Clinical exome sequencing findings in 1589 patients.

Clinical exome sequencing (CES) is important for the diagnosis of Mendelian diseases, which are clinically and etiologically heterogeneous. Sharing of large amounts of CES data associated with clinical findings will increase the accuracy of variant interpretation. We performed a retrospective study to state the diagnostic yield of CES in 1589 patients with a wide phenotypic spectrum. CES was performed using the Sophia Clinical Exome Sequencing Kit with 4493 genes, followed by sequencing on a NextSeq 500 system. The diagnosis rate was 36.8% when only pathogenic and likely pathogenic variants were included. Consanguineous unions and positive family history were associated with a high diagnostic yield. The neurological disease group had the highest number of patients. The groups with high diagnosis rates were ear, eye, and muscle disease groups. Seven candidate genes (EFHC2, HSPB3, FAAH2, ITGB1, GYG2, CD177, and CSTF2T) that are not yet associated with human diseases were identified. Owing to the high diagnostic yield of CES compared with that of other genetic tests, it can be used as a standard diagnostic test in patients with rare genetic disorders that require a wide differential diagnosis, especially in laboratories with limited resources.

ATP7B Gene Variant Profile Identified by NGS in Wilson's Disease.

Background: Wilson's disease (WD) is a copper metabolism disorder caused by ATP7B gene mutations and shows an autosomal recessive pattern of inheritance. We aimed to contribute to the mutation profile of ATP7B and show demographic and phenotypic differences in this study. Materials and methods: The clinical and demographic characteristics of patients who underwent ATP7B gene sequence analysis using next-generation sequencing were evaluated to improve genotype-phenotype correlation in WD. Results: An uncertain significance (D563N) and seven likely pathogenic (Y532D, Y715Y, T977K, K1028*, E1086K, A1227Pfs*103, and E1242K) variants were identified as associated with WD. Uniparental disomy was detected in one case. Conclusion: Our work expanded the ATP7B variant spectrum and pointed to clinical heterogeneity in ATP7B variants among patients with WD. All symptomatic patients had hepatic involvement and were clinically and/or genetically diagnosed with WD in the pediatric period. T977K, A1003V, H1069Q, E1086K, and N1270S variants were associated with hepatic failure.

Clinical features of pediatric renal glucosuria cases due to SLC5A2 gene variants.

BACKGROUND: Familial renal glycosuria (FRG) is a rare renal tubular disorder characterized by a variable loss of glucose in the urine despite normal blood glucose levels, which is seen in a condition in which other tubular functions are preserved. In this study, the molecular and clinical characteristics of pediatric FRG cases due to SLC5A2 gene variants were defined. METHODS: Demographic features, diagnostic tests, and molecular analyses of patients with a diagnosis of FRG cases due to SLC5A2 gene variants were retrospectively analyzed between 2016 and 2019. RESULTS: The data of 16 patients who were clinically and genetically diagnosed with FRG in a 4-year period were analyzed. Seven (44%) of the cases were female and 9 (56%) were male. The median age at diagnosis was 6 years old (2 months old to 17 years old). Neuromotor development was found to be appropriate for the age in each case. Systemic blood pressure was evaluated as normal. A homozygous pathogenic variant in the SLC5A2 gene was detected in 14 patients in the genetic examination. A heterozygous variant was detected in one patient. In the other patient, two different heterozygous pathological variants were found in the SLC5A2 gene. CONCLUSIONS: It was revealed that growth and development were normal in children with glucosuria due to variations in the SCL5A2 gene. Renal function tests and urinary amino acid excretion were also within normal values. In our case series, the most common genetic variation in the SCL5A2 gene was the A219T (c.655G>A) variant.

Patients with cerebrotendinous xanthomatosis diagnosed with diverse multisystem involvement.

Cerebrotendinous xanthomatosis (CTX) is a lipid storage disease caused by deficiency of sterol 27-hydroxylase enzyme encoded by CYP27A1 gene. This multicenter, cross-sectional descriptive study aimed to document clinical characteristics of CTX patients of different ages, clinical presentations of early-diagnosed patients, and responses to short-term chenodeoxycholic acid (CDCA) treatment. Seven of 11 CTX patients were diagnosed in childhood. Three patients (27%) had neonatal cholestasis, seven (63%) patients had a history of frequent watery defecation started in infantile period, and eight (72.7%) patients had juvenile cataract. Four patients in the adult age group had pyramidal signs and parkinsonism symptoms. The mean Mignarri score at diagnosis was significantly lower in the pediatric patients (267.8 ± 51.4) than in the adult patients (450.0 ± 64.0, p = 0.001). No significant difference was determined between pediatric patients and adult patients regarding plasma cholestanol concentration at diagnosis (p = 0.482). The frequency of defecation decreased with treatment in six children, who had diarrhea at admission. Compared to pretreatment values, patients' body weight and standardized body mass index significantly increased at the 12th month of treatment. In conclusion, Mignarri scores are lower in the pediatric patients than in adult patients since the most determinative signs of the CTX disease are not apparent yet in the childhood. The disease is frequently overlooked in routine practice as the disease presents itself with different clinical combinations both in adults and in children. CTX is potentially a treatable disease; thereby, enhanced awareness is critically important for early diagnosis particularly in children.

