Newborn Screening for ß-Thalassemia Identifies a Complex Genotype Involving a Novel ß-Globin Gene Mutation (HBB:c.336dup).

Newborn screening identified a Chinese-Canadian infant who was positive for possible ß-thalassemia (ß-thal). Detailed family studies demonstrated that the proband was a compound heterozygote for the Chinese Gγ(Aγδß)0-thal deletion and a novel frameshift mutation within exon 3 (HBB:c.336dup), and heterozygous for the Southeast Asian α-thal deletion (--SEA/αα). This case illustrates the importance of follow-up molecular testing of positive newborn screening results to confirm the diagnosis and define risks for future pregnancies.

ß0-Thalassemia Caused by a Novel Nonsense Mutation [HBB:c.199A > T].

We report two hemoglobinopathy cases involving a novel ß-thalassemia (ß-thal) nonsense mutation, HBB:c.199A > T. One patient had Hb S/ß-thal, and a second unrelated patient had Hb D-Punjab/ß-thal. The HBB:c.199A > T mutation introduces a premature termination codon at amino acid codon 66 (AAA→TAA) in exon 2, resulting in typical high Hb A2 ß0-thal.

Splice Acceptor Mutation [HBB:c.93-2A > T] in a Patient with Hb S/ß0-Thalassemia.

We report a case of Hb S/ß0-thalassemia (Hb S/ß0-thal) in a patient who is a compound heterozygote for the Hb Sickle mutation (HBB:c.20A > T) and a mutation of the canonical splice acceptor sequence of IVS1 (AG > TG, HBB:c.93-2A > T). This is the fifth mutation involving the AG splice acceptor site of IVS1, all of which prevent normal splicing and cause ß0-thal.

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. Herein, we describe our experience as a ClinGen Variant Curation Expert Panel to adapt the ACMG/AMP criteria for the classification of variants in three globin genes (HBB, HBA2, and HBA1) related to recessively inherited hemoglobinopathies, including five evidence categories, as use cases demonstrating the process of specification and the underlying rationale.

Outcomes of haemoglobin Bart's hydrops fetalis following intrauterine transfusion in Ontario, Canada.

OBJECTIVES: With improved access to intrauterine transfusion (IUT), more fetuses with haemoglobin Bart's hydrops fetalis (HBHF; homozygous α0-thalassaemia) will survive. DESIGN: To evaluate the long-term outcome of affected fetuses with and without IUT in Ontario, Canada, we retrospectively collected data on IUTs and pregnancy outcomes in all cases of HBHF, from 1989 to 2014. Clinical outcome and neurocognitive profiles of long-term survivors were also collected and compared with data from 24 patients with transfusion-dependent ß-thalassaemia (TDT-ß). RESULTS: Of the 99 affected pregnancies (93 prenatally diagnosed), 68 resulted in miscarriage or elective termination of pregnancy. Twelve mothers (12%) continued their pregnancies without IUT, and none of those newborns survived the first week of life. All 13 fetuses that received IUT(s) were live-born, but 3 died due to severe hydrops at birth and 1 died due to infection. The remaining nine survivors, in comparison with TDT-ß patients, had earlier iron overload requiring iron chelation therapy. Endocrinopathies and short stature were more frequent in these patients. Neurocognitive outcome was not significantly affected in five patients who were assessed, and none were diagnosed with intellectual impairment. In three patients, MRI studies demonstrated brain white matter changes in keeping with 'silent' ischaemic infarcts. CONCLUSIONS: In patients with HBHF, IUT is associated with improved survival. While acceptable neurocognitive outcome can be expected, these patients have more clinical complications compared with their TDT-ß counterparts. The clinical and neurocognitive outcomes of HBHF should be discussed in detail when counselling and offering IUT for patients.

Novel High Oxygen Affinity Hemoglobin Variant in a Patient with Polycythemia: Hb Kennisis [ß85(F1)Phe→Leu (TTT>TTG); HBB: c.258T>G].

We report the case of a 61-year-old Canadian male of Maltese descent investigated for unexplained polycythemia. Decreased p50 suggested the presence of a high oxygen affinity hemoglobin (Hb) variant. Molecular genetic testing demonstrated that he carries a novel missense mutation (HBB: c.258T>G), resulting in a Phe→Leu substitution at position 85 of the ß chain. The novel Hb variant has been designated Hb Kennisis in recognition of where the proband resides. Two other missense mutations have been reported at this position [Hb Bryn Mawr or Hb Buenos Aires, ß85(F1)Phe→Ser (HBB: c.257T>C); Hb Grantham, ß85(F1)Phe→Cys; (HBB: c.257T>G)], both of which have increased oxygen affinity.

