Probe Amplification Analysis Research Articles

Genetic instabilities as potential biomarkers for pregnancy-associated breast cancer.

e12559 Background: Pregnancy associated breast cancer (PABC), defined as breast cancer diagnosed during or within one year after pregnancy, occurs approximately once in every 3,000 pregnancies and accounts for up to 6.9% of all breast cancers in women ≤ 45 years. Compared to age-matched non-PABC patients, PABC is generally characterized by a particularly aggressive histopathologic profile (more ER-, PR- and HER2 negative tumors and tumors of higher grade) with a higher mortality rate. Whether these cancers arise before or during pregnancy, and whether their genetic profile differs from non-PABC tumors, is currently unknown. This study assesses the genetic background of PABC by detection of specific DNA copy number alterations (CNA). Methods: We assembled 29 triple-negative PABC patients from the Dutch nationwide pathology database (PALGA). From their formalin-fixed and paraffin embedded (FFPE) tumor tissue blocks, DNA was extracted. Multiplex Ligation-Dependent probe Amplification (MLPA) analysis was performed for detection of CNAs in 20 oncogenes and 2 tumor suppressor genes using the P078-D2 kit (MRC Holland). Individual gene copy number loss (MLPA ratio < 0.7), gain (ratio 1.3-2.0) and amplification (ratio > 2.0) was determined and unsupervised hierarchical cluster analysis was performed to identify differences in copy number (CN) patterns between PABC patients. Chi square statistics were used to interrogate associations with clinicopathologic characteristics. Log-Rank test was used to compare Kaplan Meier survival curves between CN subgroups. Results: Triple negative PABC tumors showed frequent copy number loss on chromosome 17q ( CPD, MED1, TOP2A, MAPT, PPM1D) and 8p ( ZNF703 and ADAM9), as well as frequent copy number gain/amplification of MYC (8q) and CCND1 (11q). Cluster analysis identified 2 clusters of PABC patients based on their tumor copy number patterns: a CN-neutral cluster and a CN-high cluster, the latter demonstrating significantly lower chromosome 17 copy numbers than the former (for PPM1D, ERBB2, CPD, MED1, TOP2A, CDC6 and MAPT). Patients with CN-neutral tumors more often showed lymph node metastases (p = 0.028), were of significantly higher stage (p = 0.031), and demonstrated a worse overall survival (p = 0.036) compared with PABC patients harboring CN-high tumors. Conclusions: We demonstrate different tumor CN patterns within a small group of triple negative PABC-patients, associated with distinct aggressiveness. This will form the basis for further in-depth genetic profiling within a larger Dutch PABC cohort, rendering insight in the disease progression and contributing to the development of more targeted and personalized treatments.

A Novel Microduplication Spanning Exons 8-16 of ATP2C1 That Was Undetectable by Standard Sanger Sequencing in a Japanese Patient With Hailey-Hailey Disease.

Hailey–Hailey disease (HHD) is genetic skin disorder characterized by repeated and exacerbated skin lesions in friction regions. ATP2C1, encoding SPCA1, was demonstrated to be the responsible gene for HHD pathogenesis. However, for some cases, no ATP2C1 mutation could be determined by standard Sanger sequencing, thereby obscuring the cause and diagnosis of HHD. In this study, we investigated the possibility that HHD is caused by complex ATP2C1 defects using multiplex ligation-dependent probe amplification (MLPA) analysis for 10 of 50 cases in our institute without ATP2C1 mutations. In one female Japanese patient and her father, who also show HHD, MLPA followed by polymerase chain reaction (PCR) analyses revealed a novel duplication of exons 8–16 of ATP2C1. The duplication was predicted to add 20,615 base pairs, 882 nt, and 294-amino-acid residues to the genome, mRNA and SPCA1 protein, respectively. By reverse transcriptase–PCR using patient skin RNA, we could confirm that a larger transcript was produced and we found that the abundance of the normal transcript was clearly reduced in the patient. Putative structures of wild-type and duplicated proteins revealed differences in arrangement of SPCA1 domains that may have functional consequences. Strikingly, the phosphorylation and the nucleotide-binding domains were interrupted by insertion of a partial actuator, transmembrane, and phosphorylation domains. The additional 294 amino acids appear to disrupt SPCA1 structure and function, causing HHD. Our study expands the spectrum of genetic defects in HHD and showed that disruption of SPCA1 structure and function by the microduplication caused HHD in the patient and her father.

