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FANCM GENE; FANCM

FANCM GENE; FANCM

Alternative titles; symbolsFANCONI ANEMIA-ASSOCIATED POLYPEPTIDE, 250-KD; FAAP250KIAA1596HGNC Approved Gene Symbol: FANCMCytogenetic location: 14q21.2 Genomi...

Alternative titles; symbols

  • FANCONI ANEMIA-ASSOCIATED POLYPEPTIDE, 250-KD; FAAP250
  • KIAA1596

HGNC Approved Gene Symbol: FANCM

Cytogenetic location: 14q21.2 Genomic coordinates (GRCh38): 14:45,135,929-45,200,889 (from NCBI)

▼ Cloning and Expression
By sequencing clones obtained from a size-fractionated fetal brain cDNA library, Nagase et al. (2000) cloned FANCM, which they designated KIAA1596. RT-PCR ELISA detected low to moderate FANCM expression in testis and ovary, lower levels in fetal liver and adult brain, skeletal muscle, kidney, and spleen, and little to no expression in fetal brain and adult spinal cord, heart, lung, liver, and pancreas. Within specific adult brain regions, low to moderate expression was detected in amygdala, with lower levels in all other brain regions examined.

Meetei et al. (2003, 2003, 2004) purified a Fanconi anemia core complex containing 7 Fanconi anemia-associated proteins each essential for monoubiquitination of FANCD2 (613984), a key reaction in the Fanconi anemia DNA damage-response pathway. Using mass spectrometry, Meetei et al. (2005) identified another component, FAAP250, as KIAA1596. Antibodies raised against KIAA1596 specifically recognized the 250-kD polypeptide of the Fanconi anemia core complex immunopurified using antibodies against FANCA (607139). FAAP250, or FANCM, has sequence similarity to DNA repair proteins, including archaeal Hef, yeast MPH1, and human ERCC4 (133520). The deduced 2,048-amino acid protein contains an N-terminal helicase domain homologous to those of DEAH box helicases (see 607570) or DNA-stimulated ATPases. It also has a C-terminal endonuclease domain homologous to that of ERCC4, but sequence degeneracy suggests that it is inactive.

By immunohistochemical analysis of human testicular tissue sections, Kasak et al. (2018) observed localization of FANCM to the cytoplasm of Sertoli cells and spermatogenic cells in the seminiferous tubules. Staining intensity was related to stage of maturation, with faint staining of spermatogonia, increased staining in primary spermatocytes to spermatids, and reduced staining in tubules with mature spermatozoa. FANCM expression was also present in the interstitial Leydig cells.

Fouquet et al. (2017) performed qRT-PCR in germ cells from human fetal ovaries and observed expression throughout ovarian development, with highest expression in ovaries at stages containing the highest proportion of germ cells progressing into meiotic prophase I. Cell-sorting experiments revealed that FANCM transcripts were predominant in oogonial cells compared to somatic cells. Immunohistochemical analysis of human fetal ovaries showed that FANCM protein was present in the nuclei of oogonia, with strongest staining in pachytene stage oocytes. In addition, staining localized along the chromosomes in pachytene cells undergoing meiotic recombination. FANCM was also observed in oocytes arrested at the diplotene stage of prophase I during the last trimester of pregnancy and in adults. Costaining with SYCP3 (604759) and DDX4 (605281) confirmed the meiotic and germinal nature, respectively, of the FANCM-positive cells.

▼ Mapping
Nagase et al. (2000) mapped the KIAA1596 cDNA, corresponding to the FANCM gene, to chromosome 14. Meetei et al. (2005) stated that 2 tightly linked flanking markers are D14S259 and D14S1027.

▼ Gene Function
Meetei et al. (2005) found that FANCM could dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM was essential for monoubiquitination of FANCD2 and became hyperphosphorylated in response to DNA damage. The data of Meetei et al. (2005) suggested an evolutionary link between Fanconi anemia-associated proteins and DNA repair. They suggested that FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.

Ciccia et al. (2007) found that FAAP24 (610884) interacted specifically with the C-terminal region of FANCM in several assays. In fractionated HeLa cells, the FAAP24/FANCM heterodimer associated with other FA core proteins in an 800-kD complex. Downregulation of FAAP24 by small interfering RNA resulted in reduced levels of monoubiquitylated FANCD2 after exposure to DNA crosslinking reagents. FAAP24 bound single-stranded DNA. Ciccia et al. (2007) suggested that FAAP24 promotes targeting of FANCM/FAAP24 dimers, and possibly other components of the FA core complex, to forked DNA intermediates generated after DNA damage.

