Principles of molecular testing for hereditary cancer.

Molecular testing for hereditary cancers has rapidly advanced over the past two decades. Next-generation sequencing has been widely adopted, which has made molecular testing increasingly accessible, and large gene panels are now routinely used in clinical care. Effectively using molecular testing as a tool for the management of patients with hereditary cancer involves understanding various basic principles. In this article, we provide an overview of general principles for molecular germline testing for hereditary cancer syndromes. We overview hereditary cancer etiology, clinical indications for molecular testing, test methodologies and limitations, interpretation and reporting of test results, the evolving nature of evidence on gene-disease relationships and penetrance, and resources related to the clinical management of hereditary cancer syndromes.

Characteristics of Adrenal Masses in Familial Adenomatous Polyposis.

BACKGROUND: Adrenal masses are a known extraintestinal manifestation of familial adenomatous polyposis. However, the literature on this association is largely confined to case reports. OBJECTIVE: This study aimed to determine the characteristics of adrenal masses in familial adenomatous polyposis and their clinical significance, as well as to estimate their prevalence. Mutational analysis was conducted to determine if any potential genotype-phenotype correlations exist. DESIGN: This is a retrospective cohort study. SETTING: Analysis included all patients meeting the criteria of classic familial adenomatous polyposis who were registered with the Familial Gastrointestinal Cancer Registry, a national Canadian database. PATIENTS: Appropriate imaging or autopsy reports were available in 311 registry patients. Patients with adrenal metastases were excluded. OUTCOME MEASURES: Data collection included demographic data, mutation genotype, adrenal mass characteristics, surgical interventions and mortality. RESULTS: The prevalence of adrenal masses was 16% (n = 48/311). The median age at diagnosis of adrenal mass was 45 years. The median diameter of adrenal mass at diagnosis was 1.7 cm (interquartile range, 1.4-3.0) with a median maximal diameter of 2.5 cm (interquartile range, 1.7-4.1) with median imaging follow-up of 48 months. The majority of adrenal masses were benign (97%, n = 61/63). Surgery was performed on 7 patients because of concerns for size, malignancy, or hormonal secretion. One adrenal-related death was due to an adrenocortical carcinoma. Mutation analysis did not identify any specific genotype-phenotype correlations. LIMITATIONS: There were incomplete or insufficient endocrinology data available in the registry to allow for the analysis of hormone secretion patterns. CONCLUSIONS: Adrenal masses are approximately twice as prevalent in the familial adenomatous polyposis population as in previous studies of the general population. Nearly all mutations led to truncation of the APC gene; however, there was no genetic signature to help predict those at increased risk. The majority of adrenal lesions identified were of benign etiology; thus, an intensive management or surveillance strategy with imaging screening is likely unwarranted. See Video Abstract at http://links.lww.com/DCR/A507.

Ethics and genomic medicine, how to navigate decisions in surgical oncology.

Using genetic information to make medical decisions and tailor treatments to individuals will likely provide major benefits and become an important part of health care. Surgical oncologists must ethically apply scientific genetic information in a complex and evolving environment to the benefit of their patients. In this review we address ethical issues associated with: indications for genetic testing, informed consent for testing and therapy, confidentiality, targeted therapy, prophylactic surgery, and genetic testing in children.

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

The spectrum of FBN1, TGFßR1, TGFßR2 and ACTA2 variants in 594 individuals with suspected Marfan Syndrome, Loeys-Dietz Syndrome or Thoracic Aortic Aneurysms and Dissections (TAAD).

