Anna Chiara Nascimbeni

Dr Marina Fanin
email: marina.fanin@unipd.it

Elisabetta Tasca

Corrado Angelini

Transcriptional and translational effects of intronic CAPN3 gene mutations

Keywords: CAPN3, LGMD2A, calpainopathy, splicing, intronic variants, pathogenetic mutations CAPN3, LGMD2A, calpainopathy, intronic variants, pathogenetic mutations, splicing

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INTRODUCTION

Autosomal recessive limb girdle muscular dystrophies (LGMD) are a group of disorders characterized by LGMD2A [MIM#253600] is the most prevalent form of LGMD in many countries, and is caused by mutations in the CAPN3 gene [MIM#114240] encoding calpain-3 protein. About 350 different CAPN3 gene mutations have so far been reported on the Leiden Muscular Dystrophy Database, most of which are private and distributed throughout the gene. Approximately 70% of mutant alleles are of missense type; the remainder are null mutations (deletion/insertion causing frame shifting, nonsense, and splice site mutations), large genomic rearrangements, and synonymous or intronic changes causing aberrant splicing.

While the truncating mutations are considered to be causative, the pathological significance of nucleotide changes localized in intronic regions is very difficult to predict, which makes distinguishing between benign and pathogenetic variants a challenge and compromises a conclusive genetic counselling. Indeed, many intronic changes remain of uncertain clinical significance (UVs), thus partially explaining the observation that, although a severe calpain-3 protein defect in muscle usually corresponds to primary LGMD2A, about 20% of biochemical defects remain without any molecular proof [Fanin et al., 2008]. Many CAPN3 intronic variants have been identified during diagnostic screening: they account for about 15% of the total variants listed at the Leiden Database (28% in splice sites, 72% in deep intronic position), and for about 25% of the mutations reported in other studies [Blazquez et al., 2008;[START_REF] Krahn | Screening of the CAPN3 gene in patients with possible LGMD2A[END_REF]. However, it is conceivable that the true frequency of intronic mutations has largely been underestimated because deep intronic sequences are not conventionally sequenced and, most aberrant transcripts are expressed at low abundance and are usually prone to degradation by nonsensemediated mRNA decay (NMD) mechanism [START_REF] Maquat | Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics[END_REF]. For the majority of intronic variants the consequences on mRNA splicing have been only inferred by in-silico analysis [START_REF] Duno | cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark[END_REF], whereas experimental demonstration of their pathogenicity has been obtained by mRNA studies for only 1% of them [Leiden Database;[START_REF] Haffner | A small in-frame deletion with in the protease domain of musclespecific calpain-3, p94 causes early-onset lib-girdle muscular dystrophy 2A[END_REF][START_REF] Krahn | Transcriptional explorations of CAPN3 identify novel splicing mutations, a largesized genomic deletion and evidence for messenger RNA decay[END_REF]Blazquez et al., 2008]. In this study we have exploited the availability of diagnostic muscle biopsies to demonstrate previously unrecognized pathogenetic effects, at both transcriptional and translational levels, of 5 different intronic mutations in the CAPN3 gene.

MATERIALS AND METHODS

Selection of patients and muscle biopsies

From a population of over 100 LGMD2A patients who had received a biochemical and genetic characterization in our Centre, 16 cases were selected because of the presence of one intronic variant in the CAPN3 gene with an unknown pathogenetic effect. In all cases except one, the second mutant allele has been identified. Patients underwent diagnostic open muscle biopsies, after written consent had been obtained. As normal controls, we used genomic DNA, cDNA from muscle tissue and calpain-3 protein from subjects who had resulted free of any neuromuscular disorders.

Protein analysis by western blotting

Semi-quantitative analysis of calpain-3 protein in muscle was conducted as reported [Fanin et al. 2008]. The quantity of immunoreactive bands at 94 kDa (full-length protein) was determined by densitometry and expressed as a percentage of control.

In-silico analysis of unclassified intronic variants (UVs)

Prediction of the potential effect of intronic variants was determined by the following splice-site prediction programs (SSPPs): Human Splicing Finder (HSF V2.4 at http://www.umd.be/HSF) [START_REF] Desmet | Human Splicing Finder: an online bioinformatics tool to predict splicing signals[END_REF], Splice Site prediction by Neural Network (NNSPLICE V0.9 at www.fruitfly.org/seq_tools/splice.html), Splice View (http://bioinfo.itb.cnr.it/oriel/splice-view.html) and NetGene2 (at www.cbs.dtu.dk/services/NetGene2). Score variation in canonical splice-site use and potential activation of cryptic splice-sites in the presence of mutations were evaluated by analysis of exon and intron sequences localized in proximity to mutations using default settings (Supp. Table S1).

RNA analysis

Total RNA was isolated from muscle biopsies using the SV Total RNA Isolation System kit (Promega, Madison, WI) including treatment with DNAsel, and reverse transcribed to cDNA with Superscript III reverse transcriptase (Invitrogen, San Diego, CA) and random hexamers. In one patient the total RNA was isolated also from blood using the PAXGene blood RNA System (PreAnalytiX, Qiagen, Hilden, Germany). cDNA was amplified with specific calpain-3 primers designed on the published human calpain-3 mRNA sequence (GenBank accession number: NM_000070). PCR products were analyzed by gel electrophoresis: aberrant bands were manually excised and processed using the QIAquick Gel Extraction kit (Qiagen) for sequencing. Furthermore, we designed splicing-specific and skipping-specific primers (sequence available on request) to selectively amplify abnormal transcripts predicted by bioinformatic tools, even when this was not detectable on RT-PCR. Each splicing-specific PCR was tested on both patients and normal controls and directly sequenced. Details of in-silico predictions of splicing are reported in Supp. Table S1.

RESULTS

We investigated the pathogenetic effect in muscle of 5 different intronic variants (one novel) in the CAPN3 gene, including 3 nucleotide substitutions in the canonical donor or acceptor splice sites and 2 more deep intronic variations, and demonstrated that they all caused abnormal mRNA splicing and abolished or impaired protein synthesis (Table 1, Table 2, Supp. Table S1). These intronic mutations accounted for 9% of the total mutant alleles and 7% of all the mutations found in our population of over 100 LGMD2A patients.

Variation c.1030-1G>A (intron 7)

This was the first time this mutation had been identified in an LGMD2A patient. The patient was a compound heterozygote for a null mutant allele (c.550delA). This mutation abolishes the canonical acceptor splice-site, but the expected skipping of exon 8 was not detected. From SSPPs analysis, this intronic variant was expected to create a novel acceptor splice site (A1) with consequent deletion of the first nucleotide of exon 8 (r.1030delG). cDNA sequencing confirmed this deletion, which resulted in the creation of a downstream premature stop codon (p.V344SfsX8). Calpain-3 protein in muscle was absent, demonstrating the deleterious effect of this mutation at translational level.

Variation c.1524+1G>C (intron 11)

This mutation was identified in one LGMD2A patient in our series, who was a compound heterozygote for a missense mutant allele (p.M252T) and showed severely reduced calpain-3 protein level in muscle (5% of control), revealing its deleterious effects at translational level. This mutation abolishes the canonical donor splice-site, but a transcript carrying the skipping of exon 11 was not detected, even when using skipping-specific primers. Following SSPPs analysis, this variant was expected to produce two different aberrant transcripts (Figure 1): one originated by the use of a cryptic donor site (D1), leading to a deletion of the last 99 basepairs in exon 11 (r.1425_1524del), and another (D2) resulting from the exonization (intron retention) of 212 basepairs in intron 11 (r.1524_1525ins1524+1_1524+31;1524+1g>c; p.E508_V509ins 508+1_508+70). D1 transcript was characterized by cDNA amplification, which showed a shortened product corresponding to the expected deletion, that was identified by sequencing (p.V476_E508del). A further confirmation was obtained by PCR with D1 splicingspecific primers. While this transcript was sufficiently expressed and stable enough to be easily detected by cDNA analysis in muscle, the D2 transcript was detected only by splicing-specific PCR.

