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Intraflagellar transport (IFT) complexes, IFT-A and IFT-B, form bidirectional trains that move along the axonemal microtubules and are essential for assembling and maintaining cilia. Mutations in IFT subunits lead to numerous ciliopathies involving multiple tissues. However, how IFT complexes assemble and mediate cargo transport lacks mechanistic understanding due to missing high-resolution structural information of the holo-complexes. Here we report cryo-EM structures of human IFT-A complexes in the presence and absence of TULP3 at overall resolutions of 3.0–3.9 Å. IFT-A adopts a “lariat” shape with interconnected core and peripheral subunits linked by structurally vital zinc-binding domains. TULP3, the cargo adapter, interacts with IFT-A through its N-terminal region, and interface mutations disrupt cargo transport. We also determine the molecular impacts of disease mutations on complex formation and ciliary transport. Our work reveals IFT-A architecture, sheds light on ciliary transport and IFT train formation, and enables the rationalization of disease mutations in ciliopathies.

BackgroundMosaicism for chromosomal structural abnormalities, other than marker or ring chromosomes, is rarely inherited.MethodsWe performed cytogenetics studies and breakpoint analyses on a family with transmission of mosaicism for a derivative chromosome 8 (der(8)), resulting from an unbalanced translocation between the long arms of chromosomes 8 and 21 over three generations.ResultsThe proband and his maternal half-sister had mosaicism for a der(8) cell line leading to trisomy of the distal 21q, and both had Down syndrome phenotypic features. Mosaicism for a cell line with the der(8) and a normal cell line was also detected in a maternal half-cousin. The der(8) was inherited from the maternal grandmother who had four abnormal cell lines containing the der(8), in addition to a normal cell line. One maternal half-aunt had the der(8) and an isodicentric chromosome 21 (idic(21)). Sequencing studies revealed microhomologies at the junctures of the der(8) and idic(21) in the half-aunt, suggesting a replicative mechanism in the rearrangement formation. Furthermore, interstitial telomeric sequences (ITS) were identified in the juncture between chromosomes 8 and 21 in the der(8).ConclusionMosaicism in the proband, his half-sister and half-cousin resulting from loss of chromosome 21 material from the der(8) appears to be a postzygotic event due to the genomic instability of ITS and associated with selective growth advantage of normal cells. The reversion of the inherited der(8) to a normal chromosome 8 in this family resembles revertant mosaicism of point mutations. We propose that ITS could mediate recurring revertant mosaicism for some constitutional chromosomal structural abnormalities.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2(G2019S). This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.

Nonallelic homologous recombination (NAHR), occurring between low-copy repeats (LCRs) >10 kb in size and sharing >97% DNA sequence identity, is responsible for the majority of recurrent genomic rearrangements in the human genome. Recent studies have shown that transposable elements (TEs) can also mediate recurrent deletions and translocations, indicating the features of substrates that mediate NAHR may be significantly less stringent than previously believed. Using >4 kb length and >95% sequence identity criteria, we analyzed of the genome-wide distribution of long interspersed element (LINE) retrotransposon and their potential to mediate NAHR. We identified 17 005 directly oriented LINE pairs located

Point mutations and genomic deletions of the CDKL5 (STK9) gene on chromosome Xp22 have been reported in patients with severe neurodevelopmental abnormalities, including Rett-like disorders. To date, only larger-sized (8-21 Mb) duplications harboring CDKL5 have been described. We report seven females and four males from seven unrelated families with CDKL5 duplications 540-935 kb in size. Three families of different ethnicities had identical 667kb duplications containing only the shorter CDKL5 isoform. Four affected boys, 8-14 years of age, and three affected girls, 6-8 years of age, manifested autistic behavior, developmental delay, language impairment, and hyperactivity. Of note, two boys and one girl had macrocephaly. Two carrier mothers of the affected boys reported a history of problems with learning and mathematics while at school. None of the patients had epilepsy. Similarly to CDKL5 mutations and deletions, the X-inactivation pattern in all six studied females was random. We hypothesize that the increased dosage of CDKL5 might have affected interactions of this kinase with its substrates, leading to perturbation of synaptic plasticity and learning, and resulting in autistic behavior, developmental and speech delay, hyperactivity, and macrocephaly.

