Turner syndrome: results of the first Tunisian study group on Turner syndrome (TuSGOT).

OBJECTIVES: Early diagnosis in Turner syndrome is desirable to optimize growth and puberty and yet, it is often made late. Here, we aim to identify age at diagnosis, clinical features at presentation and potential strategies to improve the care of TS girls. METHODS: Retrospective study, including patients from 14 care centers across Tunisia including neonatal and pediatric care units, adult endocrinology and genetics departments. RESULTS: We identified 175 patients with TS, karyotype showing 45, xmonosomy in 83(47.4 %) with mosaicism in 37(20 %). Mean ± SD, median (range) age at diagnosis available in 173 patients was 13 ± 9.2,12 (birth-48) years. The diagnosis was antenatal in 4(2.3 %), from birth-2 years in 14 (8 %)with lymphoedema (8)and dysmorphic features (9),2-12 years in 53 (35.5 %) including 35 with short stature, 13-18 years in 43(28.8 %) with short stature(28) and delayed puberty(14) and 35(23.5 %) after 18 years, related to ovarian insufficiency (20) and short stature (11). The associated malformations were cardiac in 14 (12.8 %), renal in 22 (19.6 %). A total of 56 girls (32 %) had proven gonadal dysgenesis and 13 (7 %) had otological problems. Parental height was available in 71 girls (40 %) of whom 59 were below the lower end of parental target range (LTR) (83 %). CONCLUSIONS: This first Tunisian multicenter study, the first African of its kind, reveals that more than half of Turner syndrome cases are diagnosed after the age of 12 years. Subsequently, national strategies for an earlier TS diagnosis are needed such as measuring and plotting parental heights as well as introducing a systematic height screening at 5 years in Tunisia with a view to carrying out a re-audit in five years' time.

Calcium Handling in Inherited Cardiac Diseases: A Focus on Catecholaminergic Polymorphic Ventricular Tachycardia and Hypertrophic Cardiomyopathy.

Calcium (Ca2+) is the major mediator of cardiac contractile function. It plays a key role in regulating excitation-contraction coupling and modulating the systolic and diastolic phases. Defective handling of intracellular Ca2+ can cause different types of cardiac dysfunction. Thus, the remodeling of Ca2+ handling has been proposed to be a part of the pathological mechanism leading to electrical and structural heart diseases. Indeed, to ensure appropriate electrical cardiac conduction and contraction, Ca2+ levels are regulated by several Ca2+-related proteins. This review focuses on the genetic etiology of cardiac diseases related to calcium mishandling. We will approach the subject by focalizing on two clinical entities: catecholaminergic polymorphic ventricular tachycardia (CPVT) as a cardiac channelopathy and hypertrophic cardiomyopathy (HCM) as a primary cardiomyopathy. Further, this review will illustrate the fact that despite the genetic and allelic heterogeneity of cardiac defects, calcium-handling perturbations are the common pathophysiological mechanism. The newly identified calcium-related genes and the genetic overlap between the associated heart diseases are also discussed in this review.

Expanding the genetic spectrum of mitochondrial diseases in Tunisia: novel variants revealed by whole-exome sequencing.

Introduction: Inherited mitochondrial diseases are the most common group of metabolic disorders caused by a defect in oxidative phosphorylation. They are characterized by a wide clinical and genetic spectrum and can manifest at any age. In this study, we established novel phenotype-genotype correlations between the clinical and molecular features of a cohort of Tunisian patients with mitochondrial diseases. Materials and methods: Whole-exome sequencing was performed on five Tunisian patients with suspected mitochondrial diseases. Then, a combination of filtering and bioinformatics prediction tools was utilized to assess the pathogenicity of genetic variations. Sanger sequencing was subsequently performed to confirm the presence of potential deleterious variants in the patients and verify their segregation within families. Structural modeling was conducted to study the effect of novel variants on the protein structure. Results: We identified two novel homozygous variants in NDUFAF5 (c.827G>C; p.Arg276Pro) and FASTKD2 (c.496_497del; p.Leu166GlufsTer2) associated with a severe clinical form of Leigh and Leigh-like syndromes, respectively. Our results further disclosed two variants unreported in North Africa, in GFM2 (c.569G>A; p.Arg190Gln) and FOXRED1 (c.1261G>A; p.Val421Met) genes, and we described the first case of fumaric aciduria in a Tunisian patient harboring the c.1358T>C; p.Leu453Pro FH variant. Conclusion: Our study expands the mutational and phenotypic spectrum of mitochondrial diseases in Tunisia and highlights the importance of next-generation sequencing to decipher the pathomolecular mechanisms responsible for these disorders in an admixed population.

