Bochra Ben Rhouma

A Novel Nonsense Mutation in HSD17B3 Gene in a Tunisian Patient with Sexual Ambiguity

INTRODUCTION 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) isoenzyme is present almost exclusively in the testes and converts delta 4 androstenedione to testosterone. Mutations in the HSD17B3 gene cause HSD17B3 deficiency and result in 46,XY Disorders of Sex Development (46,XY DSD). AIM This study aimed to present the clinical and biochemical features of a Tunisian patient who presented a sexual ambiguity orienting to HSD17B3 deficiency and to search for a mutation in the HSD17B3 gene by DNA sequencing. METHODS Polymerase chain reaction (PCR) amplification and subsequent sequencing of all the coding exons of HSD17B3 gene were performed on genomic DNA from the patient, her family, and 50 controls. RESULTS Genetic mutation analysis of the HSD17B3 gene revealed the presence of a novel homozygous nonsense mutation in the exon 9 (c.618 C>A) leading to the substitution p.C206X. The mutation p.C206X in the coding exons supports the hypothesis of HSD17B3 deficiency in our patient. CONCLUSION The patient described in this study represented a new case of a rare form of 46,XY DSD, associated to a novel gene mutation of HSD17B3 gene. The screening of this mutation is useful for confirming the diagnosis of HSD17B3 deficiency and for prenatal diagnosis.

Biochemical analyses and molecular modeling explain the functional loss of 17β-hydroxysteroid dehydrogenase 3 mutant G133R in three Tunisian patients with 46, XY Disorders of Sex Development

Mutations in the HSD17B3 gene resulting in 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency cause 46, XY Disorders of Sex Development (46, XY DSD). Approximately 40 different mutations in HSD17B3 have been reported; only few mutant enzymes have been mechanistically investigated. Here, we report novel compound heterozygous mutations in HSD17B3, composed of the nonsense mutation C206X and the missense mutation G133R, in three Tunisian patients from two non-consanguineous families. Mutants C206X and G133R were constructed by site-directed mutagenesis and expressed in HEK-293 cells. The truncated C206X enzyme, lacking part of the substrate binding pocket, was moderately expressed and completely lost its enzymatic activity. Wild-type 17β-HSD3 and mutant G133R showed comparable expression levels and intracellular localization. The conversion of Δ4-androstene-3,17-dione (androstenedione) to testosterone was almost completely abolished for mutant G133R compared with wild-type 17β-HSD3. To obtain further mechanistic insight, G133 was mutated to alanine, phenylalanine and glutamine. G133Q and G133F were almost completely inactive, whereas G133A displayed about 70% of wild-type activity. Sequence analysis revealed that G133 on 17β-HSD3 is located in a motif highly conserved in 17β-HSDs and other short-chain dehydrogenase/reductase (SDR) enzymes. A homology model of 17β-HSD3 predicted that arginine or any other bulky residue at position 133 causes steric hindrance of cofactor NADPH binding, whereas substrate binding seems to be unaffected. The results indicate an essential role of G133 in the arrangement of the cofactor binding pocket, thus explaining the loss-of-function of 17β-HSD3 mutant G133R in the patients investigated.

Novel cases of Tunisian patients with mutations in the gene encoding 17β-hydroxysteroid dehydrogenase type 3 and a founder effect.

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is expressed almost exclusively in the testis and converts Δ4-androstene-3,17-dione to testosterone. Mutations in the HSD17B3 gene causing 17β-HSD3 deficiency are responsible for a rare recessive form of 46, XY Disorders of Sex Development (46, XY DSD). We report novel cases of Tunisian patients with 17β-HSD3 deficiency due to previously reported mutations, i.e. p.C206X and p.G133R, as well as a case with the novel compound heterozygous mutations p.C206X and p.Q176P. Moreover, the previously reported polymorphism p.G289S was identified in a heterozygous state in combination with a novel non-coding variant c.54G>T, also in a heterozygous state, in a male patient presenting with micropenis and low testosterone levels. The identification of four different mutations in a cohort of eight patients confirms the generally observed genetic heterogeneity of 17β-HSD3 deficiency. Nevertheless, analysis of DNA from 272 randomly selected healthy controls from the same geographic area (region of Sfax) revealed a high carrier frequency for the p.C206X mutation of approximately 1 in 40. Genotype reconstruction of the affected pedigree members revealed that all p.C206X mutation carriers harbored the same haplotype, indicating inheritance of the mutation from a common ancestor. Thus, the identification of a founder effect and the elevated carrier frequency of the p.C206X mutation emphasize the importance to consider this mutation in the diagnosis and genetic counseling of affected 17β-HSD3 deficiency pedigrees in Tunisia.

