Clinical Genetics Laboratories Research Articles

Standards and requirement for technologists in clinical genetics laboratories in the United States

临床遗传学检测一直在快速地发展，而医生们均依赖于准确的实验室检测来进行疾病的诊断、预后和治疗。美国遗传检测行业强烈推荐由认证的技术人员在临床遗传实验室中进行复杂的实验操作，以达到符合行业规范的标准。这些技术员在参加权威机构如国家临床实验室科学认证局(The National Accrediting Agency for Clinical Laboratory Sciences，NAACLS)认可的培训项目后，将通过认证考试获得资质。美洲华人遗传学会组织特别工作组撰写本文，介绍美国认证遗传实验技术员培训项目的机构、培训项目获准的过程以及遗传实验技术员的培训和认证的过程，希望对中国建立一个能够确保培养合格的遗传实验检测技术人员的体系有所帮助。

Regulatory agencies of clinical genetic and genomic laboratories in the United States

美国医学遗传学资质委员会(American Board of Medical Genetics，ABMG)的成立，使医学遗传学在临床上得到了广泛的认可。在过去30年中，基因检测技术的飞跃发展，促使美国政府不断地制订和修改法律和法规，以确保临床遗传检测能够安全、有效和经济地为公众服务。联邦政府的两个主管机构，即食品药品监督管理局(U.S. Food and Drug Administration，FDA)和医疗保险和医疗补助服务中心(Centers for Medicare & Medicaid Services，CMS)已发布多项跨部门的规定，重点监督管理体外诊断(In vitro Diagnostics，IVD)产品和临检实验室的规范，包括《临床实验室改进法案(Clinical Laboratory Improvement Act，CLIA)》。联邦政府还颁布了两个健康隐私法案，即《健康保险保障和责任法案(Health Insurance Portability and Accountability Act，HIPAA)》和《反遗传信息歧视法案(Genetic Information Nondiscrimination Act，GINA)》来保护患者与公众的隐私。除政府法规外，临床遗传检测服务还受到独立的非营利性专业认证机构，如美国病理学家协会(College of American Pathologists，CAP)和医疗保健组织认证联合委员会(Joint Commission on Accreditation of Healthcare Organizations，JCAHO或TJC)的考核。专业认证机构与专业人员和专家合作，通过实施认证和能力验证，不断地改进临床实验室的质量和管理。

Introduction of standards and requirement for accredited certification of clinical genetics laboratories in the United States

临床实验遗传学的发展要求建立临床检测实验室和检测人员的相关标准和规范以及后续的审核、认证和检查过程。其中，临床实验室的认证是保证医生和患者能够得到准确可靠的检测结果的重要一步。美国国会通过的1988年临床实验室改进修正法案(Clinical Laboratory Improvement Amendments of 1988，CLIA'88)规定，临床检测实验室需要通过CLIA'88批准的认证机构的详细审核和检查。美国医疗保险和医疗补助服务中心(Centers for Medicare & Medicaid Services，CMS)则颁布了临床检测实验室的标准和认证的具体内容，要求所有的临床实验室必须通过CLIA'88认证注册，方能对血液、体液和组织等人体样本进行检测，以用于疾病的诊断、预防、评估和治疗，并得到美国政府医疗保险和医疗补助计划的付款。美国病理医师协会(College of American Pathologists，CAP)是被批准的认证机构之一。CAP认证涵盖包括临床遗传学检测在内的所有实验室专业。美洲华人遗传学会(Association of Chinese Geneticists in America，ACGA)组织特别工作组撰写此文，介绍CAP根据CLIA'88法案为临床实验室制定的要求、标准和建议，以及国际实验室为获得CLIA和CAP的认证所需要遵循的流程。我们希望这项工作能够帮助中国建立一个适当的系统来进行临床检测实验室的认证，或者直接获得国际CLIA和CAP的认证。

Venous thromboembolism laboratory testing (factor V Leiden andfactor II c.*97G>A), 2018 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

