Impact of complex NOTCH1 mutations on survival in paediatric T-cell leukaemia.

BACKGROUND: Molecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether NOTCH1 mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases. METHODS: T-ALL subtypes, status of SIL-TAL1 fusion, ectopic expression of TLX3, and mutations in FBXW7, KRAS, PTEN and NOTCH1 were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test. RESULTS: The frequencies of mutations were 43.5% for NOTCH1, while FBXW7, KRAS and PTEN exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between NOTCH1 mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. SIL-TAL1 was identified as an adverse prognostic factor. NOTCH1 mutation status was not associated with outcome, while the presence of NOTCH1 complex mutations (indels) were associated with a longer overall survival (p = 0.031) than point mutations. CONCLUSION: NOTCH1 mutations alone or in combination with FBXW7 did not impact T-ALL prognosis. Nevertheless, complex NOTCH1 mutations appear to have a positive impact on OS and the SIL-TAL1 fusion was validated as a negative prognostic marker in our series of T-ALL.

Auriculo-condylar syndrome: mapping of a first locus and evidence for genetic heterogeneity.

Auriculo-condylar syndrome (ACS), an autosomal dominant disorder of first and second pharyngeal arches, is characterized by malformed ears ('question mark ears'), prominent cheeks, microstomia, abnormal temporomandibular joint, and mandibular condyle hypoplasia. Penetrance seems to be complete, but there is high inter- and intra-familial phenotypic variation, with no evidence of genetic heterogeneity. We herein describe a new multigeneration family with 11 affected individuals (F1), in whom we confirm intra-familial clinical variability. Facial asymmetry, a clinical feature not highlighted in other ACS reports, was highly prevalent among the patients reported here. The gene responsible for ACS is still unknown and its identification will certainly contribute to the understanding of human craniofacial development. No chromosomal rearrangements have been associated with ACS, thus mapping and positional cloning is the best approach to identify this disease gene. To map the ACS gene, we conducted linkage analysis in two large ACS families, F1 and F2 (F2; reported elsewhere). Through segregation analysis, we first excluded three known loci associated with disorders of first and second pharyngeal arches (Treacher Collins syndrome, oculo-auriculo-vertebral spectrum, and Townes-Brocks syndrome). Next, we performed a wide genome search and we observed evidence of linkage to 1p21.1-q23.3 in F2 (LOD max 3.01 at theta=0). Interestingly, this locus was not linked to the phenotype segregating in F1. Therefore, our results led to the mapping of a first locus of ACS (ACS1) and also showed evidence for genetic heterogeneity, suggesting that there are at least two loci responsible for this phenotype.

An atypical deletion of the Williams-Beuren syndrome interval implicates genes associated with defective visuospatial processing and autism.

BACKGROUND: During a genetic study of autism, a female child who met diagnostic criteria for autism spectrum disorder, but also exhibited the cognitive-behavioural profile (CBP) associated with Williams-Beuren syndrome (WBS) was examined. The WBS CBP includes impaired visuospatial ability, an overly friendly personality, excessive non-social anxiety and language delay. METHODS: Using array-based comparative genomic hybridisation (aCGH), a deletion corresponding to BAC RP11-89A20 in the distal end of the WBS deletion interval was detected. Hemizygosity was confirmed using fluorescence in situ hybridisation and fine mapping was performed by measuring the copy number of genomic DNA using quantitative polymerase chain reaction. RESULTS: The proximal breakpoint was mapped to intron 1 of GTF2IRD1 and the distal breakpoint lies 2.4-3.1 Mb towards the telomere. The subject was completely hemizygous for GTF2I, commonly deleted in carriers of the classic approximately 1.5 Mb WBS deletion, and GTF2IRD2, deleted in carriers of the rare approximately 1.84 Mb WBS deletion. CONCLUSION: Hemizygosity of the GTF2 family of transcription factors is sufficient to produce many aspects of the WBS CBP, and particularly implicate the GTF2 transcription factors in the visuospatial construction deficit. Symptoms of autism in this case may be due to deletion of additional genes outside the typical WBS interval or remote effects on gene expression at other loci.

GATA1 mutations in acute leukemia in children with Down syndrome.

