Identification of the molecular etiology in rare congenital hemolytic anemias using next-generation sequencing with exome-based copy number variant analysis.

OBJECTIVES: In congenital hemolytic anemias (CHA), it is not always possible to determine the specific diagnosis by evaluating clinical findings and conventional laboratory tests. The aim of this study is to evaluate the utility of next-generation sequencing (NGS) and clinical-exome-based copy number variant (CNV) analysis in patients with CHA. METHODS: One hundred and forty-three CHA cases from 115 unrelated families referred for molecular analysis were enrolled in the study. Molecular analysis was performed using two different clinical exome panels in 130 patients, and whole-exome sequencing in nine patients. Exome-based CNV calling was incorporated into the traditional single-nucleotide variant and small insertion/deletion analysis pipeline for NGS data in 92 cases. In four patients from the same family, the PK Gypsy variant was investigated using long-range polymerase chain reaction. RESULTS: Molecular diagnosis was established in 86% of the study group. The most frequently mutated genes were SPTB (31.7%) and PKLR (28.5%). CNV analysis of 92 cases revealed that three patients had different sizes of large deletions in the SPTB and six patients had a deletion in the PKLR. CONCLUSIONS: In this study, NGS provided a high molecular diagnostic rate in cases with rare CHA. Analysis of the CNVs contributed to the diagnostic success.

The genetic spectrum of polycystic kidney disease in children.

OBJECTIVE: Autosomal dominant polycystic kidney disease is an inherited kidney disorder with mutations in polycystin-1 or polycystin-2. Autosomal recessive polycystic kidney disease is a severe form of polycystic kidney disease that is characterized by enlarged kidneys and congenital hepatic fibrosis. Mutations at PKHD1 are responsible for all typical forms of autosomal recessive polycystic kidney disease. METHODS: We evaluated the children diagnosed with polycystic kidney disease between October 2020 and May 2022. The diagnosis was established by family history, ultrasound findings, and/or genetic analysis. The demographic, clinical, and laboratory findings were evaluated retrospectively. RESULTS: There were 28 children (male/female: 11:17) evaluated in this study. Genetic analysis was performed in all patients (polycystin-1 variants in 13, polycystin-2 variants in 7, and no variants in 8 patients). A total of 18 variants in polycystin-1 and polycystin-2 were identified and 9 (50%) of them were not reported before. A total of eight novel variants were identified as definite pathogenic or likely pathogenic mutations. There was no variant detected in the PKDH1 gene. CONCLUSION: Our results highlighted molecular features of Turkish children with polycystic kidney disease and demonstrated novel variations that can be utilized in clinical diagnosis and prognosis.

The New Youngest Case of Grange Syndrome with a Novel Biallelic Pathogenic Variant in YY1AP1.

Introduction: Grange syndrome (OMIM 602531) is characterized by a constellation of symptoms of hypertension, stenosis, or occlusion of different arteries (including the cerebral, renal, abdominal, and coronary vessels) with a variable occurrence of brachysyndactyly, bone fragility, and congenital heart defects. Learning disabilities were also reported in some cases. Biallelic pathogenic variants in YY1AP1 are associated with the syndrome. Only 14 individuals with this ultra-rare syndrome (12 of them were molecularly confirmed) have hitherto been reported in the literature. Case Presentation: We herein describe a 11/2-year-old additional female case of Grange syndrome with hypertension, patent ductus arteriosus, and brachysyndactyly who was subsequently confirmed to carry a novel homozygous frameshift variant (c.2291del; p.Pro764Leufs*12) in the YY1AP1 gene through whole-exome sequencing. Conclusion: This report extends the allelic spectrum in Grange syndrome and helps provide insight into the potential role of YY1AP1 in the regulation of cellular processes.

