Genetic Analyses of the NF1 Gene in Turkish Neurofibromatosis Type I Patients and Definition of three Novel Variants.

Neurofibromatosis Type I (NF1) is a multi systemic autosomal dominant neurocutaneous disorder predisposing patients to have benign and/or malignant lesions predominantly of the skin, nervous system and bone. Loss of function mutations or deletions of the NF1 gene is responsible for NF1 disease. Involvement of various pathogenic variants, the size of the gene and presence of pseudogenes makes it difficult to analyze. We aimed to report the results of 2 years of multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) for genetic diagnosis of NF1 applied at our genetic diagnosis center. The MLPA, semiconductor sequencing and Sanger sequencing were performed in genomic DNA samples from 24 unrelated patients and their affected family members referred to our center suspected of having NF1. In total, three novel and 12 known pathogenic variants and a whole gene deletion were determined. We suggest that next generation sequencing is a practical tool for genetic analysis of NF1. Deletion/duplication analysis with MLPA may also be helpful for patients clinically diagnosed to carry NF1 but do not have a detectable mutation in NGS.

Cytotoxic Antibody Detection by Means of Flow-Cytometric Cross-Match.

BACKGROUND: Complement-dependent lymphocytotoxicity (CDC-XM) and flow-cytometric (FCXM) cross-match are analyzed individually for each donor and recipient pair, because these techniques have fundamental differences for the evaluation of histocompatibility. Lately, cytotoxic flow-cytometric cross-match (cFCXM) has been developed as an alternative to both CDC-XM and FCXM techniques. We evaluated the limits of cFCXM with the use of different positive serum dilutions. METHODS: CDC-XM, FCXM, and cFCXM tests were performed with the use of commercially available negative and positive serum samples and lymphocytes from healthy donors. RESULTS: Complement-dependent cell death was successfully detected with the use of cFCXM. Complement-dependent cell death ratios in cFCXM were similar those in CDC-XM. With cFCXM, not only complement-dependent cell death but also IgG binding could be detected within a single assay. At higher concentrations of the positive serum, IgG-fluorescein isothiocyanate (FITC) mean fluorescent intensity (MFI) values detected with the use of cFCXM were less than those of conventional FCXM. Correspondingly, for dead cells, MFI values of IgG-FITC were less than those of live cells in higher positive serum concentrations in the cFCXM assay. Moreover, our results demonstrated that in cFCXM analysis, the decreasing ratio of dead cells at increasing positive serum dilutions was not in parallel with the same decrease in IgG-FITC MFI values. CONCLUSIONS: The cFCXM technique detects complement-mediated cytotoxic cell death with the additional ability to show IgG binding in the same tube and therefore may reduce the necessary bench time and workload.

A case with Emanuel syndrome: extra derivative 22 chromosome inherited from the mother.

Emanuel syndrome (ES) is a rare chromosomal disorder that is characterized by multiple congenital anomalies and developmental disabilities. Affected children are usually identified in the newborn period as the offspring of balanced (11;22) translocation carriers. Carriers of this balanced translocation usually have no clinical symptoms and are often identified after the birth of offspring with an unbalanced form of the translocation, the supernumerary der(22) t(11;22) syndrome. We report a 3-year-old boy with the t(11;22)(q23;q11) chromosome, transmitted in an unbalanced fashion from his mother. He has several developmental delays; he is not independently ambulatory and language is significantly impaired. Using his peripheral blood, karyotyping was performed to define his multiple congenital anomalies, revealing the following chromosomal abnormality: 47, XY, +der(22)t(11;22)(q23.3;q11.2). To ascertain the origin and trait of this supernumerary marker chromosome [der(22)t(11;22)(q23.3;q11.2)], karyotyping of his parents was performed. The mother was found to be a balanced carrier: 46, XX, t(11;22) (q23.3; q11.2).

The relationship between methylenetetrahydrofolate reductase c.677TT genotype and oligozoospermia in infertile male patients living in the Trakya region of Turkey.

