Detection of Very Low-Level Somatic Mosaic COL4A5 Splicing Variant in Asymptomatic Female Using Droplet Digital PCR.

Background: Alport syndrome is a hereditary glomerulopathy featured by haematuria, proteinuria, and progressive renal failure. X-linked Alport syndrome (XLAS) due to COL4A5 disease-causing variants is the most common form. In the case of XLAS resulting from 10-18% presumed de novo COL4A5 disease-causing variants, there are only a few studies for mosaicism in the probands or parents. Very low-level (<1.0%) somatic mosaicism for COL4A5 disease-causing variants has not been published. Materials and Methods: Chinese XLAS families with suspected parental mosaicism were enrolled in the present study to evaluate the forms of mosaicism, to offer more appropriate genetic counseling. PCR and direct sequencing were used to detect COL4A5 disease-causing variants harbored by the affected probands in parental multi-tissue DNAs (peripheral blood, urine sediments, saliva, hair), and droplet digital PCR (ddPCR) was used to quantify the mutant COL4A5 allelic fractions in parental different samples such as peripheral blood, saliva, and urine sediments. Results: A Chinese asymptomatic female with suspected somatic and germline mosaicism was enrolled in the present study. She gave birth to two boys with XLAS caused by a hemizygous disease-causing variant c. 2245-1G>A in COL4A5 (NM_033380) intron 28, whereas this disease-causing variant was not detected in genomic DNA extracted from peripheral blood leukocytes in the woman using Sanger sequencing. She had multiple normal urine test results, and continuous linear immunofluorescence staining of α2 (IV) and α5 (IV) chains of skin tissue. Sanger sequencing demonstrated that COL4A5 disease-causing variant c. 2245-1G>A was not detected in her genomic DNAs isolated from urine sediments, saliva, and hair roots. Using ddPCR, the wild-type and mutant-type (c.2245-1G>A) COL4A5 was identified in the female's genomic DNAs isolated from peripheral blood, saliva, and urine sediments. The mutant allelic fractions in these tissues were 0.26% (peripheral blood), 0.73% (saliva), and 1.39% (urine), respectively. Conclusions: Germline and very low-level somatic mosaicism for a COL4A5 splicing variant was detected in an asymptomatic female, which highlights that parental mosaicism should be excluded when a COL4A5 presumed de novo disease-causing variant is detected.

Presumed COL4A3/COL4A4 Missense/Synonymous Variants Induce Aberrant Splicing.

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Interpretation of Autosomal Recessive Kidney Diseases With "Presumed Homozygous" Pathogenic Variants Should Consider Technical Pitfalls.

Background: A false interpretation of homozygosity for pathogenic variants causing autosomal recessive disorders can lead to improper genetic counseling. The aim of this study was to demonstrate the underlying etiologies of presumed homozygous disease-causing variants harbored in six unrelated children with five different genetic renal diseases when the same variant was identified in a heterozygous state in only one of the two parents from each family using direct sequencing. Methods: Peripheral blood genomic DNA samples were extracted. Six short tandem repeats were used to verify the biological relationships between the probands and their parents. Quantitative PCR was performed to detect mutant exons with deletions. Single nucleotide polymorphism analysis and genotyping with polymorphic microsatellite markers were performed to identify uniparental disomy (UPD). Results: Each proband and his/her parents had biological relationships. Patients 2, 4, and 6 were characterized by large deletions encompassing a missense/small deletion in DGKE, NPHP1, and NPHS1, respectively. Patients 1 and 5 were caused by segmental UPD in NPHS2 and SMARCAL1, respectively. In patient 6, maternal UPD, mosaicism in paternal sperm or de novo variant in NPHP1 could not be ruled out. Conclusions: When a variant analysis report shows that a patient of non-consanguineous parents has a pathogenic presumed homozygous variant, we should remember the need to assess real homozygosity for the variant, and a segregation analysis of the variants within the parental DNAs and comprehensive molecular tests to evaluate the potential molecular etiologies, such as a point variant and an overlapping exon deletion, UPD, germline mosaicism and de novo variant, are crucial.

Endoplasmic Reticulum Stress Activation in Alport Syndrome Varies Between Genotype and Cell Type.