Somatic chromosomal mosaicism arising from post-zygotic errors is known to cause several well-defined genetic syndromes as well as contribute to phenotypic variation in diseases. However, somatic mosaicism is often under-diagnosed due to challenges in detection. We evaluated 10 362 patients with a custom-designed, exon-targeted whole-genome oligonucleotide array and detected somatic mosaicism in a total of 57 cases (0.55%). The mosaicism was characterized and confirmed by fluorescence in situ hybridization (FISH) and/or chromosome analysis. Different categories of abnormal cell lines were detected: (1) aneuploidy, including sex chromosome abnormalities and isochromosomes (22 cases), (2) ring or marker chromosomes (12 cases), (3) single deletion/duplication copy number variations (CNVs) (11 cases), (4) multiple deletion/duplication CNVs (5 cases), (5) exonic CNVs (4 cases), and (6) unbalanced translocations (3 cases). Levels of mosaicism calculated based on the array data were in good concordance with those observed by FISH (10–93%). Of the 14 cases evaluated concurrently by chromosome analysis, mosaicism was detected solely by the array in 4 cases (29%). In summary, our exon-targeted array further expands the diagnostic capability of high-resolution array comparative genomic hybridization in detecting mosaicism for cytogenetic abnormalities as well as small CNVs in disease-causing genes.

Interstitial deletions involving 2q24 have been associated with a wide range of phenotypes including intellectual disability and short stature. To date, the smallest common region among reported cases of deletions in this region is approximately 2.65 Mb and contains 15 genes. In the present case report, we describe an 18-year-old male with mild intellectual disability, short stature, and mosaicism for a 0.422 Mb deletion on 2q24.2 that was diagnosed by comparative genomic hybridization and confirmed with fluorescent in situ hybridization (FISH). This deletion, which is present in approximately 61% of cells, includes three genes: TBR1, TANK, and PSMD14. The findings suggest that the critical region for intellectual disability and short stature in 2q24.2 can be narrowed to a 0.422 Mb segment. TBR1, a transcription factor involved in early cortical development, is a strong candidate for the intellectual disability phenotype seen in our patient and in patients with larger deletions in this region of the genome.

Fragile X syndrome, the most common form of X-linked intellectual disability, results from transcriptional silencing of the FMR1 gene. As of yet, the phenotypic consequences of the duplication of FMR1 have not been well characterized. In this report, we characterize the clinical features in two females with duplications involving only the FMR1 gene. In addition, we describe the phenotypes of two subjects with deletion of FMR1 and show that both loss and gain of FMR1 copy number can lead to overlapping neurodevelopmental phenotypes. Our report supports the notion that FMR1 gene dosage is important for normal neurocognitive function.

Objective To evaluate the results of prenatal chromosomal microarray analysis (CMA) on >1000 fetal samples referred for testing at our institution and to compare these data to published reports. Methods High resolution CMA was offered to women undergoing amniocentesis or chorionic villus sampling. Parental samples were obtained concurrently to exclude maternal cell contamination and assist interpretation of copy number variations. Results Clinically significant copy number variations were observed in 85/1115 cases (7.6%) overall, and in 45/1075 cases (4.2 %) if 40 abnormal cases with known chromosome abnormalities or familial genomic imbalances were excluded. Eighteen of the 1115 cases had variants of unclear clinical significance (1.6%). Indications yielding the most clinically significant findings were abnormal karyotype/fluorescence in situ hybridization (26/61, 42.6%), family history of chromosomal abnormality (13/137, 9.5%), abnormal ultrasound (38/410, 9.3%), abnormal serum screening (2/37, 5.4%) and advanced maternal age (5/394, 1.3%). Of 1075 cases having no previously known cytogenetic abnormality or family history, 18 (1.7%) had clinically significant genomic changes undetectable by conventional prenatal chromosome analysis. Conclusion Current experience confirms that the detection rate of CMA for prenatal chromosomal abnormalities surpasses that of conventional karyotype analysis and continues to improve with higher resolution arrays, while maintaining a low frequency of results of unclear clinical significance. © 2012 John Wiley & Sons, Ltd.