It has been reported that somatic mutations in the X-linked GATA1 gene are present in hematological clonal disorders in children with Down syndrome (DS). We analyzed retrospective samples of DS children with acute myeloid leukemia, transient leukemia (TL), and myelodysplastic syndrome (MDS) to test whether the specificity of GATA1 mutations can be helpful in distinguishing these hematopoietic disorders. A total of 49 samples were subjected to GATA1 mutation screening by direct sequencing and denaturing polyacrylamide gel electrophoresis (PAGE). Mutations in exon 2 of GATA1 were detected in six of eight DS-AML M7 samples and in four of six DS-TL; no mutation was detected in 13 children with acute lymphoblastic leukemia (DS-ALL), 6 with DS-AML (M0, M2, and M5), 6 with DS-MDS and in 8 DS infants without hematological disorders and 2 children with AML M7 without DS. Blast cells proportion in the sample represented a critical aspect on the sensitivity of mutation detection in GATA1, and a combination of sequence analysis and PAGE is necessary to detect mutations when blast percentage is low. The absence of detected mutations in any of the DS-MDS cases raises the question whether MDS in DS children is an intermediate stage between TL and AML M7, as previously suggested.

TCOF1 mutation database: novel mutation in the alternatively spliced exon 6A and update in mutation nomenclature.

Recently, a novel exon was described in TCOF1 that, although alternatively spliced, is included in the major protein isoform. In addition, most published mutations in this gene do not conform to current mutation nomenclature guidelines. Given these observations, we developed an online database of TCOF1 mutations in which all the reported mutations are renamed according to standard recommendations and in reference to the genomic and novel cDNA reference sequences (www.genoma.ib.usp.br/TCOF1_database). We also report in this work: 1) results of the first screening for large deletions in TCOF1 by Southern blot in patients without mutation detected by direct sequencing; 2) the identification of the first pathogenic mutation in the newly described exon 6A; and 3) statistical analysis of pathogenic mutations and polymorphism distribution throughout the gene.

A functional SNP in the promoter region of TCOF1 is associated with reduced gene expression and YY1 DNA-protein interaction.

Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial malformation caused by null mutations in the TCOF1 gene. High inter and intra familial clinical variability, ranging from mild malar hypoplasia to perinatal death due to airway collapse is observed, but, to date, no genotype-phenotype correlation has been reported. Considering haploinsufficiency as the molecular mechanism underlying the disease, we have hypothesized that mutations in the promoter region of the gene, which has never been previously characterized, in trans with a pathogenic mutation, could modulate the phenotype. Therefore, the aims of the present study were to determine the TCOF1 gene's core promoter and to identify mutations in this region that could contribute to the phenotypic variation observed in this syndrome. We have delimitated the minimal promoter to a region of less than 150 bp, with 63% of identity among 5 different species. We screened 1.2 kbp of the TCOF1 5' flanking sequence in the DNA obtained from 21 patients and 51 controls and identified four new single nucleotide polymorphisms (SNPs), one of which (-346C>T), was proved to be functional, as it decreased the promoter activity by 38%. Electrophoretic mobility shift assay (EMSA) analysis demonstrated that the -346T allele impairs DNA-binding to the YY1 transcription factor. This promoter variant represents a candidate allele to explain the clinical variability in patients bearing TCS.

Parental origin of mutations in sporadic cases of Treacher Collins syndrome.

In some autosomal dominant conditions, there is a correlation between new mutations and paternal age, with new mutations arising almost exclusively in the male germ line. To test this hypothesis in Treacher Collins syndrome, we analyzed 22 sporadic cases, determining the parental origin of the pathogenic mutation in 10 informative families. Mutations were found to be of both paternal and maternal origin, without a detectable parental age effect, confirming that a paternal age effect is not universal to all autosomal dominant disorders. A discussion on the parental origin of mutations and paternal age effect in other diseases is included.

T-cell lymphoblastic leukemia in early childhood presents NOTCH1 mutations and MLL rearrangements.

T-cell acute lymphoblastic leukemia (T-ALL) may affect children in very early age. However, the critical events leading to this brief latency is still unclear. We used standard methods to explore NOTCH1 mutations and other specific molecular markers in 15 early childhood T-ALL cases. Most of them consisted of immature differentiation subtype. Despite being found in a lower frequency than that described for overall pediatric T-ALL, NOTCH1 alterations were the most frequent ones. Other alterations included MLL(+) (n=4), SIL-TAL1(+) (n=3), FLT3 mutation (n=1) and HOX11L2(+) (n=1). Our results suggest that NOTCH1 and MLL abnormalities are primary leukemogenic hits in early T-ALL.

