PCR-RFLP sequencing and quantification in molecular diagnosis of spinal muscular atrophy: Limits and advantages

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PCR-RFLP, Sequencing, and Quantification in Molecular

Diagnosis of Spinal Muscular Atrophy: Limits and Advantages

K. Hamzi_&H. Bellayou_&M. Itri_&S. Nadifi

Received: 15 November 2012 / Accepted: 20 December 2012 / Published online: 8 January 2013

#Springer Science+Business Media New York 2013

Abstract

Spinal muscular atrophy (SMA) is a severe neuro- muscular disease. It is a common cause of infant mortality. Its incidence is estimated at 1 in 10,000. Clinically, age of onset and the symptoms can distinguish four types of SMA. The objective of this study is to make available to clinicians a reliable and reproducible test for the molecular diagnosis of SMA. We evaluate the benefits and limitations of three tests used in our laboratory (RFLP-PCR, sequencing, and qPCR).

Keywords

SMA . SMN1 . SMN2 . Quantification

Introduction

Spinal muscular atrophy (SMA) is a severe neuromuscular disease. It is a common cause of infant mortality. Its inci- dence is estimated at 1 in 10,000 (Brichta et al.

2006).

Clinically, age of onset and motor achievements allow to distinguish four types of SMA (Zerres & Rudnik- Schoneborn

1995). Type 1 is the most severe form of

SMA (Werdnig-Hoffman disease). The onset is below 6 months of age, and life expectancy does not exceed 7 years (Feldkotter et al.

2002). In type 2, clinical signs are gener-

ally present before 18 months; patients are able to sit, but do not walk without. In patients with SMA type 3 or Kugelberg–Welander disease, motor achievements are nor- mal and may loss autonomous ambulation at variable age.

SMA type 4 or Adult form appears after 30 years (Rudnik-

Schoneborn et al.

2001). The survival motor neuron (SMN)

genes are located on chromosome 5q13. There are two nearly identical copies (SMN1/telomeric and SMN2/centro- meric) (Chen et al.

1998a). The sequence homology be-

tween SMN1 and SMN2 genes is 99 % with only five nucleotide differences (Monani et al.

1999). SMA is caused

by a homozygous deletion of exon 7 (+ /

−

exon 8) of SMN1 gene. It has been shown that number of SMN2 genes is variable in the population (generally two to four copies) and is partially related to clinical severity of the disease, being higher copy number related to milder phenotypes (McAndrew et al.

1997). It has been shown that mild forms

of SMA (Feldkotter et al.

2002; Rudnik-Schoneborn et al.

2001) are associated with more than two copies of SMN2

(Wirth et al.

2006a).

The presence of SMN2, the sequence homology with SMNt, as well as the correlation between the phenotype and number of copies of SMN2 make molecular diagnosis difficult. Thus, many methods have been used for molecular diagnosis of SMA (RFLP-PCR, sequencing, quantification, ARMS-PCR, etc.).

The objective of this study is to make available to clini- cians a reliable and reproducible test for the molecular diagnosis of SMA. We evaluate the benefits and limitations of three tests used in our laboratory (RFLP-PCR, sequenc- ing, and qPCR).

Patients and Methods

Patients

Our study involved thirty-three subjects with SMA of vary- ing degrees of severity. For each one, a detailed family history was recorded, and a pedigree was established. The family was advised on genetic testing, and informed consent

K. Hamzi (*)

:

H. Bellayou

:

S. Nadifi

Medical Genetic Laboratory and Molecular Pathology, Medical School, Casablanca, Morocco

e-mail: Khalil.hamzi@yahoo.fr M. Itri

Pediatrics Department, PIII, CHU Ibn Rochd, Casablanca, Morocco

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was obtained. The fact sheet has been completed according to the diagnostic criteria defined by the International Consortium SMA with a motor and sensory neurophysio- logical assessment studies and conventional electromyogra- phy (EMG) nerve conduction.

Extraction

Blood sample of 5 ml was collected in EDTA, and DNA was extracted using standard phenol–chloroform extraction pro- tocol (Brichta et al.

2006). After extraction, molecular ge-

netic studies were done.

PCR-RFLP

Amplification of SMN was carried out with 100 ng of DNA in 25 ml reaction using 20 pmol of S7F, S7R, S8F, and S8R for amplification of SMN exons 7 and 8 as described pre- viously. The products were analyzed by electrophoresis through 1 % agarose gel in an ethidium bromide stain followed by enzymatic digestion using DraI and DdeI;

products were analyzed in 3 % agarose gel.

Sequencing

To amplify exons 7 and 8 of both the SMN1 and SMN2 genes using the same primers. This yielded a 188- bpfragment. After gel purification of the PCR product, di- rect sequencing was performed using the primary amplifi- cation primers. The sequenced products were analyzed on the automated sequencer (Model 3700, ABI).

Real Time PCR

We employed a quantitative method using SMN1and SMN2 specific probes, and we use TELO as housekeeping gene for control. DNA was diluted first to 20 ng/μl and then to a final concentration of 5 ng/μl. Quantification was performed on the RotorGene 6000 instrument.

Results

Patients

This study involved 33 subjects with 19 children (10 females, 9 males) and 14 parents. All patients were of Moroccan origin.

The mean age of patients is 10 months with an extreme of 3 months to 8 years. We selected for classification of pa- thology diagnostic criteria defined by the International SMA Consortium with neurophysiological evaluation studies and conventional EMG neuroconduction (Table

1).

PCR-RFLP

The study was performed on genomic DNA of patients and their parents. We simultaneously amplified by PCR, exons 7 and 8 of SMN1 and SMN2 gene and digested with restriction enzymes (DraI exon7) DdeI (exon 8) using the technique of (Zerres & Rudnik-Schoneborn

1995).

Analysis of the results on agarose gel 1–3 % showed no restriction site in exon 7 of SMN1 gene (188 bp band) and the presence of a restriction site on exon 7 of SMN2 gene (bands of 164 and 24 bp). For exon 8, the enzyme DdeI digested specifically SMN1 gene (123 and 65 bp) and not SMN2 188 bp (Fig.

1).

The result of the study by PCR-RFLP revealed deletion of exon 7 in 8 subjects and no deletion in 11 subjects (Table

2).

Table 1 Clinical characteristics of patients suffering from SMA Patient Sex Age Type Characteristics 1 M 2 years Type II sitting/walking with

support

2 M 1mois Type I Severe form of SMA:

Werdnig-Hoffman Syndrome, amyotrophy, hypotony, neonatal breathing distress. age of the dead: 1 year 3 F 3 months Type I Severe form of SMA:

Werdnig-Hoffman Syndrome: amyotrophy, hypotony, neonatal breathing distress.

Age of the dead:

3 months 4 F 18 months Type II sitting/walking with

support

5 M 18 months Type II sitting/walking with support

6 M 4 years Type III sitting/walking normally 7 M 7 years Type III sitting/walking normally 8 M 5 years Type III sitting/walking normally 9 F 18 months Type II sitting/walking with

support

10 F 4 years Type III sitting/ walking normally 11 M 2 years Type III sitting/walking normally 12 F 1 years Type III sitting/walking normally 13 M 18 months Type II sitting/walking with

support

14 F 4 years Type III sitting/walking normally 15 F 6 years Type III sitting/walking normally 16 F 7 years Type II sitting/walking with

support

17 M 3 years Type III sitting/walking normally 18 F 5 years Type III sitting/walking normally 19 M 6 years Type III sitting/walking normally

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The number of copies of SMN1 genes deleted was not know from this result, and conclusion on the existence of mutation was not reflected. Therefore we sequenced all the DNA of patients for whom we did not find deletion.

Sequencing

Sequencing was made with the aim to detect exons 7 and 8 punctual mutations in negative patients with typical SMA feature.

No mutation was detected for 11 patients; the sequence homology was confirmed with the five nucleotide difference between SMN1 and SMN2 sequence.

Sequencing was done to detect point mutations in patients with SMA clinical feature, but in which no deletion by PCR-RFLP (Fig.

2).

No mutation was detected in 11 patients. Furthermore, the sequence homology was confirmed by the nucleotide difference between SMN1 and SMN2 gene sequences.

Real Time PCR

For positive patients, gene quantification was done in the objective to confirm parent’s heterozygosis and to detect number of SMN2 gene copies in patients. The relative quantification showed a copy of SMN1 with parents and zero copy in patients. Concerning SMN2 gene, qPCR per- mit to detect two or three copies in patients, and two SMN2 copies among parents. For patients who have a deletion by PCR-RFLP, quantification of the SMN1 gene has been done to confirm the heterozygous parents. The number of SMN2 gene copies detected in patients is necessary in order to correlate genotype–phenotype (Table

3).

Discussion

SMA is a common cause of infant neuromuscular mortality, and its incidence is estimated at 1 in 10,000 (Zerres &

Fig. 1 PCR-RFLP results for SMN 1 and 2, Exons 7 and 8 (MT: Weight Marker,F:Father,M:Mother,P:Patient)

Table 2 PCR-RFLP results for SMA patients

PPatient,MMother,F Father

Family Subject RFLP

Exon 7 Exon 8

I P1 + −

F1 − −

M1 − −

II P2 + +

F2 − −

M2 − −

III P3 + −

F3 − −

M3 − −

IV P4 + +

F4 − −

M4 − −

V P5 + +

F5 − −

M5 − −

VI P6 + −

F6 − −

M6 − −

VII P7 + −

P8 + −

F7 − −

M7 − −

P9 − −

P10 − −

P11 − −

P12 − −

P13 − −

P14 − −

P15 − −

P16 − −

P17 − −

P18 − −

P19 − − Fig. 2 Detection of the difference sequence with T in SMN1/ C in SMN2 in exon 7

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Rudnik-Schoneborn

1995; Feldkotter et al.2002). The age

of onset and clinical presentation are widely sales person to distinguish four types of SMA. The most severe form of SMA is type 1 or Werdnig-Hoffman disease; it occurs before 6 months, and the median survival is 5–7 years.

