ALBERT

Description: Background:Reduced fertility and increased fetal and maternal complications during pregnancy have been reported in patients with Fanconi anemia, Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Such high-risk pregnancies benefit from coordinated care by hematologists and maternal-fetal medicine specialists with expertise in inherited bone marrow failure syndromes (IBMFS). There are no data on fertility and pregnancy outcomes in women with dyskeratosis congenita (DC). Objectives: To determine pubertal development, fertility, and pregnancy outcomes in women with DC. Methods: Prospective evaluation and medical record review was performed of women with DC ≥10 years of age enrolled in the NCI IBMFS cohort study. We examined ages at menarche and menopause, details regarding fertility and pregnancy, hematologic and non-hematologic complications of pregnancy, and maternal and fetal outcomes. Results: We evaluated 27 women with DC (median age 31 years, range 10-63), who all attained menarche (median age 12 years, range 9-17). The median age at natural menopause was 51 years, range 50-52. Seventeen women had 46 pregnancies (median 2 pregnancies per person, range 1-6); 1 was unable to conceive, 3 were using contraceptives and 6 were not yet sexually active. Thirty-two of 46 pregnancies (70%) in 17 women who carried to 27-42 weeks gestation (median 39 weeks) resulted in 34 live births (2 sets of twins). Thirty pregnancies (65%) in 11 women were associated with a variety of complications: 13 ended in miscarriages; 1 was an elective abortion. Maternal complications were preeclampsia (n=3), placenta previa with abruption (n=2 pregnancies in the same patient) and cesarean section for failure to progress (n=5; 4 were in 1 patient). Fetal complications were 5 preterm births (3 associated with preeclampsia) and 2 others had fetal distress. Six women had cytopenia during 8 pregnancies: in 2, mild cytopenia worsened during pregnancy but did not need treatment. One progressed to severe aplastic anemia (SAA) and 1 with SAA was transfusion-dependent throughout. Two patients had normal counts at the start of pregnancy, but developed cytopenia with preeclampsia or abruption with placenta previa, respectively. Three others with normal blood counts had macrocytosis. One was post-transplant. Four had only non-hematologic manifestations of DC. And, 3 women, with no DC-associated clinical features, were identified after diagnosis of an affected offspring. Fourteen of 32 pregnancies were cesarean deliveries performed in 8 patients for maternal complications (n=7), fetal distress (n=2) or failure to progress (n=5). Three of the 18 vaginal deliveries were in 2 mothers with mild cytopenia, and 1 preterm birth in a patient with SAA. Only 14/46 pregnancies (30%) in 6 women were uncomplicated, term vaginal deliveries; of these, 2 occurred in 1 post-hematopoietic stem cell transplant recipient, conceived by in vitro fertilization. There were 8 patients with TERC mutations, 3 TERT, 2 TINF2, 2 RTEL1and 2 were gene unknown. Fetal loss or maternal complication did not differ significantly in relation to mutated genes in mothers. Conclusions: This is the first study of fertility and pregnancy outcomes in patients with DC. We show that females with DC attain menarche and menopause at normal ages and have normal fertility. However, women with DC appear to have high rates of maternal and fetal complications resulting in miscarriage, preeclampsia, and worsening cytopenias. These complications may lead to increased likelihood of cesarean section and/or preterm delivery. Thus, similar to Fanconi anemia and Diamond-Blackfan anemia, pregnant women with DC are at high-risk for complications and should be managed by a hematologic and high risk maternal-fetal medicine team. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2361 Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome (IBMFS) classically diagnosed by the triad of dysplastic nails, reticular skin pigmentation and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure (BMF), cancer, pulmonary fibrosis, and other medical problems. Germline mutations in key telomere biology genes cause DC (DKC1, TERC, TERT, TINF2, NOP10, NHP2, WRAP53, or CTC1), although about 50% of patients lack a known mutation. Leukocyte telomere lengths

Description: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome. The spectrum of cancer susceptibility in this disorder of telomere biology has not been described. There were more than 500 cases of DC reported in the literature from 1910 to 2008; the National Cancer Institute (NCI) prospective DC cohort enrolled 50 cases from 2002 to 2007. Sixty cancers were reported in 52 literature cases, while 7 occurred among patients in the NCI DC cohort. The 2 cohorts were comparable in their median overall survival (42 years) and cumulative incidence of cancer (40%-50% by age 50 years). The most frequent solid tumors were head and neck squamous cell carcinomas (40% of patients in either cohort), followed by skin and anorectal cancer. The ratio of observed to expected cancers (O/E ratio) in the NCI cohort was 11-fold compared with the general population (P 〈 .05). Significantly elevated O/E ratios were 1154 for tongue cancer and 195 for acute myeloid leukemia. Survival after bone marrow transplantation for aplastic anemia or leukemia was poor in both cohorts. The frequency and types of cancer in DC are surpassed only by those in Fanconi anemia (FA), indicating that FA and DC have similarly high risks of adverse hematologic and neoplastic events, and patients with these diseases should be counseled and monitored similarly.

