7 Med Genet 1995;32:549-552 549

Brief papers

Mutations in Li-CAM in two families with X

linked complicated spastic paraplegia, MASA
syndrome, and HSAS
Juan Carlos Ruiz, Harry Cuppens, Eric Legius, Jean-Pierre Fryns, Thomas Glover,
Peter Marynen, Jean-Jacques Cassiman

Abstract X linked spastic paraplegia (SPG1), MASA

The suggestion that the three X linked syndrome, and X linked hydrocephalus owing
syndromes X linked spastic paraplegia to stenosis of the aqueduct of Sylvius (HSAS)
(MIM 312900), MASA syndrome (MIM were originally reported as three separate X
303350), and X linked hydrocephalus owing linked conditions.`5 However, family studies
to stenosis ofthe aqueduct of Sylvius (MIM have suggested that SPG1, MASA, and HSAS
307000) are variable clinical mani- are variable clinical manifestations of mutations
festations of mutations at the same locus at the same locus at Xq28,"9 and indeed a
at Xq28 was confirmed by the finding of series of mutations in the Li-CAM gene have
Centre for mutations in the LI-CAM gene in the three been identified in subjects with the three syn-
Human Genetics,
University of syndromes. Recently, two families in dromes. Attempts to correlate the variable
Leuven, Campus which different subjects showed a clearly phenotypic manifestations (SPG1, MASA, and
Gasthuisberg, O&N, different phenotype within the same family HSAS) with the nature or the localisation of the
Herestraat 49,
B-3000 Leuven, of the three X linked syndromes were de- different mutations have been unsuccessful.6
Belgium scribed. A reverse transcription PCR The molecular analysis of the Li-CAM gene
J C Ruiz assay was developed for the analysis ofthe in two families with subjects showing different
H Cuppens
E Legius LI-CAM cDNA in two of the members of phenotypes within one family is reported here.
J-P Fryns these families. RNA isolated from EBV
P Marynen transformed cell lines and a colon car-
J-J Cassiman cinoma derived cell line was used as a Materials and methods
Department of starting material. The Li-CAM cDNA of Blood samples for DNA extraction were ob-
Pediatrics and Human two male patients from each family was tained from affected and normal subjects from
Genetics, University sequenced. We report two new mutations
of Michigan, two previously reported families.89 Family 1
Ann Arbor, in the LI-CAM gene in these two families (fig 1) was reported by Fryns et a18 as a three
Michigan, USA showing that the three different pheno- generation family which suggested that X
T Glover types observed in different generations linked spastic paraplegia, MASA syndrome,
Correspondence to: within the same family are variable pheno- and X linked hydrocephalus owing to con-
Professor Cassiman. typic expressions of the same mutation.
Received 21 November 1994
genital stenosis of the aqueduct of Sylvius are
Revised version accepted for variable clinical manifestations of mutations at
publication 6 February 1995 (J Med Genet 1995;32:549-552) the same locus at Xq28. The second family
(fig 2) was reported by Kaepernick et al.9 In
this family similar variable manifestations of
clinical symptoms were observed, varying from
hydrocephalus to MASA syndrome in affected
members, while two obligate carrier females
[}A
413
_ _ +
5
+
LbA
6
_
7
had mild mental retardation and adducted
thumbs. In addition, EBV transfected cell lines
were available from at least two affected people
in each family. These cell lines were used for
RNA extraction using the RNAzolTm Kit
III
(TEL-TEST Inc, Texas, USA) following the
2 _1 3 4 6 7 8 9 10 recommendations of the manufacturers. A
1 human colon carcinoma cell line SW480
(ATCC CCL 228) was used as a control since
IV the expression of Li-CAM in this cell line has
5 6 7 8 been documented.'0 RNA (2,g) was reverse
_+ _
-+ - +

transcribed with M-MLV reverse transcriptase

Figure 1 Pedigree of family 1 with mutation Il 79S.8 The sequenced patients are (BRL) according to the recommendations of
indicated by an arrow. Carriers of the mutation are indicated with (+) and of the the manufacturer. The cDNA was then amp-
wildtype sequence (-). lified in four overlapping fragments by a hemi-
 550 Ruiz, Cuppens, Legius, Fryns, Glover, Marynen, Cassiman

7778

Figure 2 Pedigree offamily 2 with mutation G370R.9 The sequenced patients are indicated by an arrow. Cariers of the mutation are indicated with
(+) and of the wildtype sequence (-).

