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Background: RNA polymerase III-related or 4H leukodystrophy (POLR3-HLD) is an autosomal recessive hypomyelinating leukodystrophy characterized by neurological dysfunction, hypodontia and hypogonadotropic hypogonadism. The disease is caused by biallelic pathogenic variants in POLR3A , POLR3B , POLR1C or POLR3K . Craniofacial abnormalities reminiscent of Treacher Collins syndrome have been originally described in patients with POLR3-HLD caused by biallelic pathogenic variants in POLR1C . To date, no published studies have appraised in detail the craniofacial features of patients with POLR3-HLD. In this work, the specific craniofacial characteristics of patients with POLR3-HLD associated with biallelic pathogenic variants in POLR3A , POLR3B and POLR1C are described., Methods: The craniofacial features of 31 patients with POLR3-HLD were evaluated, and potential genotype-phenotype associations were evaluated., Results: Various craniofacial abnormalities were recognized in this patient cohort, with each individual presenting at least one craniofacial abnormality. The most frequently identified features included a flat midface (61.3%), a smooth philtrum (58.0%) and a pointed chin (51.6%). In patients with POLR3B biallelic variants, a thin upper lip was frequent. Craniofacial anomalies involving the forehead were most commonly associated with biallelic variants in POLR3A and POLR3B while a higher proportion of patients with POLR1C biallelic variants demonstrated bitemporal narrowing., Conclusion: Through this study, we demonstrated that craniofacial abnormalities are common in patients with POLR3-HLD. This report describes in detail the dysmorphic features of POLR3-HLD associated with biallelic variants in POLR3A , POLR3B and POLR1C ., Competing Interests: Competing interests: LTT currently manages sponsored clinical trials at the site level for Ionis Pharmaceuticals (Alexander disease clinical trial 2021–present), Passage Bio (Krabbe disease and GM1 gangliosidosis clinical trials, 2021–present) and Teva Pharmaceuticals (chronic and episodic migraine clinical trials, 2022–present). He also manages a GM1 gangliosidosis natural history study sponsored by the University of Pennsylvania with funding from Passage Bio. NIW is advisor and/or co-investigator for trials in Metachromatic Leukodystrophy (Shire/Takeda, Orchard, Evidera) and other leukodystrophies (Ionis, PassageBio, Vigil Neuro, Sana Biotech), without personal payment. AV receives research grants or in-kind research support without any personal compensation from Takeda, Passage Bio, Sanofi, Affynia, Orchard Therapeutics, Eli Lilly, ISD therapeutics, Illumina, Myrtelle, Homology, Sana and Ionis. She is a site investigator for the Ionis clinical trial in Alexander’s disease, SHP611 in Metachromatic leukodystrophy of Shire/Takeda and Passage Bio gene therapy in Krabbe. She serves on the scientific advisory board of the ELA foundation, the ULF Foundation and the Yaya Foundation Scientific and Clinical Advisory Council. She is a member of the Vanishing White Matter Consortium, the H-ABC Clinical Advisory Board. She receives grant funding from this RDCRN NCATS/NINDS (U01 NS106845, U54TR002823 and R21 NS123477. GB is/was a consultant for Passage Bio Inc (2020–2022) and Ionis (2019). She is/was a site investigator for the Alexander’s disease trial of Ionis (2021–present), Metachromatic leukodystrophy of Shire/Takeda (2020–2021), Krabbe and GM1 gene therapy trials of Passage Bio (2021–present), GM1 natural history study sponsored by the University of Pennsylvania with funding from Passage Bio (2021–present) and Adrenoleukodystrophy/Hematopoietic stem cell transplantation natural history study of Bluebird Bio (2019), a site sub-investigator for the MPS II gene therapy trial of Regenxbio (2021–present) and the MPS II clinical trial of Denali (2022–present). She has received an unrestricted educational grant from Takeda (2021–2022). She serves on the scientific advisory board of the Pelizaeus-Merzbacher Foundation, the Yaya Foundation Scientific and Clinical Advisory Council and is the Chair of the Medical and Scientific Advisory Board of the United Leukodystrophy Foundation. She is a member of the Vanishing White Matter Consortium, the H-ABC Clinical Advisory Board and the Chair of the POLR3-related (4H) Leukodystrophy Consortium. She is on the editorial boards of Neurology Genetics, Frontiers in Neurology – Neurogenetics and Journal of Medical Genetics., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Three deceased infants from a Pakistani consanguineous family presented with a similar phenotype of cholestatic liver disease, hypotonia, severe failure to thrive, recurrent vomiting, renal tubulopathy, and a progressive neurodegenerative course. Mitochondrial DNA depletion syndrome was considered in view of multisystem involvement. Exome sequencing, revealed a homozygous novel mutation c.1183T>C (p.F395L) in exon 1 of the C10orf2 TWINKLE gene. The hepatocerebral phenotype is well recognized in association with recessive mutations involving the C10orf2 TWINKLE gene. The feature of renal tubulopathy adds to the multisystemic presentation in our patients and further demonstrates an expansion of the phenotype in mitochondrial DNA depletion syndrome associated with TWINKLE gene