Alport syndrome (AS), a hereditary kidney disease with a high risk for renal failure, is attributed to pathogenic variants in genes COL4A3, COL4A4, and COL4A5 that encode type IV collagen. Next-generation sequencing (NGS) is increasingly applied to the diagnosis of AS, but complex genotype-phenotype correlation, that is, identifying the significance of variants, is still a huge clinical challenge. In this study, we reported the case of a 27-year-old Chinese woman with a family history of hematuria and proteinuria. Notably, the proband is the only one in her family with renal insufficiency. NGS was performed in this family, and it was revealed that the proband was a compound heterozygote for two variants in the COL4A3 gene: c.2990G>A inherited from her father and c.4981C>T inherited from her mother. We modeled the spatial structure of the corresponding protein and assumed that structural abnormalities led to the breakdown of type IV collagen networks, a major component of the glomerular basement membrane. Thus, the proband was diagnosed with autosomal recessive AS, characterized by severe defects of the glomerular basement membrane. Hence, the proband showed a loss of renal function. This case presentation emphasizes the importance of NGS for AS diagnosis and introduces a novel genotype of AS.

BACKGROUND: Cancer stem cells (CSCs) play a vital role in the occurrence, maintenance, and recurrence of solid tumors. Although, miR-145-5p can inhibit CSCs survival, poor understanding of the underlying mechanisms hamperes further therapeutic optimization for patients. Lentivirus with remarkable transduction efficiency is the most commonly used RNA carrier in research, but has shown limited tumor-targeting capability.
METHODS: We have applied liposome to decorate lentivirus surface thereby yielding liposome-lentivirus hybrid-based carriers, termed miR-145-5p-lentivirus nanoliposome (MRL145), and systematically analyzed their potential therapeutic effects on liver CSCs (LCSCs).
RESULTS: MRL145 exhibited high delivery efficiency and potent anti-tumor efficacy under in vitro and in vivo. Mechanistically, the overexpressed miR-145-5p can significantly suppress the self-renewal, migration, and invasion abilities of LCSCs by targeting Collagen Type IV Alpha 3 Chain (COL4A3). Importantly, COL4A3 can promote phosphorylating GSK-3β at ser 9 (p-GSK-3β S9) to inactivate GSK3β, and facilitate translocation of β-catenin into the nucleus to activate the Wnt/β-catenin pathway, thereby promoting self-renewal, migration, and invasion of LCSCs. Interestingly, COL4A3 could attenuate the cellular autophagy through modulating GSK3β/Gli3/VMP1 axis to promote self-renewal, migration, and invasion of LCSCs.
CONCLUSIONS: These findings provide new insights in mode of action of miR-145-5p in LCSCs therapy and indicates that liposome-virus hybrid carriers hold great promise in miRNA delivery.

UNASSIGNED: Genetic diagnosis of Alport syndrome (AS), which results from pathogenic variants in COL4A3, COL4A4, or COL4A5 genes, is hindered by large numbers of unclassified variants detected using next-generation sequencing (NGS). We examined the impact on splicing of variants of uncertain significance in COL4A3 to COL4A5.
UNASSIGNED: Nine unrelated patients with clinical diagnosis or suspicion of AS were enrolled according to the criteria. Their clinical and genetic data were collected. Blood and urine samples were obtained from the patients and their family members. Sanger sequencing was used to confirm the 9 COL4A3 to COL4A5 unclassified variants identified by NGS. COL4A3 to COL4A5 mRNAs from urine were analyzed using targeted reverse transcription polymerase chain reaction and direct sequencing.
UNASSIGNED: Nine COL4A3 to COL4A5 unclassified variants were found to alter mRNAs splicing. Skipping of an exon or an exon fragment was induced by variants COL4A3 c.828+5G>A; COL4A4 c.3506-13_3528del; and COL4A5 c.451A>G (p. [Ile151Val]), c.2042-9 T>G, c.2689 G>C (p. [Glu897Gln]) and c.1033-10_1033-2delGGTAATAAA. Retention of an intron fragment was caused by variants COL4A3 c.3211-30G＞T, and COL4A5 c.4316-20T>A and c.1033-10 G>A, respectively. The 9 families in this study obtained genetic diagnosis of AS, including 3 with autosomal recessive AS and 6 with X-linked AS.
UNASSIGNED: Our findings demonstrate that urine mRNA analysis facilitates the identification of abnormal splicing of unclassified variants in Alport genes, which provides evidence of routine use of RNA analysis to improve genetic diagnosis of AS.

