Carrier frequency of the IVS4 + 4 A--\u003eT mutation of the Fanconi anemia gene FAC in the...

1995 86: 4034-4038   

 PC Verlander, A Kaporis, Q Liu, Q Zhang, U Seligsohn and AD Auerbach anemia gene FAC in the Ashkenazi Jewish populationCarrier frequency of the IVS4 + 4 A-->T mutation of the Fanconi

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Carrier Frequency of the IVS4 + 4 A - T Mutation of the Fanconi Anemia Gene FAC in the Ashkenazi Jewish Population

By Peter c. Verlander, Athena Kaporis, Qian Liu, Qiao Zhang, Uri Seligsohn, and Arleen D. Auerbach

Fanconi anemia (FA) is a genetically and phenotypically het- erogeneous autosomal recessive disorder defined by a cellu- lar hypersensitivity to DNA cross-linking agents. One of the FA genes, FAC, has been cloned and the genomic structure of the coding region has been characterized. We have devel- oped amplification refractory mutation system (ARMS) assays for five known mutations in FAC, and have applied these assays to determine the carrier frequency of the IVS4 + 4 A - T (IVS4) mutation in an Ashkenazi Jewish popula- tion. We tested 3,104 Jewish individuals, primarily of Ash- kenazi descent, for the two most common FAC mutations, IVS4 and 322delG. Thirty-five IVS4 carriers were identified, for a carrier frequency of 1 in 89 (1.1%; 95% confidence inter-

ANCON1 ANEMIA (FA) is an autosomal recessive DNA instability syndrome that displays both clinical

and genetic heterogeneity. The clinical manifestations of FA include progressive pancytopenia, a variety of congenital malformations, and a predisposition to cancer.'.* FA patients display a wide range of clinical features: patients may be severely affected, with multiple congenital malformations, or may have a mild phenotype, with no major malformations. This variability can make diagnosis of FA difficult; the dis- tinguishing feature of FA is a cellular hypersensitivity to clastogenic agents such as diepoxybutane (DEB) and mito- mycin C ("C).'

FA includes at least five complementation groups, as de- fined by cell fusion experiment^:^ and the gene FAC, cor- recting complementation group C (FA-C), has been cloned.6 FA patients have been screened for mutations in FAC by single-strand conformation polymorphism (SSCP) analysis7 and by chemical mismatch cleavage analysis,'"' and six mu- tations that are pathogenic for FA have been identified. The most common mutation is IVS4 + 4 A -+ T, a splice site mutation that has only been found in patients with Jewish an~estry.~,'".'' The other common mutation is 322delG, a frameshift mutation found in patients of Northern European ancestry. Genotype-phenotype analysis has shown that IVS4 is always associated with a severe FA phenotype, whereas

F

From the Laboratory of Human Genetics and Hematology, The Rockefeller University, New York, NY; and the Institute of Thrombo- sis and Hemostasis, the Department of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer, Israel.

Submitted August 2, 1995; accepted September I , 1995. Supported in part by National Institutes of Health Grant No. R01

HW2987. Address reprint requests to Peter C. Verlander, PhD, Box 178,

The Rockefeller University, 1230 York Ave. New York, NY 10021- 6399.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/8611-0043$3.00/0

4034

Val 0.79% to 1.56%); no 322delG carriers were found. To determine if the IVS4 mutation was confined to the Ashke- nazi Jewish population, we tested 563 Iraqi Jews for IVS4, and no carriers were found. Because the IVS4 mutation has only been found on chromosomes of Ashkenazi Jewish ori- gin and is the only FAC mutation found on these chromo- somes, we suggest that a founder effect is responsible for the high frequency of this mutation. With a carrier frequency greater than 1% and simple testing available, the IVS4 muta- tion merits inclusion in the battery of tests routinely pro- vided to the Jewish population. 0 7995 by The American Society of Hematology.

322delG is commonly associated with the milder form of the ~yndrome.~

There are a number of genetic diseases that occur at high frequency in the Ashkenazi Jewish population, including Tay-Sachs, Gaucher, and Canavan diseases; FA-C may be considered to be another disease in this category. The IVS4 mutation apparently is unique to the Jewish population, be- cause all FA patients identified with this mutation have Jew- ish ancestry. In addition, IVS4 appears to be the only FA- C mutation in this population.

