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Biomedical subjects

M G Tibiletti

Publications and source records attributed to M G Tibiletti.

At least 19 recordsLinked to original sources

Chromosome band 6q deletion pattern in malignant lymphomas.

Deletion of chromosome arm 6q is a frequent karyotypic alteration found in a variety of cancers and lymphoproliferative disorders, including leukemia and lymphomas. We characterized 6q deletions in 35 malignant lymphomas, using conventional and molecular cytogenetic approaches, to define the deletion pattern of 6q in different histological types. Conventional cytogenetics revealed a 6q deletion in 46% of lymphomas, including two cases that showed 6q deletion as the sole chromosome anomaly. Interphase FISH analysis demonstrated allelic loss of 6q regions in 33 out of 35 cases (94.2%); the deletions were discontinuous, involving nonadjacent molecular regions. Although 6q deletion is a common event in all types of lymphomas, specific deletion patterns seem to characterize different histological types, suggesting that different tumor suppressor genes play different roles in different types of lymphomas. Two specific 6q regions deleted in diffuse large B cell lymphomas but not in follicular lymphomas may be implicated in the clinical transformation.

Chromosome Deletion↗

Molecular and genomic characterisation of cryptic chromosomal alterations leading to paternal duplication of the 11p15.5 Beckwith-Wiedemann region.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder with increased risk of paediatric tumours. The aetiology involves epigenetic and genetic alterations affecting the 11p15 region, methylation of the differentially methylated DMR2 region being the most common defect, while less frequent aetiologies include mosaic paternal 11p uniparental disomy (11patUPD), maternally inherited mutations of the CDKN1C gene, and hypermethylation of DMR1. A few patients have cytogenetic abnormalities involving 11p15.5. METHODS: Screening of 70 trios of BWS probands for 11p mosaic paternal UPD and for cryptic cytogenetic rearrangements using microsatellite segregation analysis identified a profile compatible with paternal 11p15 duplication in two patients. RESULTS: Fluorescence in situ hybridisation analysis revealed in one case the unbalanced translocation der(21)t(11;21)(p15.4;q22.3) originated from missegregation of a cryptic paternal balanced translocation. The second patient, trisomic for D11S1318, carried a small de novo dup(11)(p15.5p15.5), resulting from unequal recombination at paternal meiosis I. The duplicated region involves only IC1 and spares IC2/LIT1, as shown by fluorescent in situ hybridisation (FISH) mapping of the proximal duplication breakpoint within the amino-terminal part of KvLQT1. CONCLUSIONS: An additional patient with Wolf-Hirschorn syndrome was shown by FISH studies to carry a der(4)t(4;11)(p16.3;p15.4), contributed by a balanced translocation father. Interestingly, refined breakpoint mapping on 11p and the critical regions on the partner 21q and 4p chromosomal regions suggested that both translocations affecting 11p15.4 are mediated by segmental duplications. These findings of chromosomal rearrangements affecting 11p15.5-15.4 provide a tool to further dissect the genomics of the BWS region and the pathogenesis of this imprinting disorder.

Beckwith-Wiedemann Syndrome↗

Specificity of interphase fluorescence in situ hybridization for detection of chromosome aberrations in tumor pathology.

Interphase fluorescence in situ hybridization (IFISH) is an interesting and intriguing cytogenetic approach in the study of tumor chromosomal abnormalities when metaphases are not available. This technique can be applied to different types of tumor nuclei, including imprinted nuclei (IM), nuclei obtained from conventional cytogenetic procedures (PB), frozen nuclei, paraffin-embedded nuclei (PE), and nuclei extracted from paraffin-embedded sections (EX). IFISH is a high-sensitivity approach in tumor studies that can give evidence of genetic aberrations present in a small percentage of cells that are likely to escape detection if only molecular techniques are applied. Despite its high sensitivity and versatility, IFISH is an indirect cytogenetic method and needs controls to have adequate specificity. This study includes present data obtained in IFISH experiments using different types of probes (alpha-satellite and YAC clones) hybridized on different types of normal control nuclei, such as PB, IM, EX, and PE nuclei, to define the threshold level for monosomy and trisomy of different chromosomal regions. My findings demonstrate that the cut-off values depend both on the types of probes and on the types of target nuclei. Therefore, even if IFISH is a versatile, high-sensitivity technique for detecting chromosomal abnormalities, the lack of accurate controls may result in the misdiagnosis of some abnormalities.

