Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

Clinical Heterogeneity in Patients with Long QT Syndrome and Segregation of Single Nucleotide Variants and Clinical Symptoms in 17 Affected Families.

Introduction: Long QT syndrome (LQTS) is a disorder of ventricular myocardial repolarization characterized by a prolonged QT interval on the electrocardiogram. It increases the risk of ventricular arrhythmias, which can cause syncope or sudden cardiac death. In this study, we study the genotype-phenotype relationships of patients referred to us with suspected arrhythmia syndrome. Methods: Seventeen cases and their twenty relatives were evaluated. Next-generation sequencing analysis was performed for 17 LQTS-related genes. Results: We detected seventeen single nucleotide variants (SNVs) with potential pathogenic significance in 26 of the 36 subjects analyzed. KCNH2 c.172G>A, KCNQ1 c.1768G>A, ANK2 c.4666A>T, c.1484_1485delCT, KCNH2 c.1888G>A were reported as pathogenic or likely pathogenic in HGMD variant classification database. Conclusion: Current study pointed out that early diagnosis can be life-saving for patients and their families by taking family history and detailed examination. Also, we highlight the clinical heterogeneity of arrhythmia syndrome through a patient with a dual phenotype.

Genetic evaluation of 50 Turkish patients with neurofibromatosis type 1: 2 years experience of a single center.

BACKGROUND/AIM: Neurofibromatosis type 1 is an autosomal dominant neurocutaneous disorder. Clinical diagnosis is difficult in early childhood, and it is possible to miss a critical interval for tumour screening. In this study, we aimed to characterize the mutational spectrum of Turkish patients and discuss the benefits of molecular testing. MATERIAL AND METHODS: Fifty individuals from 35 unrelated families were included. Main referral reasons for genetic testing were as follows: to confirm a clinical diagnosis, to use in differential diagnosis and to evaluate first-degree family member of a known patient. Two-step process consisting of initial next generation sequencing of the NF1 gene and consequent multiplex ligation-dependent probe amplification were performed. RESULTS: We identified a total of 30 variants in 28 individuals. Variant detection rate was 56% in the entire study group and 71.4% within the index patients. Four novel variants were found. Truncating variants constituted 60% of the entire mutation spectrum. A deletion or duplication was not detected. The most common feature was cafe au lait macules in 70% of the patients, followed by focal areas of signal intensity on brain imaging (26%), cutaneous neurofibromas (24%) and axillary freckling (24%). CONCLUSIONS: Early sequencing in all suspected patients followed by deletion/duplication analysis in patients meeting clinical criteria and a case-to-case based consideration for RNA studies seems to be the effective algorithm for NF-1 diagnosis.

Investigation of different genomic variants in familial Mediterranean fever cases with monoallelic MEFV mutation.

OBJECTIVES: Familial Mediterranean Fever (FMF) is an inflammatory disease characterised by periodic fever and concurrent episodes of serous membrane inflammation. FMF is considered to be inherited in autosomal recessive manner and biallelic mutations in the MEFV gene are associated with the disease. However, approximately 20-25% of patients only have a single mutation in MEFV gene, which creates confusion in differential diagnosis of many patients. This study aimed to reveal rare variants that may act in conjunction with the single pathogenic MEFV variant in the pathogenesis of FMF. METHODS: We performed whole exome sequencing in 17 individuals from 5 different families who were diagnosed according to the clinical criteria, responded positively to colchicine treatment, but had no biallelic MEFV mutation. RESULTS: A disease-causing variant or a common affected cellular pathway that was shared in all index cases was not detected. When cases were examined individually, two de novo variants were identified in the BIRC2 and BCL10 genes, both of which play a role in inflammatory pathways. Functional studies are needed to confirm the physiopathological relationship of these genes with FMF. CONCLUSIONS: This study is one of the most extensive aetiological researches in FMF cases with monoallelic MEFV mutation. We have shown that genotype-phenotype correlation in these cases may not be established by rare genetic variants and discussed underlying causes. Clinical criteria with emphasis on colchicine response and family history should be the main tool and genetic results should only be used for support in FMF diagnosis.

Spondyloepimetaphyseal dysplasia-Maroteaux type due to dominant TRPV4 mutation: expanding the phenotype with a case report.

