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Supplementary Methods
DNA preparation
Biological specimens for the preparation of genomic DNA samples were obtained from
one or several of the following sources: fresh whole blood; mononuclear cells found at the
plasma/histopaque gradient interface after centrifugation of whole blood through a Histopaque1077 (Sigma) gradient or through a Leuco Prep cell separation tube (Becton-Dickinson), and
cryopreserved in 10% dimethyl sulfoxide in liquid nitrogen; granulocytes, obtained from the
layer that lies on the packed red cells after centrifugation of whole blood through a Histopaque1077 gradient or through a Leuco Prep tube and subsequently frozen in liquid nitrogen without
preservative; cultured FCLs; and, cultured LCLs. DNA samples from a small number of
registered persons were generously provided by cooperating geneticists. DNA samples were
prepared using the DNAzol extraction method (Molecular Research Center, Inc.) or the
PUREGENE kit (Gentra Systems, Inc.) according to the manufacturer’s instructions. The phenolchloroform extraction method was used to prepare some of the DNA samples, as described in
German et al., 1994.
Methods of mutation analysis
Analysis by reverse transcription PCR (RT-PCR). The coding region of the BLM mRNA
was divided into five overlapping DNA fragments (Supplementary Table S2A). First-strand
1
DNA synthesis was performed using 20 g of RNA isolated from BS cell lines. The RNA was
incubated with RNAse inhibitor in 2x RT buffer (Life Technologies), 10 mM dithiothrietol, 0.5
mM dNTPs, 10 g/ml oligoT or hexanucleotides (Boehringer Mannheim) as primers, and 400
units of reverse transcriptase (Life Technologies, Inc) at 42 oC for 1 hr followed by heat
inactivation at 70 oC for 10 min. A minus RT reaction was added as a control for the subsequent
PCR steps. PCR was then carried out with each of the five pairs of oligonucleotide primers
(Supplementary Table S2A). One l of copy DNA products was amplified in 1X PCR reaction
buffer (Boehringer Mannheim) containing 0.25 M of each oligonucleotide primer, 0.2 mM
dNTPs, and 2.5 units of Taq polymerase (Boehringer Mannheim) in a 50 l reaction volume.
PCR consisted of 20 cycles of 94 oC for 1 min, 55 oC for 1 min, and 72 oC for 1 min, with a final
incubation at 72 oC for 7 min. The amplification products were analyzed by agarose gel
electrophoresis.
DNA fragments that exhibited abnormal migration relative to fragments
obtained by RT-PCR of mRNA from normal cells were excised from the agarose gel, reamplified, and subjected to direct DNA sequencing in order to identify changes that could
explain the abnormal migration, such as exon mis-splicing. Subsequently, Southern blot analysis,
direct sequence analysis of genomic DNA, or both analyses were performed to determine the
genetic basis of the mRNA abnormality as described below. DNA fragments generated by RTPCR that exhibited normal migration based on agarose gel electrophoresis were subjected further
to mutational analysis by either a nonisotopic RNAseA mismatch cleavage assay, the protein
truncation test, or both.
Nonisotopic RNAseA mismatch cleavage assay. First-strand copy DNA synthesis and the
first round of PCR was performed using RT as described above, and this reaction was followed
by PCR using nested oligonucleotide primers. The internal pair of primers included T7 and SP6
2
RNA polymerase transcription initiation sequences attached to the 5’ end of the forward and
reverse oligonucleotide primers, respectively. The PCR was performed as described above, and
products were divided into two separate RNA transcription reactions using the T7 and SP6 RNA
polymerases. Two l of PCR product were incubated at 37 oC for 1 hr in a 10 l volume
containing 1X Transcription Buffer (Promega), 0.5 mM NTPs, and 2 units of either T7 or SP6
RNA polymerase. For each fragment of BLM, a T7-generated RNA from a BS cell line was
mixed with an SP6-generated RNA from a normal cell line and an SP6-generated RNA from a
BS cell line was mixed with a T7-generated RNA from a normal cell line. The two RNAs were
hybridized by combining empirically optimized volumes of “mutant” and “normal” transcription
reactions, and “normal” and “normal” transcription reactions as a control, heating the mix to 95
o
C for 3 min, and cooling at room temperature for 30 min. The heteroduplexes were treated with
a mixture of RNAses under three different digestion conditions using the Mismatch Detect Point
Mutation Screening Kit (Ambion, Inc.) according to the manufacturers’ instructions.
The
products generated by RNAse cleavage were separated by electrophoresis through 1% agarose
gels containing 0.05 mg/ml ethidium bromide and visualized by ultraviolet light.
RNAse
digestions of heteroduplexes were compared to the control homoduplexes prepared by mixing
RNAs generated from the normal cell line. RNA fragments with abnormal mobilities were
identified as possible mutations. The estimated size of the abnormal RNA fragment was used to
predict the site of RNAse cleavage, and therefore the site of the mutation.
Direct DNA
sequencing of the RT-PCR fragment and of the appropriately selected exon in genomic DNA
was performed to identify the putative mutation.
Protein truncation test (PTT). The coding region of the BLM mRNA was divided into
three overlapping DNA fragments (Supplementary Table S2B).
3
First-strand copy DNA
synthesis was performed and again followed by PCR using a nested PCR strategy. The internal
pair of primers included a T7 RNA polymerase transcription initiation sequence and an in-frame
initiator methionine codon attached to the 5’ end of the forward oligonucleotide primer. RNA
transcription and protein translation in the presence of
35
S-methionine (New England Nuclear)
were carried out using a Novagen Single Tube System 2 kit according to the manufacturer’s
instructions.
In vitro synthesized protein products were analyzed by SDS-PAGE and
autoradioagraphy. Proteins produced using BS cell line mRNAs were compared to proteins
produced using normal cell line mRNA. Protein products of faster-than-normal mobility were
identified. The estimated size of the abnormal protein product was used to predict the site of
premature termination, and therefore the site of the mutation. Direct DNA sequencing of the
RT-PCR fragment and of the appropriately selected exon in genomic DNA were performed to
identify the putative mutation.
