SECTION I

Introduction and Program Summary
INTRODUCTION

The Genetics Research Center that would be enabled by this grant will be a new
level of cooperative involvement of the Departments of Genetics and Pediatrics of
the Stanford University School of Medicine in research in medical genetics and
the application of such research to clinical aspects of medical genetics. We are
requesting support for an interrelated set of basic and applied research projects
involving members of both departments. Besides their importance as basic science,
these investigations relate to genetic counselling, pathogenesis and diagnosis of
genetic disorders and the mechanisms of human adaptation in genetic variation.
in preparing this application, the two departments, labelled as basic science
and clinical science respectively, have already enhanced the coordination of their
activities for the benefit of patients with genetic disorders and their families.

Since 1959, the inception of the School of Medicine on the Stanford University
campus, Genetics and Pediatrics each have been deeply involved in various aspects
of genetic research. A number of ad hoc cooperative efforts have also evolved.

Both departments are together teaching medical genetics to medical students. In-
formal communications in training graduate students and postdoctoral fellows have
been excellent. Research projects undertaken in one department have involved
consultation or collaboration with various members of the other department, and
clinical problems have stimulated basic research activities in both departments.

In accordance with its clinical mission Pediatrics has focused on patients
with genetic disorders, diagnosis, pathogenesis, and therapy; and the counselling
of such patients and their families. Pediatrics has likewise emphasized perinatal
biology, featuring a program of detection and management of high risk pregnancies
in which the potential for morbidity or mortality of mother, fetus and/or newborn
infant is high.

Genetics is well known for its basic research Programs in molecular biology,
population genetics, and immunogenetics, whose potential for clinical applications
have long been recognized by members of both departments. As knowledge in molecular
biology has increased and the necessary technology has permitted applications
to the study of man and his disorders, it has become increasingly evident that
relevant research efforts of both departments should converge on problems connected
with clinical care. Both departments nave begun to cooperatively design and carry
out research applicable to genetic medicine and to plan for the application of the
findings to the care of patients encountered on the various clinical services of
the Department of Pediatrics.

  
  
    
   
  
  

 

rT,
The underlying theme that unifies the range of specific studies outlined below =

is genetic polymorphism in man. The pediatrician views this as the source of
genetic disease; the basic scientist as an expression of gene mutation and evolu-
tionary pressures. These are roles shared within as well as among individual in-
; vestigators. To these challenges are brought a combination of clinical insights,

| experience with several aspects of basic genetics, and new analytical technologies
| -- the application of instruments like the mass-spectrometer, the computer and the
cell sorter. .

  
 

en 8 EN ONIN

 

 

Besides the specific research projects to be funded under the Center grant,
we work in an extensive context of genetic and related research -- and indispensable
aspect of our own environment, and a set of activities to which the Center organiza-
tion may also bring a new focus for developments that should advance both basic
scientific knowledge and its application to human problems.

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COMMENTS ON ORGANIZATION

The Stanford Genetics Research Center is organized as a
cooperation of the Departments of Genetics and Pediatrics. Thus,
it follows, rather than conflicts with existing departmental
authority. Phis sinimizes the need for new, formal arrangements
that might be essential in other circumstances. It does reflect a
deep-seated intention on the part of both departaents to isprove
the application of new analytical methodology and of basic
genetic science to clinical probless, and to relate clinical
studies to enhancing knowledge of genetic polymorphism in man.
The length of the following comments on organizational detail may
lead one to believe that we ar2 amore interested in sanagement
than in scientific substance.

Exactly the opposite is the case, but we cannot afford the
consequences of misunderstanding about how we will work together.
Obviously, as at any other university, the actual character of
our "organization" has a larger compass and bears little
resemblance to the forgal hierarchy of imputed power. Research is
actually done in individual laboratories under the supervision of
an autonosous faculty member. Most "direction" aust be in the
fora of intellectual stimulation and criticism, and in the
selection of key people for roles of autonomous responsibility.

Professor Joshua Lederberg, Chairman of the Department of
Genetics, will serve as Principal Investigator and Director of
the overall Genetics Center Proyram. Dr. Howard Cann, Associate
Professor of Pediatrics, will serve as Associate Program Director
with special responsibility for clinical research related to the
Center's activities. (If this application is approved, and offers
a funding basis for the step, it is anticipated that Dr. Cann
will receive a joint appointment in the Genetics Department as
well.)

The Director will have executive responsibility for the
adainistration of the prograa, including the formulation of
extended ani new projects, their budgets, and reports to the
NIGMS of progress under the grant, and internal functions of
coordination, information and criticisa.

The internal administration of the prograa is facilitated by
the Director's position as Chairman of the Genetics Department,
and by the enthusiastic support and participation of br. Irving
Schulman, Chairman of Pediatrics, and of other senior members of
both departaents. In practice, the Director will be advised by,
and will delegate subproject responsibility to, these colleagues
in accordance with their special skills and interests.

The Director will also invite an informal visiting
committee, principally from among his colleagues involved in
sisilar lines of work at other West Coast institutions, to visit
Stanford annually and to advise him of advantageous directions of
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policy. This group may also help to identify new opportunities
for inter-iastitutional cooperation and coordination, especially
to further our erphasis on building complementary rather than
competitive capabilities. These annual visits may also
advantageously coincide with a local or regional conference on
research progress concerning genetic polymorphism and disease.

The Director must play both an attractive and a critical
role. That is he aust encourage his colleagues to invest in the
effort needed to orient their activities towards the common goals
of the Center's research. At various times this attraction may be
enhanced by the fiscal support of the Center's budget; on the
other hand, it is hard to predict what the level of that support
will be, and the level of discretion that will be delegated to
the Director. Realistically speaking, then, his role is sore
inspirational than directive, at least with respect to the
tenured fellow-members of the faculty. If the Center's budget is
approved, and develops some de facto continuity, he may have
greater formal authority -- bat always with accountability to
outside review groups and to the independent authority of other
professors. The moral and intellectual influence that the
Director can exert will obviously be greatest in relation to
people in the same or closely co-functioning departrgjents who
already relate in many other ways. His main positive role will be
to help bring in new ideas -- his own and others -- into the
progress of the Center's research.

Conversely, he has responsibilities for negative functions
-- especially to discourage the growth or even survival of
projects that are inherently unsound, or become obsolete. Within
a close-knit institution there are often serious personal and
political obstacles to the exercise of objective scientific
quality judgments. These ligitations are both aggravated and
coapensated for by the limited discretion usually given to a
director, i.e., the fact that final decisions about project items
are generally made by peer review groups.

