Hongwen Zhang

Skin Biopsy Is a Practical Approach for the Clinical Diagnosis and Molecular Genetic Analysis of X-Linked Alport's Syndrome

A total of 209 unrelated patients of predominantly Han Chinese ethnicity and with X-linked Alports syndrome, a clinically heterogeneous hereditary nephritis, were enrolled in the present study to evaluate the ability to make a clinical diagnosis and perform molecular genetics analysis using skin biopsy. A negative or mosaic α5(IV) chain staining in the epidermal basement membrane was detected in 86.2% of male and 93.5% of female patients. COL4A5 mutations were identified in 85% of male patients with a negative α5(IV) chain staining pattern in the epidermal basement membrane. With use of skin biopsy and immunostaining, 16.4% of our patients were diagnosed before 3 years of age, and the youngest was diagnosed at 1 year of age. COL4A5 mutations were detected in 22 patients with normal epidermal basement membrane staining for the α5(IV) chain. Analysis of COL4A5 cDNA fragments from skin fibroblasts yielded a mutation detection rate of 83%, which was particularly valuable for identification of cryptic splicing mutations. Furthermore, 83% of COL4A5 mutations identified in the present study were novel. Thus, skin biopsy is a practical approach for the clinical diagnosis and molecular genetic analysis of X-linked Alports syndrome.

WT1 gene mutations in Chinese children with early onset nephrotic syndrome.

In Chinese children with steroid-resistant nephrotic syndrome (SRNS), it was reported that NPHS2 mutation was detected in 4.3%, which was lower than that in Caucasians (10–30%). However, there were no data on WT1 mutation in nephrotic syndrome (NS), especially in early-onset NS of Chinese children. Thus, a study, which enrolled 36 Chinese children with early-onset (before 3 y old) NS and steroid resistance if failing steroid therapy (early-group), was conducted. As control, 35 children with SRNS and with disease onset age after 3 y old were also analyzed (control-group). WT1 gene was examined by PCR and direct sequencing. The result showed that in the early-group 6/36 (16.7%) were detected with WT1 mutations. Further analysis according to different onset age revealed that the mutation detection rates of WT1 were 26.3% (5/19), 6.3% (1/16), and 0 (0/1) in children younger than 1 y, 1–2 y, and 2–3 y, respectively. In control-group, no WT1 (0/35) mutation was detected. WT1 mutation combined with NPHS2 variant was detected in a girl. In conclusion, WT1 mutations seemed more common in Chinese children with early-onset NS.

Prenatal Diagnosis and Genetic Counseling of a Chinese Alport Syndrome Kindred

Alport syndrome (AS) is a progressive renal disease characterized by hematuria and progressive renal failure. X-linked dominant AS (XLAS) is the major inheritance form, accounting for almost 80% of the cases, caused by mutations in COL4A5 gene. An accurate genetic diagnosis of AS is very important for genetic counseling and even prenatal diagnosis. In this study we detected mutation of COL4A5 by amplifying the entire coding sequence mRNA of peripheral blood lymphocytes using nested PCR in a Chinese XLAS family, and then performed the first prenatal diagnosis of AS in China. Mutation analysis of the fetus was performed on both cDNA-based level and DNA-based level of amniocytes. Fetus sex was determined by PCR amplification of SRY and karyotypes analysis. Maternal cell contamination was excluded by linkage analysis. There was a G-to-A substitution at position 4,271 in exon 46 of COL4A5 gene (c.G4271A) in the pregnant woman; this genetic variant has not been described previously and was a novel missense mutation. The fetus did not carry the same mutation as the mother. PCR amplification product of SRY and karyotypes analysis revealed a male fetus. Linkage analysis showed that there was no contamination of maternal cells in amniocytes.

Correlation Between mRNA Expression Level of the Mutant COL4A5 Gene and Phenotypes of XLAS Females

Alport syndrome (AS) is a progressive hereditary glomerulonephritis presented with hematuria, progressive renal failure, sensorineural deafness, and ocular lesions. Females with X-linked Alport syndrome (XLAS) have variable phenotypes, from asymptomatic hematuria to renal failure. In order to understand the possible mechanism of different phenotypes in female XLAS, we analyzed mRNA expression level of the mutant COL4A5 gene in fibroblasts, the X-inactivation pattern in peripheral blood DNA, and the phenotype variability of XLAS females. Total RNA was isolated from cultured skin fibroblasts in five females with XLAS and confirmed deletion mutations of COL4A5 mRNA. Reverse transcription–polymerase chain reaction (PCR) was performed to amplify the fragment, including the mutation sequences of the COL4A5 gene. The PCR products were electrophoresed with 8% polyacrylamide gel. Messenger RNA expression level of the mutant COL4A5 gene was analyzed with the optical density of PCR product revealed under polyacrylamide gel. The X-inactivation analysis was performed using HpaII predigestion of peripheral blood DNA followed by PCR of the highly polymorphic CAG repeat of the androgen receptor (AR) gene. All patients in the study had persistent microscopic hematuria. Two of them had gross hematuria. Three cases had persistent and severe proteinuria of 2+~3+, and the others had discontinuous and milder proteinuria of − ~+. The patients whose mRNA expression level of the mutant COL4A5 gene was higher had persistent and more severe proteinuria (r = 0.975, P = 0.005). None of them had skewed X inactivation. Our preliminary results demonstrate that the quantity of mRNA expression level of the mutant COL4A5 gene was correlated with the phenotypic severity of females with XLAS, and this could not be explained by X-inactivation pattern in peripheral blood leukocytes.

