Achieving sustained virologic response after interferon-free hepatitis C virus treatment correlates with hepatic interferon gene expression changes independent of cirrhosis.

Chronic hepatitis C virus (HCV) infection can now be treated with oral directly acting antiviral agents, either with or without ribavirin (RBV). Virologic relapse after treatment can occur, and in some studies was more common in cirrhotic subjects. We previously observed changes in hepatic immunity during interferon (IFN)-free therapy that correlated with favourable outcome in subjects with early liver disease. Here, we compared changes in endogenous IFN pathways during IFN-free, RBV-free therapy between cirrhotic and noncirrhotic subjects. mRNA and microRNA (miRNA) expression analyses were performed on paired pre- and post-treatment liver biopsies from genotype-1 HCV subjects treated with sofosbuvir/ledipasvir (SOF/LDV) for 12 weeks (n = 4, 3 cirrhotics) or SOF/LDV combined with GS-9669 or GS-9451 for 6 weeks (n = 6, 0 cirrhotics). Nine of ten subjects achieved a sustained virologic response (SVR), while one noncirrhotic subject relapsed. Hepatic IFN-stimulated gene expression decreased with treatment in the liver of all subjects, with no observable impact of cirrhosis. Hepatic gene expression of type III IFNs (IFNL1, IFNL3, IFNL4-ΔG) similarly decreased with treatment, while IFNA2 expression, undetectable in all subjects pretreatment, was detected post-treatment in three subjects who achieved a SVR. Only the subject who relapsed had detectable IFNL4-ΔG expression in post-treatment liver. Other IFNs had no change in gene expression (IFNG, IFNB1, IFNA5) or could not be detected. Although expression of multiple hepatic miRNAs changed with treatment, many miRNAs previously implicated in HCV replication and IFN signalling had unchanged expression. In conclusion, favourable treatment outcome during IFN-free HCV therapy is associated with changes in the host IFN response regardless of cirrhosis.

Expression profiling reveals fundamental biological differences in acute myeloid leukemia with isolated trisomy 8 and normal cytogenetics.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases. Normal cytogenetics (CN) constitutes the single largest group, while trisomy 8 (+8) as a sole abnormality is the most frequent trisomy. How trisomy contributes to tumorigenesis is unknown. We used oligonucleotide-based DNA microarrays to study global gene expression in AML+8 patients with +8 as the sole chromosomal abnormality and AML-CN patients. CD34(+) cells purified from normal bone marrow (BM) were also analyzed as a representative heterogeneous population of stem and progenitor cells. Expression patterns of AML patients were clearly distinct from those of CD34(+) cells of normal individuals. We show that AML+8 blasts overexpress genes on chromosome 8, estimated at 32% on average, suggesting gene-dosage effects underlying AML+8. Systematic analysis by cellular function indicated up-regulation of genes involved in cell adhesion in both groups of AML compared with CD34(+) blasts from normal individuals. Perhaps most interestingly, apoptosis-regulating genes were significantly down-regulated in AML+8 compared with AML-CN. We conclude that the clinical and cytogenetic heterogeneity of AML is due to fundamental biological differences.

Characterization of the cystatin B gene promoter harboring the dodecamer repeat expanded in progressive myoclonus epilepsy, EPM1.

Mutations in the gene encoding cystatin B (CSTB) are responsible for the primary defect in progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1). A novel and unique type of disease-causing mutation, an unstable dodecamer repeat expansion, accounts for the majority of EPM1 patients world-wide. This minisatellite repeat expansion, located in the putative promoter of CSTB 175 bp upstream from the translation initiation codon, appears to downregulate CSTB gene expression in vivo. We report here the characterization of the CSTB promoter using different promoter-luciferase gene constructs. Transient transfections of cultured mammalian cells suggest that the region from -670 to -1 bp from the translation initiation codon functions as the CSTB promoter. Active binding to five Sp1 and four AP1 sites as well as weak binding to an androgen response element (ARE) half site was demonstrated by electrophoretic mobility shift assays. The effect of the minisatellite expansion on the promoter activity was evaluated by comparing the activity of constructs containing wild-type and expanded alleles. An increase in the number of dodecamer units from three to 19 repeats lowered transcription in vitro by 10-fold. Northern analysis of lymphoblastoid RNA from individuals with 'premutation' length dodecamer repeat (12-17 copies) expansions showed decreased levels of CSTB mRNA expression. These data indicate that expansion of the dodecamer repeat located in the proximal promoter of CSTB severely disrupts the function of the promoter and thereby reduces transcription of CSTB.

