Chromosome Breakage Test Research Articles

The Significance of the Next Generation Targeted Sequencing in the Precise Diagnosis of Pediatric Acquire Aplastic Anemia and Inherited Bone Marrow Failure Syndromes

▪BackgroundDiagnosis of inherited bone marrow failure syndromes (IBMFs) depend on classic clinical manifestation including early onset, physical anomalies, family history of cancer and/or bone marrow failure and chromosome breakage testing (MMC and/or DEB), mutation analyses and bone marrow chromosome analyses. At present, more than 70 pathogenic gene mutations had been identified. However, in some patients, physical anomalies is absent or delayed, and were misdiagnosed as acquired aplastic anemia(AA). Genetic analysis is very important to establish a precise diagnosis, predict cancer risk, direct treatment and genetic counseling. In this study, we focus on the application of next generation targeted sequencing in precise diagnosis of pediatric acquired AA/IBMFs, and the association between genetic abnormalities and clinical and laboratory characteristics.MethodsWe designed a targeted sequencing assay to test a panel of 417 genes. The panel contain reported gene associated with IBMFs and other diseases need be differentiated. Pediatic patients (≤14 year old) with suspected diagnosis of AA/IBMFs were enrolled. Peripheral blood (PB) DNA was used to genetic analysis and oral epithelia cells or PB DNA from their parents were used to identify somatic mutations and unreported polymorphism. All the results were validated by Sanger sequencing.ResultsThe average coverage of targeted region was 98.15%, and the average sequencing depth was 315.9×. Totally, 283 patients were enrolled, including 176 clinically diagnosed acquired AA, 51 Fanconi anemia (FA), 8 dyskeratosis congenital(DKC), 30 Diamond-Blanckfan anemia(DBA), 15 congenital neutropenia(CN), and 3 congenital thrombocytopenia. Totally, 19% subjects had IBMFs related genetic mutations. In the patients who were clinically diagnosed as acquired AA patients, about 7% had IBMFs related disease-causing genetic mutations. Finally, 7 patients were genetically diagnosed as FA, 2 were DKC, 1 was WAS and 1 was SDS. In patients who were clinically diagnosed as FA, 33.4% had FANC related gene mutations. Telomere associated gene mutations were detected in 75% of clinical diagnosed DKC. For patients clinically diagnosed as DBA and CN, 36.7% and 20% were detected disease-causing mutations. After genetic screening, 2 patients who had been diagnosed as FA were modified as WAS and 1 DBA was modified as SDS.Only 26% genetic diagnosed IBMFs patients had family history of bone marrow failure, leukemia, tumor or physical anomalies. Compared with acquired AA, patients with genetic diagnosed FA were more likely to have physical anomalies of short stature and development retardation, Cafe au lait spots and finger or toe malformation(P<0.001).However, 46% patients with IBMFs did not have any type of physical anomalies. Moreover, there were only 24% patients with genetic diagnosed IBMFs had positive results of MMC induced chromosome breakage test or SCGE, and both the examinations could not differentiate subtype of IBMFs. FANCD2 mono-ubiquitination test were performed recently. However, even in the genetically confirmed FA, the positive rate was only 18% (2/11). And, there were positive results in some acquired AA patients.For FA patients with definitely genetic mutations, 62.5%(15/24) were compound heterozygous mutations,37.5%(9/24) were homozygous mutations.Mutational frequencies of FANC were: FANCA 65%, FANCD2 23%, FANCG 9%, FANCI 9% and FANCB 4%. For the mutated type, the frequencies of missense, frameshift, nonsense, splicing mutation were 42%, 26%, 16%, 16%. In our study, there were 4 undetermined patients met the clinical diagnostic criteria of FA, and having heterozygous damaged mutations in FANC genes.ConclusionIn conclusion, our IBMFs associated genes targeted sequencing assay is an effective strategy for precise diagnosis of bone marrow failure diseases, especially for those without family history or physical anomalies. However, nearly half of the clinically diagnosed IBMFs patients in our study were not detected the disease-causing mutations. This may be due to the mutations in the intron area, or large fragment deletion, which cannot be detected by targeted sequencing. And the novel gene involved in IBMFs need further study. DisclosuresNo relevant conflicts of interest to declare.

Profiling Fanconi Anemia Gene Mutations among Iranian Patients.

