Genome-wide SNP Array Analysis Research Articles

Infantile onset Sandhoff disease: clinical manifestation and a novel common mutation in Thai patients

BackgroundSandhoff disease (SD) is an autosomal recessive lysosomal storage disorder, resulting in accumulation of GM2 ganglioside, particular in neuronal cells. The disorder is caused by deficiency of β-hexosaminidase B (HEX-B), due to pathogenic variant of human HEXB gene.MethodThis study describes clinical features, biochemical, and genetic defects among Thai patients with infantile SD during 2008–2019.ResultsFive unrelated Thai patients presenting with developmental regression, axial hypotonia, seizures, exaggerated startle response to noise, and macular cherry red spot were confirmed to have infantile SD based on deficient HEX enzyme activities and biallelic variants of the HEXB gene. In addition, an uncommon presenting feature, cardiac defect, was observed in one patient. All the patients died in their early childhood. Plasma total HEX and HEX-B activities were severely deficient. Sequencing analysis of HEXB gene identified two variants including c.1652G>A (p.Cys551Tyr) and a novel variant of c.761T>C (p.Leu254Ser), in 90 and 10% of the mutant alleles found, respectively. The results from in silico analysis using multiple bioinformatics tools were in agreement that the p.Cys551Tyr and the p.Leu254Ser are likely pathogenic variants. Molecular modelling suggested that the Cys551Tyr disrupt disulfide bond, leading to protein destabilization while the Leu254Ser resulted in change of secondary structure from helix to coil and disturbing conformation of the active site of the enzyme. Genome-wide SNP array analysis showed no significant relatedness between the five affected individuals. These two variants were not present in control individuals. The prevalence of infantile SD in Thai population is estimated 1 in 1,458,521 and carrier frequency at 1 in 604.ConclusionThe study suggests that SD likely represents the most common subtype of rare infantile GM2 gangliosidosis identified among Thai patients. We firstly described a potential common variant in HEXB in Thai patients with infantile onset SD. The data can aid a rapid molecular confirmation of infantile SD starting with the hotspot variant and the use of expanded carrier testing.

Recurrent Rare Genomic Copy Number Variants and Bicuspid Aortic Valve Are Enriched in Early Onset Thoracic Aortic Aneurysms and Dissections.

Thoracic Aortic Aneurysms and Dissections (TAAD) are a major cause of death in the United States. The spectrum of TAAD ranges from genetic disorders, such as Marfan syndrome, to sporadic isolated disease of unknown cause. We hypothesized that genomic copy number variants (CNVs) contribute causally to early onset TAAD (ETAAD). We conducted a genome-wide SNP array analysis of ETAAD patients of European descent who were enrolled in the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC). Genotyping was performed on the Illumina Omni-Express platform, using PennCNV, Nexus and CNVPartition for CNV detection. ETAAD patients (n = 108, 100% European American, 28% female, average age 20 years, 55% with bicuspid aortic valves) were compared to 7013 dbGAP controls without a history of vascular disease using downsampled Omni 2.5 data. For comparison, 805 sporadic TAAD patients with late onset aortic disease (STAAD cohort) and 192 affected probands from families with at least two affected relatives (FTAAD cohort) from our institution were screened for additional CNVs at these loci with SNP arrays. We identified 47 recurrent CNV regions in the ETAAD, FTAAD and STAAD groups that were absent or extremely rare in controls. Nine rare CNVs that were either very large (>1 Mb) or shared by ETAAD and STAAD or FTAAD patients were also identified. Four rare CNVs involved genes that cause arterial aneurysms when mutated. The largest and most prevalent of the recurrent CNVs were at Xq28 (two duplications and two deletions) and 17q25.1 (three duplications). The percentage of individuals harboring rare CNVs was significantly greater in the ETAAD cohort (32%) than in the FTAAD (23%) or STAAD (17%) cohorts. We identified multiple loci affected by rare CNVs in one-third of ETAAD patients, confirming the genetic heterogeneity of TAAD. Alterations of candidate genes at these loci may contribute to the pathogenesis of TAAD.

