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Pathogenic short tandem repeat (STR) expansions cause over 20 neurodegenerative diseases. To determine the contribution of STRs in sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), we used ExpansionHunter, REviewer, and polymerase chain reaction validation to assess 21 neurodegenerative disease-associated STRs in whole-genome sequencing data from 608 patients with sporadic ALS, 68 patients with sporadic FTD, and 4703 matched controls. We also propose a data-derived outlier detection method for defining allele thresholds in rare STRs. Excluding C9orf72 repeat expansions, 17.6% of clinically diagnosed ALS and FTD cases had at least one expanded STR allele reported to be pathogenic or intermediate for another neurodegenerative disease. We identified and validated 162 disease-relevant STR expansions in C9orf72 (ALS/FTD), ATXN1 [spinal cerebellar ataxia type 1 (SCA1)], ATXN2 (SCA2), ATXN8 (SCA8), TBP (SCA17), HTT (Huntington’s disease), DMPK [myotonic dystrophy type 1 (DM1)], CNBP (DM2), and FMR1 (fragile-X disorders). Our findings suggest clinical and pathological pleiotropy of neurodegenerative disease genes and highlight their importance in ALS and FTD.

Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.

RLIM, also known as RNF12, is an X-linked E3 ubiquitin ligase acting as a negative regulator of LIM-domain containing transcription factors and participates in X-chromosome inactivation (XCI) in mice. We report the genetic and clinical findings of 84 individuals from nine unrelated families, eight of whom who have pathogenic variants in RLIM (RING finger LIM domain-interacting protein). A total of 40 affected males have X-linked intellectual disability (XLID) and variable behavioral anomalies with or without congenital malformations. In contrast, 44 heterozygous female carriers have normal cognition and behavior, but eight showed mild physical features. All RLIM variants identified are missense changes co-segregating with the phenotype and predicted to affect protein function. Eight of the nine altered amino acids are conserved and lie either within a domain essential for binding interacting proteins or in the C-terminal RING finger catalytic domain. In vitro experiments revealed that these amino acid changes in the RLIM RING finger impaired RLIM ubiquitin ligase activity. In vivo experiments in rlim mutant zebrafish showed that wild type RLIM rescued the zebrafish rlim phenotype, whereas the patient-specific missense RLIM variants failed to rescue the phenotype and thus represent likely severe loss-of-function mutations. In summary, we identified a spectrum of RLIM missense variants causing syndromic XLID and affecting the ubiquitin ligase activity of RLIM, suggesting that enzymatic activity of RLIM is required for normal development, cognition and behavior.

Most genes associated with neurodevelopmental disorders (NDDs) were identified with an excess of de novo mutations (DNMs) but the significance in case-control mutation burden analysis is unestablished. Here, we sequence 63 genes in 16,294 NDD cases and an additional 62 genes in 6,211 NDD cases. By combining these with published data, we assess a total of 125 genes in over 16,000 NDD cases and compare the mutation burden to nonpsychiatric controls from ExAC. We identify 48 genes (25 newly reported) showing significant burden of ultra-rare (MAF 0.01%) gene-disruptive mutations (FDR 5%), six of which reach family-wise error rate (FWER) significance (p 1.25E-06). Among these 125 targeted genes, we also reevaluate DNM excess in 17,426 NDD trios with 6,499 new autism trios. We identify 90 genes enriched for DNMs (FDR 5%; e.g., GABRG2 and UIMC1); of which, 61 reach FWER significance (p 3.64E-07; e.g., CASZ1). In addition to doubling the number of patients for many NDD risk genes, we present phenotype-genotype correlations for seven risk genes (CTCF, HNRNPU, KCNQ3, ZBTB18, TCF12, SPEN, and LEO1) based on this large-scale targeted sequencing effort.

We report two unrelated families with multigenerational nonsyndromic intellectual disability segregating with a recurrent de novo missense variant (c.1543C>T:p.Leu515Phe) in the alkali cation/proton exchanger gene SLC9A7 (also commonly referred to as NHE7). SLC9A7 is located on human X chromosome at Xp11.3 and has not yet been associated with a human phenotype. The gene is widely transcribed, but especially abundant in brain, skeletal muscle and various secretory tissues. Within cells, SLC9A7 resides in the Golgi apparatus, with prominent enrichment in the trans-Golgi network (TGN) and post-Golgi vesicles. In transfected Chinese hamster ovary AP-1 cells, the Leu515Phe mutant protein was correctly targeted to the TGN/post-Golgi vesicles, but its N-linked oligosaccharide maturation as well as that of a co-transfected secretory membrane glycoprotein, vesicular stomatitis virus G (VSVG) glycoprotein, was reduced compared to cells co-expressing SLC9A7 wild-type and VSVG. This correlated with alkalinization of the TGN/post-Golgi compartments, suggestive of a gain-of-function. Membrane trafficking of glycosylation-deficient Leu515Phe and co-transfected VSVG to the cell surface, however, was relatively unaffected. Mass spectrometry analysis of patient sera also revealed an abnormal N-glycosylation profile for transferrin, a clinical diagnostic marker for congenital disorders of glycosylation. These data implicate a crucial role for SLC9A7 in the regulation of TGN/post-Golgi pH homeostasis and glycosylation of exported cargo which may underlie the cellular pathophysiology and neurodevelopmental deficits associated with this particular nonsyndromic form of X-linked intellectual

Familial Adult Myoclonic Epilepsy (FAME) is characterised by cortical myoclonic tremor usually from the second decade of life and overt myoclonic or generalised tonic-clonic seizures. Four independent loci have been implicated in FAME on chromosomes (chr) 2, 3, 5 and 8. Using whole genome sequencing and repeat primed PCR, we provide evidence that chr2-linked FAME (FAME2) is caused by an expansion of an ATTTC pentamer within the first intron of STARD7. The ATTTC expansions segregate in 158/158 individuals typically affected by FAME from 22 pedigrees including 16 previously reported families recruited worldwide. RNA sequencing from patient derived fibroblasts shows no accumulation of the AUUUU or AUUUC repeat sequences and STARD7 gene expression is not affected. These data, in combination with other genes bearing similar mutations that have been implicated in FAME, suggest ATTTC expansions may cause this disorder, irrespective of the genomic locus involved., Familial cortical myoclonic tremor (FAME) has so far been mapped to regions on chromosome 2, 3, 5 and 8 and pentameric repeat expansions in SAMD12 were identified as cause of FAME1. Here, Corbett et al. identify ATTTT/ATTTC repeat expansions in intron 1 of STARD7 in individuals with FAME2.”

