ATM Gene Founder Haplotypes and ...

ATM Gene Founder Haplotypes and Associated Mutations in Polish Families with Ataxia-Telangiectasia, ATM

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doi: 10.1111/j.1529-8817.2005.00199.x
ATM Gene Founder Haplotypes and Associated
Mutations in Polish Families with Ataxia-Telangiectasia
M. Mitui
1
,E.Bernatowska
2
,B.Pietrucha
2
,J.Piotrowska-Jastrzebska
3
,L.Eng
1
,S.Nahas
1
,
S. Teraoka
4,5
,G.Sholty
1
,A.Purayidom
1
,P.Concannon
4,5
and R. A. Gatti
1,
∗
1
Department of Pathology and Laboratory Medicine, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095–
1732, USA
2
Department of Immunology, The Children’s Memorial Health Institute, Warsaw, Poland
3
Department of Pediatrics, The Medical University of Bialystok, Bialystok, Poland
4
Department of Molecular Genetics, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101, USA
5
Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA
Summary
Ataxia-telangiectasia (A-T) is an early onset autosomal recessive ataxia associated with characteristic chromosomal
aberrations, cell cycle checkpoint defects, cancer susceptibility, and sensitivity to ionizing radiation. We utilized
the protein truncation test (PTT), and single strand conformation polymorphism (SSCP) on cDNA, as well as
denaturing high performance liquid chromatography (dHPLC) on genomic DNA (gDNA) to screen for mutations
in 24 Polish A-T families. Twenty-six distinct Short Tandem Repeat (STR) haplotypes were identiﬁed. Three
founder mutations accounted for 58% of the alleles. Three-quarters of the families had at least one recurring
(shared) mutation, which was somewhat surprising given the low frequency of consanguinity in Poland. STR
haplotyping greatly improved the efﬁciency of mutation detection. We identiﬁed 44 of the expected 48 mutations
(92%): sixty-nine percent were nonsense mutations, 23% caused aberrant splicing, and 5% were missense mutations.
Four mutations have not been previously described. Two of the Polish mutations have been observed previously
in Amish and Mennonite A-T patients; this is compatible with historical records. Shared mutations shared the
same Single Nucleotide Polymorphism (SNP) and STR haplotypes, indicating common ancestries. The Mennonite
mutation, 5932 G
>
T, is common in Russian A-T families, and the STR haplovariants are the same in both Poland
and Russia. Attempts to correlate phenotypes with genotypes were inconclusive due to the limited numbers of
patients with identical mutations.
Keywords: ATM mutations, Polish, Amish, Mennonite, Haplotypes
Introduction
of cancer (Gatti & Good, 1971; Swift
et al.
1986). They
typically manifest premature aging, degeneration of the
cerebellum, thymus and gonads, growth retardation, and
telomere shortening (Gatti, 2002; Chun & Gatti 2004).
Carrier frequencies of ATM mutations have been esti-
mated as 1–1.8% and are proving signiﬁcant with regard
to breast cancer susceptibility (Swift
et al.
1987; Easton,
1994; Gatti
et al.
1999; Concannon, 2002; Buchholz
et al.
2004).
A-T is caused by mutations in the
A
taxia-
T
elangiectasia
M
utated gene (
ATM
) located at 11q23.1
(Gatti
et al.
1988; Lange
et al.
1995; Savitsky
et al.
1995).
The
ATM
gene is over 150 kb in size and includes 62
Ataxia-telangiectasia (A-T; MIM # 208900) is an auto-
somal recessive, neurological disorder with a frequency
of 1/40 000–1/100 000 (Gatti, 2002). Cerebellar ataxia,
immunodeﬁciency, oculocutaneous telangiectasia, and
radiation sensitivity are characteristic ﬁndings in A-T
patients. These patients also have a greatly increased risk
∗
Correspondence to: Richard Gatti, The David Geffen School
of Medicine, Department of Pathology, Los Angeles, CA
90095-1732, Phone (310) 825-7618, Fax (310) 825-7618.
E-mail: rgatti@mednet.ucla.edu
University College London 2005
Annals of Human Genetics (2005)
69
,657–664
657
C
Mitui
et al.
coding exons, encoding a 13 kb main transcript, with
an open reading frame of 9168 bp (Uziel
et al.
1996;
Platzer
et al.
