Variations of the Amnionless gene in recurrent spontaneous abortions
Department of Clinical Genetics, Helsinki University Central Hospital
Department of Obstetrics and Gynecology
University of Helsinki
, FIN-00014 Helsinki,
Folkhlsan Institute of Genetics, University of Helsinki
Recurrent spontaneous abortions (RSA) are estimated to affect 0.5-1% of couples trying to have a child. The causes of RSA are unknown in the majority of cases. This study aimed to determine whether homozygous mutations in the AMN gene in a fetus cause spontaneous abortions in humans, as they are known to cause spontaneous abortions in mice. The study was conducted by screening 40 couples and 5 women with three or more unexplained spontaneous abortions for heterozygous mutations in the AMN gene using denaturing high-performance liquid chromatography. Altogether, 3 exonic and 11 intronic sequence variations were found. There were no significant differences in the frequencies of the variations between the patients and a control group. One of the exonic variations was non-synonymous, and three of the variations may affect gene splicing. None of the putative phenotypeaffecting variations were found in both partners in any couple. These results indicate that RSA in the couples studied cannot be explained by homozygous AMN mutations in the fetus. However, two couples had different, potentially deleterious variations in both partners. If these variations have a phenotypic effect, the RSA experienced by these couples may be caused by mutations in the AMN gene. In addition, birthplaces of the patients' ancestors revealed some clustering, suggesting that some patients may carry a founder mutation in another gene which may contribute to RSA.
Spontaneous abortions are the most common complication of
pregnancy, affecting approximately 15% of all pregnancies. Recurrent
spontaneous abortions (RSA), defined as the occurrence of three or
more consecutive, clinically detectable pregnancy failures, are
estimated to occur in 0.51% of all couples (Tulppala et al., 1993; Katz
and Kuller, 1994). After excluding known causes of spontaneous
abortion including genetic, metabolic, endocrine, environmental and
anatomic factors, the cause remains unknown in approximately 50%
of women who experience RSA (Plouffe et al., 1992; Tulppala et al.,
1993; Clifford et al., 1994). Even though genetic factors such as
chromosome abnormalities (Stephenson et al., 2002) are one of the major
and best defined causes of spontaneous abortions, new genetic factors
associated with recurrent abortions remain to be found.
One way to identify new genes associated with RSA is to study
candidate genes required for normal development in mice. One gene
known to cause miscarriage in mice when mutated is Amnionless
(Amn). The Amn mutation is a transgene-induced insertion mutation in
the distal region of mouse chromosome 12. This mutation is
prenatally lethal, and homozygous mutant fetuses die at an early stage of
development (Wang et al., 1996). Gastrulation begins normally, but
despite the apparently normal development of extra-embryonic
structures, the embryonic ectoderm of the mutant mice remain small and
undifferentiated, and no amnion is generated (Wang et al., 1996).
Mutant embryos that survive to the tenth day of gestation have
headfolds, a beating heart and abundant posterior mesoderm, but they have
none of the mesoderms that produces the limb buds, dermis, muscle,
vertebrae and other organs of the trunk (Tomihara-Newberger et al.,
1998). At the onset of gastrulation, the proximal and distal portions of
the primitive streak are normally organized, but the middle region is
absent in the mutant mouse. The Amn protein has been shown to be
required in the visceral endoderm (VE) for normal middle streak
formation (Tomihara-Newberger et al., 1998), but its exact role in
mouse development has not yet been determined. The extracellular
region of the Amn protein contains a 70 amino acid cysteine-rich (CR)
domain similar to CR domains present in bone morphogenetic protein
(BMP)-binding proteins. Therefore, it has been proposed that Amn
functions as a part of a BMP-signalling pathway within the VE to
direct the expression of a set of genes that control the formation of the
middle streak, which is proposed to be directed by a genetic pathway
different from the pathways controlling the formation of the proximal
and distal parts of the primitive streak (Kalantry et al., 2001).
In both mouse and human, Amn is expressed in the intestine and
kidney (Tanner et al., 2003; Strope et al., 2004). Mouse Amn is also
expressed during gastrulation in the VE (Tomihara-Newberger et al.,
1998; Kalantry et al., 2001). In these tissues, Amn forms a complex
with the cubilin (Cubn) protein, a multi-ligand scavenger receptor.
