Karin Dahan

NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.

Familial idiopathic nephrotic syndromes represent a heterogeneous group of kidney disorders, and include autosomal recessive steroid-resistant nephrotic syndrome, which is characterized by early childhood onset of proteinuria, rapid progression to end-stage renal disease and focal segmental glomerulosclerosis. A causative gene for this disease, NPHS2, was mapped to 1q25–31 and we report here its identification by positional cloning. NPHS2 is almost exclusively expressed in the podocytes of fetal and mature kidney glomeruli, and encodes a new integral membrane protein, podocin, belonging to the stomatin protein family. We found ten different NPHS2 mutations, comprising nonsense, frameshift and missense mutations, to segregate with the disease, demonstrating a crucial role for podocin in the function of the glomerular filtration barrier.

A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.

The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and γ-glutamyltranspeptidase (γGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

A Cluster of Mutations in the UMOD Gene Causes Familial Juvenile Hyperuricemic Nephropathy with Abnormal Expression of Uromodulin

Familial juvenile hyperuricemic nephropathy (FJHN [MIM 162000]) is an autosomal-dominant disorder characterized by abnormal tubular handling of urate and late development of chronic interstitial nephritis leading to progressive renal failure. A locus for FJHN was previously identified on chromosome 16p12 close to the MCKD2 locus, which is responsible for a variety of autosomal-dominant medullary cystic kidney disease (MCKD2). UMOD, the gene encoding the Tamm-Horsfall/uromodulin protein, maps within the FJHN/MCKD2 critical region. Mutations in UMOD were recently reported in nine families with FJHN/MCKD2 disease. A mutation in UMOD has been identified in 11 FJHN families (10 missense and one in-frame deletion)-10 of which are novel-clustering in the highly conserved exon 4. The consequences of UMOD mutations on uromodulin expression were investigated in urine samples and renal biopsies from nine patients in four families. There was a markedly increased expression of uromodulin in a cluster of tubule profiles, suggesting an accumulation of the protein in tubular cells. Consistent with this observation, urinary excretion of wild-type uromodulin was significantly decreased. The latter findings were not observed in patients with FJHN without UMOD mutations. In conclusion, this study points to a mutation clustering in exon 4 of UMOD as a major genetic defect in FJHN. Mutations in UMOD may critically affect the function of uromodulin, resulting in abnormal accumulation within tubular cells and reduced urinary excretion.

Transcriptional and Functional Analyses of SLC12A3 Mutations: New Clues for the Pathogenesis of Gitelman Syndrome

Gitelman syndrome (GS) is a recessive salt-losing tubulopathy that is caused by mutations in the SLC12A3 gene that encodes the sodium-chloride co-transporter (NCC). GS is characterized by significant inter- and intrafamilial phenotype variability, with early onset and/or severe clinical manifestations in some patients. No correlations between the disease variability and the position/nature of SLC12A3 mutations have been investigated thus far. In this study, extensive mutational analyses of SLC12A3 were performed in 27 patients with GS, including genomic DNA sequencing, multiplex ligation-dependent probe amplification, cDNA analysis, and quantification of allele-specific transcripts, in parallel with functional analyses in Xenopus laevis oocytes and detailed phenotyping. Twenty-six SLC12A3 mutations were identified in 25 patients with GS, including eight novel (detection rate 80%). Transcript analysis demonstrated that splicing mutations of SLC12A3 lead to frameshifted mRNA subject to degradation by nonsense-mediated decay. Heterologous expression documented a novel class of NCC mutants with defective intrinsic transport activity. A subgroup of patients presented with early onset, growth retardation, and/or detrimental manifestations, confirming the potential severity of GS. The mutations that were associated with a severe presentation were the combination at least for one allele of a missplicing resulting in a truncated transcript that was downregulated by nonsense-mediated decay or a nonfunctional, cell surface-absent mutant. The most recurrent mutation on the second allele was a newly described NCC mutant that affected the functional properties of the co-transporter. These data suggest that the nature/position of SLC12A3 mutation, combined with male gender, is a determinant factor in the severity of GS and provide new insights in the underlying pathogenic mechanisms of the disease.

