Multiplex Ligation Probe Amplification Research Articles

CDH1 Germline Variants in a Tunisian Cohort with Hereditary Diffuse Gastric Carcinoma.

Mutational screening of the CDH1 gene is a standard treatment for patients who fulfill Hereditary Diffuse Gastric Cancer (HDGC) testing criteria. In this framework, the classification of variants found in this gene is a crucial step for the clinical management of patients at high risk for HDGC. The aim of our study was to identify CDH1 as well as CTNNA1 mutational profiles predisposing to HDGC in Tunisia. Thirty-four cases were included for this purpose. We performed Sanger sequencing for the entire coding region of both genes and MLPA (Multiplex Ligation Probe Amplification) assays to investigate large rearrangements of the CDH1 gene. As a result, three cases, all with the HDGC inclusion criteria (8.82% of the entire cohort), carried pathogenic and likely pathogenic variants of the CDH1 gene. These variants involve a novel splicing alteration, a missense c.2281G > A detected by Sanger sequencing, and a large rearrangement detected by MLPA. No pathogenic CTNNA1 variants were found. The large rearrangement is clearly pathogenic, implicating a large deletion of two exons. The novel splicing variant creates a cryptic site. The missense variant is a VUS (Variant with Uncertain Significance). With ACMG (American College of Medical Genetics and Genomics) classification and the evidence available, we thus suggest a revision of its status to likely pathogenic. Further functional studies or cosegregation analysis should be performed to confirm its pathogenicity. In addition, molecular exploration will be needed to understand the etiology of the other CDH1- and CTNNA1-negative cases fulfilling the HDGC inclusion criteria.

Necessity of multiplex ligation probe amplification in genetic tests: Germline variant analysis of the APC gene in familial adenomatous polyposis patients

BackgroundFamilial adenomatous polyposis (FAP) is an autosomal dominant hereditary cancer syndrome characterized by hundreds to thousands of colorectal adenomatous polyps. Without treatment, progression to colorectal cancer is inevitable. Most pathogenic mutations in the APC gene were nonsense or frameshift mutations; however, some previous studies reported large deletions or duplications. MethodWe reviewed the results of APC gene analyses from January 2010 to December 2020. We analyzed the entire coding sequence of the APC gene by Sanger sequencing to detect genetic abnormalities. Moreover, multiplex ligation-dependent probe amplification (MLPA) testing was performed starting in September 2017, and a multigene panel study that includes the APC gene was begun in July 2019. ResultsIn the 266 collected cases, pathogenic variants/likely pathogenic variants (PV/LPVs) in the APC gene were detected in 73 patients, and variants of uncertain significance were found in 13 patients. Among those variants, 14 variants were novel. We performed MLPA for 29 patients, and 7 of them (24.1%) were positive for a large duplication/deletion. Among the 73 cases in the multigene panel study, 17 cases (23.3%) of APC gene variation were detected. ConclusionWe retrospectively analyzed the APC gene in Korean patients suspected to have FAP. Variants truncated by nonsense and frame shift mutations were common PV/LPVs. However, the detection rate in the MLPA study was higher than in previous studies of Caucasian populations. We suggest that MLPA should be performed for patients likely to have FAP but in whom no variant is detected by sequencing methods.

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Spinal muscular atrophy (SMA) is one of the most common and severe genetic diseases. SMA carrier screening is an effective way to identify couples at risk of having affected children. Next-generation sequencing (NGS)-based expanded carrier screening could detect SMN1 gene copy number without extra experiment and with high cost performance. However, its performance has not been fully evaluated. Here we conducted a systematic comparative study to evaluate the performance of three common methods. 478 samples were analyzed with multiplex ligation probe amplification (MLPA), real-time quantitative polymerase chain reaction (qPCR) and NGS, simultaneously. Taking MLPA-based results as the reference, for 0 copy, 1 copy and ≥ 2 copy SMN1 analysis with NGS, the sensitivity, specificity and precision were all 100%. Using qPCR method, the sensitivity was 100%, 97.52% and 94.30%, respectively; 98.63%, 95.48% and 100% for specificity; and 72.72%, 88.72% and 100% for precision. NGS repeatability was higher than that of qPCR. Moreover, among three methods, NGS had the lowest retest rate. Thus, NGS is a relatively more reliable method for SMN1 gene copy number detection. In expanded carrier screening, compared with the combination of multiple methods, NGS method could reduce the test cost and simplify the screening process.

