ARSACS, a spastic ataxia common in northeastern Québec, is caused by mutations in a new gene encoding an 11.5-kb ORF.

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): high-resolution physical and transcript map of the candidate region in chromosome region 13q11.

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.

Location score and haplotype analyses of the locus for autosomal recessive spastic ataxia of Charlevoix-Saguenay, in chromosome region 13q11.

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.

Clinical spectrum of infantile free sialic acid storage disease.

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.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay.

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.

GAA instability in Friedreich's Ataxia shares a common, DNA-directed and intraallelic mechanism with other trinucleotide diseases.

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.

Phenotypic variability in Friedreich ataxia: role of the associated GAA triplet repeat expansion.

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.

Friedreich ataxia in Acadian families from eastern Canada: clinical diversity with conserved haplotypes.

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.

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures.

We have used peripheral blood T-lymphocyte cultures to analyze the hprt mutation in two Lesch-Nyhan syndrome males who are cousins and to confirm the carrier status of female members of the family. Both cDNA and genomic DNA sequencing studies show that this patient carries a hitherto undescribed single base deletion in the exon 5 donor splice site sequence (I5: +1, delta G, base number 31635). The largest cDNA product contained all nine hprt exons plus an insertion of 66 bases of intron 5, consistent with the use of a cryptic splice site in intron 5 (aag67/gtaagc). This splicing error would result in a chain terminating codon immediately after exon 5 (I5:2-4, taa) and predicts a polypeptide of 133 amino acids. This loss of the normal splice donor site also results in multiple hprt mRNA species, combining the use of the cryptic splice site in intron 5 and splicing errors involving exons 2-6. In addition to defining a new Lesch-Nyhan mutation (hprtHenryville), these results provide insight into aberrant splicing of hprt mRNA in T-lymphocytes.

Chronic sodium benzoate therapy in children with inborn errors of urea synthesis: effect on carnitine metabolism and ammonia nitrogen removal.

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.

Prenatal diagnosis of Smith-Lemli-Opitz syndrome is possible by measurement of 7-dehydrocholesterol in amniotic fluid.

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.

Alu-PCR combined with non-Alu primers reveals multiple polymorphic loci.

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.

Linkage disequilibrium analysis of childhood-onset spinal muscular atrophy (SMA) in the French-Canadian population.

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.

Diarrhea and autonomic dysfunction in a patient with hexosaminidase B deficiency (Sandhoff disease).

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.

Linkage study of chronic childhood-onset spinal muscular atrophy (SMA): confirmation of close linkage to D5S39 in French Canadian families.

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.

An unusual splicing mutation in the HEXB gene is associated with dramatically different phenotypes in patients from different racial backgrounds.

Sandhoff disease is caused by mutations affecting the beta subunit of lysosomal beta-hexosaminidase (EC 3.2.1.52) and displays a wide spectrum of clinical phenotypes. We report a 57-year-old patient with a very mild phenotype, although residual hexosaminidase A activity in his cultured fibroblasts was less than 3% of normal activity, a level observed in juvenile onset patients. Northern and Western blot analyses confirmed a similar low level of beta subunit-mRNA and mature beta-protein, respectively. Two mutations of the HEXB gene were identified in this patient, a partial 5' gene deletion (a null allele), and a C----T transition 8 nucleotides downstream from the intron 10/exon 11 junction affecting the splicing of the beta subunit-mRNA. In their homozygous forms, the 5' deletion has been previously shown to result in a severe infantile phenotype, and the C----T transition in a juvenile phenotype. The genotype and the low level of residual hexosaminidase A activity would be expected to produce a juvenile Sandhoff phenotype in this patient, as well as in four of his six clinically normal siblings. The biochemical basis of his mild phenotype is uncertain, but may result from genetic variations in the RNA splicing machinery.

Deletions in the dystrophin gene: analysis of Duchenne and Becker muscular dystrophy patients in Quebec.

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.