Spectrum of mutations in Gitelman syndrome

Gitelmans syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by mutations in the SLC12A3 gene, which encodes the thiazide-sensitive NaCl cotransporter (NCC). Because 18 to 40% of suspected GS patients carry only one SLC12A3 mutant allele, large genomic rearrangements may account for unidentified mutations. Here, we directly sequenced genomic DNA from a large cohort of 448 unrelated patients suspected of having GS. We found 172 distinct mutations, of which 100 were unreported previously. In 315 patients (70%), we identified two mutations; in 81 patients (18%), we identified one; and in 52 patients (12%), we did not detect a mutation. In 88 patients, we performed a search for large rearrangements by multiplex ligation-dependent probe amplification (MLPA) and found nine deletions and two duplications in 24 of the 51 heterozygous patients. A second technique confirmed each rearrangement. Based on the breakpoints of seven deletions, nonallelic homologous recombination by Alu sequences and nonhomologous end-joining probably favor these intragenic deletions. In summary, missense mutations account for approximately 59% of the mutations in Gitelmans syndrome, and there is a predisposition to large rearrangements (6% of our cases) caused by the presence of repeated sequences within the SLC12A3 gene.

Mutations in SACS cause atypical and late-onset forms of ARSACS

Background: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a complex neurodegenerative disorder caused by mutations in SACS. The phenotype consists of a childhood-onset triad of cerebellar ataxia, peripheral neuropathy, and pyramidal tract signs. Objective: To provide more insight into the prevalence of SACS mutations and the variability of the associated phenotype. Methods: Mutation screening of SACS by direct sequencing and multiplex amplicon quantification for detection of intragenic copy number variations in a cohort of 85 index patients with phenotypes suggestive for ARSACS. Additional short tandem repeat (STR) marker analysis was performed for haplotype sharing. Results: In 11 families,18 new SACS mutations were found (12.9% of total cohort). Five patients displayed onset ages in adulthood, a feature not known to be associated with ARSACS. The remaining index patients displayed a classic early onset phenotype. Initial phenotypic presentation was atypical in several patients, obscuring the clinical diagnosis. A founder mutation in SACS was identified in 3 Belgian families. In one isolated patient, an intragenic SACS deletion of exons 3–5 was detected. Partial SACS deletions were not previously described. Conclusions: In this study, we enlarge the ARSACS phenotype and the underlying genetic spectrum of SACS mutations. Patients with ARSACS are more common than previously known and risk underdiagnosis due to late onset age and unusual presentation.

Phenotype and Outcome in Hereditary Tubulointerstitial Nephritis Secondary to UMOD Mutations

BACKGROUND UMOD mutations cause familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease (MCKD), although these phenotypes are nonspecific. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We reviewed cases of UMOD mutations diagnosed in the genetic laboratories of Necker Hospital (Paris, France) and of Université Catholique de Louvain (Brussels, Belgium). We also analyzed patients with MCKD/FJHN but no UMOD mutation. To determine thresholds for hyperuricemia and uric-acid excretion fraction (UAEF) according to GFR, these parameters were analyzed in 1097 patients with various renal diseases and renal function levels. RESULTS Thirty-seven distinct UMOD mutations were found in 109 patients from 45 families, all in exon 4 or 5 except for three novel mutations in exon 8. Median renal survival was 54 years. The type of mutation had a modest effect on renal survival, and intrafamilial variability was high. Detailed data available in 70 patients showed renal cysts in 24 (34.3%) of nonspecific localization in most patients. Uricemia was >75th percentile in 31 (71.4%) of 42 patients not under dialysis or allopurinol therapy. UAEF (n = 27) was <75th percentile in 70.4%. Among 136 probands with MCKD/FJHN phenotype, UMOD mutation was found in 24 (17.8%). Phenotype was not accurately predictive of UMOD mutation. Six probands had HNF1B mutations. CONCLUSIONS Hyperuricemia disproportionate to renal function represents the hallmark of renal disease caused by UMOD mutation. Renal survival is highly variable in patients with UMOD mutation. Our data also add novel insights into the interpretation of uricemia and UAEF in patients with chronic kidney diseases.

