Absence of activating mutations in ras and gsp oncogenes in a cohort of nine patients with sporadic pediatric thyroid tumors

BACKGROUND: Characterization of the genetic background of pediatric thyroid carcinomas could aid in distinguishing between differently staged tumors with respect to treatment and prognosis. Two known genetic factors associated with thyroid carcinoma, the proto-oncogenes gsp and ras were investigated. PROCEDURE: DNA was extracted from paraffin sections from both tumor and normal thyroid tissue of nine patients (ages 9-16 years). Of these patients, eight were diagnosed with papillary carcinoma and one with follicular adenoma. The coding exons of gsp and the three known ras genes (H, K, and N-ras) were screened for mutations using SSCP-analysis. RESULTS: There were no mutations present in the ras and gsp proto-oncogenes hot spots, however, LOH of H-ras (chromosome location 11p15.5) was found in tumor tissue from one patient and a homozygous mutation in exon 12 of gsp causing a Pro-->Ser conversion was present in the thyroid tumor tissue from another patient. Two silent polymorphisms were detected, H-ras exon1, 86T-->C and gsp exon 5, 81T-->C. CONCLUSIONS: Our results indicate that the ras/gsp mutations found are probably late events in the tumorigenesis representing general oncogenic stress. In conclusion, it seems that ras/gsp activation is not a factor in the mechanism causing sporadic thyroid carcinoma in children.     

Control of beta-catenin/Tcf-directed transcription in medulloblastoma

The beta-catenin, glycogen synthase kinase 3beta (GSK-3beta), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot's syndrome and patients with Turcot's syndrome, who develop medulloblastomas, have been shown to harbor germline APC mutations. While APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the beta-catenin and GSK-3beta genes has not been evaluated in this tumor. This study shows that 3 of 67 medulloblastomas harbor beta-catenin mutations, each of which converts a GSK-3beta phosphorylation site from serine to cysteine. The beta-catenin mutation seen in the tumors was not present in matched constitutional DNA in the 2 cases where matched normal DNA was available. A loss of heterozygosity (LOH) analysis of 32 medulloblastomas with paired normal DNA samples was performed with 4 microsatellite markers flanking the GSK-3beta locus; LOH with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3beta allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the beta-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3beta gene does not appear to be a target for inactivation in this tumor.     

Control of β-catenin/Tcf-directed transcription in medulloblastoma

The β-catenin, glycogen synthase kinase 3β (GSK-3β), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot's syndrome and patients with Turcot's syndrome, who develop medulloblastomas, have been shown to harbor germline APC mutations. While APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the β-catenin and GSK-3β genes has not been evaluated in this tumor. This study shows that 3 of 67 medulloblastomas harbor β-catenin mutations, each of which converts a GSK-3β phosphorylation site from serine to cysteine. The β-catenin mutation seen in the tumors was not present in matched constitutional DNA in the 2 cases where matched normal DNA was available. A loss of heterozygosity (LOH) analysis of 32 medulloblastomas with paired normal DNA samples was performed with 4 microsatellite markers flanking the GSK-3β locus; LOH with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3β allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the β-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3β gene does not appear to be a target for inactivation in this tumor.     

Identification of a novel mutation in the human growth hormone receptor gene (GHR) in a patient with Laron syndrome

Deletions and mutations in the growth hormone receptor (GHR) gene are the underlying etiology of Laron syndrome (LS) or growth hormone (GH) insensitivity syndrome (GHIS), an autosomal recessive disease. Most patients are distributed in or originate from Mediterranean and Middle-Eastern countries. Sixty mutations have been described so far. We report a novel mutation in the GHR gene in a patient with LS. Genomic DNA sequencing of exon 5 revealed a TT insertion at nucleotide 422 after codon 122. The insertion resulted in a frameshift introducing a premature termination codon that led to a truncated receptor. We present clinical, biochemical and molecular evidence of LS as the result of this homozygous insertion.     

