Functional analysis of MMR gene VUS from potential Lynch syndrome patients.

Lynch syndrome is caused by inactivating variants in DNA mismatch repair genes, namely MLH1, MSH2, MSH6 and PMS2. We have investigated five MLH1 and one MSH2 variants that we have identified in Turkish and Tunisian colorectal cancer patients. These variants comprised two small deletions causing frameshifts resulting in premature stops which could be classified pathogenic (MLH1 p.(His727Profs*57) and MSH2 p.(Thr788Asnfs*11)), but also two missense variants (MLH1 p.(Asn338Ser) and p.(Gly181Ser)) and two small, in-frame deletion variants (p.(Val647-Leu650del) and p.(Lys678_Cys680del)). For such small coding genetic variants, it is unclear if they are inactivating or not. We here provide clinical description of the variant carriers and their families, and we performed biochemical laboratory testing on the variant proteins to test if their stability or their MMR activity are compromised. Subsequently, we compared the results to in-silico predictions on structure and conservation. We demonstrate that neither missense alteration affected function, while both deletion variants caused a dramatic instability of the MLH1 protein, resulting in MMR deficiency. These results were consistent with the structural analyses that were performed. The study shows that knowledge of protein function may provide molecular explanations of results obtained with functional biochemical testing and can thereby, in conjunction with clinical information, elevate the evidential value and facilitate clinical management in affected families.

Functional characterization of MLH1 missense variants unveils mechanisms of pathogenicity and clarifies role in cancer.

Lynch syndrome is a heritable condition caused by a heterozygous germline inactivating mutation of the DNA mismatch repair (MMR) genes, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants, for which the clinical significance is unclear in many cases. We have identified three MLH1 missense alterations (p.(Glu736Lys), p.(Pro640Thr) and p.(Leu73Pro)) in six individuals from large Tunisian families. For none of these alterations, a classification of pathogenicity was available, consequently diagnosis, predictive testing and targeted surveillance in affected families was impossible. We therefore performed functional laboratory testing using a system testing stability as well as catalytic activity that includes clinically validated reference variants. Both p.(Leu73Pro) and p.(Pro640Thr) were found to be non-functional due to severe defects in protein stability and catalytic activity. In contrast, p.(Glu736Lys) was comparable to the wildtype protein and therefore considered a neutral substitution. Analysis of residue conservation and of the structural roles of the substituted residues corroborated these findings. In conjunction with the available clinical data, two variants fulfil classification criteria for class 4 "likely pathogenic". The findings of this work clarify the mechanism of pathogenicity of two unclear MLH1 variants and enables predictive testing and targeted surveillance in members of carrier families worldwide.

Prostatic tissue in 46XX congenital adrenal hyperplasia: Case report and literature review.

The presence of prostatic tissue, in addition to uterus and adrenal tumors, is possible in 46XX patients with CAH. Lesions of these organs are usually benign. However, complications including prostate and adrenal cancer were also reported.

c.1227_1228dupGG (p.Glu410Glyfs), a frequent variant in Tunisian patients with MUTYH associated polyposis.

INTRODUCTION: Familial adenomatous polyposis (FAP) is an autosomal dominant-inherited disease caused by germline variants in the APC gene. It is characterized by the development of hundreds to thousands of adenomatous polyps in colon and rectum. Recently, biallelic germline variants in the base excision repair (BER) gene: MUTYH have been identified in patients with attenuated FAP and/or negative APC result. It can be responsible for an autosomal recessive inherited colorectal cancer syndrome (MAP syndrome: MUTYH-associated polyposis). OBJECTIVE: The aim of this study was to evaluate germline variants of MUTYH gene in Tunisian patients with attenuated FAP. METHODS: thirteen unrelated patients from Tunisia with attenuated FAP were screened for MUTYH germline variants. Direct sequencing was performed to identify point variants in this gene. RESULTS: A Biallelic MUTYH germline variant were found in all patients and showed an attenuated polyposis phenotype almost of them without extra-colic manifestations: The known pathogenic frameshift variant c.1227_1228dupGG (p. Glu410Glyfs) was found, in homozygous state, in 13 index patients. CONCLUSION: Patients with attenuated familial adenomatous polyposis (<=100) and no obvious vertical transmission of the disease should be considered for MUTYH gene testing.