The function of Amn in the complex is to ensure the proper
localization and endocytosis of Cubn and its ligand (Fyfe et al., 2004; Strope
et al., 2004). An alternative role suggested for Amn in mouse
development is that the Cubn/Amn complex may be required for
endocytosis of several ligands in the VE, and the complex would thereby
facilitate a signalling pathway during gastrulation to coordinate
primitive streak assembly (Strope et al., 2004).
In humans, the CUBN/AMN complex is required for uptake of
cobalamin (vitamin B12), and to date all known homozygous
mutations in either gene cause ImerslundGrsbeck syndrome (IGS).
Although AMN is needed for absorption of cobalamin in humans, it
may be a moonlighting protein with more than one function. High
similarity between mouse and human Amnionless genes suggests that
the genes would have the same functions. That mutations in Amn
cause fetal loss in mice (Wang et al., 1996) makes human AMN a
candidate gene for RSA. The role of AMN in the early development of
humans has not yet been investigated, and the aim of this study was to
determine whether homozygous mutations in the AMN gene of a fetus
could offer a new explanation for RSA in humans. We hypothesized
that parents with recurrent unexplained fetal losses are healthy
heterozygous carriers of mutations in AMN, and the aborted fetuses would
have the mutations in a homozygous state.
Materials and methods
Patients with RSA treated at the Department of Gynaecology and Obstetrics of
the Helsinki University Hospital during 20012004 were chosen for the study.
The inclusion criteria for the study included women aged 1840 years with
previous history of recurrent abortion, defined as three or more consecutive
abortions. In 44 women, all the abortions had taken place during the first
trimester (<13 weeks). In addition to first trimester losses, two women had
experienced second trimester (from 13 weeks to 23 weeks) abortions, and one
women a third trimester (24 weeks) intrauterine fetal death.
Uterine anomalies were checked by ultrasonography or hysterosonogram.
Maternal and paternal karyotypes were tested from peripheral blood
lymphocyte culture. No patients included in this study had chromosomal or uterine
abnormalities. A total of 85 (40 couples and 5 women) patients with
unexplained RSA were chosen for this study.
The control group used in the study consisted of 95 women who had at least
one normal pregnancy and no history of spontaneous abortion. The ethics
committee of the Department of Obstetrics and Gyneacology, Helsinki University
Central Hospital, approved this study.
Polymerase chain reaction
DNA was extracted from whole blood collected from patients and controls
using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN,
USA). The 12 exons and 3-UTR of AMN were amplified via PCR, performed
in a 25 l reaction mix containing the following reagents: 50100 ng of
genomic DNA, 1 Optimized Detergent-free EXT buffer, 2 nmol of each
dNTP, 10 pmol of forward primer, 10 pmol of reverse primer and 0.5 units of
DyNAzyme EXT DNA-polymerase (Finnzymes, Espoo, Finland).
Additionally, DMSO (final concentration 5% [v/v]) was added to some of the
amplicons. Thermocycling was perfomed in a PTC-225 DNA Engine Tetrad
thermocycler (MJ Research, Waltham, MA, USA). Initial denaturation at 95C
for 2 min was followed by 3340 cycles of denaturation at 95C, annealing at
5166C depending on the amplicon and extension at 72C. The lengths of the
denaturation, annealing and extension steps varied depending on the amplicon.
A final extension was performed at 72C for 10 min. Information concerning
the exact PCR conditions and the primers used is available on request.
Amplification of appropriately sized PCR products was confirmed by agarose gel
electrophoresis before further analysis.
DHPLC sample analysis
Denaturing high-performance liquid chromatography (DHPLC) analysis was
carried out using a Transgenomic WAVE Nucleic Acid Fragment Analysis
System (Transgenomic, Omaha, NE, USA) and the associated Navigator
software. Before analysing the samples with DHPLC, the PCR products were
denatured for 3 min at 95C and then gradually reannealed by decreasing the
temperature from 95 to 50C over a period of 50 min to enable the formation of
heteroduplexes. To obtain optimal resolution of homoduplex and heteroduplex
DNA fragments, the temperature was set for partially denaturing conditions.
The melting profile of the amplicons was predicted by the Navigator software,
but the exact temperature was determined empirically by injecting one PCR
product for each amplicon at 0.5 and 1C under, and 1 and 2C over the
optimal temperature suggested by the Navigator program. Conditions used for
DHPLC analysis for each amplicon are available on request.
