Detection of 13 Novel Variants and Investigation of Mutation Distribution by Next Generation Sequencing in Hemoglobinopathies: A Single Center Experience.

Hemoglobinopathies are the most common monogenic disorders in the world. Traditional diagnostic algorithms generated by conventional methods for thalassemia can be labor-intensive and time-consuming due to the complexities of the genes involved and the variability in disease-causing mutations. With the advantages of next-generation sequencing (NGS) technology, molecular analysis of highly complex diseases such as hemoglobinopathies has become easier. Next-generation sequencing is a highly sensitive and effective method due to its capacity to sequence many gene regions simultaneously while allowing good read depths. In this study, single nucleotide changes, small deletions and copy number variations in HBA1, HBA2 and HBB in 914 patients with suspected hemoglobinopathy were analysed with NGS. At least one HBA1, HBA2, HBB or HBD variant was detected in 483 (52.8%) patients. Ten novel variants were detected in HBA1 and HBA2, three in HBB, and one in HBD. From these variants, c.*76T > A, c.301-24 G > A, c.301-24G > C c.-41C > G, c.-37-40C > G, c.-9G > C, c. 95 + 9C > T, c.95 + 26C > A, c.95 + 38C > T and c.*18C > G variants were located in α-globin genes, c.-25T > C, c.*103T > C and c92 + 39A > G variants were located in ß-globin genes, and c.-43C > A was located in HBD. This is the first comprehensive study using NGS for the molecular diagnosis of hemoglobinopathies in Turkey. Accurate molecular diagnosis is of critical importance in hemoglobinopathies which are a public health problem due to their increased prevalence, high burden to society, and lack of curative treatment. Currently, NGS appears to be an advanced option over conventional methods to detect all variants occurring by molecular mechanisms and simultaneously analyse many genomic sequences.

Detecting a Novel NOTCH3 Variant in Patients with Suspected CADASIL: A Single Center Study.

Introduction: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of familial cerebral small vessel disease in adults and is caused by NOTCH3 variants. Clinical manifestations of CADASIL include recurrent ischemic strokes, dementia, migraine or migraineous headaches, epileptic seizures, and psychiatric disorders. The clinical-radiological phenotype of the disease is also highly variable. In this study, we investigated the variability of clinical, radiological, and genetic data in patients analyzed for NOTCH3 variant in our clinic. Methods: We performed clinical and neuropsychological examination, cerebral magnetic resonance imaging (MRI) and Doppler sonography of cerebral arteries in all patients. Next-generation sequencing test was used for detect variants in NOTCH3 gene from all CADASIL patients. Results: By using the next-generation sequencing method, heterozygous c.380C>T pathogenic variant was detected in the 4th exon of the NOTCH3 gene in 3 patients. This is a previously unreported novel variant and resulted in the replacement of the amino acid Proline at 127th position with Leucine. Discussion and Conclusion: The discovery of this novel pathogenic variant region may contribute to the expansion of the clinical and genetic spectrum of diseases associated with NOTCH3, leading to further research and treatment options for this disease in the future.

Utility of a targeted next-generation sequencing-based genetic screening panel in patients with periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome.

Objectives: This study aims to investigate a genetic panel in patients with periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome and examine its performance for an accurate differential diagnosis. Patients and methods: Between January 2021 and January 2022, a total of 104 children with PFAPA syndrome (63 males, 41 females; mean age: 4.8±2.3 years; range, 1.2 to 8.9 years) were retrospectively analyzed. Next-generation sequencing test was performed using a custom QIAGEN- QIAseq™ Targeted DNA Panel which includes six genes namely ELANE, LPIN2, MEFV, MVK, NLRP3, and TNFRSF1A. Results: Of 104 patients, 38 (36.5%) had variants in the genetic panel. The most common variants were found in the MEFV gene (n=35, 33.6%), the most frequent genotype was E148Q heterozygosity (n=16). Four and two patients were eventually diagnosed with Familial Mediterranean fever (FMF) and hyperimmunoglobulin D syndrome (HIDS), since they had confirmative biallelic pathogenic in the MEFV and MVK genes, respectively. Conclusion: A genetic panel, including MEFV and MVK genes, may be useful in patients, clinically resembling PFAPA, since they may have HIDS or FMF, but lack typical features of the exact disease. Nonetheless, we believe that distinct genetic panels should be developed for different populations.

Dicentric Recombinant Chromosome 18 due to Maternal Paracentric Inversion Analyzed by Array CGH.

