American Journal of Medical Genetics Part C (Semin. Med. Genet.) 121C:5–17 (2003) ARTICLE

Pediatric Medicine and the Genetic Disorders of the and Mennonite People of D. HOLMES MORTON,* CAROLINE S. MORTON, KEVIN A. STRAUSS, DONNA L. ROBINSON, ERIK G. PUFFENBERGER, CHRISTINE HENDRICKSON, AND RICHARD I. KELLEY

The Clinic for Special Children in Lancaster County, Pennsylvania, is a community-supported, nonprofit pediatric medical practice for Amish and Mennonite children who have genetic disorders. Over a 14-year period, 1988– 2002, we have encountered 39 heritable disorders among the Amish and 23 among the . We emphasize early recognition and long-term medical care of children with genetic conditions. In the clinic laboratory we perform amino acid analyses by high-performance liquid chromatography (HPLC), organic acid analyses by gas chromatography/mass spectrometry (GC/MS), and molecular diagnoses and carrier tests by polymerase chain reaction (PCR) amplification and sequencing or restriction digestion. Regional hospitals and midwives routinely send whole-blood filter paper neonatal screens for tandem mass spectrometry and other modern analytical methods to detect 14 of the metabolic disorders found in these populations as part of the NeoGen Inc. Supplemental Newborn Screening Program (Pittsburgh, PA). Medical care based on disease pathophysiology reduces morbidity, mortality, and costs for the majority of disorders. Among our patients who are homozygous for the same mutation, differences in disease severity are not unusual. Clinical problems typically arise from the interaction of the underlying genetic disorder with common infections, malnutrition, injuries, and immune dysfunction that act through classical pathophysiological disease mechanisms to influence the natural history of disease. ß 2003 Wiley-Liss, Inc.

KEY WORDS: genetic diseases; general pediatric medical care; metabolic diseases; genotype-phenotype correlation

INTRODUCTION [Hostetler, 1963]. McKusick recognized McKusick that spanned several decades. the importance of the book as a socio- In 1978 McKusick edited and Johns Features of the Population logical study and realized the potential Hopkins Press published Medical Gene- of Amish populations for the study of tic Studies of the Amish. [McKusick, The manuscript of John A. Hostetler’s human genetics. McKusick’s review,and 1978] Table I lists 26 genetic disorders book Amish Society was reviewed in the subsequent publication of Amish that were described in this publication, the early 1960’s for Johns Hopkins Society in 1963, marked the beginning which came from many different Amish University Press by Victor McKusick of a collaboration between Hostetler and demes throughout Pennsylvania and the midwest. Puffenberger and Francomano discuss the origins of genetic studies in D. Holmes Morton, M.D., Sc.D. (Hon.), is co-founder and director of the Clinic for Special the Mennonite and Amish populations Children. He is a pediatrician with an interest in the influence of early diagnosis and treatment upon the natural history and neurobiology of inherited metabolic disorders. in accompaning articles [Francomano, Caroline S. Morton, Ed. M., is co-founder and manager of the non-profit clinic and educational 2003; Puffenberger, 2003]. organization called the Clinic for Special Children. Although these populations are Kevin A. Strauss, M.D., is a pediatrician doing post-graduate studies at the Clinic focused upon the neurobiology of genetic disorders and the pathophysiology of metabolic injuries of the brain. unique in many ways, when compared Donna L. Robinson, R.N., C.N.P., is a pediatric intensive care nurse practitioner with special to the general population of Pennsylva- interest in developing nursing protocols for management of hospitalized patients with metabolic nia, the Amish and Mennonites are less disorders. Erik G. Puffenberger, Ph.D., is laboratory director at the Clinic for Special Children, with special distinct than is generally appreciated. In interest in molecular biology and population genetics. each of the 14 or more generations since Christine Hendrickson, RN, is a pediatric nurse who recently joined the Clinic staff to work with the early 1700s, 10–20% of the children children with genetic disorders in the outpatient setting. Richard I. Kelley, M.D., Ph.D., is Associate Professor, Department of Pediatrics, Johns Hopkins have left the Old Order communities University, and member of the Division of Metabolism, Kennedy-Krieger Institute, Baltimore MD. [Hostetler, 1993]. Lancaster County Dr. Kelley is consulting pediatrician and geneticist, and a founding board member, of the Clinic church cemeteries, public school rosters, for Special Children. *Correspondence to: D. Holmes Morton, Clinic for Special Children, 535 Bunker Hill Rd., and regional phone books include many Strasburg, PA 17579. Amish and Mennonite family names. DOI 10.1002/ajmg.c.20002 Therefore, many so-called Amish alleles

ß 2003 Wiley-Liss, Inc. 6 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE

TABLE I. Metabolic Disorders and Syndromes Found in Medical Genetic Studies of the Amish [McKusick, 1978]

Inheritance OMIM Demeb Metabolic disordersa Phenylalanine hydroxylase deficiency AR 261600 IN/PA Propionyl-CoA carboxylase deficiency AR 232000 OH/PA Pyruvate kinase deficiency AR 266200 PA(B) Syndromes Albinism, oculocutaneous, tyrosinase deficient, type 1B AR 606952 IN Spastic paraplegia AD PA(L) Arthrogryposis, dysmelination, craniosynostosis, and AR cleft palate Ataxia-telangiectasia AR 208900 IN Brittle hair, intellectual impairment, and decreased AR fertility Byler disease, progressive familiar intrahepatic cholestasis AR 216400 PA(Lw) Cardiomyopathy, asymmetric sepal hypertrophy AD IN Cardiomyopathy, nonobstructive AD OH Cartilage-hair hypoplasia AR 250250 PA,OH,IN Charcot-Marie-Tooth syndrome AR 118200 Coclear deafness, myopia, and intellectual impairment AR 221200 PA(L) Jackson Weiss syndrome AD 123150 IN Deafness, congenital AR Ellis-van Creveld dwarfism AR 22550 PA(L) Epidermolysis bullosa letalis AR 226700 OH Familial manic-depressive illness AD 136800 PA(L) Hypothyroidism and muscular hypertrophy Limb girdle dystrophy, type 2E 604286 IN/PA Mast syndrome AR 248900 OH(H) McKusick-Kaufman syndrome AR 236700 OH/PA Muscular dystrophy, limb-girdle type AR 253600 IN/PA Nanophthalmos, familial AR 251600 NR Oculocerebral syndrome with hypopigmentation AR 257800 Renal-retinal dysplasia AR 266900 Troyer syndrome AR 275900 OH(H) Weill-Marchesani syndrome AR 277600 PA(L)

aDisorders in italics are also entered in Tables II and III. bPA(B) is Belleville, PA; PA(L) is Lancaster county, PA; PA(Lw) is Lawrence county, PA, OH(H) is Holmes County, OH.

