a population-based study of genotypic and phenotypic variability in children with spinal muscular...

Acta Pædiatrica ISSN 0803–5253

REGULAR ARTICLE

A population-based study of genotypic and phenotypic variabilityin children with spinal muscular atrophyEva Arkblad ([email protected])1,3, Mar Tulinius2, Anna-Karin Kroksmark2, Mirja Henricsson1, Niklas Darin2

1.Department of Clinical Genetics, Sahlgrenska University Hospital, Goteborg, Sweden2.The Queen Silvia Children’s Hospital, Sahlgrenska University Hospital, Goteborg, Sweden3.Department of Clinical Genetics, Unilabs, Karnsjukhuset i Skovde, Skovde, Sweden

KeywordsGenotype, MLPA, Phenotype, SMA, SMN1

CorrespondenceEva Arkblad, Department of Clinical Genetics,Unilabs, Karnsjukhuset i Skovde,SE-54185 Skovde, Sweden.Tel: +46-500-432167 |Fax: +46-500-438753 |Email: [email protected]

Received30 September 2008; revised 29 November 2008;accepted 5 December 2008.

DOI:10.1111/j.1651-2227.2008.01201.x

AbstractAims: To describe the occurrence of spinal muscular atrophy (SMA) in childhood; to evaluate if any

of the genes in the SMA region on chromosome 5q13 correlates with disease severity; to make

genotype–phenotype correlations; to evaluate the variability of different disease alleles in carriers and

the sensitivity of multiplex ligation-dependent probe amplification (MLPA) for detecting carriers.

Methods: In a population-based study from Western Sweden MLPA was used to determine the

copy-numbers of several genes in the SMA region (SMN1, SMN2, BIRC1, GTF2H2 and SERF1A) in

SMA-patients and their parents.

Results: We estimated the incidence of SMN1-related SMA in childhood at 1 in 11 800 live births and

confirmed the relationship between the number of SMN2 copies and the severity of disease. No

other direct relationships were found. All but one of the analysed parents were confirmed as carriers

by MLPA analysis. A total of at least 30 different disease alleles were identified and no specific

disease allele represented more than 15% of the total.

Conclusion: The childhood incidence of SMA in the Swedish population is around 1 in 12 000 live births and it is

unlikely that there is any founder effect involved in SMA in western Sweden.

INTRODUCTIONSpinal muscular atrophy (SMA) is characterized by degener-ation of the anterior horn cells of the spinal cord, leading toprogressive muscle weakness and atrophy with highly vari-able age at onset and clinical severity (1,2). It is consideredto be the second most common autosomal recessive disease,with an overall incidence frequently cited to be around 1 in6000 to 1 in 10 000 live births and a carrier frequency prob-ably as high as 1 in 40 (3–6). There is a great variability ofdisease severity in SMA. Classification in subtypes is prac-tical for management and prognosis. SMA is divided intotypes 1, 2 and 3, based on age at onset and maximum mo-tor function. SMA type 1 (MIM 253300) usually manifestsbefore 6 months of age and afflicted children are never ableto sit unaided. SMA type 2 (MIM 253550) usually manifestsbefore 18 months of age; patients are never able to walkunaided. Patients with SMA type 3 (MIM 253400) achievethe ability to walk unaided.

The vast majority of all patients with SMA have muta-tions in the survival motor neuron gene (SMN) on chromo-some 5q13 (7,8). SMN occurs in two versions, SMN1 (MIM600354; GenBank U18423) and SMN2 (MIM 601627;GenBank NM_022875), differing by only one nucleotide in

AbbreviationsMLPA, multiplex ligation-dependent probe amplification; SMA,spinal muscular atrophy; PEG, percutaneous endoscopicgastrostomy.