Splicing Defects in the AAAS Gene Leading to both Exon Skipping and Partial Intron Retention in a Tunisian Patient with Allgrove Syndrome

Background/Aims: Allgrove syndrome is a rare autosomal recessive disorder characterized by the triad of adrenal insufficiency, achalasia, and alacrima. This syndrome is caused by mutations in the AAAS gene. A major splice site mutation c.1331+1G>A was found previously in North African families affected by Allgrove syndrome. In this study, we analyzed in vivo and in silico the effect of this mutation on the splicing process. Methods: Using reverse transcriptase-polymerase chain reaction, sequencing and bioinformatics tools, we analyzed all transcripts produced by the AAAS gene containing this splice site mutation. Results: The altered splicing of mRNA produces two aberrant transcripts: one with exon 14 skipping, the other with concurrent exon 14 skipping and retention of 99 bp of intron 14, both outcomes resulting in frameshifts with a new stop codon generation in the untranslated region of the last exon. Using in silico bioinformatics tools, we demonstrated that this mutation abolishes the splice donor site of exon 14 and activates a new intronic cryptic splice site in intron 14. Conclusion: This study demonstrated that a single splicing mutation affects the AAAS transcripts and consequently the ALADIN protein structure and function.

Mutational analysis in patients with neuromuscular disorders: Detection of mitochondrial deletion and double mutations in the MT-ATP6 gene.

Mitochondrial diseases encompass a wide variety of pathologies characterized by a dysfunction of the mitochondrial respiratory chain resulting in an energy deficiency. The respiratory chain consists of five multi-protein complexes providing coupling between nutrient oxidation and phosphorylation of ADP to ATP. In the present report, we studied mitochondrial genes of complex I, III, IV and V in 2 Tunisian patients with mitochondrial neuromuscular disorders. In the first patient, we detected the m.8392C>T variation (P136S) in the mitochondrial ATPase6 gene and the m.8527A>G transition at the junction MT-ATP6/MT-ATP8 which change the initiation codon AUG to GUG. The presence of these two variations in such an important gene could probably affect the ATP synthesis in the studied patient. In the second patient, we detected several known variations in addition to a mitochondrial deletion in the major arc of the mtDNA eliminating tRNA and respiratory chain protein genes. This deletion could be responsible of an inefficient translation leading to an inefficient mitochondrial protein synthesis in P2.

Cytogenetic monitoring of hospital staff exposed to ionizing radiation: Optimize protocol considering DNA repair genes variability

Abstract Purpose: Chronic occupational exposure to ionizing radiation (IR) induces a wide spectrum of DNA damages. The aim of this study was to assess the frequencies of micronucleus (MN), sister chromatid exchanges (SCE) and to evaluate their association with XRCC1 399 Arg/Gln and XRCC3 241 Thr/Met polymorphisms in Hospital staff occupationally exposed to IR. Materials and methods: A questionnaire followed by a cytogenetic analysis was concluded for each subject in our study. The exposed subjects were classified into two groups based on duration of employment (Group I < 15 years; Group II ≥15years). The genotypes of all individuals (subjects and controls) were determined by the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Results: DNA damage frequencies were significantly greater in IR workers compared with controls (p < .05). However, no association arised between XRCC1 399 Arg/Gln and XRCC3 241 Thr/Met polymorphisms, on one hand, and the severity of DNA damages in the studied cohort of Tunisian population, on the other hand. Conclusion: Our data provide evidence for an obvious genotoxic effect associated with IR exposure and reinforce the high sensitivity of cytogenetic assays for biomonitoring of occupationally exposed populations. These results indicate that workers exposed to IR should have periodic monitoring, along their exposure. The variants, rs25487 and rs861539, of XRCC1 and XRCC3 genes have obvious functional effects. Paradoxically, these variants are not associated with the severity of damages, according to used assays, in the studied cohort of Tunisian population, unlike other studies.

