Hemoglobin Bart's hydrops fetalis: charting the past and envisioning the future.

ABSTRACT: Hemoglobin Bart's hydrops fetalis syndrome (BHFS) represents the most severe form of α-thalassemia, arising from deletion of the duplicated α-globin genes from both alleles. The absence of α-globin leads to the formation of nonfunctional hemoglobin (Hb) Bart's (γ4) or HbH (ß4) resulting in severe anemia, tissue hypoxia, and, in some cases, variable congenital or neurocognitive abnormalities. BHFS is the most common cause of hydrops fetalis in Southeast Asia; however, owing to global migration, the burden of this condition is increasing worldwide. With the availability of intensive perinatal care and intrauterine transfusions, an increasing number of patients survive with this condition. The current approach to long-term management of survivors involves regular blood transfusions and iron chelation, a task made challenging by the need for intensified transfusions to suppress the production of nonfunctional HbH-containing erythrocytes. Although our knowledge of outcomes of this condition is evolving, it seems, in comparison to individuals with transfusion-dependent ß-thalassemia, those with BHFS may face an elevated risk of complications arising from chronic anemia and hypoxia, ongoing hemolysis, iron overload, and from their respective treatments. Although stem cell transplantation remains a viable option for a select few, it is not without potential side effects. Looking ahead, potential advancements in the form of genetic engineering and innovative therapeutic approaches, such as the reactivation of embryonic α-like globin gene expression, hold promise for furthering the treatment of this condition. Prevention remains a crucial aspect of care, particularly in areas with high prevalence or limited resources.

A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects.

ATRX is a chromatin remodelling ATPase that is involved in transcriptional regulation, DNA damage repair and heterochromatin maintenance. It has been widely studied for its role in ALT-positive cancers, but its role in neurological function remains elusive. Hypomorphic mutations in the X-linked ATRX gene cause a rare form of intellectual disability combined with alpha-thalassemia called ATR-X syndrome in hemizygous males. Clinical features also include facial dysmorphism, microcephaly, short stature, musculoskeletal defects and genital abnormalities. As complete deletion of ATRX in mice results in early embryonic lethality, the field has largely relied on conditional knockout models to assess the role of ATRX in multiple tissues. Given that null alleles are not found in patients, a more patient-relevant model was needed. Here, we have produced and characterized the first patient mutation knock-in model of ATR-X syndrome, carrying the most common causative mutation, R246C. This is one of a cluster of missense mutations located in the chromatin-binding domain and disrupts its function. The knock-in mice recapitulate several aspects of the patient disorder, including craniofacial defects, microcephaly, reduced body size and impaired neurological function. They provide a powerful model for understanding the molecular mechanisms underlying ATR-X syndrome and testing potential therapeutic strategies.

Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors.

The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4-7 fold higher than starting cell numbers within 9-12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations.

The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats.

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG)n ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes.

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog.

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6-9 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf/(+);Shh(-/-) mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf/(+) embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf/(+) limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf/(+) mutants, we sequenced approximately 20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a approximately 600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5' of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf/(+) mutants. However, we did not detect anologous deletions in cases of Laurin-Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.

Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome.

Craniofrontonasal syndrome (CFNS) is an X-linked developmental disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Females have frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures); in males, hypertelorism is the only typical manifestation. Here, we show that the classical female CFNS phenotype is caused by heterozygous loss-of-function mutations in EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases. In mice, the orthologous Efnb1 gene is expressed in the frontonasal neural crest and demarcates the position of the future coronal suture. Although EFNB1 is X-inactivated, we did not observe markedly skewed X-inactivation in either blood or cranial periosteum from females with CFNS, indicating that lack of ephrin-B1 does not compromise cell viability in these tissues. We propose that in heterozygous females, patchwork loss of ephrin-B1 disturbs tissue boundary formation at the developing coronal suture, whereas in males deficient in ephrin-B1, an alternative mechanism maintains the normal boundary. This is the only known mutation in the ephrin/Eph receptor signaling system in humans and provides clues to the biogenesis of craniosynostosis.