Adapting the ACMG/AMP variant classification framework: A perspective from the ClinGen Hemoglobinopathy Variant Curation Expert Panel.

Accurate and consistent interpretation of sequence variants is integral to the delivery of safe and reliable diagnostic genetic services. To standardize the interpretation process, in 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published a joint guideline based on a set of shared standards for the classification of variants in Mendelian diseases. The generality of these standards and their subjective interpretation between laboratories has prompted efforts to reduce discordance of variant classifications, with a focus on the expert specification of the ACMG/AMP guidelines for individual genes or diseases.

Phenotype-driven variant filtration strategy in exome sequencing toward a high diagnostic yield and identification of 85 novel variants in 400 patients with rare Mendelian disorders.

About 6000 to 7000 different rare disorders with suspected genetic etiologies have been described and almost 4500 causative gene(s) have been identified. The advent of next-generation sequencing (NGS) technologies has revolutionized genomic research and diagnostics, representing a major advance in the identification of pathogenic genetic variations. This study presents a 3-year experience from an academic genetics center, where 400 patients were referred for genetic analysis of disorders with unknown etiology.

Rapid Detection of Fetal Mendelian Disorders: Thalassemia and Sickle Cell Syndromes.

The inherited disorders of hemoglobin synthesis constitute the most common monogenic diseases worldwide. The clinical severity of β-thalassemia major and the sickle cell syndromes targets them as priority genetic diseases for prevention programs, which incorporates population screening to identify heterozygotes, with the option of prenatal diagnosis for carrier couples. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis.

Clinical Outcomes in Duchenne Muscular Dystrophy: A Study of 5345 Patients from the TREAT-NMD DMD Global Database.

Background: Recent short-term clinical trials in patients with Duchenne Muscular Dystrophy (DMD) have indicated greater disease variability in terms of progression than expected. In addition, as average life-expectancy increases, reliable data is required on clinical progression in the older DMD population.

Objective: To determine the effects of corticosteroids on major clinical outcomes of DMD in a large multinational cohort of genetically confirmed DMD patients.

Preconception carrier screening and prenatal diagnosis in thalassemia and hemoglobinopathies: challenges and future perspectives.

Hemoglobinopathies constitute the most common severe monogenic disorders worldwide, with an increasing global burden each year. The benefit of applying programmes for preconception carrier screening, with the option of prenatal diagnosis, to minimize the incidence of new cases is recognized in many countries. Areas covered: The challenges associated with identifying carrier couples using hematology-based screening, along with DNA diagnosis and prenatal diagnosis were addressed, based on a literature search and the authors expertise.

Pre-implantation genetic diagnosis.

The aim of pre-implantation genetic diagnosis (PGD) is to characterize the genetic status of the cells (usually single cells) that have been biopsied from oocytes/zygotes or embryos created in vitro during assisted reproductive treatment. PGD is a multi-step procedure that requires close collaboration between gynaecologists who are experts in assisted reproduction, embryologists who are experts in micromanipulation of germ cells and in embryo biopsy and geneticists who are experts in genetic analysis at the single-cell level. PGD can be applied as a form of early pre-natal diagnosis with the aim to establish a pregnancy unaffected by a haemoglobinopathy.

Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD).

Background: Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis.

Methods: A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs.

ESHRE PGD Consortium data collection XIII: cycles from January to December 2010 with pregnancy follow-up to October 2011.

Study Question: How do data in the 13th annual data collection (Data XIII) of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium compare with the cumulative data for collections I-XII?

Summary Answer: The 13th retrospective collection represents valuable data on PGD/PGS cycles, pregnancies and children: the main trend observed is the decrease in the routine implementation of PGS.

What Is Known Already: Since 1999, the PGD Consortium has collected, analysed and published 12 data sets and an overview of the first 10 years of data collections.

Study Design, Size, Duration: Data were collected from each participating centre using a FileMaker Pro database (versions 5-11).

EMQN Best Practice Guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies.

Haemoglobinopathies constitute the commonest recessive monogenic disorders worldwide, and the treatment of affected individuals presents a substantial global disease burden. Carrier identification and prenatal diagnosis represent valuable procedures that identify couples at risk for having affected children, so that they can be offered options to have healthy offspring. Molecular diagnosis facilitates prenatal diagnosis and definitive diagnosis of carriers and patients (especially 'atypical' cases who often have complex genotype interactions).

Prenatal diagnosis for CF using High Resolution Melting Analysis and simultaneous haplotype analysis through QF-PCR.