Variation c.1992+1G>T (intron 17)

This mutation was identified in 3 unrelated LGMD2A patients in our series; its deleterious effect at translational level was demonstrated by the observation that it was associated with absent or virtually absent protein when found in a compound heterozygote state with different mutant alleles. The mutation abolishes the canonical donor splice-site and we demonstrated that it caused the skipping of the exon 17 (r.1915_1992del; p.P639_D664del) in muscle. This result was confirmed also in blood mRNA from one patient. The resulting transcript (WTD) is probably unstable and poorly expressed, since it was detectable only with skipping-specific primers. At least 3 cryptic splicing sites in the region surrounding this mutation obtained a high score from SSPPs analysis, but only one of them was shown to be effectively activated (D1) in muscle and blood cDNA by splicing-specific primers. This cryptic splice site generated an aberrant transcript carrying the insertion of 31 basepairs in intron 17 (r.1992_1993ins1992+1_1992+31;1992+6g>u; p.D664_D665ins 664+1_664+10) (Figure 2). This mutation was identified in 6 unrelated LGMD2A patients in our series, all from the same administrative district of the Veneto Region (a possible founder effect followed by genetic isolation might have occurred). Among the 6 patients with this mutation, one was the object of a family study (Fig. 3): we conducted both a segregation analysis of the mutant allele and a study of the effect of this mutation at calpain-3 protein level in 2 different family members (a muscle biopsy was obtained from both an affected girl and her heterozygote father, who reported hyperCKemia before the diagnosis was obtained in his daughter).

F o r P e e r R e v i e w

The deleterious effect of this mutation at translational level was demonstrated by the observation that: 1) when it was associated with a null mutant allele, it produced absent or very reduced amounts of protein. This variant was not predicted to cause the loss of the canonical donor splice site, but the score for its use was reduced by all the SSPPs algorithms used. This means that the generation of a correctly spliced transcript would still be possible invivo, but our protein data suggest that, if this is the case, this might only take place to a very limited extent; 2) when it was expressed in heterozygote state, it produced one half of the amount of protein; 3) when associated with a missense mutant allele, which, as we had previously reported, caused the loss of functional autolytic activity without any quantitative defect, it produced normal protein quantities. Furthermore, following SSPPs analysis, this variation was expected to affect correct splicing by use of an alternative cryptic donor splice site (D1), resulting in the insertion of 31 basepairs at the beginning of intron 9 (r.1193_1194ins1193+1_1193+31; 1193+6u>a; p.M399X). The D1 transcript was sufficiently expressed to be detected by RT-PCR analysis: it produced an extraband which was detectable after gel electrophoresis and which was excised and sequenced (Figure 3). No previous functional characterization of this mutation has been provided. -1) who shows the WT product and one additional low-abundance product corresponding to the alternately spliced mRNA (D1). Family pedigree (case n. 7652) and western blot show that this intronic mutation (filled symbol) produced a reduction of calpain-3 protein of about 50% of control (C) after myosin normalization, as assessed in the muscle biopsy from both the heterozygous father (I-1) and his affected daughter (II-1), who was a compound heterozygote for a second missense mutation (p.E435K, dashed symbol). This mutation was identified in 6 unrelated LGMD2A patients in our series, and also in several previous reports, where its pathogenetic effects have been suggested [START_REF] Piluso | Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes[END_REF][START_REF] Hermanova | Analysis of histopathologic and molecular pathological findings in Czech LGMD2A patients[END_REF]Leiden Database], but unsupported by experimental data [Stehlikova et al., 2007;[START_REF] Krahn | Transcriptional explorations of CAPN3 identify novel splicing mutations, a largesized genomic deletion and evidence for messenger RNA decay[END_REF], or even reported as a polymorphism [START_REF] Groen | Analysis of the UK diagnostic strategy for limb girdle muscular dystrophy[END_REF]Leiden Database].

Conversely, in our study, the deleterious effect of this mutation has definitely been demonstrated at both translational (when this mutation was associated with another mutant allele, it always resulted in severely reduced protein quantity; Figure 4) and transcriptional levels. Indeed, this mutation had been predicted by the HSF algorithm to create a new acceptor splice site (A1), which was detected in muscle mRNA by splicing-specific PCR. Furthermore, numerous cryptic splicing sites in the region surrounding this mutation obtained a high score by SSPPs analysis and were expected to cause the insertion of different portions of intron 13. Three such transcripts (A2, A3, A4) were detected in our patients by cDNA analysis. However, even though none of the algorithms used predicted the loss of the canonical acceptor splice site (and only a slight decrease in its score was obtained for two of them), we identified an aberrant transcript (WTA) carrying the insertion of the entire intron 13. All these transcripts were variably expressed and detected in the patients, but while 4 aberrant transcripts (A1, A2, A3, A4) were detected only in mutant patients, suggesting their pathogenetic effect, the variant WTA was also expressed in 10 normal controls, indicating its non-pathogenicity. We attribute the occurrence of this latter transcript to the result of an alternative splicing event that takes place in normal tissues, as previously reported [START_REF] Kawabata | Newly identified exons encoding novel variants of p94/calpain-3 are expressed ubiquitously and overlap the alpha-glucosidase C gene[END_REF][START_REF] De Tullio | Characterization of a new p94-like calpain form in human lymphocytes[END_REF]. Family pedigree (case n. 6211) and western blot showing that this intronic mutation (filled symbol) produced a deleterious effect at protein level, as demonstrated by the complete loss of calpain-3 protein in the muscle from one affected patient (II-1) who was a compound heterozygote for a second missense mutation that has a deleterious effect as well (p.L204V, dashed symbol).

DISCUSSION

Despite the frequent identification of intronic changes in the CAPN3 gene, the demonstration of their pathogenetic effect has only occasionally been sought or achieved, both due to the laborious investigations involved and, also, because mRNA/protein studies in LGMD2A are often limited by the unavailability of a muscle biopsy. Although assessment of the pathogenetic effects of such variants is challenging, the effort required is justified because successful results are crucial to offer definitive diagnoses and conclusive genetic counseling, when establishing genotype-phenotype correlations and in view of future therapies.

Using in-silico predictions combined with RT-PCR analyses on muscle mRNA, we demonstrated the pathological significance of 5 UVs, showing their effects on splicing and protein translation.

We showed that CAPN3 mRNA degradation by NMD is not a drawback in muscle RT-PCR analyses, provided that these are combined with SSPPs analysis and the use of splicing-specific PCR tests. SSPPs are valuable tools to select variants that are predicted to impair correct RNA splicing, for address the subsequent variant-specific analyses at RNA level. However, they may provide contradictory or negative results, requiring the use of multiple softwares to resolve the discrepancies. Among the 4 softwares we used in this study, the most reliable and informative was the HSF, which contains also matrices for the prediction of effects on putative enhancer/silencer sequences.

Previous studies have reported different results for 3 of the 5 variants we analyzed (c.1992+1G>T, c.1524+1G>C and c.1746-20C>G). In particular, one study on blood mRNA identified only one aberrant transcript (D1) for the mutation c.1992+1G>T and failed to detect any mRNA for the mutation c.1524+1G>C [Blazquez et al., 2008]. We suggest that this discrepancy could be due to the different approach used. Indeed, using splicingspecific PCR, combined with in-silico predictions, we demonstrated that all the aberrant transcripts for the c.1992+1G>T mutation identified in muscle were expressed also in blood. Another possible explanation may be the use of a tissue other than muscle. Earlier studies reported the unreliability of RNA splicing analysis from blood samples, both because of its different splicing pattern and of the possibility that degenerated or illegitimate splicing had occurred associated with diverse storage conditions [START_REF] Wimmer | Illegitimate splicing of the NF1 gene in healthy individuals mimics mutations-induced splicing alterations in NF1 patients[END_REF]. These observations highlight how critical the choice of the tissue to be studied is. Furthermore, because of the lack of a molecular proof, some intronic variants have been reported either as polymorphisms or as "possibly pathogenetic", thus generating confusion and compromising a conclusive genetic counselling. This is the case of the variation c.1746-20C>G in intron 13 [START_REF] Hermanova | Analysis of histopathologic and molecular pathological findings in Czech LGMD2A patients[END_REF]Stehlikova et al., 2007;[START_REF] Krahn | Transcriptional explorations of CAPN3 identify novel splicing mutations, a largesized genomic deletion and evidence for messenger RNA decay[END_REF][START_REF] Groen | Analysis of the UK diagnostic strategy for limb girdle muscular dystrophy[END_REF], for which we have provided a definite demonstration of the pathogenetic effect.

