Biochemical diagnosis of Sanfilippo disorder types A and B.

BACKGROUND: One of the 11 recognized mucopolysaccharidosis (MPS) diseases is Sanfilippo. It is autosomal recessive in its mode of transmission. There are four subtypes of Sanfilippo (A, B, C, and D). The most worldwide prevalent subtypes of mucopolysaccharidosis type III (MPS III) are A and B followed by C and D subtypes. To estimate the frequency of MPS IIIA among MPS III patients, we diagnose and compare their clinical features with those of MPS IIIB and also compare the prevalence of MPS IIIB versus MPS IIIA among diagnosed cases at the Biochemical Genetic Department at NRC. For every case that was referred, the quantitative determination of urine Glycosaminoglycans (GAGs) was assessed. Two-dimensional electrophoresis (2DE) of GAGs extracted from urine was performed on all cases with high urinary GAG levels. Both N-sulphoglucosamine sulphohydrolase (MPS IIIA) and N-alpha-acetylglucosaminidase (MPS IIIB) enzyme activity were determined fluorometrically. RESULTS: From November 2019 to May 2022, 535 cases were referred to the National Research Centre's Biochemical Genetics Department. 233 (43%) MPS cases were diagnosed with high urinary GAG levels for their ages. 73 (31.3%) MPS III cases were diagnosed by 2DE out of the 233 MPS cases. Plasma N-alpha-acetylglucosaminidase enzyme assay was insufficient in 36 (49.3%) patients (Sanfilippo type B), while N-sulphoglucosamine sulphohydrolase enzyme activity was deficient in 15 (20.6%) patients. The other 22 (30.1%) patients are either Sanfilippo type C or D. CONCLUSION: N-sulphoglucosamine sulphohydrolase enzyme activity was measured for the first time in Egypt. Thirty-one percent of all diagnosed MPS cases during the last 3 years were MPS type III, making Sanfilippo the most common MPS type among the referred cases to our Biochemical Genetics Department. MPS IIIA accounts for 20.6% of MPSIII cases in this study. Still, MPS type IIIB is the commonest type among diagnosed patients.

An atypical expression of core α-Dystroglycan and Laminin-α2 in skin fibroblasts of patients with congenital muscular dystrophies.

BACKGROUND: Congenital muscular dystrophies (CMDs) result from genetically inherited defects in the biosynthesis and/or the posttranslational modification (glycosylation) of laminin-α2 and α-dystroglycan (α-DG), respectively. The interaction between both proteins is responsible for the stability and integrity of the muscle cell. We aimed to study the expression profiles of both proteins in two classes of CMDs. SUBJECTS AND METHODS: Whole-exome sequencing (WES) was done for four patients with neuromuscular manifestations. The expression of core α-DG and laminin-α2 subunit in skin fibroblasts and MCF-7 cells was assessed by western blot. RESULTS: WES revealed two cases with nonsense mutations; c.2938G > T and c.4348 C > T, in LAMA2 encodes laminin-α2. It revealed also two cases with mutations in POMGNT1 encode protein O-mannose beta-1,2-N-acetylglucosaminyltransferase mutations. One patient had a missense mutation c.1325G > A, and the other had a synonymous variant c.636 C > T. Immunodetection of core α-DG in skin fibroblasts revealed the expression of truncated forms of core α-DG accompanied by reduced expression of laminin-α2 in POMGNT1-CMD patients and one patient with LAMA2-CMD. One patient with LAMA2-CMD had overexpression of laminin-α2 and expression of a low level of an abnormal form of increased molecular weight core α-DG. MCF-7 cells showed truncated forms of core α-CDG with an absent laminin-α2. CONCLUSION: A correlation between the expression pattern/level of core α-DG and laminin-α2 could be found in patients with different types of CMD.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum.

BACKGROUND: Finding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. METHODS: In this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography-mass spectrometric analysis (GC-MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. RESULTS: The findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. CONCLUSIONS: The anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

Production of a novel laccase from Ceratorhiza hydrophila and assessing its potential in natural dye fixation and cytotoxicity against tumor cells.