the coding region. The mutational mechanism behind SMA,in around 94% of typical cases, is lack of SMN1 due to ei-ther homozygous SMN1 deletion or SMN1-to-SMN2 geneconversion. Furthermore, 2–5% of the cases are compoundheterozygotes with a point mutation on one allele and adeletion of the other. Around 30 different small, intragenicmutations have been reported (1). SMN1 is located in ahighly complex genetic region containing a variety of pseu-dogenes and repetitive elements due to an inverted duplica-tion of approximately 500 kilobases (kb) (Fig. 1). The tran-sition of a cytosine to a thymine in SMN2 exon 7 alters thesplicing pattern and gives rise to a truncated transcript (�7SMN), which encodes a non-functional protein lacking thelast 16 residues at the C-terminal end. Nevertheless, SMN2also produces a small proportion (10–20%) of full-lengthSMN transcripts and hospital-based studies have shown thatthe number of SMN2 copies present modulates and corre-lates inversely with disease severity (3,7,9). The number ofthe neighbouring BIRC1 (NAIP) (GenBank NM_004536),GTF2H2 (GenBank NM_001515) and SERF1A (GenBankNM_021967) genes and/or the size of the deletion might berelated to the severity of the disease, which may also be re-lated to other, as yet unidentified, genetic components. TheSMN protein is located in cytoplasmic and nuclear inclu-sions called gems, and appears to play an important role inspliceosomal snRNP biogenesis (10).

In this paper, the results of a population-based study fromwestern Sweden are presented, the aims of which were todescribe the incidence, prevalence and clinical presentation

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Phenotype/genotype study of SMA Arkblad et al.

of SMA in children; to evaluate which of the different genesin the SMA region on chromosome 5q13 correlate with dis-ease severity; to make genotype–phenotype correlations andto evaluate the variability of different disease alleles in car-riers.

MATERIALS AND METHODSThe study was approved by the Ethics Committee ofGoteborg University and informed consent was obtainedfrom all families.

Study populationThe geographic area studied was the Western SwedishHealth Care Region, which consists of the city of Goteborgand Halland, Bohuslan, Skaraborg and Alvsborg counties.There are seven local hospitals in the region with paediatricwards, seven local rehabilitation centres, one regional reha-bilitation centre and one university hospital which is the re-ferral hospital for the region. The total number of live birthswere 531 746 between 1980 and 2006 and 224 736 between1980 and 1990, while the total population aged <16 yearswas 340 179 on December 31, 2007.

PatientsWe intended to identify all children younger than 16 yearsof age with SMA diagnosed between 1980 and 2006 andonset of disease before 1 January 2007. Data from 1980 to2006 from residential and outpatient registers in local andregional paediatric hospitals and child rehabilitation centreswere analysed. Written inquiries were sent to all outpatientpaediatric clinics in the region. Registers at the departmentsof pathology, neurophysiology and clinical genetics at theUniversity of Goteborg, Sweden, were reviewed. The Na-tional Swedish Board of Health and Welfare’s cause of deathregister was studied. SMA was considered in children lack-ing SMN1 if there were clinical symptoms and signs com-

Table 1 Clinical features in patients with SMA type 1

Patient no. Type of SMA Age at onset Maximum motor function Nutrition/nasogastric tube Nutrition/PEG Respiratory support Life span (years)

1 1 0.2 No head control Oxygen 0.92 1 0.3 Some head control/some trunk stability 1.8 3.0 CPAP (2.0 year) 4.53 1 0.2 No head control 0.5 Oxygen + Suc ap 0.74 1 0.1 No head control 0.7 Oxygen + Suc ap 0.85 1 0.1 No head control 0.36 1 0.3 No head control 0.5 Oxygen + Suc ap 0.67 1 na No head control/no trunk stability Oxygen + Suc ap 0.88 1 0.3 Some head control Oxygen + Suc ap 0.69 1 0.1 No head control 0.5 Oxygen + Suc ap 0.710 1 0.1 No head control 0.5 0.511 1 0.1 No head control 0.4 0.912 1 0.1 No head control 0.3 0.613 1 0.1 Some head control, no trunk stability 0.4 CPAP (0.8 year) 1.414 1 0.1 No head control 0.4 Suc ap 0.815 1 0.2 Some head control/no trunk stability 0.5 Oxygen 0.716 1 0.1 No head control 0.3 Oxygen 0.4