In our study, the 3 mutations localized at conserved canonical donor/acceptor splice sites showed clearly their pathogenicity at both RNA and protein level, causing the loss of splice site and resulting in the absence of protein.

For the 2 mutations localized in less conserved regions nearby the exon/intron boundaries, SSPPs did not predict the loss of canonical splice sites, but only provided a decreased score, indicating a higher probability of use of nearby cryptic splicing sites, which indeed we experimentally demonstrated. Patients carrying these 2 latter mutations and heterozygous for a different frame-shifting mutation showed either absent or severely reduced calpain-3 protein, indicating that if a correct splicing were still possible, it might occur to a very limited extent.

The majority of the aberrant transcripts characterized in this study both contained a frame-shifting and either were expressed at very low abundance or were detectable only by splicing-specific-PCR. This result agrees with trace amounts of the corresponding transcript identified in LGMD2A patients who were compound heterozygous for 2 null mutations [Stehlikova et al., 2007]. Conversely, the transcripts originating from the c.1524+1G>C and c.1992+1G>T variants contained in-frame insertions/deletions, which were however expressed at low abundance, possibly because alternative splicing may limit the export of mRNA, making it a target for degradation. These transcripts could potentially have generated longer/truncated protein products, which were however not detected.

One conclusion from our study is that although SSPPs analysis helps in addressing the potential effects of intronic variations, experimental demonstration of this is not always easy to do by mRNA analysis (low abundance of transcripts, NMD mechanism) and might prove unsuccessful unless the individual transcripts are identified by both splicing-specific-PCR tests and sequencing. Furthermore, SSPPs analysis proved to be very valuable and reliable in predicting aberrant splicing for variants localized in the intronic 5' and 3' splice-site region, whereas it was neither conclusive nor always successful in the prediction of the consequences of such loss. For this purpose, however, it was useful to check the regions surrounding the nucleotide variant for the presence of potential cryptic splicing sites and for the loss/gain of splicing regulatory elements [START_REF] Faustino | Pre-mRNA splicing and human disease[END_REF][START_REF] Shapiro | RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression[END_REF].

We verified that a common effect of intronic variations in the CAPN3 gene, occurring in all 5 mutations studied, is the aberrant splicing caused by the activation of a series of cryptic splicing sites near the mutant nucleotide, and the creation/disruption of potential silencer/enhancer motifs. Mutation analysis by SSPPs should therefore involve the sequence context, in order to obtain more informative predictions. Conversely, the occurrence of exon skipping was observed only in one case in our series, suggesting either that this a less frequent mechanism used for the variants analysed (possibly because of the presence of a nearby cryptic splice site) or that transcripts carrying exon-skipping are more unstable and prone to degradation by NMD.

Although experimental demonstration of the pathogenetic effects of intronic mutations is difficult and laborious, we found that it is often successful when this effort is based on a preliminary SSPPs analysis. This aim should be pursued more frequently because of its important consequences for clinical and genetic counselling, for establishing genotype-phenotype correlations and providing novel insights into the complex mechanism of splicing.

AKNOWLEDGMENTS

We are very grateful to patients for their generous cooperation. This work was supported by the "Association Francaise contre le Myopathies", the Italian Telethon and the EuroBioBank Network.

  proximal limb girdle muscles, including at least 14 different genetic entities.

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  Pathogenetic effect of intronic CAPN3 gene mutations > 3

Figure 1 .

 1 Figure 1. Splicing analysis of the c.1524+1 G>C variant. Panel A. Schematic representation of the splicing patterns generated by this variant: D1, carrying the deletion of the last 99bp of exon 11 (gray box), and D2, with the retention of 212bp of intron 11 (dashed box), resulting from the use of the alternative donor splice sites D1 and D2, respectively. The arrows indicate the localization of the primers used. Panel B. Sequences from PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the co-amplification of the wild-type (WT) and the alternately spliced mRNA (D1), and from splicing-specific PCR amplification, which selectively amplifies the allele carrying the mutation (D2) in the same patient. Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (Pt.) who shows the WT product and two additional low-abundant products corresponding to the alternately spliced mRNA (D1 and D2). Calpain-3 western blot shows that this mutation caused a severe reduction of protein (Pt.) corresponding to about 5% of control (C) after myosin normalization.

Figure 2 .

 2 Figure 2. Splicing analysis of the c.1992+1 G>T variant. Panel A. Schematic representation of the two splicing patterns generated by this variant: WTD, carrying the skipping of exon 17, and D1, with the retention of 31bp of intron 17 (dashed box), resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequences from splicing-specific PCR amplification of muscle cDNA from a heterozygous mutant patient, showing the two aberrant transcripts (WTD and D1) generated by this variant. Panel C. Splicing-specific PCR amplification showing the selective amplification of the aberrant transcripts generated by this variant (WTD and D1) only in the heterozygous mutant patient (Pt.). Calpain-3 western blot in a patient (Pt.) shows that this mutation produced absent protein.

Figure 3 .

 3 Figure 3. Splicing analysis and translational effect of the c. 1193+6 T>A variant. Panel A. Schematic representation of the aberrant splicing product (D1) generated by this variant, carrying the insertion of 31bp of intron 9 (dashed box) and resulting from the use of the alternative donor splice site D1. The arrows indicate the localization of the primers used. Panel B. Sequence from PCR amplification of muscle cDNA from an heterozygous mutant patient, showing the co-amplification of the WT and the alternately spliced mRNA (D1). Panel C. RT-PCR analysis of normal control (C) and a heterozygous mutant patient (II-1) who shows the WT product and one additional low-abundance product corresponding to the alternately spliced mRNA (D1).Family pedigree (case n. 7652) and western blot show that this intronic mutation (filled symbol) produced a reduction of calpain-3 protein of about 50% of control (C) after myosin normalization, as assessed in the muscle biopsy from both the heterozygous father (I-1) and his affected daughter (II-1), who was a compound heterozygote for a second missense mutation (p.E435K, dashed symbol).

Figure 4 .

 4 Figure 4. Splicing analysis and translational effect of the c. 1746-20 C>G variant. Panel A. Schematic representation of the 5 splicing patterns generated by this variant, resulting from the use of the cryptic acceptor splice sites A1, A2, A3, A4, carrying the retention of 19, 86, 122 and 305 bp of intron 13 (dashed boxes), respectively, and WTA13, with the retention of the entire intron 13 (dashed box). The arrows indicate the localization of the primers used. Panel B. Splicing-specific PCR (designed to potentially amplify all the 5 aberrant transcripts) showing that these transcripts are variably expressed and not always detectable in the heterozygous mutant patients (Pt.1, II-1, and Pt.3). The WTA transcript is expressed also in normal control (C). Panel C.Family pedigree (case n. 6211) and western blot showing that this intronic mutation (filled symbol) produced a deleterious effect at protein level, as demonstrated by the complete loss of calpain-3 protein in the muscle from one affected patient (II-1) who was a compound heterozygote for a second missense mutation that has a deleterious effect as well (p.L204V, dashed symbol).

