Classical phenylketonuria presenting as maternal PKU syndrome in the offspring of an intellectually normal woman.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism resulting from a deficiency of phenylalanine hydroxylase (PAH). If untreated by dietary restriction of phenylalanine intake, impaired postnatal cognitive development results from the neurotoxic effects of excessive phenylalanine (Phe). Signs and symptoms include severe intellectual disability and behavior problems with a high frequency of seizures and variable microcephaly. Maternal PKU syndrome refers to fetal damage resulting in congenital abnormalities when the mother has untreated PKU during pregnancy. Here, we report an intellectually normal 32-year-old female who presented with recurrent pregnancy loss and two neonatal deaths with congenital heart disease, microcephaly, intrauterine growth restriction, and respiratory distress. She was diagnosed with PKU through exome sequencing performed for carrier testing with a homozygous pathogenic variant in the PAH gene, c.169_171del, p.(Glu57del) that is associated with classical PKU. Consistent with the genetic finding, she had a markedly increased plasma phenylalanine concentration of 1642 µmol/L (normal <100). This case demonstrates that recurrent pregnancy loss due to untreated maternal PKU may present as an initial finding in otherwise unsuspected classical PKU and illustrates that extreme degrees of variable expressivity may occur in classical PKU. Moreover, this case illustrates the value of genomic sequencing of women who experience recurrent pregnancy loss or neonatal anomalies.

Effect of RNA Interference on the Hepatitis C Virus Core Expression in HepG2/C3A Cells Using Genotype 4 Isolates from Saudi Patients.

OBJECTIVE: To explore the effects of two different methodologies of RNA interference, namely small interfering RNA, and vector-based short hairpin RNA, on the expression levels of hepatitis C virus core RNA and protein of Saudi genotype 4 isolates. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Laboratories of the College of Medicine Research Center, King Saud University, Saudi Arabia, from January to December 2018. METHODOLOGY: Hepatitis C virus core small interfering RNA molecule and short hairpin RNA vector were designed against core region. Viral RNA expression was tested by RT-PCR; whereas, core protein was tested by flow cytometry and immunofluorescence. Results were statistically analysed by Chi-square analysis to calculate the p-value. RESULTS: Both molecules caused a reduction in core RNA and protein expression in infected cells. The effect of 100-pmol of small interfering RNA was more evident. For the vector-based short hairpin RNA, inhibition of core RNA expression was quite evident after 96 hours (p = 0.007). The results of flow cytometry and immunofluorescence showed a decline in core protein expression. The most dramatic effect was observed with 100-pmol small interfering RNA treatment of cells for 24 and 48 hours, which resulted in 63.5% and 91.1% core RNA expression reduction, respectively. CONCLUSION: RNA interference of hepatitis C virus core gene efficiently stopped viral replication and offer a promising therapeutic alternative against virus infection.

Molecular characterization and epidemic history of hepatitis C virus using core sequences of isolates from Central Province, Saudi Arabia.

The source of HCV transmission in Saudi Arabia is unknown. This study aimed to determine HCV genotypes in a representative sample of chronically infected patients in Saudi Arabia. All HCV isolates were genotyped and subtyped by sequencing of the HCV core region and 54 new HCV isolates were identified. Three sets of primers targeting the core region were used for both amplification and sequencing of all isolates resulting in a 326 bp fragment. Most HCV isolates were genotype 4 (85%), whereas only a few isolates were recognized as genotype 1 (15%). With the assistance of Genbank database and BLAST, subtyping results showed that most of genotype 4 isolates were 4d whereas most of genotype 1 isolates were 1b. Nucleotide conservation and variation rates of HCV core sequences showed that 4a and 1b have the highest levels of variation. Phylogenetic analysis of sequences by Maximum Likelihood and Bayesian Coalescent methods was used to explore the source of HCV transmission by investigating the relationship between Saudi Arabia and other countries in the Middle East and Africa. Coalescent analysis showed that transmissions of HCV from Egypt to Saudi Arabia are estimated to have occurred in three major clusters: 4d was introduced into the country before 1900, the major 4a clade's MRCA was introduced between 1900 and 1920, and the remaining lineages were introduced between 1940 and 1960 from Egypt and Middle Africa. Results showed that no lineages seem to have crossed from Egypt to Saudi Arabia in the last 15 years. Finally, sequencing and characterization of new HCV isolates from Saudi Arabia will enrich the HCV database and help further studies related to treatment and management of the virus.

Hepatitis c virus genotype 4 replication in the hepatocellular carcinoma cell line HepG2/C3A.

BACKGROUND/AIMS: The lack of a reliable cell culture system allowing persistent in vitro hepatitis C virus (HCV) propagation is still restraining the search for novel antiviral strategies. HepG2 cells transfection with HCV allows for viral replication. However, the replication is weak presumably because of HepG2 lack of miRNA-122, which is essential for viral replication. Other agents such as polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) have been shown to increase the efficiency of infection with other viruses. This study included comparison of HCV genotype 4 5'UTR and core RNA levels and HCV core protein expression at different time intervals in the absence or presence of PEG and/or DMSO postinfection. MATERIALS AND METHODS: We used serum with native HCV particles in infecting HepG2 cells in vitro. HCV replication was assessed by reverse transcriptase polymerase chain reaction for detection of HCV RNA and immunofluorescence and flow cytometry for detection of HCV core protein. RESULTS: HCV 5'UTR and core RNA expression was evident at different time intervals after viral infection, especially after cells were treated with PEG. HCV core protein was also evident at different time intervals using both immunofluorescence and flow cytometry. PEG, not DMSO, has increased the HCV core protein expression in the treated cells, similar to its effect on viral RNA expression. CONCLUSIONS: These expression profiles suggest that the current model of cultured HepG2 cells allows the study of HCV genotype 4 replication and different stages of the viral life cycle.

Characterization of hepatitis C virus genotypes by direct sequencing of HCV 5'UTR region of isolates from Saudi Arabia.

The current study was designed to determine the Hepatitis C Virus (HCV) genotypes in a representative sample of HCV chronically infected patients in Saudi Arabia. All HCV isolates were genotyped by sequencing of the 5'UTR region and newly identified HCV isolates were identified. Specific universal primers targeting 5'UTR region were used for both amplification and sequencing of all isolates that resulted in 244 bp fragment which represent about 80% of 5'UTR region. Most of HCV isolates in this study were genotype 4 (76.4%) where only few isolates were recognized as genotype 1 (19.6%). All results were compared to HCV reference sequences from LOS ALAMOS HCV database, considering only the complete full genomes for the main phylogenetic analysis. Sequences that showed maximum identity (98% -100%) were selected. Most isolates were identical with HCV genotype 4 references. Some isolates were similar to different subtypes of HCV genotypes 4, 1 and 6. Phylogenetic analysis showed resemblance of most isolates to similar ones from the Far East, North America and Egypt. Using sequence Weblogo, Alignment analysis of isolated HCV genotypes 4 and 1 showed 92% and 95.5% nucleotide conservation, respectively. There was no predominant nucleotide in the varied sites, in both genotypes. All isolated sequences were submitted to GenBank database.

Salamander-derived, human-optimized nAG protein suppresses collagen synthesis and increases collagen degradation in primary human fibroblasts.

Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF- ß 1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects.