Throwing off the keratin chains: a potential therapy for hereditary podocytopathy.

In the current issue, Kuzmuk et al. offer a therapeutic option for patients with NPHS2 R138Q-associated nephrotic syndrome. For the first time in hereditary podocytopathies, this is offered by restoring the membrane targeting of a pathogenic protein. The idea that it is enough to liberate podocin from the trap of keratin 8, a key member of endoplasmic-reticulum-associated protein degradation complex, was brilliantly recognized based on former results obtained in cystic fibrosis.

Reassuring pregnancy outcomes in women with mild COL4A3-5-related disease (Alport syndrome) and genetic type of disease can aid personalized counseling.

Individualized pre-pregnancy counseling and antenatal care for women with chronic kidney disease (CKD) require disease-specific data. Here, we investigated pregnancy outcomes and long-term kidney function in women with COL4A3-5 related disease (Alport Syndrome, (AS)) in a large multicenter cohort. The ALPART-network (mAternaL and fetal PregnAncy outcomes of women with AlpoRT syndrome), an international collaboration of 17 centers, retrospectively investigated COL4A3-5 related disease pregnancies after the 20th week. Outcomes were stratified per inheritance pattern (X-Linked AS (XLAS)), Autosomal Dominant AS (ADAS), or Autosomal Recessive AS (ARAS)). The influence of pregnancy on estimated glomerular filtration rate (eGFR)-slope was assessed in 192 pregnancies encompassing 116 women (121 with XLAS, 47 with ADAS, and 12 with ARAS). Median eGFR pre-pregnancy was over 90ml/min/1.73m2. Neonatal outcomes were favorable: 100% live births, median gestational age 39.0 weeks and mean birth weight 3135 grams. Gestational hypertension occurred during 23% of pregnancies (reference: 'general' CKD G1-G2 pregnancies incidence is 4-20%) and preeclampsia in 20%. The mean eGFR declined after pregnancy but remained within normal range (over 90ml/min/1.73m2). Pregnancy did not significantly affect eGFR-slope (pre-pregnancy ß=-1.030, post-pregnancy ß=-1.349). ARAS-pregnancies demonstrated less favorable outcomes (early preterm birth incidence 3/11 (27%)). ARAS was a significant independent predictor for lower birth weight and shorter duration of pregnancy, next to the classic predictors (pre-pregnancy kidney function, proteinuria, and chronic hypertension) though missing proteinuria values and the small ARAS-sample hindered analysis. This is the largest study to date on AS and pregnancy with reassuring results for mild AS, though inheritance patterns could be considered in counseling next to classic risk factors. Thus, our findings support personalized reproductive care and highlight the importance of investigating kidney disease-specific pregnancy outcomes.

The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies.

Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease.

The dominant findings of a recessive man: from Mendel's kid pea to kidney.

The research of Mendel, born two centuries ago, still has many direct implications for our everyday clinical work. He introduced the terms "dominant" and "recessive" characters and determined their 3:1 ratio in the offspring of heterozygous "hybrid" plants. This distribution allowed calculation of the number of the phenotype-determining "elements," i.e., the alleles, and has been used ever since to prove the monogenic origin of a disorder. The Mendelian inheritance of monogenic kidney disorders is still of great help in distinguishing them from those with multifactorial origin in clinical practice. Inheritance of most monogenic kidney disorders fits to Mendel's observations: the equal contribution of the two parents and the complete penetrance or the direct correlation between the frequency of the recessive character and the degree of inbreeding. Nevertheless, beyond the truth of these basic concepts, several observations have expanded their genetic characteristics. The extreme genetic heterogeneity, the pleiotropy of the causal genes and the role of modifiers in ciliopathies, the digenic inheritance and parental imprinting in some tubulopathies, and the incomplete penetrance and eventual interallelic interactions in podocytopathies, reflect this expansion. For all these reasons, the transmission pattern in a natural setting may depend not only on the "character" but also on the causal gene and the variant. Mendel's passion for research combined with his modest personality and meticulous approach can still serve as an example in the work required to understand the non-Mendelian universe of genetics.

The pathogenic c.1171A>G (p.Arg391Gly) and c.2359G>A (p.Val787Ile) ABCC6 variants display incomplete penetrance causing pseudoxanthoma elasticum in a subset of individuals.

ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.

Identification of incompletely penetrant variants and interallelic interactions in autosomal recessive disorders by a population-genetic approach.

