Application value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma / 肿瘤研究与临床

Objective:To investigate the value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma.Methods:The mRNA sequencing data of 471 lung adenocarcinoma patients and corresponding methylation data of 413 cases were downloaded from The Cancer Genome Atlas (TCGA) database, the methylation levels of SHOX2 and RASSF1A gene promoter regions were calculated, and the difference in methy lation level between normal lung tissues and tumor tissues was analyzed. The clinical data of 54 patients with early-stage lung adenocarcinoma and 31 patients with benign lung tumors who underwent surgery at Drum Tower Hospital Affiliated to Nanjing University Medical School from January 2018 to January 2019 were retrospectively analyzed. The methylation status of SHOX2 and RASSF1A in tumor tissues and normal lung tissues (>5 cm from the edge of the tumor foci) (called clinical samples) was detect, and a positive methylation in the promoter region of either gene was considered as a combination of two genes methylation positivity. Using pathological diagnosis as the gold standard, the efficacy of gene methylation positivity in diagnosing early-stage lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve. Patients with >80% of tumor cells in paraffin samples were screened, and mRNA high-throughput sequencing was performed in their tumor tissues and normal lung tissues. The relationship between positive methylation of the two genes and clinicopathological features was analyzed, and the correlation between the promoter region methylation level of the two genes and mRNA expression levels in clinical samples and TCGA database samples was analyzed by Spearman method. Gene set variance analysis (GSVA) method was used to analyze the differences in Kyoto Encyclopedia of Genes and Genomes enrichment pathways between two-gene methylation-positive clinical lung adenocarcinoma samples and corresponding methylation-negative lung adenocarcinoma.Results:In TCGA database, the SHOX2 promoter region methylation island contained 6 sequenced methylation sites, of which sites cg04532033 and cg01557547 methylation levels were higher in lung adenocarcinoma tissues than in normal lung tissues (both P < 0.05); the RASSF1A gene promoter region methylation island contained 11 sequenced methylation sites, and the methylation levels of 6 of these sites in lung adenocarcinoma tissues were higher than those in normal lung tissues (all P < 0.05). Compared with normal lung tissues, the methylation level of SHOX2 promoter region was higher in stage Ⅰ and Ⅱ lung adenocarcinoma tissues (both P < 0.05); the methylation level of RASSF1A promoter region was higher in all stages of lung adenocarcinoma ( P < 0.001). Among 54 patients with early-stage lung adenocarcinoma, 28 were positive for SHOX2 promoter region methylation in tumor tissues, 21 were positive for RASSF1A promoter region methylation, and 40 were positive for combined methylation of both genes; 31 benign lung nodules were negative for SHOX2 and RASSF1A methylation. ROC curve analysis showed that the sensitivity of positive SHOX2 promoter region methylation for diagnosing early-stage lung adenocarcinoma was higher than that of RASSF1A promoter region methylation positivity (51.8% vs. 38.9%), and the area under the curve (AUC) for diagnosis by two-gene methylation positivity was larger than that for diagnosis by SHOX2 or RASSF1A gene methylation positivity alone (0.870 vs. 0.759 and 0.694). The circulating thresholds (Ct) of SHOX2 and RASSF1A methylation tested by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in stage Ⅰ and Ⅱ lung adenocarcinoma were lower than those in normal lung tissues (all P < 0.05); patients with two-gene methylation positivity were characterized by older age, longer tumor longest diameter and more advanced pathological stage compared with patients with two-gene methylation negativity (all P < 0.05). In clinical stage Ⅰ-Ⅱ lung adenocarcinoma samples, the Ct of SHOX2 and RASSF1A promoter region methylation tested by qRT-PCR was negatively correlated with their mRNA relative expression levels ( r=-0.43, P = 0.003; r = -0.48, P = 0.001); in TCGA database stage Ⅰ-Ⅱ lung adenocarcinoma samples, the level of SHOX2 promoter region methylation was negatively correlated with its mRNA relative expression level ( r = -0.23, P < 0.001), and the level of RASSF1A promoter region methylation was also negatively correlated with its mRNA relative expression level, but without statistical difference ( r = -0.05, P = 0.310). In two-gene promoter methylation-positive lung adenocarcinoma samples, the pathways related to folate metabolism and DNA stability were upregulated, and the pathways related to vasoconstriction and cell growth and differentiation were downregulated. Conclusions:The combined detection of SHOX2 and RASSF1A promoter region methylation can be used as an indicator for screening and diagnosis of early-stage lung adenocarcinoma. Abnormal promoter region methylation of the two genes may affect multiple tumor-related pathways and promote the occurrence and progression of early-stage lung adenocarcinoma.

