Yoshimi Nozu

Molecular analysis of digenic inheritance in Bartter syndrome with sensorineural deafness

Background: Bartter syndrome (BS) is a genetic disorder accompanied by hypokalaemic metabolic alkalosis. BS with sensorineural deafness (SND, OMIM602522) is a newly identified phenotype caused by mutations in the BSND gene that encodes barttin, a β-subunit for chloride channel ClC-Ka and ClC-Kb and classified as type IV BS. Type IV BS features the most severe phenotype entailing life-threatening neonatal volume depletion and chronic renal failure developing during infancy. A recent report described a case of BS with SND from a consanguineous family who showed homozygous mutations in the CLCNKA and CLCNKB genes. This case indicated the possibility of the occurrence of digenic inheritance in BS with SND resulting from double mutations in the CLCNKA and CLCNKB genes. Subject and results: The current report concerns a 2-year-old girl from a non-consanguineous family with BS accompanied by SND. In our case, four loss-of-function mutations, consisting of mutations in both parental alleles in both CLCNKA and CLCNKB, were identified. The paternal allele had a nonsense mutation (Q260X) in CLCNKA and a splicing site mutation (IVS17+1 g>a) in CLCNKB. The maternal allele had a large deletion mutation (about 12 kbp) extending from CLCNKA to CLCNKB. Our case provides clear evidence that loss-of-function alleles in both alleles of both CLCNKA and CLCNKB results in a phenotype indistinguishable from that of mutations in BSND (type IV BS). Conclusions: Recent advances in genetics have resulted in a better understanding of many human inherited diseases, but most of them are monogenic disorders and more complex inheritance patterns remain unresolved. Our case provides clear evidence of digenic inheritance outside the scope of Mendelian inheritance disorders.

Recurrent EIARF and PRES With Severe Renal Hypouricemia by Compound Heterozygous SLC2A9 Mutation

Renal hypouricemia (RHU) is a hereditary disease that predisposes affected people to exercise-induced acute renal failure (EIARF). In most patients with RHU, the disorder is caused by loss-of-function mutations in SLC22A12 (solute carrier family 22, member 12), which encodes urate transporter 1 (URAT1). Patients with RHU without any mutations in the URAT1 gene were recently found to have a mutation in the glucose transporter 9 (GLUT9) gene (SLC2A9 [solute carrier family 2, member 9]). Central nervous system complications seem to be rare in patients with RHU with SLC22A12 mutations. Here, we report the case of a girl with severe RHU (serum urate: 5.9 μmol/L [0.1 mg/dL]) associated with recurrent EIARF in whom the disease was caused by a compound heterozygous mutation in SLC2A9, a nonsense mutation in the paternal allele (p.G207X in exon 7), and a large duplication (c.1–2981_1204+16502) in the maternal allele detected by reverse-transcription polymerase chain reaction (PCR), semiquantitative PCR, long PCR, and direct sequencing. The episodes of EIARF were complicated by posterior reversible encephalopathy syndrome (PRES), which suggested a relationship between PRES and GLUT9 or severe hypouricemia. This is the second report of mutations of both alleles of SLC2A9 that resulted in severe hypouricemia. Our findings indicate that even a nonsense mutation responsible for the heterozygous status of SLC2A9 did not cause severe hypouricemia, and they lend support to previous speculation that mutations of both SLC2A9 alleles cause severe hypouricemia. Our case shows that GLUT9, unlike URAT1, may play a specific role in exercise-induced PRES.