Following DHPLC screening, samples showing heterozygous peaks were
sequenced in order to determine the nature of the sequence change.
Additionally, for each amplicon at least 10 samples showing only a homoduplex peak
were sequenced to confirm that no variation went undetected. The PCR
products were purified using Exonuclease I and shrimp alkaline phosphatase (USB
Corporation, Cleveland, OH, USA), and the purified products were sequenced
using BigDye version 3.1 sequencing chemistry and an ABI 3730 DNA
Analyzer (Applied Biosystems, Foster City, CA, USA).
By sequencing, four variations were detected in a homozygous state. Even
though this study focused on heterozygous variations, we determined the exact
genotype of the samples by restriction enzymes in the cases where the
variations either created or deleted a restriction site. All the samples were genotyped
for variations c.-27T>C, c.1169+42C>G and c.1362+38G>C using restriction
enzymes HpaII, BsaJI and AciI (New England Biolabs, Ipswich, MA, USA),
respectively. Variation c.-23G>C does not create or delete any restriction site,
and therefore the samples were sequenced to determine the exact genotype for
After the digestion, the restriction pattern was detected by agarose gel
Predicting the effects of variations
Exonic splicing enhancers (ESEs) are common in both alternative and
constitutive exons, where they act as binding sites for Ser/Arg-rich proteins (SR
proteins), splicing factors needed in multiple steps of the splicing pathway.
ESEfinder (http://exon.cshl.edu/ESE/) is a Web-based resource which scans
nucleotide sequences to identify putative ESEs. The ESEfinder was used to
predict whether the exonic or intronic sequence variations disrupt the sequence
of known ESE-elements.
The variations predicted to change an amino acid were analysed by the SIFT
(sorting intolerant from tolerant) program (http://blocks.fhcrc.org/sift/
SIFT.html) which predicts whether an amino acid substitution in a protein will
Fishers exact tests were used for statistical analysis of the data. The allele
frequencies of the patients and controls were compared to determine if any of the
variations were more frequent in either group. Differences were considered as
statistically significant for P-values <0.05.
All patients were given questionnaires enquiring as to the full names, dates and
places of birth of their parents and grandparents. Answers were received from
82 patients. To investigate the geographic distribution of ancestral birthplaces,
the birthplaces of the parents and grandparents were placed onto a map of
Finland, including the formerly Finnish areas of Russian Karelia. The maps
were compared with the maps of the distribution of the Finnish population at
the time the parents/grandparents of the couples were born to determine if
these birthplaces were clustered to some specific region.
We screened 85 patients (40 couples and 5 women) with a history of
unexplained spontaneous recurrent abortions and 95 controls with
DHPLC for mutations in the 12 exons and the 3-UTR region of the
AMN gene. The nature of the variations detected was confirmed by
In total, 14 sequence variations were found (Table I). Of these, one
was found only in patients. Three were found only in controls, while
*Numbering relative to adenine in the first ATG startcodon of AMN (UCSC Genome Browser, http://genome.ucsc.edu/).
Novel variations (variations which have according to our knowledge not been previously reported).
SS, start site.
(predicted amino acid change)
the remaining ten variations were detected in both patients and
controls. There were no significant differences in the frequencies of
heterozygous (or homozygous) patients and controls for any of the
sequence variations. Two of the variations were single-nucleotide
substitutions within exons. Sequence variation c.363G>A is a
singlenucleotide substitution in exon 5, which changes the last nucleotide
of codon 121 in the AMN gene (GGGGGA). This synonymous
variation, which was detected only in patient samples, does not predict a
change of the amino acid (glycine) and is therefore predicted to be a
silent polymorphism. Sequence variation c.829A>G is a
non-synonymous single-nucleotide substitution in exon 8, which changes the
first nucleotide of codon 276 (ACCGCC). The variation is
predicted to change the amino acid from threonine to alanine. According
to the SIFT program, this amino acid change would be tolerated even
though the hydroxyl group of threonine makes it much more
hydrophilic and reactive than alanine. Ten variations were
singlenucleotide substitutions within introns or the 3-UTR region, and two
were duplications. One of the duplications was a 10 bp (GCGTG
GCGTG) duplication in intron 4 and the other was a 6 bp duplication
(GCCGGG) of codons 447 and 448 in exon 12. This variation is
predicted to make the final protein product two amino acids (Ala+Gly)
All 14 sequence variations were analysed with the ESEfinder to
predict whether any affect the splicing of the gene by altering the
SF2/ASF (Thr = 1.956)
(homozygous patients) (n = 85)
(homozygous controls) (n = 95)
binding sites of the SR proteins needed for splicing. ESEfinder
searches for putative ESE motifs (six to eight nucleotides long) and
calculates a score for all sequences with a motif match. A score is
considered significant and the sequence potentially needed for splicing
when the score is greater than the threshold value defined by the
program. The results indicate that the single-nucleotide substitutions in
exon 8 (c.829A>G), intron 8 (c.843+11C>T) and intron 10
(c.11706C>T) may affect the splicing by deleting one or two binding sites for
the known SR proteins (Table II).