Adult Léri-Weill dyschondrosteosis coexists with endocrinal metabolic disorders: a report of two cases / 中华内分泌代谢杂志

Léri-Weill dyschondrosteosis (LWD) has typical triad: short middle limbs, short stature, Madelung deformity of wrist, and increased body mass index. Short stature and high body mass index are risk factors for metabolic syndrome, type 2 diabetes, cardiovascular diseases, and autoimmune thyroid diseases. However, metabolic disorders and thyroid diseases in adult LWD patients have not been elucidated. This paper reports two adult patients with LWD presented to the Department of Endocrinology and metabolism. By introducing clinical characteristics, genetic variations, and diagnostic methods, physicians can deepen their understanding of LWD, improve diagnosis, and be aware of the comorbid metabolic diseases and thyroid disorders with a view of early prevention and treatment.

Diagnostic Efficacy of SHOX2 Gene Hypermethylation for Lung Cancer: A Meta-Analysis / 中国肺癌杂志

BACKGROUND@#Lung cancer is the most common malignant tumor in clinic. The prognosis of advanced patients is poor, and the 5-year survival rate is low. Therefore, early diagnosis becomes the key to improve the prognosis of patients. In recent years, with the development of molecular biology technology, aberrant modification of some driver genes, such as methylation, has become an important method for early diagnosis of lung cancer. The purpose of the present work was to quantitatively evaluate the diagnostic value of abnormal hypermethylation in short state homeobox 2 (SHOX2) promoter region in lung cancer by evidence-based medicine.@*METHODS@#We searched MEDLINE, EMBASE, Ovid, Web of Science and CNKI for literatures related to the relationship between SHOX2 gene promoter hypermethylation and lung cancer. The data of SHOX2 promoter hymethylation in the original study were extracted. The diagnostic sensitivity, specificity and area under receiver operating characteristic (ROC) curve of SHOX2 promoter methylation were calculated.@*RESULTS@#Finally, 13 publications were included in this meta-analysis, and due to significant statistical heterogeneity among the studies (P<0.05) the data was pooled by random effect model. The diagnostic sensitivity and specificity of SHOX2 promoter hypermethylation in the diagnosis of lung cancer were 0.75 (95%CI: 0.74-0.77) and 0.89 (95%CI: 0.88-0.91), respectively; The positive likelihood ratio value was 6.75 (4.56-9.99), and the negative predictive value was 0.36 (0.25-0.52); The diagnostic odds ratio was 23.16 (11.34-47.31), and the area under the ROC curve was 0.9.@*CONCLUSIONS@#SHOX2 gene promoter hypermethylation is high in serum, broncholavage fluid and pleural effusion of lung cancer patients, which can be used as a biomarker for auxiliary diagnosis of lung cancer.

Deformidad de Madelung: presentación de un caso / Madelung deformity: a case presentation

La deformidad de Madelung es una alteración poco común de la articulación de las muñecas. Se vincula a mutaciones del gen SHOX y se caracteriza por alteraciones en el radio, carpo y cúbito, con predominio bilateral. Afecta principalmente a pacientes de sexo femenino y aparece al inicio de la adolescencia. Se presenta una paciente de 15 años de edad, con antecedentes de problemas de salud. Al entrar en la adolescencia comenzó a presentar deformidad en ambas muñecas, más marcada en el lado derecho acompañado de dolor. El diagnóstico de deformidad de Madelung se concluyó mediante la clínica asociado a la positividad de los estudios imagenológicos, basados en los criterios radiográficos de Dannenberg y otros. Se decidió tratamiento quirúrgico, mediante osteotomía doble correctora para longitud y fijación externa de la mano derecha, con la resolución completa de la deformidad y seguimiento en la Consulta Externa de Ortopedia(AU)

