Molecular Diagnosis of Fanconi Anemia and Dyskeratosis Congenita

Abstract New discoveries in cell biology, molecular biology and genetics have unveiled some of the pathophysiological mysteries of some of the bone marrow failure syndromes. Many of these discoveries have revealed why these syndromes show so much clinical overlap and some hold the potential for influencing the development of new therapies. In children and adults with pancytopenia and hypoplastic bone marrows proper differential diagnosis requires that some attention be directed toward defining molecular and cellular pathogenetic mechanisms because, once identified, some of these mechanisms will clearly suggest rational therapeutic approaches, treatment options that should be avoided, or both. In Section I, Drs. Jeffrey Lipton and Grover Bagby review the approach to diagnosis and management of patients with the inherited bone marrow failure syndromes, Fanconi anemia, dyskeratosis congenita, Diamond-Blackfan anemia, and the Shwachman-Diamond syndrome. Extraordinary progress has been made in identifying the genes bearing pathogenetically relevant mutations in these disorders, but slower progress has been made in defining the precise functions of the proteins these genes encode in normal cells, in part because it is increasingly obvious that the proteins are multifunctional. In practice, it is clear that in patients with dyskeratosis congenita and Fanconi anemia, the diagnosis must be considered not only in children but in adults as well. In Section II, Dr. Elaine Sloand outlines a very practical and evidence-based approach to diagnosis and management of acquired hypoplastic states emphasizing overlap between non-clonal and clonal hematopoiesis is such conditions. The pathogenesis of T lymphocyte–mediated marrow failure is presented as a clear-cut rationale for use of immunosuppressive therapy and stem cell transplantation. Practical management of patients with refractory disease with and without evidence of clonal evolution (either paroxysmal nocturnal hemoglobinuria [PNH] or myelodysplasia [MDS]) is presented. In Section III, the challenge of hypoplastic MDS is reviewed by Dr. Charles Schiffer. After reviewing the most up-to-date classification scheme, therapeutic options are reviewed, focusing largely on agents that have most recently shown some promising activity, including DNA demethylating agents, thalidomide and CC5013, arsenic trioxide, and immunosuppressive therapy. Here are also outlined the rationale and the indications for choosing allogeneic bone marrow transplantation, the only therapy with known curative potential.

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Head and neck cancer in Fanconi anemia and dyskeratosis congenita.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.5563 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 5563-5563

Author(s):

Blanche P Alter ◽

Neelam Giri ◽

Sharon H Savage

Keyword(s):

General Population ◽

Oral Cavity ◽

Head And Neck ◽

Fanconi Anemia ◽

Hpv Vaccine ◽

Bone Marrow Failure ◽

Time Frame ◽

Dyskeratosis Congenita ◽

Hpv Status ◽

Study Results

5563 Background: Fanconi Anemia (FA) and dyskeratosis congenita (DC) are inherited bone marrow failure syndromes (IBMFS) with extraordinarily high risks of cancer, particularly head and neck squamous cell carcinoma (HNSCC). The standardized incidence ratios (SIR) for solid tumors are 40 in FA and 10 in DC, and for HNSCC 700 and 900 respectively. In the general population, cancers of the oropharynx may be associated with human papilloma virus (HPV) more than the oral cavity. The specific locations of HNSCC in FA and DC have not been reviewed, and thus the hypothetical role of HPV in these cancers is unknown. Methods: Literature cases were reviewed through PubMed searches using the terms “Fanconi anemia” and “dyskeratosis congenita”. All papers were reviewed, and details of the specific cancers recorded. FA literature included reports from 1927 through 2011, and DC literature from 1910 through 2011. Participants in the National Cancer Institute (NCI) IBMFS retrospective/prospective protocol (NCI 02-C-0052, opened in 2002) were also analyzed for cancer. The HPV vaccine was not available during most of the time frame of this study. Results: There were 411 cases of cancer among 2190 (19%) cases of FA described individually in the literature, and 21 cancer cases out of 116 (18%) FA patients in the NCI cohort. HNSCC were reported in 112 FA literature cases (5%), and 9 (8%) in the cohort. The oropharynx was the initial site for HNSCC in 31/112 literature cases (28%) and 3/9 NCI cases (33%). With regard to DC, 51/647 (8%) cases in the literature had cancer, as did 9 of 94 (10%) NCI participants. HNSCC were reported in 21/647 (3%) DC literature cases, and in 5/94 (5%) from the NCI cohort. Four of the 21 HNSCC (19%) in the literature were oropharyngeal, and 2/5 (40%) in the NCI cohort. The cumulative incidences of any HNSCC in the NCI FA and DC cohorts were 50% and 21% by 40 years of age (censored at death from other causes). Conclusions: Patients with FA and DC have extremely high risks of HNSCC at substantially younger ages than the general population. Approximately 30% of these cancers occur in the oropharynx. The HPV vaccine could be important in prevention of these cancers. Future studies of the HPV status of oral cavity and oropharynx HNSCC tumors from patients with FA and DC will be informative.