Somatic mosaicism for a mutation of the COL4A5 gene is a cause of mild phenotype male Alport syndrome

BACKGROUND Alport syndrome is the most common form of hereditary nephritis and is mainly caused by mutations in the COL4A5 gene, which shows the X-linked form. It is well known that some male Alport syndrome cases show a relatively mild phenotype, but few molecular investigations have been conducted to clarify the mechanism of this phenotype. Methods and results. This report concerns an 8-year-old male sporadic Alport syndrome patient. While electron microscopy of the glomerular basement membrane showed typical findings for Alport syndrome, however, the immunohistochemical analysis of the glomerulus showed mosaic staining of the type IV collagen alpha 5 chain. The mutational analysis of the COL4A5 gene unexpectedly disclosed two peaks at the intron 43 splicing acceptor site (c. 3998-2 a/t) with direct sequencing. Restriction enzyme analysis demonstrated that the presence of somatic mosaicism was responsible for this mutation. mRNA extracted from the urinary sediments was analysed by RT-PCR and two PCR fragments were amplified, one consisting of a normal sequence and one with skipping of exon 44. CONCLUSIONS Our findings indicate that somatic mosaicism for COL4A5 is responsible for male X-linked Alport syndrome with an alpha 5 mosaic staining pattern. Several cases with somatic mosaicism have previously been reported, however, this is the first case where the presence of this mutation was proved with a comprehensive analysis of genomic DNA, mRNA and alpha 5 expression in the tissues. Somatic mosaicism may thus be one of the causes of the mild phenotype in Alport syndrome.

A Deep Intronic Mutation in the SLC12A3 Gene Leads to Gitelman Syndrome

Many mutations have been detected in the SLC12A3 gene of Gitelman syndrome (GS, OMIM 263800) patients. In previous studies, only one mutant allele was detected in ∼20 to 41% of patients with GS; however, the exact reason for the nonidentification has not been established. In this study, we used RT-PCR using mRNA to investigate for the first time transcript abnormalities caused by deep intronic mutation. Direct sequencing analysis of leukocyte DNA identified one base insertion in exon 6 (c.818_819insG), but no mutation was detected in another allele. We analyzed RNA extracted from leukocytes and urine sediments and detected unknown sequence containing 238bp between exons 13 and 14. The genomic DNA analysis of intron 13 revealed a single-base substitution (c.1670–191C>T) that creates a new donor splice site within the intron resulting in the inclusion of a novel cryptic exon in mRNA. This is the first report of creation of a splice site by a deep intronic single-nucleotide change in GS and the first report to detect the onset mechanism in a patient with GS and missing mutation in one allele. This molecular onset mechanism may partly explain the poor success rate of mutation detection in both alleles of patients with GS.

Differential diagnosis of Bartter syndrome, Gitelman syndrome, and pseudo–Bartter/Gitelman syndrome based on clinical characteristics

Purpose:Phenotypic overlap exists among type III Bartter syndrome (BS), Gitelman syndrome (GS), and pseudo-BS/GS (p-BS/GS), which are clinically difficult to distinguish. We aimed to clarify the differences between these diseases, allowing accurate diagnosis based on their clinical features.Methods:A total of 163 patients with genetically defined type III BS (n = 30), GS (n = 90), and p-BS/GS (n = 43) were included. Age at diagnosis, sex, body mass index, estimated glomerular filtration rate, and serum and urine electrolyte concentrations were determined.Results:Patients with p-BS/GS were significantly older at diagnosis than those with type III BS and GS. Patients with p-BS/GS included a significantly higher percentage of women and had a lower body mass index and estimated glomerular filtration rate than did patients with GS. Although hypomagnesemia and hypocalciuria were predominant biochemical findings in patients with GS, 17 and 23% of patients with type III BS and p-BS/GS, respectively, also showed these abnormalities. Of patients with type III BS, GS, and p-BS/GS, 40, 12, and 63%, respectively, presented with chronic kidney disease.Conclusions:This study clarified the clinical differences between BS, GS, and p-BS/GS for the first time, which will help clinicians establish differential diagnoses for these three conditions.Genet Med 18 2, 180–188.