Madelung's deformity is a rare alteration of the wrist joint. It is linked to mutations of the SHOX gene. It is characterized by alterations in the radius, carpus and ulna, predominantly bilateral. It mainly affects female patients; signs and symptoms are evident at the beginning of adolescence. To present a case of a patient with a diagnosis of Madelung deformity. The case of a 15-year-old female patient with a health history and family history of interest of an equine clubfoot father is presented. When she entered adolescence, she began to present deformity in both wrists, more marked in the right side accompanied by pain. This is a patient with a Madelung deformity. The diagnosis was concluded by the clinic associated with the positivity of the imaging studies(AU)

Screening of SHOX gene sequence variants in Saudi Arabian children with idiopathic short stature / 소아과

PURPOSE: Short stature affects approximately 2%–3% of children, representing one of the most frequent disorders for which clinical attention is sought during childhood. Despite assumed genetic heterogeneity, mutations or deletions in the short stature homeobox-containing gene (SHOX) are frequently detected in subjects with short stature. Idiopathic short stature (ISS) refers to patients with short stature for various unknown reasons. The goal of this study was to screen all the exons of SHOX to identify related mutations. METHODS: We screened all the exons of SHOX for mutations analysis in 105 ISS children patients (57 girls and 48 boys) living in Taif governorate, KSA using a direct DNA sequencing method. Height, arm span, and sitting height were recorded, and subischial leg length was calculated. RESULTS: A total of 30 of 105 ISS patients (28%) contained six polymorphic variants in exons 1, 2, 4, and 6. One mutation was found in the DNA domain binding region of exon 4. Three of these polymorphic variants were novel, while the others were reported previously. There were no significant differences in anthropometric measures in ISS patients with and without identifiable polymorphic variants in SHOX. CONCLUSION: In Saudi Arabia ISS patients, rather than SHOX, it is possible that new genes are involved in longitudinal growth. Additional molecular analysis is required to diagnose and understand the etiology of this disease.

Discondrosteose de Léri-Weill: importância do exame físico / Léri-Weill dyschondrosteosis: importance of physical examination

OBJETIVOS: Descrever um caso de Discondrosteose de Léri-Weill, uma displasia óssea hereditária rara, frequentemente associada a uma haploinsuficiência do gene SHOX (short stature homeobox-containing). DESCRIÇÃO DO CASO: Criança de três anos, sexo feminino, enviada à consulta de pediatria por suspeita de hipertensão arterial. Antecedentes pessoais e familiares irrelevantes. Ao exame físico apresentava aspeto dismórfico, com encurtamento dos membros superiores e inferiores, mãos e pés pequenos e pescoço curto. A hipertensão arterial não foi confirmada. O estudo genético para excluir cromossomopatia revelou mutações no gene SHOX compatíveis com Discondrosteose de Léri-Wei. CONCLUSÕES: O exame físico é fundamental na prática clínica, permitindo identificar alterações importantes que nem sempre são o motivo da consulta. Na Discondrosteose de Léri-Weill o pronto diagnóstico e a orientação adequada são fundamentais, sobretudo pelas implicações terapêuticas desse distúrbio, que apresenta evolução favorável se tratado com hormônio do crescimento.

AIMS: To report a case of Léri-Weill dyschondrosteosis, a rare hereditary bone dysplasia often associated with a haploinsufficiency of the SHOX (short stature homeobox-containing) gene. CASE DESCRIPTION: A three-year-old female had sought medical care for hypertension. Irrelevant personal and family background. The physical examination showed dysmorphic aspect with shortening of arms and legs, small hands and feet, and short neck. Hypertension was not confirmed. Genetic study for chromosomal deletion syndromes revealed mutations in the SHOX gene compatible with Léri-Weill dyschondrosteosis. CONCLUSIONS: Physical examination is essential in clinical practice, allowing the identification of majorchanges that are not always the reason for the medical appointment. Early diagnosis and proper guidance are essential in Léri-Weill dyschondrosteosis, especially because of its therapeutic implications. The outcome is favorable if the disorder is treated with growth hormone therapy.

