Rafal Przybyslaw Krol

Publications (5)13.65 Total impact

• Article: Detection of large deletion mutations in the COL4A5 gene of female Alport syndrome patients.

  Kandai Nozu, Rafal Przybyslaw Krol, Yasufumi Ohtsuka, Koichi Nakanishi, Norishige Yoshikawa, Yoshimi Nozu, Hiroshi Kaito, Kyoko Kanda, Yuya Hashimura, Yuhei Hamasaki, Kazumoto Iijima, Masafumi Matsuo
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  ABSTRACT: Alport syndrome is the most common form of hereditary nephritis, and the majority of cases are caused by mutations in the COL4A5 gene. However, direct sequencing by polymerase chain reaction (PCR), from genomic DNA, or reverse transcriptase-polymerase chain reaction (RT-PCR), from mRNA, or polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) has reportedly resulted in detection rates of 31% to 84%, but of only 20% to 71% when restricted to female patients. This report concerns two female patients with X-linked Alport syndrome. Although mutational analysis of the COL4A5 gene was conducted with direct sequencing using genomic DNA and mRNA extracted from leukocytes, the results were negative for detection of mutations. Semi-quantitative PCR using genomic DNA was therefore conducted to detect large heterozygous deletions. The results were that the first patient showed complete loss of the COL4A5 gene and the second patient showed deletion from exons 37 to 51. Our patients possessed large heterozygous deletions in the COL4A5 gene that could not be detected with the standard direct sequencing method and were identified with semi-quantitative PCR. Previously reported mutation detection rates for female patients have been lower than overall rates. Our findings indicate that this difference may, in part, be due to failure to detect this type of mutation with conventional analytical methods.
  Pediatric Nephrology 07/2008; 23(11):2085-90. · 2.52 Impact Factor
• Article: Somatic mosaicism for a mutation of the COL4A5 gene is a cause of mild phenotype male Alport syndrome.

  Rafal Przybyslaw Krol, Kandai Nozu, Koichi Nakanishi, Kazumoto Iijima, Yasuhiro Takeshima, Xue Jun Fu, Yoshimi Nozu, Hiroshi Kaito, Kyoko Kanda, Masafumi Matsuo, Norishige Yoshikawa
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  ABSTRACT: 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.
  Nephrology Dialysis Transplantation 04/2008; 23(8):2525-30. · 3.40 Impact Factor
• Article: Molecular analysis of patients with type III Bartter syndrome: picking up large heterozygous deletions with semiquantitative PCR.

  Kandai Nozu, Xue Jun Fu, Koichi Nakanishi, Norishige Yoshikawa, Hiroshi Kaito, Kyoko Kanda, Rafal Przybyslaw Krol, Ritsuko Miyashita, Hidekazu Kamitsuji, Shoichiro Kanda, Yoshiki Hayashi, Kenichi Satomura, Nobuhiko Shimizu, Kazumoto Iijima, Masafumi Matsuo
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  ABSTRACT: Type III Bartter syndrome (BS) (OMIM607364) is caused by mutations in the basolateral chloride channel CIC-Kb gene (CLCNKB). The CLCNKB gene is sometimes reported as having a large deletion mutation, but all cases reported previously were large homozygous deletions and a large heterozygous deletion is impossible to detect by direct sequencing. This report concerns a genetic analysis of five Japanese patients with type III BS. To identify the mutations, we used polymerase chain reaction (PCR) and direct sequencing. To detect large heterozygous deletion mutations of the CLCNKB gene, we conducted semiquantitative PCR amplification using capillary electrophoresis. The result was that four mutations were identified, comprising one novel 2-bp deletion mutation, an entire heterozygous deletion, and a heterozygous deletion mutation of exons 1 and 2. The nonsense mutation W610X was detected in all patients, and this mutation is likely to constitute a founder effect in Japan. Capillary electrophoresis is a new method and extremely useful for detecting large heterozygous deletions, and should be used to examine type III BS cases in whom only a heterozygous mutation has been detected by direct sequencing. This is the first report to identify large heterozygous deletion mutations in the CLCNKB gene in patients with type III BS.
  Pediatric Research 10/2007; 62(3):364-9. · 2.70 Impact Factor
• Article: A novel mutation in KCNJ1 in a Bartter syndrome case diagnosed as pseudohypoaldosteronism.

  Kandai Nozu, Xue Jun Fu, Hiroshi Kaito, Kyoko Kanda, Naoki Yokoyama, Rafal Przybyslaw Krol, Toshihiro Nakajima, Mizutaka Kajiyama, Kazumoto Iijima, Masafumi Matsuo
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  ABSTRACT: Bartter syndrome (BS) is a genetic disorder with hypokalemic metabolic alkalosis and is classified into five types. One of these, type II BS (OMIM 241200), is classified as neonatal Bartter syndrome, which is caused by mutations in the KCNJ1 gene. Transient hyperkalemia and hyponatremia are usually noted in the early postnatal period, but as type II BS is a relatively rare disease, its exact clinical course and genetic background have not yet been thoroughly characterized. This report concerns a male type II BS patient with a novel mutation in the KCNJ1 gene. The unique clinical findings of this case are that hyperkalemia (8.9 mEq/l), hyponatremia, and metabolic acidosis detected in the early postnatal period led to a diagnosis of pseudohypoaldosteronism (PHA). As an adolescent, however, the patient currently shows normal potassium levels and normal renal function, although with hypercalciuria and nephrocalcinosis, without having received any treatment. In such cases, KCNJ1 mutations should be suspected. In our case, genetic analysis of the KCNJ1 gene identified a novel homozygous 1-bp deletion mutation (c.607 del. C in exon 5).
  Pediatric Nephrology 08/2007; 22(8):1219-23. · 2.52 Impact Factor
• Article: The effect of aldosterone blockade in patients with Alport syndrome.

  Hiroshi Kaito, Kandai Nozu, Kazumoto Iijima, Koichi Nakanishi, Kunihiko Yoshiya, Kyoko Kanda, Rafal Przybyslaw Krol, Norishige Yoshikawa, Masafumi Matsuo
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  ABSTRACT: Recent studies indicate that adding the mineralocorticoid receptor antagonist spironolactone (SP) to angiotensin converting enzyme inhibitors (ACEI) or ACEI and angiotensin receptor blocker (ARB), which is known as a triple blockade, enhances the more beneficial effects on urinary protein excretion of patients with chronic kidney diseases. In this study, we explored the effects of SP on urinary protein excretion in patients with Alport syndrome featuring persistent proteinuria in spite of the long-term use of ACEI (lisinopril) or both ACEI and ARB (candesartan). Five patients with Alport syndrome were enrolled and SP treatment (25 mg/day) was started. At the start of SP administration, all patients showed good renal function and none of them suffered from hypertension. We decided to assess the effect of SP by determining the morning urinary protein/creatinine ratio (U-P/C) and estimated glomerular filtration rate (EGFR). After SP treatment was started, U-P/C was significantly reduced at 3, 6, 12 and 18 months, while EGFR did not change. The drop in systolic and diastolic blood pressure was statistically significant and serum potassium level was slightly elevated. None of the patients showed signs of severe hyperkalemia (>5.0 mEq/l). These results suggest that aldosterone receptor blockade combined with ACEI and ARB therapy offers a valuable adjuvant treatment for the reduction of proteinuria in patients with Alport syndrome as in those with other chronic kidney diseases. SP can thus be expected to constitute a good renoprotective agent for Alport syndrome. These preliminary data indicate that large-scale trials of this therapy should be done.
  Pediatric Nephrology 01/2007; 21(12):1824-9. · 2.52 Impact Factor