Hum Genet (1983) 65 : 94-98
© Springer-Verlag1983
Cystic fibrosis in the Ohio Amish: Gene frequency and founder effect
Katherine Wood Klinger
Department of Molecular Biology and Microbiology, and the Cystic Fibrosis Center,
Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA
Summary. A high incidence of cystic fibrosis (CF), 1/569 live
births (0.00176), was found among 10 816 live births in an Ohio
Amish isolate. The minimum gene frequency calculated from
this incidence was 0.042. In marked contrast, a second Amish
isolate was described with no occurrence of CF among 4448
live births. Founder effect is the most probable explanation for
the difference in the CFgene frequency between the two communities. Pedigree analysis identified a single ancestral couple
born in the 1700s common to all obligate heterozygotes.
Introduction
The reported incidence of cystic fibrosis (CF) in the United
States ranges from as frequent as 1:100-1:500 to as rare
as 1 : 8000-1 : 83 000 (Warwick 1978). The generally accepted
average incidence is 1 in 2000-2400, the highest incidence of
any lethal autosomal recessive disease among whites. The
factors that maintain the high gene frequency have not been
demonstrated. Despite the high frequency of CF, very little is
known about the disease beyond its pattern of inheritance and
its clinical course. The underlying genetic defect is unknown.
DNA analysis using molecular genetic techniques might
identify gene sequences specifically related to the basic
defect responsible for CF. The DNA sequences identified in
this manner could represent the "CFgene" or might be DNA
sequences closely linked to the CF locus. The probability of
success of these analyses can be improved by using human
population genetics to support the molecular genetic techniques. Identification of a population that is maximally informative because of inbreeding, distribution of affected
family members, and quality of genealogical records would aid
in the selection of D N A donors and in the segregation analysis
of any unique DNA fragments isolated from CF cells. Genetic
analysis of CF in inbred populations might also permit definition of the role of some of the causative factors proposed for
the high gene frequency [e.g., heterozygote advantage (Anderson et al. 1967), random drift (Wright and Morton 1968), and
high mutation rate (Goodman and Reed 1952)]. The aim of the
present study was to examine the incidence and inheritance of
cystic fibrosis in the Ohio Old Order Amish, a well-defined
inbred population. Subsequent investigations will be devoted
to molecular genetic analyses of DNA samples from this population.
The Amish are a religious group, of Swiss origin, that began
in the late 1600s. Waves of immigration to the United States
occurred from the early to middle 1700s, and again in the early
to mid-1800s. Since that time the Amish have formed a closed,
self-defined community that practices strict endogamy. There
are two major Amish populations in Ohio, one in Holmes
County, established in 1808-1810, and one in Geauga County,
settled in 1886-1889. The Geauga community was founded by
settlers from widespread Amish communities, including some
founders who immigrated from Holmes County. This pattern
of immigration is reflected by the surname distribution within
the two settlements. The surnames Miller, Troyer, and Yoder
account for 40%-50% of the heads of household in both communities. The surnames Byler, Detweiler, and Fisher are
virtually confined to Geauga County, whereas the surnames
Beachy, Eicher, Keim, and Raber are prominent in Holmes
County.
The informative nature of Amish populations for genetic
studies was previously described by McKusick et al. (1964).
The Amish avail themselves of modern medical care and,
despite the separate nature of the Amish life-style, cooperate
with genetic studies. The existence of separate Amish isolates
allows comparisons to be made, and conclusions to be drawn
regarding the establishment of the CFgene in the Ohio Amish.
Materials and methods
Probands were identified by the CF Centers at Rainbow Babies
and Childrens Hospital, Cleveland, Ohio, and Children's
Hospital Medical Center of Akron, Ohio. Additional cases
of CF, both living and deceased, within those families were
identified by review of the medical records. An individual was
classified as affected only on the basis of unequivocal diagnosis
at one of the CF centers. Diagnoses were based on verified
sweat chloride levels in excess of 60 mEq/1 in the presence of at
least one of the following: family history of CF, chronic pulmonary disease, or pancreatic insufficiency.
The following methods were used to identify cases of CF in
the Geauga County Amish, because no children from this
community have been treated at either CF center: (1) verbal
questionnaires regarding CF or CF-like symptoms in Geauga
County Amish were completed by parents of children with CF
residing in Holmes County, Amish church elders, and the
directors of treatment centers for other inherited diseases;
(2) written survey was sent to physicians in Geauga County.
Follow-up telephone contact was used as necessary.
