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.

Fetopathological examination for the fetuses with Down syndrome in Tunisia: Epidemiological study and associated malformations.

BACKGROUND: For Down syndrome (DS), traditional epidemiological studies to determine the prevalence, cause, and clinical significance of the syndrome have been conducted over the last 100 years. In Tunisia, the current work is the first in-depth study in epidemiology of DS from fetopathological data. AIM OF THE STUDY: The aim of this epidemiological study was to determine the impact of some feto-maternal characteristics in occurrence of DS and to search the frequency of associated congenital malformations with this syndrome. METHODS: Our retrospective study was realized for 144 fetuses with DS among 9321 autopsied fetuses in embryo-fetopathological service between 1994 and 2011. RESULTS: In our study, the majority of mothers (72.91%) were 35 years and older, with a statistically significant difference (p<10-6, OR=16.7, CI=8.7-32.4). The abnormalities of extremities (31%) were the most common fetal abnormalities followed by facial (23.51%) and digestive abnormalities (19.63%). CONCLUSION: One of the main conclusions of this research is that the most common risk factor for DS is maternal age. On the other hand, the type and the frequency of associated congenital anomalies with DS are still controversial.

[Marshall syndrome: Clinical, radiological and genetical features of a Tunisian family]. / Syndrome de Marshall: Aspects cliniques, radiologiques et génétiques d'une famille tunisienne.

BACKGROUND: Marshall syndrome is a rare autosomal dominant skeletal dysplasia. It associates a particular facial dysmorphism with midface hypoplasia, ocular abnormalities and sensorineural hearing loss. It is caused by heterozygous mutations in COL11A1 gene coding the 1 chain of collagen XI. Stickler syndrome is the principal differential diagnosis of Marshall syndrome. AIM: Clinical and radiological study of Marshall syndrome in a Tunisian family with a linkage study of the COL11A1 gene to this disease. METHODS: We report the clinical and the radiological findings of a Tunisian family including 8 members affected by Marshall syndrome. The linkage of the COL11A1 gene to this disease was tested using the polymorphic microsatellite markers of DNA. RESULTS: A variability of the clinical expression of Marshall syndrome was reported. Specific Marshall phenotype and an overlapping phenotype between the Marshall and Stickler syndromes were observed among the affected members of this family. The ocular manifestations were also heterogeneous. Marshall syndrome's specific radiological signs were found. The linkage study supports the linkage of the abnormal phenotype to the COL11A1 gene. CONCLUSION: There is a variability of the clinical expression among the affected members of the study's family. We will continue searching the causative mutation to establish a clear genotype- phenotype correlation.

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.

GJB2 and GJB6 screening in Tunisian patients with autosomal recessive deafness.

UNLABELLED: Autosomal recessive nonsyndromic deafness (ARNSD or DFNB) is a very common genetically heterogenous disorder. Although DFNB1 mutations are known to be the most frequent cause of this disorder, they are largely dependent on ethnic groups. The aims of our study are to specify the prevalence and the spectrum of GJB2 mutations as well as the prevalence of GJB6 large deletion in Tunisian population. PATIENTS AND METHODS: 95 unrelated patients with moderate to severe sensorineural hearing loss have been tested. The GJB2 coding region has been studied by PCR/Sequencing and the del(GJB6-D13S1830) mutation has been screened by fluorescent PCR multiplex. RESULTS: 27.36% of patients present mutations on both alleles of GJB2 gene and no one has the del(GJB6-D13S1830) mutation. The c.35delG mutation represents 86.5% of GJB2 deafness alleles and is found in homozygous state in 22 patients and in heterozygous state in one patient. Four other mutations are detected in four probands: two are compound heterozygous for the p.V37I/p.E47X and the c.35delG/p.R184P mutations, and two are homozygous for the p.E47X and the c.333-334delAA mutations. CONCLUSION: Our results showed that c.35delG is the most common but not the only GJB2 mutation and that the del(GJB6-del D13S1830) is absent in our cohort. Consequently, we propose a systematic sequencing of GJB2 coding region for ARNSD Tunisian patients and we suggest additional studies to specify the real prevalence of del(GJB6-D13S1830) in our population.