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  Pathogenetic effect of intronic CAPN3 gene mutations > 9

4 <Nascimbeni AC et al.> Table 1. CONSEQUENCES OF INTRONIC MUTATIONS AT RNA LEVEL

	Intronic sequence variant *	Alternative acceptor/donor site, position	Effect at RNA level	Theoretical effect at protein level *	Methods	Interpretation
	c.1030-1G>A	A1/ c.1030-1	r.1030delG	deletion of the first bp exon 8	p.V344SfsX8	cDNA amplification, sequencing	pathogenetic
	c.1524+1G>C	D1/c.1426 D2/c.1524+213	r.1425_1524del r.1524_1525ins1524+1_1 524+31;1524+1g>c	deletion of 99 bp exon 11 exonization of 212 bp intron 11	p.V476_E508del p.E508_V509ins 508+1_508+70	cDNA amplification, gel extraction of aberrant band, sequencing Splicing specific-PCR, sequencing	pathogenetic pathogenetic
	c.1992+1G>T	WT D/c.1992+1 D1/c.1992+32	r.1915_1992del r.1992_1993ins1992+1_1 992+31;1992+6g>u	skipping of exon 17 exonization of 31 bp intron 17	p.P639_D664del p.D664_D665ins 664+1_664+10	Splicing specific-PCR, sequencing Splicing specific-PCR, sequencing	pathogenetic pathogenetic
	c.1193+6T>A	D1/c.1193+32	r.1193_1194ins1193+1_1 193+31;1193+6u>a	exonization of 31 bp intron 9	p.M399X	cDNA amplification, gel extraction of aberrant band, sequencing	pathogenetic
		A1/ c.1746-21	r.1745_1746ins1746-19_1746-1;1746-20c>g	exonization of 19 bp intron 13	p.E582_E583ins 582+1_582+6fsX9	Splicing specific-PCR, gel extraction of aberrant band, sequencing	pathogenetic
		A2/ c.1746-88	r.1745_1746ins1746-86_1746-1;1746-20c>g	exonization of 86 bp intron 13	p.E582_E583ins582+ 1_582+28fsX41	Splicing specific-PCR, gel extraction of aberrant band, sequencing	pathogenetic
	c.1746-20C>G	A3/ c.1746-124	r.1745_1746ins1746-122_1746-1;1746-20c>g	exonization of 122 bp intron 13	p.E582_E583ins 582+1_582+30fsX53	Splicing specific-PCR, gel extraction of aberrant band, sequencing	pathogenetic
		A4/ c.1746-307	r.1745_1746ins1746-305_1746-1;1746-20c>g	exonization of 305 bp intron 13	p.E583X	Splicing specific-PCR, sequencing	pathogenetic
		WT A13/c.1537-2	r.1745_1746ins1745+1_1 746-1;1746-20c>g	exonization of entire intron 13	p.E583X	Splicing specific-PCR, gel extraction of aberrant band, sequencing	non pathogenetic
	WT: Wild type; D: donor site; D1, D2, etc: alternative cryptic donor sites; A: acceptor site; A1, A2, etc: alternative cryptic acceptor sites; exonization: intron retention; *: Human
	Genetic Variation Society (HGVS) approved guidelines (www.hgvs.org/mutnomen). GeneBank accession number AF209502.1.

Table 2 . CONSEQUENCES OF INTRONIC MUTATIONS AT PROTEIN LEVEL

 2

	Intronic sequence variant *	Case N.	Second mutant allele	Protein amount (% of control)
	c.1030-1G>A	7161	c.550 delA, p.T184RfsX36	0
	c.1524+1G>C	7555	c.755T>C, p.M252T	5
		1324	c.1193+6T>A	0
	c.1992+1G>T	1754	c.1061T>G, p.V354G	0
		5393	c.1343G>A, p.R448H	0
		1324	c.1992+1G>T	0
		3393	c.1468C>T, p.R490W	100 §
	c.1193+6T>A	5427	not identified	20
		6804	c.309+4469_1116-1204del, p.E104MfsX11	20
		7652	c.1303G>A, p.E435K	50
		8242	c. 1469 G>A, p.R490Q	100 §
		1338	c.1333G>A, p.G445R	0
		2522	c.245C>T, p.P82L	20
	c.1746-20C>G	4622	c.1061T>G, p.V354G	10
		6211	c.610C>G, p.L204V	0
		6385	c.550 delA, p.T184RfsX36	5
		7894	c.697G>C, p.G233R	10

*: Human Genetic Variation Society (HGVS) approved guidelines (www.hgvs.org/mutnomen). GeneBank accession number AF209502.1. §: protein with loss of function (autolytic activity)

John Wiley & Sons, Inc.Human Mutation

Contract grant sponsor: Association Francaise contre le Myopathies, Contract grant number: 2007.0889/12925 and 13859 to M.F. Contract grant sponsor: Telethon, Contract grant number: GTB07001 to C.A. Contract grant sponsor: EuroBioBank Network, Contract grant number: QLRT2001027769 to C.A.


MD Yvonne Hilhorst-Hofstee

Marry Eb Rijlaarsdam

Arthur Jha Scholte

Marietta Swart-Van Den Berg

Michel Im Versteegh

Iris Van Der Schoot-Van Velzen

Hans-Joachim Schäbitz

Emilia K Bijlsma

Marieke J Baars

Allgemeinmedizin Bijlsma

Wilhelmina S Kerstjens-Frederikse

Jacques C Giltay

Ben C Hamel

Martijn H Breuning

Gerard Pals

The clinical

Keywords: Marfan syndrome, fibrillin-1 (FBN1) gene, cbEGF domain, pathogenesis, autosomal recessive inheritance Marfan syndrome, fibrillin-1, FBN1 gene, autosomal recessive inheritance, pathogenesis

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ABSTRACT: Marfan syndrome (MFS) is a dominant disorder with a recognizable phenotype. In most patients with the classical phenotype mutations are found in the fibrillin-1 gene (FBN1) on chromosome 15q21. It is thought that most mutations act in a dominant negative way or through haploinsufficiency. In 9 index cases referred for MFS we detected heterozygous missense mutations in FBN1 predicted to substitute the first aspartic acid of different calcium-binding Epidermal Growth Factor-like (cbEGF) fibrillin-1 domains. A similar mutation was found in homozygous state in 3 cases in a large consanguineous family. Heterozygous carriers of this mutation had no major skeletal, cardiovascular or ophthalmological features of MFS. In the literature 14 other heterozygous missense mutations are described leading to the substitution of the first aspartic acid of a cbEGF domain and resulting in a Marfan phenotype. Our data show that the phenotypic effect of aspartic acid substitutions in the first position of a cbEGF domain can range from asymptomatic to a severe neonatal phenotype. The recessive nature with reduced expression of FBN1 in one of the families suggests a threshold model combined with a mild functional defect of this specific mutation. ©2010 Wiley-Liss, Inc.

INTRODUCTION

Human fibrillin-1 is a large protein of approximately 350 kD and member of a family of extracellular cysteinerich glycoproteins. Since 1991 mutations in the fibrillin-1 (FBN1) gene have been found to be responsible for Marfan syndrome (MFS; MIM# 134797) [START_REF] Kainulainen | Location on chromosome 15 of the gene defect causing Marfan syndrome[END_REF][START_REF] Dietz | Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene[END_REF]. Fibrillin-1 is characterized by a highly conserved modular domain organization. The most prominent domain is the Epidermal Growth Factorlike (EGF) domain present 46 times and containing six highly conserved cysteine residues stabilizing the structure by three disulfide bonds. Of these EGF domains, 43 have a consensus sequence for calcium binding (cb) in the Nterminal pocket of the domain which may mediate protein-protein interactions. The EGF domains are interrupted by seven transforming growth factor (TGF)-binding protein domains characterized by 8 cysteine residues involved in intra-domain disulfide bonds [START_REF] Pereira | Genomic organization of the sequence coding for fibrillin, the defective gene product in Marfan syndrome[END_REF][START_REF] Robinson | The molecular genetics of Marfan syndrome and related disorders[END_REF]. In the last update of the Universal Marfan Database -FBN1 (UMD-FBN; http://www.umd.be) [START_REF] Faivre | Effect of mutation type and location on clinical outcome in 1,013 probands with Marfan syndrome or related phenotypes and FBN1 mutations: an international study[END_REF] 803 different mutations are reported. Most of the mutations are missense mutations (56%) mainly substituting or creating a cysteine in a cbEGF domain. Other mutations are frameshift mutations, splice mutations and nonsense mutations. About 14% of mutations are recurring.

