Chromosome Breakage Test Research Articles

Two cases of cytopenia associated with multiple malformations

The first patient, a 10-year-old girl, presented with pancytopenia and recurrent epistaxis, along with a history of repeated upper respiratory infections, café-au-lait spots, and microcephaly. Genetic testing revealed compound heterozygous mutations in the DNA ligase IV (LIG4) gene, leading to a diagnosis of LIG4 syndrome. The second patient, a 6-year-old girl, was seen for persistent thrombocytopenia lasting over two years and was noted to have short stature, hyperpigmented skin, and hand malformations. She had a positive result from chromosome breakage test. She was diagnosed with Fanconi anemia complementation group A. Despite similar clinical presentations, the two children were diagnosed with different disorders, suggesting that children with hemocytopenia and malformations should not only be evaluated for hematological diseases but also be screened for other potential underlying conditions such as immune system disorders.

Novel homozygous mutation in the FANCA gene (c.2222G>A) in a Chinese girl of Fanconi anemia

AbstractFanconi anemia is the most common inherited bone marrow failure syndrome. Its clinical manifestations include congenital dysplasia, bone marrow hematopoietic failure and tumor susceptibility. At present, there are 23 related gene abnormalities, among which FANCA is the most common. We report a case of a Chinese girl with bone dysplasia and aplastic anemia. The next‐generation sequencing results showed a homozygous mutation in the FANCA gene (c.2222G > A), which was predicted to be a pathogenic mutation based on protein function. This mutation at this site has not been reported in the previous literature. The diagnosis of Fanconi anemia should be determined with combined clinical, chromosome breakage test and gene sequencing results.

Diagnosis status and genetic characteristics analysis of Fanconi anemia in China

Objective: To analyze the clinical and molecular diagnostic status of Fanconi anemia (FA) in China. Methods: The General situation, clinical manifestations and chromosome breakage test and genetic test results of 107 pediatric FA cases registered in the Chinese Blood and Marrow Transplantation Registry Group (CBMTRG) and the Chinese Children Blood and Marrow Transplantation Registry Group (CCBMTRG) from August 2009 to January 2022 were analyzed retrospectively. Children with FANCA gene variants were divided into mild and severe groups based on the type of variant, and Wilcoxon-test was used to compare the phenotypic differences between groups. Results: Of the 176 registered FA patients, 69 (39.2%) cases were excluded due to lack of definitive genetic diagnosis results, and the remaining 107 children from 15 hospitals were included in the study, including 70 males and 37 females. The age at transplantation treatment were 6 (4, 9) years. The enrolled children were involved in 10 pathogenic genes, including 89 cases of FANCA gene, 7 cases of FANCG gene, 3 cases of FANCB gene, 2 cases of FANCE gene and 1 case each of FANCC, FANCD1, FANCD2, FANCF, FANCJ, and FANCN gene. Compound heterozygous or homozygous of loss-of-function variants account for 69.2% (72/104). Loss-of-function variants account for 79.2% (141/178) in FANCA gene variants, and 20.8% (37/178) were large exon deletions. Fifty-five children (51.4%) had chromosome breakage test records, with a positive rate of 81.8% (45/55). There were 172 congenital malformations in 80 children.Café-au-Lait spots (16.3%, 28/172), thumb deformities (16.3%,28/172), polydactyly (13.9%, 24/172), and short stature (12.2%, 21/172) were the most common congenital malformations in Chinese children with FA. No significant difference was found in the number of congenital malformations between children with severe (50 cases) and mild FANCA variants (26 cases) (Z=-1.33, P=0.185). Conclusions: FANCA gene is the main pathogenic gene in children with FA, where the detection of its exon deletion should be strengthened clinically. There were no phenotypic differences among children with different types of FANCA variants. Chromosome break test is helpful to determine the pathogenicity of variants, but its accuracy needs to be improved.

Cytogenetic findings in Polish patients with suspected Fanconi anemia.

The high sensitivity of cells of Fanconi anemia (FA) patients to DNA cross-linking agents (clastogens), such as mitomycin C (MMC), was used as a screening tool in Polish children with clinical suspicion of FA. The aim of the study was to compare chromosome fragility between 3 groups, namely non-FA, possible mosaic FA and FA patients. The study included 100 children with hematological manifestations and/or congenital defects characteristic of FA, and 100 healthy controls. Blood samples obtained from participants were analyzed using an MMC-induced chromosomal breakage test. Patients with clinical suspicion of FA were divided into 3 subgroups based on the MMC test results, namely FA, possible mosaic FA and non-FA. Thirteen out of 100 patients had a true FA cellular phenotype. The mean value of MMC-induced chromosome breaks/cell for FA patients was higher than for non-FA patients (6.67 ±3.92 compared to 0.23 ±0.18). In addition, the percentage of cells with spontaneous aberrations was more than 9 times higher in FA patients than in non-FA patients. Our results confirmed that the MMC sensitivity test distinguishes between individuals affected by FA, those with possible somatic mosaicism, and patients with bone marrow failure for other reasons, who were classified as non-FA in the first diagnostic step. However, a definitive differential diagnosis requires follow-up mutation testing and chromosome breakage analysis of skin fibroblasts.