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Characterization of Pathogenic Variants and Clinical Phenotypes in 117 Japanese Fanconi Anemia Patients

Blood ◽

10.1182/blood-2018-99-110362 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3860-3860

Author(s):

Minako Mori ◽

Asuka Hira ◽

Kenichi Yoshida ◽

Hideki Muramatsu ◽

Yusuke Okuno ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Molecular Diagnosis ◽

Fanconi Anemia ◽

Direct Sequencing ◽

Bone Marrow Failure ◽

Whole Genome ◽

Aberrant Splicing ◽

Large Deletions ◽

Complementation Groups

Abstract Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations. Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients. In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database. We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation. In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor). Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. Disclosures Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

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Evidence for T-Cell Oligoclonal Expansion in Aplastic Anemia Associated with Telomerase Complex Mutations: Pathophysiological and Clinical Implications.

Blood ◽

10.1182/blood.v106.11.1052.1052 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1052-1052 ◽

Cited By ~ 1

Author(s):

Rodrigo T. Calado ◽

Tullia Bruno ◽

Keisha L. Wilkerson ◽

Neal S. Young

Keyword(s):

Bone Marrow ◽

T Cell ◽

Aplastic Anemia ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Interferon Γ ◽

Dyskeratosis Congenita ◽

Dominant Type ◽

Hematopoietic Stem ◽

Oligoclonal Expansion

Abstract Aplastic anemia is characterized by peripheral blood pancytopenia and a hypocellular bone marrow. In the majority of patients, bone marrow destruction is immune-mediated by a type-1 T cell response, dominated by oligoclonal expansion of CD8+ cells and targeting hematopoietic stem cells. Some patients with acquired aplastic anemia are heterozygous for mutations in genes encoding the major components of the telomerase complex, telomerase reverse transcriptase (encoded by TERT; Yamaguchi et al., N Engl J Med2005;352:1413) and the RNA component (encoded by TERC; Fogarty et al., Lancet2003;362:1628). These mutations lead to shortened telomeres of leukocytes, low telomerase activity and reduced hematopoietic function. Relatives carrying the same mutations also have short telomeres and reduced hematopoietic function, but they do not inevitably develop overt marrow failure. In contrast, dyskeratosis congenita, a constitutional type of marrow failure, is caused by mutations in TERC (autosomal dominant type) or in the DKC1 gene, which encodes an additional component of the telomerase complex, dyskerin. In addition to marrow failure, patients with dyskeratosis congenita also often have physical anomalies, such as leukoplakia, nail dystrophy, and hyperpigmentation as well as hepatic or pulmonary fibrosis. In dyskeratosis congenita, family members bearing the genetic mutation present variable degrees of physical abnormalities or organ damage. However, in patients with acquired aplastic anemia carrying telomerase mutations without physical anomalies, it is unclear whether these mutations are sufficient for the development of marrow failure. We hypothesized that telomerase complex mutations reduce the size of the hematopoietic stem cell compartment and affect its regenerative capacity, making carriers more vulnerable to environmental insults or autoimmune damage. We analyzed the distribution of the T-cell repertoire (T-cell receptor [TCR] Vβ subfamily) and expansion of particular Vβ subsets by flow cytometry in peripheral blood of six aplastic anemia patients carrying telomerase complex mutations (five with TERT mutations, one with a TERC mutation), two patients with Fanconi anemia, and 15 healthy subjects. The expression of 22 Vβ subfamilies were evaluated in CD8+CD28− cells, and expansion was defined when the percentage of a given Vβ subfamily was above two standard deviations based on the control group (Risitano et al. Lancet2004;364:355). We also evaluated interferon-γ levels in serum of nine telomerase mutant patients and 10 controls by ELISA. Expanded Vβ subsets were observed in all six aplastic patients carrying telomerase complex mutations analyzed. Five patients (with TERT mutations) had two clones expanded and one (with TERC mutation) showed three overrepresented clones. The Vβ subsets 9, 13.6, 17, and 20 were expanded in more than one patient. Oligoclonal Vβ expansion was not observed in Fanconi anemia. Telomerase mutant patients also had significantly increased interferon-γ serum levels in comparison to controls (27 pg/mL; range, 0–95 vs. 7.6, 0–16, respectively; P<0.02). These results indicate that an immune process targeting hematopoiesis may operate in patients carrying telomerase complex gene mutations. Limited responsed to immunosuppression may reflect the poor hematopoietic reserve rather than a nonimmunologic mechanism of marrow destruction.

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The First Single Center Phenotypic Comparison of Fanconi Anemia, Dyskeratosis Congenita, Diamond-Blackfan Anemia, and Shwachman- Diamond Syndrome: The NCI IBMFS Cohort.

Blood ◽

10.1182/blood.v112.11.2043.2043 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2043-2043

Author(s):

Neelam Giri ◽

Christine Mueller ◽

Sarah Weinstein ◽

Suvimol Hill ◽

John Butman ◽

...

Keyword(s):