Denaturing-high performance liquid chromatography (D-HPLC) analysis. Each of the
22 exons of BLM was amplified by PCR using oligonucleotide primers that flanked each exon
(Supplementary Table S3). Exons 3 and 7 were too large to analyze by D-HPLC as a single
exon; consequently, these exons were analyzed as two overlapping PCR fragments.
Approximately 50 bp of up- and downstream DNA sequences were included in each PCR
fragment to ensure that putative mutations in the splice-site regions were not missed by their
presence in the easily melted sequences at the end of the fragment. PCR amplification was
carried out in buffer II (Applied Biosystems, Inc.) containing 2 mM MgCl2, 0.2 mM each
oligonucleotide primer, 0.2 mM dNTPs, 25 ng genomic DNA, and 1.25 units AmpliTaq Gold
(Applied Biosystems, Inc.) in a 50 l reaction volume. PCR amplification consisted of 1 cycle
of 94 oC for 10 minutes; followed by 1 cycle at the annealing temperature (see Supplementary
4
Table S3) for 45 seconds and 72 oC for 1 minute; followed by 35 cycles of 94 oC for 30 seconds,
annealing for 54 seconds, and 72 oC for 1 minute; and finishing with 1 cycle of 72 oC for 7
minutes. Heteroduplex DNA molecules were formed by heating the DNA at 95 oC for 5 min and
cooling at 1 oC per min for 30 min. D-HPLC analysis relies on the presence of normal and
mutated DNA sequences so that heteroduplex DNAs can be formed in the population of
molecules to be analyzed. Consequently, because it was not known beforehand in most cases
whether or not a person was a genetic compound, we added an equal amount of amplified normal
DNA to the amplified BS DNA to ensure that heteroduplex DNA products would be produced in
the heteroduplexing step. This procedure was validated with DNA samples containing known
BLM mutations in both the homozygous and heterozygous state to show that mutations could be
detected under either condition. DNA fragments were subjected to chromatography using
conditions determined by Wavemaker software (Transgenomic, Inc.) and a range of melting
temperatures (Supplementary Table S3). Fragments that displayed an abnormal chromatograph
in comparison to a normal, non-variant fragment were subjected to DNA sequencing.
DNA sequencing. PCR amplification was performed for each of the BLM exons or for
selected BLM exons. Amplified DNA fragments were prepared for sequencing by column
purification (Qiagen) or by centrifugation through a Chromaspin 100 column (Clontech)
followed by ethanol precipitation.
The DNA fragment was quantified by agarose gel
electrophoresis and sequenced either by cycle sequencing with Taq polymerase and BigDye
Terminator reaction mixes (Applied Biosystems, Inc.) followed by electrophoretic analysis on an
ABI377 sequencer as described [Ellis et al., 2001] or by “manual” sequencing as originally
described [Ellis et al., 1995a].
5
Southern blot analysis. Two to 10 g of genomic DNA were digested with EcoRI or
BamHI and the DNA fragments were separated by electrophoresis through 0.8% agarose gels in
89 mM Tris, 89 mM boric acid, and 2 mM EDTA. The DNA was transferred to Hybond N+
nylon membrane (Amersham). Hybridization was performed as previously described [German et
al., 1994] with the full-length BLM cDNA H1-5’ [Ellis et al., 1995a]. Three of the four large
deletions identified were further characterized by long-range PCR followed by DNA sequencing
to identify the breakpoints of the deletion. For one deletion, which included the deletion of
sequences outside the BLM gene, this analysis was unsuccessful.
Supplementary Figure Legend
Figure S1. Northern blot analysis of mRNAs derived from selected Bloom’s syndrome cell lines.
The BLM cDNA H1-5’ was radioactively labeled with 32P and hybridized to 20 micrograms of
polyA-purified mRNA from the indicated cell lines. The BLM mRNA appears as a 4.5 kb major
form and a 4.3 kb minor form. The cell lines HG1321, HG1805, HG2709, HG1440, HG1542,
HG1544, and HG1547 are low-SCE Bloom’s syndrome lymphoblastoid cells. The cell lines
HG1304, HG1309, HG14335, HG1431, HG2118, HG2508, HG1374, HG1348, and HG1783 are
Bloom’s syndrome fibroblasts, all presumed to be high-SCE. See Supplementary Table S1 for
further details on these cell lines. HG2608 is a normal lymphoblastoid cell, and HG2635 is a
normal fibroblast line. The levels of BLM mRNA in fibroblasts are lower than in lymphoblastoid
cells. To control for Northern blot loading and mRNA quality, the blot was probed with a labeled
cDNA derived from the glucose-3-phosphate dehydrogenase gene. One preparation of RNA
6
from the normal fibroblast HG2635 (indicated by the dash) was poor quality and no signal from
the BLM mRNA was detected.
Supplementary Reference
Passarge E. 1991. Bloom's syndrome: the German experience. Ann Genet 34:179-97.
7
Supplementary Table S1. Cell lines used in the mutational analysis of persons with Bloom’s syndrome.