All of these probleas would be much worse if the Director
had the responsibility of coordinating every genetics-relevant
activity in the school. He would have neither the budget nor the
political mandate to do this well. In fact, the present proposal
encompasses a serious and soul-searching effort to initiate a
major Center program at a practically achievable and useful level
of integration. It will also be the basis for less formal but
equally important co-operations with other departments, whose
policies and programs can be influenced but not directed in the
exercise of our responsibilities.

Provision for Succession of Directorship.

Day to day deputization will be handled ad hoc in the same
fashion as the duties of the departmental chairman. The question
of possible succession, in the event of incapacity or transfer of
the Director, poses a more difficult question. The Associate
Director will serve pro-tem, in concert with the (acting)
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chairsan of each department; and a new director will be proposed
by the Dean of the school after consultation with the visiting
committee and with the participating faculty of the departments.

The Associate Director's role will be to maintain contacts
with other clinical departments for patient referals to Center
projects, and to assure a high standard of clinical
responsibility in all Center research activities that deal with
patients. Stanford has long experience with Clinical Research
Centers -- e.g. in sedicine, in cancer therapy, in cardiovascular
disease, anf in premature infant research. (These facilities
afford further research bed opportunities for protocols involving
intensive study of selected cases without burdening the present
proposal with inappropriate in-patient care charges). In
addition, they have led to the institutionalization of formal
procedures for the ethical review of research involving human
subjects. Dr. Cann will be responsible for defending the present
proposal before that review coamittee, and for assuring that ali
further research under this grant is properly sabmitted and
reviewed according to established procedures.

An Application for renewal of a Genetics Research Training
Grant was approved by Council for the period 1974-1979. fhis
action was superseded, however, by the executive decision to wind
down such programs and we are now operating on a decreasing
budget to cover only the previously enrolled trainees.

That training grant renewal had, for the first tire,
provided for clinical research in genetics as part of our
program. Lacking these funds, it is, of course, imperative that
the research activities of the Center also double as training
opportunities for graduate research assistants and postdoctoral
fellows (research associates.) We will sake every effort to fit
each fellow into employment in the support roles of the Center.
We cannot pretend, however, to be able to support the training
functions at the level previously assumed without a compensatory
expansion of the funding for these research programs. This is not
reflected in the budgets submitted at this time.

The department faculties having agreed to the programs of
the Center, there is such to.do in substance, but little in
organizational process, to coordinate our activities and insure
reeting the Center's objectives. We are already in frequent,
almost daily, communication in the pursuit of our other duties
and socially. These contacts are promoted by the fact that our
laboratories and offices are inmediately adjacent in the Medical
Center Building.

The Genetics Center Budget is intended to fund programs of
new substance and scope or those for which other support (e.g.
NASA) is no longer available. It goes into new territory beyond
many on-going research projects which are identified in the
material on each affiliated investigator. However, the
coordinated fundiny and administration of the Center allows nore
careful planning and resource-sharing than would be possible for

pms
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a disparate set of individual proposals.

We intend to minimize the indefinite prolongation of
autonomous projects merely to keep a research group intact.
Instead we will promote an innovative search for new
Opportunities and on-going mutaal scrutiny of projects.

For the continuity of a program as complex as the Center, we
are requesting an award for 5 years. Yet it is obvious that many
unforeseen obstacles and opportunities will arise in that
interval to warrant changes of tactical direction and evolution
of major strategies. The responsibility of the Director to manaye
this progression of emphasis is his most important executive
role. It will be discussed with the visiting committee and
reported to NIGMS from year to year, to assure the consistency of
his decisions with the approved mandate of the Center grant.

This is not an assertion of unwarranted latitude. To the
contrary, the political structure of a university ensures, if
anything, a great deal of conservatism in changes of direction
and support for individual programs, once approved.

The present proposal will also be a saodel for further
developments to be initiated with the Departments of Gyn-Ob.,
Medicine, Psychiatry and Dermatology -- to sention only those
with which we have had tangible discussions. For various reasons,
potential projects with them are not yet ripe for formal
submissions; these will be the subject of future requests,
depending in part on the climate for funding expansion of genetic
research. Meanwhile, we have working arrangements to assure their
awareness of our activities and the provision of patient material
for mutual advantage.

A brief description of the role of each participant in this
progras and of his research ani/or clinical interests are
presented here:

THE STANFORD ENVIRONMENT

Besides the Program Director (Professor Lederberg) and
Associate Director (Professor Cann) the faculty and professional
personnel associated with the Center, their roles and
biographical detail are spelled out in Section IX.

In addition, we have included biographical sketches of a
number of key members of the environment of the Department of
Pediatrics and Genetics, although their research projects are not
included in funding under the Center at this time.

Within one or two years after the activation of this
program, we anticipate the direct participation of one or more
colleagues from the Department of Obstetrics and Gynecology. A
search for a chairman of this department is presently under way,
and we expect that this indiviiual will renew active research

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here in fetal physiology and fetal monitoring. We look forward to
interacting in this program with our obstetrical colleagues in
various projects pertaining to antenatal detection of genetic
disorders and selective abortion.

We have also consulted with the chairsgjen of the Departments
of Medicine and of Dermatology, and have encountered great
interest in the development of the Center, and assurances of
cooperation in the referal of patients who would be pertinent to
our screening technologies. Their position is best witnessed by
the attached correspondence. At some future date, we gay expect
to forsulate sore specific proposals, as a consequence of
recruiting now under way.

The Department of Psychiatry will be chaired by Albert
Stunkard (now at University of Pennsylvania) beginning September
1973. He is personally well known to the Director (who served on
the search committee for this appointgrent, which was chaired by
Professor Schulman) and many other participants and we have
strong assurances that the Psychiatry Department's interest in
genetic etiologies of psychiatric disease will continue under his
leadership. Dr. Stunkard has also voiced his concern that
psychiatrists have not hitherto been more directly involved in
problems of genetic counseling, like those reflected in Dr.
Barnett*s proposal here; and we are looking forward to closer
cooperation on such issues after his assumption of his duties.
For some tise, Professor Cavalli-Sforza of the Genetics
Departaent has been cooperating with Professor Barchas of
Psychiatry on polymorphisas of biogenic amine metabolisa.

Genetic counseling, per se, is a responsibility mainly of
the Pediatrics Department -- diseases of adults only occasionally
present serious problers of reproductive policy of the family.
Genetic disease, of course, presents itself to all of the medical
and surgical specialities. In recent years, these departments
have not seen any requirement for a special organization to deal
with genetic aspects of internal sedicine or surgery -- and
indeed these are fully and competently integrated into their
overall teaching and practice. When special problems do arise,
there are no impedisents to consultation with pediatricians and
with basic geneticists. Many members of those departments are
already involved in immunogenetic and other genetic research.
To

From :

Sussect:

Date: May 25, 1973

Joshua Lederberg, Ph.D.
Daniel D. Federman, M.D, ‘Chairman, Department of Medicine)

Genetics Research Center

Dear Josh:

I am not really clear bout your timetable for submission of the
proposal for a Genetics Research Center, but I thought I would make
explicit several areas of potential involvement of the Department of

edicine.