Genetic, Clinical, and Pathologic Backgrounds of Patients with Autosomal Dominant Alport Syndrome

BACKGROUND AND OBJECTIVES Alport syndrome comprises a group of inherited heterogeneous disorders involving CKD, hearing loss, and ocular abnormalities. Autosomal dominant Alport syndrome caused by heterozygous mutations in collagen 4A3 and/or collagen 4A4 accounts for <5% of patients. However, the clinical, genetic, and pathologic backgrounds of patients with autosomal dominant Alport syndrome remain unclear. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We conducted a retrospective analysis of 25 patients with genetically proven autosomal dominant Alport syndrome and their family members (a total of 72 patients) from 16 unrelated families. Patients with suspected Alport syndrome after pathologic examination who were referred from anywhere in Japan for genetic analysis from 2006 to 2015 were included in this study. Clinical, laboratory, and pathologic data were collected from medical records at the point of registration for genetic diagnosis. Genetic analysis was performed by targeted resequencing of 27 podocyte-related genes, including Alport-related collagen genes, to make a diagnosis of autosomal dominant Alport syndrome and identify modifier genes or double mutations. Clinical data were obtained from medical records. RESULTS The median renal survival time was 70 years, and the median age at first detection of proteinuria was 17 years old. There was one patient with hearing loss and one patient with ocular lesion. Among 16 patients who underwent kidney biopsy, three showed FSGS, and seven showed thinning without lamellation of the glomerular basement membrane. Five of 13 detected mutations were reported to be causative mutations for autosomal recessive Alport syndrome in previous studies. Two families possessed double mutations in both collagen 4A3 and collagen 4A4, but no modifier genes were detected among the other podocyte-related genes. CONCLUSIONS The renal phenotype of autosomal dominant Alport syndrome was much milder than that of autosomal recessive Alport syndrome or X-linked Alport syndrome in men. It may, thus, be difficult to make an accurate diagnosis of autosomal dominant Alport syndrome on the basis of clinical or pathologic findings. No modifier genes were identified among the known podocyte-related genes.

Different phenotypes of HNF1ß deletion mutants in familial multicystic dysplastic kidneys.

Multicystic dysplastic kidney (MCDK) is one of the most common congenital abnormalities of the kidney and urinary tract (CAKUT), although its pathophysiology remains unknown. Familial occurrence of MCDK suggests that mutations in genes associated with nephrogenesis are involved in the pathogenesis in at least some cases. Hepatocyte nuclear factor 1β (HNF1β) is a member of the homeodomain-containing super family of transcription factors, and is known to regulate tissue-specific gene expression in a number of organs including the kidneys, pancreas and liver. It has been recently postulated to be associated with CAKUT, including MCDK. We recently encountered a family with a deletion mutant of HNF1β, of which the 2nd son, the proband, developed bilateral MCDK resulting in renal loss of function in infancy while the 1st son developed unilateral MCDK. Their father has two normal kidneys. This family confirmed that mutations in the HNF1β gene are strongly associated with the development of MCDK. Furthermore, the fact that no clear phenotype-genotype correlation exists suggests that gene(s) other than HNF1β are also involved in nephrogenesis and the development of MCDK.

Detection by multiplex ligation-dependent probe amplification of large deletion mutations in the COL4A5 gene in female patients with Alport syndrome

