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.

Multisite verification of the accuracy of a multi-gene next generation sequencing panel for detection of mutations and copy number alterations in solid tumours.

Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for information derived from RNA was lower than observed for DNA, due largely to decreased quality metrics associated with the RNA components of the assay, suggesting that the RNA portions of comprehensive NGS assays may be more vulnerable to variations in approach and workflow. Overall the results of this study support the use of the OFA for targeted sequencing for testing of clinical samples and suggest specific internal quality metrics that can be reliable indicators of assay failure. While we believe this evidence can be interpreted to support deep targeted sequencing in general, additional studies should be performed to confirm this.

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.

ATM whole gene deletion in an Italian family with hereditary pancreatic cancer: Challenges to cancer risk prediction associated with an 11q22.3 microdeletion.

Hereditary pancreatic cancer has been attributed to variants of several cancer predisposition genes including ATM. While heterozygous pathogenic variants in the ATM gene are implicated as a cause of familial breast and pancreatic cancers to our knowledge ATM whole gene deletions have not been previously reported. We describe a contiguous gene deletion of the ATM locus in a multi-generation family of Italian descent with a strong family history of pancreatic cancer. A deletion of one copy of the entire ATM gene was identified by routine panel testing and further characterized by chromosomal microarray analysis. An 11q22.3 microdeletion of approximately 960 kb was identified that is predicted to result in loss of 10 genes including ATM. The deletion was identified in two additional family members including a presymptomatic daughter and an affected sibling. A normal disomic complement of the 11q22.3 region was detected in a third family member with a history of prostate and pancreatic cancer. Additional family members were not available for testing. Given available evidence that ATM haploinsufficiency can increase cancer risk, we predict that the observed copy number loss has likely contributed to hereditary cancer in this family. However, absence of the familial microdeletion in at least one affected family member suggests that ATM deletions are unlikely the sole contributing factor influencing tumor development in affected individuals. This case highlights 11q22.3 microdeletions of the ATM gene region as a possible risk factor for hereditary cancer, including pancreatic cancer. The same case provides a further cautionary tale for over interpretation of cancer risk associated tumor suppressor microdeletions and suggests that the variant may not be sufficient for tumor development or may modify the cancer risks associated with other, yet unidentified hereditary cancer genes.

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.

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.

α(+)-Thalassemia Due to a Frameshift Mutation of the α2-Globin Gene [codons 55/56 (+T) or HBA2: c.168dup].

We report a case of α(+)-thalassemia (α(+)-thal) trait in a Chinese-Canadian family caused by a novel frameshift mutation of the α2-globin gene, specifically the duplication of a single nucleotide at amino acid codon 56 [HBA2: c.168dup]. The mutation results in substitution of a termination codon (TAA) for lysine (AAG) at amino acid position 56.

Non-thalassemic phenotype associated with the -83 (G > A) mutation of the ß-globin gene promoter (HBB: c.-133G > A).

The -83 (G > A) mutation of the ß-globin gene promoter (HBB: c.-133G > A) was first reported in an adult male patient with mild thalassemic indices, suggesting that this may be a mild ß(+)-thalassemia (ß(+)-thal) allele. In this report, we present data from several patients who are simple heterozygotes for the -83 mutation, or compound heterozygotes for -83 and Hb S (HBB: c.20A > T) or ß-thal. These cases illustrate that the -83 sequence variant is not associated with a thalassemic phenotype. This has important implications for carrier screening and genetic counseling, particularly since the -83 mutation is relatively common in African and Hispanic populations.

Mild ß(+)-thalassemia associated with two linked sequence variants: IVS-II-839 (T>C) and IVS-II-844 (C>A).

We report four unrelated families with a mild ß(+)-thalassemia (ß(+)-thal) allele consisting of two sequence variants at the 3' end of IVS-II: IVS-II-839 (T>C) (HBB: c.316-12T>C) and IVS-II-844 (C>A) (HBB: c.316-7C>A). These sequence variants alter the conserved polypyrimidine tract of the consensus splice acceptor sequence (Y11NYAG/G), which could reduce splicing efficiency. This may represent a common, yet under-diagnosed ß(+)-thal allele in African populations.

Normal Hb A2 ß-thalassemia trait: frameshift mutation (HBB: c.187_251dup) in cis with the Hb A2' δ-globin gene missense mutation (HBD: c.49G>C).

We report the case of a father and daughter who are heterozygous for a duplication of 65 bp within exon 2 of the ß-globin gene, resulting in an altered and truncated ß-globin chain that is predicted to be non functional. The ß-globin gene mutation is in cis with the common Hb A2 ' missense mutation of the δ-globin gene (HBD: c.49G>C), resulting in ß-thalassemia (ß-thal) trait with normal levels of Hb A2. This is the second report of this ß(0)-thal mutation, and both families were associated with the Hb A2 ' variant and normal levels of Hb A2. Laboratories should be aware of the rare occurrence of ß-thal trait with normal levels of Hb A2.

α(+)-Thalassemia trait caused by a frameshift mutation in exon 2 of the α2-globin gene [HBA2 c.244delT].

We report a case of α(+)-thalassemia (α(+)-thal) trait caused by a novel frameshift mutation in exon 2 of the α2-globin gene, specifically a deletion of a single nucleotide at amino acid codon 81 [HBA2 c.244delT]. The mutation results in a premature termination of translation at codon 83.

ß+-Thalassemia trait due to a novel mutation in the ß-globin gene promoter: -26 (A>C) [HBB c.-76A>C].

We report the case of a woman with ß(+)-thalassemia (ß(+)-thal) trait, in which there were two sequence variants within the ß-globin gene promoter: -54 (G>A) [HBB c.-104G>A] and -26 (A>C) [HBB c.-76A>C]. Data from other patients indicate that the -54 substitution is a non pathogenic sequence variant. Therefore, the ß-thal phenotype is most likely due to the -26 mutation that is adjacent to the conserved ATAA box.