Barnaby Clark

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.

Mutations in TJP2 cause progressive cholestatic liver disease

Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that protein-truncating mutations in the tight junction protein 2 gene (TJP2) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species.

Genome annotation of a 1.5 Mb region of human chromosome 6q23 encompassing a quantitative trait locus for fetal hemoglobin expression in adults

BackgroundHeterocellular hereditary persistence of fetal hemoglobin (HPFH) is a common multifactorial trait characterized by a modest increase of fetal hemoglobin levels in adults. We previously localized a Quantitative Trait Locus for HPFH in an extensive Asian-Indian kindred to chromosome 6q23. As part of the strategy of positional cloning and a means towards identification of the specific genetic alteration in this family, a thorough annotation of the candidate interval based on a strategy of in silico / wet biology approach with comparative genomics was conducted.ResultsThe ~1.5 Mb candidate region was shown to contain five protein-coding genes. We discovered a very large uncharacterized gene containing WD40 and SH3 domains (AHI1), and extended the annotation of four previously characterized genes (MYB, ALDH8A1, HBS1L and PDE7B). We also identified several genes that do not appear to be protein coding, and generated 17 kb of novel transcript sequence data from re-sequencing 97 EST clones.ConclusionDetailed and thorough annotation of this 1.5 Mb interval in 6q confirms a high level of aberrant transcripts in testicular tissue. The candidate interval was shown to exhibit an extraordinary level of alternate splicing – 19 transcripts were identified for the 5 protein coding genes, but it appears that a significant portion (14/19) of these alternate transcripts did not have an open reading frame, hence their functional role is questionable. These transcripts may result from aberrant rather than regulated splicing.

Novel NFKB2 mutation in early-onset CVID.

Common variable immunodeficiency (CVID) is heterogeneous, clinically, immunologically and genetically. The majority of genetic mechanisms leading to CVID remain elusive. We studied a Greek Cypriot family of non-consanguineous parents. Two children were diagnosed with CVID at an early age. Whole exome sequencing revealed 8bp deletion in the C-terminal part of NFKB2 gene associated with disease. The mutation leads to a frameshift (p.Asp865Valfs*17) altering 17 C-terminal amino acids from residue 865, and creating a premature stop-codon resulting in a truncated protein, 19 amino acids shorter than wild type (p100Δ19). We validated the results with Dye-termination sequencing and Western blot, and confirmed that the conserved residue at 866 is mutated from serine to arginine in p100Δ19, leaving the mutant protein unphosphorylated at this critical regulatory position. Consequently, NFKB2/p100 processing and nuclear translocation were abrogated. Using flow cytometry, we further demonstrated that there was a reduction in B cells (CD19+), switched memory B cells (CD27+IgD−) and T follicular helper (Tfh) cells (both CD4+CXCR5+ and CD4+CXCR5Hi) in a CVID patient with NFKB2/p100Δ19, compared to healthy controls. These data support the notion that the non-canonical NFκB pathway plays an important role in B cell differentiation and the development of Tfh cells, and may pave the way for better understanding of the pathology of CVID.

A novel 506kb deletion causing εγδβ thalassemia.

We describe a novel deletion causing εγδβ thalassemia in a Pakistani family. The Pakistani deletion is 506kb in length, and the second largest εγδβ thalassemia deletion reported to date. It removes the entire β globin gene (HBB) cluster, extending from 431kb upstream to 75kb downstream of the ε globin gene (HBE). The breakpoint junction occurred within a 160bp palindrome embedded in LINE/LTR repeats, and contained a short (9bp) region of direct homology which may have contributed to the recombination event. Characterization of the deletion breakpoints has been particularly challenging due to the complexity of DNA deletion, insertion and inversion, involving a multitude of methodologies, mirroring the changing DNA analysis technologies.

Next generation sequencing identifies a novel rearrangement in the HBB cluster permitting to-the-base characterization

Genetic testing for hemoglobinopathies is required for prenatal diagnosis, understanding complex cases where multiple pathogenic variants may be present or investigating cases of unexplained anemia. Characterization of disease causing variants that range from single base changes to large rearrangements may require several different labor‐intensive methodologies. Multiplex ligation probe amplification analysis is the current method used to detect indels, but the technique does not characterize the breakpoints or detect balanced translocations. Here, we describe a next‐generation sequencing (NGS) method that is able to identify and characterize a novel rearrangement of the HBB cluster responsible for εγδβ thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassing HBG2 exon 3, HBG1, HBD, HBB, and OR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence, HBG2 exon 1 and 2, HBE, and the β‐locus control region. Identification of reads spanning the breakpoints provided to‐the‐base resolution of the rearrangement, subsequently confirmed by gap‐PCR and Sanger sequence analysis. The same rearrangement, termed Inv‐Del English V εγδβ thalassemia (HbVar 2935), was identified in two other unrelated English individuals with a similar hematological phenotype. Our NGS approach should be applicable as a diagnostic tool for other disorders.