A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis

Ductal plate malformations (DPMs) are developmental anomalies considered to result from lack of ductal plate remodeling during bile duct morphogenesis. In mice, bile duct development is initiated by the formation of primitive ductal structures lined by two cell types, namely ductal plate cells and hepatoblasts. During ductal plate remodeling, the primitive ductal structures mature to ducts as a result from differentiation of the ductal plate cells and hepatoblasts to cholangiocytes. Here, we report this process is conserved in human fetal liver. These findings prompted us to evaluate how DPMs develop in three mouse models, namely mice with livers deficient in hepatocyte nuclear factor 6 (HNF6), HNF1β, or cystin‐1 (cpk [congenital polycystic kidney] mice). Human liver from a patient with a HNF1B/TCF2 mutation, and from fetuses affected with autosomal recessive polycystic kidney disease (ARPKD) were also analyzed. Despite the epistatic relationship between HNF6, HNF1β, and cystin‐1, the three mouse models displayed distinct morphogenic mechanisms of DPM. They all developed biliary cysts lined by cells with abnormal apicobasal polarity. However, the absence of HNF6 led to an early defect in ductal plate cell differentiation. In HNF1β‐deficient liver, maturation of the primitive ductal structures was impaired. Normal differentiation and maturation but abnormal duct expansion was apparent in cpk mouse livers and in human fetal ARPKD. Conclusion: DPM is the common endpoint of distinct defects initiated at distinct stages of bile duct morphogenesis. Our observations provide a new pathogenic classification of DPM. (HEPATOLOGY 2011;)

Improving Mutation Screening in Familial Hematuric Nephropathies through Next Generation Sequencing

Alport syndrome is an inherited nephropathy associated with mutations in genes encoding type IV collagen chains present in the glomerular basement membrane. COL4A5 mutations are associated with the major X-linked form of the disease, and COL4A3 and COL4A4 mutations are associated with autosomal recessive and dominant forms (thought to be involved in 15% and 1%-5% of the families, respectively) and benign familial hematuria. Mutation screening of these three large genes is time-consuming and expensive. Here, we carried out a combination of multiplex PCR, amplicon quantification, and next generation sequencing (NGS) analysis of three genes in 101 unrelated patients. We identified 88 mutations and 6 variations of unknown significance on 116 alleles in 83 patients. Two additional indel mutations were found only by secondary Sanger sequencing, but they were easily identified retrospectively with the web-based sequence visualization tool Integrative Genomics Viewer. Altogether, 75 mutations were novel. Sequencing the three genes simultaneously was particularly advantageous as the mode of inheritance could not be determined with certainty in many instances. The proportion of mutations in COL4A3 and COL4A4 was notably high, and the autosomal dominant forms of Alport syndrome appear more frequently than reported previously. Finally, this approach allowed the identification of large COL4A3 and COL4A4 rearrangements not described previously. We conclude that NGS is efficient, reduces screening time and cost, and facilitates the provision of appropriate genetic counseling in Alport syndrome.

Evidence of digenic inheritance in Alport syndrome

Background Alport syndrome is a clinically heterogeneous, progressive nephropathy caused by mutations in collagen IV genes, namely COL4A3 and COL4A4 on chromosome 2 and COL4A5 on chromosome X. The wide phenotypic variability and the presence of incomplete penetrance suggest that a simple Mendelian model cannot completely explain the genetic control of this disease. Therefore, we explored the possibility that Alport syndrome is under digenic control. Methods Using massively parallel sequencing, we identified 11 patients who had pathogenic mutations in two collagen IV genes. For each proband, we ascertained the presence of the same mutations in up to 12 members of the extended family for a total of 56 persons studied. Results Overall, 23 mutations were found. Individuals with two pathogenic mutations in different genes had a mean age of renal function deterioration intermediate with respect to the autosomal-dominant form and the autosomal-recessive one, in line with molecule stoichiometry of the disruption of the type IV collagen triple helix. Conclusions Segregation analysis indicated three possible digenic segregation models: (i) autosomal inheritance with mutations on different chromosomes, resembling recessive inheritance (five families); (ii) autosomal inheritance with mutations on the same chromosome resembling dominant inheritance (two families) and (iii) unlinked autosomal and X-linked inheritance having a peculiar segregation (four families). This pedigree analysis provides evidence for digenic inheritance of Alport syndrome. Clinical geneticists and nephrologists should be aware of this possibility in order to more accurately assess inheritance probabilities, predict prognosis and identify other family members at risk.