? 2007 Wiley-Liss, Inc.American Journal of Medical Genetics Part A 143A:1938–1940 (2007)
Phenylalanine Hydroxylase Deficiency Exhibits
Mutation Heterogeneity in Two Large Old Order
Heng Wang,1* Leah Nye,1Erik Puffenberger,2and Holmes Morton2
1Das Deutsch Center (DDC) Clinic for Special Needs Children, Middlefield, Ohio
2The Clinic for Special Children, Strasburg, Pennsylvania
Received 24 January 2007; Accepted 8 April 2007
How to cite this article: Wang H, Nye L, Puffenberger E, Morton H. 2007. Phenylalanine hydroxylase
deficiency exhibits mutation heterogeneity in two large old order Amish settlements.
Am J Med Genet Part A 143A:1938–1940.
To the Editor:
(OMIM 261600) results in intolerance to dietary
including phenylketonuria (PKU) and non-PKU
hyperphenylalaninemia. PAH deficiency, the most
common inborn error of amino acid metabolism in
Caucasians with an incidence of about 1/10,000, is
ing to PAHdb, a public, on-line, locus-specific
mutation database (http://www.pahdb.mcgill.ca/),
over 500 causative mutations have been identified in
this phenotypically heterogeneous metabolic disor-
der. Historically, identification of pathogenic PAH
alleles in different populations has provided unique
opportunities to study important issues related to
human population genetics, such as migration,
genetic drift, and founder effects [Scriver and Kauf-
man, 2000]. The Old Order Amish are a religious sect
that emigrated from Europe in the mid-1700s.
Individual Amish settlements throughout North
America have remained isolated through social and
religious mechanisms. The founder effect in such
isolated populations often appears pronounced, as
demonstrated by the high incidence of some other-
wise rare genetic disorders [McKusick, 1978; Morton
et al., 2003]. Here, we report on PAH mutation
analysis in PKU patients from two large Old Order
Amish settlements in the United States; the Lancaster
County, PA, and Geauga County, OH, settlements.
share many common founders, this study demon-
and within the two populations.
The PKU patients included in this study were
previously identified through state newborn screen-
ing programs; the diagnosis was confirmed by
plasma amino acid profiles. Affected individuals, as
well as parents and siblings, were recruited to
participate in the study and informed consent was
obtained from each participant. DNA was isolated
from peripheral blood with commercially available
DNA purification kits. The 13 coding exons of the
PAH gene were amplified by polymerase chain
reaction (PCR) using specific oligonucleotide pri-
mers and subsequently sequenced on an ABI 310
Genetic Analyzer (Applied Biosystems, Foster City,
CA). Multiple patients from the Lancaster County
settlement and the index case from the Geauga
County settlement were subject to complete PAH
gene sequencing. Genotyping in additional patients
and their families concentrated on the population-
specific mutations identified in each community.
Genealogical data obtained from participating
families was confirmed and expanded based on
private or published family records provided by the
local Amish communities.
The Geauga County Amish settlement was
founded in 1886, with initial immigrants from
settlements joined them, creating the extant popula-
tion in Geauga County. In this settlement,14 indivi-
families. The closest common ancestral couple (JM
(1817–1890) and LT (1819–1865)) was seven or
eight generations removed from all patients (Fig. 1).
*Correspondence to: Heng Wang, M.D., Ph.D., DDC Clinic for Special
Needs Children, P. O. Box 845, 15809 Madison Road, Middlefield, OH
44062. E-mail: email@example.com
All genotyped patients were homozygous for the
same pathogenic allele, a G to A transition 11
basepairs upstream from the intron 10 acceptor
splice site (IVS10-11G>A). No other PAH sequence
variants were detected. This mutation was originally
identified as one of the most common pathogenic
PAH alleles in southern Europe [Dworniczak et al.,
1991]. Indeed, this is the second most common
mutation reported in PAHdb. This variant activates a
cryptic splice site and results in an in-frame insertion
of 9 nucleotides preceding exon 11, leading to
protein conformational changes and abrogation of
previously identified this mutation in Old Order
Mennonite patients in Lancaster County, PA [Puffen-
berger, 2003], this is the first description of this
patients in the general population are compound
heterozygotes rather than homozygotes; therefore,
the cohort of homozygous patients in this report is
valuable for further study of the disease, such as
exploring the role of other genetic influences and
environmental factors in disease expression.