The genetic spectrum of polycystic kidney disease in children

SUMMARY OBJECTIVE: Autosomal dominant polycystic kidney disease is an inherited kidney disorder with mutations in polycystin-1 or polycystin-2. Autosomal recessive polycystic kidney disease is a severe form of polycystic kidney disease that is characterized by enlarged kidneys and congenital hepatic fibrosis. Mutations at PKHD1 are responsible for all typical forms of autosomal recessive polycystic kidney disease. METHODS: We evaluated the children diagnosed with polycystic kidney disease between October 2020 and May 2022. The diagnosis was established by family history, ultrasound findings, and/or genetic analysis. The demographic, clinical, and laboratory findings were evaluated retrospectively. RESULTS: There were 28 children (male/female: 11:17) evaluated in this study. Genetic analysis was performed in all patients (polycystin-1 variants in 13, polycystin-2 variants in 7, and no variants in 8 patients). A total of 18 variants in polycystin-1 and polycystin-2 were identified and 9 (50%) of them were not reported before. A total of eight novel variants were identified as definite pathogenic or likely pathogenic mutations. There was no variant detected in the PKDH1 gene. CONCLUSION: Our results highlighted molecular features of Turkish children with polycystic kidney disease and demonstrated novel variations that can be utilized in clinical diagnosis and prognosis.

Copy number variations in patients with idiopathic recurrent pregnancy loss: an arrayCGH approach.

BACKGROUND: It is not always possible to determine the causative basis of pregnancy losses and even today it has been reported that 50% of cases with recurrent pregnancy loss (RPL) have no reason to be detected. In our study, it is aimed to reveal the copy number variations (CNVs) of the genes which presumably have a potential effect in individuals with RPL and contribute to subsequent functional studies in the follow-up. METHODS: We retrospectively evaluated the array-comparative genomic hybridization (aCGH) data of cytogenetically 64 normal individuals (21 couples, 11 unrelated women, and 11 unrelated men) who had applied to our outpatient clinic from January 2016 to December 2017, for the history of idiopathic two or more RPL. RESULTS: A total of 83 CNVs were detected in 56 different chromosomal regions [36% (20/56) is deletion and 64% (36/56) is duplication] in 40/64 (62.5%) of the cases. Two detected deleterious CNVs encompassing 1p36.22-p36.21 and 10q11.22 chromosomal locus have been reported as pathogenic according to the Database of Genomic Variants (DGV). DISCUSSION: CNVs that may play a role in the genetic etiology of idiopathic RPL were revealed in our study and potential chromosomal loci were introduced to the literature for further analysis. The detection of CNVs and their association with reproduction such as RPL, infertility, and even other diseases will allow us to have more information about the clinical consequences and will make it possible to provide more accurate and comprehensive genetic counseling.

A New Mutation, Hb A2-Canakkale [δ10(A7)Ala→Val; HBD: c.32C>T], and Other Well-Known δ Variants Identified in a Selected Cohort with Low Hb A2 Levels.

Hemoglobinopathies are the most common single-gene disorders, and ß-thalassemia (ß-thal) imposes a tremendous health burden on Turkey. Thus, premarital carrier screening is obligatory in Turkey, as it is in some other countries. As a result of this mandatory procedure, at routine clinical checkups, many individuals who had undergone premarital screening but did not have any clinical symptoms and/or hematological findings, have compulsorily been required to undergo further evaluation due to abnormal levels of hemoglobin (Hb) fractions (Hb A, Hb A2 and Hb F). Many consequences, such as mutations in unrelated gene(s) or someone's nutritional status, have been reported to affect the Hb fractions levels. In the present study, we aimed to determine whether HBD has a molecular causative role in patients with low Hb A2 levels (below 1.8%). The study was conducted with 20 individuals with low Hb A2 levels who had applied to our outpatient clinic. All DNA samples were analyzed for the HBD gene. Nineteen of the 20 subjects were diagnosed to carry a mutation with one of four different δ-globin variants. Three of them had been described previously [Hb A2-Yialousa (HBD: c.82G>T), Hb A2-Bornova (HBD: c.350G>C) and Hb A2-Yokoshima (HBD: c.77G>A)]. The novel [δ10(A7)Ala→Val, HBD: c.32C>T] mutation was defined as a new δ variant and reported to the HbVar database as Hb A2-Canakkale. In conclusion, the molecular characterization of Hb A2 low levels has been suggested to be significant for a definite diagnosis and counseling.

