Three exonic variants in the PHEX gene cause aberrant splicing in a minigene assay.

Background: X-linked hypophosphatemia (XLH, OMIM 307800) is a rare phosphorus metabolism disorder caused by PHEX gene variants. Many variants simply classified as missense or nonsense variants were only analyzed at the DNA level. However, growing evidence indicates that some of these variants may alter pre-mRNA splicing, causing diseases. Therefore, this study aimed to use bioinformatics tools and a minigene assay to ascertain the effects of PHEX variations on pre-mRNA splicing. Methods: We analyzed 174 variants in the PHEX gene described as missense or nonsense variants. Finally, we selected eight candidate variants using bioinformatics tools to evaluate their effects on pre-mRNA splicing using a minigene assay system. The complementary DNA (cDNA) sequence for the PHEX gene (RefSeq NM_000444.6) serves as the basis for DNA variant numbering. Results: Of the eight candidate variants, three were found to cause abnormal splicing. Variants c.617T>G p.(Leu206Trp) and c.621T>A p.(Tyr207*) in exon 5 altered the splicing of pre-mRNA, owing to the activation of a cryptic splice site in exon 5, which produced an aberrant transcript lacking a part of exon 5, whereas variant c.1700G>C p.(Arg567Pro) in exon 16 led to the activation of a cryptic splice site in intron 16, resulting in a partial inclusion of intron 16. Conclusion: Our study employed a minigene system, which has a great degree of flexibility to assess abnormal splicing patterns under the circumstances of patient mRNA samples that are not available, to explore the impact of the exonic variants on pre-mRNA splicing. Based on the aforementioned experimental findings, we demonstrated the importance of analyzing exonic variants at the mRNA level.

Complex phenotype in Fanconi renotubular syndrome type 1: Hypophosphatemic rickets as the predominant presentation.

GATM-related Fanconi renotubular syndrome 1 (FRTS1) is a form of renal Fanconi syndrome (RFS), which is a disorder of solute and water reabsorption caused by defects in the function of the entire proximal tubule. Recent findings reveal the molecular basis of FRTS1: Intramitochondrial fiber aggregation triggered by mutant GATM provides a starting point for proximal tubule damage and drives disease progression. As a rare and newly recognized inherited kidney disease, the complex manifestations of FRTS1 are easily underdiagnosed or misdiagnosed. We discuss the complex phenotype of a 26-year-old woman with onset in infancy and a long history of hypophosphatemic rickets. We also identified a novel heterozygous missense variant in the GATM gene in this patient. The novel variant and phenotype we report expand the disease spectrum of FRTS1. We recommend screening for GATM in children with RFS, especially in patients with resistant rickets who have previously had negative genetic testing. In addition, we found pathological deposition of mutant GATM proteins within mitochondria in the patient's urinary sediment cells by a combination of electron microscopy and immunofluorescence. This unique urine cytology experiment has the potential to be a valuable tool for identifying patients with RRTS1.

Identification of seven variants in the col4a1 gene that alter RNA splicing by minigene assay.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

A novel heterozygous variant of the SALL1 gene with atypical Townes-Brocks syndrome phenotypes in Chinese family.

Townes-Brocks syndrome (TBS) is an autosomal dominant disorder characterised by the triad of anorectal, thumb, and ear malformations. It may also be accompanied by defects in kidney, heart, eyes, hearing, and feet. TBS has been demonstrated to result from heterozygous variants in the SALL1 gene, which encodes zinc finger protein believed to function as a transcriptional repressor. The clinical characteristics of an atypical TBS phenotype patient from a Chinese family are described, with predominant manifestations including external ear dysplasia, unilateral renal hypoplasia with mild renal dysfunction, and hearing impairment. A novel heterozygous variant c.3060T>A (p.Tyr1020*) in exon 2 of the SALL1 gene was identified in this proband. Pyrosequencing of the complementary DNA of the proband revealed that the variant transcript accounted for 48% of the total transcripts in peripheral leukocytes, indicating that this variant transcript has not undergone nonsense-mediated mRNA decay. This variant c.3060T > A is located at the terminal end of exon 2, proximal to the 3' end of the SALL1 gene, and exerts a relatively minor impact on protein function. We suggest that the atypical TBS phenotype observed in the proband may be attributed to the truncated protein retaining partial SALL1 function.

