Lorne E. Seargeant

Phenotypic variability in glutaric aciduria type I: Report of fourteen cases in five Canadian Indian kindreds

We describe 14 patients with glutaric aciduria type 1 in five Canadian Indian kindreds living in Manitoba and northwest Ontario. The patients had marked clinical variability of the disease, even within families. Eight followed the typical clinical course of normal early growth and development until the onset of neurologic abnormalities, often precipitated by infection, between 6 weeks and 7 1/2 months of age. Five patients had early developmental delay; one was thought to be normal until 8 years of age. Three patients died, seven are severely mentally and physically handicapped, and four have only mild mental retardation or incoordination. Six patients had macrocephaly in the neonatal period. Computed tomography was done for 12 patients, and findings were abnormal in 11. Glutaric acid and 3-hydroxyglutaric acid were detected in increased amounts in the urine of all patients, but the concentrations were much lower than those in most other reported patients. Glutaryl coenzyme A dehydrogenase activity in skin fibroblasts, interleukin-2-dependent lymphocytes, or both, ranged from 0% to 13% of control values. There was no correlation between clinical severity and urine glutaric acid concentration or level of residual enzyme activity. We recommend that organic acid analysis of the urine be done in patients with unexplained cerebral palsy-like disorders, especially if the computed tomographic scan is abnormal. If there is suspicion of glutaric aciduria, glutaryl-coenzyme A dehydrogenase should be measured in fibroblasts or lymphocytes even if glutaric acid is not increased in the urine.

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.

The paradox of the carnitine palmitoyltransferase type Ia P479L variant in Canadian Aboriginal populations

Investigation of seven patients from three families suspected of a fatty acid oxidation defect showed mean CPT-I enzyme activity of 5.9+/-4.9 percent of normal controls. The families, two Inuit, one First Nation, live in areas of Canada geographically very distant from each other. The CPT1 and CPT2 genes were fully sequenced in 5 of the patients. All were homozygous for the same P479L mutation in a highly conserved region of the CPT1 gene. Two patients from the first family were also homozygous for the CPT2 F352C polymorphism in the CPT2 gene. Genotyping the patients and their family members confirmed that all seven patients were homozygous for the P479L variant allele in the CPT1 gene, as were 27 of 32 family members. Three of the seven patients and two cousins had hypoketotic hypoglycemia attributable to CPT-Ia deficiency, but adults homozygous for the variant denied hypoglycemia. We screened 422 consecutive newborns from the region of one of the Inuit families for this variant; 294 were homozygous, 103 heterozygous, and only 25 homozygous normal; thus the frequency of this variant allele is 0.81. There was an infant death in one family and at least 10 more deaths in those infants (7 homozygous, 3 heterozygous) consecutively tested for the mutation at birth. Thus there is an astonishingly high frequency of CPT1 P479L variant and, judging from the enzyme analysis in the seven patients, also CPT-I deficiency in the areas of Canada inhabited by these families. Despite the deficiency of CPT-Ia which is the major rate-limiting enzyme for long chain fatty acid oxidation, clinical effects, with few exceptions, were slight or absent. One clue to explaining this paradox is that, judging from the fatty acid oxidation studies in whole blood and fibroblasts, the low residual activity of CPT-Ia is sufficient to allow a reasonable flux through the mitochondrial oxidation system. It is likely that the P479L variant is of ancient origin and presumably its preservation must have conveyed some advantage.

CDG‐IL: An infant with a novel mutation in the ALG9 gene and additional phenotypic features

We describe the second case of congenital disorder of glycosylation type IL (CDG‐IL) caused by deficiency of the ALG9 a1,2 mannosyltransferase enzyme. The female infants features included psychomotor retardation, seizures, hypotonia, diffuse brain atrophy with delayed myelination, failure to thrive, pericardial effusion, cystic renal disease, hepatosplenomegaly, esotropia, and inverted nipples. Lipodystrophy and dysmorphic facial features were absent. Magnetic resonance imaging of the brain showed volume loss in the cerebral hemispheres and cerebellum and delayed myelination. Laboratory investigations revealed low levels of multiple serum proteins including antithrombin III, factor XI, and cholesterol. Hypoglycosylation was confirmed by the typical CDG type 1 pattern of serum transferrin analyzed by isoelectric focusing. A defect in the ALG9 enzyme was suggested by the accumulation of the DolPP‐GlcNAc2Man6 and DolPP‐GlcNAc2Man8 in the patients fibroblasts and confirmed by mutation analysis: the patient is homozygous for the ALG9 mutation p.Y286C. The causal effect of the mutation was shown by complementation assays in alg9 deficient yeast cells. The child described here further delineates the clinical spectrum of CDG‐IL and confirms the significant clinical overlap amongst CDG subtypes.