Abstract P1-06-06: Direct ex vivo observation of homologous recombination defect reversal after DNA damaging chemotherapy in metastatic breast cancer patients

Abstract Introduction Better predictive biomarkers for response to Poly ADP-Ribose inhibitors (PARPi) are required, since on the one hand evidence is emerging that PARPi are also effective beyond germline BRCA mutated (gBRCAm) cancers and on the other hand gBRCAm cancers can become resistant to PARPi. Therefore, we previously developed a functional homologous recombination (HR) assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer (BrC) tissue (n=148): the REpair CAPacity (RECAP) test. The aim of the current study is to molecularly characterize real-time HR deficient (HRD) tumors and explore the utility of RECAP as a predictive biomarker for PARPi treatment in metastatic BrCs. Material and method Patients with advanced or recurrent BrC with easily accessible metastases were eligible. Fresh tissue biopsies from metastatic BrC lesions were collected in customized medium, irradiated with 5 Gy and cultured for 2 hours. Molecular characterization of functional HRD biopsies as well as platinum/PARP resistant biopsies was performed by targeted sequencing (BRCA1/2, TP53, CHEK2, PALB2), BRCA1 promoter methylation analysis and multiplex ligation-dependent probe amplification (MLPA) analysis of BRCA1 and BRCA2 to identify large rearrangements. Results 41 biopsies were derived from 38 patients with recurrent or metastatic BrC. The RECAP test had a high success rate (93%) when performed on core needle or punch biopsies, and test results were available within 1 week. HRD was detected in 13 out of 41 biopsies (32%), among which 5 were gBRCAm, indicating that the RECAP test identifies more patients who may benefit from PARPi treatment than gBRCA analysis only. Among the 8 non-gBRCAm HRD tumors was one tumor with a germline PALB2 mutation, one with BRCA1 promoter hypermethylation and two with somatic variants of unkown significance (VUSes) in BRCA2. In three gBRCAm patients BRCA reversion was detected, as the HRD tumors became HR proficient (HRP) after showing in vivo progressive disease (PD) on cisplatin/PARPi treatment. One of these patients obtained a secondary BRCA1 mutation that restored the open reading frame and led to production of full-length BRCA1 protein, while the causative molecular event in the other patients is still elusive. Conclusion The RECAP test is a robust and reproducible HRD test which identifies approximately 60% more potential candidates for PARPi treatment, as 40% of HRD tumors were caused by gBRCAm. Due to its functional character, the RECAP test reflects the real-time HR status regardless of BRCA mutational status and therefore detects HR reversal upon therapy resistance. Thus, RECAP shows great potential as a predictive biomarker for PARPi treatment of metastatic BrC. Citation Format: Meijer TG, Verkaik NS, Van Deurzen CH, Dubbink HJ, Den Toom TD, Dinjens WN, Kanaar R, Van Gent DC, Jager A. Direct ex vivo observation of homologous recombination defect reversal after DNA damaging chemotherapy in metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-06.

The –(α)5.2 Deletion Detected in a Uruguayan Family: First Case Report in the Americas

In Uruguay, α-thalassemia (α-thal) mutations were introduced predominantly by Mediterranean European immigrant populations and by slave trade of African populations. A patient with anemia with hypochromia and microcytosis, refractory to iron treatment and with normal hemoglobin (Hb) electrophoresis was analyzed for α-thal mutations by multiplex gap-polymerase chain reaction (gap-PCR), automated sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses. Agarose gel electrophoresis of the multiplex gap-PCR showed a band of unexpected size (approximately 700 bp) in the samples from the proband and mother. Automated sequencing of the amplified fragment showed the presence of the –(α)5.2 deletion (NG_000006.1: g.32867_38062del5196) [an α-thal-1 deletion of 5196 nucleotides (nts)]. The MLPA analysis of the proband’s sample also showed the presence of the –(α)5.2 deletion in heterozygous state. We report here the presence of the –(α)5.2 deletion, for the first time in the Americas, in a Uruguayan family with Italian ancestry, detected with a previously described multiplex gap-PCR.

Novel GLA Deletion in a Cypriot Female Presenting with Cornea Verticillata.

