Mutations affecting mRNA splicing are the most common molecular defect in patients with familial hemophagocytic lymphohistiocytosis type 3

Mutations of UNC13D have been described in patients affected by familial hemophagocytic lymphohistiocytosis (FHL3). The Munc13-4 protein contributes to the priming of the secretory granules. Mutation in this gene results in defective cellular cytotoxicity and the familial hemophagocytic lymphohistiocytosis clinical picture. Among reported mutations, few are predicted to impair splicing. Yet, functional impact of these mutations has not been addressed. We identified 18 out of 31 familial hemophagocytic lymphohistiocytosis families showing at least one mutation responsible for splicing error. We identified some known and three novel splicing mutations: one falls at the acceptor site of exon 11 and 2 are deep intronic mutations in IVS1 and in IVS30. We demonstrated that these deep intronic mutations affect regulatory sequences causing aberrant splicing. We report that UNC13D mutations leading to splicing errors represent the majority of mutations observed in familial hemophagocytic lymphohistiocytosis. This finding has implications for designing strategies for analysis of the families with suspected familial hemophagocytic lymphohistiocytosis.     

Compound and heterozygous mutations of DSG2 identified by Whole Exome Sequencing in arrhythmogenic right ventricular cardiomyopathy/dysplasia with ventricular tachycardia

Backgrounds

This study was designed to identify the pathogenic mutations in two Chinese families of arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) using the Whole Exome Sequencing (WES).

Characterization of PRF1, STX11 and UNC13D genotype-phenotype correlations in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare autosomal recessive lethal condition characterized by fever, cytopenia, hepatosplenomegaly and hemophagocytosis. The hallmark of FHL is defect apoptosis triggering and lymphocyte cellular cytotoxicity. Thus far three disease-causing genes (PRF1, UNC13D, STX11) have been identified. We performed a genotype-phenotype study in a large, multi-ethnic cohort of 76 FHL patients originating from 65 unrelated families. Biallelic mutations in PRF1, UNC13D and STX11 were demonstrated in 13/74 (18%), 6/61 (10%) and 14/70 (20%) patients, respectively. In 27/60 (45%) patients analyzed for all three genes, no molecular diagnosis was established. STX11 mutations were most common in Turkish families (7/28, 25%), whereas in Middle East families, PRF1 mutations were most frequent (6/13, 46%). No biallelic mutation was identified in most families of Nordic origin (13/14, 93%). Patients carrying PRF1 mutations had higher risk of early onset (age <6 months) compared to patients carrying STX11 mutations [adjusted odds ratio 8.23 (95% confidence interval [CI] = 1.20-56.40), P = 0.032]. Moreover, patients without identified mutations had increased risk of pathological cerebrospinal fluid (CSF) at diagnosis compared to patients with STX11 mutations [adjusted odds ratio 26.37 (CI = 1.90-366.82), P = 0.015]. These results indicate that the disease-causing mutations in FHL have different phenotypes with regard to ethnic origin, age at onset, and pathological CSF at diagnosis.     

Clinical and genetic profiles of patients with X-linked agammaglobulinemia from southeast Turkey: Novel mutations in BTK gene

Background

X-linked agammaglobulinemia (XLA) is characterized by absent or severely reduced B cells, low or undetectable immunoglobulin levels, and clinically by extracellular bacterial infections which mainly compromise the respiratory tract. We aimed to analyze the clinical, immunological and genetic characteristics of 22 male children with XLA.

Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations

Summary Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a defect in the iduronate-2-sulfatase gene (IDS). Alternative splicing of the IDS gene can occur and the underlying regulatory mechanism may be rather complex. Nevertheless, little information is available on the role of variations at the IDS locus in the splicing process. Here we report that splice mutations at the IDS locus are an important source of MPS II pathogenicity, accounting for almost 56% of Portuguese cases. Among 16 unrelated Portuguese MPS II patients, 15 different mutations were identified: six intronic splice mutations (c.104−2AG, c.241−2A>G, c.241−1G>A, c.418+1G>A, c.880−8AG and c.1181−1G>C); two exonic splice mutations (c.1006G>lC and c.1122C>T); five missense mutations (D269V, D69V, D148N, R88C and P86L); one nonsense mutation (Q465Ter); one total IDS gene deletion; and one rearrangement involving a IDS gene inversion. Furthermore, nine of the 15 detected mutations affected the usual splicing pattern at the locus. Some of them are responsible for dramatic changes in the splicing mechanism. For example, the substitution mutation, c.418+1G>A, revealed the presence of an exonic sequence inside intron 3. Our study provides evidence that the IDS locus is prone to splicing mutations and that such susceptibility is particularly high in exon 3 and neighbouring regions. Consequently, mutation screening of the IDS gene cannot be restricted to gDNA examination. Unless cDNA analysis is also conducted, misclassifications as silent or missense mutations can be produced and even uncharacteristic splice-site mutations can be misinterpreted as classic splicing defects that may generate severe, unconventional splicing alterations. Communicating editor: Douglas Brooks Competing interests: None declared References to electronic databases. OMIM disorder/gene accession number: 309900. EC number 3.1.6.13. HUGO-approved gene symbol: IDS. GenBank Accession number: AH002847. Vega Gene ID: TTHUMG00000022615. Ensembl Gene ID: ENSG00000010404     

Characterization and outcome of 41 patients with beta-ketothiolase deficiency: 10 years’ experience of a medical center in northern Vietnam

Beta-ketothiolase (T2) deficiency is an inherited disease of isoleucine and ketone body metabolism caused by mutations in the ACAT1 gene. Between 2005 and 2016, a total of 41 patients with T2 deficiency were identified at a medical center in northern Vietnam, with an estimated incidence of one in 190,000 newborns. Most patients manifested ketoacidotic episodes of varying severity between 6 and 18 months of age. Remarkably, 28% of patients showed high blood glucose levels (up to 23.3 mmol/L). Ketoacidotic episodes recurred in 43% of patients. The age of onset, frequency of episodes, and identified genotype did not affect patient outcomes that were generally favorable, with the exception of seven cases (five died and two had neurological sequelae). Custom-tailored acute and follow-up management was critical for a positive clinical outcome. Two null mutations, c.622C>T (p.Arg208*) and c.1006-1G>C (p.Val336fs), accounted for 66% and 19% of all identified ACAT1 mutant alleles, respectively. Most patients showed characteristic biochemical abnormalities. A newborn screening program could be expected to have a high yield in Vietnam. Investigation findings of haplotypes linked to the most common ACAT1 mutation (c.622C>T) are consistent with an ancient common founder of mutation-bearing chromosomes belonging to the Kinh ethnic population. The direct management and long-term follow-up of a large number of T2-deficient patients enabled us to study the natural history of this rare disease.     

Congenital factor XIII deficiency in Pakistan: characterization of seven families and identification of four novel mutations

Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in populations with consanguineous marriages. The aims of this study were to characterize patients and relatives from seven families with suspected FXIII deficiency from Pakistan and to identify the underlying mutations. As a first indicator of FXIII deficiency, a 5M urea clot solubility test was used. Plasma FXIII A‐ and B‐subunit antigen levels were determined by ELISA. FXIII activity was measured with an incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed, and a novel splice site defect was confirmed by RT‐PCR analysis. Genetic analysis revealed six different mutations in the F13A gene. Two splice site mutations were detected, a novel c.1460+1G>A mutation in the first nucleotide of intron 11 and a previously reported c.2045G>A mutation in the last nucleotide of exon 14. Neither of them was expressed at protein level. A novel nonsense mutation in exon 4, c.567T>A, p.Cys188X, was identified, leading in homozygous form to severe FXIII deficiency. Two novel missense mutations were found in exons 8 and 9, c.1040C>A, p.Ala346Asp and c.1126T>C, p.Trp375Arg, and a previously reported missense mutation in exon 10, c.1241C>T, p.Ser413Leu. All patients homozygous for these missense mutations presented with severe FXIII deficiency. We have analysed a cohort of 27 individuals and reported four novel mutations leading to congenital FXIII deficiency.     