FANCM displays DNA-dependent ATPase activity and promotes dissociation of DNA triplexes. Gari et al. (2008) found that recombinant human FANCM bound Holliday junctions and replication forks with high specificity and promoted migration of their junction point in an ATPase-dependent manner. FANCM dissociated large recombination intermediates via branch migration of Holliday junctions through 2.6 kb of DNA. Gari et al. (2008) concluded that FANCM has a direct role in DNA processing, consistent with the view that FA proteins coordinate DNA repair at stalled replication forks.

By copurification of epitope-tagged proteins, Collis et al. (2008) found that the checkpoint protein HCLK2 (TELO2; 611140) interacted strongly with the N-terminal region of FANCM and weakly with the C-terminal region. The interaction required the HEAT repeat structure of HCLK2. Immunoprecipitation analysis of human cell lines showed that endogenous FANCM and FAAP24 formed a stable complex with HCLK2 in the absence of other FA core complex components. Knockdown of either FANCM, FAAP24, or HCLK2 via small interfering RNA compromised ATR (601215)/CHK1 (603078)-mediated checkpoint signaling, leading to increased endogenous DNA damage and failure to efficiently invoke cell cycle checkpoint responses. Moreover, the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, was required for its role in ATR/CHK1 signaling. Collis et al. (2008) concluded that FANCM and FAAP24 couple checkpoint signaling with DNA repair via their interactions with HCLK2 and FA core complex components.

Fanconi anemia and Bloom syndrome (BS; 210900) share overlapping phenotypes, including aberrant DNA repair and cancer predisposition. Treatment of cells with DNA crosslinking agents results in association of the BS complex with the FA core complex in a supercomplex called BRAFT. Deans and West (2009) found that FANCM functioned as the bridge in the FA/BS supercomplex by binding specific FA and BS components via its highly conserved MM1 and MM2 motifs, respectively. MM1 specifically bound FANCF (603467) of the FA core complex. MM2 specifically bound the BS complex components RMI1 (610404) and topoisomerase III-alpha (TOP3A; 601243), but not the helicase BLM (RECQL3; 604610). Knockdown of FANCM using small interfering RNA eliminated FA/BS association. Alanine substitution of phe1232 and phe1236 within MM2 resulted in a FANCM protein unable to interact with the BS complex. Interaction of FANCM with RMI1 and TOP3A was required for targeting of BLM to nuclear foci after treatment with replication stalling agents, but not ionizing radiation. Deans and West (2009) concluded that the FA and BS complexes interact via FANCM in a subset of DNA repair reactions, including those at replication forks stalled at sites of DNA damage.

Blackford et al. (2012) found that cells expressing translocase-defective FANCM showed altered global replication due to increased accumulation of stalled replication forks, which subsequently degenerated into DNA double-strand breaks, leading to ATM activation, CTIP-dependent end resection, and homologous recombination repair.

▼ Molecular Genetics
Spermatogenic Failure 28

In 2 Estonian brothers with nonobstructive azoospermia and Sertoli cell-only syndrome (SPGF28; 618086), Kasak et al. (2018) identified compound heterozygosity for a 1-bp duplication (609644.0003) and a splice site mutation (609644.0004) in the FANCM gene. The authors also reported homozygosity for nonsense mutations in the FANCM gene in 2 additional men with nonobstructive azoospermia: an unrelated Estonian man (Q1701X; 609644.0005) and a Portuguese man (R1931X; 609644.0006).

In 3 Pakistani brothers with infertility due to oligoasthenospermia or azoospermia, Yin et al. (2019) identified homozygosity for a 13-bp deletion in the FANCM gene (609644.0007).

Premature Ovarian Failure 15

In 2 Finnish sisters with premature ovarian failure-15 (POF15; 618096), Fouquet et al. (2017) identified homozygosity for the Q1701X mutation (609644.0005) in the FANCM gene.

Associations Pending Confirmation

Kiiski et al. (2014) performed whole-exome sequencing of constitutional genomic DNA from 24 breast cancer patients from 11 Finnish breast cancer families. From all rare damaging variants, 22 variants in 21 DNA repair genes were genotyped in 3,166 breast cancer patients, 569 ovarian cancer patients, and 2,090 controls, all from the Helsinki or Tampere regions of Finland. A nonsense mutation in FANCM, c.5101C-T (Q1701X; rs147021911) was significantly more frequent among breast cancer patients than among controls (odds ratio (OR) = 1.86, 95% CI = 1.26-2.75; p = 0.0018), with particular enrichment among patients with triple-negative breast cancer (TNBC; OR = 3.56, 95% CI = 1.81-6.98, p = 0.0002). In the Helsinki and Tampere regions, respectively, carrier frequencies of FANCM Q1701X were 2.9% and 4.0% of breast cancer patients, 5.6% and 6.6% of TNBC patients, 2.2% of ovarian cancer patients (from Helsinki), and 1.4% and 2.5% of controls. Kiiski et al. (2014) concluded that their findings identified FANCM as a breast cancer susceptibility gene, mutations in which confer a particularly strong predisposition for TNBC.