Methylenetetrahydrofolate reductase (MTHFR), the key enzyme of the folate metabolic pathway, has been reported to be five times more active in the testicles compared to other organs in adult mice. The aim of this study was to investigate the relationship between MTHFR c.677C>T and c.1298A>C polymorphisms and infertility in nonobstructive azoospermic and oligozoospermic male patients living in the Trakya region of Turkey. The study population included 75 nonobstructive azoospermic and 62 oligozoospermic, nonconsanguineous patients who were referred to the Department of Medical Genetics of Trakya University between 01.03.2012 and 01.06.2013 due to infertility and who had been diagnosed based on clinical examinations and spermiograms. All of the patients had a normal karyotype without a Y chromosome microdeletion. Melting curve analysis with labelled probes and primers that were designed by the manufacturers and the real-time polymerase chain reaction method were used. The MTHFR c.677TT genotype frequency in the oligozoospermic infertile male patient group was greater than that of the fertile control group [odds ratio (OR) = 2.675 (95% CI: 0.979-7.305), (P < 0.048)]. The MTHFR c.677TT genotype may be a genetic risk factor for oligozoospermic infertile male patients who live in the Trakya region of Turkey.

Identification of a novel HLA-A*26 allele, HLA-A*26:01:36, in a Turkish family by sequence-based typing.

HLA-A*26:01:36 differs from the closest allele HLA-A*26:01:01 by a nucleotide change at the position 114.

An Investigation of the Relationship between the eNOS Gene Polymorphism and Diagnosed Migraine.

We investigated the phenotype-genotype association of the following endothelial nitric oxide synthase (eNOS) gene polymorphisms, rs743506, rs2070744, rs1799983, rs180079, rs3918226, rs207468799 and rs148554851, in patients suffering from migraine living in Edirne, Turkey. A total of 175 individuals, who had been diagnosed with migraine between April 2013 and December 2013, at the Neurology Department, Trakya University Medical Faculty, Edirne, Turkey, and 125 healthy controls were recruited. The above gene polymorphisms were analyzed from genomic DNA in both patient and control groups, using the pyro-sequencing method. The eNOS rs1799983 TT genotype frequency in migraine patients who had a headache duration of longer than 24 hours was statistically significantly higher than in patients who had migraine attacks that lasted under 24 hours (p = 0.047). In terms of the AGGTGGA haplotype, the severity of headache was statistically significant, and was found to be severe in 61.0% (p = 0.0001). Also in terms of the AGGTGGA haplotype, the duration of headache was statistically significant, and was >24 hours in 56.0% of patients (p = 0.008). In our study, there was no significant genotypephenotype relationship between eNOS rs743506, rs2070744, rs1799983, rs180079, rs3918226, rs207468799 and rs148554851 gene polymorphisms and migraine patients with and without aura living in Edirne, Turkey. The AGGTGGA haplotype constitutes a risk in terms of the severity and the duration of headaches in patients with migraine. This risk is significantly higher in patients with migraine with aura than patients with migraine without aura.

Novel NLRP7 mutations in familial recurrent hydatidiform mole: are NLRP7 mutations a risk for recurrent reproductive wastage?

OBJECTIVE: Familial recurrent hydatidiform mole is an exceedingly rare clinical condition. Affected women are predisposed to molar pregnancies of diploid, biparental origin rather than androgenetic origin. At present, NLRP7 and KHDC3L (C6orf221) are the only genes known to be associated with familial recurrent hydatidiform mole. This study investigated the genetic dispositions in two large Turkish families with recurring molar conceptuses. STUDY DESIGN: Copy number variation analysis was performed followed by NLRP7 gene sequencing. The finding of a mono-allelic condition in one family led to investigation of the adjacent NLRP2 gene and recently associated KHDC3L gene. Sampled molar tissues were genotyped using microsatellite markers. RESULTS: In one family, a homozygous single nucleotide insertion that caused a frameshift leading to an early stop codon, c.2940_2941insC (p.Glu981ArgfsX13), was identified in the affected sisters. In the other family, a heterozygous 60-kb deletion eliminating substantial portions of the NLRP2 and NLRP7 genes on one allele was found. Screening of NLRP2 and KHDC3L genes revealed no alterations that were considered to be pathological. Genotyping of six independent molar conceptions revealed that five were of diploid, biparental origin and one was of diandric, triploid origin. CONCLUSIONS: Two novel protein-truncating mutations in the NLRP7 gene were found to be associated with familial recurrent hydatidiform mole. Mutations in the NLRP7 gene causing recurrent biparental hydatidiform mole may also be associated with other forms of recurrent reproductive wastage.

A case of treacher collins syndrome.