Expanding the clinical spectrum of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency with Turkish cases harboring novel HMGCS2 gene mutations and literature review.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) deficiency is a very rare autosomal recessive inborn error of ketone body synthesis and presents with hypoketotic hypoglycemia, metabolic acidosis, lethargy, encephalopathy, and hepatomegaly with fatty liver precipitated by catabolic stress. We report acute presentation of two patients from unrelated two families with novel homozygous c.862C>T and c.725-2A>C mutations, respectively, in HMGCS2 gene. Affected patients had severe hypoketotic hypoglycemia, lethargy, encephalopathy, severe metabolic and lactic acidosis and hepatomegaly after infections. Surprisingly, molecular screening of the second family showed more affected patients without clinical findings. These cases expand the clinic spectrum of this extremely rare disease.

Association of GSTM1, GSTT1, GSTP1-ILE105VAL and ACE I/D polymorphisms with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease of unknown origin. The aim of this study is to clarify the relationships between susceptibility and severity of AS and GST-mu1 (GSTM1), GST-theta1 (GSTT1), GST-pi1 (GSTP1)-Ile105Val and angiotensin-converting enzyme (ACE) I/D polymorphisms in AS patients. One hundred thirty-eight AS patients and seventy-one healthy controls were enrolled in this study. Erythrocyte sedimentation rate and C-reactive protein (CRP) levels of the AS patients were recorded. The scores of the numeric rating scale (NRS) pain, the Bath Ankylosing Spondylitis Activity Index, the Bath Ankylosing Spondylitis Metrology Index and the Bath Ankylosing Spondylitis Functional Index were calculated. The genotypes distributions and allele frequencies of GSTM1, GSTT1, GSTP1-Ile105Val and ACE I/D polymorphisms were compared between patients and healthy controls. The Multiplex polymerase chain reaction (PCR) and the PCR-restriction fragment length polymorphism methods were used to detect the polymorphisms of ACE I/D, the GSTT1 and GSTM1 genes and the GSTP1-Ile105Val polymorphism, respectively. There were significantly higher levels of the GSTT1 null and the ACE II genotypes in AS patients compared to those in healthy controls (p = 0.002 and 0.005, respectively). We found significantly higher levels of CRP and the NRS pain scores in the patients with ACE ID or DD genotypes compared to those in the patients with ACE II genotypes (p = 0.005 and 0.035, respectively). The present results showed that genes involved in protection from oxidative stress and ACE gene may influence disease development and course in AS.

Molecular diagnostic results of a nephropathy gene panel in patients with suspected hereditary kidney disease.

OBJECTIVE: Clinical diagnosis of hereditary kidney disease can be difficult because of its rarity and severe phenotypic variability. Identifying mutated causative genes can provide diagnostic and prognostic information. In this study, we report the clinical application and outcome of a next-generation sequencing-based, targeted multi-gene panel test for the genetic diagnosis of patients with hereditary kidney disease. METHODS: A total of 145 patients evaluated for hereditary kidney disease who underwent a nephropathy panel with 44 different genes were retrospectively reviewed and included in the study. RESULTS: Genetic diagnosis of other hereditary kidney diseases, particularly autosomal dominant polycystic kidney disease, was made in 48% of patients. The nephropathy panel changed the preliminary diagnosis in 6% of patients. The variants in 18 (12%) patients had not been previously reported in the literature. CONCLUSION: This study demonstrates the utility of the nephropathy panel in identifying patients diagnosed with hereditary kidney disease who are referred for genetic testing. A contribution was made to the variant spectrum of genes associated with hereditary kidney disease.

NOTCH3 Variants in Patients with Suspected CADASIL.

