Marwa M. Nabhan

Genomic and clinical profiling of a national Nephrotic Syndrome cohort advocates a precision medicine approach to disease management

Steroid Resistant Nephrotic Syndrome (SRNS) in children and young adults has differing etiologies with monogenic disease accounting for 2.9-30% in selected series. Using whole exome sequencing we sought to stratify a national population of children with SRNS into monogenic and non-monogenic forms, and further define those groups by detailed phenotypic analysis. Pediatric patients with SRNS were identified via a national United Kingdom Renal Registry. Whole exome sequencing was performed on 187 patients, of which 12% have a positive family history with a focus on the 53 genes currently known to be associated with nephrotic syndrome. Genetic findings were correlated with individual case disease characteristics. Disease causing variants were detected in 26.2% of patients. Most often this occurred in the three most common SRNS-associated genes: NPHS1, NPHS2, and WT1 but also in 14 other genes. The genotype did not always correlate with expected phenotype since mutations in OCRL, COL4A3, and DGKE associated with specific syndromes were detected in patients with isolated renal disease. Analysis by primary/presumed compared with secondary steroid resistance found 30.8% monogenic disease in primary compared with none in secondary SRNS permitting further mechanistic stratification. Genetic SRNS progressed faster to end stage renal failure, with no documented disease recurrence post-transplantation within this cohort. Primary steroid resistance in which no gene mutation was identified had a 47.8% risk of recurrence. In this unbiased pediatric population, whole exome sequencing allowed screening of all current candidate genes. Thus, deep phenotyping combined with whole exome sequencing is an effective tool for early identification of SRNS etiology, yielding an evidence-based algorithm for clinical management.

Large-scale targeted sequencing comparison highlights extreme genetic heterogeneity in nephronophthisis-related ciliopathies

Background: The term nephronophthisis-related ciliopathies (NPHP-RC) describes a group of rare autosomal-recessive cystic kidney diseases, characterised by broad genetic and clinical heterogeneity. NPHP-RC is frequently associated with extrarenal manifestations and accounts for the majority of genetically caused chronic kidney disease (CKD) during childhood and adolescence. Generation of a molecular diagnosis has been impaired by this broad genetic heterogeneity. However, recently developed high-throughput exon sequencing techniques represent powerful and efficient tools to screen large cohorts for dozens of causative genes. Methods: Therefore, we performed massively multiplexed targeted sequencing using the modified molecular inversion probe strategy (MIPs) in an international cohort of 384 patients diagnosed with NPHP-RC. Results: As a result, we established the molecular diagnoses in 81/384 unrelated individuals (21.1%). We detected 127 likely disease-causing mutations in 18 of 34 evaluated NPHP-RC genes, 22 of which were novel. We further compared a subgroup of current findings to the results of a previous study in which we used an array-based microfluidic PCR technology in the same cohort. While 78 likely disease-causing mutations were previously detected by the array-based microfluidic PCR, the MIPs approach identified 94 likely pathogenic mutations. Compared with the previous approach, MIPs redetected 66 out of 78 variants and 28 previously unidentified variants, for a total of 94 variants. Conclusions: In summary, we demonstrate that the modified MIPs technology is a useful approach to screen large cohorts for a multitude of established NPHP genes in order to identify the underlying molecular cause. Combined application of two independent library preparation and sequencing techniques, however, may still be indicated for Mendelian diseases with extensive genetic heterogeneity in order to further increase diagnostic sensitivity.

Clinical characterization and NPHP1 mutations in nephronophthisis and associated ciliopathies: A single center experience

Nephronophthisis (NPHP) is a recessive disorder of the kidney that is the leading genetic cause of end-stage renal failure in children. Egypt is a country with a high rate of consanguineous marriages; yet, only a few studies have investigated the clinical and molecular characteristics of NPHP and related ciliopathies in the Egyptian population. We studied 20 children, from 17 independent families, fulfilling the clinical and the ultrasonographic criteria of NPHP. Analysis for a homozygous deletion of the NPHP1 gene was performed by polymerase chain reaction on the genomic DNA of all patients. Patients were best categorized as 75% juvenile NPHP, 5% infantile NPHP, and 20% Joubert syndrome-related disorders (JSRD). The mean age at diagnosis was 87.5 + 45.4 months, which was significantly late as compared with the age at onset of symptoms, 43.8 ± 29.7 months (P <0.01). Homozygous NPHP1 deletions were detected in six patients from five of 17 (29.4%) studied families. Our study demonstrates the clinical phenotype of NPHP and related disorders in Egyptian children. Also, we report that homozygous NPHP1 deletions account for 29.4% of NPHP in the studied families in this cohort, thereby confirming the diagnosis of type-1 NPHP. Moreover, our findings confirm that NPHP1 deletions can indeed be responsible for JSRD.