Introduction: Chromosomal abnormalities are mostly found in 0.5-0.8% of live-born infants with developmental and morphological defects. Paracentric inversions are structural intrachromosomal rearrangements resulting in a risk of chromosomally unbalanced gametes in carriers. Case Presentation: Herein, we report a patient with dicentric rearrangement of chromosome 18 due to maternal paracentric inversion of chromosome 18. The patient was a girl, aged 3 years and 11 months. She was referred due to multiple congenital abnormalities, severe intellectual disability, and motor retardation. She had microcephaly, prominent metopic suture, synophrys, epicanthic folds, telecanthus, wide-set alae nasi, wide columella, bilateral cleft lip and palate, pectus carinatum, umbilical hernia, pes planus, and anteriorly displaced anus. She had bilateral external auditory canal stenosis and mild right-sided and moderate left-sided sensorineural hearing loss. Echocardiography showed secundum-type atrial septal defect and mild tricuspid failure. Brain magnetic resonance imaging showed only thinning of posterior areas of the corpus callosum. Chromosome analysis showed 46,XX,dic rec(18) by GTG and C banding. Dicentric chromosome was confirmed by fluorescence in situ hybridization analysis. Paternal karyotype was normal 46,XY but maternal chromosome analysis showed a paracentric inversion in chromosome 18 with 46,XX,inv(18)(q11.2?q21.3?) karyotype. Array CGH was performed on a peripheral blood sample from the patient and showed duplication at 18p11.32p11.21 and 18q11.1q11.2, and deletion at 18q21.33q23. The patient's final karyotype is arr 18p11.32p11.21(64,847_15,102,598)×3,18q11.1q11.2(18,542,074_22,666,470)×3,18q21.33q23(59,784,364_78,010,032)×1. Discussion: To the best of our knowledge, this is the first report of a patient with dicentric chromosome 18 due to a parental paracentric inversion of chromosome 18. We present the genotype-phenotype correlation with literature review.

An unusual manifestation in a pediatric patient with MAFB mutation: Sacroiliitis in multicentric carpotarsal osteolysis syndrome.

Multicentric carpotarsal osteolysis (MCTO) syndrome, is typically characterized by progressive bone resorption in especially carpal and tarsal bones, in addition to abnormal facial appearance and proteinuria. This disorder is caused by monoallelic pathogenic MAFB mutations, which result in excessive osteoclastogenesis via aberrant receptor activator of nuclear factor kappa-B ligand activation. Most cases are sporadic with de-novo mutations, and it is still unclear why carpal and tarsal bones are predominantly affected. The early phases of MCTO resemble juvenile idiopathic arthritis (JIA) with ankle and wrist swelling and pain, even with inflammatory changes in magnetic resonance imaging. Herein we report a pediatric patient, previously treated with antirheumatic drugs, and eventually diagnosed with MCTO. This case was a descriptive case with exophthalmos, significant proteinuria, and total loss of carpal and tarsal bones at the time of genetic diagnosis. Similar to the literature, our case had typical radiological findings despite methotrexate and anti-tumor necrosis factor-alpha treatment. However, while arthritis affecting joints other than wrists and ankles has not been reported so far in the literature, our case had bilateral sacroiliitis which completely resolved after adalimumab treatment. We cannot be sure if sacroiliitis was incidental or occurred as a component of the disease, nonetheless, we think that sharing our experience may lead to easy and early recognition of MCTO, with more knowledge on rare manifestations of MCTO, and thus we may be able to clarify the benefits of denosumab, which is the most promising agent in early phases of the disease.

The Role of miR-26a, miR-29a and miR-448-3p in the Development of Cerebral Aneurysm.

AIM: To elucidate the role of microRNAs (miRNAs) in the development of cerebral aneurysms. MATERIAL AND METHODS: This study compared the expression levels of miR-26a, miR-29a and miR-448-3p in 50 samples each of cerebral aneurysm tissues and normal superficial temporal artery tissues. The miRNA expression levels were also compared in terms of aneurysm location and rupture status, i.e., presence or absence of rupture. RESULTS: Expression levels of miR-26a, miR-29a and miR-448-3p were increased in aneurysm tissues compared with normal vascular tissues. No significant difference was found in the miRNA expression levels with respect to aneurysm location or rupture status. CONCLUSION: This study showed that miR-26a, miR-29a and miR-448-3p overexpression could play an important role in intracranial aneurysm development independent of aneurysm location and rupture status. miR-26a, miR-29a and miR-448-3p could act as potential therapeutic targets in patients with intracranial aneurysms; however, further studies are needed on this issue.

A Rare Autoinflammatory Disorder in a Pediatric Patient with Favorable Response to Etanercept: Sideroblastic Anemia with B Cell Immunodeficiency, Periodic Fevers, and Developmental Delay Syndrome.