Background: High Resolution Melting (HRM) Analysis is a validated, robust, low-cost, high throughput CF screening method. Here, we report the development and retrospective evaluation of the diagnostic value of a novel multiplex HRM, genotyping and haplotyping method for CF prenatal diagnosis (generic HRM/haplotyping).

Methods: 80 study samples from 20 carrier couples referred for PND (whole blood in EDTA and CVS or amniotic fluid) were genotyped retrospectively using the suggested protocol.

Advances in technologies for screening and diagnosis of hemoglobinopathies.

Hemoglobinopathies constitute the most common monogenic disorders worldwide, caused by mutations in the globin genes that synthesize the globin chains of hemoglobin. Synthesis may be reduced (thalassemia) or underlie abnormal hemoglobins. Mutation interactions produce a wide range of disorders.

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study.

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles.

Preimplantation genetic diagnosis, an alternative to conventional prenatal diagnosis of the hemoglobinopathies.

Prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD) both represent highly important reproductive choices for couples with a high risk of transmitting a severe disease, such as a severe hemoglobinopathy. Conventional PND for hemoglobinopathies based on molecular analysis of trophoblast or amniocyte DNA has been applied for around 30 years, but the major disadvantages with this approach include 'invasive' fetal sampling, and the potential involvement of pregnancy termination when affected. In comparison, the major advantage of PGD over conventional PND is that it supports the initiation of unaffected pregnancies, avoiding the need to terminate affected pregnancies.

Microsatellite markers within the α-globin gene cluster for robust preimplantation genetic diagnosis of severe α-thalassemia syndromes in Mediterranean populations.

In this study we report the development of a generic protocol for preimplantation genetic diagnosis (PGD) of severe α-thalassemia (α-thal) syndromes in α-thal carrier couples of Mediterranean origin. The in silico identification and design of primers for multiplex analysis of short tandem repeats (STRs), was followed by the optimization of polymerase chain reaction (PCR) conditions for multiplexed STR analysis within the α-globin gene cluster (16p3.3) and subsequent optimization and validation of a single-cell multiplex reaction including the selected STRs.

Prenatal, noninvasive and preimplantation genetic diagnosis of inherited disorders: hemoglobinopathies.

Disorders of hemoglobin synthesis have been used as a prototype for the development of most approaches for prenatal diagnosis (PND). PND for hemoglobinopathies based on molecular analysis of trophoblast or amniocyte DNA has accumulated approximately 30 years of experience. Disadvantages with conventional PND include 'invasive' fetal sampling and the need to terminate affected ongoing pregnancies.

Novel and known microsatellite markers within the β-globin cluster to support robust preimplantation genetic diagnosis of β-thalassemia and sickle cell syndromes.

Preimplantation genetic diagnosis (PGD) for β hemoglobinopathies has become the most common application among monogenic disorders. We present the identification of microsatellite markers [short tandem repeats (STRs)] closely linked to the β-globin gene for incorporation within PGD protocols, with the aim of increasing the number of transferable embryos. Nine candidate STRs were identified in-silico, of which three were selected based on rate-of-heterozygosity, polymerase chain reaction (PCR) efficiency and size.

Rapid detection of fetal Mendelian disorders: thalassemia and sickle cell syndromes.

The inherited disorders of hemoglobin synthesis constitute the most common monogenic diseases worldwide. The clinical severity of beta-thalassemia major and the sickle cell syndromes targets them as priority genetic diseases for prevention programs, which incorporates population screening to identify heterozygotes,with the option of prenatal diagnosis for carrier couples. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis.

Real-time PCR for prenatal and preimplantation genetic diagnosis of monogenic diseases.

The provision of prenatal diagnosis requires the highest standards in laboratory practice to ensure an accurate result. In preimplantation genetic diagnosis protocols additionally have to address the need to achieve an accurate result from 1 to 2 cells within a limited time. Emerging protocols of "non-invasive" prenatal diagnosis, which are based on analysis of free fetal DNA in the circulation of the pregnant mother, also have to achieve a result from a limited quantity of fetal DNA against a high background of maternal free DNA.

Trigonocephaly and Wilson's disease in two siblings.

Trigonocephaly and Wilson's disease (WD) are two different entities. The former is a type of craniosynostosis that occurs because of fusion of the metopic suture and the latter, also called hepatolenticular degeneration, is caused by an accumulation of copper in tissues all over the body because of failure of copper excretion. No single gene has been identified for trigonocephaly whereas the ATP7B gene has been shown to be responsible for Wilson's disease.