Clinical and genetic characteristics of Tunisian children with infantile nephropathic cystinosis.

BACKGROUND: Nephropathic cystinosis is an autosomal recessive disease caused by a mutation in the CTNS gene which encodes cystinosin, a lysosomal cystine transporter. The spectrum of mutations in the CTNS gene is not well defined in the North African population. Here, we investigated twelve patients with nephropathic cystinosis belonging to eight Tunisian families in order to analyze the clinical and genetic characteristics of Tunisian children with infantile nephropathic cystinosis. METHODS: Clinical data were collected retrospectively. Molecular analysis of the CTNS gene was performed by Sanger sequencing. RESULTS: We describe a new splicing mutation c.971-1G > C in the homozygous state in 6/12 patients which seems to be a founder mutation. The reported deletion of 23nt c.771_793 Del (p.Gly258Serfs*30) was detected in a homozygous state in one patient and in a heterozygous compound state with the c.971-1G > C mutation in 3/12 patients. Two of 12 patients have a deletion of exons 4 and 5 of the CTNS gene. None of our patients had the most common 57-kb deletion. CONCLUSIONS: The mutational spectrum in the Tunisian population is different from previously described populations. Thus, a molecular diagnostic strategy must be implemented in Tunisia, by targeting as a priority the common mutations described in this country. Such a strategy will allow a cost-effective diagnosis confirmation as well as early administration of treatment with oral cysteamine. A higher resolution version of the Graphical abstract is available as Supplementary information.

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.

Distinctive findings in a boy with Simpson-Golabi-Behmel syndrome.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre and post natal overgrowth, facial malformations, and visceral, skeletal, and neurological anomalies. The physical characteristics of SGBS have been well documented; however there is a lack of description regarding the behavioral phenotype. We report the case of a 6-year-old boy, with confirmed deletion of 6-8 exons of the glypican-3 gene (GPC3) who presents three distinctive findings: the persistence of the craniopharyngeal canal, an immune-allergic specificity, and a scarcely behavioral phenotype consisting in the association of Austim Spectrum Disorder with accompanying mild intellectual disability and language impairments. He also fulfilled the criteria of Attention Deficit Hyperactivity Disorder and Oppositional Defiant Disorder according to DSM 5 criteria. The specificities of the case are discussed in the light of recent pathophysiological data.

3M syndrome: A Tunisian seven-cases series.

3M syndrome (3MS) is a rare autosomal recessive primordial growth disorder characterized by a severe pre- and post-natal growth deficiency, minor dysmorphisms and skeletal abnormalities, contrasting with normal intellect and endocrine function. Three different genes have been so far involved in the disease, with mutations in CUL7, OBSL1 and CCDC8. The CUL7 gene mutations are accountable for 77,5% of the genetically confirmed patients, with a founder mutation identified in exon 24 for the Maghreb families. The follow up is mainly orthopedic with possible GH-based treatment. The objective of this report was to carry out a clinical analysis of a series of Tunisian patients with features evoking 3MS and to perform a molecular analysis of the CLU7 exon 24. We carried out a descriptive retrospective study including Tunisian patients who consulted at the congenital disorders and hereditary diseases department of Charles Nicolle's hospital, Tunis, Tunisia, for intra-uterine onset growth retardation with normal intellect. We selected the patients having characteristic 3MS facial dysmorphia. The molecular analysis of the CUL7 exon 24 was performed using PCR and Sanger sequencing searching the founder mutation c.4451_4452delTG. Seven patients were included in this study. Consanguinity was noted for four families. The mean age at the first consult was 2.5 years. All the patients had an intra-uterine onset growth retardation with a preserved head circumference. All patients presented facial dysmorphia of 3MS, with a prominent forehead (7/7), a triangular face (6/7), an underdeveloped midface (7/7), a fleshy tipped nose (5/7), anteverted nares (6/7), a long philtrum (7/7) and full lips (4/7). All the patients presented skeletal abnormalities with various severities such as lumbar lordosis, hyperextensible joints, short thorax, square shoulders, hip dislocation, and prominent heels. Less frequent features were noted such as spina bifida occulta in one case, and single transverse palmar crease in 4 cases. One GH treatment response was reported. The molecular genetic analysis of the CUL7 gene (exon 24) revealed the founder mutation for all the patients which reinforces the hypothesis of founder effect for 3MS in the Tunisian population.