Many of the Amish parents of affected children participate
in an Amish "CF circle letter." No cases of CF in either Geauga
or Holmes County Amish in addition to those I have identified
were reported in this letter.
95
Family I
Table 1. Sibship distribution of cases of CF in Holmes County
Amish
Family 2
Family 3
Size
of sibship
No.
of sibships
Observed No. affected
5
6
12
14
2
2
1
1
5
7
3
4
Maximum likelihood estimate of P, 0.3816; SE, 0.0739
Famil:
-
_
~
5571 5657
Family 6
m
I
8000
5570
8005
8001
e~'t
Fig. 1. Distribution of CF among six Holmes County Amish sibships.
I , • affected male, female; i , ~ affected male, female, deceased;
N, ~ heterozygous male, female. The numbers assigned to each heterozygote are the respective computer identification numbers
The number of live births between 1950 and 1981 in the
Amish communities of Holmes and Geauga Counties was calculated from the 1981 Holmes County (Gingerich 1981) and
1981 Geauga County (Byler 1982) Amish directories. This is a
maximum estimate of the number of live births because all
deaths that occurred on the day of birth were treated as live
births (unless specifically marked as stillbirths). All of the
families of affected children belong to church districts included among the 134 out of 137 districts that participate in
the directory. Therefore, the number of live births derived
from the directories provides an accurate denominator for the
incidence rate.
Pedigree analyses were based primarily on the Ohio Amish
Genealogy (Cross 1966). The information relevant to families
with affected children was stored as computer files and the data
analyzed using a Vax Computer and the Ingres Data Base
Management System (Dept. Biometry, CWRU). Additional
information was obtained from other published genealogies
(Cross et al. 1970; Hostetler 1912, 1938; Mast et al., no date;
O. R. Miller 1974, 1976, and 1981; J.V. Miller 1976) through
Bluffton College, Bluffton, Ohio, and added to the data base.
6765 6764 5656
|
8004
8006 8007
Fig.2. Partial pedigree of the CF kindreds showing the family relationships of the heterozygotes. The symbols are as in Fig. 1. The
numbers within the heterozygote symbols refer to the family numbers
assigned in Fig. 1. Matching numbers denote marriage partners. Only
individuals contributing to the extended family relationship are
indicated
Results
present in the Geauga County Amish at the same frequency as
in the Holmes County Amish, seven to eight cases of CF would
be expected.
Segregation analysis of the data in Table 1 by Haldane's
maximum likelihood method for truncate ascertainment
(Haldane and Smith 1947) estimated P as 0.38+0.07
(0.08>P>0.07), i.e., compatible with an autosomal recessive
mode of inheritance, as has been reported by many other investigators. (It should be noted that the observed frequency
is not significantly different from 0.5.) It was not possible to
determine gene frequency, number of gene loci, or mode of
inheritance from cousin analysis despite the large number
(385) of first cousins of probands because of the consanguinous relationship of the parents, and the small number of kindreds (6).
Incidence
Origin of the CF allele
Nineteen cases of CF were identified in Old Order Amish
children living in Holmes County born between 1950 and 1981.
These cases were distributed among six families which contained an additional 28 unaffected siblings. The distribution of
affected children among the families is shown in Fig. 1. During
the years 1950-1981, 10816 live births were recorded in the
Holmes County 1981 Amish Directory. Thus the incidence of
the disease in this community was 1 case per 569 live births, the
gene frequency is 0.042, and the heterozygote frequency is
0.08. During the same time span 4448 live births were recorded
in the Geauga County Amish Directory. No cases of CF have
been identified among these children. If the CF gene were
Eleven generation pedigrees were constructed for the parents
of the affected children. Although the genealogical information was not complete to 11 generations for all ancestors, the
incomplete portions occurred mainly in the first three generations, and had little effect on pedigree coefficients, etc. The
relationships are complex because of the limited number of
original Amish settlers of Holmes County (70-80 adults, many
of them related) and subsequent inbreeding. Therefore, the
complete pedigrees are not presented. The obligate heterozygote (i.e., parents of affected children) relationships can be
subdivided into two extended families if the analysis is limited
to three generations. This relationship is shown in Fig. 2, and
96
5570
8001
5571 5657
6765
8000
8005
6764 56,56
[•
8004
8006
Fig.3. Partial p e d i g r e e (four g e n e r a t i o n s ) of the CF kindre ds , illustrating the e x t e n d e d family r e l a t i o n s h i p of 11 of the 12 he t e roz y g o tes . T h e
dashed line d e n o t e s a half-sibling. All o t h e r symbols are as in Fig. 2
Table 2. C o u s i n r e l a t i o n s h i p s b e t w e e n affected H o l m e s C o u n t y A m i s h a
Family
No. 1
No.2
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
Maternal b
-
2nd cousin
1 x removed
2nd c ous i n
1 × removed
1st cousin
-
-
Paternal c
-
1st cousin
1 x removed
1st c ous i n
1 × removed
--
--
-
Maternal
1st c o u s i n
1 × removed
2nd cousin
1x removed
-
2nd c ous i n
2nd c ous i n
1 × removed
-
-
Paternal
No.3
No. 4
No. 5
No. 6
m
Maternal
2nd co u s in
1 x an d 2 ×
removed
m
Paternal
1st cousin
1 × removed
2nd c o u s i n
1 × removed
2nd cous i n
-
2rid c o u s i n
1x removed
Maternal
1st c o u s i n
2rid c o u s i n
1× removed
2nd c ous i n
1 × removed
-
Paternal
.