We aimed to identify incompletely penetrant (IP) variants and interallelic interactions in autosomal recessive disorders by a population-genetic approach. Genotype and clinical data were collected from 9038 patients of European origin with ASL, ATP7B, CAPN3, CFTR, CTNS, DHCR7, GAA, GALNS, GALT, IDUA, MUT, NPHS1, NPHS2, PAH, PKHD1, PMM2, or SLC26A4-related disorders. We calculated the relative allele frequency of each pathogenic variant (n = 1936) to the loss-of-function (LOF) variants of the corresponding gene in the patient ( ACptV/ACptLOF ) and the general population ( ACgnomADV/ACgnomADLOF ) and estimated the penetrance of each variant by calculating their ratio: (ACptV/ACptLOF)(ACgnomADV/ACgnomADLOF) (V/LOF ratio). We classified all variants as null or hypomorphic based on the associated clinical phenotype. We found 25 variants, 29% of the frequent 85 variants, to be underrepresented in the patient population (V/LOF ratio <30% with p < 7.22 × 10-5 ), including 22 novel ones in the ASL, CAPN3, CFTR, GAA, GALNS, PAH, and PKHD1 genes. In contrast to the completely penetrant variants (CP), the majority of the IP variants were hypomorphic (IP: 16/18, 88%; CP: 177/933, 19.0%; p = 5.12 × 10-10 ). Among them, only the NPHS2 R229Q variant was subject to interallelic interactions. The proposed algorithm identifies frequent IP variants and estimates their penetrance and interallelic interactions in large patient cohorts.

An international cohort study of autosomal dominant tubulointerstitial kidney disease due to REN mutations identifies distinct clinical subtypes.

There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct.

EPG5 c.1007A > G mutation in a sibling pair with rapidly progressing Vici syndrome.

We report on a sibling pair with the EPG5 c.1007A > G mutation who developed a severe form of Vici syndrome and died in infancy. The c.1007A > G (p.Gln336Arg) mutation, affecting the penultimate nucleotide and the splicing of exon 2 is the most common mutation of EPG5 and is typically associated with a less devastating prognosis: cardiomyopathy and cataract are less frequent consequences and the median survival time is 78 months compared to an overall median survival of 42 months. The less severe course related to c.1007A > G was formerly explained by the preserved canonical splicing in 25% of the transcripts. In contrast, we found the messenger RNA encoded by the c.1007A > G allele to be absent, explaining the severe course of the disease. This family provides another example of phenotypic variability related to a differential splicing.

Endoplasmic reticulum-retained podocin mutants are massively degraded by the proteasome.

Podocin is a key component of the slit diaphragm in the glomerular filtration barrier, and mutations in the podocin-encoding gene NPHS2 are a common cause of hereditary steroid-resistant nephrotic syndrome. A mutant allele encoding podocin with a p.R138Q amino acid substitution is the most frequent pathogenic variant in European and North American children, and the corresponding mutant protein is poorly expressed and retained in the endoplasmic reticulum both in vitro and in vivo To better understand the defective trafficking and degradation of this mutant, we generated human podocyte cell lines stably expressing podocinwt or podocinR138Q Although it has been proposed that podocin has a hairpin topology, we present evidence for podocinR138QN-glycosylation, suggesting that most of the protein has a transmembrane topology. We find that N-glycosylated podocinR138Q has a longer half-life than non-glycosylated podocinR138Q and that the latter is far more rapidly degraded than podocinwt Consistent with its rapid degradation, podocinR138Q is exclusively degraded by the proteasome, whereas podocinwt is degraded by both the proteasomal and the lysosomal proteolytic machineries. In addition, we demonstrate an enhanced interaction of podocinR138Q with calnexin as the mechanism of endoplasmic reticulum retention. Calnexin knockdown enriches the podocinR138Q non-glycosylated fraction, whereas preventing exit from the calnexin cycle increases the glycosylated fraction. Altogether, we propose a model in which hairpin podocinR138Q is rapidly degraded by the proteasome, whereas transmembrane podocinR138Q degradation is delayed due to entry into the calnexin cycle.

The mutation-dependent pathogenicity of NPHS2 p.R229Q: A guide for clinical assessment.