Three exonic variants in the COL4A5 gene alter RNA splicing in a minigene assay.

BACKGROUND: X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. METHODS: We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. RESULTS: Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. CONCLUSION: Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.

Functional analysis of the CTNS gene exonic variants predicted to affect splicing.

Cystinosis is a severe, monogenic systemic disease caused by variants in CTNS gene. Currently, there is growing evidence that exonic variants in many diseases can affect pre-mRNA splicing. The impact of CTNS gene exonic variants on splicing regulation may be underestimated due to the lack of routine studies at the RNA level. Here, we analyzed 59 exonic variants in the CTNS gene using bioinformatics tools and identified candidate variants that may induce splicing alterations by minigene assays. We identified six exonic variants that induce splicing alterations by disrupting the ratio of exonic splicing enhancers/exonic splicing silencers (ESEs/ESSs) or by interfering with the recognition of classical splice sites, or both. Our results help in the correct molecular characterization of variants in cystinosis and inform emerging therapies. Furthermore, our work suggests that the combination of in silico and in vitro assays facilitates to assess the effects of DNA variants driving rare genetic diseases on splicing regulation and will enhance the clinical utility of variant functional annotation.

Identified eleven exon variants in PKD1 and PKD2 genes that altered RNA splicing by minigene assay.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic multisystem disease caused primarily by mutations in the PKD1 gene or PKD2 gene. There is increasing evidence that some of these variants, which are described as missense, synonymous or nonsense mutations in the literature or databases, may be deleterious by affecting the pre-mRNA splicing process. RESULTS: This study aimed to determine the effect of these PKD1 and PKD2 variants on exon splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 19 candidate single nucleotide alterations, 11 variants distributed in PKD1 (c.7866C > A, c.7960A > G, c.7979A > T, c.7987C > T, c.11248C > G, c.11251C > T, c.11257C > G, c.11257C > T, c.11346C > T, and c.11393C > G) and PKD2 (c.1480G > T) were identified to result in exon skipping. CONCLUSIONS: We confirmed that 11 variants in the gene of PKD1 and PKD2 affect normal splicing by interfering the recognition of classical splicing sites or by disrupting exon splicing enhancers and generating exon splicing silencers. This is the most comprehensive study to date on pre-mRNA splicing of exonic variants in ADPKD-associated disease-causing genes in consideration of the increasing number of identified variants in PKD1 and PKD2 gene in recent years. These results emphasize the significance of assessing the effect of exon single nucleotide variants in ADPKD at the mRNA level.

Four novel variants identified in primary hyperoxaluria and genotypic and phenotypic analysis in 21 Chinese patients.

Background: Primary hyperoxaluria (PH) is a rare genetic disorder characterized by excessive accumulation of oxalate in plasma and urine, resulting in various phenotypes due to allelic and clinical heterogeneity. This study aimed to analyze the genotype of 21 Chinese patients with primary hyperoxaluria (PH) and explore their correlations between genotype and phenotype. Methods: Combined with clinical phenotypic and genetic analysis, we identified 21 PH patients from highly suspected Chinese patients. The clinical, biochemical, and genetic data of the 21 patients were subsequently reviewed. Results: We reported 21 cases of PH in China, including 12 cases of PH1, 3 cases of PH2 and 6 cases of PH3, and identified 2 novel variants (c.632T > G and c.823_824del) in AGXT gene and 2 novel variants (c.258_272del and c.866-34_866-8del) in GRHPR gene, respectively. A possible PH3 hotspot variant c.769T > G was identified for the first time. In addition, patients with PH1 showed higher levels of creatinine and lower eGFR than those with PH2 and PH3. In PH1, patients with severe variants in both alleles had significantly higher creatinine and lower eGFR than other patients. Delayed diagnosis still existed in some late-onset patients. Of all cases, 6 had reached to end-stage kidney disease (ESKD) at diagnosis with systemic oxalosis. Five patients were on dialysis and three had undergone kidney or liver transplants. Notably, four patients showed a favorable therapeutic response to vitamin B6, and c.823_824dup and c.145A > C may be identified as potentially vitamin B6-sensitive genotypes. Conclusion: In brief, our study identified 4 novel variants and extended the variant spectrum of PH in the Chinese population. The clinical phenotype was characterized by large heterogeneity, which may be determined by genotype and a variety of other factors. We first reported two variants that may be sensitive to vitamin B6 therapy in Chinese population, providing valuable references for clinical treatment. In addition, early screening and prognosis of PH should be given more attention. We propose to establish a large-scale registration system for rare genetic diseases in China and call for more attention on rare kidney genetic diseases.

