AP2A2 mutation and defective endocytosis in a Malian family with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) comprises a large group of neurogenetic disorders characterized by progressive lower extremity spasticity. Neurological evaluation and genetic testing were completed in a Malian family with early-onset HSP. Three children with unaffected consanguineous parents presented with symptoms consistent with childhood-onset complicated HSP. Neurological evaluation found lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI showed corpus callosum thinning with cortical and spinal cord atrophy, and an EEG detected slow background in the index patient. Whole exome sequencing identified a homozygous missense variant in the adaptor protein (AP) complex 2 alpha-2 subunit (AP2A2) gene. Western blot analysis showed reduced levels of AP2A2 in patient-iPSC derived neuronal cells. Endocytosis of transferrin receptor (TfR) was decreased in patient-derived neurons. In addition, we observed increased axon initial segment length in patient-derived neurons. Xenopus tropicalis tadpoles with ap2a2 knockout showed cerebral edema and progressive seizures. Immunoprecipitation of the mutant human AP-2-appendage alpha-C construct showed defective binding to accessory proteins. We report AP2A2 as a novel genetic entity associated with HSP and provide functional data in patient-derived neuron cells and a frog model. These findings expand our understanding of the mechanism of HSP and improve the genetic diagnosis of this condition.

FANCA c.3624C>T (p.Ser1208=) is a hypomorphic splice variant associated with delayed onset of Fanconi anemia.

ABSTRACT: Fanconi anemia (FA) is a hereditary, DNA repair deficiency disorder caused by pathogenic variants in any 1 of 22 known genes (FANCA-FANCW). Variants in FANCA account for nearly two-thirds of all patients with FA. Clinical presentation of FA can be heterogeneous and include congenital abnormalities, progressive bone marrow failure, and predisposition to cancer. Here, we describe a relatively mild disease manifestation among 6 individuals diagnosed with FA, each compound heterozygous for 1 established pathogenic FANCA variant and 1 FANCA exon 36 variant, c.3624C>T. These individuals had delayed onset of hematological abnormalities, increased survival, reduced incidence of cancer, and improved fertility. Although predicted to encode a synonymous change (p.Ser1208=), the c.3624C>T variant causes a splicing error resulting in a FANCA transcript missing the last 4 base pairs of exon 36. Deep sequencing and quantitative reverse transcription polymerase chain reaction analysis revealed that 6% to 10% of the FANCA transcripts included the canonical splice product, which generated wild-type FANCA protein. Consistently, functional analysis of cell lines from the studied individuals revealed presence of residual FANCD2 ubiquitination and FANCD2 foci formation, better cell survival, and decreased late S/G2 accumulation in response to DNA interstrand cross-linking agent, indicating presence of residual activity of the FA repair pathway. Thus, the c.3624C>T variant is a hypomorphic allele, which contributes to delayed manifestation of FA disease phenotypes in individuals with at least 1 c.3624C>T allele.

Haplotype-based analysis resolves missing heritability in oculocutaneous albinism type 1B.

Oculocutaneous albinism (OCA) is a rare disorder of pigment production. Affected individuals have variably decreased global pigmentation and visual-developmental changes that lead to low vision. OCA is notable for significant missing heritability, particularly among individuals with residual pigmentation. Tyrosinase (TYR) is the rate-limiting enzyme in melanin pigment biosynthesis and mutations that decrease enzyme function are one of the most common causes of OCA. We present the analysis of high-depth short-read TYR sequencing data for a cohort of 352 OCA probands, ∼50% of whom were previously sequenced without yielding a definitive diagnostic result. Our analysis identified 66 TYR single-nucleotide variants (SNVs) and small insertion/deletions (indels), 3 structural variants, and a rare haplotype comprised of two common frequency variants (p.Ser192Tyr and p.Arg402Gln) in cis-orientation, present in 149/352 OCA probands. We further describe a detailed analysis of the disease-causing haplotype, p.[Ser192Tyr; Arg402Gln] ("cis-YQ"). Haplotype analysis suggests that the cis-YQ allele arose by recombination and that multiple cis-YQ haplotypes are segregating in OCA-affected individuals and control populations. The cis-YQ allele is the most common disease-causing allele in our cohort, representing 19.1% (57/298) of TYR pathogenic alleles in individuals with type 1 (TYR-associated) OCA. Finally, among the 66 TYR variants, we found several additional alleles defined by a cis-oriented combination of minor, potentially hypomorph-producing alleles at common variant sites plus a second, rare pathogenic variant. Together, these results suggest that identification of phased variants for the full TYR locus are required for an exhaustive assessment for potentially disease-causing alleles.

