Maddalena Gigante

The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families.

Objective—To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene. Methods and Results—Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-&bgr; high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL3 particle size were reduced in a gene–dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former. Conclusion—In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene–dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED.

Copy-Number Disorders Are a Common Cause of Congenital Kidney Malformations

We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10(-11)). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10(-58)). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay.

CD2AP mutations are associated with sporadic nephrotic syndrome and focal segmental glomerulosclerosis (FSGS)

BACKGROUND CD2-associated protein (CD2AP) is a crucial protein for the slit-diaphragm assembly and function. In spite of the fact that CD2AP knockout causes nephrotic syndrome in mice and the heterozygous +/- mouse is prone to proteinuria, little is known about the relevance of this molecule in human renal pathology. METHODS A total of 80 Italian patients with idiopathic nephrotic syndrome were enrolled and screened for changes in the CD2AP gene. A normal control group of 200 healthy donors was also studied. The coding region of the CD2AP gene was analysed by polymerase chain reaction, denaturing high-performance liquid chromatography and sequencing. Peripheral blood mononuclear cells from patients with CD2AP mutations and from healthy donors were isolated by the Ficoll-Hypaque gradient, and the CD2/CD2AP interaction was studied on T-lymphocytes by confocal laser scanning microscopy analysis. The expression levels of CD2AP, nephrin and podocin proteins were evaluated by indirect immunofluorescence on renal biopsies from a patient with p.delGlu525 mutation and from control subjects. Moreover, the effect of the p.K301M mutation on cell viability was evaluated by flow cytometry and annexin V/propidium iodide staining. RESULTS Three heterozygous mutations (c.904A>T; c.1120A>G; c.1573delAGA) producing respectively aminoacidic changes (p.K301M, p.T374A) or a deletion in functional domains (p.delGlu525) were found in three unrelated patients. One (p.K301M) produced a lysine to methionine change in the third interactive SH3 domain (position 301) and resulted in the defective CD2-CD2AP interaction and clustering; the other (c.1573delAGA) caused the deletion of the glutamic acid in position 525 in the COOH-terminal region of binding with nephrin and was associated with down-modulation of CD2AP, podocin and nephrin glomerular expression. CONCLUSIONS Our findings suggest that CD2AP mutations modify the interaction with CD2 in lymphocytes and alter the composition of the renal slit diaphragm.

Mutations in DSTYK and Dominant Urinary Tract Malformations

BACKGROUND Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood. METHODS We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies. RESULTS Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine-threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases. CONCLUSIONS We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.).

Exome sequencing identified MYO1E and NEIL1 as candidate genes for human autosomal recessive steroid-resistant nephrotic syndrome

To identify gene loci associated with steroid-resistant nephrotic syndrome (SRNS), we utilized homozygosity mapping and exome sequencing in a consanguineous pedigree with three affected siblings. High-density genotyping identified three segments of homozygosity spanning 33.6 Mb on chromosomes 5, 10, and 15 containing 296 candidate genes. Exome sequencing identified two homozygous missense variants within the chromosome 15 segment; an A159P substitution in myosin 1E (MYO1E), encoding a podocyte cytoskeletal protein; and an E181K substitution in nei endonuclease VIII-like 1 (NEIL1), encoding a base-excision DNA repair enzyme. Both variants disrupt highly conserved protein sequences and were absent in public databases, 247 healthy controls, and 286 patients with nephrotic syndrome. The MYO1E A159P variant is noteworthy, as it is expected to impair ligand binding and actin interaction in the MYO1E motor domain. The predicted loss of function is consistent with the previous demonstration that Myo1e inactivation produces nephrotic syndrome in mice. Screening 71 additional patients with SRNS, however, did not identify independent NEIL1 or MYO1E mutations, suggesting larger sequencing efforts are needed to uncover which mutation is responsible for the phenotype. Our findings demonstrate the utility of exome sequencing for rapidly identifying candidate genes for human SRNS.