Novel RAB3GAP1 Mutation in the First Tunisian Family With Warburg Micro Syndrome.

BACKGROUND AND PURPOSE: Warburg Micro syndrome (WARBM) is a rare autosomal recessive genetic disease characterized by ocular, neurologic, and endocrine anomalies. WARBM is a phenotypically and genetically heterogeneous syndrome caused by mutations in RAB3GAP1, RAB3GAP2, RAB18, and TBC1D20. Here we present the clinical and genetic characterization of a consanguineous Tunisian family with a WARBM phenotype presenting two pathogenic variations, one of which is on RAB3GAP1. METHODS: We applied whole-exome sequencing (WES) to two affected young males presenting a WARBM-compatible phenotype. RESULTS: We reveal a new variation in RAB3GAP1 (NM_012233.3: c.297del, p.Gln99fs) and another variation in ABCD1 (NM_000033: c.896A>G, p.His299Arg). Each of these mutations, which in silico predictions concluded as being pathogenic variations, affects a critical protein region. Both affected males presented a WARBM-compatible phenotype, with severe intellectual disability, severe developmental delay, postnatal growth delay, postnatal microcephaly, congenital bilateral cataracts, general hypotonia, and a thin corpus callosum without a splenium. However, intrafamilial clinical heterogeneity was present, since only the oldest child had large ears, microphthalmia, foot deformities, and a genital anomaly, and only the youngest child had microcornea. Despite the mutation identified in ABCD1, our patients did not have any X-linked symptoms of adrenoleukodystrophy disorder that are usually caused by ABCD1 mutations, which prompted our interest in clinical monitoring. CONCLUSIONS: WES analysis of a consanguineous Tunisian family with WARBM revealed a novel variation in RAB3GAP1 (NM_012233.3: c.297del, p.Gln99fs) that is most likely pathogenic and allowed us to confirm the diagnosis of WARBM.

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.

Absence of mutations in four genes encoding for congenital cataract and expressed in the human brain in Tunisian families with cataract and mental retardation.

BACKGROUND: To identify the genetic defect associated with autosomal recessive congenital cataract (ARCC), mental retardation (MR) and ARCC, MR and microcephaly present in most patients in four Tunisian consanguineous families. METHODS: We screened four genes implicated in congenital cataract by direct sequencing in two groups of patients; those affected by ARCC associated to MR and those who presented also microcephaly. Among its three genes PAX6, PITX3 and HSF4 are expressed in human brain and one gene LIM2 encodes for the protein MP20 that interact with the protein galectin-3 expressed in human brain and plays a crucial role in its development. All genes were screened by direct sequencing in two groups of patients; those affected by ARCC associated to MR and those who presented also microcephaly. RESULTS: We report no mutation in the four genes of congenital cataract and its flanking regions. Only variations that did not segregate with the studied phenotypes (ARCC associated to MR, ARCC associated with MR and microcephaly) are reported. We detected three intronic variations in PAX6 gene: IVS4 -274insG (intron 4), IVS12 -174G>A (intron12) in the four studied families and IVS4 -195G>A (intron 4) in two families. Two substitutions polymorphisms in PITX3 gene: c.439 C>T (exon 3) and c.930 C>A (exon4) in one family. One intronic variation in HSF4 gene: IVS7 +93C>T (intron 7) identified in one family. And three intronic substitutions in LIM2 gene identified in all four studied families: IVS2 -24A>G (intron 2), IVS4 +32C>T (intron 4) and c.*15A>C (3'-downstream sequence). CONCLUSION: Although the role of the four studied genes: PAX6, PITX3, HSF4 and LIM2 in both ocular and central nervous system development, we report the absence of mutations in all studied genes in four families with phenotypes associating cataract, MR and microcephaly.

Non-syndromic autosomal recessive mental retardation in Tunisian families : exclusion of GRIK2 and TUSC3 genes.