Factor V Leiden and factor II c.*97G>A (formerly referred to asprothrombin 20210G>A) are the two most common genetic variants associated withvenous thromboembolism (VTE). Testing for these variants is one of the most commonreferrals in clinical genetics laboratories. While the methodologies for testingthese two variants are relatively straightforward, the clinical implementation canbe complicated with regard to test indications, risk assessment of occurrence andrecurrence of VTE, and related genetic counseling. This document provides anoverview of VTE, information about the variants and their influence on risk,considerations before initiating genetic testing, and the clinical and analyticalsensitivity and specificity of the tests. Key information that should be included inthe laboratory report is also provided. Disease-specific statements are intended toaugment the general American College of Medical Genetics and Genomics (ACMG)technical standards for clinical genetics laboratories. Individual laboratories areresponsible for meeting the Clinical Laboratory Improvement Amendments(CLIA)/College of American Pathologists (CAP) quality assurance standards withrespect to appropriate sample documentation, assay validation, general proficiencytesting, and quality control measures. This 2018 edition of the ACMG technicalstandard updates and supersedes the 2005 edition on this topic. It is designed to bea checklist for genetic testing professionals who are already familiar with thedisease and the methods of analysis.

Standards and guidelines for canine clinical genetic testing laboratories

This publication represents a proposed approach to quality standards and guidelines for canine clinical genetic testing laboratories. Currently, there are no guidelines for laboratories performing clinical testing on dogs. Thus, there is no consensus set of protocols that set the minimal standards of quality among these laboratories, potentially causing variable results between laboratories, inconsistencies in reporting, and the inability to share information that could impact testing among organizations. A minimal standard for quality in testing is needed as breeders use the information from genetic testing to make breeding choices and irreversible decisions regarding spay, neuter or euthanasia. Incorrect results can have significant impact on the health of the dogs being tested and on their subsequent progeny. Because of the potentially serious consequences of an incorrect result or incorrect interpretation, results should be reviewed by and reported by individuals who meet a minimum standard of qualifications. Quality guidelines for canine genetic testing laboratories should include not only the analytical phase, but also the preanalytical and postanalytical phases, as this document attempts to address.

ClinVar at five years: Delivering on the promise.

The increasing application of genetic testing for determining the causes underlying Mendelian, pharmacogenetic, and somatic phenotypes has accelerated the discovery of novel variants by clinical genetics laboratories, resulting in a critical need for interpreting the significance of these variants and presenting considerable challenges. Launched in 2013 at the National Center for Biotechnology Information, National Institutes of Health, ClinVar is a public database for clinical laboratories, researchers, expert panels, and others to share their interpretations of variants with their evidence. The database holds 600,000 submitted records from 1,000 submitters, representing 430,000 unique variants. ClinVar encourages submissions of variants reviewed by expert panels, as expert consensus confers a high standard. Aggregating data from many groups in a single database allows comparison of interpretations, providing transparency into the concordance or discordance of interpretations. In its first five years, ClinVar has successfully provided a gateway for the submission of medically relevant variants and interpretations of their significance to disease. It has become an invaluable resource for the clinical genetics community seeking guidance from consensus interpretations. Building on the platform of providing transparency and leveraging aggregation of variant interpretations, ClinVar is now well positioned to help the clinical genetics community improve interpretations.

Frequency of frontotemporal dementia gene variants in C9ORF72, MAPT, and GRN in academic versus commercial laboratory cohorts.

Frontotemporal lobar degeneration (FTLD) is a leading cause of dementia, and elucidating its genetic underpinnings is critical. FTLD research centers typically recruit patient cohorts that are limited by the center's specialty and the ways in which its geographic location affects the ethnic makeup of research participants. Novel sources of data are needed to get population estimates of the contribution of variants in known FTLD-associated genes. We compared FLTD-associated genetic variants in microtubule-associated protein tau (MAPT), progranulin (GRN), and chromosome nine open reading frame 72 (C9ORF72) from an academic research cohort and a commercial clinical genetics laboratory. Pathogenicity was assessed using guidelines of the American College of Medical Genetics and Genomics and a rule-based DNA variant assessment system. We conducted chart reviews on patients with novel or rare disease-associated variants. A total of 387 cases with FTLD-associated variants from the commercial (n=2,082) and 78 cases from the academic cohort (n=2,089) were included for analysis. In the academic cohort, the most frequent pathogenic variants were C9ORF72 expansions (63%, n=49), followed by GRN (26%, n=20) and MAPT (11%, n=9). Each gene's contribution to disease was similarly ranked in the commercial laboratory but differed in magnitude: C9ORF72 (89%, n=345), GRN (6%, n=24), and MAPT (5%, n=19). Of the 37 unique GRN/MAPT variants identified, only six were found in both cohorts. Clinicopathological data from patients in the academic cohort strengthened classification of two novel GRN variant as pathogenic (p.Pro166Leufs*2, p.Gln406*) and one GRN variant of unknown significance as a possible rare risk variant (p.Cys139Arg). Differences in gene frequencies and identification of unique pathogenic alleles in each cohort demonstrate the importance of data sharing between academia and community laboratories. Using shared data sources with well-characterized clinical phenotypes for individual variants can enhance interpretation of variant pathogenicity and inform clinical management of at-risk patients and families.