Molecular autopsy and clinical family screening in a case of sudden cardiac death reveals ACTN2 mutation related to hypertrophic/dilated cardiomyopathy and a novel LZTR1 variant associated with Noonan syndrome.

BACKGROUND: Genetic cardiac diseases are the main trigger of sudden cardiac death (SCD) in young adults. Hypertrophic cardiomyopathy (HCM) is the most prevalent cardiomyopathy and accounts for 0.5 to 1% of SCD cases per year. METHODS: Herein, we report a family with a marked history of SCD focusing on one SCD young adult case and one pediatric case with HCM. RESULTS: For the deceased young adult, postmortem whole-exome sequencing (WES) revealed a missense variant in the ACTN2 gene: c.355G > A; p.(Ala119Thr) confirming the mixed hypertrophic/dilated cardiomyopathy phenotype detected in the autopsy. For the pediatric case, WES allowed us the identification of a novel frameshift variant in the LZTR1 gene: c.1745delT; p.(Val582Glyfs*10) which confirms a clinical suspicion of HCM related to Noonan syndrome. CONCLUSION: The present study adds further evidence on the pathogenicity of ACTN2: p. Ala119Thr variant in SCD and expands the mutational spectrum of the LZTR1 gene related to Noonan syndrome.

Multiallelic rare variants support an oligogenic origin of sudden cardiac death in the young.

Unexplained sudden death in the young is cardiovascular in most cases. Structural and conduction defects in cardiac-related genes can conspire to underlie sudden cardiac death. Here we report a clinical investigation and an extensive genetic assessment of a Tunisian family with sudden cardiac death in young members. In order to identify the family-genetic basis of sudden cardiac death, we performed Whole Exome Sequencing (WES), read depth copy-number-variation (CNV) screening and segregation analysis. We identify 6 ultra-rare pathogenic heterozygous variants in OBSCN, RYR2, DSC2, AKAP9, CACNA1C and RBM20 genes, and one homozygous splicing variant in TECRL gene consistent with an oligogenic model of inheritance. CNV analysis did not reveal any causative CNV consistent with the family phenotype. Overall, our results are highly suggestive for a cumulative effect of heterozygous missense variants as disease causation and to account for a greater disease severity among offspring. Our study further confirms the complexity of the inheritance of sudden cardiac death and highlights the utility of family-based WES and segregation analysis in the identification of family specific mutations within different cardiac genes pathways.

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.

WDR73-related galloway mowat syndrome with collapsing glomerulopathy.

Galloway-Mowat syndrome (GAMOS [MIM 251300]) is a rare autosomal recessive disorder that manifests as a combination of nephrotic syndrome, brain abnormalities and developmental delay. It is a clinically and genetically heterogeneous disease. The WDR73 variations are associated with GAMOS1. Here we report two consanguineous families affected by GAMOS1. In the first family, three sisters are affected and in the second family, only one index case is identified. They all show a nephrotic syndrome, a neurological involvement and a collapsing glomerulopathy. The analysis of mutations of WDR73 revealed a new homozygous missense mutation NM_032856.3 c.293T > C; p.(Leu98Pro) in two patients from the first family, and a new homozygous missense mutation NM_032856.3: c.767G > A; p.(Arg256Gln) in the second one. This study extended the clinical and molecular spectrum of GAMOS1 with other cases associated with collapsing glomerulopathy and two novel WDR73 variations that are most likely pathogenic.

Novel ALPK3 mutation in a Tunisian patient with pediatric cardiomyopathy and facio-thoraco-skeletal features.

Pediatric cardiomyopathy is a complex disease with clinical and genetic heterogeneity. Recently, the ALPK3 gene was described as a new hereditary cardiomyopathy gene underlying pediatric cardiomyopathies. Only eight patients carrying mutations in ALPK3 have been reported to date. Here, we report a 3-year-old male patient with both hypertrophic and dilated cardiomyopathy. The patient presented dysmorphic features and skeletal deformities of hands and feet, pectus excavatum, and cleft palate. The genetic investigation was performed by whole-exome sequencing in the patient and his parents. We identified a novel homozygous mutation in ALPK3 (c.1531_1532delAA; p.Lys511Argfs*12). Our work extends the phenotypic spectrum of the ALPK3-associated cardiomyopathy by reporting additional clinical features. This is the first study of a Tunisian patient with mutation in the ALPK3 gene. In conclusion, ALPK3 should be included in the list of genes to be considered in genetic studies for patients affected with pediatric syndromic cardiomyopathy.