Differential Regulation of Retinoic Acid Metabolism in Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital abnormalities and increased risk for bone marrow failure and cancer. FA is caused by mutation of any one of 23 genes, the protein products of which function primarily in the maintenance of genome stability. An important role for the FA proteins in the repair of DNA interstrand crosslinks (ICLs) has been established in vitro . While the endogenous sources of ICLs relevant to the pathophysiology of FA have yet to be clearly determined, a role for the FA proteins in a two-tier system for the detoxification of reactive metabolic aldehydes has been established. To discover new metabolic pathways linked to FA, we performed RNA-seq analysis on non-transformed FA-D2 ( FANCD2 -/- ) and FANCD2-complemented patient cells. Multiple genes associated with retinoic acid metabolism and signaling were differentially expressed in FA-D2 ( FANCD2 -/- ) patient cells, including ALDH1A1 and RDH10 , which encode for retinaldehyde and retinol dehydrogenases, respectively. Increased levels of the ALDH1A1 and RDH10 proteins was confirmed by immunoblotting. FA-D2 ( FANCD2 -/- ) patient cells displayed increased aldehyde dehydrogenase activity compared to the FANCD2-complemented cells. Upon exposure to retinaldehyde, FA-D2 ( FANCD2 -/- ) cells exhibited increased DNA double-strand breaks and checkpoint activation indicative of a defect in the repair of retinaldehyde-induced DNA damage. Our findings describe a novel link between retinoic acid metabolism and FA and identify retinaldehyde as an additional reactive metabolic aldehyde relevant to the pathophysiology of FA.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Studies of a mosaic patient with DBA and chimeric mice reveal erythroid cell-extrinsic contributions to erythropoiesis.

We follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.

Pathogenic germline IKZF1 variant alters hematopoietic gene expression profiles.

IKZF1 encodes Ikaros, a zinc finger-containing transcription factor crucial to the development of the hematopoietic system. Germline pathogenic variants in IKZF1 have been reported in patients with acute lymphocytic leukemia and immunodeficiency syndromes. Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by erythroid hypoplasia, associated with a spectrum of congenital anomalies and an elevated risk of certain cancers. DBA is usually caused by heterozygous pathogenic variants in genes that function in ribosomal biogenesis; however, in many cases the genetic etiology is unknown. We identified a germline IKZF1 variant, rs757907717 C > T, in identical twins with DBA-like features and autoimmune gastrointestinal disease. rs757907717 C > T results in a p.R381C amino acid change in the IKZF1 Ik-x isoform (p.R423C on isoform Ik-1), which we show is associated with altered global gene expression and perturbation of transcriptional networks involved in hematopoietic system development. These data suggest that this missense substitution caused a DBA-like syndrome in this family because of alterations in hematopoiesis, including dysregulation of networks essential for normal erythropoiesis and the immune system.

The causes of Fanconi anemia in South Asia and the Middle East: A case series and review of the literature.

