Gordana Raca

Trinucleotide repeats affect DNA replication in vivo

(CGG)n (CCG)n and (CTG)n (CAG)n repeats of varying length were cloned into a bacterial plasmid, and the progression of the replication fork through these repeats was followed using electrophoretic analysis of replication intermediates. We observed stalling of the replication fork within repeated DNAs and found that this effect depends on repeat length, repeat orientation relative to the replication origin and the status of protein synthesis in a cell. Interruptions within repeated DNAs, similar to those observed in human genes, abolished the replication blockage. Our results suggest that the formation of unusual DNA structures by trinucleotide repeats in the lagging-strand template may account for the observed replication blockage and have relevance to repeat expansion in humans.

The genomic landscape of hypodiploid acute lymphoblastic leukemia

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24–31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32–39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

Detailed Characterization of, and Clinical Correlations in, 10 Patients with Distal Deletions of Chromosome 9p

Purpose: Deletions of distal 9p are associated with trigonocephaly, mental retardation, dysmorphic facial features, cardiac anomalies, and abnormal genitalia. Previous studies identified a proposed critical region for the consensus phenotype in band 9p23, between 11.8 Mb and 16 Mb from the 9p telomere. Here we report 10 new patients with 9p deletions; 9 patients have clinical features consistent with 9p− syndrome, but possess terminal deletions smaller than most reported cases, whereas one individual lacks the 9p− phenotype and shows a 140-kb interstitial telomeric deletion inherited from his mother.Methods: We combined fluorescence in situ hybridization and microarray analyses to delineate the size of each deletion.Results: The deletion sizes vary from 800 kb to 12.4 Mb in our patients with clinically relevant phenotypes. Clinical evaluation and comparison showed little difference in physical features with regard to the deletion sizes. Severe speech and language impairment were observed in all patients with clinically relevant phenotypes.Conclusion: The smallest deleted region common to our patients who demonstrate a phenotype consistent with 9p− is <2 Mb of 9pter, which contains six known genes. These genes may contribute to some of the cardinal features of 9p deletion syndrome.

Whole-exome sequencing supports genetic heterogeneity in childhood apraxia of speech

BackgroundChildhood apraxia of speech (CAS) is a rare, severe, persistent pediatric motor speech disorder with associated deficits in sensorimotor, cognitive, language, learning and affective processes. Among other neurogenetic origins, CAS is the disorder segregating with a mutation in FOXP2 in a widely studied, multigenerational London family. We report the first whole-exome sequencing (WES) findings from a cohort of 10 unrelated participants, ages 3 to 19 years, with well-characterized CAS.MethodsAs part of a larger study of children and youth with motor speech sound disorders, 32 participants were classified as positive for CAS on the basis of a behavioral classification marker using auditory-perceptual and acoustic methods that quantify the competence, precision and stability of a speaker’s speech, prosody and voice. WES of 10 randomly selected participants was completed using the Illumina Genome Analyzer IIx Sequencing System. Image analysis, base calling, demultiplexing, read mapping, and variant calling were performed using Illumina software. Software developed in-house was used for variant annotation, prioritization and interpretation to identify those variants likely to be deleterious to neurodevelopmental substrates of speech-language development.ResultsAmong potentially deleterious variants, clinically reportable findings of interest occurred on a total of five chromosomes (Chr3, Chr6, Chr7, Chr9 and Chr17), which included six genes either strongly associated with CAS (FOXP1 and CNTNAP2) or associated with disorders with phenotypes overlapping CAS (ATP13A4, CNTNAP1, KIAA0319 and SETX). A total of 8 (80%) of the 10 participants had clinically reportable variants in one or two of the six genes, with variants in ATP13A4, KIAA0319 and CNTNAP2 being the most prevalent.ConclusionsSimilar to the results reported in emerging WES studies of other complex neurodevelopmental disorders, our findings from this first WES study of CAS are interpreted as support for heterogeneous genetic origins of this pediatric motor speech disorder with multiple genes, pathways and complex interactions. We also submit that our findings illustrate the potential use of WES for both gene identification and case-by-case clinical diagnostics in pediatric motor speech disorders.

