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). However, the haemoglobin disorders are unique among all genetic diseases in that identification of carriers is preferable by haematological (biochemical) tests rather than DNA analysis. These Best Practice guidelines offer an overview of recommended strategies and methods for carrier identification and prenatal diagnosis of haemoglobinopathies, and emphasize the importance of appropriately applying and interpreting haematological tests in supporting the optimum application and evaluation of globin gene DNA analysis

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw

Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome

: The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species

239-kb Microdeletion Spanning KMT2E in a Child with Developmental Delay: Further Delineation of the Phenotype

Pathogenic KMT2E variants underly O'Donnell-Luria-Rodan syndrome, a recently described neurodevelopmental disorder characterized by global developmental delay, variable degrees of intellectual disability, and subtle facial dysmorphism. Less common findings include autism, seizures, gastrointestinal (GI) problems, and abnormal head circumference. Occurrence of mostly truncating variants as well as the similar phenotype observed in individuals with deletions spanning KMT2E suggest haploinsufficiency of this gene as a common mechanism for the disorder, while a gain-of-function or dominant-negative effect cannot be ruled out for some missense variants. Deletions reported in the literature encompass several additional known or presumed haploinsufficient genes, thus leading to more complex phenotypes. Here, we describe a male with antenatal onset hydronephrosis, hypotonia, global developmental delay, prominent GI symptoms as well as facial dysmorphism. Chromosomal microarray revealed a 239-kb de novo microdeletion spanning KMT2E and LHFPL3. Clinical presentation of our proband, harboring one of the smallest deletions of the region confirms the core features of this disorder, suggests GI symptoms as a prominent finding in affected individuals while expanding the phenotypic spectrum to abnormalities of the urinary tract

Impact of α-Globin Gene Expression and α-Globin Modifiers on the Phenotype of β-Thalassemia and Other Hemoglobinopathies: Implications for Patient Management

In this short review, we presented and discussed studies on the expression of globin genes in beta-thalassemia, focusing on the impact of alpha-globin gene expression and alpha-globin modifiers on the phenotype and clinical severity of beta-thalassemia. We first discussed the impact of the excess of free alpha-globin on the phenotype of beta-thalassemia. We then reviewed studies focusing on the expression of alpha-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free alpha-globin on erythroid cells. Alternative processes controlling alpha-globin excess were also considered, including the activation of autophagy by beta-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with beta-thalassemia and other hemoglobinopathies for which reduction in alpha-globin excess is clinically beneficial

Heterozygosity of the Complex Corfu δ0β+ Thalassemic Allele (HBD Deletion and HBB:c.92+5G>A) Revisited

The Corfu δ0β+ thalassemic allele is a unique thalassemic allele consisting of the simultaneous presence in cis of a deletion of the δ-globin (Hemoglobin Subunit Delta, HBD) and a single nucleotide variant in the β-globin gene (Hemoglobin Subunit Beta, HBB). The allele has, so far, been described in individuals of Greek origin. The objectives of the study are to ascertain the prevalence of the Corfu δ0β+ allele in comparison to other β-thalassemia variants encountered in Greece using our in-house data repository of 2558 β-thalassemia heterozygotes, and to evaluate the hematological phenotype of Corfu δ0β+ heterozygotes in comparison to heterozygotes with the most common β+- and deletion α0- thalassemia variants in Greece. The results of the study showed a relative incidence of heterozygotes with Corfu δ0β+ at 1.56% of all β-thalassemic alleles, and a distinct hematological phenotype of the heterozygotes characterized by microcytic, hypochromic anemia with normal levels of HbA2 (Hemoglobin A2) and elevated HbF (Hemoglobin F) levels. The application of a specific methodology for the identification of the Corfu δ0β+ allele is important for precise prenatal and antenatal diagnosis programs in Greece