TOP3B: A Novel Candidate Gene in Juvenile Myoclonic Epilepsy?

Juvenile myoclonic epilepsy (JME) is characterized by seizures, severe cognitive abnormalities, and behavior impairments. These features could evolve over time and get worse, especially when the encephalopathy is pharmacoresistant. Thus, genetic studies should provide a better understanding of infantile epilepsy syndromes. Herein, we investigate the genetics of JME in a consanguineous family analyzing the copy number variations detected using over 700 K SNP arrays. We identified a 254-kb deletion in the 22q11.2 region, including only the TOP3B gene, detected in the patient and her father. TOP3B encodes a topoisomerase DNA (III) ß protein and has been implicated in several neurological diseases such as schizophrenia and autism. In this study, we discuss the implication of the 22q11.2 region in neurodevelopmental disorders and the association of TOP3B with epilepsy.

Novel PAX3 mutations causing Waardenburg syndrome type 1 in Tunisian patients.

Waardenburg syndrome (WS) is an auditory-pigmentary disease characterized by a clinical and genetic variability. WS is classified into four types depending on the presence or absence of additional symptoms: WS1, WS2, WS3 and WS4. Type 1 and 3 are mostly caused by PAX3 mutations, while type 2 and type 4 are genetically heterogeneous. The aims of this study are to confirm the diagnostic of WS1 by the sequencing of PAX3 gene and to evaluate the genotype phenotype correlation. A clinical classification was established for 14 patients WS, as proposed by the Waardenburg Consortium, and noted a predominance of type 1 and type 2 with 6 patients WS1, 7 patients WS2 and 1 patient WS3. A significant inter and intra-familial clinical heterogeneity was also observed. A sequencing of PAX3 gene in the 6 patients WS1 confirmed the diagnosis in 4 of them by revealing three novel mutations that modify two functional domains of the protein: the c.942delC; the c.933_936dupTTAC and the c.164delTCCGCCACA. These three variations are most likely responsible for the phenotype, however their pathogenic effects need to be confirmed by functional studies. The MLPA analysis of the 2 patients who were sequence negative for PAX3 gene revealed, in one of them, a heterozygous deletion of exons 5 to 9 confirming the WS1 diagnosis. Both clinical and molecular approaches led to the conclusion that there is a lack of genotype-phenotype correlation in WS1, an element that must be taken into account in genetic counseling. The absence of PAX3 mutation in one patient WS1 highlights the fact that the clinical classification is sometimes insufficient to distinguish WS1 from other types WS hence the interest of sequencing the other WS genes in this patient.

Testis development in the absence of SRY: chromosomal rearrangements at SOX9 and SOX3.

Duplications in the ~2 Mb desert region upstream of SOX9 at 17q24.3 may result in familial 46,XX disorders of sex development (DSD) without any effects on the XY background. A balanced translocation with its breakpoint falling within the same region has also been described in one XX DSD subject. We analyzed, by conventional and molecular cytogenetics, 19 novel SRY-negative unrelated 46,XX subjects both familial and sporadic, with isolated DSD. One of them had a de novo reciprocal t(11;17) translocation. Two cases carried partially overlapping 17q24.3 duplications ~500 kb upstream of SOX9, both inherited from their normal fathers. Breakpoints cloning showed that both duplications were in tandem, whereas the 17q in the reciprocal translocation was broken at ~800 kb upstream of SOX9, which is not only close to a previously described 46,XX DSD translocation, but also to translocations without any effects on the gonadal development. A further XX male, ascertained because of intellectual disability, carried a de novo cryptic duplication at Xq27.1, involving SOX3. CNVs involving SOX3 or its flanking regions have been reported in four XX DSD subjects. Collectively in our cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father. Translocations interrupting this region may also affect the gonadal development, possibly depending on the chromatin context of the recipient chromosome. SOX3 duplications may substitute SRY in some XX subjects.