INTRODUCTION: Dominant pathogenic mutations in the TRPV4 gene give rise to a wide spectrum of abnormal phenotypes, including bone dysplasia as well as spinal muscular atrophy and hereditary motor and sensory neuropathy. Spondyloepimetaphyseal dysplasias (SEMDs) are autosomal dominant skeletal dysplasias characterized by mild epiphyseal dysplasia, flared metaphyses, prominent joints, spondyler dysplasia, and brachydactyly with various carpal, metacarpal, and finger malformations. CASE PRESENTATION: We present a boy who has the clinical and radiological signs of SEMD-M with a dominant TRPV4 mutation. He also has some striking findings that have not been seen in these patients before, and they may be able to provide assistance to medical professionals in the process of diagnosis.These include a shorter distance between his lumbar vertebrae, congenital contractures, and an arachnoid cyst.

Prenatal diagnosis of cystic hygroma cases in a tertiary centre and retrospective analysis of pregnancy results.

The aim of this study is to retrospectively examine invasive diagnostic methods, structural anomalies accompanying cystic hygroma, and pregnancy outcomes in cystic hygroma cases admitted to a tertiary centre. The population of the study consisted of 29 live foetuses with cystic hygroma in the foetal neck only in the first or second trimester. In the study, pregnant women who applied to our centre were included. Amniocentesis or chorionic villus sampling was performed for genetic analysis according to the weeks of the pregnant women who were diagnosed with cystic hygroma by ultrasound examination by two clinicians experienced in foetal anomaly. Of the pregnant women included in the study, 10 had normal karyotype, 12 had abnormal karyotype and 13 had structural abnormality. It is very important to provide genetic counselling to the families of foetuses with cystic hygroma with a multidisciplinary team approach consisting of neonatologists, paediatric surgeons and experienced sonographers. Implications for rehabilitationWhat is already known on this subject? Cystic hygroma, also known as cystic lymphangioma, is a congenital cystic malformation often seen in the first trimester, which occurs in the foetal neck due to the failure of the connections between the cervical lymphatic vessels and the jugular venous system to develop normally. Cystic hygroma may be isolated, but highly associated with foetal aneuploidy, hydrops fetalis, abnormal foetal nuchal translucency.What do the results of this study add? Invasive prenatal diagnostic tests (CVS or amniocentesis) should be performed in all patients with cystic hygroma, as cystic hygromas can be diagnosed by first trimester foetal genetic sonogram screening and are largely accompanied by chromosomal abnormalities.What are the implications of these findings for clinical practice and/or further research? In foetuses with cystic hygroma, foetal karyotyping, detailed sonography and their documentation, genetic counselling is important to families of cystic hygroma foetuses with a multidisciplinary team approach consisting of neonatologists, paediatric surgeons and maternal foetal medicine specialists, since there is a high risk for aneuploidy and foetal malformation.

Evaluation of hereditary/familial breast cancer patients with multigene targeted next generation sequencing panel and MLPA analysis in Turkey.

Breast cancer, a worldwide leading cause of cancer in women, may occur in familial cases. Germline mutations in BRCA1/2 genes are responsible for 15% of the familial cases. With the power of next generation sequencing (NGS) analysis, it is possible to analyze genes related to hereditary susceptibility to breast cancer and investigate the genetic etiology more thoroughly. In this study, we investigated 30 genes identified frequent pathogenic alleles in Turkish population. The study includes 495 unrelated individuals diagnosed with breast cancer who are selected for genetic testing according to NCCN criteria for hereditary breast cancer. All patients were analyzed by NGS for BRCA1/2 genes. Deletion/duplication investigation by Multiplex ligation-dependent probe amplification (MLPA) and massive sequencing of 30 breast cancer-related genes (Oncorisk Gene Panel) were performed in a stepwise manner. BRCA1/2 variants are the most frequent pathogenic variants which are found in 45 of 495 (9.1%) patients. Four previously unreported, novel, pathogenic variants of BRCA2 gene are identified. In four cases, exonic deletions of BRCA1/2 genes are determined and there is no duplication of these genes. NGS panel investigation involving other moderate-high risk genes contributed genetic diagnosis in an extra 39 out of 419 (9.3%) cases. Our study presents the cost effectiveness of the gene panel approach. We suggest that gene panels should be the first-tier genetic testing for hereditary breast cancer and MLPA analysis of BRCA1/2 genes should be investigated as a complementary method of NGS analysis.