Deficiency of ATP6V1H Causes Bone Loss by Inhibiting Bone Resorption and Bone Formation through the TGF-β1 Pathway.

Vacuolar-type H +-ATPase (V-ATPase) is a highly conserved, ancient enzyme that couples the energy of ATP hydrolysis to proton transport across vesicular and plasma membranes of eukaryotic cells. Previously reported mutations of various V-ATPase subunits are associated with increased bone density. We now show that haploinsufficiency for the H subunit of the V1 domain (ATP6V1H) is associated with osteoporosis in humans and mice. A genome-wide SNP array analysis of 1625 Han Chinese found that 4 of 15 tag SNPs (26.7%) within ATP6V1H were significantly associated with low spine bone mineral density. Atp6v1h+/- knockout mice generated by the CRISPR/Cas9 technique had decreased bone remodeling and a net bone matrix loss. Atp6v1h+/- osteoclasts showed impaired bone formation and increased bone resorption. The increased intracellular pH of Atp6v1h+/- osteoclasts downregulated TGF-β1 activation, thereby reducing induction of osteoblast formation but the bone mineralization was not altered. However, bone formation was reduced more than bone resorption. Our data provide evidence that partial loss of ATP6V1H function results in osteoporosis/osteopenia. We propose that defective osteoclast formation triggers impaired bone formation by altering bone remodeling. In the future, ATP6V1H might, therefore, serve as a target for the therapy of osteoporosis.

Abstract 3293: Molecular evidence for a parent of origin effect in pediatric insulinomas

Abstract Insulinomas are well-differentiated pancreatic endocrine tumors that cause abnormal beta cell proliferation and hyperinsulinimic hypoglycemia. With an incidence of 2-4 cases per million per year, insulinomas are rare in adults and even less common in children. Since congenital focal hyperinsulinism, another developmental lesion, is associated with paternal isodisomy for the imprinted 11p15 locus, we sought to characterize the role of this region and of mutations in MEN1 on 11q13 in pediatric insulinoma cases. We identified 13 pediatric insulinoma cases, which is the largest pediatric series reported, and performed immunohistochemical analysis for the maternally expressed tumor suppressor marker p57 located in 11p15. Using blood and tumor DNA, we tested 9 candidate genes associated with congenital hyperinsulinism including ABCC8, KCNJ11 and the MEN1 tumor suppressor. We performed methylation analysis of imprint control regions (ICR) that included the paternally expressed IGF2 (ICR1) and maternally expressed CDKN1C (ICR2) located on 11p15 and genome-wide SNP array analysis with tumor DNA. MEN1 mutations were found in 4 cases and suspected in one (MEN1 group); all 5 tumors stained negative for p57 and were hypermethylated at ICR1 and hypomethylated at ICR2 indicating loss of the maternal allele. SNP arrays confirmed loss of heterozygosity (LOH) of entire chromosome 11 in 4 cases. Four out of the 6 remaining non-MEN1 cases also stained negative for p57; in confirmation, 2 of the 4 cases carried mosaic copy number abnormality of chromosome 11. SNP array data demonstrated extensive genome-wide aneuploidy in MEN1 and non-MEN1 cases; however interstitial changes were not present in any of the cases. Notably, MEN1 cases carried only entire chromosome changes, while non-MEN1 cases carried whole arm changes in addition whole chromosome changes. In conclusion, MEN1 mutations are more common in pediatric insulinomas than in adult insulinoma cases (40% in this study vs. 8% in adults). Compared to adult cases, our pediatric insulinoma cases had smaller sized tumors and a shorter median duration of symptoms, suggesting that these tumors are rapidly growing and that the most common chromosome gains and losses very likely represent early events in insulinoma development. Mechanistically, our data suggest that loss of p57 expression, in conjunction with loss of either MEN1 and/or additional imprinted genes on 11p, play a role in the pathogenesis of most insulinomas. In cases with a germ-line MEN1 mutation, we identified a previously unreported paternal parent-of-origin effect that is unique to pediatric insulinomas but not to other MEN1 endocrine tumors (e.g., prolactinoma, parathyroid adenoma). Since a congenital focal hyperinsulinism has a similar parent of origin effect this suggests a broader role for the imprinted locus, 11p15, in not just the pathogenesis of pediatric insulinomas but also in beta-cell replication. Citation Format: Arupa Ganguly, Tricia Bhatti, Karthik Ganapathy, Laura Conlin, Courtney MacMullen, Susan Becker, Eduardo Ruchelli, Charles Stanley. Molecular evidence for a parent of origin effect in pediatric insulinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3293. doi:10.1158/1538-7445.AM2015-3293