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.

Targeted high-throughput sequencing for diagnosis of genetically heterogeneous diseases: efficient mutation detection in Bardet-Biedl and Alström syndromes.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a pleiotropic recessive disorder that belongs to the rapidly growing family of ciliopathies. It shares phenotypic traits with other ciliopathies, such as Alström syndrome (ALMS), nephronophthisis (NPHP) or Joubert syndrome. BBS mutations have been detected in 16 different genes (BBS1-BBS16) without clear genotype-to-phenotype correlation. This extensive genetic heterogeneity is a major concern for molecular diagnosis and genetic counselling. While various strategies have been recently proposed to optimise mutation detection, they either fail to detect mutations in a majority of patients or are time consuming and costly. METHOD: We tested a targeted exon-capture strategy coupled with multiplexing and high-throughput sequencing on 52 patients: 14 with known mutations as proof-of-principle and 38 with no previously detected mutation. Thirty genes were targeted in total including the 16 BBS genes, the 12 known NPHP genes, the single ALMS gene ALMS1 and the proposed modifier CCDC28B. RESULTS: This strategy allowed the reliable detection of causative mutations (including homozygous/heterozygous exon deletions) in 68% of BBS patients without previous molecular diagnosis and in all proof-of-principle samples. Three probands carried homozygous truncating mutations in ALMS1 confirming the major phenotypic overlap between both disorders. The efficiency of detecting mutations in patients was positively correlated with their compliance with the classical BBS phenotype (mutations were identified in 81% of 'classical' BBS patients) suggesting that only a few true BBS genes remain to be identified. We illustrate some interpretation problems encountered due to the multiplicity of identified variants. CONCLUSION: This strategy is highly efficient and cost effective for diseases with high genetic heterogeneity, and guarantees a quality of coverage in coding sequences of target genes suited for diagnosis purposes.

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.

Molecular analysis of Bardet-Biedl syndrome families: report of 21 novel mutations in 10 genes.

PURPOSE: Bardet-Biedl syndrome (BBS) is genetically heterogeneous with 15 BBS genes currently identified, accounting for approximately 70% of cases. The aim of our study was to define further the spectrum of BBS mutations in a cohort of 44 European-derived American, 8 Tunisian, 1 Arabic, and 2 Pakistani families (55 families in total) with BBS. METHODS: A total of 142 exons of the first 12 BBS-causing genes were screened by dideoxy sequencing. Cases in which no mutations were found were then screened for BBS13, BBS14, BBS15, RPGRIP1L, CC2D2A, NPHP3, TMEM67, and INPP5E. RESULTS: Forty-three mutations, including 8 frameshift mutations, 10 nonsense mutations, 4 splice site mutations, 1 deletion, and 20 potentially or probably pathogenic missense variations, were identified in 46 of the 55 families studied (84%). Of these, 21 (2 frameshift mutations, 4 nonsense mutations, 4 splice site mutations, 1 deletion, and 10 missense variations) were novel. The molecular genetic findings raised the possibility of triallelic inheritance in 7 Caucasian families, 1 Arabian family, and 1 Tunisian patient. No mutations were detected for BBS4, BBS11, BBS13, BBS14, BBS15, RPGRIP1L, CC2D2A, NPHP3, TMEM67, or INPP5E. CONCLUSIONS: This mutational analysis extends the spectrum of known BBS mutations. Identification of 21 novel mutations highlights the genetic heterogeneity of this disorder. Differences in European and Tunisian patients, including the high frequency of the M390R mutation in Europeans, emphasize the population specificity of BBS mutations with potential diagnostic implications. The existence of some BBS cases without mutations in any currently identified BBS genes suggests further genetic heterogeneity.

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.

Detection of a frequent duplicated CYP21A2 gene carrying a Q318X mutation in a general population with quantitative PCR methods.