Description: It is now recognized that Fanconi anemia (FA) is both an inherited bone marrow failure syndrome (IBMFS) and also a highly penetrant cancer susceptibility syndrome associated with leukemias and specific solid tumors. However, the spectrum of cancer susceptibility in other IBMFS remains unclear. The National Cancer Institute (NCI) IBMFS Cohort is the first prospective/retrospective study to follow patients with diverse IBMFS using a comprehensive and unified protocol. During 2002–2007, 66 patients with FA, 55 with Dyskeratosis Congenita (DC), 63 with Diamond-Blackfan anemia (DBA), and 16 with Shwachman-Diamond syndrome (SDS) enrolled in the study and contributed a combined total of 4113 person-years of follow-up. Adverse outcomes ascertained for all patients included bone marrow failure (BMF) leading to death or bone marrow transplant, acute leukemia (AL), myelodysplastic syndrome (MDS), and development of a solid tumor (ST). For each syndrome, we calculated the ratio of observed (O) numbers of cancers versus expected (E) numbers in a demographically matched cohort from the general United States population (O/E ratio). We also calculated cause-specific hazards and cumulative incidence by age of BMF, AL, and ST. For FA, the first adverse event was BMF in 25 patients, AL in 4, and ST in 11; 8 developed MDS. The FA experience was broadly consistent with published reports from our prior North American Survey (NAS) and an independent FA cohort in Germany. In the NCI FA cohort, the O/E ratio was 37 for all ST (versus 48 and 26 respectively in previously analyzed cohorts) and 311 for AL (versus 785 and 868); these increased risks compared with the general population were statistically significant. The O/E ratio for MDS was 4910 (also significant). In time-dependent analysis, the MDS hazard was stable at 0.6%/year. For SDS, no patient has yet developed an adverse event. For DBA, the first adverse event was BMF in 4 and ST in 3 (1 colon and 2 lung cancers); no DBA patient has yet developed AL or MDS. The cumulative incidence of BMF in DBA was 10% by age 30 years. For DC, the first adverse event was BMF in 15, AML in 2, and ST in 5; 5 developed MDS. For DC, the O/E ratio was 10 for all cancers, 7 for all solid tumors, 897 for tongue cancer, and 188 for AL; these increased risks were statistically significant. The O/E ratio was also significantly elevated for MDS (2362). To gain statistical power, we compared DC patients to a pooled cohort of 458 FA from 4 cohorts (NAS, Germany, Israel, and NCI). For DC, the cumulative incidence by age 50 years was 50% for BMF, 10% for AL, and 22% for ST, broadly similar to corresponding values of 56%, 13%, and 30%, respectively, in the pooled FA cohorts. In DC, the cause-specific hazards of ST and AL increased significantly with age, but had a later rise than in FA. The hazard of BMF in DC also increased significantly with age, but lacked the early hazard peak seen in FA. This initial analysis from the NCI IBMFS cohort reveals that as for FA, DC is a highly penetrant bone marrow failure syndrome and a major cancer susceptibility syndrome with numerous events occurring in young adulthood and early middle age. This is the first study to quantify the risk in DC in this way. Continued follow-up of the cohort should clarify the spectrum of cancer susceptibility in each syndrome.