Bone Marrow↗

Exploring the clinical and epidemiological complexity of GJB2-linked deafness.

GJB2 mutation analysis was performed in 179 unrelated subjects with sporadic or familial hearing loss (HL). Among 57 families, 18 showed a vertical transmission of HL, the disease being present in two or three generations. Besides 155 nonsyndromic cases, 24 patients presenting with extra-auditory clinical signs were included in the molecular study. GJB2 mutation analysis was also performed in 19 subjects with an anamnestic history of perinatal risks factors for acquired HL. The 35delG mutation accounted for 22.1% of analyzed chromosomes in sporadic cases and 39.4% in familial cases; 35delG prevalence reached 41% in autosomal recessive and 44.4% in pseudodominant pedigrees. Two novel GJB2 mutations were identified in compound heterozygosity with 35delG allele (D159V, 284ins/dup[CACGT]). Two 35delG homozygous subjects were identified among HL cases classified as environmental in origin. Four patients 35delG heterozygous (35delG/V95M, 35delG/L90P, 35delG/167delT, and 35delG/?) and two homozygous presented with extra-auditory clinical signs involving different organs (skin, vascular system, hemopoietic lineages, and thyroid). In a high proportion of 35delG heterozygous HL patients (52%), no second GJB2 mutation was detected. The reported data highlight the complexity of the genetic epidemiology of GJB2-linked deafness, further enlarging the spectrum of situations in which GJB2 mutation analysis should be performed. The presence of extra-auditory signs in a significant portion of GJB2-mutated patients suggests the possibility that GJB2 loss of function could contribute to clinical phenotypes presenting in association with deafness. This hypothesis deserves further investigation. The failure to identify a presumed partnering GJB2 mutation in a high proportion of deaf patients remains a challenging problem to be clarified.

Amino Acid Sequence↗

Mutation analysis of the inhibin alpha gene in a cohort of Italian women affected by ovarian failure.

BACKGROUND: Premature ovarian failure (POF) is a secondary hypergonadotrophic amenorrhoea affecting 1-3% of females, whose aetiology is almost unknown. However, inhibin alpha gene (INHalpha) has recently been indicated as candidate in POF pathogenesis. METHODS: We analysed patients affected by POF (n = 157) for the missense mutation (769G-->A transition) in the exon 2 of the INHalpha gene. The same analysis was carried out on early menopause (EM) (n = 36) and primary amenorrhoea (n = 12) patients. RESULTS: The incidence of the mutation was significantly more frequent within both POF (7/157, 4.5%) (Fisher's exact test, P = 0.030) and primary amenorrhoea (3/12, 25%) (Fisher's exact test, P < 0.001) patients, compared with the control population of women (0/100), who experienced physiological menopause. No mutation was found in EM patients. Furthermore, the likelihood of finding the mutation was statistically significant in familial (5/65; 7.7%) (Fisher's exact test, P < 0.01) but not in sporadic (2/92; 2.2%) (Fisher's exact test, P = not significant) POF, compared with the control group. The analysis of pedigrees showing the inheritance of the 769G-->A mutation and POF strengthens the concept of the disease heterogeneity, since the POF phenotype was not always associated with the mutation. Moreover, a higher prevalence of the C allele of a single nucleotide polymorphism (129C-->T), located in the 5'-UTR of the INHalpha gene, was observed in POF patients (80.3%) than in the control group (66.7%) (Fisher's exact test, P = 0.014). CONCLUSION: These data strengthen the concept of the INHalpha gene as a candidate for ovarian failure.

