Eleven Cases of Hb J-Paris-I [HBA2: c.38C>A (or HBA1)]: A Stable α Chain Variant Elutes in the P3 Window on High-Performance Liquid Chromatography.

Hb J-Paris-I [HBA2: c.38C>A (or HBA1)] is a stable fast-moving hemoglobin (Hb) that elutes in the P3 window on high performance liquid chromatography (HPLC). The mutation can happen on either the α1- or α2-globin gene. Codon 12 changes from GCC to GAC to replace the alanine amino acid with aspartic acid. This change is external with no clinical significance. The elution in the P3 wave on HPLC can interfere with the glycated Hb assay by HPLC. In this study, data of 11 cases of Hb J-Paris-I were thoroughly presented. The majority of the cases were of Indian ethnicity. The mean value of Hb J-Paris-I on HPLC was 26.7 ± 2.0%. The retention time (RT) was 1.75 ± 0.03 min. The isoelectric focusing (IEF) mean value was -5.6 (range -6.1 to -4.9). Hb A2 was consistently reduced to 1.8 ± 0.3%. A fraction of 0.8% corresponding to the Hb A2-J-Paris-I (α2J-Paris-Iδ2) is likely to be concealed within the A0 peak of Hb A on HPLC. Interestingly, two cases were associated with two different polymorphisms [HBA2: c.-24C>G or Cap +14 (C>G) and HBA2: c.*136A>G polymorphism] without apparent effect on the variant expression.

Eight Cases of Hb Winnipeg [HBA2: c.226G>T (or HBA1)]: A Detailed Study.

Hb Winnipeg [α75(EF4)Asp→Tyr (α2); HBA2: c.226G>T (or HBA1)] is a stable α-globin chain variant described in a few articles. The majority of reported cases in older articles were clustered in Canada. It can occur on both α1- and α2-globin genes and in different populations. In this study, eight cases of Hb Winnipeg were characterized by DNA sequencing during a wide-spectrum study of suspected α-globin gene variants collected in the United Kingdom. All cases detected peaked in the S window between 4.4 and 4.54 min. on high performance liquid chromatography (HPLC). The isoelectric focusing (IEF) averaged at 6.21 below Hb A. All the mutations were detected on the α1-globin gene except in one case. The ethnic origin of the majority of the patients was Canadian. Only one case was associated with the common polymorphism HBA2: c.-24C>G (or HBA1) [Cap +14 (C>G)] on both α-globin genes without any apparent effect on the variant expression. All cases were detected in a heterozygous state. Hb Winnipeg expression was consistently lower than the theoretical value for α chain variants, ranging between 11.8 and 15.8% of total hemoglobin (Hb). This study gave more details about Hb Winnipeg that may help in presumptive diagnosis, especially in routine laboratories.

Hb Calgary (HBB: c.194G>T): A Highly Unstable Hemoglobin Variant with a ß-Thalassemia Major Phenotype.

We describe two unrelated patients, both heterozygous for an unstable hemoglobin (Hb) variant named Hb Calgary (HBB: c.194G>T) that causes severe hemolytic anemia and dyserythorpoietic, resulting in transfusion dependence and iron overload. The molecular pathogenesis is a missense variation on the ß-globin gene, presumed to lead to an unstable Hb. The phenotype of Hb Calgary is particularly severe presenting as transfusion-dependent anemia in early infancy, precluding phenotypic diagnosis and highlighting the importance of early genetic testing in order to make an accurate diagnosis.

Fifteen Cases of Hb J-Meerut: The Rare Association with Hb E and/or HBA1: c.-24C>G (or HBA2) Variants.

Hb J-Meerut [HBA2: c.362C>A (or HBA1)] is a rare, stable, nonpathogenic α-globin gene variant that peaks in the area between the P3 and A0 windows on high performance liquid chromatography (HPLC). Few cases from different ethnic origins have been published but the majority were Asian Indians. Coinheritance with other hemoglobin (Hb) variants are rarer and can change the Hb J-Meerut phenotype making a diagnostic dilemma. In this study, we have reported 15 cases of Hb J-Meerut, discovered during a wide spectrum study of α-globin chain variants in the UK. The diagnosis was confirmed by forward and reverse DNA sequencing of the α1- and α2-globin genes. The average of the Hb J-Meerut expression was 20.9% of total Hb and characterized by a retention time (RT) of 1.9 min. (on average) on HPLC. The median of isoelectric focusing (IEF) was 5.6 mm above Hb A. Among the 15 cases studied, one case coinherited the Hb E (HBB: c.79G>A) mutation in heterozygosity and another case was associated with the Cap +14 (C>G) [HBA1: c.-24C>G (or HBA2)] variant. We noticed that the coinheritance of the Hb E mutation reduced the Hb J-Meerut expression with the formation of a hybrid peak missed on the HPLC chromatograph. We also noticed an increased expression of Hb J-Meerut in the case showing the coinheritance of the HBA2: c.-24C>G (or HBA1) variant.