mutations. The absence of features of an epileptic encephalopathy appears to be of added interest., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Fabry disease is a complex, multisystemic and clinically heterogeneous disease, in which the urinary excretion of globotriaosylceramide (Gb3), the principal substrate of the deficient enzyme, alpha-galactosidase A, is more prominent than the increased concentrations of the lipid in the plasma of affected hemizygotes and heterozygotes. We have developed and validated a simultaneous analysis of Gb3 and creatinine in a 2.6-min run using filter paper discs saturated with urine and analyzed by LC-MS/MS. Using this method, we studied the relationship between urinary levels of total Gb3/creatinine excretion and four types of mutations in the GLA gene (missense, nonsense, frameshift, and splice-site defects) in 32 children and 78 adult patients with Fabry disease. Forty-one patients were treated by enzyme replacement therapy and 69 were untreated. Our results show that the mean recoveries of Gb3 and creatinine from the urine filter paper standards were 91% and 97%, respectively, with precision, reproducibility, and linearity within acceptable ranges. Statistical analysis using the independent variables of sex, age, types of mutations and treatment showed that the mutation factor has a statistically significant impact on urinary Gb3 excretion (p = 0.0007). This means that the levels of urinary excretion of Gb3/creatinine in children and adults with Fabry disease are directly related to the types of mutations. The same correlation was found for the sex (p < 0.0001) and treatment (p = 0.0011). In conclusion, we studied 35 mutations in 110 children and adults with Fabry disease and found a significant correlation between the types of mutations and total Gb3 excretion in Fabry patients.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is an early onset neurodegenerative disease with high prevalence (carrier frequency 1/22) in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec. We previously mapped the gene responsible for ARSACS to chromosome 13q11 and identified two ancestral haplotypes. Here we report the cloning of this gene, SACS, which encodes the protein sacsin. The ORF of SACS is 11,487 bp and is encoded by a single gigantic exon spanning 12,794 bp. This exon is the largest to be identified in any vertebrate organism. The ORF is conserved in human and mouse. The putative protein contains three large segments with sequence similarity to each other and to the predicted protein of an Arabidopsis thaliana ORF. The presence of heat-shock domains suggests a function for sacsin in chaperone-mediated protein folding. SACS is expressed in a variety of tissues, including the central nervous system. We identified two SACSmutations in ARSACS families that lead to protein truncation, consistent with haplotype analysis.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is a neurodegenerative disease frequent in northeastern Québec. In a previous study, we localized the disease gene to chromosome region 13q11 by identifying excess sharing of a marker allele in patients followed by linkage analysis and haplotyping. To create a detailed physical map of this region, we screened CEPH mega-YACs with 41 chromosome 13 sequence-tagged-sites (STSs) known to map to 13q11-q12. The YAC contig, composed of 27 clones, extends on the genetic map from D13S175 to D13S221, an estimated distance of at least 19.3 cM. A high-resolution BAC and PAC map that includes the ARSACS critical region flanked by D13S1275 and D13S292 was constructed. These YAC and BAC/PAC maps allowed the accurate placement of 29 genes and ESTs previously mapped to the proximal region of chromosome 13q. We confirmed the position of two candidate genes within the critical region and mapped the other 27 genes and ESTs to nearby intervals. Six BAC/PAC clones form a contig between D13S232 and D13S787 for sequencing within the ARSACS critical region., (Copyright 1999 Academic Press.)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a clinically homogeneous form of early-onset familial spastic ataxia with prominent myelinated retinal nerve fibers. More than 300 patients have been identified, and most of their families originated in the Charlevoix-Saguenay region of northeastern Quebec, where the carrier prevalence has been estimated to be 1/22. Consistent with the hypothesis of a founder effect, we observed excess shared homozygosity at 13q11, among patients in a genomewide scan of 12 families. Analysis of 19 pedigrees demonstrated very tight linkage between the ARSACS locus and an intragenic polymorphism of the gamma-sarcoglycan (SGCG) gene, but genomic DNA sequence analysis of all eight exons of SGCG revealed no disease-causing mutation. On the basis of haplotypes composed of seven marker loci that spanned 11.1 cM, the most likely position of the ARSACS locus was 0.42 cM distal to the SGCG polymorphism. Two groups of ARSACS-associated haplotypes were identified: a large group that carries a common SGCG allele and a small group that carries a rare SGCG allele. The haplotype groups do not appear to be closely related. Therefore, although chromosomes within each haplotype group may harbor a single ARSACS mutation identical by descent, the two mutations could have independent origins.