Bone Mineral Density ◽

Short Stature ◽

Fanconi Anemia ◽

Dyskeratosis Congenita ◽

Palpebral Fissure ◽

Single Center ◽

Mineral Density ◽

Cardiac Defects ◽

Diamond Blackfan Anemia ◽

Shwachman Diamond Syndrome

Abstract Inherited bone marrow failure syndromes (IBMFS) comprise a group of heterogeneous genetic disorders characterized by single or multilineage cytopenias, distinctive clinical features, and an increased risk of specific malignancies, with considerable overlap in hematologic and physical manifestations. Our objective was to identify specific distinguishing features for each of the major IBMFS by identical systematic evaluations, in the first comparison of all 4 disorders in a single center. Participants were: 23 with Fanconi anemia (FA), 32 with dyskeratosis congenita (DC), 23 with Diamond-Blackfan anemia (DBA) and 7 with Shwachman-Diamond syndrome (SDS). Evaluations included physical characteristics, hematologic data, and abnormalities of the skeletal, ophthalmic, otologic, renal, cardiac, central nervous system (CNS) and endocrine systems. We found a similar frequency of cytopenias (~80%) in all disorders; predominantly multilineage in FA and DC, anemia in DBA, and neutropenia in SDS. 50% of FA patients had clonal marrow cytogenetic abnormalities, of whom 24% had morphologic myelodysplastic syndrome (MDS); 25% of DC patients had clones of whom one had MDS; none with DBA had a clone or MDS; and 4/7 with SDS had clones [i(7)q in one, del(20)q in two, i(X)p in one] but no MDS. Skeletal malformations were frequent in patients with FA (68%). Anomalies unique to FA were radial ray defects (thumb or thumb and radius) in 45%, Klippel-Feil Syndrome in 45%, and fused or partial ribs in 25%. No specific defects were seen in patients with DC or DBA. 3/7 patients with SDS had metaphyseal dysplasia. 80% of FA patients had smaller than normal eye measurements, including one or more of outer canthal distance, inner canthal distance, palpebral fissure length or interpupillary distance, compared with age and sex-matched published controls, unaffected family members and patients with other IBMFS; 75% had small eyes by A-scan and 60% had microcorneas. ~15% of patients with DC, DBA and SDS had small palpebral fissure lengths but no microcorneas. Eye findings unique to DC were lacrimal duct stenosis in 27%, exudative retinopathy in one and proliferative retinopathy with retinal neovascularization in two. Hearing was decreased only in FA, in 65%, associated with abnormalities of tympanic membrane bony island and/or short malleus, with conductive hearing loss in 40%. 40% of FA patients had renal anomalies vs. 3% with DC, 8% with DBA and none with SDS. Congenital cardiac defects were more frequent in patients with DBA (40%) than in those with FA (15%), DC (3%) or SDS (none). 