Experiences in the molecular genetic and histopathological evaluation of calpainopathies.

Calpainopathy is mainly characterized by symmetric and progressive weakness of proximal muscles. Several reports showed that the most common LGMD subtype is LGMDR1 or calpainopathy, which had previously been defined as LGMD2A. Until now, more than 500 likely pathogenic/pathogenic variants in the CAPN3 gene have been reported. However, a clear genotype-phenotype association had not yet been established and this causes major difficulties in predicting the prognosis in asymptomatic patients and in providing genetic counseling for prenatal diagnosis. In this report, we aimed to add new data to the literature by evaluating 37 patients with likely pathogenic/pathogenic variants for the detected variants' nature, patients' phenotypes, and histopathological features. As a result, the general clinical presentation of the 23 different variants was presented, the high frequency of NM_000070.3:c.550delA mutation in Exon 4 was discussed, and some novel genotype-phenotype associations were suggested. We have underlined that calpainopathy can be misdiagnosed with inflammatory myopathies histopathologically. We have also emphasized that, in young or adult patients with mild to moderate proximal muscle weakness and elevated CK levels, calpainopathy should be the first suspected diagnosis.

The Efficiency of SNP-Based Microarrays in the Detection of Copy-Neutral Events at 15q11.2 and 11p15.5 Loci.

Prader-Willi, Angelman, Beckwith-Wiedemann, and Russell-Silver are imprinting syndromes. In this study, we aimed to compare the efficiency of single nucleotide polymorphism (SNP) microarray analysis with methylation-specific Multiplex ligation-dependent probe amplification (MS-MLPA) in the detection of uniparental disomy in these syndromes. The patient samples with regions of loss of heterozygosity (LOH), covering 15q11.2 and 11p15.5 critical loci, were analyzed with MS-MLPA to demonstrate the efficiency of SNP microarray in the detection of uniparental disomy (UPD). In a total of seven patients, LOH covering 15q11.2 and 11p15.5 critical loci was detected. Two (28.6%) of these seven patients showed aberrant methylation (suggesting UPD) in MS-MLPA. SNP microarray is a useful tool in the detection of LOH; however, it should be used with caution, since false-positive or false-negative LOH results can be obtained. Although methylation analysis is recommended as the first tier test in the diagnosis of most of the imprinting disorders, combining methylation analysis with SNP microarray can enhance our evaluation process.

Unexpected Coexistence of a Derivative t(21;21) and Complementary Mosaic r(21) in a Female with Multiple Miscarriages.

The case presented here describes a female patient with recurrent miscarriages and a normal microarray analysis result. However, the coexistence of a robertsonian (21;21) translocation and complementary mosaic ring chromosome 21 was detected by karyotyping and FISH analysis. Partial trisomy 21 was found with QF-PCR and microarray analysis in one of the fetuses. The aim of this report was to emphasize the diagnostic importance of conventional cytogenetics.

Impact of next-generation sequencing panels in the evaluation of limb-girdle muscular dystrophies.

INTRODUCTION: Limb-girdle muscular dystrophy (LGMD) is the fourth most common muscular dystrophy, with progressive proximal muscle weakness. However, a large number of neuromuscular conditions are similarly presented. Because of this, the use of high-throughput methods such as next-generation sequencing (NGS) is important in the evaluation of LGMD. METHODS: In this report, we applied a custom target capture-based NGS panel covering 31 LGMD-associated genes (MYOT, LMNA, CAV3, DES, DNAJB6, FLNC, CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TCAP, TRIM32, FRKP, TTN, POMT1, ANO5, FKTN, POMT2, POMGnT1, DAG1, PLEC, GAA, GMPPB, HNRNPDL, TNPO3, LIMS2, POMK, TRAPPC11, ISPD) in 74 patients suspected of LGMD. RESULTS: In 25 (33.8%) out of 74 patients analyzed, one or more pathogenic/likely pathogenic variants in 13 different genes were detected. Six of the patients had the variants that were not found in databases and literature; thus, they were interpreted as novel pathogenic variants. DISCUSSION: The diagnosis rate achieved (33.8%) is consistent with previous literature reports and underlines the efficiency and importance of NGS technology in the molecular genetic evaluation of LGMD.

Targeted fetal cell-free DNA screening for aneuploidies in 4,594 pregnancies: Single center study.

