Outcome of the first 3-years of a DNA-based neonatal screening program for glutaric acidemia type 1 in Manitoba and northwestern Ontario, Canada.

Glutaric acidemia type 1 (GA1) is overrepresented in the aboriginal population of Island Lake, Manitoba, and northwestern Ontario who speak the Ojibway-Cree (Oji-Cree) dialect. The carrier frequency in these communities has been predicted to be as high as 1 in 10 individuals. Prior to beginning newborn screening for GA1 in May 1998, 18 of 20 affected patients diagnosed at this center have been from these high-risk communities. Most have followed an acute encephalopathic course with permanent neurologic sequelae and high mortality. They excrete small amounts of glutaric acid and 3-hydroxyglutaric acid and have significant residual enzyme activity. A single homozygous mutation in glutaryl-CoA-dehydrogenase (GCDH IVS-1 + 5g right arrow t) has been identified in this population. DNA-based newborn screening targeted to our high-risk communities was begun in order to provide presymptomatic detection and treatment of affected patients. Of the first 1176 newborns screened, 4 affected infants were identified and treated with a low-protein diet, carnitine, and riboflavin. All 4 infants have required numerous hospitalizations for treatment of intercurrent illnesses. Eventually, 3 infants presented with acute dystonic encephalopathy and seizures along with permanent neurological sequelae. One of these infants died unexpectedly at home at 18 months of age. The fourth, now 9 months old, has had a gastrostomy tube placed to facilitate fluid replacement in addition to a standard treatment protocol and is doing well. The reasons for our initial disappointing outcomes in the first 3 of 4 affected babies are likely multiple. Based on our early experience and that of other centers screening newborns for GA1, current therapeutic strategies may be insufficient in preventing the occurrence of neurologic sequelae in some children. An incomplete understanding of the neurotoxic mechanisms underlying this devastating disorder hampers effective management.

Celiac disease in a patient with a congenital deficiency of intestinal enteropeptidase.

We report on a 40-yr-old man with both primary enteropeptidase deficiency and celiac disease. He suffered from severe intestinal malabsorption and growth failure as a child. Enteropeptidase deficiency was found and pancreatic enzyme replacement therapy resulted in a growth spurt. Enteropeptidase levels in his intestinal mucosa and intraluminal fluid remained very low throughout childhood and early adult life. Celiac disease was confirmed by characteristic abnormalities in tests of intestinal function and in mucosal biopsies, which recovered when he instituted a gluten-free diet. He remains clinically intolerant to gluten as an adult. Enteropeptidase levels have remained abnormally low whether or not his intestinal mucosa has been normal in response to gluten restriction. Enteropeptidase levels have previously been shown to be normal in untreated celiac patients. The relationship between the two disorders remains unclear.

Hepatic carnitine palmitoyl transferase 1 (CPT1 A) deficiency in North American Hutterites (Canadian and American): evidence for a founder effect and results of a pilot study on a DNA-based newborn screening program.

We describe six patients with hepatic carnitine palmitoyl transferase (CPT1 A) deficiency who are members of a large extended Hutterite kindred living in widely scattered communities in the United States and Canadian Prairies. Two patients have significant neurological impairment due to severe recurrent hypoglycemic crises. The remaining four patients with earlier detection and treatment have near normal outcomes. The Canadian and American Hutterite families share two common ancestors who married in 1812, about 60 years before the Hutterites arrived in North America and prior to their subdivision into the three groups (Schmiedeleut, Dariusleut, and the Lehrerleut). These patients share a common haplotype on chromosome 11q13 and are all homozygous for a common CPT1 A G710E mutation, suggesting a founder effect. The clustering of such a rare disorder of fatty acid oxidation prompted us to initiate a pilot DNA-based neonatal screening program to determine the carrier frequency of this mutation in Hutterite newborns with the participation and support of the community. To date our carrier frequency is 1/16, close to the predicted frequency based on diagnosed patients and number of births. We believe our newborn screening program for CPT1 A deficiency in the Hutterite community will serve as a prototype model for delivery of targeted genetic services to other similar unique genetic isolates.

Molecular and enzymatic characterization of a unique carnitine palmitoyltransferase 1A mutation in the Hutterite community.