.
.
.
Maternal
.
.
.
.
Paternal
-
Maternal
.
Paternal
-
-
.
1st c ous i n
F a m i l y d e s i g n a t i o n s are as d e s c r i b e d in Fig. 1
b C o u s i n r e l a t i o n s h i p b e t w e e n affected c h i l d r e n via m o t h e r ' s family
° C o u s i n r e l a t i o n s h i p b e t w e e n affected c h i l d r e n via father's family
2nd co u s in
1 x removed
2nd co u s in
2 x removed
twice
1st c ous i n
.
--
2nd cousin
1× removed
2nd co u s in
2 x removed
twice
m
-
.
m
m
2nd c ous i n
1 × a nd
2 × removed
2nd c ous i n
1× removed
2nd c ous i n
2 × removed
twice
2nd c ous i n
1× removed
2rid c ous i n
2 × removed
twice
97
indicates that three heterozygotes (no.6765, no.8001, and
no. 8007) are unrelated at this level. An extended relationship
can be constructed including all but one (no. 8007) of the obligate heterozygotes if consideration is expanded one generation, i.e., to the great-grandparental level (Fig. 3). As
diagrammed in Table 2 the family relationships among the
affected children are highly informative for linkage analyses.
The pedigrees were analyzed to determine if all of the cases
of CF could be accounted for by descent from a common
ancestor. Eleven of the 12 obligate heterozygotes shared 4 common ancestral couples. Only one of these, couple no. 0384no. 0004, was common to all twelve obligate heterozygotes;
therefore, either Jacob Hochstetler (no.0384) or his wife
(no. 0004) may have been heterozygous for CF. Descent from a
son of couple no. 0384-no. 0004 was the only ancestry the
twelfth heterozygote (no. 8007) shared with any of the remaining 11 parents.
Jacob Hochstetler and his wife were prominent eighteenth
century Amish immigrants, and their descendents have
migrated throughout the United States. The Jacob Hochstetler
family is the subject of two comprehensive books (Hostetler
1912, 1938). As a result, Amish pedigrees are frequently more
complete with respect to Hochstetler descent than with respect
to descent from some other ancestors. The variation in pedigree completeness could cause misidentification of the original heterozygote immigrant ancestor. The possibility of misidentification of the common ancestor was addressed by construction of (1) pedigrees showing the Hochstetler descent for
the Amish residents of Geauga County and (2) for the spouses
of the obligate heterozygotes' siblings. Preliminary analysis of
these data indicated that it is possible that Jacob Hochstetler
and/or his wife were heterozygous for CF. Four of Jacob
Hochstetler's children survived to adulthood. The Geauga
County Amish are descended from a set of Jacob Hochstetler's
grandchildren that are different from those that are ancestraI to
the Holmes County Amish. In a similar manner, the descent of
the siblings' spouses was not identical to the descent of the
carrier spouses. These findings support the probability that
either Jacob Hochstetler or his wife was the source of the CF
allele in the Holmes County Amish. Because of the complexity
of the pedigrees (relative to descent from Hochstetler) extensive modeling and statistical analysis will be required to
determine the probability that Jacob Hochstetler or his wife
was heterozygous for CE
Discussion
The incidence of CF in the Holmes County Amish was found
to be 0.00176, higher than would have been expected based on
data derived from the general population. Steinberg and
Brown (1960) estimated that the frequency of CF in Ohio
(whites born 1950-1953) was 0.000267. The currently accepted
(but possibly incorrect) national incidence (United States) is
0.0005 (1/2000) (Warwick 1978). There are two primary sources
of error in a population study of this nature. One is misdiagnosis of cases of CF, and the other is an incorrect determination of the size of the base population. Only diagnoses confirmed at an established CF center were accepted for the purpose of this study. Three of the CF patients died before the
availability in 1960 of the most reliable sweat test, i.e., pilocarpine io,ntophoresis and chemical analysis (Gibson and
Cooke 1959; Gibson et al. 1975). However, these patients (who
belonged to family no. 1) have two surviving siblings with
CF, and were diagnosed at the Rainbow Babies and Childrens
Hospital CF Center. It is possible that some cases of CF
were not detected, particularly in the early years included in
this study. Prevalence rates of 1/963, 1/468, and 1/516 were