50% of patients with FA, 25% with DBA and 5/7 patients with SDS had short stature (height Z-score <2SD) and microcephaly, whereas in DC only 3% had short stature, and 38% had microcephaly without short stature. Only patients with FA and DC had abnormal brain MRIs: 27% of FA patients had absent corpus callosum and/or septum pellucidum, and 57% had a small pituitary; 38% of DC patients had cerebellar hypoplasia and associated microcephaly, developmental/speech delay and ataxia. 56% of DC patients had at least two features of the diagnostic triad (nail dystrophy, oral leukoplakia, and lacey skin pigmentation). Endocrine abnormalities which were common in FA [hypothyroidism (35%), growth hormone deficiency (25%) and hypogonadism (>90% adults)] were rare in the other IBMFS. However, 90% of the patients with FA, 27% with DC, and 60% with DBA and SDS had reduced bone mineral density on Dexa scan. In this first direct, single center comparison of the 4 major IBMFS, we identified: skeletal anomalies, small eyes, abnormal hearing and endocrine deficiencies in FA; high rates of cardiac defects in DBA; CNS anomalies in FA and DC; microcephaly in all disorders; and reduced bone mineral density in all 4 disorders but highest in FA. Thus, comprehensive systematic investigations detected unique syndrome-specific abnormalities in much higher frequencies than expected based on literature reports. These findings have important clinical consequences and warrant early intervention.

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Molecular diagnosis and therapeutic measures in patients with dyskeratosis congenita

Orvosi Hetilap ◽

10.1556/oh.2010.28811 ◽

2010 ◽

Vol 151 (8) ◽

pp. 285-292

Author(s):

Zsolt Reiger ◽

Gergely Varga ◽

Beáta Tóth ◽

László Maródi ◽

Melinda Erdős

Keyword(s):

Molecular Diagnosis ◽

Splice Site ◽

Dyskeratosis Congenita ◽

Therapeutic Measures

A dyskeratosis congenita különböző öröklődésmenetet mutató ritka kórkép, amelyre csontvelő-elégtelenség és korai öregedés jellemző. A közleményben, egy felnőttkorban diagnosztizált beteg esete kapcsán, a szerzők áttekintést nyújtanak a betegség klinikumáról, patomechanizmusáról, genetikai hátteréről és a diagnosztikai, illetve terápiás lehetőségekről. A beteg esetében a dyskerint kódoló gén mutációanalízis-vizsgálata során a c.IVS2-5C>G splice site mutáció volt kimutatható. Az esetismertetéssel a szerzők felhívják a figyelmet a korai diagnózis jelentőségére, amely lehetővé teszi a súlyos, invazív fertőzések és noninfekciós szövődmények kialakulásának megelőzését, és a beteg idejekorán történő csontvelő-transzplantációs programba vételét. A genetikai vizsgálatok segítségével lehetőség nyílik az érintett családokban a hordozóállapot kiszűrésére, így a génszintű diagnosztika pótolhatatlan segítséget nyújt a családtervezésben és a praenatalis diagnosztikában is.

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