Infantile free sialic acid storage disease (ISSD) is a rare autosomal recessive metabolic disorder caused by a lysosomal membrane transport defect, resulting in accumulation of free sialic acid within lysosomes. Only a few cases have been described. We report on three new cases of ISSD with different modes of presentation: an infant with nephrotic syndrome, a case of fetal and neonatal ascites with heart failure, and a case of fetal ascites with esophageal atresia type III. From these patients and a review of the literature (27 cases total) we draw the following conclusions. 1) "Coarse facies," fair complexion, hepatosplenomegaly, and severe psychomotor retardation are constant findings in this disorder. 2) Nephrotic syndrome occurred in most cases (four in seven) in which renal evaluation was performed. Therefore, ISSD is an important cause of nephrosis in infants with a storage disorder phenotype. 3) Fetal/neonatal ascites or hydrops was the mode of presentation in 13 (60%) of 21 cases. Thus, ISSD enters in the differential diagnosis of hydrops fetalis with a storage disease phenotype. 4) Cardiomegaly was evident in nine cases. 5) Corneae were always clear, and albinoid fundi were reported in five cases. 6) Dysostosis multiplex was not prominent. 7) Bone marrow aspiration could be negative. 8) Death ensued in early infancy with a mean age of 13.1 months. All reported deaths were caused by respiratory infections.

