Andrew E. Timms

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.

A search for type 1 diabetes susceptibility genes in families from the United Kingdom

Genetic analysis of a mouse model of major histocompatability complex (MHC)-associated autoimmune type 1 (insulin-dependent) diabetes mellitus (IDDM) has shown that the disease is caused by a combination of a major effect at the MHC and at least ten other susceptibility loci elsewhere in the genome. A genome-wide scan of 93 affected sibpair families (ASP) from the UK (UK93) indicated a similar genetic basis for human type 1 diabetes, with the major genetic component at the MHC locus (IDDM1) explaining 34% of the familial clustering of the disease (λs = 2.5; Refs 3,4). In the present report, we have analysed a further 263 multiplex families from the same population (UK263) to provide a total UK data set of 356 ASP families (UK356). Only four regions of the genome outside IDDM1/MHC, which was still the only major locus detected, were not excluded at λs = 3 and lod = –2, of which two showed evidence of linkage: chromosome 10p13–p11 (maximum lod score (MLS) = 4.7, P = 3 × 10 –6, λs = 1.56) and chromosome 16q22–16q24 (MLS = 3.4, P = 6.5 × 10–5, λ s = 1.6). These and other novel regions, including chromosome 14q12–q21 and chromosome 19p13–19q13, could potentially harbour disease loci but confirmation and fine mapping cannot be pursued effectively using conventional linkage analysis. Instead, more powerful linkage disequilibrium-based and haplotype mapping approaches must be used; such data is already emerging for several type 1 diabetes loci detected initially by linkage.

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH.

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5p15. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

The Interleukin 1 Gene Cluster Contains a Major Susceptibility Locus for Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a common and highly heritable inflammatory arthropathy. Although the gene HLA-B27 is almost essential for the inheritance of the condition, it alone is not sufficient to explain the pattern of familial recurrence of the disease. We have previously demonstrated suggestive linkage of AS to chromosome 2q13, a region containing the interleukin 1 (IL-1) family gene cluster, which includes several strong candidates for involvement in the disease. In the current study, we describe strong association and transmission of IL-1 family gene cluster single-nucleotide polymorphisms and haplotypes with AS.

Support for the N -Methyl-D-Aspartate Receptor Hypofunction Hypothesis of Schizophrenia From Exome Sequencing in Multiplex Families

IMPORTANCE Schizophrenia is a complex genetic disorder demonstrating considerable heritability. Genetic studies have implicated many different genes and pathways, but much of the genetic liability remains unaccounted for. Investigation of genetic forms of schizophrenia will lead to a better understanding of the underlying molecular pathways, which will then enable targeted approaches for disease prevention and treatment. OBJECTIVE To identify new genetic factors strongly predisposing to schizophrenia in families with multiple affected individuals with schizophrenia. DESIGN We performed genome-wide array comparative genomic hybridization, linkage analysis, and exome sequencing in multiplex families with schizophrenia. SETTING Probands and their family members were recruited from academic medical centers. PARTICIPANTS We intended to identify rare disease-causing mutations in 5 large families where schizophrenia transmission appears consistent with single-gene inheritance. INTERVENTION Array comparative genomic hybridization was used to identify copy number variants, while exome sequencing was used to identify variants shared in all affected individuals and linkage analysis was used to further filter shared variants of interest. Analysis of select variants was performed in cultured cells to assess their functional consequences. MAIN OUTCOME MEASURES Rare inherited disease-related genetic mutations. RESULTS No segregating rare copy number variants were detected by array comparative genomic hybridization. However, in all 5 families, exome sequencing detected rare protein-altering variants in 1 of 3 genes associated with the N -methyl-D-aspartate (NMDA) receptor. One pedigree shared a missense and frameshift substitution of GRM5, encoding the metabotropic glutamate receptor subtype 5 (mGluR5), which is coupled to the NMDA receptor and potentiates its signaling; the frameshift disrupts binding to the scaffolding protein tamalin and increases mGluR5 internalization. Another pedigree transmitted a missense substitution in PPEF2, encoding a calmodulin-binding protein phosphatase, which we show influences mGluR5 levels. Three pedigrees demonstrated different missense substitutions within LRP1B, encoding a low-density lipoprotein receptor-related protein tied to both the NMDA receptor and located in a chromosome 2q22 region previously strongly linked to schizophrenia. CONCLUSIONS AND RELEVANCE Exome sequencing of multiplex pedigrees uncovers new genes associated with risk for developing schizophrenia and suggests potential novel therapeutic targets.

Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism

IMPORTANCE Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality. OBJECTIVE To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly. DESIGN, SETTING, AND PARTICIPANTS Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Childrens Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase-AKT (serine/threonine kinase)-mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. MAIN OUTCOMES AND MEASURES Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. RESULTS Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size. CONCLUSIONS AND RELEVANCE In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.

Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese

Objective: To test the association of interleukin 1 (IL1) gene family members with ankylosing spondylitis (AS), previously reported in Europid subjects, in an ethnically remote population. Methods: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms (SNPs) and the IL1RN.VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. Results: Association of alleles and genotypes of the markers IL1F10.3, IL1RN.4, and IL1RN.VNTR was observed with AS (p<0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN.4, and with the two-marker haplotype IL1RN.4–IL1RN.VNTR (both p = 0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 (D′ 0.4 to 0.9, p<0.01). Conclusions: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding. These authors contributed equally to the study.

Genetic testing for haemochromatosis in patients with chondrocalcinosis

Hereditary haemochromatosis (HH) is the most common lethal monogenic human disease, affecting roughly 1 in 300 white northern Europeans. Homozygosity for the C282Y polymorphism within the HFE gene causes more than 80% of cases, with compound heterozygosity of the C282Y and H63D polymorphism also increasing susceptibility to disease. The aim of this study was to determine the frequency of the C282Y and H63D polymorphisms in the disease, and to assess the risk of HH in heterozygotes for the C282Y polymorphism. 128 patients were recruited because of either radiographic chondrocalcinosis (at least bicompartmental knee disease or joints other than the knee involved) or CPPD pseudogout. Genotyping of the HFE C282Y and H63D mutations was performed using PCR/SSP and genotypes for the C282Y polymorphism confirmed by PCR/RFLP. Historical white European control data were used for comparison. Two previously undiagnosed C282Y homozygotes (1.6%), and 16 C282Y heterozygotes (12.5%), including four (3.1%) C282Y/H63D compound heterozygotes were identified. This represents a significant overrepresentation of C282Y homozygotes (relative risk 3.4, p=0.037), but the number of heterozygotes was not significantly increased. At a cost per test of £1 for each subject, screening all patients with chondrocalcinosis using the above ascertainment criteria costs only £64 for each case of haemochromatosis identified, clearly a highly cost effective test given the early mortality associated with untreated haemochromatosis. Routine screening for haemochromatosis in patients with appreciable chondrocalcinosis is recommended.

A novel rasopathy caused by recurrent de novo missense mutations in PPP1CB closely resembles Noonan syndrome with loose anagen hair

Noonan syndrome is a rasopathy caused by mutations in multiple genes encoding components of the RAS/MAPK pathway. Despite its variable phenotype, limited genotype–phenotype correlations exist. Noonan syndrome with loose anagen hair (NS‐LAH) is characterized by its distinctive hair anomalies, developmental differences, and structural brain abnormalities and is caused by a single recurrent missense SHOC2 mutation. SHOC2 forms a complex with protein phosphatase 1 (PP1C). Protein phosphatases counterbalance kinases and control activation of signaling proteins, such as the mitogen‐activated protein kinases of the RAS/MAPK pathway. Here we report four patients with de novo missense mutations in protein phosphatase one catalytic subunit beta (PPP1CB), sharing a recognizable phenotype. Three individuals had the recurrent PPP1CB c.146G>C, p.Pro49Arg mutation, the fourth had a c.166G>C, p.Ala56Pro change. All had relative or absolute macrocephaly, low‐set and posteriorly angulated ears, and developmental delay. Slow growing and/or sparse hair and/or an unruly hair texture was present in all. Three individuals had feeding difficulties requiring feeding tubes. One of two males had cryptorchidism, another had pectus excavatum. Short stature was present in three. A female with the recurrent mutation had a Dandy–Walker malformation and optic nerve hypoplasia. Mild ventriculomegaly occurred in all, cerebellar tonsillar ectopia was seen in two and progressed to Chiari 1 malformation in one individual. Based on the combination of phenotypic findings and PPP1CBs effect on RAF dephosphorylation within the RAS/MAPK pathway, this novel condition can be considered a rasopathy, most similar to NS‐LAH. Collectively, these mutations meet the standardized criteria for pathogenicity.