Childhood Apraxia of Speech (CAS) in two patients with 16p11.2 microdeletion syndrome

We report clinical findings that extend the phenotype of the ∼550 kb 16p11.2 microdeletion syndrome to include a rare, severe, and persistent pediatric speech sound disorder termed Childhood Apraxia of Speech (CAS). CAS is the speech disorder identified in a multigenerational pedigree (‘KE’) in which half of the members have a mutation in FOXP2 that co-segregates with CAS, oromotor apraxia, and low scores on a nonword repetition task. Each of the two patients in the current report completed a 2-h assessment protocol that provided information on their cognitive, language, speech, oral mechanism, motor, and developmental histories and performance. Their histories and standard scores on perceptual and acoustic speech tasks met clinical and research criteria for CAS. Array comparative genomic hybridization analyses identified deletions at chromosome 16p11.2 in each patient. These are the first reported cases with well-characterized CAS in the 16p11.2 syndrome literature and the first report of this microdeletion in CAS genetics research. We discuss implications of findings for issues in both literatures.

NSD1 analysis for Sotos syndrome: Insights and perspectives from the clinical laboratory

Purpose: Sotos syndrome is a genetic disorder characterized primarily by overgrowth, developmental delay, and a characteristic facial gestalt. Defects in the NSD1 gene are present in approximately 80% of patients with Sotos syndrome. The goal of this study was to determine the incidence of NSD1 abnormalities in patients referred to a clinical laboratory for testing and to identify clinical criteria that distinguish between patients with and without NSD1 abnormalities.Methods: Deletion or mutation analysis of the NSD1 gene was performed on 435 patients referred to our clinical genetics laboratory. Detailed clinical information was obtained on 86 patients with and without NSD1 abnormalities, and a clinical checklist was developed to help distinguish between these two groups of patients.Results: Abnormalities of the NSD1 gene were identified in 55 patients, including 9 deletions and 46 mutations. Thus, in the clinical laboratory setting, deletions were found in 2% and mutations in 21% of samples analyzed, because not all patients had both tests. Thirty-three previously unreported mutations in the NSD1 gene were identified. Clinical features typically associated with Sotos syndrome were not found to be significantly different between individuals with and without NSD1 abnormalities. The clinical checklist developed included poor feeding, increased body mass index, and enlarged cerebral ventricles, in addition to the typical clinical features of Sotos syndrome, and was able to distinguish between the two groups with 80% sensitivity and 70% specificity.Conclusions: The dramatic decrease in the frequency of finding NSD1 abnormalities in the clinical laboratory is likely because of the heterogeneity of the patient population. Our experience from a diagnostic laboratory can help guide clinicians in deciding for whom NSD1 genetic analysis is indicated.

Next generation sequencing in research and diagnostics of ocular birth defects.

Sequence capture enrichment (SCE) strategies and massively parallel next generation sequencing (NGS) are expected to increase the rate of gene discovery for genetically heterogeneous hereditary diseases, but at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. Our study assessed whether array based target enrichment followed by re-sequencing on the Roche Genome Sequencer FLX (GS FLX) system could be used for novel mutation identification in more than 1000 exons representing 100 candidate genes for ocular birth defects, and as a control, whether these methods could detect two known mutations in the PAX2 gene. We assayed two samples with heterozygous sequence changes in PAX2 that were previously identified by conventional Sanger sequencing. These changes were a c.527G>C (S176T) substitution and a single basepair deletion c.77delG. The nucleotide substitution c.527G>C was easily identified by NGS. A deletion of one base in a long polyG stretch (c.77delG) was not registered initially by the GS Reference Mapper, but was detected in repeated analysis using two different software packages. Different approaches were evaluated for distinguishing false positives (sequencing errors) and benign polymorphisms from potentially pathogenic sequence changes that require further follow-up. Although improvements will be necessary in accuracy, speed, ease of data analysis and cost, our study confirms that NGS can be used in research and diagnostic settings to screen for mutations in hundreds of loci in genetically heterogeneous human diseases.