Alport syndrome (AS) is a genetic kidney disease of basement membrane collagen disorder accounting for approximately 2% of ESRD patients. Next-generation and whole-exome sequencing methods are increasingly frequently used as an efficient tool not only for the diagnosis of AS but also for the establishment of genotype-phenotype correlation. We herein report the identification of a novel heterozygous missense mutation in COL4A3 gene (c.G3566A: p.G1189E) causing variable phenotypes in an ADAS Family based on the combination of clinical, histologic, pedigree, and genetic sequencing information. The proband is a 48-year-old Chinese woman suffering from persistent subnephrotic proteinuria and intermittent hematuria without renal function impairment over a 10-year time-span. Renal biopsy showed diffuse thin basement membrane and focal interstitial foam cell infiltration. The proband\'s mother progressed to end-stage renal failure and the proband\'s sister presented with subnephrotic proteinuria and intermittent hematuria as well. AS was highly suspected and confirmed by exome sequencing which revealed a novel heterozygous missense mutation in COL4A3 gene (c.G3566A: p.G1189E) in all the affected family members, although their current medical conditions vary significantly. Our present finding emphasizes the significance of next-generation sequencing technology for genetic screening which gives us an accurate clinical diagnosis of ADAS patients. The identification of c.G3566A as a new ADAS-related mutation contributes to both genetic diagnosis of ADAS and further functional study of COL4A3. The variable phenotypes from the same genotype of our case also provide more information to genotype-phenotype correlation study.

UNASSIGNED: The incorrect interpretation of missense and synonymous variants can lead to improper molecular diagnosis and subsequent faulty genetic counselling. The aim of this study was to evaluate the pathogenicity of presumed COL4A3/COL4A4 missense and synonymous variants detected by next-generation sequencing to provide evidence for diagnosis and genetic counselling.
UNASSIGNED: Patients\' clinical findings and genetic data were analysed retrospectively. An in vitro minigene assay was conducted to assess the effect of presumed COL4A3/COL4A4 missense and synonymous variants on RNA splicing.
UNASSIGNED: Five unclassified COL4A3/COL4A4 variants, which were detected in five of 343 patients with hereditary kidney diseases, were analysed. All of them were predicted to affect splicing by Human Splicing Finder. The presumed COL4A3 missense variant c.4793T > G [p. (Leu1598Arg)] resulted in a loss of alternative full-length transcript during the splicing process. The COL4A3 transcript carried synonymous variant c.765G > A [p. (Thr255Thr)], led to an in-frame deletion of exon 13. Nevertheless, variants c.3566G > A [p. (Gly1189Glu)] in COL4A3 and c.3990G > A [p. (Pro1330Pro)], c.4766C > T [p. (Pro1589Leu)] in COL4A4 exhibited no deleterious effect on splicing. Among the five patients harbouring the abovementioned COL4A3/COL4A4 variants, three patients were genetically diagnosed with autosomal recessive Alport syndrome, one patient was highly suspected of having thin basement membrane nephropathy, and the other patient was clinically diagnosed with Alport syndrome.
UNASSIGNED: COL4A3 presumed missense variant p. (Leu1598Arg) and synonymous variant p. (Thr255Thr) affect RNA splicing, which highlights the prime importance of transcript analysis of unclassified exonic sequence variants for better molecular diagnosis and genetic counselling. Meanwhile, the reliability of splicing predictions by predictive tools for exonic substitutions needs to be improved.

BACKGROUND: Alport syndrome (ATS) is a rare hereditary disease caused by mutations in genes such as COL4A3, COL4A4, and COL4A5. ATS involves a spectrum of phenotypes ranging from isolated hematuria that is nonprogressive to progressive renal disease with extrarenal abnormalities. Although ATS can be combined with other diseases or syndromes, ATS combined with lupus nephritis has not been reported before.
METHODS: A Chinese family with ATS was recruited for the current study. Clinical characteristics (including findings from renal biopsy) of ATS patients were collected from medical records, and potential causative genes were explored by whole-exome sequencing. A heterozygous substitution in intron 22 of COL4A3 (NM_000091 c.2657-1G>A) was found in the patients, which was further confirmed by quantitative polymerase chain reaction.
CONCLUSIONS: Heterozygous substitution of a COL4A3 gene splice site was identified by whole-exome sequencing, revealing the molecular pathogenic basis of this disorder. In general, identification of pathogenic genes can help to fully understand the molecular mechanism of disease and facilitate precise treatment.

