Gene 518 (2013) 470–475

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Short Communication

Identification of a novel large intragenic deletion in a family with Fanconi anemia:First molecular report from India and review of literature

Pallavi Shukla, Anita Rao, Kanjaksha Ghosh, Babu Rao Vundinti ⁎Department of Cytogenetics, National Institute of Immunohaematology (ICMR), 13th Floor, New Multistoried Building, K.E.M. Hospital Campus, Parel, Mumbai-400012, India

Abbreviations: FA, Fanconi anemia; MLPA, multipamplification; DNA, deoxyribonucleic acid; OMIM, OnlineDEB, diepoxybutane; MMC, mitomycin C; NCHS, NationANC, absolute neutrophil count; HB, hemoglobin; EDTA, ePHA, phytohemagglutinin; RPMI, Roswell Park Memoribovine serum; Tris–HCl, tris–hydrochloride; NACL, sodichain reaction.⁎ Corresponding author at: Department of Cytoge

Immunohaematology (NIIH), 13th Floor, New MultistoCampus, Parel, Mumbai-400012, India. Tel.: +91 22 2424111161; fax: +91 22 24138521.

E-mail address: vbaburao@hotmail.com (B.R. Vundin

0378-1119/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.gene.2013.01.016

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 10 January 2013Available online 29 January 2013

Keywords:Fanconi anemiaIntragenic deletionMLPAFANCA

We report here an Indian case with Fanconi anemia (FA) presented with fever, pallor, short stature,hyperpigmentation and upper limb anomaly. Chromosome breakage analysis together with FANCD2Westernblot monoubiquitination assay confirmed the diagnosis as FA. Multiplex ligation-dependent probe amplifica-tion (MLPA) revealed a novel homozygous large intragenic deletion (exons 8–27 del) in the FANCA gene inthe proband. His sib and parents were also analyzed and found to be heterozygous for the same mutation.We also reviewed the literature of FANCA large intragenic deletions found in FA patients from different coun-tries and the mechanism involved in the formation of these deletions. To the best of our knowledge, this is thefirst molecular report from India on FA. The finding expands the mutation spectrum of the FANCA gene.Identification of the mutation confirms the diagnosis of FA at DNA level and helps in providing proper geneticcounseling to the family.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Fanconi anemia (FA) (OMIM ID 227650) is a clinically heteroge-neous disorder with incidence of 1 in 350,000 births and is character-ized by bone marrow failure (aplastic anemia), developmental delay,physical abnormalities and increased incidence of solid tumors andleukemias (Auerbach et al., 2001; D'Andrea and Grompe, 1997). Thediagnosis of FA can be done by cytogenetic testing for the increasedchromosomal breakages or rearrangements in the presence of DNAinterstrand cross-linking agents such as diepoxybutane (DEB) or mito-mycin C (MMC). However, molecular analysis is still required for char-acterization of FA patients and demonstrating pathogenic mutations inthe FA genes. So far 15 genes (FAA, B, C, D1/BRACA2, FANCD2, E, F, G, I, J,L, M, N/PALPB2, FANCO/RAD51C and FANCP/SLX4) have been identifiedthat can be mutated in FA (Matsushita et al., 2011). Among all comple-mentation groups, FAA accounts for the majority of FA patients(60–65%) followed by FAC (10%) and FAG (9%).

lex ligation-dependent probeMendelian Inheritance in Man;al Center for Health Statistics;thylenediaminetetraacetic acid;al Institute medium; FBS, fetalum chloride; PCR, polymerase

netics, National Institute ofried Building, K.E.M. Hospital138518, +91 24138519, +91

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l rights reserved.

FA proteins are known to play an important role in DNA damagerepair. When DNA damage occurs, the eight FA proteins, A, B, C, E, F,G, L, and M that form nuclear core complex at upstream of the path-way monoubiquitinate two other FA proteins FANCD2 and FANCI,resulting in targeting of FANCD2 protein in nuclear foci (Longerichet al., 2009). FANCD2 then interacts with BRCA1 and other DNA dam-age response proteins downstream of the FA pathway such as BRCA2,RAD51 and NBS and repair the damage (Wang et al., 2004). Mutationin the genes at upstream of the FA/BRCA pathway disrupts the path-way and results in non-ubiquitinated FANCD2 protein in the Westernblot (Garcia-Higuera et al., 2001).