Clinico-molecular study of synchronous head and neck squamous cell carcinoma (HNSCC) and esophageal squamous cell carcinoma (ESCC).

e17080 Background: The prognoses of HNSCC and ESCC are poor, especially when two primary tumors occur at the same time. In an earlier study, we found 12.4% of all HNSCC patients had a synchronous ESCC at the time of diagnosis. To confirm whether these second tumors were of metastatic origin or a separate second primary tumor (SPT), we employed genome-wide SNP array analysis to analyze the differential LOH pattern of each synchronous tumor pair. The clinical information of the patients with synchronous tumors was also summarized. Methods: Twenty-one synchronous HNSCC formalin-fixed paraffin-embedded (FFPE) tissue samples and their ESCC counterparts were subjected to DNA extraction, quality assessment, and subsequent SNP array experiments using Illumina HumanCytoSNP12 FFPE BeadChip, which contains 300,000 probes. Signal intensity and B-allele frequency data of each pair were analyzed to identify Copy Number Variation (CNV) and LOH using Nexus Copy Number software suite. Results: Comparing the LOH pattern of each primary HNSCC to its esophageal tumor counterpart, we found 20 out of 21 pairs had a low number of concordant LOHs (LOH at the same genomic location seen in both HNSCC and ESCC) and a high number of discordant LOHs (LOH identified solely in primary HNSCC, but not in second ESCC at the same genomic location). Only one of the cases fell into the undetermined category. We conclude that the second esophageal tumors in our patients were indeed individual SPTs. Synchronous HNSCC and ESCC are considered to be of worse prognostic value compared with non synchronous tumors. Conclusions: We propose here the use of a genome-wide SNP array as a tool to differentiate metastatic tumors from SPT. The SNP array offers genome-wide LOH information that earlier microsatellite analysis studies lack. The ability to accurately identify SPT should contribute to a better treatment plan and follow-up care of this group of patients. Clinical correlation using a larger study group may help clarify the clinical characteristics of SPT.

Near-haploidization significantly associates with oncocytic adrenocortical, thyroid, and parathyroid tumors but not with mitochondrial DNA mutations.

Mitochondrial-rich oncocytic thyroid tumors frequently show near-haploidization and endoreduplication (masked haploidization), which manifests as a near-homozygous genome (NHG). We now extend this investigation to include adrenocortical cancer and parathyroid carcinoma (PaTC), which we studied for a NHG in association with mitochondrial DNA mutations. Sixty endocrine tumors from 59 patients were studied, including 46 thyroid tumor samples of varying histology, 11 adrenocortical cancers, and 3 PaTCs. Genome-wide SNP array analysis and DNA content analysis were combined to determine the chromosomal dosage (allelic state). The entire mitochondrial genome was also studied for mutations. In addition, tumors were characterized for somatic mutations in a subset of genes that are directly or indirectly implicated in cellular metabolism. In addition to a subset of thyroid cancers (n = 5), a NHG was also observed in 1 of 3 PaTCs and 6 of 11 adrenocortical cancers. All but one of the tumors with a NHG (n = 12) showed oncocytic metaplasia (P = 0.0001, two-tailed Fisher's exact). One or more damaging or disrupting mtDNA mutations were found in 68% (41/60) of tumor samples. No correlation was found between mtDNA mutations and the oncocytic phenotype or a NHG, and none of the mutations in nuclear encoded genes correlated with the oncocytic phenotype or a NHG. A subset of oncocytic tumors of the thyroid, parathyroid, and adrenocortical carcinomas carries a NHG. Although damaging/disrupting mtDNA mutations are frequently found in oncocytic and nononcocytic endocrine tumors, neither correlates with a NHG phenotype nor with an oncocytic phenotype.