Fanconi anemia (FA) is a rare genetic syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. FA is usually inherited as an autosomal recessive condition. This disease is genetically heterogeneous and mutations in 16 different genes have been identified in FA patients to date. An accurate diagnosis needs detection of pathogenic variations in the FA genes along with positive results from chromosome breakage test. In this study, 48 families with at least 2 affected FA patients and positive chromosome breakage test were enrolled from the Iranian population. Molecular analysis of FA genes was performed using Next Generation Sequencing (NGS) method and Multiple Ligation Dependent Probe Amplification (MLPA). Causal mutations for 30 (63%) patients were identified in homozygous or compound heterozygous forms. FANCA had the highest mutation frequency rate (83%) followed by FANCG (10%), FANCD2 (3%) and FANCL (3%). A significant proportion (44%) of FANCA mutations were large rearrangements. Genetic testing for FA patients improves the accuracy of diagnosis and also will be essential for genetic counselling and prenatal diagnosis for future pregnancies in the family. Availability of NGS technology has made the screening of all known FA genes at once more practical and affordable.

Diagnosis of Fanconi Anemia By Chromosome Breakage Tests Using 3 Different Scoring Systems and Whole Genome Sequencing Among Patients with Aplastic Anemia in Korean

BACKGROUD: Fanconi anemia (FA), an inherited bone marrow failure syndrome with impaired DNA repair system, is characterized by cytopenias, congenital abnormalities, and predisposition to malignancy as a consequence of chromosomal instability and hypersensitivity to DNA interstrand cross-linking agents. Differential diagnosis of FA and aplastic anemia requires integrated work-up including physical findings, bone marrow histologic findings and chromosome breakage test. Yet, there have been no consensus criteria for chromosome breakage test, which depend on each laboratory's own decision. The aim of our study was 1) to investigate the incidence of FA showing positive results for chromosome breakage test among patients diagnosed with aplastic anemia, and 2) to investigate the frequency of the gene mutations related to inherited bone marrow failure syndrome in patients with aplastic anemia. In addition to chromosome breakage test, we performed whole genome sequencing with bone marrow mononuclear cells in 18 pediatric patients with aplastic anemia whose bone marrow specimen was available.METHOD: We reviewed total 79 chromosome breakage tests from 67 patients who had been on suspicion of aplastic anemia between May 2005 and April 2015. MMC and DEB stress test were performed at concentration of 50ng/mL and 100ng/mL both on peripheral blood of suspicious patients and normal controls, respectively. The scoring of chromosome breakages test was performed, based on widely used 3 different scoring systems: those proposed by Jean Soulier, Barch MJ, and Arleen D. Auerbach. In each cases, we applied 3 different scoring systems and compared the concordance rate. In 16 among 67 patients, we performed whole genome sequencing.RESULTS: The median age of the pediatric patients was 11years (range, 7months - 19 years) and the male-to-female ratio was 1.39:1. Of 67 enrolled patients, 8 had been tested twice or 3 times because of ambiguous results. In these cases, we chose the last results. Five of 67 patients satisfied the all three criteria mentioned above, which shows 7.5% (5/67) of positive rates. Other 3 of 67 patients met only one or more of Soulier's prerequisites. Among those, one fulfilled both Barch MJ's system and Aeurbach's, and remain 2 patients were positive in each system, respectively. Mutation variants of BMF syndrome related genes were detected in 25% (4/16 patients); RPS19 (1 patient), PAX5 (1 patient), and FANC (3 patient). Inherited predisposition to myeloid leukemia related genes were detected in 56.3% (9/16 patients) and gene variants were MSH6 (5 patients), ATM (2 patients), PMS2 (1 patients), and MLH1 (1 patients). Coexisting somatic mutations of oncogene (ERB2) was detected in 6.3% (1/16 patients). Among 3 patients with fanconi anemia gene mutations, 2 patients showed positive results for chromosome breakage test and the other1patient showed negative results for chromosome breakage test.CONCLUSION: The frequency of FA based on chromosome breakage test among patients with pancytopenia suspicious of aplastic anemia was 7.5% by Soulier's prerequisites, but 9.0% when based on either of 3 different criteria. Molecular testing can additionally detect FA in 4 (25.0%) among 16 patients showing negative result by chromosome breakage test. Our study shows it is necessary to standardize a diagnostic scoring system as well as to develop complementary molecular test for accurate diagnosis of FA. DisclosuresNo relevant conflicts of interest to declare.

Guidelines for the diagnosis and management of adult aplastic anaemia.