BACKGROUND: Mental retardation is one of the most frequent major handicap, with a 1-3 % frequency in the general population, it appear a major problem of public health. The recent progress of molecular biology and cytogenetic allowed to identify new genes for non syndromic autosomal recessive mental retardation (NSAR-MR). AIM: Genetic analysis of NSAR-MR: the GRIK2 gene (6q16.3-q21) and the TUSC3 gene (8p22). METHODS: Four Tunisian families with NSAR-MR were included in this study. Genotyping was made using polymorphic microsatellite markers and statistical analysis was validated using the Fast Link programme of the Easy linkage software (V4:00beta). RESULTS: Genotyping and linkage analysis excluded linkage of the GRIK2 gene and TUSC3 gene. CONCLUSION: Our results confirm the extreme genetic heterogeneity of NSAR-MR.

Spectrum of Genetic Diseases in Tunisia: Current Situation and Main Milestones Achieved.

Genetic diseases in Tunisia are a real public health problem given their chronicity and the lack of knowledge concerning their prevalence and etiology, and the high rates of consanguinity. Hence, we performed systematic reviews of the literature in order to provide a more recent spectrum of these disorders and to expose the challenges that still exist to tackle these kinds of diseases. A manual textual data mining was conducted using MeSH and PubMed databases. Collected data were classified according to the CIM-10 classification and the transmission mode. The spectrum of these diseases is estimated to be 589 entities. This suggests remarkable progress through the development of biomedical health research activities and building capacities. Sixty percent of the reported disorders are autosomal recessive, which could be explained by the high prevalence of endogamous mating. Congenital malformations (29.54%) are the major disease group, followed by metabolic diseases (22%). Sixty percent of the genetic diseases have a known molecular etiology. We also reported additional cases of comorbidity that seem to be a common phenomenon in our population. We also noticed that epidemiological data are scarce. Newborn and carrier screening was only limited to pilot projects for a few genetic diseases. Collected data are being integrated into a database under construction that will be a valuable decision-making tool. This study provides the current situation of genetic diseases in Tunisia and highlights their particularities. Early detection of the disease is important to initiate critical intervention and to reduce morbidity and mortality.

Identification of two additional novel mutations in the AR gene associated with severe forms of androgen insensitivity syndrome.

The Androgen insensitivity syndrome (AIS) in its complete form (CAIS) is a disorder in abnormal male development characterized by a complete female phenotype in a 46,XY individual. The most frequent cause of this disorder is a hemizygous mutation in androgen receptor (AR) gene located in X chromosome. The first aim of this study was to confirm the clinical diagnosis in a series of Tunisian patients with a typical phenotype of CAIS by molecular genetic analysis. The second aim was to determine the AR mutational profile in the local population. The entire coding region and the exon-intron junctions of the AR gene were sequenced in a series of ten patients. AR defects were found in nine patients. Despite the small number of cases, two of the nine identified mutations were novel. The first novel mutation was an 8-bp deletion in exon 1 (c.862_869del) resulting in a frameshift (p.A288Qfs*14). The second was a splice site mutation c.1885 + 1G > T (IVS3 + 1G > T). In this study, genetic testing has confirmed the diagnosis of most CAIS patients and has revealed two novel mechanisms responsible for the pathogenesis of AIS, as well as seven other reported mutations.

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.

Autosomal recessive acro-fronto-facio-nasal dysostosis associated with genitourinary anomalies: a third case report.

We report on a 22-day-old Tunisian boy born to consanguineous (first-cousin) parents (F = 1/16). The patient presents wide forehead with frontal encephalocele, wide anterior fontanel, marked hypertelorism, coloboma of the upper lids, proptosis, congenital glaucoma, broad nose, syndactyly between fingers 3 and 4, hypoplastic 3rd, 4th and 5th toes with nail dysplasia, hypospadias with cleft glans, bifid scrotum. Brain MRI showed right frontal encephalocele with anomalies of the cortical gyration without any corpus callosum abnormality. Normal chromosomes and parents' consanguinity are suggestive of autosomal recessive inheritance. Facial midline anomalies associated with limb and genitourinary anomalies is very uncommon. We present the third case reported in the literature.