Hepatic carnitine palmitoyltransferase 1 (CPT1A) deficiency is a rare disorder of mitochondrial fatty acid oxidation inherited as an autosomal recessive trait. Symptomatology comprises attacks of hypoketotic hypoglycemia with risk of sudden death or neurological sequelae. Only one CPT1A mutation has been reported so far. Identification of the disease-causing mutations allows both insights into the structure-function relationships of CPT1A and management of the patients and their relatives. The molecular analysis of CPT1A deficiency in a large Hutterite kindred illustrates this point. Both cDNA and genomic DNA analysis demonstrate that the affected patients are homozygous for a 2129G>A mutation predicting a G710E substitution. Studies in fibroblasts from one patient as well as heterologous expression of the mutagenized CPT1A in yeast show that the G710E mutation alters neither mitochondrial targeting nor stability of the CPT1A protein. By contrast, kinetic studies conclusively establish that the mutant CPT1A is totally inactive, indicating that the G710E mutation dramatically impairs the catalytic function of CPT1A. Finally, due to a strongly suspected founder effect for the origin of CPT1A deficiency in this Hutterite kindred, identification of this disease-causing mutation allows the setup of a targeted DNA-based newborn screening in this at-risk population.

Amerindian pyruvate carboxylase deficiency is associated with two distinct missense mutations.

We characterized the pyruvate carboxylase (PC) gene by PCR amplification, subcloning, and sequencing. The coding region has 19 exons and 18 introns spanning approximately 16 kb of genomic DNA. Screening both the cDNA and the gene of individuals with the simple A form of PC deficiency revealed an 1828G-->A missense mutation in 11 Ojibwa and 2 Cree patients and a 2229G-->T transversion mutation in 2 brothers of Micmac origin. Carrier frequency may be as high as 1/10 in some groupings. The two point mutations are located in a region of homology conserved among yeast, rat, and human PC, in the vicinity of the carboxylation domain of the enzyme. These data provide the first characterization of the human PC gene structure, the identification of common pathogenic mutations, and the demonstration of a founder effect in the Ojibwa and Cree patients.

Two novel mutations in a Canadian family with aspartylglucosaminuria and early outcome post bone marrow transplantation.

Aspartylglucosaminuria (AGU) is a lysosomal storage disease caused by deficiency of aspartylglucosaminidase. The disease is overrepresented in the Finnish population, in which one missense mutation (Cys163Ser) is responsible for 98% of the disease alleles. The few non-Finnish cases of AGU which have been analyzed at molecular level have revealed a spectrum of different mutations. Here, we report two new missense mutations causing AGU in two Canadian siblings. The patients were compound heterozygotes with a G299-->A transition causing a Gly100-->Gln substitution and a T404-->C transition resulting in a Phe135-->Ser change in the cDNA coding for aspartylglucosaminidase. The younger patient recently underwent bone marrow transplantation.

Blood lactate and pyruvate concentrations, and their ratio during exercise in healthy children: developmental perspective.

Blood concentrations of lactate normally increase during and after intense exercise as does the ratio of concentrations of lactate to pyruvate (L:P). Since there appear to be differences in blood lactate concentrations on exercise, in muscle metabolic enzyme activities, and in anaerobic capacity between children and adults, we speculated that there would be age related differences in lactate and pyruvate concentrations, and their ratio among children. Whole blood concentrations of lactate and pyruvate were measured in 28 healthy children aged 7-17 years, split into three age groups: less than 11, 11-14, and 15-17 years. Blood was drawn at rest, immediately after 6 min of exercise at one-third and two-thirds of maximum work capacity (Wmax), and 20 min after completion of work. Lactate and pyruvate concentrations increased significantly from rest to exercise at two-thirds Wmax [approximately 72% of peak oxygen consumption (VO2peak)]. Whereas greater increments in lactate concentration were seen with groups of increasing age, exercise-related increments in pyruvate concentrations were no different among age groups. There was a significant rise in L:P ratio on exercise, with greater increments found from the youngest to the oldest group. There were no sex differences. We concluded that in healthy children exercising at approximately 70% of VO2peak there is a rise in blood lactate concentration in excess of that of pyruvate, such that the L:P ratio rises to a degree determined by age. This suggests age dependent changes, perhaps coincident with puberty, in pathways involved in lactate production and/or elimination.

Glycogen storage disease in adults.

OBJECTIVE: To identify complications amenable to prevention in adults with glycogen storage disease (GSD) types Ia, Ib, and III and to determine the effect of the disease on social factors. DESIGN: Case series and clinical review. SETTING: Referral medical centers in the United States and Canada. PATIENTS: All patients with GSD-Ia (37 patients), GSD-Ib (5 patients), and GSD-III (9 patients) who were 18 years of age or older. MEASUREMENTS: Ultrasound or radiographic studies identified liver adenomas, nephrocalcinosis, or kidney stones. Radiographic studies identified osteopenia. Reports of the clinical examination, serum chemistry results, and social data were obtained. RESULTS: For patients with GSD-Ia, problems included short stature (90%), hepatomegaly (100%), hepatic adenomas (75%), anemia (81%), proteinuria or microalbuminuria (67%), kidney calcifications (65%), osteopenia or fractures or both (27%), increased alkaline phosphatase (61%) and gamma-glutamyltransferase (93%) activities, and increased serum cholesterol (76%) and triglyceride (100%) levels. Hyperuricemia was frequent (89%). Patients with GSD-Ib had severe recurrent bacterial infections and gingivitis. In patients with GSD-III, 67% (6 of 9) had increased creatinine kinase activity. Four of these patients had myopathy and cardiomyopathy. CONCLUSIONS: For GSD-Ia, hyperuricemia and pyelonephritis should be treated to prevent nephrocalcinosis and additional renal damage. For GSD-Ib, granulocyte-colony-stimulating factor may prevent bacterial infections. For GSD-III, more data are required to determine whether the myopathy and cardiomyopathy can be prevented. Most of the patients with GSD-I and GSD-III had 12 or more years of education and were either currently in school or employed.

