Panayiota Demosthenous

Familial C3 Glomerulopathy Associated with CFHR5 Mutations: Clinical Characteristics of 91 Patients in 16 Pedigrees

BACKGROUND AND OBJECTIVES Complement factor H and related proteins (CFHR) are key regulators of the alternative complement pathway, where loss of function mutations lead to a glomerulopathy with isolated mesangial C3 deposits without immunoglobulins. Gale et al. (12) reported on 26 patients with the first familial, hematuric glomerulopathy caused by a founder mutation in the CFHR5 gene in patients of Cypriot descent living in the United Kingdom. CFHR5 nephropathy is clinically characterized by continuous microscopic hematuria whereas some patients present with additional episodes of synpharyngitic macrohematuria, associated with infection and pyrexia. A subgroup of patients, particularly men, develop additional proteinuria, hypertension, and chronic renal disease or ESRD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We herewith expand significantly on the study by Gale et al., reporting on histologic, molecular, and clinical findings in 91 patients from 16 families with the same founder mutation. RESULTS Eighty-two patients (90%) exhibited microscopic hematuria; 51 (62%), exhibited only microscopic hematuria, whereas the remaining 31 additionally had proteinuria (38%); 28 proteinuric patients developed chronic renal failure (CRF). Among carriers of CFHR5 mutation aged >50 years, 80% of the men and 21% of the women developed CRF; 18 developed ESRD (14 men [78%], 4 women [22%]). CONCLUSIONS The diagnosis of CFHR5-related, isolated C3 glomerulopathy was established in 2009 using newly described mutation analysis after decades of follow-up with unclear diagnoses, occasionally confused with IgA nephropathy. This larger patient cohort establishes the clinical course, significant variable expressivity, and marked gender difference regarding the development of CRF and ESRD.

Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies.

X-linked Alport syndrome in Hellenic families: phenotypic heterogeneity and mutations near interruptions of the collagen domain in COL4A5.

Demosthenous P, Voskarides K, Stylianou K, Hadjigavriel M, Arsali M, Patsias C, Georgaki E, Zirogiannis P, Stavrou C, Daphnis E, Pierides A, Deltas C, Hellenic Nephrogenetics Research Consortium. X‐linked Alport syndrome in Hellenic families: Phenotypic heterogeneity and mutations near interruptions of the collagen domain in COL4A5.

Genotype–phenotype correlation in X‐linked Alport syndrome patients carrying missense mutations in the collagenous domain of COL4A5

To the Editor : Alport syndrome (AS) is a hereditary progressive glomerulopathy in which kidney function drops progressively and it can finally lead to end-stage kidney disease (ESKD). We herewith attempted a genotype–phenotype correlation of missense mutations within the collagenous domain of X-linked-COL4A5. We searched the public human gene mutation database (1) to locate papers that reported such missense mutations in males. We performed linear regression and Spearman correlation using mutations for which an exact age-at-onset of ESKD was given (72 [Correction added after online publication on 28 February 2012. The correct number of mutations should be 72, not 772 as was originally stated.] mutations). Recurrent mutations, reported more than once, were counted once, although the clinical severity was based on the information provided for all patients. Analysis was performed with GraphPad Prism (version 5.00-Windows, GraphPad Software, San Diego, CA: www.graphpad.com). Statistical significance was for p-value <0.05. For odds ratio analysis, subjects were split amongst two groups: severe phenotype (ESKD ≤31 years old) and mild phenotype (ESKD ≥35 years old). The grey area in between, i.e. 32–34 years, was treated as severe if there was hearing loss, ocular changes, or characteristic glomerular basement membrane findings, along with their ESKD. If ESKD was the only sign amongst these patients, they were classified as ‘mild’. Table 1 illustrates results from the analysis that did not reach significance. Nonparametric correlation examined whether the difference in the number of side-chain carbon atoms introduced by the mutation correlated to disease phenotype. A correlation was detected with a Spearman r-value of −0.3545 (95% confidence interval: −0.5492 to −0.1234) and a p-value of 0.0026. In addition, linear regression showed statistically significant correlation with age-at-onset of ESKD decreasing with increasing number of side-chain carbon atoms in the substituting residue (r2: 0.1362; p: 0.0017) (Fig. 1). Jais et al. (2) found that missense mutations tend to give phenotypes that are milder compared with others, in terms of both age-at-onset of ESKD and probability of developing hearing loss. Gross et al. (3) found that glycine substitutions in the first 20 exons resulted in a milder phenotype than glycine substitutions in exons 21–47. Bekheirnia et al. (4), however, failed to show an association between missense mutation position and age at ESKD. In addition, according to Persikov et al. (5) the identity of the amino acid that substitutes glycine in AS and other connective tissue diseases is not random. They report that the distribution of substituting residues is different than that predicted by gene-based mutation rates. One reason could be that only certain amino acid substitutions cause a severe enough phenotype, which, however, is compatible with life and therefore capable of being detected. This apparent non-random substitution was replicated in our results (data not shown). Here we provide evidence that the larger the side chain of the amino acid substituting a glycine, the more severe the disease is. Our results agree with Beck et al. (6) concerning patients with Osteogenesis Imperfecta who showed that the identity of the residue replacing glycine is important in determining the phenotype. Interestingly, linear regression of distance of mutation from closest interruption against age at ESKD gave a p-value of 0.2532. Although this is statistically insignificant, it contradicts recent evidence from our group that mutations close to certain interruptions might give a milder phenotype (7). Therefore, one could presume that different interruptions in the collagenous domains have different functions and biological significance. It is not appropriate to predict prognosis solely based on our single finding; however, Gross et al. (3) showed that glycine substitutions in exons 1–20 associate with milder phenotype, while Bekheirnia et al. (4) showed the same when all kinds of mutations were taken into consideration. Our data, without reaching significance, showed a trend in the same direction (Table 1). Therefore, a reasonable concluding algorithm could be that glycine substitutions with bulkier residues closer to the 7S domain might be associated with worst prognosis.