Cell line
BSR IDa
HG46
HG149
HG369
HG1013
HG1052g
HG1179
HG1232
HG1289
HG1304
HG1309
HG1321h
HG1348
HG1374
HG1431
HG1440h
HG1435
HG1514
HG1525
HG1542g
HG1544g
HG1547h
HG1584
HG1619
HG1624
HG1626
HG1647
HG1742
HG1751j
HG1782
HG1783
HG1785
HG1805l
HG1824
HG1926
HG1929
HG1947
HG1972m
HG1974
BSR3
BSR5
BSR26
BSR71
BSR50
BSR6
BSR44
BSR20
BSR59
BSR86
BSR59
BSR31
BSR128
BSR87
BSR87
BSR54
BSR15
BSR81
BSR11
BSR11
BSR11
BSR92
BSR27
BSR113
BSR93
BSR32
BSR107
BSR126
BSR110
BSR129
BSR100
BSR86
BSR169
BSR97
BSR17
BSR53
BSR140
BSR121
Cell Typeb SCE ratec Northernd
FCL
FCL
FCL
FCL
LCL
FCL
FCL
FCL
FCL
FCL
LCL
FCL
FCL
FCL
LCL
FCL
LCL
LCL
LCL
LCL
LCL
LCL
LCL
LCL
LCL
FCL
LCL
LCL
FCL
FCL
LCL
LCL
FCL
LCL
FCL
LCL
LCL
LCL
–
–
–
+
–
Low
High
High
High
High
Low
High
High
Low
Low
Low
High
–
–
–
+
+/–
–
+/–
+
+/–
–
–
+
+
+
–i
High
High
+
–i
High
Low
High
+
–
–
+
–
–
–
Low
Low
High
Mutation 1e
Mutation 2 e
blmAsh
c.2098C>T
blmAsh
c.1544_1545dupA
blmAsh
c.2923delC
blmAsh
del exon 15f
c.2098C>T
c.1544_1545dupA
c.2098C>T
c.960-2A>G
c.557_559delCAA
blmAsh
blmAsh
blmAsh
blmAsh
c.1784C>A
blmAsh
c.2098C>T
c.3261delT
c.2254C>T
del exons 20-22f
c.2923delC
blmAsh
c.2923delC
del exons 11-12f
blmAsh
c.3164G>C
c.1544_1545dupA
blmAsh
blmAsh
blmAsh
c.557_559delCAA
c.1544_1545dupA
c.1544_1545dupA
c.1544_1545dupA
c.1933C>T
c.557_559delCAA
c.1968_1969dupG
blmAsh
c.1544_1545dupA
c.2015A>G
c.557_559delCAA
c.3163T>G
c.2672G>A
blmAsh
c.1784C>A
c.1933C>T
c.1933C>T
c.1933C>T
del exons 11-12f
blmAsh
c.3164G>C
c.1544_1545dupA
blmAsh
blmAsh
c.98+1G>Tk
c.557_559delCAA
c.1544_1545dupA
c.557_559delCAA
c.1933C>T
c.557_559delCAA
c.1968_1969dupG
blmAsh
+
blmAsh
8
blmAsh
HG1987j
BSR30
LCL
Low
+
c.2695C>T
c.3028delG
HG1992
BSR133
FCL
–
c.1933C>T
c.1933C>T
g
HG2045
BSR111
LCL
Low
+
c.2695C>T
del exons 11-12f
g
HG2118
BSR65
LCL
Low
+
c.2488_2489dupA
c.3681delA
c.1933C>T
HG2122
BSR61
LCL
c.1933C>T
g
Ash
HG2166
BSR26
LCL
Low
+
blm
c.3261delT
l
HG2169
BSR40
LCL
Low
+
c.2702G>A
HG2193
BSR170
LCL
High
–
c.1933C>T
c.3261delT
HG2225
BSR22
LCL
–
c.3261delT
c.3261delT
i
HG2231
BSR139
LCL
+
c.2015A>G
c.3163T>C
HG2252
BSR80
FCL
+/–
c.3164G>C
c.311C>A
HG2508
BSR105
FCL
–
c.1933C>T
c.2725C>T
HG2510
BSR112
LCL
High
–
c.814A>T
c.1220+3A>Gn
HG2522o
BSR42
FCL
High
–
blmAsh
blmAsh
HG2703
BSR171
LCL
High
–i
blmAsh
blmAsh
h
Ash
HG2709
BSR54
LCL
Low
+
blm
HG2721
BSR177
LCL
Low
+
c.1933C>T
c.2695C>T
HG2726
BSR70
FCL
HG2820
BSR142
LCL
High
–i
blmAsh
blmAsh
HG2829
BSR98
LCL
High
+
c.1090A>T
c.1701G>A
HG2830
BSR51
LCL
High
–
c.2098C>T
c.2098C>T
HG2896
BSR191
FCL
–
c.2695C>T
c.2695C>T
HG2912
BSR193
LCL
–
c.2250_2251insAAAT c.3261delT
HG2915
BSR197
LCL
c.3197G>A
blmAsh
HG2917
BSR175
LCL
–
HG2940
BSR232
FCL
c.582delT
c.582delT
a
Bloom’s Syndrome Registry designation of the person from whom the cell line(s) was
established.
b
FCL, fibroblast cell line; LCL, lymphoblastoid cell line.
c
The rate of sister-chromatid exchanges (SCEs) is indicated for those cell lines for which
cytogenetic information was available. Low indicates that the average numbers of SCE per
metaphase was <15; High indicates that the average numbers of SCE per metaphase was >35.
The majority of FCLs were not tested (blanks in the column), because each of the FCLs studied
to date has exhibited a high level of SCEs; consequently, it is assumed that all FCLs employed
here have a high-SCE phenotype. Certain of the LCLs, however, have a low-SCE phenotype.
The two genetic mechanisms responsible for the low-SCE LCLs are somatic intragenic
recombination, which was described in Ellis et al. 1995b, and back mutation, which was
described in Ellis et al. 2001. The arrangement of mutations in low-SCE cell lines was inferred
from molecular haplotype and mutational analyses, as indicated in footnotes g and h.
d
Summary of the results of Northern blot analyses performed on RNA samples prepared from
the different cell lines. A plus sign (+) indicates that the signal from the mRNA detected in the
BS cell line was 50% or more of the signal detected in the RNA from a normal cell line. A minus
sign (–) indicates that the signal from the mRNA detected in the BS cell line was less than 20%
of the signal detected in the RNA from a normal cell line. A plus/minus (+/–) indicates that the
signal from the mRNA detected in the BS cell line was between 20 and 50% of the signal
9
detected in the RNA from a normal cell line. Steady-state levels of BLM mRNAs are ten fold
higher in LCLs than in FCLs; consequently, the sensitivity of Northern analysis is less for FCLs
than for LCLs. A blank in the column indicates that the RNA sample was not analyzed by
Northern analysis.
e
Mutations and their putative effects on protein structure are delineated using conventional
mutation nomenclature (cf. http://www.hqcv.org/mutnomen/), wherein +1 corresponds to the A
of the ATG translation initiation codon in the reference cDNA sequence (accession number
U39817) and the initiation codon is amino acid residue 1 in the protein sequence. The two
mutations present in each cell line are indicated, and the putative effects on the gene products are
shown in Tables 1 and 2. If the cell line has a low-SCE phenotype, then a normal allele is
inferred to be present in the cell line (see footnotes g and h). Consistent with this claim, all the
low-SCE cell lines have normal levels of steady-state BLM mRNAs. For a few cell lines, the BScausing mutation putatively present at BLM on one or both chromosomes is not known (blanks).