Background: Cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL) is the most common hereditary form of cerebral small vessel disease. It is clinically, radiologically, and genetically heterogeneous and is caused by NOTCH3 mutations. Methods: In this study, we analyzed NOTCH3 in 368 patients with suspected CADASIL using next-generation sequencing. The significant variants detected were reported along with the clinical and radiological features of the patients. Results: Heterozygous NOTCH3 changes, mostly missense mutations, were detected in 44 of the 368 patients (~12%). Conclusions: In this single-center study conducted on a large patient group, 30 different variants were detected, 17 of which were novel. CADASIL, which can result in mortality, has a heterogeneous phenotype among individuals in terms of clinical, demographic, and radiological findings regardless of the NOTCH3 variant.

Recently defined epileptic encephalopathy related to WWOX gene mutation: six patients and new mutations.

Purpose: Pathogenic variants of the WWOX gene have been linked to sexual differentiation disorders, spinocerebellar ataxia, and epileptic encephalopathy (EE). We evaluated the clinical and molecular data from six newly diagnosed patients with WWOX-related EE.Methods: Clinical and molecular findings in six patients with EE were investigated, and biallelic pathogenic variants in the WWOX gene were identified. Clinical exome sequencing and Sanger sequencing were performed.Results: Three variations, as well as two novel mutations, in the WWOX gene were detected.Conclusion: Pathogenic WWOX mutations are associated with early-onset EE. Here, we report the case of six children with WWOX-related EE.

Recurrent c.776T>C mutation in CHST3 with four other novel mutations and a literature review.

The chondrodysplasia with congenital joint dislocations, CHST3 type, which was distinguished by predominantly contractures, marked vertebral changes, and normal facial appearance. Although, some clinical clues can be used for differential diagnosis, it is mostly too difficult to discriminate one type from another on basis of clinical findings only. Eight patients with multiple dislocations from five unrelated families were included in this study to elucidate molecular diagnoses. Clinical exome sequencing (CES) was performed on one patient from each family. Variable degree vertebral changes, pes equinovarus, and kyphoscoliosis accompanied multiple dislocations and short stature. In CES analyses, all mutations showed in CHST3. Previously reported c.776T>C homozygous mutations were detected in two families, compound heterozygous novel c.740G>C and c.881T>C mutations were found in one family, and homozygous novel c.564C>A and c.963G>A mutations were also determined in remaining two families, separately. Biallelic CHST3 c.776T>C mutations are most frequent mutation in CHST3 and have been reported predominantly in Turkish patients which may be remarkable for genotype-ethnicity correlation in chondrodysplasia with congenital joint dislocations, CHST3 type. It is suggested that c.776T>C mutation can be accepted as a recurrent mutation in CHST3 for Turkish patients who are suspected of having chondrodysplasia with congenital joint dislocations, CHST3 type.

Associations analysis of GSTM1, T1 and P1 Ile105Val polymorphisms with carpal tunnel syndrome.

Oxidative stress was related with carpal tunnel syndrome (CTS). We aimed to clarify the associations between glutathione S-transferase (GST)M1, GSTT1 and GSTP1-Ile105Val polymorphisms and CTS. One hundred-forty patients with CTS and 97 healthy controls were enrolled in this study. Tinel and Phalen signs were noted as positive or negative. Functional and clinical status of patients was evaluated by the Boston Questionnaire. The intensity of hand and/or wrist pain was evaluated on 10 cm visual analog scale (VAS). We applied the polymerase chain reaction (PCR) to determine the polymorphisms of the GSTM1 and GSTT1 and the PCR-restriction fragment length polymorphism method for detecting the GSTP1-Ile105Val polymorphism. The M1 null genotype was significantly higher in patients with CTS compared to healthy controls, and the M1 null genotype seemed to increase the risk of CTS approximately two-fold (P = 0.011; odds ratio (OR) = 1.98; 95 % confidence interval (CI) 1.17-3.36). The M1 null, T1 present combined genotype was significantly higher in patients with CTS compared to healthy controls (P = 0.043); however, it seemed not to increase the risk of CTS (P = 0.14; OR = 0.62; 95 % CI 0.33-1.76). We found significantly higher levels of the VAS, Boston Symptom Severity Scale and Phalen sign in patients with the Ile/Val or the Val/Val genotypes compared to those in patients with the Ile/Ile genotype (P = 0.003, 0.004 and 0.044, respectively). We proposed that genes involved in the protection from oxidative stress may influence the susceptibility, clinical and functional status of CTS. The GSTM1 null genotype may be related with the development of CTS, whereas the Val allele of GSTP1-Ile105Val polymorphism may be associated with worse functional and clinical status in CTS.