Table A Pr^imers used for the amplification and sequencing of Li-CAM cDNA of cDNA reaction. For fragments A and B, 30
Fragment A (1-1273 bp) cycles of amplification were performed with
PLL A5 5'-CGCGGTGCCGCCGGGAAA-3' one minute denaturation at 95°C, one minute
PL1.A3 5'-AACGTAGATGTAGGCATTGGC-3'
PBL1 .A5* 5'-Biotin-CCGGGAAAGATGGTCGTG-3' annealing at 55°C, and an extension step at
PFLI .A3at
PFLI .A3bt
5'-FITC-CCTGGAACCTCTGAGCAA-3' 72'C for three minutes. For fragments C and
5'-FITC-GCGTGGCAGATGTAGTCT-3' D, 40 cycles were performed with an annealing
PFL1 .A3ct 5'-FITC-TGAGTTCTCGGCCAGGCA-3'
PFLI .A3dt 5'-FITC-GTCACCATTGTGTCACTG-3' temperature of 60°C. All the amplifications
Fragment B included an initial denaturing step of five min-
(993-2229 bp) utes at 95°C and a final extension time of
PL1.B5 5'-TACTATGTCACCGTGGAGGCT-3'
PL1.B3 5'-GCTTCCACGTGATGACCATAT-3' 10 minutes at 72°C. All PCR products were
PBL1 .B5*
PFL1 .B5at
5'-Biotin-GTCTCATTTCCTTCCCCCTTC-3' sequenced using a solid phase approach (Dy-
5'-FITC-CAGAGGTCACCTGGAGAA-3'
PFL1 .B5bt 5'-FITC-TGGACGAGGATGGGACAA-3' nalR, Norway and Pharmacia, Auto ReadT
PFL1.B5ct
PFLI .B3dt
5'-FITC-GATGTGGTGGAGAGTAGG-3' Sequencing Kit, Sweden) with an internal
5'-FITC-ACCTGGCTCTGCGTCAG-3' FITC labelled oligonucleotide primer. When
Fragment C ambiguities were present in the sequence, the
(2127-3265 bp)
PLI .C5 5'-GTCTCTGAGACTGTGGTCACA-3' complementary strand was also sequenced.
PL1.C3 5'-GGATCTCGTAGTCAGTGTCAG-3' All DNA sequences were numbered ac-
PBL1 .C5* 5'-Biotin-AGGCAGCCCCAGAGAAGAA-3'
PFL1 .C5at 5'-FITC-ACTCTGGAGAGACTACC-3' cording to the published sequence (GEN-
PFL1 .C3bt 5'-FITC-TTGACCAGCACGGCACTT-3' BANK: M77640); the amino acid numbering
PFL1 .C5ct 5'-FITC-CTTTAACGGGCGAGGATC-3'
PFL1 .C3dt 5'-FITC-CTGACATACTGTGGCGAA-3' starts at the first methionine.
To identify the mutation in other subjects a
Fragment D dot blot assay was performed using an end
(2949-3955 bp)
PL1.D5 5'-ACACACAACCTGAACCGATCTC-3' labelled oligonucleotide containing the mu-
PL1.D3 5'-GCAGCTGGGTTTTGGCAATAA-3'
PBL1 .D3* 5'-Biotin-CGGGTGGTAAGGAAGGGGAT-3' tated sequence or the wild type sequence as a
PFL1 .D5at 5'-FITC-GCAACATCTCAGCCACAG-3' probe (table 1B). PCR products containing
PFL1.D5bt 5'-FITC-TGACAACGAGGAGAAGGC-3' exons 4, 5, and 6 for mutation I179S, and
PFL1 .D3ct 5'-FITC-TGAACATCCACGCTGCCC-3'
PCR products containing exons 8, 9, and 10
*
Nested biotinylated primer.
t FITC labelled sequencing primers. for mutation G370R (table 2) were used in the
dot blot procedure. After hybridisation with
the labelled probe, a stringent wash was per-
Table lB Oligonucleotides used for dot blotting formed in 2 x SSC/0-5% SDS at 55°C for 10
S179 5'-TTGCACAGCAAGCAGG-3'
minutes.
I179 5'-TT-GCACATCAAGCAGG-3'
R370 5'-GAATCAACAGGATCCC-3'
G370 5'-GAATCAACGGGATCCC-3'
Results
In all the subjects analysed and in the control
cell line several differences from the published
nested PCR. Primers used for amplification sequence of the LI -CAM gene were detected."
were elaborated using the published Li-CAM A 15 bp deletion was found at coding position
cDNA sequence" and are shown in table 1A. 97 of the cDNA compared to the published
The amplification was performed in 100 p1 so- sequence and a 12bp deletion at bp3551.
lution containing 16-6 mmol/l (NH4)2S04, The latter has already been described as an
67 mmol/l Tris-HCl pH8-8, 10 mmol/l P-mer- alternative splicing form of Li-CAM. 12 The
captoethanol, 6-7 ,umol/l EDTA, 0-17 ptg BSA/ deletion at bp 97 might therefore also be the
lOl, 1 mmol/l MgCl2, 0-2 mmol/l dNTP, 10% result of an alternative splicing event in white
DMSO, 2-5 U Taq DNA polymerase, and 20 ,ul blood cells. At nucleotide position 875 a T to
Mutations in Li-CAM in two families with X linked complicated spastic paraplegia, MASA syndrome, and HSAS 551