Vitamin D deficiency in a Manitoba community.

OBJECTIVE: Using a cross-sectional survey, to investigate the vitamin D status of a random sample of 80 mother-child pairs (child age 3-24 months) in a Manitoba community with a high incidence of rickets. METHOD: A questionnaire on feeding habits, gestational history, maternal diet and vitamin supplements was administered to mothers in their homes with the assistance of a local interpreter. Venous blood was collected from both mother and child for serum 25-hydroxyvitamin D levels. RESULTS: Of 91% babies initially breastfed, 36% received no formula or milk after weaning and 40% received no vitamin supplements. 24% of mothers took no vitamin supplements during pregnancy and lactation. Knowledge about rickets was poor. In 43% of children and 76% of mothers, serum 25-hydroxyvitamin D levels were below normal range. CONCLUSIONS: Vitamin D levels are low in this population due to lack of fortified dairy products and vitamin D supplements. A public health program should include counseling on rickets and vitamin D supplementation for all infants and pregnant or lactating women.

PIP: In the isolated Island Lake area of northern Manitoba, which has a high incidence of rickets, interviews were conducted with 80 mothers, each with a child at least 2 years old, living in St. Theresa Point and Garden Hill in their homes during June-July 1987 to determine their knowledge and attitudes towards rickets. Nurses obtained blood samples from the women and their young children so the researchers could determine the vitamin D status of both. The mother-child pairs were native Canadians from the Ojibway linguistic group that speaks its own dialect of Ojibway-Cree. Mothers initially breast fed 91% of the children. After weaning, 1/3 of infants received neither infant formula nor milk. No vitamin supplements were given to 40%. Many of the children who did receive vitamin supplements did not receive them regularly. 70% of the mothers did not drink any milk. 24% were milk-intolerant. 24% took no vitamin supplements during pregnancy and lactation. Mothers who did take supplements did not do so regularly. 17% claimed that their skin was sensitive to sunlight. 84% of mothers in one community had never heard of rickets. Most did not know its cause. Neither mothers nor the children were exposed to the sunlight in the summer. When outside, almost all small infants were completely covered to protect them from the elements. The mean 25-hydroxy-vitamin D level was 26.2 nmol/l for the children and 19.8 nmol/l for the mothers. 43% of children and 76% of mothers had a 25-hydroxy-vitamin D level below the normal range. These high levels of vitamin D insufficiency were even more troublesome given that the blood was taken in late June and July when vitamin D levels would be likely to be at their highest. The dearth of vitamin D fortified dairy products and vitamin supplements greatly contributed to the low level of vitamin D status in this area. The findings show a need for public health officials to include education on rickets and vitamin D supplementation for all infants and pregnant or lactating women.

A homoallelic Gly317-->Asp mutation in ALPL causes the perinatal (lethal) form of hypophosphatasia in Canadian mennonites.

We have discovered a single homoallelic nucleotide substitution as the putative cause of the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Previous linkage and haplotype analysis in this population suggested that a single mutational event was responsible for this autosomal recessive form of hypophosphatasia. The mutation is a guanosine-to-adenosine substitution at nucleotide position 1177 in exon 10 of the tissue nonspecific (liver/bone/kidney) alkaline phosphatase gene. This Gly317-->Asp mutation segregates exclusively with the heterozygote phenotype we previously assigned by biochemical testing (maximum combined lod score of 18.24 at theta = 0.00). This putative disease-causing mutation has not been described in controls nor in other non-Mennonite probands with both lethal and nonlethal forms of hypophosphatasia studied to date. This Gly317-->Asp mutation changes a polar glycine to an acidic aspartate at amino acid position 317 within the highly conserved active site region of the 507-amino-acid polypeptide. Carrier screening for this lethal mutation in our high-risk population is now feasible.

Symptomatic and asymptomatic methylenetetrahydrofolate reductase deficiency in two adult brothers.