Turner syndrome presented with tall stature due to overdosage of the SHOX gene

Turner syndrome is one of the most common chromosomal disorders. It is caused by numerical or structural abnormalities of the X chromosome and results in short stature and gonadal dysgenesis. The short stature arises from haploinsufficiency of the SHOX gene, whereas overdosage contributes to tall stature. This report describes the first Korean case of Turner syndrome with tall stature caused by SHOX overdosage. The patient presented with primary amenorrhea and hypergonadotropic hypogonadism at the age of 17 years. Estrogen replacement therapy was initiated at that time. She displayed tall stature from childhood, with normal growth velocity, and reached a final height of 190 cm (standard deviation score, 4.3) at the age of 30 years. Her karyotype was 46,X, psu idic(X)(q21.2), representing partial monosomy of Xq and partial trisomy of Xp. Analysis by multiplex ligation-dependent probe amplification detected a duplication at Xp22.3-Xp22.2, encompassing the PPP2R3 gene near the 5'-end of the SHOX gene through the FANCD gene at Xp22.2.

A functional analysis of short stature homeobox (SHOX) gene promoter mutation in idiopathic short stature / 中华内分泌代谢杂志

Objective To investigate the effect of short stature homeobox (SHOX) gene promoter-372G →A mutation on the promoter activity and its mechanism.Methods The luciferase report gene vectors containing human SHOX gene promoter-372G or -372A were contructed.Their transcription activities were detected in chicken chondrocytes.Double-stranded DNA probes containing-372G or-372A were produced by PCR,and used for detecting the affinity with nuclear transcription factors by electrophoretic mobility shift assay(EMSA).Results The transcription activity in a-372A promoter construct was significantly higher than that in the wild type-372G (P<0.01).The result of EMSA showed that-372A gene mutation resulted in loss of the binding affinity to nuclear transcription factors.Conclusion The-372A mutation increases SHOX promoter activity with decreased DNA binding affinity to transcription factors,which may contribute to impaired long bone growth in patients with idio pathic short stature.

An illustrative case of Léri-Weill dyschondrosteosis

We report on a girl presenting Léri-Weill dyschondrosteosis (LWD) due to deletion of the SHOX gene. Her family included individuals with short stature alone or with both short stature and mesomelia or Madelung's deformity. The deletion was demonstrated through detection of hemizygosity for microsatellite markers SHOX-CA repeat, DXYS10092, DXYS10093 and DXYS10091 localized around the SHOX gene, with retention of paternal alleles in the proband and three of her sisters who had short stature as the only clinical feature. Hemizygosity for these loci was also observed in their mother, who had short stature too. The deletion in the proband was however larger, including locus DXY10083. The proband's only sister with normal height did not carry the deletion. Family history suggests transmission of the deletion from the proband's maternal great-grandfather to her grandfather via the Y chromosome, and from the grandfather to the proband's mother via the X chromosome after crossing-over in the pseudoautosomal region proximal to the SHOX gene.

Cryptic intragenic deletion of the SHOX gene in a family with Léri-Weill dyschondrosteosis detected by Multiplex Ligation-Dependent Probe Amplification (MLPA) / Deleção críptica intragênica do gene SHOX em uma família com discondrosteose de Léri-Weill detectada por Multiplex Ligation-Dependent Probe Amplification (MLPA)

LWD is associated to SHOX haploinsufficiency, in most cases, due to gene deletion. Generally FISH and microsatellite analysis are used to identify SHOX deletion. MLPA is a new method of detecting gene copy variation, allowing simultaneous analysis of several regions. Here we describe the presence of a SHOX intragenic deletion in a family with LWD, analyzed through different methodologies. Genomic DNA of 11 subjects from one family were studied by microsatellite analysis, direct sequencing and MLPA. FISH was performed in two affected individuals. Microsatellite analysis showed that all affected members shared the same haplotype suggesting the involvement of SHOX. MLPA detected an intragenic deletion involving exons IV-VIa, which was not detected by FISH and microsatellite analysis. In conclusion, the MLPA technique was proved to be the best solution on detecting this small deletion, it has the advantage of being less laborious also allowing the analysis of several regions simultaneously.