Clinical, cytogenetic, and molecular characterization of six patients with ring chromosomes 22, including one with concomitant 22q11.2 deletion

We report here on six patients with a ring chromosome 22 and the range of cytogenetic and phenotypic features presented by them. Genomic analysis was carried out using classical and molecular cytogenetics, MLPA (Multiplex Ligation-dependent Probe Amplification) and genome-wide SNP-array analysis. The ring was found in all patients, but Patient 6 displayed constitutional mosaicism with a normal cell line. Five patients had deletions in the ring chromosome 22, and in four of them the breakpoints--unique for each patient--could be identified by genome-wide SNP-array analysis. One patient presented with a 22q11.2 deletion concomitant with the deletion caused by the ring formation. Common phenotypic features included autism, speech delay and seizures, as previously reported for individuals with r(22) and/or 22q13.3 deletions. Investigation of the genes within the deletions revealed multiple genes related to development of the central nervous system, psychomotor delay, severe language impairment, hypotonia, and autistic symptoms. There was no clear correlation between the severity of clinical features and the size of the deleted segment. This study underscores the variability in ring structure and clinical presentation of the r(22) and adds information to the limited literature on this rare disorder.

Behavioural phenotype of a patient with a de novo 1.2 Mb chromosome 4q25 microdeletion

A female patient, 20 years of age, is reported with a history characterized by developmental and psychomotor delay, and during grammar-school period increasing learning problems, ritualistic behaviours and social withdrawal. Subsequently, challenging and autistic-like behaviours became prominent. The patient showed mild facial dysmorphisms, long thin fingers with bilateral mild short V metacarpals, and hyperlaxity of the joints. Neuropsychiatric examination disclosed obsessive, ritualistic behaviours and vague ideas of reference. Neuropsychological assessment demonstrated mild intellectual disability, mental inflexibility and incongruent affect. MRI-scanning of the brain showed no relevant abnormalities. Genome wide SNP array analysis revealed a 1.2 Mb de novo interstitial microdeletion in 4q25 comprising 11 genes, that was considered to be causative for the developmental delay, perseverative cognitive phenotype and dysmorphisms.To the authors knowledge, this is the first report of a de novo 4q25 microdeletion that presents with a specific behavioural phenotype.

545 – Paranoid schizophrenia in kallmann syndrome: genetics and psychopathology

Introduction Kallmann syndrome (KS) is a genetically heterogenous and rare disorder characterized by the combination of hypothalamic hypogonadism and anosmia/hyposmia. In about one third of the patients, mutations have been identified in at least seven different genes. Apart from one published case report, virtually no data are available about possible neuropsychiatric symptoms in KS. Objectives Studying the putative relationship between KS and schizophrenia. Aims Investigating the genetic etiology in a patient with a clinical diagnosis of KS and paranoid schizophrenia. Methods Detailed diagnostic evaluation including genetic analysis of an adult male patient with paranoid schizophrenia, hypogonadism and anosmia. Results Psychiatric examination of the 28-years-old male patient disclosed negative psychotic symptoms only, following an acute paranoid schizophrenic episode one year before. He used 15mg aripiprazole daily and testosterone gel 20mg/gr. Hormonal panel showed extremely low levels of LH and FSH. MRI scanning of the brain demonstrated complete absence of the olfactory bulbs. Otolaryngeal examination disclosed complete anosmia. Neuropsychological assessment revealed subaverage intelligence distinctly disconcordant with premorbid functioning, markedly lowered speed of information processing, and impaired executive and social cognitive functions. Genome wide SNP array analysis and mutation analyses for the KAL1, FGFR1, PROK2, PROKR2, FGF8 and CHD7 genes, disclosed no genetic imbalance or any pathogenic mutation. Conclusions This first report on a patient with KS and paranoid schizophrenia in whom extensive genetic analyses were performed, stipulates the need for studying a possible increased risk for psychiatric symptoms in patients with KS.