or Mennonite alleles have diffused out found within the Amish and Menno- the general population. The gene muta- into the general population. The Old nites, the alleles are not unique, but these tions carried by the founders of the Order Amish and Mennonite popula- alleles are at different frequencies than in demes of Pennsylvania are not distribut- tions of Pennsylvania are new compared ed evenly, either within or among the to the time of origin of these gene muta- many subpopulations of the state. For 18 tions. All three of the mutations causing The gene mutations carried of the disorders seen regularly at the phenylketonuria (PKU) in the Old by the founders of the demes clinic, the incidences are high, approxi- Order Amish and Mennonites are found mately 1/250 to 1/500 births. Although throughout Europe. The so-called Amish of Pennsylvania are not there is a wide variety of heritable mutation for glutaric aciduria type 1 distributed evenly, either disorders among the Plain people (GA1) is one of the more common (Table I), the majority of founder dis- mutations found throughout Europe within or among the many orders are clustered in a few families, and and the [Biery et al., subpopulations of the state. the overall disease prevalence among 1996]. Therefore, for most disorders Plain people is relatively low. Examples ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 7 of lower-frequency founder disorders ders, who had laboratory facilities to ciency [Morton et al., 2002a]. Clearly, include PKU and medium-chain acyl diagnose and monitor biochemical con- the burden of genetic disease is high and dehydrogenase deficiency (MCADD), trol during infectious illnesses, and who the need for specialized and compre- which are probably less common in the were interested in general pediatric hensive care is essential to the health of Plain populations of Lancaster County practice [Morton et al., 1991; Morton, the community. In the last 14 years we than in the general Caucasian popula- 1994; Morton et al., 2002b]. Because the tion. Some common genetic diseases early symptoms of these serious disor- were apparently excluded by founder ders are difficult to distinguish from Clearly, the burden of sampling and/or genetic drift. For common diseases, we often evaluate genetic disease is high example, cystic fibrosis is not seen in patients who turn out not to have a the Plain people of Lancaster County genetic disorder common to the Plain and the need for specialized but is found in some other western people. Infants who present with failure and comprehensive care Pennsylvanian demes. This uneven dis- to thrive and irritability or who become tribution reflects different founders and unusually ill during otherwise common is essential to the health a 300-year separation of the various infections most often do not have under- of the community. subgroups prior to, and after, settlement lying genetic disorders. After the clinic in North America. In the Plain people opened in 1989, it was apparent the of Pennsylvania, maple syrup urine practice would not be limited to patients have cared for patients from the Amish disease (MSUD) is found only in those with MSUD and GA1. The first ill and Mennonite populations with more of Mennonite descent and GA1 is neonate sent to the clinic to rule out than 60 different single-gene disorders, found only in the Amish populations. MSUD did have a family history of as shown in TablesII and III. These tables Although eight disorders are found in MSUD, but instead had Hirschsprung summarize a clinical experience with both populations, only Crigler-Najjar disease with bowel obstruction and 400–500 patients since 1988. Tables II disease and propionic acidemia are sepsis. One of the more recently identi- and III are not complete lists of genetic currently known to arise from the fied infants with MSUD also had a disorders found in the Amish and same mutations in both populations. second rare recessive disorder, severe Mennonite populations of Pennsylvania. MCADD, 3-methylcrotonylglycinuria combined immune deficiency. Two of However, we have seen patients from [Gibson et al., 1998], and at least two our patients with MSUD have first other settlements in central and north- forms of osteogenesis imperfecta are cousins with MCADD. Before 1988, western Pennsylvania where different found in both populations but are caused all Amish children with GA1 were said founder genes presumably explain the by different mutations in the Amish and to have cerebral palsy. Many children prevalence of different sets of genetic Mennonites [Puffenberger, 2003]. with so-called Amish cerebral palsy did disorders. have GA1, but others did not, and their evaluations led to the descriptions MEDICAL CARE AND CULTURAL AND of troponin T1 myopathy [Johnston THE CLINIC FOR ECONOMIC BACKGROUND et al., 2000], Amish lethal microcephaly SPECIAL CHILDREN OF THE CLINIC [Kelley et al., 2002; Rosenberg et al., The early genetic studies of the Plain 2002; Strauss et al., 2002], and an auto- The Clinic for Special Children was people indicated unique needs for the somal recessive Pelizaeus-Merzbacher- registered in Pennsylvania as an inde- medical care of people who were like central dysmyelination syndrome pendent nonprofit organization in 1989. geographically and culturally isolated. [Morton et al., unpublished results]. We The original board of directors included The Clinic for Special Children was recently evaluated a neonate with a three of the authors of this paper organized to integrate general medical sibling with GA1 who fortunately did (D.H.M., C.S.M., R.I.K.) and the late care and modern genetics [Hostetler, not have GA1, but unfortunately, as we Dr. John A. Hostetler, a descendant of 1993; Morton, 1994]. An initial goal learned from a neonatal screening test, Amish parents and a sociologist of rural of the clinic founders was to diagnose his hypospadias was a sign of another culture. In 1990, several parents of MSUD and GA1 in asymptomatic neo- recessive syndrome, 3-b hydroxyster- children with genetic disorders from nates and prevent the metabolic crises in oid dehydrogenase deficiency. Another the Amish and Mennonite communities order to prevent brain degeneration that family has three children with GA1 and joined the board. Both communities characterized these conditions. Because a fourth with PKU. Over the years, were aware of significant numbers of common childhood infections often failure to thrive workups have led to the children with MSUD, GA1, and other provoke metabolic crisis in children with diagnoses of Byler disease, three new genetic disorders. The goal of these these disorders, we reasoned that med- disorders of bile salt transport [Morton parents was for the clinic to provide ical care for affected children should be et al., 2000], Bartter syndrome, aldoster- comprehensive medical care for child- directly provided by individuals who one deficiency [Mitsuuchi et al., 1993], ren with complex and unstable disor- were knowledgeable about the disor- and primary growth hormone defi- ders. The Clinic was viewed as a way to MRCNJUNLO EIA EEIS(EI.MD EE. ARTICLE GENET.) MED. (SEMIN. GENETICS MEDICAL OF JOURNAL AMERICAN 8