PEG = percutaneous endoscopic gastrostomy; Suc ap = suction apparatus; na = not available; CPAP = continuous positive airway pressure.

patible with the diagnosis. Before 1996, when the underly-ing gene was identified, the diagnosis was defined accordingto the European Neuromuscular Centre (ENMC) criteria(11) and was considered in children with clinical and elec-trophysiological and/or histopathological evidence. Bloodand/or muscle tissue were saved from all patients with a di-agnosis made before 1996. We could confirm the diagnosisin all patients by genetic analysis. All children had homozy-gous deletion of SMN1. The number of patients diagnosedfrom 1980 to 1993 (n = 24) was approximately the same asthe number of patients diagnosed from 1994 to 2006 (n =22) (Tables 1 and 2). The patients with SMA II were classi-fied according to the Hammersmith functional motor scalefor children with SMA (12).

AnalysesCumulative childhood incidence was defined as the numberof children aged <16 years and born in western Swedenwho were diagnosed with SMA between 1980 and 2006, inrelation to the total number of live births in the study areaduring the same period. For comparison the incidence wasalso calculated for the patients born between 1980 and 1990in the region where the youngest had reached the age of16 years at the census date. Point prevalence was definedas the number of affected individuals aged <16 years in thepopulation who lived in the study area and were known to bealive on December 31, 2006, in relation to the total numberof individuals who were at risk at that time, that is the entirepopulation aged <16 years. Confidence intervals (CI) werecalculated with a simplified method, utilizing tabular valuesbased on the Poisson distribution (13). Pearson’s correlationcoefficients (r) and linear regressions were calculated usingmicrosoft excel.

Multiplex ligation-dependent probe amplificationPatients and carriers were analysed with multiplex ligation-dependent probe amplification (MLPA) to evaluate their

866 C©2009 The Author(s)/Journal Compilation C©2009 Foundation Acta Pædiatrica/Acta Pædiatrica 2009 98, pp. 865–872

Arkblad et al. Phenotype/genotype study of SMA

Table 2 Clinical features and number of SMN2 copies in children with SMA type 2 and 3

Patient no. Number of SMN2 copies Type of SMA Age at onset Maximum motor function SMA scale Ambulation

17 3 2 0.5 Sat and crawled 2.718 3 2 1.0 Crawled, stood and walked with assistance 2.819 3 2 0.7 Rolled over and sat when placed in position 2.220 3 2 1.0 Crawled, stood and walked with assistance 2.821 3 2 0.5 Sat when placed in position 2.522 3 2 0.7 Squatted 2.323 3 2 0.9 Stood and walked with assistance 2.824 3 2 0.5 Could sit unassisted 2.025 3 2 0.7 Sat and stood with assistance 2.326 3 2 0.5 Sat when placed in position 2.327 4 2 1.0 Walked with assistance 2.828 3 3 1.5 Walked independently, lost walking ability at age 17 HA29 3 3 2.5 Walked and ran CA30 3 3 1.0 Walked independently, lost walking ability at age 2.5 HA31 4 3 6.0 Walked and ran CA32 4 3 14.0 Walked and ran CA33 4 3 16.0 Walked and ran CA34 4 3 1.5 Walked independently, lost walking ability at age 2 HA35 4 3 1.2 Walked independently, lost walking ability at age 1.6 HA36 4 3 13.0 Walked and ran CA37 4 3 2.0 Walked and ran CA38 4 3 1.4 Walked CA39 4 3 1.5 Walked HA40 4 3 1.0 Walked and ran CA41 3 3 1.6 Walked CA

CA = community ambulator; HA = household ambulator.