BACKGROUND: Next-generation sequencing (NGS) and discovery of fetal cell-free DNA (cfDNA) in the maternal circulation render possible prenatal screening for trisomy 21 (Down syndrome), trisomy 18, trisomy 13, and sex chromosome aneuploidies. The approach is called "fetal cfDNA screening" and in contrast to noninvasive conventional serum screening, it provides the identification of 98%-99% of fetuses with Down syndrome. METHODS: Retrospective analysis of targeted noninvasive prenatal testing (NIPT) (Clarigo Test) pregnancies with moderate risk, which we have reported between 2016 and 2018 years is presented. Two separate laboratory workflows and NGS platforms are used for the same targeted NIPT analysis. RESULTS: In total, 4,594 pregnant women were investigated. Initial 3,594 cases are studied by MiSeq platform, the last 1,000 cases by NextSeq. Failure rate for MiSeq platform is 10.9% and for NextSeq is 8.7%. Automatically reported cases constitute 75% of the MiSeq group and 87% of the NextSeq group. CONCLUSIONS: Targeted NIPT results suggest that MiSeq platform could be used for NIPT which would be an essential option particularly for laboratories with low sample flow. And, the NextSeq platform has easier wet lab process and also increased success rate in automatic reporting which is suitable for centers with high number of NIPT cases.

Molecular genetic evaluation of NLRP3, MVK and TNFRSF1A associated periodic fever syndromes.

Periodic fever syndromes (PFSs) are a family of clinical disorders, which are characterized by recurrent episodes of fever in the absence of microbial, autoimmune or malign conditions. Most common types of PFSs are associated with four genes: MEFV, MVK, TNFRSF1A and NLRP3. This paper aims to add new data to the genotype-phenotype association of MVK-, TNFRSF-1A- and NLRP3-associated PFSs. A total number of 211 patients were evaluated. Two different approaches were used for the molecular genetic evaluation of MVK-, TNFRSF-1A- and NLRP3-associated PFSs. For the first 147 patients, Sanger sequence analysis of selected exons of MVK, TNFRSF1A and NLRP3 genes was done. For subsequent 64 patients, targeted NGS panel analysis, covering all exons of MVK, TNFRSF1A and NLRP3 genes, was used. A total number of 48 variants were detected. The "variant detection rate in index patients" was higher in the NGS group than Sanger sequencing group (19% vs. 15,1%). For the variant positive patients, a detailed genotype-phenotype table was built. In PFSs, lack of correlation exists between genotype and phenotype in the general population and even within the families. In some cases, mutations behave differently and yield unexpected phenotypes. In this study, we discussed the clinical effects of eight different variants we have detected in the MVK, TNFRSF1A and NLRP3 genes. Four of them were previously identified in patients with PFS. The remaining four were not reported in patients with PFS. Thus, we had to interpret their clinical effects by analysing their frequencies and in silico analysis predictions. We suggest that new studies are needed to evaluate the effects of these variants more clearly. To be able to demonstrate a clearer genotype-phenotype relationship, all PFS-related genes should be analysed together and the possibility of polygenic inheritance should be considered.

cfDNA in exhaled breath condensate (EBC) and contamination by ambient air: toward volatile biopsies.

Exhaled breath is a source of volatile and nonvolatile biomarkers in the body that can be accessed non-invasively and used for monitoring. The collection of lung secretions by conventional methods such as bronchoalveolar lavage, induced sputum collection, and core biopsies is limited by the invasive nature of these methods. Non-invasive collection of exhaled breath condensate (EBC) provides fluid samples that are representative of airway lining fluids. Various volatile and nonvolatile biomarkers can be detected in volatile condensates, such as H2O2, nitric oxide, lipid mediators, cytokines, chemokines, DNA, and microRNAs. Studies have examined cell-free DNA (cfDNA) in plasma samples from non-small-cell lung cancer patients, offering to new insights and fostering development of the liquid biopsy. However, few studies have examined cfDNA in EBC samples. This study examined whether EBC is an appropriate source of cfDNA using housekeeping-gene-specific primer probes and quantitative real-time polymerase chain reaction in healthy subjects. Ambient (room) air is contaminated with DNA, so caution is needed. Preliminary studies indicated that volatile biopsies are becoming an important diagnostic tool in lung cancer.