Special Issue: Abstracts of the ISPD 22nd International Conference on Prenatal Diagnosis and Therapy, Antwerp, Belgium, 8-11 July 2018.

Objectives: In most countries, non-invasive prenatal testing (NIPT) has been introduced commercially without any governmental guidance. In the Netherlands, prenatal screening for fetal anomaly is subject to a governmental license. NIPT has been implemented as part of the TRIDENT studies (Trial by Dutch laboratories for Evaluation of NIPT). TRIDENT-2 aims at offering NIPT to all pregnant women (∼174,000 women/year) within the national prenatal screening program. Since April 2017, women can choose NIPT as a contingent test after first-trimester combined testing (FCT), but may also choose NIPT as first-tier screening test. TheTRIDENT studies evaluate implementation and women's perspectives. Methods: All pregnant women in the Netherlands are offered prenatal screening and are counselled by certified counselors, generally midwives. A first-tier NIPT costs women € 175, comparable to the costs of FCT (∼€ 168). NIPT is performed by three Dutch university clinical genetic laboratories using an in-house validated test. Women can choose to have analysis of chromosomes 21, 18, and 13 without or with a report of incidental findings (findings other than trisomy 21, 13, 18) on the remaining autosomes, respectively, using the 'targeted' or 'whole genome' WISECONDOR pipeline. Sex chromosomes are not analyzed. Results: After 8 months of study, 48,234 tests have been performed (nationwide uptake of prenatal screening by NIPT as first-tier test was 40%), and 98.3% reports successfully issued. Failure rate was less than 2%. Mean turnaround time was 7 working days. 80% of women chose to have all autosomes analyzed. A total of 152 cases of T21 (0.3%), 32 cases of T18 (0.1%), 41 cases of T13 (0.1%), and 158 (0.3%) other chromosomal aberrations were found. First year results (and available follow-up) will be presented at the meeting. Conclusions: The Netherlands are the first country where NIPT is incorporated as a first-line test into a governmentally supported and health care funded prenatal aneuploidy screening program. The incorporation of the test in a university hospital laboratory and clinical service guarantees appropriate counselling and allows for proper follow-up. This 3-year study aims to provide all necessary information for a successful introduction of NIPT within the Dutch National prenatal screening program.

Spectrum of lysosomal disorders at a medical genetics center in the central part of the United States

Disclaimer: These ACMG Standards and Guidelines are developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these Standards and Guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient’s record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Galactosemias are inherited disorders of galactose metabolism due to deficiency in one of the three enzymes involved in the Leloir pathway: galactose-1-phosphate uridyltransferase, galactokinase, and uridine diphosphate (UDP)-galactose-4′-epimerase. Galactose-1-phosphate uridyltransferase deficiency, or classic galactosemia, is the most frequent and the most severe of the three enzyme deficiencies; it is characterized by failure to thrive, liver failure, susceptibility to sepsis, and death, if untreated. Newborn screening for classic galactosemia has been implemented in all of the United States, while screening for galactokinase deficiency and UDP-galactose-4′-epimerase deficiency is not universal. Early identification and treatment of galactosemia leads to improved outcome. This document reviews the laboratory methods and best practices for the diagnosis of galactosemia.