Persistent Mosaicism for 12p Duplication/Triplication Chromosome Structural Abnormality in Peripheral Blood

We present a rare case of mosaicism for a structural abnormality of chromosome 12 in a patient with phenotypic features of Pallister-Killian syndrome. A six-month-old child with dysmorphic features, exotropia, hypotonia, and developmental delay was mosaic for both a normal karyotype and a cell line with 12p duplication/triplication in 25 percent of metaphase cells. Utilization of fluorescence in situ hybridization (FISH) identified three copies of probes from the end of the short arm of chromosome 12 (TEL(12p13) locus and the subtelomere (12p terminal)) on the structurally abnormal chromosome 12. Genome-wide SNP array analysis revealed that the regions of duplication and triplication were of maternal origin. The abnormal cell line in our patient was present at 25 percent at six months and 19 months of age in both metaphase and interphase cells from peripheral blood, where typically the isochromosome 12p is absent in the newborn. This may suggest that the gene(s) resulting in a growth disadvantage of abnormal cells in peripheral blood of patients with tetrasomy 12p may not have the same influence when present in only three copies.

Mosaic segmental uniparental isodisomy and progressive clonal selection: a common mechanism of late onset -thalassemia major

Genomic DNA of 3 patients, born as healthy carriers and developing a late-onset severe transfusion-dependent beta-thalassemia major was studied by high-density genome wide SNP array analysis. A mosaic loss of heterozygosity for almost the entire 11p was found, not attributable to deletions but involving mosaicism for segmental paternal isodisomy of 11p. Mitotic recombination leading to mosaic segmental uniparental isodisomy on chromosome 11p in multiple tissues has been described as a molecular disease mechanism for a subset of sporadic Beckwith-Wiedemann syndrome cases. A similar mechanism also seems to be involved in causing late-onset disease in carriers of recessive mutations in other genes located in 11p, such as late-onset beta-thalassemia major and sickle cell disease. We suggest that the loss of maternally imprinted IGF-2 and H19 genes may account for the selective advantage of hematopoietic cells containing this segmental paternal isodisomy of 11p carrying the β-thalassemia mutation.

A de novo 3.57 Mb microdeletion in 8q12.3q13.2 in a patient with mild intellectual disability and epilepsy

A female patient, nine years of age, is reported with a history characterized by delay of psychomotor and speech development, mild to moderate intellectual disability and persistent sleep disturbances since the age of two. The patient showed facial dysmorphisms, a pectus excavatum and a sandal gap. Apart from lowered intelligence, neuropsychological functioning disclosed impaired attentional capacities and executive control as well as weak motor skills. Genome wide SNP array analysis revealed a 3.57 Mb de novo microdeletion in band q12.3 of chromosome 8. The long lasting sleep disorders turned out to originate from a rare juvenile epilepsy, continuous spike-waves during slow sleep (CSWS) syndrome, that includes the electrical status epilepticus in sleep (ESES) phenomenon. MRI-scanning of the brain showed no abnormalities.To the authors knowledge, this is the first report of a de novo 8q12.3q13.2 microdeletion syndrome that presents with ESES/CSWS.

Mosaic trisomy 17: Variable clinical and cytogenetic presentation

Mosaic trisomy 17 is rare with only 28 cases reported and the clinical presentation is highly variable. The diagnosis is most commonly made by prenatal karyotype and in most cases is followed by a normal postnatal karyotype on blood lymphocytes.We present two cases of mosaic trisomy 17 diagnosed prenatally,with follow up in multiple tissues at birth. In the first case,trisomy 17 was identified in all amniocytes, and at birth standard results of chromosome analysis in peripheral blood were normal,but mosaic trisomy 17 was identified (50–75%) in skin fibroblasts by genome-wide SNP array analysis. This patient presented with congenital heart disease, asymmetry, intestinal malrotation, and other anomalies and died on day 9 of life. In the second patient amniocentesis after ultrasound finding of tetralogy of Fallot showed mosaic trisomy 17. Postnatally, results of a SNP array were normal in blood, buccal mucosa, and skin. It is possible that the cardiac defect is related to trisomy 17 in key tissues during heart development, although at birth the aneuploidy could not be identified in tissues that are routinely analyzed for diagnosis. These cases add to our understanding of mosaic trisomy 17, highlighting the failure to diagnose this aneuploidy in peripheral blood.