All cbEGF domains start with a highly conserved aspartic acid, which is crucial for binding of a positively charged Ca 2+ ion [START_REF] Whiteman | Cellular and molecular studies of Marfan syndrome mutations identify co-operative protein folding in the cbEGF12-13 region of fibrillin-1[END_REF]. We have identified 10 index cases with a substitution of the first aspartic acid substitution of a cbEGF domain and reviewed a further 14 published cases. Most of them exhibit a complete MFS phenotype. Surprisingly, in one family the substitution only led to MFS in homozygous state in three family members, whereas 13 family members carrying the heterozygous mutation do not have Marfan syndrome after thorough clinical examination.

There is still a lot of debate how mutations in FBN1 result in the MFS phenotype, but increasing evidence for different models is emerging. Possible explanations for the observed extreme variation in expression of the substitution of the first aspartic acid of a cbEGF domain are discussed. The observation of recessive inheritance of an expected dominant mutation also underscores the fact that mutations which are predicted to have a pathogenic effect, may not always lead to clinical symptoms.

PATIENTS AND METHODS

Patients

The patients were referred for DNA analysis of the fibrillin-1 gene to confirm the clinical diagnosis of MFS. Case 1, 2, 5, 7, 9 and 10 fulfilled the clinical Ghent criteria [START_REF] De Paepe | Revised diagnostic criteria for the Marfan syndrome[END_REF] for the diagnosis MFS. All index patients fulfilled the Ghent criteria when the finding of a pathogenic mutation in FBN1 was included.

Case 9 belongs to a large Turkish pedigree (Figure 1, III-1). She was examined at the age of 22 years. At the age of 6 weeks she was operated on a right sided hernia inguinalis. She was diagnosed with bilateral subluxation of the lens when she was 3 years old. From that time on she has been operated several times for retinal detachments and lens luxation. At the age of 14 years an aortic root replacement was performed for progressive aortic root dilatation and aortic valve regurgitation. A spontaneous pneumothorax occurred at the age of 16 years.

Clinical examination at the age of 18 years showed a marfanoid habitus, slight downslanting of palpebral fissures and a high and gothic palate. Despite long fingers, wrist and thumb signs were negative. She exhibited limited extension of her elbows, mild asymmetry of the chest, and bilateral flat feet. Her skin showed several striae on the chest, shoulders, hips and lower back. Her length was 179.5 cm (+3,7SD for Turkish descent) and an arm span of 175 cm (within normal limits). The brother of case 9 (III-3) was a 13 year old boy with Marfan syndrome. He had mild skeletal manifestations of Marfan (pes planus), a mildly dilated aortic root, ectopia lentis and dural ectasia with an anterior sacral meningocele. Furthermore he suffered from recurrent episodes of intracranial hypertension treated by drainage of cerebrospinal fluid. He has been described in a case report [START_REF] Hilhorst-Hofstee | Intracranial hypertension in 2 children with marfan syndrome[END_REF].

The third patient (II-15) died at the age of 22 years. His case history was obtained from the medical records. At the age of 2 years bilateral subluxation of lenses was diagnosed. He developed severe aortic and mitral valve regurgitation with an aneurysm of the aortic root. He had skeletal involvement and an anterior sacral meningocele. When he was 17 years of age an aortic root replacement was performed with reconstruction of the aortic and mitral valve. Due to progressive aortic regurgitation, a re-operation was performed a year later. He died at the age of 22 after a second episode of ventricular fibrillation.

All heterozygous family members had a thorough skeletal, cardiologic and ophthalmologic examination including anthropometric measurements, echocardiography and slit lamp evaluation (Table 1). Only the mother and father of case 9 had an MRI evaluation for dural ectasia. The obtained clinical data of all family members are summarized in Table 1. The father of case 9 (II-6) had no clinical signs of Marfan syndrome. The mother (II-7) was tall, with a height on +2.5 SD but with normal body proportions. She had no other skeletal, ocular or cardiovascular involvement, but had several striae on the lumbar region and around the knees. Furthermore she suffered from spontaneous pneumothorax at the age of 21 years. An MRI-scan showed a dural ectasia at S2 with otherwise a normal dural sac. The father and mother of II-15 did not exhibit any signs of Marfan syndrome. None of the nine other heterozygous family members had a major criterion in one of the organ systems. Some were found to have a non-specific or minor sign. Individual II-1 has an arm span to height ratio of 1.06 and a mild dilatation of the abdominal aorta. II-4 had recurrent inguinal hernias but this was during a period of performing heavy physical labor. II-8 had an arm span to height ratio of 1.07 and bilateral flat feet. II-9 had reduced extension of the elbows.

The clinical data of cases 1-10 and published cases are summarized in Table 2 together with the molecular data.

Figure 1. Pedigree of the family of case 9. Squares, male subjects; circles, female subjects. Affected subjects with a homozygous mutation (c.7454A>T) are represented by solid symbols. Presence or absence of the heterozygous mutation is represented by an open symbol with a black dot or a minus symbol respectively.

F o r P e e r R e v i e w

Mutations of First Aspartic Acid of cbEGF-like Domains in FBN1 5 F female; M male; involve involvement; np not performed; arr aortic root replacement; hom homozygous c.7454A>T FBN1 mutation; het heterozygous c.7454A>T FBN1 mutation; Z-score related to body surface area and age according to Roman et al. [START_REF] Roman | Two-dimensional echocardiographic aortic root dimensions in normal children and adults[END_REF]; 1) aortic root measurement at the age of 14 years just before aortic root replacement; 2) no exact measurement available; 3) recurrent inguinal hernias during a period of heavy duty; 4) major criterion on the basis of a first degree affected family member or the presence of a pathogenic FBN1 mutation.

) 3,7 1,6 -1,2 2,5 -0,2 -1,7 -0,6 -1,4 -1,1 0,1 1,1 2,5 0,

Molecular studies

DNA was extracted from peripheral blood or paraffin embedded tissue, using standard techniques, analyzed by DHPLC and direct DNA sequencing as described previously [START_REF] Matyas | Evaluation and application of denaturing HPLC for mutation detection in Marfan syndrome: Identification of 20 novel mutations and two novel polymorphisms in the FBN1 gene[END_REF].

The reference sequence used to describe the mutations is the FBN1 cDNA GenBank reference sequence: NM_000138.3. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen).

Skin biopsies of case 1, 9 and her mother and case 10 were available. Fibroblasts were cultured and mRNA was isolated from confluent monolayers. For each individual two RNA isolations were performed: one cell culture of each was incubated with cycloheximide (0.25 mg/ml) for 4.5 hours prior to RNA isolation, to prevent possible nonsense mediated decay (NMD) of aberrantly spliced mRNA.

RNA was isolated using the RNA isolation minikit (Qiagen) according to the manufacturers instructions. Full length single stranded cDNA was prepared with oligo-dT-primer and Superscript TM II RT reverse trancriptase (Invitrogen). To detect possible splice errors, the complete coding sequence of FBN1 was analyzed by direct sequencing in 24 overlapping PCR fragments. The primers for overlapping fragments were positioned in different exons, to avoid allele dropout in case of exon skipping. For analysis of the mutation in exon 60, primers in exon 55 (forward) and 63 (reverse) were used.

Primer sequences are given in Supp. Table S1.

RESULTS

Heterozygous mutations leading to a substitution of the first aspartic acid of a cbEGF domain were found in nine index cases and a homozygous mutation was found in 1 index case. These mutations and 14 comparable mutations described in the literature or in the UMD-FBN1 (UMD-FBN; http://www.umd.be) are listed in Table 2. The phenotypes were classical MFS in 15 cases, neonatal MFS in two cases, thoracic aortic aneurysm in one case and lens luxation with striae in one case. In four cases described in the literature the phenotype is not clear.

The mutation was de novo in cases 2, 4, 5, and 8. In case 1, 3 and 6 parental DNA was not available. In case 1 five sibs were tested negative for the mutation. In case 6 the parents and seven sibs had no clinical symptoms of MFS. Case 7 has an affected sister with the same mutation. Their father died as a consequence of his third aortic dissection when he was 52 years of age. He was thought to have MFS. The mother of case 10 is probably affected but was not molecularly tested. Of the published mutations three were de novo, three were familial and in eight inheritance was unknown (Table 2).

