Unbiased next generation sequencing analysis confirms the existence of autosomal dominant Alport syndrome in a relevant fraction of cases.

The mode of inheritance of Alport syndrome (ATS) has long been controversial. In 1927, the disease was hypothesized as a dominant condition in which males were more severely affected than females. In 1990, it was considered an X-linked (XL) semidominant condition, due to COL4A5 mutations. Later on, a rare autosomal recessive (AR) form due to COL4A3/COL4A4 mutations was identified. An autosomal dominant (AD) form was testified more recently by the description of some large pedigrees but the real existence of this form is still questioned by many and its exact prevalence is unknown. The introduction of next generation sequencing (NGS) allowed us to perform an unbiased simultaneous COL4A3-COL4A4-COL4A5 analysis in 87 Italian families (273 individuals) with clinical suspicion of ATS. In 48 of them (55%), a mutation in one of the three genes was identified: the inheritance was XL semidominant in 65%, recessive in 4% and most interestingly AD in 31% (15 families). The AD form must therefore be seriously taken into account in all pedigrees with affected individuals in each generation. Furthermore, a high frequency of mutations (>50%) was shown in patients with only 1 or 2 clinical criteria, suggesting NGS as first-level analysis in cases with a clinical suspicion of ATS.

Two patients with history of STEC-HUS, posttransplant recurrence and complement gene mutations.

Hemolytic uremic syndrome (HUS) is a disease of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. About 90% of cases are secondary to infections by Escherichia coli strains producing Shiga-like toxins (STEC-HUS), while 10% are associated with mutations in genes encoding proteins of complement system (aHUS). We describe two patients with a clinical history of STEC-HUS, who developed end-stage renal disease (ESRD) soon after disease onset. They received a kidney transplant but lost the graft for HUS recurrence, a complication more commonly observed in aHUS. Before planning a second renal transplantation, the two patients underwent genetic screening for aHUS-associated mutations that revealed the presence of a heterozygous CFI mutation in patient #1 and a heterozygous MCP mutation in patient #2, and also in her mother who donated the kidney. This finding argues that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation. Complement gene sequencing should be performed before kidney transplantation in patients who developed ESRD following STEC-HUS since they may be undiagnosed cases of aHUS, at risk of posttransplant recurrence. Furthermore, genetic analysis of donors is mandatory before living-related transplantation to exclude carriers of HUS-predisposing mutations.

Genetic analysis of the complement factor H related 5 gene in haemolytic uraemic syndrome.

Several mutations in the CFH gene have been described in non-Shiga-toxin-associated haemolytic uraemic syndrome (non-Stx-HUS), a rare syndrome characterized by haemolytic anaemia, thrombocytopenia and acute renal failure. Mutations in genes encoding other complement regulatory proteins, membrane cofactor protein (CD46) and complement factor I (CFI), were also involved in the pathogenesis of the disease. Anyway, mutations in the three genes account for no more than 50% of cases of non-Stx-HUS. Human complement factor H related 5 (CFHR5) is a recently characterised member of the human complement factor H (CFH) family that has been found as a component of immune deposits in human kidney with sclerotic lesions from different causes. CFHR5 possesses cofactor activity and has been proposed to play a role in complement regulation in the glomerulus. We screened CFHR5 gene for variations potentially involved in the aetiology of HUS. Forty-five patients with HUS and 80 controls were analysed. Altogether, 5 genetic variants in CFHR5 were found in overall 9/45 HUS patients and in 4/80 controls. Statistical analysis showed that allelic variants in CFHR5 were prefentially associated with HUS. Based on these data, we conclude that, though not causative, CFHR5 genetic alterations may play a secondary role in the pathogenesis of HUS.

Rapid DNA-based prenatal diagnosis by genetic linkage in three families with Alport's syndrome.

Alport's syndrome (AS) is a clinically and genetically heterogeneous progressive inherited glomerulonephritis characterized by hematuria, sensorineural hearing loss, ocular lesions, and specific alterations of the glomerular basement membrane. Typically, AS shows an X-linked dominant pattern of inheritance, with mutations affecting the collagen type IV alpha5 chain gene (COL4A5) at Xq22. Rarely, AS is caused in some families by mutations of the COL4A3/A4 genes on chromosome 2q, showing an autosomal recessive transmission. Very few families have been described with possible autosomal dominant AS, but no mutations in any of the COL4 genes have been found. We describe three unrelated families affected with a severe AS phenotype in which DNA-based prenatal diagnosis by linkage analysis was made in fetuses at risk for the disease. In two families, the pedigree structure and the clinical picture were consistent with typical X-linked dominant AS. In these families, autosomal inheritance was also ruled out molecularly. In one family, despite careful clinical and molecular evaluation, the mode of transmission could not be firmly established. We used tightly linked and intragenic COL4A5 markers, as well as COL4A3/A4-linked markers. A chromosome Y-specific marker for fetal sex determination was simultaneously used. In all the families, before the fetal analysis, the putative at-risk X haplotype was identified with high diagnostic accuracy. We diagnosed a healthy male fetus in one family, and female but carrier fetuses in the other two kindreds, who decided not to terminate their pregnancies. We used rapid nonisotopic polymerase chain reaction-based methods, and the results were available within 2 to 3 days. The genetic results significantly affected the reproductive decisions of the parents. This report illustrates the application of genetic linkage analysis as an additional tool for molecular diagnosis in AS, and also addresses the issue of the attitudes of the families toward prenatal testing. To our knowledge, prenatal diagnosis of AS using a genetic linkage approach has not been previously reported.