Homozygous NPHP1 deletions in Egyptian children with nephronophthisis including an infantile onset patient

Sirs, Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that constitutes the most frequent genetic cause for end-stage renal disease (ESRD) in the first three decades of life. Homozygous deletions in the NPHP1 gene account for approximately 21% of all NPHP cases, whereas the other genes contribute less than 3% each. Interestingly, positional cloning of the nine genes (NPHP 1 through 9) and functional characterization of their encoded proteins (nephrocystins) have contributed to a unifying theory that defines cystic kidney diseases as “ciliopathies” [1]. Since there are no clinical or molecular data about NPHP in Egyptian children, we investigated the prevalence of homozygous NPHP1 deletion among a cohort of children with presumptive diagnosis of NPHP at Center of Pediatric Nephrology & Transplantation (CPNT), Cairo University. We studied 20 children, from 17 independent families, fulfilling the clinical and the ultrasonographic criteria of NPHP. All patients had full ophthalmologic examination, brain MRI was carried out in children with neurological involvement. Analysis for a homozygous deletion of the NPHP1 gene was performed by multiplex polymerase chain reaction (PCR) approach on genomic DNA of patients described earlier [2]. Sex distribution among affected patients showed a slight preponderance of females, with a ratio of 1.2:1 (11 females and 9 males). Seventy five percent of our study patients were the products of consanguineous marriages, the percentage of affected siblings was strikingly high 65% (13/20 patients) and 40% (8/20 patients) had a history of sibling death due to a similar condition. Fifteen of 20 patients (75%) presented with signs of ESRD. All the patients suffered from anemia and growth retardation when they first came to medical attention. Nineteen of 20 patients (95%) had a history typical of nephronophthisis, with symptoms of polydipsia, polyuria, and secondary enuresis. Four of 20 patients (20%) were hypertensive with elevated blood pressure above the 95th percentile for age, gender, and height. Clinically, study patients were best categorized as: 13/20 (65%) patients with isolated juvenile NPHP, 3/20 (15%) patients as infantile NPHP, while the remaining 4/20 (20%) patients had extrarenal associations (molar tooth sign on brain MRI, ataxia, mental retardation, retinal dystrophy, oculomotor apraxia, and facial dysmorphy) hence were clinically categorized as Joubert Syndrome Related Disorder (JSRD). The mean age at diagnosis 87.5 + 45.4 months was significantly late as compared to the age of onset of symptoms 43.8 ± 29.7 months (P <0.01). Homozygous deletions in the NPHP1 gene were identified in 6 patients from 5 independent families out of the 17 studied families (29.4%), thereby confirming the diagnosis of type 1 nephronophthisis (Fig. 1). This does not seem to differ from reported figures in Western countries. Five patients had isolated NPHP whereas the sixth patient was among the JSRD group with the distinct MTS detected in his brain MRI images, ataxia, retinitis pigmentosa and dysmorphic facies. This confirms that NPHP1 deletions can indeed be responsible for JSRD as previously reported [3]. Figure 1 Screening for homozygous NPHP1 deletions. 20 patients (lane 1–20) from 17 independent families were analyzed using a multiplex PCR approach amplifying 5 different markers. Two control markers (a, e) and 3 NPHP1 exon markers [arrow “d” ... Moreover we report one patient with the homozygous NPHP1 deletion presenting with an infantile onset phenotype. In the literature, Infantile NPHP frequently caused by mutations in the NPHP2/inversin gene differs from the other types of NPHP in the early age of onset of ESRD usually <5 years in all reported cases, whereas the median age of ESRD in juvenile NPHP (NPHP type 1 or type 4) is about 13 years [4]. Renal cortical microcysts is another criterion where detailed analysis of a murine model of NPHP2/inversin demonstrates cystic dilatation of Bowman’s capsule, proximal tubule, thick ascending limb and collecting duct [5]. Although homozygous NPHP1 deletions is the most frequent NPHP1 mutation known, nevertheless, in more than 6% of all NPHP1 cases the underlying mutation has been found to be a heterozygous deletion combined with a single point mutation [2]. As for the patients with no homozygous NPHP1 deletion we will investigate for heterozygous deletions in a future study. Further analysis of all other NPHP loci for potential homozygosity in consanguineous families and sequencing all exons of NPHP genes located within regions of significant homozygosity is crucial to identify the underlying genetic defect. Our infantile NPHP patient showed most of the clinical and histopathological features of NPHP type 2 previously reported in the literature [4]: (1) the characteristic cortical cysts in a normal sized hyperechogenic kidneys on renal sonogram, (2) distinct renal microcysts in the kidney histopathological examination, (3) clinical manifestations started in the first year of life and progressed to ESRD at the age of 32 months. In conclusion, homozygous deletions in the NPHP1 gene do not seem to differ in Egypt from reported figures in Western countries. Most likely this is the first report of a homozygous NPHP1 deletion in an infantile onset NPHP patient.