Earlier we had reported a large prevalence of the Q318X mutation in the CYP21A2 gene with 35.3% in Tunisian patients with a classical form of 21-hydroxylase deficiency, in contrast with 0.5% to 13.8% as described in other populations. Here we present the analysis of the Q318X mutation in a healthy Tunisian population. We screened 136 individuals by the polymerase chain reaction (PCR)/random fragment length polymorphism method, which was confirmed by direct sequencing. Surprisingly, 17 Q318X carriers were identified, for a carrier frequency of 12.5% (95% confidence interval: 7.86-19.20). To explain this unexpectedly high rate we suggest that the haplotype with Q318X mutation and duplicated CYP21A2 gene could be very frequent in the Tunisian population. To test our hypothesis, we used 2 different quantitative PCR methods, that is, multiplex ligation-dependent probe amplification and real-time PCR. The molecular studies showed the presence of a duplicated CYP21A2 gene in all 17 heterozygous Q318X mutation carriers. In addition, both quantitative PCR methods used in this study represent a sensitive and useful approach to detecting copy number variations of the CYP21A2 gene. We have identified a very high frequency of carriers with duplicated CYP21A2 gene haplotype in a healthy Tunisian population. This finding complicates the molecular diagnosis of 21-hydroxylase deficiency and we recommend that, whenever a Q318X is identified, the structure of the CYP21A2 region should be determined to discriminate between the severe Q318X mutation and the normal Q318X variant.

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.

Coexistence of Behçet's disease with ankylosing spondylitis and familial Mediterranean fever: a rare occurrence.

Behçet's disease (BD) and familial Mediterranean fever (FMF), which are two separate diseases sharing some clinical features, may also coexist in the same patient. Further investigations are needed to understand whether this coexistence is due to either chance or geographical distribution patterns of these diseases or to common etiopathogenetic characteristics. Spondylarthritis as part of the clinical picture in these two diseases has been questioned and probably it is not a prominent characteristic of any of them. We report a 35-year-old Tunisian man who had an association of BD, FMF and Human Leukocyte Antigen (HLA) B27 positive ankylosing spondylitis. Although that spondylarthritis is an infrequent joint involvement of FMF and BD, it must be looked for in case of association of these diseases.

Subtelomeric microduplications in three sisters with moderate mental retardation.

Copy number changes of subtelomeric regions are a common cause of mental retardation, occurring in approximately 5% of mentally retarded patients. New molecular techniques allow the identification of subtelomeric microduplications. We report a Tunisian family of three sisters with moderate mental retardation, facial dysmorphism, cardiopathy, and bilateral clinodactyly of the third and fourth toes, explored by MLPA, showing the same associated microduplications, 15q and Xq, without a concurrent deletion.

Pallister-Killian syndrome with additional manifestations of cleft palate and sacral appendage.

AIM: Reppor of a rare congenital abnormalities. OBSERVATION: We report a rare case of Pallister-Killian syndrome in a 33 weeks gestation infant. In addition to the characteristic phenotype, this patient had a cleft palate, diaphragmatic hernia and sacral appendage. These additional manifestations are not among the Pallister-Killian syndrome's features. The diagnosis was made in antenatal period by cytogenetic studies and showed mosaic 47, XY+i (12p). Presence of diaphragmatic hernia makes this syndrome, prenatally letal, similar to the Fryns syndrome and then requires skin biopsy and fibroblast chromosome examination for cytogenetic diagnosis.

Exploring the 7p22.1 chromosome as a candidate region for autism.

A high incidence of de novo chromosomal aberrations in a population of persons with autism suggests a causal relationship between certain chromosomal aberrations and the occurrence of autism. A previous study on a Tunisian boy carrying a t(7;16) translocation identified the 7p22.1 as a positional candidate region for autism on chromosome 7. The characterization of the chromosomal breakpoints helped us to identify new candidate regions on chromosome 16p11.2 which contain no known genes and the other one on 7p22.1 containing a portion of genes (NP 976327.1, RBAK, Q6NUR6 also called RNF216L and MMD2). We proposed Q6NUR6 (RNF216L) as a candidate gene for autism due to its vicinity to the translocation breakpoint on the chromosome derivative 7. Q6NUR6 is predicted to be an E3ubiquitin-ligase. Quantitative PCR demonstrates that Q6NUR6 gene has an ubiquitous expression and that it is strongly expressed in fetal and adult brain. The Q6NUR6 expression is increased in the patient blood cells in comparison to controls. This is the first report of Q6NUR6 gene (E3 ubiquitin ligase TRIAD3 EC 6.3.2) increasing blood levels in a patient with autism. It's probably caused by a position effect involving this gene and modifying its expression.