f
The formal names for the gross deletions are as follows: del exons 11-12 (BSR92), c.2308953_2555+4719del6126; del exons 11-12 (BSR111), c.2308-117_2555+7420del7811; del
exon15, c.2824-1077_2999+310del1583; del exons 20-22, c.3752-?_4442+?del. See Tables 1
and 2.
g
The cell line exhibits low levels of SCEs. Consequently, a normal allele at BLM is presumed to
be present on one chromosome No. 15. Molecular haplotype and mutational analyses indicate
that a normal BLM gene is present on one chromosome No. 15 and a mutated BLM gene that
contains both constitutional mutations is present on the other chromosome No. 15. These lowSCE cells lines were described in Ellis et al. 1995b along with their high-SCE counterparts.
h
The cell line exhibits low levels of SCEs. Consequently, a normal allele at BLM is presumed to
be present on one chromosome No. 15. Molecular haplotype and mutational analyses indicate
that a normal BLM gene is present on one chromosome No. 15 and a mutated BLM that contains
a single constitutional mutation—the more distal mutation within the BLM gene—is present on
the other chromosome No. 15. These low-SCE cells lines were described in Ellis et al. 1995b
along with their high-SCE counterparts.
i
Northern analysis of these cell lines was shown in Ellis et al. 1995a.
j
Two different mutations of BLM that cause premature protein-translation termination were
uncovered in the cell line, however the LCL is low-SCE or presumed to be low-SCE based on
the presence of normal levels of steady-state BLM mRNAs by Northern blot analysis.
Consequently, it is probable that a normal BLM gene is present on one chromosome No. 15 and a
mutated BLM gene that contains both constitutional mutations is present on the other
chromosome No. 15. Molecular haploptype analyses have not been performed on DNA from
these cell lines.
k
RT-PCR products of smaller-than-normal sizes were detected by agarose gel electrophoresis.
The consequence of this mutation of the 5’ splice site of exon 2 was a deletion in the mRNA
product of exon 2, nucleotides –4 to 98, as determined by DNA sequencing of the abnormallysized cDNA fragment. Splicing out exon 2 removes the initiator methionine for BLM. Use by the
ribosome of the next downstream AUG would result in a small out-of-frame peptide.
l
A normal allele at BLM is present on one chromosome No. 15 due to a back mutation at BLM.
See Ellis et al. 2001.
m
The Northern analysis suggests that at least one normal BLM allele probably is present.
Sequencing of the cDNA from this cell line and sequencing and Southern analysis of the
10
genomic DNA from the person with BS from whom this cell line was developed failed to
uncover mutation of BLM.
n
RT-PCR products with a smaller-than-normal size were detected by agarose gel
electrophoresis. The consequence of this mutation in the 5’ splice site of exon 6 was a deletion in
the mRNA product of exon 6, nucleotides 1088 to 1220, as determined by DNA sequencing of
the abnormally-sized cDNA fragment. The effect on BLM translation is a frameshift beginning
at codon 363, which normally codes for alanine.
o
This widely used cell line is also known as GM08505; it was transformed with simian virus 40
and propagated through crisis to produce an indefinitely proliferating cell line.
11
Supplementary Table S2. Oligonucleotide primers used in the mutational analysis of mRNA from Bloom’s syndrome cells.
A. RNAseA Mismatch Cleavage Analysis Primers
Segment
Primer 1 name (cDNA Nos.)
Sequence primer 1
SR1 Outside Y180 (–71 TO –56)
CGGCGGCCGTGGTTGC
SR1 Nested
1F (–47 TO –32)
AAGTTTGGATCCTGGT
SR2 Outside BC16 (586-610)
GCACAGCTTTATACAACAAACACAG
SR2 Nested
2F (752-767)
ATGCTCAGGAAAGTGA
SR3 Outside BC21 (1634-1657)
GAGAAACCCAACCTTCCTATGATA
SR3 Nested
3F (1697-1712)
ACTGGGAAGACATAAT
SR4 Outside BC27 (2307-2328)
GATCTGTGCAAGTAACAGACTC
SR4 Nested
4F (2566-2581)
AGCTTTAACAGACATA
SR5 Outside BC2 (3477-3499)
ATATATCAATGCCAATGACCAGG
SR5 Nested
5F (3449-3464)
CAATTTAAAGGTAGAC
B. Protein Truncation Test Primers
Segment
Primer 1 name (cDNA Nos.)
SP1 Outside Y189 (–46 to –27)
SP1 Nested
PTT1 (–26 to –7)
SP2 Outside
SP2 Nested
Y204 (969-990)
PTT2 (1128-1148)
SP3 Outside
SP3 Nested
BC27 (2307-2328)
PTT3 (2482-2501)
Primer 2 name (cDNA Nos.)
BC17 (963-942)
1R (902-887)
BC7 (1935-1914)
2R (1800-1785)
BC3 (2842-2821)
3R (2688-2673)
HG5 (3690-3767)
4R (3624-3609)
5R (4324-4309)
5R (same)
Sequence primer 1
AGTTTGGATCCTGGTTCCGT
TAATACGACTCACTATAGGAACAGACCAC
C-ATG-CCGCTAGGAGTCTGCGTGCG
GGACCTTGACACATCTGACAGA
TAATACGACTCACTATAGGAACAGACCAC
CATGGAGCACATCTGTAAATTAAT
GATCTGTGCAAGTAACAGACTC
TAATACGACTCACTATAGGAACAGACCAC
C-ATG-GCTCTTACGGCCACAGCTAA
12
Sequence primer 2
CGTACTAAGGCATTTTGAAGAG
ACAAAATCCGTATCAT
TTGGAAACGCTCATGTTTCAGA
AAGTCTTTCTGATACT
CAATTGTAGCACAGATAACCTG
GAGGCAGTAAATTATC
CCCCAGAGATTTGCAGACTTCTGT
CACTTTTGCTACTAAC
GCTTTATAGTCACAGA
Same
Primer 2 name (cDNA Nos.)