Sirolimus and proteinuria in renal transplant patients: evidence for a dose-dependent effect on slit diaphragm-associated proteins.

Background. The mechanisms underlying the development of proteinuria in renal-transplant recipients converted from calcineurin inhibitors to sirolimus are still unknown. Methods. This is a single-center cohort study. One hundred ten kidney transplant recipients converted from calcineurin inhibitors to sirolimus in the period from September 2000 to December 2005 were included in the study. All patients underwent a graft biopsy before conversion (T0) and a second protocol biopsy 2 years thereafter (T2), according to our standard clinical protocol. On the basis of the changes observed in proteinuria between T0 and T2 (median 70%), the patients were divided into two groups: group I (<70%) and group II (>70%). The authors blinded the sirolimus blood trough levels. We investigated in vivo the effects of sirolimus on nephrin, podocin, CD2ap, and actin protein expression. Slit diaphragm (SD)-associated protein expressions were evaluated in T0 and T2 biopsies. The same analysis was performed in cultured human podocytes treated with different doses of sirolimus (5, 10, 20, and 50 ng/mL). Results. The SD protein expression in group II T2 biopsies was significantly reduced compared with the T0 biopsies and with T2 group I biopsies. In addition, sirolimus blood trough levels directly and significantly correlated with the SD protein expression at T2 graft biopsies. Group II patients presented significantly higher sirolimus blood levels than group I. In vitro study confirmed that sirolimus effect on podocytes was dose dependent. Conclusions. Our data suggest that sirolimus-induced proteinuria may be a dose-dependent effect of the drug on key podocyte structures.

TRPC6 Mutations in Children with Steroid-Resistant Nephrotic Syndrome and Atypical Phenotype

BACKGROUND AND OBJECTIVES Mutations in the TRPC6 gene have been recently identified as the cause of late-onset autosomal-dominant focal segmental glomerulosclerosis (FSGS). To extend the screening, we analyzed TRPC6 in 33 Italian children with sporadic early-onset SRNS and three Italian families with adult-onset FSGS. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS TRPC6 mutation analysis was performed through PCR and sequencing. The effects of the detected amino acid substitutions were analyzed by bioinformatics tools and functional in vitro studies. The expression levels of TRPC6 and nephrin proteins were evaluated by confocal microscopy. RESULTS Three heterozygous missense mutations (c.374A>G_p.N125S, c.653A>T_p.H218L, c.2684G>T_p.R895L) were identified. The first new mutation, p.H218L, was found in a 18-year-old boy who presented a severe form of FSGS at the age of 8 years. The second, p.R895L, a new de novo mutation, was identified in a girl with collapsing glomerulosclerosis at the age of 2 years. The former mutation, p.N125S, was found in two siblings with early-onset steroid-resistant nephrotic syndrome (SRNS) at the ages of 4 and 14 years. Renal immunofluorescence revealed upregulated expression of TRPC6 and loss of nephrin in glomeruli. The intracellular calcium concentrations were significantly higher in the cells expressing all mutant TRPC6 channels compared with cells expressing wild-type TRPC6. CONCLUSIONS Our findings suggest that TRPC6 variants can also be detected in children with early-onset and sporadic SRNS (4 of 33 patients). Moreover, in one patient a new de novo TRPC6 mutation was associated with a rare severe form of childhood collapsing glomerulosclerosis with rapid progression to uremia.

WT1 mutations in nephrotic syndrome revisited. High prevalence in young girls, associations and renal phenotypes

