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. 2012 Jun 21;2012:603253. doi: 10.1155/2012/603253

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

Johan J P Gille 1,*, Karijn Floor 1, Lianne Kerkhoven 1, Najim Ameziane 1, Hans Joenje 1, Johan P de Winter 1
PMCID: PMC3388349  PMID: 22778927

Abstract

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

1. Introduction

Fanconi anemia (FA) is a rare inherited syndrome with diverse clinical symptoms including developmental defects, short stature, bone marrow failure, and a high risk of malignancies. Fifteen genetic subtypes have been distinguished: FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, and -P. [1–4]. The majority of patients (~85%) belong to the subtypes A (~60%), C (~10–15%), or G (~10%), while a minority (~15%) is distributed over the remaining 12 subtypes, with relative prevalences between <1 and 5%. These percentages may differ considerably within certain ethnic groups, due to founder effects. All subtypes seem to fit within a “classical” FA phenotype, except for D1 and N (mutated in BRCA2/FANCD1 and PALB2/FANCN), which are associated with a distinct, more severe, syndromic association. The mode of inheritance for all subtypes is autosomal recessive, except for FA-B, which is X-linked. These two different modes of inheritance have important consequences for the counseling of FA families. The relative prevalence of FA-B amongst unselected FA patients is estimated at 1.6% [5]. For all genetic subtypes disease genes have been identified (Table 1). Many mutations found in the various subtypes are private, but recurrent mutations are known, particularly in specific ethnic backgrounds (Table 2).

Table 1.

Fanconi anemia complementation groups, genes, and proteins.

Group Gene symbol(s)a Cytogenetic location Protein (amino acids) Domain structure (references)
A FANCA 16q24.3 1455 HEAT repeats [8]
B FANCB Xp22.31 859 —
C FANCC 9q22.3 558 HEAT repeats [8]
D1 b BRCA2 13q12.3 3418 RAD51- and DNA-binding motifs [9]
D2 FANCD2 3p25.3 1451 —
E FANCE 6p21.3 536 —
F FANCF 11p15 374 —
G FANCG 9p13 622 Tetratricopeptide repeats (TPR) [10]
I FANCI 15q26.1 1328 —
J b BRIP1 17q22 1249 DNA helicase [11, 12]
L FANCL 2p16.1 375 RING finger motif (E3 ligase) [7, 8]
M FANCM 14q21.3 2014 DNA helicase, nuclease [13]
N b PALB2 16p12.1 1186 —
O b RAD51C 17q25.1 376 —
P b SLX4 16p13.3 1834 Endonuclease scaffold [3, 4]
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aFor gene nomenclature see http://www.genenames.org/.

bThe proteins defective in groups D1, J, N, O, and P (boldface) act downstream or independent of the monoubiquitination of FANCD2; all other FA proteins act upstream of this process.

Table 2.

Major recurrent mutations in FA.

Gene Mutation* Geographic/ethnic background Comment References
FANCA c.3788_3790del (p.Phe1263del) European, Brazilian Relatively mild [14, 15]
c.1115_1118delTTGG (p.Val372fs) European Relatively mild [16]
Exon 12–17del
Exon 12–31del		 South-African Relatively common in Afrikaners [17]
c.295C>T (p.Gln99X) Spanish Gypsy population Worldwide highest prevalence of mutant FANCA allele [18]
FANCC c.711+4A>T (originally reported as IVS4+4A>T) Homozygous in 80% of Ashkenazi Jewish FA; relatively common in Japan. Severe phenotype in Jews, milder in Japanese. [19–22]
c.67delG (originally reported as 322delG) Homozygous in approx. 50% of Dutch FA patients Like other exon 1 mutations, relatively mild phenotype. [19, 23–25]
FANCD2 c.1948-16T>G Turkish Founder mutation [26]
FANCG c.313G>T (p.Glu105X) European 44% of mutated FANCG alleles in Germany. [27]
c.1077-2A>G Portuguese/Brazilian Founder mutation [27, 28]
c.1480+1G>C French-Canadian Founder mutation [28]
c.307+1G>C Japanese Founder mutation [28, 29]
c.1794_1803del (p.Trp599fs) European [28]
c.637_643del (p.Tyr213fs) Sub-Saharan Africa 82% of all black FA patients [30]
FANCJ c.2392C>T (p.Arg798X) Found in ca. 50% of FA-J patients of diverse ancestry; ancient mutation or hot spot. [11, 12]
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Nucleotide numbering based on ATG = +1.

Published sequence variations in FA genes, with their descriptions conforming to the current nomenclature rules, are listed at http://www.rockefeller.edu/fanconi/.

Most FA genes encode orphan proteins with no known molecular function. At least eight FA proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) assemble into a nuclear multiprotein core complex, which is required to activate FANCD2 and FANCI by monoubiquitination [6]. FANCL, which carries a RING finger domain, is supposed to represent the ubiquitin E3 ligase in this activation [7]. FANCM probably acts as a sensor of DNA damage and recruits the FA core complex to the site of damage, but FANCM also interacts with other proteins including Blm [6]. Monoubiquitination of FANCD2 and FANCI directs these proteins to areas of damaged chromatin where they interact with other proteins, resulting in repair of the damage [6]. The remaining FA proteins function downstream of or parallel to the FANCD2 activation step [6]. The exact nature of the DNA damage response, which when defective causes FA, remains to be defined. FANCJ/BRIP1 and FANCM possess DNA helicase motifs, which strongly suggests that the FA pathway acts through a direct interaction with DNA, presumably to resolve or remodel blocked DNA replication forks resulting from DNA interstrand cross-link damage [6]. This idea is strengthened by the recent extension of the FA pathway with SLX4, a scaffold protein for structure-specific endonucleases involved in unhooking the DNA cross-link [3, 4].