Evaluation of copy number variants for genetic hearing loss: a review of current approaches and recent findings.

Structural variation includes a change in copy number, orientation, or location of a part of the genome. Copy number variants (CNVs) are a common cause of genetic hearing loss, comprising nearly 20% of diagnosed cases. While large deletions involving the gene STRC are the most common pathogenic CNVs, a significant proportion of known hearing loss genes also contain pathogenic CNVs. In this review, we provide an overview of currently used methods for detection of CNVs in genes known to cause hearing loss including molecular techniques such as multiplex ligation probe amplification (MLPA) and digital droplet polymerase chain reaction (ddPCR), array-CGH and single-nucleotide polymorphism (SNP) arrays, as well as techniques for detection of CNVs using next-generation sequencing data analysis including targeted gene panel, exome, and genome sequencing data. In addition, in this review, we compile published data on pathogenic hearing loss CNVs to provide an up-to-date overview. We show that CNVs have been identified in 29 different non-syndromic hearing loss genes. An understanding of the contribution of CNVs to genetic hearing loss is critical to the current diagnosis of hearing loss and is crucial for future gene therapies. Thus, evaluation for CNVs is required in any modern pipeline for genetic diagnosis of hearing loss.

ASO Author Reflections: Germline Testing for All Patients With Breast Cancer: Has the Time Finally Come?

For the young breast cancer population in India, the burden of hereditary breast cancer is not well defined. Moreover, genetic testing criteria (National Comprehensive Cancer Network and Mainstreaming Cancer Genetics (MCG) plus) have never been validated for the Indian population. Therefore, this study tested 236 consecutive breast cancer patients for germline pathogenic mutations using next-generation sequencing and reflex Multiplex Ligation Probe Amplification (MLPA). The findings showed a high prevalence of pathogenic/likely pathogenic (P/LP) mutations (18.64%), with 34% mutations in non BRCA genes. The sensitivity of the testing criteria was inadequate (88.6% for NCCN and 86.36% for MCG plus criteria), reiterating the need to expand the criteria. The uptake of cascade testing was low (10% of eligible previvors), highlighting this as an area of unmet need. Multicentric studies to validate these data and provide further insight into the hereditary cancer burden in India are the need of the hour.

Pendred Syndrome, or Not Pendred Syndrome? That Is the Question.

Pendred syndrome (PDS) is the most common form of syndromic Hearing Loss (HL), characterized by sensorineural HL, inner ear malformations, and goiter, with or without hypothyroidism. SLC26A4 is the major gene involved, even though ~50% of the patients carry only one pathogenic mutation. This study aims to define the molecular diagnosis for a cohort of 24 suspected-PDS patients characterized by a deep radiological and audiological evaluation. Whole-Exome Sequencing (WES), the analysis of twelve variants upstream of SLC26A4, constituting the “CEVA haplotype” and Multiplex Ligation Probe Amplification (MLPA) searching for deletions/duplications in SLC26A4 gene have been carried out. In five patients (20.8%) homozygous/compound heterozygous SLC26A4 mutations, or pathogenic mutation in trans with the CEVA haplotype have been identified, while five subjects (20.8%) resulted heterozygous for a single variant. In silico protein modeling supported the pathogenicity of the detected variants, suggesting an effect on the protein stabilization/function. Interestingly, we identified a genotype-phenotype correlation among those patients carrying SLC26A4 mutations, whose audiograms presented a characteristic slope at the medium and high frequencies, providing new insights into PDS. Finally, an interesting homozygous variant in MYO5C has been identified in one patient negative to SLC26A4 gene, suggesting the identification of a new HL candidate gene.

Clinical and Molecular Features of First Mexican Friedreich's Ataxia Patients with Compound Heterozygous FXN Mutations.