The 40 couples included in this study were analysed to
determine whether some variations existed in both partners of a couple.
The only variations detected in both partners were the four most
common variations. In no couples were the same exonic or
putative splice-site affecting variations found in both partners. In two
of the couples, both partners had a different exonic or putative
splice site affecting variation. One couple had variations
c.363G>A and c.1170-6C>T, and another couple had variations
c.843+11C>T and c.1339_1344dup.
The birthplaces of the parents and grandparents (data concerning
grandparents not shown) of the patients were mapped to determine
whether these were clustered. Compared to the reference map, the
geographical distribution of the birthplaces is somewhat uneven
(Figure 1), and the results suggest that there may be an eastern
enrichment (in the province of Kuopio) of the ancestral birthplaces.
SC35 (Thr = 2.383)
SRp55 (Thr = 2.267)
Threshold (Thr) values defined by the ESEfinder and values obtained for normal and mutant alleles for sequence variations predicted to affect the binding sites of
The high similarity between human AMN and mouse Amn (Kalantry
et al., 2001), and the fact that homozygous mutations in Amn are
known to cause fetal death in the mouse (Wang et al., 1996), makes
AMN a candidate gene for spontaneous abortions in humans. In this
study, we have examined whether there is a connection between RSA
and mutations in the AMN gene. To our knowledge, there are no
published data concerning the role of AMN in human development. As a
result of screening 85 patients using DHPLC, 3 exonic and 11 intronic
sequence variations were detected. All the detected variations in AMN
can be defined as polymorphisms, and there were no significant
differences in the frequencies of the variations between patients and
controls. Three variations were predicted to affect exon splicing. In two of
the 40 couples screened, both partners had different exonic or
potential splice site-disrupting variations, but in no couple was the same
putative protein-altering variation detected in both partners. These
results suggest that RSA in the couples studied is not explained by
homozygous mutations in AMN. However, the genealogic studies
showed clustering in the birthplaces of the parents and grandparents,
indicating that the patients may carry a founder mutation in another
gene which may contribute to RSA.
The polymorphisms detected in AMN may not directly affect protein
function but may have effects on the protein indirectly by changing the
function of regulatory sequences that control gene expression, by
altering the stability of the mRNA or by disrupting the splicing mechanisms
of the gene. Splice site mutations may result in exon skipping,
activation of cryptic splice sites, creation of a pseudo-exon within an intron
or intron retention (Krawczak et al., 1992; Nakai and Sakamoto, 1994).
We attempted to predict whether the variations detected in AMN would
disrupt the splicing of the gene using ESEfinder, a Web-based splicing
motif recognition program. The program predicted that three sequence
variations, c.829A>G in exon 8, c.843+11C>T in intron 8 and
c.11706C>T in intron 10, may affect splicing. These variations are predicted
to abolish one or more of the binding sites for known SR proteins and
may therefore result in exon skipping or intron retention. The ESE
finder-software, however, only makes predictions about the effect of a
variant on the splicing of the mRNA. The presence of a high-score
motif in a sequence does not necessarily make the sequence an ESE,
and the sequence with maximum score is not necessarily the most
effective ESE (Cartegni et al., 2003). Further studies are required to
determine whether the variations predicted to affect splicing actually
disrupt the splicing mechanisms.
Two couples in this study had different exonic or putative splice
site variations in both partners. Variations c.363G>A in exon 5 and
c.1170-6C>T in intron 10 were detected in one couple and the other
couple had variations c.843+11C>T in intron 8 and c.1339_1344dup
in exon 12. The variation detected in exon 5 is a synonymous
(Gly121Gly) change and is unlikely to affect the protein function.