Description: Telomeres protect the ends of chromosomes, shorten with age, and are very short in dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS) associated with mutations in telomere biology genes. “Short telomeres” were reported in Fanconi Anemia (FA), Diamond-Blackfan Anemia (DBA) and Shwachman-Diamond Syndrome (SDS) using telomere restriction fragment length or Q-FISH assays of total leukocyte or mononuclear cell DNA. These reports focused on group averages, not results from individual patients. Our objective was to determine which categories of IBMFS patients have very short telomeres, and in which leukocyte subsets, using a more sensitive and specific assay. Telomere length was measured in granulocytes, lymphocytes, naïve T-cells, memory T-cells, B-cells, and NK cells using automated multicolor flow fluorescence in situ hybridization (FISH). We previously showed that very short telomeres (

Description: Abstract 197 Dyskeratosis congenita (DC) is a multisystem disorder characterized by the diagnostic triad of nail dysplasia, abnormal skin pigmentation, and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure, cancer, pulmonary fibrosis, and other complications. All patients with DC have very short telomeres; approximately 60% have a mutation in one of 6 telomere biology genes (DCK1, TERC, TERT, TINF2, NOP10 or NHP2). Telomerase (TERT) is the reverse transcriptase which, with its RNA component TERC, extends telomeric repeats to offset the telomere shortening that occurs with DNA replication. Mutations in the catalytic core component of TERT (autosomal dominant or recessive) or TERC (autosomal dominant) cause DC and related telomere biology disorders. The function of the telomerase enzyme in vivo requires additional components including dyskerin (DKC1), which is mutated in patients with X-linked recessive DC. Dyskerin binds the H/ACA sequence within TERC and is required for the biogenesis of telomerase, as well as the biogenesis and function of other RNP complexes, including small nucleolar RNAs (snoRNAs) and small Cajal body RNAs (scaRNAs), involved in the modification of ribosomal RNAs and splicing RNAs, respectively. We recently identified TCAB1 (gene names WDR79, WRAP53) as a novel telomerase component through biochemical purification of telomerase complexes. TCAB1 is a WD40-repeat containing protein that binds the CAB box sequence within TERC. It is a constituent of the active telomerase holoenzyme and inhibition of TCAB1 prevents telomerase from localizing to Cajal bodies where RNA-protein complexes are assembled and modified. Since TCAB1 is required for telomerase trafficking, we evaluated mutations in TCAB1 as a potential cause of DC in 16 mutation-negative patients who were participants in the NCI's prospective Inherited Bone Marrow Failure Syndromes (IBMFS) study. Nine patients had classic DC, defined by the diagnostic triad, or the combination of 1 of the triad plus BMF and 2 other DC-related complications, and leukocyte telomeres

Description: Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure syndrome (IBMFS) in which germline mutations in telomere biology genes account for approximately 70% of known families. DC is clinically diagnosed by the presence of the triad of nail dysplasia, lacy skin pigmentation, and oral leukoplakia. However, not all patients have the triad and multiple other medical problems may include, stenosis of the esophagus, urethra and/or lacrimal ducts, avascular necrosis of the hips or shoulders, developmental delay, head and neck squamous cell cancer, and/or leukemia. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients have features of DC but also have microcephaly, cerebellar hypoplasia, and intrauterine growth retardation, and may present with severe immunodeficiency and enteropathy. Telomere lengths (in blood leukocyte subsets analyzed by flow FISH) less than the 1st percentile for age are diagnostic of any form of DC, including HH. We identified a germline autosomal recessive (AR) mutation (p.Arg1264His) in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The minor allele frequency of this variant is ∼0.0001 in public databases of 9600 individuals. The affected individuals in these families are homozygous for this mutation, which affects three isoforms of RTEL1. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In both families, each parent was a healthy, heterozygous carrier of one mutant allele. Haplotypes were reconstructed from twelve common SNPs based on allele sharing in the unaffected siblings and parents. No recombinants were seen in either family and the segregating risk haplotype was identical in affected individuals from both families. Thus, p.Arg1264His is carried on a common haplotype, likely from a common AJ founder. We determined the carrier frequency of the p.Arg1264His mutation, as well as three other mutations, p.Gly763Val, p.Met516Ile and p.Arg998Ter, which were recently reported and possibly found in individuals of AJ ancestry. DNA was derived from 1,048 self-described AJ individuals enrolled in the Dor Yeshorim program. Consent form information included that patient material would be used for clinical testing and that excess material would be de-identified and used for research purposes. The mutations were genotyped by TaqMan assays and heterozygous carrier samples were confirmed by Sanger sequencing with stringent quality control. No individuals in this study carried the p.Gly763Val or p.Arg998Ter minor alleles. Two individuals (0.19%) were carriers of the p.Met516Ile mutation. Notably, 1% (10 of 1,032) of AJ individuals in this study were carriers of the p.Arg1264His mutation in RTEL1. This carrier frequency of 1 in 100 is similar to that of the FANCC AJ mutation and many other FA founder populations, such as FANCA in South African Afrikaners, Spanish Gypsies, Brazilians, Tunisians, and Moroccans, as well as FANCG in Sub-Saharan Blacks, and BRCA2 (FANCD1) in the US general population. A carrier frequency of 1 in 100 is similar to that of genetic disorders found in the AJ population recommended for screening by the American College of Medical Genetics. Based on this, we suggest that genetic counseling and RTEL1 p.Arg1264His carrier screening for the HH variant of DC be offered to individuals of AJ ancestry. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3192 Poster Board III-129 Introduction Bone marrow fibrosis has been reported in many benign and malignant disorders, and it may be associated with a poor prognosis in patients with chronic idiopathic myelofibrosis and adult myelodysplastic syndrome (MDS). There are no data on the incidence or significance of bone marrow fibrosis in patients with the inherited bone marrow failure syndromes (IBMFS), genetic disorders characterized by cytopenias, distinctive clinical features, varied molecular pathways and high risks of MDS and acute myeloid leukemia. We have now studied marrow fibrosis in the four most common IBMFS: Fanconi anemia (FA), Diamond-Blackfan anemia (DBA), dyskeratosis congenita (DC), and Shwachman-Diamond syndrome (SDS). Patients and Methods Blinded bone marrow biopsies were analyzed from 42 patients: 12 FA, 13 DBA, 13 DC, and 4 SDS. Reticulin fibrosis was graded on a scale of 1-4 according to the quantity and pattern of distribution of reticulin. The frequencies of abnormalities in marrow fibrosis, cellularity, MDS, cytogenetic clones, blood counts, erythropoietin levels and treatment were compared between the disorders. Fisher exact test was used to compare frequencies and two-sided Wilcoxon rank sum test was used to compare continuous variables. P value of less than 0.05 was considered statistically significant for all tests. Results See Table. Patients with FA, DBA and DC were older than those with SDS; there was an excess of females with FA and males with DC and DBA. Patients with FA, DC and SDS had multilineage cytopenias, while most patients with DBA had only anemia. All patients with FA and DC had bone marrow hypocellularity; it was less frequent in those with DBA or SDS (P

Description: This study was designed to evaluate the utility of flow-FISH telomere length measurement in white blood cells (WBC) as a screening test for Dyskeratosis congenita (DC). We studied 26 patients: 17 with DC, 1 silent carrier (clinically normal; mutation in TERC), 4 with the Hoyeraal-Hreidarsson variant (HH), and 4 with Revesz Syndrome. Five had mutations in DKC1, 5 in TERC, and 2 in TERT. 23 had hematologic abnormalities, 19 had 2 or 3 of the DC diagnostic triad (lacey pigmentation, dyskeratotic nails, and leukoplakia), and 4 had soft signs of DC. We evaluated 54 first-degree relatives of DC patients, 16 Fanconi Anemia patients (FA), 14 with Diamond-Blackfan Anemia (DBA), 5 with Shwachman Diamond Syndrome (SDS), and 10 with other possibly inherited cytopenias (Other). Telomere length was measured in granulocytes and lymphocyte subsets by automated multicolor flow-FISH; results were compared with age-matched values from 