respective numbers of SMN1, SMN2, BIRC1, GTF2H2 andSERF1A copies. The MLPA technique is a gene-dosagemethod that can be used to detect both deletions and dupli-cations (14). Genomic DNA was extracted from blood us-ing the PuregeneTM Kit from Gentra systems (Minneapolis,MN, USA). The MLPA analysis was carried out accordingto the manufacturer’s recommendations (www.mlpa.com)and, as previously described (15), using the SALSA SMNregion test kit, P021, from MRC-Holland (the Netherlands).This kit contains specific probes for SMN1 exons 7 and8, SMN2 exons 7 and 8, SMN (1 and 2) exons 1, 4, 6

Figure 1 Overview of the gene area involved in SMA. The locations of the MLPA-probes are marked by asterisks.

and 8, BIRC1 (and NAIPψ), GTF2H2 (BTFFp44) and anN-Cadherin-like probe located approximately 10 kb fromSERF1A. Approximate locations of the probes are shown inFigure 1. Data were analysed using the Seq-Pilotsoftware (JSI medical systems GmbH, Kippenheim,Baden-Wurrtemberg, Germany).

RESULTSIn western Sweden, 47 children aged <16 years were di-agnosed with SMA between 1980 and 2006. Two chil-dren with SMA type 2 were born outside the region



Figure 2 Number of patients with SMA diagnosed each year between 1980and 2006.

and were thus excluded from the epidemiological survey.Of the remaining 45 patients, the proportion of boys to girlswas 22/23. SMA type 1 was diagnosed in 19 patients (m/f =6/13), type 2 was diagnosed in 11 patients (m/f = 8/3) andtype 3 was diagnosed in 15 patients (m/f = 8/7). The num-ber of patients diagnosed from 1980 to 1993 (n = 24) wasapproximately the same as the number of patients diagnosedfrom 1994 to 2006 (n = 22) (Fig. 2). The estimated cumula-tive childhood incidence was 1 in 11 800 live births (8.5 ×10−5, 95% CI: 6.2–11.3), while the estimated point preva-lence was 1 in 31 250 children aged <16 years (3.2 × 10−5,95% CI: 1.6–5.8). The estimated cumulative incidence wasalso calculated for children born during 1980–1990, in or-der for all children to have reached the age of 16 years atthe census date. All in all 17 patients were diagnosed duringthis period giving an identical incidence of 8.5 × 10−5.

Four children (three with SMA type 1 and one with SMAtype 3) were either lost to follow-up or did not want to partic-ipate in the MLPA study. Thus, 41 children from 39 familieswere included for further clinical and genetic analyses.

A summary of the clinical data for all the patients is pre-sented in Table 1.

SMA type 1 was diagnosed in 16 participating children.The median age at onset was 0.1 year, ranging from 0.1 to0.3 years, while the median life span was 0.7 year, rangingfrom 0.3 to 4.5 years. Patients 2 and 13 had a prolongedlife span, compared to the others: 1.4 and 4.5 years, re-spectively. These were the only patients with SMA type 1treated with continuous positive airway pressure (CPAP).Five children were treated with nasogastric tubes or percu-taneous endoscopic gastrostomy (PEG), but this treatmentdid not seem to prolong their life span.

SMA type 2 was diagnosed in 11 participating children.The median age at onset was 0.7 year, ranging from 0.5to 1.0 years. All 11 children with SMA type 2 developedscoliosis during childhood. Eight were treated with spinaljackets and three with spinal fusion. One child was treatedwith PEG due to undernutrition problems. Three childrenstarted nocturnal ventilation during childhood.

SMA type 3 was diagnosed in 14 participating children.The median age at onset was 1.6 years, ranging from 1.0 to16.0 years. Four of the children with SMA type 3 lost theirwalking ability at the respective ages of 1.6, 2.0, 2.5 and17 years. Four developed scoliosis during childhood; allwere treated with spinal jackets and three with spinalfusion.

Two pairs of siblings were included in the study. Patients34 and 36 with SMA type 3 were brothers, while patient25 with SMA type 2 and patient 41 with SMA type 3 werebrother and sister. Patient 9 and patient 31 were first cousins.The cousins had different numbers of SMN2 copies (2 and4, respectively) and, as expected, had very different diseasecourses. Each sibling pair had the same genotype. The pairs’internal differences in degree of severity could thereforenot be explained in terms of the genotypes analysed in thisstudy.