5' Untranslated Regions↗

Germline mutation in the juxtamembrane domain of the kit gene in a family with gastrointestinal stromal tumors and urticaria pigmentosa.

BACKGROUND: Gain-of-function mutations of the c-kit protooncogene, mainly clustered in the juxtamembrane domain, have been reported in a significant fraction of gastrointestinal (GI) stromal tumors (GISTs) that represent the most common mesenchymal tumor of the GI tract. Two families also have been described with a GIST predisposition syndrome with a germline c-kit mutation affecting either the juxtamembrane domain or the tyrosine kinase domain. Here, the authors report on a family in which the dominantly inherited trait of hyperpigmented spots was inherited from an individual who developed multiple GISTs with diffuse hyperplasia of the myenteric plexus by his son, who was affected with urticaria pigmentosa. METHODS: Screening for the c-kit mutation was performed by means of polymerase chain reaction-based denaturing gradient gel electrophoresis/constant denaturing gel electrophoresis followed by direct sequencing of abnormal conformers. Expression of KIT and CD34 was determined by immunohistochemistry. RESULTS: In peripheral blood DNA samples, both affected family members showed a previously undescribed c-kit mutation in the juxtamembrane domain, resulting in the substitution of alanine for valine(559). Mutation and polymorphic marker analyses on DNA samples from three GISTs and two skin biopsy specimens evidenced the same mutation in the heterozygous condition. Immunohistochemical examination showed coexpression of CD117 (c-kit) and CD34 in all independent GISTs and CD117 positivity in mast cells from the skin lesions. CONCLUSIONS: Comparative analysis of clinical presentation and mutation mapping in the families described to date point to the peculiar association of mast cells, melanocytic dysfunction, and GIST predisposition in carriers of c-kit mutations within the juxtamembrane domain.

DNA Mutational Analysis↗

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

Animals↗

Nine novel APC mutations in Italian FAP patients.

Familial adenomatous polyposis (FAP) is a common hereditary syndrome characterized by early development of colorectal cancer consequent to extensive adenomatous polyps of the colon. In addition to the colonic manifestations the syndrome presents several extracolonic features including polyps of the upper gastrointestinal tract, congenital hypertrophy of the retinal pigment, jaw cysts, osteomata and desmoid tumors. In this study the entire APC coding region has been analysed for mutation in a panel of one Turcot and 33 unrelated Italian FAP patients using SSCP analysis, PTT and DNA sequencing. We detected APC mutations in 23 of them and identified nine which, to our knowledge were not previously reported. All of these novel mutations are in exon 15, including two nonsense mutations, 6 deletions or insertions leading to premature termination of the protein and one missense mutation (7697G>A). This last mutation occurs in the EB1-binding domain of the APC protein and segregates in four relatives of the patient with three of them presenting 2-3 adenomatous polyps.

Adenoma↗

Immunohistochemical pattern of hMSH2/hMLH1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences.

Alterations of DNA mismatch repair (MMR) genes are involved in carcinogenesis of sporadic and inherited human cancers characterised by instability of DNA microsatellite sequences (MSI). MSI tumours are usually identified using molecular analysis. In the present investigation, hMLH1 and hMSH2 immunohistochemistry was tested in order to evaluate the utility of this method in predicting MMR deficiency. Colorectal (72), gastric (68), endometrial (44) and ovarian (17) carcinomas were independently evaluated for familial history, histological type of tumour, MSI status and immunohistochemical results. Loss of expression of either hMLH1 or hMSH2 was observed in 51 of 55 (92.8%) MSI tumours, while 145 of 146 microsatellite stable (MSS) tumours expressed both the hMLH1 and hMSH2 gene products. Independently of tumour site, an overall agreement between immunohistochemical and molecular results was observed in 15 hereditary non-polyposis colorectal cancer-related tumours. Among sporadic tumours, only 2 of 60 colorectal and 2 of 66 gastric carcinomas, displaying MSI, expressed both hMLH1 and hMSH2 gene products. All 39 endometrial and 16 ovarian tumours presented a concordant molecular and immunohistochemical profile. These data show that immunohistochemistry is an accurate and rapid method to predict the presence of defective DNA MMR genes and to identify both sporadic and familial MSI tumours.