1997). The ATM protein is 370 kDa, is
found predominantly in the cell nucleus, and is a pro-
tein serine/threonine kinase (Shiloh, 2003; Bakkenist &
Kasdan, 2003).
A-T patients are typically compound heterozygotes
carrying unique mutations, and no “hot spots” in the
ATM
gene have been found (Mitui
et al.
2003). There-
fore, the entire gene must be screened to determine
the two disease-causing mutations for each patient. Our
strategy for ATM mutation screening has been to ﬁrst
perform SNP and STR haplotyping (Mitui
et al.
2003;
Coutinho
et al.
2004), followed by PTT (Telatar
et al.
1996; Den Dunnen
et al.
1999), SSCP (Castellvi-Bel
et al.
1999) or dHPLC (Bernstein
et al.
2003). Finally,
each relevant genomic region is sequenced to identify
the mutation.
Previous studies have shown that STR haplotyping
can greatly increase mutation detection in ethnic pop-
ulations by associating founder mutations with their
STR haplotypes (Uhrhammer
et al.
1995; Telatar
et al.
1998; Laake
et al.
1998; Ejima
et al.
1998; Campbell
et al.
2003; Mitui
et al.
2003; Coutinho
et al.
2004;
Babaei
et al.
2005; Birrell
et al.
2005). Haplotyping is
also useful for prenatal testing and occasionally for het-
erozygote identiﬁcation within A-T families (Gatti
et al.
1993). Herein we studied twenty-four Polish families
with A-T and found that three founder mutations re-
curred (were shared) in 58% of the families, and nine
recurring founder haplotypes accounted for 83% of
the families. Mutations were identiﬁed for all founder
haplotypes.
followed approved Human Subject Protection protocols
in Poland and the United States.
Haplotype Analysis
STR haplotyping was used to ﬁrst determine whether
founder mutations were present in the Polish popula-
tion, thus minimizing the number of mutations that
would have to be screened. As previously described
(Mitui
et al.
2003), STR haplotypes were identiﬁed us-
ing four markers: S1819 (Rotman
et al.
1994), NS22
(Udar
et al.
1999), S2179 (Vanagaite
et al.
1995), and
S1818 (Rotman
et al.
1994). Markers NS22 and S2179
are located within the
ATM
gene; markers S1819 and
S1818 ﬂank the gene within 1.4 cM on the proxi-
mal and distal ends, respectively. PCR ampliﬁed end-
radiolabelled fragments were run on 6% polyacrylamide
gel (National Diagnostics, Atlanta, Georgia) and com-
pared with a known control (CEPH1347-2) (Mitui
et al.
2003). This control has allowed the allele sizes to be stan-
dardized so that haplotypes from various ethnic popu-
lations could be compared (Mitui
et al.
2003; Coutinho
et al.
2004; Birrell
et al.
2005). Haplotype phase was
deﬁned using parents or by comparing haplotypes of
patients with the same mutation.
SNP haplotyping was carried out by SSCP (Castellvi-
Bel
et al.
1999), using three SNP markers: IVS17-
56G
>
C, 5557G
>
A, and IVS62-55T
>
C. These three
SNPs deﬁned the three most common SNP haplotypes
across the
ATM
region (H2, H3 and H4), which encom-
pass 91% of SNP haplotypes worldwide (Thorstenson
et al.
2001; Campbell
et al.
2003). The more uncom-
mon Haplotype H1 was also identiﬁed in two families.
Mutation Detection
Materials and Methods
Mutation screening was performed with PTT (Telatar
et al.
1996) followed by SSCP (Castelvi-Bel
et al.
1999) and dHPLC (Bernstein
et al.
2003). PTT de-
tects truncating mutations, such as nonsense mutations,
frameshifts caused by small insertions or deletions, or
aberrant splicing (Telatar
et al.
1996; Teraoka
et al.
1999). SSCP was used to further identify abnormal
regions in the cDNA of the
ATM
gene. Sequencing
changes were revealed by a measurable difference in
mobility through a gel, due to differences in the sec-
ondary structure of single stranded cDNA or genomic
DNA (Castellvi-Bel
et al.
1999). This technique used 34
Subjects
Tw enty-four unrelated A-T families from Poland com-
prised this study group. All patients displayed classical
A-T phenotypes. Lymphoblastoid cell lines (LCLs) were
established for most of the probands. The diagnosis was
conﬁrmed by the absence of the ATM protein by im-
munoblotting and the ﬁnding of radiosensitivity by a
colony survival assay (Sun
et al.