PCR-RFLP has been widely used by different laborato- ries (Zerres & Rudnik-Schoneborn

1995). It can detect

homozygous deletion of SMN1 gene (Feldkotter et al.

2002). But does not detect heterozygosity (especially among

parents who are carriers). She did not inform either the number of copies of SMN2 (Rudnik-Schoneborn et al.

2001). It remains a standard technique for initial screening

(Chen et al.

1998b; Monani et al.1999).

The sequencing is done in order to detect point muta- tions in patients with negative PCR-RFLP and whose clinical SMA is evident (McAndrew et al.

1997; Wirth

et al.

2006b).

However, the limits of sequencing are the absence of information about the copy number for SMN1 and SMN2.

Absence of deletion and punctual mutation with typi- cal SMA can be explained by the presence of mutation in other exons out of exons 7 and 8 has identified five novel missense mutations localized in: exon 2 88 G>A and 131A >T; exon 3 283 G>C and 332C>G; and exon 6 c.784A > G (Zerres & Rudnik-Schoneborn

1995).

Before that, D.W. Found two other mutations (800ins11 and 542delGT).

The qPCR is a sensitive and rapid method for the diag- nosis of SMA (Table

4). Specific hybridization of SMN1

and SMN2 probes detects also deletion and punctual muta- tions with possibility of quantification of gene copy number;

SMN1 number in patients and parents and SMN2 in patients. (Feldkotter et al.

2002) report that the specificity Table 3 results of

quantification concerning SMA patients and parents

PPatient,MMother,F Father

Family Subject SMN1 Copy

SMN2 Copy

I P1 0 3

F1 1 2

M1 1 2

II P2 0 2

F2 1 2

M2 1 2

III P3 0 2

F3 1 2

M3 1 2

IV P4 0 3

F4 1 2

M4 1 2

V P5 0 3

F5 1 2

M5 1 2

VI P6 0 3

F6 1 2

M6 1 2

VII P7 0 3

P8 0 3

F7 1 2

M7 1 2

Table 4 Comparison of results of PCR-RFLP, sequencing, and qPCR in the diagnosis of SMA

Family Subject RFLP qPCR Sequencing

mutation Exon 7 Exon 8 SMN1

Copy number

SMN2 Copy number

I P1 + − 0 3

F1 − − 1 2

M1 − − 1 2

II P2 + + 0 2

F2 − − 1 2

M2 − − 1 2

III P3 + − 0 2

F3 − − 1 2

M3 − − 1 2

IV P4 + + 0 3

F4 − − 1 2

M4 − − 1 2

V P5 + + 0 3

F5 − − 1 2

M5 − − 1 2

VI P6 + − 0 3

F6 − − 1 2

M6 − − 1 2

VII P7 + − 0 3

P8 + − 0 3

F7 − − 1 2

M7 − − 1 2

P9 − − −

P10 − − −

P11 − − −

P12 − − −

P13 − − −

P14 − − −

P15 − − −

P16 − − −

P17 − − −

P18 − − −

P19 − − −

+: presence of deletion;−: absence of deletion

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of the quantification is 100 %, whereas the sensitivity is 96.2 %.

Quantitative analysis of SMN2 copies in patients with Type I, Type II, or Type III SMA showed a significant correlation between SMN2 copy number and type of SMA with the survival time. Thus, 80 % of patients with type I SMA carrying two SMN2 copies, and 82 % of patients with type II SMA carry three SMN2 copies. Among patients with SMA type I, patients with two SMN2 copies lived 21 months in general, and those with three SMN2 copies lived 33–

66 months; on the basis of the number of SMN2 copies.

However, in a prenatal test, qPCR was used to estimate the posterior probability that the child in the absence of SMN1 homozygous could develop Type I, Type II, or Type III SMA [32] and also give appropriate genetic counseling [33].

However, the presence of mutations in other exons of SMN1 explains the reduction of the sensitivity of the test. About 4 % of patients have a combination of the removal or conversion of one allele and an intragenic mutation on the second [34].

In our study, PCR-RFLP allowed us to have 42 % (8/19) of positivity as a first screening of SMA, confirmed by the qPCR, which in addition give the copy number of SMN1 and SMN2. In this field, qPCR confirmed the presence of only two copies of SMN2 in patients P2 and P3 with severe SMA and the presence of three copies in the other cases. The absence of deletion in 11 patients of 19 tested by PCR-RFLP and confirmed by sequencing of the exons 7 and 8 allowed us to think about mutations in other exons on SMN1 which will be tested in a second time.

We conclude that real time PCR is a rapid, sensitive, and robust method for SMA diagnosis; the study of correlation genotype–phenotype and the parent’s genetic profile allowed us to give an appropriate genetic counseling.

Acknowledgments We are deeply grateful to the ten families for their invaluable collaboration to this study. We are also grateful to Mr. M El Fahim of CNRST-Morocco; Prof. Giovanni Neri, Danilo Tiziano, and Stefania Fiori from Institute of Medical Genetics at Catholic University in Roma (Italy), for accepting our training and article reviewing.

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