Prenatal Diagnosis of HbE-β-Thalassemia: Experience of a Center in Western India

The clinical presentation of HbE-β-thalassemia is extremely variable, however, many cases are severe and transfusion dependent. We offered prenatal diagnosis to 108 couples, 20 of whom came prospectively. CVS was done in 93 cases (9.5–13 weeks of gestation) while amniocentesis/cordocentesis was done for 15 cases in the second trimester. Diagnosis was done by reverse dot blot hybridization, ARMS, DNA sequencing and in a few cases by HPLC analysis of fetal blood. The genetic combinations in the couples at-risk were the following: HbE trait/β-thal trait-95, HbE-thal/HbE trait-5, HbE homozygous/β-thal trait-3, HbE-thal/β-thal trait-3, HbE Lepore/β-thal trait-1, HbE trait/HbD^Punjab trait-1. IVS1-5(G>C) was the commonest β-thalassemia mutation followed by codon15(G>A), codon30(G>C), codons41/42(-CTTT), the 619 bp deletion and codon8/9(+G) in the β-thalassemic parent. However, several rare mutations seen in India like -90(C>T), -88(C>T),codon15(-T), IVS1-129(A>C), IVS1-130(G>C), IVSII-1(G>A), IVSII-837(C>T) and IVSII 848(C>A) were also encountered. Twenty-one fetuses were affected (HbE-β-thal-20, β-thal major-1) and all the couples opted for termination of the pregnancies. Couples with affected children wish to undergo prenatal testing for HbE-β-thal in subsequent pregnancies. More regional centers are needed for these services, particularly in West Bengal and the North-East where HbE is very common.     

Description of a rare β-globin gene mutation,IVS-II-848 (C>A) (HBB: c.316-3C>A) in association with IVS-I-1 (G>A) (HBB: c.92 + 1G>A), observed in a Syrian family: a case report

Abstract

β-Thalassemia (β-thal) is a hereditary and heterogeneous group of disorders caused by mutations on the β-globin gene that result in the reduced or non production of β-globin chains. We report a rare β-globin mutation, IVS-II-848 (C>A) (HBB: c.316-3C>A), which was found in a female Syrian patient. This mutation was associated with the IVS-I-1 (G>A) (HBB: c.92+1G>A) mutation, and the genotype is a compound heterozygote for IVS-I-1(G>A)/IVS-II-848(C>A). This combination was found for the first time in Syria.     

Compound Heterozygosity for Hb Alperton (HBB: c.407C>T) and IVS-I-5 (G>C) (HBB: c.92+5G>C) Mutations Presenting as a Moderate Anemia in an Indian Family

While knowledge of HBB gene mutations is necessary for offering prenatal diagnosis (PND) of β-thalassemia (β-thal), a genotype-phenotype correlation may not always be available for rare variants. We present for the first time, genotype-phenotype correlation for a compound heterozygous status with IVS-I-5 (G>C) (HBB: c.92+5G>C) and HBB: c.407C>T (Hb Alperton) mutations on the HBB gene in an Indian family. Hb Alperton is a very rare hemoglobin (Hb) variant with scant published information about its clinical presentation, especially when accompanied with another HBB gene mutation. Here we provide biochemical as well as clinical details of this variant.     

Molecular Analysis of β-Thalassemia Patients: First Identification of Mutations HBB:c.93-2A>G and HBB:c.114G>A in Brazil

The various clinical phenotypes in β-thalassemias have stimulated the study of genetic factors that could modify the manifestations of these diseases. We examined 21 patients with β-thalassemia (β-thal) in order to identify some genetic modifying factors: β-thalassemia mutations, HBG2:g.–158C>T polymorphism, α-globin gene deletions and (AT)xNz(AT)y motif within the hypersensitive site 2-locus control region (HS2-LCR). In the 42 alleles analyzed, the most frequent mutations observed were HBB:c.92+6T>C (30.9%), HBB:c.118C>T (16.7%), HBB:c.93-21G>A (11.9%) and HBB:c.92+1G>A (4.8%); this finding is in accordance with previous data of the Brazilian population. The other genetic factors analyzed showed no relation with the severity of the disease. For the first time in Brazil, we report HBB:c.93-2A>G and HBB:c.114G>A mutations on the β-globin gene, both in a heterozygous state. This is also the first study to analyze the HS2-LCR in β-thalassemic individuals in the Brazilian population.     

