Tomoko Horinouchi

Cryptic exon activation in SLC12A3 in Gitelman syndrome

Gitelman syndrome (GS) is an autosomal recessive renal tubulopathy characterized by hypokalemic metabolic alkalosis with hypocalciuria and hypomagnesemia. GS clinical symptoms range from mild weakness to muscular cramps, paralysis or even sudden death as a result of cardiac arrhythmia. GS is caused by loss-of-function mutations in the solute carrier family 12 member 3 (SLC12A3) gene, but molecular mechanisms underlying such a wide range of symptoms are poorly understood. Here we report cryptic exon activation in SLC12A3 intron 12 in a clinically asymptomatic GS, resulting from an intronic mutation c.1669+297 T>G that created a new acceptor splice site. The cryptic exon was sandwiched between the L3 transposon upstream and a mammalian interspersed repeat downstream, possibly contributing to inclusion of the cryptic exon in mature transcripts. The mutation was identified by targeted next-generation sequencing of candidate genes in GS patients with missing pathogenic SLC12A3 alleles. Taken together, this work illustrates the power of next-generation sequencing to identify causal mutations in intronic regions in asymptomatic individuals at risk of developing potentially fatal disease complications, improving clinical management of these cases.

Diagnostic strategy for inherited hypomagnesemia

BackgroundHereditary hypomagnesemia is difficult to diagnose accurately because of its rarity and the variety of causative genes. We established a flowchart for identifying responsible genes for hypomagnesemia, and we confirmed its diagnostic efficacy in patients with suspected inherited hypomagnesemia.MethodsWe established a flowchart and applied it to five index cases with suspected inherited hypomagnesemia. Direct sequence analysis was used to detect the causative gene variants in four cases, and targeted sequencing analysis using next-generation sequencing (NGS) of all causative genes for hypomagnesemia was used in one.ResultsExpected pathogenic variants were detected in the HNF1B, TRPM6, CLDN16, CASR, or SLC12A3 gene in all five cases. The results of all genetic analyses were consistent with the clinical diagnostic results using the flowchart.ConclusionsAccurate genetic diagnosis is crucial for estimating the prognosis, detecting complications in organs other than the kidneys, and for directing genetic counseling. The developed flowchart for identifying responsible genes for hypomagnesemia was useful for diagnosing inherited hypomagnesemia. In addition, NGS analysis will help to resolve clinical difficulties in making an accurate diagnosis and thus improve the diagnostic strategy for inherited hypomagnesemia.

Detection of Splicing Abnormalities and Genotype-Phenotype Correlation in X-linked Alport Syndrome

BACKGROUND X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the COL4A5 gene. Genotype-phenotype correlation in male XLAS is relatively well established; relative to truncating mutations, nontruncating mutations exhibit milder phenotypes. However, transcript comparison between XLAS cases with splicing abnormalities that result in a premature stop codon and those with nontruncating splicing abnormalities has not been reported, mainly because transcript analysis is not routinely conducted in patients with XLAS. METHODS We examined transcript expression for all patients with suspected splicing abnormalities who were treated at one hospital between January of 2006 and July of 2017. Additionally, we recruited 46 males from 29 families with splicing abnormalities to examine genotype-phenotype correlation in patients with truncating (n=21, from 14 families) and nontruncating (n=25, from 15 families) mutations at the transcript level. RESULTS We detected 41 XLAS families with abnormal splicing patterns and described novel XLAS atypical splicing patterns (n=14) other than exon skipping caused by point mutations in the splice consensus sequence. The median age for developing ESRD was 20 years (95% confidence interval, 14 to 23 years) among patients with truncating mutations and 29 years (95% confidence interval, 25 to 40 years) among patients with nontruncating mutations (P=0.001). CONCLUSIONS We report unpredictable atypical splicing in the COL4A5 gene in male patients with XLAS and reveal that renal prognosis differs significantly for patients with truncating versus nontruncating splicing abnormalities. Our results suggest that splicing modulation should be explored as a therapy for XLAS with truncating mutations.

Detection of copy number variations by pair analysis using next-generation sequencing data in inherited kidney diseases

BackgroundComprehensive genetic approaches for diagnosing inherited kidney diseases using next-generation sequencing (NGS) have recently been established. However, even with these approaches, we are still failing to detect gene defects in some patients who appear to suffer from genetic diseases. One of the reasons for this is the difficulty of detecting copy number variations (CNVs) using our current approaches. For such cases, we can apply methods of array-based comparative genomic hybridization (aCGH) or multiplex ligation and probe amplification (MLPA); however, these are expensive and laborious and also often fail to identify CNVs. Here, we report seven cases with CNVs in various inherited kidney diseases screened by NGS pair analysis.MethodsTargeted sequencing analysis for causative genes was conducted for cases with suspected inherited kidney diseases, for some of which a definitive genetic diagnosis had not been achieved. We conducted pair analysis using NGS data for those cases. When CNVs were detected by pair analysis, they were confirmed by aCGH and/or MLPA.ResultsIn seven cases, CNVs in various causative genes of inherited kidney diseases were detected by pair analysis. With aCGH and/or MLPA, pathogenic CNV variants were confirmed: COL4A5 or HNF1B in two cases each, and EYA1, CLCNKB, or PAX2 in one each.ConclusionWe presented seven cases with CNVs in various genes that were screened by pair analysis. The NGS-based CNV detection method is useful for comprehensive screening of CNVs, and our results revealed that, for a certain proportion of cases, CNV analysis is necessary for accurate genetic diagnosis.