Minigene splicing assays reveal new insights into exonic variants of the SLC12A3 gene in Gitelman syndrome.

BACKGROUND: Gitelman syndrome (GS) is a type of salt-losing tubular disease, most of which is caused by SLC12A3 gene variants, and missense variants account for the majority. Recently, the phenomenon of exon skipping, in which variants disrupt normal pre-mRNA splicing, has been related to a variety of diseases. Therefore, we hypothesize that a certain proportion of SLC12A3 variants can result in disease via interfering with the normal splicing process. METHODS: We analyzed 342 previously presumed SLC12A3 missense variants using bioinformatics programs and identified candidate variants that may alter the splicing of pre-mRNA through minigene assays. RESULTS: Our study revealed that, among ten candidate variants, six variants (c.602G>A, c.602G>T, c.1667C>T, c.1925G>A, c.2548G>C, and c.2549G>C) led to complete or incomplete exon skipping by affecting exonic splicing regulatory elements and/or disturbing canonical splice sites. CONCLUSION: It is worth mentioning that this is the largest study on pre-mRNA splicing of SLC12A3 exonic variants. In addition, our study emphasizes the importance of detecting splicing function at the mRNA level in GS and indicates that minigene analysis is a valuable tool for splicing functional assays of variants in vitro.

Systemic immune-inflammation index is associated with diabetic kidney disease in Type 2 diabetes mellitus patients: Evidence from NHANES 2011-2018.

Objective: Diabetic kidney disease (DKD) is the most common chronic kidney disease (CKD) and has the highest prevalence of end-stage kidney disease (ESKD) globally, owing mostly to the rise in Type 2 diabetes mellitus (T2DM) correlated with obesity. Current research suggested that the immune response and inflammation may play a role in the pathophysiology of T2DM. The systemic immune-inflammation index (SII) is a novel and integrated inflammatory biomarker that has not yet been linked to DKD. We aimed to identify the potential relationship between SII and DKD. Methods: In the National Health and Nutrition Examination Survey (NHANES) between 2011 and 2018, the current cross-sectional study was conducted among adults with T2DM. SII was calculated as the platelet count × neutrophil count/lymphocyte count. DKD was diagnosed with impaired glomerular filtration rate (< 60 mL/min/1.73 m2 assessed by using the Chronic Kidney Disease Epidemiology Collaboration algorithm), albuminuria (urine albumin to creatinine ratio ≥ 30 mg/g), or both in T2DM patients. To investigate the independent association between SII and DKD, weighted univariate and multivariable logistic regression analyses and subgroup analyses were performed. Results: The study involved 3937 patients in total, of whom 1510 (38.4%) had DKD for the diagnosis. After adjustment for covariates, multivariable logistic regression revealed that a high SII level was associated with increased likelihood of DKD (OR = 1.42, 95% CI: 1.10-1.83, P = 0.01). Subgroup analyses and interaction tests revealed that age, gender, estimated glomerular filtration rate (eGFR), urine albumin-to-creatinine ratio (ACR), body mass index (BMI), hypertension, hyperlipidemia, anti-inflammation therapy (yes or no), metformin use (yes or no), and insulin use (yes or no) had no significant dependence on this positive relationship (all p for interaction >0.05). Conclusions: Our results indicate that the higher SII level is associated with DKD in T2DM patients. The SII could be a cost-effective and straightforward approach to detecting DKD. This needs to be verified in further prospective investigations.

A novel WDR60 variant contributes to a late diagnosis of Jeune asphyxiating thoracic dystrophy in a Chinese patient: A case report.