1. Pharmacogenetics - We have a slot in Clinical Pharmacology
for a new Assistant Professor. Stan Cohen and I have discussed finding
someone with a particular interest in the area of pharmacogenetics, but
at the present time, no particular individual has been chosen.

2. Endocrinology -

A. Hormonal control of gene expression. We will have at
least one new person in the Endocrine Division in the next
year and have placed as our first interest finding someone
working in the area of the effective hormone on the expres-
sion of genetic information. This would provide an obvious
link to a genetic center.

3. Early detection of hereditary endocrine disease. There are

at least 80 endocrinopathies which show Mendelian segregation and a
number more which probably have a major genetic control. Early de-
tection of assymptomatic patients can be achieved by suitable pro-
vocative testing. This opens the way to family studies in which
a) the hereditary mechanism can be defined, b) preventive or therapeutic

intervention can be tried before irreversible damage has occurred, and
c) the effects that intervention can be compared to the natural history
of the disorder. The latter approach has important application to the
management of “patients" discovered by multiphasic screening techniques.

4. Immunogenetics - This area of course is strongly represented in
our Department and we could, I believe, contribute significantly to the
activities of the genetic center in this area.

I should have liked to provide more specific information, but until
particular people have been identified, it would be difficult to do so.
I hope this is of some help -- I am certainly anxious for an active col-
laboration.

Sincerely, p-?

DF
CLINIC FOR CHLLDREN WITH PSOREASTS: AND OTHER INUERTTABLE SKIN DISEASES
Directors:

. Eugene M. Farber, M.D,
Professor and Chairman, Department of Dermatology

Alvin H. Jacobs, M.D.
Professor of Dermatology and Pediatrics

_In the clinics of the Department of Dermatology there are about 12,000
total patient visits annually,of these approximately 3,000 are children seen
in the Pediatric Dermatology Clinic. Over half of these children have either
psoriasis or atopic dermatitis, both conditions with important heritable
factors in their etiology. In addition many pattents are seen with the Tess
common genodermatoses, such as, the varjous types of ichthyosis, epidermolysis
bullosa, and the many types of heritable neurocutaneous disorders. At the
present time a special cltnic is being established for the care and study
of children with psoriasis and other inheritable skin diseases. The purpose
of this clinic is not only to offer more complete care for these types of
patients, but to study the genetic aspects of these conditions and offer
genetic councelling to the patients and their families.

The Department of Dermatology already has an unmatched reservoir of
material for genetic study in its Psoriasis Life History Research files; a
data-bank of approximately 8000 patient histories. Not only is this computerized
information a source of information on the epidemiology of psoriasis, but
has served as the basis for several published follow-up studjes on the genetics
of psoriasis including family and twin studies.

At present several genetic studies are under way. For example, Dr. Farber
and associates are studying the association between H-LA antigens and psoriasis.
Drs. Jacobs and Chan are studying and developing techniques for accurate counting
of melanocytes in pigmented and depigmented macules in order to predict the

development of certain genodermatoses, such as neurofibromatosis, tuberous

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sclerosis etc.

Studies are also under way to properly classify the varjous genetic
types of ichthyosis and in cooperatton with Dr. Howard Cann we are planning
to investigate the x-linked blood type in the families with x-linked
ichthyosis.

It is hoped that in the future, with the vast resources avajlable
in Genetics, Pediatrics, and Dermatology at Stanford, an investigation
could be launched into the genetic aspects of atopic dermatitis, one of

the commonest skin problems of childhood.
Continvat’-n poge Cesaniment ot coe

CLINICAL FACILITIES

The Stanford Medical Center, located midway between tne cities of San
Francisco and San Jose on tne campus of Stanford University, cousists of the

School of Medicine, the University Hospital and the Stanford Clinics. Stanford

is the only university hospital in the Regional Nedical Program (@@) Arca Ili

and functions as a tertiary care center. RIP Area III consists of eleven counties

in mid California consisting of 2.6 million people. There are 60 acute hospital
facilities, and 4000 physicians practice in RIP Area III. Approximately 90 percent
of patients admitted to Stanford University Hospital live in this area, Additicnaliy,
patients for tne clinics and university hospital are also dravm from other areas in
and out of California. RIP Area III and its.population form a base from waich is
drawn numerous patients with diseases resulting from major genes or chromosomal
abnormalities or with a significant genetic component. ilost of these patients a
seen primarily on one of the services of the Department of Pediatrics or are
referred for diagnostic evaluation and/or genetic counselling from other clinical
services at the medical center. Clinical teaching services at other hospitals*
affiliated with the Department of Pediatrics add to the source of patients with
medical genetic problems.

re

As the Genetics Center activities evolve and research developrents beccne
available for clinical application, we anticipate that more patients will be
referred to this medical center for genetic counselling and evaluation and
management of genetic disease. The numver of patients seen annually by the compinea
clinical facilities of the Department of Pediatrics for genetic disorders is
estimated to be about 200. We predict that this number will dousle during tus course
of the Genetics Center grant.

Other clinical divisions of the Medical Center, of course, also see manv
adult patients with genetic disorders. Historically, Genetics has had weaker ties
with them tnan with Pediatrics, although several joint projects connect with
Medicine (H. McDevitt) and with Psveniatrv (J. Barchas; S. Kessler). The aptointment
(effective February 1) of a "genetic endocrinoiogist", Dr. Daniel Federman, as new
head of Medicine should remove administrative obstacles to extensions of Center-
related programs.

Patients with genetic disorders are seen in the following pediatric sub-
specialty clinics at the Stanford Medical Center.

 

*The Children's Hospital at Stanford and the pediatric services at the Santa
Clara County Hospital (San Jese) and at the Kaiser-Permanente Medical Center
in Santa Clara.

 

 

 

PHS 2495-1 12-65

Poll
 

Continuali-n page

Genetic Counselling Clinic (Director, Dr. Howard Cann, Associate Professor
of Pediatrics)

Approximately fifty patients and their families are referred annually for
genetic counselling. These include patients from outside of the Stanford Medical
Center and from various clinical services at the Medical Center. About 50% of
families seen are counselled for disorders determined by major genes at single
loci. The remaining 50% of patients represent more complex counselling problems,
e.g. possible sporadic cases and phenocopies, multifactorial threshold traits,
undiagnosed familial disorders, and excessive exposure to radiation. Counselling
includes confirmation of the diagnosis of the disorder and identification of
heterozygotes among asymptomatic relatives at risk (autosomal recessive and
X-linked recessive disorders). Families are always screened for the possibility
of applying ante-natal diagnostic techniques. The motivation for and expectations
from genetic counselling are explored by Dr. Cann and, for selected families,
by a social worker, in order to plan for effective communication with each family.