BC32 (2037-2020)
BC7 (1935-1914)
Sequence primer 2
TGCAGCATTGATCGCCTC
TTGGAAACGCTCATGTTTCAGA
BC34 (3200-3183)
BC25 (2999-2978)
CAGCAATTATCACAAGAA
CTGGTCACATCATGATAGGTAT
HG8 (4319-4294)
HG2 (4252-4233)
ATAGTCACAGATGGTCAGATGCTGAC
ATGAGAATGCATATGAAGGC
Supplementary Table S3. Oligonucleotide primers used in the mutational analysis of genomic DNA from persons with Bloom’s syndrome.
Exon
Primer 1
Sequence Primer 1
Primer 2
Sequence Primer 2
2
3
3
2A5’
3A5’
BC41
AGTCCTTCCTCCCCTCAAA
GGATTCTTTGCTCAGTTGGGATAC
CGGGATACTGCTCTCAAGAAAT
2A3’
BC15
3A3’
4
5
6
7
7
8
9
10
11-12
13
14
15
16
17
18
19
20
21
22
4D5’
5A5’
6A5’
7A5’
BC20
8A5’
9A5’
Y209
11A5’
13A5’
14D5’
15A5’
16A5’
17A5’
18A5’
19A5’
20A5’
21A5’
22A5’
GACTTAATCCACTTGACTCA
TATTGTCTGATCAGTGGTAGAA
CTGGGATTACAGTCATGAGCC
TGTGGCCTACCAGAGTAAACTAC
GAAAGGCCTTTATTCAATACCCA
CTTTCACTGTATTCATGTACTG
TGCCTTGGTGTCCTATTAATGAT
CTTTGATAGGTTTGATATGTG
CGACCTCGGATGATCCAC
CTCATTCATAACTCCACCCCTC
TGGTCTTCCAGCAGTATAAG
GGACCAAGTTAAGATATTAGGT
GTCTTACTATAGTCTTCATCTC
GGTTATGATGAATCTACTATAG
AGAAATGAGTGTCTGTGCCGGG
CCTGTATGGTACAAGTGCACAT
TGGGTTTTCTATGGGTGATAA
GTGTCTCTTCATATACACTAAA
GAAGTGGTATTGTAGCTCTGTGC
4D3’
5A3’
6A3’
BC19
7A3’
8A3’
9A3’
10A3’
12B3’
13B3’
14A3’
15A3’
16A3’
17A3’
18A3’
19B3’
20A3’
21A3’
22A3’
TCTCTCAGCTTCATTTCCTCATCTG
GTCATCCATATCATCCCAATC
TGACTATTCCCAATGGCTAGCTTT
G
AACAATTTAAAGTATCCCAG
CACAATCTTTGTGTTACGTTGT
TCCTTATGCCATTTCCAGGCCAG
TTGTGAGAACATTTCCTGGGAA
CAGGCAATGATGATTTGCTATG
GAGCTTTCATTTAACATCTGCC
AAAGGTTATGCAGAGGACTGAA
ATATGTATTCTAGTGGTCTTTAAT
CTCTGGCAGTCACTGC
AGACAGCTAATCACATGGCTG
GTTTGCATTCTACATGTGCATG
CATGAGGCTGAAGATGACAG
GTTAATCTGTCGGAGACCACCT
CAATTGAAGTAACTCAAAC
TTTGGTTCACTCATTGTGAGAT
CTTTCGGCCTATTAATCTGTGC
AGTGTGACGAGGTGTAGGAAGC
TTCAAAGCAAGGCAGAGCTGTTG
TAAAAAGAAGAACTATCACCCC
13
PCR
(oC)
55
55
50
Size
(bp)
297
506
478
HPLC (oC)
57, 58
57, 58
57, 58
HPLC
Gradient (%A)
52/47/38
48/43/34
49/44/35
43
55
60
50
55
55
55
50
60
55
50
55
50
50
55
55
55
50
50
413
304
336
418
515
302
289
281
687
303
373
384
324
288
387
301
285
358
429
54, 57
56, 58
52, 57, 58
57, 58, 60
55, 57, 58
55, 57
56, 59
53, 54, 55
58, 59, 60
57, 58
55, 57, 60
57, 58, 60
55, 57
56, 57
54, 58, 59
56, 58, 59
55, 58, 59
59, 60
56, 58, 59
52/47/38
52/47/38
51/46/37
50/45/36
48/43/34
52/47/38
53/48/39
53/48/39
47/42/33
52/47/38
50/45/36
50/45/36
52/47/38
53/48/39
50/45/36
52/47/38
53/48/39
51/46/37
49/44/35
Supplementary Table S4. Foundredsa identified in the Bloom's Syndrome Registry as result of the analysis of the syndrome-causing
mutations.