Sally B. Killick (Writing Group Chair), Nick Bown, Jamie Cavenagh, Inderjeet Dokal, Theodora Foukaneli, Peter Hillmen, Robin Ireland, Austin Kulasekararaj, Ghulam Mufti, John A Snowden, Sujith Samarasinghe, Anna Wood (BCSH Task Force Member), Judith C.W. Marsh on behalf of the British Society for Standards in Haematology. The Royal Bournemouth and Christchurch Hospitals NHS Foundation Trust, Bournemouth, Northern Genetics Service, Newcastle upon Tyne, St Bartholomew’s Hospital, Barts Health NHS Trust, London, Barts and The London School of Medicine and Dentistry, Queen Mary University of London and Barts Health NHS Trust, London, Addenbrooks Hospital, University of Cambridge, Cambridge, Leeds Teaching Hospitals, Leeds, Kings College Hospital NHS Foundation Trust, London, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield Great Ormond Street Hospital for Children NHS Foundation Trust, London, West Hertfordshire NHS Trust, Watford.

Sensitivity to chromosomal breakage as risk factor in young adults with oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) may develop in young adults. In contrast to older patients, the well-known etiological factors, exposure to tobacco and alcohol, play a minor role in the carcinogenesis in this patient group. It has been suggested that an intrinsic susceptibility to environmental genotoxic exposures plays a role in the development of OSCC in these patients. The hypothesis was tested whether young OSCC patients have an increased sensitivity to induced chromosomal damage. Fourteen OSCC patients with an average age of 32 years (range 20-42) were selected. Peripheral blood lymphocytes and skin fibroblasts of patients and 14 healthy controls were subjected to the chromosome breakage test with Mitomycin C. This test is routinely used to identify Fanconi anemia patients, who are well-known for their inherited high sensitivity to this type of DNA damage, but also for the high risk to develop OSCC. Human papilloma virus status of the carcinomas was also determined. None of the 14 young patients with OSCC had an increased response in the MMC-chromosomal breakage test. All tumors tested negative for human papilloma virus. No evidence was obtained for the existence of a constitutional hypersensitivity to DNA chromosomal damage as a potential risk factor for OSCC in young adults.

Current insights into the diagnosis and treatment of inherited bone marrow failure syndromes in China.

Inherited bone marrow failure syndromes (IBMFs) account for 20% of pediatric BMFs. Although recommendations for the diagnosis and treatment of IBMFs in China have been published recently, improvements are still needed in making precise diagnoses and properly treating pediatric patients with IBMFs. This review provides current insights into IBMFs in China. The data of our single institution data showed that pediatric patients with IBMFs accounted for 7.4% of BMFs. However, the number of reported cases with IBMFs may be underestimated than the actual morbidity in China because of limitations in the detection approaches and lacking of awareness of these diseases in local hospitals. Although patients with IBMFs are candidates for bone marrow transplantation or gene therapy, their phenotypic heterogeneity can delay or incompetent diagnosis. The golden standard test for Fanconi anemia is the chromosome breakage test, but it can be completed by few hospital and diagnostic companies in China. In addition, there are still no consistent standardized testing methods for other rare IBMFs. Recently, the combined application of targeted capture and next-generation sequencing (NGS) provides and accurate and efficient diagnostic method for IBMFs.

Comparison of rabbit antithymocyte globulin and Jurkat cell-reactive anti-t lymphocyte globulin as a first-line treatment for children with aplastic anemia

The purpose of this study was to investigate the effects of rabbit antihuman thymocyte globulin (R-ATG) and Jurkat cell-reactive anti-T lymphocyte globulin (ATG-F) in the treatment of childhood aplastic anemia (AA) and compare their efficacy and side effects. A total of 53 children with AA were analyzed in the present study, including 32 cases of severe AA, 10 cases of very severe AA and 11 cases of transfusion-dependent nonsevere AA. While receiving immunosuppressive therapy (IST), 29 and 24 patients, all of whom received long-term oral supplement with cyclosporin A (CSA), androgen, and traditional Chinese medicines, were treated with R-ATG and ATG-F, respectively. If necessary, the patients were also given supportive care such as component transfusion and/or infection control. Absolute counts of peripheral blood lymphocyte at various time points were dynamically measured after ATG therapy. According to the International AA Treatment and Effect standards, we found that there were no statistically significant differences in the response rate (70.83% vs. 68.97%, p > 0.05) and the overall survive rate (83.33% vs. 82.76%, p > 0.05) between the ATG-F and R-ATG groups. In addition, no obvious differences were observed between these two groups in the response time, efficacy in severe AA and very severe AA, or the incidence rates of ATG-related adverse reactions. After ATG treatment, the extent of peripheral blood lymphocyte reduction and duration in peripheral blood were similar between the ATG-F and R-ATG groups. The results of this study showed that ATG-F and R-ATG had similar efficacy and adverse reactions in the first-line treatment of childhood AA, despite being derived from different immunogens.