Birth Defects Clinics (Director, Dr. Luigi Luzzatti, Professor of Pediatrics)

Approximately 100 new patients are seen in the Birth Defects Clinics
annually for diagnostic evaluaticn and comprehensive and interdisciplinarv
management of congenital defects. There are about 400 return visits per annum
in the clinics. In addition, about 50 newborn infants are seen annually for
evaluation of congenital defects in the Stanford Hospital Nurseries. Most children
seen have multiple congenital defects of unclear etiology, about 50% of these
are screened for chromosomal abnormalities, about 25% are patients with disorders
determined by major genes at single loci, and about one third are seen for
structural malformationswaicn are multifactorially inherited threshold traits.
This is the clinic where most of the patients with mucopolysaccharidoses,
mucolipidoses, simply inherited disorders of bone, Down's syndrome and other
chromosomal abnormalities, cleft lip and/or palate and congenital defects or
neural tube development are seen. Where appropriate, genetic counselling is
provided to families of patients enrolled in the Birth Defects Clinics. In
addition about 50 families are referred each year specifically for genetic
counselling. An average of about 109 karyotype analyses are done annually in tha

Cytogenetics Laboratory in probands with congenital defects and/or in fami ly nenbers
as indicated. To date diagnostic amniocentesis has been performed at 12-16 weeks
gestation on approximately 25 women referred to the clinic for either advanced
maternal age or a documented family history for an X linked gene.

Pediatric Hematology Clinic (Clinic Director, Dr. Herbert C. Schwartz,
: Professor of Pediatrics)

In this clinic approximately 100 new patients are seen annually for various
disorders of blood. Between five and ten percent of these are children with
inherited disorders -- sickle cell anemia and other hemoglobinopathies,
thalassemia, congenital spherocytosis, glucose-6-phosphate dehydrogenase
deficiency and other instances of congenital non-spherocytic hemolytic anemia
and hemolytic disease of the newborn (feto-maternal blood group incompatibility).
In this clinic workup of patients with genetic disorders includes identification
of heterozygotes and homozygotes among first degree relatives of probands and
subsequent genctic counselling.
 

Conlisuation cage Depsntont ¢ Ceo

The Stanford Nurseries (Director, Dr. Philip Sunshine, Associate Professor
of Pediatrics)

The newborn nurseries of the Stanford Medical Center include facilities
for uncomplicated births, an intensive care nursery and the Premature Infant
Research Center. The research and clinical emphasis of these newborn units is on
perinatal biology of the fetus, newlyborn infant and the mother. This emchasis
has led to the development of a program dealing with high risk pregnancies, in
which mother, infant or both are at high risk for morbidity and mortality. "hen=
ever appropriate the fetus is monitored and such monitoring includes diagnostic
amniocentesis for evaluation of fetal maturity, for fetomaternal Rh incompacibility
and early antenatal detection of genetic disorders. A study conducted in California
in 1971 by the Bureau of Maternal and Child Health indicated that the StanZord
Medical Center is the only facility in RMP Area III with an intensive care aurserv
(based on fairly ridgid criteria, e.g. full time neonatologists and basic infant ,
monitoring equipment available at all times), and this is evident by referrals of
fetuses and their mothers and newborn infants. Approximately 700 infants arz
admitted per year to either the intensive care nursery of the Premature Research
Center at Stanford and about 30% of these are born at other facilities. Anoroximatcly
54 of newborn infants receiving intensive care at Stanford are ill because they
are heterovygous or homozygous for a major mutant gene, because of a chromosomal
abnormality or because of a malformation with a significant genetic contribution.
We anticipate a major expansion of perinatal activities involving detecticn and
management of high risk mothers and fetuses with the appointment in the near
future of a Chairman of tne Department of Obstetrics and Gynecology. The scarch
is presently underway, and one of the criteria for selection is comoetence in t:
areas of fetal monitoring and evaluation and fetal and maternal physiologic in
actions.

rom
3
m

Pediatric Neurology Clinic (Clinic Director, Dr. Judith Koehler, Assistant Professor
, of Pediatrics and Neurology)

Approximately 500-600 patients with various neurologic disorders are seen
annually in this clinic. Many of these patients have genetically determined or
inherited disease, e.g. learning disorders, familial myopathies, spinoceresellar
degeneration, familial spastic paraparesis and other metabolic or degenerative
disorders of the central and peripheral nervous system. The Division of Pediatric
Neurology has special interest in clinical treatments of and researcn into the basic
mechanisms of epilepsy. Some patients with seizure disorders have heredofemnilial
determinants underlving their basic seizure problems. Additionally, response to
medication and drug metabolism may be genetically determined. The Divisicn of
Pediatric Neurology also has special clinical and research interest in neuro-
muscular disease, much of which is hereditofamilial. Specifically for this purpose,
a neuromuscular clinic is being established in conjunction with the Muscular
Dystrophy Association. This clinic will see 200-300 patients, mostly children,
annually. A diagnostic neuromuscular histochemistry laboratory is already in
function to process nerve and muscle biopsies from these patients. Active researcn
in neuromuscular disease is being carried out by several workers in the Dagartoars
which incorporates ultrastructure and basic physiologic studies. weuTOLegy

 

 

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Hemophilia Program (Clinic Director, Dr. John Gribble)

The Hemophilia Program provides diagnostic evaluation and comprehensive
management for patients with disorders of blood coagulation. Approximately 109
new patients are seen annually; these patients represent the clinical spectrum of
disorders of blood coagulation, the most frequent being hemophilia A, hemophilia 8B
and von Willebrand's disease. The families of all patients with inherited disorders
are provided with genetic counselling. A comprehensive care clinic is located
at the Children's Hospital at Stanford in order to provide continuing care for
patients with hemophilia and includes prophylactic treatment of patients with
hemophilia A with Factor VIII preparations.

