Anemia

Volume 2012 (2012), Article ID 603253, 13 pages

doi:10.1155/2012/603253

Research Article

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

Johan J. P. Gille, Karijn Floor, Lianne Kerkhoven, Najim Ameziane, Hans Joenje, and Johan P. de Winter

Department of Clinical Genetics, VU University Medical Center, Van der Boechorsttraat 7, 1081 BT Amsterdam, The Netherlands

Received 22 December 2011; Accepted 21 March 2012

Academic Editor: Stefan Meyer

Copyright © 2012 Johan J. P. Gille et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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]

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]

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

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

(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

(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

(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

(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

(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

PCR conditions:

PCR was performed in Applied Biosystems PE9700 system using 96-well plates. PCR reactions (final volume 25 l) 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.

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