Clinica Chimica Acta 429 (2014) 96–103

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A simplified approach for FSHD molecular testing Frantzeskos Papanikos a, Christina Skoulatou a, Paraskevi Sakellariou b, Kyriaki Kekou b, Theodore K. Christopoulos c,d, Emmanuel Kanavakis b, Jan Traeger-Synodinos b, Penelope C. Ioannou a,⁎ a

Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece Laboratory of Medical Genetics, Athens University, Athens 11527, Greece Department of Chemistry, University of Patras, Patras 26500, Greece d Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras 26504, Greece b c

a r t i c l e

i n f o

Article history: Received 8 November 2013 Accepted 25 November 2013 Available online 7 December 2013 Keywords: FSHD Biomarkers Molecular testing

a b s t r a c t Background: Facioscapulohumeral muscular dystrophy (FSHD) is characterized by complex genetics linked to DNA rearrangements in a polymorphic genomic region of tandemly repeated D4Z4 segments. A panel of FSHD biomarkers including contracted D4Z4 array repeat combined with the 4qA(159/161/168)PAS haplotype has been proposed as molecular signature for defining alleles causally related to FSHD. The aim of the present study was to develop a simple approach for FSHD molecular testing in order to extend studies related to the applicability of FSHD molecular signature in Greek population. Methods and results: The method comprises: (i) visual genotyping of the common 4qA and 10qA subtelomeric haplotypes by a multiplex assay in a dipstick format. (ii) Detection of 4qA161 haplotype in D4Z4 contracted alleles by tri-primer PCR. (iii) Detection of PAS SNP in PLAM region and GNC SNP in the first proximal D4Z4 unit by tri-primer PCR. The method was evaluated by analysing DNA from monoallelic sources representing common 4q and 10q haplotypes, samples from 3 FSHD families, 36 unrelated probands and 38 control individuals of Greek origin. Conclusions: The proposed method could be a very useful tool for FSHD testing making it more accessible to clinical diagnostic laboratories and the wider FSHD community. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Facioscapulohumeral muscular dystrophy (FSHD, OMIM #158900) is the third common myopathy characterized by progressive weakness and atrophy of facial, shoulder and upper arm muscles. FSHD affects 1 in 20,000 people and is considered to be an autosomal dominant inherited disease [1–4]. FSHD genetic locus has been linked to chromosome 4q35 and the causal defect has been genetically associated to the contraction of the macrosatellite D4Z4 repeat unit [4]. D4Z4 is highly polymorphic in normal population ranging from 11 to 150 repeats (N38 kb, EcoR1 alleles), whereas patients carry less than 11 units (b38 kb). Two subtelomeric variations distal to D4Z4, named 4qA and 4qB, with an equal distribution in the general population, were identified and FSHD disease expression was exclusively associated with the 4qA variant [5–8]. Α highly identical (~ 98% homology) and equally polymorphic D4Z4 repeat array on chromosome 10q26 has not been associated with the disease [3]. Since the mapping of the FSHD genetic locus [9], numerous research efforts have been made towards revealing the complex FSHD genetics. Biomarkers proximal to the D4Z4 repeat named simple sequence length

⁎ Corresponding author. Tel.: +30 210 7274574; fax: +30 210 7274750. E-mail address: [email protected] (P.C. Ioannou). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.11.032

polymorphism (SSLP) haplotypes and haplotype specific single nucleotide polymorphisms (SNPs) in the D4F104S1 sequence region were reported [10,11]. In addition, a polymorphism, AT(C/T)AAA, named poly(A) signal (PAS) in the chromosomal region distal to the last D4Z4 repeat of FSHD patients, in the adjacent pLAM sequence of permissive 4qA alleles was found to potentially affect polyadenylation of the distal DUX4 transcript [12]. Non-permissive 4qB chromosomes lack pLAM altogether, including poly(A) site, whereas, poly(A) signals were not identified in non-permissive 10qA chromosomes. Contracted D4Z4 array repeat combined with the 4qA(159/161/168)PAS haplotype has been proposed as molecular signature for defining alleles causally related to FSHD. Exploitation of 4qA161 haplotype specific SNP for molecular testing of FSHD either by direct sequencing of PCR amplified products or by detecting restriction fragment length polymorphisms indicative of 4q161 alleles was reported [13]. Finally, guidelines on FSHD genetic diagnosis based on a panel of molecular biomarkers were also proposed [14]. This panel comprises: (i) sizing of the polymorphic macrosatellite repeat D4Z4 on chromosome 4q35 (1–10 repeats for FSHD1 patients) by Southern blotting after digestion of genomic DNA with specific set of restriction enzymes and hybridization with p13E-11 probe, (ii) Southern blot discrimination between D4Z4 homologs (4qA/4qB) distal to D4Z4 repeat based on specific hybridization probes and (iii) analysis of SSLP haplotypes by PCR amplification followed by capillary electrophoresis (CE).