We describe two brothers with 5,10-methylene tetrahydrofolate reductase (MTHFR) deficiency. The younger patient first developed limb weakness, incoordination, paresthesiae, and memory lapses at age 15 years, and by his early twenties he was wheelchair bound. His older brother remains asymptomatic at age 37 years. Both had homocystinuria and homocystinemia and low plasma levels of methionine. MTHFR activities in cultured skin fibroblasts of both patients were < 10% control and residual enzyme activities were markedly reduced on heating. The parents had intermediate enzyme activities and the reductase in the father (who had unexplained paraparesis and homocystinemia), but not in the mother, was also thermolabile. Both patients were treated with oral folate and betaine which improved, but did not totally correct, their biochemical abnormality. MTHFR deficiency should be considered in the differential diagnosis of unexplained neurologic disease in adolescents and adults.

Atypical features of the hepatic form of carnitine palmitoyltransferase deficiency in a Hutterite family.

We describe hepatic carnitine palmitoyltransferase (CPT I) deficiency in three children (a brother and sister and their second cousin) from an extended inbred Hutterite kindred. The patients were first seen between 8 and 18 months of age with recurrent episodes of hypoketotic hypoglycemia accompanied by a decreased level of consciousness and hepatomegaly. One patient had two Reye syndrome-like episodes. Abnormal organic acids were rarely detected in urine. Serum total and free carnitine levels were elevated in all three patients. Fibroblast acyl-coenzyme A dehydrogenase activities were normal in all, but palmitic acid oxidation, performed in fibroblasts from one patient, was less than 10% of control values. Activity of CPT I in cultured skin fibroblasts from the three patients was 10% to 15% of control levels; CPT II activity was normal. Activity of CPT I and CPT II in muscle from one patient was normal. Atypical features in two of these patients were greatly elevated levels of liver enzymes and creatine kinase during acute episodes. The patients have recently been successfully treated with medium-chain triglycerides and avoidance of fasting. Early identification and treatment of this disorder may avert potentially fatal episodes of hypoglycemia.

Carrier detection in glutaric aciduria type I using interleukin-2-dependent cultured lymphocytes.

Cultured interleukin 2 (IL-2)-dependent leukocytes from 13 patients with glutaric aciduria type I, 12 obligate carriers, 105 family members and 31 normal controls were assayed for glutaryl-CoA dehydrogenase activity. Of the 13 affected patients, 10 (all Ojibway Indian) had residual enzyme activity (2-13% of control) and 3 patients (all non-Indian) had undetectable enzyme activity. There was partial overlap between the distribution of enzyme activity in obligate heterozygotes and in normal controls (mean values +/- SD: 6.29 +/- 0.94 and 10.75 +/- 2.58 nmol/h per mg protein respectively). Using an arbitrary cutoff level of < 7 nmol/h per mg protein as presumptive evidence of carrier status, the observed frequency of carriers did not differ significantly from that expected from their a priori risk of carrier status. Thirteen per cent of the family members had inconclusive status (activity between 7 and 8.5 nmol/h per mg protein). The method appears suitable for carrier detection, although definitive carrier assignment awaits identification of the mutation(s) responsible for glutaric aciduria type I.

Increased prevalence of hereditary metabolic diseases among native Indians in Manitoba and northwestern Ontario.

OBJECTIVE: To compare the prevalence of hereditary metabolic diseases in the native and non-native populations of Manitoba and northwestern Ontario. DESIGN: Retrospective analysis. SETTING: Children's Hospital, Winnipeg. PATIENTS: Patients were selected by three methods: laboratory tests designed to screen patients suspected of having a metabolic disease, laboratory investigation of newborn infants with abnormalities detected through screening, and investigation of near relatives of probands with disease. RESULTS: A total of 138 patients with organic acid, amino acid and carbohydrate disorders were seen from 1960 to 1990. Of these, 49 (36%) were native Indians (Algonkian linguistic group). This was in sharp contrast to the proportion of native Indians in the total study population (5.8%). Congenital lactic acidosis due to pyruvate carboxylase deficiency (13 patients), glutaric aciduria type I (14 patients) and primary hyperoxaluria type II (8 patients) were the most common disorders detected. Other rare disorders included glutaric aciduria type II (one patient), 2-hydroxyglutaric aciduria (one patient) and sarcosinemia (one patient). Underreporting, especially of glutaric aciduria type I and hyperoxaluria type II, was likely in the native population. CONCLUSIONS: Hereditary metabolic diseases are greatly overrepresented in the native population of Manitoba and northwestern Ontario. We recommend that native children who present with illnesses involving disturbances of acid-base balance or with neurologic, renal or liver disease of unknown cause by investigated for a possible metabolic disorder.