Twelve Cases of Hb Manitoba [α102(G9)Ser→Arg]: the Fluctuation in the Variant Expression.

Hb Manitoba [α102(G9)Ser→Arg] is a rare α chain variant with diverse ethnic origins. It is mildly unstable with an expression of around 10.0-14.2% in the heterozygous state in most literature. In this study, 12 cases of Hb Manitoba [11 cases carried Hb Manitoba II (HBA1: c.309C>A) and one case carried Hb Manitoba IV (HBA1: c.307A>C)] were detected during a wide-spectrum study of α chain variants in the UK. Fluctuation in variant expression from 6.9 to 15.2% of total Hb on high performance liquid chromatography (HPLC) would pose a diagnostic dilemma in routine laboratories. Focusing on the variant expression, the median of Hb Manitoba was around 11.5% of total Hb in three cases, apparently with normal hemoglobin (Hb), and normal red blood cell (RBC) indices. Two cases showed a higher expression (13.9 and 15.2%) and five cases showed a lower expression (6.9-9.9%). The common α-thalassemia (α-thal) -α3.7 (rightward) deletion coexisted with one case of increased Hb Manitoba expression. Iron (or other nutrient) deficiency was likely the cause of decreased Hb Manitoba percentage in this study. The α73(EF2)Val→Val (α2) (HBA2: c.222G>T) polymorphism is published for the first time and coexisted with two cases. The Cap +14 (C>G) (HBA2: c.-24C>G) polymorphism coexisted with another case in a heterozygous state. In conclusion, the fluctuation in variant expression can cause a diagnostic dilemma, especially in routine laboratories. Screening for the common -α3.7 deletion and iron deficiency is recommended when an α chain variant is suspected.

A Wide Spectrum Study of α-Globin Chain Variants: Cases from the UK.

Over many years, cases of suspected α-globin chain variants were collected from different parts of the UK. The suspicion was based on the clinical picture, high performance liquid chromatography (HPLC) variant percentage, retention time (RT) and isoelectric focusing (IEF). DNA sequencing and the restriction enzyme EaeI were used for definitive diagnosis. One hundred and forty-eight variants were confirmed on one or both of the two α-globin genes (HBA2, HBA1). These cases were identified as 46 different α-globin chain variants. The most common variants were Hb J-Meerut [HBA2: c.362C>A (or HBA1)] (10.1%) and Hb Q-India (HBA1: c.193G>C) (8.1%), followed by Hb J-Paris-I [HBA2: c.38C>A (or HBA1)] and Hb Manitoba II (HBA1: c.309C>A) (7.4% for each). Other α variants were detected at lower frequencies. Two novel alleles were also detected: Hb Walsgrave [α116(GH4)Glu→Val (HBA2: c.350A>T)] and Hb Coombe Park [α127(H10)Lys→Glu (HBA2: c.382A>G)]. The majority of the ethnic origin was Indian. The positive predictive value for α variant identification by HPLC-RT analysis was 65.9%, 41.9% by IEF, and using both RT and IEF, the value was 72.1%. The number of variants was higher in HBA1 than in HBA2 genes and in exons 1 and 2 than in exon 3. There was no clustering of mutations in consecutive codons. This study, the characterization of a wide spectrum of α-globin chain variants, can facilitate the presumptive diagnosis of these variants prior to screening by a panel of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a definitive diagnosis by DNA sequencing.

Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project.