Novel sequence variation of AIRE and detection of interferon‐ω antibodies in early infancy

Objective Autoimmune polyendocrine syndrome type I (APS I) is a rare primary immunodeficiency disorder characterized by chronic mucocutaneous candidiasis, multi‐organ autoimmunity and ectodermal dysplasia. Autoantibodies to parathyroid and adrenal glands and type I interferons (IFN) are hallmarks of APS I, which results from mutations in the autoimmune regulator (AIRE) gene. We wished to study clinical, immunological and genetic features of APS I in Hungarian patients, and to correlate anti‐IFN‐ω serum concentration with APS I and other multi‐organ autoimmune diseases. Design Detailed analysis of patients with APS I and multi‐organ autoimmune diseases. Patients Seven patients with APS I and 11 patients with multi‐organ autoimmune diseases were studied. Measurements Mutational analysis was performed by bidirectional sequencing of AIRE. Antibodies against IFN‐ω and endocrine organ‐specific autoantigens were studied with radioimmunoassay. RFLP was performed by digestion of DNA with Hin6I restriction enzyme. Results AIRE sequence analysis revealed homozygous c.769C>T mutations in three patients and compound heterozygous sequence variants (c.769C>T/c.44_66dup26bp; c.769C>T/c.965_977del13bp; c.769C>T/c.1344delC) in four patients with APS I. All the six live patients tested had markedly elevated IFN‐ω antibodies, which were not found in heterozygous siblings or parents. One of the identified patients was negative for antibodies against IFN‐ω at 6 weeks of age, but became positive at 7 months. At age 1, he is still without symptoms of the disease. In contrast to patients with APS I, no AIRE mutation or elevation of IFN‐ω antibodies were detected in patients with multi‐organ autoimmune diseases. Conclusion This is the first overview of patients diagnosed with APS I in Hungary. A novel c.1344delC mutation in AIRE was detected. Anti‐IFN‐ω antibodies seem to appear very early in life and are helpful to differentiate APS I from other multi‐organ autoimmune diseases.     

Haplotype Analysis of Three Common β-Thalassemia Mutations in Syrian Patients

Abstract

β-Globin haplotypes were used to investigate the origin of three common β-globin mutations, IVS-I-110 (G>A); HBB: c.93-21G>A, IVS-I-1 (G>A); HBB: c.92?+?1G>A and codon 39 (C>T); HBB: c.118C?> T in Syrian patients. Haplotype analysis was done for 49 unrelated patients with β-thalassemia (β-thal) and 20 unrelated healthy subjects by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) for the β-globin gene cluster of the following polymorphic restriction sites: HincII 5' to ε, HindIII 5' to ^Gγ, HindIII 5' to ^Aγ, HincII in ψβ, HincII 3' to ψβ, AvaII in β, and HinfI 3' to β. The IVS-I-110 mutation was associated with three haplotypes: I [+ – – – – + +] (79.4%), V [+ – – – – + –] (5.9%) and VII [+ – – – – – +] (14.7%), while, the two mutations IVS-I-1 and codon 39 were be linked to a single haplotype V (100.0%) and II [– + + – + + +] (100.0%), respectively. The normal chromosomes (β^A/β^A) were associated with four haplotypes, I (50.0%), II (7.5%), V (32.5%) and VII (10.0%). In the Syrian population, the IVS-I-110 mutation was associated with multi haplotypes, whereas the IVS-I-1 and codon 39 mutations have a single origin. More studies for the other mutations will be very useful for genetic epidemiological studies in Syria.     