Diagnosis of Fanconi Anemia: Mutation Analysis by Next-Generation Sequencing

Fanconi anemia (FA) is a rare genetic instability syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. Fifteen genetic subtypes have been distinguished. The majority of patients (≈85%) belong to the subtypes A (≈60%), C (≈15%) or G (≈10%), while a minority (≈15%) is distributed over the remaining 12 subtypes. All subtypes seem to fit within the “classical” FA phenotype, except for D1 and N patients, who have more severe clinical symptoms. Since FA patients need special clinical management, the diagnosis should be firmly established, to exclude conditions with overlapping phenotypes. A valid FA diagnosis requires the detection of pathogenic mutations in a FA gene and/or a positive result from a chromosomal breakage test. Identification of the pathogenic mutations is also important for adequate genetic counselling and to facilitate prenatal or preimplantation genetic diagnosis. Here we describe and validate a comprehensive protocol for the molecular diagnosis of FA, based on massively parallel sequencing. We used this approach to identify BRCA2, FANCD2, FANCI and FANCL mutations in novel unclassified FA patients.

Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing.

Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed.

Fanconi Anemia

Fanconi anemia (FA) is the most frequent inherited cause of BM failure (BMF). Fifteen FANC genes have been identified to date, the most prevalent being FANCA, FANCC, FANCG, and FANCD2. In addition to classical presentations with progressive BMF during childhood and a positive chromosome breakage test in the blood, atypical clinical and/or biological situations can be seen in which a FA diagnosis has to be confirmed or eliminated. For this, a range of biological tools have been developed, including analysis of skin fibroblasts. FA patients experience a strong selective pressure in the BM that predisposes to clonal evolution and to the emergence in their teens or young adulthood of myelodysplasia syndrome (MDS) and/or acute myeloid leukemia (AML) with a specific pattern of somatic chromosomal lesions. The cellular mechanisms underlying (1) the hematopoietic defect which leads to progressive BMF and (2) somatic clonal evolutions in this background, are still largely elusive. Elucidation of these mechanisms at the molecular and cellular levels should be useful to understand the physiopathology of the disease and to adapt the follow-up and treatment of FA patients. This may also ultimately benefit older, non-FA patients with aplastic anemia, MDS/AML for whom FA represents a model genetic condition.

High Incidence of Fanconi Anemia in Patients with a Morphological Picture Consistent with Refractory Cytopenia of Childhood

Abstract 2780 Introduction:Refractory cytopenia in childhood (RCC) is the most common subtype of myelodysplastic syndrome (MDS) in children. Differential diagnosis from inherited bone marrow failure (IBMF) such as Fanconi anemia (FA) remains an intriguing challenge, because most patients with RCC have a hypocellular bone marrow (BM) and dysplastic features in haematopoiesis are observed in both RCC and IBMF. Moreover the spectrum of phenotypic findings in FA is extremely wide. Some FA patients have a mild phenotype without malformation. The purpose of this study is to estimate the incidence of FA in an RCC cohort without a full clinical feature of FA, but subsequently diagnosed by chromosome breaking test.Patients and Methods: Between 01/2007 and 12/2010 reference pathologists of the European Working Group of MDS in Childhood (EWOG-MDS) provided a morphological report consistent with RCC in 137 children studied in Germany. Seventeen patients with hypercellular BM or abnormal karyotype, 2 patients, in whom dyskeratosis congenital was diagnosed after initial inclusion and one patient, in whom chromosome breaking test was not performed, were excluded. Results:Seven of remaining 117 patients had facial and/or skeletal anomalies typically noted in FA and one patient had a brother with FA. In these 8 patients, FA had been suspected by their local physicians (group FA-1). Nine patients (8.3%) without these typical anomalies were subsequently diagnosed of FA by chromosome breakage test (group FA-2). The diagnosis of RCC was finally made in the remaining 100 patients with negative chromosomal breakage test (group RCC). The clinical features of patients in each group are summarized in the Table. The mean corpuscular volume of red cells (MCV) was elevated (> +2SD) for ages in all patients with FA, but only 42 % in patient with RCC. In some children of group FA-2 additional non-haematological abnormalities were also observed. However, they were not evident and or typical to prompt the treating physicians to suspect FA. A few patients in the group RCC also had some physical anomalies, not specific for any of the known IBMF disorders. Possibly that other known or not yet described IBMF disorders remain uncovered in children with “de novo” RCC. Conclusion:Our results illustrate that the same haematological features and congenital anomalies can be noted in FA and RCC. More importantly, they indicate that the exclusion of FA by a chromosomal breakage test or other methods is mandatory in all patients prior to diagnosis RCC. Chromosomal breakage analysis may identify patients with FA in 8% of patients with a morphological description of RCC without a full clinical picture of FA.TableClinical characteristic of patients with Fanconi anemia (FA) and refractory cytopenia in childhood (RCC)FA-1 (n=8)FA-2 (n=9)RCC (n=100)Age of diagnosis (years): median (range)5 (2–12)9 (5–16)11 (1–18)ANC (x109/L): median (range)1.2 (0.3–2.8)0.6 (0.3–1.6)0.6 (0.01–4.1)MCV: low/normal/elevated0/0/80/0/90/41/30 (n=71)Non-haematological anomaliesDysmorphic face4—0Hand or arm anomalies3—2Microcephaly541Small stature744Skin pigmentation or depigmentation540Hearing loss111Mental retardation113Renal and urogenital anomalies340Gastrointestinal malformations200Other1 (spastic hemiparasis)ANC: absolute neutrophil count, MCV: mean corpuscular volume of red cells, FA-1: FA with typical facial and/or skeletal anomalies for FA (n=7) or with a family history of FA (n=1), FA-2: FA without these features Disclosures:No relevant conflicts of interest to declare.