The individuals with Bloom's syndrome comprising the foundredsb
The mutations at BLM
The mutations carried
by the founders - the
founder mutationsc
The mutations at BLM on the homologous
chromosomesd
Identical (i.e.,
homozygous)
Identificatione
Parental
consanguinityf
Noteworthy parental ancestryg
Places of birth and/or
principal residence
Different (i.e., heterozygous)
Jews
Foundred 1 (n = 35)
blmAsh
blmAsh
BSR2
AA
New York
blmAsh
blmAsh
BSR3
AA
New York
blmAsh
blmAsh
BSR9
AA
Israel
blmAsh
blmAsh
BSR14
AA
New York
blmAsh
blmAsh
BSR15
AA
California
blmAsh
blmAsh
BSR16
AA
New York
blmAsh
BSR26
A-
Indiana
blmAsh
blmAsh
c.3261delT (Foun. 10)
BSR27
AA
Ohio
blmAsh
blmAsh
BSR32
AA
New Jersey
blmAsh
blmAsh
BSR34
AA
Illinois
blmAsh
blmAsh
BSR42
AA
Israel
blmAsh
blmAsh
BSR44
AA
Israel
blmAsh
blmAsh
BSR45
AA
Israel
blmAsh
blmAsh
BSR47
AA
New York
BSR50
A-
Ontario
BSR53
AA
New Jersey
BSR54
A-
New York
blmAsh
blmAsh
blmAsh
c.3752-?_4442+?del (Foun. 5)
blmAsh
c.2672G>A
14
blmAsh
blmAsh
BSR56
AA
Ohio
blmAsh
blmAsh
BSR57
AA
Israel
blmAsh
blmAsh
BSR79
AA
Connecticut
BSR87
A-
Michigan
blmAsh
c.3163T>G
+
blmAsh
blmAsh
BSR106
AA
Florida
blmAsh
blmAsh
BSR107
JA
New York
blmAsh
blmAsh
BSR119
AA
New York
blmAsh
blmAsh
BSR121
AA
Israel
c.98+1G>T
BSR126
AJ
Brazil
c.2407_2408dupT (Foun. 2)
blmAsh
blmAsh
BSR141
AA
Israel
blmAsh
blmAsh
BSR142
AA
Israel
blmAsh
blmAsh
BSR171
AA
District of Columbia
blmAsh
blmAsh
BSR172
AA
Michigan
blmAsh
blmAsh
BSR178
AA
New York
blmAsh
blmAsh
BSR182
AA
Belgium
blmAsh
c.3510T>A
BSR225
A-
Minnesota
blmAsh
c.2407_2408dupT (Foun. 2)
BSR237
AA
New York
BSR238
AA
Israel
blmAsh
blmAsh
Foundred 2 (n = 2)
c.2407_2408dupT
blmAsh (Foun. 1)
[BSR141]
[AA]
[Israel]
c.2407_2408dupT
blmAsh
[BSR237]
[AA]
[New York]
Spanish via Mexico
Colorado
Mexican, Texan, New Mexican
Utah
(Foun. 1)
Mexicans and Americans of Spanish Ancestry
Foundred 3 (n = 5)
blmAsh
blmAsh
blmAsh
BSR127
c.2506_2507delAG (Foun. 4)
BSR130
15
+
blmAsh
blmAsh
blmAsh
blmAsh
BSR179
c.3197G>A
blmAsh
Mexico
BSR197
El Salvadoran and Ecuadoran
Texas
BSR200
El Salvadoran
Maryland
[BSR130]
[Mexican, Texan, New Mexican]
[Utah]
Foundred 4 (n = 2)
blmAsh
c.2506_2507delAG
c.2506_2507delAG
c.2506_2507delAG
BSR173
Mexico
Portuguese and Brazilians
Foundred 5 (n = 4)
blmAsh (Foun. 1)
c.3752-?_4442+?del
[BSR50]
[A-]
[Vancouver]
Portuguese
Switzerland
c.3752-?_4442+?del
c.3752-?_4442+?del
BSR181
c.3752-?_4442+?del
c.3752-?_4442+?del
BSR195
Brazil
c.3587delG (Foun. 6)
BSR216
Brazil
c.3752-?_4442+?del (Foun. 5)
[BSR216]
[Brazil]
c.3752-?_4442+?del
Foundred 6 (n = 2)
c.3587delG
c.3587delG
c.3587delG
BSR217
+
Brazil
Non-Italian Europeans and North Americans
Foundred 7 (n = 18)
c.1933C>T
c.1933C>T
c.1933C>T
c.1933C>T
c.1933C>T
c.2250_2251insAAAT (Foun. 15)
c.1933C>T
BSR11
Ontario
BSR61
California
BSR64
+
Ohio
BSR67
The Netherlands
c.1933C>T
c.1284G>A (Foun. 16)
BSR91
The Netherlands
c.1933C>T
c.2725C>T
BSR105
Texas
c.1933C>T
c.3223_3224dupA
BSR114
Germany
c.1933C>T
c.2098C>T (Foun. 17)
BSR115
Florida
16
c.1933C>T
c.1933C>T
c.1284G>A (Foun. 16)
c.1933C>T
c.1933C>T
c.1933C>T
c.1933C>T
c.2923delC (Foun. 9)
c.1933C>T
BSR118
Germany
BSR133
Germany
BSR147
Belgium
BSR164
New Brunswick
BSR169
Ontario
c.1933C>T
c.3261delT (Foun. 10)
BSR170
New York
c.1933C>T
c.2695C>T (Foun. 8)
BSR177
Michigan
c.1933C>T
c.2695C>T (Foun. 8)
BSR183
Utah
c.1933C>T
c.1933C>T
BSR188
+
Belgium
c.1933C>T
c.1933C>T
BSR189
c.2695C>T
BSR21
+
California
+
Germany
California
Foundred 8 (n = 9)
c.2695C>T
c.2695C>T
c.3028delG (Foun. 11)
BSR30f
c.2695C>T
c.3415C>T
BSR109
New York
c.2695C>T
c.2308-117_2555+7420del7811
BSR111
Massachusetts
BSR136
Maryland
c.2695C>T
c.2695C>T
c.1933C>T (Foun. 7)
[BSR177]
[Michigan]
c.2695C>T
c.1933C>T (Foun. 7)
[BSR183]
[Utah]
c.2695C>T
c.2695C>T
BSR185
El Salvadoran
Ontario
c.2695C>T
c.2695C>T
BSR191
South Carolina
c.2923delC
BSR6
Maine
BSR20
Maryland
c.2923delC
BSR69
Washington
c.2923delC
BSR137
Australia
Foundred 9 (n = 7)
c.2923delC
c.2923delC
c.2824-1077_2999+310del1583bp
17
c.2923delC
c.2098C>T (Foun. 17)
BSR146
Minnesota
c.2923delC
c.1933C>T (Foun. 7)
[BSR164]
[New Brunswick]
c.2923delC
c.2923delC
BSR176
+
BSR22
+
French Canadian
Quebec
Foundred 10 (n = 4)
c.3261delT
c.3261delT
Illinois
c.3261delT
blmAsh
c.3261delT
c.1933C>T (Foun. 7)
[BSR170]
[New York]
c.3261delT
c.2250_2251insAAAT (Foun. 15)
BSR194
California
c.2695C>T (Foun. 8)
[BSR30]g
(Foun. 1)
[BSR26]
[A-]
[Indiana]
Foundred 11 (n = 3)
c.3028delG
c.3028delG
[Germany]
BSR52
Germany
c.991_995delAAAGA (Foun. 14)
BSR207
Kentucky
c.1642C>T
c.991_995delAAAGA (Foun. 14)
BSR167
Adopted (American)
Virginia
c.1642C>T
c.1882+5G>A
BSR202
Adopted (German)
Germany
c.2015A>G
c.1088-2A>G
BSR31
Belgium
c.2015A>G
c.3163T>C
BSR139
Illinois
c.991_995delAAAGA
c.1642C>T (Foun. 12)
[BSR167]
c.991_995delAAAGA
c.3028delG (Foun. 11)
[BSR207]
[Kentucky]
c.2250_2251insAAAT
c.1933C>T (Foun. 7)
[BSR64]
[Ohio]
c.2250_2251insAAAT
c.3261delT (Foun. 10)
[BSR194]
[California]
