[Clinical and genetic analysis of a patient with primary distal renal tubular acidosis due to variants of ATP6V0A4 gene].

OBJECTIVE: To explore the clinical features and genetic etiology of a patient with primary distal renal tubular acidosis (dRTA). METHODS: A child who was diagnosed with primary dRTA at the Xi'an Children's Hospital in April 2021 due to poor appetite and persistent crying was selected as the study subject. Clinical data of the patient was collected. Whole exome sequencing (WES) was carried out for the child. Candidate variants were validated by Sanger sequencing of his family members. RESULTS: The child, a 1-month-and-18-day male, had featured poor appetite, persistent crying, poor weight gain and dehydration. Laboratory examination has suggested metabolic acidosis, hyperchloremia, hypokalemia, abnormal alkaline urine and anemia. Ultrasonographic examination of the urinary system revealed calcium deposition in renal medulla. DNA sequencing revealed that he has harbored compound heterozygous variants of the ATP6V0A4 gene, namely c.1363dupA (p.M455NfsX14) and c.2257C>T (p.Q753X), which were respectively inherited from his father and mother. Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were classified as pathogenic (PVS1+PM3+PM2_Supporting). CONCLUSION: The compound heterozygous variants of c.1363dupA (p.M455NfsX14) and c.2257C>T (p.Q753X) of the ATP6V0A4 gene probably underlay the pathogenesis of primary dRTA in this patient. Discovery of the c.2257C>T (p.Q753X) variant has also expanded the mutational spectrum of the ATP6V0A4 gene.

[Clinical and genetic analysis of an infant with permanent neonatal diabetes mellitus due to novel variant of insulin gene].

OBJECTIVE: To explore the genetic basis for an infant with permanent neonatal diabetes mellitus (PNDM). METHODS: Clinical data of the child was collected. Targeted capture-next generation sequencing was carried out to identify the potential variants. Candidate variant was verified by Sanger sequencing of her family members. RESULTS: The child was a 4-month-and-26-day female featuring onset of ketoacidosis accompanied with fasting blood glucose of 24.4 mmol/L, positive urine glucose, decreased serum C-peptide, HbA1c of 9.58%, and negative diabetes autoantibody. Genetic testing revealed that she has carried a heterozygous c.314T>G (p.L105R) variant of the INS gene. Sanger sequencing verified that neither of her parents has carried the same variant, which was also unreported in the literature. The variant was classified as likely pathogenic based on the ACMG guidelines. CONCLUSION: The c.314T>G (P.L105R) variant of the INS gene probably underlay the genetic etiology in this child. Genetic testing should be conducted for children with suspected PNDM for early diagnosis and appropriate treatment.

[Identification of a novel variant of SRD5A2 gene in a child featuring steroid 5α-reductase type 2 deficiency].

OBJECTIVE: To explore the clinical characteristics and genetic basis of a child with 5α-reductase type 2 deficiency. METHODS: Clinical data of the child was retrospectively analyzed. Targeted capture-next generation sequencing and Sanger sequencing were carried out to detect potential variants. RESULTS: The patient's main features included micropenis and hypospadia. He was found to harbor compound heterozygous c.680G>A (p.R227Q) and c.3G>T (p.M1I) variants of the SRD5A2 gene. Among these, c.680G>A (p.R227Q) was inherited from his father and was a known pathogenic mutation, while c.3G>T (p.M1I) was inherited from his mother and was unreported previously. CONCLUSION: The compound heterozygous variants of the SRD5A2 gene probably underlay the disease in this child, who was eventually diagnosed with 5α-reductase 2 deficiency.

FRP-Confined Recycled Coarse Aggregate Concrete: Experimental Investigation and Model Comparison.

The in situ application of recycled aggregate concrete (RAC) is of great significance in environmental protection and construction resources sustainability. However, it has been limited to nonstructural purposes due to its poor mechanical performance. External confinement using steel tubes and fiber-reinforced polymer (FRP) can significantly improve the mechanical performance of RAC and thus the first-ever study on the axial compressive behavior of glass FRP (GFRP)-confined RAC was recently reported. To have a full understanding of FRP-confined RAC, this paper has extended the type of FRP and presents a systematic experimental study on the axial compressive performance of carbon FRP (CFRP)-confined RAC. The mechanical properties of CFRP-confined RAC from the perspective of the failure mode, ultimate strength and strain, and stress⁻strain relationship responses were analyzed. Integrated with existing experimental data of FRP-confined RAC, the paper compiles a database for the mechanical properties of FRP-confined RAC. Based on the database, the effects of FRP type (i.e., GFRP and CFRP) and the replacement ratio of recycled coarse aggregate were investigated. The results indicated that the stress⁻stain behavior of FRP-confined RAC depended heavily on the unconfined concrete strength and the FRP confining pressure instead of the replacement ratio. Therefore, this study adopted eleven high-performance ultimate strength and strain models developed for FRP-confined normal aggregate concrete (NAC) to predict the mechanical properties of FRP-confined RAC. All the predictions had good agreement with the test results, which further confirmed similar roles played by FRP confinement in improving the mechanical properties of RAC and improving those of NAC. On this basis, this paper finally recommended a stress⁻strain relationship model for FRP-confined RAC.