Next generation genomic technologies have made a significant contribution to the understanding of the genetic architecture of human neurodevelopmental disorders. Copy number variants (CNVs) play an important role in the genetics of intellectual disability (ID). For many CNVs, and copy number gains in particular, the responsible dosage-sensitive gene(s) have been hard to identify. We have collected 18 different interstitial microduplications and one microtriplication of Xq25. There were 15 affected individuals from 6 different families and 13 singleton cases, 28 affected males in total. The critical overlapping region involved the STAG2 gene, which codes for a subunit of the cohesin complex that regulates cohesion of sister chromatids and gene transcription. We demonstrate that STAG2 is the dosage-sensitive gene within these CNVs, as gains of STAG2 mRNA and protein dysregulate disease-relevant neuronal gene networks in cells derived from affected individuals. We also show that STAG2 gains result in increased expression of OPHN1, a known X-chromosome ID gene. Overall we define a novel cohesinopathy due to copy number gain of Xq25 and STAG2 in particular. ispartof: Human Molecular Genetics vol:24 issue:25 pages:7171-81 ispartof: location:England status: published

International audience; To identify genetic causes of intellectual disability (ID), we screened a cohort of 986 individuals with moderate to severe ID for variants in 565 known or candidate ID-associated genes using targeted next-generation sequencing. Likely pathogenic rare variants were found in ß11% of the cases (113 variants in 107/986 individuals: ß8% of the individuals had a likely pathogenic loss-of-function [LoF] variant, whereas ß3% had a known pathogenic missense variant). Variants in SETD5, ATRX, CUL4B, MECP2, and ARID1B were the most common causes of ID. This study assessed the value of sequencing a cohort of probands to provide a molecular diagnosis of ID, without the availability of DNA from both parents for de novo sequence analysis. This modeling is clinically relevant as 28% of all UK families with dependent children are single parent households. In conclusion, to diagnose patients with ID in the absence of parental DNA, we recommend investigation of all LoF variants in known genes that cause ID and assessment of a limited list of proven pathogenic missense variants in these genes. This will provide 11% additional diagnostic yield beyond the 10%–15% yield from array CGH alone.

Item does not contain fulltext Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities.

Background Trichothiodystrophy (TTD) is a group of rare autosomal recessive disorders that variably affect a wide range of organs derived from the neuroectoderm. The key diagnostic feature is sparse, brittle, sulfur deficient hair that has a ‘tiger-tail’ banding pattern under polarising light microscopy. Patients and methods We describe two male cousins affected by TTD associated with microcephaly, profound intellectual disability, sparse brittle hair, aged appearance, short stature, facial dysmorphism, seizures, an immunoglobulin deficiency, multiple endocrine abnormalities, cerebellar hypoplasia and partial absence of the corpus callosum, in the absence of cellular photosensitivity and ichthyosis. Obligate female carriers showed 100% skewed X-chromosome inactivation. Linkage analysis and Sanger sequencing of 737 X-chromosome exons and whole exome sequencing was used to find the responsible gene and mutation. Results Linkage analysis localised the disease allele to a 7.75 Mb interval from Xq23–q25. We identified a nonsense mutation in the highly conserved RNF113A gene (c.901 C>T, p.Q301*). The mutation segregated with the disease in the family and was not observed in over 100 000 control X chromosomes. The mutation markedly reduced RNF113A protein expression in extracts from lymphoblastoid cell lines derived from the affected individuals. Conclusions The association of RNF113A mutation with non-photosensitive TTD identifies a new locus for these disorders on the X chromosome. The extended phenotype within this family includes panhypopituitarism, cutis marmorata and congenital short oesophagus.

Although de novo missense mutations have been predicted to account for more cases of autism than gene-truncating mutations, most research has focused on the latter. We identified the properties of de novo missense mutations in patients with neurodevelopmental disorders (NDDs) and highlight 35 genes with excess missense mutations. Additionally, 40 amino acid sites were recurrently mutated in 36 genes, and targeted sequencing of 20 sites in 17,688 patients with NDD identified 21 new patients with identical missense mutations. One recurrent site substitution (p.A636T) occurs in a glutamate receptor subunit, GRIA1. This same amino acid substitution in the homologous but distinct mouse glutamate receptor subunit Grid2 is associated with Lurcher ataxia. Phenotypic follow-up in five individuals with GRIA1 mutations shows evidence of specific learning disabilities and autism. Overall, we find significant clustering of de novo mutations in 200 genes, highlighting specific functional domains and synaptic candidate genes important in NDD pathology.

Item does not contain fulltext Gene-disruptive mutations contribute to the biology of neurodevelopmental disorders (NDDs), but most of the related pathogenic genes are not known. We sequenced 208 candidate genes from >11,730 cases and >2,867 controls. We identified 91 genes, including 38 new NDD genes, with an excess of de novo mutations or private disruptive mutations in 5.7% of cases. Drosophila functional assays revealed a subset with increased involvement in NDDs. We identified 25 genes showing a bias for autism versus intellectual disability and highlighted a network associated with high-functioning autism (full-scale IQ >100). Clinical follow-up for NAA15, KMT5B, and ASH1L highlighted new syndromic and nonsyndromic forms of disease.