3D-FISH analysis reveals chromatid cohesion defect during interphase in Roberts syndrome.

BACKGROUND: Roberts syndrome (RBS) is a rare autosomal recessive disorder mainly characterized by growth retardation, limb defects and craniofacial anomalies. Characteristic cytogenetic findings are "railroad track" appearance of chromatids and premature centromere separation in metaphase spreads. Mutations in the ESCO2 (establishment of cohesion 1 homolog 2) gene located in 8p21.1 have been found in several families. ESCO2, a member of the cohesion establishing complex, has a role in the effective cohesion between sister chromatids. In order to analyze sister chromatids topography during interphase, we performed 3D-FISH using pericentromeric heterochromatin probes of chromosomes 1, 4, 9 and 16, on preserved nuclei from a fetus with RBS carrying compound heterozygous null mutations in the ESCO2 gene. RESULTS: Along with the first observation of an abnormal separation between sister chromatids in heterochromatic regions, we observed a statistically significant change in the intranuclear localization of pericentromeric heterochromatin of chromosome 1 in cells of the fetus compared to normal cells, demonstrating for the first time a modification in the spatial arrangement of chromosome domains during interphase. CONCLUSION: We hypothesize that the disorganization of nuclear architecture may result in multiple gene deregulations, either through disruption of DNA cis interaction -such as modification of chromatin loop formation and gene insulation - mediated by cohesin complex, or by relocation of chromosome territories. These changes may modify interactions between the chromatin and the proteins associated with the inner nuclear membrane or the pore complexes. This model offers a link between the molecular defect in cohesion and the complex phenotypic anomalies observed in RBS.

3-Phosphoglycerate dehydrogenase deficiency: description of two new cases in Tunisia and review of the literature.

3-Phosphoglycerate dehydrogenase (3-PGDH) deficiency is a rare autosomal recessive disorder of serine biosynthesis. It is typically characterized by congenital microcephaly, intractable seizures of infantile onset, and severe psychomotor retardation. Diagnosis is suspected on decreased l-serine levels in plasma and cerebrospinal fluid (CSF) and confirmed by genetic study. Early diagnosis in index cases allows supplementation in serine and prevention of fixed lesions. Prenatal diagnosis and genetic counseling allows prevention of secondary cases. We report on the two first unrelated Tunisian families with 3-PGDH deficiency confirmed by biochemical and genetic study. We discuss clinical, biochemical, imaging, electroencephalographic, and therapeutic aspects and review the literature.

Epidemiologic and clinical characteristics of 458 Tunisian patients with intellectual deficiency and a reconsidered diagnostic strategy.

Intellectual Deficiency (ID) is a common neuropsychiatric disorder whose etiopathogenesis still insufficiently understood. In the last decade, several surveys, assessing epidemiologic, clinical and etiologic parameters of ID, have been performed but none of them is realized in a Tunisian population. In this retrospective survey, we propose to study these parameters, in a Tunisian cohort of 458 patients with constitutional ID, and to assess our diagnostic strategy. Data analyses, by the SPSS program, reveal a male predominance, a high level of consanguinity, an advanced mean age of patients, a rare frequentation of specialized institutions by the severely affected patients, and a high frequency of familial forms with predominance of the recessive autosomal ones. The study of clinical parameters and investigations' results shows that 72.1% of our patients present a syndromic ID. For these patients, chromosomal anomalies are rarely described, EEG anomalies were usually non-specific in patients without clinical evidence of epilepsy, and brain anomalies are common in patients with severe ID, neurological symptoms or history of seizures. Aetiology is identified in 13.1% of them whereas it is still unknown in 100% of patients with non-specific ID. This study allows us to better characterize, epidemiologically and clinically, the first large Tunisian cohort of patients with ID and to assess our diagnostic strategy in order to propose a revised one that will improve the diagnostic lead, the care chain and the preventive resources of ID.

Constitutional NRAS mutations are rare among patients with Noonan syndrome or juvenile myelomonocytic leukemia.

Recently, germline mutations of NRAS have been shown to be associated with Noonan syndrome (NS), a relatively common developmental disorder characterized by short stature, congenital heart disease, and distinctive facial features. We report on the mutational analysis of NRAS in a cohort of 125 French patients with NS and no known mutation for PTPN11, KRAS, SOS1, MEK1, MEK2, RAF1, BRAF, and SHOC2. The c.179G>A (p.G60E) mutation was identified in two patients with typical NS, confirming that NRAS germline mutations are a rare cause of this syndrome. We also screened our cohort of 95 patients with juvenile myelomonocytic leukemia (JMML). Among 17 patients with NRAS-mutated JMML, none had clinical features suggestive of NS. None of the 11 JMML patients for which germline DNA was available had a constitutional NRAS mutation.

