Alessandra Splendore

High mutation detection rate in TCOF1 among Treacher Collins syndrome patients reveals clustering of mutations and 16 novel pathogenic changes

Twenty‐eight families with a clinical diagnosis of Treacher Collins syndrome were screened for mutations in the 25 coding exons of TCOF1 and their adjacent splice junctions through SSCP and direct sequencing. Pathogenic mutations were detected in 26 patients, yielding the highest detection rate reported so far for this disease (93%) and bringing the number of known disease‐causing mutations from 35 to 51. This is the first report to describe clustering of pathogenic mutations. Thirteen novel polymorphic alterations were characterized, confirming previous reports that TCOF1 has an unusually high rate of single‐nucleotide polymorphisms (SNPs) within its coding region. We suggest a possible different mechanism leading to TCS or genetic heterogeneity for this condition, as we identified two families with no apparent pathogenic mutation in the gene. Furthermore, our data confirm the absence of genotype–phenotype correlation and reinforce that the apparent anticipation often observed in TCS families is due to ascertainment bias. Hum Mutat 16:315–322, 2000.

Screening of TCOF1 in patients from different populations: confirmation of mutational hot spots and identification of a novel missense mutation that suggests an important functional domain in the protein treacle

Treacher Collins syndrome (TCS, MIM *154500) is an autosomal dominant craniofacial disorder characterised by malar hypoplasia, micrognathia, downward slanting palpebral fissures, lower eyelid coloboma, malformed auricles, conductive deafness, and cleft palate. The estimated incidence is 1/50 000, with 60% of the cases resulting from sporadic mutations.1 There is marked phenotypic variability among patients, ranging from perinatal death because of a compromised airway to those that go undetected by medical examination. The gene underlying this condition, TCOF1 , was mapped in 19962 and since then mutation detection studies have concluded that: (1) the majority of pathogenic mutations are small deletions and insertions causing frameshifts that are predicted to result in a truncated protein; (2) mutations (both polymorphic and pathogenic) can be found throughout the 25 coding exons of the gene; (3) most mutations are family specific with the exception of a commonly occurring 5 bp deletion in exon 24 (found in approximately 16% of families); and (4) there is no correlation between type and/or localisation of the mutation and phenotypic expression.3–6 Furthermore, the observation that more than 50% of all described pathogenic mutations known to date are clustered in five exons (10, 15, 16, 23, and 24) has led to the hypothesis that these five exons are mutational hot spots, suggesting that any effort to identify mutations in TCOF1 would benefit from testing these five exons before extending the analysis to the rest of the gene.6 The protein encoded by the TCOF1 gene, treacle, has repetitive motifs encoded by exons 7-16, which are subject to phosphorylation by casein kinase II7 and a nucleolar localisation signal on its C-terminus.8 It bears weak similarity to a family of nucleolar phosphoproteins and its precise function remains unknown.9,10 In the present work, screening for TCOF1 mutations …

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.

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

BackgroundMolecular 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.MethodsT-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.ResultsThe 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.ConclusionNOTCH1 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 θ=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.

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.

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.