Newly identified transcripts of UL4 and UL5 genes of human cytomegalovirus.

Human cytomegalovirus (HCMV) UL4 and UL5 genes are two members of the RL11 gene family. In an earlier study, three UL4 transcripts of about 1.7, 1.5 and 1.4 kb were found in early and late classes after infection by the Towne strain by nuclease protection and primer extension analyses. In the present study, two UL4 transcripts (1.5 and 1.7 kb) were found by cDNA library screening, Northern blot, 3' and 5' RACE analyses to appear initially in the immediate early phase and one UL4 transcript (1.4 kb) in the late phase in a low-passage clinical isolate. Furthermore, two novel low-abundance UL5 transcripts with the same 3' terminus as the identified UL4 transcripts in the UL4-UL5 gene region were found in late class RNAs.

Validation of three splice donor and three splice acceptor sites for regulating four novel low-abundance spliced transcripts of human cytomegalovirus UL21.5 gene locus.

In a previous study, one spliced transcript of human cytomegalovirus (HCMV), named UL21.5 was identified. UL21.5 has been found to be one of the viral transcripts packaged within HCMV particles. The UL21.5 mRNA is translated into a secreted glycoprotein, which is a viral chemokine decoy receptor specifically interacting with regulated upon activation normal T cell expressed and secreted (RANTES). In the present study, four novel low-abundance 3'-coterminal spliced transcripts were identified to be transcribed from the UL21.5 gene region of a low-passage HCMV strain during the late infection phase by cDNA library screening, northern blot hybridization, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR. Three splicing donor and three splicing acceptor sites found in the UL21.5 gene region were validated to be functional in an in vitro expression system. In addition, the determinant regulatory region that is necessary for the splice donor site at nucleotide (nt) 25533 was located in a 9-bp sequence around the site; the regulatory regions for the splice acceptor sites at nt 26597 and nt 26633 were located in a 20-bp sequence upstream of the site at nt 26597 and in a 10-bp sequence from nt 26641 to nt 26650 downstream of the site at nt 26633, respectively.

Characterization of a novel group of antisense transcripts in human cytomegalovirus UL83 gene region.

The rapid advances in research on antisense transcripts are gradually changing our understanding of the expression of the Herpesviridae genome. In this study, the transcripts of the human cytomegalovirus (HCMV) UL83 antisense strand were investigated in three clinical isolates. Three cDNA clones containing sequences with an antisense orientation to the UL83 gene were identified in a late HCMV cDNA library. The UL83 antisense transcripts (UL83asts) were then shown to be transcribed only in the late infection phase of the three clinical HCMV strains, using rapid amplification of cDNA ends (RACE) and northern blotting. These UL83asts were identical at their 3' termini but different at 5' ends. Two open reading frames were predicted in the UL83asts.

Analysis and mapping of a 3' coterminal transcription unit derived from human cytomegalovirus open reading frames UL30-UL32.

BACKGROUND: It has been predicted that the UL31 gene originates from the positive strand of the human cytomegalovirus (HCMV) genome, whereas the UL30 and UL32 genes originate from the complementary strand. Except for the UL32 gene, the transcription of this gene region has not been investigated extensively. METHODS: Northern blotting, cDNA library screening, RACE-PCR,and RT-PCR were used. RESULTS: At least eight transcripts of the antisense orientation of UL31 were transcribed from the UL30-UL32 region during the late phase of HCMV infection. The 3' coterminus of these transcripts was located within the predicted UL30 gene. The longest 6.0-kb transcript was initiated upstream of the predicted UL32 gene. Other transcripts were derived from the predicted UL30 and UL31 gene region. Except for the previously predicted UL32 open reading frame (ORF), three novel ORFs, named UL31anti-1, UL31anti-2 and UL31anti-3, were located in the transcripts from the UL31anti-UL32 transcription unit. No transcription was found in UL31. CONCLUSION: A family of novel 3' coterminal transcripts was transcribed from the UL30-UL32 gene region.

Overlapping transcription structure of human cytomegalovirus UL140 and UL141 genes.