NPHS2, encoding podocin, is the major gene implicated in steroid-resistant nephrotic syndrome. Its c.686G>A, p.R229Q variant is the first human variant with a mutation-dependent pathogenicity; it is only pathogenic when trans-associated to specific mutations. Secondary to its high allele frequency in the European, South Asian, African, and Latino populations, its benign trans-associations can be accidentally identified in affected patients. Distinguishing pathogenic and benign p.R229Q associations can be challenging. In this paper, we present the currently known pathogenic and benign associations, and show that a rare p.R229Q association can be considered pathogenic if the variant in trans meets the following criteria; it affects the 270-351 residues and alters but does not disrupt the oligomerization, its p.R229Q association is found in a family with slowly progressing focal segmental glomerulosclerosis, but is expected to be rare in the general population (<1:106 ). We show that >15% of the p.R229Q associations identified so far in patients are benign.

C-terminal oligomerization of podocin mediates interallelic interactions.

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3' mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): principally through the first C-terminal helical region (H1, 283-313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332-348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r2 = 0.68, P = 9.2 × 10-32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.

Comprehensive genetic testing in children with a clinical diagnosis of ARPKD identifies phenocopies.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is genetically one of the least heterogeneous ciliopathies, resulting primarily from mutations of PKHD1. Nevertheless, 13-20% of patients diagnosed with ARPKD are found not to carry PKHD1 mutations by sequencing. Here, we assess whether PKHD1 copy number variations or second locus mutations explain these cases. METHODS: Thirty-six unrelated patients with the clinical diagnosis of ARPKD were screened for PKHD1 point mutations and copy number variations. Patients without biallelic mutations were re-evaluated and screened for second locus mutations targeted by the phenotype, followed, if negative, by clinical exome sequencing. RESULTS: Twenty-eight patients (78%) carried PKHD1 point mutations, three of whom on only one allele. Two of the three patients harbored in trans either a duplication of exons 33-35 or a large deletion involving exons 1-55. All eight patients without PKHD1 mutations (22%) harbored mutations in other genes (PKD1 (n = 2), HNF1B (n = 3), NPHP1, TMEM67, PKD1/TSC2). Perinatal respiratory failure, a kidney length > +4SD and early-onset hypertension increase the likelihood of PKHD1-associated ARPKD. A patient compound heterozygous for a second and a last exon truncating PKHD1 mutation (p.Gly4013Alafs*25) presented with a moderate phenotype, indicating that fibrocystin is partially functional in the absence of its C-terminal 62 amino acids. CONCLUSIONS: We found all ARPKD cases without PKHD1 point mutations to be phenocopies, and none to be explained by biallelic PKHD1 copy number variations. Screening for copy number variations is recommended in patients with a heterozygous point mutation.

[Trisomy 9p and clinical heterogeneity: case report of an unusual presentation]. / 9p triszómia és a klinikai sokszínuség: egy váratlan megjelenésu eset ismertetése.

Whole or partial trisomy of the short arm of chromosome 9 (9p) is considered to be one of the more frequent chromosome abnormalities compatible with life. The duplication may affect various organs, however the most common symptoms are certain specific facial dysmorphisms and abnormalities of the fingers, toes and nails. A one month old boy presented with failure to thrive, jaundice, ventricular septal defect (VSD) and dysmorphic face. He displayed symptoms of heart failure. The cardiologic examination revealed a significant VSD, hypoplasia of the aortic arch, pulmonary hypertension, decompensated circulatory failure and moderate left ventricle dysfunction. Routine cytogenetic analysis revealed a supernumerary marker chromosome. Fluorescence in situ hybridization (FISH) identified this as the short arm of chromosome 9. The child's karyotype was determined as 47,XY,+der(9)dup(9)(p10p24)dn. Due to his worsening condition and the high risk of the operation, it was decided to forego the procedure. After a short palliative care the child passed away. The child's clinical presentation and the uncharacteristic severity of his condition show that chromosome abnormalities involving duplicated genetic material are extremely heterogeneous. Thus treatment of each child should be individualized and may also involve difficult ethical considerations. Orv Hetil. 2018; 159(47): 1994-2000.

Selective measurement of α smooth muscle actin: why ß-actin can not be used as a housekeeping gene when tissue fibrosis occurs.

BACKGROUND: Prevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms. RESULTS: Real-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO. CONCLUSION: We developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur.

QMPSF is sensitive and specific in the detection of NPHP1 heterozygous deletions.