2. Laboratory Diagnostics in FA

Cells derived from FA patients are—by definition—hypersensitive to chromosomal breakage induced by DNA cross-linking agents such as mitomycin C (MMC) or diepoxybutane (DEB) [31]. This cellular phenotype is ascertained using stimulated blood T lymphocytes. The indications for FA laboratory testing are rather broad [32]. As a consequence, in only a small proportion of patients (about 10%) the chromosomal breakage test is positive, and an FA diagnosis is established. Since mutation testing by Sanger sequencing and MLPA is rather laborious, time consuming and therefore expensive, a positive chromosomal breakage test is a prerequisite for starting mutation screening. Confirmation of the FA diagnosis at the DNA level is important in patients in whom the chromosomal breakage test was inconclusive. Furthermore, knowledge about the FA subtype is relevant for the treatment and prognosis of the patients. In addition, identification of mutations allows carrier testing in the family and will enable prenatal DNA testing and preimplantation genetic diagnosis (PGD) in future pregnancies. Finally, this information can be used to rule out FA in potential donors for bone marrow transplantation.

Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories worldwide. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative [33]. The strategy outlined below has been developed at our DNA diagnostics laboratory to provide a molecular diagnosis of FA. It is recognized that mutations in FANCA account for 60–70% of all FA cases and that about 15–20% of the mutations in this gene are large deletions [33, 34]. Therefore, DNA testing usually starts with a screen for deletions in FANCA. However, depending on the circumstances strategies may differ from case to case.

2.1. Materials

Genomic DNA (from e.g., leukocytes or fibroblasts derived from the proband or the parents) is adequate for most mutation screening assays. Screening on cDNA is more efficient but has several drawbacks: for high-quality cDNA, growing cells (stimulated leukocytes, lymphoblastoid cell lines, or fibroblasts) are necessary. In addition, common alternative splice variants will hamper the evaluation of DNA sequences. Therefore, screening on gDNA is the preferred method for mutation screening. However, during the diagnostic process, growing cells from the proband will be helpful in a couple of situations. Growing cells are indispensable for studying the effect of unclassified variants on splicing or to verify the disease gene by functional complementation of the cellular phenotype with a construct expressing a wild type copy of the suspected gene [35–37]. Finally, if no mutations can be de detected, growing cells can be used to reconfirm the diagnosis FA by checking MMC sensitivity in cell growth or G2-arrest assays [38, 39].

2.2. Mutation Screening Strategy

2.2.1. Hints from Ethnic Background or Phenotype

Information on the ethnic background of the proband may provide a clue for a specific pathogenic mutation that most likely causes the disease, such as c.711 + 4A > T (IVS4 + 4A > T) in FANCC, a mutation present in homozygous state in 80% of all FA cases of Ashkenazi Jewish ancestry, and c.295C > T in FANCA, which was present homozygously in all 40 FA cases of Spanish Gypsy ancestry so far investigated. More examples of recurrent mutations are shown in Table 3. The distinct clinical phenotype of D1 and N patients (severely affected, often combined with leukemia or solid tumors below the age of 5 years) may provide a clue to favor BRCA2/FANCD1 and PALB2/FANCN as the first gene to be screened [40–44]. This is especially worthwhile if confirmed by the cellular phenotype: in contrast to cells from all other known FA subtypes, cells from D1, N and O patients are unable to form RAD51 foci upon exposure to X rays or MMC [43–45].

Table 3.

Mutations detected in a cohort of 54 patients by screening FANCA, FANCC, FANCE, FANCF and FANCG.

Allele 1 Allele 2
Country of origin1 Gene DNA change Effect Number of database entries DNA change Effect Number of database entries
1 ES FANCA ex16_17del del 12x c.1115_1118del p.Val372fs 62x
2 PT FANCA c.718C>T p.Gln240X 2x c.2870G>A W957X 1x
3 NL FANCA ex15del del 3x ex15del del 3x
4 NL FANCA c.3788_3790del p.Phe1263del 215x c.3788_3790del p.Phe1263del 215x
5 CA FANCA c.718C>T pGlnx240X 2x c.1085T>C p.Leu362Pro novel
6 PT FANCA c.3788_3790del p.Phe1263del 215x c.4130C>G p.Ser1377X 1x
7 IE FANCA c.2812_2830dup p.Asp944fs 3x c.2812_2830dup p.Asp944fs 3x
8 AU FANCA c.2303T>C p.Leu768Pro 5x c.2303T>C p.Leu768Pro 5x
9 NL FANCA c.862G>T p.Glu288X 1x c.862G>T p.Glu288X 1x
10 NL FANCA ex11_33del del 1x c.2121delC p.Asn707fs novel
11 DK FANCA ex1_8del del 1x c.3788_3790del p.Phe1263del 215x
12 UK FANCA c.337_338del p.Ala114fs 1x c.3349A>G p.Arg1117Gly 2x
13 UK FANCA c.3568C>T p.Gln1190X novel c.3568C>T p.Gln1190X novel
14 NL FANCA c.487delC p.Arg163fs 1x c.2851C>T p.Arg951Trp 11x
15 SE FANCA c.88delG p.Val30fs novel c.100A>T p.Lys34X 2x
16 NL FANCA c.862G>T p.Glu288X 9x c.1771C>T p.Arg591X 9x
17 PT FANCA c.1709_1715+4del p.Glu570fs novel c.3430C>T p.Arg1144Trp novel
18 NO FANCA c.100A>T p.Lys34X 2x c.1378C>T p.Arg460X novel
19 PT FANCA ex15_17del del 2x ex15_17del del 2x
20 NL FANCA c.2982-192A>G splice2 novel ex7_31del del
21 AU FANCA c.427-8_427-5del splice novel c.1771C>T p.Arg591X 9x
22 AU FANCA c.3491C>T p.Pro1164Leu novel c.3491C>T p.Pro1164Leu novel
23 CA FANCA ex4_29del del novel ex31del del 6x
24 NL FANCA c.3391A>G p.Thr1131Ala 15x c.3391A>G p.Thr1131Ala 15x
25 GR FANCA c.2T>C p.Met1? 1x c.3788_3790del p.Phe1263del 215x
26 IE FANCA c.851dup p.Val285fs novel c.2534T>C p.Leu845Pro 4x
27 NL FANCA c.2852G>A p.Arg951Gln 6x c.3624C>T p.= (splice) 2x
28 AU FANCA c.331_334dup p.Leu112fs novel ex22_29del del novel
29 NL FANCA c.862G>T p.Glu288X 9x c.3920delA p.Gln1307fs 2x
30 IR FANCA ex21del del novel ex21del del novel
31 SE FANCA ex1_12del del novel ex22_29del del novel
32 NL FANCB c.755_767del p.Leu252fs novel — — —
33 NL FANCC c.67delG p.Asp23fs 50x c.553C>T p.Arg185X 14x
34 NL FANCC c.67delG p.Asp23fs 50x c.67delG p.Asp23fs 50x
35 CA FANCC c.67delG p.Asp23fs 50x c.553C>T p.Arg185X 14x
36 NL FANCC c.67delG p.Asp23fs 50x c.1155-1G>C splice novel
37 NL FANCC c.67delG p.Asp23fs 50x c.67delG p.Asp23fs 50x
38 NL FANCC c.67delG p.Asp23fs 50x c.467delC p.Ser156fs novel
39 PT FANCE c.1111C>T p.Arg371Trp 6x c.1111C>T p.Arg371Trp 6x
40 UK FANCF c.496C>T p.Gln166X 4x c.496C>T p.Gln166X 4x
41 UK FANCG c.307+2delT splice novel c.307+2delT splice novel
42 UK FANCG c.1471_1473delinsG p.Lys491fs novel c.1471_1473delinsG p.Lys491fs novel
43 NL FANCG c.65G>C p.Arg22Pro 6x c.65G>C; p.Arg22Pro 6x
44 IR FANCG c.307+1G>C splice 21x c.307+1G>C splice 21x
45 NL FANCG c.85-1G>A splice novel c.85-1G>A splice novel
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1Country of origins: AU: Australia; CA: Canada; DK: Denmark; ES: Spain; GR: Greece; IE: Ireland; IR: Iran; NL: Netherlands; PT: Portugal; SE: Sweden; UK: United Kingdom