Fabry disease is an X-linked lysosomal storage disorder resulting from a deficiency of the hydrolytic enzyme α-galactosidase A (α-Gal-A). It is characterized by progressive lysosomal accumulation of globotriaosylceramide (Gb3) and multisystem pathology, affecting the skin, nervous and cerebrovascular systems, kidneys, and heart. Heterozygous females typically exhibit milder symptoms and a later age of onset than males. Rarely, they may be relatively asymptomatic throughout a normal life span or may have symptoms as severe as those observed in males with the classic phenotype. We report on a 17-year-old female in whom cornea verticillata was found during a routine ophthalmological examination but with no other clinical symptoms. Leucocyte α-galactosidase activity was within the overlap range between Fabry heterozygotes and normal controls. Sanger sequencing of the GLA gene failed to reveal any pathogenic variants. Multiplex Ligation-dependent Probe Amplification (MLPA) analysis revealed a deletion of exon 7. Using a long-range PCR walking approach, we managed to identify the deletion breakpoints. The deletion spans 1182 bp, with its 5′ end located within exon 6 of the GLA gene and its 3′ end located 612 bp downstream of exon 7. This finding represents a novel deletion identified in the first reported Cypriot female carrier of Fabry disease.

Abstract 3944: High incidence of somatic BAP1 alterations in sporadic malignant mesothelioma

Abstract Background: Malignant mesothelioma (MM) is a lethal tumor whose pathogenesis results from complex interactions between host genetics and environmental carcinogens. BAP1 mutations were reported in 22% and 23% of sporadic MM in two US Caucasian studies whereas BAP1 mutations were found in 61% of sporadic MM in a Japanese study. The significant discrepancy in frequency of BAP1 mutations suggested either a possible influence of ethnicity or differences related to the methodological approaches. Methods: To investigate this apparent discrepancy, we carried out comprehensive genomic analyses using multiple techniques in order to detect somatic alterations of the BAP1 gene in tumors of 22 US Caucasian MM patients. Results: Six mutations were detected by Sanger sequencing, gross gene alterations were found in 7 MM specimens by Multiplex Ligation-Dependent Probe Amplification and copy number analysis, and a new splice variant was identified by cDNA sequencing. Changes in methylation were not observed in our study. Immunohistochemistry (IHC) revealed nuclear BAP1 staining in the 8 MM tumors containing wild-type BAP1 while tumor cells in the 14 biopsies with mutated BAP1 did not show any nuclear staining. We extended our BAP1 IHC analysis to an independent cohort of MM samples from the National Virtual Mesothelioma Bank and we found loss of BAP1 nuclear staining in 46 out of 67 biopsies. Moreover we found similar frequency of BAP1 loss across different MM histological subtypes. Conclusions: We identified somatic alterations of the BAP1 gene in 73.7% of Caucasian MM tumors in two independent cohorts of somatic MMs, underscoring the need to apply integrated genomic analyses to scrutinize the BAP1 gene for its alterations. Immunohistochemistry was the most reliable technique to detect BAP1 mutations in MM biopsies. The high incidence of alterations establishes BAP1 as the most commonly mutated gene in MM, regardless of ethnic background or histological subtype. Citation Format: Masaki Nasu, Sandra Pastorino, Hugh Luk, Erin Flores, Francine Baumann, Amy Powers, Shreya Kanodia, Giovanni Gaudino, Yu-an Zhang, Adi Gazdar, Haining Yang, Harvey I. Pass, Mitsuru Emi, Michele Carbone. High incidence of somatic BAP1 alterations in sporadic malignant mesothelioma. [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 3944. doi:10.1158/1538-7445.AM2015-3944

Subtelomeric chromosomal anomalies in infantile epileptic encephalopathies

The infantile epileptic encephalopathies are a heterogeneous group of disorders where in about 30% of cases the cause remains unknown. Chromosomal aberrations, identified by karyotype analysis, constitute one of the major causes for these disorders. The new molecular cytogenetic techniques such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification (MLPA) and single nucleotide polymorphism array analyses may allow us to discover additional submicroscopic chromosomal rearrangements in this group of patients. Herein, we describe a girl with infantile epileptic encephalopathy. Fluorescence in situ hybridization, MLPA and single nucleotide polymorphism array analyses revealed a microdeletion and microduplication at the 1p36 region. Because of that, we further screened a cohort of 38 patients with infantile epileptic encephalopathy by using MLPA analysis to exclude the presence of subtelomeric microdeletions or microduplications. One patient was shown to harbor a microduplication at 11q25, which is however most likely a benign copy number variant derived from the healthy mother. No disease-causing dose changes were identified, indicating that unbalanced rearrangements at the subtelomeric regions may not be a common cause for infantile epileptic encephalopathy. However, when the patient has infantile epileptic encephalopathy and other features such as dysmorphic facies or major congenital anomalies, molecular cytogenetic analysis is warranted.

