COL4A5 and LAMA5 variants co-inherited in familial hematuria: digenic inheritance or genetic modifier effect?

BACKGROUND: About 40-50% of patients with familial microscopic hematuria (FMH) caused by thin basement membrane nephropathy (TBMN) inherit heterozygous mutations in collagen IV genes (COL4A3, COL4A4). On long follow-up, the full phenotypic spectrum of these patients varies a lot, ranging from isolated MH or MH plus low-grade proteinuria to chronic renal failure of variable degree, including end-stage renal disease (ESRD). METHODS: Here, we performed Whole Exome Sequencing (WES) in patients of six families, presenting with autosomal dominant FMH, with or without progression to proteinuria and loss of renal function, all previously found negative for severe collagen IV mutations. Hierarchical filtering of the WES data was performed, followed by mutation prediction analysis, Sanger sequencing and genetic segregation analysis. RESULTS: In one family with four patients, we found evidence for the contribution of two co-inherited variants in two crucial genes expressed in the glomerular basement membrane (GBM); LAMA5-p.Pro1243Leu and COL4A5-p.Asp654Tyr. Mutations in COL4A5 cause classical X-linked Alport Syndrome, while rare mutations in the LAMA5 have been reported in patients with focal segmental glomerulosclerosis. The phenotypic spectrum of the patients includes hematuria, proteinuria, focal segmental glomerulosclerosis, loss of kidney function and renal cortical cysts. CONCLUSIONS: A modifier role of LAMA5 on the background of a hypomorphic Alport syndrome causing mutation is a possible explanation of our findings. Digenic inheritance is another scenario, following the concept that mutations at both loci more accurately explain the spectrum of symptoms, but further investigation is needed under this concept. This is the third report linking a LAMA5 variant with human renal disease and expanding the spectrum of genes involved in glomerular pathologies accompanied by familial hematurias. The cystic phenotype overlaps with that of a mouse model, which carried a Lama5 hypomorphic mutation that caused severely reduced Lama5 protein levels and produced kidney cysts.

Diversity of kidney care referral pathways in national child health systems of 48 European countries.

Background: Primary, secondary and tertiary healthcare services in Europe create complex networks covering pediatric subspecialties, sociology, economics and politics. Two surveys of the European Society for Paediatric Nephrology (ESPN) in 1998 and 2017 revealed substantial disparities of kidney care among European countries. The purpose of the third ESPN survey is to further identify national differences in the conceptualization and organization of European pediatric kidney health care pathways during and outside normal working hours. Methods: In 2020, a questionnaire was sent to one leading pediatric nephrologist from 48 of 53 European countries as defined by the World Health Organization. In order to exemplify care pathways in pediatric primary care nephrology, urinary tract infection (UTI) was chosen. Steroid sensitive nephrotic syndrome (SSNS) was chosen for pediatric rare disease nephrology and acute kidney injury (AKI) was analyzed for pediatric emergency nephrology. Results: The care pathways for European children and young people with urinary tract infections were variable and differed during standard working hours and also during night-time and weekends. During daytime, UTI care pathways included six different types of care givers. There was a shift from primary care services outside standard working hours to general outpatient polyclinic and hospital services. Children with SNSS were followed up by pediatric nephrologists in hospitals in 69% of countries. Patients presenting with community acquired AKI were admitted during regular working hours to secondary or tertiary care hospitals. During nights and weekends, an immediate shift to University Children's Hospitals was observed where treatment was started by intensive care pediatricians and pediatric nephrologists. Conclusion: Gaps and fragmentation of pediatric health services may lead to the risk of delayed or inadequate referral of European children with kidney disease to pediatric nephrologists. The diversity of patient pathways outside of normal working hours was identified as one of the major weaknesses in the service chain.

Epidemiology and risk factors for resistance to treatment of Kawasaki disease in Cyprus.

Kawasaki disease (KD) is one of the most common vasculitides of early childhood. There are no previous studies on KD in Cyprus. The aim of this study was to evaluate the epidemiology of KD in Cyprus, risk factors for resistance to treatment and the development of cardiac complications. This is a retrospective multicenter study of pediatric patients with KD hospitalized between January 2000 and-December 2019. The data were collected from medical records. A total of 136 patients with KD were included in the study. 83% of patients were < 5 years of age and 10% were < 6 months. Thirty patients (22%) developed coronary artery lesions. Serum sodium ≤ 133 mmol/L, albumin ≤ 3.2 g/dl, ALT ≥ 80 U/L and neutrophils percentage ≥ 80% at diagnosis, were identified as risk factors for resistance to IVIG. Clinical and epidemiological characteristics of KD in Cyprus population were similar to those reported in the literature. Although the majority of cases received appropriate treatment in time, cardiac complications still occurred.

Evidence that NPHS2-R229Q predisposes to proteinuria and renal failure in familial hematuria.