Adaptor Proteins, Signal Transducing↗

Expression of acidic fibroblast growth factor (aFGF) and fibroblast growth factor receptor 4 (FGFR4) in breast fibroadenomas.

BACKGROUND/AIM: Fibroadenomas are benign tumours composed of both glandular and fibrous tissue. The mechanisms regulating the growth of these tumours and the relation between the stromal and epithelial cells are poorly understood. Acidic fibroblast growth factor (aFGF) is a well known fibroblast activator, which acts through four specific cell surface receptors, among which, fibroblast growth factor receptor 4 (FGFR4) is highly specific. The aim of this study was to evaluate the distribution of aFGF and FGFR4 in specific cell types of fibroadenomas to understand their possible role in the growth of these breast lesions. METHODS: Formalin fixed and paraffin wax embedded tissues from 15 fibroadenomas and peritumoral normal breasts were investigated for the expression of aFGF and FGFR4 using immunohistochemistry. The presence of aFGF mRNA was also investigated using in situ hybridisation. RESULTS: Immunoreactivity for aFGF and FGFR4 was seen in epithelial cells, but it was lacking in myoepithelial cells of both normal tissues and fibroadenomas. Strong FGFR4 immunoreactivity was found in stromal fibroblasts, which were also weakly positive for aFGF. aFGF mRNA was detected in epithelial cells and in some stromal fibroblasts. CONCLUSIONS: These results suggest a paracrine/autocrine modulation of epithelial and stromal cells of fibroadenomas through an aFGF-FGFR4 interaction. This interaction might regulate various cell functions and the growth of fibroadenomas.

Adolescent↗

Chromosome 6 abnormalities in ovarian surface epithelial tumors of borderline malignancy suggest a genetic continuum in the progression model of ovarian neoplasms.

PURPOSE: We used conventional cytogenetics, molecular cytogenetics, and molecular genetic analyses to study the pattern of allelic loss on chromosome 6q in a cohort of borderline epithelial ovarian tumors. EXPERIMENTAL DESIGN: Fifteen tumor samples were collected from patients undergoing surgery for ovarian tumors. The tumors of borderline malignancy, classified according to the standard criteria, included 4 mucinous and 11 serous tumors. Cytogenetic and molecular cytogenetic (with yeast artificial chromosome clones from 6q26-27) studies were performed on tumor areas contiguous to those used for histological examination ensuring the appropriate sampling. Moreover loss of heterozygosity analysis was performed using PCR amplification of eight microsatellite markers mapping on 6q27 (D6S193, D6S297), 6q26 (D6S305, D6S415, D6S441), 6q21 (D6S287), 6q16 (D6S311), and 6q14 (D6S300). RESULTS: Deletions of this chromosome arm, in particular of 6q24-27, were the most frequent lesions found in this set of tumors. In a tumor with a normal karyotype the only detectable alteration was a deletion of approximately 300 kb within the D6S149-D6S193 interval at band 6q27. This is, to date, the smallest deletion described for borderline tumors. CONCLUSION: Alterations in the above-mentioned interval are a common finding in advanced ovarian carcinomas but also in benign ovarian cysts, implying that some tumors of borderline malignancy may arise from benign tumors and that malignant ones may evolve from tumors of borderline malignancy. Genes located in 6q27 seem to be crucial for this mechanism of early events in ovarian tumorigenesis.

Adenocarcinoma, Mucinous↗

A tumor suppressor locus in familial and sporadic chordoma maps to 1p36.