A form of autosomal recessive spastic ataxia unique to the Charlevoix-Saguenay area was clinically identified 20 years ago in patients from that region. This region of Québec, Canada, was once considered a genetic isolate. First noted at gait initiation, signs of ataxia slowly progress along with spasticity of the four limbs, slurred speech, and followed by distal amyotrophy. Early diagnosis relies on the presence of prominent myelinated fibers embedding retinal blood vessels at funduscopy and marked saccadic alteration of ocular smooth pursuit. Imaging of the posterior fossa shows cerebellar vermis atrophy and nerve conduction studies reveal loss of sensory and reduced motor conduction velocities. The clinical features are consistent with a developmental defect in myelination of both retinal and peripheral nerve fibers. The cause of this defect and the progressive axonal degeneration in the corticospinal and spinocerebellar tracts, as well as in the peripheral nerves is still unknown. Results of recent molecular genetic linkage analysis have located the gene locus to chromosome 13q12. Further research is needed to define where this hereditary spastic ataxia stands in the classification of the early onset spinocerebellar degenerations.

We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreich's Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.

We studied genotype-phenotype correlations in a group of 100 patients with typical Friedreich ataxia (FRDA), and in three groups of patients with atypical clinical presentations, including 44 Acadian FRDA, 8 late-onset FRDA (LOFA), and 6 FRDA with retained reflexes (FARR). All patients, except 3 with typical FRDA, carried two copies of the FRDA-associated GAA triplet repeat expansion. Overall, the phenotypic spectrum of FRDA appeared to be wider than defined by the currently used diagnostic criteria. Our study indicated the existence of several sources of variability in FRDA. Patients with larger GAA expansions tended to have earlier onset and were more likely to show additional manifestations of the disease. Mitotic instability of the expanded GAA repeats may partially account for the limited degree of correlation between expansion sizes as determined in lymphocytes and clinical parameters. Some clinical variants associated with specific FRDA haplotypes, such as Acadian FRDA and FARR, turned out to be unrelated to expansion sizes. No polymorphism in the frataxin coding sequence could be associated with these clinical variants.

The gene for Friedreich ataxia (FRDA), an autosomal-recessive neurodegenerative disease, remains elusive. The current candidate region of about 150 kb lies between loci FR2 and F8101 near the D9S15/D9S5 linkage group at 9q13-21.1. Linkage homogeneity between classical FRDA and a milder, slowly progressive Acadian variant (FRDA-Acad) has been demonstrated. An extended D9S15-D9S5 haplotype (C6) predominates in FRDA-Acad chromosomes from Louisiana. We studied 10 Acadian families from New Brunswick, Canada. In eight families, affected individuals conformed to the clinical description of FRDA-Acad; in one, 2 sibs presented with spastic ataxia (SPA-Acad). In the last family, 2 sibs had FRDA-Acad, and one had SPA-Acad. We found that SPA-Acad is linked to the FRDA gene region. The C6 haplotype and a second major haplotype (B7) were identified. The same ataxia-linked haplotypes segregated with both FRDA-Acad and SPA-Acad in two unrelated families. The parental origins of these haplotypes were different. Our observation of different phenotypes associated with the same combination of haplotypes may point to the influence of the parent of origin on gene expression, indicate the effect of modifier genes, or reflect the presence of different mutations on the same haplotypes. Our findings underline the need to investigate families with autosomal-recessive ataxias for linkage to the FRDA region, despite lack of key diagnostic manifestations such as cardiomyopathy or absent deep-tendon reflexes.

Sodium benzoate (SB) therapy is known to increase ammonia (NH3) nitrogen elimination via conjugation with glycine and excretion as urinary hippurate. In 16 children with inborn errors of urea synthesis we studied two issues: (1) the effect of chronic SB administration upon carnitine metabolism and (2) the efficacy of chronic SB therapy as measured by the molar ratio of hippurate excretion to SB intake. Measurements were performed during elective hospitalizations when the patients were in stable metabolic condition. We found that chronic SB therapy is not associated with a constant level of hippurate elimination and that interindividual and intraindividual variability may result in irregular removal of NH3 nitrogen. This variability may be due to various factors including the formation of small quantities of benzoylcarnitine, which was detected in the plasma of three of four patients receiving SB and carnitine therapy and in one of two patients on SB therapy without carnitine supplementation. The ratios of acyl to free carnitine were elevated in both plasma and urine in patients not receiving carnitine supplementation, but were normal in patients receiving supplementation.

Amniocentesis was performed at 17.3 weeks in a pregnancy with severe intrauterine growth retardation. Cytogenetic studies on amniocytes were normal, 46,XX, and the pregnancy was continued. The diagnosis of Smith-Lemli-Opitz syndrome was suspected in the neonatal period and confirmed by the presence of 7-dehydrocholesterol (7-DHC) in the plasma (0.4 mmol/l, normal = not detectable) associated with a low total cholesterol concentration (0.4 mmol/l, normal = 2.56 +/- 0.23). Retrospective analysis of the amniotic fluid sample revealed an elevated level of 7-DHC (0.022 mmol/l; normal = undetectable). Therefore measurement of 7-DHC levels in amniotic fluid during the second trimester of pregnancy is useful for the prenatal diagnosis of Smith-Lemli-Opitz syndrome in families at risk and should be considered in cases of severe growth retardation of unknown aetiology for which amniotic fluid is available and in which a normal chromosomal pattern in amniocytes is present.