Pediatric Metabolic and Endocrine Clinic (Clinic Director, Dr. R.O. Christiansen,
Assistant Professor of Pediatrics)

Seventy to eighty new patients are seen annually in this clinic which
logs about 709 patient visits each year. Many of the patients may be classified
as inborn metabolic errors and these include phenylketonuria, galactosemia,
the various syndromes associated with adrenal hyperplasia, alcaptcnuria, the
various glycogenoses and defects in thyroxine piosynthesis. Diet therapy of
patients with phenylketonuria and galactosemia is an ongoing activity of these
clinics. Furthermore, a number of patients with hypophosphatemia (X-linked)
are enrolled in this clinic. Genetic counselling is provided for the families of
all patients with hereditary metabolic and endocrine diseases, and this includes
attempts to designate heterozygotes among unaffected siblings of probands with
inborn errors of metabolism.

Growth and Development Clinic (Director, Dr. Norman Kretchmer, Professor of
Pediatrics)

This clinic deals with genetic and environmental problems in infants and
children which affect their growth and development. Usually children are referred
to this clinic because of failure to grow or abnormally slow growth.

Thus children
with hereditary conditions which result in Significant growth ‘failure in the
phenotype (e.g. isolated growth hormone deficiency, various aminoacidurias and
various chondrodystrophies) comprise a proportion of this clinic's patient
population. Approximately 150 new patients are seen annually.

 
payee cee 48 ee 8
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The Children's Hospital at Stanford, located approximately 1/2 mile from
the Medical Center on the Stanford campus, is affiliated with the Department of
Pediatrics for teaching and postdoctoral clinical and research traiainz.

There are three clinical services relevant to a medical genetics program at this
hospital: 1) Cystic Fibrosis Service (Director, Dr. Birt Harvey, Clinicai
Associate Professor of Pediatrics), 2) Clinical Immunology Service (Director,
Dr. Vincent Marinkovich, Assistant Professor of Pediatrics) and 3) Pediatric
Oncology Service (Director, Dr. Jordan Wilbur, Clinical Associate Professor of
Pediatrics). These services have in-patient and out-patient facilities at the
Children's Hospital at Stanford. Here patients are evaluated, diagnosed and
treated for cystic fibrosis, hereditary immune defects and hereditary malignant

tumors (e.g. retinoblastoma). Genetic counselling is provided by the staff of these
services.

Research Facilities

Research facilities in the Departments of Genetics and Pediatrics available
to this Genetics Center Program include some 15,000 square feet of laboratcries,
research support areas and offices. Tne research and administrativ2 ireas for each
department are contiguous on the third floor (the Joseph P. Kennedy Jr.
Laboratories for Molecular Medicine) of the Joseph D. Grant Building of the
School of Medicine. In addition the Instrumentation Research Laboratory (Desartmant
of Genetics), in the basement of this building,comprises about 7,990 square feet
of offices, laboratories, and snops. Tnus biochemical laboratories, fully deveioned
tissue culture areas, heavy eauipment areas, cold and warm rooms and an electron
microscope facility are available for this program. A cell separator, a ga
chromatograph, a mass spectrometer and an electron microscope lead the iong list
of equipment which is available for the research projects to be carried out ia
this program. Other equipment also includes centrifuges, scintillation counters,
lyophilizers, spectrophotometers, incubators, microscopes and coulter counters.
Rather good computer facilities are available.

 

 

PHS 2439-1 12-65 PAS
EPP E. BS PPR fT Pine "UDINE CAPA ANI

Continuation page

 

Bee UNE Ne E-

I.

II.

IIl.

IV.

Vv.

 

THE RESEARCH PROGRAM

The Center program consists of research projects which have direct relevance

to medical genetics. Some of these projects will require blood, urine, amniotic
fluid and cells from patients and others will require the patients themselves.
In none of these research projects are we ready to test methods for use in the
actual clinical situation; rather, we are developing methodology. The general
areas of research are as follows:

Screening and Characterization of Inborn Errors of Metabolism by Gas
Chromatography/Mass Spectrometry Analysis of Body Fluids.
(Drs. Lederberg, Kretchmer, Cann and Duffield).

Maternal Blood Stream - Another Source of Fetal Tissue for Pre-Natal
Diagnosis of Genetic Disorders.
(Drs. Herzenberg and Cann).

Polymorphic Genetic Markers in Amniotic Fluid.
(Drs. Cann and Tsuboi).

A Search for Genetic Polymorphisms and Variances of Specific Binding
Proteins in Blood.
(Dr. Cavalli-Sforza).

The Impact of Genetic Counseling Practices on Family Decisions and Behavior.
(Drs. Barnett, Cann and Luzzatti).

The arrangement is also reflected in the budget presentation.

 

PHS-398
Rev. 2-69

Page
GPO : 1969 © - 350-360

Batl

 
SECTION II

Screening and Characterization of Inborn Errors of
Metabolism by Gas Chromatography/Mass Spectrometry
Analysis of Body Fluids

Drs. Lederberg, Kretchmer, Cann, and Duffield

P-7
Screening and Characterization of Inborn Errors of
Setabolisa with Cosputerized Gas Chromatography
And Mass Spectrosetry

Dr. J. Lederberg, Principal Investigator
Des. N. Kretchager, H. Cann, and A. Duffield,
Associate Investigators

A. INTRODOCTION
A.1. Objectives

The objectives of this work are to develop the uses of
gas-liguid chromatography (GC) and sass spectrometry (AS)
instrumentation, under computer managesent, for the screening,
diagnosis (pre and postnatal), and study of inborn errors of
metabolisr. The efficacy of these analytical tools has been
deronstrated when applied to lisited populations of urine saaples
in the research laboratory environment. We propose to enlarge the
clinical investigative applications of GC/MS technology and to
desonstrate its utility for sore economical and routine diagnosis
and screening of disease.

Specific goals include the application of GC/MS analysis
capabilities to larger and more diversified populations to
establish better defined norms, deviations, and control
parameters necessary to relate GC/HS analysis results to
identifiable disease states. In order to ease the problemas in
analyzing the prodigious amounts of information expected in this
research, we will augment the existing GC/MS data handling systea
to provide for increased throughput and automation.

Two other on-going or pending research projects relate to
the present application, each with distinctive aias:

1. DEWDRAL (NIH: BR-00612; Principal Investigator, E. A.
Feigenbaan) is concerned with the advancement of artificial
intelligence (computer software) techniques for the autosated
interpretation of sass spectrometry data. These prograas
atteapt to eaulate human reasoning processes in constructing
explanations for sass spectra froa basic principles.

2. SUMEX (NIH: RR-00785 pending; Principal Investigator, J.
Lederberg) is a coaprehensive resource grant to establish a
national facility for develsping applications of artificial
intelligence in sedicine. Our own use of this facility will
include the integration of DENDRAL software with HIGH
RESOLUTION mass spectrometry instrumentation. Genetic
screening can be made even sore sophisticated by using these
techniques for the corroboration of the structures of newly
discerned aetabolites. However, the present program can also
operate stand-alone, if necessary, using low resolution MS on
line with the GC.