The molecular basis of low activity levels of coagulation factor VII: a Brazilian cohort

Inherited factor VII (FVII) deficiency is the most common among the rare bleeding disorders. It is transmitted as an autosomal recessive inheritance, due to mutations in the FVII gene (F7). Molecular studies of FVII deficiency are rare in non‐Caucasian populations. The aim of the study was to evaluate the molecular basis behind low levels of FVII activity (FVII:C) levels in a cohort of Brazilian patients. A total of 34 patients with low FVII levels were clinically evaluated and submitted to laboratory tests, among these, prothrombin time and FVII:C, with different thromboplastins. All exons and intron/exon boundaries of F7 were amplified and sequenced. A total of 14 genetic alterations were identified, of which six were described previously, c.1091G>A, c.1151C>T, c.‐323_‐313insCCTATATCCT, c.285G>A, c.525C>T, c.1238G>A and eight (54.0%) and eight were new, c.128G>A, c.252C>T, c.348G>A, c.417G>A, c.426G>A, c.745_747delGTG, c.843G>A and c.805+52C>T. In addition to the mutation c.1091G>A, known as FVII Padua, the mutation c.1151C>T also presented discrepant FVII:C levels when tested with human and rabbit brain thromboplastin. There was no association between phenotype and genotype. Most of the identified genetic alterations found were polymorphisms. Low levels of FVII:C in this population were mostly related to polymorphisms in F7 and associated with a mild clinical phenotype. Mutation c.1151C>T was associated with discrepant levels of FVII:C using different thromboplastins, such as reported with FVII Padua.     

Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients

Familial hemophagocytic lymphohistiocytosis (FHL) is typically an early onset, fatal disease characterized by a sepsislike illness with cytopenia, hepatosplenomegaly, and deficient lymphocyte cytotoxicity. Disease-causing mutations have been identified in genes encoding perforin (PRF1/FHL2), Munc13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In contrast to mutations leading to loss of perforin and Munc13-4 function, it is unclear how syntaxin-11 loss-of-function mutations contribute to disease. We show here that freshly isolated, resting natural killer (NK) cells and CD8(+) T cells express syntaxin-11. In infants, NK cells are the predominant perforin-containing cell type. NK cells from FHL4 patients fail to degranulate when encountering susceptible target cells. Unexpectedly, IL-2 stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared with FHL2 and FHL3 patients. Since the effector T-cell compartment is still immature in infants, our data suggest that the observed defect in NK-cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK-cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities.     

Molecular Heterogeneity of β-Thalassemia in Algeria: How to Face Up to a Major Health Problem

This study concerns the molecular characterization of β-thalassemia (β-thal) alleles in 210 chromosomes. In the studied population, mutations were detected in 98% of the β-thalassemic chromosomes. Twenty-one molecular defects have been found, where the five dominant mutations, IVS-I-110 (G>A), nonsense mutation at codon 39 (C>T), the frameshift codon (FSC) 6 (?A), IVS-I-1 (G>A), and IVS-I-6 (T>C), account for 80% of the independent chromosomes. Among the remaining alleles, 16 different mutations were identified, half of them being described for the first time in Algeria. These include the ?101 (C>T) and the ?90 (C>T) mutations in the distal and proximal promoter elements, respectively, the FSC 8 (?AA), IVS-I-5 (G>T), IVS-I-128 (T>G), FSC 47 (+A), IVS-II-1 (G>A), and the substitution in the polyadenylation signal (poly A) site AATAAA>AATGAA. Haplotype analyses on rare variants were performed. The possible origin of these mutations either by founder effect or by migrations is discussed, and raises the question of an adequate strategy to be used adapted to socio-economical status.     

Novel and Recurrent Mutations of STK11 Gene in Six Chinese Cases with Peutz–Jeghers Syndrome

Background

The serine/threonine kinase 11 (STK11) gene is the main causal gene in Peutz–Jeghers syndrome (PJS). Abnormal STK11 may increase cancer risk of PJS patients via affecting its target proteins such as P53, AMPK, and PTEN. In this study, we investigated the molecular basis of six Chinese PJS patients.

Materials and Methods

Blood samples were collected from four Chinese PJS families and two sporadic patients. The entire coding region of the STK11 gene was amplified by polymerase chain reaction and analyzed by direct sequencing. Functions of mutants were assessed by PolyPhen-2, Swiss-Model software, and luciferase reporter assay.