Novel mutations in the CDKL5 gene in complex genotypes associated with West syndrome with variable phenotype: First description of somatic mosaic state

INTRODUCTION West syndrome is a rare epileptic encephalopathy of early infancy, characterized by epileptic spasms, hypsarrhythmia, and psychomotor retardation beginning in the first year of life. METHODS The present study reports the clinical, molecular and bioinformatic investigation in the three studied West patients. RESULTS The results revealed a complex genotype with more than one mutation in each patient including the known mutations c.1910C>G (P2, P3); c.2372A>C in P3 and c.2395C>G in P1 and novel variants including c.616G>A, shared by the three patients P1, P2 and P3; c.1403G>C shared by P2 and P3 and c.2288A>G in patient P1. CONCLUSIONS All the mutations were at somatic mosaic state and were de novo in the patients except ones (c.2372A>C). To our knowledge; the somatic mosaic state is described for the first time in patients with West syndrome. Five identified mutations were located in the C-terminal domain of the protein, while the novel mutation (c.616G>A) was in the catalytic domain. Bioinformatic tools predicted that this latter is the most pathogenic substitution affecting 3D protein structure and the secondary mRNA structure. Complex genotype composed of different combinations of mutations in each patient seems to be related to the phenotype variability.

ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling

Significance Sex determination involves antagonistic interactions between the testis-determining (SRY-SOX9-FGF9) and ovary-promoting (RSPO1-WNT/β-catenin-FOXL2) pathways, but the underlying molecular mechanisms remain unclear. We show that ZNRF3, an E3 ubiquitin ligase that inhibits WNT signaling and is a direct target of RSPO1-mediated membrane clearance, is testis-determining in mice. Testis determination defects in the absence of ZNRF3 arise due to ectopic canonical WNT signaling in XY gonads at the sex-determining stage. We identify human ZNRF3 sequence variants in cases of 46,XY disorders of sex development with XY female presentation. In vitro functional assays show that these variants disrupt ZNRF3 function. Our data reveal a sex-determining role for ZNRF3 and indicate that interactions between ZNRF3 and RSPO1 regulate mammalian sex determination. Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-β-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/β-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

Clinical, Molecular, and Computational Analysis Showed a Novel Homozygous Mutation Among the Substrate-Binding Site of ARSA Protein in Consanguineous Family with Late-Infantile MLD

Metachromatic leukodystrophy (MLD) is a neurodegenerative disorder characterized by progressive demyelination resulting from impaired degradation and thus the accumulation of cerebroside-3-sulfate (sulfatide). It is caused by the deficiency of arylsulfatase A (ARSA) enzyme which is encoded by the ARSA gene. The present study reports the clinical, molecular, and bioinformatic investigation of three patients belonging to a consanguineous family with late-infantile MLD disorder. The results revealed a novel homozygous missense mutation c.699C>A (p.His231Gln) in exon 4 of ARSA gene in the three patients inherited from their heterozygous parents. Interestingly, this novel mutation is the second mutation identified in the substrate-binding site of ARSA protein and it was classified as damaging and deleterious by several bioinformatics tools. The c.699C>A (p.His231Gln) leads to changes in the pre-mRNA secondary structure and in the ARSA protein 3D structure with a significant root mean square deviation value which could probably affect its stability and function.

A duplex polymerase chain reaction-restriction fragment length polymorphism for rapid screening of methylenetetrahydrofolate reductase gene variants: Genotyping in acute leukemia

Methylenetetrahydrofolate reductase (MTHFR; NM_005957.4) is the key enzyme for folate metabolism which plays in DNA biosynthesis and the epigenetic process of DNA methylation. MTHFR gene polymorphisms, the c. 677C>T and c. 1298A>C have been implicated as risk factors for several types of cancers as the acute leukemia.