Diagnostic Overlap between Fanconi Anemia and the Cohesinopathies: Roberts Syndrome and Warsaw Breakage Syndrome

Fanconi anemia (FA) is a recessively inherited disease characterized by multiple symptoms including growth retardation, skeletal abnormalities, and bone marrow failure. The FA diagnosis is complicated due to the fact that the clinical manifestations are both diverse and variable. A chromosomal breakage test using a DNA cross-linking agent, in which cells from an FA patient typically exhibit an extraordinarily sensitive response, has been considered the gold standard for the ultimate diagnosis of FA. In the majority of FA patients the test results are unambiguous, although in some cases the presence of hematopoietic mosaicism may complicate interpretation of the data. However, some diagnostic overlap with other syndromes has previously been noted in cases with Nijmegen breakage syndrome. Here we present results showing that misdiagnosis may also occur with patients suffering from two of the three currently known cohesinopathies, that is, Roberts syndrome (RBS) and Warsaw breakage syndrome (WABS). This complication may be avoided by scoring metaphase chromosomes—in addition to chromosomal breakage—for spontaneously occurring premature centromere division, which is characteristic for RBS and WABS, but not for FA.

Changing to a New Donor for Second Hematopoietic Stem Cell Transplantation in Fanconi Anemia Seems to Improve Survival.

Abstract 2294Poster Board II-271This retrospective analysis of the EBMT ProMISe database was designed to investigate the role of donor choice for second hematopoietic stem cell transplantation (HSCT) in Fanconi Anemia (FA). Our hypothesis was that using a different donor for the second HSCT would increase the probability of survival, the patient not being sensitized to donor antigens as a result of the first HSCT. We conducted a survey of the EBMT ProMISe database in order to identify FA patients transplanted more than once for bone marrow failure and retrospectively analyzed their data to determine overall survival and presence of acute GvHD. A donor for the second HSCT was defined as the “same” as for the first if both donors had identical: birth date, relation to patient (identical sibling or not), gender and family relation or donor number. A donor was “different” when this information indicated that a different person donated for second HSCT. One hundred and three patients were eligible for the study by the following criteria: diagnosis of Fanconi Anemia (confirmed by chromosome breakage test), presentation with aplastic anemia (AA) and having undergone two HSCTs between August, 1980 and December, 2007. FA patients with myelodysplasia or acute leukemia were excluded. After retracing data in the database, additional questionnaires were sent out to 44 EBMT centers that had performed second HSCTs for FA-AA, asking for supplementary data. In 80 cases (study population), enough information was collected to identify first and second donors. Forty four subjects (25 males and 19 females) received a second HSCT from the “same donor” (SD) and 36 patients (15 males and 21 females) from a “different donor” (DD). The mean age of the study population at second HSCT was 10.7 years (range, 1.8-34.5). Median interval between first and second HSCT was 63 days.At first HSCT patient gender, age at HSCT, use of cyclophosphamide and irradiation in the conditioning regimen and type of rejection were not differently distributed in the SD and DD group whereas significantly more HLA identical sibling donors were used in the SD (31%) vs. DD (11%) group (p=0.027). In 68.2% of the SD group bone marrow was used vs. in 52.7% of the DD group (p=0.002). Median cell dose in SD group was 9×106 CD34+/kg and in DD group 1.8×106 CD 34+/kg (p=0.002). At second HSCT age at HSCT, use of cyclophosphamide and irradiation in the conditioning regimen, dose of CD34+ cells and cell source were not differently distributed between SD and DD groups whereas significantly more HLA identical sibling donors were used in SD (31%) vs. DD (11%) group (p=0.027).Overall survival of all 80 patients at one year from second transplant is 36%. Probability of overall survival after 7 years is 35%. An interval between first and second HSCT of >80 days resulted in better survival (p=0.007). Overall survival at 7 years is 41% in DD and 26% in SD patients (Cox regression, HR 0.483, p=0.017). This difference remained significant after correction for the confounding effects of irradiation in first HSCT and relation donor/patient (identical sibling or not). Rejection rate after second HSCT was similar in SD and DD group (54.5%). Death occurred in 73% of SD vs. 56% of DD. Causes of death included infections (70% in SD vs. 48% in DD) and rejection (39% in SD and 38% in DD). Noteworthy more than one major cause of death was often found in the same patient. Only 2 secondary tumors were reported in the whole group. Acute GvHD gr II-IV was not significantly different in SD vs DD groups (multivariate logistic regression models, OR 1.642, p=0.47).Our data suggest that after having failed a first transplant, using the same or a different donor does not affect the occurrence of aGVHD, whereas change of donor would improve the survival of FA patients in need of a second HSCT for AA. Mechanisms underlying rejection of a first HSCT should be studied more in depth. International centers should collaborate in finding the most appropriate regimen for first and second transplants in FA. Disclosures:No relevant conflicts of interest to declare.