Clinical Molecular Genetic Laboratory Practice, Where We Stand in 2018

Clinical Molecular Genetic Laboratory Practice, Where We Stand in 2018

Clinical implications and considerations for evaluation of in silico algorithms for use with ACMG/AMP clinical variant interpretation guidelines

Clinical genetics laboratories have recently adopted guidelines for the interpretation of sequence variants set by the American College of Medical Genetics (ACMG) and Association for Molecular Pathology (AMP). The use of in silico algorithms to predict whether amino acid substitutions result in human disease is inconsistent across clinical laboratories. The clinical genetics community must carefully consider how in silico predictions can be incorporated into variant interpretation in clinical practice.Please see related Research article: https://doi.org/10.1186/s13059-017-1353-5

Laboratory diagnosis of galactosemia: a technical standard and guideline of the American College of Medical Genetics and Genomics (ACMG).

Disclaimer: These ACMG Standards and Guidelines are developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these Standards and Guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Galactosemias are inherited disorders of galactose metabolism due to deficiency in one of the three enzymes involved in the Leloir pathway: galactose-1-phosphate uridyltransferase, galactokinase, and uridine diphosphate (UDP)-galactose-4'-epimerase. Galactose-1-phosphate uridyltransferase deficiency, or classic galactosemia, is the most frequent and the most severe of the three enzyme deficiencies; it is characterized by failure to thrive, liver failure, susceptibility to sepsis, and death, if untreated. Newborn screening for classic galactosemia has been implemented in all of the United States, while screening for galactokinase deficiency and UDP-galactose-4'-epimerase deficiency is not universal. Early identification and treatment of galactosemia leads to improved outcome. This document reviews the laboratory methods and best practices for the diagnosis of galactosemia.

Allelic spectrum of formiminotransferase-cyclodeaminase gene variants in individuals with formiminoglutamic aciduria.

BackgroundElevated plasma and urine formiminoglutamic acid (FIGLU) levels are commonly indicative of formiminoglutamic aciduria (OMIM #229100), a poorly understood autosomal recessive disorder of histidine and folate metabolism, resulting from formiminotransferase‐cyclodeaminase (FTCD) deficiency, a bifunctional enzyme encoded by FTCD.MethodsIn order to further understanding about the molecular alterations that contribute to FIGLU‐uria, we sequenced FTCD in 20 individuals with putative FTCD deficiency and varying laboratory findings, including increased FIGLU excretion.ResultsIndividuals tested had biallelic loss‐of‐function variants in protein‐coding regions of FTCD. The FTCD allelic spectrum comprised of 12 distinct variants including 5 missense alterations that replace conserved amino acid residues (c.223A>C, c.266A>G, c.319T>C, c.430G>A, c.514G>T), an in‐frame deletion (c.1373_1375delTGG), with the remaining alterations predicted to affect mRNA processing/stability. These included two frameshift variants (c.990dup, c.1366dup) and four nonsense variants (c.337C>T, c.451A>T, c.763C>T, c.1607T>A).ConclusionWe observed additional FTCD alleles leading to urinary FIGLU elevations, and thus, providing molecular evidence of FTCD deficiency in cases identified by newborn screening or clinical biochemical genetic laboratory testing.

SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population

Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.

Clinical Validation of Copy Number Variant Detection from Targeted Next-Generation Sequencing Panels

Next-generation sequencing (NGS) technology has rapidly replaced Sanger sequencing in the assessment of sequence variations in clinical genetics laboratories. One major limitation of current NGS approaches is the ability to detect copy number variations (CNVs) approximately >50 bp. Because these represent a major mutational burden in many genetic disorders, parallel CNV assessment using alternate supplemental methods, along with the NGS analysis, is normally required, resulting in increased labor, costs, and turnaround times. The objective of this study was to clinically validate a novel CNV detection algorithm using targeted clinical NGS gene panel data. We have applied this approach in a retrospective cohort of 391 samples and a prospective cohort of 2375 samples and found a 100% sensitivity (95% CI, 89%-100%) for 37 unique events and a high degree of specificity to detect CNVs across nine distinct targeted NGS gene panels. This NGS CNV pipeline enables stand-alone first-tier assessment for CNV and sequence variants in a clinical laboratory setting, dispensing with the need for parallel CNV analysis using classic techniques, such as microarray, long-range PCR, or multiplex ligation-dependent probe amplification. This NGS CNV pipeline can also be applied to the assessment of complex genomic regions, including pseudogenic DNA sequences, such as the PMS2CL gene, and to mitochondrial genome heteroplasmy detection.