BACKGROUND: Flavonoid natural dyes have gained attention because they are nontoxic and eco-friendly. However, they do not work effectively with artificial fibers and require the use of mordants, which are considered as hazardous chemicals. Laccase enzyme catalyzes the oxidation of phenols, forming phenoxyl radicals that undergo a further polymerization process. So, laccase can oxidize flavonoid dyes, and it can be used instead of harmful mordants in flavonoid dye fixation on cotton fabrics. Laccases also are involved in a variety of metabolic processes, and they have anti-proliferative effects toward HepG2 and MCF-7 tumor cells. RESULTS: Among fifteen fungal isolates, the fungus Ceratorhiza hydrophila isolated from the submerged plant Myriophyllum spicatum was selected as the most potent laccase producer. Optimization of the production medium resulted in a 9.9-fold increase in laccase productivity. The partially purified Ceratorhiza hydrophila laccase could successfully improve the affinity of cotton fabrics toward quercetin (flavonoid) dye with excellent color fastness properties. The partially purified laccase also showed anti-proliferative activity against HepG2 and MCF-7 tumor cells. However, high laccase concentration is required to estimate IC50. CONCLUSIONS: Ceratorhiza hydrophila MK387081 is an excellent laccase producer. The partially purified laccase from Ceratorhiza hydrophila can be used in textile dyeing and printing processes as a safer alternative to the conventional hazardous mordants. Also, it can be used in preparation of cancer treatment drugs. However, further studies are needed to investigate IC50 for both cell types at higher laccase concentrations.

Prenatal diagnosis of mucopolysaccharidoses type II by two-dimensional electrophoresis and mass spectrometry in amniotic fluid.

AIM: To introduce a quantitative determination of heparan sulfate and dermatan sulfate by mass spectrometry and to compare it with two-dimensional electrophoresis of the glycosaminoglycans in the amniotic fluid for the prenatal diagnosis of mucopolysaccharidoses type II (MPS II). METHODS: Thirty pregnancies each with single fetus were subjected to amniocentesis at 16 weeks: 10 with a previously affected MPS II infant and 20 as controls. Prenatal diagnosis was done by both mass spectrometry two two-dimensional electrophoresis. RESULTS: Two-dimensional electrophoresis showed four affected with MPS II and six unaffected fetuses. Mass spectrometry verified these results. CONCLUSION: Two-dimensional electrophoresis of the glycosaminoglycans in amniotic fluid is a good qualitative method and mass spectrometry is a new accurate quantitative method for prenatal diagnosis of MPS II. Quantitative determination of glycosaminoglycans in amniotic fluid by mass spectrometry is both rapid and accurate. Prenatal diagnosis is recommended for at risk pregnancies and mass spectrometry offers speed and quantitation.

Quaternary diagnostics scheme for mucolipidosis II and detection of novel mutation in GNPTAB gene.

BACKGROUND: Mucolipidosis II (ML II α/ß) is an inherited lysosomal storage disorder caused by deficiency of GlcNAc-phosphotransferase enzyme and results in mis-targeting of multiple lysosomal enzymes. Affected patients are characterized by skeletal deformities and developmental delay. Homozygous or compound heterozygous mutations in GNPTAB gene are associated with the clinical presentation. This is the first study to characterize the underlying genetics of ML among a cohort of Egyptian patients. ML II diagnosis established by clinical assessment, biochemical evaluation of enzymes, electron microscopy examination of gingival inclusion bodies, and molecular study of GNPTAB gene using targeted next-generation sequencing panel in 8 patients form 8 unrelated Egyptian families. RESULTS: Sequencing revealed 3 mutations in GNPTAB gene; 1 novel frame-shift mutation in exon 19 (c.3488_3488delC) and 2 previously reported mutations (c.1759C>T in exon 13 and c.3503_3504delTC in exon 19). All patients were homozygous for their corresponding mutations and the parents were consanguineous. CONCLUSIONS: According to the established quaternary diagnostic scheme, ML II was the final diagnosis in eight patients. The most common mutation was the frame shift c.3503_3504delTC mutation, found in 5 patients and associated with a severe phenotype.