Biallelic ZNF335 mutations cause basal ganglia abnormality with progressive cerebral/cerebellar atrophy.

To date, less than 10 pedigrees have been reported with ZNF335 mutations since it was discovered in 2012 and little is known about ZNF335-related clinical spectrum. We describe a 12 years old male patient who is only child of nonconsanguineous Turkish parents. Trio whole genome sequencing identified previously unreported compound heterozygous variants in ZNF335, namely, c.3889T > A p.(Ser1297Thr) and c.758G > A p.(Arg253Gln) where transmitted by his father and mother, respectively. Patient' magnetic resonance imaging findings were overlapping to those observed in the previous cases with ZNF335 mutations. Here we report the oldest patient with biallelic ZNF335 mutations. We recommend screening for ZNF335 defects in patients with basal ganglia anomaly, secondary white matter abnormalities and microcephaly.

A novel compound heterozygous variant in CYP19A1 resulting in aromatase deficiency with normal ovarian tissue.

BACKGROUND: Aromatase deficiency leading to virilization in mother and female fetuses during pregnancy is a rare disease. It is characterized by impaired estrogen production, increased gonadotropins, and ovarian cysts. CASE: Herein, we report a clinical phenotype of the virilized female due to a novel compound heterozygous variant in CYP19A1 [IVS10 + 1 G > A; c.344 G > A (p.R115Q)], with normal gonadotropin levels at the time of admission and histologically normal ovarian tissues. CONCLUSION: Aromatase deficiency should also be considered even if the initial follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels are normal, and ovarian cysts are lacking.

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.

Phenotypic spectrum of CHARGE syndrome based on clinical characteristics

Background/aim: CHARGE syndrome is a rare autosomal dominant disease with multiple congenital anomalies and cognitive impairment, which is caused by mutations in the CHD7 gene. This study aimed to disclose the mild end of the phenotypic spectrum of CHARGE syndrome, which has a highly variable expressivity. Materials and methods: Twenty-one patients who had at least one of the major symptoms of CHARGE syndrome (coloboma, choanal atresia, characteristic ear anomalies, semicircular canal hypoplasia, and cranial nerve anomalies) were included in the study. All patients were tested for karyotype analysis and CHD7 gene mutation/deletion. Results: In the study population, 6 different mutations were detected in 5 patients, and 2 different polymorphisms were detected in the CHD7 gene in 3 patients. MLPA analysis of all coding exons of the CHD7 gene revealed no pathogenic deletion/duplication. Conclusion: CHARGE syndrome should be considered as a differential diagnosis to detect the mild end of the spectrum, even if the patient does not fit the criteria.

Localization of the Werner Protein Together with H2AX in γ-Irradiation-Induced Neoplastic Transformed Human Mesenchymal Stem Cells.

The H2A histone family, member X (H2AX), and Werner (WRN) are important proteins for genome and telomere maintenance. WRN has a major role in genome stability, particularly during DNA replication, transcription, recombination, and repair of DNA double-stranded breaks (DSBs) via base excision repair, homologous recombination, or nonhomologous end joining. H2AX plays a part in the rapid, sensitive, cellular response to the ionizing radiation or DNA-damaging chemotherapeutic agents that cause DSBs. This occurs when radiation-induced DSBs trigger the activation of H2AX and begin the damage-repair process. In this study, we investigate the role and localization of WRN together with DNA damage marker H2AX at the radiation-induced damaged sides of both the telomere-immortalized human mesenchymal stem cells (hMSCs) and hMSC-telomere 1 (hMSC-telo1) and in control primary hMSCs. Phosphorylated H2AX and WRN immune staining enabled evaluation of overall genomic integrity and damage/repair. We used peptide nucleic acid-fluorescent in situ hybridization to visualize telomeric damage as a short-term effect. A high-level WRN signal was observed in both primary hMSCs and telomerase-immortalized hMSCs after the cells had been subjected to infrared radiation. Afterward, the irradiation level of the WRN signals decreased considerably, especially in later passages, and WRN was nondetectable in the latest passages of the hMSC Telo1 cells. Contrary to this finding, we found that levels of H2AX phosphorylation in hMSC-telo1 cells increased with time, especially at telomere sides, suggesting that cells with long telomeres and high telomerase activity have the advantage of maintaining genomic integrity. Evaluation of localization of WRN signals demonstrated that WRN does not leave the nucleolus after irradiation. We did not detect the WRN signal at the telomere sides, but we could detect H2AX at the telomeric sides. Thus, our overall data suggest that the WRN protein is not involved in irradiationinduced DNA damage/repair, even at telomeric sides in hMSC and hMSC-telo1.