Diagnostic cytogenetic testing following positive noninvasive prenatal screening results: a clinical laboratory practice resource of the American College of Medical Genetics and Genomics (ACMG).

Disclaimer: ACMG Clinical Laboratory Practice Resources are developed primarily as an educational tool for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these practice resources is voluntary and does not necessarily assure a successful medical outcome. This Clinical Laboratory Practice Resource should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with this Clinical Laboratory Practice Resource. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Noninvasive prenatal screening (NIPS) using cell-free DNA has been rapidly adopted into prenatal care. Since NIPS is a screening test, diagnostic testing is recommended to confirm all cases of screen-positive NIPS results. For cytogenetics laboratories performing confirmatory testing on prenatal diagnostic samples, a standardized testing algorithm is needed to ensure that the appropriate testing takes place. This algorithm includes diagnostic testing by either chorionic villi sampling or amniocentesis samples and encompasses chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray.

CAP/ACMG proficiency testing for biochemical genetics laboratories: a summary of performance

PurposeTesting for inborn errors of metabolism is performed by clinical laboratories worldwide, each utilizing laboratory-developed procedures. We sought to summarize performance in the College of American Pathologists’ (CAP) proficiency testing (PT) program and identify opportunities for improving laboratory quality. When evaluating PT data, we focused on a subset of laboratories that have participated in at least one survey since 2010.MethodsAn analysis of laboratory performance (2004 to 2014) on the Biochemical Genetics PT Surveys, a program administered by CAP and the American College of Medical Genetics and Genomics. Analytical and interpretive performance was evaluated for four tests: amino acids, organic acids, acylcarnitines, and mucopolysaccharides.ResultsSince 2010, 150 laboratories have participated in at least one of four PT surveys. Analytic sensitivities ranged from 88.2 to 93.4%, while clinical sensitivities ranged from 82.4 to 91.0%. Performance was higher for US participants and for more recent challenges. Performance was lower for challenges with subtle findings or complex analytical patterns.ConclusionUS clinical biochemical genetics laboratory proficiency is satisfactory, with a minority of laboratories accounting for the majority of errors. Our findings underscore the complex nature of clinical biochemical genetics testing and highlight the necessity of continuous quality management.

European registration process for Clinical Laboratory Geneticists in genetic healthcare

Tremendous progress in genetics and genomics led to a wide range of healthcare providers, genetic tests, and more patients who can benefit from these developments. To guarantee and improve the quality of genetic testing, a unified European-based registration for individuals qualified in biomedicine was realized. Therefore a Europe-wide recognition of the profession ‘European registered Clinical Laboratory Geneticist (ErCLG)’ based on a syllabus of core competences was established which allows for harmonization in professional education. The ‘European Board of Medical Genetics division − Clinical Laboratory Geneticist’ provides now since 3 years the possibility to register as an ErCLG. Applicants may be from all European countries and since this year also from outside of Europe. Five subtitles reflect the exact specialty of each ErCLG, who can reregister every 5 years. A previously not possible statistics based on ~300 individuals from 19 countries as holders of an ErCLG title provides interesting insights into the professionals working in human genetics. It could be substantiated that there are around twice as many females than males and that a PhD title was achieved by 80% of registered ErCLGs. Also most ErCLGs are still trained as generalists (66%), followed by such ErCLGs with focus on molecular genetics (23%); the remaining are concentrated either on clinical (6%), tumor (4%) or biochemical genetics (1%). In conclusion, besides MDs and genetic counselors/nurses an EU-wide recognition system for Clinical Laboratory Geneticist has been established, which strengthens the status of specialists working in human genetic diagnostics in Europe and worldwide.

Laboratory diagnosis of creatine deficiency syndromes: a technical standard and guideline of the American College of Medical Genetics and Genomics.