Transcription of human cytomegalovirus UL/b' region has been studied extensively for some genes. In this study, transcripts of the UL140 and UL141, two of the UL/b' genes, were identified in late RNAs of three HCMV isolates using Northern blot hybridization, cDNA library screening and RACE-PCR. At least three transcripts with length of 2800, 2400 and 1700 nt, as well as a group of transcripts of about 1000-1300 nt, were found in this gene region with an accordant 3' ends. Among the transcripts, two initiated upstream of the start code of the UL140 gene and contained the UL140 and UL141 open reading frame (ORF), one initiated in the middle of the UL140 gene, and could encode short ORFs upstream of the UL141 ORF. A group of transcripts initiated upstream or downstream of the start code of the UL141 gene, and could encode 'nested' ORFs, including the UL141 ORF. These 'nested' ORFs possess different initiation sites but the same termination site as that of the UL141 ORF.

Transcription characteristics of the human cytomegalovirus UL13 gene.

The human cytomegalovirus (HCMV) UL13 gene is located in the unique long (UL) region of its genome. The transcript structure of UL13 gene has not been investigated to date. By using cDNA library screening, northern blot, and rapid amplification of cDNA ends (RACE), the HCMV UL13 gene was demonstrated to be transcribed from the immediate early (IE) to the late (L) phase of infection, and at least one 1602-nt unspliced transcript was identified in the present study from three clinical isolates.

Human CMV transcripts: an overview.

The human CMV (HCMV) genome consists of an approximately 230-kb dsDNA and is predicted to contain over 165 open reading frames. Although the entire sequence of the laboratory-adapted AD169 strain of HCMV was first available in 1991, the precise number and nature of viral genes and gene products are still unclear. Fewer than 100 predicted genes have been convincingly elucidated with respect to their expression patterns, transcript structure and transcription characteristics. The high gene number of HCMV creates a crowded genome with many overlapping transcriptional units. 3´- or 5´-coterminal overlapping polycistronic transcripts could use a common promoter element or a poly-A signal. 3´-coterminal monocistronic transcripts could encode 'nested' open reading frames, which possess different initiation but the same termination sites. As a virus with eukaryotic cells as the host, HCMV has the capacity to splice out introns during transcription. Major alternately spliced mRNA species of HCMV originate primarily, but not exclusively, from the immediate early gene regions. Alternate splicing patterns of the mRNAs could encode a number of gene products with different sizes. In recent years, some antisense and noncoding transcripts of HCMV have been reported. These RNAs probably have functions in genomic replication or the regulation of gene expression.

Characterization of human cytomegalovirus UL146 transcripts.

BACKGROUND: The genome of human cytomegalovirus (HCMV) has been studied extensively, particularly in the UL/b' region. In this study, transcripts of one of the UL/b' genes, UL146, were identified in 3 HCMV isolates obtained from urine samples of congenitally infected infants. METHODS: Northern blot hybridization, cDNA library screening, and RACE-PCR were used. RESULTS: All sequences of clones from a cDNA library were about 3225 bp in length with the same 5' and 3' ends. The results were accordant with that analyzed by Northen-blot and RACE-PCR in three HCMV clinical isolates. The transcript initiated from the region upstream of UL146 flanking region and terminated just downstream of UL132 including UL146, UL147, UL147A, UL148, and UL132 ORFs. Treatment of the infected cells with phosphonoacetic acid inhibited its transcription. CONCLUSIONS: UL146 ORF was transcribed with 4 downstream ORFs from UL147 to UL132 at true late kinetics. The transcript of UL146 initiated at 73nt upstream of UL146 and terminated just downstream of UL132 in the 3 clinical isolates.

An antisense transcript in the human cytomegalovirus UL87 gene region.

BACKGROUND: Rapid advances in research on antisense transcripts are gradually changing our comprehension of genomic and gene expression aspects of the Herpesviridae. One such herpesvirus is the human cytomegalovirus (HCMV). Although transcription of the HCMV UL87 gene has not been specifically investigated, cDNA clones of UL87 antisense transcripts were found in HCMV cDNA libraries previously. In this study, the transcription of the UL87 antisense strand was investigated in three clinically isolated HCMV strains. RESULTS: First, an 800 nucleotides transcript having an antisense orientation to the UL87 gene was found in a late HCMV cDNA library. Then, the UL87 antisense transcript was confirmed by Rapid amplification of cDNA ends (RACE) and Northern blot in three HCMV clinical strains. Two ORFs were predicted in the antisense transcript. The putative protein of ORF 1 showed a high degree of conservation among HCMV and other CMV strains. CONCLUSION: An 800nt antisense transcript in the UL87 gene region exists in HCMV clinical strains.

Transcriptional features and transcript structure of UL145 in different strains of human cytomegalovirus.