Table 2 Primers used to amplify genomic DNA detected. Dot blot analyses of genomic DNA
Family 1 (exons 4, 5, and of the other members of the family showed that
6) I1 79S the mutation segregated in all female carriers
PG1.5 5'-GTCCACTTCAAACCCAAG-3'
PG1.3 5'-GCGTGGCAGATGTAGTCT-3' (II-3, IIIl,1,III9, IV-5, IV-6, and IV-8) as
determined previously by linkage analysis,8 and
Family 2 (exons 8, 9, and all affected males (II-5, spastic gait and bor-
10) G370R
PG2.5 5'-CCGTACTGGCTGCACAAGCC-3' derline intelligence; III3, spastic paresis, men-
PG2.3 5'-GTCACCATTGTGTCACTG-3' tally retarded, and adducted thumbs; III8
spastic paresis without involvement of the
thumbs).
A different point mutation was found in the
two patients of family 2 (fig 2), a G to A
transversion at nucleotide position 1128 of the
Li-CAM gene sequence, resulting in the
change of glycine into arginine at position 370
of the amino acid sequence (G370R) (fig 4).
In family 2 the mutation G370R was detected
by dot blot analysis of genomic DNA in the
two partially expressing females (samples 6 and
30), in all obligate female carriers (samples 10,
13, 20, 26, and 51), in the females predicted
to be carriers by linkage analysis (samples 45,
53, 54, and 62), and all the affected males
(samples 16, 17, 23, 24, 31, 33, 40, 41, 60,
79, and 80). No G370R mutation was found
in the normal males (patients 12, 14, 19, 25,
27, 35, 42, 26, 47,55, and 84) and non-carriers
as determined previously by linkage analysis"3
(patients 15, 28, 44, 48, 49, 52, 61, 63, 64,
65, 68, and 77). Neither I179S nor G370R
mutations were present in 100 X chromosomes
Figure 3 Sequence of part of exon 6 of Li-CAM gene showing the T to G mutation at from normal controls including two samples
bp position 556 (I179S) (indicated by an arrow) in the family reported by Fryns et al.8 which had been completely sequenced and used
(A) Control sequence SW480, (B) mutated sequence. as negative controls.

Figure 4 Sequence of part of exon 9 of Li-CAM gene showing the G to A mutation at
bp position 1128 (G370R) (indicated by an arrow in the family reported by Kaepernick
et al.9 (A) Control sequence SW480, (B) mutated sequence.
C transition was observed, which at the protein
level would be a silent mutation, and an in-
sertion of a cytosine at nucleotide position 3806
within the 3' untranslated region of the Li-
CAM transcript was detected.
In the two patients of family 1 (fig 1) a T
to G transition at nucleotide position 556,
resulting in a change of isoleucine into serine
at amino acid position 179 (1179S) (fig 3), was
Discussion
LI-CAM belongs to the immunoglobulin
superfamily of proteins. These proteins can be
structurally divided into three segments: (1) an
extracellular portion consisting of six im-
munoglobulin-like (Ig) domains and five
fibronectin III (Frn) domains,'415 (2) a trans-
membrane domain, and (3) a cytoplasmic
domain.
We present here two new mutations in the
LI-CAM gene in affected subjects from two
different families: an I179S mutation located
within the loop ofthe first Ig-like domain of L1 -
CAM and a G370R mutation located within
the fourth Ig-like domain. Dot blot analyses
showed that neither of the mutations were
polymorphisms found in the general population
and confirmed that they segregated with the
clinical syndrome. Moreover, hybridisation to
both the mutated oligonucleotide and the wild
type oligonucleotide showed the presence of
one affected and one normal allele in all carrier
females.
It is interesting to note that these mutations
were observed in affected subjects from two
families where we observed the three syn-
dromes, complicated spastic paraplegia, MASA
syndrome, and X linked hydrocephalus owing
to stenosis of the aqueduct of Sylvius as ap-
parent variable expression of mutations at the
same locus at Xq28.8913 All the reports pub-
lished so far identified mutations in the Li-
CAM gene in families with one of the three
phenotypes. These data further illustrate the
552
fact that the three clinical entities are pheno-
typic variants of the same genetic defect and
not separate entities, as originally suggested by
Straussberg et al.'6
The finding of a single point mutation in
each of the members of the same family with
variable clinical phenotypes suggests that the
phenotypic expression of the Li-CAM gene is
influenced by other genetic or environmental
factors. This observation may then explain the
absence of a definitive phenotype-genotype cor-
relation in these X linked syndromes.
The authors wish to thank Dr Susan Kenwrick for kindly
providing us with the genomic sequence of the Li-CAM gene.
Peter Marynen is a "Bevoegdverklaard Navorser" of the "Na-
tionaal Fonds voor Wetenschappelijk Onderzoek", Belgium.
This investigation was supported by the Interuniversity Network
for Fundamental Research sponsored by the Belgian Gov-
ernment (1991-1996).
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