Screening for congenital adrenal hyperplasia: Distribution of 17α-hydroxyprogesterone concentrations in neonatal blood spot specimens

In a retrospective analysis of 24 cases of congenital adrenal hyperplasia (CAH) in neonates born in the province of Manitoba during the last 20 years, we set out to determine whether patients, in particular male infants with salt-losing CAH, were being missed by the usual forms of clinical ascertainment. Although the overall incidence of 1/14,500 live births was similar to that found in several screening surveys, a skewed female/male sex ratio of 2.2:1 suggested probable death among male infants with unrecognized adrenal insufficiency. These results led to a prospective analysis of 17 alpha-hydroxyprogesterone (17-OHP) levels in 1194 neonatal blood specimens by a solid-phase direct radioimmunoassay procedure to determine whether this method would be suitable for CAH screening. In 1103 neonates weighing greater than 2500 gm at birth, all 17-OHP values were less than 30 nmol/L (approximately 1000 ng/dl), with a mean of 8.2 nmol/L; values in male infants were slightly higher than in female infants. In 89 neonates with a birth weight less than 2500 gm, 17-OHP values were skewed, with nine having levels greater than 30 nmol/L and two greater than 50 nmol/L. Postnatal age (1 to 24 days) at the time of specimen collection had no effect on 17-OHP levels, although higher values occur during the first 24 hours. One unsuspected case of CAH in a male infant was discovered during the trial period. We conclude that neonatal CAH screening can permit diagnosis and therapy of affected male infants who are being missed by normal clinical evaluation. This radioimmunoassay method is relatively simple and inexpensive, and it has the specificity and sensitivity necessary to provide such mass screening.

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.

Infantile hypophosphatasia—Linkage with the RH locus

Linkage analysis of six nuclear families with infantile hypophosphatasia which were informative for the Rh blood group locus was performed. The maximum combined lod score was 4.76 with the recombinant distance (theta) of 0.04. These preliminary data provide evidence for linkage between the genes for infantile hypophosphatasia and the Rh blood group and provisionally assign the gene locus for infantile hypophosphatasia (designated HOPS) to chromosome 1p.

Increased plasma pyridoxal-5′-phosphate levels before and after pyridoxine loading in carriers of perinatal/infantile hypophosphatasia

SummaryWe measured plasma levels of pyridoxal-5′-phosphate (PLP), a cofactor form of vitamin B6 and apparent natural substrate for alkaline phosphatase (ALP), in carriers and in non-carriers of the severe perinatal and infantile forms of hypophosphatasia, both before and after an oral load of pyridoxine (i.e. 1/3 mg/kg body weight). The assignment of carrier status was determined by serum ALP activity, level of serum inorganic phosphate, and if necessary urinary phosphoethanolamine excretion. Plasma PLP levels were significantly increased in the carriers both before and especially after B6 loading.

A simple screening test for fatty acid oxidation defects using whole-blood palmitate oxidation

We report that measurement of whole-blood palmitate oxidation is a rapid and inexpensive screening test for fatty acid oxidation defects. The assay has been adapted from published assays using cultured fibroblasts or isolated white blood cells. Micro whole-blood samples are incubated with tritiated palmitic acid as substrate. The tritiated water produced is proportional to the mitochondrial β-oxidation of palmitic acid. Patients with confirmed β-oxidation defects show low whole-blood palmitate oxidation.

A rare case of Niemann–Pick disease type C without neurological involvement in a 66-year-old patient

The case of a 66 year-old female — the oldest known living patient with Niemann–Pick disease type C (NP-C) who remains free of any neurological or psychiatric manifestations 18 years after presentation — is presented. An incidental finding of massive splenomegaly was detected during a routine pelvic ultrasound. The pathology report after splenectomy showed the presence of lipid-laden macrophages. Fibroblasts cultured in LDL-enriched medium revealed abnormal filipin staining consistent with cholesterol-filled vesicles and the rate of cholesterol esterification in response to stimulation of LDL-cholesterol uptake was significantly depressed at 6% of that seen in cells from normal controls, but at a level similar to that observed in an NP-C positive control. Molecular genetic testing later revealed a compound heterozygous mutant NP-C genotype comprising two previously described disease-causing mutations in the NPC1 gene, one in exon 8 (c.1133T>C [V378A]) and one in exon 13 (c.1990G>A [V664M]). These findings confirmed the diagnosis of NP-C. Only three patients with this disorder aged > 53 years have previously been reported, all of whom presented with neurological or neuropsychiatric manifestations. Our patient is the first reported NP-C patient, now in her seventh decade of life, who has to date only manifested splenomegaly. This case highlights the extreme clinical variability of NP-C, and the need to consider this disease in the differential diagnosis of organomegaly, even in the absence of neurological, psychiatric and related clinical signs. Synopsis An elderly female patient with confirmed NP-C and isolated splenomegaly has remained asymptomatic for neurological, cognitive, psychiatric or ophthalmologic abnormailities into her seventh decade of life.