Detection of a Splice Site Variant in a Patient with Glomerulopathy and Fibronectin Deposits

Background/Aims: Glomerulopathy with fibronectin deposits (GFND; OMIM: 601894) is a very rare inherited kidney disease caused by pathogenic variants in the FN1 gene. Only 9 exonic pathogenic variants in FN1, 9 at the heparin-binding site, and 1 at the integrin-binding site have been reported. No intronic variants in FN1 have been detected. Methods: We found a pathogenic intronic variant in intron 36 (c.5888–2A>G) located at the heparin-binding site. To determine whether this mutation influences splicing processes, we conducted RT-PCR analysis and an in vitro splicing assay using minigene construction. Results: RT-PCR using RNA extracted from leukocytes of the proband failed because of the low expression of FN1 mRNA in leukocytes. We conducted in vitro functional splicing analysis using minigenes and found that c.5888–2A>G caused a 12 bp deletion at exon 37 by the activation of a novel splicing acceptor site within exon 37. We were able to detect the same abnormal transcript in mRNA extracted from the patient’s urinary sediment and confirmed the pathogenicity of c.5888–2A>G by both RT-PCR using the patient sample and an in vitro splicing assay. Conclusion: Intronic variants can cause GFND. Minigene analysis is useful for determining the pathogenicity of the intronic variants and could be used for all inherited kidney diseases.

Clinical and Genetic Characteristics in Patients With Gitelman Syndrome

Introduction Gitelman syndrome (GS) is a tubulopathy exhibited by salt loss. GS cases are most often diagnosed by chance blood test. Aside from that, some cases are also diagnosed from tetanic symptoms associated with hypokalemia and/or hypomagnesemia or short stature. As for complications, thyroid dysfunction and short stature are known, but the incidence rates for these complications have not yet been elucidated. In addition, no genotype–phenotype correlation has been identified in GS. Methods We examined the clinical characteristics and genotype–phenotype correlation in genetically proven GS cases with homozygous or compound heterozygous variants in SLC12A3 (n = 185). Results In our cohort, diagnostic opportunities were by chance blood tests (54.7%), tetany (32.6%), or short stature (7.2%). Regarding complications, 16.3% had short stature, 13.7% had experienced febrile convulsion, 4.3% had thyroid dysfunction, and 2.5% were diagnosed with epilepsy. In one case, QT prolongation was detected. Among 29 cases with short stature, 10 were diagnosed with growth hormone (GH) deficiency and GH replacement therapy started. Interestingly, there was a strong correlation in serum magnesium levels between cases with p.Arg642Cys and/or p.Leu858His and cases without these variants, which are mutational hotspots in the Japanese population (1.76 mg/dl vs. 1.43 mg/dl, P < 0.001). Conclusion This study has revealed, for the first time, clinical characteristics in genetically proven GS cases in the Japanese population, including prevalence of complications. Patients with hypokalemia detected by chance blood test should have gene tests performed. Patients with GS need attention for developing extrarenal complications, such as short stature, febrile convulsion, thyroid dysfunction, epilepsy, or QT prolongation. It was also revealed for the first time that hypomagnesemia was not severe in some variants in SLC12A3.

Strong Association of the HLA-DR/DQ Locus with Childhood Steroid-Sensitive Nephrotic Syndrome in the Japanese Population

Background Nephrotic syndrome is the most common cause of chronic glomerular disease in children. Most of these patients develop steroid-sensitive nephrotic syndrome (SSNS), but the loci conferring susceptibility to childhood SSNS are mainly unknown.Methods We conducted a genome-wide association study (GWAS) in the Japanese population; 224 patients with childhood SSNS and 419 adult healthy controls were genotyped using the Affymetrix Japonica Array in the discovery stage. Imputation for six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1) was conducted on the basis of Japanese-specific references. We performed genotyping for HLA-DRB1/-DQB1 using a sequence-specific oligonucleotide-probing method on a Luminex platform. Whole-genome imputation was conducted using a phased reference panel of 2049 healthy Japanese individuals. Replication was performed in an independent Japanese sample set including 216 patients and 719 healthy controls. We genotyped candidate single-nucleotide polymorphisms using the DigiTag2 assay.Results The most significant association was detected in the HLA-DR/DQ region and replicated (rs4642516 [minor allele G], combined Pallelic=7.84×10-23; odds ratio [OR], 0.33; 95% confidence interval [95% CI], 0.26 to 0.41; rs3134996 [minor allele A], combined Pallelic=1.72×10-25; OR, 0.29; 95% CI, 0.23 to 0.37). HLA-DRB1*08:02 (Pc=1.82×10-9; OR, 2.62; 95% CI, 1.94 to 3.54) and HLA-DQB1*06:04 (Pc=2.09×10-12; OR, 0.10; 95% CI, 0.05 to 0.21) were considered primary HLA alleles associated with childhood SSNS. HLA-DRB1*08:02-DQB1*03:02 (Pc=7.01×10-11; OR, 3.60; 95% CI, 2.46 to 5.29) was identified as the most significant genetic susceptibility factor.Conclusions The most significant association with childhood SSNS was detected in the HLA-DR/DQ region. Further HLA allele/haplotype analyses should enhance our understanding of molecular mechanisms underlying SSNS.