An evaluation of genetic causes and environmental risks for bilateral optic atrophy.

PURPOSE: To assess the clinical utility of next-generation sequencing (NGS) for the diagnosis of patients with optic atrophy (OA). DESIGN: Retrospective cohort study. METHODS: 97 patients were referred to the McMaster University Medical Center (Hamilton, Ontario) for evaluation of bilateral OA. All patients were sent for NGS including a 22 nuclear gene panel and/or complete mitochondrial DNA (mtDNA) sequencing. Positive genetic test results and abnormal vibration sensation were compared in patients +/- environmental exposures or a family history. RESULTS: 19/94 (20.2%) had a positive nuclear variant, of which 15/19 (78.9%) were in the OPA1 gene. No positive mtDNA variants were identified. The detection of a positive genetic variant was significantly different in patients who reported excessive ethanol use, but not in patients who smoke (0/19 (0%) vs. 19/78 (24.4%), P = 0.0164 and 4/22 (18.2%) vs. 15/74 (20.3%), P = 0.829, respectively). Patients with a positive family history were more likely to have a positive genetic variant compared to patients with a negative family history (P = 0.0112). There were significantly more excessive drinkers with an abnormal vibration sensation (P = 0.026), and with a similar trend in smokers (P = 0.074). CONCLUSIONS: All positive genetic variants were identified in nuclear genes. We identified a potential independent pathophysiological link between a history of excessive ethanol consumption and bilateral OA. Further investigations should evaluate and identify potential environmental risk factors for OA.

A Novel Human ß-Globin Gene Variant [Hb London-Ontario, HBB: c.332T>G] is Associated with Transfusion-Dependent Anemia in a Patient with a Hemoglobin Electrophoresis Pattern Consistent with ß-Thalassemia Trait.

We present the case of a novel ß-globin gene variant associated with early-onset transfusion-dependent anemia compatible with a ß-thalassemia major (ß-TM) phenotype in a patient of British descent. As a child, our patient developed chronic symptomatic anemia with hemoglobin (Hb) nadirs of 3.0 g/dL. She started receiving occasional transfusions by the age of 13 years and became transfusion-dependent by the age of 32 years. Work-up performed at our center showed a Hb electrophoresis compatible with ß-thalassemia (ß-thal) trait. Polymerase chain reaction (PCR) of the ß-globin gene detected a novel mutation situated at codon 110 (CTG). This missense mutation led to a substitution of the thymine nucleotide (nt) base for guanine (CGG) at position 332 (HBB: c.332T>G). We have named this new mutation Hb London-Ontario. The majority of previously described dominant allelic mutations of the ß-globin gene led to a ß-thal intermedia (ß-TI) phenotype. The heterozygous mutation which was detected in our patients is unique at it leads to a more severe ß-TM phenotype. We suspect this is a de novo mutation of which the mother of our patient, who was reported to have a form of thalassemia, was the proband.

Hepatoblastoma in a Child With Early-onset Cirrhosis.

Hepatoblastoma is the most common hepatic malignancy of childhood with known genetic predispositions and perinatal risk factors, with rare case reports occurring in the setting of cirrhosis. This case describes a young patient with cirrhosis attributed to early-onset hereditary hemochromatosis who was diagnosed with hepatoblastoma with uncommon histologic findings, evidence of chemotherapy resistance who ultimately succumbed to her disease. It is important to consider diagnoses beyond hepatocellular carcinoma in this scenario and consider early biopsy. With atypical histology, the tumor may respond poorly to conventional treatment and aggressive surgery or intensive therapy should be contemplated.

Data sharing as a national quality improvement program: reporting on BRCA1 and BRCA2 variant-interpretation comparisons through the Canadian Open Genetics Repository (COGR).

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.

Multiplex Allele-Specific PCR for Simultaneous Detection of H63D and C282Y HFE Mutations in Hereditary Hemochromatosis.

BACKGROUND: Hereditary hemochromatosis (HH) is characterized by excessive iron absorption in the intestine, which can lead to failure of vital organs such as the heart, liver, and pancreas. Among northern Europeans, HH is most often associated with the C282Y and H63D mutations of the HFE gene. We developed a test that allows screening for both mutations in a single reaction. METHODS: A multiplex allele-specific PCR was developed for simultaneous genotyping of the H63D and C282Y HFE mutations. PCR fragments were designed such that the resulting PCR product can be analyzed in a single polyacrylamide gel lane. RESULTS: Test results from our multiplex assay were concordant with genotypes of 55 Canadian patients with suspected hemochromatosis, which had previously been established by allele-specific PCRs that targeted H63D and C282Y in separate reactions. CONCLUSIONS: Molecular diagnostic detection of H63D and C282Y mutations can be achieved by a variety of methods, but these are not necessarily time-efficient or economical. Multiplex allele-specific PCR is an excellent tool for molecular diagnostic screening for H63D and C282Y mutations in patients with suspected hemochromatosis. This method is inexpensive, accurate, and highly efficient in terms of labor, throughput, and turnaround time.