Disclaimer: These ACMG Standards and Guidelines are intended as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of others that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, clinical laboratory geneticists should apply their professional judgment to the specific circumstances presented by the patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Cerebral creatine deficiency syndromes are neurometabolic conditions characterized by intellectual disability, seizures, speech delay, and behavioral abnormalities. Several laboratory methods are available for preliminary and confirmatory diagnosis of these conditions, including measurement of creatine and related metabolites in biofluids using liquid chromatography-tandem mass spectrometry or gas chromatography-mass spectrometry, enzyme activity assays in cultured cells, and DNA sequence analysis. These guidelines are intended to standardize these procedures to help optimize the diagnosis of creatine deficiency syndromes. While biochemical methods are emphasized, considerations for confirmatory molecular testing are also discussed, along with variables that influence test results and interpretation.Genet Med 19 2, 256-263.

A Novel Micro RNA Signature Identifies a Patient Subset with Poor Prognosis in Core Binding Factor AML

Disruption of core binding factor (CBF) in acute myeloid leukemia (AML) has been identified as a favourable prognostic biomarker in AML. Consequently, CBF AML patients are typically treated less intensively than AML patients in most other prognostic groups. Nevertheless, a subset of CBF AML patients fail initial therapy and treatment of these patients after relapse is challenging. Few prognostic markers are available to stratify risk in CBF AML other than t(8;21) vs. inv(16) where t(8;21) is the unfavourable marker and KIT mutation status in t(8;21). In this study, we aimed to utilize miRNA expression profiles in CBF AML to identify patients with favourable vs. unfavourable prognoses.Patients and MethodsWe analyzed small RNA sequencing data from a discovery cohort of 188 de novo AML patients, of which 19 were CBF AML patients, from The Cancer Genome Atlas study (NEJM, 2013). We selected miRNAs with expression that satisfied a mean of greater than 10 read/106 miRNA mapped and a coefficient of variance greater than 2. Forty-eight miRNAs met these criteria and were further used for model-based clustering. As a validation cohort, we enrolled 38 CBF AML patients diagnosed from 1998 to 2014 at the Prince Margaret Cancer Center (PMCC). Diagnosis of CBF AML was confirmed with conventional cytogenetic analyses at a clinical genetics laboratory at the PMCC. PerfeCTa microRNA assays were applied to quantify the expression of miRNA normalized to RNU6.ResultsDiscovery of the poor prognosis group in CBF AML patientsModel-based clustering with 48 miRNAs from TCGA AML data set identified 4 distinct patient clusters (Figure A). Cluster 2 (C2) contained exclusively acute promyelocytic leukemia (APL) patients with high expression of all miRNAs and was excluded from further analysis. Notably, Cluster 1 (C1) was characterized by low expression of most of the 48 miRNAs of the list (Figure A). CBF AML patients were present in C1 (C1-CBF group; n=10) and C3 (C3-CBF group; n=9) only, with none in cluster 4. Figures B and C illustrate survival analyses of C1-CBF and C3-CBF patients compared to several other groups. Notably, C1-CBF patients had significantly worse overall survival (OS) (p=0.001) and disease free survival (DFS) (p=0.005) compared to C3-CBF, with survival statistics comparable to intermediate or poor risk AML subgroups, as measured by Kaplan-Meier analysis.Validation of C1-CBF and C3-CBF group in PMCC patientsWe identified 3 miRNAs, miR-127, miR-494, and miR-495, from the original 48, that were sufficient to perfectly reproduce the C1 and C3 subgroupings when clustering on them alone. We performed real time qPCR to measure the expression of these 3 miRNAs in the 38 CBF AML patients from the PMCC cohort and assigned them to the clusters. The PMCC C1-CBF (n=13) patients had significantly worse 3-yr OS and 3-yr DFS than the C3-CBF (n=25) patients (OS: C1-CBF, 23.1±11.7% vs C3-CBF, 72±9%; p=0.0062 and DFS: C1-CBF,27.8±13.6% vs C3-CBF 79.1± 8.3%; p=0.0092, Figures D & E).Using multivariate analysis with covariates including age, KIT mutation and translocation status, we demonstrated that these 3 miRNAs form an independent prognostic biomarker in CBF-AML for both OS and DFS (Table).ConclusionWe demonstrate that 3 miRNAs (miR-127, miR-494, miR-495) can be used to stratify CBF-AML patients into favourable and unfavourable prognostic subgroups. We show that expression levels of this trio of miRNAs can be used as a clinical tool and we propose that CBF AML patients in the unfavourable miRNA group, C1-CBF, should no longer be treated as favourable risk patients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.