Upward (local growth and invasion of the base of skull), downward (distant metastasis) and mixed progressing types of nasopharyngeal carcinoma (NPC) have been identified and are distinctly different with respect to clinical symptoms, therapeutic strategies and prognosis. The present study aimed to identify the genetic difference and collagen expression levels in the upward and downward progressing types of NPC. Whole exon sequencing (WES) was used to detect genes differentially mutated between the upward and downward progressing types of NPC. Collagen deposition in the upward and downward progressing types of NPC was determined using Masson trichromatic staining, while the protein expression level of COL4A3 was detected using immunohistochemistry. Survival analysis was also performed using the Kaplan-Meier Plotter database to examine the role of COL4A3 expression level in the prognosis of head and neck squamous cell carcinoma. Knockdown of COL4A3 was performed using short interfering (si)RNA-COL4A3 in a 5-8F NPC cell line. Reverse transcription-quantitative PCR and western blot analyses were utilized to analyze the mRNA and protein expression levels of COL4A3, respectively. The roles of COL4A3 in the migration and invasion of the 5-8F cell line were examined using wound-healing Transwell and Matrigel assays, respectively. A total of 21 genes were differentially mutated between the upward and downward progressing types of NPC. The COL4A3 was investigated further, as it was found to be associated with extracellular matrix deposition and cancer metastasis. The COL4A3 gene was markedly downregulated in the downward progressing type compared with that in the upward progressing type (2.161±1.306 vs. 5.077±3.619; P<0.05). In addition, the deposition of collagen in the downward progressing type was also significantly decreased compared with that in the upward progressing type (5.63±6.83 vs. 10.94±9.60; P<0.05). Kaplan-Meier analysis indicated that high expression level of COL4A3 was positively associated with a favorable prognosis of head and neck squamous cell carcinoma (HR, 0.69; 95% CI, 0.49- 0.97; P=0.031). To confirm the role of COL4A3, the expression level of COL4A3 was knocked down using siRNA in the 5-8F cell line and the results showed that the invasion and migration was significantly increased when the expression of COL4A3 was inhibited (P<0.0001). In conclusion, the gene mutation patterns were significantly different between the upward and downward progressing types of NPC. In addition, the expression level of the COL4A3 gene was decreased in the downward progressing type, which might promote NPC metastasis through the downregulation of extracellular collagen expression.

High-altitude polycythemia (HAPC) is a chronic high-altitude disease that can lead to an increase in the production of red blood cells in the people who live in the plateau, a hypoxia environment, for a long time. The most frequent symptoms of HAPC include headache, dizziness, breathlessness, sleep disorders, and dilation of veins. Although chronic hypoxia is the main cause of HAPC, the fundamental pathophysiologic process and related molecular mechanisms responsible for its development remain largely unclear yet.
This study aimed to explore the related hereditary factors of HAPC in the Chinese Han and Tibetan populations. A total of 140 patients (70 Han and 70 Tibetan) with HAPC and 60 healthy control subjects (30 Han and 30 Tibetan) were recruited for a case-control association study. To explore the genetic basis of HAPC, we investigated the association between HAPC and both phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit delta gene (PIK3CD) and collagen type IV α3 chain gene (COL4A3) in Chinese Han and Tibetan populations.
Using the unconditional logistic regression analysis and the false discovery rate (FDR) calculation, we found that eight SNPs in PIK3CD and one SNP in COL4A3 were associated with HAPC in the Tibetan population. However, in the Han population, we did not find any significant association. Our study suggested that polymorphisms in the PIK3CD and COL4A3 were correlated with susceptibility to HAPC in the Tibetan population.

Focal segmental glomerulosclerosis (FSGS) is a histologically identifiable glomerular injury often leading to proteinuria and renal failure. To identify its causal genes, whole-exome sequencing and Sanger sequencing were performed on a large Chinese cohort that comprised 40 FSGS families, 50 sporadic FSGS patients, 9 independent autosomal recessive Alport\'s syndrome (ARAS) patients, and 190 ethnically matched healthy controls. Patients with extrarenal manifestations, indicating systemic diseases or other known hereditary renal diseases, were excluded. Heterozygous COL4A3 mutations were identified in five (12.5%) FSGS families and one (2%) sporadic FSGS patient. All identified mutations disrupted highly conserved protein sequences and none of them was found in either public databases or the 190 healthy controls. Of the FSGS patients with heterozygous COL4A3 mutations, segmental thinning of the glomerular base membrane (GBM) was only detected in the patient with electronic microscopy examination results available. Five ARAS patients (55.6%) had homozygous or compound-heterozygous mutations in COL4A3 or COL4A4. Serious changes in the GBM, hearing loss, and ocular abnormalities were found in 100%, 80%, and 40% of the ARAS patients, respectively. Overall, a new subgroup of FSGS patients resulting from heterozygous COL4A3 mutations was identified. The mutations are relatively frequent in families diagnosed with inherited forms of FSGS. Thus, we suggest screening for COL4A3 mutations in familial FSGS patients.