Molecular and computational characterization of ABCB11 and ABCG5 variants in Tunisian patients with neonatal/infantile low-GGT intrahepatic cholestasis: Genetic diagnosis and genotype-phenotype correlation assessment.

Many inherited conditions cause hepatocellular cholestasis in infancy, including progressive familial intrahepatic cholestasis (PFIC), a heterogeneous group of diseases with highly overlapping symptoms. In our study, six unrelated Tunisian infants with PFIC suspicion were the subject of a panel-target sequencing followed by an exhaustive bioinformatic and modeling investigations. Results revealed five disease-causative variants including known ones: (the p.Asp482Gly and p.Tyr354 * in the ABCB11 gene and the p.Arg446 * in the ABCC2 gene), a novel p.Ala98Cys variant in the ATP-binding cassette subfamily G member 5 (ABCG5) gene and a first homozygous description of the p.Gln312His in the ABCB11 gene. The p.Gln312His disrupts the interaction pattern of the bile salt export pump as well as the flexibility of the second intracellular loop domain harboring this residue. As for the p.Ala98Cys, it modulates both the interactions within the first nucleotide-binding domain of the bile transporter and its accessibility. Two additional potentially modifier variants in cholestasis-associated genes were retained based on their pathogenicity (p.Gly758Val in the ABCC2 gene) and functionality (p.Asp19His in the ABCG8 gene). Molecular findings allowed a PFIC2 diagnosis in five patients and an unexpected diagnosis of sisterolemia in one case. The absence of genotype/phenotype correlation suggests the implication of environmental and epigenetic factors as well as modifier variants involved directly or indirectly in the bile composition, which could explain the cholestasis phenotypic variability.

Identification of mutations that causes glucose-6-phosphate transporter defect in tunisian patients with glycogenosis type 1b.

BACKGROUND: Glycogen storage disease type 1b (GSD1b) is an autosomal recessive lysosomal storage disease caused by defective glucose-6-phosphate transporter encoded by SLC37A4 leading to the accumulation of glycogen in various tissues. The high rate of consanguineous marriages in Tunisian population provides an ideal environment to facilitate the identification of homozygous pathogenic mutations. We aimed to determine the clinical and genetic profiles of patients with GSD1b to evaluate SLC37A4 mutations spectrum in Tunisian patients. METHODS: All exons and flanking intron regions of SLC37A4 gene were screened by direct sequencing to identify mutations and polymorphisms in three unrelated families with GSD1b. Bioinformatics tools were then used to predict the impacts of identified mutations on the structure and function of protein in order to propose a function-structure relationship of the G6PT1 protein. RESULTS: Three patients (MT, MB and SI) in Families I, II and III who had the severe phenotype were homoallelic for the two identified mutations: p.R300H (famillies I, II) and p.W393X (Family III), respectively. One of the alterations was a missense mutation p.R300H of exon 6 in SLC37A4 gene. The analysis of the protein structure flexibility upon p.R300H mutation using DynaMut tool and CABS-flex 2.0 server showed that the reported mutation increase the molecule flexibility of in the cytosol region and would probably lead to significant conformational changes. CONCLUSION: This is the first Tunisian report of SLC37A4 mutations identified in Tunisia causing the glycogenosis type Ib disease. Bioinformatics analysis allowed us to establish an approximate structure-function relationship for the G6PT1 protein, thereby providing better genotype/phenotype correlation knowledge.

A Case of Infantile Cataract and Neonatal Hypoglycemia.

Purpose: To describe a case of lamellar cataract in a child with a history of neonatal hypoglycemia and illustrate the importance of meticulous history-taking in children with cataracts, especially before planning surgery. Methods: A single case report. Results: A 7-year-old girl with a history of neonatal hypoglycemia and ketoacidosis with high suspicion of a primary defect in ketone body utilization who presented with bilateral progressive blurred vision. Her best-corrected visual acuity (BCVA) was 20/200 in her right eye (OD) and 20/400 in her left eye (OS). Slit-lamp examination showed the presence of bilateral dense lamellar cataract. Fundus examination was unremarkable in both eyes (OU). She underwent cataract surgery with intraocular lens implantation under general anesthesia without prolonged fasting. Final BCVA was 20/82 in OD and 20/63 in OS. Conclusion: Our aim through this case is to emphasize the importance to screen for episodes of hypoglycemic attacks in children with infantile cataracts.

Nonketotic Hyperglycinemia in Tunisia. Report upon a Series of 69 Patients.