Two Cases with Ring Chromosome 13 at either End of the Phenotypic Spectrum.

Ring chromosome 13 is a rare genetic condition with an incidence of 1/58,000 in live births. Major clinical features of patients with ring chromosome 13 include growth and developmental retardation, microcephaly, facial dysmorphism, ambiguous genitalia, anal atresia, eye malformations, retinoblastoma, and hand, foot, and toe abnormalities. The severity of the phenotype depends on the amount of genetic material lost during ring chromosome formation. Here, we report 2 cases with ring chromosome 13 at either end of the phenotypic spectrum.

Assessment of sleep problems in children with familial Mediterranean fever.

AIMS: This study aimed to investigate sleep patterns, sleep disturbances and possible factors that are associated with sleep disturbances among children with familial Mediterranean fever (FMF). PATIENTS AND METHODS: Fifty-one patients with FMF and 84 age- and sex-matched healthy controls were enrolled in the study. The patients who had an attack during the last 2 weeks were not included. Demographic data, FMF symptoms, disease duration, dose of colchicine, disease severity score, number of attacks in the last year, MEFV mutation and serum C-reactive protein (CRP) levels were recorded for each patient. A Children's Sleep Habits Questionnaire was performed. RESULTS: The total sleep scores of the patients with FMF were significantly higher than the control group. Total sleep durations were similar between FMF patients and controls. Children with FMF had significantly higher scores regarding sleep-onset delay, sleep anxiety, night wakings and sleep-disordered breathing when compared to healthy controls. There was a significant positive correlation between number of attacks in the last year and sleep onset delay, night wakings and sleep disordered-breathing. Disease severity score and CRP levels were not associated with any of the subscale scores. The patients with exertional leg pain had significantly higher total sleep scores than the ones without. Furthermore, patients with exertional leg pain had significantly higher subscale scores regarding sleep onset delay, parasomnias and sleep-disordered breathing. CONCLUSION: This study showed for the first time that children with FMF had more sleep disturbances than their healthy peers. Higher numbers of attacks and exertional leg pain were associated with poor sleep quality. In conclusion, this study underlines the need to assess and manage sleep problems in children with FMF.

Analysis of first-trimester combined test results in preparation for a cell-free fetal DNA era.

OBJECTIVE: To survey experience with the first-trimester combined test (FCT) for trisomy 21 (T21) in different risk score groups to determine the most useful clinical application of cell-free fetal DNA (cffDNA) screening. METHODS: In a retrospective study, the records of FCT results obtained at a center in Turkey between January 2009 and January 2014 were reviewed. The FCT results and rates of uptake of invasive diagnostic testing were compared among different risk score groups. RESULTS: FCT results were available for 4804 pregnancies; 276 (5.7%) had IDT results. Ten (72.7%) of 11 cases of T21 had a risk score of 1:300 or more. The IDT uptake rates were 54.5%, 51.9%, and 47.4% at risk scores of 1:100 or more, 1:200 or more, and 1:300 or more, respectively. In the group at intermediate risk (1:1001-1:3000), no pregnancy had an FCT result of both low pregnancy-associated plasma protein A and high free ß-human chorionic gonadotropin, but 30 (3.9%) of 766 pregnancies had both advanced maternal age and high ß-human chorionic gonadotropin. CONCLUSION: cffDNA screening should be used to optimize IDT uptake in pregnancies with a risk score of 1:101-1:1000. The selective power of the FCT diminishes beyond the 1:1001 score and cffDNA screening cannot yet be recommended routinely.

Identification of a novel frameshift heterozygous deletion in exon 8 of the PAX6 gene in a pedigree with aniridia.