P-18
A.2 Background and Rationale

The Instrumentation Research Laboratory was established in
the Genetics Department under NASA auspices in 1961. Its task was
to define and improve microanalytical methods for the detection
of living processes that might be useful for the biological
exploration of the planets. Many of the concepts that we explored
have been esbodied in NASA's planetary mission plans. However, we
have not undertaken to design and build hardware for such
missions. Instead, we have served as experienced advisors to the
experiment teams responsible for scientific studies on the
Mariner and Viking Mars programs. Our work on GC/MS is one of
several lines of instrumentation effort.

Our original mandate from NASA included generous
encouragement to seek health-related applications as a spin-off
of the development work they were supporting. However, they have
not been able to support the full fledged extension of
space-related technology to genetic disease research per se. The
present application also comes at a time when overall funding for
basic research by NASA is declining rapidly and may disappear
within the next year. We have already begun to reduce our GC/MS
laboratory staff in response to these cutbacks. It is therefore
appropriate that we seek NIH support to help maintain this
existing laboratory to apply its capabilities to probleas of
characterizing genetic metabolic disease.

Our focus on mass spectrometry (ref 3c) originally steased
from the exquisite sensitivity, speed, and specificity of this
technigue for the identification of organic molecules. We have
had some experience with instruments (like the Bendix
Time-of-Flight Mass Spectrometer) which can generate a coamplete
low resolution mass spectrum in 100 microseconds and whose
sensitivity is limited by the statistics of the number of ionized
fragments, and by the data handling problems of averaying
repetitive spectra emerging at a rate of 10,000 frames per
second. We have also been led to look at the computational
challenges of MS from another standpoint - namely the
mechanization of the scientific thinking that is entailed by the
interpretation of a mass spectrum. This task has been the focus
of the research in "artificial intelligence" of the DENDRAL
project.

The application of these instruments to genetic research
requires another dimension, nasely the separation of complex
bixtures, e.g., from body fluiis, into individual components.
These are then available for identification by the mass
spectrometer. Gas chromatography has proven to be a useful
companion to the mass spectrometer - the output gas stream can be
fed directly to the inlet of the spectrometer and much of the
carrier gas (helium) selectively deviated by a semi-permeable
membrane. (Automated, continuous flow into a mass spectrometer of
other chromatographic streams, e.g., high pressure liquid
chromatography, is a speculation that may eventually materialize
to great advantage but is not yet available for applications like

ours).

For some time then we have been developing the means to
integrate GC with MS under computer management. The present
project represents the systematic application of these skills to
the recognition and identification of metabolic variations,
viewed both as genetic polymorphisms and as clinical problems of

genetic disease in man.

The sample populations comprise mainly healthy (control) and
problematical newborns already under intensive study in the

Stanford Pediatrics Departsent.

Other samples will be furnished

by collaborative arrangements with physicians elsewhere and with
other Departments at Stanford (e.g., Medicine, Psychiatry,
Dermatology, etc.). These inputs will have been prescreened for
conditions Likely to relate to possible genetic etiologies or
otherwise to exercise the analytical utility of GC/MS screening.

The Techniques of MS and GC

The technique of mass spectrometry gives information about
the structure of a molecular species by measuring the
characteristic mass abundance pattern of fragments resulting from
ionizing the parent molecule. Lonization is usually accomplished
by electron bombardment. The compound under analysis must have a
measurable vapor pressure at about 200 degrees C. (This
temperature and a pressure of 0.01 microbars are the normal
operating conditions of a GC-coupled mass spectrometer ion
source). The ionizing electron beam (70 eV energy) removes one
electron from some of the molecules of the sample vapor to yield

excited positive molecalar ions:

M + @ ----} M(+) + 22

The molecular ion, M(+), is generally unstable (especially
if at a high energy of excitation) and may decompose within a few
microseconds to yield a series of positively charged fragment
ions. Each fragment ion can in turn decompose to ions of lesser

ion where they are separated an :
ratio. In sector instruments

in quadrupole instruments by an

ght instruments by an

y. The mass spectrum of an
a table of positive ions of
olecular structure (for
teroatoms) determines the

pture subsequent to ionization,

Tt, a characteristic mass

rical isomers may show subtle

@ mass spectra owing to the

7a

o.

Twa

spectrometer into the analyzer reg
according to their mass-to-charge

this is done by a magnetic field,

electric field, and in time-of-fli
adjustable ion detection time dela
organic cormpound thus consists of

different rmasses and abundances. M
instance number and location of he
frequency with which bonds will ru
thereby producing, for the most pa
spectrum for each compound. Geomet
differences within their respectiv
-4-

influence of the geometry of nzighboring groups. Optical
enantiomers yield identical spectra.

Although the technique of mass spectrosetry was extensively
used by petroleum chemists from the 1940's, it was not widely
utilized in organic chemistry until the late 1950's. The first
extensive rgonograph on biochemical applications of mass
spectrometry has just appeared ("Biochemical Applications of Mass
Spectroaetry," edited by G. R. Waller, Wiley-interscience, New
York, 1972). Our colleagues and close collaborators in the
Stanford Chemistry Department, led by Professor Carl Djerassi,
have been agong the pioneers in the development of MS for natural
product chergistry, especially as applied to steroids (4 books and
in excess of 200 papers on various aspects of the theory and
application of MS have been published by Prof. Dijerassi's group
since 1961). During the 1960's, mass Spectrometry was applied to
many different types of organic compounds. The accumulation of
these reference mass spectra was necessary to establish
fragmentation rules for the interpretation of unknown mass
spectra. The experienced sass spectroscopist becomes adept at
recognizing the mass spectral signatures of those types of
compounds with which he works but he cannot encompass within his
memory all the relevant information contained in the literature.
In addition, many reference spectra determined by mass
spectrometry laboratories have not been published. To overcome
this problea, libraries of mass spectra are being compiled for
computer storage and retrieval so that they will eventually be
available for matching by computer against the gass spectra of
unknown compounds. Progress is now being made toward compiling
libraries of mass spectra relevant to general metabolic studies.
These will match the accuaulation already available for special
classes of organic rgolecules and for some drugs whose spectra are
important for emergency toxicological analyses (ref 2).

Instrurgentation advances in mass spectrometry during the
past decade like improved sensitivity, direct coupling with GC
and the use of computers for the routine recording and
presentation of mass spectra, all facilitate the large scale
application of mass spectrometry to biomedical probleas.