A marker suitable for genetic mapping and genomic fingerprinting is characterized by high polymorphic information content (PIC) and high "multiplex ratio" (defined as the number of loci that can be simultaneously typed). Towards this goal, we combined an Alu-specific with a non-Alu primer in a single PCR amplification targeting genomic regions where length polymorphisms are abundant. Three loci were revealed with the variable number of (AAT), (TAAA), (AG), and/or (AAAGG) motifs, and PIC values between 0.7 and > 0.94. Their location on Chromosomes (Chrs) 19q12, 17q12-q24, and 5q31.2-33.3 was determined by multipoint analysis with markers from CEPH database. The most common genotype for this three-locus marker, estimated from the occurrence of the most frequent alleles, is of the order of 2 x 10(-4), while the combined PIC value of a single typing experiment is 2.37. The use of a similar primer pair, as well as examples from the literature, indicates the general nature of this approach when a non-Alu oligonucleotide, presumably with "random" priming sites downstream of Alu repeats, is combined with an Alu-specific one. Clustering of DNA length variants in the regions adjacent to interspersed repeats provides opportunity to develop other highly informative multiple-locus markers similar to that described here.

Spinal muscular atrophy (SMA) is, after Duchenne muscular dystrophy, the most common neuromuscular disorder in childhood. The gene responsible for childhood SMA has been mapped to the q11.2-q13.3 region of chromosome 5. We have extended our linkage studies of SMA in the French-Canadian population to include microsatellite markers at the D5S125, D5S351, D5S435, JK53CA1/2 and MAP1B loci. These markers span about 4 cM of the SMA candidate region. We observed significant evidence for linkage between SMA and all the markers tested. The analysis of recombinant chromosomes provide evidence for the following genetic order: D5S125-D5S435-MAP1B-3'-JK53CA1/2 and places D5S351 proximal to JK53CA1/2. Furthermore, we confirm the current localization of the SMA gene distal to D5S435. Finally, we provide demonstration of significant linkage disequilibrium between childhood-onset SMA and four of the five marker loci, D5S125, D5S435, D5S351 and JK53CA1/2. Analysis of SMA-region haplotypes suggests that there may be a predominant SMA allele that is present on about 17% of SMA chromosomes in this sample of the French-Canadian population. We conclude that the observed linkage disequilibrium is likely due to genetic drift among regions of Quebec, consistent with this population's early history.

The causal factors and the physiopathology of motor diarrhea are still unclear. This case report describes a 60-year-old white man with severe diarrhea for more than 10 years and minor signs of autonomic dysfunction. Extensive investigation showed that small intestinal motility and absorption were normal but that accelerated colon transit precluded water and solute absorption from the large bowel. Orthostatic hypotension, sexual dysfunction, and loss of sweating suggested dysfunction of the autonomous nervous system, which was confirmed by reduced plasma concentrations of norepinephrine and dopamine. Rectal biopsy specimens showed enlarged enteric ganglion cells filled with lipidic material. Levels of total hexosaminidase and hexosaminidase B in plasma, white blood cells, and fibroblasts were decreased, as found in Sandhoff disease. The pedigree of the proband's family showed several affected and heterozygous individuals, detected by examination of total hexosaminidase and hexosaminidase B levels in plasma. Among the five homozygous subjects, three had a clinical picture of diarrhea and orthostatic hypotension since the age of 50. Therefore, hexosaminidase B deficiency should probably be regarded as a cause for dysautonomia; dysfunction of the gastrointestinal tract, manifested by motor diarrhea or esophageal dysmotility, could be the initial and prevalent presentation of dysautonomia.

Chronic childhood-onset spinal muscular atrophy (SMA) is, after Duchenne muscular dystrophy, the most common neuromuscular disorder in childhood. Recent linkage analyses have mapped this disease to 5q12-5q14. We show that chronic SMA (Types II and III) is tightly linked to the marker locus D5S39 (Zmax = 5.47 at theta = 0.02) in eight French Canadian families. In contrast to previously published results, we do not observe close linkage between chronic SMA and D5S6 (Zmax = 0.34 at theta = 0.18) or D5S78 (Zmax = 0.25 at theta = 0.21). Last, we present a family that appears to be discordant for this localization but may represent the first example of an incompletely penetrant individual.