Thalassemia Intermedia Caused by 16p13.3 Sectional Duplication in a β-Thalassemia Heterozygous Child

Thalassemia intermedia is an inherited hemoglobin disorder characterized by a significant genetic and clinical heterogeneity. A wide spectrum of different genotypes—homozygous, heterozygous, and compound heterozygous—have been found to be responsible for it. The authors describe a Chinese child of β-thalassemia heterozygote with the mutation IVS2-654 (C→T) (HBB:c.316-197C→T) presenting with severe thalassemia intermedia. Multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (CGH) analyses of the α gene cluster revealed an approximate 146-kb duplication at 16p13.3 including the complete α gene cluster. The duplicated allele and the normal allele in trans result in a total of 6 active α genes. The severe clinical phenotype seemed to be related to the considerable excess of the α-globin and the β-globin deficit caused by the presence of the β-thalassemia. The α gene duplication should be considered in patients heterozygous for β-thalassemia who show a more severe phenotype than β-thalassemia trait.

Association of an α-Globin Gene Cluster Duplication and Heterozygous β-Thalassemia in a Patient with a Severe Thalassemia Syndrome

We describe a new case of a β-thalassemia (β-thal) heterozygote with the mutation IVS-II-654 (C>T) presenting with a transfusion-dependent phenotype. Multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (CGH) analyses of the α-globin gene cluster revealed a full duplication of the α-globin genes including the upstream regulatory element. The duplicated allele and the normal allele in trans resulted in a total of six active α-globin genes. The severe clinical phenotype seemed to be related to the considerable excess of the α- and β-globin deficit caused by the presence of the β-thal. α-Globin cluster duplication should be considered in patients heterozygous for β-thal who show a more severe phenotype than β-thal trait.

UNUSUAL SPECTRUM OF GENETIC PATHOLOGIES AND NOVEL MUTATIONS IN PWS AND AS PATIENTS DETECTED BY A WIDE CLUSTER OF METHODS

Prader-Willi and Angelman syndromes are clinically distinct neurodevelopmental genetic disorders that map to 15q11.2-q13 locus. The common phenotypes are attributable to loss of expression of parentally specific imprinted genes inside this region, where the gene function is dependent on parental origin. Initial diagnosis was proved for the years by methylation pattern analyses of the SNRPN exon 1/promoter region within the PWS/AS critical domain. Apart from unifying methylation-specific PCR and allele specific real-time PCR with melt-curve analysis as the fundamental methods for suspected diagnosis confirmation, we combined several specifically methods used to clarify the molecular cause. In our study we had identified and genotyped 24 PWS and AS patients from 450 suspected. Applied cluster of methods-microsatellite analysis of SNPs within the chromosome 15, Methylation-specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) and UBE3A gene sequence analysis, enable us to determined atypical deletion that does not include common breakpoints, novel highly likely to be pathologic UBE3A mutation, uniparental heterodisomy together with partial isodisomy and epimutation without any deletions in the imprinting centre. We present genotype-phenotype correlation of all positive cases. In addition, we estimate the incidence for Slovakian population at 1 in 20,000 for PWS and 1 in 40,000 for AS.

Copy number variations in KCNQ1 gene were frequently identified in the pediatric patients of long QT syndrome and caused exercise related QT prolongation

Purpose: More than ten genes have been reported as the cause of long QT syndrome (LQTS). However, approximately 30 percent of the patients have not been genotyped yet. Among non-genotyped LQTS patients, copy number variations (CNV) in KCNQ1 and KCNH2, the causative genes of LQTS1 and LQTS2, were recently reported, though the frequency of the CNV carriers or clinical characteristics of the carriers have not been fully elucidated yet. Therefore, we aimed to clarify the frequency of CNV in the pediatric patients with non-genotyped LQTS and to characterize their clinical symptoms. Methods: The subject of this study consisted of 172 LQTS patients (92 boys) who were less than 20-year-old (mean age was 12.3±0.3 year-old) and carried no gene mutation in LQTS1 through LQTS6. They were performed clinical evaluations including resting ECG and exercise stress test. To identify the CNV in KCNQ1 and KCNH2, we introduced multiplex ligation-dependent probe amplification (MLPA) analysis. Results: Among 172 patients, we identified 5 patients (2.9%) who carried CNV in KCNQ1 and no one in KCNH2. Table 1 summarizes the results of CNV and clinical characteristics. Two patients were asymptomatic and three patients experienced syncope while exercise. The mean QTc was prolonged from 453.6±12.9 ms½ at rest to 542.6±6.6 ms½ after exercise (p<0.01). View this table: Summary of the patients with CNV Conclusions; In the pediatric LQTS patients, especially with exercise induced QT prolongation, CNV in KCNQ1 may be the cause of the disease. Therefore screening of CNV in pediatric LQTS patients is indispensable to perform appropriate treatment and to prevent unexpected sudden cardiac death.