PURPOSE: Fresh-frozen (FF) tissue is the optimal source of DNA for whole-genome sequencing (WGS) of cancer patients. However, it is not always available, limiting the widespread application of WGS in clinical practice. We explored the viability of using formalin-fixed, paraffin-embedded (FFPE) tissues, available routinely for cancer patients, as a source of DNA for clinical WGS. METHODS: We conducted a prospective study using DNAs from matched FF, FFPE, and peripheral blood germ-line specimens collected from 52 cancer patients (156 samples) following routine diagnostic protocols. We compared somatic variants detected in FFPE and matching FF samples. RESULTS: We found the single-nucleotide variant agreement reached 71% across the genome and somatic copy-number alterations (CNAs) detection from FFPE samples was suboptimal (0.44 median correlation with FF) due to nonuniform coverage. CNA detection was improved significantly with lower reverse crosslinking temperature in FFPE DNA extraction (80 °C or 65 °C depending on the methods). Our final data showed somatic variant detection from FFPE for clinical decision making is possible. We detected 98% of clinically actionable variants (including 30/31 CNAs). CONCLUSION: We present the first prospective WGS study of cancer patients using FFPE specimens collected in a routine clinical environment proving WGS can be applied in the clinic.

Clinical applicability and cost of a 46-gene panel for genomic analysis of solid tumours: Retrospective validation and prospective audit in the UK National Health Service.

BACKGROUND: Single gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing. METHODS AND FINDINGS: We validated a 46-gene hotspot cancer panel assay allowing multiple gene testing from small diagnostic biopsies. From 1 January 2013 to 31 December 2013, solid tumour samples (including non-small-cell lung carcinoma [NSCLC], colorectal carcinoma, and melanoma) were sequenced in the context of the UK National Health Service from 351 consecutively submitted prospective cases for which treating clinicians thought the patient had potential to benefit from more extensive genetic analysis. Following histological assessment, tumour-rich regions of formalin-fixed paraffin-embedded (FFPE) sections underwent macrodissection, DNA extraction, NGS, and analysis using a pipeline centred on Torrent Suite software. With a median turnaround time of seven working days, an integrated clinical report was produced indicating the variants detected, including those with potential diagnostic, prognostic, therapeutic, or clinical trial entry implications. Accompanying phenotypic data were collected, and a detailed cost analysis of the panel compared with single gene testing was undertaken to assess affordability for routine patient care. Panel sequencing was successful for 97% (342/351) of tumour samples in the prospective cohort and showed 100% concordance with known mutations (detected using cobas assays). At least one mutation was identified in 87% (296/342) of tumours. A locally actionable mutation (i.e., available targeted treatment or clinical trial) was identified in 122/351 patients (35%). Forty patients received targeted treatment, in 22/40 (55%) cases solely due to use of the panel. Examination of published data on the potential efficacy of targeted therapies showed theoretically actionable mutations (i.e., mutations for which targeted treatment was potentially appropriate) in 66% (71/107) and 39% (41/105) of melanoma and NSCLC patients, respectively. At a cost of £339 (US$449) per patient, the panel was less expensive locally than performing more than two or three single gene tests. Study limitations include the use of FFPE samples, which do not always provide high-quality DNA, and the use of "real world" data: submission of cases for sequencing did not always follow clinical guidelines, meaning that when mutations were detected, patients were not always eligible for targeted treatments on clinical grounds. CONCLUSIONS: This study demonstrates that more extensive tumour sequencing can identify mutations that could improve clinical decision-making in routine cancer care, potentially improving patient outcomes, at an affordable level for healthcare providers.

A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.

Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next-generation-sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical-grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically-reliable diagnostic test and minimize false-negative results we developed an open-source tool (CoverMi) to accurately determine base-coverage and the 'discoverability' of known mutations for every sample. We validated our 33-gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS-based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.

Ten Years of Routine α- and ß-Globin Gene Sequencing in UK Hemoglobinopathy Referrals Reveals 60 Novel Mutations.