A functional variant in NEPH3 gene confers high risk of renal failure in primary hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in the general population

Background Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms. Methods We looked in silico for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as “Severe” or “Mild”, based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the NEPH3-V353M variant has any renal effect in the general population. Results and conclusions Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant NEPH3-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10-3 adjusting for patients’ kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a “rare variant-strong effect” role for NEPH3-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria.

Founder Mutations in the ATP6V1B1 GeneExplain Most Cypriot Cases of Distal Renal Tubular Acidosis: First Prenatal Diagnosis

Aims: To investigate clinically and genetically all the distal renal tubular acidosis (dRTA) cases in Cyprus, to study one more family from Greece and to perform the first dRTA prenatal diagnosis. We also tried to find any association with sensorineural hearing loss (SNHL) onset and particular mutations. Methods: Nine dRTA families from Cyprus and one from Greece were analyzed for mutations in ATP6V1B1 gene by DNA resequencing and PCR-RFLPs. Clinical diagnosis was performed by standard criteria. Prenatal diagnosis was performed for one Cypriot family. Results: Results show that 7/9 dRTA cases in Cyprus are caused by 229+1G>T and R157C founder mutations in ATP6V1B1 gene. 229+1G>T mutation was estimated to be older than 400 years. No genotype- phenotype correlation was found with SNHL. A known (L81P) and a novel mutation (912delT) were found in the Greek family. Prenatal diagnosis was performed for one Cypriot family, after parents’ demand, showing that the embryo was a heterozygous carrier. Conclusion: Existence of only two ATP6V1B1 mutations in the Cypriot population is a diagnostic advantage. The age of onset of SNHL varies in our patients and probably is not related to ATP6V1B1 genotypes. Effective therapy for most of the syndrome symptoms is not satisfactory for some parents who choose prenatal diagnosis to ensure their child’s health.

Epistatic Role of the MYH9/APOL1 Region on Familial Hematuria Genes

Familial hematuria (FH) is explained by at least four different genes (see below). About 50% of patients develop late proteinuria and chronic kidney disease (CKD). We hypothesized that MYH9/APOL1, two closely linked genes associated with CKD, may be associated with adverse progression in FH. Our study included 102 thin basement membrane nephropathy (TBMN) patients with three known COL4A3/COL4A4 mutations (cohort A), 83 CFHR5/C3 glomerulopathy patients (cohort B) with a single CFHR5 mutation and 15 Alport syndrome patients (cohort C) with two known COL4A5 mild mutations, who were categorized as “Mild” (controls) or “Severe” (cases), based on renal manifestations. E1 and S1 MYH9 haplotypes and variant rs11089788 were analyzed for association with disease phenotype. Evidence for association with “Severe” progression in CFHR5 nephropathy was found with MYH9 variant rs11089788 and was confirmed in an independent FH cohort, D (cumulative p value = 0.001, odds ratio = 3.06, recessive model). No association was found with APOL1 gene. Quantitative Real time PCR did not reveal any functional significance for the rs11089788 risk allele. Our results derive additional evidence supporting previous reports according to which MYH9 is an important gene per se, predisposing to CKD, suggesting its usefulness as a prognostic marker for young hematuric patients.

A hardware-efficient lowpass filter design for biomedical applications

A hardware-efficient lowpass filter design technique based on an exponentially weighted moving average (EWMA) filter architecture is proposed for the detection of general action potentials and nerve spikes in noisy signals. The EWMA VLSI architecture is compared with a basic moving average (MA) architecture and it is found that the EWMA technique is the most economical in terms of space of the two. In addition, a rule of thumb is given for converting a MA filter to the proposed filter. In the comparison, it was found that an EWMA filter is almost 85% more hardware-efficient than an MA filter.

Infrared Fluorescence-Based Cancer Screening Capsule for the Small Intestine

Infrared fluorescence endoscopy (IRFE), in conjunction with an infrared fluorescent-labelling contrast agent, is a well known technique used for efficient early-stage cancer detection. In this paper we present a cost-effective ( <;

Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae

500) screening capsule prototype, which is able to detect infrared (IR) fluorescence emitted by indocyanine green (ICG) fluorophore dye. Rather than image, the capsule works as a high-sensitivity fluorometer that records fluorescence levels throughout the small intestine. The presented mixed-signal system has a small size, consumes very little power ( ≈6.3 mA) and does not require an external belt and hardware for data collection. By determining fluorescence levels in the intestine, rather than collecting images, we avoid the need for labour intensive video analysis. The whole system is contained within a compact ingestible capsule, that is sized so as to come into close contact with the intestine walls during peristalsis. Ex-vivo experiments, on ICG-impregnated swine intestine, have shown that the prototype system is able to detect low concentrations of ICG in the nanomolar and micromolar region, which is required to detect early cancer in the small intestine.