In the Lancaster County settlement, we have
identified two mutations, namely 782G>A (R261Q)
and 283_285delATC (I94del) in five patients with
PKU and/or hyperphenylalaninemia, Based on
PAHdb, the 782G>A (R261Q) mutation, found in a
CpG dinucleotide in exon 7 as previously reported
[Abadie et al., 1989], is one of the most common
PAH mutations. The 3-base pair in-frame deletion
(283_285delATC) was initially reported in a con-
sanguineous Portuguese family and was subse-
quently found in other European patients as well.
This PAH mutation was shown to exhibit reduced
affinity for phenylalanine [Caillaud et al., 1991].
We have ascertained 14 patients with PKU in the
Geauga County settlement, where the Amish popu-
lation numbers between 12,000 and 15,000. These
greater than 1/1,000, which is an order of magnitude
higher than the population incidence of 1/10,000.
This higher incidence also exceeds that reported for
any other population [Scriver and Kaufman, 2000].
We postulate that a population bottleneck followed
by random genetic drift (i.e., founder effect) is
responsible for the high incidence of disease in this
community. Conversely, PKU in the Lancaster
County settlement demonstrates allelic heterogene-
ity and an incidence of approximately 1/10,000,
comparable to the incidence in the general popula-
tion. This example highlights two important points:
(1) rare genetic diseases in isolated populations
are not uniformly caused by single mutations
(i.e., homoallelic) and (2) genetic drift is random.
Sampling effects can initially lead to a decrease in
mutation heterogeneity and an increase in the
relative carrier frequency within a founder popula-
tion, yet population-wide increases in mutation
frequency (and thus, incidence) are not inevitable.
Mutations introduced into a sub-population by one
(or more) founders are as likely to decrease in
frequency as they are to increase. Thus, sampling
effects and genetic drift can lead to a paucity of
disease (vis-a `-vis the general population), as we
suspect is the case for cystic fibrosis in the Old Order
Amish of Lancaster County, where no cases of cystic
of unique, isolated populations bound together by a
common social structure. The individual settlements
are often affected by rare autosomal recessive gene-
tic disorders due to the founder effect [McKusick,
1978; Morton et al., 2003]. Studying these genetic
disorders in different settlements often yields inter-
esting information regarding population migration
and related historical and social events. More
importantly, it provides valuable information for
important for public health in the community. This
information can be utilized for newborn screening
and diagnostic molecular genetic testing.
FIG. 1. Partial pedigree for PKU in the Geauga County Amish settlement.
PHENYLALANINE HYDROXYLASE DEFICIENCY IN AMISH
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
Our work has revealed that the genetic disease
profile in each Amish settlement is quite distinct
although shared diseases do exist. For instance,
many relatively common genetic disorders in the
Lancaster County settlement, such as glutaric acid-
uria, type I, [Strauss et al., 2003] and Amish lethal
microcephaly [Kelley et al., 2002], have not been
found in the Geauga County settlement. Similarly,
several diseases recently reported in the Geauga
County settlement, such as Cohen syndrome [Falk
et al., 2004] and prolidase deficiency [Wang et al.,
2006], have not been identified in patients from
Consistent with the genealogical evidence, several
rare genetic diseases have been found in both
was first reported in the Pennsylvania Amish in 1965
[Bowman et al., 1965]. Many years later, it was found
in the Geauga County settlement [Muir et al., 1984].
Some newly defined genetic disorders reported in
one Amish settlement have been rapidly discovered
in another settlement. Examples include nemaline
rod myopathy [Johnston et al., 2000], ganglioside
GM3 synthase deficiency [Simpson et al., 2004], and
cortical dysplasia and focal epilepsy associated with
mutant contactin-associated protein-like 2 [Strauss
et al., 2006]. These three disorders have been
identified in multiple Amish settlements and all
patients have been homoallelic for the published
pathogenic variant. As a result of mutation homo-
geneity, targeted DNA-based testing can be readily
employed for diagnostic purposes. However, as
shown here, phenylalanine hydroxylase deficiency
is an exception to this simple rule.
We thank the Amish families in this report for their
participation in the study and staffs of Rainbow
Babies & Children’s Hospital, Cleveland Clinic for
providing outstanding care for patients and families.
This study is partially supported by The Mid-Atlantic
Connection for PKU and Allied Disorders, Inc.
(MACPAD) to both DDC Clinic for Special Needs
Children and The Clinic for Special Children.
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