Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects.

Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Molecular mechanisms underlying the defects of two novel mutations in the HSD17B3 gene found in the Tunisian population.

17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) converts Δ4-androstene-3,17-dione (androstenedione) to testosterone. It is expressed almost exclusively in the testes and is essential for appropriate male sexual development. More than 70 mutations in the HSD17B3 gene that cause 17ß-HSD3 deficiency and result in 46,XY Disorders of Sex Development (46,XY DSD) have been reported. This study describes three novel Tunisian cases with mutations in HSD17B3. The first patient is homozygous for the previously reported mutation p.C206X. The inheritance of this mutation seemed to be independent of consanguineous marriage, which can be explained by its high frequency in the Tunisian population. The second patient has a novel splice site mutation in intron 6 at position c.490 -6 T > C. A splicing assay revealed a complete omission of exon 7 in the resulting HSD17B3 mRNA transcript. Skipping of exon 7 in HSD17B3 is predicted to cause a frame shift in exon 8 that affects the catalytic site and results in a truncation in exon 9, leading to an inactive enzyme. The third patient is homozygous for the novel missense mutation p.K202M, representing the first mutation identified in the catalytic tetrad of 17ß-HSD3. Site-directed mutagenesis and enzyme activity measurements revealed a completely abolished 17ß-HSD3 activity of the p.K202M mutant, despite unaffected protein expression, compared to the wild-type enzyme. Furthermore, the present study emphasizes the importance of genetic counselling, detabooization of 46,XY DSD, and a sensitization of the Tunisian population for the risks of consanguineous marriage.

A novel de novo splicing mutation c.1444-2A>T in the TSC2 gene causes exon skipping and premature termination in a patient with tuberous sclerosis syndrome.

Tuberous sclerosis complex (TSC) syndrome is a neurocutaneous syndrome that affects the brain, skin, and kidneys that has an adverse impact on the patient's health and quality of life. There have been several recent advances that elucidate the genetic complex of this disorder that will help understand the basic neurobiology of this disorder. We report a Tunisian patient with clinical manifestations of TSC syndrome. We investigated the causative molecular defect in this patient using PCR followed by direct sequencing. Subsequently, in silico studies and mRNA analysis were performed to study the pathogenicity of the new variation found in the TSC2. Bioinformatics tools predicted that the novel mutation c.1444-2A>T have pathogenic effects on splicing machinery. RT-PCR followed by sequencing revealed that the mutation c.1444-2A>T generates two aberrant transcripts. The first, with exon 15 skipping, is responsible for the loss of 52 amino acids, which causes the production of an aberrant protein isoform. The second, with the inclusion of 122 nucleotides of intron 14, is responsible for the creation of new premature termination codons (TGA), which causes the production of a truncated TSC2 protein. This study highlighted the clinical features of a Tunisian patient with TSC syndrome and revealed a splicing mutation c.1444-2A>T within intron 14 of TSC2 gene, which is present for the first time using Sanger sequencing approach, as a disease-causing mutation in a Tunisian patient with TSC syndrome.

ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling.

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-ß-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/ß-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.

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

BACKGROUND: 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. AIM: We have optimized a duplex polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) for the simultaneous detection of both variants in acute leukemia patients, from Tunisia. METHODS: Genomic DNA was extracted from EDTA-anticoagulant blood samples from a total of 50 patients suffering from acute leukemia (AL). After DNA extraction, the polymerase chain reaction using specific primers, designed using Primer 3 Software. Restriction Fragment Length Polymorphism (RFLP) was performed in two separate tubes followed by agarose gel electrophoresis. CONCLUSION: This new method has proved to be a rapid, simple, and reliable method that should facilitate high throughput genotyping of MTHFR polymorphisms in acute leukemia.

Association study of apoptosis gene polymorphisms in mitochondrial diabetes: A potential role in the pathogenicity of MD.

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia and is maternally transmitted. Syndromic MD is a subgroup of MD including diabetic microangiopathy and macroangiopathy, in addition to extrapancreatic disorder. MD is caused by genetic mutations and deletions affecting mitochondrial DNA. This mitochondrial damage initiates apoptosis. In this study, we hypothesized that functional polymorphisms in genes involved in apoptotic pathway could be associated with the development of apoptosis in MD disease and increased its risk. Detection of apoptosis was confirmed on muscle biopsies taken from MD patients using the TUNEL method and the Cytochrome c protein expression level. We genotyped then 11 published SNPs from intrinsic and extrinsic apoptotic pathway and assessed the signification of these polymorphisms in 43 MD patients and 100 healthy controls. We found 10 selected polymorphisms (p53 (rs1042522 and rs17878362), BCL2 (rs2279115), BAX (rs1805419), BAK1 (rs210132 and rs2227925), CASP3 (rs1405937), CASP7 (rs2227310), CASP8 (rs1045485) and CASP10 (rs13006529)) with a potential apoptosis effect in MD patients compared to control population. Specifically, SNPs involved in the intrinsic pathway (p53, BCL2, BAK1 and CASP3) presented the highest risk of apoptosis. Our result proved that apoptosis initiated by mtDNA mutations, can be emphasized by a functional apoptotic polymorphisms associated with high expression of cytochrome c protein and more myofibers with apoptosis in syndromic MD subgroup compared with non-syndromic MD subgroup.

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

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

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is expressed almost exclusively in the testis and converts Δ4-androstene-3,17-dione to testosterone. Mutations in the HSD17B3 gene causing 17ß-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 17ß-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 17ß-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 17ß-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.

A novel nonsense mutation in HSD17B3 gene in a Tunisian patient with sexual ambiguity.

INTRODUCTION: 17ß-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.