We have developed amplification refractory mutation sys- tem (ARMS) assays for five FAC mutations to provide a means for rapid, nonradioactive testing, and we have applied these assays to screen a healthy Jewish population for IVS4 and 322delG, the two most common FA-C alleles. In addi- tion, we have applied these ARMS assays to (1) assign newly diagnosed FA patients to group C, (2) provide rapid carrier testing for FA-C families, and (3) provide prenatal diagnosis for FA-C families. We conclude that the carrier frequency in the Jewish population screened in this study is greater than 1 in 100.

MATERIALS AND METHODS

Study Sample ARMS assay development andprenatal diagnosis. DNA samples

used for development of the ARMS assay were from FA families enrolled in the International Fanconi Anemia Registry (IFAR), main- tained at The Rockefeller University since 1982. FA patients with mutations in FAC were previously identified by SSCP analysis.' Prenatal diagnosis was performed for an FA-C family enrolled in the IFAR that was known to carry the IVS4 + 4 A + T mutation. DNA samples were isolated from peripheral blood (PB) or from chorionic villus samples as described below.

Carrier testing. DNA samples from 3,104 Jewish individuals ascertained anonymously were supplied by Dor Yeshorim, an organi- zation that provides genetic testing services to the religious Jewish community in New York. Dor Yeshorim tests 8,000 to 9,000 individ- uals annually; the samples used in this study were obtained between December 1993 and March 1995. DNA samples from 563 Jewish individuals born in Iraq were obtained in Israel during a recent study.''

DNA Isolation DNA was isolated from PB with a modification of the salting out

technique." Three hundred microliters of whole blood was combined

Blood, Vol 86, No 11 (December I ) , 1995: pp 4034-4038

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FAC CARRIER FREQUENCY IN ASHKENAZI JEWS 4035

Table 1. ARMS Assay Primers ~-

Primer

RU6 RUlO RUl2 RU12c RU14 RU15 E1F E4F E6F E13F E13R E14F

~

Primer Sequence

5"AGCTTCTGCATCCAAAACTfi-3' 5"AAAGTGGAAGCCTGATACA-3' 5 ' - A n n C A m C A A A A G T G A T ~ T A l T A A A ~ C A - 3 ' 5 ' - A A A C A T T T C A A A A G T G A T ~ T A l T A n n ~ ~ - 3 '

5'-AATAAGTGGGACACAAACOCA-3' 5'-ACCATTTCClTCAGTGCTGG-37

5"ClTCTAGACTTGAGlTCACG-3'

5"GTAGGCATTGTACATAAAAG-3' 5"GTCCTTAAlTATGCATGGCTC-3' 5"CCTAGAAGTATGTCTGTCCTG-3' 5"CTCTCClTGACTAGGATGCTG-3' 5'-GGATAGGGCTTCmCAGGG-3'

Boldface type indicates a base that is mismatched when annealed to the wild-type sequence. Underline indicates a base that matches a mutation.

with 900 pL of red blood cell (RBC) lysis buffer (10 mmoVL Tris- Cl, 125 mmoVL NKCI, 1 mmom EDTA, pH 8.0). mixed, and held at room temperature for 10 minutes. White blood cells were pelleted and resuspended in 300 pL of TE (IO mmovL Tris-Cl, 1 mmoVL EDTA, pH 8.0). Ten microliters of proteinase K (20 mg/mL in 1% sodium dodecyl sulfate [SDS], 2 mmoVL EDTA), 20 pL 10% SDS, and 2 pL of RNAse A (10 mg/mL in W20) were added, mixed, and incubated at 56°C for 15 minutes. One hundred fifty microliters of 7.5 mom NKOAc was then added, mixed, and the resulting precipitate was pelleted by centrifugation in a microfuge for 3 minutes at 4°C. The supernatant was transferred to a clean tube, genomic DNA precipitated by addition of 300 pL of isopropanol, and DNA was pelleted for 3 minutes. The pellet was rinsed with 500 pL of 70% ethanol, air dried, and resuspended in 100 pL TE. DNA was extracted from chorionic villus sample (CVS) cultures essentially as described above,with the omission of the RBC lysis step. Cells from one confluent T25 flask were removed by trypsiniza- tion, washed with phosphate-buffered saline, resuspended in 300 pL TE, and DNA was extracted as described.