Genome-wide Profiling in AML Patients Relapsing after Allogeneic Hematopoietic Cell Transplantation

Molecular pathogenesis of relapse after allogeneic hematopoietic cell transplantation is poorly understood. Data regarding relapse mechanisms after transplantation is scarcely available. We investigated genomic aberrations (GAs) in 21 patients undergoing related and unrelated HLA-matched transplantation in leukemic blasts before transplant and at relapse after transplantation. We found a higher number of GAs after transplantation, suggesting increased genomic instability during relapse. Two of 21 patients showed a large homozygous region spanning the whole HLA-locus on chromosome 6p in the relapse sample. In both patients sequence-based HLA typing of the blasts revealed a loss of the patient-specific allele at the mismatched locus leading to homozygosity for the HLA haplotype shared by the patient and the donor. In addition, GAs were found in critical regions such as 12p13, 13q12.2, and 17p13. Our results suggest that escape from immunologic surveillance may be a relevant mechanism of relapse after transplantation in patients with GAs on chromosome 6p. A combination of continuous immunologic pressure mediated by donor T cells and clonal evolution of myeloid leukemia may result in acquired GAs after transplantation.

Mutations in Frizzled 6 Cause Isolated Autosomal-Recessive Nail Dysplasia

Inherited and isolated nail malformations are rare and heterogeneous conditions. We identified two consanguineous pedigrees in which some family members were affected by isolated nail dysplasia that suggested an autosomal-recessive inheritance pattern and was characterized by claw-shaped nails, onychauxis, and onycholysis. Genome-wide SNP array analysis of affected individuals from both families showed an overlapping and homozygous region of 800 kb on the long arm of chromosome 8. The candidate region spans eight genes, and DNA sequence analysis revealed homozygous nonsense and missense mutations in FZD(6), the gene encoding Frizzled 6. FZD(6) belongs to a family of highly conserved membrane-bound WNT receptors involved in developmental processes and differentiation through several signaling pathways. We expressed the FZD(6) missense mutation and observed a quantitative shift in subcellular distribution from the plasma membrane to the lysosomes, where the receptor is inaccessible for signaling and presumably degraded. Analysis of human fibroblasts homozygous for the nonsense mutation showed an aberrant response to both WNT-3A and WNT-5A stimulation; this response was consistent with an effect on both canonical and noncanonical WNT-FZD signaling. A detailed analysis of the Fzd(6)(-/-) mice, previously shown to have an altered hair pattern, showed malformed claws predominantly of the hind limbs. Furthermore, a transient Fdz6 mRNA expression was observed in the epidermis of the digital tips at embryonic day 16.5 during early claw morphogenesis. Thus, our combined results show that FZD6 mutations can result in severe defects in nail and claw formation through reduced or abolished membranous FZD(6) levels and several nonfunctional WNT-FZD pathways.

Abstract 976: Hypoxic microenvironment promotes breast cancer stem cells through regulation of Polycomb protein expression