Till date, no work has been done on molecular analysis of FApatients in India. We here report the first FA case from India withnovel large intragenic deletion (exons 8–27 del) in the FANCA geneusing multiplex ligation-dependent probe amplification (MLPA),which has been proved to be a rapid diagnostic tool for the identifica-tion of gross deletions in many disorders including FA.

2. Materials and methods

2.1. Case

A 3-year and 6-month old male child was referred to ourlaboratory for chromosome breakage analysis. The proband was firstborn of second degree consanguineous marriage. Mother's age was19 years and fathers's age was 22 years at the time of child's birth.The mother had a history of spontaneous abortion at 4.5 monthsof gestational age. The proband has a 9-month old male sib whowas asymptomatic at the time of diagnosis. The proband presented

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with low weight (9 kg, less than 5th percentile NCHS data), pallor,short stature (height 84 cm, less than 5th percentile NCHS data),hyperpigmentation and upper limb anomaly (extra digit thumb).His hematological profile revealed decreased platelets (15,000×109/L)and absolute neutrophil count (ANC) with hemoglobin 9 g/dL. Ultraso-nography of abdomen showed multiple minimally enlarged mesentericlymph nodes with preserved hilar echoes largest of which measures(1.0×0.6 cm). Bone marrow biopsy revealed hypocellular marrowwith decreased myeloid and erythroid series.

The study was approved by the institutional ethics committee.After obtaining informed consent from the family, 8 mL heparinblood for chromosome breakage test and FANCD2 Western blot and2 mL EDTA blood were collected in vacutainers from proband, siband the parents for mutation analysis.

2.2. Chromosome breakage test

Chromosome breakage test was carried out using peripheral bloodcultures stimulated with phytohemagglutinin and induced with MMC(20 ng/mL) andDEB (0.1 μg/mL) using standard procedure as previouslydescribed (Gregory et al., 2001).

2.3. FANCD2 monoubiquitination detection by Western blot

For FANCD2 Western blot, peripheral blood mononuclear cells(PBMC) were isolated using 5 mL of heparinized blood from patientsby centrifugation over a Histopaque1077 (Sigma). The cells werewashed twice and cultured in RPMI 1640 containing 15% heat-inactivated fetal bovine serum (FBS; Sigma) and 2 mM glutamine,penicillin and streptomycin. Cultures were stimulated with phytohe-magglutinin (PHA). The cells were induced with or without DNAinterstrand cross-linking agent, MMC (20 ng/mL) at the 0 h andincubated for 72 h. Cell cultures were maintained in a humidifiedincubator containing 5% carbon dioxide at 37 °C. After 72 h of culture,lysate was prepared. Sample was heated for 5 min at 96 °C andloaded on 3–8% gradient tris–acetate gel for electrophoresis. Proteinswere transferred to nitrocellulose using iBlot® Dry Blotting System(Invitrogen). The nitrocellulose membrane was blocked with 5% non-fat dried milk in TBS-T (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween20) and incubated for 2 h with primary anti-FANCD2 mouse mono-clonal antibody diluted 1:200 followed by incubation with the respec-tive secondary antibody linked to horseradish peroxidase. Detectionwas done using ECL PLUS kit (GE Healthcare). The short or smallband (S) of 155 kDa refers to the non-ubiquitinated protein and thelong or large (L) band of 162 kDa refers to the monoubiquitinatedFANCD2 protein.

Table 1Chromosomal breakage and radial forms frequency in a child with Fanconi anemia.

Spontaneous MMC induced DEB induced

A. Patient Total cells 50 cells 50 cells 50 cells% cells with breaks 24% 98% 80%Breaks/cell 0.25 br/cell 6.5 br/cell 3.2 br/cell% cells with radials None 52% 24%

B. Control Total cells 50 cells 50 cells 50 cells% cells with breaks 0% 4% 3%Breaks/cell 0.0 br/cell 0.05 br/cell 0.03 br/cell% cells with radials None None None

2.4. Mutation detection using MLPA

Genomic DNA was extracted from peripheral blood of the patientusing kit method (Qiagen). MLPA was performed using FANCA SALSAMLPA kits P031/P032 (MRC Holland, Netherlands, http://www.mlpa.com) according to the manufacturer's instructions. Briefly, targetDNA was denatured for 5 min at 98 °C, probe mix was added, afterwhich the mixture was heated for 1 min at 98 °C and incubated at60 °C overnight (16 h); after addition of ligase the mixture was incu-bated at 54 °C for 15 min. Ligase was subsequently inactivated at98 °C for 5 min. Next, ligation product was transferred to PCR mix.The PCR reaction was carried out for 35 cycles (30 s at 95 °C, 30 s at60 °C, and 60 s at 72 °C). The fragments were analyzed on an ABImodel 3130 capillary sequencer (Applied Biosystems) using genescan-TAMRA 500 size standard (Applied Biosystems). MLPA result was con-firmed by real time PCR. No attempts were made to determine theexact breakpoints of deletions at the genomic DNA level.

3. Results

Chromosomal breakage analysis using DEB and MMC induced cul-tures revealed a high frequency of chromosome breakages comparedto controls (Table 1, Fig. 1A). The Western blot pattern of FANCD2showed nonubiquitinated protein (absence of large (L) band)suggesting defect in the upstream of the FA/BRCA pathway (Fig. 1B).Detection of mutation in the FANCA gene using MLPA revealed ahomozygous large intragenic deletion (exons 8–27 del) (Figs. 2A, B).Real time PCR confirmed the deletion (ΔΔCt control=0.000 and ΔΔCtproband=−3.814). MLPA analysis of sib and parents showed hetero-zygous deletion of the same mutation (Figs. 3A, B, C) (Data not shownfor deletion of even exons).

4. Discussion and review of literature

Fanconi anemia is a rare hereditary disorder and characterized bybone marrow failure, chromosome breakage and development ofcancer. We report a case of FA with classical presentation. The chro-mosome breakage investigation is a gold standard for diagnosis ofFA. In our case, lymphocyte culture induced with MMC and DEBexhibited a significantly high frequency of chromosomal breakages,confirming the diagnosis of FA. In FA patients, the monoubiquitinationpattern of FANCD2 plays an important role in understanding the defectsin the upper/lower stream genes of the FA pathway. For accessing theintegrity of the FA pathway, Shimamura and group have developed anovel diagnostic test that provides a rapid subtyping assay for the pa-tients diagnosed with FA (Shimamura et al., 2002). This test is basedon FANCD2 Western blot pattern where both isoforms of FANCD2(monoubiquitinated and non-ubiquitinated) can be detected. Absenceof the monoubiquitinated band (L band) suggests defect in the up-stream genes of the FA pathway (FANCA, B, C, E, F, G, I, L, M). In ourcase, the Western blot pattern for FANCD2 showed absence of upperband (L) suggesting defect in the upstream genes of the pathwayincluding FANCA. Since FANCA is a primary candidate gene for screeningof FA as it accounts formajority (60–65%) of FA patients and deletions inFANCA gene are more frequent (http://www.rockefeller.edu/fanconi/genes/jumpa), we analyzed FANCA gene mutations using MLPA. MLPA,a PCR based technique is a rapidmethod to identify deletions and dupli-cations, enabling faster identification of mutations in FA. We have iden-tified a novel and homozygous large intragenic deletion (exons 8–27) inthe probandwhereas sib and parentswere found to be heterozygous forthe same mutation.

A large spectrum of mutations has been reported in the FANCAgene, including microdeletions, large deletions, microinsertions andpoint mutations (Centra et al., 1998; Levran et al., 1997; Morgan etal., 1999; Tachibana et al., 1999;Wijker et al., 1999). However, severalreports indicate that intragenic large deletions are common in theFANCA (Centra et al., 1998; Levran et al., 1997; Wijker et al., 1999).Centra and group have described two large intragenic deletionsin the FANCA gene, one of 5.0 kb and another of at least 120 kb(Centra et al., 1998). In the present study, a large intragenic deletion

Fig. 2. Multiplex ligation-dependent probe amplification analysis revealing novel homozy(A) Deletion of FANCA exons 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 using MLPA kit P031. (

Fig. 1. (A) Chromosome breakage analysis showing high frequency of breaks and radialformation in the proband. (B) FANCD2 monoubiquitination assay by Western blotanalysis showing absence of upper band (L) in presence and absence of MMC (Lanes1 and 2) suggesting non-ubiquitinated FANCD2 protein and defect in the upstreamgenes of the FA/BRCA pathway.

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(exons 8–27 del) was identified in the FANCA gene in FA patient. Tothe best of our knowledge, it is a novel deletion. The study of the fam-ily including parents and sib showed heterozygous pattern of thesame mutation. Mutational analysis of FA patients from Europerevealed that among 31 different mutations being detected, eight(26%) were large intragenic deletions (Wijker et al., 1999). Morganand group studied the FANCA gene mutations in 26 cell lines fromFA complementation group A using a two-step fluorescence-basedmutation-screening strategy and detected 33 different mutations.Forty percent of these mutations were large intragenic deletionsthat removed up to 31 exons from the gene, suggesting that thismight be the most prevalent form of mutation in the FANCA gene(Morgan et al., 1999). Two large intragenic deletions involvingexons 24–28 and exon 38 were observed in Japanese FA patients(Tachibana et al., 1999). In Afrikaner population of South Africa,an intragenic deletion of exons 12–31 in FANCA gene is the mostcommon. It accounts for 60% of FA chromosomes in 46 unrelatedAfrikaner FA patients and results in founder effect, explaining veryhigh incidence of FA in this population (Tipping et al., 2001). Highprevalence of large intragenic deletions in the FANCA gene was alsoobserved in Spanish FA patients (Callén et al., 2004). A total of eightheterozygous deletions involving from one to more than 26 exonswere detected in 25 Spanish FAA patients (non-gypsies) which ac-counts for one third of the patients who carried a large intragenicdeletion (Callén et al., 2004). Analysis of mutations in the FANCA genein different tribal groups (Cales, Roma, Sinti, Irish travelers) of Spanishgypsies revealed a large intragenic homozygous exons 11–14 deletionin Irish travelers (Callén et al., 2005). Castella and group in 2011reported 20 large deletions in 130 alleles (15.4%) of Spanish FA patientspreviously subtyped as FAA and reported 5 novel large deletions found

gous large intragenic deletion (exons 8–27 del) in the FANCA gene in the proband.B) Deletion of FANCA exons 8, 12, 14, 16, 18, 20, 22, 24 and 26 using MLPA kit P032.

Fig. 3. Multiplex ligation-dependent probe amplification analysis revealing novel heterozygous large intragenic deletion (exons 8–27 del) in the FANCA gene in (A) sib, (B) fatherand (C) mother of the proband.

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in several FAA patients (Castella et al., 2011). In another report, 29different mutations including eight large deletions were found in theFANCA gene of 35 unrelated Japanese FA patients (Yagasaki et al.,2004). High frequency of large intragenic deletions was also evidentin the report by Chandra and group who found 9 large deletions in 20FAA patients (Chandra et al., 2005). Similarly a high frequency of largedeletions was also observed in the mutation reports published by vari-ous other groups (Adachi et al., 2002; Ameziane et al., 2008; Savino etal., 2003). Table 2 summarizes all the large intragenic deletions foundin FANCA gene in FAA patients from different population.

It has been repeatedly demonstrated that the majority of thesedeletions are the product of a recombination between Alu-repeatsin cis (Callén et al., 2004; Centra et al., 1998; Levran et al., 2005;Morgan et al., 1999). Centra et al. characterized intragenic deletionof 5.0 kb that removed exons 18–21 and showed that breakpointslie in between introns 17 and 21. Further characterization of the dele-tion revealed several Alu-repeats and suggested a recombinationevent likely to have occurred between the right arm of Alu (Centraet al., 1998). It was statistically proved that deletion breakpoint corre-lated strongly with the presence of Alu-repeats (Morgan et al., 1999).Similarly, Alu medicated recombination was also shown to be involvedin the generation of intragenic deletion of exons 12–31 in Africanpopulation (Tipping et al., 2001) and of FANCA deletion exons 6–31(Tamary et al., 2004). In another study, characterization of two dele-tions that removed exons 24_28 and exons 12_26 revealed involvementof diversemechanisms in the generation of large deletions in FANCA in-cluding nonhomologous recombination and Alu-mediated homologousrecombination respectively (Yagasaki et al., 2004). Tachibana and groupalso reported non-homologous recombination as a likely cause of dele-tion from intron 37 to exon 38 (Tachibana et al., 1999). They reported a

breakpoint in intron 37 exactly at the 3′ end of an Alu element. ThisAlu element can pair with an Alu element in an opposite direction inintron 38 to form a stem-loop structure. It can be speculated that thegeneration of DNA nicks by a structure-specific nuclease(s) or withtopoisomerase I may predispose to nonhomologous recombination.Castella and group characterized the FANCA exons 1–20 deletion anddemonstrated that the deletion was a result of interstitial deletionwith two breakpoints, the first between exons 20 and 21 of the FANCAgene and the second within a region of 276 kb at 5′ of the FANCA gene(Castella et al., 2011). This deletion spans 112.5 kb and involves 3genes, FANCA, SPIRE2, and TCF revealing that long distance Alu-Alu re-combination in cis, results in large pathogenic deletion, suggestingthat not all large FANCA deletions are intragenic. Thus, diverse mecha-nisms are involved in the generation of large deletions in FANCA.

Our case is a classical presentation of FA phenotype. In FA,genotyping is done with complementation analysis of all FA relatedgenes. However, since defects in the FANCA gene are reported inhigh frequency of FA patients and large deletions are the most com-mon mutations in the gene, MLPA has been found to be very usefultechnique for the rapid detection of genetic changes in FA. Identifica-tion of the deletion expands the mutational spectrum of FA, facilitatesprenatal diagnosis and helps in taking decisions on treatment andmanagement of the disease.

Acknowledgments

We thank the patient and family for their voluntary participationin this study. The work was supported by the post doctoral fellowshipfrom the Indian Council of Medical research (ICMR), India.

Table 2Large intragenic deletions identified in FANCA gene in different population of the world.

Mutation Exon Population Method of detection used References

5′ UTR-522del 5′ UTR-5 USA Dosage using quantitative fluorescent multiplex PCR Morgan et al. (1999)5′ UTR-1900del 5′ UTR-21 USA Dosage using quantitative fluorescent multiplex PCR Morgan et al. (1999)5′ UTR-3066del 5′ UTR-31 USA, Europe Dosage using quantitative fluorescent multiplex PCR Morgan et al. (1999), Adachi et al. (2002)

and Wijker et al. (1999)1–2981del 1–30 Europe, Iran Consistent lack of amplification of the relevant exons Wijker et al. (1999) and Tipping et al. (2001)c.-32-?_5481del 1_43 Germany, Europe, Italy Loss of heterozygosity for selected SNPs, MLPA Centra et al. (1998), Chandra et al. (2005),

Ameziane et al., 2008, Centra et al. (1998)and Savino et al. (2003)

c.190-?_596+?del 3_6 Japan PCR-based gene dosage assay Yagasaki et al. (2004)c.190-?_4368+?del 3_3′ UTR Japan PCR-based gene dosage assay Yagasaki et al. (2004)c.284-?_522+?del 4_5 Europe MLPA Ameziane et al. (2008)523–1359del 6–14 USA Dosage using quantitative fluorescent multiplex PCR Morgan et al. (1999)c.523-?_792+?del 6_8 Europe MLPA Ameziane et al. (2008)c.523-?_3066+?del 6–31 Israeli Arabs By the consistent lack of amplification of the relevant exons Tamary et al. (2004)597–3066del 7–31 India, Europe Consistent lack of amplification of the relevant exons Wijker et al. (1999) and Tipping et al. (2001)597–1826del 7–20 USA, Europe Dosage using quantitative fluorescent multiplex PCR, MLPA Morgan et al. (1999), Savino et al. (2003)