Results

Novel mutations (c.842_843insC, c.804_805insG, and c.922T>G) and recurrent mutations (c.526G>A, c.180C>G, and c.1062C>G) were identified. Missense mutation c.922T>G and c.526G>A were predicted as probably damaging by PolyPhen-2, while c.1062C>G was benign. Mutation c.108C>G was a nonsense mutation. The 284Ter mutants of c.842_843insC and c.804_805insG significantly diminished the capacity of P53 activity in 293FT cells.

Conclusions

Our results support that STK11 gene mutations underlie Chinese patients with PJS. Mutation involving partial kinase domain disrupts normal function of STK11. Our results also enlarge the spectrum of STK11 variants in PJS patients.     

The Spectrum of β-Thalassemia Mutations in Siirt Province,Southeastern Turkey

Abstract

β-Thalassemia (β-thal) is the most common hereditary genetic blood disorder. The aims of this study were: (i) to determine the mutation types and the frequency of these mutations in β-thal patients to obtain the ethnic origins of the population in Siirt Province; (ii) to evaluate the pathogenicity of these mutations by performing in silico analysis; (iii) to reveal the genotype-phenotype correlation by comparing the clinical manifestation of our patients to the specific mutations in this population. This study included 34 patients (18 males and 16 females) with a mean age of 9.1?±?3.6 years (range 3–16 years). All mutations were determined using sequence analysis methods, and the mutations were analyzed using bioinformatics tools. Thirteen different mutations were detected in the patients: IVI-I-110 (G>A) (HBB: c.93-21G>A) (38.9%); IVS-II-1 (G>A) (HBB: c.315_1G>A) (11.1%); –30 (T>A) (HBB: c.-80T>A) (9.25%) and IVS-I-1 (G>A) (HBB: c.92?+?1G>A) (9.25%), were the most common, and these mutations constituted 68.5% of the cases. Missense codon 6 (A>T) (HBB: c.20A>T) was not pathogenic; however, all the intronic mutations (IVS-I-1, IVS-I-110, IVS-II-1) and frameshift mutations [codon 44 (–C) (HBB: c.135delC) and codons 36/37 (–T) (HBB: c.112delT)] resulted in disease. These mutations can be used to determine the ethnic origin of the Siirt population and, in affected pregnant women, to develop prenatal strategies. A fatal phenotype can be identified by in silico analysis; however, mutations that are unknown prior to marriage, pregnancy, and childbirth or new mutations can be less accurately identified.     

Impact of 3′UTR variation patterns of the KRAS gene on the aggressiveness of pancreatobiliary tumors with the KRAS G13D mutation in a Turkish population

ObjectiveSeveral studies have demonstrated the importance of mutations in codons 12, 13 and 61 and variations in the 3′ untranslated region (3′UTR) of the KRAS gene, frequently observed genetic events in the progression of pancreatobiliary tumors (PBT). However, limited data exist on the clinical effect of these alterations. The aim of the current study was to clarify the frequency of relevant alterations of the 3′UTR regions of the KRAS gene and the effect of KRAS 3′UTR polymorphisms on the prognosis of patients with codon 12, 13 and 61 mutations in a Turkish population with PBT.MethodsCodons 12, 13, and 61 and 3′UTRs of the KRAS gene were screened by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing in 43 patients and 10 controls. Chi-squared and independent sample T tests were used to evaluate the results of the mutation analysis and clinical features of the patients.ResultsWe defined the c.38G > A (rs112445441, p.G13D) (39.54%) mutation and two 3′UTR variations, c.*4066delA (rs560890523) (23.26%) and c.*4065_*4066delAA (rs57698689) (6.98%), in the KRAS gene of Turkish patients. There was a statistically significant relationship between the c.*4066delA (rs560890523) and c.*4065_*4066delAA (rs57698689) variations and invasion and lymph node metastasis status of the patients (p < 0.001). Compared to patients with c.38G > A (rs112445441, p.G13D), patients with c.*4066delA (rs560890523) and c.38G > A (rs112445441, p.G13D) presented more aggressive tumors with highly invasive features. The present study contributes findings regarding the clinical effects of KRAS alterations in PBT. Based on our study, further investigations are required.