Ella J. Wilkins

Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

Novel RUNX1 mutations in familial platelet disorder with enhanced risk for acute myeloid leukemia: clues for improved identification of the FPD/AML syndrome.

Novel RUNX1 mutations in familial platelet disorder with enhanced risk for acute myeloid leukemia: clues for improved identification of the FPD/AML syndrome

Multiple Sclerosis Susceptibility-Associated SNPs Do Not Influence Disease Severity Measures in a Cohort of Australian MS Patients

Recent association studies in multiple sclerosis (MS) have identified and replicated several single nucleotide polymorphism (SNP) susceptibility loci including CLEC16A, IL2RA, IL7R, RPL5, CD58, CD40 and chromosome 12q13–14 in addition to the well established allele HLA-DR15. There is potential that these genetic susceptibility factors could also modulate MS disease severity, as demonstrated previously for the MS risk allele HLA-DR15. We investigated this hypothesis in a cohort of 1006 well characterised MS patients from South-Eastern Australia. We tested the MS-associated SNPs for association with five measures of disease severity incorporating disability, age of onset, cognition and brain atrophy. We observed trends towards association between the RPL5 risk SNP and time between first demyelinating event and relapse, and between the CD40 risk SNP and symbol digit test score. No associations were significant after correction for multiple testing. We found no evidence for the hypothesis that these new MS disease risk-associated SNPs influence disease severity.

It wasn't a disaster or anything: Parents' experiences of their child's uncertain chromosomal microarray result.

Chromosomal microarray is an increasingly utilized diagnostic test, particularly in the pediatric setting. However, the clinical significance of copy number variants detected by this technology is not always understood, creating uncertainties in interpreting and communicating results. The aim of this study was to explore parents’ experiences of an uncertain microarray result for their child. This research utilized a qualitative approach with a phenomenological methodology. Semi‐structured interviews were conducted with nine parents of eight children who received an uncertain microarray result for their child, either a 16p11.2 microdeletion or 15q13.3 microdeletion. Interviews were transcribed verbatim and thematic analysis was used to identify themes within the data. Participants were unprepared for the abnormal test result. They had a complex perception of the extent of their childs condition and a mixed understanding of the clinical relevance of the result, but were accepting of the limitations of medical knowledge, and appeared to have adapted to the result. The test result was empowering for parents in terms of access to medical and educational services; however, they articulated significant unmet support needs. Participants expressed hope for the future, in particular that more information would become available over time. This research has demonstrated that parents of children who have an uncertain microarray result appeared to adapt to uncertainty and limited availability of information and valued honesty and empathic ongoing support from health professionals. Genetic health professionals are well positioned to provide such support and aid patients’ and families’ adaptation to their situation as well as promote empowerment.

Poor prognosis in familial acute myeloid leukaemia with combined biallelic CEBPA mutations and downstream events affecting the ATM, FLT3 and CDX2 genes