Next-generation sequencing reveals novel variants and large deletion in FANCA gene in Polish family with Fanconi anemia

BackgroundFanconi anemia (FA) is the most common inherited bone marrow failure syndrome. However, establishing its molecular diagnosis remains challenging. Chromosomal breakage analysis is the gold standard diagnostic test for this disease. Nevertheless, molecular analysis is always required for the identification of pathogenic alterations in the FA genes.ResultsWe report here on a family with FA diagnosis in two siblings. Mitomycin C (MMC) test revealed high level of chromosome breaks and radial figures. In both children, array—Comparative Genomic Hybridization (aCGH) showed maternally inherited 16q24.3 deletion, including FANCA gene, and next generation sequencing (NGS) disclosed paternally inherited novel variants in the FANCA gene—Asn1113Tyr and Ser890Asn. A third sibling was shown to be a carrier of FANCA deletion only.ConclusionsAlthough genetic testing in FA patients often requires a multi-method approach including chromosome breakage test, aCGH, and NGS, every effort should be made to make it available for whole FA families. This is not only to confirm the clinical diagnosis of FA in affected individuals, but also to enable identification of carriers of FA gene(s) alterations, as it has implications for diagnostic and genetic counselling process.

P814: CLINICAL CHARACTERISTICS AND GENE MUTATION ANALYSIS OF 148 CHILDREN WITH FANCONI ANEMIA IN CHINA

Background: Fanconi anemia (FA) is a rare autosomal recessive, X-linked (FANCB) or autosomal dominant (FANCR/RAD51) disease of bone marrow failure. Pathogenesis is mainly caused by gene mutation related to the FA pathway. At present, at least 23 genes have been discovered to play a role in the FA pathway. The gene mutations of FA have been reported in more and more countries. In China, there no large-scale cases have been reported. Aims: In this study, we analyzed the clinical characteristics of 148 FA children from 20 provinces in China from October 2003 to October 2021. We explored the relationship between genotype and phenotype in 105 children with FA genotyping results. This is the largest series of subtyped Chinese Fanconi anemia patients to date, and the results will be helpful for future clinical management. Methods: A total of 148 Fanconi anemia patients from 140 families were diagnosed by clinical phenotype, family history, chromosome breakage test induced by mitomycin C, Single cell gel electrophoresis experiment, targeted - sequence, whole exon sequencing, and MLPA method from October 2003 to October 2021. Since there were no genetic test results for the children before 2010, only the 105 patients after 2010 had gene sequencing results, which allowed genotyping. DNA damage repair defects were detected by MMC-induced Single cell gel electrophoresis and chromosome breakage experiments. Targeted-seq was used to identify the FA mutations of the patients. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large fragment deletions in patients with negative results in the targeted-seq but conforming to the FA by MMC-induced chromosome breakage experiments test and clinical manifestations. We performed WES of DNA samples from five patients and their parents. Results: 1) The most common subtype of FA in China is FA-A, followed by FA-D2 and FA-P。2) The most common deformities in FA-A patients are finger deformities and skin pigmentation, while heart deformities are more common in FA-D2/B/G/I/P subtypes. 3) The common mutations of FANCA are exon23c.2101A>G, exon28c.2778 + 1G>A, exon34c.3348 + 1G>A, exon21c.1844delC, and exon30 C2941T>G, which are different from Korea and Japan. 4) Homozygous nonsense mutation of exon32 c.3188G>A and exon29 c.2851C>T in FA-A likely benign, but the homozygous splicing mutation in exon29c.2852 + 1G>T is harmful. 4) Children with splicing and Del mutations of FANCD1 gene had poor clinical prognosis, while children with homozygous mutations of exon10c. 1792A>G is likely benign. 5) Cases of initial diagnosis of MDS or poor disease progression tend to occur in FA-B/C/E/G/J/L/M and FA-S subtypes. 6)Chromosome 1, 3, 7, and 8 abnormalities and mutations of SF3B1 P53 are related to disease progression. Summary/Conclusion: We made a comprehensive description and analysis of the clinical characteristics and mutant genes of Children with FA in China. We proposed the mutation sites related to clinical prognosis and the cytogenetic abnormalities of disease progression, providing new data for our comprehensive understanding of FA. Our data will be useful for FA future menagement.