We report a Chinese patient with JATD presenting a mild skeletal phenotype and with renal insufficiency as the initial symptom of the disease. A novel homozygous c.2789C>T (p.S930L) variant in the WDR60 gene was identified. Our report will help to improve awareness and diagnosability for this disease.

Twelve exonic variants in the SLC12A1 and CLCNKB genes alter RNA splicing in a minigene assay.

Background: Bartter syndrome (BS) is a rare renal tubular disease caused by gene variants in SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND or MAGED2 genes. There is growing evidence that many exonic mutations can affect the pre-mRNA normal splicing and induce exon skipping by altering various splicing regulatory signals. Therefore, the aim of this study was to gain new insights into the consequences of exonic mutations associated with BS on pre-mRNA splicing. Methods: We analyzed all the missense, nonsense and synonymous variants described in six pathogenic genes by bioinformatics programs and identified candidate mutations that may promote exon skipping through a minigene system. Results: Results of the study showed that 12 of 14 candidate variants distributed in SLC12A1 (c.728G>A, C.735C>G, c.904C>T, c.905G>A, c.1304C>T, c.1493C>T, c.2221A>T) and CLCNKB (c.226C>T, c.228A>C, c.229G>A, c.229G>C, c.1979C>A) were identified to induce splicing alterations. These variants may not only disrupt exonic splicing enhancers (ESEs) but also generate new exonic splicing silencers (ESSs), or disturb the classic splicing sites. Conclusion: To our knowledge, this is a comprehensive study regarding alterations in pre-mRNA of exonic variants in BS pathogenic genes. Our results reinforce the necessity of assessing the consequences of exonic variants at the mRNA level.

Particulate matter of air pollution may increase risk of kidney failure in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by deposition of galactose-deficient IgA1 (Gd-IgA1) in glomerular mesangium associated with mucosal immune disorders. Since environmental pollution has been associated with the progression of chronic kidney disease in the general population, we specifically investigated the influence of exposure to fine particulate matter less than 2.5 µm in diameter (PM2.5) on IgAN progression. Patients with biopsy-proven primary IgAN were recruited from seven Chinese kidney centers. PM2.5 exposure from 1998 to 2016 was derived from satellite aerosol optical depth data and a total of 1,979 patients with IgAN, including 994 males were enrolled. The PM2.5 exposure levels for patients from different provinces varied but, in general, the PM2.5 exposure levels among patients from the north were higher than those among patients from the south. The severity of PM2.5 exposure in different regions was correlated with regional kidney failure burden. In addition, each 10 µg/m3 increase in annual average concentration of PM2.5 exposure before study entry (Hazard Ratio, 1.14; 95% confidence interval, 1.06-1.22) or time-varying PM2.5 exposure after study entry (1.10; 1.01-1.18) were associated with increased kidney failure risk after adjustment for age, gender, estimated glomerular filtration rate, urine protein, uric acid, hemoglobin, mean arterial pressure, Oxford classification, glucocorticoid and renin-angiotensin system blocker therapy. The associations were robust when the time period, risk factors of cardiovascular diseases or city size were further adjusted on the basis of the above model. Thus, our results suggest that PM2.5 is an independent risk factor for kidney failure in patients with IgAN, but these findings will require validation in more diverse populations and other geographic regions.

Identification of two novel variants of BCS1L gene in a patient with classical GRACILE syndrome.

BCS1L pathogenic variants cause widely different clinical phenotypes. Disease phenotypes can be as mild as Björnstad syndrome, characterized by pili torti (abnormal flat twisted hair shafts) and sensorineural hearing loss, or as severe as GRACILE syndrome, characterized by growth restriction, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death. BCS1L pathogenic variants are also linked to an undefined complex III deficiency, a heterogeneous condition generally involving renal and hepatic pathologies, hypotonia, and developmental delays. So far, all patients with GRACILE syndrome carry a homozygous p.Ser78Gly variant in BCS1L gene by reviewing articles. A 24-day-old boy presented with typical clinical phenotype of GRACILE syndrome. The Whole Exome Sequencing confirmed that the patient had a missense variant (c.245C > T, p.Ser82Leu) and a small deletion (c.231_232delCA, p. Ser78Cysfs*9) in BCS1L gene inherited from his father and mother separately, he died at 5 months of age. We reported a patient with GRACILE syndrome and identified two novel variants in BCS1L gene. Our study expands the mutational spectrum of BCS1L gene associated with GRACILE syndrome and will be beneficial for genetic diagnosis.

Two novel variants in a Bardet-Biedl syndrome type 5 patient with severe renal phenotype.

Bardet-Biedl syndrome type 5 (BBS5) has never been reported in Chinese populations. The aim of this study is to report the first BBS5 case in China, explore the phenotype and genotype correlation. The case was male, Han nationality, born with polydactyly and gained weight after birth, accompanied by polydipsia, polyuria and nocturia. He was found to have low vision at the age of 7 years, and having insufficient renal function at the age of 20 years. After hospitalization, he was found to have suffered from atrophy of the whole layer of macular retina, and end stage of kidney disease, presenting with shrinking and cyst-like changes of bilateral kidneys. Whole-exome sequencing was performed among the proband and his parents (Trios), further validated using Sanger sequencing and quantitative polymerase chain reaction. Two novel compound heterozygous variants of BBS5 gene [a missense variant NC_000002.12, NM_152384.3:c.1A>G(p.Met1?) & a large deletion c.(?_-60)_(386 + 1_387-1)del] were detected. BBS is rare, whereas BBS5 is rarer. Herein, we reported a Chinese BBS5 patient with severe renal phenotype and identified two novel BBS5 variants.

Double synonymous mutations in exon 9 of the Cullin3 gene restore exon inclusion by abolishing hnRNPs inhibition.

All mutations in exon 9 of the Cullin3 gene associated with pseudohypoaldosteronism type II (PHA II) contribute to exon skipping to different degrees, but the specific molecular mechanism of this aberrant splicing is still unclear. The aims of this study were to investigate the regulatory mechanism underlying two synonymous splicing events, c.1221A > G (p. Glu407Glu) and c.1236G > A (p. Leu412Leu), and to discover a therapeutic strategy for correcting this aberrant splicing by targeting potential regulatory sites. Through a series of RNA pulldown, silver staining, western blotting, small interfering RNA knockdown, in vitro overexpression and single or double site-directed mutagenesis experiments, we first explored the pathogenesis of exon 9 skipping caused by mutations in the CUL3 gene and verified that the main splicing regulators associated with the synonymous c.1221A > G and c.1236G > A mutations were heterogeneous nuclear ribonucleoproteins. In addition, we verified that introducing another synonymous mutation, c.1224A > G (A18G), significantly rescued the abnormal splicing caused by c.1221A > G and c.1236G > A, highlighting the therapeutic potential for the treatment of PHA II.

Role of abnormal energy metabolism in the progression of chronic kidney disease and drug intervention.

Chronic kidney disease (CKD) is a severe clinical syndrome with significant socioeconomic impact worldwide. Orderly energy metabolism is essential for normal kidney function and energy metabolism disorders are increasingly recognized as an important player in CKD. Energy metabolism disorders are characterized by ATP deficits and reactive oxygen species increase. Oxygen and mitochondria are essential for ATP production, hypoxia and mitochondrial dysfunction both affect the energy production process. Renin-angiotensin and adenine signaling pathway also play important regulatory roles in energy metabolism. In addition, disturbance of energy metabolism is a key factor in the development of hereditary nephropathy such as autosomal dominant polycystic kidney disease. Currently, drugs with clinically clear renal function protection, such as Angiotensin II Type 1 receptor blockers and fenofibrate, have been proven to improve energy metabolism disorders. The sodium-glucose co-transporter inhibitors 2 that can mediate glucose metabolism disorders not only delay the progress of diabetic nephropathy, but also have significant protective effects in non-diabetic nephropathy. Hypoxia-inducible factor enhances ATP production to the kidney by improving renal oxygen supply and increasing glycolysis, and the mitochondria targeted peptides (SS-31) plays a protective role by stabilizing the mitochondrial inner membrane. Moreover, several drugs are being studied and are predicted to have potential renal protective properties. We propose that the regulation of energy metabolism represents a promising strategy to delay the progression of CKD.