Abstract The microenvironment of solid tumors contains regions of poor oxygenation as a result of hypoxia, which is associated with high grade breast cancer and poor prognosis. However, critical regulatory mechanism linking tumor microenvironment to cancer progression remains unclear. Cancer stem cells (tumor initiating cells) are suggested to be responsible for initiation and aggressive progression of cancer. It is recently proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genomic/genetic mutations and drives cancer progression. Additionally, high expression of a Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to aggressive progression of breast cancer. Here, we discovered that hypoxic microenvironment upregulates HIF1α-mediated transcriptional activation of EZH2 in CD44+CD24−/low breast tumor initiating cells (BTICs) isolated from several human primary tumor samples. Enhanced EZH2 expression was required for the hypoxia-promoted BTICs, where hypoxia significantly increased BTIC abundance, tumor sphere formation and tumor growth in mammary fat pads implanted with less than 500 primary BTICs. Using cDNA expression array and genome wide SNP array analysis, we further identified a novel mechanism in which enhanced expression of EZH2 by hypoxia downregulates DNA damage repair proteins through H3K27 methylation and thereby allows the acquisition of recurrent genomic abnormalities such as RAF1 gene amplification in BTICs. Using proteomic analysis, we demonstrated that RAF1 amplification highly activates its downstream ERK-β-catenin signaling, which leads to enhanced survival and proliferation of the BTICs. Furthermore, using immunohistochemistry analysis in a cohort of breast tumor samples, we found that EZH2 expression is positively correlated with RAF1, p-ERK and β-catenin expression in the high grade, poorly-differentiated tumors but not in the low grade, well-differentiated tumors, suggesting EZH2-amplified RAF1-ERK-β-catenin signaling may contribute significantly to promote BTICs and aggravate breast cancer malignancy. Our data also reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, can significantly suppress EZH2-promoted BTICs. To the best of our knowledge, this is the first study that provides evidences linking a specific genomic aberration mediated by EZH2-impaired DNA damage response to expansion of BTICs. Together, this study elucidates a previously unidentified therapeutic effect of RAF1-ERK signaling inhibitors to prevent breast cancer progression by eliminating BTICs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 976. doi:10.1158/1538-7445.AM2011-976

A patient with a de novo distal 22q11.2 microdeletion and anxiety disorder

We report on a young female with normal intelligence evaluated for long-term anxiety. Her history includes prematurity, neonatal feeding problems, surgical correction of congenital heart defects, recurrent upper airway and urinary tract infections, and delayed motor and developmental milestones. Physical examination disclosed small stature and minor dysmorphisms. Chromosome analysis, 22q11.2 FISH analysis, and subtelomeric MLPA testing did not detect any abnormalities. Genome wide SNP Array analysis showed a de novo deletion in 22q11.21q11.22, the so-called distal 22q11 microdeletion that involves the MAPK1 gene. A diagnosis of panic disorder was made and the patient was successfully treated with a daily dose of 20 mg citalopram. To our knowledge, this is the first adolescent patient with a long history of complaints about anxiety and a distal 22q11 microdeletion. We speculate that genes from the deleted region, especially MAPK1, increase the neurobiological susceptibility to anxiety disorders that may be a part of the psychopathological phenotype of the distal 22q11.2 microdeletion syndrome.

Genome-Wide SNP Array Analysis in Patients with Features of Sotos Syndrome

Background: Sotos syndrome is characterized by overgrowth, facial dysmorphism and learning impairment. Haploinsufficiency of NSD1 accounts for approximately 60–90% of the patients. Consequently, a considerable number of patients with features of Sotos syndrome remain without a molecular diagnosis. To date, target-gene approaches in these patients have not been successful. Methods: Twenty-six Sotos syndrome-like patients were analyzed with a high-resolution whole-genome SNP array, and segregation was studied in the parents. Results: Four possible pathogenic copy-number variants including deletions of 10p12.32-p12.31, 14q13.1, Xq21.1-q21.31 and a duplication of 15q11.2-q13.1 were detected. They varied in size from 155 kb to 13.36 Mb. The 10p12.32-p12.31 deletion revealed a candidate gene (PLXDC2) for overgrowth. The 14q13.1 deletion affected only the NPAS3 gene and the patient carrying this deletion displayed mental retardation as the main feature. The Xq21.1-q21.31 deletion and the 15q11.2-q13.1 duplication encompassed multiple genes of which several could be associated with phenotypic expression. Conclusion: The high-resolution genome-wide SNP array approach resulted in a detection rate of 15% of novel abnormalities and is therefore a powerful method to attain a molecular diagnosis in Sotos syndrome-like patients. Identified candidate genes provide directions for future screening of larger patient cohorts.