c.3028delG
c.3028delG
[+]
Foundred 12 (n = 2)
Foundred 13 (n = 2)
Foundred 14 (n = 2)
[Adopted (American)]
[Virginia]
Foundred 15 (n = 2)
Foundred 16 (n = 2)
18
c.1284G>A
c.1933C>T (Foun. 7)
[BSR91]
[The Netherlands]
c.1284G>A
c.1933C>T (Foun. 7)
[BSR118]
[Germany]
Italians and North Americans
Foundred 17 (n = 8)
c.2098C>T
c.2098C>T
BSR5
+
Colorado
c.2098C>T
c.2098C>T
BSR51
+
Kentucky
c.2098C>T
BSR59
Connecticut
Italy (Sicily)
c.2098C>T
c.3191A>T
BSR102
c.2098C>T
c.3475_3476delTT
BSR103
c.2098C>T
c.1933C>T (Foun. 7)
[BSR115]
[Florida]
c.2098C>T
c.3210+3A>T
BSR134
Missouri
c.2098C>T
c.2923delC (Foun. 9)
[BSR146]
[Minnesota]
c.311C>A
BSR80
Italian (Sicilian)
Switzerland
Foundred 18 (n = 3)
c.3164G>C
+
Italian
Massachusetts
Italian
New York
c.3164G>C
c.3164G>C
BSR113
c.3164G>C
c.3164G>C
BSR222
Italy
BSR78
Japan
BSR97
Japan
Japanese
Foundred 19 (n = 5)
c.557_559delCAA
c.557_559delCAA
c.2074+1G>T
c.557_559delCAA
c.557_559delCAA
c.1544_1545dupA (Foun. 20)
BSR100
c.557_559delCAA
c.557_559delCAA
BSR110
c.557_559delCAA
c.557_559delCAA
BSR128
+
Japan
Japan
+
Japan
Foundred 20 (n = 5)
c.1544_1545dupA
c.2254C>T
BSR71
19
West African
Pennsylvania
c.1544_1545dupA
c.1544_1545dupA
BSR86
Japan
c.1544_1545dupA
c.1544_1545dupA
BSR93
Japan
[BSR100]
[Japan]
c.1544_1545dupA
c.1544_1545dupA
c.557_559delCAA (Foun. 19)
c.1544_1545dupA
BSR129
+
Japan
a
The term foundred (abbreviated foun.) refers to a group of persons with BS each of whom has a particular BS-causing mutation, all
of whom, therefore, are descended from a person who carried that mutation—the founder. n, the number of persons comprising the
foundred.
Note. Dividing the 20 foundreds broadly into 6 groups as attempted in this table -- Jews, Non-Italian Europeans and North Americans,
Italians and North Americans, et cetera - is arbitrary and imperfect, exceptions readily noted being the following: In Foun. 1, four nonJewish parents, identifiable by "A-" in Col 6; in Foun. 5, a person with an Ashkenazi Jewish father and a non-Jewish North American
mother ("A-" in Col. 6), whereas all others in Founs. 5 + 6 are Portuguese or Brazilian; in Foun. 8. an El Salvadoran homozygous for a
mutation limited otherwise to North Americans and Europeans; in Foun. 9, an Australian; and, in Foun. 20, a North American of West
African ancestry living in the United States, all others in Founs. 19 + 20 being Japanese living in Japan.
Note. Only two of the 153 families represented in the survey knew of any "blood" relationship. The mothers of BSR9 and BSR44,
two young men with BS in Foun. 1, shared two of their great grandparents, i.e., were 2nd cousins twice removed. Both families lived
in Israel.
b
Their mutations assign 20 persons not to just 1 but to 2 foundreds. To facilitate counting, square bracketing is used in Cols. 4-7 to
identify such persons (and those data that pertain to them) who had been listed earlier in the table.
c
Mutations and their putative effects on protein structure are delineated using conventional mutation nomenclature (cf.
http://www.hqcv.org/mutnomen/), wherein +1 corresponds to the A of the ATG translation initiation codon in the reference cDNA
sequence (accession number U39817) and the initiation codon is amino acid residue 1 in the protein sequence. The mutation referred
to as blmAsh is a 6-bp deletion and 7-bp insertion at nucleotide 2207 described as c.2207_2212delATCTGAinsTAGATTC. For
reasons given in the Ellis et al. [1998], blmAsh appears in this table as the founding mutation in both Foun. 1 and Foun. 3.
d1
A blank in both Cols. 2 and 3 for a given individual indicates that although DNA was available and successfully analyzed, only a
single BLM mutation could be identified. The six such individuals are in Founs. 7, 8, 9, and 17.
d2
For a mutation that is in another foundred also, that other foundred is indicated in parenthesis in Col. 3.
20
d3
Re the possibility of identifying mutational hot spots: the only instances in either Table 1 or 2 of the same nucleotide's being
mutated in more than one way are in individuals BSR87 in Foun. 1 in whom a T becomes a G and BSR139 in Foun. 13 (also listed in
Table 2 in whom the same T becomes a C.
e
The identification of persons with BS accessioned to the Registry has since the early 1960s (see German [1969] and German and
Passarge [1989]) been by number (signifying the order in which they came to the Registry’s attention) plus the two (or three) first
letters in the given and the surname. E.g., if the 225th person to be registered has the name Boaz Strizinski, he would be identified
255(BoSt), or 255(BoStr) if a family with a surname beginning with “St” was already registered. In the present report, on the direction
of the administrative director of the Weill Medical College IRB (Institutional Review Board), the initials are deleted, each individual
now being identified simply by a Bloom’s Syndrome Registry number, e.g., BSR1, BSR2, …BSR238.