The clinically and genetically heterogenous foetal akinesias have low rates of genetic diagnosis. Exome sequencing of two siblings with phenotypic lethal multiple pterygium syndrome identified compound heterozygozity for a known splice site mutation (c.691+2T>C) and a novel missense mutation (c.956A>G; p.His319Arg) in glycogen branching enzyme 1 (GBE1). GBE1 mutations cause glycogen storage disease IV (GSD IV), including a severe foetal akinesia sub-phenotype. Re-investigating the muscle pathology identified storage material, consistent with GSD IV, which was confirmed biochemically. This study highlights the power of exome sequencing in genetically heterogeneous diseases and adds multiple pterygium syndrome to the phenotypic spectrum of GBE1 mutation.

The clinical significance of chromosomal microdeletions and microduplications was predicted based on their gene content, de novo or familial inheritance and accumulated knowledge recorded on public databases. A patient group comprised of 247 cases with epilepsy and its common co-morbidities of developmental delay, intellectual disability, autism spectrum disorders, and congenital abnormalities was reviewed prospectively in a diagnostic setting using a standardized oligo-array CGH platform. Seventy-three (29.6%) had copy number variations (CNVs) and of these 73 cases, 27 (37.0%) had CNVs that were likely causative. These 27 cases comprised 10.9% of the 247 cases reviewed. The range of pathogenic CNVs associated with seizures was consistent with the existence of many genetic determinants for epilepsy. © 2012 Wiley Periodicals, Inc.

Intellectual disability (ID) is a clinically complex and heterogeneous disorder, which has variable severity and may be associated with additional dysmorphic, metabolic, neuromuscular or psychiatric features. Although many coding variants have been implicated in ID, identification of pathogenic non-coding regulatory variants has only been achieved in a few cases to date. We identified a duplication of a guanine on chromosome X, NC_000023.10:g.69665044dupG 7 nucleotides upstream of the translational start site in the 5' untranslated region (UTR) of the known ID gene DLG3 that encodes synapse-associated protein 102 (SAP102). The dupG variant segregated with affected status in a large multigenerational family with non-syndromic X-linked ID and was predicted to disrupt folding of the mRNA. When tested on blood cells from the affected individuals, DLG3 mRNA levels were not altered, however, DLG3/SAP102 protein levels were. We also showed by dual luciferase reporter assay that the dupG variant interfered with translation. All currently known pathogenic DLG3 variants are predicted to be null, however the dupG variant likely leads to only a modest reduction of SAP102 levels accounting for the milder phenotype seen in this family. Refereed/Peer-reviewed

Summary Background Bilateral perisylvian polymicrogyria (BPP), the most common form of regional polymicrogyria, causes the congenital bilateral perisylvian syndrome, featuring oromotor dysfunction, cognitive impairment, and epilepsy. The causes of BPP are heterogeneous, but only a few genetic causes have been reported. The aim of this study was to identify additional genetic causes of BPP and characterise their frequency in this population. Methods Children (aged ≤18 years) with polymicrogyria were enrolled into our research programme from July, 1980, to October, 2015, at two centres (Florence, Italy, and Seattle, WA, USA). We obtained samples (blood and saliva) throughout this period at both centres and did whole-exome sequencing on DNA from eight trios (two parents and one affected child) with BPP in 2014. After the identification of mosaic PIK3R2 mutations in two of these eight children, we performed targeted screening of PIK3R2 by two methods in a cohort of 118 children with BPP. First, we performed targeted sequencing of the entire PIK3R2 gene by single molecule molecular inversion probes (smMIPs) on 38 patients with BPP with normal to large head size. Second, we did amplicon sequencing of the recurrent PIK3R2 mutation (Gly373Arg) in 80 children with various types of polymicrogyria including BPP. One additional patient had clinical whole-exome sequencing done independently, and was included in this study because of the phenotypic similarity to our cohort. Findings We identified a mosaic mutation (Gly373Arg) in a regulatory subunit of the PI3K-AKT-mTOR pathway, PIK3R2 , in two children with BPP. Of the 38 patients with BPP and normal to large head size who underwent targeted next-generation sequencing by smMIPs, we identified constitutional and mosaic PIK3R2 mutations in 17 additional children. In parallel, one patient had the recurrent PIK3R2 mutation identified by clinical whole-exome sequencing. Seven of these 20 patients had BPP alone, and 13 had BPP in association with features of the megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome. 19 patients had the same mutation (Gly373Arg), and one had a nearby missense mutation (Lys376Glu). Mutations were constitutional in 12 patients and mosaic in eight patients. In patients with mosaic mutations, we noted substantial variation in alternate (mutant) allele levels, ranging from ten (3%) of 377 reads to 39 (37%) of 106 reads, equivalent to 5–73% of cells analysed. Levels of mosaicism varied from undetectable to 37 (17%) of 216 reads in blood-derived DNA compared with 2030 (29%) of 6889 reads to 275 (43%) of 634 reads in saliva-derived DNA. Interpretation Constitutional and mosaic mutations in the PIK3R2 gene are associated with developmental brain disorders ranging from BPP with a normal head size to the MPPH syndrome. The phenotypic variability and low-level mosaicism, which challenge conventional molecular methods, have important implications for genetic testing and counselling. Funding US National Institutes of Health.