Friedreich's ataxia (FRDA) is caused by homozygous GAA repeat expansions or compound heterozygous (CH) mutations in FXN gene. Its broad clinical spectrum makes it difficult to identify, thus an accurate diagnosis can only be made by genetic testing. This study aims to present data on FXN variants observed in patients with sporadic or recessive ataxia, including detailed data of the first CH Mexican patients. One hundred and eight patients with recessive or sporadic cerebellar ataxia were referred to our institution between 2009 and 2019 for FXN molecular testing. This was achieved using a combined methodology of triplet repeat-primed PCR (polymerase chain reaction), long PCR, FXN sequencing and multiplex-ligation probe-amplification. Eighteen patients had a homozygous FXN genotype; whereas five were CH patients with a slow progression and phenotypic variability, including a late-onset case with spastic paraparesis, and a Charcot-Marie-Tooth-like case. These first Mexican CH patients pose important implications for genetic counseling and FRDA management.

PDX Models Recapitulate Genetics and Epigenetics of Pediatric T-Cell Leukemia

Patient-derived xenografts (PDX) emerged as important tools to study leukemia biology and potential therapeutic regimens. Here, we compared cellular, molecular and developmental features of PDX-derived blasts with primary pediatric precursor T-cell acute leukemia (T-ALL) cells. The cells were obtained at the times of initial diagnosis and relapse. We analyzed the samples at the genomic level (whole genome sequencing (WGS), whole exome sequencing (WES), multiplex ligation probe amplification (MLPA), targeted sequencing) and the epigenetic level (DNA methylation, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq)).Altogether 75% of our samples engrafted successfully. Of these 12 were matched pairs of initial diagnosis and relapse as well as corresponding PDX. Of further 12 matched pairs either initial diagnosis (2/12) or relapse (7/12) or neither (3/12) engrafted indicating that relapse samples tend to engraft more successfully (p= 0.1824 (McNemar's test)), which is in agreement with observations in other cancer entities.In 11 out of 12 matched pairs (initial and relapse) of primary and PDX genomic stability of the PDXs was assessed using WGS and WES. Ninety-two % of the mutations with an allele frequencies (AF) >30% were conserved. Of altogether 609 SNVs and InDels (AF>10%) detected in primary samples 68 % (417) were also detected in the corresponding PDXs. In some PDXs, mutations not identified by targeted sequencing in the primary sample (NOTCH1, PHF6, DNA11, USP9X) occured at a high AF (22-59%), which may have been the result of selection of a subclone or of an acquisition of the mutation in the process of PDX generation.Thirty of 35 (86%) large CNA assessed by WGS were preserved in corresponding PDX models indicating that CNAs are likely earlier events in the leukemogenesis that are present at high AF in the primary samples. CNA analysis with MLPA confirmed that PDXs retained 96% (80/83) of the deletions and amplifications of primary leukemia.In 18 out of the matched 22 PDXs all clonal mutations (AF >30%) detected in the primary sample have been preserved in the PDX. Seventeen out of 22 contained more than 1 clone. Fifteen out of these 17 retained the complexity of primary sample with different subclones successfully engrafted.When considering the 11 matched pairs, we observed 4 different engraftment types of initial disease. Type 1 engrafted in a similar way in which they relapsed preserving all or nearly all mutations found in patients' sample (4 samples). Type 2: the relapsing clone that was not detected in initial diagnosis was present in PDX derived of initial diagnosis (PDX_INI) but it was not dominant (3). Type 3: the relapsing clone that was present at initial diagnosis did not engraft into PDX_INI (2). Type 4 preserved the relapsing clone as well as the mutations specific for initial diagnosis (2). Out of 11 PDXs of relapse 9 preserved all or nearly all mutations detected in primary sample. In 2/11 PDXs of relapse one of the clones preexisting in primary relapse was selected for, while the other one was lost.Differential methylation analysis by EPIC array of 21,711 promoters of primary samples and PDXs revealed that the average methylation in primary samples (β=0.43) did not differ from that in PDXs (β=0.45). Principal Component Analysis and unsupervised hierarchical clustering showed that matched pairs, both of the primary and the PDX samples cluster together (ρ=0.9912). Similarly, analysis of chromatin accessibility by ATAC-seq showed that more than 92% of peaks of a height > 20 and more than 75% of the peaks of a height of > 10 were preserved between primary samples and corresponding PDXs.In conclusion, PDX models largely reflect the heterogeneity and the complexity of the primary leukemic sample at the genomic and epigenomic level, although some clonal and particularly subclonal mutations can be lost. Differences in engraftment and various engraftment scenarios of initial diagnosis in contrary to relapse samples suggest that relapse samples are more preselected and stable in engraftment. Altogether, this overall genomic and epigenomic stability of the primary T-ALL in the PDX renders the models as a suitable source for material when primary samples are limiting and also to reduce uncontrollable pre-analytical changes in primary samples. Further, these models are suitable for the preclinical development of new treatment strategies for pediatric T-ALL. DisclosuresSchrappe:Baxalta: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding.