Pseudoaminopterin syndrome.

Pseudoaminopterin syndrome or aminopterin syndrome-like sine aminopterin (ASSA syndrome--OMIM 600325] is a rare autosomal recessive syndrome defined by characteristic dysmorphic features, skeletal defects, limb anomalies, cryptorchidism, and growth retardation. The syndrome owes its name to the fact that patients resemble the children exposed to aminopterin or to methotrexate, two dihydrofolate reductase inhibitors used for chemotherapy, or as an abortificient in early pregnancy. Ten patients have been described with pseudoaminopterin syndrome. Their phenotype is variable, and differs from the phenotype resulting from folic acid deprivation, leading to the notion that the pathogenesis may be more complex than simple vitamin deficiency. We report on an Algerian patient with pseudoaminopterin syndrome, review the previously reported cases and confirm that pseudoaminopterin syndrome does not result from a detectable contiguous gene imbalance as high resolution CGH array was normal in this child.

Hexasomy of the Prader-Willi/Angelman critical region, including the OCA2 gene, in a patient with pigmentary dysplasia: case report.

Derivatives of chromosome 15, often referred to as inv dup(15), represent the most common supernumerary marker chromosome (SMC). SMC(15)s can be classified into two major groups according to their length: small SMC(15) and large ones. Depending on the amount of euchromatin, the carriers may either present with a normal phenotype or with a recognizable syndrome. Here we describe a patient with severe mental retardation, epilepsy, dysmorphic features and pigmentary dysplasia. His karyotype was 47,XY,+mar[41]/46,XY[9]. Chromosomal fluorescence in situ hybridization (FISH) showed the SMC to be originating from chromosome 15, dicentric and containing four copies of the Prader-Willi/Angelman Syndrome Critical Region (PWACR), including the OCA2 gene. Molecular studies indicated that it is maternally derived. This report supports the previous observations assuming that severity of phenotype in patients with SMC(15) depends on the dosage of the PWACR and that skin pigmentation is correlated to OCA2 gene copy number.

A novel frame shift mutation in the PQBP1 gene identified in a Tunisian family with X-linked mental retardation.

Mental retardation (MR) is the most frequent cause of serious handicap in children and young adults. Despite recent progress, in most cases the molecular defects underlying this disorder remain unknown. Linkage studies followed by mutational analysis of known X-chromosomal genes related to mental retardation (MRX genes) localized within defined genetic intervals represent a rational strategy to identify a genetic cause of the disorder. Here, we report a Tunisian family including 3 males with severe to mild mental retardation, short stature, lean body and microcephaly; we mapped the disease to a unique interval encompassing Xp21.1-Xq21.33 (with a maximum LOD score of 0.90). Subsequent mutation analysis of genes located in this interval allowed us to identify a truncating mutation in the PQBP1 gene. This mutation is an insertion of an adenosine residue in exon 5 (c.631insA). This frameshift insertion causes premature stop codon at amino acid position 226. The observed mutation was found in all males with MR in this family. Together with previously reported observations, our data further confirm that PQBP1 gene should be tested for males showing mental retardation, short stature, lean body and microcephaly.

Germinal mosaicism in Noonan syndrome: A family with two affected siblings of normal parents.

Noonan syndrome (NS; OMIM 163950) is an autosomal dominant disorder with variable clinical expression and genetic heterogeneity. Clinical manifestations include characteristic facial features, short stature, and cardiac anomalies. Mutations in protein-tyrosine phosphatase, non-receptor-type 11 (PTPN11), encoding SHP-2, account for about half of NS patients. We report on a Moroccan family with two children with NS and apparently unaffected parents. The molecular studies showed the heterozygous mutation c.922A>G of PTPN11 gene in the two affected sibs. Neither the parents, nor the oldest brother carries this mutation in hematologic cells. The mutation was also absent in buccal epithelial cells and fingernails of both parents. We believe this is the first report of germ cell mosaicism in NS and suggest an empirical risk for recurrence of that is less than 1%.