Note. The numbers that have been assigned to the 238 persons in the Bloom's Syndrome Registry signify nothing more than the order
in which the persons were accessioned. In this table,within each foundred, persons are listed in numerical order (Col. 4).
f
A second cousin or closer relationship was reported by the family in all those indicated by a plus sign except in BSR30 whose
parents are 4th cousins. Re BSR30: his parents did not themselves know that they were related, but they were found to be 4th cousins
in an exhaustive search of church records in the valley in Germany's Sauerland where their families long had dwelt [Passarge, 1991];
BSR30 is the only persons with known parental consanguinity who is not homozygous at BLM.
g1
"Noteworthy" in the sense that the ancestry differs from that expected from the individual's place of birth or principal dwelling place
shown in Col. 7.
g2
For those who were adopted, the ancestry of their biological parents is shown in parenthesis.
g3
A, an Ashkenazi Jewish parent; J, a Jewish parent but not Ashkenazi [see (g4) below]; -, a non-Jewish parent in a union in which the
other parent is Jewish; the father's designation appears before the mother's, so that A- indicates that the father was Ashkenazi Jewish
and the mother non-Jewish.
g4
The non-Ashkenazi but Jewish father of BSR107 in Foun. 1 was Bulgarian, and he emphasized that he was Sephardi; he, therefore,
provides evidence that blmAsh is segregating in the Sephardi Jewish population of Eastern Europe. The Jewish but non-Ashkenazi
mother of BSR126 in Foun. 1, who transmitted to BSR126 the unique mutation c.98+1G>T, was born to a Sephardi Jewish father and
an Egyptian Jewish, but non-Sephardi, mother.
21
Supplementary Table S5. The 64 Bloom’s syndrome-causing mutations from the Bloom’s
Syndrome Registry as of December 31, 1999.
Person
a
Mutation
Exon(s)
Effect on protein
or foundredb
c.98+1G>T
2
p.0
BSR126
c.275delA
3
p.Asn92fs
BSR186
c.311C>A
3
p.Ser104X
BSR80
c.557_559delCAA
3
p.Ser186X
Foundred 19
c.582delT
3
p.Phe194fs
BSR232
c.772_773delCT
3
p.Leu257fs
BSR211
c.814A>T
4
p.Lys272X
BSR112
c.991_995delAAAGA
5
p.Lys331fs
Foundred 14
c.1088-2A>G
6
p.Ala363fs
BSR31
c.1090A>T
6
p.Arg364X
BSR98
c.1220+3A>G
6
p.Ala363fs
BSR112
c.1284G>A
7
p.Trp428X
Foundred 16
c.1346delC
7
p.Ser449fs
BSR74
c.1544_1545dupA
7
p.Asn515fs
Foundred 20
c.1628T>A
7
p.Leu543X
BSR192
c.1642C>T
7
p.Gln548X
Foundred 12
c.1701G>A
7
p.Trp567X
BSR98
c.1784C>A
7
p.Ser595X
BSR81
c.1882+5G>A
7
p.Arg408fs
BSR202
c.1933CT
8
p.Gln645X
Foundred 7
c.1968_1969dupG
8
p.Lys657fs
BSR17
c.2015A>G
8
p.Gln672Arg
Foundred 13
c.2074+1G>T
8
p.628_691del
BSR78
c.2098C>T
9
p.Gln700X
Foundred 17
c.2193+2T>G
9
p.Gly692fs
BSR205
c.2207_2212delATCTGAinsTAGATTCc 10
p.Tyr736fs
Foundreds 1 & 3
c.2250_2251insAAAT
10
p.Leu751fs
Foundred 15
c.2254C>T
10
p.Gln752X
BSR71
c.2308-953_2555+4719del6126
11-12
p.Ile770fs
BSR92
c.2308-117_2555+7420del7811
11-12
p.Ile770fs
BSR111
c.2406+2T>G
11
p.770_802del
BSR218
c.2407_2408dupT
12
p.Trp803fs
Foundred 2
c.2488_2489dupA
12
p.Thr830fs
BSR65
c.2506_2507delAG
12
p.Arg836fs
Foundred 4
c.2643G>A
13
p.Trp881X
BSR149
c.2672G>A
14
p.Gly891Glu
BSR54
c.2695C>T
14
p.Arg899X
Foundred 8
c.2702G>A
14
p.Cys901Tyr
BSR40
c.2725C>T
14
p.Gln909X
BSR105
c.2824-1077_2999+310del1583
15
p.Val942fs
BSR20
c.2855G>T
15
p.Gly952Val
BSR223
c.2887C>T
15
p.His963Tyr
BSR123
22
c.2923delC
15
p.Gln975fs
Foundred 9
c.3028delG
16
p. Asp1010fs
Foundred 11
c.3118C>T
16
p.Gln1040X
BSR10
c.3163T>G
16
p.Cys1055Gly
BSR87
c.3163T>C
16
p.Cys1055Arg
BSR139
c.3164G>C
16
p.Cys1055Ser
Foundred 18
c.3191A>T
16
p.Asp1064Val
BSR102
c.3197G>A
16
p.Cys1066Tyr
BSR197
c.3210+3A>T
16
p.Met1007fs
BSR134
c.3223_3224dupA
17
p.Arg1075fs
BSR114
c.3255_3256insT
17
p.Arg1086X
BSR166
c.3261delT
17
p. Phe1087fs
Foundred 10
c.3278C>G
17
p.Ser1093X
BSR215
c.3415C>T
18
p.Arg1139X
BSR109
c.3475_3476delTT
18
p.Lys1159fs
BSR103
c.3510T>A
18
p.Tyr1170X
BSR225
c.3558+1G>A
18
p.Ser1121fs
BSR198
c.3587delG
19
p.Ser1196fs
Foundred 6
c.3681delA
19
p.Lys1227fs
BSR65
c.3727_3728dupA
19
p.Thr1243fs
BSR104
c.3752-?_4442+?del
20-22
p.Glu1251fs
Foundred 5
c.3847C>T
20
p.Gln1283X
BSR7
a
Mutations and their putative effects on protein structure are delineated using conventional
mutation nomenclature (cf. http://www.hqcv.org/mutnomen/), wherein +1 corresponds to the A of
the ATG translation initiation codon in the reference cDNA sequence (accession number U39817)
and the initiation codon is amino acid residue 1 in the protein sequence.
b
See Tables 1 and 2 for further information on foundreds and persons with BS.
c
The blmAsh mutation.
23