TABLE II. Genetic Disorders of Pennsylvania Amish

Illness History Neo gen Serum Urine or Amino acids and Diagnosed during and or PA amino acid (serum) organic acyl carnitines by mutation infancy exam screen analysis acids by GC/MS by MS/MS detectiona Clinical notes Metabolic disorders Adenosine deaminase deficiency þ Milroy Amish. Severe combined immune deficiency. Lethal w/t bone marrow transplant Adrenal hyperplasia II 3ß-hydroxysteroid þþ þ þNeonatal hyponatremia/hyperkalemia. Males have dehydrogenase def. ambiguous genitalia but severity variable Congenital growth hormone deficiency þ Poor growth in infancy with anorexia and low IGF-1 and GH Corticosterone methyl oxidase II def. þþþPoor growth, hyponatremia/hyperkalemia, variable Aldosterone def. phenotype Crigler-Najjar syndrome type 1 þ þ Kernicterus w/t neonatal diagnosis and phototherapy. Lethal w/t liver transplant Galactosemia, Duarte and Duarte/classical þþ þþCarriers of classical gene identified by NeoGen. variants Common in Maryland Amish Glutaryl CoA dehydrogenase deficiency þþ þþþBasal ganglial degeneration with cerebral palsy-like dystonia GM1 gangliosidosis, beta-galactosidase þþ Lethal. Found in Milroy Amish deme but not in deficiency Lancaster County Medium chain acyl-CoA dehydrogenase þþ þþþRare. High morbidity and mortality w/t neonatal deficiency screenings. Infection induced crisis 3-Methylcrotonyl-CoA carboxylase þþ þþþMild phenotype, several asymptomatic adults and deficiency children Phenylalanine hydroxylase deficiency þþþþþþVariable phenotype, severe to mild hyperphe Propionyl-CoA carboxylase deficiency þþ þþþVariable Phenotype. Basal ganglial injury, cardiomyopathy Pyruvate kinase deficiency þþ þCommon in Belleville deme. Erthroblastosis, hepatopathy and hemolysis Short chain acyl dehydrogenase deficiency þþ þþ Infants asymptomatic. Increased butyrylcarnitine and urine ethylmalonate Sitosterolemia þþCoronary artery disease in childhood. High cholersterol and sitosterol Syndromes Arterial venous malformation syndrome þ Common in Lancaster Amish. Intracerebral hemorrhages in all age groups. Dx by MRA Blank syndrome, familiar hypercholanemia þ þ Poor growth, pruritis, vitamin K, and D deficiencies in infancy Byler disease, familiar intrahepatic þ þ Poor growth, pruritis, vitamin K, and D deficiencies in cholestasis infancy. Lethal w/t liver transplant RIL MRCNJUNLO EIA EEIS(EI.MD EE. 9 GENET.) MED. (SEMIN. GENETICS MEDICAL OF JOURNAL AMERICAN ARTICLE

Cartilage-hair hypoplasia dwarfism þþ þVariable phenotype for growth, hair, immune deficiency, and Hirschsprungs disease Cerebellar ataxia with preserved reflexes þþ Gross motor and language delays. Ataxia and abnormal gait variable Cockayne syndrome þ Lethal. Typical phenotype, infantile form. Seen in Amish in Belleville area Lethal microcephally with 2-ketoglutaric þþ þCommon in Lancaster. Lethal with brain aciduria malformations and progressive encephalopathy Congenital supraventricular arrhythmias þ Severe arrhythmias. Life-threatening cardiac events in the neonate. Dx by EKG Ectrodactyly, omphalocele and cardiac þþ Rare, lethal syndrome. Some infants have ectodactyly malformations only Ellis-van Creveld dwarfism þþ þCommon in Lancaster. Pulmonary hypoplasia, cardiac disease in 25–40% Fuchs endothelial dystrophy of the cornea þ Presents in young adults as early glaucoma and cataracts Mckusick-Kauffman syndrome þþ þFemale infertility without early detection and repair Moyamoya disease þ One case in Belleville Amish. The child also had pyruvate kinase deficiency Muscular dystrophy, limb-girdle type þ Beta-sarcoglycan glycoprotein defect Neurofibromatosis Uncommon. Two cases with: periobital and facial tumors; obstructive hydrocephalis Osteogenesis imperfecta with opalescent þ þ Common. Dominant variant. Fractures later onset teeth, fractures and osteoporosis Polycystic kindey disease, adult type Onset renal failure age 30–50 years. No liver disease. Renal transplant Rett syndrome þþ Typical progressive microcephaly in females. Sporatic, full disability Riehl syndrome, Pelizaeus-Merzbacher-like þþ Autosomal recessive variant. Congenital nystagmus, dysmylination motor delays. Dx by MRI Swarey syndrome, fetal gonadal failure, þþ Common in Belleville area. Lethal. Not sudden infant death endocrinopathy or Kennedy syndrome Troponin type 1 myopathy, infantile nemalin þþ þCommon, lethal. Tremors in utero and at birth. myopathy ‘‘Chicken breast disease’’ aGene and mutation identification Puffenberger, 2003. 0AEIA ORA FMDCLGNTC SMN E.GNT)ARTICLE GENET.) MED. (SEMIN. GENETICS MEDICAL OF JOURNAL AMERICAN 10