Submicroscopic deletions involving chromosome 1q43-q44 result in cognitive impairment, microcephaly, growth restriction, dysmorphic features, and variable involvement of other organ systems. A consistently observed feature in patients with this deletion are the corpus callosal abnormalities (CCAs), ranging from thinning and hypoplasia to complete agenesis. Previous studies attempting to delineate the critical region for CCAs have yielded inconsistent results. We conducted a detailed clinical and molecular characterization of seven patients with deletions of chromosome 1q43-q44. Using array comparative genomic hybridization, we mapped the size, extent, and genomic content of these deletions. Four patients had CCAs, and shared the smallest region of overlap that contains only three protein coding genes, CEP170, SDCCAG8, and ZNF238. One patient with a small deletion involving SDCCAG8 and AKT3, and another patient with an intragenic deletion of AKT3 did not have any CCA, implying that the loss of these two genes is unlikely to be the cause of CCA. CEP170 is expressed extensively in the brain, and encodes for a protein that is a component of the centrosomal complex. ZNF238 is involved in control of neuronal progenitor cells and survival of cortical neurons. Our results rule out the involvement of AKT3, and implicate CEP170 and/or ZNF238 as novel genes causative for CCA in patients with a terminal 1q deletion.

In order to understand the inflammatory mechanisms related to rabbit interleukin-15 (RIL-15), we cloned and expressed RIL-15 cDNA gene. A cDNA encoding RIL-15 was cloned from heart mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using hIL-15 primers. The RIL-15 cDNA contains an open reading frame (ORF) of 162 amino acids (aa) with a 48 aa leader sequence. The predicted molecular weight of the encoded protein (12.5 kDa) matched the size of recombinant IL-15 on Western blotting in an Escherichia coli (pET32a) expression system. Amino acid and nucleotide sequence analyses of RIL-15 revealed 82.7% and 87% homology with human IL-15 (hIL-15), respectively. RIL-15 is similar to the hIL-15 (hIL-15) in that it contains seven cysteine residues. RT-PCR showed that IL-15 is expressed in many tissues in the rabbit, including heart, spleen, lung, liver, muscle and kidney. Expressed and purified recombinant RIL-15, in the absence of the 48 aa leader sequence, stimulated the proliferation of cells of the mouse T cell line, CTLL-2, and its activity is comparable to hIL-15. Western blotting demonstrated that recombinant RIL-15 can be recognized by anti-IL-15 neutralization antibody. Western blotting also confirmed that IL-15 is present in many tissues including heart, spleen, lung, liver, muscle and kidney.

Background Recurrent rearrangements of the human genome resulting in disease or variation are mainly mediated by non-allelic homologous recombination (NAHR) between low-copy repeats. However, other genomic structures, including AT-rich palindromes and retroviruses, have also been reported to underlie recurrent structural rearrangements. Notably, recurrent deletions of Yq12 conveying azoospermia, as well as non-pathogenic reciprocal duplications, are mediated by human endogenous retroviral elements (HERVs). We hypothesized that HERV elements throughout the genome can serve as substrates for genomic instability and result in human copy-number variation (CNV). Results We developed parameters to identify HERV elements similar to those that mediate Yq12 rearrangements as well as recurrent deletions of 3q13.2q13.31. We used these parameters to identify HERV pairs genome-wide that may cause instability. Our analysis highlighted 170 pairs, flanking 12.1% of the genome. We cross-referenced these predicted susceptibility regions with CNVs from our clinical databases for potentially HERV-mediated rearrangements and identified 78 CNVs. We subsequently molecularly confirmed recurrent deletion and duplication rearrangements at four loci in ten individuals, including reciprocal rearrangements at two loci. Breakpoint sequencing revealed clustering in regions of high sequence identity enriched in PRDM9-mediated recombination hotspot motifs. Conclusions The presence of deletions and reciprocal duplications suggests NAHR as the causative mechanism of HERV-mediated CNV, even though the length and the sequence homology of the HERV elements are less than currently thought to be required for NAHR. We propose that in addition to HERVs, other repetitive elements, such as long interspersed elements, may also be responsible for the formation of recurrent CNVs via NAHR. Electronic supplementary material The online version of this article (doi:10.1186/s12915-014-0074-4) contains supplementary material, which is available to authorized users.