CEBPA is implicated in the delicate balance between myeloid cell proliferation and differentiation. Loss of CEBPA gene function, through biallelic point mutation, has been identified in up to 14% of patients with sporadic acute myeloid leukaemia (AML), and germline mutations have been reported in several families with autosomal dominant inheritance of AML and eosinophilia (Pabst et al, 2001; Gombart et al, 2002; Smith et al, 2004; Sellick et al, 2005). In both sporadic and familial AML, CEBPA point mutations are commonly biallelic (one germline and one acquired in the familial cases), consisting of a dominant negative N-terminal and a loss of DNA-binding C-terminal leucine zipper mutation (Preudhomme et al, 2002; Barjesteh van Waalwijk van DoornKhosrovani et al, 2003). Interestingly, in families carrying CEBPA mutations, the germline mutation is always N-terminal, whilst the acquired mutation is C-terminal, suggesting a role in leukaemia predisposition for the N-terminal mutation only. In 1978 a large familial aggregation of AML, with 14 diagnosed individuals over four generations, was reported (Gunz et al, 1978). The family is of Irish and English extraction, with the majority of documented members living in and around Sydney, Australia at the time of diagnosis. Contact with the family was lost in 1980, however at that time an additional three leukaemia cases had been diagnosed. Figure 1A shows the affected arm of the pedigree with numbering as in the original publication. Anticipation is notable: while diagnosis occurred at 66 years for individual II-13, the mean age at diagnosis decreased to 20 and 11 years in generations III and IV, respectively. Of the 17 diagnosed leukaemia cases, seven occurred in children under the age of 10 years, including one aged just three months (V-63a) whose mother (IV-89) had AML through the pregnancy. In addition, only five of the 17 individuals with leukaemia were known to be alive in 1980, three of whom were diagnosed shortly before contact was lost. The majority of the patients documented in the original article died within weeks to months of diagnosis, despite various treatment regimens (1968 onwards) including prednisone, cyclophosphamide and vincristine (Gunz et al, 1978). This suggests that the leukaemia predisposition in this family is both incredibly precocious and highly aggressive. Due to the high morbidity of disease, and the length of time that has lapsed since contact was lost, only one somatic DNA sample was available for analysis; individual III-45. This individual presented at age 50 years with a 2-month history of worsening health with unexplained fevers and colds. Peripheral blood showed 406 · 10/l leucocytes with 99% blasts and a platelet count of 74 · 10/l, and cytogenetic analysis del(11q23) in all analysable metaphases. He was given a diagnosis of AML and treated with leucapheresis followed by chemotherapy including daunorubicin and cytosine arabinoside. One year after diagnosis he died from a relapse. Additional sample information is provided in Appendix S1. DNA sequencing of CEBPA revealed a single heterozygous base pair deletion (c.68delC) (Fig 1B). The affected poly-C string is a mutational hot spot and this identical mutation has been reported in both familial and sporadic AML cases (Barjesteh van Waalwijk van Doorn-Khosrovani et al, 2003; Smith et al, 2004). It leads to a p.P22fsX158 frameshift, causing truncation of the wild type 42 kDa protein and increased production of a 30 kDa dominant negative isoform (Pabst et al, 2001; Gombart et al, 2002). We also identified a probable acquired three base pair duplication in the C-terminal end of CEBPA (c.937_939dupAAG) (Fig 1C) in this same individual. The mutation was found in 25% of cloned alleles, indicative of it being an acquired mutation and only present in a proportion of peripheral blood cells. Long-range polymerase chain reaction demonstrated that the wild type CEBPA allele was affected. This mutation has previously been described in patients with sporadic AML and leads to an in-frame p.K313dup that is predicted to disrupt the leucine zipper domain (bZIP) (Gombart et al, 2002). (Method and primer sequence information is provided in Appendix S1). As discussed in the original publication (Gunz et al, 1978), the penetrance of the AML predisposition in this family differs across the pedigree, with the branch studied here having the highest penetrance. In addition, the high morbidity and aggressive nature of the disease in this family is in contrast to the good prognosis normally associated with sporadic and familial CEBPA mutations (Preudhomme et al, 2002; Smith et al, 2004; Sellick et al, 2005). Environmental as well as germline and somatic genetic events in this current family might be contributing to the high penetrance, early onset and mortality observed. To further investigate a possible additional genetic contribution, we performed a genomic single nucleotide polymorphism chip copy number analysis. Excluding copy number aberrations that were present in normal unrelated DNA samples of European, Asian or American ancestry (Redon et al, 2006), and considered to be polymorphic, we Correspondence

A DNA resequencing array for genes involved in Parkinson’s disease

Parkinsons disease (PD) is aetiologically complex with both familial and sporadic forms. Familial PD results from rare, highly penetrant pathogenic mutations whereas multiple variants of low penetrance may contribute to the risk of sporadic PD. Common variants implicated in PD risk appear to explain only a minor proportion of the familial clustering observed in sporadic PD. It is therefore plausible that combinations of rare and/or common variants in genes already implicated in disease pathogenesis may help to explain the genetic basis of PD. We have developed a CustomSeq Affymetrix resequencing array to enable high-throughput sequencing of 13 genes (44 kb) implicated in the pathogenesis of PD. Using the array we sequenced 269 individuals, including 186 PD patients and 75 controls, achieving an overall call rate of 96.5% and 93.6%, for two respective versions of the array, and >99.9% accuracy for five samples sequenced by capillary sequencing in parallel. We identified modest associations with common variants in SNCA and LRRK2 and a trend suggestive of an overrepresentation of rare variants in cases compared to controls for several genes. We propose that this technology offers a robust and cost-effective alternative to targeted sequencing using traditional sequencing methods, and here we demonstrate the potential of this approach for either routine clinical investigation or for research studies aimed at understanding the genetic aetiology of PD.

Multiplex ligation-dependent probe amplification (MLPA) genotyping assay for mouse models of down syndrome.

The Ts65Dn mouse is the most widely investigated segmentally trisomic mouse model of Down syndrome. Quantitative PCR based methods are the preferred way of detecting the trisomic segment for genotyping purposes. However, identification of a 1.5 fold difference in target DNA is at the limit of detection of most quantitative PCR based methods, and in practice this can lead to difficulties in assigning genotypes. We report a 100% accurate multiplex ligation-dependent probe amplification (MLPA) assay for genotyping the Ts65Dn mouse that is also applicable to all other segmentally trisomic mouse models of Down syndrome.

RUNX1 mutations are rare in chronic phase polycythaemia vera.

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