INTRODUCTION: In this study, patients suspected of having a clinical diagnosis of Marfan Syndrome (MFS), Loeys-Dietz Syndrome (LDS) and Thoracic Aortic Aneurysms and Dissections (TAAD) were referred for genetic testing and examined for mutations in the FBN1, TGFßR1, TGFßR2 and ACTA2 genes. METHODS: We examined 594 samples from unrelated individuals and different combinations of genes were sequenced, including one or more of the following: FBN1, TGFßR1, TGFßR2, ACTA2, and, in some cases, FBN1 was analyzed by MLPA to detect large deletions. RESULTS: A total of 112 patients had a positive result. Of those, 61 had a clinical diagnosis of MFS, eight had LDS, three had TAAD and 40 patients had clinical features with no specific diagnosis provided. A total of 44 patients had an inconclusive result; of these, 12 patients were referred with a clinical diagnosis of MFS, 4 with LDS and 9 with TAAD and 19 had no clinical diagnosis. A total of 89 mutations were novel. CONCLUSION: This study reveals the rate of detection of variants in several genes associated with MFS, LDS and TAAD. The evaluation of patients by individuals with expertise in the field may decrease the likelihood of ordering unnecessary molecular testing. Nevertheless, genetic testing supports the diagnosis of MFS, LDS and TAAD.

HLA Variation and SARS-CoV-2 Specific Antibody Response.

Differences in SARS-CoV-2-specific immune responses have been observed between individuals following natural infection or vaccination. In addition to already known factors, such as age, sex, COVID-19 severity, comorbidity, vaccination status, hybrid immunity, and duration of infection, inter-individual variations in SARS-CoV-2 immune responses may, in part, be explained by structural differences brought about by genetic variation in the human leukocyte antigen (HLA) molecules responsible for the presentation of SARS-CoV-2 antigens to T effector cells. While dendritic cells present peptides with HLA class I molecules to CD8+ T cells to induce cytotoxic T lymphocyte responses (CTLs), they present peptides with HLA class II molecules to T follicular helper cells to induce B cell differentiation followed by memory B cell and plasma cell maturation. Plasma cells then produce SARS-CoV-2-specific antibodies. Here, we review published data linking HLA genetic variation or polymorphisms with differences in SARS-CoV-2-specific antibody responses. While there is evidence that heterogeneity in antibody response might be related to HLA variation, there are conflicting findings due in part to differences in study designs. We provide insight into why more research is needed in this area. Elucidating the genetic basis of variability in the SARS-CoV-2 immune response will help to optimize diagnostic tools and lead to the development of new vaccines and therapeutics against SARS-CoV-2 and other infectious diseases.

Unique Capabilities of Genome Sequencing for Rare Disease Diagnosis.

Background: Causal variants underlying rare disorders may remain elusive even after expansive gene panels or exome sequencing (ES). Clinicians and researchers may then turn to genome sequencing (GS), though the added value of this technique and its optimal use remain poorly defined. We therefore investigated the advantages of GS within a phenotypically diverse cohort. Methods: GS was performed for 744 individuals with rare disease who were genetically undiagnosed. Analysis included review of single nucleotide, indel, structural, and mitochondrial variants. Results: We successfully solved 218/744 (29.3%) cases using GS, with most solves involving established disease genes (157/218, 72.0%). Of all solved cases, 148 (67.9%) had previously had non-diagnostic ES. We systematically evaluated the 218 causal variants for features requiring GS to identify and 61/218 (28.0%) met these criteria, representing 8.2% of the entire cohort. These included small structural variants (13), copy neutral inversions and complex rearrangements (8), tandem repeat expansions (6), deep intronic variants (15), and coding variants that may be more easily found using GS related to uniformity of coverage (19). Conclusion: We describe the diagnostic yield of GS in a large and diverse cohort, illustrating several types of pathogenic variation eluding ES or other techniques. Our results reveal a higher diagnostic yield of GS, supporting the utility of a genome-first approach, with consideration of GS as a secondary or tertiary test when higher-resolution structural variant analysis is needed or there is a strong clinical suspicion for a condition and prior targeted genetic testing has been negative.

The clinical implementation of whole genome sequencing: a conversation with seven scientific experts.

