroscopic heterozygous interstitial deletions on chromosome 1, 11, 7, 13 with a total loss of 21.1 Mb of genetic material in regions close to those, designated as breakpoints by conventional cytogenetic analysis. The described case clearly illustrates that high-resolution molecular genetic analysis should be combined with conventional cytogenetic techniques to exclude subtle chromosomal abnormalities in CCR cases detected prenatally.

DOES THE PATTERN OF CLONAL EVOLUTION IN THE KARYOTYPE OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES DEPEND ON THE TYPE OF THE PRIMARY CHROMOSOMAL ABERRATIONS?

The aim of our study was to define if the type of primary chromosomal aberrations (CA) of the karyotype of patients with Acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) determines the way and the rate of karyotype development. Conventional cytogenetic analysis was carried out on 248 AML and 105 MDS patients at diagnosis. Clonal evolution (CE) was found in 40% (51 of 128) of AML patients and in 47.5% (19 of 40) of MDS patients having CA in their karyotype. The first pattern we established was for the most frequent CA which initiate CE in 28 patients with a complex karyotype. These CA were non-balansed rearrangements in the following regions: 5q, 7q, 11q, 3q, monosomy 5, monosomy 7. The second pattern of CE was regarding the most frequent aneuploidias (+8, +11, +21, -Y, and the third pattern concerned balanced CA. We found significant difference in the distribution of karyotypes in different stages of progression between the first and the other two groups (p < 0.001). No statistical difference was found between the patterns in the second and the third group CA (p > 0.5).

Chromosome mis-segregation and cytokinesis failure in trisomic human cells.

Cancer cells display aneuploid karyotypes and typically mis-segregate chromosomes at high rates, a phenotype referred to as chromosomal instability (CIN). To test the effects of aneuploidy on chromosome segregation and other mitotic phenotypes we used the colorectal cancer cell line DLD1 (2n = 46) and two variants with trisomy 7 or 13 (DLD1+7 and DLD1+13), as well as euploid and trisomy 13 amniocytes (AF and AF+13). We found that trisomic cells displayed higher rates of chromosome mis-segregation compared to their euploid counterparts. Furthermore, cells with trisomy 13 displayed a distinctive cytokinesis failure phenotype. We showed that up-regulation of SPG20 expression, brought about by trisomy 13 in DLD1+13 and AF+13 cells, is sufficient for the cytokinesis failure phenotype. Overall, our study shows that aneuploidy can induce chromosome mis-segregation. Moreover, we identified a trisomy 13-specific mitotic phenotype that is driven by up-regulation of a gene encoded on the aneuploid chromosome.

Direct mapping of symbolic DNA sequence into frequency domain in global repeat map algorithm.

The main feature of global repeat map (GRM) algorithm (www.hazu.hr/grm/software/win/grm2012.exe) is its ability to identify a broad variety of repeats of unbounded length that can be arbitrarily distant in sequences as large as human chromosomes. The efficacy is due to the use of complete set of a K-string ensemble which enables a new method of direct mapping of symbolic DNA sequence into frequency domain, with straightforward identification of repeats as peaks in GRM diagram. In this way, we obtain very fast, efficient and highly automatized repeat finding tool. The method is robust to substitutions and insertions/deletions, as well as to various complexities of the sequence pattern. We present several case studies of GRM use, in order to illustrate its capabilities: identification of α-satellite tandem repeats and higher order repeats (HORs), identification of Alu dispersed repeats and of Alu tandems, identification of Period 3 pattern in exons, implementation of 'magnifying glass' effect, identification of complex HOR pattern, identification of inter-tandem transitional dispersed repeat sequences and identification of long segmental duplications. GRM algorithm is convenient for use, in particular, in cases of large repeat units, of highly mutated and/or complex repeats, and of global repeat maps for large genomic sequences (chromosomes and genomes).

Identification of novel suggestive loci for high-grade myopia in Polish families.

PURPOSE: Myopia is the most common human eye disorder with complex genetic and environmental causes. To date, several myopia loci have been identified in families of different geographic origin. However, no causative gene(s) have yet been identified. The aim of this study was the characterization of Polish families with high-grade myopia, including genetic analysis. METHODS: Forty-two multiplex Polish families with non-syndromic high-grade myopia participated in the study. All family members underwent detailed ophthalmic examination and high-grade myopia was defined as ≤-6.0 diopters (D) based on the spherical refractive error. A genome-wide single nucleotide polymorphism (SNP)-based high-density linkage scan was performed using Affymetrix Human SNP Array 6.0 on a selected family (HM-32) with multiple affected individuals. RESULTS: Nonparametric linkage analysis identified three novel loci in family HM-32 at chromosome 7p22.1-7p21.1 ([NPL] 8.26; p=0.006), chromosome 7p12.3-7p11.2 ([NPL] 8.23; p=0.006), and chromosome 12p12.3-12p12.1 ([NPL] 8.02; p=0.006), respectively. The effect of linkage disequilibrium on linkage due to dense SNP map was addressed by systematically pruning SNPs from the linkage panel. CONCLUSIONS: Haplotype analysis with informative crossovers in affected individuals defined a 12.2; 10.9; and 9.5 Mb genomic regions for high-grade myopia spanned between SNP markers rs11977885/rs10950639, rs11770622/rs9719399, and rs4763417/rs10842388 on chromosomes 7p22.1-7p21.1, 7p12.3-7p11.2, and 12p12.3-12p12.1, respectively.

