Multiplex Ligation Probe Amplification Research Articles

Three novel splicing mutations at 5' terminal of DMD gene corresponding to different phenotypes

To study the correlation of splicing mutations at the 5' end of the DMD gene with their phenotypes. DMD gene mutations were analyzed using Multiplex Ligation Probe Amplification (MLPA) and Sanger sequencing. Co-segregation analysis was performed for the pedigrees of the probands. Influence of mutations on protein function was predicted by bioinformatic analysis. Three novel splicing mutations were identified in three patients with different phenotypes. Patient 1 carried a c.31+3insT mutation and presented primarily with dilated cardiomyopathy (XLDC). There was no clinical signs of skeletal myopathy. Bioinformatic analysis predicted that the mutation may inactivate the splicing donor of intron 1 and lead to premature termination of protein translation. Patient 2 carried a c.264_264+4delTGTAA mutation, which led to loss of splicing donor site for intron 4, and manifested Becker muscular dystrophy (BMD). The mutation was predicted to result in skipping of exon 4. The defective protein may still retain most of its function. Patient 3 had Duchenne muscular dystrophy (DMD) and carried a c.832-3C>T mutation which was predicted to decrease the activity of splicing acceptor of intron 8, resulting in usage of alternative acceptor site or retain of intron 8. All related transcripts may cause premature termination of protein translation and complete loss of protein function. The three mutations were all inherited from the mothers of the patients. Three novel splicing mutations were discovered at the 5' end of DMD gene in three patients with different disease phenotypes. Our study may facilitate understanding of the influence of splicing mutations at the 5' end of the DMD gene on dystrophin function and the correlation between genotypes and phenotypes.

27-OR: Simplifying Detection of Large Scale Deletions Causing MODY5

Maturity Onset Diabetes of the Young (MODY) 5, also known as renal cysts and diabetes syndrome, is a rare autosomal dominant form of monogenic diabetes caused by mutations in the HNF1B gene encoding hepatocyte nuclear factor 1 homeobox beta. Whole gene deletions of one copy of HNF1B are the most common mutation underlying MODY5, with more extensive deletions encompassing HNF1B and several surrounding genes, known as 17q12 deletion syndrome, present in a sub-set of individuals with MODY5, who present with additional clinical features. Historically, these large scale deletions are not identified using Sanger or targeted next generation sequencing (NGS) panels, and require other tools, such as microarray-based genomic hybridization or multiplex ligation probe amplification to detect. Incorporating such tests to targeted sequencing adds significantly to cost and labor. Recent advances in bioinformatics allow for identification of large-scale copy number variation (CNV) from NGS output data without requiring additional laboratory methods. The NGS data from 57 patients suspected to have MODY by their physicians but who were negative for pathogenic mutations using a targeted NGS approach were thus re-examined using a CNV calling tool (CNV Caller, VarSeq v1.4.3). Three unrelated patients were found to have large scale deletions spanning HNF1B. These deletions were verified using whole exome sequencing data as well as microarray analysis (CytoScan, Affymetrix), which confirmed the presence of 17q12 deletion in all three individuals. The deletions varied in size from 1.46 to 1.85 million base pairs. Clinical correlation revealed a history of genitourinary abnormalities in all three probands. These results confirm the utility of the CNV Caller tool in identifying large-scale deletions causing MODY5, as well as 17q12 deletion syndrome. This may represent an easier, more cost-effective approach to making this diagnosis, ultimately improving case detection and allowing for more personalized care for affected individuals. Disclosure A.J. Berberich: None. J. Wang: None. H. Cao: None. A.D. McIntyre: None. J.F. Robinson: None. P. Yang: None. J. Knoll: Stock/Shareholder; Self; Cytognomix Inc. R. Hegele: Consultant; Self; Aegerion Pharmaceuticals, Akcea Therapeutics, Amgen Inc., Regeneron Pharmaceuticals, Sanofi.