We review and report here the genotypes and phenotypes of 60 novel thalassemia and abnormal hemoglobin (Hb) mutations discovered following the adoption of routine DNA sequencing of both α- and ß-globin genes for all UK hemoglobinopathy samples referred for molecular investigation. This screening strategy over the last 10 years has revealed a total of 11 new ß chain variants, 15 α chain variants, 19 ß-thalassemia (ß-thal) mutations and 15 α(+)-thalassemia (α(+)-thal) mutations. The large number of new thalassemia alleles confirms the wide racial heterogeneity of mutations in the UK immigrant population. Eleven of the new variants ran with Hb A on high performance liquid chromatography (HPLC), demonstrating the value of routine sequencing of both α- and ß-globin genes for all hemoglobinopathy investigations. The new ß chain variants are: Hb Bury [ß22(B4)Glu → Asp (HBB: c.69A > T)], Hb Fulwood [ß35(C1)Tyr → His (HBB: c.106T > C)], Hb Little Venice [ß42(CD1)Phe → Cys (HBB: c.128T > G)], Hb Cork [ß57(E1)Asn → Ser (HBB: c.173A > G), Hb Basingstoke [ß118(GH1)Phe → Ser (HBB: c.356T > C)], Hb Howden [ß20(B2)Val → Ala (HBB: c.62T > C)], Hb Wilton [ß41(C7)Phe → Leu (HBB: c.126C > A)], Hb Belsize Park [ß120(GH3)Lys → Asn (HBB: c.363A > T)], Hb Hampstead Heath [ß2(NA2)His → Gln;ß26(B8)Glu → Lys (HBB: c.[6C > G;79G > A])], Hb Grantham [ß85(F1)Phe → Cys (HBB: c.257T > G)] and Hb Calgary [ß64(E8)Gly → Val (HBB: c.194G > T). The new α chain variants are: Hb Edinburgh [α70(E19)Val → Gly (HBA2: c.212T > G)], Hb Walsgrave [α116(GH4)Glu → Val (HBA2: c.350A > T)], Hb Wexham [α117(GH5) and 118(H1) insertion Ser (HBA1: c.354-355insTCA)], Hb Coombe Park [α127(H10)Lys → Glu (HBA2: c.382A > G)], Hb Oxford [α17(A15)Val → Asp (HBA2: c.53T > A)], Hb Bridlington [α32(B13)Met → Thr (HBA1: c.98T > C), Hb Wolverhampton [α81(F2)Ser → Tyr (HBA2: c.9245C > A)], Hb Little Waltham [α13(A11)Ala → Asp (HBA2: c.41C > A)], Hb Derby [α61(E10)Lys → Arg (HBA1: c.185A > G)], Hb Uttoxter [α74(EF3)Tyr → Asp (HBA2: c.223G > T)], Hb Harehills [α124(H7)Ser → Cys (HBA1: c.374C > G)], Hb Hekinan II [α27(B8)Glu → Asp (HBA1: c.84G > T)], Hb Manitoba IV [α102(G9)Ser → Arg (HBA1: c.307A > C), Hb Witham [α139(HC1)Lys → Arg (HBA2: c.419A > G) and Hb Farnborough [α9(A7)Asn → Asp (HBA1: c.28A > G). In addition, 10 more paralogous α-globin chain variants have been discovered. The novel ß-thal alleles are: HBB: c.-138C > G, HBB: c.-121C > T, HBB: c.-80T > G, HBB: c.18_19delTG, HBB: c.219_220insT, HBB: c.315 + 2_315 + 13delTGAGTCTATGGG, HBB: c.316-70C > G, HBB: c.345_346insTGTGCTG, HBB: c.354delC, HBB: c.376-381delCCAGTG, HBB: c.393T > A, HBB: c.394_395insA, HBB: c.375_376insA, HBB: c.*+95_*+107delTGGATTCTinsC, HBB: c.* + 111_*+112delAA, HBB: c.*+112A > T, HBB: c.394C > T, HBB: c.271delG and HBB: c.316-3C > T. The novel α (+ )-thal alleles are: HBA1: c.95+1G > C, HBA1: c.315C > G [Hb Donnington, α104(G11)Cys → Trp], HBA1: c.327delC, HBA1: c.333_345del, HBA1: c.*+96G > A, HBA2: c.2T > G, HBA2: c.112delC, HBA2: c.143delA, HBA2: c.143_146delACCT, HBA2: c.156_157insG, HBA2: c.220_223delGTGG, HBA2: c.305T > C [Hb Bishopstown, α101(G8)Leu → His], HBA2: c.169_170delAA, HBA2: c.1A > T and HBA2: c.-3delA.

Impact of isolated germline JAK2V617I mutation on human hematopoiesis.