We have analyzed patient DNA samples in 77 unrelated Duchenne (DMD) and Becker (BMD) muscular dystrophy families, 73 of which were of French Canadian origin. We show that the frequency (68%) and distribution of deletions within the dystrophin gene was neither random nor unique in this population. We localized 33% of the deletions to the proximal portion of the dystrophin gene while 63% involved the exons spanning introns 43 through 55 with breakpoint clusters occurring within introns 44 and 50. Whether the dystrophin open reading frame (ORF) is maintained constrains the distribution of DMD/BMD deletions such that BMD deletions tend to be strikingly homogeneous. Finally, the conservation of the dystrophin ORF and the severity of the clinical phenotype were concordant in 95% of the DMD/BMD deletions documented by this work.

We have studied the plasma and urinary levels of free and esterified carnitine in 18 patients affected by a primary defect of ammonia metabolism, which had been managed with or without a therapy of sodium benzoate. None of these patients presented with any acute neurologic or digestive symptoms during the study. Our group of non-treated patients showed an increase in the levels of plasma esterified carnitine and an elevation of urinary concentration of free carnitine, while the levels of urinary esterified carnitine clearly approached the superior limits of normal values. The group treated with sodium benzoate showed a more profoundly disturbed plasma and urinary carnitine profile: a significantly lower plasma and urinary free carnitine, accompanied by a clearly increased esterified/free carnitine ratio. We did not find any evidence of a relationship between the plasma levels of free or esterified carnitine and the protein intake or the plasma ammonia concentration. We are proposing a hypothesis to explain the hypocarnitinemia seen in our patients being treated with benzoate, along with other modifications observed in the carnitine profile. We believe that a supplement of carnitine could be beneficial in the management of some of these patients.

We have measured the activity of malic enzyme NADP+ dependent in the nuclear, mitochondrial, lysosomal and cytosolic fractions of cultured skin fibroblasts from twelve patients with Friedreich's ataxia and nine control subjects. Hexosaminidase, cytochrome-C-oxidase, lactate dehydrogenase and malic enzyme NAD+ dependent were used as marker enzymes. The activity of malic enzyme NADP+ dependent was not significantly reduced in the mitochondrial fraction of patients with Friedreich's ataxia as compared with controls. When corrected for possible contamination between mitochondrial and cytosolic fractions, malic enzyme NADP+ dependent activity was still not significantly reduced in patients with Friedreich's ataxia. Unless critical methodological differences were overlooked in this or previously published studies, we conclude that mitochondrial malic enzyme deficiency is not the primary genetic defect underlying Friedreich's ataxia.

The radiation inactivation method was used to determine the molecular size of the two enzymes that participate in the synthesis of the phosphomannosyl recognition marker of lysosomal proteins. The determinations were carried out in situ, in Golgi membranes isolated from normal human placenta and cultured skin fibroblasts. A molecular size of 228 +/- 29 kDa was found for placental N-acetylglucosaminyl-phosphotransferase, and 129 +/- 11 kDa for placental alpha-N-acetylglucosaminyl phosphodiesterase. The values for the fibroblast enzymes were about 20% higher, 283 +/- 27 kDa and 156 +/- 14 kDa for the transferase and phosphodiesterase respectively. Triton X-100 had no effect on the molecular size of these enzymes.

Amniotic fluid cells obtained by transabdominal amniocentesis at 15-17 weeks of gestation and cultured for 15 and 21 days were separated into three fractions by density gradient centrifugation in Percoll. Each fraction (or peak) corresponded to the following densities: peak A, 1.02-1.03 g/ml; peak B, 1.04-1.05; peak C, 1.05-1.06. Peak A was composed of both non-viable and viable cells; the latter adopted the morphology of epithelial cells in culture. Peak B contained a mixture of fibroblasts and epithelioid cells and peak C had only epithelioid cells. The variability of N-acetyl-beta-hexosaminidase and alkaline phosphatase activities was reduced in peaks B and C as compared to that of peak A and of unseparated cells suggesting that more defined and homogeneous cell types for enzymatic determinations can be obtained by centrifugation in Percoll density gradient. In prenatal diagnosis of biochemical defects, the separation of cells would permit a more precise diagnosis by eliminating enzyme variability due to the presence of different cell types or non-viable cells.