2002); at least one ATM
mutation was also identiﬁed for each proband. Family
WA R49included two affected sibs. Blood collection
658
Annals of Human Genetics (2005)
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University College London 2005
C
ATMMutations in Polish Families
300 nt of coding sequence. dHPLC
was performed on samples that still had one mutation
unidentiﬁed after the PTT and SSCP screening. Candi-
∼
date regions were sequenced in both directions and the
mutated site identiﬁed using genomic DNA.
Results
Haplotypes
Tw enty-six distinct STR haplotypes were observed in
24 ostensibly unrelated Polish families. The incidence
of homozygosity was very low; only two probands had
homozygous haplotypes and mutations, WAR 12 and
WA R31(Figure 1). Nine haplotypes were observed in
more than one family, encompassing 83% of the families.
The Polish A-T population proved to be more homo-
geneous than expected, as 83% of the families carried at
least one founder haplotypes (Figure 1A). Indeed, the
three most common haplotypes, [A], [B], and [D], oc-
curred in 14 of the 24 families (58%).
With few exceptions, STR haplotypes are con-
served between A-T patients with the same mutation
(Campbell
et al.
2003). We observed this to be true
for markers NS22 and S2179. However, some variation
was found for S1819, and to a lesser degree S1818. This
most likely reﬂects the instability of STR markers or
could be due to polymerase slippage in some cases. Oc-
casional recombination outside the gene is also possible,
although recombination within the gene is very un-
common (Bonnen
et al.
2000; Thorstenson
et al.
2001).
Va r iant STR haplotypes (haplovariants) were observed
for some patients carrying the same mutation. For ex-
ample, haplotype [G] of WAR2 and WAR15, carried
the 3085 3086insA mutation. Another example can be
seen with WAR 31, where the S1818 alleles differ in a
family with consanguineous history. WAR33 was more
difﬁcult to interpret: the mutation (IVS53-2A
>
Figure 1
Haplotypes and mutations in 24 Polish A-T families.
A. Recurring haplotypes are colour shaded. The
indicate
that phase has not been deﬁned. B. Mutations corresponding to
the affected haplotypes in A. Bold mutations have not been
previously reported. Conserved amino acids are underlined.
Superscripts: a, ﬁrst allele; b, second allele; h: homozygous.
Asterisk denotes a primary premature termination codon
(PTC). Nucleotide numbering is based on
+
1 being the A of
the initiation start codon.
University College London 2005
Annals of Human Genetics (2005)
69
,657–664
659
overlapping fragments to cover the gene, with each frag-
ment spanning
C) and
the H3 SNP haplotype remained unchanged as com-
pared to other patients (e.g., WAR 24), while the STR
haplotype was different for S2179, similar to changes
observed by Campbell
et al.
(2003). It was not possible
to deﬁne phase in WAR16.
SNP haplotypes H1, H2, H3, and H4 were identiﬁed
in the Polish population. SNP haplotype frequencies in
Poland were similar to previously described worldwide
SNP haplotype frequencies (Throstenson
et al.
2001).
H2 was the most common haplotype (41%). H3 was
found in 33%; this is higher than the global frequency
of 12%. The H4 frequency was lower than expected
C
Mitui
et al.
(20% vs 40%). H1 was seen in association with two
distinct STR haplotypes (WAR 23 and WAR 49) and
two distinct mutations. This haplotype is uncommon
outside of Africa (Thorstenson
et al.
2001; Coutinho
et al.
2004).
Table 1
Genotype/phenotype comparisons
Haplotype
A
B
D All
Number of patients (N) 7 5
∗
5
∗
Male: Female 4:3 3:2 3:2 10:14
Ataxia onset (years old) 2.0 3.1 1.3 2 (0.9–7 yrs)
Progression of ataxia
†
1.7 1.4 2 1.6
Wheelchair (%N) 57 100 100 67
Wheelchair (years old) 15 14 12 14 (9–19 yrs)
Telangiectasia onset (years old) 3.6 6 3.4 4 (2–7 yrs)
Growth Retardation (%N) 57 20 50 50
Mental Retardation (%N) 43 20 20 29
Bronchiectasia (%N) 43 60 20 29
Cancer (%N) 0 0 0 8.7
IVIg therapy (%N) 57 80 60 50
AFP elevated (%N) 100 100 100 100
AFP (IU/ml) 276 107 147 208
IgM
↑
(%N)
‡
16 0 25 6
IgG
↓
(%N)
‡
0 3 5 5
IgA
↓
(%N)
‡
66 40 100 45
IgE
↓
(%N)
‡
80 100 67 96
†
Progression of ataxia 1
=
slow, 2
=
moderate, 3
=
rapid
∗
The family of WAR 49 includes two affected children.