Targeted next generation sequencing screening of Lynch syndrome in Tunisian population.

A high colorectal cancer (CRC) incidence is observed in Tunisia, with a relatively high proportion of patients developing CRC before the age of 40. While this suggests a genetic susceptibility, only a few Tunisian Lynch Syndrome families have been described. In this study we aimed to identify the underlying genetic cause in 32 patients with early onset CRC and/or a positive family history. Of twenty-four patients' tumor or biopsies could be analyzed with immunohistochemical staining to detect loss of expression of one of the MMR proteins. Ten tumors showed loss of expression, of which one tumor was from a patient where a germline pathogenic MSH2 variant was detected previously with Sanger sequencing. Next generation sequencing of the MMR, POLE and POLD1 genes was performed in leukocyte and tumor DNA of the remaining nine patients, as well as in two patients with MMR-proficient tumors, but with severe family history. In six of 11 patients a germline variant was detected in MLH1 (n = 5) or MSH2 (n = 1). Two of six patients were from the same family and both were found to carry a novel in-frame MLH1 deletion, predicted to affect MLH1 function. All MLH1 variant carriers had loss of heterozygosity with retention of the variant in the tumors, while a somatic pathogenic variant was detected in the patient with the germline MSH2 variant.

Genetic Analysis of Mu and Kappa Opioid Receptor and COMT Enzyme in Cancer Pain Tunisian Patients Under Opioid Treatment.

BACKGROUND: Pain and its opioid treatments are complex measurable traits. Responses to morphine in terms of pain control is likely to be determined by many factors, including the underlying pain sensitivity of the patient, along with nature and extent of the painful process, concomitant medications, genetic and other clinical and environmental factors. This study investigated genetic polymorphisms implicated in the inter-individual pain response variability to opioid treatment in the Tunisian population. METHODS: This prospective association study investigated seven variations in the OPRM1, OPRK1 and COMT gene, which encode Mu and KAPPA opioid receptors, and Catechol-O-methyltransferase enzyme respectively, in a cohort of 129 Tunisian cancer pain patients under oral morphine treatment. Genotyping was performed by simple probe probes on Light Cyler for rs17174629, rs1799972, rs1799971, rs1051659, rs1051660 and rs4680 and by PCR assay for the indel in the promoter region of OPRK1 (rs35566036). A statistical associations study between dose (continuous), dose escalation (yes/no) and SNP or haplotypes were investigated using linear multiple regressions and logistic regressions respectively adjusted on metastases and pain covariates in the R software. RESULTS: We detected significant association of the rs1051660 adjusted on metastasis and pain (P=0.02), no other association has been detected between the 7 polymorphisms screened and the dose of morphine needed for pain relief. CONCLUSION: This can be explained by the strong genetic heterogeneity in the cosmopolitan areas where our patients were recruited for this study, compared to more homegenous population recruited in other studies.

Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

Association of the OPRM1 and COMT genes' polymorphisms with the efficacy of morphine in Tunisian cancer patients: Impact of the high genetic heterogeneity in Tunisia?

BACKGROUND: Genetic causes for inter-individual variability response to opioids are clinical difficulties for treatment efficiency. The aim of the present study was to investigate the possible association of opioid treatment outcome with single nucleotide polymorphisms (SNPs) in the mµ opioid receptor (OPRM1) and catechol-o-methyltransferase (COMT) genes, in Tunisian cancer pain patients. METHODS: We genotyped one hundred and twenty-nine cancer patients treated with different doses of morphine for 3 SNPs in OPRM1 gene (rs17174629, rs1799972 and rs1799971) and one in the COMT gene (rs4680). Associations between dose (continuous), dose escalation (yes/no) and SNP or haplotypes were investigated. RESULTS: Unlike other studies on Caucasian and Chinese populations, no significant association were found between the 4 polymorphisms screened and the dose of morphine needed for pain relief. CONCLUSION: This result can be explained by the genetic heterogeneity and cosmopolitan areas of our Tunisian patients compared to the others homogenous population.

Analysis of FMR1 gene premutation and X chromosome cytogenetic abnormalities in 100 Tunisian patients presenting premature ovarian failure.