[Interest of studying subtelomeric abnormalities by in situ fluorescent hybridization to explore idiopathic mental retardation]. / Intérêts de l'étude des remaniements subtélomériques par la technique d'hybridation fluorescente (FISH) dans l'exploration des retards mentaux idiopathiques.

Nowadays, the genetic basis of mental retardation is a huge field of investigations. Genetic abnormalities frequently give rise to a mental retardation phenotype and are observed in 10 to 40% of known etiologies. New syndromes have identified (chromosome 1p, 22q, 3q29 and 9q34) but for 60% of patients there is no etiology because there is no characteristic phenotype. Many studies involve subtelomeric duplications and deletions in idiopathic mental retardation. The autours describe and discuss the interest and the limits of telomeric FISH [Chromoprobe Multiprobe T System] in exploring mental retardation.

[Molecular diagnosis of fragile X syndrome]. / Diagnostic moleculaire du sydrome de l'X fragile.

BACKGROUND: The fragile X syndrome was the most frequent etiology of hereditary mental retardation but the clinical diagnosis is not easy and the individual clinical symptoms were not specific so the confirmation will be made par molecular study of the gene of the fragile X syndrome. The aim of our study is to realise the molecular diagnosis of the fragile X syndrome in 200 Tunisian boys with mental retardation. Our results shows that the frequency of the fragile X syndrome is 7,6%. In the most cases there is a family history of mental retardation with medium age at 11 years. All the boys with the full mutation have mental retardation, dysmorphic features and macro-orchidism (pubescent boy) CONCLUSION: The screening of the molecular abnormalities of FMRI gene must be realised in every boy with mental retardation or boy with delayed speech without any identified etiology. The earlier diagnosis is important for genetic counselling.

MEFV mutations in Tunisian patients suffering from familial Mediterranean fever.

OBJECTIVES: To identify the frequency and distribution of familial Mediterranean fever (FMF) gene (MEFV) mutations in Tunisian patients. PATIENTS AND METHODS: This study was performed in the Genetic Department of Tunis University Hospital. A clinical diagnosis of FMF was made according to published criteria. Mutation screening of the MEFV gene was performed in the Human Genetic Laboratory of the "Faculté de Medecine de Tunis" for 8 mutations including the 5 most common known mutations M694V, V726A, M694l, M680l, and E148Q. The tests performed were polymerase chain reaction (PCR) restriction-digestion for M694V, V726A, M680l, R761H, E148Q; amplification refractory mutation system for A744S, M694l; and PCR-electrophoresis assay for l692del. RESULTS: Of the 139 unrelated patients investigated, 61 (44%) had 1 or 2 mutations. In 78 (56%) probands no mutation was identified: 28 patients were homozygous; 16 were compound-heterozygous; 2 had complex alleles; and 17 had only 1 identifiable mutation. Of the mutations, M680l, M694V, M694l, V726A, A744S, R761H, l692DEL, and E148Q accounted for 32, 27, 13, 5, 3, 1, 1, and 18%, respectively. CONCLUSION: The profile of the MEFV gene mutations in the Tunisian population is concordant with other Arab populations but with some differences. M680l is the most common mutation, while V726A, the commonest mutation among Arabs, is rare in our population.

[Treacher-Collins syndrome: clinical and genetic aspects apropos of 4 cases of which 1 is familial]. / Le syndrome de Treacher Collins: aspects clinique et génétique à propos de 4 observations dont une familiale.

Treacher Collins syndrome was first mentioned by Thompson in 1847, and described by Treacher Collins in 1900, then it was called mandibulo-facial dysostosis and well defined by Franceschetti in 1949. It is a very rare affection occurring lin 50.000 live births, which includes facial and auricular anomalies leading to functional, morphological and psychological difficulties due to related handicaps. Treacher Collins syndrome is inherited as autosomal dominant pattern with a variable expressivity and incomplete penetrance of "TCOF1" gene localized at 5q31.3q32. Today the gene is well identified and several mutations have been reported. In this paper we report the case of 4 Tunisian unrelated girls with Treacher Collins syndrome. One of them was born from an affected father. Clinical diagnostic was performed between age 12 days and 2 years demonstrating the large dysmorphic expression. Main clinical features were present in all reported cases. Family at risk might have genetic counselling and probably prenatal diagnostic in some situations. Out of our observations, we gave genetic counselling and proposed ultrasound prenatal diagnosis for two families without molecular study.