F. Papanikos et al. / Clinica Chimica Acta 429 (2014) 96–103

Recently, two large studies based on the FSHD molecular signature were conducted including samples from unrelated FSHD families, normal individuals unrelated to any FSHD patients and samples of unrelated FSHD patients [15,16]. According to the findings it was concluded that the current FSHD molecular signature is a common polymorphism carried only by half of FSHD probands and suggested revisiting of genetic basis of FSHD. In an attempt to extend studies revealing the applicability of the 4A161PAS molecular signature in molecular testing and/or predicting of disease in Greek population, we developed a simplified FSHD molecular testing approach using as tools PCR and electrophoresis. Briefly, this approach comprises (i) an initial screening of the DNA sample for the presence of 4qA161 and PAS SNP, (ii) confirmation of the presence/or absence of 4qA161 SNP in contracted D4Z4 alleles (EcoRI alleles) and (iii) SSLP analysis for the presence/or absence of rare permissive haplotypes that could be differentiated only by size. In addition, we propose a simultaneous detection of SNPs related to the four most common 4qA161, 4qB163, 4qB168 and 10qA166 alleles by primer extension (PEXT) reaction-dipstick assay as an additional tool to the SSLP haplotyping method. 2. Materials and methods 2.1. Materials and instrumentation Materials and instrumentation are provided in the Supplementary information. 2.2. Subjects and DNA clones For the method evaluation, samples from 36 unrelated probands, 3 FSHD families (4 members each) and 38 controls (5 healthy relatives and 33 unrelated controls) that had been clinically confirmed and molecularly diagnosed were included in this study after informed consent [17]. DNA from phage clone λ260201, monochromosomal rodent somatic cell hybrid 4L10 and cosmid C85 representing 4qA161, 4qB163 and 10qA166 haplotypes, respectively, were also included in this study. 2.2.1. DNA isolation Genomic DNA was obtained from peripheral blood lymphocytes by “salting out” procedure. 10 μg of genomic DNA was digested with 20 U of EcoRI and incubated at 37 °C overnight. PFGE was performed

according to [17]. The desired bands (between 10 and 38 kb) representing pathogenic fragments, were sliced out and gel extracted using the UltraClean™ GelSpin DNA Purification Kit (MO BIO, Laboratories, Inc.) according to manufacturer's instructions. 2.2.2. Genotyping of 4qA161, 4qB163, 4qA168 and 10qA166 haplotypes by multiplex (PEXT) reaction and visual multi-allele dipstick assay Four SNPs in the D4F104S1 sequence region (Accession Number AF117653.2) specific for the most common 4qA161 (g.4519TNC), 4qB163 (g.4761TNA), 4qB168 (g.4796GNC) and 10qA166 (g.4790ANC) haplotypes (Fig. 1) were detected simultaneously by a rapid PEXTdipstick assay (Fig. 2a). The haplotype related SNPs were selected according to their specificity and relative abundance in the European population [11]. Briefly, the method consists of: (i) PCR amplification of a 414 bp D4F104S1 fragment flanking haplotype specific SNPs. (ii) A multiplex (8-plex) PEXT reaction in unpurified PCR product in the presence of allele-specific primers (two primers per SNP, one for the common allele and the second specific for the haplotype allele) and biotinmodified nucleotide. The 3′ end of each allele-specific primer is complementary to the D4F104S1 allele, whereas the 5′ end contains a unique segment that enables subsequent capture of the primer on the test spot of the dipstick through hybridization with complementary capture oligonucleotides. Allele-specific primer is extended and incorporates biotin only if it is perfectly complementary to the target sequence. (iii) Visual detection of the extended products using a multiallele dipstick assay as described previously [18,19]. Capture probes corresponding to the variant alleles (specific for each of the four haplotypes) were spotted on the right side of the membrane, whereas those corresponding to the four common alleles on the left side (Fig. 2b). The haplotype is assigned by observing each pair of spots. A single spot on the top of the strip (control spot) ensures the proper performance of the dipstick assay. Detailed information on oligonucleotide sequences (Table S1), PCR, PEXT reaction and dipstick assay protocols are given in the Supplementary information. 2.2.3. Genotyping of 4qA161 SNP in D4Z4-contracted alleles by nested triprimer PCR Genotyping of the TNC substitution (AF117653.2: g.4519TNC) in the D4F104S1 sequence comprises a triprimer PCR of a DNA sample isolated either from whole blood or from EcoRI digest (D4Z4-contracted alleles). To increase the yield of the amplification reaction products in D4Z4contracted alleles, nested triprimer PCR was used. A 643 bp fragment

D4Z4 SNP (G/C) Haplotype specific SNPs

4q35 or 10q26

D4F104S1

SSLP

157-180 bp

4qA161 (g.4519T>C)

97

AT(T/C)AAA SNP pLAM

A/B or null

D4Z4 (1-100units)

10qA166 (g.4790A>C)

4qB163 4qB168 (g.4761T>A) (g.4796G>C) Fig. 1. Schematics of 4q and 10q subtelomeric region and the polymorphic markers comprising (i) simple sequence length polymorphism (SSLP) 3.5 kb proximal to D4Z4 repeat array, (ii) D4F104S1 sequence immediately proximal to D4Z4 tandem repeat, (iii) D4Z4 SNP in the first D4Z4 repeat, (iv) pLAM region and (v) qA/qB distal variants. In larger scale the D4F104S1 sequence with the relative position and the exact location of the haplotype specific SNPs according to AF117653.2 reference sequence is shown.

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b

a

>control >4qB168

G C extension C C no extension

c

>10qA166 biotin

>4qB163 >4qA161

λ260201

4L10

161

163

FW

4qA161

4qB163

C85)

166 FW

FW

g.4761T>A (4qB163)

g.4519T>C (4qA161)

10qA166

g.4790A>C (10qA166)

Fig. 2. (a) Schematic representation of the PEXT-dipstick assay. An 8-plex PEXT reaction is performed in the presence of four pairs of allele-specific primers (two primers per SNP, one for the common allele and the second for the haplotype specific allele) and biotin-dUTP. Biotin is incorporated in the extended product. The 3′ end of each allele-specific primer is complementary to the D4F104S1 allele, whereas the 5′ end contains a unique segment enabling subsequent capture of the primer on the test spot of the dipstick through hybridization. An allelespecific primer is extended only if it is perfectly complementary to the target sequence. Immobilised biotinylated PEXT products are visually detected by interaction with antibiotin labelled gold nanoparticles (red colour). (b) Position of the dots on the membrane corresponds to haplotype specific SNPs and genotyping of 4qA161 (λ260201), 4qB163 (4L10) and 10qA166 (C85) monoallellic sources. (c) Confirmation of the genotyping results by (1) DNA sequencing (polymorphic base is shown in bold and underlined) and (2) SSLP fragment analysis.

flanking the polymorphic site is first is amplified using D4F104S1-F2 and D4F104S1-R2 primers. Next, a nested PCR is performed with a set of three primers, two forward and one reverse. The relative positions of the primers are presented in Fig. 3a. The outer forward (D4F104S1F3) and reverse (D4F104S1-RV) primers amplify a 500 bp (long) product that spans the 4qA161 SNP and serves as internal control. The inner forward AS-161 (+) is specific for the D4F104S1 TNC substitution. In the absence of 4qA161 allele only the long product (500 bp) is formed. If the 4qA161 allele is present, the pair of the inner forward and reverse primers generates an additional fragment of 362 bp (short product). Thus, both the long and the short products are formed only when the 4qA161 SNP is present (Fig. 3a). Detection of 4qA161 SNP in genomic

3’

C (4499-4519)

5’

362 bp

S1

3’

FF44

5’

A 4qA161

S3

S4

S5

S1

S2

S3

S4

D4Z4-R3 AS(C)-R (6061-6045) (6189-6172)

3’

514 bp

3’

C (5548-5569) FF44

642bp

10qA166

137bp

S2

5’

AS-pLAM pLAM-RV (8067-8048) (8172-8146)

3’ 242bp

4qA161 10qA166

(AF117653:g.6045G>C)

T

(5548-5569)

(4860-4843)

G 500 bp

5’

D4F104S1-RV

AS-161F(+)

3’

c

(FJ439133.1:g.8048C>T)

(AF117653:g.4519T>C) (4361-4380) D4F104S1-F3

2.2.4. Triprimer PCR for PAS polymorphism Triprimer PCR assay for the AT(C/T)AAA polymorphism (FJ439133. 1: g.8048CNT) (PAS SNP) was performed with a set of two reverse and one forward primers (Fig. 3b). The forward (pLAM-FW) and the outer reverse (pLAM-RV) primer amplify a 242 bp fragment that spans the SNP and serves as internal control. The inner reverse primer (AS-pLAM) is specific for the PAS SNP. A sample that does not contain the PAS SNP gives only a 242 bp product whereas, in the presence of PAS SNP

b

a 5’

DNA could be performed by single triprimer PCR. Detailed information on oligonucleotide sequences (Table S1) and PCR protocols are given in the Supplementary information.

5’

G 4qA161 G

4qB163

C

G

F62

S5 G

10qA166 C

G

F209 C

G

C

F219 C

G

C

Fig. 3. Upper panel: schematic representation of the relative positions and the location of primers used for triprimer PCR genotyping reactions for 4qA161 SNP (a), PAS SNP (b) and D4Z4 SNP (c). In general, the outer common primers amplify a long fragment spanning the SNP of interest and serves as internal control. The pair of an inner allele-specific primer with one of the outer primers amplifies a short fragment only in the presence of the corresponding SNP. Therefore, the formation of two fragments (of the desired length) is indicative for the presence of the SNP. The formation of only one (long) fragment indicates the absence of the SNP. Lower panel: electropherograms of triprimer PCR products for monoallelic sources and selected for each SNP samples are also provided.

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the pair of forward and reverse (inner) primers generates an additional fragment of 137 bp. Both products (242 bp and 137 bp) are formed when the PAS SNP is present (Fig. 3b). For detailed information on oligonucleotide sequences (Table S1) and PCR protocol see the Supplementary information.

99

System and ABI Prism 3500 Genetic Analyzer. For details see the Supplementary information.

3. Results 3.1. PEXT-dipstick assay for visual detection of four common 4q and 10q haplotypes in genomic DNA samples

2.2.5. Analysis of D4Z4 SNP (GNC) The method developed for the detection of the D4Z4 GNC SNP (AF117653.2: g.6045GNC) consisted of: (i) Amplification of a 1892 bp fragment in the first proximal D4Z4 unit with a pair of a forward primer (D4F104S1-F2) complementary to a D4F104S1 probe region (proximal to the D4Z4 repeat) and a reverse primer (D4Z4-R2) complementary to an interior region of the D4Z4 unit. (ii) Nested triprimer PCR for G or C allele with a set of one forward (FF44) and two reverse (outer and inner). The forward and the outer reverse (D4Z4-R3) primer amplify a 642 bp fragment that spans the SNP and serves as internal control. The two inner reverse allele-specific primers (AS(G)-R and AS(C)-R) carry at their 3′-end a nucleotide complementary to G or C SNP, respectively. The pair of the forward (common) and reverse (allele-specific) primers generates an additional fragment of 514 bp. Both products (642 bp and 514 bp) are formed when G or C SNP is present (Fig. 3c). For detailed information on oligonucleotide sequences (Table S1) and PCR protocol see the Supplementary information.

PEXT-dipstick assay was evaluated by genotyping DNA samples from monoallelic sources λ260201 (4qA161), 4L10 (4qB163) and C85 (10qA166) and the results are presented in Fig. 2b and Table 1. As it is shown in Fig. 2b, red spots at the right side of the membrane are observed only where probes enabling the detection of the corresponding variant alleles are spotted. Red spots at the left side correspond to the common for all haplotypes alleles. The presence of allele-specific SNPs was confirmed by DNA sequencing and the size of the haplotypes by SSLP fragment analysis (Fig. 2c). Next, genomic DNA samples from 3 FSHD families (4 members each) where analysed by the proposed assay. An example of the genotyping in four individuals from FSHD family 1 is presented in Figs. 4 and 5. Figs. 4a,d and 5a,d show genotyping results by PEXT-dipstick assay; Figs. 4b,e and 5b,e, DNA sequencing for each of the four polymorphic sites and Figs. 4c,f and 5c,f, SSLP fragment analysis. Genotyping results were found in concordance with those obtained by DNA sequencing and, with some exceptions, with those obtained by SSLP haplotyping. D4F104S1 SNP genotyping in combination with SSLP fragment analysis where used to determine the inheritance of haplotypes within the family members. Genotyping results for all three FSHD families are summarized in Table 1.

2.2.6. SSLP fragmentation The SSLP region proximal to D4Z4 repeat array that is localized between 1532 and 1694 positions of AF117653 was studied by PCR. SSLP size determination was performed using MegaBACE 1000 Sequencing

Table 1 Genotyping results for D4F104S1 SNPs by PEXT-dipstick assay, D4Z4, pLAM and 4qA161 SNPs by triprimer PCR, Southern blot analysis of EcoRI and EcoRI/AvRII alleles and SSLP fragment analysis for monoallelic sources λ260201, 4L10 and C85 and for individuals of three FSHD families. Sample

Haplotypesa λ260201 4L10 C85 Family 1 F40 (M)b F41 (D1) F42 (F) F45 (D2)

Family 2 F31 (D)b F243 (M) F244 (F)

F245 (S)

Family 3 F64 (S)b F324 (F) F325 (M) F326 (D) a b

D4F104S1 SNPs Haplotype related (g.DNA)

D4Z4 G/C SNP (g.DNA)

pLAM SNP (g.DNA)

4qA161 SNP (g.DNA)

4qA161 4qB163 10qA166

+/− +/− +/−

+ − −

+ − −

+

+

1 ~ 37, 1 ~ 34, 2 N 48

1 ~ 34, 1 ~ 31, 1 N 48

161, 163, 166

+

+

1 ~ 37, 1 ~ 34, 1 ~ 50

1 ~ 34, 1 ~ 31, 1 ~ 47

161, 163, 166

+

−

2 N 48, 1 ~ 50

1 N 48, 1 ~ 47

163, 166

+

+

1 ~ 37, 1 ~ 34, 1 N 48

1 ~ 34, 1 ~ 31

161, 166

4qA161 4qB163 10qA166 4qB168 4qA161 4q\B163 10qA166 4qB168 4qB163 10qA166 4qA161 10qA166 4qB168

4qA161 4qB163 10qA166 4qA161 4qB163 10qA166 4qA161 4qB163 10qA166 4qA161 4qB163 10qA166

4qA161 4qB163 10qA166 4qB163 10qA166 4qA161 4qB163 10qA166 4qA161 4qB163 10qA166

4qA161 SNP (EcoRI digest)

Southern blot analysis (EcoRI alleles)

Southern blot analysis (EcoRI/AvRII alleles)

SSLP haplotypes (g.DNA)

161 163 166

+/+

+

+

1 N 48, 1 = 45, 1 = 33

1 ~ 30, 1 ~ 57

161, 163, 66

+/+

+

ND

1 N 48

1 ~ 57

161, 163, 166

+/−

+

+

1 N 48, 1 = 40, 1 = 33

1 ~ 30

161, 166

+/−

+

+

1 = 40, 1 = 33

1v~ 29

161, 163, 166

+

+

1 ~ 35, 1 N 48, 1 ~ 20

1 ~ 17

161, 163, 166

−

−

3 N 48

3 N 48

161, 163, 166

+

+

1 N 48, 1 ~ 43, 1 ~ 35

1 N 48, 1 ~ 40

161, 163, 166

+

+

2 N 48, 1 ~ 35

2 N 48

161, 163, 166

Monoallelic sources: λ260201 (4qA161); 4L10 (4qB163); C85 (10qA166). Affected individual; M (mother), F (father), D (daughter), S (son).

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c

a

b

F40

FW

FW

FW

g.4761T>A (4qB163)

g.4519T>C (4qA161)

g.4790A>C (10qA166)

g.4796G>C (4qB168)

f

d e

F42 FW

RV

g.4519T>C (4qA161)

FW

g.4761T>A (4qB163)

g.4790A>C (10qA166)

g.4796G G>C (4qB168)

Fig. 4. Examples of genotyping/haplotyping comprising SNPs in D4F104S1 sequence and SSLP fragment analysis in genomic DNA of F40 (affected mother) and F42 (non-affected father) individuals (FSHD family1). (a, d) Genotyping of SNPs related to the most common haplotypes by PEXT-dipstick assay. (b, e) Confirmation of genotyping results by DNA sequencing. (c, f) SSLP fragment analysis. More specifically, individual F40 (affected mother) carries SNPs related to 4qA161, 4qB163, 10qA166 and 4qB168 haplotypes (a, b). SSLP profile of the sample (c) consists of 161 (double height), 163 and 166 bp fragments and two shorter fragments at 159 bp (possibly 4qA159 permissive haplotype sharing the 4qA161 SNP) and 156 bp. The presence of three 4q alleles in a sample is not compatible assuming that 4qB163 SNP is related to 10qB161T haplotype sharing the same SNP. Individual F42 (asymptomatic father) carries SNPs related to 4qB163 and 10qA166 haplotypes (d, e). SSLP profile for this sample is represented by a double peak of approximately the same height at 163 and 164 bp and a peak of double height at 166 bp (f). Altogether, this analysis demonstrates that individual F40 carries 4qA159/4qA161 (alternatively, two 4qA161 alleles) and 10qA166/10qB161 alleles; individual F42 carries 4B163/4qA166 and 10qA166/10qA164 alleles (10qA164 shares the same SNP as 10qA166 haplotype). The detection in sample F40 of SNP related to 4qB168 haplotype is discussed in the legend of Fig. 5.

3.2. Identification of the 4qA161 haplotype in D4Z4-contracted alleles by triprimer PCR The phage clone λ260201 (4qA161) and the cosmid C85 (10qA166) representing the positive and negative control for 4qA161 SNP, respectively, were first analysed by triprimer PCR (Fig. 3b). EcoRI digested samples from FSHD family 2 members (n = 4) and 30 samples from unrelated FSHD patients were analysed by the proposed assay. The results are summarized in Tables 1 and 2, respectively. All samples, except for F243 that did not carry contracted alleles (Table 1), were found positive for 4qA161 SNP. The method was also applied to genomic DNA samples from FSHD families 1 and 3 (n = 8). All samples, except for F42 (family 1) and F324 (family 3), were found positive for 4qA161 SNP. These results were in concordance with those obtained by PEXTdipstick assay and DNA sequencing (Fig. 4d,e). All samples were D4Z4sized by Southern blotting (Tables 1 and 2). 3.3. Detection of the polyadenylation signal (PAS) polymorphism The assay was applied to genomic DNA samples from monoallelic DNA sources, 3 FSHD families (n = 12), 36 FSHD patients (Tables 1 and 2) and 38 control individuals. 30 out of 36 patient's samples were analysed for the presence of 4qA161 haplotype, as described above. All, except one patient sample (35/36, 97.2%) were found positive for

the AT(C/T)AAA polymorphism. 29 out of 30 (96.7%) patient samples carrying contracted EcoRI/AvRII alleles were found positive for both 4qA161 and PAS SNPs. On the other hand, a quite high percentage of samples from control individuals (33/38, 86.8%) was found positive for the PAS SNP. Four out of 38 control samples, that were found negative for PAS SNP, were also analysed for the 4qA161 SNP by triprimer PCR. Three out of four samples were found negative and one was positive for 4qA161 SNP. The accuracy of the proposed genotyping method, was confirmed by DNA sequencing of pLAM SNP in (i) 4qA161 monoallelic source, (ii) samples found positive for pLAM and 4qA161 SNP (F325, F41, F40, F45) and (iii) samples found negative for pLAM and 4qA161 SNP (SA, F42, F324 and 10qA166 monoallelic source) (Fig. S1 in the Supplementary information). 3.4. Analysis of the D4Z4 SNP The proposed nested triprimer PCR assay for D4Z4 SNP was first evaluated by analysing the monoallelic sources λ260201 (4qA161), 4L10 (4qB163) and C85 (10qA166). As it was expected [10] these samples carried “G” SNP (Fig. 3c, and Table 1). 51 genomic DNA samples from FSHD patients and control individuals were also analysed for G and C SNP. 17 out of 51 samples (33.3%) were found positive for “G” and “C” SNP (due to the presence of both 4q and 10q chromosomes) and 34 samples were found positive only for “G” SNP. The lower than

F. Papanikos et al. / Clinica Chimica Acta 429 (2014) 96–103

101

c

a b

F41

FW

FW

FW

g.4519T>C (4qA161)

d

g.4761T>A (4qB163)

g.4790A>C (10qA166)

g.4796G>C C (4qB168)

e

F45 RV

RV

FW

g.4519T>C (4qA161)

f

g.4761T>A (4qB163)

g.4790A>C (10qA166) FW

g.4796G>C (4qB168)

Fig. 5. Genotyping/haplotyping analysis in genomic DNA of individuals F41 and F45 (non-affected daughters) (FSHD family1). (a) Four spots on the right side of the membrane (from the bottom) represent 4qA161, 4qB163, 10qA166 and 4qB168 alleles in sample F41. SSLP profile of the sample (c) consists of 161 (double height), 163 and 166 bp fragments and two shorter fragments at 159 (possibly 4qA159 permissive haplotype sharing the 4qA161 SNP) and 156 bp. (d) Three spots on the right side of the membrane represent 4qA161, 4qB168 and 10qA166 alleles in sample F45. SSLP profile of the sample (f) consists of 161 and 166 bp (double height). Altogether, this analysis demonstrates that individual F41 carries 4qA159 or 4qA161/10qB161 alleles inherited from the mother and 4qB163/10qA166 inherited from the father (Fig. 4). Individual 45 carries 4qA161/10qA166 alleles inherited from the mother and 4qA166/10qA164 inherited from the father. The presence of SNP related to 4qB168 haplotype in samples F40, F41 and F45 (Figs. 4 and 5) was confirmed by sequencing, but not by SSLP analysis. Because the inheritance of two 4q or 10q chromosome alleles from one parent to the daughters is incompatible, it is assumed that, in particular case, this SNP might not be related to 4qB168 or other haplotypes (4qB170/172, 4qA168, 10qA176/180) sharing the same SNP.

expected allele frequency could be explained by the fact that the common 4qB163 allele carries the G SNP. 4. Discussion The flow chart of the proposed methodology is presented in Fig. 6. Initially, genomic DNA is analysed for the presence of SNPs related to the four common haplotypes (including 4qA161) in the D4F104S1 sequence region and the pLAM SNP. Alternatively, for screening purposes, detection of 4qA161 and PAS SNPs could be simply performed by triprimer PCR. The results of the initial screening determine the further analysis steps as follows. If the sample is found negative for both 4qA161 and pLAM SNP, no further analysis (including Southern blotting) is required. In the absence of 4qA161 SNP, other rare haplotypes, sharing the same SNP such as 4qA159 (permissive) and 4qB161 (nonpermissive), are also excluded. If the sample is found positive for 4qA161 and PAS SNP, it is suggested to confirm the presence/or absence of 4qA161 permissive allele in EcoRI digested sample by triprimer PCR. Sample found positive for PAS SNP but negative for 4qA161 and, consequently, negative for 4qA159 permissive haplotype [11] should be analysed by SSLP for the presence/or absence of 4qA168 permissive haplotype. The same applies to a sample found positive for 4qA161 and PAS SNP but negative for 4qA161 SNP in EcoRI alleles. A supporting

indication for the presence/absence of the 4qA168 permissive haplotype might be the detection of 4qB168 SNP in the initial screening of the sample by PEXT-dipstick assay. Altogether, FSHD confirmation (by molecular testing) should be done if sample is found positive for either 4qA161/168/159 haplotype in EcoRI contracted alleles and PAS SNP. Single nucleotide polymorphisms constitute one of the most important groups of biomarkers in molecular testing of diseases. The main limitation, however, in using D4F104S1 SNPs as FSHD biomarkers is their specificity. More specifically, 4qA159 (permissive) and 4qB161 (non-permissive) haplotypes share the same 4qA161 SNP. The 4qB163 SNP is shared by 10qB161T haplotype (prevalence 4.6%) and the 10qA166 SNP by 10qA164T haplotype (prevalence 4.4%). The 4qB168 SNP is shared by a number of rare haplotypes such as 4qB162/170/172, 4qA168 (permissive), and 10qA176/178. Similar limitations apply also to the current SSLP fragment analysis despite the method enables discrimination of most of the haplotypes differentiated by size and peak heights. For example, 161 bp fragment corresponds to 3 haplotypes (4qA161, 4qB161, 10qB161); fragment 168 bp corresponds to 2 haplotypes (4qB168 and 4qA168), with the second one being “permissive”, and fragment 166 bp to two haplotypes (10qA166 and 4qA166). On the other hand, combination of SNP genotyping with SSLP fragmentation analysis seems to be more efficient means to elucidate the sample's haplotypes than SSLP or SNP genotyping alone. From another point of

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Table 2 Genotyping of D4Z4, pLAM and 4qA161 SNPs by triprimer PCR, Southern blot analysis of EcoRI and EcoRI/AvRII alleles and SSLP fragment analysis for samples of FSHD affected individuals. A/A

Sample

D4Z4 SNP G/C (g. DNA)

pLAM SNP (g. DNA)

4qA161 SNP (EcoRI digest)

Southern blot analysis (EcoRI alleles, kb)

Southern blot analysis (EcoRI/AvRII alleles)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

F39 F49 F50 F62 F63 F76 F83 F86 F90 F91 F95 F96 F111 F119 F131 F134 F208 F209 F211 F217 F219 F224 F236 F247 F251 F253 F257 F261 F276 F283 F287 F306 F336 F346 F349 F354

+/+ +/− +/− +/− +/− +/− +/− +/− +/− +/− +/− +/+ +/− +/− +/− +/− +/+ +/− +/− +/− +/+ +/+ +/+ +/− +/+ +/+ +/− +/− +/− +/− +/+ +/− +/− +/− +/+ +/+

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − + + + + +

+ +

1 = 26 1 = 13 1 = 13, 3 N 48 1 = 30, 1 = 37, 2 N 48 3 N 48, 1 = 37 2 N 48, 1 = 30, 1 = 37 1 N 48, 1 = 48, 1 = 20 2 N 48, 1 = 35, 1 = 37 1 N 48, 1 = 30 1 = 30 2 N 48, 1 = 27, 1 = 23 2 N 48, 1 = 26, 1 = 23 1 N 48, 1 ~ 45, 1 = 34,1 = 23 2 N 48, 1 = 48, 1 = 14 1 = 14, 1 = 34, 2 N 48 2 N 48, 1 = 45, 1 = 38 1 N 48, 1 = 39, 1 = 23 1 = 30, 1 = 23, 1 N 48 3 N 48, 1 = 20 1 N 48, 1 = 45, 1 = 29 2 N 48, 1 = 35, 1 = 19 1 N 48, 1 = 40, 1 = 32 1 N 48, 1 = 33, 1 = 30 1 N 48, 1 = 37, 1 = 30 1 = 29, 1 = 48 1 = 33,1 = 19 2 N 48, 1 = 48, 1 = 29 1 N 48, 1 = 45, 1 = 26 1 N 48, 1 = 43, 1 = 33 1 = 39 3 N 48, 1 = 33 2 N 48, 1 = 25, 1 N 48 1 N 48, 1 = 50, 1 = 27 1 ~ 30, 1 ~ 22 3 N 48, 1 = 34 3 N 48, 1 = 22

1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 0 1

+ + + + + + + + + + + + + + + + + + + + + +

+ + + + + +

= 23 = 10 = 10, 1 N 48 N 48, 1 = 34 = 34 = 25 = 17, 1 N 48 N 48, 1 = 34 N 48, 1 = 27 = 26 N 48, 1 = 24 N 48, 1 = 24 N 48, 1 = 20 = 10 = 11, 1 N 48 N 48, 1 = 50 N 48, 1 = 20 = 20 = 17, 1 N 48 = 26, 1 N 48 = 16, 1 N 48 = 29 = 30, 1 = 27 N 48, 1 = 34 = 45, 1 = 26 = 16 = 26, 1 N 48 = 23 N 48, 1 = 30 = 37 N 48, 1 = 30 = 22, 1 N 48 = 24 ~ 19 = 19, 1 N 48

SSLP haplotypes

161, 163, 166 161, 163, 166 161, 166 161, 166

161, 166 161, 163, 166 161, 163, 166

161, 163, 166 161, 163, 166 161, 163, 166

161, 163, 166, 168 161, 166 161, 163, 166 161, 163

163, 166 161, 163, 166, 168

view, the lack of specificity of SNPs representing particular haplotypes might be advantageous. For example, in a sample negative for 4qA161 SNP, the 4qA159 permissive haplotype should be excluded; in a sample negative for 4qB168 SNP, haplotypes such as 4qB170/172, 4qA168 (permissive) and 10qA176/180 should also be excluded. To conclude, SNP genotyping may contribute to elucidation of a sample haplotyping profile by SSLP fragmentation and reveal discrepancies between haplotypes defined by size and haplotypes defined by specific SNPs. In addition, two subtelomeric variations distal to D4Z4 (4qA/4qB) represent another biomarker used in current FSHD molecular testing, because FSHD was uniquely associated with the 4qA variant [5]. For 4qA/4qB typing, DNA is digested with specific restriction enzymes and serially hybridized with probes specific for 4qA and 4qB variants. Typing an additional biomarker in the very first proximal D4Z4 unit represents another approach to discriminate 4qA/4qB variants. The so called D4Z4 SNP (or GNC SNP) specifies the non-permissive 4qB168 haplotype and rare 4qB alleles, except for the 4qB163 allele that carries the ‘G’ D4Z4 variant [10]. ‘G’ variant is also carried by all 4qA and the most common 10qA166 alleles. Triprimer PCR for D4Z4 SNP combined with genotyping results by PEXT-dipstick assay enables the discrimination of 4qA/4qB alleles in only few hours without the need for laborious and time-consuming Southern blotting.

FSHD molecular signature. This approach is based on the targeted identification of SNPs in D4F104S1 region, PAS SNP in pLAM region and GNC SNP in the first proximal D4Z4 unit. All biomarkers used, except for the fragmentation of the 4q35 polymorphic D4Z4 macrosatellite repeat, are single nucleotide polymorphisms proximal or distal to D4Z4 repeat. The main advantages of the proposed strategy are as follows: (i) Initial screening of the sample for the presence of 4qA161/PAS SNP which determines the necessity/or not to proceed with D4Z4 macrosatellite repeat fragmentation and Southern blotting; (ii) Simple means for SNP detection; (iii) Elimination of the false negative genotyping results using triprimer PCR; (iv) Elucidation of the sample's genotype by SSLP fragment analysis and genotyping of SNPs related to the common 4q and 10q haplotypes. Genotyping of a panel of biomarkers related to FSHD seems to be a simpler alternative to Southern blotting molecular testing. The proposed approach might be useful for families wishing to proceed with prenatal or preimplantation genetic diagnosis [20].

5. Conclusions

We sincerely thank Dr Richard Lemmers (Dept of Human Genetics, Leiden University Medical Center) for providing DNA samples from monoallelic sources. We also thank Dimitris Petichakis for sequencing analysis of D4F104S1 and pLAM fragments and CE analysis of the SSLP fragments. Funding: None.

Molecular testing of FSHD by Southern blotting remains the most common method for diagnostic laboratories up today. Here, we present a less laborious and time-consuming FSHD molecular testing approach to facilitate more extensive population studies on the applicability of

Conflict of interest The authors declare no conflict of interest. Acknowledgements

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103

Genotyping of SNPs related to common FSHD haplotypes (genomic DNA) by PEXT-Dipstick Assay (4qA161, 4qB163, 4qB168 & 10qA166) 4qA161 (-)

4qB168 (+)

PAS SNP genotyping by PAS (-) Tri-primer PCR (genomic)

4qB163 (+) 4qA161 (+) and 4qB168 (-) PAS SNP genotyping by PAS (-) Tri-primer PCR (genomic)

FSHD exclusion PAS (+)

PAS (+)

SSLP analysis for other permissive haplotypes (4qA168, 4qA159)

4qA168 (-) 4qA159 (-)

4qA161 (-)

4qA168 (+) or 4qA159 (+)

FSHD exclusion

4qA161 genotyping by Tri-primer PCR (Eco RI digest)

4qA161 (+)

FSHD Confirmation

Fig. 6. Flow chart of the proposed FSHD molecular testing approach. Initially, genomic DNA sample is subjected to a screening assay for the presence of the most abundant 4q35 and 10q26 subtelomeric haplotypes by multiplex PEXT reaction of PCR amplified D4F104S1 fragment followed by multiallele visual dipstick assay. Genotyping/haplotyping results will determine the further analysis steps as follows: (i) sample found positive (+) for 4qA161 SNP should be further analysed for PAS SNP (in genomic DNA) by triprimer PCR. If sample is found 4qA161 (+)/PAS (+), the presence/or absence of permissive 4qA161 haplotype should be confirmed in EcoR1 digested sample (contracted alleles) by nested triprimer PCR. Sample found 4qA161 (+)/PAS (+)/4qA161 (+) (in digest) should be categorised as FSHD. Sample found 4qA161 (+)/PAS (+)/4qA161 (−) (in digest) means that the sample does not carry 4qA161 contracted alleles. However, before excluding FSHD, the sample has to be analysed by SSLP for rare permissive 4q haplotypes (4qA159 or 4qA168). (ii) Sample found 4qA161 (+)/PAS (−) is not pathogenic and no further examination is required; (iii) sample found 4qA161 (−)/PAS (+) is possibly not pathogenic. SSLP analysis in genomic DNA and/or in digest is recommended to confirm the absence of rare permissive 4qA haplotypes (4qA159 or 4qA168); (iv) sample found 4qA161 (−)/4qB163 (+)/PAS (−) or 4qA161 (−)/4qB168 (+)/PAS (−) is not pathogenic and further examination is not required.

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