Over 1200 recessive disease genes have been described in humans. The prevalence, allelic architecture, and per-genome load of pathogenic alleles in these genes remain to be fully elucidated, as does the contribution of DNA copy-number variants (CNVs) to carrier status and recessive disease. We mined CNV data from 21,470 individuals obtained by array-comparative genomic hybridization in a clinical diagnostic setting to identify deletions encompassing or disrupting recessive disease genes. We identified 3212 heterozygous potential carrier deletions affecting 419 unique recessive disease genes. Deletion frequency of these genes ranged from one occurrence to 1.5%. When compared with recessive disease genes never deleted in our cohort, the 419 recessive disease genes affected by at least one carrier deletion were longer and located farther from known dominant disease genes, suggesting that the formation and/or prevalence of carrier CNVs may be affected by both local and adjacent genomic features and by selection. Some subjects had multiple carrier CNVs (307 subjects) and/or carrier deletions encompassing more than one recessive disease gene (206 deletions). Heterozygous deletions spanning multiple recessive disease genes may confer carrier status for multiple single-gene disorders, for complex syndromes resulting from the combination of two or more recessive conditions, or may potentially cause clinical phenotypes due to a multiply heterozygous state. In addition to carrier mutations, we identified homozygous and hemizygous deletions potentially causative for recessive disease. We provide further evidence that CNVs contribute to the allelic architecture of both carrier and recessive disease-causing mutations. Thus, a complete recessive carrier screening method or diagnostic test should detect CNV alleles.

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ∼70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles. © 2013 The American Society of Human Genetics.

Chromosomal microarray analysis enables the detection of microdeletions/duplications and has become the standard in clinical diagnostic testing for individuals with congenital anomalies and developmental disabilities. In the era of genomic arrays, the value of traditional chromosome analysis needs to be reassessed.We studied 3,710 unrelated patients by chromosomal microarray analysis and chromosome analysis simultaneously and compared the results.We found that chromosomal microarray analysis detected the chromosomal imbalances that were identified by chromosome analysis with the exception of six cases (0.16%) that had mosaic abnormalities. Of note, one case showed mosaicism for two abnormal cell lines, resulting in a balanced net effect and a normal chromosomal microarray analysis. Further structural abnormalities such as unbalanced translocations, rings, and complex rearrangements were subsequently clarified by chromosome analysis in 18% of the cases with abnormal chromosomal microarray analysis results. Apparently balanced rearrangements were detected by chromosome analysis in 30 cases (0.8%).Our data demonstrate that although chromosomal microarray analysis should be the first-tier test for clinical diagnosis of chromosome abnormalities, chromosome analysis remains valuable in the detection of mosaicism and delineation of chromosomal structural rearrangements.

Clinically significant cardiovascular malformations (CVMs) occur in 5–8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be detected in patients with CVMs plus extracardiac anomalies (ECAs). Through a genome-wide survey of 203 subjects with CVMs and ECAs, we identified 55 CNVs >50 kb in length that were not present in children without known cardiovascular defects (n=872). Sixteen unique CNVs overlapping these variants were found in an independent CVM plus ECA cohort (n=511), which were not observed in 2011 controls. The study identified 12/16 (75%) novel loci including non-recurrent de novo 16q24.3 loss (4/714) and de novo 2q31.3q32.1 loss encompassing PPP1R1C and PDE1A (2/714). The study also narrowed critical intervals in three well-recognized genomic disorders of CVM, such as the cat-eye syndrome region on 22q11.1, 8p23.1 loss encompassing GATA4 and SOX7 and 17p13.3-p13.2 loss. An analysis of protein-interaction databases shows that the rare inherited and de novo CNVs detected in the combined cohort are enriched for genes encoding proteins that are direct or indirect partners of proteins known to be required for normal cardiac development. Our findings implicate rare variants such as 16q24.3 loss and 2q31.3-q32.1 loss, and delineate regions within previously reported structural variants known to cause CVMs.

Haploinsufficiency of the gene encoding the insulin-like growth factor 1 receptor (IGF1R), either caused by telomeric 15q26 deletions, or by heterozygous point mutations in IGF1R, segregate with short stature and various other phenotypes, including microcephaly and dysmorphic facial features. Psychomotor retardation and behavioral anomalies have been seen in some cases. Here we report small, intragenic deletions of IGF1R, identified by chromosome microarray analysis in two unrelated families affected primarily with neuropsychiatric phenotypes including developmental delay, intellectual disability and aggressive/autoaggressive behaviors. The deletions are in frame, and both wild-type and mutant mRNAs are expressed as measured by quantitative real-time PCR. While short stature is considered a phenotypic hallmark of IGF1R haploinsufficiency, the present report suggests that in frame exon deletions of IGF1R present predominantly with cognitive and neuropsychiatric phenotypes.