obtained when the Holmes County Amish data were subdivided by decade (1950s, 1960s, 1970s), indicating that there may
have been a slight deficit of cases in the 1950s. This would make
the incidence of 1/569 a minimum estimate of the frequency
of CF in the Holmes County Amish. The denominator of the
incidence rate was determined from the Holmes County directory. The number of live births is a maximum estimate,
because of the treatment of possible stillbirths (see "Material
and methods"). Again, this would generate an underestimate
of the frequency of CF in Holmes County Amish. The minimum gene frequency for CF in Holmes County Amish, 0.042,
is among the highest reported. The only higher gene frequency
known (0.0515) was reported for the years 1946-1972 in Plouzevade, Brittany (Bois et al. 1978). High CF incidence values have
been reported for other small, semi-defined populations, e.g.,
1/1192 (0.0084) white births in the Republic of South-West
Africa (Namibia) (Super 1978).
The data indicate that large deviations both above and
below the mean national incidence of CF occur in subisolates
of the Ohio Amish. The incidence of CF in Holmes County
Amish is 3-4 times the national average, and 6-7 times higher
than the frequency for Ohio reported by Steinberg and Brown
(1960), whereas CF has not been found to occur in Geauga
County Amish. Founder effect is the most probable explanation for the marked difference in the incidence of CF within
the two communities.
There was no significant difference in the level of consanguinity between the two Amish communities, which was
estimated by sampling to be approximately 0.016. There were,
however, differences between the two communities in the
degree of relationship to founding ancestors (e.g., Beachy,
Hochstetler, Beiler, Detweiler). This finding is in agreement
with earlier genetic studies of the Amish that suggested that
separate Amish isolates exist (McKusick et al. 1964; B. Schacter, personal communication). Thus the high frequency of CF
in Holmes County Amish is the result of the original enrichment of the gene in the founding population, coupled with
subsequent endogamy. The absence of CF in Geauga County
Amish can be attributed to the absence of the CFgene in the
original settlers. Other explanations (e.g., high mutation rate,
heterozygote advantage, etc.) of the difference in prevalence
between the two communities require the assumption of a differential effect on the two communities. A differential influence is unlikely because of the similarity of overall genetic
makeup and socioeconomic and occupational conditions between the two groups.
Founder effect is clearly the cause of the high CF gene
frequency in the Holmes County Amish. Determining which
founder ancestor established the gene in the community is difficult, but the data implicate Jacob Hochstetler or his wife
as possible CF heterozygotes. Comparative analysis of the
pedigrees of the spouses of siblings of CFheterozygotes and
of the Geauga County Amish (both groups presumably lacking
the CFgene) do not rule out the CFheterozygote assignment of
Jacob Hochstetler and/or his wife. Further studies are necessary to confirm this assignment. These studies will include
probability analysis, risk assessment, and analysis of CFin the
Indiana Amish (two families currently identified).
The profound consequences of founder effect on the frequency of CF in these two Amish isolates will facilitate both
98
classical and molecular genetic linkage studies. T h e failure to
observe the CF gene in the G e a u g a C o u n t y A m i s h provides
n o r m a l control donors w h o are genetically similar to the C F
patients, but w h o are unlikely to be u n d e t e c t e d heterozygotes.
T h e high frequency of the CF gene in the H o l m e s C o u n t y
A m i s h and the family relationships of the affected children
provide a large n u m b e r of informative donors. T h e availability
of such u n i q u e populations and powerful n e w t e c h n i q u e s for
D N A comparisons should make possible an approach to defining the CF gene.
Acknowledgments. This work was supported in part by a grant from the
Cystic Fibrosis Foundation (Rainbow Chapter) and United Torch
Services of Cleveland. I thank Dr. V. A. McKusick for funishing helpful information, Dr. C. W. Shuster for help in establishing the data
base management system, and Dr. A. G. Steinberg for many helpful
discussions.
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Received June 10 / Revised August 8, 1983