We found a clear correlation (Pearson’s correlation, r =0.94) between SMN2 copy numbers and the severity of thedisease (Fig. 3A). All 15 children with SMA type 1 had twocopies of SMN2. Of the children with SMA type 2, 11 hadthree copies of SMN2 and one had four copies. The childwith four SMN2 copies (patient 27 in Table 2) was consid-erably stronger in his upper body than the rest of the SMAtype 2 patients. Of the children with SMA type 3, 10 hadfour copies of SMN2, while three had three copies. The pa-tient assigned the diagnosis of SMA type 4 in Figure 3 is theasymptomatic mother of patient 37 in Table 2.

BIRC1 is located immediately upstream, telomeric, ofSMN1 (Fig. 1). Homozygous absence of BIRC1 was mostcommon in SMA type 1 (Fig. 3B). Twelve of the 15 chil-dren with SMA type 1 had no BIRC1 copy. Only one ofthe 12 children with SMA type 2 and five of the 13 chil-dren with SMA type 3 had no copy of BIRC1. No corre-lation between the number of BIRC1 copies and diseaseseverity was found when comparisons were made amongchildren with the same number of SMN2 copies (Figs. S1A,S1B, S2A, S2B, S3A and S3D in supporting informationonline).

GTF2H2 is the next gene upstream of SMN1 (Fig. 1).No correlation between the type of SMA and deletion ofGTF2H2 could be found among our patients (Fig. 3C). Nocorrelation between the number of GTF2H2 copies and dis-ease severity could be found when comparisons were madeamong children with the same number of SMN2 copies(Figs. S1C, S1D, S2C, S2D, S3B and S3E in supporting in-formation online).

More central in the inverted repeat are the SERF1A genes,represented by the N-Cadherin-like probe in the MLPA-kit.A correlation (Pearson’s r = 0.63) was found between SMAtype (number of SMN2 copies) and the number of SERF1Acopies (Fig. 3D). All children with SMA type 1 had at leastone deletion of SERFIA, while half of the children withSMA type 2 had one deletion of SERFIA and only 31% ofthe children with SMA type 3 had one deletion of SERFIA.

No correlation between the number of SERF1A copiesand disease severity was found when comparisons weremade among children with the same number of SMN2copies



Figure 3 Correlation between number of copies of SMN2, BIRC1, GTF2H2 and SERF1A and the disease severity, as indicated by the type of SMA (A–D).

(Figs. S1E, S1F, S2E, S2F, S3C and S3F in supportinginformation online).

MLPA analysis was performed on 71 parents from 36 fam-ilies. The remaining parents declined to participate or wereunavailable. Of the 71 analysed parents, 69 had one copyof SMN1, one had two copies of SMN1 and one had noSMN1. The mother with no SMN1 had five copies of SMN2and was asymptomatic, as reported earlier (15).

The polymorphism consisting of an extra exon 1–6 frag-ment was present in heterozygous form in seven of the anal-ysed parents (10%). This concurs with our previously re-ported population frequency of 8% (15). However, none ofthe children had this polymorphism.

The families were analysed for different kinds of diseasealleles. A total of at least 30 different disease alleles wereidentified, 17 of which were unique for one person. The fourmost common disease alleles were carried by 30 parents(43%). The two most common disease alleles were (genename (copy number)): GTF2H2c (1), NAIPψ (1), SMN2(1), SERF1A (2), SMN1 (0), BIRC1 (0), GTF2H2t (1) andGTF2H2c (1), NAIPψ (1), SMN2 (1), SERF1A (1), SMN1(0), BIRC1 (0), GTF2H2t (1), each carried by 10 parents.