Discondrosteose de Léri-Weill (DLW) está associada à haploinsuficiência do gene SHOX resultante, principalmente, de deleções. Geralmente, o FISH e a análise de microssatélites são os métodos utilizados para a identificação destas deleções. MLPA é um novo método para detectar variações do número de cópias gênicas, permitindo uma análise simultânea de várias regiões. Aqui, descrevemos uma pequena deleção intragênica no SHOX em uma família com DLW analisada por diferentes metodologias. DNA genômico de 11 membros de uma família foram estudados por microssatélites, seqüenciamento direto e MLPA. FISH foi realizado em dois indivíduos afetados. Os microssatélites demonstraram que todos os membros afetados apresentavam o mesmo haplotipo, sugerindo o envolvimento do SHOX. MLPA identificou uma deleção intragênica envolvendo os éxons IV-VIa, que não foi detectada pelo FISH e pelos microssatélites. Conclui-se que o MLPA demonstrou melhor resolução para detectar esta pequena deleção, com a vantagem de ser menos trabalhoso e permitir a análise de várias regiões simultaneamente.

Tall stature and poor breast development after estrogen replacement in a hypergonadotrophic hypogonadic patient with a 45,X/46,X,der(X) karyotype with SHOX gene overdosage / Estatura alta e hipodesenvolvimento mamário após reposição estrogênica em paciente com hipogonadismo hipergonadotrófico e cariótipo 45,X/46,X, der(X) com superdosagem do gene SHOX

SHOX is exclusively expressed in the developing distal limb bones of human embryos and in the first and second pharyngeal arches. It works as a promoter for linear growth and as a repressor of growth plate fusion. It was reported, recently, that SHOX overdosage and gonadal estrogen deficiency have led to tall stature due to continued growth. We report, in the present study, a female patient with 45,X/46,X, psu idic(X)(pter→q21::q21→pter) karyotype, tall stature, and hypergonadotrophic hypogonadism without Turner stigmas. She did not present breast development even after long term therapy with high estrogen doses. Fluorescence in situ hybridization depicted the presence of three copies of SHOX gene. Microsatellite studies showed paternal origin of der(X). Further studies in similarly affected patients will clarify if the absence of breast development, despite previous high-dose estrogen treatment, is associated to triple copy of SHOX gene.

O gene SHOX é expresso, exclusivamente, no primeiro e no segundo arcos faríngeos, assim como nas extremidades dos ossos dos membros em embriões humanos. SHOX normalmente atua como um promotor para o crescimento linear e como um repressor do fechamento da placa de crescimento. Recentemente, foi descrito que o excesso da proteína SHOX associada à deficiência estrogênica gonadal leva à estatura alta devido ao contínuo crescimento. Neste estudo descrevemos uma paciente do sexo feminino com cariótipo 45,X/46,X,psu idic(X)(pter→q21::q21→pter), estatura alta, hipogonadismo hipergonadotrófico e sem estigmas de Turner. A paciente não apresentou desenvolvimento de mamas, mesmo depois do tratamento prolongado com altas doses de estrógenos. FISH evidenciou a presença de três cópias do SHOX. Estudo de microssatélites demonstrou a origem paterna do der(X). Estudos futuros em pacientes com semelhanças clínicas esclarecerão se a ausência de desenvolvimento de mamas, apesar do tratamento com altas doses de estrógenos, está associada à tripla cópia do SHOX.

Aspectos genéticos de la talla baja: Revisión

La talla baja es una condición que afecta el crecimiento lineal en el proceso de desarrollo del individuo es ocasionada por múltiples factores pero con un fuerte componente genético. En los últimos años, se ha incrementado el conocimiento de las causas genéticamente determinadas de talla baja debido a reporte de pacientes con características especiales, quienes han ofrecido una excelente oportunidad para estudiar genes que juegan un papel crucial en el crecimiento. En esta revisión se delinea, desde la perspectiva de un médico genetista, un flujograma diagnóstico a ser considerado en todo paciente con talla baja.

Short stature is a condition affecting the body growth in the development process of an individual which is caused by multiples factors, but with a strong component genetic. In the last few years, our knowledge of genetically determined causes of short stature has greatly increased by reports of challenging patients, who offered the opportunity to study genes that play a role in growth. In this review, a diagnostic flow chart is delinead to consider in all patients with short stature from the perspective of a medical geneticist.