Targeted next generation sequencing in patients with maturity-onset diabetes of the young (MODY).

Background Maturity-onset diabetes of the young (MODY) is a common form of monogenic diabetes. Fourteen genes have been identified, each leading to cause a different type of MODY. The aims of this study were to reveal both known and novel variants in MODY genes in patients with MODY using targeted next generation sequencing (NGS) and to present the genotype-phenotype correlations. Methods Mutation analysis of MODY genes (GCK, HNF1A, HNF4A, HNF1B, ABCC8, INS and KCNJ11) was performed using targeted NGS in 106 patients with a clinical diagnosis of MODY. The variants were evaluated according to American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines recommendations. Results A total of 18 (17%) variants were revealed among all patients. Seven variants in GCK, six in HNF4A, four in HNF1A and one in ABCC8 genes were found. Eight of them were previously published and 10 of them were assessed as novel pathogenic or likely pathogenic variants. Conclusions While the most frequent mutations are found in the HNF1A gene in the literature, most of the variants were found in the GCK gene in our patient group using the NGS method, which allows simultaneous analysis of multiple genes in a single panel.

Fetal HLA-G alleles and their effect on miscarriage.

BACKGROUND: Immunosuppression at the feto-maternal interface is crucial for a successful pregnancy outcome. Human leukocyte antigen-G (HLA-G) seems to be a major contributor to fetal tolerance. The HLA-G expression is seen in cytotrophoblasts and in maternal blood. Fetal HLA-G acts on decidual antigen-presenting cells (APCs), natural killers (NKs) and T cells. Recent findings revealed that defects in placentation and their consequences are associated with maternal HLA-G variants and their expression levels. OBJECTIVES: The objective of this article is to investigate the relationship between fetal HLA-G alleles and miscarriage, which has not been investigated to date. MATERIAL AND METHODS: The present study includes 204 recurrent miscarriage (RM) cases who were admitted to our clinic between 2012 and 2016. Twenty-eight miscarriage products without maternal cell contamination and any known pathology were analyzed by HLA-G typing. In addition, 3' untranslated region (UTR) 14-base pair (bp) insertion/deletion polymorphism was also investigated by Sanger sequencing. RESULTS: For our population, the most frequent HLA-G type was G*01:01, both in the study group (30.3%) and in the control group (47%). The study revealed that the G*01:04 allele was significantly associated with miscarriage (p = 0.007). The 3' UTR 14bp deletion was more frequent in the miscarriage group, but there was no significant correlation. CONCLUSIONS: HLA-G alleles seem to be related with miscarriage and should be considered in RM cases.

EVALUATION OF PREIMPLANTATION GENETIC ANEUPLOIDY SCREENING CASES AT A REFERENCE GENETICS CENTER: 10 YEARS' EXPERIENCE.

The aim of this study is to review and evaluate our preimplantation genetic screening (PGS) records in terms of their demographic data, indications, cytogenetic results, pregnancy outcomes and discuss these findings in different aspects. PGS was performed in a total of 84 couples (87 cycles) between the period 2005 to 2015. Biopsied blastomeres from embryos on day 3 were fixed and fluorescence in situ hybridization was carried out for chromosomes 13, 16, 18, 21, 22, X and Y depending on the indication. The diagnostic and clinical data were retrospectively evaluated. A total of 450 blastomeres were biopsied. Ninety-eight of them were found to be suitable for transfer. They were transferred to 72 patients in 75 cycles resulting in 23 pregnancies and 20 healthy births. The most common indication was unexplained infertility. The implantation rate was calculated as 23.4% whereas the take-home baby rate was 26.6% per transfer. The highest rate of healthy living births is achieved in patients having low grade maternal mosaic sex chromosomal aneuploidy. All living births achieved by PGS had normal chromosomal structure which we can propose it as an alternative test for couples at risk to select normal embryos to improve the outcomes of assisted reproductive procedures and to avoid the transfer of chromosomally unbalanced and multiple embryos.

Dentofacial changes from fan-type rapid maxillary expansion vs traditional rapid maxillary expansion in early mixed dentition.