alpha-Glucosidase, beta-glucosidase and beta-galactosidase were studied in cell-free amniotic fluid samples using corresponding 4-methylumbelliferyl-glycosides and a series of disaccharides (maltose, sucrose, trehalose, turanose, cellobiose, gentiobiose and lactose) as substrates. The glycosidases exhibited several properties of intestinal disaccharidases such as pH optimum between 5.2 and 6.4, more activity towards the disaccharides than the artificial substrates, tight association of the activities with sedimentable complexes and beta-glucosidase and beta-galactosidase activities exerted by a single catalytic site. With the disaccharides as substrates, the amniotic fluid glycosidase activities were well correlated to those reported in the literature for fetal intestine of corresponding gestational ages. The presence of intestinal disaccharidases in amniotic fluid indicates that the fetal intestine contributes to the protein and enzymes of amniotic fluid.

A 38-month-old apparently healthy male has been followed for three years because of a massive glutamic and aspartic aminoaciduria detected shortly after birth in a neonatal screening program. Amino acid clearance studies revealed the presence of renal wastage of dicarboxylic amino acids. Intestinal transport and in vitro oxidation of dicarboxylic amino acids were found to be intact. Clinical and metabolic data obtained on a previously described patient and the present case suggest that some patients with dicarboxylic aminoaciduria might have a selective renal conservation defect without clinical abnormalities, whereas others might demonstrate an additional defect in intestinal transport associated with fasting hypoglycemia.

Comparison of relative disaccharidase activities in jejunal mucosa, amniotic fluid, and meconium samples taken from the ileum, proximal and distal colon of 5 human fetuses, indicates that trehalase and lactase are more sensitive to the denaturation conditions existing in the fetal intestinal lumen than maltase, sucrase, palatinase and turanase . Relative activities of sensitive disaccharidases (trehalase and lactase) were also low in the amniotic fluid of these fetuses as compared to the jejunal mucosa. These results suggest that the in vivo stability of disaccharidases, exposed to proteolytic digestion and other denaturing conditions in the fetal intestinal lumen, governs the relative activities of these enzymes released into the amniotic fluid.

Lipoamide dehydrogenase was measured in cultivated skin fibroblasts from twelve patients with Friedreich's ataxia and nine normal controls. No difference in specific activity, subcellular distribution and Vmax or Km was observed between patients and controls.

We report relatively high trehalase activity in the amniotic fluid of two fetuses affected with the congenital nephrotic syndrome of the Finnish type. The results suggest that prenatal detection of this condition can be done on this basis in conjunction with the alpha-fetoprotein and acetylcholinesterase tests in amniotic fluid.

Though frequently noted, orofacial anomalies in poly X syndromes have been seldom reviewed. In a study of patients with an X chromosome aneuploidy (XXXXX, XXXX, XXXXY, XXXY) the following facial and dental defects were found to be characteristic features of these syndromes: mainly bifid uvula and macroglossia, enamel defects, dental taurodontism and abnormal roots. These observations are discussed in the light of the possible role of X-linked genes.

The size of the mutant N-acetylglucosamine 1-phosphotransferase in Golgi membranes from fibroblasts of patients with I-cell disease and classical pseudo-Hurler polydystrophy, which comprised one complementation group characterized by deficiency towards both artificial and natural acceptor substrates, was significantly smaller than the normal enzyme, 151-174 kDa compared with 225-278 kDa. The size of the mutant enzyme from cell lines of patients with variant forms of pseudo-Hurler polydystrophy, which comprised another complementation group characterized by normal activity towards mono- and oligo-saccharide substrates, was significantly larger than the normal enzyme, ranging from 321 to 356 kDa in two families and from 528 to 547 kDa in a third family. These findings suggest that the mutations in I-cell disease and classical pseudo-Hurler polydystrophy result in a missing enzyme component, which renders the enzyme catalytically inefficient toward any type of acceptor substrate. In contrast, the mutations in the variant forms of pseudo-Hurler polydystrophy produce a larger enzyme molecule which is active toward small substrates but is incapable of binding natural lysosomal glycoprotein substrates.

Lipoamide dehydrogenase was identified in cultured skin fibroblasts of normal individuals and patients with Friedreich's ataxia. The optimum conditions for its assay were defined. Data disclosed a normal range of 36--122 mumol/min/mg protein in control fibroblasts and 61--112 mumol/min/mg protein in patients fibroblasts. Numerous precautions should be taken in handling fibroblast cultures for lipoamide dehydrogenase determination.