Citrin缺陷导致的新生儿肝内胆汁淤积症家系SLC25A13基因突变研究

目的探讨一个来自中国的Citrin缺陷导致的新生儿肝内胆汁淤积症（NICCD,MIM# 605814）家系的基因诊断过程。方法从先证者及其所在家系其他9名成员的血样中提取DNA,PCR扩增后行琼脂糖凝胶电泳,初步发现2个突变,并用本实验室建立的基因扫描法进一步证实,然后行DNA测序,最终确定突变位置和性质。结果先证者为851—854del和1638—1660dup两种突变的复合杂合子,两种突变分别位于SLC25A13基因外显子9和外显子16。母亲及哥哥为851—854del携带者,父亲、一个姑姑及其子为1638—1660dup携带者。结论该家系中SLC25A13基因外显子9和16分别发生了缺失突变851—854del和插入突变1638—1660dup。     

Thalassemias in Sardinia: molecular pathology, phenotype-genotype correlation, and prevention

This article reviews the molecular bases of alpha- and beta-thalassemias in Sardinia. In addition, it describes the characteristics and the effects of a genetic program designed to prevent homozygous beta-thalassemia. In the large majority of the cases (95.7%), beta-thalassemia is caused by the nonsense mutation at codon 39, followed by frameshifts at codon 6 (2.1%). Homozygous beta-thalassemia most commonly results in thalassemia major, but in a small proportion this genotype produces milder forms referred to as thalassemia intermedia. The reasons for the attenuated forms were determined only in a small proportion of the cases. The reduced clinical severity was due to either of two factors: (a) There could be the presence of cytosine-thymidine (C----T) substitution of position - 158 G gamma, which is able to increase the G gamma chain output (as in homozygotes for frameshift at codon 6 or compound heterozygote for frameshift at codon 6 and codon 39 nonsense mutation). (b) Reduced clinical severity could be the result of co-inheritance of alpha-thalassemia in the form of two alpha-globin gene deletions of functional loss of the alpha 2-globin gene (homozygote for codon 39 nonsense mutation). The most prominent clinical form of alpha-thalassemia is hemoglobin H disease, which may result from the compound heterozygous state for deletion alpha 0- and alpha(+)-thalassemia (in 83.1% of cases) or deletion and nondeletion alpha-thalassemia (in 16.9%). Deletion Hb disease shows a milder clinical picture as compared to the nondeletion form. The most common nondeletion alpha-thalassemia is the ATG----ACG substitution at the initiation codon of the alpha 2 gene. Double heterozygotes of alpha (-alpha/-alpha) and beta-thalassemia or delta- and beta-thalassemia are relatively common and may cause confusion in carrier identification. The preventive program aimed to control beta-thalassemia is based on voluntary carrier screening., counselling, and prenatal diagnosis. Prenatal diagnosis is carried out by dot blot analysis with allelic specific oligonucleotides on amplified trophoblast DNA. This procedure gave very reliable results; in fact, no misdiagnosis has occurred so far. The preventive program was highly effective, resulting in a decline of the incidence of thalassemia major from 1:250 to 1:1,000 live births.     

A glycine 375-to-cysteine substitution in the transmembrane domain of the fibroblast growth factor receptor-3 in a newborn with achondroplasia

Achondroplasia, the most common form of chondrodysplasia, has been associated with mutations in the gene of the fibroblast growth factor receptor-3 (FGFR-3) on chromosome 4p. All 39 achondroplasia alleles studied so far carried point mutations which caused the same amino acid exchange, a substitution of glycine by arginine at position 380 (G380R) in the transmembrane domain of the receptor. We report on a newborn with achondroplasia who does not carry a G380R mutation but has a mutation causing substitution of a nearby glycine with a cysteine (G375C). This observation indicates allelic heterogeneity and confirms the role of mutations in the transmembrane domain of FGFR-3 in the pathogenesis of achondroplasia.     

Recurrence of a nonsense mutation in the conserved domain of SRY in a Brazilian patient with 46,XY gonadal dysgenesis

We tested a female patient with 46,XY karyotype and pure gonadal dysgenesis for the presence of the SRY gene and for mutations within the SRY conserved domain. A point mutation was identified at nucleotide position 209 with respect to the first ATG. The base substitution is a G-->A transition in the first nucleotide of codon 70 which changes a tryptophan (TGG) to a stop codon (TAG). Even though the father was not available for investigation we assumed that it is a de novo mutation, since it probably generates a nonfunctional truncated protein.     

