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Hypertrophic cardiomyopathy is a relatively frequent disease with a prevalence of 0.2% worldwide and a remarkable genetic heterogeneity, with more than 30 causative genes reported to date. Current PCR-based strategies are inadequate for genomic investigations involving many candidate genes. Here, we report a next-generation sequencing procedure associated with DNA sequence capture that is able to sequence 202 cardiomyopathy-related genes simultaneously. We developed a complementary data analysis pipeline to select and prioritize genetic variants. The overall procedure can screen a large number of target genes simultaneously, thereby potentially revealing new disease-causing and modifier genes. By using this procedure, we analyzed hypertrophic cardiomyopathy patients in a shorter time and at a lower cost than with current procedures. The specificity of the next-generation sequencing–based procedure is at least as good as other techniques routinely used for mutation searching, and the sensitivity is much better. Analysis of the results showed some novel variants potentially involved in the pathogenesis of hypertrophic cardiomyopathy: a missense mutation in MYH7and a nonsense variant in INS-IGF2(patient 1), a splicing variant in MYBPC3and an indel/frameshift variant in KCNQ1(patient 2), and two concomitant variations in CACNA1C(patient 3). Sequencing of DNA from the three patients within a pool allowed detection of most variants identified in each individual patient, indicating that this approach is a feasible and cost-effective procedure.

Emerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants — referred as qualifying variants (QVs) — of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease.

Long-chain polyphosphates inhibit SARS-CoV-2 infection by targeting a host receptor and a viral RNA polymerase.

The essential sequences needed for Alu repeat transcription by RNA polymerase III were mapped. Experimental evidence is presented showing that the Alu repeat promoters are organised in a bipartite structure similar to the split tRNA promoters as suggested by DNA sequence homology. Furthermore, by combining fragments from efficiently and inefficiently transcribed natural Alu repeats in several recombinant clones, it was possible to investigate the regions responsible for their differences. It is clear that, apart from the short stretches of homology with the tRNA consensus sequence, there is very little constraint in the promoter sequences. However, our studies indicate that some influence on the efficiency of transcription may be exerted by regions outside the accepted promoter components.

The essential sequences needed for Alu repeat transcription by RNA polymerase III were mapped. Experimental evidence is presented showing that the Alu repeat promoters are organised in a bipartite structure similar to the split tRNA promoters as suggested by DNA sequence homology. Furthermore, by combining fragments from efficiently and inefficiently transcribed natural Alu repeats in several recombinant clones, it was possible to investigate the regions responsible for their differences. It is clear that, apart from the short stretches of homology with the tRNA consensus sequence, there is very little constraint in the promoter sequences. However, our studies indicate that some influence on the efficiency of transcription may be exerted by regions outside the accepted promoter components.

Summary The mapping of the gene coding for human aldolase C has been studied using a specific cDNA probe and genomic blots from a panel of human-hamster somatic cell hybrids. The results show that the aldolase C gene is on chromosome 17. In situ experiments have restricted the mapping to the region 17cen→q21.1. Using the same panel of human-hamster somatic cell hybrids, we have confirmed the localization of aldolase A and B on chromosomes 16 and 9, respectively.

A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of hisIE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared.

Peripheral blood DNA was hybridized to the full-length cDNA and the cloned structural gene of human aldolase B. With PvuII endonuclease a restriction fragment length polymorphism was detected that was present in the heterozygous state in about 21% of the individuals tested. A map of the human aldolase gene was constructed for the two groups of individuals found to produce different fragments after PvuII digestion. This allowed the localization of the polymorphic site within the gene, which was found to be due to the loss of a PvuII site in the last intron upstream from the 3' end. This polymorphism may be used as a genetic marker to study individuals affected by hereditary fructose intolerance.

The structure of two alternatively spliced regions, ED-A and ED-B, of human fibronectin gene, was determined, in order to show whether any similarity was present between the two. Although some interesting features are present in each, no obvious common structure or sequence homology was found. Functional analysis of the alternative splicing events was carried out by transient expression in Hela cells. A hybrid gene was constructed by inserting the ED-B region into the third exon of the human α1-globin gene. The transfected hybrid gene is expressed and produces, in Hela cells, two alternatively spliced RNAs, showing a pattern very similar to that observed for the endogenous fibronectin gene in fibroblasts. Cotransfection of this gene with a similar gene containing the ED-A region, shows that no interference is present between the two alternative splicing processes.

A fast general purpose DNA handling program has been developed in BASIC and machine language. The program runs on the Apple II plus or on the Apple IIe microcomputer, without additional hardware except for disk drives and printer. The program allows file insertion and editing, translation into protein sequence, reverse translation, search for small strings and restriction enzyme sites. The homology may be shown either as a comparison of two sequences or through a matrix on screen. Two additional features are: (i) drawing restriction site maps on the printer; and (ii) simulating a gel electrophoresis of restriction fragments both on screen and on paper. All the operations are very fast. The more common tasks are carried out almost instantly; only more complex routines, like finding homology between large sequences or searching and sorting all the restriction sites in a long sequence require longer, but still quite acceptable, times (generally under 30 s).

A microcomputer program which locates tRNA genes within long DNA sequences is described. The search is performed either by identifying tRNA-like secondary structures or by locating eukaryotic RNA polymerase III promoter consensus sequences. The program is also useful in finding inverted repeats allowing the formation of stem-loop secondary structures in tRNA. The program has been developed in BASIC and 6502 Assembler and runs on the Apple II plus and He microcomputers. The execution is quite fast; all the operations are carried out in 1–90 s, depending on the required task and on the sequence length.