This commentary is based on the key note address given by Jordan Lerner-Ellis at the annual symposium of the Society for the Study of Inborn Errors of Metabolism, held in Geneva, Switzerland in August 2011. The content of the address was developed from a series of discussions with seven clinicians and medical geneticists, all having a long-time interest in genetic testing and genomic medicine (the interviews were not intended to be a forum for the introduction of new data). All participants were asked to offer their views on five questions: the benefits of using whole genome sequencing (WGS) in the clinic; the corresponding risks; the limitations on its wider use; the interviewees' particular interests in using WGS in their practice; and projected timelines for successful adoption in clinical medicine.

Gene identification for the cblD defect of vitamin B12 metabolism.

BACKGROUND: Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. METHODS: We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. RESULTS: The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. CONCLUSIONS: Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder.

Spectrum of mutations in MMACHC, allelic expression, and evidence for genotype-phenotype correlations.

Methylmalonic aciduria and homocystinuria, cblC type, is a rare disorder of intracellular vitamin B(12) (cobalamin [Cbl]) metabolism caused by mutations in the MMACHC gene. MMACHC was sequenced from the gDNA of 118 cblC individuals. Eleven novel mutations were identified, as well as 23 mutations that were observed previously. Six sequence variants capture haplotype diversity in individuals across the MMACHC interval. Genotype-phenotype correlations of common mutations were apparent; individuals with c.394C>T tend to present with late-onset disease whereas patients with c.331C>T and c.271dupA tend to present in infancy. Other missense variants were also associated with late- or early-onset disease. Allelic expression analysis was carried out on human cblC fibroblasts compound heterozygous for different combinations of mutations including c.271dupA, c.331C>T, c.394C>T, and c.482G>A. The early-onset c.271dupA mutation was consistently underexpressed when compared to control alleles and the late-onset c.394C>T and c.482G>A mutations. The early-onset c.331C>T mutation was also underexpressed when compared to control alleles and the c.394C>T mutation. Levels of MMACHC mRNA transcript in cell lines homozygous for c.271dupA, c.331C>T, and c.394C>T were assessed using quantitative real-time RT-PCR. Cell lines homozygous for the late onset c.394C>T mutation had significantly higher levels of transcript when compared to cell lines homozygous for the early-onset mutations. Differential or preferential MMACHC transcript levels may provide a clue as to why individuals carrying c.394C>T generally present later in life.

Epigenetic modification of the gene for the vitamin B(12) chaperone MMACHC can result in increased tumorigenicity and methionine dependence.

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines and some tumors in situ. Most cell lines proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line, MeWo. MeWo-LC1 has a cellular phenotype identical to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin-dependent enzymes methionine synthase and methylmalonylCoA mutase. Inability of cblC cells to complement the defect in MeWo-LC1 suggested that it was caused by decreased activity of the MMACHC gene. However, no potentially disease causing mutations were detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression was detected in MeWo-LC1 by quantitative or non-quantitative PCR. There was virtually complete methylation of a CpG island at the 5'-end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation. The CpG island was partially methylated (30-45%) in MeWo and only lightly methylated (2-11%) in control fibroblasts. Infection of MeWo-LC1 with wild type MMACHC resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

Phenotypic variability in deficiency of the α subunit of succinate-CoA ligase.

Succinyl-CoA synthetase or succinate-CoA ligase deficiency can result from biallelic mutations in SUCLG1 gene that encodes for the alpha subunit of the succinyl-CoA synthetase. Mutations in this gene were initially associated with fatal infantile lactic acidosis. We describe an individual with a novel biallelic pathogenic mutation in SUCLG1 with a less severe phenotype dominated by behavioral problems. The mutation was identified to be c.512A>G corresponding to a p.Asn171Ser change in the protein. The liquid chromatography tandem mass spectrometry-based enzyme activity assay on cultured fibroblasts revealed a markedly reduced activity of succinyl-CoA synthetase enzyme when both ATP and GTP were substrates, affecting both ADP-forming and GDP-forming functions of the enzyme.

Mutations in the MMAA gene in patients with the cblA disorder of vitamin B12 metabolism.