Exclusion Studies

In a large population-based exome-sequencing study of 3,000 Finnish individuals, Lim et al. (2014) did not find a deficit of individuals homozygous for predicted loss-of-function variants in the FANCM gene (5 expected, 7 observed). Examination of hospital records for homozygous carriers did not provide any evidence for blood diseases, increased cancer events, or other chronic diseases in these individuals compared to those without these variants. The findings did not support the hypothesis that FANCM is a gene associated with Fanconi anemia (see, e.g., FANCA, 227650).

▼ Animal Model
Bakker et al. (2009) generated Fancm-deficient mice by deleting exon 2. Fancm deficiency caused hypogonadism in mice and hypersensitivity to crosslinking agents in mouse embryonic fibroblasts (MEFs), similar to other FA mouse models. Fancm -/- mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm -/- mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, Fancm appeared to have a stimulatory rather than essential role in Fancd2 (227646) monoubiquitination. Bakker et al. (2009) suggested that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.

McNairn et al. (2019) found that mutations in the heterohexameric minichromosome maintenance (MCM; see 116945) complex that cause genomic instability render female mouse embryos markedly more susceptible than males to embryonic lethality. XX embryos could be rescued by transgene-mediated sex reversal or testosterone administration. The ability of exogenous or endogenous testosterone to protect embryos was related to its antiinflammatory properties. Ibuprofen, a nonsteroidal antiinflammatory drug, rescued female embryos that contained mutations in not only the Mcm genes but also the Fancm gene; similar to Mcm mutants, Fancm mutant embryos have increased levels of genomic instability (measured as the number of cells with micronuclei) from compromised replication fork repair.

▼ ALLELIC VARIANTS ( 7 Selected Examples):

.0001 RECLASSIFIED - VARIANT OF UNKNOWN SIGNIFICANCE
FANCM, SER724TER
This variant, formerly titled FANCONI ANEMIA, COMPLEMENTATION GROUP M based on the report of Meetei et al. (2005), has been reclassified based on the findings of Singh et al. (2009).

In a cell line derived from a patient (EUFA867) with Fanconi anemia, Meetei et al. (2005) identified compound heterozygous variants in the FANCM gene: a c.2171C-A transversion in exon 13 resulting in a ser724-to-ter (S724X) substitution, and a 2,554-bp deletion (609644.0002), spanning part of intron 14 and almost all of exon 15, resulting in absence of the sequence encoded by exon 15. A sib with Fanconi anemia was reported to carry the same 2 mutations. The nonsense mutation was inherited from the healthy mother; the deletion was not detected in the father but he was thought to be gonadal mosaic for it. Immunoblotting of patient cells showed absence of the FANCM protein.

In cell lines derived from the 2 sibs originally reported by Meetei et al. (2005), Singh et al. (2009) identified biallelic mutations in the FANCA gene (607139.0011 and 607139.0012). In addition, Singh et al. (2009) noted that only 1 of the sibs had clinical features of the disorder and that the clinically affected sib carried only 1 of the FANCM variants. The clinically unaffected sib (EUFA867) carried both biallelic FANCA mutations and biallelic FANCM variants. Singh et al. (2009) reclassified the affected sib as having FANCA, and suggested that FANCM deficiency in the unaffected sib may have overruled the FANCA defect and changed the clinical outcome, possibly even attenuating the phenotype. Cellular studies of EUFA867 by Singh et al. (2009) showed that expression of FANCA could rescue the FANCD2 (613984) monoubiquitination defect. After correction of the FANCA defect, the FANCM-deficient cells remained hypersensitive to the cross-linking agent mitomycin C; they were also sensitive to the topoisomerase inhibitor camptothecin and to UV light. FANCM-null cells had some residual monoubiquitinating FANCD2. The findings suggested that FANCM is involved in different steps of the DNA damage response, including efficient FANCD2 monoubiquitination and DNA repair steps later in the Fanconi anemia pathway.

.0002 RECLASSIFIED - VARIANT OF UNKNOWN SIGNIFICANCE
FANCM, 2,554-BP DEL
This variant, formerly titled FANCONI ANEMIA, COMPLEMENTATION GROUP M based on the report of Meetei et al. (2005), has been reclassified based on the findings of Singh et al. (2009).

For discussion of the 2,554-bp deletion identified by Meetei et al. (2005), see 609644.0001.