‡
Immunoglobulins were increased on at least two occasions.
Mutations
A, were
each found in 5 and 4 chromosomes (10% and 8%), in
association with Haplotypes [D] and [B], respectively.
Only three mutations were novel; however, our labora-
tory has also previously published other unique Polish
mutations (Telatar
et al.
1998).
Mutation 5932G
>
T and 6095G
>
T
AA or prema-
ture termination condon. However, a small proportion
of the transcript is missing all of exon 42 (88nt), suggest-
ing that the mutation may also lead to aberrant splicing.
This mutation was observed in ﬁve of the families, on
three haplovariants. Two of the haplovariants have been
observed in Russian A-T families as well (Birrell
et al.
2005). .
>
Tresults in
G
AA
>
eraging 15 years of age when they became wheelchair
dependent. No other phenotype differences were ap-
parent.
Haplotype [B], carrying 6095G
>
A, affected ﬁve pa-
tients (two of whom were sibs in family WAR 49; only
one is shown in Fig 1). The average age of ataxia onset
was 3.1 years, with greater variation than for haplotype
[A]. Within this group, the WAR 49 sibs did not have
telangiectasia at ages 14 and 15. The average age of onset
for the other three patients was 6 years. All ﬁve patients
required a wheelchair by an average age of 14.
Haplotype [D], carrying 5932G
Genotypes versus Phenotypes
Serum alphafetoprotein (AFP) levels were elevated in all
24 patients. Similarly, all patients tested lacked detectable
intranuclear levels of ATM protein. None of the geno-
type/phenotype comparisons were signiﬁcantly differ-
ent, due to the limited number of patients in each group
and the paucity of homozygous patients; none of the pa-
tients carrying haplotypes [A], [B] or [D] were homozy-
gous. A summary of the clinical dataset is presented in
Ta b l e 1 .
Haplotype [A], carrying IVS53-2A
T, affected 5 pa-
tients; the average age of ataxia onset was 1.3 years. This
average was almost identical to that of the Russian A-T
families (Birrell
et al.
2005).
>
C, was observed
in seven patients; none were homozygous. The average
age of onset of ataxia was 2.0 years and the average age
of onset of telangiectasia was 3.6 years. Of the seven
patients with this mutation, three were not yet conﬁned
to a wheelchair (all were under 10 years old); the other
four patients were wheelchair bound but were older, av-
>
Discussion
Due to the large size of the
ATM
gene and the broad
spectrum of ATM mutations, mutation detection is not
yet cost-effective for establishing a diagnosis of A-T. In
this study, the diagnosis was conﬁrmed by a lack of ATM
protein western blotting and radiosensitivity on by CSA,
in all patients. Serum AFP levels were elevated in all
660
Annals of Human Genetics (2005)
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,657–664
University College London 2005
Mutations were detected in 44 of 48 alleles (92%). Of
21 different mutations, 15 were truncating, 5 aberrant
splicing, and 1 missense (Figure 1B). The most com-
mon mutation, IVS53-21
<
C,asplicing mutation, was
found in 7 of 48 chromosomes (14.6%) and was asso-
ciated with Haplotype [A]. The second and third most
common mutations, 5932G
C
ATMMutations in Polish Families
Table 2
observed in German, Turkish and Hispanic-American
patients (Mitui
et al.
2003; Eng
et al.
2004); the standard-
ized STR haplotypes of the Hispanic-American families
differ slightly from those of the Old World (Polish, Ger-
man, Turkish) A-T families (Eng
et al.
2004), provid-
ing further evidence that many
ATM
mutations predate
STR haplotypes, but not the common SNP haplotypes
(Thorstenson
et al.
2001; Campbell
et al.
2003)
Most
ATM
mutations are associated with speciﬁc
STR and SNP haplotypes (Campbell
et al.