OBJECTIVE: To test the null hypothesis that there is no difference between the effects of fan-type rapid (FRME) and rapid maxillary expansion (RME) used with an acrylic bonded expansion appliance on dentofacial structures in early occlusal stages. MATERIALS AND METHODS: This was a prospective clinical trial. The FRME group had an anterior constricted maxillary width with a normal intermolar width, and the RME group had bilateral constricted maxillary width. The FRME group consisted of 20 patients (mean age, 8.96 ± 1.19 years), and the RME group consisted of 22 patients (mean age, 8.69 ± 0.66 years). Lateral and frontal cephalometric radiographs and dental casts were taken before and after expansion and 3 months after completing treatment for each patient. The data were compared using repeated-measures analysis of variance. The paired-samples t-test was used to evaluate treatment and retention effects, and the independent samples t-test was used to consider the differences between the two groups. RESULTS: The maxilla moved downward and forward in both groups. The nasal cavity and maxillary width were expanded more in the RME group, and there were only a few relapses in this group during the retention period. There was significant labial tipping of the upper incisors in the FRME expansion group. The expansion of intercanine width was similar in both groups, but the expansion of intermolar width was significantly greater in the RME group. CONCLUSION: The null hypothesis was rejected. There was a difference between the effects of FRME and RME used with an acrylic bonded expansion appliance on dentofacial structures in the early occlusal stages.

The effects of Trolox treatment on experimental strangulation ileus.

BACKGROUND: Intestinal ischemia-reperfusion injury is a serious and widespread clinical problem. Trolox effectively prevents lipid peroxidation in oxidative stress and protects cell injury. The aim of this study is to investigate the effect of Trolox alone on the intestinal ischemia-reperfusion injury in strangulation ileus model, which has not been investigated previously. METHODS: Twenty-eight Sprague-Dawley rats randomly divided into four groups were used. Group Laparotomy + Physiological Saline underwent laparotomy and was administered physiological saline; Group Laparotomy + Trolox was administered Trolox. Group Strangulation + Physiological Saline was administered physiological saline before reperfusion following strangulation ileus; Group Strangulation + Trolox was administered Trolox. RESULTS: Histopathological study was evaluated and catalase, malondialdehyde, superoxide dismutase measurements were performed in intestinal samples. Serum biochemistry parameters were evaluated. The higher grade (grade > or = 2) was significantly observed to decrease in Group Strangulation + Trolox when compared with Group Strangulation + Physiological Saline (p = 0.04). In Group Laparotomy + Trolox, when compared with Group Laparotomy + Physiological Saline, the higher grade was found to be significantly lower (p = 0.03). The catalase values were found to be significantly lower in Group Strangulation + Trolox, when compared with Group Strangulation + Physiological Saline, and in Group Laparotomy + Trolox, when compared with Group Laparotomy + Physiological Saline (p < 0.01). CONCLUSIONS: Trolox is a powerful antioxidant as well as effectively prevents ischemia-reperfusion injury of the strangulated intestine segment.

Preconditioning-like amelioration of erythropoietin against laparoscopy-induced oxidative injury.

BACKGROUND: Laparoscopic surgery has gained wide acceptance for almost every kind of surgical procedure, although it has produced significant oxidative injury to intraabdominal organs depending on the pressure level and the kind of the gas used. The literature describes several preventive measures for decreasing the postlaparoscopic oxidative injury such as low intraabdominal pressure, gasless laparoscopy, and laparoscopic preconditioning. Erythropoietin was shown previously to decrease ischemia-reperfusion injury to the liver. The current study evaluated the effect of erythropoietin against laparoscopy-induced oxidative injury, as compared with laparoscopic preconditioning. METHODS: For this study, 64 male Spraque-Dawley rats were randomly assigned to one of the following groups. The control group was subjected to a sham operation. The laparoscopy group was subjected to 60 min of pneumoperitoneum. The laparoscopic preconditioning plus laparoscopy group was subjected to 5 min of insufflation and 5 min of desufflation followed by 60 min of pneumoperitoneum. The erythropoietin plus laparoscopy group was subjected to a subcutaneous injection of erythropoietin as a single 1,000-U/kg dose followed by 60 min of pneumoperitoneum. After 45 min of desufflation subsequent to cessation of pneumoperitoneum, blood, liver, and kidney samples were obtained from half of the rats. The other half of the rats were observed for a reperfusion period of 24 h. Tissue and blood samples also were obtained after this period. RESULTS: Laparoscopy produced significant oxidative injury, as compared with the sham treatment. Laparoscopic preconditioning produced significant amelioration of the ischemic injury. Although erythropoietin administration during the prelaparoscopic period decreased the pneumoperitoneum-induced oxidative injury, the beneficial effect of laparoscopic preconditioning was more pronounced. CONCLUSION: Laparoscopic preconditioning is more effective than the preischemic administration of erythropoietin in reducing laparoscopy-induced oxidative injury.