A number of genes in the 9q34.11 region may be haploinsufficient. However, studies analyzing genotype-phenotype correlations of deletions encompassing multiple dosage-sensitive genes in the region are lacking.We mapped breakpoints of 10 patients with 9q34.11 deletions using high-resolution 9q34-specific array comparative genomic hybridization (CGH) to determine deletion size and gene content.The 9q34.11 deletions range in size from 67 kb to 2.8 Mb. Six patients exhibit intellectual disability and share a common deleted region including STXBP1; four manifest variable epilepsy. In five subjects, deletions include SPTAN1, previously associated with early infantile epileptic encephalopathy, infantile spasms, intellectual disability, and hypomyelination. In four patients, the deletion includes endoglin (ENG), causative of hereditary hemorrhagic telangiectasia. Finally, in four patients, deletions involve TOR1A, of which molecular defects lead to early-onset primary dystonia. Ninety-four other RefSeq genes also map to the genomic intervals investigated.STXBP1 haploinsufficiency results in progressive encephalopathy characterized by intellectual disability and may be accompanied by epilepsy, movement disorders, and autism. We propose that 9q34.11 genomic deletions involving ENG, TOR1A, STXBP1, and SPTAN1 are responsible for multisystemic vascular dysplasia, early-onset primary dystonia, epilepsy, and intellectual disability, therefore revealing cis-genetic effects leading to complex phenotypes.

Constitutional deletions of distal 9q34 encompassing the EHMT1 (euchromatic histone methyltransferase 1) gene, or loss-of-function point mutations in EHMT1, are associated with the 9q34.3 microdeletion syndrome, also known as Kleefstra syndrome [MIM#610253]. We now report further evidence for genomic instability of the subtelomeric 9q34.3 region as evidenced by copy number gains of this genomic interval that include duplications, triplications, derivative chromosomes and complex rearrangements. Comparisons between the observed shared clinical features and molecular analyses in 20 subjects suggest that increased dosage of EHMT1 may be responsible for the neurodevelopmental impairment, speech delay, and autism spectrum disorders revealing the dosage sensitivity of yet another chromatin remodeling protein in human disease. Five patients had 9q34 genomic abnormalities resulting in complex deletion-duplication or duplication-triplication rearrangements; such complex triplications were also observed in six other subtelomeric intervals. Based on the specific structure of these complex genomic rearrangements (CGR) a DNA replication mechanism is proposed confirming recent findings in Caenorhabditis elegans telomere healing. The end-replication challenges of subtelomeric genomic intervals may make them particularly prone to rearrangements generated by errors in DNA replication.

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner.

To evaluate the results of prenatal chromosomal microarray analysis (CMA) on1000 fetal samples referred for testing at our institution and to compare these data to published reports.High resolution CMA was offered to women undergoing amniocentesis or chorionic villus sampling. Parental samples were obtained concurrently to exclude maternal cell contamination and assist interpretation of copy number variations.Clinically significant copy number variations were observed in 85/1115 cases (7.6%) overall, and in 45/1075 cases (4.2 %) if 40 abnormal cases with known chromosome abnormalities or familial genomic imbalances were excluded. Eighteen of the 1115 cases had variants of unclear clinical significance (1.6%). Indications yielding the most clinically significant findings were abnormal karyotype/fluorescence in situ hybridization (26/61, 42.6%), family history of chromosomal abnormality (13/137, 9.5%), abnormal ultrasound (38/410, 9.3%), abnormal serum screening (2/37, 5.4%) and advanced maternal age (5/394, 1.3%). Of 1075 cases having no previously known cytogenetic abnormality or family history, 18 (1.7%) had clinically significant genomic changes undetectable by conventional prenatal chromosome analysis.Current experience confirms that the detection rate of CMA for prenatal chromosomal abnormalities surpasses that of conventional karyotype analysis and continues to improve with higher resolution arrays, while maintaining a low frequency of results of unclear clinical significance.