2003; Mi-
tui
et al.
2003; Eng
et al.
2004). This held true with-
out exception for the SNP haplotypes associated with
Polish mutations. In general, this was also true for the
association of these mutations with STR haplotypes,
with two exceptions: in WAR33 [A][D] and WAR 19
[B][E]. WAR33 carries the Haplotype [A] mutation,
IVS53-2A
Mutation
Also found in
381delA Iranian
742C
>
T Japanese
1563 1564delAG Amish, Turkish, Italian,
German, Brazilian
IVS20–579˙IVS20–582delAAGT German, American-Hispanic
5188C
>
T
Spanish
5712 5713insA
Phillippino, Turkish,
5932G
>
T
Norwegian, Danish,
Mennonite,
American-Hispanic,
German, Russian
6095G
>
A
Swedish, German, French
7010 7011delGT
English/Irish
IVS53-2A
>
C
Danish, American-Hispanic,
Brazilian, Portuguese
8545C
>
T
Italian
C; however, the S2179 allele appears to have
changed from ‘141’ to ‘139’. The H3 SNP haplotype
background remains the same as that observed for all [A]
haplovariants in this study. Haplovariants were also ob-
served for the 5932 G
patients. Thus, these aspects of the A-T phenotype were
not inﬂuenced by genotype in any apparent way.
We observed that 58% of the A-T families in Poland
shared one of three founder mutations (Haplotype [A],
[B], and [D]), and 83% of the families carried at least
one of eight Polish founder haplotypes. We were sur-
prised to ﬁnd this degree of genetic homogeneity, con-
sidering that Polish population migrations have not been
restricted by geographical features such as large bodies of
water or high mountain ranges. Nonetheless, our previ-
ous studies of ATM haplotypes and mutations strongly
suggest that shared, recurring mutations predate modern
ethnicities and nationalities (Campbell
et al.
2003), and
STR haplotypes such as [A], [B], and [D] may reﬂect
inﬂuences on ancient migrations rather than on mod-
ern ones. Eleven Polish
ATM
mutations have also been
found in other ethnic groups (Table 2).
Splicing mutations comprised 23% of the mutations
found in this study, a proportion not unlike those in
previous studies (Teraoka
et al.
1999; Mitui
et al.
2003).
Splicing mutations typically involve the highly con-
served canonical 3
or 5
splice sites, as is the case for
IVS 53-2A
Tmutation on Haplotype [D]
(see below) and for the 6095G
>
Amutation on Haplo-
type [B]. Both long and short forms of this Haplotype
[B] were observed (Fig 1A), with only a single allele
(S2179 ‘137’) shared by all four chromosomes (WAR
6, 19, 22, and 49–3). Taken together, these data suggest
that the longest variant (eg: WAR 49–3) is the older, an-
cestral haplotype for this mutation, although alternative
interpretations are possible.
The mutation on Haplotype [F], 1563 1564delAG, is
perhaps the most commonly observed
ATM
mutation
worldwide and always occurs on a SNP H
2
background.
It was observed in three Polish families in association
with SNP haplotype (H2), but with several STR haplo-
variants. As previously described (Campbell
et al.
2003),
1563 1564delAG is associated with STR haplovariant
1 (in Turkish, Polish and Amish A-T patients), haplo-
variant 2 (in a Brazilian patient), and haplovariant 3 (in
Turkish and Italian patients). In all of these families, the
allele for S1818 was ‘160’, as is also observed in two of
the Polish families; however, in WAR 46, a new haplo-
variant 4 was deﬁned by allele S1818 ‘158’ (instead of
‘160’). These ﬁndings are compatible with the historical
origins of the Amish of Pennsylvania (U.S.A) from Ger-
manic settlers, descendants of an Anabaptist movement
in northern Europe (1525–1536) (Hostetler, 1983a).
>
ConPolish haplotype [A]. Three other
splicing mutations were noted on non-recurring Polish
haplotypes. IVS20–597delAAGT is a ‘masked’ muta-
tion that causes Type II splicing with pseudoexon for-
mation (Eng
et al.
2004). The mutation occurs deep
within intron 20, and disrupts the U6 portion of a U1
snRNA binding site (Pagani
et al.
2002). It has also been
>
University College London 2005
Annals of Human Genetics (2005)
69
,657–664
661
>
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