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. Refereed/Peer-reviewed

X-linked intellectual disability (XLID), defined as clinical ID combined with a pedigree consistent with X-linked inheritance, is a genetically heterogeneous condition that affects more than 10% of males with ID. Currently there are at least 92 genes known to cause XLID,1–3 yet a large proportion of XLID cases remain unexplained, as each of the XLID genes identified so far only accounts for a small fraction (< 1%) of affected individuals. Given that about one third of mutations affect gene expression levels,4 we reasoned that transcriptome profiling of lymphoblast cell lines from XLID patients may highlight genes harboring disease-causing mutations and may be an efficient follow-up method for rare sequence variants of unknown functional significance. We analyzed expression profiles of lymphoblast cell lines from 64 XLID patients, including 13 cases that were part of a recent X-chromosome exon re-sequencing study5 (Supplementary Methods, Supplementary Table 1). We found polyglutamine-binding protein 1 (PQBP1), a gene previously implicated in XLID,6,7 to be significantly downregulated in two cases (Supplementary Table 2), and confirmed an exon 4 (AG)2 deletion as the cause of mRNA downregulation in both instances. PQBP1 mutations cause a sydromic form of XLID commonly referred to as Renpenning syndrome.8 The specific mutation we describe here has been previously shown to cause XLID,6 and has also been proven to decrease mRNA levels by nonsense-mediated mRNA decay,7 thus being likely to be detected by assessment of gene expression. The cases for which we identified PQBP1 mutations were not part of the cohort studied by Tarpey et al. (Supplementary Table 1). We further asked whether any of the non-recurrent sequence variants identified by the exon re-sequencing study in the 13 overlapping cases was associated with a significant alteration of mRNA levels. We found that CCDC22, which encodes a coiled-coil domain protein of unknown function, was significantly downregulated (Figure 1c, Supplementary Figure 1) in one of our XLID patients. A CCDC22 non-recurrent sequence variant c.49A > G/p.T17A had been identified in a proband from the same family by the Sanger re-sequencing study, but its functional significance had not been determined.5 The proband belongs to a large family (55 individuals) with six affected males over three generations (Figure 1a). Notably, female carriers in this family show highly skewed X-chromosome inactivation (data not shown). Linkage analysis delineated a 58.4-Mb linkage interval (logarithm in base 10 of odds (LOD) score = 2.7). Whole X-chromosome exon re-sequencing identified four non-recurrent mis-sense variants within the linkage interval: CCDC22 c.49A> G/p.T17A, FAM121A (APOOL) c.40G> T/p.A14S, LOC402414 c.453T> A/p.S151R, ABCB7 c.941G> A/p.R314Q.5 Although these sequence variants were predicted to be neutral for protein function (Polyphen, data not shown), we found that the CCDC22 change was associated with a fivefold decrease in mRNA level (Figure 1c, Supplementary Figure 1 and Supplementary Table 3). No other gene in the 58.4-Mb linkage interval showed significant downregulation of mRNA levels (Supplementary Figure 2a). We sequenced CCDC22 in all available family members and found that the c.49A>G change segregates with the disease consistent with its location within the linkage interval. Figure 1 CCDC22 mutation causes X-linked intellectual disability (XLID) in a large pedigree. (a) The pedigree of the IGOLD #586 family shows six affected males over three generations. The case analyzed by expression profiling in this study (ID11) is the case from ... The c.49A >G sequence variant is located within exon 1, two base pairs away from the 5′ splice site of intron 1 of CCDC22 (Figure 1b) and is predicted to significantly decrease the splicing efficiency at the corresponding 5′ splice site (splice site score decrease from 0.9 to 0.7, using NNsplice9; http://www.fruitfly.org/seq_tools/splice.html). We found abnormally spliced transcripts retaining intron 1 to be much more abundant in cases harboring the c.49A >G mutation than in controls (Figure 1c, Supplementary Methods, Supplementary Figure 1), demonstrating that the mutation impedes efficient alternative splicing at the 5′ splice site. The abnormally spliced transcripts with intron 1 retention contain several in-frame premature terminal codons, which would likely cause the transcript to be degraded by nonsense-mediated mRNA decay surveillance (NMD). Alternatively, the splicing defect could have a negative impact on the transcription efficiency of CCDC22.10–12 Inhibition of NMD by cyclohexamide did not significantly affect the total CCDC22 mRNA level or the abundance of the abnormally spliced isoform (Supplementary Figure 1), indicating that NMD did not have a major role in CCDC22 mRNA downregulation. Recent studies have suggested that abnormal transcripts with retained intron(s), having failed the nuclear quality control mechanism, are not exported into the cytoplasm and thus are not degraded by NMD.13–17 In addition, splicing events proximal to the transcription start sites have been shown to be important for efficient recruitment of basal transcription factors.10–12 We thus suspect that the c.49A >G change near the first splice site removes the positive feedback necessary for efficient transcription of CCDC22. To test whether CCDC22 downregulation occurred frequently in the general population we analyzed CCDC22 expression level in 52 age-matched control males using genome-wide expression data from lymphoblast cell lines from an AGRE cohort (Methods). None of the controls showed significant downregulation of CCDC22 mRNA level (Supplementary Figure 2b). The phenotype of the IGOLD #586 family is consistent with syndromic XLID. In addition to intellectual disability, affected individuals have cardiac abnormalities (atrial septal defect, ventricular septal defect, dextrocardia), skeletal abnormalities (hypoplastic distal phalanges, syndactyly, hip subluxation, scoliosis) and specific facial features (Table 1). Table 1 Phenotype characterization of the IGOLD 586 family CCDC22 is a ubiquitously expressed coiled-coil domain protein (Supplementary Figure 3). Although the function of CCDC22 is currently poorly defined, CCDC22 has been shown to interact in vitro with copines, a family of calcium-dependent membrane-binding proteins, via its coiled-coil domain18 as well as the Nance–Horan syndrome protein.19 In the rat brain, CCDC22 is expressed in multiple regions including the prefrontal and somatosensory cortex, dentate gyrus and thalamus,20 and CCDC22-specific antibodies stain primarily axons.20,21 In the rat spinal cord, CCDC22 is primarily expressed in the dorsal columns, as well as in ipsilateral motor neurons after sciatic nerve trans-section,21 suggesting a role for this gene in neuronal injury response. To gain further insight into the function of CCDC22, we took a bioinformatics approach. To identify genes that are functionally related to, or potentially interact with CCDC22 in the developing human brain, we used a recently published human fetal brain transcriptome dataset22 and queried which are the nearest neighbors of CCDC22 by co-expression topological overlap.23 Figure 1d shows the genes co-expressed with CCDC22 in the human fetal brain. Remarkably, this module contains genes that have been implicated in hereditary cardiac and skeletal disorders, the main classes of extra-central nervous system pathological changes observed in family IGOLD #586. The IGOLD #586 case was the only one in the X-chromosome re-sequencing cohort of 208 with a CCDC22 mutation, suggesting that mutations of CCDC22 are a rare cause of XLID. Here we highlight CCDC22 as a novel XLID candidate gene for future targeted re-sequencing studies and propose that the mRNA downregulation associated with the described mutation likely results from reduced transcriptional efficiency rather than nonsense-mediated mRNA decay.