Aniridia is a congenital, panocular abnormality which is characterized by partial or complete absence of iris and various degrees of iris hypoplasia. Mutations in the PAX6 gene are found in ~90% of cases with aniridia. The human PAX6 gene is located at chromosome 11p13 and encodes a transcriptional regulator that has crucial roles in the development of the eyes, central nervous system and pancreatic islets. The present study performed a clinical and genomic analysis of two families containing multiple cases of aniridia. All exons of the PAX6 gene of the probands were sequenced using the Sanger sequencing technique. A heterozygous non­stop mutation in exon 14 was identified in the first family, which has been previously reported for a different ophthalmological pathology. This mutation causes on­going translation of the mRNA into the 3'­untranslated region. In the second family, a novel frameshift heterozygous deletion in exon 8 was identified.

Termination of pregnancy for fetal abnormalities: main arguments and a decision-tree model.

INTRODUCTION: By looking through our ethical committee cases, we demonstrate the main arguments we use for making a judgment in face of fetal abnormalities. Our decision making model is a simplified algorithm of the arguments and concepts we use in scientific-ethic discussion. MATERIALS AND METHODS: A retrospective analysis was conducted from single, tertiary referral center of patients evaluated for fetal abnormalities from 2004 to 2014. We hypothesized that all our judgments would fit into a decision-tree model. RESULTS: 553 fetal abnormality cases were discussed, 348 (63%) were given termination of pregnancy (TOP) proposal. When detected <24 weeks, fetuses with chromosomal abnormality/genetic disorders (n:100) and with mental retardation risk (n:93) ended up with TOP proposal. For incompatibility with life cases (n:111) and the multimorbidity cases (n:44) the committee suggest TOP, regardless of gestational age. The highest family approval ratios were in chromosomal abnormalities/genetic disorders group (93%), and the lowest figures were in mental retardation risk group (80%). DISCUSSION: Continuously changing literature on prenatal and postnatal therapy options and the long term outcome of various fetal abnormalities influence committee decisions. Theoretical high success rates and inconsistent data on long term prognosis of some anomaly groups resulted in heterogenous decisions and various approval ratios.

Risk factors for subclinical inflammation in children with Familial Mediterranean fever.

Familial Mediterranean fever (FMF) is the most common autosomal recessive inherited inflammatory disease characterized by attacks of painful inflammation. Some patients with FMF have subclinical inflammation persisting between the attacks. We aimed to identify the demographic, clinical and genetic risk factors for subclinical inflammation in children with FMF. The medical records of the children with FMF were evaluated retrospectively for acute-phase response along with gender, age at the onset of symptoms and at the time of diagnosis, clinical signs and symptoms, the presence of amyloidosis and MEFV genotype. Patients with persistently elevated acute-phase response between the attacks were considered to have subclinical inflammation. Patients with or without subclinical inflammation (Group 1 and Group 2, respectively) were compared for the parameters defined above. Independent risk factors for subclinical inflammation were identified by multivariate logistic regression analysis. There were 105 children (male/female: 52/53) who were compliant on colchicine treatment. Subclinical inflammation was detected in 22 (20 %) patients. Group 1 had significantly higher rate of myalgia, arthritis/arthralgia, erysipelas like erythema, amyloidosis, protracted febrile myalgia and M694V mutation compared with Group 2. However, only the presence of myalgia and erysipelas like erythema were found to be independent risk factors for subclinical inflammation (OR 9.8 and 5.9, respectively). Children with FMF who have myalgia and erysipelas like erythema during the attacks are particularly at risk of ongoing inflammation and should be closely monitored for subclinical inflammation even during attack-free periods.

A novel mutation in the mitochondrial DNA cytochrome b gene (MTCYB) in a patient with Prader Willi syndrome.

In recent years, it has been suggested that defects in energy metabolism may accompany Prader Willi syndrome. Mutations in the mitochondrial cytochrome b gene have been commonly associated isolated mitochondrial myopathy and exercise intolerance, rarely with multisystem disorders. The authors describe a novel mutation (mt. 15209T>C) in mitochondrial cytochrome b gene in a 2-year-old girl with Prader-Willi syndrome with a clinical history of lactic acidosis attacks, renal sodium loss, hepatopathy, progressive cerebral atrophy, and sudden death. The authors suggest that atypical clinical findings in patients with Prader-Willi syndrome should direct the physician to search for a mitochondrial disease.