Abstract 4702: Chromosome 2p gain and N-Myc amplification predict poor outcome of patients with LIN28 negative supratentorial primitive neuroectodermal tumors of the central nervous system (CNS-PNET)..

Abstract Primitive neuroectodermal tumors of central nervous system (CNS-PNET) which represents 3-7% of all pediatric brain tumors, are a rare group of neoplasms occurring in the CNS and include supratentorial PNETs (s-PNET), medulloepitheliomas and ependymoblastomas. Whereas ependymoblastomas harbour chromosome 19q13.41 amplification, express the neural stem cell marker LIN28 and show aggressive clinical behaviour, the biological mechanisms and molecular alterations contributing to the pathogenesis of s-PNETs remain so far poorly understood. Expression analyses and limited CGH studies indicated that s-PNETs may represent genetically a heterogeneous group with frequent but multiple copy number aberrations. In order to find possible specific molecular and cytogenetic alterations suitable for molecular risk stratification and predictive for the overall survival, we performed a multiplex ligation-dependent probe amplification (MLPA) and molecular inversion probe (MIP) analysis on DNA samples of 25 s-PNET cases of patients (median age 5.35 years; range, 2.41 - 17.28 years). The majority of patients were enrolled in the clinical GPOH HIT2000 trial. MLPA and MIP analysis revealed large losses of genomic material of chromosomes 3, 4, 5, 13, while frequent gains were observed at chromosomes 1, 17, 19, 20 and 22. Recurrent regions of gains were found at chromosome 7q11.2, 7q22.1, 12q13 and losses at 16q23.1, 6p21.1, and 11q11. High-copy number gains (amplifications) were found in particular at chromosome 2p24.3 (N-Myc) (in 6 cases) and 4q21 (in 2 cases). None of the chromosomal alterations identified was found significantly associated with the age of the patients at diagnosis, presence of tumor metastases or presence of postoperative residual tumor. In contrast, patients with tumors harbouring 2p gain or N-Myc amplification showed shorter overall survival (OS) (p=0.0029 and p=0.0012, respectively).These parameters were independent from the presence of metastases, which was indeed the most important clinical factor for unfavourable OS (p=0.0112) in our series. In the era of the personalized neuro-oncology, the identification of molecular marker associated to patients outcome may represent the further significant step towards the patients's risk stratification and the improvement of therapeutic strategies also for patients suffering from s-PNETs. Citation Format: Marco Gessi, Andre' von Bueren, Andras Treszl, Anja zur Muehlen, Stefan Rutkowski, Torsten Pietsch. Chromosome 2p gain and N-Myc amplification predict poor outcome of patients with LIN28 negative supratentorial primitive neuroectodermal tumors of the central nervous system (CNS-PNET). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2013-4702

Sistani Population: a Different Spectrum oF β-Thalassemia Mutations From other Ethnic Groups of Iran

This study aimed to characterize the molecular spectrum of β-thalassemia (β-thal) mutations and evaluate the services available for prenatal diagnosis (PND) among the Sistani population of Iran. Mutations were analyzed with amplification refractory mutation system (ARMS), gap-polymerase chain reaction (gap-PCR), multiplex ligation-dependent probe amplification (MLPA) analysis and direct sequencing. Fetal diagnosis was also confirmed by linkage analysis. Over a 9-year period (2002-2011), 405 at-risk Sistani couples were referred for mutation analysis and/or PND. Of the referred couples, 18.5% had one to three affected children with β-thal major (β-TM) and the remainder had no children or were not married. Most of the couples (73.3%) lived in urban areas and the rate of consanguineous marriage was 76.8%. Twenty-one mutations were identified, of which the most frequent ones were IVS-I-5 (G>C) with a frequency of 74.1%, followed by codon 15 G>A (5.0%), codon –88 (C>T) (3.8%), IVS-II-1 (G>A) (3.4%), codons 8/9 (+G) (2.9%) and IVS-I-1 (G>T) (2.7%), which accounted for about 91.9% of the total β-thal mutations for this region. Furthermore, fetal DNA was obtained from chorionic villus sampling (CVS) for 266 pregnant women and 68 (25.5%) fetuses were diagnosed as affected. In summary, β-thal mutations are very heterogeneous and significantly different from those found in other parts of Iran and are similar to those of Pakistani and Indian populations. These results could greatly facilitate timely and accurate PND.

