Retrospective mutational analysis of NPHS 1 , NPHS 2 , WT 1 and LAMB 2 in children with steroid-resistant focal segmental glomerulosclerosis – a single-centre experience

Th e aim of our study was to examine NPHS, NPHS, WT and LAMB mutations, previously reported in two thirds of patients with nephrotic syndrome with onset before the age of one year old. Genomic DNA samples from Polish children (n=) with Steroid-Resistant Nephrotic Syndrome (SRNS) due to focal segmental glomerulosclerosis (FSGS), manifesting before the age of  years old, underwent retrospective analysis of NPHS, NPHS, WT (exons ,  and adjacent exon/intron boundaries) and LAMB. No pathogenic NPHS or LAMB mutations were found in our FSGS cohort. SRNS-causing mutations of NPHS and WT were detected in  of  patients (), including those with nephrotic syndrome manifesting before one year old: fi ve of seven patients. Four patients had homozygous c.G>A (p.ArgGln) NPHS mutations; one subject was homozygous for c.G>A (p.ValMet) NPHS. A phenotypic female had C>T transition at position + of the WT intron  (c.+C>T) splice-donor site, and another phenotypic female was heterozygous for G>A transition at position + (c.+G>A). Genotyping revealed a female genotypic gender (, XX) for the fi rst subject and male (, XY) for the latter. In addition, one patient was heterozygous for c.dup (p.ArgProfs*) NPHS; two patients carried a c.G>A (p.ArgGln) NPHS non-neutral variant. Results indicate possible clustering of causative NPHS mutations in FSGS-proven SRNS with onset before age one year old, and provide additional evidence that patients with childhood steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis should fi rst undergo analysis of NPHS coding sequence and WT exons  and  and surrounding exon/intron boundary sequences, followed by gender genotyping. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Genetically-heterogenous Steroid-Resistant Nephrotic Syndrome (SRNS) aff ects approximately  of children with idiopathic nephrotic syndrome, progresses toward End-Stage Renal Disease (ESRD), and typically shows Focal Segmental GlomeruloSclerosis (FSGS) in renal histology [, ].To the best of our knowledge, mutations of at least  genes have been associated with hereditary SRNS [].In , Kes-tila et al. [] discovered that the NPHS gene, encoding the podocytic protein nephrin, is mutated in the Finnish type of congenital nephrotic syndrome (CNS).Th is fi nding was the fi rst proof of concept for the heredity of childhood nephrotic syndrome.In , Hinkes et al. [] found that mutations in NPHS, NPHS (encoding podocin), WT (exons  and ) and LAMB (encoding laminin-β) are causes of two thirds of cases of nephrotic syndrome with onset in the fi rst year of life.Mutational analysis of seven podocyte genes (NPHS, NPHS, WT, CDAP, ACTN, TRPC and PLCE) in  non-familial childhood-onset, steroid resistant, biopsy-proven FSGS patients revealed variants of NPHS, NPHS, WT and CDAP that could be the cause of the disease in four subjects ().In addition, these results have suggested the role of combinations of genetic variants (bigenic heterozygosity) in the pathogenesis of steroid-resistant FSGS [].Santin et al. [] proved  respectively [].Th e NPHS mutations identifi ed by Al-Hamed et al. [] and McCarthy et al. [] in congenital nephrotic syndrome have also been detected in early childhoodonset SRNS [] and late childhood-onset SRNS [].In addition, the NPHS mutations were also identifi ed in adult-onset SRNS [], including SRNS due to FSGS [].Th erefore, the lack of NPHS mutations in our group is somewhat surprising.However, the homogeneity of ethnic origin (of Central European/ Polish descent) and renal histology (FSGS), as well as the absence of children from consanguineous marriages, should be taken into consideration in relation to this lack of NPHS mutation.In addition, Hinkes et al. reported that NPHS mutations in patients of Central European descent were solely found in nephrotic syndrome with congenital onset (- months) [], which would only apply to two patients in our cohort (Table ).Note also that, in one of these two patients, the homozygous NPHS mutation found confirms previous reports that CNS may be caused by mutations in