Characterization of Two Novel Deletions Involving the 5' Region of the ß-Globin Gene.

We report two novel ß-thalassemia (ß-thal) deletions involving the 5' region of the ß-globin gene (HBB). The first deletion spans 538 bp and removes the ß-globin promoter, 5' untranslated region (5'UTR) and most of exon 1. This deletion was identified in a 3-year-old Vietnamese boy with non transfusion dependent Hb E (HBB: c.79G>A)/ß0-thal. The second deletion spans 1517 bp and removes the ß-globin gene promoter, 5'UTR, and exons 1 and 2. This deletion was identified in two unrelated adults of European descent who had ß-thal trait with unusually high Hb A2 levels. Deletions such as these are generally associated with higher levels of Hb A2 and Hb F than typical ß-thal alleles, which may ameliorate the severity of the disease.

α0-Thalassemia Due to a 90.7 kb Deletion (- -NFLD).

We report an α0-thalassemia (α0-thal) trait in Newfoundlanders caused by a novel 90.7 kb deletion. The deletion, designated the Newfoundland deletion (- -NFLD), removes both the HBA2 and HBA1 genes, while leaving the HBZ gene intact. The 5' deletion endpoint is within the HBAP1 pseudogene, approximately 3.7 kb upstream of the HBA2 gene. The 3' deletion endpoint is approximately 82.5 kb downstream of the HBA1 gene, within the second intervening sequence (IVS-II) of the FAM234A gene. This is the second α0-thal deletion reported in Newfoundland families of northern European descent.

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.

Clinical evaluation of a hemochromatosis next-generation sequencing gene panel.

BACKGROUND: Next-generation sequencing of an iron metabolism gene panel could identify pathogenic mutations, improving on standard hemochromatosis genetic testing and providing a molecular diagnosis in patients with suspected iron overload. METHODS: A next-generation sequencing panel of 15 genes with known roles in iron metabolism was constructed. A total of 190 patients were sequenced: 94 from a tertiary hemochromatosis clinic and 96 submitted for HFE testing with biochemical evidence of iron overload [elevated ferritin (>450 µg/L) or transferrin saturation (>55%)] obtained from a chart review. RESULTS: From the hemochromatosis clinic cohort, six patients were diagnosed with non-HFE hemochromatosis due to homozygous hemojuvelin (HFE2) mutations. Ten additional heterozygous pathogenic mutations were observed. From the chart review cohort, a C-terminus ferritin light chain (FTL) frameshift mutation was observed consistent with neuroferritinopathy. Heterozygous deletion of HFE2 and four additional rare pathogenic or likely pathogenic heterozygous mutations were identified in seven patients. CONCLUSIONS: An iron metabolism gene panel provided a molecular diagnosis in six patients with non-HFE iron overload and is suitable for diagnostic purposes in exceptional cases in specialized clinics. Further research will be required to assess the modifier effect of rare heterozygous mutations in iron metabolism genes.

Novel Mutation of the Translation Initiation Codon of the α1-Globin Gene (ATG>AAG or HBA1:c.2T>A).

We report two Italian-Canadian families with α+-thalassemia (α+-thal) trait caused by a novel mutation of the translation initiation codon of the α1-globin gene (ATG>AAG or HBA1:c.2T>A). This is the tenth reported α-thal mutation involving the translation initiation codon or the conserved Kozak consensus sequences of the HBA2 or HBA1 genes.

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis.

Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.

Sudanese (뫧)0-Thalassemia: Identification and Characterization of a Novel 9.6 kb Deletion.

We report a case of δß-thalassemia (δß-thal) trait in an adult male originally from Sudan. Multiplex ligation-dependent probe amplification (MLPA) was used to localize the approximate boundaries of the deletion, followed by polymerase chain reaction (PCR) amplification and sequence analysis of the junction fragment to determine the precise deletion endpoints. The deletion spans 9594 bp, with the 5' deletion endpoint located 1560 bp upstream of the δ-globin gene and the 3' endpoint within the second intervening sequence (IVS-II) of the ß-globin gene.