Number of database entries refers to the FA database at: http://www.rockefeller.edu/fanconi/.

The pathogenic state of novel missense mutations is based upon in silico prediction algorithms (SIFT, POLYPHEN2, Align GVGD), the presence of a second clearly pathogenic mutation in the same gene and segregation in the family.

2Effect c.2982-192A>G: by studying cDNA it was shown that the mutation created a new splice donor site resulting in an aberrant mRNA.

2.2.2. No Clues Available

  1. In the absence of any clue to the disease gene, mutation screening starts with a search for deletions in FANCA, as this type of mutation accounts for 40% of all mutant FANCA alleles. The quantitative multiplex ligation-dependent probe amplification (MLPA) method [46] is used for this initial screen, which identifies FANCA as the most likely disease gene in 1 out of 4 patients by the detection of a—usually hemizygous-deletion. In parallel, the FANCA gene is completely sequenced. The combination of these two approaches identifies 60–70% of all FA patients as FA-A.

  2. Next, FANCC, -E, -F, and -G are screened by DNA sequencing.

  3. Only if the proband is a male, FANCB is screened by MLPA and DNA sequencing,

In Table 4, optimized conditions are provided for the PCR amplification of FANCA, -C, -E, -F, -G, and -B. Most PCRs can be performed under standard conditions. The PCR primers have M13 extensions which allow sequencing of all fragments with universal sequencing primers. MLPA was performed according to the instructions of the supplier. Detailed information about the sequences of the MLPA probes is available from the website of the supplier (http://www.mlpa.com). In a well-equipped laboratory with sufficient dedicated personal, testing of FANCA, -C, -E, -F, -G and -B can be completed within 1-2 weeks.

Table 4.

Primers and conditions for PCR on genomic DNA of the coding sequence plus intron/exon boundaries of FANCA, FANCC, FANCE, FANCF, FANCG, and FANCB.

(a)