The association between somatic JAK2 mutation and myeloproliferative neoplasms (MPNs) is now well established. However, because JAK2 mutations are associated with heterogeneous clinical phenotypes and often occur as secondary genetic events, some aspects of JAK2 mutation biology remain to be understood. We recently described a germline JAK2V617I mutation in a family with hereditary thrombocytosis and herein characterize the hematopoietic and signaling impact of JAK2V617I. Through targeted sequencing of MPN-associated mutations, exome sequencing, and clonality analysis, we demonstrate that JAK2V617I is likely to be the sole driver mutation in JAK2V617I-positive individuals with thrombocytosis. Phenotypic hematopoietic stem cells (HSCs) were increased in the blood and bone marrow of JAK2V617I-positive individuals and were sustained at higher levels than controls after xenotransplantation. In signaling and transcriptional assays, JAK2V617I demonstrated more activity than wild-type JAK2 but substantially less than JAK2V617F. After cytokine stimulation, JAK2V617I resulted in markedly increased downstream signaling compared with wild-type JAK2 and comparable with JAK2V617F. These findings demonstrate that JAK2V617I induces sufficient cytokine hyperresponsiveness in the absence of other molecular events to induce a homogeneous MPN-like phenotype. We also provide evidence that the JAK2V617I mutation may expand the HSC pool, providing insights into both JAK2 mutation biology and MPN disease pathogenesis.

Characterization of Hb Lepore variants in the UK population.

A molecular study of Hb Lepore heterozygotes identified by the UK population screening program has revealed four out of the five known Lepore variants. The region of homologous δ- and ß-globin gene sequence was determined in 58 unrelated Hb Lepore heterozygotes referred for confirmation of their carrier status by DNA analysis through the national thalassemia and sickle cell screening program over a period of 10 years. The most common variant found was Hb Lepore-Boston-Washington (Hb LBW, HBD: c.265 C > c.315 + 7 C) observed in 46 carriers (79.0%). Hb Lepore-Hollandia (HBD: c.69 A > c.92 + 16 A) was found in nine cases (16.0%); Hb Lepore-Baltimore (HBD: c.208 G > c.254 C) in two cases (4.0%) and Hb Lepore-ARUP (HBD: c.97 C > c.150 C) in one carrier (2.0%). Analysis of the hematological findings showed no significant differences between the four groups. The wide range of Hb Lepore variants observed in this study confirms the very diverse range of α- and ß-globin gene mutations observed in the UK population by previous studies.

Monitoring chronic lymphocytic leukemia progression by whole genome sequencing reveals heterogeneous clonal evolution patterns.

Chronic lymphocytic leukemia is characterized by relapse after treatment and chemotherapy resistance. Similarly, in other malignancies leukemia cells accumulate mutations during growth, forming heterogeneous cell populations that are subject to Darwinian selection and may respond differentially to treatment. There is therefore a clinical need to monitor changes in the subclonal composition of cancers during disease progression. Here, we use whole-genome sequencing to track subclonal heterogeneity in 3 chronic lymphocytic leukemia patients subjected to repeated cycles of therapy. We reveal different somatic mutation profiles in each patient and use these to establish probable hierarchical patterns of subclonal evolution, to identify subclones that decline or expand over time, and to detect founder mutations. We show that clonal evolution patterns are heterogeneous in individual patients. We conclude that genome sequencing is a powerful and sensitive approach to monitor disease progression repeatedly at the molecular level. If applied to future clinical trials, this approach might eventually influence treatment strategies as a tool to individualize and direct cancer treatment.

A study of δ-globin gene mutations in the UK population: identification of three novel variants and development of a novel DNA test for Hb A'2.

We report here the spectrum of δ-globin gene mutations found in the UK population. Nine different δ chain variants and two δ-thalassemia (δ-thal) mutations were characterized in a study of 127 alleles in patients with either a low Hb A2 value or a split Hb A2 peak on high performance liquid chromatography (HPLC). The most common δ chain variant was Hb [Formula: see text] (or Hb B2) [δ16(A13)Gly → Arg; HBD: c.49G > C] (77.0%), followed by Hb A2-Yialousa [δ27(B9)Ala → Ser; HBD: c.82G > T] (12.0%), Hb A2-Babinga [δ136(H14)Gly → Asp; HBD: c.410G > A] (3.0%), Hb A2-Troodos [δ116(G18)Arg → Cys; HBD: c.349C > T] (1.0%), Hb A2-Coburg [δ116(G18)Arg → His; HBD: c.350G > A] (2.0%) and Hb A2-Indonesia [δ69(E13)Gly → Arg; HBD: c.208G > C] (1.0%). Three novel variants were identified: Hb A2-Calderdale [codon 2 (CAT > AAT), His → Asn; HBD: c.7C > A], Hb A2-Walsgrave [codon 52 (GAT > CAT), Asp → His; HBD: c.157G > C] and Hb A2-St. George's [codon 81 (CTC > TTC), Leu → Phe; HBD: c.244C > T]. In addition, two known δ-thal mutations were observed: -68 (C > T); HBD: c.-118C > T and codon 4 (ACT > ATT); HBD: c.14C > T. Amplification refractory mutation system (ARMS) primers were developed to provide a simple molecular diagnostic test for the most common variant, Hb [Formula: see text]. Three of the variants had a characteristic HPLC retention time that can be used for a presumptive diagnosis.