BACKGROUND: Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with characteristic dysmorphology primarily caused by biallelic pathogenic germline variants in any of 22 different DNA repair genes. There are limited data on the specific molecular causes of FA in different ethnic groups. METHODS: We performed exome sequencing and copy number variant analyses on 19 patients with FA from 17 families undergoing hematopoietic cell transplantation evaluation in Pakistan. The scientific literature was reviewed, and we curated germline variants reported in patients with FA from South Asia and the Middle East. RESULTS: The genetic causes of FA were identified in 14 of the 17 families: seven FANCA, two FANCC, one FANCF, two FANCG, and two FANCL. Homozygous and compound heterozygous variants were present in 12 and two families, respectively. Nine families carried variants previously reported as pathogenic, including two families with the South Asian FANCL founder variant. We also identified five novel likely deleterious variants in FANCA, FANCF, and FANCG in affected patients. CONCLUSIONS: Our study supports the importance of determining the genomic landscape of FA in diverse populations, in order to improve understanding of FA etiology and assist in the counseling of families.

Association of clinical severity with FANCB variant type in Fanconi anemia.

Fanconi anemia (FA) is the most common genetic cause of bone marrow failure and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry. Those with FANCB deletion or truncation demonstrate earlier-than-average onset of bone marrow failure and more severe congenital abnormalities compared with a large series of FA individuals in published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization were associated with 2 missense variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays tended to show earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is associated with level of residual FANCD2 monoubiquitination.

A founder variant in the South Asian population leads to a high prevalence of FANCL Fanconi anemia cases in India.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease-causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.

Somatic mosaicism of an intragenic FANCB duplication in both fibroblast and peripheral blood cells observed in a Fanconi anemia patient leads to milder phenotype.

BACKGROUND: Fanconi anemia (FA) is a rare disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer. Patients harboring X-linked FANCB pathogenic variants usually present with severe congenital malformations resembling VACTERL syndrome with hydrocephalus. METHODS: We employed the diepoxybutane (DEB) test for FA diagnosis, arrayCGH for detection of duplication, targeted capture and next-gen sequencing for defining the duplication breakpoint, PacBio sequencing of full-length FANCB aberrant transcript, FANCD2 ubiquitination and foci formation assays for the evaluation of FANCB protein function by viral transduction of FANCB-null cells with lentiviral FANCB WT and mutant expression constructs, and droplet digital PCR for quantitation of the duplication in the genomic DNA and cDNA. RESULTS: We describe here an FA-B patient with a mild phenotype. The DEB diagnostic test for FA revealed somatic mosaicism. We identified a 9154 bp intragenic duplication in FANCB, covering the first coding exon 3 and the flanking regions. A four bp homology (GTAG) present at both ends of the breakpoint is consistent with microhomology-mediated duplication mechanism. The duplicated allele gives rise to an aberrant transcript containing exon 3 duplication, predicted to introduce a stop codon in FANCB protein (p.A319*). Duplication levels in the peripheral blood DNA declined from 93% to 7.9% in the span of eleven years. Moreover, the patient fibroblasts have shown 8% of wild-type (WT) allele and his carrier mother showed higher than expected levels of WT allele (79% vs. 50%) in peripheral blood, suggesting that the duplication was highly unstable. CONCLUSION: Unlike sequence point variants, intragenic duplications are difficult to precisely define, accurately quantify, and may be very unstable, challenging the proper diagnosis. The reversion of genomic duplication to the WT allele results in somatic mosaicism and may explain the relatively milder phenotype displayed by the FA-B patient described here.

A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families.

Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two-thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA-null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype-phenotype correlations.

Assessing the spectrum of germline variation in Fanconi anemia genes among patients with head and neck carcinoma before age 50.