Detection of mutations in human genes by a new rapid method: cleavage fragment length polymorphism analysis (CFLPA).

Cleavage fragment length polymorphism analysis with silver staining visualization (CFLPA-SS) was used for the detection of mutations previously detected by single strand conformation (SSCA) or heteroduplex analyses (HA); in order to assess this new method for mutation screening. The analysed mutations include single nucleotide transitions, transversions, a deletion and a duplication in the following genes: CFTR (cystic fibrosis transmembrane conductance regulator), COL4A5 (collagen type 4 alpha 5 chain), PKD1 (polycystic kidney disease 1), and FGFR3 (fibroblast growth factor receptor 3). Peripheral blood leukocyte genomic DNA was isolated, amplified by polymerase chain reaction (PCR), and then cleaved by Cleavase I enzyme at different temperatures. Electrophoresis of the fragments on denaturing polyacrylamide gel was followed by silver staining for 1 min. All 13 mutations investigated were reproducibly detected. CFLPA-SS proved to be a reliable method for mutation detection and more rapid than SSCA and HA.

A common polymorphism in exon 46 of the human autosomal dominant polycystic kidney disease 1 gene (PKD1).

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common single gene diseases in humans. We have identified a synonymous T to C transition polymorphism in exon 46 of the PKD1 gene (12838T-->C, Pro4209Pro). The polymorphism was present with similar frequencies in ADPKD patients and unaffected individuals. The heterozygosity, determined in 89 Italian individuals, was 0.347. The frequency of the rarer allele was 0.222. This polymorphism is easy to determine as it abolishes a naturally occurring Ddel restriction site. The availability of an additional intragenic marker in the PKD1 gene will improve the accuracy of linkage studies in ADPKD families.

Autosomal dominant polycystic kidney disease (ADPKD) in an Italian family carrying a novel nonsense mutation and two missense changes in exons 44 and 45 of the PKD1 Gene.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the 3' unique region of the PKD1 gene, using heteroduplex DNA analysis. Novel aberrant bands were detected in 3 patients from the same family. DNA sequencing showed a C to T transition in exon 44 (C12269T), resulting in a premature stop codon (R4020X), predicted to impair the synthesis of the putative intracytoplasmic C-terminus tail of the PKD1 protein, polycystin. The mutation also generates a novel DdeI restriction site, and the abnormal restriction pattern was observed both on genomic DNA and on cDNA from the affected relatives, indicating that this is indeed the pathogenetic molecular lesion. Reverse transcriptase-polymerase chain reaction (RT-PCR) performed on lymphocyte mRNA showed that the mutant transcript is normally present and stable. No aberrantly spliced mRNAs were detected. Interestingly, the mutant PKD1 chromosome in this family also bears two missense mutations downstream (A12341G and C12384T), not found in the other ADPKD families studied.

Detection of two different nonsense mutations in exon 44 of the PKD1 gene in two unrelated Italian families with severe autosomal dominant polycystic kidney disease.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the PKD1 gene. We used PCR, heteroduplex and single-strand conformation polymorphism DNA analysis, and automated DNA sequencing for exons 35, 36, 38, 44 and 45. We detected abnormal heteroduplexes in affected individuals from two unrelated families with clinically severe ADPKD phenotype. These changes were absent in other, unaffected members, as well as in the probands of the other families studied. DNA sequencing revealed in both cases different C to T transitions in exon 44, which created premature stop codons. Both mutations altered restriction sites, and the abnormal patterns were observed in all the affected family members. RT-PCR performed on lymphocyte mRNA showed that both the mutant and the normal transcript are represented. To our knowledge these are the first nonsense mutations described in the PKD1 gene.

A novel nonsense mutation in the PKD1 gene (C3817T) is associated with autosomal dominant polycystic kidney disease (ADPKD) in a large three-generation Italian family.

We have looked for disease-causing mutations in the PKD1 gene in 20 unrelated ADPKD probands from northern Italy, all members of families in which our previous studies had indicated linkage to PKD1. Using PCR with primer pairs located in the 3' unique region of the gene and heteroduplex DNA analysis, we have detected novel aberrant bands in five affected individuals from the same family, which were absent in 13 other unaffected family members. Cloning and automated DNA sequencing revealed a C to T transition at nucleotide position 3817 of the published cDNA sequence, which created a premature stop codon. The mutation destroyed a MspA1I restriction site, and the abnormal restriction pattern was observed on genomic DNA from all the affected family members. RT-PCR and restriction analysis performed on peripheral white blood cell mRNA showed that in the affected members, both the mutant and the normal transcript are represented. This mutation was not found in the probands of the other families studied. To our knowledge, this is the first nonsense mutation described in the PKD1 gene.