OBJECTIVE: To evaluate the prevalence of FMR1 premutations and X chromosome cytogenetic abnormalities in a large cohort of Tunisian women with premature ovarian failure (POF). PATIENTS AND METHODS: The cohort consisted of 127 Tunisian women with POF referred by endocrinologists and gynecologists for genetic investigation in the context of idiopathic POF and altered hormonal profiles. Clinical information concerning the reproductive function in the family, previous hormonal measurements and/or possible fertility treatment were collected. Karyotype, FISH analyses, FMR1 and FMR2 testing were performed for all patients. RESULTS: Fifteen patients (11.81%) presented structural or numerical X chromosomal abnormalities. Moreover, we detected in 12 patients (10.71%) a high level of X mosaicism. Analysis of FMR1 gene in the 100 patients without X chromosomal abnormalities showed that five percent of the patients carried a FMR1 premutation allele. On the other hand, the FMR2 screening did not reveal any deletion. CONCLUSION: Our study confirms the major role of X chromosome abnormalities in POF and highlights the importance of karyotype analyses and FMR1 screening. These investigations provide valuable information for diagnosis and genetic counseling for these women who still have a 5% chance of spontaneous conception.

Frequency of HNF4A-P.I463V Variant in the Tunisian North-African Population and Its Relation with Diabetes Mellitus.

BACKGROUND: HNF4A-p.I463Vvariant, reported previously in two distinct families suspected of MODY-1, is assessed in this report to determine whether it is a mutation or a polymorphism (frequency >1%). METHODS: 200 Tunisian healthy people were screened for the presence of HNF4A-p.I463V variant, using RFLP-PCR technique and sequencing. Then, the frequency of this variant was estimated in the Tunisian population and compared to other populations registered in genetic databases. We also performed in-silico analysis using PolyPhen2 and Mutation T@sting softwares to assess the probable effect of HNF4A-p.I463V variant. RESULTS: HNF4A-p.I463V had a rare frequency in different populations and was found in 3 control subjects (1.5%) of the studied population. PolyPhen2 predicted that it is a polymorphism, whereas mutation T@sting suggested a probably affected mutant protein. CONCLUSION: HNF4A-p.I463V has a relatively high frequency (>1%) in our control cohort. It is also present in different ethnicities and in- silico analysis showed conflicting results. For these reasons, HNF4A-p.I463V should not be considered as a mutation responsible for MODY-1.

Partial KCNQ1OT1 hypomethylation: A disguised familial Beckwith-Wiedemann syndrome as a sporadic adrenocortical tumor.

Beckwith-Wiedemann syndrome has a wide spectrum of complications such as embryonal tumors, namely adrenocortical tumor. Tumor predisposition is one of the most challenging manifestations of this syndrome. A 45-day old female with a family history of adrenocortical tumor presented with adrenocortical tumor. The case raised suspicion of a hereditary Beckwith-Wiedemann syndrome, therefore molecular analysis was undertaken. The results revealed partial KCNQ1OT1 hypomethylation in the infant's blood DNA which was associated with a complete loss of methylation in the infant's adrenocortical tumor tissue. It is unique for familial Beckwith-Wiedemann syndrome caused by KCNQ1OT1 partial hypomethylation to manifest solely through adrenocortical tumor. Incomplete penetrance and specific tissue mosaicism could provide explanations to this novel hereditary Beckwith-Wiedemann syndrome presentation.

Assessment of MYCN amplification status in Tunisian neuroblastoma: CISH and MLPA combining approach.

BACKGROUND: Neuroblastoma (NB) shows a complex combination of genetic aberrations. Some of them represent poor genetic prognosis factors that require specific and intensive chemotherapy. MYCN amplification consists of the major bad outcome prognostic factor, it is indeed frequently observed in aggressive neuroblastomas. To date different methods are used for MYCN status detection. OBJECTIVES: The primary aim of our study was to provide a critical assessment of MYCN status using 2 molecular techniques CISH and MLPA. We also focused on the correlation between neuroblastoma genetic markers and patient's clinical course among 15 Tunisian patients. METHODS: we developed a descriptive study that includes 15 pediatric Tunisian patients referred to our laboratory from 2004 to 2011. We reported the analysis of fresh and FFPE NB tumors tissues. RESULTS: No significant correlation was found between COG grade and patients overall survival. Assessment of NMYC gene copy number by kappa statistic test revealed high concordance between CISH and MLPA tests (kappa coefficient = 0.02). CONCLUSION: Despite misdiagnosing of MYCN status fewer than 5 copies, MLPA remains an effective molecular technique that enables a large panel of genomic aberrations screening. Thus combining CISH and MLPA is an effective molecular approach adopted in our laboratory. Our results allow pediatric oncologists to set up the first Neuroblastoma therapeutic strategy based on molecular markers in Tunisia.

Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma.

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin-binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin-binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.

NLRP7 mutation analysis in sporadic hydatidiform moles in Tunisian patients: NLRP7 and sporadic mole.

CONTEXT: Hydatidiform mole, an aberrant human pregnancy, is commonly a nonrecurrent disease. Recently, a rare autosomal recessive form of familial and/or recurrent molar pregnancies was associated with mutations in the NLRP7 gene. OBJECTIVE: To investigate whether NLRP7 mutations exist in Tunisian women with sporadic hydatidiform moles. DESIGN: Genomic DNA from 38 unrelated Tunisian patients with sporadic hydatidiform moles were screened by sequencing all NLRP7 exons. A high-resolution melting curve analysis was performed on 170 DNA controls to analyze new sequence variants. RESULTS: More than 13% of these patients were heterozygous for NLRP7 mutations. We found 2 novel missense mutations in the heterozygous state, c.544G>A (p.Val182Met) in 1 patient and c.1480G>A (p.Ala494Thr) in 2 patients, and 2 already reported mutations, c.1532A>G (p.Lys511Arg) and c.2156C>T (p.Ala719Val), in 2 patients. None of these mutations were identified in 170 controls except for 1 woman who was heterozygous for p.Val182Met. CONCLUSION: As homozygous NLRP7 mutations are associated with recurrent hydatidiform mole or conception loss, the heterozygous state could represent a risk factor for nonrecurrent mole.

The usefulness of p57(KIP2) immunohistochemical staining and genotyping test in the diagnosis of the hydatidiform mole.

Classification of molar gestations into complete hydatidiform mole (CHM) and partial hydatidiform mole (PHM) and their differentiation from nonmolar hydropic abortions (HA) are traditionally accomplished by morphology alone. Sometimes, the process may be inaccurate or inconclusive especially in early diagnosed cases. With the availability of p57(KIP2) immunostaining (the product of a strongly paternally imprinted and maternally expressed gene), it may be possible to classify these lesions objectively. P57(KIP2) immunostaining is absent in CHM because it lacks a maternal genome, whereas PHM and HA show positive staining. The aims of this study were to evaluate the results of routine histopathological examination and p57(KIP2) immunoreactivity in a large series of molar and nonmolar HA in Tunisia, and to compare the accuracy of p57(KIP2) immunohistochemistry with that of nuclear DNA microsatellite polymorphism in identifying CHM. The immunohistochemical expression of p57(KIP2) protein was investigated in 220 specimens of first trimester hydropic abortuses, and it was compared with the original diagnosis based on morphology, including 132 CHM, 49 PHM, and 39 HA. Concordant results were obtained in 210 cases. In 9 of 10 cases with a discordant diagnosis (negative immunostaining in 8 cases morphologically diagnosed as PHM and one case diagnosed as HA), microsatellite DNA genotyping analysis agreed with the results of p57(KIP2) staining, confirming the diagnosis of CHM in these cases. Twenty cases of CHM with negative p57(KIP2) immunostaining were also analyzed by genotyping and indicated the absence of maternal contribution and the homozygosity for a single paternal allele in concordance with the androgenetic and monospermic origin of CHM in these cases. We confirm that for distinguishing CHM from its mimics, p57(KIP2) immunohistochemistry can be used as successfully as DNA microsatellite genotyping. However, molecular techniques are still required for the evaluation of some difficult cases with discordant positive p57(KIP2) staining.

Screening for NLRP7 mutations in familial and sporadic recurrent hydatidiform moles: report of 2 Tunisian families.

A familial or sporadic recurrent hydatidiform mole is a rare autosomal recessive condition that has been associated with biallelic mutations in the nucleotide-binding, leucine-rich repeat, pyrin domain 7 (NLRP7) gene (19q13.42). Cases from different ethnic origins have been reported earlier. Here we report the first Tunisian patients: 2 sisters with homozygous NLRP7 mutations (p.E570X) and 1 sporadic case with no mutation in NLRP7. Our results extend the number of familial recurrent reproductive wastages due to mutations in NLRP7. We suggest that mutations screening of NLRP7 could be proposed more systematically in women with recurrent pathologic pregnancy outcomes of unknown origin. The rare cases with a typical clinical picture, which were not related to NLRP7 mutation as in our sporadic case, should be investigated more to identify the causative gene.

