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alport syndrome ( as ) is a hereditary nephropathy characterized by a family history of hematuria , progressive renal failure , sensorineural hearing loss , and ocular abnormalities ( 1 ) .
progression to renal failure is predictable in men , whereas the course of renal involvement is much more variable in women , remaining mild in the majority of patients .
approximately 80% of as is inherited in an x - linked manner and is caused by mutations in the col4a5 gene .
nearly all male patients will develop end - stage renal disease ( esrd ) , whereas heterozygous females exhibit a wide variability in disease severity ( 2,3 ) .
to date , more than 700 different col4a5 mutations have been identified ( 4,5 ) .
we herein report a novel mutation in a japanese family with an x - linked as mutation in col4a5 .
the patient ( patient 1 ) was a 2-year - old japanese girl who was the first child of healthy non - consanguineous parents .
her maternal aunt had an episode of nephritis , and a renal biopsy was performed during childhood , but the findings were uncertain . at 14 months of age ,
patient 1 was referred to our hospital because of microscopic hematuria that had persisted from the neonatal period . she had no clinically detectable hearing loss or ocular abnormalities . a urine culture revealed no infection . at 15 months of age ,
enalapril malate was initiated for proteinuria , with a urinary protein / creatinine ratio ( p / cr ) of 1.3 - 3.6 g / gcr . at 21 months of age
a urinalysis showed 2 + proteinuria ( p / cr , 1.3 g / gcr ) and 3 + hematuria , with the urine sediment containing > 100 red cells per high - power field .
her blood urea nitrogen ( bun ) level was 17.7 mg / dl , serum creatinine level was 0.22 mg / dl , serum total protein level was 5.9 g / dl , and albumin level was 3.7 g / dl .
serum c3 and c4 levels were 118.2 mg / dl and 24.8 mg / dl , respectively .
her mother ( patient 2 ) also had a history of proteinuria and hematuria without renal dysfunction , deafness , or ocular abnormalities .
, she was admitted to our hospital along with her daughter ( patient 1 ) for a renal biopsy .
a urinalysis showed 3 + proteinuria ( p / cr , 1.7 g / gcr ) and 2 + hematuria , with the sediment containing 10 - 19 red cells per high - power field .
her bun level was 20.0 mg / dl , serum creatinine level was 0.59 mg / dl , serum total protein level was 6.8 g / dl , and albumin level was 4.0 g / dl .
serum c3 and c4 levels were 134.4 mg / dl and 34.2 mg / dl , respectively .
unaffected individuals with kidney diseases are denoted by empty squares ( men ) and circles ( women ) .
affected individuals with x - linked alport syndrome are denoted by blackened circles ( patients 1 and 2 ) . affected individuals with biopsy - unproven kidney disease
genetic analyses revealed that both patients had a heterozygous mutation ( c. 2,767g > c ) in exon 32 .
( b ) x chromosome inactivation assays for our patients . in methylation - sensitive enzymes ,
patient 1 and 2 both showed a random pattern with ratios of 77 : 23 and 31 : 69 , respectively .
it was impossible to distinguish whether the mutated allele was possessed by an activated or inactivated x chromosome because both had the same number of cag repeats . 
 
, 29 glomeruli were observed on light microscopy ; the glomerulus , tubules , and interstitium showed no significant alterations .
immunofluorescence ( if ) staining for alpha 5 chains of type iv collagen showed segmental and mosaic patterns in the glomerular basement membrane ( gbm ) .
electron microscopy ( em ) demonstrated diffusely thinned - out gbms ( 139 - 143 nm ) with focal lamellation and splitting . in patient 2 , 40 glomeruli were observed on light microscopy , two of which were globally sclerotic .
if staining for alpha 5 chains of type iv collagen showed segmental and mosaic patterns in the gbm , bowman 's capsule , and distal tubular basement membrane ( tbm ) .
em demonstrated diffusely thinned - out gbms ( 149 - 166 nm ) with dense granules and splitting . in both patients , the merged if staining images for alpha 2 and 5 chains of type iv collagen clarified the findings of segmental and mosaic patterns in the gbm . 

patient 1 shows ( a ) a normal pattern of the alpha 2 chain of type iv collagen in the gbm and ( b ) segmental and mosaic patterns of the alpha 5 chain of type iv collagen in the gbm and the absence of staining in bowman s capsule and the tbm .
( c ) the merged findings of the alpha 2 and 5 chains of type iv collagen are shown .
( d ) electron microscopy reveals a split lamina densa ( arrows ) and thin gbm .
patient 2 shows ( e ) a normal pattern of the alpha 2 chain of type iv collagen in the gbm and ( f ) a mosaic pattern of the alpha 5 chain of type iv collagen in the gbm and tbm .
( g ) the merged findings of the alpha 2 and 5 chains of type iv collagen are shown .
gbm : glomerular basement membrane , tbm : tubular basement membrane 
  a sequence analysis of col4a5 in the index patient and her mother was performed .
the study was approved by the institutional review board of kobe university school of medicine , and written informed consent was obtained .
genomic dna was isolated from each patient 's peripheral blood leukocytes using the quick gene mini 80 system ( kurabo industries , tokyo , japan ) , according to the manufacturer 's instructions .
mutational analyses of col4a5 were performed using polymerase chain reaction ( pcr ) and direct sequencing of genomic dna of all exons and exon - intron boundaries .
the pcr - amplified products were then purified and subjected to direct sequencing using a dye terminator cycle sequencing kit ( amersham biosciences , piscataway , usa ) with an automatic dna sequencer ( abi prism 3130 ; perkin elmer applied biosystems , foster city , usa ) .
the analysis revealed that both patients had a heterozygous mutation ( c.2767g > c ) in exon 32 ( fig .