BACKGROUND: Patients with Fanconi anemia (FA) have an increased risk for head and neck squamous cell carcinoma (HNSCC). The authors sought to determine the prevalence of undiagnosed FA and FA carriers among patients with HNSCC as well as an age cutoff for FA genetic screening. METHODS: Germline DNA samples from 417 patients with HNSCC aged <50 years were screened for sequence variants by targeted next-generation sequencing of the entire length of 16 FA genes. RESULTS: The sequence revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, 9 indels, 1 large deletion, and 1 synonymous variant that was predicted to effect splicing. One hundred eight patients (26%) had at least 1 rare variant that was predicted to be damaging, and 57 (14%) had at least 1 rare variant that was predicted to be damaging and had been previously reported. Fifteen patients carried 2 rare variants or an X-linked variant in an FA gene. Overall, an age cutoff for FA screening was not identified among young patients with HNSCC, because there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, an increased burden, or mutation load, of FA gene variants was observed in carriers of the genes FA complementation group D2 (FANCD2), FANCE, and FANCL in the HNSCC patient cohort relative to the 1000 Genomes population. CONCLUSIONS: FA germline functional variants offer a novel area of study in HNSCC tumorigenesis. FANCE and FANCL, which are components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. In the current cohort, the increased mutation load of FANCD2, FANCE, and FANCL variants among younger patients with HNSCC indicates the importance of the FA pathway in HNSCC. Cancer 2017;123:3943-54. © 2017 American Cancer Society.

Deficiency of Interleukin-1 Receptor Antagonist (DIRA): Report of the First Indian Patient and a Novel Deletion Affecting IL1RN.

PURPOSE: Deficiency of interleukin-1 receptor antagonist (DIRA) is a rare life-threatening autoinflammatory disease caused by autosomal recessive mutations in IL1RN. DIRA presents clinically with early onset generalized pustulosis, multifocal osteomyelitis, and elevation of acute phase reactants. We evaluated and treated an antibiotic-unresponsive patient with presumed DIRA with recombinant IL-1Ra (anakinra). The patient developed anaphylaxis to anakinra and was subsequently desensitized. METHODS: Genetic analysis of IL1RN was undertaken and treatment with anakinra was initiated. RESULTS: A 5-month-old Indian girl born to healthy non-consanguineous parents presented at the third week of life with irritability, sterile multifocal osteomyelitis including ribs and clavicles, a mild pustular rash, and elevated acute phase reactants. SNP array of the patient's genomic DNA revealed a previously unrecognized homozygous deletion of approximately 22.5 Kb. PCR and Sanger sequencing of the borders of the deleted area allowed identification of the breakpoints of the deletion, thus confirming a homozygous 22,216 bp deletion that spans the first four exons of IL1RN. Due to a clinical suspicion of DIRA, anakinra was initiated which resulted in an anaphylactic reaction that triggered desensitization with subsequent marked and sustained clinical and laboratory improvement. CONCLUSION: We report a novel DIRA-causing homozygous deletion affecting IL1RN in an Indian patient. The mutation likely is a founder mutation; the design of breakpoint-specific primers will enable genetic screening in Indian patients suspected of DIRA. The patient developed anaphylaxis to anakinra, was desensitized, and is in clinical remission on continued treatment.

Whole-Exome Sequencing for Diagnosis of Turner Syndrome: Toward Next-Generation Sequencing and Newborn Screening.

Context: Turner syndrome (TS) is due to a complete or partial loss of an X chromosome in female patients and is not currently part of newborn screening (NBS). Diagnosis is often delayed, resulting in missed crucial diagnostic and therapeutic opportunities. Objectives: This study sought to determine if whole-exome sequencing (WES) as part of a potential NBS program could be used to diagnose TS. Design, Setting, Patients: Karyotype, chromosomal microarray, and WES were performed on blood samples from women with TS (n = 27) enrolled in the Personalized Genomic Research study at the National Institutes of Health. Female control subjects (n = 37) and male subjects (n = 27) also underwent WES. Copy number variation was evaluated using EXCAVATOR2 and B allele frequency was calculated from informative single nucleotide polymorphisms. Simulated WES data were generated for detection of low-level mosaicism and complex structural chromosome abnormalities. Results: We detected monosomy for chromosome X in all 27 TS samples, including 1 mosaic for 45,X/46,XX and another with previously unreported material on chromosome Y. Sensitivity and specificity were both 100% for the diagnosis of TS with no false-positive or false-negative results. Using simulated WES data, we detected isochromosome Xq and low-level mosaicism as low as 5%. Conclusion: We present an accurate method of diagnosing TS using WES, including cases with low-level mosaicism, isochromosome Xq, and cryptic Y-chromosome material. Given the potential use of next-generation sequencing for NBS in many different diseases and syndromes, we propose WES can be used as a screening test for TS in newborns.