The introduction of clinical genetic testing in Ethiopia: Experiences and lessons learned.

Limited data are available on genetic testing laboratories in low- and middle-income countries including those in sub-Saharan Africa (SSA). To characterize the need for genetic testing in SSA we describe the experience of MRC-ET Advanced Laboratory, a genetic testing laboratory in Ethiopia. Test results were analyzed based on indication(s) for testing, referral category, and diagnostic yield. A total of 1311 tests were run using the full MRC-Holland catalogue of Multiplex-Ligation Probe Amplification assays. Of all samples, 77% were postnatal samples, 15% products of conception (POC), and 8% amniotic samples. Of postnatal samples, the most common testing categories were multiple congenital anomalies (32%), disorders of sex development (17%), and Obstetrics/Gynecology (16%). Forty-three percent of postnatal samples were diagnostic, 11% were variants of uncertain significance (VUS), and 46% were normal with Trisomy 21 the most common diagnosis. Of POC samples, 10% were diagnostic, 34% revealed VUSs, and 55% were normal with Trisomy 18 the most common diagnosis. Of amniotic samples 17.5% were diagnostic, 3% revealed VUSs, and 79% were normal with Trisomy 18 the most common diagnosis. There is increasing demand for genetic testing in Ethiopia. Diagnostic genetic testing in SSA deserves increased attention as testing platforms become more affordable.

The spectrum of FVIII gene variants detected by next generation sequencing in 236 Chinese non-inversion hemophilia A pedigrees

IntroductionThe reported variants of hemophilia A are mainly from European subjects and American subjects of European descent, and limited data are available from more diverse ethnic backgrounds. This study was performed to identify the causative variants in a large HA cohort from Chinese population. Materials and methodsA total of 236 HA pedigrees were included. Molecular analysis of F8 gene was performed using next-generation sequencing (NGS) and then validated by Sanger sequencing and multiplex ligation probe amplification (MLPA) results. Variants were classified as pathogenic, likely pathogenic, variant of unknown significance, likely benign, and benign according to the American College of Medical Genetics and Genomics guidelines. ResultsA total of 186 F8 variants were identified, with 139 (139/186, 74.73%) point mutations, 44 (44/186, 23.66%) small insertions/deletions (InDels), and 3 (3/186, 1.61%) large deletions, they included 80 pathogenic and 84 likely pathogenic variants. Of these variants, 119 had been reported previously, and 67 were novel. No potentially causative mutations were found in the targeted F8 region in seventeen HA pedigrees. ConclusionsThe spectrum of F8 variants identified in this study provides additional information about HA and enriches our knowledge of the variant spectrum in a wider range of ethnic backgrounds.