Using DHPLC for mutation scanning of all 65 exons of FBN1, initially no mutation was found in case 9. As the parents are consanguineous, a recessive mutation was suspected. Testing one of the parents, a heterozygous mutation was detected in exon 60: c.7454A>T, leading to the amino acid substitution p.Asp2485Val. This mutation was found in a homozygous state in the patients III-1, III-3 and II-15. The parents (II-6 and II-7) of patients III-1 and III-3 are first cousins. The parents of patient II-15 are distantly related and both are related to II-6 and II-7 (Figure 1). In the parents of patients III-1, III-3 and II-15 and 9 family members the mutation was heterozygous. The mutation was not detected in 1000 Caucasian and 60 Turkish control chromosomes.

Sequence analysis of cDNA, made from mRNA from cultured fibroblasts of case 1, case 9 and her mother, and case 10 showed no evidence of erroneous splicing of exon 60. Treatment with cycloheximide, to prevent possible nonsense mediated decay of erroneously spliced mRNA, gave the same results. Fibrobasts of the other patients were not available.

DISCUSSION

We identified a heterozygous substitution of the first aspartic acid of a cbEGF domain in FBN1 in nine index patients and a homozygous substitution in one index patient with MFS. Reviewing the literature we found 12 reports of substitution of the first aspartic acid, and a further two unpublished cases are quoted in the UMD database (UMD-FBN; http://www.umd.be) [START_REF] Collod-Beroud | Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphism database[END_REF] as summarized in Table 2. All 10 index patients found in our center fulfilled the Ghent criteria when the finding of a pathogenic mutation was included. Of the 14 published cases, eight were reported to have a classical Marfan phenotype, one was a neonatal Marfan and one had a thoracic aortic aneurysm. Of four cases the phenotype was not reported. In all cases the acidic amino acid aspartic acid is replaced by a nonpolar or noncharged polar amino acid, apart from one mutation where aspartic acid is replaced by another acidic amino acid (p.Asp2127Glu) [START_REF] Kainulainen | Mutations in the fibrillin gene responsible for dominant ectopia lentis and neonatal Marfan syndrome[END_REF]. The codons of the first aspartic acids in the cbEGF domains always contain the last base of one exon and the first two bases of the next. Consequently, mutations of these codons may affect splicing. Aberrant splicing was excluded in cases 1, 9, and 10 and in one of the published cases [START_REF] Milewicz | Fibrillin-1 (FBN1) mutations in patients with thoracic aortic aneurysms[END_REF]. The mutation c.4210G>T was shown to destroy a donor splice site with abnormal splicing as a consequence [START_REF] Hayward | Mutation screening of all 65 exons of the fibrillin-1 gene in 60 patients with Marfan syndrome: report of 12 novel mutations[END_REF]. Of the 19 cases in which no cDNA analysis was performed, prediction software predicted aberrant splicing in eight cases (Table 2). Exon skipping, as a result of these mutations, may have more severe effects than missense mutations, because the exons are all in frame and consequently, skipping will lead to a shorter protein that may exert a dominant negative effect [START_REF] Robinson | Mutations of FBN1 and genotype-phenotype correlations in Marfan syndrome and related fibrillinopathies[END_REF].

There are several reasons to argue that a substitution of the first aspartic acid of a cbEGF domain will lead to a MFS phenotype in the heterozygous state. Calcium binding of cbEGF domains is necessary for stabilization of the secondary structure, prevention of proteolytic degradation and for protein-protein interaction [START_REF] Dietz | Four novel FBN1 mutations: significance for mutant transcript level and EGF-like domain calcium binding in the pathogenesis of Marfan syndrome[END_REF][START_REF] Handford | Key residues involved in calciumbinding motifs in EGF-like domains[END_REF][START_REF] Cooke | The solution structure of human epidermal growth factor[END_REF]. The first aspartic acid of a cbEGF domain is highly conserved in evolution and in the human fibrillin-1 gene all cbEGF domains start with an aspartic acid, which is crucial for binding of a positively charged Ca 2+ ion (Figure 2) [START_REF] Whiteman | Cellular and molecular studies of Marfan syndrome mutations identify co-operative protein folding in the cbEGF12-13 region of fibrillin-1[END_REF]. Furthermore mutations of the first amino acid of a cbEGF domain of coagulation factor IX in haemophilia B have been proven to reduce calcium binding even if the aspartic acid is replaced by the acidic amino acid glutamic acid [START_REF] Handford | Key residues involved in calciumbinding motifs in EGF-like domains[END_REF][START_REF] Winship | Identification of haemophilia B patients with mutations in the two calcium binding domains of factor IX: importance of a beta-OH Asp 64----Asn change[END_REF]. The finding of 23 MFS or MFS-like cases with a heterozygous substitution of an aspartic acid in this position of the cbEGF domain underscores the crucial role of this amino acid. In this view the recessive nature of the mutation p.Asp2485Val in the family of case 9 came as a surprise. The family of case 9 (Figure 1) has been thoroughly investigated. Patients III-1 (case 9), III-3 and II-15 have the classical type of Marfan syndrome according to the Ghent criteria [START_REF] De Paepe | Revised diagnostic criteria for the Marfan syndrome[END_REF]. Based on the pedigree with three affected patients and healthy consanguineous parents recessive inheritance could be expected. This was confirmed by finding a homozygous missense mutation in all three affected patients. The four unaffected parents and nine other unaffected relatives were found to be carriers of the mutation. Unexpectedly in none of the investigated heterozygous carriers obvious signs of Marfan syndrome could be found. Only after thorough clinical examination one of them (II-7) was found to have a dural ectasia at S2, which as yet is considered a major symptom in the Ghent criteria. Together with the family history and some minor signs (pneumothorax, striae and reduced extension of the elbows) this classifies II-7 as having Marfan syndrome. However, compared to the homozygous cases, the cardinal Marfan features in the skeletal, cardiac and ophthalmological systems are absent. [START_REF] Downing | Solution structure of a pair of calciumbinding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders[END_REF]. (B) cbEGD like domain [START_REF] Handford | Key residues involved in calciumbinding motifs in EGF-like domains[END_REF]. The arrow points to the first aspartic acid residue. Solid lines are the disulphide bridges between cysteine residues.

To our knowledge very little is known about recessive FBN1 mutations. Only one family has been reported in which Marfan syndrome was found in two affected cousins homozygous for a FBN1 mutation while the four normal parents were heterozygous carriers, indicating recessive inheritance of the syndrome (De [START_REF] Vries | Homozygosity for a FBN1 missense mutation: clinical and molecular evidence for recessive Marfan syndrome[END_REF]. The mutation is located in exon 11 and leads to an amino-acid substitution creating a cysteine. Like us the authors expected this mutation to have a dominant effect in the heterozygous state. Two of the parents were sibs and exhibited minor signs of Marfan syndrome (increased arm span to height ratio and a highly arched palate). No other family members have been investigated. Only one other probably recessively inherited form of Marfan syndrome has been described but could not be proven by molecular analysis as the gene was not yet known [START_REF] Fried | Probable autosomal recessive Marfan syndrome[END_REF].

Three families have been described in the literature in which both parents are affected with more severely affected children. The first is an Italian couple of first cousins, both affected with Marfan syndrome, who had 4 affected children. Two of the four affected children showed more severe manifestations than other affected family members, presumably due to homozygosity [START_REF] Capotorti | Contribution to the study of the heredity of Marfan's syndrome: description of a family tree of 4 generations with marriage between consanguineous parents[END_REF]. In 1984, Chemke described a family with Marfan syndrome. Two sibs suffered from a severe phenotype reminiscent of neonatal Marfan syndrome. Their parents were cousins and had a much milder phenotype. Remarkable is that the probably homozygous sibs were the only patients in the family with ectopia lentis [START_REF] Chemke | Homozygosity for autosomal dominant Marfan syndrome[END_REF]. In 1988 a severely affected boy has been described [START_REF] Schollin | Probable homozygotic form of the Marfan syndrome in a newborn child[END_REF]. Both parents were affected and were found to carry a missense mutation in FBN1. Compound heterozygosity was identified in the severely affected child [START_REF] Karttunen | A compound-heterozygous Marfan patient: two defective fibrillin alleles result in a lethal phenotype[END_REF].