A rare case of pancytopenia

Pancytopenia is a reduction in all the three peripheral blood cell lineages and presents as anemia, leukopenia, and thrombocytopenia. Aplastic anemia is pancytopenia with bone marrow hypocellularity. Aplastic anemia can be constitutional or acquired. Genetic diseases such as Fanconi anemia and dyskeratosis congenita usually present as pancytopenia with typical physical anomalies and are usually seen in early childhood. Fanconi anemia is a rare cause of pancytopenia, which is an autosomal recessive disorder and manifests as progressive pancytopenia with congenital developmental anomalies and an increased risk of malignancy. Here, we present a case of Fanconi anemia who presented with pancytopenia, short stature, and hypoplastic thumb of the right hand and was diagnosed on the basis of bone marrow biopsy and chromosomal breakage test.

An update on Fanconi anemia: Clinical, cytogenetic and molecular approaches (Review).

Fanconi anemia is a genetic syndrome clinically characterized by congenital malformations that affect several human systems, leads to progressive bone marrow failure and predisposes an individual to cancer, particularly in the urogenital area as well as the head and neck. It is commonly caused by the biallelic compromise of one of 22 genes involved in the FA/BRCA repair pathway in most cases. The diagnosis is based on clinical suspicion and confirmation using genetic analysis, where the chromosomal breakage test is considered the gold standard. Other diagnostic methods used include western blotting, multiplex ligation-dependent probe amplification and next-generation sequencing. This genetic condition has variable expressiveness, which makes early diagnosis difficult in certain cases. Although early diagnosis does not currently allow for improved cure rates for this condition, it does enable healthcare professionals to perform a specific systematic follow-up and, if indicated, a bone marrow transplantation that improves the mobility and mortality of affected individuals. The present review article is a theoretical revision of the pathophysiology, clinical manifestations and diagnosis methods intended for different specialists and general practitioners to improve the diagnosis of this condition.

Excellent Outcome of Alternative Donor HSCT in Fanconi Anemia Using Chemotherapy Only Regimen: Single Korean Institution Experience

Title) Excellent outcome of alternative donor HSCT in Fanconi anemia using chemotherapy only conditioning regimen: single Korean institution experience.Introduction) Fanconi anemia (FA) is a rare autosomal (or X linked) recessive disorder characterized by bone marrow failure, secondary malignancies, and skeletal anomalies. Hematopoietic stem cell transplantation (HSCT) remains the standard treatment of hematologic disease. Although HSCT from a matched sibling donor (MSD) is the optimal mode of transplant, many patients lack such a donor necessitating alternative donor HSCT (AD-HSCT). Radiation treatment during the conditioning regimen results in a high incidence of post-HSCT complications in FA patients, while serotherapy with alemtuzumab has been reported to increase infectious toxicities. Here, we report on the excellent outcome of 15 FA patients who received AD-HSCT with a fludarabine-based conditioning regimen with in vivo T cell depletion using rabbit anti-thymocyte globulin (ATG).Methods and Results) We reviewed the medical records of FA patients diagnosed by chromosomal breakage test and who received AD-HSCT between September 2007 and December 2016. Median age at transplant was 10.8 years (4.2~18.6) and 7 were male (46.7%). Eleven of 14 patients who received genetic study for FA were found to have mutations (6 with FANC-A mutations (40%), and 5 with FANC-G mutations (33.3%)). Hematologic reasons for HSCT were severe aplastic anemia in 13 (93.3%) and myelodysplastic syndrome in 2 patients (13.3%). One patient received HSCT from a mismatched related donor, while the remaining patients received unrelated HSCT. Seven transplants were human leukocyte antigen (HLA) fully matched (13.3%). Cell sources were as follows: peripheral blood stem cells 9 (60%), cord blood 4 (26.7%), bone marrow 2 (13.3%). Conditioning regimen consisted of fludarabine 30mg/BSA/day x5days (D-8,-7,-6,-5,-4), cyclophosphamide 5mg/kg/day x4days (D-5,-4,-3,-2), rabbit ATG 2.5mg/kg/day x3days (D-3,-2,-1). Graft-versus-host disease prophylaxis consisted of cyclosporine and mini-dose methotrexate. Medians of infused cell doses were as follows: TNC 16.51 x108/kg (5.17~42.4), CD34+ 15.49x106/kg(3.49~33.7).Twelve patients (80%) showed initial engraftment and full donor chimerism according to short tandem repeat polymerase chain reaction (STR-PCR) analysis. Of the 3 patients who showed primary graft failure, one showed autologous recovery, while the remaining 2 received additional HSCs from the same donor and a haploidentical family donor respectively, leading to engraftment. Acute GVHD grade II or above was diagnosed in 8 patients (53.5%) and resolved with steroid therapy. Chronic GVHD developed in two patients, one mild and one of moderate severity. The patient with moderate oral chronic GVHD was diagnosed with squamous cell carcinoma of the tongue 5years after HSCT. She received surgical resection and remains disease-free for 40 months. After transplantation, 5 patients (33.3%) experienced infectious complications including viral reactivation, bacteremia and fungemia but all recovered without complications. At a median follow-up of 59.6 months (6.3~107.9) all 15 FA patients are alive and remain free of hematologic disease.Conclusion) FA patients who received AD-HSCT with fludarabine-based conditioning with in vivo T cell depletion using rabbit ATG showed excellent outcome with a lower rate of infectious toxicities than has been reported with conditioning incorporating alemtuzumab. The incidence of primary graft failure should be monitored in a larger cohort of patients. DisclosuresNo relevant conflicts of interest to declare.