AIM: The aim of the study is to report on epidemiological, clinical, and biochemical characteristics of nonketotic hyperglycinemia (NKH) in Tunisia. METHODS: Patients diagnosed with NKH in Laboratory of Biochemistry at Rabta hospital (Tunis, Tunisia) between 1999 and 2018 were included. Plasma and cerebrospinal fluid (CSF) free amino acids were assessed by ion exchange chromatography. Diagnosis was based on family history, patient's clinical presentation and course, and increased CSF to plasma glycine ratio. RESULTS: During 20 years, 69 patients were diagnosed with NKH, with 25 patients originating from Kairouan region. Estimated incidences were 1:55,641 in Tunisia and 1:9,684 in Kairouan. Consanguinity was found for 73.9% of the patients and 42% of the families have history of infantile death due to a disease of similar clinical course than the propositus. Clinical symptoms initiated within the first week of life in 75% of the patients and within the first 3 months in 95.7% ones. The phenotype was severe in 76.8% of the patients. Main symptoms were hypotonia, feeding difficulties, coma, apnea, and seizures. Most patients died within few days to months following diagnosis. CSF to plasma glycine ratio was increased in all patients. CSF and plasma glycine levels were negatively correlated with age of disease onset and severity. CONCLUSION: NKH is quite frequent in Tunisia. Kairouan region has the highest NKH incidence rate, worldwide. However, due to lack of confirmatory enzymatic and genetic tests, NKH diagnosis was based on first-line biochemical tests. Characterization of causal mutations is needed for accurate diagnosis and prenatal diagnosis of this devastating life-threatening disease.

A severe clinical phenotype of Noonan syndrome with neonatal hypertrophic cardiomyopathy in the second case worldwide with RAF1 S259Y neomutation.

Noonan syndrome and related disorders are a group of clinically and genetically heterogeneous conditions caused by mutations in genes of the RAS/MAPK pathway. Noonan syndrome causes multiple congenital anomalies, which are frequently accompanied by hypertrophic cardiomyopathy (HCM). We report here a Tunisian patient with a severe phenotype of Noonan syndrome including neonatal HCM, facial dysmorphism, severe failure to thrive, cutaneous abnormalities, pectus excavatum and severe stunted growth, who died in her eighth month of life. Using whole exome sequencing, we identified a de novo mutation in exon 7 of the RAF1 gene: c.776C > A (p.Ser259Tyr). This mutation affects a highly conserved serine residue, a main mediator of Raf-1 inhibition via phosphorylation. To our knowledge the c.776C > A mutation has been previously reported in only one case with prenatally diagnosed Noonan syndrome. Our study further supports the striking correlation of RAF1 mutations with HCM and highlights the clinical severity of Noonan syndrome associated with a RAF1 p.Ser259Tyr mutation.

Molecular and biochemical characterization of a novel intronic single point mutation in a Tunisian family with glycogen storage disease type III.

Genetic deficiency of the glycogen debranching enzyme causes glycogen storage disease type III, an autosomal recessive inherited disorder. The gene encoding this enzyme is designated as AGL gene. The disease is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy and cardiomyopathy. In the present study, we present clinical features and molecular characterization of two consanguineous Tunisian siblings suffering from Glycogen storage disease type III. The full coding exons of the AGL gene and their corresponding exon-intron boundaries were amplified for the patients and their parents. Gene sequencing identified a novel single point mutation at the conserved polypyrimidine tract of intron 21 in a homozygous state (IVS21-8A>G). This variant cosegregated with the disease and was absent in 102 control chromosomes. In silico analysis using online resources showed a decreased score of the acceptor splice site of intron 21. RT-PCR analysis of the AGL splicing pattern revealed a 7 bp sequence insertion between exon 21 and exon 22 due to the creation of a new 3' splice site. The predicted mutant enzyme was truncated by the loss of 637 carboxyl-terminal amino acids as a result of premature termination. This novel mutation is the first mutation identified in the region of Bizerte and the tenth AGL mutation identified in Tunisia. Screening for this mutation can improve the genetic counseling and prenatal diagnosis of GSD III.

First report of a molecular prenatal diagnosis in a tunisian family with lysinuric protein intolerance.

Lysinuric protein intolerance (LPI, MIM# 222700) is an inherited aminoaciduria caused by defective transport of cationic amino acids (CAAs; arginine, lysine, ornithine) at the basolateral membrane of epithelial cells in the intestine and kidney. We report the first prenatal diagnosis by direct mutational analysis of LPI performed in a Tunisian family. An amniotic fluid sample was carried out at 16 weeks of gestation in a 32-year-old Tunisian woman who consulted for prenatal diagnosis. The 1471 delTTCT mutation at homozygous state was identified indicating that the fetus was affected by LPI. The identification of this specific mutation provides a tool, which can be easily applied in Tunisia for molecular diagnosis, genetic counseling, and prenatal diagnosis of LPI.