Drugs controlling proteinuria of patients with Alport syndrome

BackgroundProteinuria is one of the risk factors for the progression of renal diseases including Alport syndrome (AS), a hereditary glomerular renal disease. This study aimed to evaluate the efficacy of angiotensin converting enzyme inhibitors (ACEIs) and/or tripterygium, a Chinese herbal medicine widely used in Chinese patients with hematuria and proteinuria, on proteinuria in patients with AS.MethodsTwenty-nine children were enrolled into this retrospective study. Patients were divided into 3 therapy groups: ACEI group, tripterygium group, and ACEI plus tripterygium group.ResultsIn the 29 children, 23 were male and 6 female. In the ACEI group and the tripterygium group, the effective rate was 87.5% and 25.0%, respectively and in the ACEI plus tripterygium group was 42.9%.ConclusionsACEI is effective in controlling proteinuria of AS patients. Tripterygium should be carefully administered in controlling proteinuria of AS patients.

Steroid-resistant nephrotic syndrome caused by co-inheritance of mutations at NPHS1 and ADCK4 genes in two Chinese siblings

Hereditary nephrotic syndrome often presents with steroid-resistance and onset within the first year of life. Mutations in genes highly expressed in podocytes have been found in two thirds of these patients, especially NPHS1 and NPHS2 among at least 29 genetic causes that have been discovered. We reported two siblings with steroid-resistant nephrotic syndrome caused by co-inheritance of mutations at NPHS1 (c.1339G>A, p.E447K) and ACDK4 (c.748G>C, p.D250H) genes. The siblings presented with steroid-resistant nephrotic syndrome and pathological lesions of focal segmental glomerulosclerosis (FSGS), while the elder sister also developed hypertension, renal failure and cardiac dysfunction.

LAMB2 mutation with different phenotypes in China .

BACKGROUND Mutations of the LAMB2 gene mainly cause Pierson syndrome (OMIM) #609049), characterized by congenital nephrotic syndrome (CNS) and complex ocular involvements with microcoria as the most prominent clinical feature. However, the phenotypic spectrum of LAMB2-associated disorders is broader, isolated congenital or infantile nephrotic syndrome can also be seen. The aim of this study was to explore the phenotypes of different LAMB2 mutations in China. METHODS LAMB2 mutations were analyzed in three Chinese childhood steroid-resistant nephrotic syndrome cases, two of them with ocular abnormalities. RESULTS LAMB2 mutations were confirmed in all the three cases, two presented with Pierson syndrome, while one presented with isolated infantile steroid-resistant nephrotic syndrome. CONCLUSIONS The phenotypes caused by LAMB2 mutation were variable, mainly Pierson syndrome, as well as isolated nephrotic syndrome without ocular involvement. Mutational analysis of LAMB2 should be considered in all steroid-resistant nephrotic syndrome patients, with or without any ocular abnormalities.
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Attitudes toward genetic diagnosis and prenatal diagnosis of X-linked Alport syndrome in China

Aim:  Alport syndrome (AS) is a progressive renal disease characterized by hematuria and progressive renal failure. X‐linked dominant AS (XLAS) is the predominant inheritance form caused by mutations in COL4A5 gene. Attitudes toward genetic diagnosis and prenatal diagnosis for Chinese AS families were investigated. Attitudes toward genetic diagnosis and prenatal diagnosis in Chinese XLAS families were evaluated in the current study.

Mutations in TTC21B cause different phenotypes in two childhood cases in China

The TTC21B gene is now known as causative of nephronophthisis‐related ciliopathies (NPHP‐RC). We reported two Chinese paediatric cases with end‐stage renal disease and other phenotypes caused by the TTC21B gene mutations.

The ratio of urinary α1-microglobulin to microalbumin can be used as a diagnostic criterion for tubuloproteinuria

Low-molecular-weight proteinuria is one of the characteristic clinical manifestations of renal tubular and interstitial diseases. Low-molecular-weight proteinuria is defined as excessive urinary loss of α1-microglobulin, β2-microglobulin, or other low-molecular-weight plasma proteins. The current study examined the ratio of urinary α1-microglobulin to microalbumin in 24 Chinese pediatric patients with renal tubular and interstitial diseases, including 10 patients with Dent disease, 2 patients with Lowe syndrome, 6 patients with acute tubulointerstitial nephritis (ATIN), 4 patients with acute tubulointerstitial nephritis with uveitis syndrome (TINU), and 2 patients with nephronophthisis (NPHP). Patients with steroid-sensitive nephrotic syndrome, IgA nephropathy, Henoch-Schonlein purpura nephritis, or lupus nephritis served as control groups. In all of the patients with tubular and interstitial disease, urinary α1-microglobin increased 10-300-fold above the upper limit of the normal range, the ratio of urinary α1-microglobulin to microalbumin was greater than 1, and the percentage of low-molecular-weight plasma proteins (LMWP) in urine was greater than 50% according to urine protein electrophoresis. There was close correlation between the ratio of urinary α1-microglobulin to microalbumin and the percentage of LMWP in urine according to urine protein electrophoresis (r = 0.797, p = 0.000). We suggested firstly that the ratio of urinary α1-microglobulin to microalbumin, greater than 1, can be used as a diagnostic criterion for tubuloproteinuria.