Induced chromosome deletions cause hypersociability and other features of Williams-Beuren syndrome in mice.

The neurodevelopmental disorder Williams-Beuren syndrome is caused by spontaneous approximately 1.5 Mb deletions comprising 25 genes on human chromosome 7q11.23. To functionally dissect the deletion and identify dosage-sensitive genes, we created two half-deletions of the conserved syntenic region on mouse chromosome 5G2. Proximal deletion (PD) mice lack Gtf2i to Limk1, distal deletion (DD) mice lack Limk1 to Fkbp6, and the double heterozygotes (D/P) model the complete human deletion. Gene transcript levels in brain are generally consistent with gene dosage. Increased sociability and acoustic startle response are associated with PD, and cognitive defects with DD. Both PD and D/P males are growth-retarded, while skulls are shortened and brains are smaller in DD and D/P. Lateral ventricle (LV) volumes are reduced, and neuronal cell density in the somatosensory cortex is increased, in PD and D/P. Motor skills are most impaired in D/P. Together, these partial deletion mice replicate crucial aspects of the human disorder and serve to identify genes and gene networks contributing to the neural substrates of complex behaviours and behavioural disorders.

Detection of mutations in GATA1 gene using automated denaturing high-performance liquid chromatography and direct sequencing in children with Down syndrome.

Denaturing high-performance liquid chromatography (dHPLC) was developed to screen DNA variations by separating heteroduplex and homoduplex DNA fragments by ion-pair reverse-phase liquid chromatography. In this study, we have evaluated the dHPLC screening method and direct sequencing for the detection of GATA1 mutations in peripheral blood and bone marrow aspirates samples from children with Down syndrome (DS). Cases were ascertained consecutively as part of an epidemiological study of DS and hematological disorders in Brazil. A total of 130 samples corresponding to 115 children with DS were analysed using dHPLC and direct sequencing methods to detect mutations in GATA1 exons 2, 3 and 4 gene sequences. The overall detection rate of sequencing and dHPLC screening methods was similar. Twenty mutations were detected in exon 2 and one mutation in exon 3 (c.231_232 dupGT) sequences of acute megakaryoblastic leukemia and transient leukemia samples. Four GATA1 mutations were newly described [c.155C > G; c.156_178 del23 bp; c.29_30 del GG; c.182C > A and c.151A > T,c.153_162 del 10 bp). Out of four, three had single nucleotide change. In conclusion, our results indicate that dHPLC is an efficient and valuable tool for GATA1 mutational analysis.

Whole-genome array-CGH identifies novel contiguous gene deletions and duplications associated with developmental delay, mental retardation, and dysmorphic features.

Cytogenetic imbalances are the most frequently identified cause of developmental delay or mental retardation, which affect 1-3% of children and are often seen in conjunction with growth retardation, dysmorphic features, and various congenital anomalies. A substantial number of patients with developmental delay or mental retardation are predicted to have cytogenetic imbalances, but conventional methods for identifying these imbalances yield positive results in only a small fraction of these patients. We used microarray-based comparative genomic hybridization (aCGH) to study a panel of 20 patients predicted to have chromosomal aberrations based on clinical presentation of developmental delay or mental retardation, growth delay, dysmorphic features, and/or congenital anomalies. Previous G-banded karyotypes and fluorescence in situ hybridization results were normal for all of these patients. Using both oligonucleotide-based and bacterial artificial chromosome (BAC)-based arrays on the same panel of patients, we identified 10 unique deletions and duplications ranging in size from 280 kb to 8.3 Mb. The whole-genome oligonucleotide arrays identified nearly twice as many imbalances as did the lower-resolution whole-genome BAC arrays. This has implications for using aCGH in a clinical setting. Analysis of parental DNA samples indicated that most of the imbalances had occurred de novo. Moreover, seven of the 10 imbalances represented novel disorders, adding to an increasing number of conditions caused by large-scale deletions or duplications. These results underscore the strength of high-resolution genomic arrays in diagnosing cases of unknown genetic etiology and suggest that contiguous genomic alterations are the underlying pathogenic cause of a significant number of cases of developmental delay.

Transient neonatal myeloproliferative disorder without Down syndrome and detection of GATA1 mutation.