Glutathione S-transferase T1, M1 and P1 Genetic Polymorphisms and Susceptibility to Colorectal Cancer in Turkey.

Colorectal cancer (CRC) is reported to be the third most common cancer worldwide and the fourth most common cause of cancer related deaths. CRC is considered to be a multifactorial disease whose risk varies due to the complex interaction between individual genetic basis and exposure to multiple endogenous factors. Glutathione S-transferases are pro-carcinogenic in CRC and are required for the conjugation between chemotherapeutics and broad spectrum xenobiotics. One hundred and eleven patients with CRC and 128 control subjects without any cancer history were enrolled in this study. Multiplex PCR was applied to determine polymorphisms for the GSTT1 and M1 genes, and PCR-RFLP was applied for the GSTP1 (Ile105Val) gene polymorphism. Values <0.05 were defined as statistically significant. We detected a significant high correlation between predisposition for CRC and presence of the Ile/Ile genotype of the GSTP1 (IIe105Val) gene polymorphism, but we did not find a significant relationship between predisposition for CRC and GSTT1 and M1 deletion polymorphisms. In addition, we did not determine a relationship between GSTT1, M1 and P1 gene polymorphisms and any clinicopathological features of CRC. GSTT1 null/GSTM1 positive and GSTT1 null/GSTM1 positive/GSTP1 Ile/ Ile genotypes were significantly higher in the patient group. Our results revealed that there is no relationship among CRC, its clinicopathologic features, and GSTT1 M1 gene polymorphisms. However, there was a significant correlation between CRC and the GSTP1 Ile/Ile genotype. Further studies with larger patient groups are required to delineate the relationships between GST gene polymorphisms and the clinicopathologic features of CRC in Turkey.

Spectrum of EGFR gene mutations and ALK rearrangements in lung cancer patients in Turkey.

The EGFR gene and ALK rearrangements are two genetic drivers of non-small cell lung cancer (NSCLC). The frequency of EGFR mutations and ALK rearrangement varies according to not only ethnicity but also gender, smoking status and the histological type of NSCLC. In the present study, we demonstrated the distribution of EGFR mutations in 132 NSCLC patients by using a pyrosequencing technique and the distribution of ALK rearrangements in 51 NSCLC patients by using fluorescent in situ hybridization technique in Turkey. Additionally, we compared the clinicopathological data of NSCLC patients with the mutation status of EGFR in their cancerous tissues. Both EGFR mutations and ALK rearrangements were identified in 19 (14.39 %) and 1 (1.96 %) patients, respectively. We found EGFR mutations in codon 861, 719 and 858 with the ratios of 10.52 % (2/19), 10.52 % (2/19) and 31.58 % (6/19), respectively, and deletion of exon 19 in 47.37 % (9/19) of the patients. We found the frequency of EGFR mutations to be significantly higher in female patients and nonsmokers (p = 0.043, p = 0.027, respectively). Consequently, we found EGFR mutations to be more frequent in female patients and nonsmokers. Future studies on larger patient groups would provide more accurate data to exhibit the relationship between EGFR mutations and ALK rearrangements and the clinicopathological status.

Association of the ACE I/D gene polymorphisms with JAK2V617F-positive polycythemia vera and essential thrombocythemia.

The renin-angiotensin system contributes to cell growth, proliferation, and differentiation in the bone marrow. We investigated the role of the ACE I/D gene polymorphism in 108 polycythemia vera (PV) and essential thrombocytosis (ET) patients who were positive for the JAK2V617F mutation, with a thrombosis group (TG) of 95 patients who had a history of vascular events, but did not have a history of myeloproliferative neoplasms and compared these to a healthy control group (CG) of 72 subjects. In the patients, II genotype and I allele frequency (p=0.009, odds ratio [OR]=9.716, 95% confidence interval [CI]=1.242-76.00, p=0.004, OR=2.019, 95% CI=1.243-3.280, respectively) were found to be higher than those in the controls. The DD genotype (p=0.021, OR=0.491, 95% CI=0.268-0.899) and D allele (p=0.004, OR=0.495, 95% CI=0.305-0.805) were found to be correlated with a decreased risk of a myeloproliferative neoplasm. These findings support the hypothesis that the ACE II genotype and I allele may be related to increased risk of ET and PV. Conversely, the DD genotype and D allele may be related to decreased risk of ET and PV. The results also indicated that the ACE I/D gene polymorphism was independent of thrombosis formation.

Association and Prognostic Significance of the Functional -1562C/T Polymorphism in the Promoter Region of MMP-9 in Turkish Patients with Gastric Cancer.