Intrafamilial Variability and Clinical Heterogeneity in Two Siblings with NPHP4 loss of Function Mutations

Joubert syndrome–related disorders (JSRDs) are a group of clinically and genetically pleiotropic conditions that share a midbrain-hindbrain malformation, the pathognomonic molar tooth sign (MTS) visible on brain imaging, with variable involvement of other organs and systems mainly the eyes and the kidneys. Nevertheless, the definition of JSRDs remained problematical due to the extreme phenotypic heterogeneity, often with intrafamilial variability, and the significant clinical overlap among distinct forms. Here we describe two siblings with nephronophthisis (NPHP), the elder is best categorized as JSRD. Nevertheless, his younger sibling lacked the characteristic molar tooth sign; hence best categorized as NPHP- related ciliopathy. Both siblings had NPHP as the common renal phenotype, yet with variable neurological and ocular involvement. Genetic linkage and mutation analysis revealed a novel homozygous, potential loss of function mutation (c.2618dupA, pH is 873Glnfs*14) in the gene NPHP4 in both siblings. This finding extends the phenotypic spectrum associated with NPHP4 mutations, with discernible clinical heterogeneity and intrafamilial variability.

Cystic kidneys in fetal Walker–Warburg syndrome with POMT2 mutation: Intrafamilial phenotypic variability in four siblings and review of literature

Walker–Warburg syndrome (WWS) is a severe form of congenital muscular dystrophy secondary to α‐dystroglycanopathy with muscle, brain, and eye abnormalities often leading to death in the first weeks of life. It is transmitted in an autosomal recessive pattern, and has been linked to at least 15 different genes; including protein O‐mannosyltransferase 1 (POMT1), protein O‐mannosyltransferase 2 (POMT2), protein O‐mannose beta‐1,2‐N acetylglucosaminyltransferase (POMGNT1), fukutin (FKTN), isoprenoid synthase domain‐containing protein (ISPD), and other genes. We report on a consanguineous family with four consecutive siblings affected by this condition with lethal outcome in three (still birth), and termination of the fourth pregnancy based on antenatal MRI identification of brain and kidney anomalies that heralded proper and deep clinical phenotyping. The diagnosis of WWS was suggested based on the unique collective phenotype comprising brain anomalies in the form of lissencephaly, subcortical/subependymal heterotopia, and cerebellar hypoplasia shared by all four siblings; microphthalmia in one sibling; and large cystic kidneys in the fetus and another sibling. Other unshared neurological abnormalities included hydrocephalus and Dandy‐Walker malformation. Whole exome sequencing of the fetus revealed a highly conserved missense mutation in POMT2 that is known to cause WWS with brain and eye anomalies.In conclusion, the heterogeneous clinical presentation in the four affected conceptions with POMT2 mutation expands the current clinical spectrum of POMT2‐associated WWS to include large cystic kidneys; and confirms intra‐familial variability in terms of brain, kidney, and eye anomalies.

Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection

BACKGROUND An essential milestone in pediatric transplantation is to find noninvasive biomarkers to monitor acute rejection (AR). In this retrospective (Case-control) study, we examined the role of Fas -670A/G and Fas Ligand (FasL) -843C/T gene polymorphisms in allograft nephropathy in pediatric renal transplant recipients. METHODS In 47 pediatric kidney transplant recipients and 20 healthy controls, Fas -670A/G and FasL -843C/T gene polymorphisms as well as serum soluble Fas Ligand level (sFasL) were measured. RESULTS Serum sFasL levels were significantly higher in transplant recipients children than that in controls (548.25±298.64pg/ml vs 143.17±44.55pg/ml, p=0.0001). There was no significant difference between patients with AR and those without AR in regards to serum sFasL levels (567.70±279.87pg/ml vs 507.85±342.80pg/ml, p=0.56). Fas -670A/G genotypes or alleles were not significantly different between controls and transplant recipients and among transplant recipients with and without AR. (P>0.05 for all). FasL -843C/T genotypes were not different between transplant recipients and controls and among transplant recipients with and without AR (P>0.05 for all). However, Frequency of C allele in transplant patients was significantly higher than that in the control group (44.68% vs 25%, P=0.03). FasL -843C/T alleles were significantly different between patients with and without AR (P=0.03). The percentages of C allele were higher in children with AR (58.82% vs 36.67%). We found that serum FasL and serum creatinine were variables that were independently associated with AR. CONCLUSION This study suggests that FasL gene polymorphisms in peripheral blood might be accurate in detecting cellular AR.

Clinical and ultrasonographical characterization of childhood cystic kidney diseases in Egypt

Abstract Background: Renal cystic disorders (RCD) constitute an important and leading cause of end-stage renal disease (ESRD) in children. It can be acquired or inherited; isolated or associated with extrarenal manifestations. The precise diagnosis represents a difficult clinical challenge. Methods: The aim of this study was to define the pattern of clinical phenotypes of children with renal cystic diseases in Pediatric Nephrology Center, Cairo University. We have studied the clinical phenotypes of 105 children with RCD [45 (43%) of them had extrarenal manifestations]. Results: The most common disorders were the presumably inherited renal cystic diseases (65.7%) mainly nephronophthisis and related ciliopathies (36.2%), as well as polycystic kidney diseases (29.5%). Moreover, multicystic dysplastic kidneys accounted for 18% of study cases. Interestingly, eight syndromic cases are described, yet unclassified as none had been previously reported in the literature. Conclusion: RCD in this study had an expanded and complex spectrum and were largely due to presumably inherited/genetic disorders (65.7%). Moreover, we propose a modified algorithm for clinical and diagnostic approach to patients with RCD.

Visual acuity, fundus changes, and electroretinographic findings in Egyptian children with Bardet-Biedl syndrome

Purpose The aim of the present study was to assess visual acuity, measure the degree of visual impairment, and examine fundus changes in Egyptian children with Bardet-Biedl syndrome (BBS), and to correlate these findings with the results of flash electroretinography (ERG). Materials and methods A cross-sectional study was conducted on infants and children with BBS. Diagnosis was carried out by a specialized pediatrician. Full ophthalmological examination, including slit-lamp examination, refraction, and indirect ophthalmoscopy, was carried out. Best-corrected visual acuity was measured and full-field flash ERG was performed. Results This study included 20 patients with BBS. The median age was 9 years (ranged from 1 day to 18 years). The mean age of onset of symptoms in the whole group was 6.5 ± 4.3 years. Visual impairment, defined as a best-corrected visual acuity in the better seeing eye of 20/60 (6/18) or less, was present in 11 patients (55% of the cases), and night blindness as a symptom was present in nine patients (45%); pigmentary retinopathy was clinically detected in 35% of the patients and retinal dystrophy as an ERG finding was detected in 60% of the patients. Conclusion Visual impairment in BBS is mainly caused by rod-cone dystrophy, which is one of the major criterion of diagnosis, which manifests with age. Before the age of 6, children rarely present with ocular manifestations, and usually have normal fundus examination; on the other hand, retinitis pigmentosa-like picture is usually seen in children older than 6. However, the electrophysiological changes may precede the fundus changes.