FANCA
Primer name Sequence (5′ > 3′) Product length (bp)
FANCA_ex1F gtaaaacgacggccag GCGCCTCCCCCAGGACCAACA 362
FANCA_ex1R caggaaacagctatga AGGCTCTGGCGGGAAGGGATCGG
FANCA_ex2F gtaaaacgacggccag CTCTTCGGGAGGGTGTCGCTGGT 328
FANCA_ex2R caggaaacagctatga CTCTTCGGGAGGGTGTCGCTGGT
FANCA_ex3F gtaaaacgacggccag GCCTGGCCTGGAGCTTGAAT 392
FANCA_ex3R caggaaacagctatga CGCAGGTTGAATCAGACGCTGTT
FANCA_ex4F gtaaaacgacggccag TAAGGCATTTTAAACAGCAAGTC 430
FANCA_ex4R caggaaacagctatga TGCCAATAAATACTGAGCAAACT
FANCA_ex5F gtaaaacgacggccag AGTATTGTTTCAGGTAATTTGTT 356
FANCA_ex5R caggaaacagctatga TGAAGGTACTTCTTTCCAATCCA
FANCA_ex6F gtaaaacgacggccag AGATGTGTTTCAGGCTCTAAGTT 402
FANCA_ex6R caggaaacagctatga GCAATGCAATCTAGTCTAGTACA
FANCA_ex7F gtaaaacgacggccag TGGGATTTAGTTGAGCCTTACGTCTGC 421
FANCA_ex7R caggaaacagctatgaAAGGTGAATGGAAACACTTAAACTCATGTCA
FANCA_ex8F gtaaaacgacggccag GTGGTCAGGTGAGCAGTAACTTC 401
FANCA_ex8R caggaaacagctatga TAAATAGGTACAAACAGCACGTT
FANCA_ex9F gtaaaacgacggccag TTCTCTTGTGTGATGCAGGTATC 332
FANCA_ex9R caggaaacagctatga TGACCCACAGATTCATGAGGTAT
FANCA_ex10F gtaaaacgacggccag TTTTGATTAAGGCCTACAGATTG 406
FANCA_ex10R caggaaacagctatga CCTCCTCCTCACGCACGTTATCG
FANCA_ex11F gtaaaacgacggccag TTTCAAGTCTGTGGTTATAGTGG 410
FANCA_ex11R caggaaacagctatga AGACGTAAAAGAGGTCCTAGAAT
FANCA_ex12F gtaaaacgacggccag CTGTGGGGCTGGTCCTTAACAAA 236
FANCA_ex12R caggaaacagctatga AGGCAGCATGACAAGTACTAGGC
FANCA_ex13F gtaaaacgacggccag ACATTGGTTTGCTTGGATATTGA 377
FANCA_ex13R caggaaacagctatga CTGACAAAGAATGTTCCATCGAC
FANCA_ex14F gtaaaacgacggccag TGCTGTAATTGCTGTGTAGTCTT 411
FANCA_ex14R caggaaacagctatga ACTCACATGACAGAGAATCAGGT
FANCA_ex15F gtaaaacgacggccag ACTACAGCAGCCGCCCGGACACT 430
FANCA_ex15R caggaaacagctatga GCAGATCTGCAGGAGGCTCTTGG
FANCA_ex16F gtaaaacgacggccag TCCCAGGCAGTTCCCAGACTAAC 312
FANCA_ex16R caggaaacagctatga AGCTGATGACAAATCCTCGTAGA
FANCA_ex17F gtaaaacgacggccag ACCGCTCCCTCCTCACAGACTAC 334
FANCA_ex17R caggaaacagctatga AAGGCTGAAAAACTCAACTCAAG
FANCA_ex18F gtaaaacgacggccag GCGCACAGCATGTGGGCCTTTAC 397
FANCA_ex18R caggaaacagctatga GCAGCTGCTAGAGGCCTTTTCGG
FANCA_ex19F gtaaaacgacggccag GTGCACAAGAAGACTTCATAATG 284
FANCA_ex19R caggaaacagctatga AGTCCTTGCTTTCTACACAACTG
FANCA_ex20F gtaaaacgacggccag CTTCTCTGTGTTGCAGCATATTC 298
FANCA_ex20R caggaaacagctatga AGAAGAAACCTGGAAGTAGTCAT
FANCA_ex21F gtaaaacgacggccag ATAATAGATTTGGGGATTGTAAT 255
FANCA_ex21R caggaaacagctatga CAACAGACACTCAAGGTTAGGAA
FANCA_ex22F gtaaaacgacggccag TGCAGTGAAGAGTCCTGTTGAGT 305
FANCA_ex22R caggaaacagctatga ACACACCAGCCTGATGTCACTAT
FANCA_ex23F gtaaaacgacggccag CAGTCAGCAGGATCCGTGGAATC 416
FANCA_ex23R caggaaacagctatga GGCCCTGGAACATCTGATACGAC
FANCA_ex24F gtaaaacgacggccag CCTTCCTGCTGCTCCCGTCC 229
FANCA_ex24R caggaaacagctatga CAGACTTGGCCCAGCAAGAG
FANCA_ex25F gtaaaacgacggccag CCGCTGGTGGTTGGATTAGCTGT 296
FANCA_ex25R caggaaacagctatga TTTCCAGGGCACTGAAGACGAAT
FANCA_ex26F gtaaaacgacggccag AGCTTGGAAGAGGGCAGTCTGCT 347
FANCA_ex26R caggaaacagctatga CTCTTCTAATTTTATCAAACGAG
FANCA_ex27F gtaaaacgacggccag AGACTGTCTCACAACAAACGAAC 356
FANCA_ex27R caggaaacagctatga CGGTCCGAAAGCTGCGTAAAC
FANCA_ex28F gtaaaacgacggccag GTTGATGGTCTGTTTCCACCTGA 401
FANCA_ex28R caggaaacagctatga GAAGGAACGGTCACCTACGTGCT
FANCA_ex29F gtaaaacgacggccag GACATGGAGGACTGCGTATGAGA 411
FANCA_ex29R caggaaacagctatga GTGGCTGTGATGACTGGAACGTG
FANCA_ex30F gtaaaacgacggccag CCCGAGCCGCCAGTCTCAACCCA 411
FANCA_ex30R caggaaacagctatga AAAGGCAGACCCACCCTAAGCTA
FANCA_ex31F gtaaaacgacggccag GATAAGCCTCCTTGGTCATGGTA 406
FANCA_ex31R caggaaacagctatga TGGCAATAAATATCTTAATAGCA
FANCA_ex32F gtaaaacgacggccag TTCCTGTGCCAGCATACTGCTCT 359
FANCA_ex32R caggaaacagctatga GGGTGGGGACACACAGACAAGTA
FANCA_ex33F gtaaaacgacggccag TGGGTTTCAGGGTGGTGGTTGCT 356
FANCA_ex33R caggaaacagctatga GAACCCTTTCCTCAGTAATTCAC
FANCA_ex34F gtaaaacgacggccag CGCCCAGGGAAGCCGTTAAGTTT 333
FANCA_ex34R caggaaacagctatga GCGTTCTGAGAAGGCCACGAGAG
FANCA_ex35F gtaaaacgacggccag TTCCTTCACTCTACTAGTTGTGG 311
FANCA_ex35R caggaaacagctatga TGAGATGGTAACACCCGTGATGG
FANCA_ex36F gtaaaacgacggccag CCATCTCAGCCACCCTCATCTGT 350
FANCA_ex36R caggaaacagctatga AGGCGCCCACCACCACGAGAACT
FANCA_ex37F gtaaaacgacggccag GACTTGGTTTCTATGGCGTGGTT 310
FANCA_ex37R caggaaacagctatga CCCAGAGAAATAGCACTGATTGA
FANCA_ex38F gtaaaacgacggccag GTTTTCTAAGATCCACTTAAAGG 362
FANCA_ex38R caggaaacagctatga CTCACTCACACTTCCGCAAACAC
FANCA_ex39F gtaaaacgacggccag CTGTCCAGAGGCCCAGTATTACC 387
FANCA_ex39R caggaaacagctatga AGGAGGGCTCGTTCTTAACCATT
FANCA_ex40F gtaaaacgacggccag GGTGTCCCCAGCACTGATAATAG 353
FANCA_ex40R caggaaacagctatga AGACATAGTGACAAATGGCTACA
FANCA_ex41F gtaaaacgacggccag CCCTTGGCATCACCTGCTACCTT 403
FANCA_ex41R caggaaacagctatga AACAGGCAAACTCACAGGTTAGA
FANCA_ex42F gtaaaacgacggccag ACCAGCCCTGTTTCTGTATGTCT 248
FANCA_ex42R caggaaacagctatga ACATGGCCCAGGCAGCTGTCAAT
FANCA_ex43F gtaaaacgacggccag TGTGGGGGACATGAGAATTGACA 378
FANCA_ex43R caggaaacagctatga GTAATCCACTTTTTAGTGCAACA
FANCAIVS10F gtaaaacgacggccag TTTACATGTGCATCAGTTAGCTT 184
FANCAIVS10R caggaaacagctatga CATGAAGACACAGAAAAAGTAGGT
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(b)