400 normal controls. “Very low (VL)” telomere length was defined as a mean telomere length below the normal first percentile for age and specific WBC type. We observed VL telomeres in all subsets in the silent carrier, all HH and Revesz patients, and 15/17 with DC. Eight of 51 DC relatives had VL telomeres in granulocytes versus 2/54 with VL telomeres in lymphocytes. The sensitivities for distinguishing a DC patient from an unaffected relative were 92% in lymphocytes and 96% in granulocytes; the specificities were 96% and 98%, respectively; the sensitivity and specificity for VL telomeres in both cell types were 96% and 96%. The silent carrier with a TERC mutation developed thrombocytopenia, hypocellular marrow, and a cytogenetic clone during follow-up. The 2 DC relatives with VL telomeres in lymphocytes were from a family without a known mutant gene; they may also be silent carriers. The latter possibility disqualified an HLA-matched sibling as a donor for DC-related aplastic anemia, because of engraftment concerns; another sibling donor with normal telomere length was selected. VL granulocyte telomeres were observed in 5/16 FA, 3/14 DBA, 1/5 SDS, and 1/10 Other patients, versus 2/16 FA, 1/14 DBA, 1/5 SDS, and 0/10 Other in lymphocytes, and in both lineages in only 1 each of FA, DBA, and SDS. The sensitivity and specificity for distinguishing DC from non-DC patients using VL telomeres in both lineages were 96% and 93%, respectively. Only DC patients had consistently VL telomeres in all cell subsets. Flow-FISH telomere length measurement provides a sensitive and specific method for identifying patients with DC among families, regardless of mutation status, and distinguishes patients with DC from those with other inherited or acquired marrow failure syndromes. It may also help to detect silent carriers, and facilitate identification of mutations in other telomere biology genes. Our data suggest that the diagnostic triad, soft physical findings and/or bone marrow failure may not be required for the diagnosis of DC. Correct diagnosis of DC will enhance genetic counseling and hematologic management.

Description: Abstract 511 Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red blood cell aplasia, variable physical anomalies, and increased risk of leukemia, myelodysplastic syndrome, lymphoma, and certain solid tumors, including osteosarcoma. DBA has been considered to be a disorder of ribosomal biogenesis because approximately 50% of cases are due to a mutation or deletion in 1 of 9 ribosomal protein genes (RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, or RPS26). However, mutations in GATA1, a hematopoietic transcription factor, have recently been reported to also cause DBA. DBA is inherited in an autosomal dominant manner, but de novo germline mutations have also been reported. Our IBMFS cohort study conducts detailed clinical evaluations and medical record review of patients with DBA and their family members. We evaluated a large family with DBA in which mutation testing for the 10 known genes was negative. The male proband had steroid-responsive anemia as a child; he was in remission until he was treated with chemotherapy for squamous cell lung cancer at age 55 years. His healthy sister is an obligate carrier because her daughter was diagnosed with DBA as a child; her daughter had steroid-responsive anemia. The proband's maternal 1st cousin had steroid-responsive DBA as a child which relapsed during pregnancy. Her 3 children (the proband's 2nd cousins) had DBA. One died due to complications of transfusion-related iron overload, one had successful hematopoietic stem cell transplantation for steroid-refractory and transfusion-dependent anemia at age 26 years, and one has been off treatment and in remission for over 20 years. Except for the proband with lung cancer and a transfusion-dependent individual, all affected individuals had elevated red blood cell adenosine deaminase consistent with DBA. We performed whole-exome sequencing on the 5 clinically affected individuals (a male proband, his affected first cousin, 2 affected second cousins, and an affected niece), his obligate carrier sister, and the unaffected father of the proband's niece. Genomic DNA was used to create an enriched multiplexed sequencing library (Nimblegen v2); this was followed by paired-end sequencing using an Illumina HiSeq™. We detected a total of 229,024 exonic nucleotide variants across this family, including 2,484 uncommon (minor allele frequency