Mutations in the MMAA gene on human chromosome 4q31.21 result in vitamin B12-responsive methylmalonic aciduria (cblA complementation group) due to deficiency in the synthesis of adenosylcobalamin. Genomic DNA from 37 cblA patients, diagnosed on the basis of cellular adenosylcobalamin synthesis, methylmalonyl-coenzyme A (CoA) mutase function, and complementation analysis, was analyzed for deleterious mutations in the MMAA gene by DNA sequencing of exons and flanking sequences. A total of 18 novel mutations were identified, bringing the total number of mutations identified in 37 cblA patients to 22. A total of 13 mutations result in premature stop codons; three are splice site defects; and six are missense mutations that occur at highly conserved residues. Eight of these mutations were common to two or more individuals. One mutation, c.433C>T (R145X), represents 43% of pathogenic alleles and a common haplotype was identified. Restriction endonuclease or heteroduplex diagnostic tests were designed to confirm mutations. None of the sequence changes identified in cblA patients were found in 100 alleles from unrelated control individuals.

Evaluation of second-generation sequencing of 19 dilated cardiomyopathy genes for clinical applications.

Medical sequencing for diseases with locus and allelic heterogeneities has been limited by the high cost and low throughput of traditional sequencing technologies. "Second-generation" sequencing (SGS) technologies allow the parallel processing of a large number of genes and, therefore, offer great promise for medical sequencing; however, their use in clinical laboratories is still in its infancy. Our laboratory offers clinical resequencing for dilated cardiomyopathy (DCM) using an array-based platform that interrogates 19 of more than 30 genes known to cause DCM. We explored both the feasibility and cost effectiveness of using PCR amplification followed by SGS technology for sequencing these 19 genes in a set of five samples enriched for known sequence alterations (109 unique substitutions and 27 insertions and deletions). While the analytical sensitivity for substitutions was comparable to that of the DCM array (98%), SGS technology performed better than the DCM array for insertions and deletions (90.6% versus 58%). Overall, SGS performed substantially better than did the current array-based testing platform; however, the operational cost and projected turnaround time do not meet our current standards. Therefore, efficient capture methods and/or sample pooling strategies that shorten the turnaround time and decrease reagent and labor costs are needed before implementing this platform into routine clinical applications.

Late-onset combined homocystinuria and methylmalonic aciduria (cblC) and neuropsychiatric disturbance.

We report on the case of a 36-year-old Hispanic woman with a spinal cord infarct, who was subsequently diagnosed with methylmalonic aciduria and homocystinuria, cblC type (cblC). Mutation analysis revealed c.271dupA and c.482G > A mutations in the MMACHC gene. The patient had a past medical history significant for joint hypermobility, arthritis, bilateral cataracts, unilateral hearing loss, anemia, frequent urinary tract infections, and mental illness. There was no significant past history of mental retardation, failure to thrive, or seizure disorder as reported in classic cases of cblC. Prior to the thrombotic incident, the patient experienced increased paresthesia in the lower extremities, myelopathy, and impaired gait. Given her previous psychiatric history, she was misdiagnosed with malingering until hemiplegia and incontinence became apparent. The authors would like to emphasize the recognition of a neuropsychiatric presentation in late onset cblC. Ten other reported late onset cases with similar presentations are also reviewed.

Combined methylmalonic aciduria and homocystinuria (cblC): phenotype-genotype correlations and ethnic-specific observations.