We report the identification of a recurrent, 520-kb 16p12.1 microdeletion associated with childhood developmental delay. The microdeletion was detected in 20 of 11,873 cases compared with 2 of 8,540 controls (P = 0.0009, OR = 7.2) and replicated in a second series of 22 of 9,254 cases compared with 6 of 6,299 controls (P = 0.028, OR = 2.5). Most deletions were inherited, with carrier parents likely to manifest neuropsychiatric phenotypes compared to non-carrier parents (P = 0.037, OR = 6). Probands were more likely to carry an additional large copy-number variant when compared to matched controls (10 of 42 cases, P = 5.7 × 10 5, OR = 6.6). The clinical features of individuals with two mutations were distinct from and/or more severe than those of individuals carrying only the co-occurring mutation. Our data support a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity indicates that this two-hit model might be more generally applicable to neuropsychiatric disease. © 2010 Nature America, Inc. All rights reserved.

Children with intellectual disability, dysmorphic features, malformations and/or growth abnormalities frequently display normal karyotypes. Recent studies have shown that genome-wide single nucleotide polymorphism (SNP) arrays can be effective in detecting abnormalities involving copy number variation (CNV), deletions, duplications and loss of heterozygosity (LOH) that routine cytogenetic tests fail to identify. Five patients with various degrees of intellectual disability and/or dysmorphic features and other malformations were whole-genome genotyped using the Human-1 Genotyping BeadChip – Exon-Centrix 100K SNP arrays (Illumina). All patients had undergone routine cytogenetic testing; four patients had normal karyotypes, while one patient had an apparently balanced complex translocation involving chromosomes 1q25, 1q32, 2q23, 7q22 and 16q24. We detected deletions on chromosome 1q44 and 13q31.1 in one patient, and LOH of the entire chromosome 2 in another patient, both with cytogenetically normal karyotypes. The patient with the complex translocation had a deletion on chromosome 7q22.2-22.3, which is in conjunction with one of the translocation breakpoints. Our findings provide further evidence of there being a critical region for the development of microcephaly and corpus callosum abnormalities in children with distal 1q deletions. We have also shown that apparently balanced complex translocations might not be balanced at the DNA level, and we report the fourth case of paternal uniparental disomy of chromosome 2. The results of this study suggest that it may be desirable to investigate idiopathic mental retardation using genome-wide SNP arrays, in conjunction with other cytogenetic and molecular techniques.

We report two familial cases of 22q11.2 duplication detected using multiplex ligation-dependent probe amplification (MLPA). In the first case, eight individuals from a three-generation family were found to carry a 3-Mb 22q11.2 duplication. The individuals carrying the duplication show phenotypic variation. This phenotypic variation includes heart defect (1 in 8 individuals, 1/8), submucous cleft palate (2/8), intellectual disability (2/8), speech delay (2/8), behaviour problems (3/8) and brachydactyly (3/8). In the second case, a 1.5-Mb 22q11.2 duplication was detected in a neonate and her normal mother. The neonate presented with severe laryngomalacia causing intermittent stridor. Cranial ultrasound showed small subependymal cysts bilaterally. There was no heart defect or cleft palate, her chest X ray and renal ultrasound were normal. Review at 2 months of age revealed normal growth and development. Our findings broaden the understanding of 22q11.2 duplication syndrome and demonstrate that MLPA is sensitive for detection and sizing of 22q11.2 microduplications.

In the course of systematic screening of the X-chromosome coding sequences in 250 families with nonsyndromic X-linked mental retardation (XLMR), two families were identified with truncating mutations in BRWD3, a gene encoding a bromodomain and WD-repeat domain–containing protein. In both families, the mutation segregates with the phenotype in affected males. Affected males have macrocephaly with a prominent forehead, large cupped ears, and mild-to-moderate intellectual disability. No truncating variants were found in 520 control X chromosomes. BRWD3 is therefore a new gene implicated in the etiology of XLMR associated with macrocephaly and may cause disease by altering intracellular signaling pathways affecting cellular proliferation.

The Aristaless-related homeobox (ARX) gene is implicated in intellectual disability with the most frequent pathogenic mutations leading to expansions of the first two polyalanine tracts. Here, we describe analysis of the ARX gene outlining the approaches in the Australian and Portuguese setting, using an integrated clinical and molecular strategy. We report variants in the ARX gene detected in 19 patients belonging to 17 families. Seven pathogenic variants, being expansion mutations in both polyalanine tract 1 and tract 2, were identifyed, including a novel mutation in polyalanine tract 1 that expands the first tract to 20 alanines. This precise number of alanines is sufficient to cause pathogenicity when expanded in polyalanine tract 2. Five cases presented a probably non-pathogenic variant, including the novel HGVS: c.441_455del, classified as unlikely disease causing, consistent with reports that suggest that in frame deletions in polyalanine stretches of ARX rarely cause intellectual disability. In addition, we identified five cases with a variant of unclear pathogenic significance. Owing to the inconsistent ARX variants description, publications were reviewed and ARX variant classifications were standardized and detailed unambiguously according to recommendations of the Human Genome Variation Society. In the absence of a pathognomonic clinical feature, we propose that molecular analysis of the ARX gene should be included in routine diagnostic practice in individuals with either nonsyndromic or syndromic intellectual disability. A definitive diagnosis of ARX-related disorders is crucial for an adequate clinical follow-up and accurate genetic counseling of at-risk family members. Unit for Multidisciplinary Research in Biomedicine, UMIB, ICBAS-UP, Porto, Portugal was funded by FEDER funds of the Operational Program for Competitiveness Factors – COMPETE through FCT – Foundation for Science and Technology under the project: Fcomp-01-0124-FEDER-015896. The Neurogenetics research program in the Department of Paediatrics, University of Adelaide, Australia was funded by the Australian National Health and Medical Research Council (Grant No. 1063025). C. S. is supported Australian Research Council (Future Fellowship FT120100086)