DISCUSSIONThe childhood incidence of SMA in our study from Swedenwas 1 in 11 800 live births. This is similar to what was found

by John Pearn in England in the early 1970s when he de-scribed an incidence of 1 in 12 468 (16,17). Our incidencefigures should be viewed as minimal figures since we couldnot account for patients born in the study area that latermoved out and where diagnosed elsewhere. There is alsoa possibility that we could have missed patients that didnot come for diagnostic evaluation, that is very severe earlyonset patients that died shortly after birth or minimally af-fected children. We have had a focus on population-basedstudies of neuromuscular disorders in Western Sweden fora long time (18) and there is a general awareness of SMAin the region based on prior studies (19). We are thereforeconfident that we have found all patients with the commonphenotypes. Our estimated incidence is lower than the esti-mated incidence of between 1 in 6000 and 1 in 10 000 livebirths included in most recent articles in the field (3–6). Acomparison is not possible to make since the incidence fig-ures stated in these articles have been extrapolated from theworks by Pearn (16,17).

In our study, the proportions of SMA types 1, 2 and 3were 42%, 24% and 33%, respectively. Although gender dis-tribution was similar in the whole group, we found a higherproportion of girls among those with severe SMA. We haveno satisfactory explanation for this difference but speculatethat it could be due to chance, since the total number of pa-tients in this study is quite low. In comparison, a large study



by Rudnik-Schoneborn did not identify any significant sexdifference in SMA (20). The clinical course was very homo-geneous among the patients with SMA type 1. Treatmentwith CPAP, but not nasogastric tube/PEG, seemed to pro-long survival.

Two pairs of siblings were included in the study. Both pairsdiffered considerably in the clinical severity of their disor-der despite having the same number of SMN2 copies. In-trafamilial variability is rare in SMA but has been described(20–24). The pair of brothers (patients 34 and 36) bothhad four copies of SMN2. Although both cases were classi-fied as SMA type 3, patient 34 had onset at 1.5 years of ageand lost the ability to walk at age 2, while his brother hadonset at 13 years of age and could both walk and run. Thebrother (patient 25)/sister (patient 41) pair were classifiedas SMA type 2 and 3, respectively. Part of the explanationin this case may be that the less affected sibling is a girl.Gender-biased SMA severity has been reported; females areusually affected less severely (21,22,25,26), possibly througha gender-specific effect of the modifier PLS3 (27). The num-ber of BIRC1 copies, the number of copies of nearby genesor the very size of the deletion are hypothetic explanationsfor the discordance in sibling pairs, since these factors arenormally not analysed and differences might escape detec-tion. When it comes to the analyses in this study, both pairsof siblings are internally identical and our results can thusnot explain the substantial clinical variability. The observeddifference in clinical severity between the siblings indicatesthat genetic factors other than those analysed in this studyand/or environmental factors can also influence the clinicalexpression of SMA.

In this population-based study of SMA, we have con-firmed the relationship between the number of SMN2 copiesand disease severity found in previous hospital-based stud-ies (3,7,9,28). The outlier among the SMA type 2 patientswho had four copies of SMN2 differed from the other SMAtype 2 patients in that he was considerably stronger in hisupper body. In this aspect, he resembled the type 3 pheno-type more. Another interesting outlier was the asymptomaticmother with five copies of SMN2 that seemed to protect heragainst disease.

We did not find any direct relationship between dis-ease severity and the involvement of BIRC1, GTF2H2 orSERF1A. The discussion of whether BIRC1 (NAIP) has animpact on disease severity has never been concluded. Thereis probably a mechanistic relationship between a disease al-lele with only one copy of SMN2 and a missing BIRC1. Dele-tions instead of conversions and unequal crossing-overs aremore likely to both involve BIRC1 and result in a disease al-lele with only one SMN2. The next, analysed, gene upstreamof SMN1 – GTF2H2 may also be co-deleted in SMA alleles.There is a large variability in the number of GTF2H2 copiesan individual has, both in affected and normal populations.The SERF1A genes, central in the repeat, are also likely tobe co-deleted with both SMN1 and SMN2. There is a clearcorrelation between the number of copies of the SERF1Agene (N-Cadherin-like gene probe) and a deletion of either