The widespread clinical utilization of array comparative genome hybridization, has led to the unraveling of many new copy number variations (CNVs). Although some of these CNVs are clearly pathogenic, the phenotypic consequences of others, such as those in 16p13.11 remain unclear. Whereas deletions of 16p13.11 have been associated with multiple congenital anomalies, the relevance of duplications of the region is still being debated. We report detailed clinical and molecular characterization of 10 patients with duplication and 4 patients with deletion of 16p13.11. We found that patients with duplication of the region have varied clinical features including behavioral abnormalities, cognitive impairment, congenital heart defects and skeletal manifestations, such as hypermobility, craniosynostosis and polydactyly. These features were incompletely penetrant. Patients with deletion of the region presented with microcephaly, developmental delay and behavioral abnormalities as previously described. The CNVs were of varying sizes and were likely mediated by non-allelic homologous recombination between low copy repeats. Our findings expand the repertoire of clinical features observed in patients with CNV in 16p13.11 and strengthen the hypothesis that this is a dosage sensitive region with clinical relevance. European Journal of Human Genetics (2011) 19, 280-286; doi:10.1038/ejhg.2010.184; published online 8 December 2010

Application of array comparative genomic hybridization (aCGH) has allowed an unprecedented high-resolution analysis of cancer genomes. We developed a custom genome-wide oligonucleotide microarray interrogating 493 genes involved in hematological disorders. We analyzed 55 patients with hematological neoplasms by using this microarray. In 33 patients with apparent normal conventional cytogenetic analysis, aneuploidy or isochromosomes were detected in 12% (4 of 33) of the patients by aCGH. The chromosomal changes included trisomy of chromosomes 10, 14, and 15, tetrasomy 11, and isochromosome 17q. In 17 patients with chronic lymphocytic leukemia who were initially investigated by using a panel of standard fluorescence in situ hybridization probes, additional copy number changes that were not interrogated by the fluorescence in situ hybridization (FISH) panel were detected in 47% (8 of 17) of the patients by aCGH. Important copy number changes included gain on 2p16 involving REL and BCL11A genes, rearrangements of chromosomes 8 and 15, and trisomy of chromosomes 19 and 22. In five patients with known abnormal karyotypes, aCGH identified the origin of two marker chromosomes and detected microdeletions at five breakpoints involved in three apparent balanced translocations. Our results suggest that a subset of potentially significant genomic alterations is missed by the currently available cytogenetic techniques. This pilot study clearly demonstrates high sensitivity of oligonucleotide aCGH for potential use in diagnosis and follow-up in patients with hematological neoplasms.

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.

Two novel CD18 mutations were identified in a patient who was a compound heterozygote with type 1 leukocyte adhesion deficiency and whose phenotype was typical except that he exhibited hypertrophic scarring. A deletion of 36 nucleotides in exon 12 (1622del36) predicted the net loss of 12 amino acid (aa) residues in the third cysteine-rich repeat of the extracellular stalk region (mut-1). A nonsense mutation in exon 15 (2200G>T), predicted a 36-aa truncation of the cytoplasmic domain (mut-2). Lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen-1 (Mac-1) containing the mut-1 β2 subunit were expressed at very low levels compared with wild-type (wt) β2. Mac-1 and LFA-1 expression with the mut-2 β2 subunit were equivalent to results with wt β2. Binding function of Mac-1 with mut-2 β2 was equivalent to that with wt β2. However, binding function of LFA-1 with the mut-2 β2 subunit was reduced by 50% versus wt β2. It was concluded that (1) the portion of the CD18 stalk region deleted in mut-1 is critical for β2 integrin heterodimer expression but the portion of the cytoplasmic domain truncated in mut-2 is not; and (2) the mut-2 cytoplasmic domain truncation impairs binding function of LFA-1 but not of Mac-1. Studies with the patient's neutrophils (PMNs) were consistent with functional impairment of LFA-1 but not of Mac-1. (Blood. 2004;103:1105-1113)