Novel and known ribosomal causes of Diamond-Blackfan anaemia identified through comprehensive genomic characterisation.

BACKGROUND: Diamond-Blackfan anaemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterised by erythroid hypoplasia. It is associated with congenital anomalies and a high risk of developing specific cancers. DBA is caused predominantly by autosomal dominant pathogenic variants in at least 15 genes affecting ribosomal biogenesis and function. Two X-linked recessive genes have been identified. OBJECTIVES: We aim to identify the genetic aetiology of DBA. METHODS: Of 87 families with DBA enrolled in an institutional review board-approved cohort study (ClinicalTrials.gov Identifier:NCT00027274), 61 had genetic testing information available. Thirty-five families did not have a known genetic cause and thus underwent comprehensive genomic evaluation with whole exome sequencing, deletion and CNV analyses to identify their disease-associated pathogenic variant. Controls for functional studies were healthy mutation-negative individuals enrolled in the same study. RESULTS: Our analyses uncovered heterozygous pathogenic variants in two previously undescribed genes in two families. One family had a non-synonymous variant (p.K77N) in RPL35; the second family had a non-synonymous variant (p. L51S) in RPL18. Both of these variants result in pre-rRNA processing defects. We identified heterozygous pathogenic variants in previously known DBA genes in 16 of 35 families. Seventeen families who underwent genetic analyses are yet to have a genetic cause of disease identified. CONCLUSIONS: Overall, heterozygous pathogenic variants in ribosomal genes were identified in 44 of the 61 families (72%). De novo pathogenic variants were observed in 57% of patients with DBA. Ongoing studies of DBA genomics will be important to understand this complex disorder.

iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations.

Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs-whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines. Targeted deep sequencing of parental fibroblasts revealed that most variants detected in clonal iPSCs and fibroblast subclones were rare variants inherited from the parental fibroblasts. Only a small number of variants remained undetectable in the parental fibroblasts, which were thus likely to be de novo. Importantly, the clonal iPSCs and fibroblast subclones contained comparable numbers of de novo variants. Collectively, our data suggest that iPSC reprogramming is not mutagenic.

Paternal or Maternal Uniparental Disomy of Chromosome 16 Resulting in Homozygosity of a Mutant Allele Causes Fanconi Anemia.

Fanconi anemia (FA) is a rare inherited disorder caused by pathogenic variants in one of 19 FANC genes. FA patients display congenital abnormalities, and develop bone marrow failure, and cancer susceptibility. We identified homozygous mutations in four FA patients and, in each case, only one parent carried the obligate mutant allele. FANCA and FANCP/SLX4 genes, both located on chromosome 16, were the affected recessive FA genes in three and one family respectively. Genotyping with short tandem repeat markers and SNP arrays revealed uniparental disomy (UPD) of the entire mutation-carrying chromosome 16 in all four patients. One FANCA patient had paternal UPD, whereas FA in the other three patients resulted from maternal UPD. These are the first reported cases of UPD as a cause of FA. UPD indicates a reduced risk of having another child with FA in the family and has implications in prenatal diagnosis.

Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia.

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.

Comprehensive analysis of pathogenic deletion variants in Fanconi anemia genes.

Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution comparative genome hybridization arrays, single-nucleotide polymorphism arrays, and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by nonallelic homologous recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants.