 1996 Oxford University Press
Human Molecular Genetics, 1996, Vol. 5 Review
1505–1514
The factor IX gene as a model for analysis of human
germline mutations: an update
Steve S. Sommer* and Rhett P. Ketterling
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905 USA
Received June 25, 1996
The variation generated by germline mutation is essential for evolution, but individuals pay a steep price in the
form of Mendelian disease and genetic predisposition to complex disease. Indeed, the health of a species is
determined ultimately by the rate of germline mutation. Analysis of the factor IX gene in patients with hemophilia
B has provided insights into the human germline mutational process. Herein, seven topics will be reviewed with
emphasis on recent advances: (i) proposed mechanisms of deletions, inversions, and insertions; (ii) discordant
sex ratios of mutation and associated age effects; (iii) somatic mosaicism; (iv) founder effects; (v) mutation rates;
(vi) the factor IX gene as a germline mutagen test; and (vii) cancer as a possible mechanism for maintaining a
constant rate of germline mutation.
INTRODUCTION
The human factor IX gene has highly advantageous properties for
the study of germline mutations (see Table 1). By careful attention
to the possible biases [e.g., see (1–5)], it has been possible to
deduce the underlying pattern of mutation at this locus. Recent
observations in this system will be emphasized; more detail on
earlier work may be found in previous reviews (3,6,7). For
overviews of human germline mutation, see Cooper and Krawczak (8) and the ‘Mutation Update’ section of the journal Human
Mutation. An annual database of factor IX germline mutations
currently lists 1380 point mutations, microdeletions and/or
microinsertions (9). The wealth of missense mutations can now
be located within a recently available crystal structure (10).
Since many independent point mutations are available, the
factor IX gene has been utilized to develop and/or validate
multiple methods of mutation detection (e.g., PASA, double
PASA, and SNuPE) (11–13) or mutation screening (chemical
cleavage, rSSCP, ddF, Bi-ddF, REF, and heteroduplex analysis)
[(14–20), respectively]. In addition, recent reviews document that
study of the factor IX gene has provided insight into other fields: e.g.,
structure-function analyses (21), genotype-to-phenotype analyses
(22), gene regulation (23), molecular diagnosis/genetic counseling
(24), and gene therapy (25–27).
I. MECHANISMS OF INSERTIONS AND DELETIONS:
INTERACTING ALTERNATING PURINE-PYRIMIDINE
HAIRPIN LOOPS AS A NOVEL MECHANISM FOR
CONCERTED DELETIONS
Microinsertions/microdeletions (20 bp) are more common
than large deletions or insertions (see Table 4 in ref. 28,).
Microinsertions presumably derive from polymerase slippage
events since a direct repeat is generated in nearly every reported
factor IX microinsertion (Ketterling et al., in preparation). In
contrast, most factor IX microdeletions do not eliminate repeat
*To whom correspondence should be addressed
units. Most of the reported microdeletions are unique, but
hotspots have been detected at two sites (29). These microdeletion hotspots generate perfect hairpin loops from imperfect, or
quasipalindromic, sequences. Such quasipalindromic sequences
were found previously to be hotspots of microdeletions in E. coli and
yeast (30,31).
Genes with either highly homologous segments (e.g., steroid
sulfatase and α-globin) or excessive gene length (e.g., dystrophin) are predisposed to large deletions (i.e., >20 bp) (32–34).
However, in the great majority of genes, large deletions generally
account for only 5–10% of the observed mutations. In the factor
IX gene, large deletions account for 6% of observed, independent
mutations. Insertions and duplications are much rarer than large
deletions (35). Only one transposition of an Alu1 sequence and
only one partial gene duplication have been described (36,37).
While pure insertions are rare, deletions are often associated with
insertions. Of the nine large deletions with junctions that have
been defined in the 33 kb factor IX gene, five are associated with
insertions (28). The insertions can be inversions of segments
within the deleted regions, orphan sequences (i.e., novel sequences of unknown origin), or both. Two factor IX deletions
with inversions but no orphan sequences fit a proposed ‘doubleloop model’ (38). In brief, a ‘double-loop’ structure forms when
two short (5–7 bp) sets of complementary sequences anneal
within single-stranded DNA, presumably during DNA replication or repair. Subsequent endonuclease cleavage, DNA repair,
and mismatch correction delete segments in both loops and invert
the loop connector. Since the sequence complementarity required
for the double-loop mechanism is substantial, the likelihood is
very small (p <10–8) that the required sequences are present at the
deletion-inversion junctions by random chance. Recently, other
replication-based models for deletions with inversions have been
proposed (39). These models are distinct from recombination at
highly homologous repeats, a mechanism responsible for the high
frequency of inversions in severe hemophilia A (40,41).
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Long segments of alternating purine and pyrimidine segments
[RY(i)] in the factor IX gene occur in intron 1 and in the 3′
untranslated region (42,43). These RY(i) are ‘cryptic’ repeats
because the simple nature of the repeat unit is only apparent when
the sequences are categorized into purines and pyrimidines.
These RY(i) are hotspots of microdeletions/microinsertions with
mutation rates, such that race-specific alleles tend to occur
(43,44). The eight different alleles found at the long cryptic repeat
in intron 1 seem to result from a combination of polymerase
slippage events and recombination (43).
These cryptic RY(i) sequences may well predispose to large
deletions (28). Of the 17 defined factor IX deletion junctions
reviewed, RY(i) were present at one or both of the junctions 18%
of the time, although such segments constitute only 0.7% of the
factor IX gene sequence. The mutation in one patient, HB128, is
of particular interest. The patient initially was thought to have a
24.5 kb deletion without insertions, but subsequent analysis
revealed a downstream 50 bp deletion at a distance of 2.3 kb (Fig.
1A). Haplotype, sequence, and paternity analyses determined that
these ‘two’ deletions occurred in the germline of the maternal
grandfather (45).
Long hairpin loops composed of the RY(i) in the factor IX gene
are present at the flanking deletion junctions in HB128 (Fig. 1A).
Are seemingly simple deletions, in which one of the junctions occurs
in a hairpin sequence, associated with a deletion in a neighboring
hairpin region? Surprisingly, few deletion junctions have been
sequenced in genes without known highly homologous repeats. The
junction sequences for 18 large simple deletions (i.e., without
insertions) from multiple genes were analyzed to identify potential
nucleic acid secondary structures. A deletion junction with a large
RY(i) hairpin was found in the type III procollagen gene from a
patient with Ehlers-Danlos type 4 (46) (Fig. 1B). Thus, another
ostensibly simple deletion has a signature RY(i) hairpin. The
presence of another RY(i) hairpin at one of these 18 deletion
junctions is a highly nonrandom event, since only 0.1% of human
genomic sequence contains RY(i), and the overwhelming majority
of RY(i) are not self-complementary (42). Unfortunately, neither
intronic nor flanking intragenic sequence was available for the type
III procollagen gene so neighboring RY(i) hairpins could not be
identified to determine if a second deletion is present.
How might interactions between RY(i) initiate concerted
deletions? In this regard, it is intriguing that stable four-stranded
structures spontaneously result from the association of RY(i)
sequences (47). These structures can spontaneously form from
duplex DNA in the absence of protein. Furthermore, gel-retardation assays demonstrate that the four-stranded structures can
associate specifically with nuclear-high-mobility-group proteins
1 and 2. Perhaps illegitimate recombination at such structures
initiates concerted deletions.
II. MALE:FEMALE SEX RATIOS OF MUTATION (SRM)
AND MATERNAL AGE EFFECTS
In an analysis of 15 families from Sweden with sporadic
hemophilia B in which the mother was either a carrier (n = 12) or
a noncarrier (n = 3), an aggregate SRM of 11 was calculated
(48,49). These non-Bayesian calculations did not take the
pedigree structure of the ascertained families into account and
assumed an equilibrium between selection and mutation. Other
non-Bayesian analyses in which germline origins in mothers and
grandmothers were pooled and compared to origins in grandfathers yielded aggregate SRM values of 2 (50,51). A Bayesian
analysis of 29 factor IX origins, which considered pedigree
structure, estimated an overall SRM for factor IX germline
origins of 3.5 (95% confidence interval of 1.5–8.4) (4). Recently,
this Bayesian analysis was extended to include a total of 59
defined origins of mutation. Based on these data, the SRMs were
estimated for six types of mutation (Table 2). The estimated
aggregate SRM was 3.75, but the SRMs ranged from 6.65 and
6.10 for transitions at CpG and A:T→G:C transitions at non-CpG
dinucleotides, respectively, to 0.57 and 0.42 for microinsertions/
microdeletions and deletions (>20 bp), respectively. Surprisingly,
the two subtypes of non-CpG transitions possessed markedly
different SRMs (6.10 for A:T→G:C vs. 0.80 for G:C→A:T). In
contrast, inversion mutations in the X-linked factor VIII gene,
which are presumptively due to homologous recombination, are
almost exclusively paternal in origin (estimated SRM of 302)
(52,53).
Table 1. Advantages of the Hemophilia B/factor IX model for studying human germline mutation
Hemophilia B
Common Mendelian disease for which care is centralized
X-linked recessive disease of historically low reproductive fitness; i.e., most mutations have occurred within the past 150 years.
The clinical diagnosis implies a mutation at the factor IX locus
Independent mutations occur in 95+% of all families with severe/moderate disease
Almost all cases in the population are diagnosed
A small reduction in enzymatic activity produces disease
Epidemiological data is available on the incidence of de novo disease
Fetal survival is uncompromised
Little, if any, heterozygote advantage occurs in females
Factor IX Gene
Analysis of 2.2 kb detects the mutation in 96% of patients
Full gene sequence (33 kb) is available when needed
No repetitive domains with similar function in the protein
The known mutations cause only one disease phenotype
Few polymorphisms and most residues are critical or spacers—there is seldom doubt about whether a sequence change is the causative mutation
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Figure 1. (A) Large hairpin structures that can form in the RY(i) sequences located in intron 1 and in the 3′ UTR of the factor IX gene. The RY(i) deletion breakpoints
found in patient HB128 are indicated by brackets and are labeled. (B) Large RY(i) hairpin structure that can form within intron 24 of the type III procollagen gene.
The deletion breakpoints found in a patient with Ehlers-Danlos syndrome type 4 are indicated by brackets (46).
Shimmin et al. (54) estimated the aggregate SRM during
primate evolution by examining intronic regions from homologous zinc finger genes on both the X and Y chromosomes. Their
data consisted of point mutation analyses and suggested an
overall ‘male-driven evolution’ (estimated SRM = 6). A similar
analysis in rodents suggests a SRM of only two (55,56).
Does parental age at conception affect the risk of mutation?
Oocytes arrest in meiosis at five months gestation while
spermatocytes undergo mitosis approximately every 14 days after
puberty. By age 30, about 15-fold more divisions occur in the
male germline than in the female germline (57). If mutations
occur during DNA replication, the male germline should have a
much higher mutation rate than the female germline due to
continual post-pubertal mitotic divisions. In addition, the likelihood
of germline mutation should increase in older males (paternal age
effect).
While studies of some Mendelian X-linked and autosomal
dominant diseases generally found an advanced paternal age
effect [reviewed in (57)], studies of other diseases found only a
mild or absent paternal age effect (58–61). In these studies, the
causative mutation generally was not defined. In addition, the
influence of imprinting on autosomal genes may play an
important role and affect interpretation of the age analyses.
Studies of age effects on X-linked genes avoids the bias of
imprinting. Mutation screening coupled with direct genomic
sequencing facilitates the detection of germline origins. However,
appropriate interpretation of the data depends critically on at least
six ascertainment biases, i.e., the biases of: pedigree structure,
family pursuit, generational proximity, gender-based inferences,
mutation detection, and sex ratio [see (4); Ketterling et al.,
submitted].
Paternal origins of mutation have been defined for 22
single-base substitutions and three deletions in the factor IX gene
(Ketterling et al., submitted). The average age percentiles for the 18
Caucasian paternal origins were not elevated for any mutation
subtype or in total (51.4%) (Fig. 2A). In contrast, the age percentiles
of the 23 Caucasian maternal origins were elevated for non-CpG
transitions (68.7%) and transversions (78.5%) and in total (68%)
(Fig. 2B). The markedly unequal distribution seen for the maternal
age percentiles was statistically significant (p = 0.03).
The absence of a paternal age effect suggests that errors during
DNA replication do not play the dominant role in the generation
of germline mutations. Perhaps certain types of mutations occur
preferentially in the male germline because of less efficient DNA
repair, higher levels of reactive cellular metabolites, or differences in DNA or chromatin structure. For at least one type of
mutation with a high SRM, transitions at the dinucleotide CpG,
the known mechanism of mutation strongly suggests that the
SRM is due to profound deficiency in DNA methylation in
oocytes (62), rather than replication errors.
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Table 2. Sex Ratios of Mutation (SRM) estimated from 59 germline origin families with hemophilia Ba
Mutation type
# Origins defined
Mg
CpG transitions
Non-CpG transitions (G:C → A:T)
Non-CpG transitions (A:T → G:C)
Transversionsf
Microdel./microins. (<20 bp)c
Large deletions (>20 bp)
All mutations
3
3d
2
7
5
3
23
MMg
MFg
Estimated
SRM
95% Confidence
Intervals
0
4e
1
1
2d
2
6
2d
5
11b
1
1
6.65
0.80
6.10
3.61
0.57
0.42
1.67–32.74
0.11–3.46
1.43–30.0
1.36–10.19
0.03–3.24
0.02–2.90
10
26
aFrom
Ketterling et al., submitted. See [(4); Methods] for detailed explanation of how the SRMs were calculated.
two paternal origins in families with female hemophiliacs.
cIncludes one microinsertion and one combined microdeletion/microinsertion; all others are microdeletions. dIncludes one somatic mosaic.
eIncludes two somatic mosaics.
fThe only transversion subtype with large enough numbers to calculate a SRM was G:C→T:A, 2.42 (0.52–12.26).
gM = mother; MM = maternal grandmother; MF = maternal grandfather.
bIncludes
Why is there an increased age effect in females? Does the
cellular machinery become error prone after being in stationary
phase for many years? The generality of the maternal age effect
will need to be assessed as germline origins for point mutations
accumulate in other Mendelian disorders.
III. MOSAICISM: A HIGH RATE OF G:C→A:T NON-CPG
TRANSITIONS EARLY IN EMBRYOGENESIS
Mosaicism has been documented for chromosomal aberrations,
mitochondrial mutations, triplet repeat diseases and a growing
number of dominant and X-linked single gene disorders. Somatic
mosaicism is sometimes responsible for mild phenotypic manifestations of a new mutation (63) and could allow the survival of a
normally lethal phenotype as hypothesized for the McCune-Albright
syndrome (64). As a result of numerous cell divisions during life,
each individual displays some degree of mosaicism, but most of
these mutations are phenotypically silent [see calculations in (65)].
The effect of a mutation that causes a loss of gene function depends
on the stage of development at which it occurs: a mutation in an
embryo might result in malformations (66) or inborn errors of
metabolism. In an adult, mutations can cause malfunction (as in
aging) or overgrowth (as in neoplasia) (63). The phenotypic
manifestation also depends on the tissues involved. An early
mutation before lineage allocation results in both germline and
somatic mosaicism. Isolated germline mosaicism arises from a
mutation in a precursor cell committed to only germline development. Additionally, a mutant allele might survive only in certain
tissues (i.e. germline) because it is lethal for other cell types. Detailed
studies in Duchenne’s muscular dystrophy (DMD) have illustrated
the clinical importance of germline mosaicism in a human disease
(67,68). Bakker et al. (67) found that six of 41 (15%) DMD families
had detectable germline mosaicism while van Essen (68) found a
recurrence risk of 20% for subsequent siblings due to maternal
germline mosaicism. Other than the above studies on deletions,
reports of germline or somatic mosaicism have historically been
limited to single case reports.
Of the factor IX germline origins determined in 59 families by
haplotype and sequence analyses, no examples were identified in
which the mutant allele was transmitted to more than one of the
children tested. However, in four origin individuals (Ketterling et
al., submitted), somatic mosaicism and, by implication, germline
mosaicism occurred. In one additional family, a mosaic female
with an approximately 50:50 ratio of wildtype:mutant signal in
leukocytes had one carrier and four noncarrier daughters who
inherited the haplotype associated with the mutation (69). Thus,
in total, of the 45 origins which could be visualized directly by
sequence analysis, five (11%) exhibited mosaicism due to a
mutation within a few cell divisions after fertilization. Seventeen
families were analyzed recently by methods which should detect
mosaicism at frequencies of 1–0.01% (70). Early embryogenesis
may be a highly mutagenic period while later embryogenesis is
less mutagenic, since no additional somatic mosaicism was
identified in these families.
Four of these five mosaics had G:C→A:T non-CpG transitions
(p = 0.01). Is this non-CpG transition subtype truly associated
with somatic mosaicism? Is early embryogenesis a highly
mutagenic period of development, particularly for G:C→A:T
non-CpG transition mutations? While there are multiple
examples of somatic mosaicism associated with germline deletions [e.g., (67,71–73)], only five other examples of somatic
mosaicism associated with single-base germline substitutions
were found in the literature (74–78). It is intriguing that three of
these five changes were G:C→A:T non-CpG transitions. Thus,
seven of ten germline, single-base substitutions associated with
somatic mosaicism were G:C→A:T non-CpG transitions.
IV. HEMOPHILIA B FOUNDER EFFECTS: A SMALL
AMINO ACID TARGET FOR MILD DISEASE
Four founder effects are prevalent in U.S. Caucasian hemophiliacs (79–83). The founder mutations are responsible for about
30% of all disease cases (84/276 mutations in one study) (83).
The G60S and T296M mutations reproducibly cause clinically
mild hemophilia, while the R248Q and I397T mutations cause
borderline mild/moderate disease. The origins of the G60S,
R248Q, T296M, and I397T founders appear to be English/Dutch,
Irish, German/Amish, and French, respectively. In addition to the
predominant founder mutation, haplotype analysis reveals occasional independent mutations for G60S, R248Q and T296M
which occur at a CpG dinucleotide (83).
Putative founder effects at R248Q and T296M in MexicanHispanic patients with hemophilia B account for 19% of the
observed mutations in the Mexican-Hispanic population (83).
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of two known mutations in patients with mild disease (84). Thus,
a significant number of the mutations causing mild or mildmoderate disease result from mutations at G60S, R248Q, and
T296M. This conclusion is supported by previous populationbased studies of factor IX mutations which show that 37% of
patients have mild disease (85). Of these, approximately twothirds are due to founder effects in the U.S. Caucasian population
(82), suggesting that 13% of all independent disease causing
mutations in the factor IX gene result in mild hemophilia B. Thus,
the factor IX gene generally behaves like an ‘on-off switch’, i.e.,
most missense mutations will not cause any substantial loss of
factor IX activity, while the majority of the remaining missense
mutations will cause a near complete loss (>20-fold) of protein
function. Mutations at very few sites produce mild disease in
which there is a 4-fold to 20-fold loss of factor IX coagulant
activity (85). Since the small target size for mutations causing
mild disease includes three CpG transitions, known hotspots for
mutation, missense changes at these sites account for a significant
fraction of all independent mutations.
V. MUTATION RATES
Figure 2. (A) Bar graph distribution of age percentiles for 18 paternal origins.
These include only those Caucasian families in which both the age at
conception and the year in which the first carrier/hemophiliac was born are
known. (B) Bar graph distribution of age percentiles for 23 Caucasian, maternal
origins.
However, haplotype analysis demonstrates that the Mexican
founders are different than those in the U.S. Caucasian population. In Turks, an independent T296M mutation accounts for one
The mutations observed in patients with Mendelian diseases
result from: the underlying pattern of mutation, the biology of the
gene, the biology of the disease, and ascertainment biases. The
underlying mutational pattern is often heavily distorted by the
latter factors. The dystrophin gene illustrates the point. The 2.2
Mb gene is a huge target for deletions, larger than the complete
genome of H. influenza. In addition, most of the dystrophin
protein product is composed of repetitive domains. Most
missense mutations do not cause disease and even deletions of
multiple domains may cause only mild disease if the proper
reading frame is maintained (86). Thus, the enhancement of large
deletions observed in most patients with Duchenne muscular
dystrophy reflects the biology of the gene rather than the
underlying pattern of mutation at that locus.
The rate of germline mutation per basepair per generation has
been estimated by many different methods and these estimates
vary from 2.5–90 × 10–9 (1,57,87–90). The two most direct
methods yielded estimates of 10 × 10–9 (from electrophoretic
variants of polypeptides) (88) and 8.6 × 10–9 (from a meta-analysis of 40 cases worldwide of de novo mutations producing
unstable hemoglobin or hemoglobin M) (89). For the reasons
outlined in Table 1, the analysis of patients with hemophilia B has
provided an ideal model for deducing the underlying pattern of
germline mutation within the past 150 years [see (3) for details].
A compilation of the underlying mutation rates in the factor IX
gene (Table 3) suggests that transitions and transversions at
non-CpG dinucleotides occur 15-fold and 7-fold more frequently
than microdeletions/microinsertions, respectively. Transitions
and transversions at CpG dinucleotides are enhanced 17-fold and
5-fold relative to transitions and transversions at non-CpG
dinucleotides, respectively. In combination, transitions and
transversions should account for 96.1% of the types of de novo
mutations amenable to analysis with this system. If the underlying
mutation rates in the factor IX gene are typical of the genome, the
aggregate mutation rate is about 3.6 × 10–9.
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Table 3. Absolute and relative rates of germline mutations in the gene encoding factor IX and estimation of the number of mutations per genome per generationa
Mutation
type
Mutation
rates for
the factor IX
gene (× 10–10)
Rates relative
to microdeletions
or
microinsertions
Estimated genomic
target size
(× 109 bp)
Estimated number
of de novo mutations
per diploid genome
Transitions at CpGb
Transversions at CpGb
Transitions at non-CpGc
Transversions at non-CpGc
Microdeletions and
microinsertionsd
Deletionse
Totalf
Overall mutation ratef
360
45.4
20.6
8.89
1.36
265
33.4
15.1
6.54
1
0.0384
0.0384
2.96
2.96
3.0
2.76
0.35
12.2
5.26
0.82
0.037
0.03
3.0
0.02
21.41
35.7 × 10–10
aThe
relative rates are likely more accurate than the absolute rates. The mutation rates are calculated per basepair per generation. From (7).
will be lower at partially methylated sites.
cValues were calculated from all missense mutations that cause hemophilia B.
dValues were calculated for microdeletions and microinsertions of 1–20 bp (Ketterling et al., unpublished).
eValues were calculated for deletions of 200–200 000 bp as the likelihood that the junction of the deletion would occur at a given base (28). The rate of deletions
for the range 21–200 bp is unknown, but the mutation rate is unlikely to be greater than 0.256 × 10–10 (p <0.05).
fThe overall mutation rate = total number of mutations divided by 6 × 109 bp. The total number of mutations is calculated by multiplying the mutation rate by the
estimated target size for each type of mutation, multiplying by two to give the number of mutations per diploid individual, and summing the values.
bValues
When these mutation rates are extrapolated to the genome, each
person should have about 21 de novo germline mutations [12.2
non-CpG and 2.8 CpG transitions, 5.3 non-CpG and 0.4 CpG
transversions, 0.8 microdeletions/microinsertions, and 0.02 large
deletions (>20 bp)]. Some of these de novo mutations are more
likely to produce functional defects than others, i.e., while only
0.1% of all underlying mutations are deletions, deletions account
for 5% of all observed mutations causing hemophilia B.
VI. THE FACTOR IX GENE AS A ‘GERMLINE
MUTAGEN TEST’
At least four lines of evidence suggest that endogenous processes
are overwhelmingly the cause of germline mutations [reviewed
in (3,7)]: (i) about 1/4 of all independent mutations causing
hemophilia B are transitions at CpG (1,2,91) which arise
secondary to spontaneous deamination of endogenously methylated cytosines; (ii) the germline mutational pattern found in all but
one ethnic population (see below) is compatible with the
postulated ancient mutational pattern that fashioned the G+C
content of 40% and the nonrandom dinucleotide frequencies
within the factor IX locus (92); (iii) the pattern of mutation
observed in factor IX deficient patients has remained constant
when compared with the evolutionary pattern deduced from
direct sequence analysis of factor IX introns in primates (Grouse
et al., manuscript in preparation); and (iv) the pattern of
polymorphism in human factor IX intronic sequences and the
pattern of sequence changes fixed during primate evolution are
highly similar to the mutational pattern observed in patients with
hemophilia B (Feng et al., manuscript in preparation). With the
exception of ubiquitous mutagens such as cosmic rays, the pattern
of germline mutation in hemophiliacs should vary between
populations if environmental mutagens predominated because:
(i) different environments, diets, or lifestyles should lead to
differential mutagen exposures, and (ii) mutagens tend to produce
highly recognizable patterns or ‘fingerprints’ of mutation (93).
Extensive data on somatic mutations in the p53 gene illustrate the
highly specific patterns of mutation that are produced by
mutagens such as ultraviolet radiation, aflatoxin, and cigarette
smoke (94).
Deviation from the germline mutational pattern is expected if
a population is exposed to physiologically important germline
mutagens or if there are relevant genetic differences. Even small
differences in the overall mutation rate can alter the pattern of
mutation significantly. Thus, the factor IX gene can serve as a
molecular epidemiological screen for the presence of a significant
germline mutagen within a population. Previously, the pattern of
germline mutation in the factor IX gene was found to be similar
in a small Asian (mostly Korean) hemophilia B sample and a U.S.
Caucasian sample of mostly Western European descent (95).
Subsequently, similar mutational patterns were observed in a
larger study in which direct sequence analysis was coupled with
haplotype analysis to determine recurrent, independent mutations
from common ‘founder’ mutations, in the following hemophilia
B populations: (i) 22 Asians; (ii) 22 U.S. minorities (Hispanics,
Blacks, and AmerIndians); (iii) 24 patients whose mutations
originated outside of the United States, Canada, and Europe; (iv)
127 Caucasians of Western European descent; and (v) 32
Mexican-Hispanics (83,96).
Recently, 36 independent sequence changes were defined in
Chinese hemophilia B patients from the Peoples Republic of
China (PRC) (Liu et al., submitted). The pattern of mutation in the
PRC Chinese population differs significantly from the pattern
previously described in U.S. Caucasians (p = 0.01; Fisher exact
test) (Table 4). Similar results were obtained when the categories
of mutation were divided further to examine each of the six types
of transitions and transversions at non-CpG dinucleotides.
Pairwise analysis for each mutation type indicates that, in
comparison with the U.S. Caucasian hemophilia B sample, the
PRC hemophilia B patients have fewer non-CpG transitions (p =
0.008 for this category of mutation versus all other mutations) and
a moderate increase in microdeletions/microinsertions (p = 0.09
for this category versus all others).
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Table 4. Pattern of independent germline mutationsa
Sample
U.S. Caucasians
Total # independent
Transitions
mutations
at CpG
158
27%
Transversions
not at CpG
34%
at CpG
3%
not at CpG
20%
Microdeletions
Large
/microinsertions
deletions
(20 bp)
(>20 bp)
11%
7%
PRCb
36
36%
11%
0%
31%
22%
0%
U.S. Caucasian, Severe Disease
101
20%
35%
1%
20%
15%
10%
PRCb, Severe Disease
26
46%
12%
0%
15%
27%
0%
Chinese from Taiwan and Hong Kongc
26
27%
27%
4%
31%
12%
0%
aFrom
Liu et al., submitted.
= People’s Republic of China.
cOther Chinese mutations published previously (105–107).
bPRC
To control for the possibility that mild and moderate hemophilia B may be underrepresented in the PRC sample, the patterns
of mutation were compared for families with severe hemophilia
B (Table 4). Comparison of the 26 PRC patients and the 101 U.S.
Caucasian patients with severe disease confirm that the observed
patterns of mutation differ significantly (p = 0.01). These
different mutational patterns may reflect genetic differences
between the two populations. However, while the previously
analyzed Asian population, (predominantly Korean) is genetically similar to the Chinese, the mutational pattern is similar to
U.S. Caucasians (p = 0.73). In addition, Chinese patients from
Taiwan and Hong Kong have an intermediate pattern which is not
significantly different from the U.S. Caucasian or PRC patterns.
While these data are intriguing, additional study is needed to
distinguish between mutagen exposure, genetic differences and
ascertainment biases/statistical artifact.
VII. DOES CANCER PROTECT THE INTEGRITY
OF THE GENOME?
Since multiple lines of evidence support endogenous control of
recent germline mutations in the factor IX gene at a level
compatible with the ancient pattern that shaped the mammalian
genome (see above), it has been speculated that an optimal
germline mutation rate might exist in humans and other mammals
at a level just sufficient to maintain adequate biological variation
(97). In brief, sexual reproduction and recombination enhance
variation, but ultimately new germline mutation is required to
replenish variant alleles that are lost by negative selection, genetic
drift, and population bottlenecks. If selection maintains an
optimal rate of mutation, cancer may be the most important
mediator (98). Early onset cancer can result if somatic cells are
affected by germline mutations that generate high levels of
mutagenic byproducts or compromise DNA repair or replication.
The multiple mutations necessary for neoplasia serve to amplify
any differences in the mutation rate, thereby providing an
efficient selection against individuals that have even small
increases in the rate of germline mutation. From this viewpoint,
the similar fraction of humans, dogs, or outbred mice that develop
early-onset cancer (99,100) (before the end of the female
reproductive period) include individuals who: (i) experience the
misfortune of developing early onset somatic mutations in the
requisite tumor suppressor genes and oncogenes; (ii) inherit a
germline defect in a tumor suppressor gene (or oncogene) such as
individuals with familial retinoblastoma or Li-Fraumeni syndrome; or (iii) inherit germline defects in any of hundreds of genes
that affect the rate of germline mutation. For the unfortunate
individuals who inherit germline defects that could increase the
rate of germline mutation, early onset cancer serves to guard the
integrity of the genome and thereby the overall health of the
species.
Hereditary nonpolyposis colorectal cancer (HNPCC), an
autosomal dominant disease associated with colon and multiple
other types of cancer, illustrates how ‘mutator’ mutations can be
eliminated efficiently from the germline often before there is any
increase in the rate of germline mutation. Patients with the disease
carry a germline defect in one allele of MSH2, or another gene
critical for mismatch repair (101). While the somatic mutation
rate in heterozygotes may not be detectably elevated, a spontaneous, deleterious somatic mutation in the second allele is
associated with a dramatic increase in the rate of additional point
mutations in other genes (102). In addition, marked instability of
microsatellite sequences also has been observed (103). Germline
mutations in the MSH2 gene are eliminated rapidly from the
population within a few generations, because a substantial
fraction of individuals with these mutations (∼30%) contract
cancer between ages 10–40 (104).
In conclusion, study of the factor IX gene continues to
illuminate the germline mutational process. It will be important
to assess locus-specific differences by performing similar detailed analyses in additional Mendelian diseases for which the
underlying pattern of mutation can be inferred.
ACKNOWLEDGEMENTS
We thank the many Hemophilia Centers who provided the patient
samples essential to these studies. We thank Mary Johnson for
excellent secretarial assistance. R.P.K. is a Howard Hughes
Medical Institute Medical Student Research Training Fellow.
This work was supported by HL39762. Dedicated to Walter
Bowie, M.D., whose initial support and encouragement made a
great difference.
1512
Human Molecular Genetics, 1996, Vol. 5, Review
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