Abstract 2543 aCGH has become the state-of art tool for assaying copy number changes of the entire genome in recent years. It offers a high resolution, high throughput and an unbiased view of detecting copy number changes in a genome compared to standard cytogenetics. In fact, aCGH has become the standard of care for detection of genomic/cytogenetic abnormalities in a postnatal setting recently (PMID # 20466091). In contrast, the application of aCGH in a clinical setting is lagging behind for acquired disease such as cancer, although aCGH has contributed greatly in our understanding of these disorders. Here we show the utility of aCGH in detecting prognostic significant genomic alterations in multiple myeloma compared to standard chromosome and targeted FISH analysis. Multiple myeloma (MM) is a proliferation of malignant clonal plasma cells (PC) which accumulate in the bone marrow. It is a heterogeneous disease in which the genetic features of the malignant cells are the major contributing factor. Multiple myeloma is broadly divided into two biological genetic categories, hyperdiploid MM harboring multiple chromosome trisomies and a low frequency of IgH translocations and a non-hyperdiploid MM which have a high frequency of IgH translocations. There are also secondary genetic events that have been characterized in MM. Both the above genetic events in MM have major prognostic influence. The current clinical standard for identifying these genetic events in MM is conventional cytogenetics and targeted FISH analysis. However chromosome abnormalities are only detected in 30–40% of malignant PCs due to a low mitotic index in culture and FISH analysis only targets a few loci and therefore limiting in its use. In this study we compared the standard chromosome analysis of clinically and morphologically diagnosed MM to aCGH using a custom designed cancer genes targeted 44K Agilent array. It specifically interrogates 493 genes involved in carcinogenesis with evenly distributed probes mapped to each gene at an average spacing of 1 probe/10 kb, or at least 10 probes/gene for genes less than 100 kb in size [PMID: 20724749]. In this study a total of 53 patients with indication of MM, 9% cases (5/53) showed chromosome abnormalities by standard karyotype analysis as compared to 40% (21/53) of the cases that demonstrated a genomic change by aCGH. Furthermore only 3 of the cytogenetic abnormal cases were consistent with a MM profile of hyperdiplody, the other 2 cases had single chromosome events. While analysis of the genomic profile obtained from aCGH analysis clearly showed biological and prognostic stratification. A total of 69% (11/16) showed a hyperdiploid MM profile. In addition, 44% (7/16) cases had a gain of chromosome 1q which has been associated with a poor prognosis. All cases with the 1q gain had normal chromosome analysis. Recently a new mechanism of genomic instability termed chromothripsis has been described in 2–3 % of cancer genomes (PMID: 21215367). This is an acquired single catastrophic event during cancer development. This event has been described in multiple myeloma and has been associated to a poor outcome (PMID:21628407). We observed chromothripsis in 2 chromosomes in 2 cases. Based on this preliminary study it is quite compelling that aCGH provides a more accurate biological or prognostic profile in 69% of cases (11/16) vs 19% (3/21) by conventional cytogenetics. Therefore we conclude that the first line standard testing in Multiple myeloma should be aCGH and FISH for the IgH rearrangements and not conventional cytogenetics and FISH. Disclosures: No relevant conflicts of interest to declare.

Oligonucleotide-based array CGH (aCGH) technique enables detection of submicroscopic copy number changes in cancer. This technique offers the flexibility to increase robustness by selecting best performing probes maximize the gene coverage while excluding repetitive sequences through a combination of bioinformatics and computation and increased resolution that allows for detection of smaller region of change. We designed a high resolution (44,000 probes) custom array using an Agilent platform and specifically targeted ~500 genes implicated in leukemogenesis. The disease gene regions have an average spatial resolution of ~1 oligo per 7.5 kb whereas the backbone regions have an average resolution of 1 oligo per 78 kb. A total of 13 samples from patients with AML have been studied to date. aCGH was performed in a blinded fashion and the results were compared to the results from routine cytogenetic analysis. A GAIN in copy number on the long arm of chromosome 8 at band 8q22.2 encompassing 2.05 Mb was observed in one of 7 cases with normal chromosome analysis. Of the remaining 6 cases, the results from aCGH were concordant with the abnormality detected by chromosome analysis in three although the size of the copy number change was determined with greater precision by aCGH. Additional copy number changes were observed in the remaining two cases. In one case, in addition to the deletion of 5q14q34 detected by cytogenetic analysis, a loss in copy number on the long arm of 17q at band 17q11.2 encompassing 1.8 Mb including the NF-1 gene was detected by aCGH. Two additional copy number changes in 3q26.1 and 8q24.1 were observed in another case in which monosomy 7 was observed cytogenetically. aCGH failed to detect a clone (2 cells) with a deletion on 5 from band 5p13q13 in one case. In total, 30% of the patients with chromosome analysis displayed an additional cryptic change by aCGH. Our results suggest that a subset of potentially significant genomic alteration may be missed by the conventional chromosome analysis. This pilot study demonstrates that aCGH offers high sensitivity and specificity for routine screening of copy number changes in acute myeloid leukemia.