ARMS Assays Primer selection. ARMS assays were developed for each of the

known mutations in FAC. Primers are listed in Table 1, and primer combinations are listed in Table 2. The PRUlER program was used to predict melting temperatures, and Ah4PLIFYI4 was used to predict potential polymerase chain reaction (PCR) products. Mutation-spe- cific primers were designed so that the 3' base matched the point of the mutation. All mutation-specific primers anneal to the plus strand. The second primer for each assay was the forward primer for the exon containing the m~tation. '~ Primers were designed to have a predicted annealing temperature of 56°C. To reduce back- ground, a second mismatch was introduced at the third base from the 3' end in some primers, so that two of the last three nucleotides would be mismatched in an individual lacking the mutation. The test for the normal allele at the position of the IVS4 mutation (see Fig 2) was performed using primer RU12c in place of primer RU12; RU12c differs from RU12 only in the 3' base, making it specific for the nucleotide normally found at that position.

PCR Reactions PCR reactions were performed in a Perkin-Elmer model 9810

thermal cycler (Perkin-Elmer, Norwalk, CT). PCR reactions con- tained 20 mmoVL (NH&S04, 20 mmoVL NaCl, 80 mmoVL Tris (pH 9.0), 2 mmom MgC12, 250 pmoVL spermidine, 1.25% for-

mamide, 200 pmoVL m s , 5 pm01 of each primer and 0.6 U Taq DNA polymerase in a 15-pL final volume. PCR was initiated with a 7-minute denaturation at 95"C, followed by 30 cycles of 30 seconds at 94"C, 30 seconds at 56"C, and 40 seconds at 72°C and concluded with a 5-minute extension at 72°C. PCR reactions were set up using a hot-start procedure; 5.5 pL of an initial mix containing buffer, spermidine, formamide, dNTPs, and primers was combined with 1 .O pL genomic DNA and overlaid with 25 pL mineral oil. After heating to 95°C 8.5 pL of a hot-start mix containing buffer, spermidine, dNWs, and Taq DNA polymerase was added to initiate the reaction. PCR products were separated by electrophoresis on 2.5% agarose gels in 0.5X TridborateEDTA (TBE) for 30 minutes at 180 V.

Statistical Analysis The exact binomial method was used to calculate 95% confidence

intervals.I6

RESULTS

ARMS Assay Development

ARMS assays were developed on genomic DNA samples from individuals with known mutations. Primer sets were tested in different combinations to empirically determine which of the mutations could be assayed together in multi- plex reactions. Assay A included primers for IVS4 + 4 A + T and 322delG (Fig lA), while Assay B included prim- ers for Q13X, R185X, and L554P (Fig 1B). Each assay includes a set of primers for FAC exon 13 as a positive control for the PCR reaction.

IVS4 + 4 A + T Carrier Frequency

Genomic DNA isolates from 3,104 Jewish individuals, primarily of Ashkenazi descent, were tested for IVS4 + 4 A -+ T and 322delG (Table 3). 35 IVS4 carriers were identi- fied, for a carrier frequency of greater than 1%; no 322delG carriers were found. To determine if the IVS4 mutation was also present in the Iraqi Jewish population, which represents the original gene pool of Jews," genomic DNA isolates from 563 Iraqi Jews were tested for IVS4, and no carriers were found (Table 3).

Prenatal Diagnosis

A chorionic villus sample from an FA-C family known to carry the IVS4 mutation was tested using ARMS assay A for the presence of the mutation (Fig 2A). To distinguish carriers from affected individuals, a second assay was per- formed for detection of the wild-type allele (Fig 2B). The assay established the fetus as homozygous for IVS4, and

Table 2. ARMS Assay Primers

Mutation Primers Product

Assay A 322delG ElF, RUlO 147 bp IVS4 + 4A + T E4F. RU12 220 bp Control E13F, E13R 303 bp

Q1 3X ElF, RU6 121 bp R185X E6F. RU15 111 bp

Control E13F. E13R 303 bp

Assay B

L554P E14F. RU14 258 bp

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4036 VERLANDER ET AL

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7

A exon 13

1 * 322delG c/ IVS4

B T Q 1 3 X

R1 85X

Fig 1. ARMS assays for detection of FAC mutations. Assay (A) tests for the IVS4 and 322delG mutations, assay (B1 for the 013X. R185X.and L554P mutations. Samples are: (l) pBR322-Msp I markers, (2) dH,O control, (3) normal control, (4) IVS4/IVS4 homozygote, (5) 322delGIR185X compound heterozygote, 16) L554P carrier, (7) Q13Xl 013X homozygote.

this result was confirmed by DEB testing. The pregnancy was terminated at the request of the family. and examination of the abortus found multiple congenital malformations, in- cluding bilateral ahsent thumbs.