Functional Analysis of Mutations at Codon 127 of the SRY Gene Associated with 46,XY Complete Gonadal Dysgenesis.

Complete gonadal dysgenesis (CGD) is characterized by an incomplete differentiation of the genital organs in a patient with a 46,XY karyotype. It is induced by mutations in the sex-determining region Y (SRY) gene which plays a key role in testis-determining pathways. The aim of this study was to investigate the possible pathogenic nature of a novel SRY mutation (p.Y127H) identified in a 46,XY female patient. To determine the effect of this mutation on SRY function, we studied its impact on DNA interaction by electrophoretic mobility shift assays. Since tyrosine 127 is close to the C-terminal nuclear localization signal of SRY, we conducted HA-SRY protein expression to observe the impact of the mutation on the nuclear import function in transfected cells. Our results showed that the Y127H mutation nearly abolishes the DNA-binding capacity of SRY and strongly impairs the nuclear localization of the mutated protein. Together with a previously described mutation analyzed in parallel in this paper (p.Y127C), our results highlight this tyrosine residue as a crucial structural determinant of the high mobility group box domain. This is the first report to explain the molecular mechanism of the Y127H mutation causing sex reversal and gives new insights for clinical practice to benefit patients with disorders of sex development.

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.

Screening of the eight BBS genes in Tunisian families: no evidence of triallelism.

PURPOSE: To study Bardet-Biedl syndrome (BBS) in the Tunisian population and determine the presence of triallelism in the eight identified BBS genes. METHODS: DNA samples were collected from 19 consanguineous Tunisian families with BBS. Genome-wide scans were performed with microsatellite markers in 12 families, and two-point linkage analyses were performed. Direct sequencing was used to screen patients with BBS for mutations in all eight identified BBS genes. RESULTS: Mutations in the BBS genes were identified in nine families. In addition, a large consanguineous family (57004) showed linkage to the BBS7 locus, although no mutation was identified. Five novel mutations were present in the nine families: one in BBS2 (c.565C>T, p.ArgR189Stop), one in BBS5 (c.123delA, p.Gly42GlufsX11), one in BBS7 (g.47247455_47267458del20004insATA, p.Met284LysfsX7), and two in BBS8 (c.459+1G>A, p.Pro101LeufsX12 and c.355_356insGGTGGAAGGCCAGGCA, p.Thr124ArgfsX43). CONCLUSIONS: All families in which mutations were identified show changes in both copies of the mutant gene, and inheritance patterns in all families are consistent with autosomal recessive inheritance excluding any evidence of triallelism in the BBS genes in Tunisia.

Molecular analysis of the SMN1 and NAIP genes in 60 Tunisian spinal muscular atrophy patients.

In this study we examined the deletion of SMN and NAIP genes in 60 Tunisian families. There were 35 patients with type I SMA. 18 with type II SMA. 6 with type III SMA and I with type IV SMA. The age of onset was before 6 months for type I, between 6 months and 2 years for type II, between 2 years and 17 years for type III and 30 years for type IV. Exon 7 of SMNI gene was homozygously deleted in 95% (57/60) of SMA patients. There was a higher frequency of homozygous absence of SMN1 in type I and type II (100% and 94% respectively) than in type III (66,7%). SMN1 exon 8 was undetectable in 88% (53/60) of patients. The case type II patient with homozygous deletion of SMNI exon 7 and not exon 8 was tested for the presence of a hybrid SMN gene. This patient showed in the second PCR a SMN1 exon 8 product by restriction site assay indicating that a gene conversion event had occurred. All parents' individuals retained one copy of their SMN1 gene. Exon 5 of NAIP gene was homozygously deleted in 58% (35/60) of patients (77% in type I (27/35), 27,7% in type II (5/18), 50% (3/6) in type III. No patient had a deletion in NAIP gene without a deletion in the SMN1 gene. Homozygous deletion of NAIP exon 5 was detected in 1 parent. Our results show that the incidence of NAIP deletion is higher in the more severe SMA cases.