Esophageal cancer as initial presentation of Fanconi anemia in patients with a hypomorphic FANCA variant.

Fanconi anemia (FA) is a clinically heterogenous and genetically diverse disease with 22 known complementation groups (FA-A to FA-W), resulting from the inability to repair DNA interstrand cross-links. This rare disorder is characterized by congenital defects, bone marrow failure, and cancer predisposition. FANCA is the most commonly mutated gene in FA and a variety of mostly private mutations have been documented, including small and large indels and point and splicing variants. Genotype–phenotype associations in FA are complex, and a relationship between particular FANCA variants and the observed cellular phenotype or illness severity remains unclear. In this study, we describe two siblings with compound heterozygous FANCA variants (c.3788_3790delTCT and c.4199G > A) who both presented with esophageal squamous cell carcinoma at the age of 51. The proband came to medical attention when he developed pancytopenia after a single cycle of low-dose chemotherapy including platinum-based therapy. Other than a minor thumb abnormality, neither patient had prior findings to suggest FA, including normal blood counts and intact fertility. Patient fibroblasts from both siblings display increased chromosomal breakage and hypersensitivity to interstrand cross-linking agents as seen in typical FA. Based on our functional data demonstrating that the c.4199G > A/p.R1400H variant represents a hypomorphic FANCA allele, we conclude that the residual activity of the Fanconi anemia repair pathway accounts for lack of spontaneous bone marrow failure or infertility with the late presentation of malignancy as the initial disease manifestation. This and similar cases of adult-onset esophageal cancer stress the need for chromosome breakage testing in patients with early onset of aerodigestive tract squamous cell carcinomas before platinum-based therapy is initiated.

Fanconi Anemia Gene Associated Germline Predisposition in Aplastic Anemia and Hematologic Malignancies