[Li Fraumeni syndrome: a case with multiple primary cancers and presenting a germline p53 mutation]. / Le syndrome de Li Fraumeni: à propos d'un cas familial avec cancers multiples et présentant une mutation germinale du gène p53.

Li Fraumeni Syndrome (LFS) is a rare autosomal disorder characterized by a familial clustering of tumors. Analysis of several series of LFS families have shown that 70% of such families are attributable to germ-line mutations in TP53. We report the case of a patient who had a first degree family antecedent of cancer in young ages. At the age of 31 years, the patient was operated of bladder papillary superficial carcinoma; five years later, he was treated for a high grade pleomorphe sarcoma of the left thigh and treated by surgery, adjuvant chemotherapy and radiotherapy. At the age of 38 years, after abdominal pain, radiologic examination reveled pancreatic tumor with bone and lymphatic metastases. The patient died one month later from pulmonary embolism. Sequencing revealed a germiline mutation of this patient that was confirmed in a member of his family in codon 1009C>T, protein Arg337Cys, exon 10 of TP53 gene this mutation was revealed in his nephew (died at the age of 20 from bone sarcoma).

An autosomal dominant hypophosphatemic rickets phenotype in a Tunisian family caused by a new FGF23 missense mutation.

Autosomal dominant hypophosphatemic rickets (ADHR) is a rare disease, characterized by isolated renal phosphate wasting, hypophosphatemia, and inappropriately normal 1,25-dihydroxyvitamin D(3) (calcitriol) levels. This syndrome involves rickets with bone deformities in childhood and osteomalacia, osteoporosis, articular and para-articular pain, and fatigue in adulthood. It is caused by mutations in a consensus sequence for proteolytic cleavage of the FGF23 protein. Normally, this protein actively regulates phosphate homeostasis. Here we report a Tunisian family in which one parent and three children show clinical and biological features of ADHR. Mutation analysis of the FGF23 gene finds a heterozygous substitution of the C at position 526 by a T (526 C --> T), leading to an amino acid replacement of the FGF23 protein (R176W) at position 176. This causative new mutation is located in the consensus sequence for the proteolytic cleavage domain. These results confirm the importance of this site in FGF23 function and its essential role in ADHR physiopathology.

Assessment of the role of histopathology and DNA image analysis in the diagnosis of molar and non-molar abortion: a study of 89 cases in the center of Tunisia.

This study retrospectively evaluated the histopathological criteria commonly used in the literature on the diagnosis of hydatidiform mole, in correlation with the diagnosis rendered previously. The molar and non-molar cases seen in the first-trimester of pregnancy were separately reviewed by two pathologists. The correlation between the consensual histological diagnosis and the ploidy status was then evaluated. We retrospectively studied 89 specimens of abortus conception, including 35 complete hydatidiform moles (CHM), 12 partial hydatidiform moles (PHM), and 42 hydropic abortions (HA). The final histopathological diagnosis was compared with the results of DNA content detected by imaging analyzer (Samba 200), studying all cases of molar pregnancy and 4 cases of HA (initially diagnosed as molar pregnancies). In the consensus histological diagnosis, the cases were reclassified as follows: 30 CHM (initial diagnosis (ID): 27 CHM and 3 PHM), 12 PHM (ID: 6 PHM and 6 CHM), and one case with a persistent problem in differentiating PHM from HA and 46 HA (ID: 42 HA, 2 CHM, and 2 PHM). An agreement between the two pathologists was reached in 77 cases (K=0.72, 0.52, and 0.9, respectively, for CHM, PHM, and HA). The ploidy study demonstrated diploidy in 56.6% (17/30) of CHM and triploidy in 58.3% (7/12) of PHM. In the 4 cases of HA studied, 3 were diploid and 1 case was aneuploid. Our study demonstrated that several histopathological criteria could be used for the distinction between PHM, CHM, and HA. However, the study of DNA cannot be the technique of choice to distinguish between these entities. Some cases remain problematic since the morphological criteria are not easily reproducible. New sensitive techniques might resolve these dilemmas.