BACKGROUND: Nephronophthisis, an autosomal recessive nephropathy, is responsible for 10% of childhood chronic renal failure. The deletion of its major gene, NPHP1, with a minor allele frequency of 0.24% in the general population, is the most common mutation leading to a monogenic form of childhood chronic renal failure. It is challenging to detect it in the heterozygous state. We aimed to evaluate the sensitivity and the specificity of the quantitative multiplex PCR of short fluorescent fragments (QMPSF) in its detection. METHODS: After setting up the protocol of QMPSF, we validated it on 39 individuals diagnosed by multiplex ligation-dependent probe amplification (MLPA) with normal NPHP1 copy number (n=17), with heterozygous deletion (n=13, seven parents and six patients), or with homozygous deletion (n=9). To assess the rate of the deletions that arise from independent events, deleted alleles were haplotyped. RESULTS: The results of QMPSF and MLPA correlated perfectly in the identification of 76 heterozygously deleted and 56 homozygously deleted exons. The inter-experimental variability of the dosage quotient obtained by QMPSF was low: control, 1.05 (median; range, 0.86-1.33, n = 102 exons); heterozygous deletion, 0.51 (0.42-0.67, n = 76 exons); homozygous deletion, 0 (0-0, n = 56 exons). All patients harboring a heterozygous deletion were found to carry a hemizygous mutation. At least 15 out of 18 deletions appeared on different haplotypes and one deletion appeared de novo. CONCLUSIONS: The cost- and time-effective QMPSF has a 100% sensitivity and specificity in the detection of NPHP1 deletion. The potential de novo appearance of NPHP1 deletions makes its segregation analysis highly recommended in clinical practice.

Defects in the IFT-B component IFT172 cause Jeune and Mainzer-Saldino syndromes in humans.

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome.

Cerebello-oculo-renal syndrome (CORS), also called Joubert syndrome type B, and Meckel (MKS) syndrome belong to the group of developmental autosomal recessive disorders that are associated with primary cilium dysfunction. Using SNP mapping, we identified missense and truncating mutations in RPGRIP1L (KIAA1005) in both CORS and MKS, and we show that inactivation of the mouse ortholog Rpgrip1l (Ftm) recapitulates the cerebral, renal and hepatic defects of CORS and MKS. In addition, we show that RPGRIP1L colocalizes at the basal body and centrosomes with the protein products of both NPHP6 and NPHP4, known genes associated with MKS, CORS and nephronophthisis (a related renal disorder and ciliopathy). In addition, the RPGRIP1L missense mutations found in CORS individuals diminishes the interaction between RPGRIP1L and nephrocystin-4. Our findings show that mutations in RPGRIP1L can cause the multiorgan phenotypic abnormalities found in CORS or MKS, which therefore represent a continuum of the same underlying disorder.

Genotype-phenotype associations in WT1 glomerulopathy.

WT1 mutations cause a wide spectrum of renal and extrarenal manifestations. Here we evaluated disease prevalence, phenotype spectrum, and genotype-phenotype correlations of 61 patients with WT1-related steroid-resistant nephrotic syndrome relative to 700 WT1-negative patients, all with steroid-resistant nephrotic syndrome. WT1 patients more frequently presented with chronic kidney disease and hypertension at diagnosis and exhibited more rapid disease progression. Focal segmental glomerulosclerosis was equally prevalent in both cohorts, but diffuse mesangial sclerosis was largely specific for WT1 disease and was present in 34% of cases. Sex reversal and/or urogenital abnormalities (52%), Wilms tumor (38%), and gonadoblastoma (5%) were almost exclusive to WT1 disease. Missense substitutions affecting DNA-binding residues were associated with diffuse mesangial sclerosis (74%), early steroid-resistant nephrotic syndrome onset, and rapid progression to ESRD. Truncating mutations conferred the highest Wilms tumor risk (78%) but typically late-onset steroid-resistant nephrotic syndrome. Intronic (KTS) mutations were most likely to present as isolated steroid-resistant nephrotic syndrome (37%) with a median onset at an age of 4.5 years, focal segmental glomerulosclerosis on biopsy, and slow progression (median ESRD age 13.6 years). Thus, there is a wide range of expressivity, solid genotype-phenotype associations, and a high risk and significance of extrarenal complications in WT1-associated nephropathy. We suggest that all children with steroid-resistant nephrotic syndrome undergo WT1 gene screening.

Examination of Subbasal Nerve Plexus and Central Corneal Stromal Microstructure in Subjects With Congenital Aniridia, Using in Vivo Confocal Laser Scanning Microscopy.

PURPOSE: During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM. METHODS: 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually. RESULTS: The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia. CONCLUSIONS: Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.