Introduction Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome with a surged cancer incidence, especially in hematologic malignancies, which has been listed in the entity of myeloid neoplasms with germline predisposition in the 2016 revision to the World Health Organization classification of tumors of hematopoietic and lymphoid tissues.Whether FA heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is crucial but unsettled. We therefore retrospectively analyzed rare possibly significant variations (PSVs) in the five most obligated FA genes, BRCA2, FANCA, FANCC, FANCD2, and FANCG, in 788 aplastic anemia (AA) and hematologic malignancies patients to address this issue. Methods Patients diagnosed as AA, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL) from April 2015 to December 2018 in Hebei Yanda Lu Daopei Hospital were enrolled with the ones diagnosed as FA via chromosome breakage test and/or genetic test excluded. Detailed disease histories and workup files were retrieved from the electronic medical record. Amplicon-based high throughput sequencing of aforementioned five genes were performed. The significance of the germline missense variants was assessed by in silico prediction algorithms, including SIFT, Polyphen2, PROVEAN, FATHMM, MutationTaster, and MutationAssessor. Variant predicted as deleterious/possibly deleterious by ≥ 3/6 scoring tools was defined as rare PSV and further included in statistical analysis. For splice site mutations, GeneSplicer, Human Splicing Finder, NetGene2, and FSPLICE were employed and only when ≥2/4 algorithms predict to affect/possibly affect splicing, would the variant be regarded as possibly significant. Same criteria were adopted when stratifying variants recorded in the ExAC. All reported variants in this study were confirmed germline variants by Sanger sequencing with fingernail specimens and/or pedigree analysis. WFisher exact two-tailed test was adopted for variant frequency comparison. Development of disease was analyzed by cumulative incidence method and Kaplan-Meier method. p < 0.05 was considered as statistically significant. Results A total of 788 patients, who were all of the East Asian ancestry, were enrolled, including 341 females and 447 males (Figure 1A). The median age of onset was 10 (1-63), 27 (1-65), 14.5 (1-65), and 6 (1-53) years in AA, MDS, AML, and ALL subgroup, respectively. Sixty-eight variants were identified in 66 patients (8.38%). FANCA is the most frequently mutated gene (n = 29), followed by BRCA2 (n = 20) (Figure 1B). When compared to the ExAC East Asian dataset, there was an overall higher rare PSVs incidence in our cohort (p = 0.016). BRCA2 PSVs showed a higher frequency in ALL (p = 0.038), and FANCA PSVs were significantly enriched in AA and AML subgroups (p = 0.020; p = 0.008). The patients with FANCA heterozygotes also tended to show an increased risk for developing MDS (p = 0.075) (Table 1). No impact of FA-PSV status was found neither on cumulative disease incidence (Figure 1C). FA-PSV + MDS/AML patients have a heavier tumor mutation burden, higher rate of cytogenetic abnormalities, and less epigenetic regulation and spliceosome gene mutations than those of FA-PSV - MDS/AML patients (p = 0.024, p = 0.029, p = 0.024, and p = 0.013) (Figure 1D). Discussion The overall PSVs enrichment in our cohort buttresses that heterozygous mutations of FA genes abate the capacity of DNA homologous recombination repair pathway and contribute to hematopoietic failure. Furthermore, we present the first evidence that BRCA2 heterozygotes have a significantly higher risk of developing into ALL. Instead of all or nothing, the impact of different variant imposed on protein might be seen as a continuous variation. Therefore, contributions of the FA pathway defect could be latent and subtle but be profound as time goes by. We further firstly observed a higher incidence of cytogenetic abnormalities and somatic mutations with statistical significance, and lower frequency of epigenetic regulation and spliceosome gene mutations in FA-PSV + myeloid malignancies. This provides evidence that these FA-PSV carriers are prone to accumulate chromosomal structural abnormalities, and confer the congenital susceptibility of myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

Diagnostic Whole Exome Sequencing for 166 Patients with Inherited Bone Marrow Failure Syndrome

BACKGROUND: Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of genetic disorders characterized by bone marrow failure, physical anomalies, and various kinds of organ complications. In addition to classical IBMFSs, such as Fanconi anemia, Diamond-Blackfan anemia, Dyskeratosis congenita, Shwachman-Diamond syndrome, and familial platelet disorders, many types of unclassified IBMFSs are reported. Over 100 genes are considered causative genes; however, the precise genetic diagnosis of IBMFSs remains challenging. We developed a capture-based target sequencing method for IBMFSs that covers more than 180 associated genes. Our system achieved genetic diagnosis for 225 (35%) of 738 patients between 2013 and 2018. However, the causative gene remained unknown for 513 (65%) patients, and further genetic analysis of these "target-negative" cases was necessary to achieve a precise diagnosis. METHODS: We performed whole exome sequencing (WES) for patients who were "target-negative" but strongly suspected of having IBMFS based on the following clinical characteristics: physical or organ anomalies (skin, nail, hair, skeletal, growth, cardiac, lung, liver, or genitourinary), family history of hematological disorder, young age (≤2 years), short telomere length (<-2.0 SD), and hyper sensitivity to the chromosome breakage test. A sequencing library was prepared using the SureSelect Human All Exon 50Mb kit (Agilent Technologies, Santa Clara, CA, USA) and it was sequenced using the HiSeq2000 platform (Illumina, San Diego, CA, USA), according to manufacturers' instructions. The candidate germline variants were detected through our Genomon-exome analysis pipeline. With mean coverage of 100×, ≥ 85% of all protein coding bases were covered at 20× or more. RESULTS: Among the 513 "target-negative" cases, 166 patients were evaluated, of whom 17 patients' parents were also analyzed in a trio-based analysis. New pathogenic variants were identified in 18 of the 166 (11%) patients according to the American College of Medical Genetics (ACMG) guidelines, of which 5 variants were revealed to be de novo. Diagnostic variants were identified in FANCF, SRP54, RPL19, RPL5, RTEL1, RUNX1, MECOM, CDC42, GNE, SLNF14 (all n = 1). In addition to IBMFS-associated genes, causative genes for congenital hemolytic anemia (G6PD, PKLR), inborn error of metabolism (SLC46A1), and primary immune deficiency (NFKB2, LRBA) are also identified (all n = 1). Moreover, loss-of-function mutation of ADH5 gene are identified in three patients that seems to be associated to novel IBMFSs. On the other hand, no pathogenic variant in GATA2, ERCC6L2, LIG4, and SAMD9/SAMD9L genes that are reported as unclassified IBMFSs in Europe and United States are identified in our cohort. CONCLUSION: Our findings support the utility of WES (especially trio-based analysis) as a diagnostic tool for IBMFSs. Furthermore, genetic background of IBMFSs in East Asia seems to be different from that of Europe and United States. Disclosures No relevant conflicts of interest to declare.