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development with an incidence of 1/50,000 live births. Mutations of the TCOF1 gene have been found to be responsible for most cases of this mandibulofacial disorder. Here we report TCS in an individual who has a heterozygous c.1021_1022delAG deletion in exon 7 of the TCOF1 gene (NG_011341.1). This is the second Turkish patient with a severe TCS phenotype resulting from a de novo c.1021_1022delAG mutation.

Flow cytometric evaluation of pregnancy-induced anti-donor immunization.

BACKGROUND: Living donor kidneys from spouses and children (from offspring to parents) are currently considered to be important organ sources. However, pregnancy-induced alloimmunization may provoke acute rejection episodes after kidney transplantation. being flow cytometry cross-match (FCXM) we studied donor-specific antibodies (DSAs) in the sera of recipients planned for living kidney transplantation from their spouse or children. When the FCXM was positive, we confirmed the existence of anti-human leukocyte antigen (HLA) antibodies using flow cytometry panel-reactive antibody (flow-PRA). PATIENTS AND METHODS: Between March 2005 and November 2010, we tested 85 pretransplantation sera from renal transplant recipients for DSAs. The recipients included 37 wives (group I) and 48 husbands (group II). FCXM-positive sera were tested using a flow-PRA screening method using HLA class I and class II antigen-coated beads. The mean recipient age was 48.1 ± 9.8 (range, 28-69) years and the mean donor age was 45.1 ± 11.1 (range, 23-69 years). RESULTS: Among group I were 18 (48.6%) FCXM-positive cross-matches; for group II, 5 (10.4%) cases (P = .001). Sensitized patients were 37.9% FCXM-positive, whereas nonsensitized patients were 3.7% positive (P = .001). FCXM-positive patients were re-evaluated for anti-HLA antibodies using flow-PRA. Seventeen of 18 group I tests (94.4%) were FCXM-positive, whereas 3 of 5 (60%) were positive among group II. CONCLUSIONS: We concluded that flow cytometry-based cross-match and PRA techniques can be used to detect anti-HLA antibodies using spousal or children donors for kidney transplantation.

The presence of donor-specific antibodies in renal transplantation.

Determining the presence of anti-HLA antibodies before transplantation is an important factor to prevent loss of function among renal transplantations. In addition, recent studies have shown that not only the pretransplantation existence of anti-HLA antibody but also posttransplantation donor-specific antibodies (DSA) and non-donor-specific antibodies are significantly associated with allograft rejection or loss of graft function. This study presented DSA among patients after renal transplantation together with graft function and survival.

Is flow cytometry crossmatch analysis using sera with different dilutions important for pretransplant analysis? A case report.

The most effective form of treatment for chronic renal failure is kidney transplantation from a cadaver or a living donor. For a kidney transplant to be successful, tissue compatibility and a lack of donor-specific anti-human leukocyte antigen (HLA) antibodies in the circulation of the patient are vital, in addition to ABO blood group compatibility. The presence of anti-HLA antibodies is assayed before transplantation using various methods, but because organ rejections have been observed in previous studies, different techniques are required to detect anti-HLA antibodies. Today, flow cytometry crossmatching is one of the most important and effective techniques in testing for donor-specific anti-HLA antibodies (DSAs). If weakly positive serum is assayed after serial dilution, it can yield high positivity. Herein, we describe the differences between the results for diluted and undiluted weakly positive sera studied using the flow cytometry crossmatch (FCXM) technique. In a recent study, the sera of weakly FCXM-positive patients were diluted 1/50, and the FCXM test was repeated. The use of diluted serum eliminated the effect of the prozone so that the DSAs could be detected.

No association of KIR genes with Behcet's disease.

Behcet's disease (BD) is thought to be caused by multiple genetic, environmental and immunological factors, one of the most prominent being the strong association with human leucocyte antigen (HLA)-Bw51, an HLA-Bw4-associated allele. We examined the presence/absence of 14 killer cell immunoglobulin-like receptors (KIRs) and their ligands in 134 Turkish individuals with BD and compared the results with those of 154 ethnically matched controls. Although KIR3DL1 with its ligand (HLA-Bw4) was significantly increased in the patients with BD (P = 0.0003), this no longer applied when the patients and controls were categorised by HLA-Bw51 status. Thus, no association was identified between presence or absence of any of the 14 KIR genes studied and BD. In addition, we did not find any associations of KIR with various manifestations of the disease nor with gender or age of onset.