TABLE III. Genetic Disorder of Pennsylvania Mennonites

Urine or Serum or (serum) Amino acids Illness History Neo gen (urine) organic and acyl Diagnosed during and or PA amino acid acids by carnitines by mutation infancy exam screen analysis GC/MS by MS/MS detectiona Clinical notes Metabolic disorders Crigler-Najjar syndrome type 1 þþ þKernicterus w/t neonatal diagnosis and phototherapy Lethal w/t liver transplant Cystinuria þ (þ) þ Renal stones in early childhood with risk of renal damage. Common. Four mutations Gilbert syndrome þ Common in families with Crigler-Najjar disease Homozygotes for TATA box polymorphisms Glycogen storage disease, type 6 þ Abnormal liver enzymes and high cholesterol Irritability and hepatomegaly after weaning Immune deficiency, SCID variant þþ ALC < 100/mm, no thymic shadow or T-lymph. Bone marrow transplant. ?IL7 recept defect Branched chain 2-keto acid þþþþþþClassical MSD. Liver transplant was effective therapy, bone marrow dehydrogenase deficiency transplant was not Medium chain acyl-CoA þþ þþþHigh morbidity and mortality without neonatal screenings. Infection dehydrogenase deficiency induced crisis 3-methylcrotonyl-CoA þþ þþþMild phenotype, several asymptomatic adults and children carboxylase deficiency Mevalonate kinase deficiency þþþNeonatal jaundice, erythoblastosis. Compound heterozygote Phenylalanine hydroxylase þþþþþþVariable phenotype, mild hyperphe to severe deficiency Propionyl-CoA carboxylase þþ þþþSame mutation as Amish. Variable Phenotype deficiency Tyrosinemia type 3 þþþþ þOne case, no clinical disease Syndromes Dystonias DYT-1 þþChildhood onset, Israeli mutation, one case. DYT6 reported in midwestern Menn. group Familial periodic fever (TRAPS) þþ þ/ Tumor necrosis factor receptor mutation in 1 family. TRAPS and FMF genes nl in another family Fragile x syndrome þþCommon disorder. Variable phenotype makes clinical diagnosis difficult Hereditary spherocytosis þ Hemolysis, hyperbilirubinemia, abnormal blood smear. RBC fragility test diagnostic Hirschsprung disease þþ þ/ Neonatal bowel obstruction and sepsis. Polygenic disease. Highly variable phenotype Nephrotic syndrome, congenital þþEdema and infections. Lethal w/t renal transplant Osteogenesis imperfecta, þþ Congenital of retinaes. Fractures, late onset. Unknown detached retinaes mutation ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 11

allow the communities to care for their tion waned, the direct costs of care paid own. This desire was in response to the by the Mennonite community in- creased sharply. By 1988, the initial care for a newly diagnosed neonate The goal of these parents with MSUD in regional pediatric hos- was for the clinic to pitals was $40,000–$60,000 or more [Morton et al., 2002b]. Amino acid provide comprehensive analyses cost $450, and when coupled medical care for children with physician charges and other testing charges, the total cost of an outpa- with complex and unstable tient evaluation was typically $1,000– disorders, as a way to $1,500. These expenses were a major allow the communities difficulty for the Plain people, as they historically have not participated in to care for their own. medical insurance programs. Approxi- mately half of the families contribute to a form of major medical insurance many difficulties encountered by the called Amish Aid or Mennonite Aid, Plain people in getting access to, and which is administered by businessmen Plexin A2. Unknown mutation been seen in one family unknown paying for, specialized medical care. within the churches. Prior to the open- Common syndrome. AXPC1, Mapped to 1q31-32. No mutation in Fractures, later onset Typical progressive microcephaly in females Sporatic, full disability Common, lethal SMN1 exon 7 deletion. A milder clinical variant has Variable phenotype, common. Childhood onset does occur. Mutation The origins of community support and ing of the clinic, benefit auctions were the economic and medical impacts of the held to help pay hospital bills of $50,000 clinic can be understood in relation to or higher. Other church districts collect the history of MSUD in the Mennonite alms to help families with large medical people. The collection of essays and let- bills. Members of the Old Orders are ters What Is Wrong with Our Baby?, com- not eligible for Medicare or Medicaid piled and published by John and Verna benefits because of an exemption from Martin, parents of two MSUD children, Social Security taxes. When young is an interesting and sometimes sobering adults join the Old Order Amish or look at modern health care from a Mennonite churches, they sign an IRS parent’s perspective [Martin and Martin, Form 4029, which indicates that they 1995]. The oldest surviving Mennonite are ‘‘a member of a religious body that is patient with MSUD was born in 1967, conscientiouslyopposed to social secur- but many undiagnosed or untreated in- ity benefits but that makes reasonable fants died beforeshe was born. She was in provision for its own dependent mem- a hospital in Philadelphia for 6 months, bers’’ [Hostetler, 1993]. The exemption did not regain her birth weight for from the tax also means that those who 4 months, and today has severe disability sign IRS Form 4029 agree not to accept and retardation. Her sister, in whom money from state or federal programs MSUD was recognized by 3 days of age, that are paid for by Social Security became severely intoxicated, remained þ þ funds. This cultural practice is one of hospitalized for many weeks, and also many issues community members face has profound retardation. when obtaining care for their children. þþ þþ þþ þ FINANCIAL DIFFICULTIES CHALLENGES IN FOR PLAIN PEOPLE THE MEDICAL IN TRADITIONAL MANAGEMENT OF MEDICAL SETTINGS THESE DISORDERS Between 1966 and 1988, 36 Earlier diagnosis and improved man- Mennonitechildrenwerebornwith agement did, in many cases, allow MSUD. Over these 22 years, the normal growth and development, yet management of the neonates improved several otherwise healthy children with sensory ataxia variant infantile and hospitalization times shortened. MSUD died unexpectedly from acute Rett syndrome Retinitis pigmentosa, areflexia, Osteogenesis imperfecta, Amish Werdnig-Hoffman disease, Familial manic-depressive illness Gene and mutation identification Puffenberger, 2003.