X linked mental retardation.

BACKGROUND: Mental retardation (MR) is a group of heterogeneous clinical conditions. There are more than 900 genetic disorders associated with MR and it affects around 3% of the general population. Many MR conditions described are syndromic, fragile X syndrome being the most common clinical entity among them. X linked mental retardation (XLMR) is subdivided in two categories: syndromic XLMR (MRXS) when MR is associated with clinical features and non-syndromic XLMR (MRX) when MR is isolated. AIM: The aim of this systematic review of the literature was to join together the results of several studies related to X linked mental retardation and to present various genes implicated in this disease. In this review, focus has been given on genes implicated in mental retardation, the clinical data and on phenotype-genotype correlations. METHODS: An exhaustive electronic and library research of the recent literature was carried out on the Web sites "Science Direct" and "Interscience Wiley". The key words used were "mental retardation", "X chromosome", "gene", "syndromic mental retardation", "non-syndromic mental retardation". RESULTS: In this review a number of X linked genes, the clinical features associated with the gene abnormality, and the prevalence of the disease gene are discussed. We classified these genes by order of their first implication in MR. A table presented on the XLMR Update Web site who list the 82 known XLMR genes is available as XLMR Genes and corresponding proteins.

Tolllike receptor 4 (TLR4) polymorphisms in Tunisian patients with Crohn's disease: genotype-phenotype correlation.

BACKGROUND: The immune responses to bacterial products through the pattern recognition receptor (PRR) play a pivotal role in pathogenesis of Crohn's disease. A recent study described an association between CD and some gene coding for bacterial receptor like NOD2/CARD15 gene and TLR4. In this study, we sought to determine whether TLR4 gene was associated with Crohn's disease (CD) among the Tunisian population and its correlation with clinical manifestation of the disease. METHODS: 90 patients with CD and 80 healthy individuals are genotyped for the Asp299Gly and Thr399Ile polymorphisms by restriction fragment length polymorphism analysis. RESULTS: The allele and genotype frequency of the TLR4 polymorphisms did not differ between patients and controls. The genotype-phenotype correlation permitted to show that the Thr399Ile polymorphism was associated with early onset disease. CONCLUSION: this study reported the absence of association between CD and TLR4 gene in the Tunisian population, but this gene could play a role in clinical expression of the disease.

MLPA subtelomere analysis in Tunisian mentally retarded patients.

Subtelomeric rearrangements significantly contribute to idiopathic mental retardation and result in several mental retardation syndromes; however, most subtelomeric defects lack a characteristic phenotype. Thirty patients with unexplained mental retardation, a normal R banded karyotype at the 550 band, and no clinically recognizable syndrome were screened by Multiplex ligation-dependent probe amplification (MLPA). Four anomalies were identified: deletion 17q, duplications (4q), and associated duplications 15q and Xq. This duplication was found in two sisters of the proband. Anomalies were unidentified by the conventional technique. The prevalence of subtelomeric imbalances in our cohort of moderate to severe mental retardation is around 13% and is consistent with the literature. The sensitivity of the MLPA technique was characterized on cytogenetically verified positive and negative controls. MLPA is a fast, reliable, and relatively inexpensive technique to detect subtelomeric rearrangement in comparison with the fluorescence in situ hybridization (FISH) technique.

A 24-Mb deletion in 14q in a girl with corpus callosum hypoplasia.

Interstitial deletions of 14q including band 14q31 are uncommon. We report on a 3 year-old Tunisian girl who had a de novo interstitial deletion of the long arm of chromosome 14. The molecular cytogenetic study has identified the deletion as a del(14)(q24.3q32.2) covering nearly 24Mb. This abnormality was associated to phenotypic manifestations, mainly peculiar face, developmental delay and hypoplastic corpus callosum.