Background: Most patients with pure nonprogressive congenital cerebellar ataxia have a sporadic form of unknown heredity and etiology. Several small families have been reported with a dominantly inherited nonprogressive congenital ataxia (NPCA).Methods: The authors ascertained and clinically characterized a four-generation pedigree segregating an autosomal dominant type of congenital nonprogressive cerebellar ataxia associated with cognitive impairment. Following the exclusion of several SCA localizations (SCA-1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 17, IOSCA, and DRPLA), a genome-wide linkage study was performed.Results: Examination of the family showed that all affected members had gait ataxia and cognitive disability with variable features of dysarthria, dysmetria, dysdiadochokinesia, nystagmus, dystonic movements, and cerebellar hypoplasia on imaging. Clinical signs of pyramidal tract dysfunction and sensory changes were absent. A genome-wide search in this family detected linkage to chromosome 3p with a maximum two-point lod score of 4.26 at D3S3630. This localization to the pter is distal to D3S1304, as defined by a recombination event. This overlaps with the SCA15 locus, with the critical overlapping region between the microsatellite markers, D3S1304 and D3S1620 (approximately 8 cM).Conclusion: Autosomal dominant congenital nonprogressive cerebellar ataxia with or without cerebellar hypoplasia overlaps with the SCA15 locus on chromosome 3pter.

Isolated mitochondrial respiratory chain complex III deficiency has been described in a heterogeneous group of clinical presentations in children and adults. It has been associated with mutations in MT-CYB, the only mitochondrial DNA encoded subunit, as well as in nine nuclear genes described thus far: BCS1L, TTC19, UQCRB, UQCRQ, UQCRC2, CYC1, UQCC2, LYRM7, and UQCC3. BCS1L, TTC19, UQCC2, LYRM7, and UQCC3 are complex III assembly factors. We report on an 8-year-old girl born to consanguineous Iraqi parents presenting with slowly progressive encephalomyopathy, severe failure to thrive, significant delays in verbal and communicative skills and bilateral retinal cherry red spots on fundoscopy. SNP array identified multiple regions of homozygosity involving 7.5% of the genome. Mutations in the TTC19 gene are known to cause complex III deficiency and TTC19 was located within the regions of homozygosity. Sequencing of TTC19 revealed a homozygous nonsense mutation at exon 6 (c.937C > T; p.Q313X). We reviewed the phenotypes and genotypes of all 11 patients with TTC19 mutations leading to complex III deficiency (including our case). The consistent features noted are progressive neurodegeneration with Leigh-like brain MRI abnormalities. Significant variability was observed however with the age of symptom onset and rate of disease progression. The bilateral retinal cherry red spots and failure to thrive observed in our patient are unique features, which have not been described, in previously reported patients with TTC19 mutations. Interestingly, all reported TTC19 mutations are nonsense mutations. The severity of clinical manifestations however does not specifically correlate with the residual complex III enzyme activities.

Copy number variants (CNVs) are associated with many neurocognitive disorders; however, these events are typically large, and the underlying causative genes are unclear. We created an expanded CNV morbidity map from 29,085 children with developmental delay in comparison to 19,584 healthy controls, identifying 70 significant CNVs. We resequenced 26 candidate genes in 4,716 additional cases with developmental delay or autism and 2,193 controls. An integrated analysis of CNV and single-nucleotide variant (SNV) data pinpointed 10 genes enriched for putative loss of function. Follow-up of a subset of affected individuals identified new clinical subtypes of pediatric disease and the genes responsible for disease-associated CNVs. These genetic changes include haploinsufficiency of SETBP1 associated with intellectual disability and loss of expressive language and truncations of ZMYND11 in individuals with autism, aggression and complex neuropsychiatric features. This combined CNV and SNV approach facilitates the rapid discovery of new syndromes and genes involved in neuropsychiatric disease despite extensive genetic heterogeneity.

Summary The aim of this study was to determine prospectively the frequency of pathogenic chromosomal microdeletions and microduplications in a large group of referred patients with developmental delay (DD), intellectual disability (ID) or autism spectrum disorders (ASD) within a genetic diagnostic service. First tier testing was applied using a standardised oligo-array comparative genomic hybridization (CGH) platform, replacing conventional cytogenetic testing that would have been used in the past. Copy number variants (CNVs) found to be responsible for the clinical condition on the request form could all be subdivided into three groups: well established pathogenic microdeletion/microduplication/aneuploidy syndromes, predicted pathogenic CNVs as interpreted by the laboratory, and recently established pathogenic disease susceptibility CNVs. Totalled from these three groups, with CNVs of uncertain significance excluded, detection rates were: DD (13.0%), ID (15.6%), ASD (2.3%), ASD with DD (8.2%), ASD with ID (12.7%) and unexplained epilepsy with DD, ID and ASD (10.9%). The greater diagnostic sensitivity arising from routine application of array CGH, compared with previously used conventional cytogenetics, outweighs the interpretative issues for the reporting laboratory and referring clinician arising from detection of CNVs of uncertain significance. Precise determination of any previously hidden molecular defect responsible for the patient’s condition is translated to improved genetic counselling.