Beta thalassaemia intermedia due to co-inheritance of three unique alpha globin cluster duplications characterised by next generation sequencing analysis

Co-inheritance of a thalassaemia reduces chain imbalance and disease severity in b thalassaemia homozygotes, while increasing a globin output in heterozygotes increases chain imbalance, converting a typically asymptomatic carrier state to that of thalassaemia intermedia. The outcome depends on the number of a globin genes inherited as one or two copies of triplicated (/aaa), or quadruplicated (/aaaa) a globin complexes, and the type of b thalassaemia mutation (b or b) (Thein, 2008). Another mechanism of increasing a globin output is through segmental duplication of the whole a globin complex (Harteveld et al, 2008) but breakpoints of the reported duplications have not been fully characterised due to technological limitations. Here, we applied a previously described next generation sequencing (NGS) methodology (Shooter et al, 2015a,b) to characterise three a globin cluster duplications, permitting to-the-base resolution in two of the three cases. Patient samples were referred for work-up of unusually severe phenotype in b thalassaemia carriers. In Family 1 (Fig 1A), the proband was a 54-year-old Chinese male with hypochromic microcytic anaemia since infancy. His partner (Anglo-Saxon English) and two older sons had normal haematological profiles; the youngest son (aged 14 years) had a haematological profile similar to that of the father. Both father and son had

Haemoglobin Variant Screening in Jamaica: Meeting Student's Request

The Jamaican Cohort Study, established by the screening of 100 000 consecutive, non-operative deliveries at the main Government maternity hospital, Kingston, Jamaica between 1973 and 1981, provided the frequency of major haemoglobin variants in that population (Serjeant et al, 1986). However, newborn samples are not optimal for the diagnosis of beta thalassaemia or hereditary persistence of fetal haemoglobin and these had to be implied by extrapolation from other genotypes. Following a recent education programme on sickle cell disease to senior students of secondary schools across the island, there was demand from the students to learn their haemoglobin genotypes. In conjunction with the Jamaican Ministry of Health and Ministry of Education, the Parish of Manchester in south central Jamaica was selected for a pilot programme which targeted the 5th and 6th forms (mostly aged 15–19 years), screening 16 636 students of 15 secondary schools in the 6 academic years from 2007/8. It was assumed that greater precision would be available in the laboratory 40 years later and would allow comparison of gene frequency with the earlier study; compliance would test the interests of students in learning their haemoglobin genotypes, and, most important of all, would provide an informed cohort where the effects of genotype knowledge could be assessed on subsequent reproductive decisions. After discussions with the school staff, Parent–Teachers Associations, and illustrated lectures on sickle cell disease and its genetics, all students were given letters for their parents outlining the objectives and the option of declining for their child to be tested. At pre-arranged times, a physician, clerical assistants and 3–4 phlebotomists visited the school, the students completed a data form providing personal details and one 5 ml EDTA sample was taken by venepuncture. The turn-round time for individuals was about 20 min; 150–250 students could be screened within 2–21⁄2 h, with most schools requiring 2–5 visits. Permanent laminated cards with haemoglobin genotype were given to all students, and counselling was offered to carriers of haemoglobin variants. Compliance with screening increased over the 6 years, from 56% to 92%, testimony to the cooperation of the teaching staff and the motivation of students. Haemoglobin bands in the position of HbS were confirmed by the slide sickling test and in the position of HbC by agar gel electrophoresis at pH 6 2. Samples with mean cell haemoglobin ≤ 26 pg and red blood cell distribution width (coefficient of variation) [RDW(CV)] < 18 0 were candidates for the beta thalassaemia trait and had estimations of HbA2 performed, which if ≥3 5% underwent direct sequencing of polymerase chain reaction-amplified b-globin DNA (Vetter et al, 1997). Samples with >8% HbF, measured by high performance liquid chromatography (HPLC), were investigated by multiplex ligation-dependent probe amplification (MLPA, MRC-Holland, Amsterdam, The Netherlands) to identify deletional hereditary persistence of fetal haemoglobin (HPFH) or by DNA sequencing of gamma globin promoter regions to identify non-deletional HPFH at the Red Cell Centre, Kings College Hospital, London. Samples with negative confirmatory tests for HbS and HbC were investigated by genomic sequence analysis of HBB, HBA1 and HBA2 (Thein & Hinton, 1991; Viprakasit et al, 2002) at the Weatherall Institute of Molecular Medicine, Oxford, UK and identified using the syllabus of haemoglobin variants (http://globin.cse.psu.edu). Haemoglobin genotypes were similar to those in the Jamaican Cohort Study (Table I), which identified SS disease in every 300 births and SC disease in every 500 births,

First reported duplication of the entire beta globin gene cluster causing an unusual sickle cell trait phenotype

effect of T12 on lymphocytosis pattern is dominant; this is consistent with the prior observation that up-regulation of integrin signalling in T12 is not modulated by additional del (11q) or del(17p) (Riches et al, 2014). We did not have confirmatory integrin expression data. In contrast to the effect on lymphocytosis pattern, the presence of T12 in addition to del (17p) did not attenuate the adverse impact of del(17p) on outcomes. Further studies are required to ascertain the mechanisms underlying the abbreviated lymphocytosis in T12 CLL and its relationship, if any, to therapeutic efficacy.

Next-generation sequencing as a tool for breakpoint analysis in rearrangements of the globin gene clusters

Next‐generation sequencing (NGS), now embedded within genomic laboratories, is well suited to the detection of small sequence changes but is less well adapt for detecting structural variants (SV), mainly due to the relatively short sequence reads. Of the available target enrichment methods, bait capture or whole‐genome sequencing appears better suited to detecting SV as there is less PCR amplification and is therefore more representative of the genome being sequenced.