WT1 mutations have been considered a rare cause of nephrotic syndrome but recent reports challenge this assumption. Exclusion of inherited forms is a basic point in any therapeutic strategy to nephrotic syndrome since they do not respond to drugs. We screened for WT1 mutations in 200 patients with nephrotic syndrome: 114 with steroid resistance (SRNS) and 86 with steroid dependence (SDNS) for whom other inherited forms of nephrotic syndrome (NPHS2, CD2AP) had been previously excluded. Three girls out of 32 of the group with steroid resistance under 18 years presented classical WT1 splice mutations (IVS9+5G>A, IVS9+4C>T) of Frasier syndrome. Another one presented a mutation coding for an amino acid change (D396N) at exon 9 that is typical of Denys-Drash syndrome. All presented resistance to drugs and developed end stage renal failure within 15 years. Two girls of the Frasier group presented a 46 XY karyotype with streak gonads while one was XX and had normal gonad morphology. In the two cases with IVS9+5G>A renal pathology was characterized by capillary wall thickening with deposition of IgG and C3 in one that was interpreted as a membrane pathology. Foam cells were diffuse in tubule-interstitial areas. In conclusion, WT1 splice mutations are not rare in females under 18 years with SRNS. This occurs in absence of a clear renal pathology picture and frequently in absence of phenotype change typical of Frasier syndrome. In adults and children with SDNS, screening analysis is of no clinical value. WT1 hot spot mutation analysis should be routinely done in children with SRNS; if the molecular screening anticipates any further therapeutic approach it may modify the long term therapeutic strategy.

NPHS2 mutations in steroid-resistant nephrotic syndrome: A mutation update and the associated phenotypic spectrum

Mutations in the NPHS2 gene encoding podocin are implicated in an autosomal‐recessive form of nonsyndromic steroid‐resistant nephrotic syndrome in both pediatric and adult patients. Patients with homozygous or compound heterozygous mutations commonly present with steroid‐resistant nephrotic syndrome before the age of 6 years and rapidly progress to end‐stage kidney disease with a very low prevalence of recurrence after renal transplantation. Here, we reviewed all the NPHS2 mutations published between October 1999 and September 2013, and also all novel mutations identified in our personal cohort and in international genetic laboratories. We identified 25 novel pathogenic mutations in addition to the 101 already described. The mutations are distributed along the entire coding region and lead to all kinds of alterations including 53 missense, 17 nonsense, 11 small insertions, 26 small deletions, 16 splicing, two indel mutations, and one mutation in the stop codon. In addition, 43 variants were classified as variants of unknown significance, as these missense changes were exclusively described in the heterozygous state and/or considered benign by prediction software. Genotype–phenotype analyses established correlations between specific variants and age at onset, ethnicity, or clinical evolution. We created a Web database using the Leiden Open Variation Database (www.lovd.nl/NPHS2) software that will allow the inclusion of future reports.

Clinical Features and Long-Term Outcome of Nephrotic Syndrome Associated with Heterozygous NPHS1 and NPHS2 Mutations

BACKGROUND AND OBJECTIVES Mutations in nephrin (NPHS1) and podocin (NPHS2) genes represent a major cause of idiopathic nephrotic syndrome (NS) in children. It is not yet clear whether the presence of a single mutation acts as a modifier of the clinical course of NS. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We reviewed the clinical features of 40 patients with NS associated with heterozygous mutations or variants in NPHS1 (n = 7) or NPHS2 (n = 33). Long-term renal survival probabilities were compared with those of a concurrent cohort with idiopathic NS. RESULTS Patients with a single mutation in NPHS1 received a diagnosis before those with potentially nongenetic NS and had a good response to therapies. Renal function was normal in all cases. For NPHS2, six patients had single heterozygous mutations, six had a p.P20L variant, and 21 had a p.R229Q variant. Age at diagnosis and the response to drugs were comparable in all NS subgroups. Overall, they had similar renal survival probabilities as non-NPHS1/NPHS2 cases (log-rank chi(2) 0.84, P = 0.656) that decreased in presence of resistance to therapy (P < 0.001) and in cases with renal lesions of glomerulosclerosis and IgM deposition (P < 0.001). Cox regression confirmed that the only significant predictor of dialysis was resistance to therapy. CONCLUSIONS Our data indicate that single mutation or variant in NPHS1 and NPHS2 does not modify the outcome of primary NS. These patients should be treated following consolidated schemes and have good chances for a good long-term outcome.