SMN1 or SMN2 as has also been described previously (29).Among the analysed carriers, 47% had three or fewer allelesof SERF1A; 63% of the children had three or fewer alle-les of SERF1A. Ninety-nine unrelated controls who wereneither SMA patients nor SMA carriers were analysed, re-vealing that 15 individuals had 3 or less alleles of SERF1A(all had 2 copies of SMN1). Of these 15 individuals, onlyone had two copies of SMN2, four had one copy of SMN2and 10 had homozygous deletion of SMN2. This can bean aid in the analysis of MLPA results since the results ofthe N-Cadherin-like gene probe analysis can be used as anindicator of the total number of SMN1 and SMN2 copies(estimated sensitivity for deletion 33% and specificity 98%).

Only one of 71 parents (1.4%) could not be confirmed asa carrier by MLPA analysis. This is in contrast to the ap-proximately 7–10% of the population that has one or twochromosomes 5 with two copies of SMN1 and in whom wewould expect to fail to confirm carrier status (6,30). This dis-crepancy can be due to population differences or to the rel-atively low number of individuals analysed in this study. Inour analysed control material of 99 non-patient, non-carrierindividuals, eight had three or more copies of SMN1, in-dicating that there is no large population difference. Thereis also a relatively high rate of rearrangements in the SMAregion and an estimated 2% of the SMA cases are due tode novo mutations (31). One way of determining which al-ternative was the case in the father with two copies of SMN1would have been to analyse his parents — but they declinedto participate (false paternity was ruled out by linkage analy-sis). No effort was made to analyze this father’s chromosome5s separately.

The high number of different disease alleles identified andthe high proportion of unique disease alleles also supportsthe theory of high rearrangement rates. No specific diseaseallele represented more than 15% of the total number. Itis therefore unlikely that there is any large founder effectinvolved in SMA in western Sweden.

CONCLUSIONWe conclude that the childhood incidence of SMA in theSwedish population is likely to be around 1 in 12 000 livebirths. While there is a direct relationship between the num-ber of SMN2 copies and disease severity, this is not true forBIRC1, GTF2H2 or SERF1A. The different clinical expres-sions in siblings with similar genotypes indicate the presenceof other genetic and/or environmental factors. Identifyingsuch factors is important to improve our understanding ofthe pathogenesis of SMA.

ACKNOWLEDGEMENTSWe are very grateful to the families for participating in thisstudy. This work was partially supported by grants fromStiftelsen Samariten, Wilhelm and Martina Lundgren’s sci-ence foundation and the Health and Medical Care ExecutiveBoard of the Vastra Gotaland Region.



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SUPPORTING INFORMATIONAdditional Supporting Information may be found in the on-line version of this article:

Figure S1 Children with SMA type 1 (all have 2 copies ofSMN2). Graphs showing age at onset and life span ver-sus the number of copies of BIRC1, GTF2H2 and SERF1A.Patients represented by bold circles were treated withCPAPFigure S2 Age at onset and SMA scale versus the number ofcopies of BIRC1, GTF2H2 and SERF1A. Grey circles haveSMA type 2, clear circles have SMA type 3. All patients



shown, except the dark grey, have three copies of SMN2.The dark grey circle represents a patient with SMA type 2and four copies of SMN2Figure S3 Graphs A, B and C illustrated the ambulatoryability of children with SMA types 2 and 3 versus the numberof copies of BIRC1, GTF2H2 and SERF1A. Graphs D, Eand F show the age at onset of children with SMA type 3only versus the number of copies of BIRC1, GTF2H2 and

SERF1A. Dark grey circles have three copies of SMN2, lightgrey circles have four copies of SMN2

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials sup-plied by the authors. Any queries (other than missing mate-rial) should be directed to the corresponding author for thearticle.


a population-based study of genotypic and phenotypic variability in children with spinal muscular...

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