Four novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase gene among Egyptian patients with Morquio A disease.

Morquio A disease (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient activity of the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) encoded by the GALNS gene. This deficiency leads to a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing their accumulation within the lysosomes and consequently prominent skeletal and visceral abnormalities. Clinical evaluation and biochemical GALNS enzyme activity determination were carried out for the patients from four unrelated Egyptian families. Mutational analysis was performed to PCR products by sequencing of the 14 exons and exon-intron boundaries of GALNS gene for the 4 patients. Sequence analysis revealed four novel mutations; three nonsense mutations (p.Q12X, p.Q220X, p.Y254X) and one missense mutation, p.D40G. All four patients were offspring of consanguineous marriages and were homozygous for the corresponding mutation. The activity of the GALNS enzyme was below normal reference range in all of them. The p.Q12X and p.Y254X were associated with severe MPS IVA phenotype. Molecular analysis of GALNS gene revealed four novel mutations in four different Morquio A Egyptian patients.

Study of nonstandard auto-antibodies as prognostic markers in auto immune hepatitis in children.

BACKGROUND: Antibodies to chromatin and soluble liver antigen have been associated with severe form of autoimmune hepatitis and/or poor treatment response and may provide guidance in defining subsets of patients with different disease behaviors. The major clinical limitation of these antibodies is their lower individual occurrence in patients with autoimmune hepatitis. AIM: To estimate the value of detection of these non-standard antibodies in autoimmune hepatitis as prognostic markers. METHODS: Both antibodies were tested by enzyme immunoassay in 20 patients with autoimmune hepatitis. RESULTS: Antibodies to soluble liver antigen were not detected in any of our patients. On the other hand anti chromatin antibodies were present in 50% (10/20). Antibodies to chromatin occurred more commonly in females than males (8/14 versus 2/6). Of the 14 patients who relapsed 8(57%) had antichromatin antibodies while they were present in only 2 out of 6(33.3%) non relapsers. Antichromatin antibodies were found more in patients with antinuclear (3/4) and anti smooth muscle antibodies (9/13) more than in those with liver kidney microsomal antibodies (1/4) and those seronegative (1/4) i.e. they were +ve in patients with type I (8/12(66.6%)) more than those with type II (1/4(25%)) and those seronegative (1/4(25%)). Antibodies to chromatin are associated with high levels of gamma globulin but yet with no statistical difference between seropositive and seronegative counterparts (p = 0.65). CONCLUSION: Antibodies to chromatin may be superior than those to soluble liver antigen in predicting relapse and may be useful as prognostic marker. Further studies with larger number of patients and combined testing of more than one antibody will improve the performance parameters of these antibodies and define optimal testing conditions for them before they can be incorporated into management algorithms that project prognosis.

Flock dynamics of desert Barki sheep in relation to age structure.

Reproduction data of 8689 ewe records spread over 40 years (from 1960 to 2000) representing 2952 breeding Barki ewes were used in this study. The flock belonged to the Desert Research Center in Egypt. Flock dynamics of nine age groups (2-10 yrs) were assessed. Two parameters were used to evaluate flock dynamics, net reproduction rate (R(o)) (number of ewe- lambs reaching joining age and produced by each ewe during its lifetime in the flock) and intrinsic rate of increase (r(m)) (flock growth when no resource is limiting). Age of ewe had a highly significant (P < 0.01) effect on number of ewes lambing per ewe joined (E(PJ)), number of lambs born per ewe joined (L(BJ)), number of lambs weaned per ewe joined (L(WJ)) and number of ewe lambs reaching joining age per ewe joined (L(EJ.J)). All estimates tended to increase with dams age up to four years and decreased thereafter. The results of R(o) and r(m) showed that the studied flock must consist of 5 age groups to maintain its size and replace itself. It may be recommended to cull the breeding ewe at the age of 6 years to accelerate genetic improvement.