RPA1 Gain of Function Causes Human Short Telomere Syndrome with Revertant Somatic Mosaicism

Dyskeratosis congenita (DC) is a short telomere syndrome with bone marrow failure (BMF), mucocutaneous fragility and predisposition to malignancy due to inherited mutations in telomere associated genes. The genetic cause remains unknown in 10-20% of DC patients. Here, we describe a new DC candidate gene, RPA1, which encodes the largest subunit of the Replication Protein A (RPA) complex, a single stranded DNA (ssDNA) binding protein essential for DNA replication, damage repair and telomere maintenance. The patient presented at the age of 5 years with pancytopenia and hypocellular marrow with morphology resembling refractory cytopenia of childhood, mucocutaneous triad and a congenital retinal anomaly. Further workup revealed telomeres in blood below 1st percentile, normal female karyotype and a negative chromosomal breakage test. After excluding known genetic causes for DC, family whole exome sequencing identified a de novoRPA1 c.718G>A (p.E240K) mutation with a predicted pathogenic CADD score of 23 and absent in >140,000 gnomAD population controls. The mutation had 50% allelic frequency in fibroblasts in comparison to a reduced mutation burden of 27% in bone marrow (BM) cells. Using bulk and single cell sequencing of BM, we identified 2 somatic compensatory events that likely explain patient's stable hematologic phenotype over a period of 20 years: i) an acquired RPA1 p.E579X mutation (in cis with p.E240K) was found at 10% frequency, which resulted in RNA degradation of the mutated allele as confirmed by RNA sequencing; ii) a founder clone with uniparental isodisomy on chromosome 17p encompassing RPA1 locus causing loss of germline E240K mutation. Analysis of serial BM samples using SNP arrays and deep sequencing demonstrated UPD17p clonal expansion concurrent with a decline of germline p.E240K and increase of acquired p.E579X mutation. This is consistent with a toxic, likely gain-of-function (GOF) effect of germline p.E240K mutation leading to acquired loss-of-function (LOF) escape mechanisms. RPA1 is essential to all DNA metabolism transactions that encounter ssDNA, including binding to telomeric 3' overhangs during late S-phase to unfold G-quadruplexes and facilitate telomerase activity. Several RPA1 genetic variants impart telomere shortening and an S-phase defect in yeast and human cell lines. However, the molecular mechanisms are not well understood and thus far, RPA1 gene has not been linked to human disease. In an iPSC-based disease model with homozygous RPA1 p.E240K knock-in, we discovered severe telomere shortening in RPA1 mutant iPSCs and iPSC derived hematopoietic progenitors (HPs) compared to WT counterparts. Furthermore, decreased erythroid differentiation capacity was noted in RPA1-mutant HPs. Because the germline p.E240K mutation is located in DNA binding domain A of RPA1, we performed electromobility shift assay to assess the DNA binding affinity of mutant RPA1 protein. The p.E240K heterotrimeric RPA complex shows increased ssDNA binding compared to wild type RPA. In summary, we describe the first human disease manifesting as short telomere syndrome secondary to a de novoRPA1 mutation with likely GOF effect, which forces the development of multiple revertant mosaic events, similar to the newly described SAMD9/SAMD9L disorders. Our studies also have implications for the mechanistic understanding of the role of RPA1 in telomere maintenance and hematopoiesis. Disclosures Niemeyer: Celgene: Consultancy; Novartis: Consultancy.