a However, as funding for research in cerebral edema after the newborn biochemical causes of mental retarda- period. Of the 36 children with MSUD 12 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE

gram did not substantially improve mat- 5 days with a hospital cost of $1,000– Earlier diagnosis and ters for these patients. Until the Guthrie 2,000 per patient per day. Over a 12- improved management did, method was replaced by the tandem year period, developmental outcomes mass spectrometry program in 1993, have been good [Morton et al., 2002]. in many cases, allow normal false positive rates were unacceptably We currently follow more than 70 growth and development, high [Morton et al., 2002b]. The state patients with MSUD, most of whom program made no provisions to test and are of Mennonite descent. Between yet several otherwise healthy provide immediate results for high-risk 1988 and 2002 we had 137 admissions children with MSUD died infants. The three Mennonite infants to our hospital for acute MSUD in- who were found by the state program toxication; however, the average an- unexpectedly from acute were 5 and 6 days old and already ill. nual rate of hospitalization for our cerebral edema after More important, the state follow-up MSUD patients is less than 1 day per the newborn period. program made no provisions to pay for patient-year of follow-up. Between the care of ill neonates, had no plans 1988 and 2002, there were, however, for the assessment and care of acutely no deaths from cerebral edema. The born between 1966 and 1988, 14 (36%) ill older children, did not address the current total annual formula cost for died of cerebral edema before the age of problem of cerebral edema, and did not our MSUD patients is about $115,000. 10 years. The typical course of the support care for patients over the age Again, most Mennonite families do not children was that they became ill with a of 21 years. The state program did accept formula or reimbursement for common infection, had poor appetite, offer to pay for several amino acid levels costs from the Pennsylvania MSUD vomiting, and irritability, which pro- per year and offered formula to those follow-up program. The clinic pays half gressed to lethargy,and then to coma. By who qualified for, and would accept, of these costs for families who have the time arrangements were made to Medicaid, but the program required limited resources, and the families pay travel from Lancaster to the regional that Mennonite families obtain these the balance. The charge for an out- hospital, the biochemical intoxication services at the hospitals in distant re- patient visit is $35, and medical care was severe. Emergency efforts to reverse gional centers. and laboratory testing are provided the intoxication by intravenous fluids, In 1990, an amino acid analyzer at the clinic regardless of a family’s glucose, and dialysis failed, the cerebral was given to the Clinic for Special ability to pay. In summary, a com- edema worsened, and within 24 hr after Children by two Mennonite churches. prehensive health care system was entering the hospital, the child died of To date, the clinic has performed designed to provide sophisticated brain stem herniation. Physicians and 16,402 amino acid analyses on this medical care of patients with MSUD parents knew that common infectious instrument. Because of our nonprofit within their community. By combining illnesses provoked serious metabolic in- status and community support, we community support and other dona- toxication, but little was known about were able to reduce the patient test tions with knowledgeable and sophisti- how to control the metabolic response charge from $450 to $50. We also cated laboratory and clinical care, the to such illnesses in the outpatient set- decreased the analysis and reporting morbidity, mortality, and costs have ting. Local services for children were time for plasma branched-chain amino been reduced. limited. Specialists and equipment that acids to 20 min, and we routinely were needed to study the early stages of quantify plasma amino acids during an CARE FOR PATIENTS cerebral edema were in research labora- outpatient clinic visit. Between 1989 WITH DISORDERS tories far from the patients. The available and 2003, we managed 42 neonates OTHER THAN MSUD biochemical testing was of no utility for with MSUD. Twenty-one of these the rapid treatment necessary to avert neonates were from high-risk couples We believe that the cost of operat- cerebral edema because results took as recognized by family histories orcarrier ing the Clinic for Special Children long as 2 weeks. Routine outpatient tests, and the infants were tested for could be fully justified if we cared for visits to metabolic clinics in Philadelphia MSUD and diagnosed before 24 hr of no other patients than Mennonite could only be made on one afternoon age. With timely diagnosis, the neonates children with MSUD. However, the each week, and outpatient services to were never ill and could be managed medical and economic impacts of care assess acutely ill children were not avail- without hospitalization. The diagnosis for patients with GA1, MCADD, able. Instead, ill children were referred to of MSUD was made in 20 additional Crigler-Najjar disease, the bile salt emergency rooms, and the medical staff neonates diagnosed because of clinical disorders, and many other disorders that of the ER usually knew less about the illnesses; 3 of these infants were found affect Amish and Mennonite children management of MSUD than did the by the Pennsylvania screening pro- provide equally compelling justification child’s parents. Newborn screening for gram.These20illinfantsweremanaged for the clinic. More than 95% of the MSUD was started by Pennsylvania in at Lancaster General Hospital by the disorders we see cause clinical problems 1988, but the state-administered pro- clinic staff and had a typical stay of 3– during infancy. Ten of the syndromes ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 13