and Ameziane et al. (2008)c.793-?_1715+?del 9–18 Germany Loss of heterozygosity for selected SNPs Chandra et al. (2005)c.827-?_1083+?del 10–12 Africa Multiplex fluorescent dosage analysis Tipping et al. (2001)827–1225del 10–13 Europe (Spain) Consistent lack of amplification of the relevant exons Wijker et al. (1999) and Tipping et al. (2001)c.827-?_1626+?del 10–17 Africa Multiplex fluorescent dosage analysis Tipping et al. (2001)894–1359del 11–14 Europe, Spanish Gypsies

(Irish travelers)Consistent lack of amplification of the relevantexons, Quantitative fluorescence multiplex PCR

Wijker et al. (1999) and Callén et al. (2005)

c.894-?_1626+?del 11–17 Africa Multiplex fluorescent dosage analysis Tipping et al. (2001)c.894-?_1900+?del 11_21 Europe MLPA Ameziane et al. (2008)c.894-?_3348+?del 11_33 Europe MLPA Ameziane et al. (2008)c.1007-10_2504+209del

12_26 Japan PCR-based gene dosage assay Yagasaki et al. (2004)

1007–3066del 12–31 USA, Africa Multiplex fluorescent dosage analysis Morgan et al. (1999) and Tipping et al. (2001)1360–1826del 15–20 USA RT-PCR/dosage using quantitative fluorescent

multiplex PCRMorgan et al. (1999)

c.1360-?_1900+?del 15_21 Japan PCR-based gene dosage assay Yagasaki et al. (2004)1471–1626del 16–17 USA, Europe RT-PCR/dosage using quantitative fluorescent

multiplex PCRMorgan et al. (1999), Adachi et al. (2002),Wijker et al. (1999) and Morgan et al. (1999)

c.1471-?_2151+?del 16–23 Germany Loss of heterozygosity for selected SNPs Levran et al. (2005) and Chandra et al. (2005)c.1627-?_1900+?del 18–21 Italy, New Mexico, Europe RNA-SSCP and direct sequencing Savino et al. (2003), Savino et al. (2003),

Centra et al. (1998), Adachi et al. (2002),Wijker et al. (1999) and Morgan et al. (1999)

c.1627-?_2151+?del 18_23 Europe MLPA Ameziane et al. (2008)1827–2778del 21–28 USA Dosage using quantitative fluorescent

multiplex PCR/exon PCRMorgan et al. (1999)

c.1901-?_2014+?del Ex 22 Germany Loss of heterozygosity for selected SNPs Levran et al. (2005) and Chandra et al. (2005)c.1901-?_2778+?del 22–28 Germany, Europe Loss of heterozygosity for selected SNPs, MLPA Chandra et al. (2005) and Ameziane et al.

(2008)c .2151+328_2778+1085 del

24_28 Japan PCR-based gene dosage assay Yagasaki et al. (2004)

c.2152-?_3348+?del 24_33 Germany Loss of heterozygosity for selected SNPs Chandra et al. (2005) and Chandra et al. (2005)c.2505-?_3626+?del 27_36 Europe MLPA Ameziane et al. (2008)2779–3066del 29–31 USA Dosage using quantitative fluorescent multiplex PCR/RT-PCR Morgan et al. (1999)2779–3348del 29–33 Europe Consistent lack of amplification of the relevant exons Wijker et al. (1999)2982-?_3066+?del 31 Europe, Pakistan Sequencing Wijker et al. (1999) and Tipping et al. (2001)3061–3154del 31–32 Europe Consistent lack of amplification of the relevant exons Wijker et al. (1999)2982-?_3348+?del 31_33 Europe MLPA Ameziane et al. (2008)2982–4365del 31–43 USA Dosage using quantitative fluorescent multiplex PCR Morgan et al. (1999)c.3514-?_3626+?del 36 Germany Loss of heterozygosity for selected SNPs Chandra et al. (2005)

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