Functional alterations due to amino acid changes and evolutionary comparative analysis of ARPKD and ADPKD genes.

A targeted customized sequencing of genes implicated in autosomal recessive polycystic kidney disease (ARPKD) phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified four potential pathogenic variants in PKHD1 gene [c.4870C > T, p.(Arg1624Trp), c.5725C > T, p.(Arg1909Trp), c.1736C > T, p.(Thr579Met) and c.10628T > G, p.(Leu3543Trp)] among 12 out of 18 samples. However, one variant c.4870C > T, p.(Arg1624Trp) was common among eight patients. Some patient samples also showed few variants in autosomal dominant polycystic kidney disease (ADPKD) disease causing genes PKD1 and PKD2 such as c.12433G > A, p.(Val4145Ile) and c.1445T > G, p.(Phe482Cys), respectively. All causative variants were validated by capillary sequencing and confirmed the presence of a novel homozygous variant c.10628T > G, p.(Leu3543Trp) in a male proband. We have recently published the results of these studies (Edrees et al., 2016). Here we report for the first time the effect of the common mutation p.(Arg1624Trp) found in eight samples on the protein structure and function due to the specific amino acid changes of PKHD1 protein using molecular dynamics simulations. The computational approaches provide tool predict the phenotypic effect of variant on the structure and function of the altered protein. The structural analysis with the common mutation p.(Arg1624Trp) in the native and mutant modeled protein were also studied for solvent accessibility, secondary structure and stabilizing residues to find out the stability of the protein between wild type and mutant forms. Furthermore, comparative genomics and evolutionary analyses of variants observed in PKHD1, PKD1, and PKD2 genes were also performed in some mammalian species including human to understand the complexity of genomes among closely related mammalian species. Taken together, the results revealed that the evolutionary comparative analyses and characterization of PKHD1, PKD1, and PKD2 genes among various related and unrelated mammalian species will provide important insights into their evolutionary process and understanding for further disease characterization and management.

Next-generation sequencing for molecular diagnosis of autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD) a rare genetic disorder, described by formation of cysts in the kidney. A targeted customized sequencing of genes implicated in ARPKD phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified likely pathogenic disease causing variants during the validation process. Four potential pathogenic variants [c.4870C>T, p.(Arg1624Trp)], [c.5725C>T, p.(Arg1909Trp)], c.1736C>T, p.(Thr579Met)] and [(c.10628T>G), p.(Leu3543Trp)] were observed in PKHD1 gene among 12 out of 18 samples. The rest of the patient samples also showed few variants in ADPKD (Autosomal Dominant Polycystic Kidney Disease) disease causing genes PKD1 and PKD2 i.e. [c.12433G>A, p.(Val4145Ile)] and [c.1445T>G, p.(Phe482Cys)], respectively. All causative variants were validated by capillary sequencing, confirming the presence of a novel homozygous variants [c.10628T>G, p.(Leu3543Trp)] found in exon 61 of a male proband. All potentially deleterious variants identified in PKHD1, PKD1, and PKD2 gene, also exhibited pathologically or clinically significance based on the computational predictions involved in predicting the impact of non-synonymous SNPs (nsSNPs) on protein function such as Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen2). SIFT classified 50% of our nsSNPs as "deleterious", while PolyPhen2 identified 45% of our nsSNPs as "Probably damaged" and the results from both programs were largely complementary. Taken together, these results suggest that the NGS strategies provide a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in targeted genes sequence analysis.

Changes in the levels of cytokines in both diabetic/non-diabetic type I children living in a moderate altitude area in Saudi Arabia.

The aim of the present study was to investigate the possible effects of living in moderate altitude area on pro/anti-inflammatory cytokines profile (IFN-γ, TNF-α, IL-6, IL-1ß, IL-10, and IL-4) among type I diabetic (T1D) and non- T1D children compared with those living at sea level area. A prospective clinical study was carried out at pediatric outpatient endocrine clinics in Taif City, which is a moderate altitude area in Saudi Arabia, that stands about 1800-2000 meters above sea-level; and in Mecca City, which is a sea level area, that lies in the middle west of Saudi Arabia. Hemoglobin A1c (HbA1c) percentage was estimated and cytokine measurements were performed in sera by flow cytometry using Cytometric Bead Array (CBA) technology. In this study we included 600 children who were consecutively enrolled (sex and age were matched). The HbA1c was statistically significantly higher in children living in moderate altitude compared to those living at sea level (overall p<0.001). Furthermore, T1D patients had higher values of serum cytokine levels (IFN-γ, TNF-α, IL-6, IL-1ß, IL-4, and IL-10) in comparison to non-T1D control group (overall p<0.001). In conclusion, the data of the present study clearly showed that in both T1D and non-T1D children, moderate altitude-natives expressed high HbA1c and both pro-and anti-inflammatory cytokines. Type I diabetic children living in moderate altitude or at sea level showed elevated levels of IFN-γ, TNF-α, IL-6, IL-1ß, IL-4, and IL-10 than control subjects. Glycemic control in non-diabetic children was affected by living in moderate altitude, however, HbA1c significantly increased in diabetic children living in moderate altitude.

Rituximab for refractory cases of childhood nephrotic syndrome.

Rituximab has been used over the last decade as a rescue therapy for refractory cases of nephrotic syndrome (NS). Here we report the use of rituximab in four children with idiopathic steroid-resistant nephrotic syndrome (SRNS) with various histological backgrounds (two cases with focal segmental glomerulosclerosis, one case with IgM nephropathy, and one case with minimal change disease), who failed to respond to other immunsuppressions. Their median age (range) was 10 (8-11) years. NPHS2 genetic mutation was negative in all of them. All patients received a single dose of rituximab (375 mg/m(2)) and achieved complete B cell depletion as CD19 was <1% for 3 months following rituximab infusion. Only one patient achieved non-sustained remission as he relapsed after 4 months despite zero CD19 level. Patients received no further doses of rituximab as B cell was depleted in the peripheral circulation. We conclude that a single dose of rituximab was not effective in inducing sustained remission in children with idiopathic SRNS, despite complete B cell depletion in the peripheral circulation. Further doses might be indicated to deplete non-circulating B cells.