FANCC
Primer name Sequence (5′ > 3′) Product length (bp)
FANCC_ex1F
FANCC_ex1R		 gtaaaacgacggccag ACCATTTCCTTCAGTGCTGGACA
caggaaacagctatga CCATCGGCACTTCAGTCAATACC		 378
FANCC_ex2F
FANCC_ex2R		 gtaaaacgacggccag CTAAACAAGAAGCATTCACGTTC
caggaaacagctatga GGAGAAAGGTTCATAATGTAAGC		 303
FANCC_ex3F
FANCC_ex3R		 gtaaaacgacggccag TCAGCAGAAAGAGAATGTGCAAA
caggaaacagctatga AACATCATAGAACTGGATTCCAC		 405
FANCC_ex4F
FANCC_ex4R		 gtaaaacgacggccag TGTACATAAAAGGCACTTGCATT
caggaaacagctatga TCCCATCTCACATTTCTTCCGTA		 380
FANCC_ex5F
FANCC_ex5R		 gtaaaacgacggccag AGAACTGATGTAATCCTGTTTGC
caggaaacagctatga TTACTGCTCTGTGAGAGTTGAGA		 367
FANCC_ex6F
FANCC_ex6R		 gtaaaacgacggccag GTCTTTGACCTTTTTAGCATGAA
caggaaacagctatga AACGTTTGGACACTGCTGTCGTA		 387
FANCC_ex7F
FANCC_ex7R		 gtaaaacgacggccag ATTAGTGATTGCATTTTGAACTT
caggaaacagctatga CAAAAATAAAATGTAAATACACG		 422
FANCC_ex8F
FANCC_ex8R		 gtaaaacgacggccag CTCCTTTGGCTGATAATAGCAAG
caggaaacagctatga CTGATTTTTGAGTTTTTACCTCT		 336
FANCC_ex9F
FANCC_ex9R		 gtaaaacgacggccag ATACTGCTGAAGCTTATGGCACA
caggaaacagctatga TAACCTTTGTTGGGGCACTCATT		 400
FANCC_ex10F
FANCC_ex10R		 gtaaaacgacggccag TATGAGGTTATTGGGAGCTTATT
caggaaacagctatga CTGTCTCCCTCATGCTGTAGATA		 382
FANCC_ex11F
FANCC_ex11R		 gtaaaacgacggccag GAACCAGAAGTAAAGGGCGTCTC
caggaaacagctatga CTGACCTGCTCCAAGCCATCCGT		 416
FANCC_ex12F
FANCC_ex12R		 gtaaaacgacggccag AAGTACAATTTAAGCCAACCGTT
caggaaacagctatga AGGTTGCCATGACATATGCCATC		 451
FANCC_ex13F
FANCC_ex13R		 gtaaaacgacggccag CCTCTCTCAGGGGCCAGTGCTTA
caggaaacagctatga AGACCCTCGGACAGGTAACCCAC		 435
FANCC_ex14F
FANCC_ex14R		 gtaaaacgacggccag ACTTGCTATGCTAATCACCTTGC
caggaaacagctatga AATGCGTGGCCACAGGTCATCAC		 437
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(c)

FANCE
Primer name Sequence (5′ > 3′) Product length (bp)
FANCE_ex1F
FANCE_ex1R		 gtaaaacgacggccag CGCCTCCCTCCTTCCCTTTC
caggaaacagctatga CCCGCCTCCCATACCTGCTAA		 540
FANCE_ex2aF
FANCE_ex2aR		 gtaaaacgacggccag GCTCTGCCCAGTCTGCCTTGTGC
caggaaacagctatga CTCTGAGTCCTTTCTGCGTTTCC		 469
FANCE_ex2bF
FANCE_ex2bR		 gtaaaacgacggccag GCCAGAGACAGCTCCAAAGTCTA
caggaaacagctatga CAGCCTTCCCCATGGATAAAGCC		 479
FANCE_ex3F
FANCE_ex3R		 gtaaaacgacggccag GCCTCTTGACTTTCTTGAATCAT
caggaaacagctatga ACTGTCCTCAGACCTTTACTCCA		 352
FANCE_ex4F
FANCE_ex4R		 gtaaaacgacggccag TTGAACCAAGTGTAGACTTACCA
caggaaacagctatga GGGAAGGAACCAAGGGCTAAAAG		 436
FANCE_ex5F
FANCE_ex5R		 gtaaaacgacggccag GTATCTTTTAGCCCTTGGTTCCT
caggaaacagctatga GAATCCCCTCTCTCAAGTACCAC		 431
FANCE_ex6F
FANCE_ex6R		 gtaaaacgacggccag TTTCCTTTGTAACATGTATCATC
caggaaacagctatga AGCAGAAAGCAGGGAGGCGGTAA		 433
FANCE_ex7F
FANCE_ex7R		 gtaaaacgacggccag ACAGGCTGGGCATTCTGTTACCG
caggaaacagctatga AGTGAGACACAAGGATCCCCTAA		 425
FANCE_ex8F
FANCE_ex8R		 gtaaaacgacggccag TTGGAGCAGCAGATAGATACTCA
caggaaacagctatga AGAGGTGGAGCTGAAGTGACCAT		 380
FANCE_ex9F
FANCE_ex9R		 gtaaaacgacggccag GTTACCTGCCCAGGGTCACCTAG
caggaaacagctatga CTGGCCAGCACTCAGGGTTTTAT		 388
FANCE_ex10F
FANCE_ex10R		 gtaaaacgacggccag TGGCCTCCTCTCTCCTCAATAGA
caggaaacagctatga AACAGGGAGGCAGTTGCAATCTG		 369
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(d)