Tumour angiogenesis and mast cell density in the prognostic assessment of colorectal carcinomas.

BACKGROUND: Tumour angiogenesis is essential for the growth, invasion and metastasis of solid tumours. There are several lines of evidence that the mast cells play an important role in tumour angiogenesis. AIMS: The study focused to determine the correlation between the microvessel and mast cell densities, and to evaluate whether tumour angiogenesis and mast cell density could predict recurrence following curative surgery in patients with colorectal carcinomas. PATIENTS: Microvessel and mast cell densities were investigated in tumour specimens from 60 patients with colorectal carcinoma. METHODS: Microvessels were stained by immunohistochemical method using a monoclonal antibody anti-CD34. The routine Giemsa blue staining method was used to assess the mast cells. Microvessels and mast cells were counted in a x400 field. RESULTS: The mean microvessel and mast cell counts were higher in patients with recurrence compared with those patients who were disease-free for at least 24 months (p<0.001). The Spearman's correlation coefficient revealed a significant correlation between mast cell and microvessel counts in colorectal carcinomas (r=0.684; p<0.001). Kaplan-Meier plots of survival showed that the high microvessel (>28) and mast cell (>6) counts correlated with a shorter disease-free survival (p=0.0003 and p=0.0013, respectively). Multivariate analysis showed that the depth of penetration (T4 versus T2) (p=0.004), liver metastasis (p=0.04) and microvessel density (p=0.003) were independent predictors of recurrence. In multivariate analysis, mast cell density did not reach significance. CONCLUSIONS: Our results suggest that the microvessel density of the primary tumour may be an important independent predictor of tumour recurrence and time to recurrence in colorectal carcinomas. The significant correlation between mast cell and microvessel counts suggest that the mast cells may have a role in tumour progression via promoting angiogenesis.

Perioperative immunonutrition ameliorates the postoperative immune depression in patients with gastrointestinal system cancer (prospective clinical study in 42 patients).

Cancer surgery is a major challenge for patients to develop immune depression in postoperative period. Several cytokines can depress immune cell subpopulations. Increased cytokine response after surgery is assumed to arise mainly from lipooxygenase pathway acting on membrane arachidonic acid. Therefore; investigators focused their efforts to alter the membrane fatty acid profile by changing the nutritional regimen with epsilon-3 fatty acid supplementation and encouraging results were obtained after surgery. Despite the theoretical and clinical advantage of enteral nutrition many surgeons remain committed to parenteral nutrition for feeding of patients due to maintain bowel rest and fear of anastomosis leakage at the postoperative period. Several studies investigating role of the postoperative immunonutrition reported that beneficial immunological changes were associated with reduction of infectious complications. Interestingly; these findings were observed at least five days after the surgery in which the highest incidence of complications was seen. In this prospective study including 42 patients eligible for curative gastric or colon cancer surgery; we investigated the beneficial effect of enteral immunonutrition (EEN) compared to total parenteral hyperalimentation (TPN) beginning from the preoperative period. Cortisol and CRP levels as stress parameters significantly increased one day after surgery in both groups but they rapidly returned to (on POD1) preoperative baseline level in EEN group whereas these values remained high in the TPN group. Additionally a significant decrease in natural killer (NK) cells and CD8+ levels were observed in both groups. However they recovered on POD3 in EEN group and on POD6 in TPN group. CD4+ subset remained almost same as preoperative value in the TPN group whereas it increased from (%) 40.14 to 46.40, 51.29 and 54.7 on PO 6th hr, POD3 and POD6 in the EEN group. Our findings suggest that preoperative nutrition via the enteral route provided better regulation of postoperative immune system restoration than parenteral nutrition. On the basis of our findings we recommend enteral immunonutrition to be started at the preoperative period rather than postoperatively before a major operation whenever the enteral route is feasible.