Previous cytogenetic/FISH data have demonstrated 1p36 deletions in a relapsing familial clivus chordoma developed by a patient who has 2 daughters, respectively affected with childhood astrocytoma and clivus chordoma. Using an approach that combined the LOH (loss of heterozygosity) study of the father chordoma and the daughter astrocytoma and a segregation analysis from parents to sibs using 17 CA-repeats spanning 1p36.32-1p36.11, we mapped the cancer susceptibility locus in this family to the 1p36 region. The LOH and haplotype information was elaborated using a pairwise linkage analysis that gave a maximum lod score of 1.2. Additional LOH data relating to 6 sporadic chordomas allowed us to define an SRO (the smallest region of overlapping loss) of about 25 cM from D1S2845 (1p36.31) to D1S2728 (1p36.13). Our overall findings converge on mapping to 1p36 a tumor-suppressor gene involved in familial and sporadic chordoma.

Adult↗

Genetic progression in microsatellite instability high (MSI-H) colon cancers correlates with clinico-pathological parameters: A study of the TGRbetaRII, BAX, hMSH3, hMSH6, IGFIIR and BLM genes.

Colon carcinomas with microsatellite mutator phenotype exhibit specific genetic and clinico-pathological features. This report describes the analysis of 63 "microsatellite instability-high" (MSI-H) tumors for the presence of mutations in microsatellites located in the coding regions (CDRs) of 6 genes: TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR, and BLM. The following frequencies of mutations were detected: TGFbetaRII (70%), BAX (54%), hMSH3 (36.5%), IGFIIR (22%), hMSH6 (17.5%), and BLM (16%). The overall picture revealed combinations of mutations suggestive of a progressive order of accumulation, with mutations of TGFbetaRII and BAX first, followed by frameshifts in hMSH3, hMSH6, IGFIIR, and BLM. Correlations with 12 clinico-pathological parameters revealed that tumors with frameshifts in 1 or 2 CDRs were significantly better differentiated than tumors with frameshifts in more than 2 CDRs. We also found that mutations in the hMSH3 gene were significantly associated with decreased wall invasiveness and aneuploidy, and frameshifts in the BLM gene were significantly associated with the mucinous histotype. A trend toward an association between hMSH3 and IGFIIR with the medullary and conventional adenocarcinoma histotypes, respectively, was seen. Our results strengthen the concept that mutations in target genes have a role in the tumorigenic process of MSI-H tumors, and indicate that frameshifts in microsatellites located in CDRs occur in a limited number of combinations that could determine distinct clinico-pathological traits.

Adenosine Triphosphatases↗

Premature ovarian failure.

Secondary amenorrhoea with elevated gonadotrophins occurring under the age of 40 (premature ovarian failure (POF)), and at the age between 41 and 44 years (early menopause (EM)), respectively, affects 1-2% and 5% of women in the general population. Objective of this study was to evaluate the prevalence of familial cases of POF and EM and to assess the clinical and genetic characteristics of these patients. One hundred and sixty women with idiopathic secondary amenorrhoea before the age of 45 and serum follicle-stimulating hormone (FSH) levels greater than or equal to 40 IU/l were included in the study. Tests performed on patients included complete medical history, pedigree's analysis, clinical pelvic examination, gonadotrophins and thyroid assessment, chromosomal analysis. The 160 patients included in the study showed idiopathic POF (n=130) or EM (n=30). Following pedigree assessment, we were able to identify an incidence of familial cases of 28.5% in the POF group (n=37) and of 50% in the EM group (n=15). POF and EM condition were often present in the same family. There were no differences between POF and EM patients and between familial and sporadic cases regarding age at menarche, personal history, gynaecological history, weight, height and diet habits. There was a statistically significant difference between sporadic and familial cases in age at POF onset: 32.0+/-7.3 years (12-40) compared to 35. 0+/-5.8 (18-40), respectively (P<0.05). The POF and EM families identified showed two or more affected females and transmission through either maternal or paternal relatives; in four families both maternal and paternal transmission was observed. This study suggests that idiopathic POF and EM conditions, differing only in age of menopause onset, may represent a variable expression of the same genetic disease. The different age of menopause onset in these patients may be explained by genetic heterogeneity and/or by different environmental factors. Our results indicate a high rate of familial transmission of the condition. Pedigree's analysis suggests an autosomal or an X-linked dominant sex-limited pattern of inheritance for POF and EM.