Complex or monosomal karyotype and not blast percentage is associated with poor survival in acute myeloid leukemia and myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2): a Bone Marrow Pathology Group study

Acute myeloid leukemia and myelodysplastic syndrome with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) have a poor prognosis. Indeed, the inv(3)(q21q26.2)/t(3;3)(q21;q26.2) has been recognized as a poor risk karyotype in the revised International Prognostic Scoring System. However, inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is not among the cytogenetic abnormalities pathognomonic for diagnosis of acute myeloid leukemia irrespective of blast percentage in the 2008 WHO classification. This multicenter study evaluated the clinico-pathological features of acute myeloid leukemia/myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and applied the revised International Prognostic Scoring System to myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2). A total of 103 inv(3)(q21q26.2)/t(3;3)(q21;q26.2) patients were reviewed and had a median bone marrow blast count of 4% in myelodysplastic syndrome (n=40) and 52% in acute myeloid leukemia (n=63) (P<0.001). Ninety-one percent of patients showed characteristic dysmegakaryopoiesis. There was no difference in overall survival between acute myeloid leukemia and myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) (12.9 vs. 7.9 months; P=0.16). Eighty-three percent of patients died (median follow up 7.9 months). Complex karyotype, monosomal karyotype and dysgranulopoiesis (but not blast percentage) were independent poor prognostic factors in the entire cohort on multivariable analysis. The revised International Prognostic Scoring System better reflected overall survival of inv(3)(q21q26.2)/t(3;3)(q21;q26.2) than the International Prognostic Scoring System but did not fully reflect the generally dismal prognosis. Our data support consideration of myelodysplastic syndrome with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) as an acute myeloid leukemia with recurrent genetic abnormalities, irrespective of blast percentage.

Array comparative genomic hybridization analysis in patients with anophthalmia, microphthalmia, and coloboma

Purpose: The goal of our study was to determine whether genomic copy number abnormalities (deletions and duplications) affecting genes involved in eye development contributed to the etiology of anophthalmia, microphthalmia, and coloboma.Methods: The affected individuals were evaluated for the presence of deletions and duplications in genomic DNA by a very high-resolution array comparative genomic hybridization.Results: Array analysis of 32 patients detected one case with a deletion encompassing the renal-coloboma syndrome associated gene PAX2. Nonpolymorphic copy number changes were also observed at several candidate chromosomal regions, including 6p12.3, 8q23.1q23.2, 13q31.3, 15q11.2q13.1, 16p13.13, and 20q13.13.Conclusions: This study identified the first patient with the typical phenotype of the renal-coloboma syndrome caused by a submicroscopic deletion of the coding region of the PAX2 gene. The finding suggests that PAX2 deletion testing should be performed in addition to gene sequencing as a part of molecular evaluation for the renal-coloboma syndrome. Array comparative genomic hybridization testing of 32 affected individuals showed that genomic deletions and duplications are not a common cause of nonsyndromic anophthalmia, microphthalmia, or coloboma but undoubtedly contribute to the etiology of these eye anomalies. Therefore, array comparative genomic hybridization testing represents an important and valuable addition to candidate gene sequencing in research and diagnostics of ocular birth defects.

The advantage of using SNP array in clinical testing for hematological malignancies--a comparative study of three genetic testing methods.

Cytogenetic methods, including G-banded chromosome analysis and fluorescence in situ hybridization (FISH) analysis, serve as a critical part of routine clinical testing for hematological malignancies and provide important diagnostic and prognostic information; however, the limitations of cytogenetic methods, including the requirement for actively dividing cells and lower resolution of G-banded chromosome analysis as well as the inability of both G-banded chromosome analysis and FISH to detect copy number neutral loss of heterozygosity (CN-LOH), can result in a failure to detect genomic abnormalities with diagnostic and prognostic significance. Here, we compared the abnormality detection rate of clinically requested testing (i.e., G-banded chromosome analysis and FISH) with high-resolution oligo (i.e., array comparative genomic hybridization (aCGH)) and single-nucleotide polymorphism (SNP)/oligo hybrid (i.e., SNP-CGH) arrays in a series of patients, in an effort to assess the ability of newer technologies to overcome these limitations. This series found the detection rate for SNP-CGH to be 62.5% for myelodysplastic syndrome (MDS) cases and 72.7% for chronic lymphocytic leukemia (CLL) cases, which are significantly higher than the detection rates of aCGH (31.3% for MDS and 54.5% for CLL) and G-banding and/or FISH (43.8% for MDS and 54.5% for CLL). This demonstrates the advantages of combining SNP-CGH with conventional cytogenetics to provide comprehensive clinical information by detecting clonality, large balanced rearrangements, copy number aberrations, and CN-LOH.