572: The use of multiplex ligation-dependent probe amplification (MLPA) versus karyotype analysis in the investigation of pregnancy loss

amplification (MLPA) versus karyotype analysis in the investigation of pregnancy loss Danielle O’Leary, Keelin O’Donoghue Cork University Maternity Hospital, University College Cork, Obstetrics and Gynecology, Cork, Ireland OBJECTIVE: Karyotyping is important in pregnancy loss as about 6% of stillborn babies will have a chromosomal abnormality, while in couples with recurrent miscarriage, chromosomal abnormalities of the embryo account for 30-60% of further miscarriages. Some abnormalities are potentially recurrent and can be tested for in future pregnancies. Traditionally cytogenetic investigation of pregnancy loss is performed by culture of chorionic villi and karyotype analysis on fetal material. This technique has a high failure rate (reported at 1040%), associated with misdiagnosis due to overgrowth of maternal tissue and is also labour intensive. The technique of multiplex ligation-dependent probe amplification (MLPA) reportedly has a higher success rate, ranging from 85-95%, and a quicker reporting time. We aimed to compare the success rate and time taken for reporting with MLPA and karyotype analysis and to compare the number of normal and abnormal results and indications for testing for both techniques. STUDY DESIGN: Tissue samples sent for MLPA testing between October 2011-June 2012 and for karyotype analysis in the corresponding months in 2010-2011 were recorded. Reports were examined and time taken for processing and results calculated. RESULTS: 102 tissue samples were sent for MLPA testing, compared to 60 samples for karyotype analysis. Indications included investigation of unexplained stillbirth (26/162), late miscarriage (53/162) and recurrent miscarriage (76/162). The success rate of MLPA analysis was higher at 89%, in contrast to 73% for karyotype analysis. MLPA had a quicker reporting time, on average 9.2 days compared to 16.3 days. CONCLUSION: Our findings confirm the higher success rates and quicker reporting time with MLPA analysis versus karyotype by tissue culture. The lower cost per sample is also advantageous in times of sparse resources. From these data, we recommend the continued use of MLPA analysis as the cytogenetic investigation of choice in pregnancy loss. 573 Extremely high risk of fetal death and severe perinatal complications associated with simultaneously elevated AFP and very low unconjugated estriol David Krantz, Burton Rochelson, Jon Carmichael, Terrence Hallahan, Nidhi Vohra, Stephanie Augustine PerkinElmer, NTD Labs, Melville, NY, North Shore Long Island Jewish Heath System, Maternal Fetal Medicine, Manhassett, NY OBJECTIVE: To determine the risk of severe anomalies and perinatal complications in pregnancies associated with a simultaneously elevated AFP and very low unconjugated estriol (UE3). STUDY DESIGN: A total of 6,365 second trimester ONTD/Down syndrome screening tests from a single hospital performed between March 2010 and February 2012 were analyzed. Outcomes were obtained from pregnancies with simultaneously elevated AFP ( 2 MoM) and a very low UE3 ( 1 %ile). RESULTS: There were 139 elevated AFP results (2.2%) and 72 (1.1%) very low UE3 results. After excluding 1 case of anencephaly, 9 patients had simultaneously elevated AFP and very low UE3 results. There were 3 cases of fetal loss and 2 cases of pregnancy termination due to severe fetal anomalies or severe pregnancy complications. In the remaining 4 cases, 3 pregnancies were associated with IUGR and admittance to the NICU and one pregnancy was associated with oligohydramnios and small placenta but did not require admittance to NICU. CONCLUSION: Both elevated AFP and low UE3 are known to be associated with increased risk of fetal anomalies and pregnancy complications. Based on this case series, when both of these results occur simultaneously the pregnancy is at significant risk for severe fetal anomalies, fetal death and NICU admission at birth. Even if severe anomalies are not immediately diagnosed in these patients, continued close monitoring of the pregnancy until delivery is still warranted.

Genetic analysis of PAX3 for diagnosis of Waardenburg syndrome type I

Conclusion: PAX3 genetic analysis increased the diagnostic accuracy for Waardenburg syndrome type I (WS1). Analysis of the three-dimensional (3D) structure of PAX3 helped verify the pathogenicity of a missense mutation, and multiple ligation-dependent probe amplification (MLPA) analysis of PAX3 increased the sensitivity of genetic diagnosis in patients with WS1. Objectives: Clinical diagnosis of WS1 is often difficult in individual patients with isolated, mild, or non-specific symptoms. The objective of the present study was to facilitate the accurate diagnosis of WS1 through genetic analysis of PAX3 and to expand the spectrum of known PAX3 mutations. Methods: In two Japanese families with WS1, we conducted a clinical evaluation of symptoms and genetic analysis, which involved direct sequencing, MLPA analysis, quantitative PCR of PAX3, and analysis of the predicted 3D structure of PAX3. The normal-hearing control group comprised 92 subjects who had normal hearing according to pure tone audiometry. Results: In one family, direct sequencing of PAX3 identified a heterozygous mutation, p.I59F. Analysis of PAX3 3D structures indicated that this mutation distorted the DNA-binding site of PAX3. In the other family, MLPA analysis and subsequent quantitative PCR detected a large, heterozygous deletion spanning 1759–2554 kb that eliminated 12–18 genes including a whole PAX3 gene.