Novel FANCA mutation in the first fully-diagnosed patient with Fanconi anemia in Polish population \u2013 case report

BackgroundFanconi anemia is a rare genetic disorder caused by mutations in genes which protein products are involved in replication, cell cycle control and DNA repair. It is characterized by congenital malformations, bone marrow failure, and high risk of cancer. The diagnosis is based on morphological and hematological abnormalities such as pancytopenia, macrocytic anaemia and progressive bone marrow failure. Genetic examination, often very complex, includes chromosomal breakage testing and mutational analysis.Case presentationWe present a child with clinical diagnosis of Fanconi anemia. Although morphological abnormalities of skin and bones were present from birth, diagnosis was only suspected at the age of 8. Chromosome breakage test in patient’s lymphocytes showed increased level of aberrations (gaps, chromatid breaks, chromosome breaks, radial figures and rearrangements) compared to control. Next generation sequencing revealed presence of two pathogenic variants in FANCA gene, one of which was not previously reported.ConclusionsThe article provides additional supportive evidence that compound biallelic mutations of FANCA are associated with Fanconi anemia. It also illustrates the utility of combination of cytogenetic and molecular tests, together with detailed clinical evaluation in providing accurate diagnosis of Fanconi anemia. This report, to the best of our knowledge, describes the first fully diagnosed FA patient in Polish population.

From Surgery to Hematology: Fanconi Anemia about a Case of a Senegalese Child

Fanconi anaemia (FA) is a rare genetic abnormality. Most of FA reported from the sub-Saharan population came from southern Africa, with many patients linked to a mutation in the FANC G gene. Classic triads of this disorder are: a varied malformation, bone marrow failure, and short stature. This disorder is also associated with predisposition to malignancies. These multiple manifestations, sometimes not uniform, often cause diagnostic delay. We here report a 13-year-old Senegalese boy with FA. He was followed up for polydactyly-repair surgery. Importantly, pre-operative blood check-up revealed severe anaemia, which prompted us to perform bone marrow aspiration: examination revealed dysmyelopoiesis. Being triggered by this, systematic examinations were performed, which revealed other signs indicative of FA; i.e., radial spine abnormalities, triangular face, etc. The diagnosis of FA was strongly suggested, which prompted genetic examination. The chromosomal breakage test detected cellular hypersensitivity to DNA, which confirmed the diagnosis. He is receiving transfusion support, and androgens use is being considered. This case highlights the importance of preoperative examination. Physicians must be aware that FA, although its incidence is low, can be hidden behind infantile anaemia.

Fanconi Anemia: A Paradigm for Understanding DNA Repair During Replication.

Fanconi anemia, the most common hereditary bone marrow failure disorder, results from defective repair of DNA interstrand crosslinks (ICLs), which covalently link complementary DNA strands causing replication stalling. Mutations in 22 different genes (FANCA-FANCW) have been shown to result in Fanconi anemia. Their protein products work at different stages of DNA repair leading to considerable heterogeneity in human phenotypes. The majority of the FANC gene mutations are recessively inherited with the exceptions of FANCB and FANCR/RAD51. FANCB is X-linked, and all FANCR/RAD51 mutations arise de novo, affect only one allele, and the mutant protein acts as a dominant negative against the wild type protein. Despite advances in the molecular diagnosis of Fanconi anemia, if Fanconi anemia is suspected, chromosome breakage (DEB or MMC) testing on patient cells is essential. We have seen a number of patients referred to the International Fanconi Anemia Registry (http://lab.rockefeller.edu/smogorzewska/ifar/) who are misdiagnosed with Fanconi anemia based solely on the presence of a FANC gene variant in gene panel or whole exome sequencing. Conversely, blood mosaicism may lead to a negative blood chromosome breakage test. If there is a high suspicion of Fanconi anemia, but blood breakage results are negative, breakage test on patient fibroblasts should be performed. Diagnosis of Fanconi anemia should also be entertained in young adults presenting with squamous cell carcinoma of the aerodigestive tract, since this may be their initial presentation of Fanconi anemia and conventional chemotherapy dose would precipitate bone marrow failure in these patients. In my talk, I will discuss the mechanism of the Fanconi anemia repair pathway during DNA replication. Then, I will concentrate on the mechanism of bone marrow failure and tumorigenesis in Fanconi anemia. I will explore the hypothesis that the endogenously produced aldehydes including some that are still unknown, contribute to disease development. Fanconi anemia-deficient hematopoietic stem cells have an autonomous DNA repair defect. Accumulation of DNA damage leads to apoptosis due to the activation of p53. If cells escape death, mutagenesis may lead to the development of leukemia. The sources of endogenous DNA damage are poorly understood. Cell cycle induction of Fanconi anemia pathway-deficientmouse hematopoietic stem cells results in DNA damage and bone marrow failure, which implies that the DNA lesions encountered during replication are the culprit. There is mounting evidence that the endogenous aldehydes, including acetaldehyde and formaldehyde,may cause those DNA lesions. To identify other metabolites that may induce bone marrow failure in Fanconi anemia, we used a library of CRISPR guides to target Cas9 to metabolic genes to screen for and identify synthetic lethality with Fanconi anemia deficiency. We have identifiedALDH9A1as the most significantly depleted gene in FANCD2-/- cells. The synthetically lethal interaction was validated using single gene editing in human umbilical cord-derived hematopoietic stem progenitor cells. We propose a model in which aldehydes that are metabolized by ALDH9A1 accumulate in the absence of this enzyme and cause DNA damage that requires the Fanconi anemia pathway proteins for repair, survival, and suppression of tumorigenesis. We are testing this model using Fanca-/-Aldh9a1-/-mice. Disclosures No relevant conflicts of interest to declare.