BACKGROUND: Familial hematuria (FH) is associated with at least two pathological entities: thin basement membrane nephropathy (TBMN), caused by heterozygous COL4A3/COL4A4 mutations, and C3 nephropathy caused by CFHR5 mutations. It is now known that TBMN patients develop proteinuria and changes of focal segmental glomerulosclerosis when biopsied. End-stage kidney disease (ESKD) is observed in 20% of carriers, at ages 50-70. A similar progression is observed in CFHR5 nephropathy. Recent evidence suggests that NPHS2-R229Q, a podocin polymorphism, may contribute to proteinuria in TBMN and to micro-albuminuria in the general population. CASE-DIAGNOSIS/TREATMENT: NPHS2-R229Q was screened in a Cypriot FH cohort. 102 TBMN patients with three known COL4 mutations and 45 CFHR5 male patients with a single mutation were categorized as "Mild" or "Severe", based on the presence of microhematuria only, or proteinuria and chronic kidney disease. Nine R229Q carriers were found in the "Severe" category and none in the "Mild" (p=0.010 for genotypic association; p=0.043 for allelic association, adjusted for patients' relatedness), thus supporting the possible contribution of 229Q allele in disease progress. CONCLUSIONS: Our results offer more evidence that in patients with FH, NPHS2-R229Q predisposes to proteinuria and ESKD. R229Q may be a good prognostic marker for young hematuric patients.

Founder mutations in the ATP6V1B1 gene explain 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.

Clinico-pathological correlations in 127 patients in 11 large pedigrees, segregating one of three heterozygous mutations in the COL4A3/ COL4A4 genes associated with familial haematuria and significant late progression to proteinuria and chronic kidney disease from focal segmental glomerulosclerosis.

BACKGROUND: Heterozygous mutations in the COL4A3/ COL4A4 genes are currently thought to be responsible for familial benign microscopic haematuria and maintenance of normal long-term kidney function. METHODS: We report on 11 large Cypriot pedigrees with three such mutations. A total of 236 at-risk family members were genetically studied, and 127 (53.8%) carried a heterozygous mutation. Clinico-pathological correlations were available in all of these patients. Renal biopsies in 21 of these patients all showed various stages of focal, segmental glomerulosclerosis (FSGS). Thirteen of these biopsies were also studied with EM and showed thinning of the glomerular basement membrane. RESULTS: Mutation G1334E (COL4A3) was found in six pedigrees, mutation G871C (COL4A3) in four and mutation 3854delG (COL4A4) in one pedigree. Clinical and laboratory correlations in all 127 mutation carriers (MC) showed that microscopic haematuria was the only urinary finding in patients under age 30. The prevalence of 'haematuria alone' fell to 66% between 31 and 50 years, to 30% between 51 and 70 and to 23% over age 71. Proteinuria with CRF developed on top of haematuria in 8% of all MC between 31 and 50 years, to 25% between 51 and 70 years and to 50% over 71 years. Altogether 18 of these 127 MC (14%) developed ESRD at a mean age of 60 years. Two members with different mutations married, and two of their children inherited both mutations and developed adolescent, autosomal recessive Alport syndrome (ATS), confirming that these mutations are pathogenic. CONCLUSIONS: Our data confirm for the first time a definite association of heterozygous COL4A3/COL4A4 mutations with familial microscopic haematuria, thin basement membrane nephropathy and the late development of familial proteinuria, CRF, and ESRD, due to FSGS, indicating that the term 'benign familial haematuria' is a misnomer, at least in this cohort. A strong hypothesis for a causal relationship between these mutations and FSGS is also made. Benign familial haematuria may not be so benign as commonly thought.

Hemophagocytic lymphohistiocytosis associated with Epstein Barr virus and Leishmania donovani coinfection in a child from Cyprus.

We present a case of a 9-month-old girl from Cyprus with hemophagocytic lymphohistiocytosis associated with Epstein Barr virus and Leishmania donovani coinfection. Treatment with liposomal amphotericin B resulted in a dramatic resolution of clinical and laboratory abnormalities. To our knowledge, this is the first reported case of a coinfection-associated hemophagocytic lymphohistiocytosis and the first clinical report of visceral leishmaniasis infection in Europe by L. donovani.

Molecular and Clinical Investigation of Cystinuria in the Greek-Cypriot Population.

BACKGROUND AND AIMS: Cystinuria represents 3% of nephrolithiasis in humans. Two genes have been identified as the main genetic causes of cystinuria, SLC3A1 and SLC7A9, with an autosomal recessive mode of inheritance. In the present study, we studied for the first time, genetically and clinically, all the cystinuric families identified so far in the Greek-Cypriot population. METHODS: Discovery of mutations was performed through polymerase chain reaction (PCR)-single analysis and DNA resequencing. New families were investigated through PCR-RFLPs. Clinical data were collected through the hospital patients' records and analytical follow-up of the families. RESULTS AND DISCUSSION: We found a total of five mutations in 28 Greek-Cypriot cystinuric patients belonging in 12 families. The most frequent mutation among the 28 Greek-Cypriot patients is the SLC3A1-p.T216M, which is also the second most frequent mutation in Europe, representing a genetic founder effect. Sixteen of the 28 patients are homozygous for this mutation. Even though a consanguinity loop was obvious in only one family, other patients were from families in small villages where endogamy was practiced for many centuries. Timely clinical and genetic diagnosis, accompanied by early treatment, is significant for the good health of most of our patients. Only ∼14% of them developed chronic renal failure, and only one reached end-stage renal disease (ESRD). CONCLUSION: Five SLC3A1 and SLC7A9 mutations appear to be responsible for the genetic basis of cystinuria in the Greek-Cypriot patients; having such a limited number of causative mutations will simplify diagnostics for this population.

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.

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.