Methylmalonic aciduria and homocystinuria, cblC type (MIM 277400), is the most frequent inborn error of vitamin B12 (cobalamin, Cbl) metabolism, caused by an inability of the cell to convert Cbl to both of its active forms (MeCbl, AdoCbl). Although considered a disease of infancy, some patients develop symptoms in childhood, adolescence, or adulthood. The gene responsible for cblC, MMACHC, was recently identified. We studied phenotype-genotype correlations in 37 patients from published case-reports, representing most of the landmark descriptions of this disease. 25/37 had early-onset disease, presenting in the first 6 months of life: 17/25 were found to be either homozygous for the c.271dupA mutation (n=9) or for the c.331C>T mutation (n=3), or compound heterozygotes for these 2 mutations (n=5). 9/12 late-onset cases presented with acute neurological symptoms: 4/9 were homozygous for the c.394C>T mutation, 2/9 were compound heterozygotes for the c.271dupA and c.394C>T mutations, and 3/9, for the c.271dupA mutation and a missense mutation. Several observations on ethnic origins were noted: the c.331C>T mutation is seen in Cajun and French-Canadian patients and the c.394C>T mutation is common in the Asiatic-Indian/Pakistani/Middle Eastern populations. The recognition of phenotype-genotype correlations and the association of mutations with specific ethnicities will be useful for identification of disease-causing mutations in cblC patients, for carrier detection and prenatal diagnosis in families where mutations are known, and in setting up initial screening programs in molecular diagnostic laboratories. Further study into disease mechanism of specific mutations will help to understand phenotypic presentations and the overall pathogenesis in cblC patients.

Mutation and biochemical analysis of patients belonging to the cblB complementation class of vitamin B12-dependent methylmalonic aciduria.

Methylmalonic aciduria, cblB type (OMIM 251110) is an inborn error of vitamin B(12) metabolism that occurs due to mutations in the MMAB gene. MMAB encodes the enzyme ATP:cobalamin adenosyltransferase, which catalyzes the synthesis of the coenzyme adenosylcobalamin required for the activity of the mitochondrial enzyme methylmalonyl CoA mutase (MCM). MCM catalyzes the isomerization of methylmalonyl CoA to succinyl CoA. Deficient MCM activity results in methylmalonic aciduria and a susceptibility to life-threatening acidotic crises. The MMAB gene was sequenced from genomic DNA from a panel of 35 cblB patients, including five patients previously investigated. Nineteen MMAB mutations were identified, including 13 previously unknown mutations. These included 11 missense mutations, two duplications, one deletion, four splice-site mutations, and one nonsense mutation. None of these mutations was identified in 100 control alleles. Most of the missense mutations (9/11) were clustered in exon 7; many of these affected amino acid residues that are part of the probable active site of the enzyme. One previously described mutation, c.556C >T (p.R186W), was particularly common, accounting for 33% of pathogenic alleles. It was seen almost exclusively in patients of European background and was typically associated with presentation in the first year of life.

Identification of the gene responsible for the cblB complementation group of vitamin B12-dependent methylmalonic aciduria.

The methylmalonic acidurias are metabolic disorders resulting from deficient methylmalonyl-CoA mutase activity, a vitamin B(12)-dependent enzyme. We have cloned the gene for the cblB complementation group caused by deficient activity of a cob(I)alamin adenosyltransferase. This was accomplished by searching bacterial genomes for genes in close proximity to the methylmalonyl-CoA mutase gene that might encode a protein with the properties of an adenosyltransferase. A candidate was identified in the Archaeoglobus fulgidus genome and was used to probe the human genome database. It yielded a gene on chromosome 12q24 that encodes a predicted protein of 250 amino acids with 45% similarity to PduO in Salmonella enterica, a characterized cob(I)alamin adenosyltransferase. A northern blot revealed an RNA species of 1.1 kb predominating in liver and skeletal muscle. The gene was evaluated for deleterious mutations in cblB patient cell lines. Several mutations were identified including a 5 bp deletion (5del572gggcc576), two splice site mutations (IVS2-1G>T, IVS3-1G>A), andt several point mutations (A135T, R186W, R191W and E193K). Two additional amino acid substitutions (R19Q and M239K) were found in several patient cell lines but were found to be common polymorphisms (36% and 46%) in control alleles. The R186W mutation, which we suggest is disease-linked, is present in four of the six patient cell lines examined (homoallelic in two) and in 4 of 240 alleles in control samples. These data confirm that the identified gene, MMAB, corresponds to the cblB complementation group and has the appearance of a cob(I)alamin adenosyltransferase, as predicted from biochemical data.