Matrix metalloproteinases (MMPs) are a group of zinc-dependent peptidases that participate in matrix turnover in solid malignancies. The aim of this study was twofold. First, we sought to investigate under a case-control design the association between the functional -1562C/T polymorphism in the promoter region of MMP-9 and gastric cancer (GC) in a Turkish sample. Second, we examined its prognostic significance in GC patients. A total of 144 subjects were enrolled in the case-control study (79 GC cases and 65 controls). Overall survival (OS) and progression-free survival (PFS) served as the main outcome measures in the longitudinal study. The MMP-9 -1562C/T polymorphism was genotyped using a polymerase chain reaction-restriction fragment length polymorphism method. The odds ratio (OR) of GC for the CC genotype relative to the CT+TT genotypes was not significant (OR = 0.89, 95 % confidence interval [CI] = 0.44-1.82, P = 0.75). These results did not change after allowance for age and sex in multivariable regression analysis (OR = 0.81, 95 % CI = 0.40-1.94, P = 0.84). When the MMP-9 -1562C/T polymorphism was analyzed among GC patients in relation to OS and PFS, we found no significant differences between subjects with the CC and CT+TT genotypes. In conclusion, the results of our study did not point toward a major role of the MMP-9 -1562C/T polymorphism in the pathogenesis and clinical course of GC in Turkish subjects.

MMP2 gene-735 C/T and MMP9 gene -1562 C/T polymorphisms in JAK2V617F positive myeloproliferative disorders.

BACKGROUND: Myeloproliferative disorders (MPDs) are clonal hematologic malignancies originating at the level of the pluripotent hematopoietic stem cell. Matrix metalloproteases (MMPs) are proteolytic enzymes that contribute to all stages of malignancy progression. Genetic variants in the MMP genes may influence the biological function of these enzymes and change their role in carcinogenesis and progression. To our knowledge, this is the first investigation of associations between the -735 C/T and -1562 C/T polymorphisms in the MMP2 and MMP9 genes, respectively, and the risk of essential thrombocytosis (ET), and polycythemia vera (PV). MATERIALS AND METHODS: The case-control study included JAK2V617F mutation positive 102 ET and PV patients and 111 controls. Polymorphisms were determined by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and electrophoresis. RESULTS: No statistically significant differences were detected between patient (ET+PV) and control groups regarding genotype distribution for MMP2 gene-735 C/T and MMP9 gene -1562 C/T polymorphisms and C/T allele frequency (p>0.050). Statistically borderline significance was observed between PV and control groups regarding genotype distribution for the MMP9 gene -1562 C/T polymorphism (p=0.050, OR=2.26, 95%Cl=0.99-5.16). CONCLUSIONS: Consequently this study supported that CC genotype of MMP9 gene -1562 C/T polymorphism may be related with PV even if with borderline significance.

Mesenchymal stem cell: does it work in an experimental model with acute respiratory distress syndrome?

We hypothesized that bone marrow-derived mesenchymal stem cells (BM-MSCs) would have a possible role in the treatment of acute respiratory distress syndrome (ARDS). ARDS disease model was developed in Wistar albino male rats by intratracheal instillation of physiological saline solution. Anesthezied and tracheotomized rats (n = 8) with ARDS were pressure-controlled ventilated. Isolated and characterized rat (r-) BM-MSCs were labeled with GFP gene, and introduced in the lungs of the ARDS rat-model. After applying of MSCs, the life span of each rat was recorded. When rats died, their lung tissues were removed for histopathological examination. Also the tissue sections were analyzed for GFP labeled rBM-MSCs and stained for vimentin, CK19, proinflammatory (MPO, IL-1ß, IL-6 and MIP-2) and anti-inflammatory [IL-1ra and prostaglandin E2 receptor (EP3)] cytokines. The histopathological signs of rat-model ARDS were similar to the acute phase of ARDS in humans. rBM-MSCs were observed to home in lung paranchyma. Although the infiltration of neutrophils slightly decreased in the interalveolar, peribronchial and perivascular area, a notable improvement was determined in the degree of hemorrhage, edema and hyaline membrane formation in rats treated with rBM-MSCs. Also decreased proinflammatory cytokines levels and increased the intensity of anti-inflammatory cytokines were established. Therefore MSCs could promote alveolar epithelial repair by mediating of cytokines from a proinflammatory to an anti-inflammatory response. As a novel therapeutic approach, mesenchymal stem cell treatment with intratracheal injection could be helpful in the management of critically ill patients with ARDS.