Variant Profiling of a Large Cohort of 138 Chinese Families With Autosomal Dominant Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy, and 15–25% of RP is transmitted as an autosomal dominant (ad) trait. The objectives of this study were to establish the variant profile in a large cohort of adRP families and to elucidate the variant spectrum of each adRP gene in Chinese patients. A total of 138 probands clinically diagnosed with RP as a presumed autosomal dominant trait were recruited. All probands underwent ophthalmic examinations by specialists. A combination of molecular screening methods, including targeted next-generation sequencing, Sanger DNA sequencing, and multiplex ligation probe amplification assay, was used to detect variants. We identified heterozygous variants of 11 adRP genes in 73 probands, hemizygous, or heterozygous variants of X-linked RP genes in six patients, compound heterozygous variants of autosomal recessive RP genes in three pseudodominant families, and one heterozygous variant of one ad cone and rod dystrophy gene in one proband. One proband was found carrying both variants in RPGR and FAM161A. The overall detection rate was 59.4% (82/138). We detected 72 distinct disease-causing variants involving 16 RP genes and one cone-rod dystrophy gene; 33 of these variants have not been reported previously. Disease-causing variants were identified in the adRP genes in 52.9% of the families, followed by 4.3% in the X-linked RP genes, and 2.2% in the autosomal recessive genes. The most frequent mutant genes were RHO, PRPF31, RP1, SNRNP200, and PRPF8, which explained up to 78.0% of the genetically diagnosed families. Most of the variants identified in adRP genes were missense, and copy number variations were common (7/20) in the PRPF31 gene. We established the profile of the mutated genes and the variant spectrum of adRP genes in a large cohort of Chinese patients, providing essential information for genetic counseling and future development of therapeutics for retinal dystrophy inherited as a dominant trait.

High-throughput screening reveals novel mutations in spinal muscular atrophy patients

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive hereditary disease associated with severe muscle atrophy and weakness in the limbs and trunk. The discovery of mutated genes is helpful in diagnosis and treatment for SMA.MethodsEighty-three whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was performed. Afterwards, the complete gene sequence of SMN1 gene was detected. Furthermore, 20 SMA patients were selected from the 28 probands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing.ResultsTwenty-two probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. Six SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C > T], c.[271C > T], c.[−39A > G] and g.[70240639G > C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[−41_-40insCTCT], FUT5 SNV c.[1001A > G], and MCCC2 SNV c.[−117A > G] were the 3 most frequent mutations in SMA group (95, 85 and 75%, respectively).ConclusionsWe identified some mutations in both SMN1 and other genes, and c.[271C > T], c.[−41_-40insCTCT], c.[1001A > G] and c.[−117A > G] might be associated with the onset of SMA.

Lights and Shadows in the Genetics of Syndromic and Non-Syndromic Hearing Loss in the Italian Population.

Hearing loss (HL), both syndromic (SHL) and non-syndromic (NSHL), is the most common sensory disorder, affecting ~460 million people worldwide. More than 50% of the congenital/childhood cases are attributable to genetic causes, highlighting the importance of genetic testing in this class of disorders. Here we applied a multi-step strategy for the molecular diagnosis of HL in 125 patients, which included: (1) an accurate clinical evaluation, (2) the analysis of GJB2, GJB6, and MT-RNR1 genes, (3) the evaluation STRC-CATSPER2 and OTOA deletions via Multiplex Ligation Probe Amplification (MLPA), (4) Whole Exome Sequencing (WES) in patients negative to steps 2 and 3. Our approach led to the characterization of 50% of the NSHL cases, confirming both the relevant role of the GJB2 (20% of cases) and STRC deletions (6% of cases), and the high genetic heterogeneity of NSHL. Moreover, due to the genetic findings, 4% of apparent NSHL patients have been re-diagnosed as SHL. Finally, WES characterized 86% of SHL patients, supporting the role of already know disease-genes. Overall, our approach proved to be efficient in identifying the molecular cause of HL, providing essential information for the patients’ future management.

Blood functional assay for rapid clinical interpretation of germline TP53 variants

BackgroundThe interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a...

Pathogenicity reclassification of two BRCA1/BRCA2 exonic duplications after identification of genomic breakpoints and tandem orientation

The genomic consequence and clinical interpretation of large duplications are difficult to infer without determining the location and orientation of the duplicated sequence. We aimed to characterize two intragenic duplications detected in two hereditary breast and ovarian cancer syndrome (HBOC) families, namely BRCA1 exon 4 to 6 and BRCA2 exon 17 to 18, previously detected by multiplex ligation probe amplification and initially classified as variants of unknown significance. Using long range PCR, with duplication-specific primers, we were able to ascertain the genomic breakpoints and observed that the two rearrangements occurred in tandem and in direct orientation. The BRCA1 c.134+440_441+870dup and BRCA2 c.7806-2083_8332-1512dup duplications here identified are predicted to cause frameshifts that create a premature stop codon and were reclassified as pathogenic. Furthermore, both families present phenotypic traits typical of HBOC syndrome. We also observed that the genomic breakpoints of these two duplications occurred within highly homologous Alu elements. Concluding, we characterized two in tandem BRCA1 and BRCA2 duplications that likely occurred by Alu-mediated homologous recombination, allowing identification of the underlying cause of the HBOC syndrome in these families.