To assess the relative contributions of trans-acting factors (replication and repair functions) and cis-acting elements (repeat and flanking DNA composition) to the mechanism of trinucleotide repeat sequence mutation we have analysed the distribution of copy number polymorphisms at 12 loci associated with dynamic mutations in 15 populations of different ethnic origins. Genome wide instability of repeats in a particular population would be evidence of trans-acting factor instigation of the mutation process, whereas instability at a particular locus (perhaps even in several populations) would be evidence that the composition of the particular locus was the most significant factor contributing to mutation. The FRA16A locus is highly polymorphic in only the European population. Some other loci exhibit distinct distributions of alleles between different populations. Therefore sequences in the vicinity of the repeat -- the cis component of a particular locus -- appear(s) to be more important in the mutation mechanism than sporadic genome-wide instability induced by trans-acting factors such as the DNA mismatch repair enzymes.

Using a combination of linkage mapping and massively parallel sequencing of the X-chromosome exome, we identified an 18-bp deletion in exon 8 of the oral-facial-digital syndrome type 1 (OFD1) gene in a family with X-linked Joubert syndrome (JBTS10). The deletion results in an in-frame deletion of six amino acids. New features not noted in the two previously reported cases of X-linked Joubert syndrome include the presence of polycystic kidney disease, polymicrogyria and hydrocephalus. Our study further underlines the power of genetic mapping combined with massively parallel sequencing as a powerful tool for novel disease gene and mutation discovery.

The molecular characterisation of the dystrophin gene, mutations in which are responsible for X linked Duchenne and Becker muscular dystrophies, has led to an array of strategies for the diagnosis of affected subjects and carriers. Initially these were based on blotting and hybridisation technologies but have recently been largely superseded by PCR based techniques which afford greater speed and sensitivity. We describe the use of single strand conformation polymorphism to detect heterozygosity in regions of the dystrophin locus which are deleted in affected males, to determine the carrier status of their female relatives.

Epilepsy and mental retardation limited to females (EFMR) is a disorder with an X-linked mode of inheritance and an unusual expression pattern. Disorders arising from mutations on the X chromosome are typically characterized by affected males and unaffected carrier females. In contrast, EFMR spares transmitting males and affects only carrier females. Aided by systematic resequencing of 737 X chromosome genes, we identified different protocadherin 19 (PCDH19) gene mutations in seven families with EFMR. Five mutations resulted in the introduction of a premature termination codon. Study of two of these demonstrated nonsense-mediated decay of PCDH19 mRNA. The two missense mutations were predicted to affect adhesiveness of PCDH19 through impaired calcium binding. PCDH19 is expressed in developing brains of human and mouse and is the first member of the cadherin superfamily to be directly implicated in epilepsy or mental retardation.

Epilepsy and Mental Retardation limited to Females (EFMR) which links to Xq22 has been reported in only one family. We aimed to determine if there was a distinctive phenotype that would enhance recognition of this disorder. We ascertained four unrelated families (two Australian, two Israeli) where seizures in females were transmitted through carrier males. Detailed clinical assessment was performed on 58 individuals, using a validated seizure questionnaire, neurological examination and review of EEG and imaging studies. Gene localization was examined using Xq22 microsatellite markers. Twenty-seven affected females had a mean seizure onset of 14 months (range 6 36) typically presenting with convulsions. All had convulsive attacks at some stage, associated with fever in 17 out of 27 (63%).Multiple seizure types occurred including tonic-clonic (26), tonic (4), partial (11), absence (5), atonic (3) and myoclonic (4). Seizures ceased at mean 12 years.Developmental progress varied from normal (7), to always delayed (4) to normal followed by regression (12). Intellect ranged from normal to severe intellectual disability (ID), with 67% of females having ID or being of borderline intellect. Autistic (6), obsessive (9) and aggressive (7) features were prominent. EEGs showed generalized and focal epileptiform abnormalities. Five obligate male carriers had obsessional tendencies. Linkage to Xq22 was confirmed (maximum lod 3.5 at theta = 0).We conclude that EFMR is a distinctive, under-recognized familial syndrome where girls present with convulsions in infancy, often associated with intellectual impairment and autistic features.The unique inheritance pattern with transmission by males is perplexing. Clinical recognition is straightforward in multiplex families due to the unique inheritance pattern; however, this disorder should be considered in smaller families where females alone have seizures beginning in infancy, particularly in the setting of developmental delay. In single cases, diagnosis will depend on identification of the molecular basis. Refereed/Peer-reviewed

Neither the molecular basis for common fragile site DNA instability nor the contribution of this form of chromosomal instability to cancer is clearly understood. Fragile site FRA16D (16q23.2) is within regions of frequent loss-of-heterozygosity (LOH) in breast and prostate cancers, is associated with homozygous deletions in various adenocarcinomas and t(14;16) chromosomal translocations in multiple myeloma. The FOR (WWOX) gene spans FRA16D and encodes a partner of p53 that also has a role in apoptosis. Previously untested 53 cancer cell lines were screened for deletions within the FOR/WWOX gene. Deletions were detected in Co115, KM12C and KM12SM. Homozygous deletions in these and two previously identified tumour cell lines were intragenic on both alleles, indicating a distinct mutation mechanism from that causing LOH. Identical FRA16D deletions in two cell lines (one derived from the primary carcinoma and the other from a secondary metastasis) demonstrate that FRA16D DNA instability can be an early, transient event. Sequence analysis across one deletion locates one endpoint within a polymorphic AT-dinucleotide repeat and the other adjacent to an AT-rich mini-satellite repeat implicating AT-rich repeats in FRA16D DNA instability. Another deletion is associated with de novo repetition of the 9 bp AT-rich sequence at one of the deletion endpoints. FRA16D deleted cells retain cytogenetic fragile site expression indicating that the deletions are susceptible sites for breakage rather than regions that confer fragility. Most cell lines with FRA16D homozygous deletions also have FRA3B deletions, therefore common fragile sites represent highly susceptible genome-wide targets for a distinct form of mutation.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In similar to 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.