Genome-wide analysis of genetic susceptibility to language impairment in an isolated Chilean population.

Specific language impairment (SLI) is an unexpected deficit in the acquisition of language skills and affects between 5 and 8% of pre-school children. Despite its prevalence and high heritability, our understanding of the aetiology of this disorder is only emerging. In this paper, we apply genome-wide techniques to investigate an isolated Chilean population who exhibit an increased frequency of SLI. Loss of heterozygosity (LOH) mapping and parametric and non-parametric linkage analyses indicate that complex genetic factors are likely to underlie susceptibility to SLI in this population. Across all analyses performed, the most consistently implicated locus was on chromosome 7q. This locus achieved highly significant linkage under all three non-parametric models (max NPL = 6.73, P = 4.0 × 10(-11)). In addition, it yielded a HLOD of 1.24 in the recessive parametric linkage analyses and contained a segment that was homozygous in two affected individuals. Further, investigation of this region identified a two-SNP haplotype that occurs at an increased frequency in language-impaired individuals (P = 0.008). We hypothesise that the linkage regions identified here, in particular that on chromosome 7, may contain variants that underlie the high prevalence of SLI observed in this isolated population and may be of relevance to other populations affected by language impairments.

Duplication and relocation of the functional DPY19L2 gene within low copy repeats.

BACKGROUND: Low copy repeats (LCRs) are thought to play an important role in recent gene evolution, especially when they facilitate gene duplications. Duplicate genes are fundamental to adaptive evolution, providing substrates for the development of new or shared gene functions. Moreover, silencing of duplicate genes can have an indirect effect on adaptive evolution by causing genomic relocation of functional genes. These changes are theorized to have been a major factor in speciation. RESULTS: Here we present a novel example showing functional gene relocation within a LCR. We characterize the genomic structure and gene content of eight related LCRs on human Chromosomes 7 and 12. Two members of a novel transmembrane gene family, DPY19L, were identified in these regions, along with six transcribed pseudogenes. One of these genes, DPY19L2, is found on Chromosome 12 and is not syntenic with its mouse orthologue. Instead, the human locus syntenic to mouse Dpy19l2 contains a pseudogene, DPY19L2P1. This indicates that the ancestral copy of this gene has been silenced, while the descendant copy has remained active. Thus, the functional copy of this gene has been relocated to a new genomic locus. We then describe the expansion and evolution of the DPY19L gene family from a single gene found in invertebrate animals. Ancient duplications have led to multiple homologues in different lineages, with three in fish, frogs and birds and four in mammals. CONCLUSION: Our results show that the DPY19L family has expanded throughout the vertebrate lineage and has undergone recent primate-specific evolution within LCRs.

Maternal and paternal chromosomes 7 show differential methylation of many genes in lymphoblast DNA.

Genomic imprinting, the differential expression of paternal and maternal alleles, involves many chromosomal regions and plays a role in development and growth. Differential methylation of maternal and paternal alleles is a hallmark of imprinted genes, and thus methylation assays are widely used to support the identification of novel imprinted genes. Either blood or lymphoblast DNAs are most often used in these assays, even though methylation levels may change in cell culture. We undertook a systematic survey of parent-of-origin-specific methylation of chromosome 7 genes and ESTs by comparing DNA samples from cases of maternal and paternal uniparental disomy for chromosome 7 using DNA from fresh blood and lymphoblast cell lines. Our results revealed that up to 41% of genes and ESTs show parent-of-origin-specific methylation differences in lymphoblast DNA after only a short time in culture, whereas methylation differences were not seen in blood DNA. The methylation changes occurred most commonly on paternal chromosome 7, whereas alterations on maternal chromosome 7 were more infrequent and weaker. These findings indicate that methylation patterns may change significantly during cell culture in a parent-of-origin-dependent manner and suggest that methylation is maintained differently on maternal and paternal chromosomes 7.

Good maps are straight.

This paper proposes a simplified approach to the assembly of large physical genome maps. The approach focuses on two key problems: (i) the integration of diverse forms of data from numerous sources, and (ii) the detection and removal of errors and anomalies in the data. The approach simplifies map assembly by dividing it into three phases-overlap, linkage and ordering. In the first phase, all forms of overlap data are integrated into a simple abstract structure, called clusters, where each cluster is a set of mutually-overlapping DNA segments. This phase filters out many questionable overlaps in the mapping data. In the second phase, clusters are linked together into a weighted intersection graph. False links between widely separated regions of the genome show up as crooked, branching structures in the graph. Removing these false links produces graphs that are straight, reflecting the linear structure of chromosomes. From these straight graphs, the third phase constructs a physical map. Graph algorithms and graph visualization play key roles in implementing the approach. At present, the approach is at an early stage of development: it has been tested on real and simulated mapping data, and the results look promising. This paper describes the first two phases of the approach in detail, and reports on our progress to date.