Adolescent↗

Molecular definition of Xq common-deleted region in patients affected by premature ovarian failure.

High-resolution cytogenetic analysis of a large number of women with premature ovarian failure (POF) identified six patients carrying different Xq chromosome rearrangements. The patients (one familial and five sporadic cases) were negative for Turner's stigmata and experienced a variable onset of menopause. Microsatellite analysis and fluorescent in situ hybridization (FISH) were used to define the origin and precise extension of the Xq anomalies. All of the patients had a Xq chromosome deletion as the common chromosomal abnormality, which was the only event in three cases and was associated with partial Xp or 9p trisomies in the remaining three. Two of the Xq chromosome deletions were terminal with breakpoints at Xq26.2 and Xq21.2, and one interstitial with breakpoints at Xq23 and Xq28. In all three cases, the del(X)s retained Xp and Xq specific telomeric sequences. One patient carries a psu dic(X) with the deletion at Xq22.2 or Xq22.3; the other two [carrying (X;X) and (X;9) unbalanced translocations, respectively] showed terminal deletions with the breakpoint at Xq22 within the DIAPH2 gene. Furthermore, the rearranged X chromosomes were almost totally inactivated, and the extent of the Xq deletions did not correlate with the timing of POF. In agreement with previous results, these findings suggest that the deletion of a restricted Xq region may be responsible for the POF phenotype. Our analysis indicates that this region extends from approximately Xq26.2 (between markers DXS8074 and HIGMI) to Xq28 (between markers DXS 1113 and ALD) and covers approximately 22 Mb of DNA. These data may provide a starting point for the identification of the gene(s) responsible for ovarian development and folliculogenesis.

Adolescent↗

Association between idiopathic premature ovarian failure and fragile X premutation.

A total of 106 women affected by premature ovarian failure (POF) were evaluated for fragile X (FRAXA) premutation. The POF patients were classified as having a familial condition (33 women), at least one relative with early menopause (12 women), or a sporadic condition (61 women). The FRAXA premutation was only detected in patients with familial (four out of 33) or sporadic POF (two out of 61). In general, the results obtained indicated that the prevalence [six out of 106, 6%, 95% confidence interval (CI) 3-11%] of FRAXA premutation is significantly higher in women affected by POF than expected (P = 1.24x10(-3)), suggesting a phenotype consequence of the premutation alleles. This relationship is more convincingly derived from the observation in two analysed pedigrees of a co-segregation between FRAXA and POF. These findings suggest a possible involvement of premutated alleles in ovarian failure, and indicate the utility of POF families screening for FRAXA premutation in order to prevent the transmission of mental retardation syndrome.

Adolescent↗

Ovarian metastases from colorectal carcinoma. Clinicopathologic profile, immunophenotype, and karyotype analysis.

Ovarian metastases from colorectal carcinoma frequently mimic primary ovarian carcinomas. The present study was performed to identify possible criteria helpful in differential diagnosis. Twenty-three ovarian metastases from colorectal carcinomas and 23 primary ovarian carcinomas were evaluated clinicopathologically and immunostained with antigastric M1 antigen, cathepsin E, CA125, vimentin, estrogen and progesterone receptors, cytokeratins 7 and 20, and alpha-inhibin antibodies. We performed a conventional and molecular cytogenetic study on 5 ovarian metastases from colorectal carcinoma using direct preparation, Q banding techniques, and fluorescence in situ hybridization. Integration of clinicopathologic, immunohistochemical, and cytogenetic features is helpful for the differential diagnosis of metastases of colorectal carcinomas from primary ovarian carcinomas. Bilaterality, extrapelvic spreading, high mitotic index, and cytokeratin 20 immunoreactivity, and lack of M1, CA125, and cytokeratin 7 immunoreactivity favor the diagnosis of ovarian metastases from colon carcinomas. The identification of 13q gain as a peculiar, sensitive, and specific marker of colorectal carcinomas seems relevant.

Adult↗