Discovery of specific mutations in spinal muscular atrophy patients by next-generation sequencing.

Spinal muscular atrophy (SMA) is a type of autosomal recessive genetic disease, which seriously threatens the health and lives of children and adolescents. We attempted to find some genes and mutations related to the onset of SMA. Eighty-three whole-blood samples were collected from 28 core families, including 28 probands with clinically suspected SMA (20 SMA patients, 5 non-SMA children, and 3 patients with unknown etiology) and their parents. The multiplex ligation probe amplification (MLPA) was performed for preliminary diagnosis. The high-throughput sequencing technology was used to conduct the whole-exome sequencing analysis. We analyzed the mutations in adjacent genes of SMN1 gene and the unique mutations that only occurred in SMA patients. According to the MLPA results, 20 probands were regarded as experimental group and 5 non-SMA children as control group. A total of 10 mutations were identified in the adjacent genes of SMN1 gene. GUSBP1 g.[69515863G>A], GUSBP1 g.[69515870C>T], and SMA4 g.[69515738C>A] were the top three most frequent sites. SMA4 g.[69515726A>G] and OCLN c.[818G>T] have not been reported in the existing relevant researches. Seventeen point mutations in the DYNC1H1 gene were only recognized in SMA children, and the top two most common mutations were c.[2869-34A>T] and c.[345-89A>G]; c.[7473+105C>T] was the splicing mutation that might change the mRNA splicing site. The mutations of SMA4 g.[69515726A>G], OCLN c.[818G>T], DYNC1H1 c.[2869-34A>T], DYNC1H1 c.[345-89A>G], and DYNC1H1 c.[7473+105C>T] in the adjacent genes of SMN1 gene and other genes might be related to the onset of SMA.

Intelligent Ratio: A New Method for Carrier and Newborn Screening in Spinal Muscular Atrophy.

Aim: Spinal muscular atrophy (SMA) is an inherited, autosomal recessive neuromuscular disease that causes high morbidity and mortality. The prevalence is 1-2/100,000, while the incidence is 1/6000-1/10,000 among live births. Due to the high carrier frequency (1/40-1/60) of SMA-associated alleles, screening can prevent new cases. The aim of the current study was to present the development of a new, quantitative, real-time, polymerase chain reaction (PCR)-based screening test that uses an intelligent ratio (IR) for analyses, as well as a comparison of the results with the gold standard. Materials and Methods: Included in the study were 100 patients with various risk genotypes for survivor motor neuron 1 (SMN1) and SMN2 genes whose genetics had been previously investigated using multiplex ligation probe amplification (MLPA). A combination of the 5' nuclease assay and allele-specific PCR was used to quantify the SMN1 deletion mutation with real-time PCR using the FII gene as a reference. All of the optimized standards were adapted to software that provided automated analyses. The approval number of the institutional ethics committee for the study is 2012-KAEK-15/1497. Results: The results of the screening test were completely compatible with the MLPA results; it achieved 100% sensitivity and specificity compared with the gold standard. The use of the IR in the analyses provided a user-independent method that quickly and accurately provided results, regardless of the amount of DNA used of the extraction method. Conclusion: Carrier or newborn screening of SMA is essential in countries that have high rates of consanguineous marriages. The screening test presented in this study that uses FII as a reference gene proved to be low-cost, reliable, applicable, accurate, and amenable to use in an automated system for SMA screening.

Genetic Counseling, Testing, and Management of HBOC in India: An Expert Consensus Document from Indian Society of Medical and Pediatric Oncology.