Y-chromosome haplogroup architecture confers susceptibility to azoospermia factor c microrearrangements: a retrospective study

AimTo assess the association between azoospermia factor c microrearrangements and semen quality, and between Y-chromosome background with distinct azoospermia factor c microrearrangements and semen quality impairment.MethodsThis retrospective study, carried out in the Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov,” involved 486 men from different ethnic backgrounds referred for couple infertility from 2002-2017: 338 were azoospermic/oligozoospermic and 148 were normozoospermic. The azoospermia factor c microrearrangements were analyzed with sequence tagged site and sequence family variant markers, quantitative fluorescent polymerase chain reaction, and multiplex ligation probe amplification analysis. The Y-haplogroups of all participants were determined with direct single nucleotide polymorphism typing and indirect prediction with short tandem repeat markers.ResultsOur participants had two types of microdeletions: gr/gr and b2/b3; three microduplications: b2/b4, gr/gr, and b2/b3; and one complex rearrangement gr/gr deletion + b2/b4 duplication. Impaired semen quality was not associated with microrearrangements, but b2/b4 and gr/gr duplications were significantly associated with haplogroup R1a (P < 0.001 and P = 0.003, respectively) and b2/b3 deletions with haplogroup E (P = 0.005). There were significantly more b2/b4 duplication carriers in Albanians than in Macedonians with haplogroup R1a (P = 0.031).ConclusionEven though azoospermia factor c partial deletions/duplications and Y-haplogroups were not associated with impaired semen quality, specific deletions/duplications were significantly associated with distinct haplogroups, implying that the Y chromosome background may confer susceptibility to azoospermia factor c microrearrangements.

Clinical characteristics of HNF1B-related disorders in a Japanese population.

Hepatocyte nuclear factor 1β (HNF1B), located on chromosome 17q12, causes renal cysts and diabetes syndrome (RCAD). Moreover, various phenotypes related to congenital anomalies of the kidney and urinary tract (CAKUT) or Bartter-like electrolyte abnormalities can be caused by HNF1B variants. In addition, 17q12 deletion syndrome presents with multi-system disorders, as well as RCAD. As HNF1B mutations are associated with different phenotypes and genotype-phenotype relationships remain unclear, here, we extensively studied these mutations in Japan. We performed genetic screening of RCAD, CAKUT, and Bartter-like syndrome cases. Heterozygous variants or whole-gene deletions in HNF1B were detected in 33 cases (19 and 14, respectively). All deletion cases were diagnosed as 17q12 deletion syndrome, confirmed by multiplex ligation probe amplification and/or array comparative genomic hybridization. A retrospective review of clinical data was also conducted. Most cases had morphological abnormalities in the renal-urinary tract system. Diabetes developed in 12 cases (38.7%). Hyperuricemia and hypomagnesemia were associated with six (19.3%) and 13 cases (41.9%), respectively. Pancreatic malformations were detected in seven cases (22.6%). Ten patients (32.3%) had liver abnormalities. Estimated glomerular filtration rates were significantly lower in the patients with heterozygous variants compared to those in patients harboring the deletion (median 37.6 vs 58.8ml/min/1.73 m2; p = 0.0091). We present the clinical characteristics of HNF1B-related disorders. To predict renal prognosis and complications, accurate genetic diagnosis is important. Genetic testing for HNF1B mutations should be considered for patients with renal malformations, especially when associated with other organ involvement.

Molecular and clinical study on homozygous or heterozygous large deletion of CYP21A2 gene in 21-OHD patients

Objective: To explore the relationship between homozygous or heterozygous large deletion of CYP21A2 gene and clinical manifestation in 21-hydroxylase deficiency (21-OHD) patients. Methods: The records of 100 patients with 21-OHD were collected between June 2016 and December 2017 in Peking Union Medical College Hospital. Large deletion of CYP21A2 gene was detected by multiplex ligation probe amplification (MLPA). The biochemical results and clinical symptoms of patients with homozygous or heterozygous large deletion were analyzed in order to investigate the influence of complete or single allele deletion of CYP21A2 gene on 21-OHD patients. Results: Large deletion of CYP21A2 gene was detected in 33 patients by MLPA, including 13 males and 20 females, with an median age of 10 (6,16) years. Two of them had simultaneous deletions of two alleles. Among 31 patients with heterozygous deletion, 16 were combined with gene mutations that severely affected 21-hydroxylase enzyme activity (I2G and Q318X), 15 with mutations that retained part enzyme activity (I172N and P30L). Two patients with complete deletion of CYP21A2 gene had no significant difference in biochemical and clinical manifestations compared with those with single allele deletion combined with another gene mutation that severely affected enzyme activity. Both kinds of patients above were manifested as severe salt-wasting type. Patients with a single allele deletion and a mutation retaining part enzyme activity were manifested as mild simple-viralizing type. Conclusion: Large deletion of CYP21A2 gene could appear in 21-OHD patients and the phenotype is similar to that of salt-wasting patients with heterozygous large deletion.