Amniotic fluid samples from four fetuses with intestinal obstructions (two anal imperforations, one duodenal atresia and one multiple intestinal atresia) were deficient in disaccharidase activities. These results indicate that disaccharidase assay in amniotic fluid can be used as a rapid and simple test for the prenatal detection of fetal intestinal obstructions.

Serum lipoamide dehydrogenase activity and kinetics were studied in nine patients with Friedreich's ataxia before and three months after therapy with oral lecithin. Results disclosed a significant reduction in LAD inhibition by NADH in all patients after therapy. Three patients normalized their increased Km for lipoamide and one patient showed the opposite results after therapy. Two patients ceased lecithin after one month. All seven patients who remained in the trial group and one additional patient, showed subjective and objective signs of improvement in physical resistance. This study has offered some biochemical basis for the apparent clinical improvement in patients with Friedreich's ataxia who undergo lecithin therapy.

A twin pregnancy with trisomy 10 p due to a paternal 10;12 translocation is reported. The prenatal diagnosis steps followed in twin pregnancies are reviewed and the concordant features of trisomy 10 p seen in both fetuses confirm previous reports on the clinical features of this chromosomal defect.

The significance of neuraminidase deficiency reported to be the primary defect in mucolipidosis II has been evaluated by determination of this enzyme activity in cultured fibroblasts, culture medium, and leucocytes from homozygote and heterozygous carriers of the disease. A new and sensitive fluorometric assay of neuraminidase was used with sodium (4-methylumbeliferyl-alpha-D-N-acetylneuraminate) as substrate. We report: 1) nearly total deficiency of neuraminidase in mucolipidosis fibroblasts, 2) partial deficiency of this enzyme in leucocytes of one patient, 3) this decreased activity ceases to exist following Triton X-100 treatment, and 4) intermediary mean neuraminidase activity in fibroblasts and leucocytes from obligate heterozygotes. Although these results would be consistent with the suggestion that neuraminidase deficiency is the primary defect in this disease, evidence from the work of other authors suggests that the enzyme deficiency results from a secondary effect of the mucolipidosis II mutation.

The intestinal microvilli of fetal origin in human amniotic fluid were purified by Ca2+ precipitation of contaminating organelles followed by differential centrifugation of microvillar membranes. In the purified preparation, the specific activity of the microvillar marker-enzymes maltase and sucrase increased about 77-fold over that in cell-free amniotic fluid. Significant contamination of the purified preparation by endoplasmic reticulum (microsomes) and lysosomes was ruled out on the basis of a low content of the marker enzymes glucose-6-phosphatase (microsomes) and acid phosphatase (lysosomes). Amniotic fluid microvilli contain typical enzymes of the fetal intestine including maltase, sucrase, trehalase, alkaline phosphatase and gamma-glutamyltransferase, and their morphology by electron microscopy resembles that of vesiculated intestinal microvilli. Prenatal detection of genetic diseases due to a deficiency of a protein expressed in these membranes or associated to abnormal microvilli seems feasible.

We describe two patients with methylmalonic aciduria and homocystinuria (Cbl C). The disorder was not diagnosed in patient 1 until 4 1/2 years of age; he had a history of fatigue, anorexia, delirium, and spasticity. Moderate megaloblastic bone marrow changes were observed, and there was hyperreflexia of the lower limbs. His condition improved clinically with hydroxycobalamin therapy. Patient 2 was hospitalized at 6 weeks of age because of lethargy and poor feeding. She was found to have macrocytosis. Despite an initial good clinical response to hydroxycobalamin, she developed a striking pigmentary retinopathy. Methylmalonic aciduria persisted in both patients, and homocystinuria persisted in patient 1 despite therapy. The diagnosis of Cbl C disease has been confirmed in both patients by biochemical studies of cultured fibroblasts, including complementation studies. The differences in age of onset and clinical findings together with the similar biochemical findings in these two patients demonstrate the heterogeneity of phenotypic expression in patients with apparently identical abnormalities of vitamin B12 metabolism.