Molecular characterization of Turkish patients with demyelinating Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) disease represents a distinct subgroup of inherited peripheral neuropathies with a significant prevalence throughout the world and manifests both phenotypic and genetic heterogeneity. Electrophysiological studies subclassify CMT mainly as demyelinating or axonal types. In this study, we investigated the molecular characteristics of a Turkish cohort of 23 probands out of 34 symptomatic demyelinating CMT individuals from January 2019 to December 2021. In order to identify the underlying genetic cause, we applied a rational algorithm: PMP22 gene was initially analyzed for duplication, if PMP22-duplication testing was negative, other most causative genes (GJB1, MPZ) and PMP22 were then sequenced and if no variant was detected at aforementioned tests, whole exome sequencing (WES) test was finally performed. A total of 17 patients (≅ 74%; n = 23) were found to harbor a disease-causing variant in demyelinating CMT-related genes and among the variants, PMP22-duplication was the most frequent (≅ 41%). CMT1, CMTX, and CMT4 subtypes were manifested in ten, five, and two individuals respectively. GJB1 and SBF2 genes were the only detected genes associated with the CMTs other than CMT1. We also reported totally five novel variants: c.379A > C (p.Ile127Leu) and c.548G > T (p.Arg183Leu) variants in GJB1, c.988G > T (p.Glu330Ter) variant in NEFL, c.765_770delCCCTAT (p.Pro256_Ile257del) and c.2552A > C (p.His851Pro) variants in SBF2. As the understanding of pathophysiology and molecular mechanisms of CMT continues to evolve rapidly, many therapeutic strategy options including promising small-molecular compounds, gene replacement therapy, or disease-modifying therapies will soon be implemented in the clinical setting.

Whole-exome sequencing reveals new potential genes and variants in patients with premature ovarian insufficiency.

PURPOSE: Premature ovarian insufficiency (POI) is a heterogeneous disorder characterized by the cessation of menstrual cycles before the age of 40 years due to the depletion or dysfunction of the ovarian follicles. POI is a highly heterogeneous disease in terms of etiology. The aim of this study is to reveal the genetic etiology in POI patients. METHODS: A total of 35 patients (mean age: 27.2 years) from 28 different families diagnosed with POI were included in the study. Karyotype, FMR1 premutation analysis, single nucleotide polymorphism (SNP) array, and whole-exome sequencing (WES) were conducted to determine the genetic etiology of patients. RESULTS: A total of 35 patients with POI were first evaluated by karyotype analysis, and chromosomal anomaly was detected in three (8.5%) and FMR1 premutation was detected in six patients (17%) from two different families. A total of 29 patients without FMR1 premutation were included in the SNP array analysis, and one patient had a 337-kb deletion in the chromosome 6q26 region including PARK2 gene, which was thought to be associated with POI. Twenty-nine cases included in SNP array analysis were evaluated simultaneously with WES analysis, and genetic variant was detected in 55.1% (16/29). CONCLUSION: In the present study, rare novel variants were identified in genes known to be associated with POI, which contribute to the mutation spectrum. The effects of detected novel genes and variations on different pathways such as gonadal development, meiosis and DNA repair, or metabolism need to be investigated by experimental studies. Molecular etiology allows accurate genetic counseling to the patient and family as well as fertility planning.

Identification of a novel pathogenic variant in CKAP2L and literature review in a child with Filippi syndrome and congenital talipes equinovarus.

Filippi syndrome (MIM #272440), one of the craniodigital syndromes, is a rare genetic entity with autosomal recessive inheritance and characterized by pre- and postnatal growth retardation, microcephaly, distinctive facial appearance, developmental delay/intellectual disability, and variable syndactylies of the fingers and toes. In this report, a further female patient of Filippi syndrome who additionally had a unilateral congenital talipes equinovarus (CTEV), a feature not previously recorded, is described. Genetic testing revealed a novel homozygous frameshift pathogenic variant (c.552_555delCAAA, p.Asn184Lysfs*8) in CKAP2L and thus confirmed the diagnosis of Filippi syndrome. We hope that the newly recognized feature (CTEV) will contribute to expand the clinical spectrum of this extremely rare condition. In view of the paucity of reported cases, the full spectrum of clinical findings of Filippi syndrome necessitates obviously further affected individuals/pedigrees delineation in order to elucidate the etiological and phenotypic aspects of this orphan disease appropriately.