Transient myeloproliferative disorder is a form of self-limited leukemia that occurs almost exclusively in neonates with Down syndrome. The authors report an unusual case of a newborn without constitutional trisomy 21 who developed undifferentiated leukemia and subsequently achieved clinical and molecular remission without chemotherapy. Cytogenetics and molecular analysis have shown trisomy 21 and GATA1 mutation restricted to leukemic cells. G-to-T transversion was detected, which is predicted to result in a premature stop codon (c.119G>T; pGlu67X) in diagnosis samples. These findings emphasize that there must be a powerful interaction between GATA1 and trisomy 21 in leukemogenesis process.

Para que existem as regras de nomenclatura genética? / Why are there rules for genetic nomenclature?

Na comunicacão científica, precisamos respeitar os princípios de clareza, precisão, comunicabilidade e consistência. Para isso, existe um comitê de nomenclatura genética, cuja funcão é assegurar que cada gene humano tenha um nome e símbolo únicos que sejam usados consistentemente na literatura científica. Apesar dos esforcos, ainda encontramos textos onde o autor se refere a um gene usando um símbolo obsoleto, ou não faz a distincão adequada entre o gene e a proteína, prejudicando a compreensão por parte do leitor. Com isso em mente, apresento uma revisão das regras de nomenclatura de genes e diversos recursos disponíveis através da Internet para encontrar os nomes adequados dos genes, enfatizando aqueles envolvidos em câncer.

Influência da metileno-tetrahidrofolato redutase na patogênese das leucemias agudas infantis / Influence of the methylenetetrahydrofolate reductase in the pathogenesis of the childhood acute leukemia

As leucemias resultam de uma interação adversa entre gene-ambiente, com susceptibilidade condicionada, pelo menos em parte, por polimorfismos de múltiplos genes. Como a metilenotetrahidrofolato redutase (MTHFR) possui papel chave no metabolismo do folato, alterações na sua atividade (resultante de polimorfismos) ou alterações na suplementação de folato, podem influenciar na síntese, reparo e metilação de DNA. Neste estudo, determinamos a frequência dos polimorfismos 677C maior que T e 1298A maior que C do gene MTHFR em 177 crianças menores de 16 anos com leucemias agudas (LA) de diversas regiões brasileiras e em 249 amostras de indivíduos normais como o grupo controle. O método utilizado para genotipagem foi PCR-RFLP. As frequências dos genótipos 677CC, 677CT e 677TT foram 51 por cento, 40 por cento e 9 por cento nos controles, 57 por cento, 33 por cento e 10 por cento para os casos de leucemia linfoblástica aguda (LLA), e 49 por cento, 39 por cento e 12 por cento para os casos de leucemia mielóide aguda (LMA), respectivamente. Já as frequências dos genótipos 1298AA, 1298AC e 1298CC foram respectivamente 62 por cento, 31 por cento e 7 por cento nos controles, 51 por cento, 41 por cento e 8 por cento nos casos de LLA e, 67 por cento, 31 por cento e 2 por cento nos casos de LMA, respectivamente. Quando comparamos casos de LLA com os controles, para o polimorfismo 677C maoir que T, encontramos um OR igual a 0,7 (0,5 - 1,2) e OR igual a 1,0 (0,4 - 2,1) para os genótipos CT e TT, respectivamente. Para o polimorfismo 1298A maior que C, o OR foi 1,6 (1,0 - 2,5) e 1,3 (0,6-3,0) para os genótipos AC e CC, respectivamente. Para os casos de LMA, o OR foi 1,0 (0,5 - 2,1) para o genótipo CT, e 1,3 (0,5 - 3,9) para o genótipo TT. Para o genótipo AC, encontramos um OR igual a 0,9 (0,4 - 1,9); para o genótipo CC, o OR foi 0,3 (0,04 - 2,3). Não houve diferenças entre as frequências do MTHFR 677C maior que T e1298A maior que C em ambos os grupos na análise global. Os resultados indicam que MTHFR 677C maior que T e 1298A maior que C não conferem efeito protetor contra a LA infantil.

Sindrome de Treacher Collins: aspectos clinicos, geneticos e moleculares / Treacher Collins syndrome: clinical, genetic and molecular aspects

A sindrome de Treacher Collins (STC) e um disturbio do desenvolvimento craniofacial de heranca autossomica dominante que afeta 1 em 50.000 recem-nascidos, sendo que aproximadamente 60 por cento dos casos sao resultantes de mutacoes novas...