genes other than NPHS [-].Benoit et al. [] has even recommended that patients with nephrotic syndrome occurring later than the congenital period (and with FSGS in renal histology) should first undergo screening for NPHS mutations followed by NPHS analysis.Lipska et al. reported recessive pathogenic NPHS mutations in  of  () Polish children with steroid-resistant nephrotic syndrome [].The detection rate of NPHS mutations was very similar to that revealed in our study ().If from the study of Lipska et al. [] the c.delT NPHS variant is excluded, as it is found solely in Kashubian patients who were not enrolled in our study, then the c.G>A (p.ArgGln) NPHS mutation was the most frequently detected genetic variant both by us and by Lipska et al. [], and is the most common in populations of European descent.Lipska et al. [] also reported that NPHS mutations were to be found in the highest prevalence ( , /) in patients with SRNS diagnosed within the fi rst year of life, and it is of some interest that in our study  out of  patients with FSGS-proven SRNS caused by NPHS mutations were found with age of onset within the fi rst year of life (Table ).Both McCarthy et al. [] and Al-Hamed et al. [] found no LAMB or WT mutations in their studied cohorts.The lack of LAMB mutations in our FSGS cohort, in patients from the UK Renal Registry [] and in Saudi Arabian SRNS children [] fully justifi es removal of laminin-β genetic analysis from the algorithm introduced by Santin et al. [] for molecular genetic diagnostics of non-syndromic childhood-onset primary steroid-resistant nephrotic syndrome.Despite a suggestion to consider PLCE testing only in children with diff use mesangial sclerosis [], Al-Hamed et al. [] showed the clinical utility of PLCE analysis in childhood-onset steroid resistant FSGS, and this could be further considered (but was not analysed in our study screening based on sequencing of NPHS and WT exons  and , with the adjacent exon/intron boundaries, also for childhood-onset steroid-resistant FSGS [].In addition, although the series of FSGS patients in their study was rather small (n=), the results strongly suggest that both recessive homozygous CDAP mutations or combined haploinsufficiency in NPHS and CDAP might cause primary steroid-resistant FSGS in children [].Therefore, it seems to reasonable to consider the screening of PLCE, as discussed earlier, and CDAP as second-choice targets in a genetic testing approach for the diagnosis of primary steroid-resistant FSGS occurring before adolescence [, , ].

CONCLUSION
In conclusion, our results indicate possible clustering of causative NPHS mutations in FSGS-proven SRNS with onset in the fi rst year of life, and provide additional evidence that children with steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis, in whom NS occurs before the age of  years, should fi rst undergo analysis of the NPHS coding sequence and the WT gene, especially focused on exons  and  and the surrounding exon/intron boundary DNA sequences, followed by, in cases with WT KTS intronic splice site variants, genotyping for gender.

DECLARATION OF INTEREST
Th e authors declare no confl ict of interest.
AGNIESZKA BIŃCZAKKULETA ET AL.: RETROSPECTIVE MUTATIONAL ANALYSIS OF NPHS1, NPHS2, WT1 AND LAMB2 IN CHILDREN WITH STEROIDRESISTANT FOCAL SEGMENTAL GLOMERULOSCLEROSIS  A SINGLECENTRE EXPERIENCE the clinical utility of a genetic testing algorithm, for SRNS occurring before adolescence, based on an analysis of NPHS, NPHS, WT and PLCE genes.Recently, Rood et al. [] suggested that there might be no need for PLCE analysis, for steroid-resistant FSGS occurring before adolescence, in this genetic testing algorithm.On the other hand, Hasselbacher et al. [] and Matejas et al. [] indicated that analysis of the LAMB gene, which is mutated in Pierson syndrome (OMIM ), could be included in the diagnostics of early onset nephrotic syndrome with absence of extrarenal abnormalities.Th erefore, to further address this issue we decided to analyse retrospectively NPHS, NPHS, WT (exons  and  and the adjacent exon/intron boundaries) and LAMB genes in DNA samples from  patients with steroid-resistant focal segmental glomerulosclerosis manifesting before adolescence.