The minisatellite expansion mutation in EPM1: resolution of an initial discrepancy. Mutatations in brief no. 186. Online.

Mutations in the cystatin B (CSTB) gene underlie progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) (Pennacchio et al., 1996). We previously described an unstable minisatellite expansion mutation in the putative promoter region of CSTB that accounts for the majority of EPM1 patients. Sequencing of a genomic lambda clone, generated from a Finnish EPM1 patient homozygous for an enlarged restriction fragment, revealed a 15- to 18-mer minisatellite repeat expansion (Virtaneva et al., 1997). Later, sequencing of plasmid clones generated from Swiss and French patients revealed a dodecamer repeat expansion (Lalioti et al., 1997a). By restriction enzyme analysis of our original patient clone and a clone generated from an Italian patient, we now show that the expansion is neither a 15-mer nor an 18-mer contrary to our initial results. Moreover, direct sequencing of the Finnish patient clone with Pfu exo polymerase confirmed that the expanded repeat is a dodecamer. Based on this finding and additional experiments, we suggest that the discrepancy between the two studies was due to errors caused by the combination of native Pfu polymerase and modified guanosine deaza-7-dGTP used in the PCR reaction.

Unstable minisatellite expansion causing recessively inherited myoclonus epilepsy, EPM1.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1; MIM 254800) is an autosomal recessive disorder that occurs with a low frequency in many populations but is more common in Finland and the Mediterranean region. It is characterized by stimulus-sensitive myoclonus and tonic-clonic seizures with onset at age 6-15 years, typical electroencephalographic abnormalities and a variable rate of progression between and within families. Following the initial mapping of the EPM1 gene to chromosome 21 (ref. 6) and the refinement of the critical region to a small interval, positional cloning identified the gene encoding cystatin B (CST6), a cysteine protease inhibitor, as the gene underlying EPM1 (ref. 10). Levels of messenger RNA encoded by CST6 were dramatically decreased in patients. A 3' splice site and a stop codon mutation were identified in three families, leaving most mutations uncharacterized. In this study, we report a novel type of disease-causing mutation, an unstable 15- to 18-mer minisatellite repeat expansion in the putative promoter region of the CST6 gene. The mutation accounts for the majority of EPM1 patients worldwide. Haplotype data are compatible with a single ancestral founder mutation. The length of the repeat array differs between chromosomes and families, but changes in repeat number seem to be comparatively rare events.

Progressive myoclonus epilepsy EPM1 locus maps to a 175-kb interval in distal 21q.

The EPM1 locus responsible for progressive myoclonus epilepsy of Unverricht-Lundborg type (MIM 254800) maps to a region in distal chromosome 21q where positional cloning has been hampered by the lack of physical and genetic mapping resolution. We here report the use of a recently constituted contig of cosmid, BAC, and P1 clones that allowed new polymorphic markers to be positioned. These were typed in 53 unrelated disease families from an isolated Finnish population in which a putative single ancestral EPM1 mutation has segregated for an estimated 100 generations. By thus exploiting historical recombinations in haplotype analysis, EPM1 could be assigned to the approximately 175-kb interval between the markers D21S2040 and D21S1259.

Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1)

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is an autosomal recessive inherited form of epilepsy, previously linked to human chromosome 21q22.3. The gene encoding cystatin B was shown to be localized to this region, and levels of messenger RNA encoded by this gene were found to be decreased in cells from affected individuals. Two mutations, a 3' splice site mutation and a stop codon mutation, were identified in the gene encoding cystatin B in EPM1 patients but were not present in unaffected individuals. These results provide evidence that mutations in the gene encoding cystatin B are responsible for the primary defect in patients with EPM1.

Expression and characterization of a B cell growth promoting polypeptide derived from the 12 kDa B cell growth factor gene (BCGF 1).

The expression and partial purification of recombinant 12 kDa B cell growth factor are reported. The polypeptide was derived from the genomic sequence of the gene (BCGF 1) which is here shown to be a single copy gene that localizes to human chromosome 16. When expressed as a glutathione S-transferase fusion protein in E. coli, the protein appears as a 38 kDa polypeptide in Western blot analysis using a peptide antibody. The purified fusion protein stimulates the proliferation of activated human B cells in a dose-dependent manner, and the active site resides within the 104 carboxy-terminal amino acids. The availability of biologically active recombinant 12 kDa B cell growth factor will enable its evaluation in B cell growth regulation, and provides a new means of in vitro culturing of human B lymphocytes.

BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2.

The Bmx sequence was identified and cloned during our search for novel tyrosine kinase genes expressed in human bone marrow cells. Bmx cDNA comprises a long open reading frame of 675 amino acids, containing one SH3, one SH2 and one tyrosine kinase domain, which are about 70% identical with Btk, Itk and Tec and somewhat less with Txk tyrosine kinase sequences. The amino terminal sequences of these four tyrosine kinases are about 40% identical and each contains a so-called pleckstrin homology domain. The 2.7 kb Bmx mRNA was expressed in endothelial cells and several human tissues by Northern blotting and an 80 kD Bmx polypeptide was detected in human endothelial cells. Immunoprecipitates of COS cells transfected with a Bmx expression vector and NIH3T3 cells expressing a Bmx retrovirus contained a tyrosyl phosphorylated Bmx polypeptide of similar molecular weight. The BMX gene was located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci. Interestingly, chromosome X also contains the closest relative of BMX, the BTK gene, implicated in X-linked agammaglobulinemia. The BMX gene thus encodes a novel nonreceptor tyrosine kinase, which may play a role in the growth and differentiation of hematopoietic cells.

Chromosomal localization of three human genes coding for A15, L6, and S5.7 (TAPA1): all members of the transmembrane 4 superfamily of proteins.

The A15, L6, and S5.7(TAPA1) proteins are members of the transmembrane 4 superfamily (TM4SF). The A15 is expressed in immature human T cells and in the human brain. The MXS1(CCG-B7) gene which codes for A15 contains triplet nucleotide repeats which have been associated with neuropsychiatric diseases such as Huntington's chorea, fragile X syndrome, and myotonic dystrophy. The L6 antigen is mainly expressed in lung, breast, colon, ovarian carcinomas, and healthy epithelial tissue in humans. The S5.7(TAPA1) antigen is expressed in most human cell lines and is shown to be associated with B-cell surface molecules CD19 and Leu-13. In this study we have used interspecies specific somatic cell hybrids and human-specific cDNA probes to localize the A15 (MXS1), L6 (M3S1), and TAPA1 genes to Xq11, 3q21-25, and 11p15.5, respectively.

The genes for CD37, CD53, and R2, all members of a novel gene family, are located on different chromosomes.

CD37, CD53, and R2 leukocyte surface antigens are members of a novel family of structurally related proteins. They all have four transmembrane-spanning domains with a single major extracellular loop. The CD37 is expressed on B cells and on a subpopulation of T cells. The CD53 is known as a panleukocyte marker. The R2 protein is an activation antigen of T cells. The CD37, CD53, and R2 genes were assigned with the help of human/rodent somatic cell hybrids and human-specific probes to human chromosomes 19, 1, and 11, respectively. For the regional assignment, various deletion hybrids were used to map CD37 to 19p13-q13.4, CD53 to 1p12-p31, and R2 to 11p12.