DISCUSSION

ARMS assays specific for known mutations in FAC pro- vide rapid means for assignment of FA patients to group C. for prenatal diagnosis in FA-C families. and for identification of carriers. both in FA-C families and in populations at risk. We have developed assays for five FAC mutations and have combined these assays into multiplex reactions. which we are currently using for each of these applications.

Of the mutations identified in FAC. two are found at sig- nificantly higher frequency than the others: IVS4 + 4 A -, T. found in patients with Ashkenazi Jewish ances- try.7."'." and 311delG. found in patients with Northern Euro- pean ancestry.' IVS4 + 4 A -+ T is the only FAC mutation

Table 3. IVS4 Carrier Frequency in Jews

Ashkenazi lraai

Carriers 35 0 Noncarriers 3,069 563 Total 3,104 563 Frequency 1.1% 0% 95% Cl 0.79%-1.56% 0%-0.65%

Abbreviation: Cl, confidence interval.

dexon l3 t normal

Fig 2. Prenatal diagnosis in an FA-C family. Parallel assays were run testing for (A) the IVS4 mutation and (B) the normal allele at the position of the IVS4 mutation. Samples are: (1) pBR322-Msp I mark- ers, (2) dH,O control, (3) normal control, (4) father, (5) mother, (61 affected sibling, ( 7 ) carrier sibling, (8) fetus.

that has been found on Jewish chromosomes. Twenty-one probands from the IFAR for whom both parents have Ash- kenazi Jewish ancestry have been screened for FAC muta- tions. and 20 are homozygous for IVS4; the I remaining patient has n o mutation in FAC detectable by SSCP.7 All of these patients have a severe phenotype, with multiple congenital malformations. Four additional probands with Ashkenazi ancestry on only one side of their family have also been tested. and none carry IVS4. Given the high fre- quency of IVS4 i n the Ashkenazi population, the lack of compound heterozygotes with this allele suggests that FAC mutations are extremely rare outside the Ashkenazi popula- tion.

Whitney et all' found ;I carrier frequency of I in 157 (0.64%; 95% confidence interval 0.08% to 2.3%); however. that estimate was based on a relatively small sample size ( n = 314). and the 95% confidence interval is quite broad rela- tive to the frequency found. The carrier frequency of I in 89 ( 1 . 1 % : 95% confidence interval 0.79% to 1.56%) found in this study is almost twice a s high a s was found by Whitney et a l . with ;I much narrower 95% confidence interval. Our point estimate is more likely to reflect the true figure in the population. because o f the larger sample size tested; it is not excluded by the previous study. while the previous point estimate of 0 . 6 4 % is excluded from the 95% confidence interval of our study. This is consistent with the greater statistical power of this study compared with the study of Whitney et a l .

We can confirm the accuracy o f the higher frequency by analyzing the expected numbers of FA patients in the New

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FAC CARRIER FREQUENCY IN ASHKENAZI JEWS 4037

York metropolitan area, for which reasonable estimates can be made. The Jewish population of the New York metropoli- tan area (excluding New Jersey) numbers 1.4 million, of whom 304,000 are under 18 years old.’”’9 The proportion of Ashkenazi Jews in this population is estimated to be 90% to 95%, and both parents are Jewish in approximately 85% of the households. If one uses the upper limits of these estimates for calculations, the New York metropolitan area includes roughly 235,000 individuals under the age of 18 in families where both parents have Ashkenazi Jewish ancestry. Given a carrier frequency of 1 in 89, 7 or 8 affected FA-C individuals are predicted in this population. In fact, the IFAR includes 7 affected FA-C individuals of Jewish ancestry born in the last 18 years in the New York metropolitan area.