Bilateral Wilms tumor in a boy with severe hypospadias and cryptochidism due to a heterozygous mutation in the WT1 gene

Mutations in the WT1 gene causing Wilms tumors were first reported in WAGR syndrome (Wilms tumor, Aniridia, Genitourinary malformation, mental Retardation) and Denys Drash syndrome (pseudohermaphroditism, Wilms tumor, nephropathy), but only in a few patients with hypospadias and cryptorchidism without other signs of Denys Drash (DDS) or WAGR syndrome WT1 mutations were identified. We report a boy, who was born in 1989 with hypospadias and bilateral cryptorchidism. Previous karyotyping and endocrine studies had ruled out any known cause of male pseudohermaphroditism. Subsequently, he developed a bilateral Wilms tumor, which was detected by palpation at the age of 15 months during a routine visit by the general pediatrician. Because of its extensive size, surgery and chemotherapy were needed for treatment. Analysis of the WT1 gene was performed 5 y after diagnosis and revealed a C to T transition in one allele generating a stop codon at codon 362 and subsequently leading to a truncated protein with loss of its ability to bind to DNA. No signs of DDS or WAGR syndrome are present in the boy. The work up of this patient and the so far known few comparable cases from the literature lead to the conclusion that in newborns with severe urogenital malformations not due to known chromosomal or endocrine disorders mutational screening of the WT1 gene should be performed, to evaluate the high risk of developing a Wilms tumor. We favor mutational screening in these patients as an easy tool for investigation, because in the future it will probably decrease the necessity of frequent control visits in patients without a WT1 mutation.     

Cystic fibrosis mutations in Romania

We have investigated the genotype in 32 children with cystic fibrosis from Romania. The diagnosis of cystic fibrosis was made on the basis of typical clinical findings and sweat electrolyte levels using the pilocarpine iontophoresis method. Conclusion Genetic analysis of 32 children with cystic fibrosis from Romania showed a 25% incidence of DeltaF508 mutation and a 64.5% incidence of unknown mutations, 5 other known mutations and 1 new mutation 1717–2(A > G). Received: 26 December 1995 and in revised form: 14 May 1996 / Accepted: 24 May 1996     

Novel deletion mutations of the DAX1 (NR0B1) gene in two Taiwanese families with X-linked adrenal hypoplasia congenita

OBJECTIVE: To analyze the DAX1 (NROB1) gene in Taiwanese families with adrenal hypoplasia congenita. PATIENTS AND METHODS: Two unrelated Taiwanese patients were followed up at our pediatric endocrine clinic. Both patients presented with adrenal crisis. One patient entered puberty spontaneously. However, arrest of puberty was noted in the following years and hypogonadotropic hypogonadism was confirmed by GnRH test. The other patient was still prepubertal. Sequencing of the DAX1 (NROB1) gene was carried out in both patients and their respective family members. RESULTS: Two different novel mutations were identified. The first patient had one base (G) deletion at nucleotide 159, resulting in a frame-shift and a premature stop codon at position 84. The other patient had one base (G) deletion at nucleotide 831, leading to a frameshift and a premature stop codon at position 371. Family studies revealed that their mothers and sisters were heterozygotes for the mutations while their maternal grandmothers did not carry the mutations. CONCLUSIONS: Two Taiwanese patients with adrenal hypoplasia congenita were detected to have novel mutations of the DAX1 (NR0B1) gene. Family studies suggested that such mutations resulted from de novo mutation of the DAX1 (NROB1) gene in their mothers. These data indicate that molecular analysis of the DAX1 (NR0B1) gene is important for the diagnosis and genetic counseling of children with primary adrenal insufficiency.     

Mutations in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance genes, and treatment outcomes in Ghanaian children with uncomplicated malaria

The association between the clinical outcome of chloroquine treatment and mutations in the putative Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene at codon 76 and multidrug resistance gene 1 (Pf mdr1) at codon 86 were investigated among 406 children with uncomplicated malaria presenting at five sentinel health centres in Ghana. Presence of mutations in isolates taken at pre-treatment and on day of recurrence of parasites was detected using PCR followed by RFLP techniques. The prevalence of Pfcrt T76 mutants was 80% at Hohoe, 46% at Navrongo, 98% at Tarkwa, 61% at Sunyani and 46% at Yendi. The prevalence of the mutant Pfmdr1 at Hohoe, Navrongo, Tarkwa, Sunyani and Yendi were 78, 58, 95, 53 and 42%, respectively. Significant association between the Pfcrt mutation and treatment outcome was observed at Hohoe and Sunyani (p < 0.05), but not at Navrongo, Tarkwa or Yendi (p > 0.05). Similarly, a statistical significant association between Pfmdr1 86 and treatment failures was observed at Hohoe and Sunyani (p < 0.05) but not at the other three sites. A positive correlation was found between mutant Pfcrt prevalence only and treatment failures with a Spearman's rho-value of 0.872 and a p-value = 0.027. All parasite isolates from samples taken at recrudescence from patients with chloroquine treatment failures were found to have both Pfcrt and Pfmdr mutations.     