Natural Course of Neonatal Progeroid Syndrome

Several progeroid disorders presenting a specific "old-man" appearance since birth or childhood have been described. Here, five patients with a history of severe intrauterine and postnatal growth retardation and pseudohydrocephaloid cranium noted after birth that were suggestive of neonatal progeroid syndrome (NPS) or Wiedemann-Rautenstrauch syndrome are reported. We discuss the natural course of the syndrome. A series of anthropometric measurements, imaging, dual energy X-ray absorptiometry, and endocrine investigations to assess metabolic complications such as hyperinsulinemia and dyslipidemia were performed on these five patients who were followed for 1-7 years. Screening of inborn errors, karyotyping, chromosomal breakage rates and DNA mutational studies with direct sequencing of LMNA, ERCC8 and ZMPSTE24 genes were also performed. Generalized lipodystrophy was noted in all patients except for regions such as the cheeks, hands and feet. All cases had failure to thrive, microcephaly, ear dysplasia, laryngomalacia, hearing impairment, gastro-esophageal reflux disease, constipation, abnormal dentition, dermatitis/acrodermatitis enteropathica, hyperpigmentation of the skin, very low insulin-like growth factor I levels with delayed bone age, relative hypolipidemia, initial camptodactyly/joint contracture, progressive kyphoscoliosis, osteoporosis with loose joints, ventriculomegaly, and generalized organic aciduria. Other findings included inguinal hernia, hypothyroidism or persistent hyperthyrotropinemia, cryptorchidism, hip dysplasia, growth hormone deficiency, cloudy cornea with congenital glaucoma, neonatal teeth, cardiac defects, basal ganglia calcification and seizure disorder. These patients with NPS did not show hyperinsulinemia or dyslipidemia. Their karyotypes were all normal, while the chromosomal breakage test showed markedly increased breakage rates in four patients. LMNA, ERCC8, or ZMPSTE24 gene mutations could not account for the disorders in these patients. Four patients died after sepsis or aspiration pneumonia at the age of 1.1, 4, 6.2 and 7.5 years. Increased chromosomal breakage and the presence of basal ganglia calcification after early childhood suggest that DNA repair defects are involved in the pathogenesis of this disorder. This rare disorder represents a complex of symptoms with unknown cause and pathogenesis, and more than one disease may account for the clinical variability of NPS.

FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L-) was not detected in 77 patients (91.7%). In 2 patients (2.4%), there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L-) and 5 patients (5.9%) had both isoforms (FANCD2S+/FANCD2L+). This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84) and specificity of 100% (98/98). This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L-) or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-), to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+) require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

Pregnancy after Allogeneic Hematopoietic Stem Cell Transplantation in Fanconi Anemia Patients: Multi-Institution Cases Report of Female Fertility Recovery