In the recessive family described here the heterozygous mutation does not exert an important effect on the phenotype. Only in the homozygous state the abnormal fibrillin causes the classical clinical phenotype of Marfan syndrome. This observation together with the few other described families with bi-allelic inheritance, may support both alternative pathogenetic models of Marfan syndrome. A dominant negative effect of FBN1 mutations has been the leading hypothesis for the pathogenesis of Marfan syndrome for a long time. However, several manifestations of Marfan syndrome like bone overgrowth, craniofacial features, lung disease, and muscle and fat hypoplasia could not be explained by a structurally abnormal protein. The observation that fibrillin interacts with a variety of proteins, including the latent TGFβ binding proteins (LTBP's) has lead to several investigations indicating that fibrillin-1 can interact with TGFβ signaling [START_REF] Annes | Making sense of latent TGFbeta activation[END_REF][START_REF] Azhar | Transforming growth factor beta in cardiovascular development and function[END_REF][START_REF] Isogai | Latent transforming growth factor beta-binding protein 1 interacts with fibrillin and is a microfibril-associated protein[END_REF][START_REF] Hubmacher | Fibrillins: from biogenesis of microfibrils to signaling functions[END_REF][START_REF] Kaartinen | Fibrillin controls TGF-beta activation[END_REF][START_REF] Loeys | Aneurysm syndromes caused by mutations in the TGF-beta receptor[END_REF][START_REF] Neptune | Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome[END_REF][START_REF] Ng | TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome[END_REF][START_REF] Dijke | Extracellular control of TGFbeta signalling in vascular development and disease[END_REF].

According to splice site prediction software (Alamut mutation interpretation software version 1.5; Interactive Biosoftware, Rouen, France) the c.7454A>T mutation, found in the family described here, is not predicted to cause erroneous splicing. This was confirmed by cDNA studies, showing no evidence of splice error. The position of the mutation (exon 60) may explain the lack of expression as mutations in the more C-terminal end of the gene are expected to give a milder phenotype [START_REF] Faivre | Effect of mutation type and location on clinical outcome in 1,013 probands with Marfan syndrome or related phenotypes and FBN1 mutations: an international study[END_REF][START_REF] Robinson | Mutations of FBN1 and genotype-phenotype correlations in Marfan syndrome and related fibrillinopathies[END_REF]. However the mutation in case 10 is even more terminal and still leads to a classical MFS phenotype.

Hutchinson et al. [START_REF] Hutchinson | Allelic variation in normal human FBN1 expression in a family with Marfan syndrome: a potential modifier of phenotype?[END_REF] hypothesized that variable expression of the normal FBN1 allele could moderate the phenotypic effect of the mutant allele. A compensatory higher level of FBN1 expression from the normal allele would explain a milder phenotype. As the normal alleles are inherited from 5 different parents in our recessive family, this mechanism is highly unlikely.

We hypothesize that the p.Asp2485Val mutation acts as a hypomorphic allele with a minimal dominant negative effect. Reduction of gene expression of both alleles could be the main determinant of the phenotype in homozygotes. The observation of only one major clinical sign in one of the heterozygotes (dural ectasia in II-7, Figure 1) and no major clinical signs in 12 other heterozygotes could be explained by sufficient gene expression with only a mild functional defect of the mutant allele product. This was also shown in a mouse model, however in this model the mutation had a severe dominant negative effect [START_REF] Pereira | Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome[END_REF]. In this model with a deletion of 272 amino acids in the central part of fibrillin-1, a tenfold reduction in expression of the mutant allele was shown in heterozygous mice, resulting in a normal phenotype. Homozygous mutant mice however died shortly after birth due to severe vascular complications. The other mouse model of Pereira was a targeted FBN1 mutation leading to 15% expression of a normal product with no abnormal phenotype in heterozygous mice, while mice homozygous for this mutation have severe abnormalities comparable with the neonatal MFS phenotype. These mouse models suggests that there is a threshold of expression of the normal allele below which the abnormal phenotype will develop [START_REF] Pereira | Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1[END_REF][START_REF] Dietz | Mouse models of genetic diseases resulting from mutations in elastic fiber proteins[END_REF].

To understand the exact pathogenetic mechanism expression studies and studies on protein synthesis, secretion and matrix incorporation of the Asp2486Val mutation are necessary For comprehensive studies of this type, a mouse model should be created.

The finding of a homozygous substitution of A>T, as described here, has implications for mutation screening in MFS. Homozygous substitutions will not have an effect on denaturation of double stranded DNA, because the basepair remains the same. Consequently, heteroduplex based testing, such as DHPLC, working with the principle of differential denaturation of double stranded heteroduplex DNA, cannot detect this mutation in homozygous state. Formerly, based on the presumed dominant inheritance mode, only heterozygous mutations were expected. Now it is clear that recessive inheritance is also possible and mutations may have been missed in similar cases, because heteroduplex based testing has been used until recently in many large diagnostic centers. Most laboratories nowadays use direct sequencing, which avoids this problem.

In conclusion we have shown that the first aspartic acid of a cbEGF domain in FBN1 is important for the function of fibrillin-1, but may not always lead to a clinical effect in the heterozygous state. This underscores that missense mutations must be interpreted with care.

  Mutations of First Aspartic Acid of cbEGF-like Domains in FBN1 7

Figure 2 .

 2 Figure 2. Class I cbEGF domain showing the position of the first Asp in relation to the calcium molecule. (A) 3D picture of a cbEGF domain of fibrillin-1. The yellow arrow points to the first Asp. Picture derived from the NCBI database (http://www.ncbi.nlm.nih.gov/)[START_REF] Downing | Solution structure of a pair of calciumbinding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders[END_REF]. (B) cbEGD like domain[START_REF] Handford | Key residues involved in calciumbinding motifs in EGF-like domains[END_REF]. The arrow points to the first aspartic acid residue. Solid lines are the disulphide bridges between cysteine residues.

  Mutations of First Aspartic Acid of cbEGF-like Domains in FBN1 11

Table 1 . Summary of the clinical features in the family of case 9

 1

		III-1
		Case	III-3	II-6	II-7	II-15	I-5	I-6	I-1	II-1	II-2	II-3	II-4	II-5	II-8	II-9	II-12
		9
	Age at examination (years)	22	10	42	43	9	57	56	61	44	41	37	35	33	55	48	37
	Sex	F	M	M	F	M	M	F	M	M	M	F	M	F	M	F	F
	height (cm)	180,9	150,5	167,1	174,4	135,0	164,0	157,5	166,0	167,5	174,5	167,0	188,5	161,0	181,0	164,7	161,0
	height SDS (for Turkish
	descent

Table 1 .