Terminal 3p deletions in two families—Correlation between molecular karyotype and phenotype

The 3p deletion syndrome is a rare disorder caused by deletions of different sizes in the 3p25-pter region. It is characterized by growth retardation, developmental delay, mental retardation, dysmorphism, microcephaly, and ptosis. The phenotype of individuals with deletions varies from normal to severe. Most cases occur de novo, but a few familial cases have been reported. We describe two families with terminal 3p deletions and extremely variable clinical features. In family A, the mother and daughter were extremely mildly affected whereas the son had more severe clinical features. In family B, the mother was normal and her son was affected, having some symptoms that had not been described in the 3p deletion syndrome before. The deletions were characterized by genome-wide SNP array analysis and were 9 and 1.1 Mb in size. Sequencing analysis of the CHL1, CNTN4, and CRBN genes did not reveal any masked recessive alleles that might explain the more severe phenotypes in the probands. In family A, the 9 Mb deletion can be considered causal for the 3p deletion syndrome in the proband, but the extremely mild phenotype in the other family members remains unexplained. In family B, the 1.1 Mb terminal deletion encompasses only the CHL1 gene, which is insufficient to cause 3p deletion symptoms; thus the clinical features observed in this family may have a different cause. The variable penetrance of 3p deletions creates challenges in genetic counseling, as the phenotype of the offspring cannot be predicted based on chromosomal and/or genome-wide array analytical findings.

Dissection of Submicroscopical Aberrations and Clonal Evolution from Birth To Diagnosis and Relapse in a Childhood AML FLT3-ITD Positive Patient by RQ-PCR and SNP Array Analyses.

We have performed a combined Real Time Quantitative-PCR and single nucleotide polymorphisms array analyses for dissecting the clonal evolution in a childhood AML patient who experienced two relapses and for whom we had the availability of the cord blood (CB) sample. The patient was diagnosed at 6 years of age with an AML-M1 and showed normal karyotype and a FLT3-ITD mutation as a sole abnormality. She underwent autologous-BMT; however, 3.5 months later the patient relapsed, and 4 months after an allogeneic-BMT she suffered from a second relapse and died for disease progression. Highly sensitive (10−4) monitoring of FLT3-ITD was performed by patient-specific RQ-PCR, and showed a progressive decrease of minimal residual disease (MRD) during induction therapy. MRD was below the detection limit before auto-BMT, but the same FLT3-ITD clone re-emerged three months after auto-BMT, and preceded the clinical relapse. Thus, the same FLT3-ITD mutation was detected at the time of relapse, suggesting that the leukemic clone responsible for the first diagnosis was still present and could be potentially used as a marker for backtracking the leukemia into the CB. When tested by highly sensitive RQ-PCR, the DNA from CB resulted negative for the FLT3-ITD mutation. Although the relatively limited sensitivity of the technique might impair the interpretation, the FLT3-ITD negative result in CB is consistent with the hypothesis that FLT3-ITD mutations are secondary events, not sufficient by themselves to induce leukemia transformation in hematopoietic stem cells without a necessary primary event. With the aim to find additional submicroscopical genetic changes associated to the highly aggressive nature of the patient disease, we performed a genome wide SNP array analysis on the patient DNA through the clinical evolution of the disease, from birth to relapse. This new SNP array strategy is emerging as a powerful method to detect loss of heterozygosity (LOH) and/or copy number changes in a DNA sample with high resolution. The Affymetrix GeneChip® Mapping 10K platform has been used, which allows the scanning of more than 10.000 SNPs. SNP array analysis on DNA from the first relapse showed the deletion of the long arm of chromosome 9, a recurring chromosomal aberration in AML, and LOH on the whole chromosome 13 not associated with copy number changes. This latter has been confirmed by FISH, and it is consistent with uniparental isodisomy (UPD) as a responsible mechanism for the somatically acquired homozygosity of FLT3-ITD at 13q14. This mechanism is emerging as a frequent way of disease progression and represents a subsequent event to FLT3-ITD heterozygous mutation. The deletion of the wild type FLT3 allele has been confirmed by PCR. 10K SNP array analysis failed to reveal LOH or copy number changes in the diagnostic and in CB samples. These findings are compatible with a somatic post-natal origin of the FLT3-ITD positive AML subtype. Additional abnormalities can be responsible for the disease progression, via different mechanisms, including UPD. Other methods must be applied to find the primary event(s) giving rise to leukemia in association with FLT3-ITD mutation.