wide studies, including the cerebral found to have MCADD at the clinic was More than 95% of the arteriovenous malformation syndrome, 14 years old and had been hospitaliz- disorders we see cause clinical manic depressive illness, several conge- ed several times because of vomiting and nital deafness syndromes, and a common ketotic hypoglycemia. Her sibling died problems during infancy. seizure/mental retardation syndrome. at age 6 months with hypoglycemia and a fatty liver, which was diagnosed as Reye syndrome in 1976. THE IMPORTANCE OF can be recognized by history and Here and elsewhere, some genetic PRESYMPTOMATIC CARE physical examination, including the disorders have been misdiagnosed as common disorders, Amish lethal micro- In our clinic, we have emphasized the child abuse. Approximately 10% of cephaly [Kelley et al., 2002; Strauss et al., importance of diagnosis of asympto- patients with GA1 whom we have seen 2002], Swarey syndrome [Morton et al., matic infants. For example, the diagnosis were originally thought to be victims of unpublished results], a variant of cere- of GA1 after degeneration of the basal child abuse. Infants with GA1 may bellar ataxia [Morton et al., unpublished ganglia allows no opportunity for effec- present with subdural hemorrhages after results], and a newly described syndrome tive treatment [Morton et al., 1991; minor head trauma, because 75% of the with infantile retinitis pigmentosa and Strauss and Morton, 2003a; Strauss et al., infants have open subdural and subar- sensory ataxia [Higgins et al., 1999]. Five 2003b]. The prospective management of achnoid spaces that are crossed by bridg- disorders are primarily diagnosed by GA1 prevents basal ganglial degenera- ing veins [Strauss and Morton, 2003a]. amino acid analysis, and eight disorders tion in at least 75% of cases. The clinical These intracranial hemorrhages are by organic acid analysis with gas chro- importance of recognizing asympto- usually large, and retinal hemorrhages matography/mass spectrometry (GC/ matic infants with MCADD, propionic have developed spontaneously in asso- MS). Genomic DNA mutation analysis acidemia, Crigler-Najjar disease, adrenal ciation with large intracranial hemor- is performed in the clinic [Puffenberger insufficiencies, Bartter disease, the bile rhages [Hymel et al., 2002]. In the and Morton, 2002; Puffenberger, 2003] salt transport disorders, and galactosemia Amish population of Lancaster County, and is now used to diagnose 19 disorders, is readily apparent to a pediatrician we follow 28 children with three dif- including Byler disease and two other who has cared for infants with these ferent hepatic bile salt transport disor- disorders that disrupt bile salt transport, disorders who presented undiagnosed ders. Nine of these infants (30%) Crigler-Najjar disease, fragile-X syn- and severely ill. presented before 6 months of age with drome, glycogen storage disease type VI We have also found it helpful to be bruising and bleeding secondary to [Chang et al., 1998], Amish osteogenesis able to immediately diagnose lethal vitamin K deficiency. One such infant imperfecta, infantile spinal muscular disorders in neonates. GM1 gangliosi- presented in a coma with a full fontanel, atrophy, and methylenetetrahydrofolate dosis can be diagnosed by physical exam retinal hemorrhages, and bruising on reductase deficiency [Puffenberger and and a single inexpensive test of urine bony prominences. Shaken baby syn- Morton, 2002]. Two common lethal dis- for increased urine oligosaccharide drome was the suspected diagnosis. The orders, troponin T1 myopathy [Johnston excretion. Infantile spinal muscular atro- infant was subsequently found to have et al., 2000] and infantile spinal muscular phy and troponin T1 deficiency are both a vitamin K-responsive coagulopathy, atrophy, can be difficult to diagnose in a routinely diagnosed in neonates by high serum bile salts, a deep, intracer- newborn by examination, and we now molecular methods at a charge of less ebral hemorrhage, and unilateral retinal routinely confirm these diagnoses by than $50 [Puffenberger and Morton, hemorrhages, which were atypical of mutation analyses. Neonatal metabolic 2002]. Because of easy access to the shaken baby syndrome. This infant and screenings done by Neo Gen Inc. clinic, prolonged hospitalizations invol- her sibling are now known to be identify using tandem mass spectrometry ving multiple invasive and expensive homozygotes for a mutation in the tight routinely identify 14 of the disorders that diagnostic tests for lethal disorders can junction protein 2 gene [Carlton et al., we see. The preliminary biochemical usually be avoided. 2003]. Fractures caused by relatively diagnoses for most of these disorders is In the primary care setting, undiag- mild types of osteogenesis imperfecta confirmed in our laboratory on the nosed genetic syndromes present differ- found in the Plain populations have also original specimen through polymerase ently than in genetic clinics at referral been mistaken for child abuse. chain reaction (PCR)-based mutation centers. Most often, the infants who are detection. The molecular basis of 17 seen at the clinic are irritable, feed THE UTILITY OF disorders seen at the clinic remains to be poorly, gain weight slowly, and in other A DETAILED identified. Seven of these disorders less well defined ways seem to the parents UNDERSTANDING should yield to candidate gene sequenc- to be ‘‘just not right.’’ Many teenage OF THE NATURAL ing, including GM1 gangliosidosis, patients and adults in these populations HISTORY OF DISEASE Bartter disease, and polycystic kidney with underlying genetic syndromes had disease, whereas 10 syndromes without not been evaluated by modern diagnos- We have had a unique opportunity to candidate genes will require genome- tic methods. The first Mennonite girl observe the natural history of these 14 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE disorders in many patients. The Men- deficiencies. Our impression is that measurements of plasma amino acids and nonite variant of MSUD arises from a prolonged deficiencies of valine in the changes to a different treatment regimen stop codon in exon 1 of the BCKDHA brain during the critical period of rapid to prevent prolonged or progressive gene. Thus far, all Mennonite patients brain growth between birth and 2 years metabolic intoxication. The ultimate have been homozygous for this mutation of age may be a major factor in mental impact of these illnesses on clinical out- and all have the classical form of the retardation seen in children with poorly come is influenced by the rapid avail- disorder. In spite of the mutational treated MSUD [Morton et al., 2002b]. ability of outpatient and inpatient homogeneity, the phenotype ranges The mainstay of therapy for MSUD services, including amino acid analysis, from patients who are profoundly dis- has been the dietary restriction of the MSUD hyperalimentation solution, and abled to those who are apparently branched-chain amino acids. Unfortu- medical care by physicians and nurses normal. Poor outcomes in patients with nately, in many follow-up programs, who have experience with the manage- MSUD can usually be understood in leucine levels are measured infrequently ment of the metabolic crisis and cerebral terms of one or more of the following and isoleucine, valine, and the other edema. problems: delays in neonatal diagnosis essential