Introduction: Sideroblastic anemia with B cell immunodeficiency, periodic fevers, and developmental delay (SIFD) syndrome is caused by biallelic TRNT1 mutations. TRNT1 gene encodes a CCA-adding tRNA nucleotidyl transferase enzyme. Mutant TRNT1 results in immunodeficiency and anemia in various degrees, accompanied by several organ involvement. Case Presentation: We present here a 15-month old male, demonstrated brittle hair, growth hormone deficiency, recurrent fever, arthritis, recurrent infections, mild anemia, and hypogammaglobulinemia. The patient did not respond to colchicine treatment, and after establishing SIFD diagnosis with the presence of homozygote c.948-949delAAinsGG (p.Lys317Glu) mutation in TRNT1 gene, we commenced monthly intravenous immunoglobulin replacement and weekly subcutaneous etanercept. A rapid resolution of fever episodes and infections occurred after initiation of this treatment regimen. Afterward, both anemia and growth parameters have improved during follow-up. Conclusion: SIFD syndrome should be considered in patients with recurrent fever, arthritis, and growth retardation even in the absence of severe anemia and prominent hypogammaglobulinemia.

Clinical presentation of children with Deficiency of Adenosine deaminase 2: A case series.

Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic inflammatory disease, caused by mutations in ADA2 gene, which encodes an extracellular enzyme acting as a monocyte differentiation factor. DADA2 is first described with the clinical picture resembling polyarteritis nodosa, including livedo racemose, recurrent fever, musculoskeletal complaints. Besides, some patients have cytopenia, lymphoproliferation and mild to moderate immunodeficiency. The most crucial complication of DADA2 is neurological involvement, especially arterial stroke, which necessitates continuous treatment with anti-tumor necrosis factor α (anti-TNFα) treatment for preventing further stroke attacks. Herein, we report 5 DADA2 patients from 5 unrelated families, all had G47R mutation in at least one allele. All patients had livedo racemose, and 4 patients suffered from recurrent fever. Besides, musculoskeletal complaints and gastrointestinal symptoms were present in 4 and 3 patients, respectively. One patient had chronic arthritis and only one patient had a history of recurrent stroke without any sequela. Hematological and immunological involvement occurred in 3 and 4 patients, respectively, whereas only one had significant panhypogammaglobulinemia, requiring replacement therapy. We started etanercept treatment to all patients, which resulted the complete resolution of systemic inflammatory attacks and skin lesions and provided neurologically symptom free during their follow-up. With this report, we emphasize the importance of early referral of the patients with suspected livedo racemose to avoid the delay of DADA2 diagnosis for favorable outcome.

A rare cause of microcephaly, thin corpus callosum and refractory epilepsy due to a novel SLC1A4 gene mutation.

L-serine is an important amino acid that ensures neuronal differentiation and development. The SLC1A4 gene encodes proteins that transport amino acids such as serine, alanine, threonine and glutamate into neurons. Pathogenic variants in SLC1A4 gene interneuron transport of L-serine impaired and a severe global developmental delay occurs, characterized by microcephaly and refractory seizures. In this article, we would like to describe the demographic, clinical, electroencephalography (EEG) and magnetic resonance imaging (MRI) features of a patient with a novel pathogenic variant in the 6th exon of the SLC1A4 gene (p.Gly374Arg) detected by whole-exome sequencing, which is extremely rare (there have been twenty patients reported in the literature). It is emphasized that SLC1A4 gene variants should be kept in mind if the patients have microcephaly, global developmental delay, refractory seizures, and there are no abnormalities in basal metabolic investigations, and the thin corpus callosum and myelination delay is seen on the MRI.

Congenital Central Hypothyroidism Caused by a Novel Thyroid-Stimulating Hormone-Beta Subunit Gene Mutation in Two Siblings.

Congenital central hypothyroidism (CCH) is a very rare disease. Alterations in pituitary development genes as well as mutations of immunoglobulin superfamily member 1 and transducin ß-like protein 1 can result in CCH and multiple pituitary hormone deficiencies. However, mutations of the thyrotropin-releasing hormone receptor or thyroid-stimulating hormone-beta (TSHB) gene are responsible for isolated CCH. In this paper, we present the cases of two siblings with a novel mutation of TSHB. Direct sequencing of the coding regions and exon/intron boundaries of the TSHB gene revealed two homozygous nucleotides changes. One of them was c.40A>G (rs10776792) which is a very common variation that is also seen in healthy individuals, the other was c.94G>A at codon 32 of exon 2 which resulted in a change from glutamic acid to lysine (p.E32K). Both patients were homozygous and the parents were heterozygous.