From exome analysis in idiopathic azoospermia to the identification of a high-risk subgroup for occult Fanconi anemia

PurposeIn about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these “occult” FA cases is often a solid cancer and cancer treatment–related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing. Methods(1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test. ResultsES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell–only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test. ConclusionWe report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for “occult” FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance.

Functional analysis of Fanconi anemia mutations in China

Fanconi anemia (FA) is a rare recessive disease characterized by progressive bone marrow failure, congenital abnormalities, and increased incidence of cancers. To date, mutations in 22 genes can cause FA or an FA-like phenotype. In China, in addition to clinical information, FA diagnosis primarily relies on genetic sequencing because the chromosome breakage test is rarely performed. Here, we employed multiple genetic diagnostic tools (DNA sequencing, multiplex ligation-dependent probe amplification, and chromosome microarray) and a variant-based functional assay platform to investigate the genetic cause in 25 Chinese suspected FA patients. A total of 45 distinct candidate variants were detected in six FA genes (FA-A, FA-B, FA-C, FA-D2, FA-G, and FA-J), of which 36 were novel. Eight missense variants and one indel variant were unable to restore FANCD2 mono-ubiquitination and mitomycin C resistance in a panel of FA indicator cell lines, indicating that these mutations are deleterious. Three missense variants (FANCA-L424V, FANCC-E273K, and FANCG-A153G) were harmless. Finally, 23 patients were molecularly diagnosed with FA, consistent with their clinical phenotype. In the FA-A subgroup, large deletions accounted for 14% of the disease-causing variants. We have established a comprehensive molecular diagnostic workflow for Chinese FA patients that can substitute for standard FA cytogenetic analysis.

Inherited bone marrow failure syndrome: management and diagnostic advances utilizing next-generation sequencing

Inherited bone marrow failure syndromes (IBMFS) are part of a heterogeneous disease category in which at least one hematopoietic cell lineage is reduced in the bone marrow owing to a pathogenic genetic mutation. IBMFS comprise >25 defined disease entities, including Fanconi anemia (FA), Diamond-Blackfan anemia, and dyskeratosis congenita. The diagnosis is based on hematological and physical findings with the aid of several disease-specific diagnostic tests, such as the chromosomal breakage test for FA. With recent advances in clinical molecular studies that have revealed a considerable amount of pathognomonic molecular lesions in IBMFS, the role of genetic tests has become more important in the diagnosis of these diseases. Comprehensive genetic diagnostic systems using next-generation sequencing has been developed in Japan and other countries, making it technically possible to simultaneously analyze and evaluate >100 causative genes of IBMFS, and it is expected to be widely used in clinical settings.

Heterogeneous Diagnoses Underlying Radial Ray Anomalies.

To review perinatal Radial Ray Anomaly (RRA) cases born at the National Institute of Perinatology, Mexico, and to reveal the heterogeneous diagnoses of these patients. All patients with RRA over a 18mo period were included; 4/15 were detected prenatally and 11/15 postnatally. Karyotype was performed for all patients with bilateral RRA; and chromosomal breakage analysis, when the karyotype was normal. Fifteen RRA patients were identified: one with trisomy 18, three with an isolated defect, six with monogenic disease, four with a genetic association and one with diabetic embryopathy. Five were stillborn and two died during the early neonatal period; all of whom presented with multiple defects. Three of the live born patients and one stillborn with multiple defects had Fanconi anemia. RRAs carry a high perinatal mortality rate (47%) when they occur in association with other defects. The assessment of these patients needs to involve the combined use of ultrasound, clinical, genetic, cytogenetic and molecular testing. The present results indicate that the chromosome breakage test should always be performed to rule out Fanconi anemia in this group.