Development of ultra-deep targeted RNA sequencing for analyzing X-chromosome inactivation in female Dent disease

The pattern of X-chromosome inactivation (XCI) can affect the clinical severity of X-linked disorders in females. XCI pattern analysis has been conducted mainly by HUMARA assay, a polymerase chain reaction-based assay using a methylation-sensitive restriction enzyme. However, this assay examines the XCI ratio of the androgen receptor gene at the genomic DNA level and does not reflect the ratio of either targeted gene directly or at the mRNA level. Here, we report four females with Dent disease, and we clarified the correlation between XCI and female cases of Dent disease using not only HUMARA assay but also a novel analytical method by RNA sequencing. We constructed genetic analysis for 4 female cases showing high level of urinary low-molecular-weight proteinuria and their parents. Their XCI pattern was analyzed by both HUMARA assay and an ultra-deep targeted RNA sequencing of the CLCN5 gene using genomic DNA and mRNA extracted from both leukocytes and urine sediment. All four cases possessed pathogenic variants of the CLCN5 gene. XCI analysis revealed skewed XCI in only two cases, while the other two showed random XCI. All assay results of HUMARA and targeted RNA sequencing in both leukocytes and urinary sediment were clearly identical in all four cases. We developed a novel XCI analytical assay of ultra-deep targeted RNA sequencing and revealed that skewed XCI explains the mechanism of onset of female Dent disease in only half of such cases.

Congenital chloride diarrhea needs to be distinguished from Bartter and Gitelman syndrome

Pseudo-Bartter/Gitelman syndrome (p-BS/GS) encompasses a clinically heterogeneous group of inherited or acquired disorders similar to Bartter syndrome (BS) or Gitelman syndrome (GS), both renal salt-losing tubulopathies. Phenotypic overlap frequently occurs between p-BS/GS and BS/GS, which are difficult to diagnose based on their clinical presentation and require genetic tests for accurate diagnosis. In addition, p-BS/GS can occur as a result of other inherited diseases such as cystic fibrosis, autosomal dominant hypocalcemia, Dent disease, or congenital chloride diarrhea (CCD). However, the detection of the variants in genes other than known BS/GS-causing genes by conventional Sanger sequencing requires substantial time and resources. We studied 27 cases clinically diagnosed with BS/GS, but with negative genetic tests for known BS/GS genes. We conducted targeted sequencing for 22 genes including genes responsible for tubulopathies and other inherited diseases manifesting with p-BS/GS symptoms. We detected the SLC26A3 gene variants responsible for CCD in two patients. In Patient 1, we found the SLC26A3 compound heterozygous variants: c.354delC and c.1008insT. In Patient 2, we identified the compound heterozygous variants: c.877G > A, p.(Glu293Lys), and c.1008insT. Our results suggest that a comprehensive genetic screening system using targeted sequencing is useful for the diagnosis of patients with p-BS/GS with alternative genetic origins.

A review of clinical characteristics and genetic backgrounds in Alport syndrome

Alport syndrome (AS) is a progressive hereditary renal disease that is characterized by sensorineural hearing loss and ocular abnormalities. It is divided into three modes of inheritance, namely, X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, while ADAS and ARAS are caused by those in COL4A3/COL4A4. Diagnosis is conventionally made pathologically, but recent advances in comprehensive genetic analysis have enabled genetic testing to be performed for the diagnosis of AS as first-line diagnosis. Because of these advances, substantial information about the genetics of AS has been obtained and the genetic background of this disease has been revealed, including genotype–phenotype correlations and mechanisms of onset in some male XLAS cases that lead to milder phenotypes of late-onset end-stage renal disease (ESRD). There is currently no radical therapy for AS and treatment is only performed to delay progression to ESRD using nephron-protective drugs. Angiotensin-converting enzyme inhibitors can remarkably delay the development of ESRD. Recently, some new drugs for this disease have entered clinical trials or been developed in laboratories. In this article, we review the diagnostic strategy, genotype–phenotype correlation, mechanisms of onset of milder phenotypes, and treatment of AS, among others.