Introduction. Fanconi anemia (FA) is a rare autosomal recessive syndrome characterized by chromosome instability. Main clinical features include progressive bone marrow failure, skeletal defects, increased susceptibility to malignancy and reduced fertility. Moreover, most recipients of allogeneic hematopoietic stem cell transplantation (HSCT) suffer from secondary infertility owing to gonadal damage from myeloablative conditioning. We report a rare clinical situation of FA patients pregnancy after allogeneic HSCT.Methods. Retrospective analysis of transplanted FA female patients from 1982 to 2008. Five centers participated in this study on behalf of Aplastic Anaemia Working Party-EBMT. Medical records were reviewed and data collected on a standard case report form including detailed information on diagnosis, transplant procedure, gynecological and obstetrics follow-up.Results. Among 387 transplanted FA patients we identified 202 females who performed a HSCT with a median age of 10,5 years. Five patients became pregnant after the procedure and one of them, twice. They all had their FA diagnosis confirmed by chromosomal breakage test and a bone marrow aspirate with severe hypoplasia/aplasia. Median age at transplantation was 12 years (range 5–17 years). All patients received myeloablative conditioning regimens (cyclophosphamide with or without thoraco-abdominal irradiation) before a bone marrow transplantation, 4 patients from HLA matched sibling donors and 1 from unrelated donor. During follow-up, 4 patients presented signs of ovarian failure (amenorrhea, low levels of FSH/LH and high levels of estradiol). Apart from 1 patient who spontaneously recovered regular menses, the other three received hormonal replacement therapy (HRT) for this purpose. Pregnancy occurred from 3,5 to 17 years after transplant. One patient had an early interruption with a caesarian section at 27 weeks because of an imminent HELLP syndrome. Other pregnancies were uneventful. Among the newborns, there were no FA positive tests, no congenital anomalies and all of them had normal growth and development. Patients remain alive with a median follow-up of 12 years after transplantation with normal hematological status.Conclusion. Fertility recovery after HSCT can result from incomplete depletion of the ovarian follicle reserve. HRT should begin promptly to prevent the early and late unwanted effects related to oestrogen deficiency after HSCT. Recovery of normal ovarian function and a viable pregnancy, is a realistic possibility even in Fanconi anemia patients following allogeneic SCT.

A Case of Fanconi Anemia Diagnosed by a Chromosome Breakage Test with Skin Fibroblasts

Fanconi anemia is an autosomal recessive disease that's characterized by congenital anomalies, defective hematopoiesis and a high risk of developing acute myeloid leukemia and certain solid tumors. The clinical phenotype is extremely variable; therefore, the diagnosis is frequently delayed until the pancytopenia appears. Chromosomal instability, especially on exposure to an alkylating agent, may be seen in affected patients and it is the basis for a diagnostic test. This cellular phenotype can be demonstrated in cultured T cells, B cells, fibroblasts and fetal cells cultured from both amniotic fluid and chorionic villi. But so- matic mosaicism may make the diagnosis of Fanconi anemia difficult because of inconclusive chromosome breakage studies. If the test is negative in lymphocytes and yet the clinical setting is highly suspicious, then the skin fibroblasts must be assessed. Because skin fibroblasts are somatic cells, a definitive test can be performed on primary skin fibroblasts. In this report we describe a case of Fanconi anemia that was diagnosed with the use of cultured skin fibroblasts, and this was despite the normal breakage studies in the peripheral blood. (Korean J Hematol 2008;43:62-67.)

CURRENT DIAGNOSIS OF INHERITED BONE MARROW FAILURE SYNDROMES

Prompt and accurate diagnosis is required for optimal treatment and genetic counseling of patients with inherited bone marrow failure syndromes (IBMFS). However, the diverse clinical picture of these syndromes and their rareness is often associated with diagnostic difficulties. Recently, an improved diagnostic approach is possible by the cloning of many of the causative genes. Fanconi anemia (FA) patients belong to at least 12 complementation groups, of which 11 genes have been cloned. An approach combining an induced chromosomal breakage test, detection of FANCD2-L by Western blot analysis, complementation group analysis, and detailed mutation analysis enables unraveling the causative mutation in the majority of patients. With the use of such strategies, genotype/phenotype correlations in FA are evolving. In dyskeratosis congenita mutations in DCK1, TERC, and TERT genes have been identified, but mutations have been found in less than half of these patients. In patients with Shwachman-Diamond syndrome, mutations in the SBDS gene were found in approximately 90% of patients. In Diamond-Blackfan anemia the RSP19 gene is mutated in 20–25% of patients. Heterozygote ELA2 mutations are found in 60–80% of severe congenital neutropenia patients. All patients with congenital amegakaryocytic thrombocytopenia have mutations in the thrombopoietin receptor gene c-Mpl.

The Challenge of Diagnosing Fanconi Anemia in Patients with Bone Marrow Failure (BMF): A Study in 82 BMF Patients.