Sirs, Recently, we reported on two female subjects with Xlinked Alport syndrome (XLAS) whose genetic test findings were negative by the direct sequencing method [1]. However, we could detect their large heterozygous deletion mutations with the semi-quantitative polymerase chain reaction (PCR) method, which we frequently use these days because the technique is very simple, consisting of PCR and capillary electrophoresis [1–3]. On the other hand, this method needs sophisticated equipment for capillary electrophoresis, and every exon analysis must be conducted separately. In connection with the first submission of our last article, one of the reviewers suggested we should validate the results with the multiplex ligation-dependent probe amplification (MLPA) method, because the MLPA kit for XLAS has become commercially available. Although, at that time, there were no reports on the use of MLPA for XLAS, Hertz et al. have recently reported on the usefulness of this method [4]. This prompted us to conduct the MLPA analysis according to Hertz’s recommendation, using the SALSA P191/192 Alport MLPA assay (MRC-Holland, Amsterdam, The Netherlands) for the patients we reported on last time, and this confirmed our results. Peaks for each PCR product were obtained after capillary sequences, and the peak of every sample was determined from the relative peak area (PRA). The individual peak areas in relation to the total of all peak areas of several other parts on the X-chromosome, except for the COL4A5 and COL4A6 genes, were calculated, and the resultant values were compared to those of normal controls. We obtained values for the normal controls by calculating separately the average values for three healthy male and female subjects. A reduction in the ratio to 0 in male subjects and 0.5 in female subjects indicates a deletion in hemizygous and heterozygous form, respectively. A normal MLPA ratio is >0.7 and <1.3 [4]. We have previously detected with semi-quantitative PCR that patient 1 had an exon1-51 entire heterozygous deletion of the COL4A5 gene, patient 2 an exon 37 to 51 hemizygous deletion, and the mother of patient 2 a heterozygous deletion [1]. The MLPA results were exactly the same as those for semi-quantitative PCR (The schema is shown in Fig. 1). Moreover, the deletion in patient 1 extended to COL4A6 exons 1 and 2. XLAS associated with diffuse leiomyomas [DL-AS; online Mendelian inheritance in man (OMIM) catalogue no. 308940] has been reported previously in more than 30 cases. This phenotype is caused by deletion of the COL4A5 and extends into the second intron of the COL4A6 gene, and it should be noted that defects in the COL4A6 gene alone do not appear to cause AS or leiomyomas [5, 6]. COL4A5 and COL4A6 are arranged head-to-head at Xq22.3 Pediatr Nephrol (2009) 24:1773–1774 DOI 10.1007/s00467-009-1122-0

Natural History and Genotype-Phenotype Correlation in Female X-Linked Alport Syndrome

Introduction X-linked Alport syndrome (XLAS) is a hereditary disease characterized by progressive nephritis, hearing loss, and ocular abnormalities. Affected male patients usually progress to end-stage renal disease in early or middle adulthood, and disease severity is strongly correlated with genotype. However, the clinical course in female patients has rarely been reported. Methods We conducted a retrospective analysis of females with genetically proven XLAS (n = 275) and their affected female family members (n = 61) from 179 Japanese families. Patients suspected to have Alport syndrome from pathologic findings or a family history who were referred from anywhere in Japan for genetic diagnosis between 2006–2015 were included in this study. Clinical and laboratory data were collected from medical records at the time of registration for genetic analysis. Results Proteinuria was detected in 175 genetically proven patients (72.6%), and the median age for developing proteinuria was 7.0 years. Fifty-two of 336 patients developed end-stage renal disease with a median renal survival age of 65.0 years. No obvious genotype–phenotype correlation was observed. Additionally, targeted sequencing for podocyte-related genes in patients with severe phenotypes revealed no obvious variants considered to be modifier genes except for 1 patient with a COL4A3 gene variant. Discussion This study revealed that phenotypes in female XLAS patients may be severe, but genotype does not help to predict the disease severity. Clinicians must therefore pay careful attention to the clinical course and appropriate treatment in females with XLAS.

X-linked Alport syndrome associated with a synonymous p.Gly292Gly mutation alters the splicing donor site of the type IV collagen alpha chain 5 gene

BackgroundX-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the type IV collagen alpha chain 5 gene (COL4A5). Although many COL4A5 mutations have previously been identified, pathogenic synonymous mutations have not yet been described.MethodsA family with XLAS underwent mutational analyses of COL4A5 by PCR and direct sequencing, as well as transcript analysis of potential splice site mutations. In silico analysis was also conducted to predict the disruption of splicing factor binding sites. Immunohistochemistry (IHC) of kidney biopsies was used to detect α2 and α5 chain expression.ResultsWe identified a hemizygous point mutation, c.876A>T, in exon 15 of COL4A5 in the proband and his brother, which is predicted to result in a synonymous amino acid change, p.(Gly292Gly). Transcript analysis showed that this mutation potentially altered splicing because it disrupted the splicing factor binding site. The kidney biopsy of the proband showed lamellation of the glomerular basement membrane (GBM), while IHC revealed negative α5(IV) staining in the GBM and Bowman’s capsule, which is typical of XLAS.ConclusionsThis is the first report of a synonymous COL4A5 substitution being responsible for XLAS. Our findings suggest that transcript analysis should be conducted for the future correct assessment of silent mutations.