A carrier frequency of 1 .l% for a severe, usually fatal disease, warrants the inclusion of the IVS4 mutation of FAC as one of the genetic diseases screened routinely in the Ash- kenazi Jewish population. Whitney et a1 concluded other- wise, suggesting that the carrier frequency was not high enough to warrant this expense. We have based our recom- mendation on the fact that the carrier frequency is in fact significantly higher than originally estimated, and on the fact that testing for IVS4 does not require the creation of an entirely new program. Because the Ashkenazi population is already screened for a variety of genetic diseases, the mar- ginal cost of testing for an additional mutation is relatively low; although the carrier frequency of the IVS4 mutation might not be high enough to justify the initiation of a totally new screening program, it is certainly high enough to justify including this mutation in the battery of tests already being performed. Approximately 35,000 individuals are tested for Tay-Sachs disease annually in the United States’”; an Ash- kenazi population of this size is expected to include 395 IVS4 carriers (95% confidence interval 276 to 546), and 4 to 5 marriages of IVS4 carriers (95% confidence interval 2.2 to 8.5) During the course of this study, one marriage of IVS4 carriers was detected; the cost of performing the study was significantly less than the cost of caring for a child affected with FA.

The high carrier frequency of the IVS4 mutation in Ash- kenazi Jews places FA-C in the group of so-called “Jewish” diseases that include Tay-Sachs, Gaucher, and Canavan dis- ease, among others.“ For most of these diseases, there is a single common mutation that accounts for the majority of the affected chromosomes. For those diseases for which direct mutation screening is possible, carrier frequencies range from 1% to 2% for Niemann-Pick disease” up to 4% to 6% for Gaucher.*’,’’ Debate continues on the relative roles of genetic drift and selective advantage in generating the high carrier frequencies of these diseases in the Ashkenazi Jewish population. A variety of observations have lent support to the theory that selective advantage of heterozygotes is the primary force in fixing some of these mutations in the popu- lation; one argument is that the carrier frequency of some of these diseases, such as Gaucher, is too high to be ex- plained by genetic The observation that several of these diseases involve lysosomal enzymes (Tay-Sachs, Gaucher, Niemann-Pick) has been suggestive of a possible selective mechanism, as is the fact that multiple mutations are found in each of these diseases.”

Conversely, a founder effect followed by random genetic drift has been invoked for those diseases in which there is marked linkage disequilibrium and in which a single muta- tion is thought to account for virtually all of the affected chromosomes. Bloom syndrome and idiopathic torsion dys- tonia are diseases for which linkage disequilibrium has been shown, and persuasive arguments in favor of a founder effect followed by genetic drift have been made for each of the~e .~‘~~’ In this context, genetic drift is the most likely explanation for the high incidence of the IVS4 mutation of FA-C in the Ashkenazi population. The carrier frequency for this allele is not so high that selection need be invoked as an explanation, and the fact that 100% of affected FA-C chromosomes of Jewish origin carry the IVS4 mutation is strong evidence for a founder effect. We are currently en- gaged in haplotype analysis of IVS4 carriers to see if the expected linkage disequilibrium exists.

The lack of the IVS4 mutation in the Iraqi Jewish popula- tion suggests that the mutation originated in the Ashkenazi population after its separation from other Jewish populations. Iraqi Jews are considered to represent the original gene pool of the Jews who lived in Babylon over 2,500 years ago,l7 whereas the present day Ashkenazi population is thought to have expanded from a small group of individuals as recently as 500 years ago.27 The only other Ashkenazi mutations that have been screened for among Iraqi Jews are the type I1 and I11 mutations for factor XI deficiency.” Factor XI deficiency, an autosomal recessive bleeding disorder, is the most com- mon hereditary disorder among Ashkenazi Jews, with a het- erozygote frequency of 8.1 %. Of the four mutations identi- fied, type I1 and I11 are the most common, accounting for 98.4% of the mutant alleles in a study of Ashkenazi Jewish probands. Although the type I11 mutation was not found in the Iraqi Jewish population, the type I1 mutation was found to be present at a frequency comparable to its frequency in the Ashkenazi population.” The type I1 mutation apparently originated before the divergence of the Iraqi and Ashkenazi Jewish populations, whereas both the factor XI deficiency type I11 mutation and the FAC IVS4 mutation appear to have originated in the Ashkenazi population after its divergence from the original gene pool.

The IVS4 mutation of FAC appears to be unique to the Ashkenazi Jewish population, and places FA in the group of genetic diseases occurring at high frequency in this popu- lation. Carrier screening programs have been implemented for Ashkenazi Jews for several of these diseases; with a carrier frequency greater than 1% and simple testing avail- able, the IVS4 mutation merits inclusion in the battery of tests routinely provided to this population.

ACKNOWLEDGMENT

We thank Steven B. Auerbach, MD, MPH (US Public Health Service, Health Resources and Service Administration) for advice on statistical analysis of our data and for performing the calculations of 95% confidence intervals.

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