Low TSH congenital hypothyroidism: identification of a novel mutation of the TSH beta-subunit gene in one sporadic case (C85R) and of mutation Q49stop in two siblings with congenital hypothyroidism

Isolated congenital hypothyroidism resulting from mutation of the TSH beta-subunit gene, has rarely been reported. In the present article, we report a new mutation (C85R) in exon 3 of the TSH beta-subunit gene in one sporadic case and the mutation Q49stop in two siblings with congenital hypothyroidism. The novel mutation is a T to C transition at codon 85, resulting in a change of cysteine to arginine (C85R) of the ss-subunit. Because the cysteine residues of all glycoproteins are highly conserved, this mutation is expected to result in conformational changes of the ss-subunit, rendering it incapable to form a functional heterodimer with the alpha-subunit. The second mutation described is a C to T transition resulting in a premature stop at codon 49 (Q49stop), leading to the formation of a truncated protein. Although the two siblings reported herein carried the same mutation, they had slightly modified clinical and biochemical phenotype. The mutation C85R and the previously described E11stop have, thus far, exclusively been detected in Greek patients. The Q49stop mutation initially detected in Greek patients was subsequently identified in an Egyptian girl and most recently in two Turkish siblings. These three reports possibly indicate the presence of a mutational hot spot on the TSH beta-subunit gene. Hence, with the novel mutation herein reported, a total of five mutations of the TSH beta-subunit gene are recognized as a cause of low-TSH congenital hypothyroidism worldwide.     

儿童肾母细胞瘤WT1基因突变四例报道

目的 研究儿童肾母细胞瘤患者WT1基因的突变类型及突变频率.方法应用聚合酶链反应(PCR)扩增出54例儿童肾母细胞瘤患者WT1基因全部10个外显子及其相邻内含子序列,经纯化后进行PCR产物直接测序.结果 4例患者WT1基因分别存在3个杂合无义突变及1个纯合错义突变.例1患者WT1基因7号外显子第1006位碱基A→T杂合突变,造成第336号氨基酸由赖氨酸转变为终止密码子,即K336X.例2患者WT1基因9号外显子第1168位碱基c→T杂合突变,造成第390号氨基酸由精氨酸转变为终止密码子,即R390X.例3患者WT1基因6号外显子第814位碱基G→T杂合突变,造成第272号氨基酸由谷氨酸转变为终止密码子,即E272X.例4患者WT1基因10号外显子第1228位碱基A→G纯合突变,造成第410号氨基酸由丝氨酸转变为甘氨酸,即S410G.结论 散发的中国儿童肾母细胞瘤患者WT1基因外显子突变的发生率与国外报道相近,检测到的4例突变患者中3例为无义突变、1例为错义突变.     

The prevalence of mitochondrial gene mutations in childhood diabetes in Japan.

To investigate the prevalence of mitochondrial DNA mutations among Japanese children with IDDM as well as in those with NIDDM, a total of 155 patients with IDDM and 30 patients with NIDDM who were younger than 15 years of age at onset were studied for the following mtDNA mutations: 1) the A-->G mutation at position 3243 of mitochondrial leucine transfer RNA (3243 mutation); 2) the G-->A mutation at position 3316 of mitochondrial leucine transfer RNA (3316 mutation), and 3) The T-->C mutation at position 3394 of the mitochondrial NADH dehydrogenase subunit (3394 mutation). None of the 155 IDDM patients had the 3243 mutation. Although two of the 155 IDDM patients had homoplasmy of 3316 and five had 3394 mutations, these frequencies were not significant compared with healthy controls. None of the 30 NIDDM patients had the 3243, 3316 or 3394 mutation. The presence of these mutations even in control subjects suggests that the effect of the 3316 or 3394 mutation on mitochondrial function is relatively mild. It seems that 3316 and 3394 mutations contribute to the manifestation of diabetes together with other genetic and/or environmental factors.     