PURPOSEHereditary breast and ovarian cancer (HBOC) syndrome is primarily characterized by mutations in the BRCA1/2 genes. There are several barriers to the implementation of genetic testing and counseling in India that may affect clinical decisions. These consensus recommendations were therefore convened as a collaborative effort to improve testing and management of HBOC in India.DESIGNRecommendations were developed by a multidisciplinary group of experts from the Indian Society of Medical and Pediatric Oncology and some invited experts on the basis of graded evidence from the literature and using a formal Delphi process to help reach consensus. PubMed and Google Scholar databases were searched to source relevant articles.RESULTSThis consensus statement provides practical insight into identifying patients who should undergo genetic counseling and testing on the basis of assessments of family and ancestry and personal history of HBOC. It discusses the need and significance of genetic counselors and medical professionals who have the necessary expertise in genetic counseling and testing. Recommendations elucidate requirements of pretest counseling, including discussions on genetic variants of uncertain significance and risk reduction options. The group of experts recommended single-site mutation testing in families with a known mutation and next-generation sequencing coupled with multiplex ligation probe amplification for the detection of large genomic rearrangements for unknown mutations. Recommendations for surgical and lifestyle-related risk reduction approaches and management using poly (ADP-ribose) polymerase inhibitors are also detailed.CONCLUSIONWith rapid strides being made in the field of genetic testing/counseling in India, more oncologists are expected to include genetic testing/counseling as part of their clinical practice. These consensus recommendations are anticipated to help homogenize genetic testing and management of HBOC in India for improved patient care.

Complex Characterization of Germline Large Genomic Rearrangements of the BRCA1 and BRCA2 Genes in High-Risk Breast Cancer Patients-Novel Variants from a Large National Center.

Large genomic rearrangements (LGRs) affecting one or more exons of BRCA1 and BRCA2 constitute a significant part of the mutation spectrum of these genes. Since 2004, the National Institute of Oncology, Hungary, has been involved in screening for LGRs of breast or ovarian cancer families enrolled for genetic testing. LGRs were detected by multiplex ligation probe amplification method, or next-generation sequencing. Where it was possible, transcript-level characterization of LGRs was performed. Phenotype data were collected and analyzed too. Altogether 28 different types of LGRs in 51 probands were detected. Sixteen LGRs were novel. Forty-nine cases were deletions or duplications in BRCA1 and two affected BRCA2. Rearrangements accounted for 10% of the BRCA1 mutations. Three exon copy gains, two complex rearrangements, and 23 exon losses were characterized by exact breakpoint determinations. The inferred mechanisms for LGR formation were mainly end-joining repairs utilizing short direct homologies. Comparing phenotype features of the LGR-carriers to that of the non-LGR BRCA1 mutation carriers, revealed no significant differences. Our study is the largest comprehensive report of LGRs of BRCA1/2 in familial breast and ovarian cancer patients in the Middle and Eastern European region. Our data add novel insights to genetic interpretation associated to the LGRs.

Simplifying Detection of Copy-Number Variations in Maturity-Onset Diabetes of the Young

ObjectivesCopy-number variations (CNVs) are large-scale deletions or duplications of DNA that have required specialized detection methods, such as microarray-based genomic hybridization or multiplex ligation probe amplification. However, recent advances in bioinformatics have made it possible to detect CNVs from next-generation DNA sequencing (NGS) data. Maturity-onset diabetes of the young (MODY) 5 is a subtype of autosomal-dominant diabetes that is often caused by heterozygous deletions involving the HNF1B gene on chromosome 17q12. We evaluated the utility of bioinformatic processing of raw NGS data to detect chromosome 17q12 deletions in MODY5 patients. MethodsNGS data from 57 patients clinically suspected to have MODY but who were negative for pathogenic mutations using a targeted panel were re-examined using a CNV calling tool (CNV Caller, VarSeq version 1.4.3). Potential CNVs for MODY5 were then confirmed using whole-exome sequencing, cytogenetic analysis and breakpoint analysis when possible. ResultsWhole-gene deletions in HNF1B, ranging from 1.46 to 1.85 million basepairs in size, were detected in 3 individuals with features of MODY5. These were confirmed by independent methods to be part of a more extensive 17q12 deletion syndrome. Two additional patients carrying a 17q12 deletion were subsequently diagnosed using this method. ConclusionsLarge-scale deletions are the most common cause of MODY5 and can be detected directly from NGS data, without the need for additional methods.