Foetal akinesia/hypokinesia is an absence or decrease of movement in utero resulting in a spectrum of anomalies characterised by intrauterine growth retardation, contractures, craniofacial anomalies, limb anomalies, pulmonary hypoplasia and polyhydramnios. Despite a large number of disease genes (>30) being associated with this heterogenous group of disorders, the majority of cases are without a genetic diagnosis. The aim of this study was to investigate the use of whole exome sequencing (WES) to identify the causative gene/s in a preliminary set of four families with foetal akinesia. In three families presenting with foetal akinesia and nemaline bodies, all the known nemaline myopathy genes were excluded by WES. The fourth family presented with lethal multiple pterygium syndrome. WES of DNA from two affected sibs revealed compound heterozygous mutations in the glycogen branching enzyme-1 gene ( GBE1 ): a known splice site mutation (c.691+2T>C) and a novel missense mutation (c.956A>G; p.His319Arg) at a highly conserved residue. Each parent carried one of the mutations. GBE1 mutations are known to cause glycogen storage disease IV (GSD IV), a subset of which present with a severe neuromuscular foetal akinesia phenotype. In light of the genetic findings, the muscle pathology was reviewed and PAS positive, diastase resistant faintly refractile material was observed, consistent with GSD IV. Our data highlight that novel nemaline myopathy gene/s remain to be identified and that WES can be used successfully for the diagnosis of foetal akinesias. This study expands the genotype–phenotype correlation of GBE1 mutations: (1) since GSD IV should now be considered in cases presenting as lethal multiple pterygium syndrome and (2) since the vast majority of known foetal akinesia GSD IV cases are due to large indels or frameshift mutations and it is generally considered that the type of the mutations corresponds with clinical severity.

Abstract LBA-3 Mutations in the transcription factor genes, RUNX1 and CEBPA, can lead to an autosomal dominant familial predisposition to MDS/AML. Using a candidate gene approach, we have detected domain specific heterozygous mutations in the GATA2 gene in 4 MDS/AML families which predispose to MDS/AML. The same novel heterozygous T354M missense mutation was observed in 3 families and a 355delT mutation in 1 family, all with multigenerational transmission of MDS and/or MDS/AML. Importantly, these genetic variants segregate with all affected members in each of the families. The 2 mutated threonine residues are in 5 consecutive highly conserved threonine residues at the DNA-binding, protein-protein interacting second zinc finger (ZF2) of GATA2. Neither these mutations, nor any other variants in the GATA2 coding sequence, were seen in a population screen of 695 normal individuals. Haplotype analysis suggests that the T354M mutation has multiple ancestral origins. While mutations in RUNX1 and CEBPA, can also lead to familial predisposition to MDS/AML, these patients with GATA2 mutations are unique in that there is no obvious pre-MDS or pre-leukaemic phenotype such as thrombocytopenia (RUNX1) and eosinophilia (CEBPA) in predisposed carriers. Most patients in these families have had a rapid disease course “appearing out of the blue” leading to death, with a variety of ages of onset from teenagers to early 40s. Yet remarkably, there are still asymptomatic carriers in their 60s. One of these carriers, and his 2 children, has had bone marrow prophylactically stored over 15 years ago in case of disease onset. No pathogenic GATA2 coding sequence changes were found in 268 sporadic MDS/AML patient samples. Additionally, GATA2 mutations were not found in germline samples from 35 other families predisposed to AML and various other hematological malignancies. Both the T354M and 355delT mutants appear to localize appropriately to the nucleus and maintain at least some DNA binding in electrophoretic mobility shift assays. We used the known murine Gata3 ZF2 structure bound to DNA to model the effects of the observed mutations and demonstrated that the T354 residue does not contact DNA but makes polar contact with the adjacent threonines, and via its amino group, with C349 which coordinates the zinc atom. Replacement of the T354 side-chain with the bulky methionine moiety may affect the zinc contacts and is predicted to alter the overall structure of this ZF2. In contrast, 355delT will shorten the conserved threonine string which is predicted to impact on the orientation and position of L359 which directly contacts DNA. Thus, 355delT is likely to have an effect on DNA binding. Luciferase reporter assays indicate that T354M and 355delT greatly reduce the transactivation ability of GATA2 on multiple response elements, impacting on downstream target genes such as RUNX1 and CD34. Of note, T354M shows a markedly lesser synergistic effect than wildtype (WT) GATA2 with PU.1 on the CSF1R promoter. Competition assays show that these mutations may be acting in a dominant negative fashion in some biological contexts. In stable promyelocytic HL-60 cell lines expressing regulatable GATA2 (WT or T354M), T354M allows proliferation to proceed even under stimulus to differentiate with all-trans retinoic acid. Microarray studies indicated that the down regulation of proapoptotic BCL-xS by T354M, but not WT, may be responsible for this phenotype. GATA2 is considered to be a hematopoietic “stemness” gene, highly expressed in haematopoietic stem cells and is required for megakaryocyte and mast cell production. GATA2 is down regulated during myeloid differentiation and forced overexpression prevents such differentiation. Discovery of GATA2 mutants in MDS/AML predisposed families provides new tools for probing the mechanism of GATA2 induced leukemogenesis, and possibly also for clarifying its role in maintenance of stemness. Our findings highlight the power of investigating familial predispositions to cancer identifying specific mutations with unique biological effects. They have immediate implications for diagnostic genetic testing, and longer term therapeutic implications through identification of drugable biological pathways such as apoptosis. The poor outcome associated with these mutations may suggest that an aggressive strategy is appropriate in the treatment of affected individuals in families found to be carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

In a large kindred in which the gene for central core disease is segregating, we have demonstrated linkage between the disorder and a marker on chromosome 19q. Marker D19S9 (p1J2) was linked to central core disease with a lod score of 6.4 at theta = 0.03 (support interval 0.01-0.14) thus localizing the gene for this disorder in or very close to 19q12-q13.2.