.0003 SPERMATOGENIC FAILURE 28
FANCM, 1-BP DUP, 1491A (rs761250416)
In 2 Estonian brothers with nonobstructive azoospermia and Sertoli cell-only syndrome (SPGF28; 618086), Kasak et al. (2018) identified compound heterozygosity for a 1-bp duplication (c.1491dupA; chr14.45,159,189dupA, NCBI36) in exon 9 of the FANCM gene, causing a frameshift predicted to result in a premature termination codon (Gln498ThrfsTer7) within the conserved helicase domain, and a splice site mutation (c.4387-10A-G; 609644.0004) in intron 16, predicted to activate an intronic cryptic acceptor site and extend exon 17 by 9 base pairs, resulting in a premature termination codon (Arg1436_Ser1437insLeuLeuTer). The duplication was inherited from their mother, and the splice site mutation from their father. The mutations were identified by whole-exome sequencing and confirmed by Sanger sequencing. The duplication was present at low frequency in the gnomAD database (minor allele frequency, 7.22 x 10(-5); no homozygotes). Immunohistochemistry revealed absent or only faint staining for FANCM in patient seminiferous tubules compared to control.

.0004 SPERMATOGENIC FAILURE 28
FANCM, IVS16AS, A-G, -10
For discussion of the splice site mutation (c.4387-10A-G) in intron 16 of the FANCM gene, predicted to activate an intronic cryptic acceptor site and extend exon 17 by 9 basepairs, resulting in a premature termination codon (Arg1436_Ser1437insLeuLeuTer), that was found in compound heterozygous state in 2 Estonian brothers with spermatogenic failure-28 (SPGF28; 618086) by Kasak et al. (2018), see 609644.0003.

.0005 SPERMATOGENIC FAILURE 28
PREMATURE OVARIAN FAILURE 15, INCLUDED (1 family)
FANCM, GLN1701TER (rs147021911)
Spermatogenic Failure 28

In a 52-year-old Estonian man with small testes, nonobstructive azoospermia, elevated gonadotropic hormones, and low testosterone (SPGF28; 618086), Kasak et al. (2018) identified homozygosity for a c.5101C-T transition (chr14.45,189,123C-T, NCBI36) in exon 20 of the FANCM gene, resulting in a gln1701-to-ter (Q1701X) substitution. The variant was present at low frequency in the gnomAD database (minor allele frequency, 1.34 x 10(-3); 1 homozygote).

Premature Ovarian Failure 15

In 2 Finnish sisters with premature ovarian failure-15 (POF15; 618096), Fouquet et al. (2017) identified homozygosity for the Q1701X mutation in the FANCM gene. Their unaffected parents and brother were heterozygous for the mutation, which was present in the ExAC database at minor allele frequencies of 0.0013 in the general population and 0.0089 in the Finnish population, including 1 homozygote. Immunoblot analysis of patient cells confirmed expression of a truncated FANCM protein, which the authors noted would lack the C-terminal endonuclease and FAAP24 (610884)-interaction domain. In cells from the heterozygous mother, the mutant protein was present at significantly reduced levels compared to wildtype, consistent with nonsense-mediated decay. Analysis of patient lymphocytes exposed to mitomycin C (MMC) showed higher occurrence of chromosome breakages and rearrangements in patient cells than in those of their mother. MMC-treated patient lymphocytes also showed reduced monoubiquitination of FANCD2 (613984); however, the cells maintained detectable monoubiquitination capability in response to the replication-inhibiting agents hydroxyurea and aphidicolin. Patient lymphoblasts transiently complemented with wildtype FANCM recovered significant resistance to MMC and showed improved monoubiquitination of FANCD2.

.0006 SPERMATOGENIC FAILURE 28
FANCM, ARG1931TER (rs144567652)
In a Portuguese man with nonobstructive azoospermia (SPGF28; 618086), Kasak et al. (2018) identified homozygosity for a c.5791C-T transition (chr14.45,198,718C-T, NCBI36) in exon 22 of the FANCM gene, resulting in an arg1931-to-ter (R1931X) substitution. The variant was present at low frequency in the gnomAD database (minor allele frequency, 1.03 x 10(-3); no homozygotes).

.0007 SPERMATOGENIC FAILURE 28
FANCM, 13-BP DEL, NT1948
In 3 Pakistani brothers with infertility due to oligoasthenospermia or azoospermia (SPGF28; 618086), Yin et al. (2019) identified homozygosity for a 13-bp deletion (c.1946_1958del) in exon 11 of the FANCM gene, causing a frameshift predicted to result in a premature termination codon (Pro648LeufsTer16). Their unaffected first-cousin parents were each heterozygous for the mutation. Western blot analysis confirmed absence of full-length FANCM protein from blood samples of all 3 infertile brothers. Patient lymphocytes treated with mitomycin C displayed a dose-dependent increase in chromosomal breaks per cell, which averaged 3 to 17 times the number observed in similarly treated lymphocytes from the unaffected father.

Tags: 14q21.2