Analysis of 15q11.2 CNVs in an Indian population with schizophrenia.

Copy number variants (CNVs) of 15q11.2 yielded conflicting reports on their association with schizophrenia (SZ), indicating the need for replication studies. Because there are no 15q11.2 CNV studies on Indian patients, we began by testing 307 SZ patients and 359 age- and sex-matched controls from South India. Using an improved multiplex ligation probe amplification, six deletions were found in patients and three in controls (p=0.31), whereas one duplication was found in patients and three in controls (p=0.63). Analysis of families of two patients and two controls with deletions indicated that the mutations were de novo. In conclusion, there seems to be no significant difference in the frequencies of 15q11.2 CNVs among the controls and patients studied here. Future studies involving a larger number of controls and patients are expected to provide better clarity on the relationship between 15q11.2 CNVs and SZ patients from India.

A novel heterozygous duplication of the SLC12A3 gene in two Gitelman syndrome pedigrees: indicating a founder effect

Gitelman syndrome is an autosomal recessive salt-wasting tubulopathy caused by mutations in the SLC12A3 gene. A female and a male sibling from two unrelated Greek-Cypriot families presenting with a severe salt-wasting tubulopathy due to compound heterozygous mutations of a novel duplication and a previously reported missense mutation in the SLC12A gene are described. Sanger sequencing was used to identify possible mutations in the SLC12A3 gene. For the detection of duplications/conversions and deletions in the same gene, Multiplex ligation probe amplification (MLPA) analysis was performed. Direct sequencing and MLPA analysis of the SLC12A3 gene identified two compound heterozygous mutations in both unrelated probands. Both probands were identified to carry in compound heterozygosity the known p.Met581Lys and a novelheterozygous duplication of exons 9-14 (E9_E14dup). The diagnosis of Gitelman syndrome was made through clinical assessment, biochemical screening and genetic analysis. The identification of the novel SLC12A3 duplication seems to be characteristic of Greek-Cypriot patients and suggests a possible ancestral mutational event that has spread in Cyprus due to a possible founder effect. Testing for Gitelman syndrome probable variants can be performed before proceeding to a full gene sequencing dropping the diagnostic cost. In addition, this report adds to the mutational spectrum observed.

Rapid detection of copy number variations and point mutations in BRCA1/2 genes using a single workflow by ion semiconductor sequencing pipeline.

Molecular analysis of BRCA1 (MIM# 604370) and BRCA2 (MIM #600185) genes is essential for familial breast and ovarian cancer prevention and treatment. An efficient, rapid, cost-effective accurate strategy for the detection of pathogenic variants is crucial. Mutations detection of BRCA1/2 genes includes screening for single nucleotide variants (SNVs), small insertions or deletions (indels), and Copy Number Variations (CNVs). Sanger sequencing is unable to identify CNVs and therefore Multiplex Ligation Probe amplification (MLPA) or Multiplex Amplicon Quantification (MAQ) is used to complete the BRCA1/2 genes analysis. The rapid evolution of Next Generation Sequencing (NGS) technologies allows the search for point mutations and CNVs with a single platform and workflow. In this study we test the possibilities of NGS technology to simultaneously detect point mutations and CNVs in BRCA1/2 genes, using the OncomineTM BRCA Research Assay on Personal Genome Machine (PGM) Platform with Ion Reporter Software for sequencing data analysis (Thermo Fisher Scientific). Comparison between the NGS-CNVs, MLPA and MAQ results shows how the NGS approach is the most complete and fast method for the simultaneous detection of all BRCA mutations, avoiding the usual time consuming multistep approach in the routine diagnostic testing of hereditary breast and ovarian cancers.

Genetic Imbalances in Argentinean Patients with Congenital Conotruncal Heart Defects

Congenital conotruncal heart defects (CCHD) are a subset of serious congenital heart defects (CHD) of the cardiac outflow tracts or great arteries. Its frequency is estimated in 1/1000 live births, accounting for approximately 10–30% of all CHD cases. Chromosomal abnormalities and copy number variants (CNVs) contribute to the disease risk in patients with syndromic and/or non-syndromic forms. Although largely studied in several populations, their frequencies are barely reported for Latin American countries. The aim of this study was to analyze chromosomal abnormalities, 22q11 deletions, and other genomic imbalances in a group of Argentinean patients with CCHD of unknown etiology. A cohort of 219 patients with isolated CCHD or associated with other major anomalies were referred from different provinces of Argentina. Cytogenetic studies, Multiplex-Ligation-Probe-Amplification (MLPA) and fluorescent in situ hybridization (FISH) analysis were performed. No cytogenetic abnormalities were found. 22q11 deletion was found in 23.5% of the patients from our cohort, 66% only had CHD with no other major anomalies. None of the patients with transposition of the great vessels (TGV) carried the 22q11 deletion. Other 4 clinically relevant CNVs were also observed: a distal low copy repeat (LCR)D-E 22q11 duplication, and 17p13.3, 4q35 and TBX1 deletions. In summary, 25.8% of CCHD patients presented imbalances associated with the disease.

DUCHENNE MUSCULAR DYSTROPHY - GENETICS: P.214Genetic profile of Chilean patients with Duchenne muscle dystrophy

Duchenne muscle dystrophy (DMD), is a progressive muscular disease, inherited as a X link. Is caused by the mutations of the dystrophin gene. Deletions of one or more exons account for approximately 50-65%, duplications 5-10% and point mutations 20-25%. Analyze the genetic profile of patients with DMD through regular checkups at the pediatric neurology department of the San Borja Arriarán Hospital. Retrospective descriptive study. Analysis of clinical records of 41 patients in evaluation by DMD, excluding patients diagnosed prior to the year 2010, with only biopsy and/ or PCR (polymerase chain reaction) without alterations. 41 patients with clinical manifestations and/or muscle biopsy with markedly reduced or no detectable dystrophin immunostaining, were studied by PCR, MLPA (multiplex ligation probe amplification) and/or Sanger sequencing techniques. 27/41 (66%) of patients had large deletions, 20/27 had compromised exons 44-55, and 3/26 had compromised exons 3-7. The most frequent deletion was exon 50, followed by exon 49. 3/41 (7,3%) presented duplications, which all affected exon 2. 10/41(24,4%) of patients had a negative MLPA, but a compatible muscle biopsy. In 1/41, sequencing was performed, showing nonsense mutation, in exon 41. Our study of the genetic profile of chileans patients is similar to previously reported, deletions are the most frequent alterations. It should be noted that biopsy continues to be an important tool for confirming diagnostics, due to our countries limited access to sequencing techniques. Because of this, we can extrapolate and conclude that the large percentage of patients with compatible muscle biopsy and negative MLPA, could correspond to point mutations, which would then coincide with the published. Therefore, it is important to make genetic diagnosis advances thus giving us the possibility of integrating new therapies and the ability to offer genetic counseling.

BRCA mutation carrier detection. A model-based cost-effectiveness analysis comparing the traditional family history approach and the testing of all patients with breast cancer

BackgroundIdentification of BRCA mutation carriers among patients with breast cancer (BC) involves costs and gains. Testing has been performed according to international guidelines, focusing on family history (FH) of breast and/or ovarian cancer. An alternative is testing all patients with BC employing sequencing of the BRCA genes and Multiplex Ligation Probe Amplification (MLPA).Patients and methodsA model-based cost-effectiveness analysis, employing data from Oslo University Hospital, Ullevål (OUH-U) and a decision tree, was done. The societal and the healthcare perspectives were focused and a lifetime perspective employed. The comparators were the traditional FH approach used as standard of care at OUH-U in 2013 and the intervention (testing all patients with BC) performed in 2014 and 2015 at the same hospital. During the latter period, 535 patients with BC were offered BRCA testing with sequencing and MLPA. National 2014 data on mortality rates and costs were implemented, a 3% discount rate used and the costing year was 2015. The incremental cost-effectiveness ratio was calculated in euros (€) per life-year gained (LYG).ResultsThe net healthcare cost (healthcare perspective) was €40 503/LYG. Including all resource use (societal perspective), the cost was €5669/LYG. The univariate sensitivity analysis documented the unit cost of the BRCA test and the number of LYGs the prominent parameters affecting the result.Diagnostic BRCA testing of all patients with BC was superior to the FH approach and cost-effective within the frequently used thresholds (healthcare perspective) in Norway (€60 000–€80 000/LYG).

Comprehensive Molecular Screening in Chinese Usher Syndrome Patients.

Usher syndrome (USH) refers to a group of autosomal recessive disorders causing deafness and blindness. The objectives of this study were to determine the mutation spectrum in a cohort of Chinese patients with USH and to describe the clinical features of the patients with mutations. A total of 119 probands who were clinically diagnosed with USH were recruited for genetic analysis. All probands underwent ophthalmic examinations. A combination of molecular screening methods, including targeted next-generation sequencing, Sanger-DNA sequencing, and multiplex ligation probe amplification assay, was used to detect mutations. We found biallelic mutations in 92 probands (77.3%), monoallelic mutations in 5 patients (4.2%), and 1 hemizygous mutation in 1 patient (0.8%), resulting in an overall mutation detection rate of 78.2%. Overall, 132 distinct disease-causing mutations involving seven USH (ABHD12, CDH23, GPR98, MYO7A, PCDH15, USH1C, and USH2A) genes; 5 other retinal degeneration genes (CHM, CNGA1, EYS, PDE6B, and TULP1); and 1 nonsyndromic hearing loss gene (MYO15A) were identified, and 78 were novel. Mutations of MYOA7 were responsible for 60% of USH1 families, followed by PCDH15 (20%) and USH1C (10%). Mutations of USH2A accounted for 67.7% of USH2 families, and mutation c.8559-2A>G was the most frequent one, accounting for 19.1% of the identified USH2A alleles. Our results confirm that the mutation spectrum for each USH gene in Chinese patients differs from those of other populations. The formation of the mutation profile for the Chinese population will enable a precise genetic diagnosis for USH patients in the future.

Hypomyelinating disorders in China: The clinical and genetic heterogeneity in 119 patients.

ObjectiveHypomyelinating disorders are a group of clinically and genetically heterogeneous diseases characterized by neurological deterioration with hypomyelination visible on brain MRI scans. This study was aimed to clarify the clinical and genetic features of HMDs in Chinese population.Methods119 patients with hypomyelinating disorders in Chinese population were enrolled and evaluated based on their history, clinical manifestation, laboratory examinations, series of brain MRI with follow-up, genetic etiological tests including chromosomal analysis, multiplex ligation probe amplification, Sanger sequencing, targeted enrichment-based next-generation sequencing and whole exome sequencing.ResultsClinical and genetic features of hypomyelinating disorders were revealed. Nine different hypomyelinating disorders were identified in 119 patients: Pelizaeus-Merzbacher disease (94, 79%), Pelizaeus-Merzbacher-like disease (10, 8%), hypomyelination with atrophy of the basal ganglia and cerebellum (3, 3%), GM1 gangliosidosis (5, 4%), GM2 gangliosidosis (3, 3%), trichothiodystrophy (1, 1%), Pol III-related leukodystrophy (1, 1%), hypomyelinating leukodystrophy type 9 (1, 1%), and chromosome 18q deletion syndrome (1, 1%). Of the sample, 94% (112/119) of the patients were genetically diagnosed, including 111 with mutations distributing across 9 genes including PLP1, GJC2, TUBB4A, GLB1, HEXA, HEXB, ERCC2, POLR3A, and RARS and 1 with mosaic chromosomal change of 46, XX,del(18)(q21.3)/46,XX,r(18)(p11.32q21.3)/45,XX,-18. Eighteen novel mutations were discovered. Mutations in POLR3A and RARS were first identified in Chinese patients with Pol III-related leukodystrophy and hypomyelinating leukodystrophy, respectively.SignificanceThis is the first report on clinical and genetic features of hypomyelinating disorders with a large sample of patients in Chinese population, identifying 18 novel mutations especially mutations in POLR3A and RARS in Chinese patients, expanding clinical and genetic spectrums of hypomyelinating disorders.

STRC Gene Mutations, Mainly Large Deletions, are a Very Important Cause of Early-Onset Hereditary Hearing Loss in the Czech Population.

Hearing loss (HL) is the most common sensory deficit in humans. HL is an extremely heterogeneous condition presenting most frequently as a nonsyndromic (NS) condition inherited in an autosomal recessive (AR) pattern, termed DFNB. Mutations affecting the STRC gene cause DFNB type 16. Various types of mutations within the STRC gene have been reported from the U.S. and German populations, but no information about the relative contribution of STRC mutations to NSHL-AR among Czech patients is available. Two hundred and eighty-eight patients with prelingual NSHL, either sporadic (n = 207) or AR (n = 81), who had been previously tested negative for the mutations affecting the GJB2 gene, were included in the study. These patients were tested for STRC mutations by a quantitative comparative fluorescent polymerase chain reaction (QF-PCR) assay. In addition, 31 of the 81 NSHL-AR patients were analyzed by massively parallel sequencing using one of two different gene panels: 23 patients were analyzed by multiplex-ligation probe amplification (MLPA); and 9 patients by SNP microarrays. Causal mutations affecting the STRC gene (including copy number variations [CNVs] and point mutations) were found in 5.5% of all patients and 13.6% of the 81 patients in the subgroup with NSHL-AR. Our results provide strong evidence that STRC gene mutations are an important cause of NSHL-AR in Czech HL patients and are probably the second most common cause of DFNB. Large CNVs were more frequent than point mutations and it is reasonable to test them first by a QF-PCR method-a simple, accessible, and efficient tool for STRC CNV detection, which can be combined by MLPA.

BRCA1 and BRCA2 germline variants in breast cancer patients from the Republic of Macedonia.

We aimed to establish the spectrum of BRCA1/2 mutations among the breast cancer (BC) patients from the Republic of Macedonia. We used targeted next-generation sequencing (NGS), Sanger DNA sequencing, and multiplex ligation probe amplification analysis (MLPA) to search for point mutations and deletions/duplications involving BRCA1 and BRCA2-coding regions. We have analyzed a total of 313 BC patients, enriched for family history of cancer, early age of onset and bilateral and/or triple negative (TN) BC. A total of 26 pathogenic mutations were observed in 49 unrelated BC patients (49/313, 15.7%). BRCA2 mutations (27/49, 55.1%) were more common than BRCA1 mutations (22/49, 44.9%). We identified five novel point mutations, one in BRCA1 (c.4352_4356delA) and four in BRCA2 (c.151G>T, c.4707_4708delCA, c.7811_7814delTGTG, and c.9304_9305delG), as well as two novel deletions involving parts of the BRCA1 gene (c.81-?_593+?del and c.5470-?_5530+?del). The most common mutations were c.181T>G, c.5266dupC, and c.3700_3704del5 in BRCA1 and c.7879A>T, c.8317_8330del14 and c.5722_5723delCT in BRCA2 gene. Thus far, BRCA2 c.7879A>T and c.8317_8330del14 mutations have been described in several isolated cases; however, our study is the first one showing that they have a founder effect among Macedonian population. Nine recurrent mutations account for 65.3% of all of the detected mutations allowing for implementation of a fast first-step BRCA1/2 mutational screening strategy in our country. This study provides a comprehensive view of known and novel BRCA1/2 mutations in BC patients from the Republic of Macedonia and contributes to the global spectrum of BRCA1/2 mutations in breast cancer.

Abstract A43: Genetic and epigenetic disorders in one cohort of acute lymphoblastic leukemia

Abstract B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease. Approximately 60% of BCP-ALL present aberrations involving chromosome 21 (chr 21), including hyperdiploid (HeH), ETV6-RUNX1 fusion, and intrachromosomal amplification of the chromosome 21 (iAMP21). On the other hand, epigenetic mechanisms could regulate the transcription and induce the leukemogenesis. Polymorphisms in folate genes could influence in this aberrant methylation. This study aims to characterize the genetic and epigenetic profile of BCP-ALL with chr21 aberrations, as well as identify the epigenetic signatures of different ALL subgroups. A series of 373 BCP-ALL were selected for CNA analysis concerning the chromosome 21. Multiplex ligation probe amplification (MLPA, SALSA P327_A1, and P327_B1) was performed according to manufacturer instructions. FISH was performed using the LPH012 TEL/AML1 translocation, dual fusion probe, and centromere probes to the chr 4, 8, 10, 14, 17, 18, X, and Y (Cytocell, Cambridge, UK). Additionally, to the DNA from cases of BCP-ALL, controls and remission samples were modified with EZ DNA Methylation™ Kit (Zymo Research, Irvine, CA) and analyzed by the HumanMethylation450 Infinium Assay (Illumina, San Diego, CA). For the array validation and global (LINE-1) methylation we analyzed not just the samples with chr 21 gains, but also BCP-ALL from different subtypes using pyrosequencing. The MTHFR rs1801133 was identified by PCR-RFLP. The statistical analysis was performed using RStudio software with Bioconductor packages. We found evidence of gains in chr21 in 82 samples analyzed by MLPA. Most gains were verified by FISH and 11 samples had ≥5 RUNX1 signals. The centromere probes characterized 53 samples as HeH. The extra chr21 was confirmed in all cases in addition to other gains: chr4 (58%), 10 (57%), 14 (84%), 17 (53%), 18 (60%), X (86%), and Y (46%). Losses of the chr 17 (2%) and Y (4%) were also identified. Two BCP-ALL with ETV6/RUNX1 had an extra copy of the chr 21. One patient had iAMP21 identified by MLPA and confirmed by FISH with telomere probe targeted to chr13 and 21. The case vs. control analysis regarding the DNA methylation showed 5031 differentially methylated CpG sites (adjusted p &amp;lt;0.001). We observed that the methylation profiles of cases and controls were distinct, while the controls and remission samples had similar profiles. In the validation analysis, the ARID3A gene was hypermethylated in controls when compared with BCP-ALL and remission samples. The HeH group presents lower methylation for the ERG and ARID3A genes while the BCP-ALL with KTM2A rearrangements had a higher methylation level for ARID3A, PRDM9, and PRDM15 genes, the ETV6-RUNX1 had a higher ARID3A methylation and lastly, the TCF3-PBX1 had a higher significant PRDM15 methylation when compared against the other BCP-ALL subtypes. According to the LINE-1 methylation, the HeH group present a distinct profile of increased methylation level in comparison with the other BCP-ALL subtypes and healthy controls. Genetic susceptibility conferred by MTHFR rs1801133 has not contributed to methylation changes in settings. To discriminate the iAMP21 to HeH subgroups, it is recommended to use centromere and/or telomere FISH probes. The BCP-ALL presents a distinct global and gene-specific DNA methylation signature; these signatures are subtype-specific. Citation Format: Tállita Meciany Farias Vieira, Sheila Coelho Sores Lima, Franciane Gomes Andrade, Alython Araújo Chung-Filho, Claire Schwab, Christine Harrison, Maria do Socorro Pombo-de-Oliveira. Genetic and epigenetic disorders in one cohort of acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A43.

Evaluation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Mutation Detection

The efficiency of a novel targeted next-generation sequencing (NGS) test, the Devyser BRCA kit, for a comprehensive analysis of all 48 coding exons of the high-risk breast/ovarian cancer susceptibility genes BRCA1 and BRCA2 has been assessed. The new assay intended to detect nucleotide substitutions, small deletions/insertions, and large deletions/duplications. To document the false-negative and false-positive rates of the NGS assay in the hands of end users, 48 samples with previously identified 444 small variants and seven gross rearrangements were analyzed, showing 100% concordance with gold standards. Furthermore, all other 43 variants (42 single-nucleotide variation or insertion/deletion variation and one copy number variation, whose significance is or may be of clinical value), which were called by the NGS assay in a prospectively analyzed 179-sample set, were confirmed by Sanger sequencing or multiplex ligation probe amplification, according to their nature. We conclude that the Devyser BRCA kit performed satisfactorily for use in a clinical laboratory.

Genomic imbalances in syndromic congenital heart disease

ObjectiveTo identify pathogenic genomic imbalances in patients presenting congenital heart disease (CHD) with extra cardiac anomalies and exclusion of 22q11.2 deletion syndrome (22q11.2 DS). Methods78 patients negative for the 22q11.2 deletion, previously screened by fluorescence in situ hybridization (FISH) and/or multiplex ligation probe amplification (MLPA) were tested by chromosomal microarray analysis (CMA). ResultsClinically significant copy number variations (CNVs ≥300kb) were identified in 10% (8/78) of cases. In addition, potentially relevant CNVs were detected in two cases (993kb duplication in 15q21.1 and 706kb duplication in 2p22.3). Genes inside the CNV regions found in this study, such as IRX4, BMPR1A, SORBS2, ID2, ROCK2, E2F6, GATA4, SOX7, SEMAD6D, FBN1, and LTPB1 are known to participate in cardiac development and could be candidate genes for CHD. ConclusionThese data showed that patients presenting CHD with extra cardiac anomalies and exclusion of 22q11.2 DS should be investigated by CMA. The present study emphasizes the possible role of CNVs in CHD.

Combined genetic analyses can achieve efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

We evaluated combined genetic analyses with targeted next-generation sequencing (NGS), multiplex ligation probe amplification (MLPA) of Jagged1 (JAG1) genes and microarray comparative genomic hybridisation (CGH) in subjects with Alagille syndrome, incomplete clinical features of Alagille syndrome and biliary atresia. Subjects recruited from April 2013 to December 2015 underwent a targeted NGS analysis, including JAG1 and Notch homolog 2 (NOTCH2). If no mutations were detected in JAG1 or NOTCH2, or if copy number variations were suggested by the NGS analysis, we performed an MLPA analysis of JAG1. We also performed a microarray CGH analysis with whole-exon deletion detected by the MLPA analysis. We analysed 30 subjects with Alagille syndrome, nine with incomplete Alagille syndrome and 17 with biliary atresia and detected pathogenic mutations in JAG1 or NOTCH2 in 24/30 subjects with Alagille syndrome and in 4/9 subjects with incomplete Alagille syndrome. No pathogenic mutations were detected in subjects with biliary atresia. The frequency of JAG1 mutations was as follows: single nucleotide variants (51.9%), small insertion or deletion (29.6%) and gross deletion (18.5%). Combined genetic analyses achieved efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

VITRECTOMY-ASSISTED BIOPSY FOR MOLECULAR PROGNOSTICATION OF CHOROIDAL MELANOMA 2 MM OR LESS IN THICKNESS WITH A 27-GAUGE CUTTER.

Fine-needle aspiration biopsy of small choroidal melanomas 2 mm or less in thickness may be associated with a low biopsy yield. This report describes our center's experience using the 27-gauge vitreous cutter to perform tumor sampling in these small melanomas. This was a retrospective analysis of all patients who underwent a 27-gauge vitreous cutter biopsy for molecular prognostication at the time of iodine-125 plaque placement for local treatment of choroidal melanoma ≤2.0 mm in height. Seventeen consecutive patients with a mean baseline tumor height of 1.67 mm (range, 1.31-2.03 mm) and median follow-up time of 7.0 months (range, 3.3-19.5 months) were included. The 27-gauge cutter biopsy yielded sufficient material for multiplex-ligation probe amplification (MLPA) or gene expression profiling (GEP) in 17/17 (100%) patients. Complications were limited to focal vitreous hemorrhage in 13/17 patients and diffuse vitreous hemorrhage in 1/17 patients, with no instance of retinal detachment or nonclearing vitreous hemorrhage. Tumor sampling of small choroidal melanoma with a 27-gauge vitreous cutter is safe and offers excellent biopsy yield for molecular prognostication when cancer prognosis is desired by patients.