Bosn J Basic Med Sci 2014; 14 (2): 90-93 NPHS, NPHS, WT and LAMB genes were analyzed in genomic DNA samples of  Polish children with primary SRNS due to FSGS.Th e study cohort ( phenotypic males and  phenotypic females) consisted of seven children with SRNS with onset in the fi rst year of life,  subjects with early childhood-onset SRNS ( to  months) and  subjects with late childhood-onset SRNS ( to  months) (Table).Th e median age of NS onset was  months (range:  to  months); for males:  months (range:  to  months); for females:  months (range:  to  months).No pathoon searching for mutations in panels of genes, were published by Al-Hamed et al.[]and Mc-Carthy et al.[].Al-Hamed et al. detected disease-causing NPHS and NPHS variants in ( of )and (  of )of Saudi Arabian families with childhood nephrotic syndrome, respectively.The NPHS and NPHS mutations were exclusively detected in children of consanguineous parents[].McCarthy et al. sequenced  hereditary ia@pum.edu.pl].Subsequently, PCR amplifi cation products were purifi ed using a GenElute PCR Clean-Up Kit (Sigma-Aldrich, Poznan, Poland).Sequencing of the products used BigDye® Terminator v. Cycle Sequencing Kits(Applied  Biosystems, Life Technologies Polska, Warsaw, Poland).Electrophoresis and analysis were performed using an ABI PRISM -Avant machine (Data Collection Software v.,Sequencing Analysis Software v.; Applied Biosystems).RESULTSTh e genic NPHS or LAMB variants were found in our FSGS cohort.The SRNS-causing mutations of NPHS and WT were detected in  of  patients (), and in those with nephrotic syndrome manifesting within the fi rst year of life: five out of seven patients.Five patients had homozygous missense NPHS mutations; four of these with c.G>A (p.ArgGln) mutations, and the fifth with a c.G>A (p.ValMet) mutation.DNA sequencing revealed a C>T transition at position + of the splice-donor site in WT intron  (c.+C>T, previous nomenclature: IVSds+C>T) from a phenotypic female (subject P) and a G>A transition at position + of the splice-donor site in WT intron  (c.+G>A, previous nomenclature: IVSds+G>A) from another phenotypic female (subject P) (Table).No parental DNA samples were available for analysis.Genotyping using the AmpFℓSTR NGM PCR Amplifi cation Kit and AmpFℓSTR Yfi ler PCR Amplifi cation Kit revealed a female origin (, XX) for the DNA sample from subject P and a male origin (, XY) for the DNA sample from subject P.In addition, the P subject was heterozygous for c.dup (p.ArgProfs*) (previous nomenclature: c._insG) without any additional pathogenic NPHS variant and two patients (P and P subjects) were heterozygous for the c.G>A (p.ArgGln) NPHS non-neutral polymorphism.DISCUSSIONResults of our study revealed that one fi fth of cases of steroid-resistant FSGS which occurred before adolescence were caused by mutations in either the NPHS or WT gene.Neither NPHS nor LAMB mutations were found in our FSGS cohort.It has also not escaped of our notice that  out of  from the NPHS or WT disease-causing mutations were detected in a small subgroup of children with SRNS (n=) manifesting in the first year of life.Recent results, from similarly-designed studies with SRNS children which focused SRNS-associated genes, including the NPHS, NPHS, WT and LAMB genes, in  SRNS patients from the UK Renal Registry.Th ese patients were ethnically-heterogeneous and between  month and  years of age at onset of disease.Molecular analysis revealed causative NPHS and NPHS mutations in  ( of ) and  ( of ) of the patients,

TABLE 1 .
Background characteristics and genetic variants in children with steroid-resistant FSGS (patients listed in order of age of NS onset).
*Blank spaces indicate that reference sequences were detected for the genes analysed.
).As well as NPHS, NPHS and PLCE sequencing, Santin et al. also proposed the analysis of WT exons  and  in molecular genetic diagnostics of childhood onset SRNS[].To the best of our knowledge,Wasilewska et al. in   []identifi ed the fi rst Polish patient with Frasier syndrome caused by a WT splice-site mutation (c.+G>A), andLipska et al. (in )[], in a SRNS cohort from the international PodoNet registry (including  patients from Poland), found one Polish patient with classical Frasier syndrome caused by a de novo c.+G>A WT mutation.Th e prevalence of WT splice-site mutations with sporadic steroid-resistant FSGS in our study () was similar to results published previously by other authors[, , -].-,-,, ], and we can assume that this also applied to the WT splice-site mutations identifi ed in our FSGS cohort.Note, however, that although Lipska et al.[]recently reported WT mutations in  (/) of sporadic SRNS cases in the PodoNet registry, only one third of these ( out of  with FSGS) were due to intronic KTS splice-site variants.
of patients with adolescent-onset sporadic SRNS from the international PodoNet registry, and recommended routine screening of WT, in addition to NPHS, in children with isolated, sporadic SRNS because of the high prevalence of "truly or apparently isolated SRNS".Lowik et al. indicated the cost-eff ectiveness and the clinical value of genetic