Insights for precision oncology from the integration of genomic and clinical data of 13,880 tumors from the 100,000 Genomes Cancer Programme.

The Cancer Programme of the 100,000 Genomes Project was an initiative to provide whole-genome sequencing (WGS) for patients with cancer, evaluating opportunities for precision cancer care within the UK National Healthcare System (NHS). Genomics England, alongside NHS England, analyzed WGS data from 13,880 solid tumors spanning 33 cancer types, integrating genomic data with real-world treatment and outcome data, within a secure Research Environment. Incidence of somatic mutations in genes recommended for standard-of-care testing varied across cancer types. For instance, in glioblastoma multiforme, small variants were present in 94% of cases and copy number aberrations in at least one gene in 58% of cases, while sarcoma demonstrated the highest occurrence of actionable structural variants (13%). Homologous recombination deficiency was identified in 40% of high-grade serous ovarian cancer cases with 30% linked to pathogenic germline variants, highlighting the value of combined somatic and germline analysis. The linkage of WGS and longitudinal life course clinical data allowed the assessment of treatment outcomes for patients stratified according to pangenomic markers. Our findings demonstrate the utility of linking genomic and real-world clinical data to enable survival analysis to identify cancer genes that affect prognosis and advance our understanding of how cancer genomics impacts patient outcomes.

Ten novel mutations in the erythroid transcription factor KLF1 gene associated with increased fetal hemoglobin levels in adults.

We investigated whether mutations in the KLF1 gene are associated with increased Hb F levels in ethnically diverse patients referred to our laboratory for hemoglobinopathy investigation. Functionally effective KLF1 mutations were identified in 11 out of 131 adult samples with an elevated Hb F level (1.5-25.0%). Eleven different mutations were identified, 9 of which were previously unreported. KLF1 mutations were not identified in a matched cohort of 121 samples with normal Hb F levels (<1.0%). A further novel KLF1 mutation was also found in a sickle cell disease patient with a Hb F level of 20.3% who had a particularly mild phenotype. Our results indicate KLF1 mutations could make a significant contribution to Hb F variance in malarial regions where hemogobinopathies are common. All the mutations identified were heterozygous providing further in vivo evidence that a single altered KLF1 allele is sufficient to increase Hb F levels.

Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition.

It is well established that all of the cis-acting sequences required for fully regulated human alpha-globin expression are contained within a region of approximately 120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the alpha-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the alpha-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of alpha-globin genes, affects the pattern of NME4 expression by altering the competition for the shared alpha-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.

Prenatal diagnosis of hemoglobinopathies by pyrosequencing: a more sensitive and rapid approach to fetal genotyping.

Prenatal diagnosis of the hemoglobinopathies by fetal DNA analysis is currently performed in most countries, either by DNA sequencing, restriction enzyme polymerase chain reaction (RE-PCR) or the amplification refractory mutation system (ARMS). These methods are time consuming and prolong the turnaround time for diagnosis. We here describe a method utilizing pyrosequencing for the prenatal diagnosis of 12 common nondeletional α- and ß-globin gene mutations in the UK population. In particular, it replaced the diagnosis of sickle cell disease by RE-PCR and for the diagnosis of ß-thalassemia (ß-thal) by Sanger DNA sequencing. We have genotyped 148 chorionic villi and 29 uncultured amniotic fluid DNA samples by pyrosequencing and found 100% concordance with the fetal diagnosis result obtained by ARMS-PCR or DNA sequencing. Pyrosequencing was more robust, revealing an 83% decrease in diagnostic failures using uncultured amniocyte DNA samples, and also quantitative, revealing one case of allelic imbalance due to maternal DNA contamination. Overall, we found pyrosequencing to be simpler, more robust, quicker, and less expensive than conventional sequencing and RE-PCR, making it a good choice for rapid and cost-effective prenatal diagnosis of thalassemia and sickle cell disease.