p53 gene mutations in pediatric brain tumors

We investigated the frequency of p53 mutations in 47 pediatric brain tumors of various histologic subtypes that were collected over a period of 5 years. The specimens included 15 primitive neuroectodermal tumors (PNETs), 17 low grade astrocytomas, one anaplastic astrocytoma, three glioblastomas (GBMs), one mixed glial tumor, eight ependymomas, one choroid plexus carcinoma, and one gangliocytoma/ganglioneuroma. Mutations were identified by single strand conformation polymorphism analysis of exons 4–8 and verified by sequencing. Mutations were present in 2 of 3 cases of GBM, but not in 17 low grade astrocytomas (P = 0.02, Fisher's exact test). One GBM demonstrated a germline GGC to AGC transition (gly to ser) at codon 245 with loss of the wild-type allele. A second GBM contained a CGG to TGG transition (arg to trp) at codon 248, also with loss of the wildtype allele, but normal tissue was not available for comparison. In addition, one of 15 PNETs retained heterozygosity but demonstrated a somatic CGT to TGT transition (arg to cys) at codon 273. p53 mutations were absent in other histologic subtypes and in two cases with multiple primary cancers. These data are consistent with earlier findings that p53 mutations are rare in PNETs, which are primarily pediatric tumors. In contrast to adult gliomas, p53 mutations in pediatric gliomas appear restricted to the GBMs. The lack of p53 mutations in pediatric low grade astrocytomas suggests not only histological differences, but also a different molecular pathogenesis in adult and pediatric patients. © 1995 Wiley-Liss, Inc.     

Expression and prognostic examination of heat shock proteins (HSP 27, HSP 70, and HSP 90) in medulloblastoma

Expression of heat shock proteins (HSPs) is of prognostic significance in several tumor types. HSP expression levels were determined in medulloblastomas and tested whether HSPs expression was associated with prognostic parameters. Expression of antiapoptotic HSP 27, HSP 70, and HSP 90 was investigated by immunohistochemistry, on paraffin-embedded sections from 65 patients. Expression of HSPs was validated on internal vascular controls and by Western blotting analysis. Sample evaluation was based on the estimated percentage of HSP positive tumor cells. For survival analysis Kaplan-Meier method, for statistical analysis chi2 test, univariate analysis, and log rank test were applied. Expression of HSPs varied in medulloblastomas. On the basis of the average expression rate of HSPs, at HSP 27 and HSP 90 with a 10% cut off, and at HSP 70 with a 70% cut off 2 groups were created. The amount of expression of any of the HSP types was not significantly associated with known prognostic factors (age of patient, extent of resection, presence of metastasis) and histologic subtype. After an average follow-up period of 4.30 years, no significant difference was observed in survival depending on the expression of HSP 27 or HSP 70 or HSP 90. The high expression of HSPs indicates that these proteins are potential therapeutic targets.     

迟发型甲基丙二酸血症二家系的临床及基因突变分析

目的 对2个迟发型甲基丙二酸血症家系临床特点及MMACHC基因突变情况进行研究.方法 对2例患儿的临床特点、诊疗情况进行分析.应用聚合酶链反应与DNA直接测序法对2个家系共6名成员进行MMACHC基因突变检测.结果 2例患儿发病年龄分别为13岁和12岁,均以神经系统异常就诊,表现为运动、语言、智力发育落后及倒退.例1伴有贫血,例2表现为类似病毒性脑炎的症状,出现脑萎缩.尿有机酸分析示甲基丙二酸大量升高,检测血清同型半胱氨酸浓度也增高明显.经维生素B12治疗后症状迅速改善.随访至今,2例明显好转.例1被检出两个突变c.482G>A和c.658_660delAAG,前者来源于患儿母亲,后者来自父亲;例2被检出c.482G>A及c.609G>A,患儿父母分别携带这两个突变.所有突变均发生在第4外显子上,均为杂合子.c.482G>A为错义突变,导致精氨酸→谷氨酰胺(R161Q);c.609G>A为无义突变,由色氨酸→终止密码(W203X);c.658_660delAAG为缺失突变,造成一个赖氨酸的缺失.结论 迟发型甲基丙二酸血症临床表现多样性,两例患儿均以神经系统异常发病,1例伴有血液系统的改变.2例患儿均为维生素B12治疗有效型.基因突变均发生在第4外显子上,c.482G>A可能与迟发型有一定的相关性.