Once diagnosed with Fanconi anemia (FA), identification of the mutations remains an arduous task at present. The current screening process is a sequential, multi-step approach and successful identification of mutations may be delayed or hindered at any of these steps: establishing cell lines, growing and transducing cells for complementation, and procuring an efficient transducing vector for all FA genes. FA genes are large, with multiple exons, and harbor a wide spectrum of compound heterozygous mutations spread throughout the gene. Multi-exon size genomic deletions of FA genes are also well documented, and therefore, PCR amplification of exons and Sanger sequencing may not yield both the mutations. Therefore, there is a need for an efficient approach that scans the entire length of all the FA genes, and detects wide spectrum of changes. The next-gen sequencing (NGS) technologies allow sequencing large (megabase) regions of the genome rapidly. This enables identification of mutations, directly from DNA, with no prior requirement for establishment of cell lines and determination of the complementation group. We have targeted 13 FA and 11 additional genes that are associated with DNA repair pathways for next-gen sequencing. We employed MIP (Molecular Inversion probe) selection approach for enrichment of the genomic regions of the targeted 24 genes. Essentially, probes were designed to capture 5136 regions, and each test DNA was subjected to the MIP selection. A library of the enriched material was sequenced using a sequencing instrument (Illumina GAII). As an initial step, we tested six DNAs, each with one (or both) previously known mutation in a different FA gene. The MIP selection and sequencing helped identify all the known mutations in the DNAs we tested. We then chose 12 additional DNAs with no assigned group and thus no known mutations. We were able to find the gene and the inactivating mutations in 11 of them, and thus allowing identification of the mutations without a prior knowledge of the complementation group. One sample did not harbor mutations in any of the FA genes, and this may belong to a small number of individuals diagnosed with FA but the complementation test could not assign them to any known group, suggesting that there may be additional FA gene(s) to be discovered. Application of whole exome sequencing on these samples should help identify additional FA gene(s). Though FA patients can carry mutations in any of the 15 known FA genes, about two-thirds are affected by mutation in a single gene, FANCA. Thus, for all FA individuals with no assigned complementation group, checking for FANCA mutations by Sanger sequencing method will serve as an efficient initial step. We sequenced DNA from 88 such patients, and as anticipated, we found 58/88 to carry mutations in the FANCA gene. The non-FANCA individuals can be subjected to next-gen sequencing, and in fact, eight of the twelve samples chosen for next-gen sequencing were from the non-FANCA group, and found that each harbored mutations in a distinct FA gene. In addition to the 88 patients, we sequenced another 110 FA individuals who have been assigned to the FANCA group by complementation test, but either one or both of the disease-causing mutations were not yet known. Sequencing of 200 FA individuals carrying mutations in FANCA gene has allowed us to discover several novel (39 point, 14 indel and 18 splice) mutations. Multi-exon size genomic deletions of FA genes are well documented, and such deletions account for more than a quarter of the mutations in the FANCA gene. At present, it is difficult to discern deletions from the next-gen sequencing data. Comparative genomic hybridization (CGH) using high-resolution, high-density oligo arrays allows for efficient identification and precise mapping of genomic deletions and duplications. A CGH array was developed with 135,000 oligonucleotides, representing 37 genes that included all the FA genes, and several others known to participate in a DNA repair pathway. The arrays helped identify several deletions. Realizing that a good proportion of deletions go beyond the gene, we have designed another CGH array that queries the genomic regions up to 200kb on either side of the gene. We have now determined the precise boundaries for nearly 65 FANCA deletions with 28 extending beyond the gene region. We found FANCC gene deletion in seven patients with five sharing the same deletion, and FANCB and FANCD2 deletions in one sample each. For even larger deletions, we employ high-density SNP chips, and these chips scan the entire genomic region and reveal any other chromosomal variations, in addition to deletions and duplications The next-gen sequencing, CGH and SNP array technologies, along with the Sanger sequencing, will take us towards our goal of determining both the disease-causing mutations in all the FA samples in our collection. In addition to FA individuals, these technologies can be employed to explore the role of FA genes in pancreatic and other cancers.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
National Human Genome Research Institute
Zip Code
Chen, Y-C; Sudre, G; Sharp, W et al. (2018) Neuroanatomic, epigenetic and genetic differences in monozygotic twins discordant for attention deficit hyperactivity disorder. Mol Psychiatry 23:683-690
Kimble, Danielle C; Lach, Francis P; Gregg, Siobhan Q et al. (2018) A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families. Hum Mutat 39:237-254
Malicdan, May Christine V; Vilboux, Thierry; Ben-Zeev, Bruria et al. (2018) A novel inborn error of the coenzyme Q10 biosynthesis pathway: cerebellar ataxia and static encephalomyopathy due to COQ5 C-methyltransferase deficiency. Hum Mutat 39:69-79
Mendonca, Leonardo O; Malle, Louise; Donovan, Frank X et al. (2017) Deficiency of Interleukin-1 Receptor Antagonist (DIRA): Report of the First Indian Patient and a Novel Deletion Affecting IL1RN. J Clin Immunol 37:445-451
Demirkaya, Erkan; Zhou, Qing; Smith, Carolyne K et al. (2017) Brief Report: Deficiency of Complement 1r Subcomponent in Early-Onset Systemic Lupus Erythematosus: The Role of Disease-Modifying Alleles in a Monogenic Disease. Arthritis Rheumatol 69:1832-1839
Kwon, Erika M; Connelly, John P; Hansen, Nancy F et al. (2017) iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations. Proc Natl Acad Sci U S A 114:1964-1969
Mirabello, Lisa; Khincha, Payal P; Ellis, Steven R et al. (2017) Novel and known ribosomal causes of Diamond-Blackfan anaemia identified through comprehensive genomic characterisation. J Med Genet 54:417-425
Dewan, Ramita; Pemov, Alexander; Dutra, Amalia S et al. (2017) First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation. BMC Cancer 17:127
Murdock, David R; Donovan, Frank X; Chandrasekharappa, Settara C et al. (2017) Whole-Exome Sequencing for Diagnosis of Turner Syndrome: Toward Next-Generation Sequencing and Newborn Screening. J Clin Endocrinol Metab 102:1529-1537
Chandrasekharappa, Settara C; Chinn, Steven B; Donovan, Frank X et al. (2017) Assessing the spectrum of germline variation in Fanconi anemia genes among patients with head and neck carcinoma before age 50. Cancer 123:3943-3954

Showing the most recent 10 out of 44 publications