The Bone Marrow Failure (BMF) syndromes comprise a variety of distinct acquired or inherited clinical entities. Early distinction between syndromes has clear implications in the disease management and outcome. Fanconi anemia (FA), the most frequent cause of inherited BMF, is usually associated with congenital abnormalities, progressive cytopenia, chromosome fragility, and cancer susceptibility. However, due to a high clinical variability and/or the potential emergence of revertant hematopoietic cells (somatic mosaicism), identifying patients with FA or ruling out this diagnosis can be challenging. If undiagnosed, FA patients who initially present with bone marrow failure will die of toxicity after standard-dose conditioning regimens for HSCT. In this study, we evaluated FA diagnosis in patients with BMF but no clear initial evidence of FA, using of a combination of classical and innovative tests in blood and fibroblasts. A cohort of 82 patients with BMF and no strong clinical evidence of FA was analysed (patients with a clear FA diagnosis were not included). Based on the likelihood of an underlying inherited condition associated with the BMF, we classified patients in 3 groups: those likely to have idiopathic aplastic anemia (IAA) [n=38, group 1], those likely to have a constitutional condition other than FA [n=26, group 2], and those likely to have IAA but who had isolated clinical findings which could also be present in FA [n=18, group 3]. Chromosome breakage test and analysis of the FA/BRCA pathway by FANCD2 immunoblot were performed in PBL in all patients [n= 82]. To overcome potential somatic mosaicism, skin primary fibroblasts were analysed [n= 52]. Also, to rule out FA/BRCA downstream groups, we developed a new flow cytometry test based on MMC-sensitivity in fibroblasts. In total, 6 patients with FA were identified: 1/38 in group 1 (aplastic anemia at 10 yo, no positive clinical findings), 2/26 in group 2 (one with MDS at 48 yo with precocious menopause and vocal cord neoplasia at 38 yo; one with hypoplastic MDS at 50 yo), and 3/18 in group 3 (one with short stature and aplastic anemia at 37 yo; one with MDS and borderline physical abnormalities at 26 yo; and one with a single cafe-au-lait spot and aplastic anemia at 10 yo). Chromosomal breakage tests in PBL were sufficient to diagnose 4 of these FA patients (further classified as FA core using FANCD2 immunoblot). Additional fibroblast analyses were necessary to identify 2 more FA patients (both with complete somatic mosaicism) and, importantly, to definitely exclude FA diagnosis in other patients. In conclusion, underdiagnosing FA is rare if careful history and physical exam are done together with chromosome breakage test in PBL. However, in clinical situations where the suspicion of FA persists despite negative breakage tests, then fibroblasts should be tested. Because no cases of FA were found among patients with IAA and negative breakage tests in PBL, we suggest that FA screening can be limited to this technique in this population (group 1). The strategy here presented allowed us to identify a few unexpected FA cases in a cohort of BMF patients, and importantly, to definitely rule out FA in others, with clear clinical impact for patients who undergo HSCT.

Unexpected Fanconi Anemia Diagnosis in Patients with Bone Marrow Failure.

Early diagnosis of Fanconi Anemia (FA) in patients with bone marrow failure is critical for optimal clinical management. However, the remarkably high clinical variability and the potential emergence of revertant hematopoietic cells (somatic mosaicism) can obscure and delay the diagnosis of FA. Here we addressed FA diagnosis in a prospective series of adult and pediatric patients who presented with bone marrow failure without clear overall clinical picture of FA.Sixty-six patients were classified into three groups: (1) bone marrow failure likely to be congenital, based on dysmorphic features or a family history [n=18], (2) aplastic anemia likely to be idiopathic [n=32], (3) patients with intermediate clinical features not classified into the former groups [n=16]. Of note, FA patients with typical clinical features were not included in the present study. FA diagnosis was evaluated using chromosome breakage test and FANCD2 immunoblot in PHA-stimulated-PBL. In addition, skin primary fibroblasts were analysed in order to overcome potential hematopoietic FA reversion. For that purpose, and considering that chromosome breakage tests are barely efficient in fibroblasts, we used FANCD2 immunoblot and also developped a new flow cytometry test based on MMC-sensitivity in fibroblasts (to detect downstream FA/BRCA groups). Using these approaches, we detected FA in 4 previously undiagnosed patients: a 35-years old patient from the congenital-like group; a 10-years old patient presenting as an idiopathic aplastic anemia without any FA signs; and two patients from the intermediate group: a 10-years old patient with an isolated thrombocytopenia, and a 50-years old patient presenting with pancytopenia/MDS and complete hematopoietic reversion. Importantly, FA diagnosis was definitely excluded in all other patients. In conclusion, we could identify a few unexpected FA cases in a series of patients with bone marrow failure. Therefore, the comprehensive use of a large set of tests is useful for accurate FA diagnosis. Classical chromosomal breakage tests in PBL appeared to be sufficient to exclude FA in idiopathic aplastic anemia, whereas fibroblast analysis can be necessary to definitely diagnose or exclude FA in other patients.