Body fluids and other materials encountered in biomedical
research are complex mixtures. For example, urine is known to
contain several hundred organic compounds at levels exceeding on
the order of 1 nanogram per milliliter. The gas chromatograph is
indispensable for the separation of such mixtures into discrete
components. With medius resolution instruments, the mass
spectrometer can be scanned repeatedly once every 2-4 seconds.
The gas chromatographic separation of a urine mixture may require
40-50 minutes: the resalt is the accumulation of over 700 mass
spectra per analysis. The simplest way to identify these mass
spectra is to search a library of known compounds. Even if the
mass spectrum of a test compound does not reside in the library,
the best match found may be a related compound. This can
facilitate the manual interpretation based on the chemist!s
Knowledge of and guesses about the rules of fragmentation. The

Prat
-5-

problew of computerizing the identification of compounds whose
mass spectra are not in a library is addressed by the DENDRAL
project. Corgputer programs have been developed to interpret mass
spectra of unknown comapounds from first principles (i.e., to
emulate the reasoning processes of organic chemists).

Frequently compounds of biochemical interest occur in small
agounts in biological fluids. (By definition, many frontier
problems concern comspounds at the limit of easy detection by
existing techniques). Thus, the effectiveness of GC/MS as a
detector of biological materials is directly related to its
sensitivity. Current systems routinely operate with sensitivities
such that mixture components with as little as 50-300 nanograms
of material can be measured. This limitation is imposed by the
following instrument-related factors. In order to record an
interpretable mass spectrua, a low resolution mass spectrometer
must have input to its source on the order of 5 nanograms of
material per second. Since a gas chromatographic peak lasts for
approximately 5 to 30 seconds, in GC/MS operation some 25-150
nanograms per GC peak are required for mass spectral analysis.
Inherent in the gas chromatographic column and in the
semi-permeable membrane separator (used to preferentially remove
the helium carrier gas from the effluent stream) are losses of up
to 50% which increase the input sample requirements in a
practical sense to about 50-300 nanograms of material per GC peak
analysis.

Another limiting factor in the application of the GC/MS
technique to biological extracts concerns the volatility of the
material to be assayed. before the system can detect many
non-volatile components (e.g., carbohydrates, amino acids, etc.),
they must be converted into volatile derivatives which will pass
through the gas chromatograph at a maximum oven temperature of
300 degrees C. Above this temperature, column bleed from the gas
chromatographic phase will tend to enter the ion source and
complicate the recorded spectra. Thus the GC/MS technique is
restricted to those organic compounds which can be converted to
volatile derivatives and is not, in general, applicable to
inorganic compounds. A recent report (ref 1) describes the
analysis by GC/MS of ketose diphosphates (as their trimethylsilyl
(TMS) derivatives) while aldose diphosphates (also as their TMS
derivatives) proved to be too unstable to analyze. It is safe to
assert, however, based on our own experience and the literature,
that a broad spectrum of organic compounds of biological
significance will be amenable to analysis by GC/MS methods.

There is another sode of operation of a GC/MS system which
enables greater sensitivities to be attained for the quantitation
of KNOWN metabolites. If the mass spectrum and the gas
chromatographic retention time of the compound to be quantitated
are known, the mass spectrometer can be used as a specific
detecting system for this compound. This technigue is called mass
fragmentography. Under these experimental conditions, the mass
spectrometer is not scanned over the ENTIR#& mass range but is
directed to measure one or two SPECIFIC masses known to be
-6-

characteristic of the compound(s) being quantitated.
Consequently, there is an appreciable increase in sensitivity
since the mass Spectrometer samples only the significant data
points and can integrate the signal longer.

In this mode, existing GC/MS instrumentation matches the new
fluorescent reagents for amines (reported to detect approximately
10 picomoles). It also embodies the specificity of the mass
spectrua at individual mass numbers. At greater cost and
cumbersoreness, the MS can be extended to a quantum-counting
Tange of sensitivity. These methods are therefore likely to be
complementary to the special purpose methods, like
fluorescimetry, which are often cheaper and more efficient for
well defined classes of compounds. On the other hand, the history
of pesticide analysis shows how the GC can also be made
ultrasensitive at the cost of some loss of specificity.

Using deuterated analogs as standards for the test compound,
quantitation can also be achieved at sub-nanograa levels. We have
recently exploited certain characteristics of the quadrupole amass
spectrometer and its data system to develop a method for the
quantitation of ten amino acids in soil extracts (ref 3a, copy
attached) and subsequently for the amino acid content of
biological fluids (ref 3b). This represents an advance in the
technique of mass fragsentography since the sector mass
spectrorgeters used up to this time, have been severely limited in
the number of ions and the mass range they could monitor for any
one experiment. Our technique of quadrupole mass fraygmentography
was used for the quantitation of the amino acids in the urine of
a patient with suspected branch chain amino aciduria. The results
are discussed later (see Methods of Procedure and figure 5).

The overall management of the system and the reduction and
selective presentation of the large volume of data emanating from
the analytical instruments is an important task of the control
computer. Our experience in instrumentation comprises a good deal
of computational software embracing real time instrument
management, automated data reduction, and artificial intelligence
(ref 4, appended to this application). It also requires
considerable effort in electronic and vacuum technology for the
instrumentation hardware, and a coherent system approach for the
overall integration of these components.

Present GC/MS systeas are designed mainly for laboratory
research, incorporating great analysis flexibility but the
ability to handle only a small number of samples. Such systems
are not practical for large volume screening, but they can be
adapted for the pilot studies contemplated here. A properly
designed automated system could reduce these costs by as much as
an order of magnitude, as would be essential for cost-effective
applications to general health care.

The routine screening of normal and abnormal body

metabolites, including drugs, in human body fluids (ref 5) is
currently the object of several research programs. Various

P-aS
-~7J-

non-specific methods, including thin layer (ref 6,7), ion
exchange (ref 8,10), liquid (ref 9), and gas chromatoyraphy (ref
11-14 and 17b), are used primarily with the goal of separating a
large number of unnamed constituent materials. Using these
techniques, compound “identification” is made by a Comparison of
the migration, under identical conditions, of the unknown spot
with reference compounds. This approach can lead to erroneous
identifications, however. This point is illustrated by a recent
article (15) which describes the use of mass Spectrometry in the
identification of a case of isovaleric acidemia. Previously, the
Same patient was diagnosed as having butyric and hexanoic
acidemia on the basis of chromatographic evidence alone. This
type of error is especially important when analyzing a "new"
(previously undescribed) metabolic error where rigorous
identification of relevant metabolites in various body fluids aud
tissues is essential.