FANCF
Primer name Sequence (5′ > 3′) Product length (bp)
FANCF_ex1aF
FANCF_ex1aR		 gtaaaacgacggccag TTTCGCGGATGTTCCAATCAGTA
caggaaacagctatga CTGCACCAGGTGGTAACGAGCTG		 449
FANCF_ex1bF
FANCF_ex1bR		 gtaaaacgacggccag AGTGGAGGCAAGAGGGCGGCTTT
caggaaacagctatga GCTATCACCTTCAGGAAGTTGTT		 456
FANCF_ex1cF
FANCF_ex1cR		 gtaaaacgacggccag CCCAAATCTCCAGGAGGACTCTC
caggaaacagctatga TTTCTGAAGGTCATAGTGCAAAC		 444
FANCF_ex1dF
FANCF_ex1dR		 gtaaaacgacggccag GCTTTTGACTTTAGTGACTAGCC
caggaaacagctatga ATTTGGTGAGAACATTGTAATTT		 456
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(e)

FANCG
Primer name Sequence (5′ > 3′) Product length (bp)
FANCG_ex1F
FANCG_ex1R		 gtaaaacgacggccag AGCCTGGGCGGGTGGATTGGGAC
caggaaacagctatga TCATTTCTGGCTCTTTGGTCAAG		 389
FANCG_ex2F
FANCG_ex2R		 gtaaaacgacggccag CAGGCCAAGGTAACACGGTTGCT
caggaaacagctatga CCAGTCTCCTCTGTGCCTTAAAC		 460
FANCG_ex3F
FANCG_ex3R		 gtaaaacgacggccag TATTGTAGCTGTTTTGGTTGGAG
caggaaacagctatga GGTGACAGATGTTGTTTATCCTC		 362
FANCG_ex4F
FANCG_ex4R		 gtaaaacgacggccag GGAGATGGAGGATGAGGTGCTAC
caggaaacagctatga CGACCACCAACCCAGCCGCCTGT		 411
FANCG_ex5F
FANCG_ex5R		 gtaaaacgacggccag AGATGGAGATAGGAGAAGACGAG
caggaaacagctatga GCTTCATGAAGGCTGCTTAGTGC		 454
FANCG_ex6F
FANCG_ex6R		 gtaaaacgacggccag CAGTTCCATGGGCTTCTTAGACC
caggaaacagctatga TCAGGGCTGCAACCAAGTACAAC		 393
FANCG_ex7F
FANCG_ex7R		 gtaaaacgacggccag GCACTGGGGTCCTGTCACCGTAA
caggaaacagctatga ATAATCTTTGGGAGCCATACTTC		 418
FANCG_ex8F
FANCG_ex8R		 gtaaaacgacggccag GCTTGTGATGGGGTGACTTGACT
caggaaacagctatga AGTTCAGGTCTAGAAGCAAGGTA		 438
FANCG_ex9F
FANCG_ex9R		 gtaaaacgacggccag CCTCCTCAGGGCCCATGAACATC
caggaaacagctatga GCAGTGTCTTGAAAGGCATGAGC		 400
FANCG_ex10F
FANCG_ex10R		 gtaaaacgacggccag CAGGACTCTGCATGGTACCAG
caggaaacagctatga CCAATCAGAAAATCATCCCTC		 460
FANCG_ex11F
FANCG_ex11R		 gtaaaacgacggccag AGCTCCATGTTCACCTACTTACC
caggaaacagctatga CAGTGCCGCATCTGACTTACATC		 397
FANCG_ex12F
FANCG_ex12R		 gtaaaacgacggccag AGGATTTGGGGTTTTGGTGACTG
caggaaacagctatga AACTCTTGGGAGCCCTGCATACA		 445
FANCG_ex13F
FANCG_ex13R		 gtaaaacgacggccag CCGCTTCCATATGTGAGTGTAGG
caggaaacagctatgaC CACAATAGGTCCAAGGACTCTA		 340
FANCG_ex14F
FANCG_ex14R		 gtaaaacgacggccag CCAAACTAAGGGGTCACATGAAG
caggaaacagctatga GATGGTGAAGCAGAAAGCCCTCC		 405
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(f)

FANCB
Primer name Sequence (5′ > 3′) Product length (bp)
FANCB_ex3AF
FANCB_ex3AR		 gtaaaacgacggccag GATATGGTTATTTGAATTCTTAGCAcaggaaacagctatga GCCATCCTTCATCTCATAGCCTAGT 721
FANCB_ex3BF
FANCB_ex3BR		 gtaaaacgacggccag ATTAACCTCCCTTACATTGTGATAGcaggaaacagctatga CAATAAGACTCCAGAATGAACTCTA 811
FANCB_ex4F
FANCB_ex4R		 gtaaaacgacggccag TTTACAAATGACAACTACATGAcaggaaacagctatga TTAAGTATAAAACCACCAATAT 391
FANCB_ex5F
FANCB_ex5R		 gtaaaacgacggccag ACTGCATCTGGCCTATAGTTcaggaaacagctatga AATACCATTTTTACCCAAGC 411
FANCB_ex6F
FANCB_ex6R		 gtaaaacgacggccag GTATTTCCTGAATTATTGGTATGTC
caggaaacagctatga CATAAAAGTCCACCATTATAACCTC		 395
FANCB_ex7F
FANCB_ex7R		 gtaaaacgacggccag TGTTTGGGCCATAAGCCCTA
caggaaacagctatga TTCTGGAGCATCAAGACAGT		 355
FANCB_ex8F
FANCB_ex8R		 gtaaaacgacggccag GTTGTTTGTATGACATTTAATCATC
caggaaacagctatga ATCATTAAACTCTGCCCATTATCAG		 636
FANCB_ex9F
FANCB_ex9R		 gtaaaacgacggccag AGGTAATTTTGTTGGCACTT
caggaaacagctatga ATGCGTTCATTCATGCTAGG		 531
FANCB_ex10F
FANCB_ex10R		 gtaaaacgacggccag AATTGGTTCTGTTTATCATTATGGT
caggaaacagctatga CTACTACAGTAAGCCTCGGTGTTTA		 686
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PCR conditions:

PCR was performed in Applied Biosystems PE9700 system using 96-well plates. PCR reactions (final volume 25 μl) contained 0.5 units Platinum Taq polymerase (Invitrogen), 1,5 mM MgCl2, 0.2 mM NTPs (Invitrogen), and 10 pmol primer.

For the large majority of amplicons, standard PCR conditions were used: preheat 95°C, 5 min, denaturation 95°C, 30 sec, annealing 60°C, 30 sec., elongation 72°C, 1 min, number of cycles: 33.

Fragments with a different annealing temperature were FANCA exons 5, 7, 13, 21, 26, 31, and 38, FANCC exon 7, FANCF fragment 1d and FANCE exon 1 : 55°C; FANCA exon 1 : 64°C. For FANCE exon 1 the PCR mix was supplemented with 10% DMSO.

For FANCB different PCR conditions were used: preheat 95°C, 5 min, denaturation 95°C, 1 min, annealing 50°C, 1 min., elongation 72°C, 1 min., number of cycles 30. For FANCB exon 7 and 9 the annealing temperature was 55°C. For sequencing of exon 7 forward, a special sequencing primer was used: 5′-TTTTTAGAAGGAATGTCTTG-3′.

FA gene specific part of the primer is indicated in capitals. Primers are extended with M13 sequence (indicated in normal letter type), which is used for the sequencing reaction.

After screening FANCA, -C, -E, -F, -G, and –B, a molecular diagnosis is obtained for ~85% of the patients [34]. In our cohort of 54 patients, referred to our diagnostic service since 2008, mutations were detected in 45 patients (83%). FANCA mutations were found in 31 of the patients (57%), FANCC mutations in 6 patients (11%), and FANCG mutations in 5 patients (9%). FANCB, FANCE, and FANCF mutations were found in single families (Table 3). In the small group of patients without mutations no complementation analysis or FANCD2 western blotting was performed. Therefore, we do not know if we missed FANCA, -C, -E, -F, -G, and -B mutations in these patients or that these patients have mutations in other FA genes. Table 3 does not include prenatal cases, because prenatal testing is only offered in couples in which the FA-causing mutations are already established. Testing was offered as a diagnostic service for which a fee was charged.

For the patients negative for FANCA, -C, -E, -F, -G, and -B mutations, next generation sequencing can be used to analyze all other FA genes. If this technique is not available, further analysis will depend on the availability of growing cells from the proband. In that case a western blot should reveal whether both FANCD2 isoforms are present at normal levels.

  1. If both FANCD2 bands are absent or very weak, FANCD2 is sequenced. Because of the presence of FANCD2 pseudogene sequences in the genome, this testing must be performed on cDNA or gDNA using specially designed primers [26].

  2. If only the short isoform of FANCD2 is present, FANCL and FANCM are sequenced. If no mutations are found, the patient may be mutated in FANCI or in another unidentified FA gene acting upstream of FANCD2.

  3. If both isoforms are present, and if the clinical phenotype is compatible with FA-D1 or FA-N, BRCA2/FANCD1 and PALB2/FANCN are screened by MLPA and DNA sequencing.

  4. If negative, BRIP1/FANCJ, PALB2/FANCN, RAD51C/FANCO, and SLX4/FANCP are sequenced.

  5. If negative again, the patient should be screened for mutations in NBS1, ESCO2 and DDX11 to test for Nijmegen breakage syndrome, Roberts syndrome and Warsaw Breakage syndrome, respectively [47, 48]. The latter two syndromes can also be excluded by analyzing metaphase spreads for sister chromatid cohesion defects. If again negative, the patient is likely to be mutated in a novel FA gene acting downstream of FANCD2 ubiquitination.

3. Notes

3.1. Mutation Screening in Mosaic Patients

If an available lymphoblastoid cell line from an FA patient is phenotypically normal due to genetic reversion at the disease locus, mutation screening is still possible in the reverted cell line, since at least one mutation will be present [49–51]. The second mutation may be identified through investigating the parents.

3.2. Unclassified Variants

Missense mutations or in-frame deletions or insertions should be judged using in silico prediction algorithms (SIFT, POLYPHEN2, Align GVGD). Alternatively, they can be tested for pathogenicity in a cellular transfection assay to check the ability of the variant gene product to complement the cellular FA defect in a deficient cell line (see e.g., [10, 35, 52]). Generally, these tests are only feasible in a setting where a diagnostic laboratory is equipped with a research laboratory with all necessary technology.

3.3. Functional Assignment to Genetic Subtypes

Retroviral constructs have been used to identify the FA subtype by functional complementation, as an intermediate step before a mutation screen is undertaken [36]. Although knowing the disease gene facilitates mutation screening, retroviral transduction has some drawbacks in comparison to direct mutation screening: (i) growing, MMC-sensitive cells either from a cell line or fresh blood sample are required, which are not always easy to obtain; (ii) overexpression of some FA proteins (e.g., FANCM and FANCP) may be toxic for cells; (iii) novel genetic subtypes that emerge after all known groups have been excluded and cannot be readily distinguished from false negatives, that is, transductions that for some unknown reason have failed to cause complementation; (iv) the method requires relatively advanced laboratory facilities and technology. However, functional assignment of complementation group can rapidly be provided by laboratories with capability for this type of analysis [37], which has greatly facilitated reliable genotyping for over 95% of FA patients for which viral constructs are available.