P1-09-03: Prevalence of Germline TP53 Mutations in Young Women with HER2−Positive Breast Cancer.

Abstract Background: Li Fraumeni syndrome is a rare inherited cancer susceptibility condition associated with germline mutations in the TP53 gene, in which breast cancer (BC) is the most frequent tumor. The prevalence of TP53 mutations in population-based series of very young onset BC (&amp;lt;30 years at diagnosis) ranges from &amp;lt;1% to approximately 7%1-4. Recent data show that BC in patients carrying a germline TP53 mutation are commonly HER2 amplified (63-83%)5-7. In this study, we assessed the prevalence of germline TP53 mutations in women with HER2 positive BC diagnosed age ≤ 50 years. Material &amp; Methods: We identified 347 women with invasive HER2 positive BC diagnosed at age ≤ 50 years using the Clinical Operations and Research Information System (CORIS) at the Dana Farber Cancer Institute. Information on age at diagnosis, histology, hormone receptor and HER2 status as well as personal and family cancer history was confirmed from medical records. 129 patients were excluded for various reasons, including a cancer diagnosis prior to the BC and a documented BRCA1/2 mutation. A combination of Exon Grouping Analysis (EGAN) and Sanger sequencing for detection of TP53 mutations in exons 2–11 including surrounding intronic sequence was performed on 218 germline DNA samples. Multiplex Ligation-dependent Probe Amplification (MLPA) analysis for the detection of TP53 deletions or duplications is ongoing. Results: A germline TP53 mutation was identified in 4 women diagnosed at age ≤ 50 years (1.8%, 95%CI 0.5−4.6). At BC diagnosis, they were 23, 32, 44 and 50 years. Two BC were ER+/PR+, HER2+ and 2 were ER-/PR-, HER2+. Estimate of prevalence of germline TP53 mutations by age at BC diagnosis are: age ≤ 35, 2/41 (4.9%, 95%CI 0.6−16.6), and age ≤ 45 3/168 (1.8%, 95%CI 0.4−5.1). Among the women with germline TP53 mutations, 2 met the Chompret criteria8 and none the classic LFS criteria. Discussion: TP53 mutations were identified in a cohort of women with HER2+ BC at young age. As expected, the frequency is higher in younger women, but mutations were seen in all age groups that were evaluated. None of these women met classic LFS criteria by family history. Consideration of TP53 testing should be given to women diagnosed below age 35 who are negative for BRCA1/2 mutations regardless of family history. Analysis of other series will be helpful in reaching more stable estimates of the prevalence of mutation carriers among patients with HER2+ BC at young age. 1. Sidransky D et al. Cancer Res. 1992; 52:2984–2986. 2. Borresen AL et al. Cancer Res.1992; 52:3234–3236. 3. Lalloo F et al. Lancet 2003; 361:1101–02 4. Gonzalez KD et al. J Clin Oncol 2009;27(8):1250–6 5. Wilson JR et al. J Med Genet 2010;47(11):771–774. 6. Melhem-Bertrandt A et al: San Antonio Breast Cancer Symposium 2010: P3-12-01. 7. Masciari S et al: J Clin Oncol 29: 2011 (suppl; abstr 1519) 8. Tinat J et al. J Clin Oncol. 2009;27(26):e108–9 Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-09-03.

Implementation and Experience of an Alternative QF-PCR and MLPA Diagnostic Strategy to Detect Chromosomal Abnormalities in Fetal and Neonatal Pathology Samples