For positive identification using mass spectrometry, the
separated components must be transferred from the chromatographic
medium to the mass spectrometer. The unknown spot can be leached
from paper or thin layer chromatoyjrams, or in the case of liquiu
chromatography, the solvent removed, and a mass spectrum recorded
on the residual material. Trapping the various effluent
components From a gas chromatograph, with subsequent introduction
into the mass spectrometer, has been used. This approach requires
considerable time and is inefficient when applied to complex
separations. It has been superseded by the direct coupling of the
gas chromatograph and mass spectrometer.

Gas chromatography is unquestionably the most convenient
separation technique to couple to the mass spectrometer because
the carrier jas can be removed efficiently and easily as the
analysis proceeds. For recent examples of the use of the GC/MS
technique for the analysis of body fluids see refs 15-18. sased
on the work cited, as well as our own on-going programs, the
ability of the GC/MS technique for the analysis of body fluids is
well established. We have drawn upon the published literature in
helping to design our experimental protocols (ref 18).

As mentioned earlier, urine and other body fluids (e.y.,
Serum, amniotic fluid, etc.) are complex mixtures, The Separation
(by gas chromatography) and subsequent identification (by mass
spectrumetry) of these components can be a difficult task. To
sinplify the separation problema, the body fluid under analysis is
chemically separated into a number of fractions which permit
analysis for acids, amino acids, and carbohydrates present in
free or in conjugated form. Drugs and hormones, as well as their
metabolites, will also appear in the chromatographic separations.
The gas chromatographic analysis of each class of compounds
presents a metabolic protile. Abnormal profiles (containing
either novel peaks or peaks deviating from their expected
amplitudes) are then assayed by mass spectrometry. The mass
spectra recorded during the elution of each gas chromatographic
peak then serve to identify the constituents present Ln that
peak.
-8-

The importance of this diagnostic technology may be
illustrated by a family seen for a fatal inherited disease at the
Stanford Medical Center. Three male infants have been born to
this couple and each of the offspring died in a very similar
manner by 10 days of age. The first infant was not seen at
Stanford, but the second infant was transferred from another
hospital, in extremis. Aside from the striking family history,
there were no diagnostic clues, and the second child was dead
within 6 hoacs of arrival. During the period of observation,
blood and urine were collected from the proband, and analysis of
the latter by gas chromatography (at another institution)
revealed an excess of orotic acid. This diagnostic clue, coupled
with the clinical picture of relatively rapid demise after
protein (breast milk) teedings were well established, resulted in
a diagnosis of a hereditary deficiency of hepatic ornithine
transcarbasylase, with resulting hyperammonemia (ref 19). fhis
diagnosis was eventually confirmed when the third male infant of
this couple behaved in a manner identical to that of his two
deceased brothers and, despite attempts at therapeutic
intervention which included low protein feedings, he developed
documented hyperammonemia and died in this medical center. Study
of the liver revealed the complete absence of ornithine
transcacbamylase. It gust be stressed that clinical observations
of the first two infants and post mortem examinations were not
helpful diagnostically, and it was the chemical identification of
orotic acid which led to the understanding of the familial
pattern of neonatal sortality in this family.

Most medical centers have access to amino acid analyzers in
order to screen patients for metabolic abnormalities of the
principal amino acids, but unless a special research interest
exists, other inborn errors of metabolism cannot easily be
studied. At this institution the GC/MS system provides us the
opportunity to detect a wide variety of inborn errors which show
accumulation of amino acids, fatty acids, and many other
metabolites in urine, blood, amniotic fluid, and other biological
fluids and tissues.

Another application of GC/MS pertains to pre-natal diagnosis
of hereditacy inborn errors of metabolism. Fetal urine
contributes to the amniotic fluid by the twelfth week of
gestation and it should provide information which is
diagnostically relevant to the fetus (ref 20). Surprisingly
little is known about the origin, fate, and components of normal
amniotic fluid, although information is accumulating because of
interest in pre-natal diagnosis and evaluation of the fetus (ret
21). The fetal cells of the aaniotic fluid, cultivated IN VITRO,
are used routinely for pre-natal diagnosis of jenetic disorders
{ref 22). The fluid itself has been used infrequently for
diagnostic purposes, although there is increasing evidence for
the utility of direct assay of this component of amniotic tluid
for pre-natal diagnosis of heritable metabolic errors. Mahoney ct
al. (ref 23) have recently reported the first successtul
pre-natal diagnosis of methylmalonic aciduria (confirmed ty stucy
of the fetus and performed in time to elect abortion). Their
-~Q-

observations suggest that at 12 weeks of yestation, wethylmalonic
acid is undetectable in the amniotic fluid surrounding a normal
fetus and, probably, a fetus who is heterozygous for the
(recessive) gene (ref 24). Gooiman, et al. (ref 25) have
successfully diagnosed argininosuccinic aciduria in a 16 week
fetus. This involved detection of argininosuccinic acid in the
amniotic fluid plus enzymatic studies on cultured amniotic fluid
cells. Normally this compound is not detected in amniotic fluid
at 16 weeks of gestation. There are reports of other conditions
being diagnosed in the fetus by direct assay of amniotic fluid
(ref 26) as well.

The accuracy of diaynostic procedures probing amniotic fluid
for soluble constituents has been questioned because of the
possibility of contamination with maternal blood and because of
lack of information on the normal state at various tirges of
gestation. [t the tluid component could be used for pre-natal
diagnostic purposes, the phenotype of the fetus could be detected
relatively capidly as compared to the time required to culture
sufficient amniotic cells. Hereditary diseases which are
potentially amenable to diagnosis by analysis of the soluble
constituents of amniotic fluid are those in which the
accumulating metabolite is not cleared by the placenta but is
expected to appear in fetal urine. Defects in epithelial
transport, e.g., cystinuria ani Hartnup disease, are exaaples of
such conditions. However, it is clear that this class of
metabolic errors is not the only one which might be detectable by
direct assay of amniotic fluid. The examples provided above
Suggest that fetuses with overflow type metabolic errors may also
be detected.

The development of diagnostic and screening techniques
Suitable for various inborn errors of getabolisa will require a
suitable coaputer based methodology for screening a large
selected saaple of subjects with the subsequent resolution of
data into classifications describing normal states and ranyes as
well as specific correlations of GC/MS analysis abnormalities
with disease states. With a modest augmentation of existing
instrumentation facilities, we can accomplish these analytical
tasks on increasing numbers of patients.

Be SPECIFIC AIMS

a) We plan to use GC/MS to screen urine and plasma froa
normal individuals ot various ages, including premature and
newborn infants, in order to establish adequate control data
and to anderstand variations encountered.

b) We plan to use GC/MS in the diagnosis of inherited
metabolic abnormalities and in the detection and study of
previously unrecognized metabolic disorders.