3.4. Genetic Counseling

All patients with a diagnosis of FA confirmed by mutation analysis should be referred for genetic counselling, together with their parents and siblings. Mutation testing should be performed in all sibs regardless of any clinical symptoms. A complete pedigree, including a cancer history anamnesis, should be prepared. Mutation carriers might be at increased cancer risk (see Section 3.7) whose aspect should be included in the counseling (see Section 3.7).

FA patients themselves usually have decreased fertility. Women usually have late menarche, irregular menses, and early menopause. However, pregnancies in women with FA have been described, and therefore women should be adequately informed about the risks for their offspring, which is mainly related to an increase in pregnancy-related complications [53].

Sibs of the parents of an FA patient often request carrier screening to assess their risk of getting a child with FA. If a sib appears to be carrier, this risk is still minimal because of the very low carrier frequency in the population. In the US the carrier frequency has been estimated to be about 1 in 181 [54]. The risk of a proven carrier to get a child with FA is therefore about 1 in 724. However, in small communities or in consanguineous couples this risk is much higher, and mutation screening in spouses of proven carriers may be indicated.

3.5. Prenatal Diagnosis

Prenatal diagnosis of FA is relatively straightforward after the pathogenic mutations in a given family have been identified. Fetal cells can be obtained by chorionic villus sampling (CVS) during weeks 10–12 of the pregnancy or by amniocentesis, which is performed between weeks 14 and 16. However, CVS may be preferred as the diagnosis will be known at an earlier stage. If the mutation is not known, a chromosomal breakage test on fetal material may be performed [55], but this test may be considered less reliable than screening for mutations in the fetal material. Alternatively, flow cytometric testing of MMC sensitivity in amniotic cell cultures might be an option; however this technique is only available in a limited number of specialized laboratories [56]. Occasionally, FA may be suspected by fetal ultrasound imaging and confirmed by parental carrier testing when the family is not yet known to carry a risk for FA [57].

3.6. Genotype-Phenotype Correlation

FA is considered as one disease, and the question may be raised whether all fifteen genetic subtypes equally conform to the clinical FA phenotype. Genotype-phenotype correlation studies comparing the 3 most common groups A, C, and G indicated modest phenotypic differences, which were rather correlated with the relative severity of the mutations [23]. However, bias due to the ethnic distribution of the studied population is very well possible. Other studies reported significant differences between FA-A/G versus FA-C [58]. Cases in group FA-D1 (mutated in BRCA2) and FA-N (mutated in PALB2) present with a distinct, relatively severe, phenotype that is characterized by the development of leukemia at very young age (median 2.2 years) and by pediatric cancers such as nephroblastoma (Wilms tumor) or medulloblastoma [40–44]. The observations that one of the pathogenic mutations in BRCA2 in FA-D1 patients is hypomorphic and that mice with biallelic null alleles in Brca2 are embryonic lethals suggest that the BRCA2 protein serves a function that is essential for survival.

Different mutations in the same gene may be associated with divergent phenotypes, as illustrated by the two FANCC mutations, c.711+4A>T and c.67delG. The former (splice-site) mutation is associated with a relatively severe phenotype in Ashkenazi Jewish people [19] although the associated phenotype was reportedly less severe in patients of Japanese ancestry [20]. The carrier frequency for this mutation in the Ashkenazi population is relatively high (1 in 87), which has led to the recommendation of carrier detection to prevent disease [59]. In the Netherlands more than 50% of FA cases are homozygous for the FANCC frameshift mutation c.67delG. The phenotype associated with this mutation, like other exon 1 mutations, seems relatively mild, as these patients rarely have skeletal abnormalities and show a relatively late age of onset of their marrow failure [24]. Awareness of such genetically determined phenotypic differences may help in clinical decision making, including the counselling of patients and families.

3.7. Cancer Risk in Heterozygous Mutation Carriers

An important issue is whether FA mutation carriers are at increased risk to develop cancer or other types of disease. Overall, there is no increased risk for cancer among FA heterozygotes [60, 61]. However, the situation is different in some of the less prevalent FA subtypes. The FA-D1 subtype is caused by mutations in BRCA2 [62] which is a well-known breast and ovarian cancer predisposition gene [63]. In FA-D1 one of the mutations will be hypomorphic because biallelic “severe” mutations are supposed to be lethal [26]. Therefore, one of the parents of a FA-D1 patient will be a heterozygous carrier of a “severe” inactivating BRCA2 mutation and may thus have an increased risk for breast cancer and other BRCA2-associated cancers. Whether the parent with the hypomorphic mutation is also at increased risk is unknown: in breast cancer families these hypomorphic mutations are considered as variants with unknown clinical significance. Two other genes involved in FA and related to breast or ovarian cancer predisposition are PALB2/FANCN [64, 65] and RAD51C/FANCO [66]. Although cancer patients have been identified with germ-line mutations in these genes, an accurate estimate of the relative cancer risk for mutation carriers is still lacking.

Another special case is represented by female FANCB mutation carriers, who are supposed to consist of 50% FA-like cells due to silenced expression of the wild type FANCB allele by the random process of X inactivation that occurs during early embryonic development. Nevertheless, in the few female FANCB mutation carriers studied so far, inactivation appeared strongly skewed towards the mutated allele [67]. This suggests that FA cells have a poor chance to survive next to unaffected cells in the same tissue, and these FA cells may therefore not give an increased cancer risk. However, the data are scarce at present so that no firm conclusions can be drawn regarding the cancer risk of female FANCB mutation carriers [60].

Conflict of Interests

The authors do not declare any conflict of interests related to this study.

Acknowledgments

The authors thank the Fanconi Anemia Research Fund, Inc., Eugene, OR, the Netherlands Organization for Health and Development, and the Dutch Cancer Society, for financial support.

References

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