Alport syndrome is a hereditary progressive chronic kidney disease caused by mutations in type IV collagen genes COL4A3/4/5. X-linked Alport syndrome (XLAS) is caused by mutations in the COL4A5 gene and is the most common form of Alport syndrome. A strong correlation between the type of COL4A5 mutation and the age developing end-stage renal disease in male patients has been found. Mutation to the α (IV) chain causes retention of the protein to the endoplasmic reticulum lumen, which causes endoplasmic reticulum stress (ERS) and subsequent exertion of deleterious intracellular effects through the activation of ERS. The exact time point that mutant type IV collagen α chain exerts its deleterious effects remains elusive. In this study, we explored the relationship between the COL4A5 genotype and cell type in ERS activation. We obtained skin fibroblasts from Alport syndrome patients with different COL4A5 mutation categories [i.e., a missense mutation (c.4298G > T, p.Gly1433Val) in exon 47, a splicing mutation (c.1949-1G > A) in intron 25 and an insertion (c.573_c.574insG, p. Pro193Alafs*23) in exon 10], and then reprogrammed these fibroblasts into induced pluripotent stem cells (iPSCs). Interestingly, no significant dysregulation of ERS pathway markers was observed for the three COL4A5 mutant iPSCs; however, significant activation of ERS in COL4A5 mutant fibroblasts was observed. In addition, we found that the activation levels of some ERS markers in fibroblasts varied among the three COL4A5 mutation types. Mutant COL4A5 proteins were demonstrated to have different effects on cells at different stages of ontogenesis, providing a theoretical basis for choosing the timing of intervention. The observed differences in activation of ERS by the COL4A5 mutant fibroblasts may contribute to the intracellular molecular mechanisms that describe the correlation between genotype and clinical features in XLAS.

Phenotypic spectrum and antialbuminuric response to angiotensin converting enzyme inhibitor and angiotensin receptor blocker therapy in pediatric Dent disease.

BACKGROUND: To characterize the phenotypic spectrum and assess the antialbuminuric response to angiotensin converting enzyme (ACE) inhibitor and/or angiotensin receptor blocker (ARB) therapy in a cohort of children with Dent disease. METHODS: The patients' clinical findings, renal biopsy results, genetic and follow-up data were analyzed retrospectively. Mutations in CLCN5 or OCRL were detected by next-generation sequencing or Sanger sequencing. RESULTS: Of 31 Dent disease boys, 24 carried CLCN5 and 7 carried OCRL mutations. Low molecular weight proteinuria and albuminuria were detected in all cases. Nephrotic-range proteinuria and severe albuminuria were identified in 52% and 62% of cases, respectively; by 7 years of age, 6 patients had hematuria and nephrotic-range proteinuria, and 7 patients had hematuria and moderate to severe albuminuria. In addition to disease-related renal features, patients with Dent-1 disease also presented with congenital cataract (1/9) and developmental delay (2/7). Seventeen of 31 patients underwent renal biopsy. Glomerular changes included mild glomerular lesions, mesangial proliferative glomerulonephritis and focal segmental glomerular sclerosis. Thirteen of the 31 patients had follow-up records and received ACE inhibitor and/or ARB treatment for more than 3 months. After a median 1.7 (range 0.3-8.5) years of treatment, a reduction in the urinary microalbumin-to-creatinine ratio was observed in 54% of children. CONCLUSIONS: Hematuria with nephrotic-range proteinuria or moderate to severe albuminuria was common in Dent disease patients. Extrarenal manifestations were observed in Dent-1 patients, which extends the phenotypic spectrum. In addition, ACE inhibitors and ARBs are well tolerated, and they are partially effective in controlling albuminuria.

Diverse phenotypes in children with PAX2-related disorder.

BACKGROUND: The aim of this study was to analyze the diverse phenotypes of children with PAX2-related disorder so as to improve our understanding of this disease. METHODS: The clinical data of ten children with PAX2 mutations, detected by targeted region capture sequencing or whole-exome sequencing, were retrospectively analyzed. Family members of index cases were verified by Sanger sequencing and family segregation analysis was performed. RESULTS: The age of first symptom of 10 unrelated children (six girls and four boys) was 6.4 (ranged from postnatal day to 14.8) years old. Proteinuria, abnormal renal function, and structure were found in all patients. Renal hypoplasia and renal cysts were found in 10 of 10 and five of 10 cases, respectively. Three patients progressed to chronic kidney disease stage 5 and the onset age of end-stage renal disease was 9.8-16.4 years old. PAX2-related ocular abnormalities were found in five of seven cases and three patients were observed to have more than one ocular findings involved. In addition to diverse renal and ocular findings, new phenotypes including congenital ventricular septal defect, skeletal deformity (fourth metatarsal microsomia), ovarian teratoma, and relatively rare extrarenal manifestations such as growth retardation, gout, and microcephaly were also found. Three novel mutations were reported for the first time. De novo mutations occurred in all patients who were carried out segregation analysis. Patients with the same mutation had different manifestations. PAX2-related disorder showed remarkable clinical variability and phenotypic heterogeneity. CONCLUSION: We firstly reported skeletal deformity (fourth metatarsal microsomia), ovarian teratoma, and congenital ventricular septal defect as new phenotypes of PAX2-related disorder which enlarged the phenotypic spectrum. Gout was firstly reported as the onset symptom of PAX2-related disorder. The diagnosis of PAX2-related disorder should be considered without family history due to a much higher percentage of De novo mutations.