amino acids are not monitored and treatment, chronic essential amino at all. It is not possible to understand the acid deficiencies, ineffective manage- relationship of day-to-day metabolic The ultimate impact of these ment of metabolic intoxication during control and neurological outcome when infectious illnesses, and acute vascular only a few plasma leucine levels are illnesses on clinical outcome brain injuries caused by cerebral edema. measured per year. Intermittent changes is influenced by the rapid Neonates with MSUD who are diag- in tolerance related to normal variation availability of outpatient nosed before 24–36 hr of age are not ill, in growth velocity or illnesses will not be and progressive metabolic intoxication appreciated. Deficiencies of branched- and inpatient services, can be prevented by rational manage- chain amino acids and the unbalanced including amino acid analysis, ment [Morton et al., 2002b]. Plasma transport of the other neutral amino amino acid quantification allows diag- acids that compete with leucine for MSUD hyperalimentation nosis of the disorder as early as 12 hr of entry into the brain cannot be assessed solution, and medical care by age if high-performance liquid chroma- by isolated measurements of leucine. physicians and nurses who tography (HPLC) or tandem mass spec- The effects of dietary therapy on intel- trometry is used. It is important to note lectual outcome can only be understood have experience with the that the Guthrie bacterial inhibition by reference to the cumulative effects management of the metabolic assay is not sufficiently sensitive to be of amino acid metabolism throughout used for specimens collected before the critical period of brain growth crisis and cerebral edema. 24 hr of age [Morton et al., 2002b]. All and development. In recent years, we infants with classical MSUD identified have monitored blood amino acid by newborn screening programs will levels in rapidly growing infants twice MEDICAL CARE AND THE be very ill if screening results are not weekly and gained an appreciation of OUTCOME OF OTHER available before 4 days of age. Infants how quickly amino acid concentrat- GENETIC DISORDERS identified and treated between 4 and 10 ions change, especially during otherwise days of age can have good neurological minor infectious illnesses. Eleven of the disorders that affect the outcomes, but the cost of initial treat- Over a 13-year period, we have Amish and Mennonites are lethal or ment is higher than those diagnosed admitted patients with MSUD to the invariably cause full disability. Although within 24 hr. Neonatal screening pro- hospital for management of metabolic these 11 disorders are lethal, affected grams for MSUD wherein specimens are crisis 137 times. More than 120 of these children may live months or years and not collected until 72 hr of age and admissions were necessary because of require many human and medical ser- results are not reported before 14 days of catabolic intoxication caused by infec- vices. The clinic routinely provides age can expect high initial costs of tious illnesses such as gastroenteritis, medical care for children who have treatment and high morbidity and mor- otitis media, streptococcal pharyngitis, lethal diseases. Most of these children tality. After MSUD is diagnosed, the sinusitis, and pulmonary infections. are cared for, and die, at home. Much of prognosis for the patients depends on Appendicitis, cholecystitis, torsion and the community support for the clinic several variables, including the time re- infarction of the ovary, and fractures also comes from families who need and quired to lower the plasma and tissue cause metabolic crises. In addition, we appreciate this care. The other 51 meta- leucine levels to normal, prevention of have 100–200 MSUD outpatient visits bolic disorders and syndromes present the vascular compression and ischemic per year to manage infectious illnesses with a range of clinical problems that injuries caused by critical brain edema, and other stressors. These illnesses cause require timely diagnosis and ongoing and prevention of prolonged or severe significant metabolic changes in patients medical care. Our daily practice in- brain and systemic essential amino acid with MSUD and require one or more cludes management of episodic crises ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 15 in patients with the adrenal insufficiency many genetic disorders is directly deter- and chronic effects of high blood and syndromes, Crigler-Najjar syndrome, mined by how effectively the interaction tissue concentrations of leucine on and various organic acidemias; and the of infectious disease and underlying protein turnover in muscle and liver, chronic malnutrition and fat-soluble genetic disorder is understood and cell volume regulation, and brain amino vitamin deficiencies of the bile salt managed. acid homeostasis as it relates to neuro- disorders, as well as the cardiac and transmitter function, and the devel- pulmonary problems of the patients with opment and maintenance of brain BIOLOGY OF GENETIC Ellis-van Creveld syndrome and the structure. MSUD arises from a complex DISORDERS immune and gastrointestinal disorders derangement of interorgan amino acid in patients with cartilage-hair hypopla- Our daily clinical work within a transport. sia. The early diagnosis of genetic population with high incidences of Our impression is that scientists syndromes and anticipation of predict- genetic diseases provides a unique who work as physicians and care for able problems is medically important opportunity to study the biology of several patients with the same genetic and often limits the adverse effects of genetic disorders. Because of the allelic disorder over long periods of time will the disorder on the life of the patient, homogeneity of many disorders among develop a different understanding of especially for metabolic disorders, but the Plain people, we observe a consis- genetic disease than scientists who study also for many other nonmetabolic tent, or at least much more limited, disease mechanisms only in the labora- syndromes. spectrum of variation at the primary tory. Laboratory scientists implicitly Immune deficiency states are disease-causing locus than do most assume that a genetic disease can be known to be associated with several of physicians. These observations have adequately characterized by molecular, the disorders that affect the Plain people. given us a different perspective on geno- enzymatic, biochemical, and cellular In infants with cartilage-hair hypoplasia, type-phenotype correlation. MSUD experiments that are done at one point we have encountered severe varicella exemplifies the complex relationship of in time and are independent of experi- infections, Pneumoncystis carinii pneumo- phenotype and genotype and, in addi- ence with an individual patient. Our nia, Hemophilus influenza meningitis in tion, the manner in which health care experience suggests that descriptions of an immunized 4-month-old, and persis- delivery affects clinical outcome. The disease mechanisms based on in vitro tent parvovirus infection with a chronic description of gene mutations is useful studies are necessarily incomplete and inflammatory disease similar to juve- for the diagnosis of many disorders, but may lead to an oversimplification of nile rheumatoid arthritis. Our young mutation analysis alone has seldom models of genetic