The human cytomegalovirus UL145 gene is located between the highly variable genes UL144 and UL146 in the UL/b' region. However, unlike its neighboring genes, UL145 is relatively conserved among strains isolated from patients. The transcriptional features and transcript structure of UL145 has not yet been examined. In this study, the transcriptional features and structure of UL145 were characterized using RNA preparations from three HCMV strains isolated from patients, designated H, C, and X, respectively. Two transcripts were identified by cDNA library screening. Two main clusters of transcripts, one located between 500 and 700 nt, and the other between 1,400 and 1,700 nt, were confirmed by Northern blot analysis. Abundant transcripts were detected at 96 h post-infection by Northern blot, suggesting that UL145 was a late gene. The terminal sequences of transcripts obtained by 3' rapid amplification of cDNA ends demonstrated the presence of polyadenylation. Transcripts identified in the RNA of H, C, and X strains 96 h post-infection had the same 3' end but different 5' ends. In addition to polycistronic transcripts with upstream genes, UL145 could be transcribed as a single transcript during late strain infections.

Human cytomegalovirus RL13 gene transcripts in a clinical strain.

Human cytomegalovirus (HCMV) RL13 gene is a member of the RL11 gene family. To study the expression kinetics and transcript structures of the gene, screening of cDNA library, Northern blot, 3' and 5' RACE analyses were performed with a low-passaged clinical strain. The results showed that the RL13 gene was mainly transcribed in the late expression phase with at least seven forms of transcripts of 3628, 3114, 2515-2443, 1737, 1240, 1029 and 846 nt, respectively. All these transcripts were unspliced with an identical 3' terminal and the same typical polyA signal "AATAAA". Except for the transcript of 846 nt, RL13 open reading frames (ORFs) were 909 bp and completely identical in all of the transcripts. The sequence of the RL13 ORF in the examined clinical strain had a higher similarity with that of the UL153 ORF in the Towne strain than those of RL13 ORF in any other HCMV strains.

Characterization of the transcripts of human cytomegalovirus UL144.

BACKGROUND: The genome of human cytomegalovirus (HCMV) has been studied extensively, particularly in the UL/b' region. In this study, transcripts of one of the UL/b' genes, UL144, were identified in 3 HCMV isolates obtained from urine samples of congenitally infected infants. METHODS: Northern blot hybridization, cDNA library screening, and RACE-PCR were used. RESULTS: We identified at least 4 differentially regulated 3'-coterminal transcripts of UL144 in infected cells of 1,300, 1,600, 1,700, and 3,500 nucleotides (nt). The 1600 nt transcript was the major form of UL144 mRNA. The largest transcript initiated from the region within the UL141 open reading frame (ORF) and included UL141, UL142, UL143, UL144, and UL145 ORFs. CONCLUSIONS: These findings reveal the complex nature of the transcription of the UL144 gene in clinical isolates.

Characterization of 3' termini of human cytomegalovirus UL138-UL145 transcripts in a clinical strain.

The functions of some proteins encoded by human cytomegalovirus (HCMV) UL/b' genes have been studied; however, systematic analysis of the transcripts for this region is still insufficient. The results of both rapid amplification of cDNA ends (RACE) and cDNA library screening in this study proved that 3' termini of all transcripts in the UL138-UL145 region were located approximately 20 bp downstream from each potential poly (A) signal, which were at the positions of nucleotides 7184, 9954 and 12848 in the UL/b' sequence of the H strain, respectively. Thus, there were at least two large families of polycistronic transcripts in this gene region. The first family of 3'-coterminal transcripts contained UL139, UL140 and UL141 genes, and the second one consisted of UL142, UL143, UL144 and UL145 genes. The 3'-coterminal characterization further confirmed that multiple uses of polyadenylation signals were commonly used by HCMV to utilize genetic information.

Transcription pattern of UL131A-128 mRNA in clinical strains of human cytomegalovirus.

Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by sequencing. Meanwhile, clones containing UL131A-128 transcripts in an HCMV cDNA library of a clinical strain were selected and sequenced. It was demonstrated that UL131A-128 mRNA was expressed with immediately early, early and late kinetics. Sequences obtained by RT-PCR showed that the UL131A gene consisted of two exons and the coding region of the UL130 gene was not interrupted by any intron in the region as reported earlier. However, the transcript of the UL128 gene showed two patterns: one pattern consisted of three exons as reported earlier; the other contained the three exons and also the first intron. Moreover, the above characteristics of UL131A-128 spliced transcripts were confirmed by the sequences of clones selected from the HCMV cDNA library. Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with the 3'-coterminal, although the initiation points of their mRNA may be different. The variation in the transcripts found in our study indicated the complex nature of transcription of UL131A-128 genes in clinical strains of HCMV.