Molecular cloning of genomic DNA and chromosomal assignment of the gene for human aromatic L-amino acid decarboxylase, the enzyme for catecholamine and serotonin biosynthesis.

Aromatic L-amino acid decarboxylase (AADC) catalyzes the decarboxylation of both L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are major mammalian neurotransmitters and hormones belonging to catecholamines and indoleamines. This report describes the organization of the human AADC gene. We proved that the gene of human AADC consists of 15 exons spanning more than 85 kilobases and exists as a single copy in the haploid genome. The boundaries between exon and intron followed the AG/GT rule. The sizes of exons and introns ranged from 20 to 400 bp and from 1.0 to 17.7 kb, respectively, while the sizes of four introns were not determined. Untranslated regions located in the 5' region of mRNA were encoded by two exons, exons 1 and 2. The transcriptional starting point was determined around G at position -111 by primer extension and S1 mapping. There were no typical "TATA box" and "CAAT box" within 540 bp from the transcriptional starting point. The human AADC gene was mapped to chromosome band 7p12.1-p12.3 by fluorescence in situ hybridization. This is the first report on the genomic structure and chromosomal localization of the AADC gene in mammals.

Chromosomal localization of a human cDNA containing a DIDS binding domain and demonstrating high homology to yeast omnipotent suppressor 45.

We recently have identified a full-length cDNA (TB3-1) from a human adenocarcinoma cell line T84 cDNA library that encodes a 47.8-kDa protein. TB3-1 shares identity with the putative yeast translation termination factor omnipotent suppressor 45. Using human-mouse somatic cell panel analysis, a family of sequences with high homology to the TB3-1 cDNA clone were localized to human chromosomes 5, 6, 7, and X. Southern analysis of a panel of mammalian and chicken genomic DNA demonstrates that TB3-1 is well conserved in higher vertebrates.

Polysomy of chromosome 7 is associated with amplification and overexpression of the EGF-receptor gene in a human carcinoma cell line derived from a brain metastasis.

Overexpression of the EGF-receptor gene is associated with the malignant nature of some tumors. We have recently reported the establishment of a human carcinoma cell line (T-CAR1), derived from a brain metastasis, that had 7 million EGF receptors per cell and was growth inhibited by EGF. The present study was carried out in order to further characterize the EGF-receptor protein in T-CAR1 cells, and to see if the overexpression of the EGF-receptor gene in these cells was associated with abnormalities at the genomic level. We have compared the T-CAR1 cells with the human glioblastoma cell line T-MG1, which has 135,000 EGF-receptors and is growth stimulated by EGF. The MW of the EGF receptors in T-CAR1 cells and T-MG1 cells was estimated to be 170 kDa, equal to the normal EGF-receptor. However, in T-CAR1 cells an additional protein reacted with the monoclonal antibody directed against the internal domain of the EGF receptor. The levels of EGF receptor-related RNAs in T-CAR1 cells and T-MG1 cells reflected the number of EGF receptors in these cell lines. The EGF-receptor gene was amplified ten-fold in T-CAR1 cells, while it was not amplified in T-MG1 cells. No restriction fragment length polymorphism of DNA digested with various restriction enzymes was seen in either of the cell lines. Chromosomal analysis of T-CAR1 cells showed polysomy of chromosome 7 and marker chromosomes derived partly from chromosome 7. Thus, in the T-CAR1 cell line it was an association between polysomy of chromosome 7 and EGF-receptor gene amplification.

Human ornithine decarboxylase-encoding loci: nucleotide sequence of the expressed gene and characterization of a pseudogene.

Previous studies have shown that human ornithine decarboxylase (ODC)-encoding sequences map to two chromosome regions: 2pter-p23 and 7cen-qter. In the present work we have cloned the expressed human ODC gene from a genomic library of myeloma cells that overproduce ODC protein due to selective gene amplification and determined its entire nucleotide sequence. The gene comprises 12 exons and 11 introns and spans about 8 kb of chromosome 2 DNA. The organization of the human gene is very similar to that of the mouse and rat, with the major difference being the presence of longer intronic sequences in the human gene. Some of these differences can be accounted for by the insertion of four Alu sequences in the human gene. Several potential regulatory elements are present in the promoter region and in 5'-proximal introns, including a TATA box; GC boses; AP-1-, AP-2- and NF-1-binding sites; and a cAMP-responsive element. The 5'-untranslated sequence of ODC mRNA is extremely GC-rich, and computer predictions suggest a very stable secondary structure for this region, with an overall free energy of formation of -225.4 kcal/mol. In addition to the active ODC gene on chromosome 2, ODC gene-related sequences were isolated from human chromosome 7-specific libraries and shown to represent a processed ODC pseudogene.