disorders. MSUD, patients with Ellis-van Creveld dwarfism provided us with insight into the phe- GA1, and the majority of the other who have cardiac lesions and pulmonary notypic diversity that we encounter in disorders managed at the clinic can only hypoplasia are particularly vulnerable to our patients who share the same disease be understood in terms of the patho- infections with respiratory syncytial genotypes. Our understanding of varia- physiological mechanisms that unfold in virus. Our first encounter with the tions in the natural history of genetic individuals over significant periods of Amish-Mennonite variant of pro- disorders is more often based on the time and in relationship to many dif- pionic acidemia was an undiagnosed concepts and language of cell biology, ferent disease-causing factors, many of 12-month-old who presented with a pathophysiology, and critical periods of which are not genetic in origin. For viral respiratory tract infection and brain development than on specific GA1, MSUD, and PKU, the neurologi- developed acute striatal necrosis and a molecular lesions. MSUD provides a cal syndromes caused by single-gene dilated cardiomyopathy with endo- useful example of the complex relation- mutations arise from mechanisms of cardial fibroelastosis. Infants with the ship between gene and disease. Descrip- injury and dysfunction that are linked Mennonite variant of nephrotic syn- tions of mutations in branched-chain to critical periods of brain development. drome [Bolk et al., 1999] typically die keto-acid dehydrogenase as an under- In patients with GA1, episodes of from the immune deficiency state caused lying cause of MSUD do not help us metabolic illness that cause acute striatal by the loss of immune globulins and understand the multisystem regulators of necrosis in an infant are well tolerated in complement rather than from the loss of protein anabolism or catabolism that the 6-year-old or adult. Unbalanced albumin or renal failure. Gram-negative cause marked changes in leucine toler- amino transport that leads to mental sepsis must be anticipated in the ill ance. These perturbations of anabolism retardation in the infant with PKU and neonate with galactosemia or Hirsch- and catabolism are more important MSUD causes attention deficit disorder, sprung disease. Based on our experience influences on day-to-day metabolic mood disorders in older patients, and in Lancaster County, the practice of control and illness than are the under- presenile dementia in adults. The single- medical genetics cannot be separated lying mutations. Nor do the mutations gene mutations of MSUD and PKU give from general pediatrics, and much of our explain leucine-induced encephalopa- rise to diseases by disruptions of complex work in general pediatrics is focused thy, acute cerebral edema, or mental biological processes such as the meta- on the prevention and treatment of in- retardation. The biology of MSUD can bolic adaptations to fasts and illnesses, fectious illnesses. The disease course of only be understood in terms of the acute cell volume regulation, brain amino acid 16 AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) ARTICLE homeostasis, and postnatal organ growth Around the world, clusters of cases of Amish: Historical Perspective. Am J Med and development. Description of a GA1 caused by founder genes have been Genet 00:1–14. Gibson KM, Bennett MJ, Naylor EW, disease process without reference to such identified in Ireland, Spain, Israel, Saudi Morton DH. 1998. 3-Methylcrotonyl- biological processes and only by refer- Arabia, Chile, and two Native American coenzyme A carboxylase deficiency in ence to point mutations in genes or populations. Regardless of the under- Amish/Mennonite adults identified by de- tection of increased acylcarnitines in blood changes in enzyme activities seems to us, lying mutation, diagnosis and treatment spots of their children. J Pediatr 132:519– as clinicians, to be inadequate. Assump- of MSUD and GA1 before the onset of 523. tions about the nature of genetic disease cerebral edema are essential. The med- Goldstein JL, Brown MS. 1997. The clinical investigator: bewitched, bothered, and and the terms that are used to character- ical care of a patient with a genetic bewildered—but still beloved. J Clin Invest ize these disorders influence how phy- disorder is no different than the care of a 99:2803–2812. sician-scientists are trained and how, patient with other problems that are Higgins JJ, Morton DH, Loveless JM. 1999. Posterior column ataxia with retinitis pig- throughout their careers, these scientists medically complex, such as diabetes mentosa (AXPC1) maps to chromosome ask and answer questions. A recent mellitus, manic depressive illness, leu- 1q31-q32. Neurology 52:146–150. editorial in the Journal of Clinical Investi- kemia, congenital heart disease, renal Hostetler JA. 1993. Amish society. Baltimore: Johns Hopkins Press. p 1–435. gation indicated that research support in failure, rheumatoid arthritis, seizures, Hymel KP,Jenny C, Block RW.2002. Intracranial the past 20 years has overwhelmingly congenital and acquired immune defi- hemorrhage and rebleeding in suspected directed physician-scientists to the lab- ciency syndromes, drug dependency, victims of abusive head trauma: addressing the forensic controversies. Child Maltreat 7: oratory and away from patients, showing and many other chronic medical trou- 329–348. that 97% of physician-scientists who bles. Rare genetic disorders are like the Johnston JJ, Kelley RI, Crawford TO, Morton received the Howard Hughes clinical majority of diseases treated by physi- DH, Agarwala R, Koch T, Scha¨ffer AA, Francomano CA, Biesecker LG. 2000. A research awardees elected to do research cians: few curable diseases exist. None- novel nemaline myopathy in the Amish that did not involve patients [Goldstein theless, accessible and effective medical caused by a mutation in troponin T1. Am J and Brown, 1997]. Our experiences at care can limit suffering, improve func- Hum Genet 67:814–821. Kelley RI, Robinson D, Puffenberger EG, Strauss the clinic over the past 14 years suggest tion, reduce dependency, and otherwise KA, Morton DH. 2002. Amish lethal many important aspects of genetic dis- limit the effect of disease, genetic and microcephaly: a new metabolic disorder eases will only become apparent to acquired, on the life of a patient. William with severe congenital microcephaly and 2-ketoglutaric aciduria. Am J Med Genet physician-scientists who care for indivi- Osler said, ‘‘Such is our work.’’ 112:318–326. dual patients over long periods and who Martin J, Martin VM. 1995. What is wrong with are interested in patients, pathophysiol- our baby? Ephrata, PA: Grace Publishing. REFERENCES p 1–160. ogy, and biology in the most general McKusick VA. 1978. Medical genetic studies of sense. Baric I, Zschocke J, Christensen E, Duran M, the Amish. Selected papers. Baltimore: Goodman SI, Leonard JV, Muller E, Johns Hopkins University Press. p 1–525. 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Inherited 870. disorders in the Amish and Mennonite more common inherited metabolic dis- Francomano CA, McKusick VA, Biesecker L. demes of Pennsylvania. Am J Hum Genet orders [Strauss and Morton, 2003]. 2003. Medical Genetic Studies in the 71:A2436. ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS (SEMIN. MED. GENET.) 17

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