Variations c.1170-6C>T and c.843+11C>T are predicted to disrupt
the splicing mechanisms of the gene. The exonic duplication in exon
12 is predicted to make the protein two amino acids longer (Ala+Gly).
This variation may affect the structure of the cytoplasmic region
of the protein. The other AMN variations (c.-27T>C, c.-23G>C,
c.1169+42C>G and c.1362+38G>C) found in both partners of a
couple are previously reported common variations, also found in
homozygous states in both patients and controls, indicating that these
variations are neutral polymorphisms. In the two aforementioned
couples, there is a 25% chance for every conceptus to be a compound
heterozygote with different potentially deleterious AMN mutations in the
two alleles. Unfortunately, there are no fetal samples available to
study whether these variations may be the cause for spontaneous
abortions experienced by these couples. If these variations had a phenotypic
effect, the RSA in these couples could be explained by mutations in
the AMN gene. However, at present there is no evidence for a
phenotypic effect. Further studies are needed to determine whether the
function of AMN in human and mouse differs significantly or whether
AMN is needed both for the uptake of vitamin B12 and normal
embryonic development in humans. When considering the data from the
Human Genome Project, the human genome sequence appears to
encode far fewer proteins than predicted, indicating that some genes
may encode a protein with more than one function (Jeffery, 2003, 2004).
Even though we found no clear-cut mutations in the AMN gene
explaining the RSA in the couples studied, the result of the genealogic
studies are interesting and suggest the possibility of an unknown gene
causing the spontaneous abortions in some of the patients. The
birthplaces of the parents and grandparents of a subset of the patients
showed clustering to a region previously known as the province of
Kuopio. That the region around Helsinki is overrepresented compared
with the reference map is explained by the fact that all patients were
treated in Helsinki, but this does not explain the clustering around
Kuopio. This clustering suggests that some of these patients may have
ancestors in common, resulting in an enhanced susceptibility for
spontaneous abortion. Such a phenomenon would not be exceptional in
Finland due to the population history. A small number of original
founders, followed by isolation and rapid expansion in regional
subisolates, have resulted in enrichment of some otherwise rare
disease alleles and the absence of others. This has increased the local
incidence of rare recessive disorders, and regional clustering of cases
can still be observed in some Finnish diseases (Norio, 2003a,b). For
example, mutations in the FSH receptor (FSHR) gene cause infertility
due to ovarian failure. FSH-resistant ovaries (FSH-RO) is an
autosomal recessive trait which shows geographical enrichment of
ancestral birthplaces (Aittomki et al., 1995, 1996).
One limitation of the study may be the number of couples studied.
The aetiology of RSA is likely to be very heterogenous, and mutations
in AMN may cause fetal loss in only a small subpopulation of couples
with spontaneous abortions. Mutations in AMN seem to be rare, and
therefore one may argue that more couples would be needed to determine
the exact role of these genes in RSA. Another limitation is the fact that
some sequence variations in the studied samples may have gone
undetected. Even though DHPLC has been shown to be a highly sensitive
and specific method for mutation detection (sensitivity and specificity
of DHPLC exceed 96%), there are also rare instances in which
mutations may be missed (Xiao and Oefner, 2001). Mutations located in a
high-melt GC-rich pocket in a fragment with otherwise normal
nucleotide ratios may in some cases not be detected, and some variations
can only be detected at one unique temperature.
In conclusion, the results of this study suggest that homozygous
mutations in the AMN gene are not a major cause of RSA. However,
the phenotypic effects of the potentially deleterious variations in
homozygous or compound heterozygous fetuses cannot be determined
without further investigations. The genealogic studies of our patients
ancestral birthplaces indicate that there may be a subset of patients
with an underlying genetic cause for spontaneous abortions.
Therefore, other candidate genes need to be considered. It is, however,
particularly complicated to study conditions in which homozygous
mutations are expected to be lethal in early pregnancy because of the
need to obtain DNA from spontaneously aborted fetuses.
Furthermore, there are no clinical features by which subgroups of affected
pregnancies could be identified for further studies. While classical
linkage studies cannot be performed, further candidate genes
identified in animal models should be screened to identify new genetic
factors contributing to spontaneous abortions.
We thank Dr Stephan Tanner (Group of Prof. Albert de la Chapelle, The Ohio
State University) for providing primer sequences and the couples with RSA for
participating in this study. The study was supported by the Sigrid Juselius