1a ) . to investigate x chromosome inactivation , the human androgen receptor ( humara ) assay was performed in both patients .
genomic dna was digested by a methylation - sensitive enzyme , hpaii , at 37c for 18 hours followed by pcr using dna with humara primers , as described previously ( 6 ) .
a dna fragment analysis was performed on a 310 genetic analyzer ( thermo fisher scientific , waltham , usa ) .
fragment data analyses were performed using the gene mapper software program ( thermo fisher scientific ) .
the humara assay for patients 1 and 2 revealed that the x chromosome inactivation pattern was 77:23 and 31:69 , respectively ( fig .
the index case was a 2-year - old girl with x - linked as who had inherited a col4a5 novel mutation ( c.2767g > c ) in exon 32 from her mother .
both patients had histories of hematuria and proteinuria without sensorineural hearing loss or ocular abnormalities .
renal biopsy findings indicated hereditary glomerulonephritis , and genetic analyses were useful in making a final diagnosis .
more than 700 disease - causing mutations have already been reported in col4a5 ( 2,5 ) . in men with
x - linked as , clinical features can be predicted from the location of col4a5 mutations ( 3 ) .
pathologically , patient 1 had less signals in bowman 's capsule and the tbm compared with patient 2 .
it reported that the genotype - phenotype correlations are not observed in women , even among family members ( 2,7 ) .
although heterozygous female patients with x - linked as have a normal col4a5 allele , differences in x chromosome inactivation may influence the disease severity ( 2,8 ) .
however , the humara assay in our cases did not confirm non - random x chromosome inactivation , so - called skewed x , since it is usually considered that the non - random inactivation pattern is > 80:20 or < 20:80 ( 9 ) .
reported that 90% of x chromosomes with a normal col4a5 allele were inactivated in the kidney of a woman with a severe as phenotype ( 10 ) .
the different patterns of x chromosome inactivation in the gbm may cause the variable phenotypes in women with x - linked as .
therefore , the inactivation of a high proportion of normal x chromosomes in critical tissues could be clinically severe ( 11 ) . in this study ,
skewed x was not detected in the peripheral blood cells of both patients 1 and 2 .
however , it remains possible that skewed x operates in specific tissue , namely the gbm , more strongly in patient 1 than in patient 2 .
the mutation identified in our patient 's family was c.2767g > c , resulting in a glycine to arginine substitution at position 923 in exon 32 .
glycine substitutions are likely to alter the folding of the triple helix of collagen ; however , solid evidence is absent regarding type iv collagen ( 12,13 ) .
gross et al . proposed the following classification to link the phenotype and genotype ( 14 ) : i. severe as .
genotype alterations in col4a5 include large rearrangement , premature stop codons , frameshift and donor splice site mutations , and mutations involving the nc1 domain .
the phenotype is associated with the early onset of end - stage renal failure ( esrf ) at -20 years of age , with 80% presenting with hearing loss and 40% presenting with ocular lesions .
the genotype is characterized by non - glycine - x - y missense , glycine - x - y substitutions involving exons 21 - 47 , and in - frame and acceptor splice site mutations .
the phenotype is associated with esrf at -26 years of age , with 65% presenting with hearing loss and 30% presenting with ocular lesions .
the phenotype is associated with esrf at -30 years of age , with 70% presenting with hearing loss , and 30% presenting with ocular lesions . according to this classification ,
our patients were classified as having moderate - severe as . taken together with no detection of skewed x ,
however , it would be clinically difficult to predict the prognosis of kidney function in women with as due to the lack of genotype - phenotype correlations and the intra - familial heterogeneity of the phenotype ( 2 ) .
therefore , our patients should be longitudinally followed while paying careful attention to a progressive increase in proteinuria , as previously suggested ( 2 ) .
there is no specific treatment available for as ; however , early intervention with angiotensin - converting enzyme inhibitors ( acei ) significantly delayed esrd progression and improved life expectancy in x - linked as ( 15,16 ) . in women with x - linked as , treatment with either acei or angiotensin ii receptor blockers ( arbs ) inhibits progression to esrd ( 15 ) .
therefore , it is recommended that patients with x - linked as are annually followed by a nephrologist , and treatment with aceis / arbs should be initiated for patients with microalbuminuria , proteinuria , or hypertension ( 15,17 ) . in our patients ,
acei ( enalapril malate ) was administered to patient 1 but not to patient 2 , in whom a renal biopsy was performed 3 months after the delivery of her second child .
we had to consider the potential high risk of fetal and newborn morbidity and mortality , which may be associated with acei / arb fetopathy in pregnant women ( 18,19 ) . in conclusion , the identification of mutations and characteristic pathological findings were useful in making the diagnosis of as .
for a close long - term follow - up , the early detection and treatment of women with x - linked as are important .
we herein report a novel mutation in a japanese family with an x - linked alport syndrome ( as ) mutation in col4a5 . 
 patient 1 was a 2-year - old japanese girl . 
 she and her mother ( patient 2 ) had a history of proteinuria and hematuria without renal dysfunction , deafness , or ocular abnormalities . 
 pathological findings were consistent with as , and a genetic analysis revealed that both patients had a heterozygous mutation ( c.2767g > c ) in exon 32 . in summary , the identification of mutations and characteristic pathological findings was useful in making a diagnosis of as . for a close long - term follow - up , the early detection and treatment of women with x - linked as are important 
 .