Chromosomal abnormalities are a significant cause of pregnancy loss. Solid tissue fetal and neonatal pathology samples are routinely examined by karyotype analysis after cell culture. However, there is a high failure rate, and this approach is expensive and labor intensive. We have therefore evaluated a new molecular strategy involving quantitative fluorescent polymerase chain reaction (QF-PCR) and subtelomere multiplex ligation-dependent probe amplification (MLPA) analysis. A retrospective audit showed that less than 4% of abnormal cases may not be detected by this molecular strategy. We validated this strategy in parallel with cytogenetic analysis on 110 patient samples, which included cases of fetal loss, still birth, neonatal death, termination of pregnancy, recurrent miscarriage, and sudden unexpected death in infancy. This validation showed that 55 of the 57 samples that gave a result for both strategies were concordant. During the 1st year of diagnostic testing, we analyzed 382 samples by the molecular strategy. A 16% abnormality rate was observed. These included trisomies 13, 18, 21, monosomy X, and triploidy detected by QF-PCR (77%), and 23% were other trisomies and subtelomere imbalances detected by MLPA. This strategy had a 92% success rate in contrast to the 20%-30% failure rate observed with cell culture and cytogenetic analysis. We conclude that QF-PCR and subtelomere MLPA is a suitable strategy for analysis of the majority of fetal and neonatal pathology samples, with many advantages over conventional cytogenetic analysis.

Clinical Phenotype and Mutation Spectrum of the CYP21A2 Gene in Patients with Steroid 21-Hydroxylase Deficiency

Steroid 21-hydroxylase deficiency is caused by inactivating mutations in the CYP21A2 gene. This paper reports on the mutation spectrum and the genotype-phenotype correlation of 21-hydroxylase deficiency. 72 unrelated patients with congenital adrenal hyperplasia (CAH) were included. Molecular analysis of CYP21A2 was performed, via the multiplex ligation-dependent probe amplification (MLPA) analysis and sequence-specific differenzial PCR amplification of the CYP21A2 and CYP21A1P genes, using 4 pair-wise sequence-specific primers, followed by sequencing of the entire CYP21A2 gene. Large gene deletions were identified in 45 (31.3%) of the 144 unrelated CAH alleles, whereas the most frequent point mutations were intron 2 splice mutations (c.293-13A>G) (41/144, 28.5%). The MLPA analysis successfully identified 23 of 72 patients (31.9%) with single copy deletion in CYP21A2. This paper describes a rapid and accurate method for the molecular diagnosis of 21-hydroxylase deficiency, which relies on the identification of point mutations and structural rearrangements within the CYP21A2 gene.

Multiplex Ligation-Dependent Probe Amplification Analysis Is Useful for Diagnosing Congenital Adrenal Hyperplasia but Requires a Deep Knowledge of CYP21A2 Genetics

To the Editor: We read with great interest the recent report in Clinical Chemistry by Canturk et al. (1). These authors affirmed that the CYP21A1P 1 (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) genotype interferes with quantitative multiplex ligation-dependent probe amplification (MLPA) analysis of the CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) gene. They also reported that the p.I172N and p.Q318X mutations were absent in 3.6% and 8.5%, respectively, of the CYP21A1P alleles (200 unrelated individuals examined). Because CYP21A2 MLPA-specific probes contain the wild-type sequences for the Del8bp, p.I172N, Cluster E6, and p.Q318X mutations, Canturk et al. stated that “without a parallel CYP21A1P [sequencing] analysis, one would falsely infer a heterozygous duplication of CYP21A2 with a risk of 3.6%” (p.I172N) with an MLPA method, …

22q11.2 Microduplication in Two Patients with Bladder Exstrophy and Hearing Impairment

Purpose Bladder exstrophy is a congenital malformation of the bladder occurring in about 1 in 35 000 newborns. A few cases of bladder exstrophy patients have been described to carry chromosomal aberrations and in addition the recurrence risk in higher among relatives of bladder exstrophy cases than in the general population. This speaks in favour of a genetic contribution to the pathogenesis of bladder exstrophy. Material and Methods We have performed array-CGH analysis of 36 Swedish bladder exstrophy patients in order to look for submicroscopic chromosomal aberrations. Results The analysis revealed a 22q11.21 microduplication in two unrelated cases with bladder exstrophy and hearing impairment consistent with the recently described 22q11.2 microduplication syndrome. This finding was confirmed by multiplex ligation-dependent probe amplification (MLPA) and FISH analysis. Subsequent MLPA analysis of this chromosomal region in 33 bladder exstrophy patients did not reveal any deletion/duplication within this region. MLPA analysis of 171 anonymous control individuals revealed one individual carrying this microduplication. Conclusions Chromosomal rearrangements of 22q11.2 are implicated in several genomic disorders i.e. DiGeorge/velocardiofacial- and cat-eye syndrome. Deletions within this chromosomal region are relatively common while duplications of 22q11.2 are much less frequently observed. An increasing number of reports of microduplications of this region describe a highly variable phenotype. This is the first report of 22q11.2 microduplication associated with bladder exstrophy and hearing impairment. The finding of one carrier among a cohort of normal controls further highlights the variable phenotype linked to this microduplication syndrome.