 1 Continued

	8	1,2	0,7	0,2

Family or genetic history major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major 4 major

	1st degree relative with Marfan syndrome yes pathogenic mutation in FBN1 no yes yes hom hom het het	hom	het	het	het	het	het	het	het	het	het	het	het

Table 2 . Published and observed missense mutations leading to the substitution of the first aspartic acid of a cbEGF domain

 2

	Nucleotide change	Amino acid change	Exon	cbEGF domain	Diagnosis	Phenotype	Reference	Aberant mRNA splicing	Predicted aberant splicing a mRNA	Inheritance
	c.1468G>T p.Asp490Tyr	11	#3	Classical MFS	ard, el, sk	(Hayward and Brock, 1997)	np	yes	Unknown
	c.2168A>C p.Asp723Ala	18	#7	Severe classical MFS	ard, mvp, el, myop, sk (Dietz et al., 1993)	np	no	De novo
	c.2168A>T p.Asp723Val	18	#7	Classical MFS	ard, mvp, el, sk, myop (Katzke et al., 2002)	np	no	De novo
	c.2728G>A p.Asp910Asn	22	#10 Classical MFS	unknown	UMD	np	yes	Unknown
	c.3209A>G p.Asp1070Gly	26	#12 Neonatal MFS	unknown	UMD	np	no	Unknown
	c.3338A>G p.Asp1113Gly	27	#13 Phenotype unknown	unknown	(Liu et al., 1997)	np	no	Unknown
	c.3463G>A p.Asp1155Asn	27	#14 Thoracic aortic aneurysm ard, mvp, diss	(Milewicz et al., 1996)	no	yes	De novo
	c.3464A>G p.Asp1155Gly	28	#14 Classical MFS	ard, el	Case 1	no	no	Parents not available, 5 sibs neg for mutation
	c.3712G>A p.Asp1238Asn	29	#16 Phenotype unknown	unknown	(Yuan et al., 1999)	np	no	Unknown
	c.3713A>G p.Asp1238Gly	30	#16 Classical MFS	ard, mvp, sk	(Tiecke et al., 2001)	np	no	Familial
	c.3964G>A p.Asp1322Asn	31	#18 Neonatal MFS	ard, mi, ti, myop, sk	Case 2	np	yes	De novo
	c.4210G>T p.Asp1404Tyr	33	#20 Classical MFS	ard, el, sk	(Hayward et al., 1997)	yes	yes	Familial
	c.5422G>C p.Asp1808His	43	#26 Lens luxation and striae	el, str	Case 3	np	no	Parents not available
	c.5671G>A p.Asp1891His	45	#28 Classical MFS	ard, sk	Case 4	np	no	De novo
	c.5788G>C p.Asp1930His	45	#29 Classical MFS	ard, el, sk	Case 5	np	yes	De novo
	c.5788G>A p.Asp1930Asn	46	#29 Phenotype unknown	unknown	(Liu et al., 1997)	np	yes	Unknown
	c.5788G>A p.Asp1930Asn	46	#29 Classical MFS	ard, el, sk, de, str	Case 6	np	yes	Parents not available
	c.6037G>T p.Asp2013 Tyr	48	#31 Classical MFS	ard, el, sk, de, str	Case 7	np	yes	Familial
	c.6379G>T p.Asp2127Tyr	51	#32 Classical MFS	ard, el	(Matsukawa et al., 2001)	np	yes	Familial
	c.6381T>A p.Asp2127Glu	52	#32 Classical MFS	ard, sk	(Kainulainen et al., 1994)	np	no	Familial
	c.7331A>G p.Asp2444Gly	59	#38 Classical MFS	ard, sk	Case 8	np	no	De novo
	c.7454A>T p.Asp2485Val	60	#39	Classical MFS in homozygous state	ard, el, sk, str, her, pn Case 9	no	no	Familial
	c.7819G>A p.Asp2607Asn	62	#42 Classical MFS	ard, mvp, sk	Case 10	no	no	Mother suspect for MFS
	c.7820A>G p.Asp2607Gly	63	#42 Phenotype unknown	unkown	(Liu et al., 1997)	np	no	Unknown
	UMD Universal Marfan Database -FBN1 (UMD-FBN; http:/www.umd.be); cbEGF calcium binding Epidermal Growth Factor domain; bp basepair; np not performed; neg
	negative; pos positive; ard aortic root dilatation; diss aortic dissection; mvp mitral valve prolapse, mi mitral valve insufficiency; el ectopia lentis; pal high arched palate; ti
	tricuspid valve insufficiency; myop high myopia; ar arachnodactyly; hm hypermobility; contr contractures; str striae; her hernia; sk skeletal involvement; de dural ectasia; pn
	pneumothorax; unknown. The gray row represents the recessive mutation described in this article.

Mutation numbering refers to the FBN1 cDNA GenBank reference sequence: NM_000138.3, with the A of the ATG translation initiation codon as nucleotide +1 (www.hgvs.org/mutnomen). a ) Alamut mutation interpretation software version 1.5; Interactive Biosoftware, Rouen France.

Mutations of First Aspartic Acid of cbEGF-like Domains in FBN1 9

Table S1 . Primers for cDNA sequencing of FBN1: F=forward; R=reverse; numbers refer to position in cDNA sequence

 S1

	Page
	fragment	primername	Sequence 5' > 3'	Length bp
	1	FBN1F1	ATGCGTCGAGGGCGTCTGCT	384
		FBN1R384	GCTACCTCCATTCATACAGCGA
	2	FBN1F318	GATAGCTCCTTCCTGTGGCTCC	406
		FBN1R728	CCGTGCGGATATTTGGAATG
	3	FBN1F657	CCCCTGTGAGATGTGTCCTG	407
		FBN1R1064	TTGGTTATGGACTGTGGCAGC
	4	FBN1F1012	ACAGCTCTGACAAACGGGCG	384
		FBN1R1396	TGCAGCGTCCATTTTGACAG
	5	FBN1F1358	GCCAGTTGGTCCGCTATCTC	330
		FBN1R1688	ACATGAAAGCCCGCATTACAC
	6	FBN1F1609	AATGGCCGGATCTGCAATAA	398
		FBN1R2007	CTGGCCTCTCTTGTATCCACCA
	7	FBN1F1927	CTGGCTGTGGGTCTGGATGG	362
		FBN1R2289	GCAGTTTTTCCCAGTTGAATCC
	8	FBN1F2212	ATCTGTGAAAACCTTCGTGGGA	399
		FBN1R2611	AGGTGGCTCCATTGATGTTGA
	9	FBN1F2458	GTCTGCAAGAACAGCCCAGG	357
		FBN1R2815	TGGGACACTGACACTTGAATGA
	10	FBN1F2699	TACTCAAGAATTAAAGGAACA	525
		FBN1R3224	CGGCATTCGTCAATGTCTGTGC
	11	FBN1F3141	CATTGGCAGCTTTAAGTGCAGG	460
		FBN1R3621	ACCACCATTCATTATGCTGCA
	12	FBN1F3558	CCATTCAACTCCCGATAGGCT	335
		FBN1R3893	TTTTCACAGGTCCCACTTAGGC
	13	FBN1F3783	CAGGTGCTTGTGTTATGATG	392
		FBN1R4173	GCACAGACAGCGGTAAGA
	14	FBN1F4062	GATTGGAGATGGCATTAAGTGC	451
		FBN1R4513	TGTTGACACAGTTCCCACTGA
	15	FBN1F4425	CTACGAACTGGACAGAAGCGG	593
		FBN1R5018	ATACAGGTGTAGTTGCCAACGG
	16	FBN1F4910	ACTACCTGAATGAAGATACACG	626
		FBN1R5536	GACCTGTGGAGGTGAAGCGGTAG
	17	FBN1F5348	TCAACATGGTTGGCAGCTTCC	476
		FBN1R5824	AAAGATTCCCATTTCCACTTGC
	18	FBN1F5722	ACAATTGGTTCCTTCAACTG	356
		FBN1R6074	GCACAAATTTCTGGCTCTT
	19	FBN1F5973	CTTGGATGGGTCCTACAGATGC	579
		FBN1R6552	CACATTCTTGCAGGTTCCATT
	20	FBN1F6466	GGTTATACTCTAGCGGGAATG	450
		FBN1R6937	TCCCACGGGTGTTGAGGCAGCG
	21	FBN1F6842	AGCGGAGACCTGATGGAGAGG	645
		FBN1R7487	CAGTTGTGTTGCTTGGTTGCA
	22	FBN1F7429	CAAGAGGATGGAAGGAGCTGC	476
		FBN1R7905	GAACTGTTCATACTGGAAGCCG
	23	FBN1F7785	CTACCTCCAGCACTACCAGTGG	384
		FBN1R8169	GTAGCCATTGATCTTACACTCG
	24	FBN1F8024	CACCTGGTTACTTCCGCATAGG	672
		FBN1R8696	ATGATTCTGATTGGGGGAAAA

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John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGMENTS

We thank the patients and their families for their very kind cooperation, Prof. Dr. E. Bakker for his helpful suggestions and Jacqueline Egthuijsen for technical assistance.