Academic achievement in individuals with infantile nephropathic cystinosis

The present study examined academic skills in children and young adults with infantile nephropathic cystinosis. Cystinosis is a genetic metabolic disorder in which the amino acid cystine accumulates in various tissues and organs, including the kidney, cornea, thyroid, and brain. Individuals with cystinosis have normal intelligence but subtle visual processing impairments. Subjects were 19 children and young adults with cystinosis and 19 age-, sex-, and IQ-matched controls. All subjects had IQs within the normal range. On a test of academic achievement, mean standard scores for cystinosis and control subjects, respectively, were as follows: arithmetic 89.95 ± 13.77 vs. 102.16 ± 9.62; spelling 90.68 ± 18.81 vs. 98.00 ± 10.96; reading 97.47 ± 15.59 vs. 98.58 ± 12.41. Multivariate analysis of variance revealed a significant main effect for Group (P = .009); there was no main effect for Sex, nor was there a Group x Sex interaction. Univariate follow-up tests indicated that the cystinosis group performed significantly more poorly than did controls on the arithmetic subtest (P = .001) and that there was a trend (P = .085) toward poorer performance by the cystinosis group on the spelling subtest. Regression analyses revealed no evidence of a developmental lag or deterioration of function with age. The visual processing deficits previously identified in these individuals may underlie the academic difficulties observed here. It is possible that both visual processing and academic difficulties may reflect a common mechanism of selective cortical damage by this genetic defect. Am. J. Med. Genet. 74:157–161, 1997. © 1997 Wiley-Liss, Inc.     

Identification and detection of the common 65-kb deletion breakpoint in the nephropathic cystinosis gene (CTNS)

The most common mutation in the cystinosis gene, CTNS, is a 65-kb deletion thought to have originated in Germany. Although homozygotes for this deletion are detectable by the absence of the D17S829 polymorphic marker, no method exists to identify heterozygotes. We identified the 65-kb deletion breakpoints and used flanking PCR primers to amplify a 423-bp fragment present only in the deletion alleles. Using this method, we determined that 121 of 216 (56%) cystinosis alleles examined bore the 65-kb deletion. We found no non-Europeans with the deletion, and the deletion size and breakpoints appeared identical in all patients studied, supporting the concept of a founder effect. The addition of D17S829 primers (266 bp apart) to the PCR created a multiplex PCR system useful for diagnosing cystinosis patients homozygous and heterozygous for the 65-kb deletion.     

Maternal uniparental heterodisomy of chromosome 17 in a patient with nephropathic cystinosis

We report maternal uniparental disomy of chromosome 17 (mat UPD17) in a 2.5-year-old girl presenting infantile cystinosis. This patient was homozygous for the 57 kb deletion encompassing the CTNS gene, frequently found in patients from the European origin. The proband''s mother was heterozygous for the deletion and the father did not carry the deletion. We carried out haplotype analysis with polymorphic markers spanning the whole chromosome 17. Informative markers showed the presence of two maternal alleles but no paternal allele for regions spanning the 17q arm and the proximal half of 17p, and only one maternal allele on the distal 17p arm. As deletion of half of 17p is probably not viable, these results suggest mat UPD17 with heterodisomy of 17q and proximal 17p and isodisomy of distal 17p. This is the first demonstration of mat UPD17, in particular of isodisomy 17p, in cystinosis.     

Functional analysis of the CTNS gene exonic variants predicted to affect splicing

Cystinosis is a severe, monogenic systemic disease caused by variants in CTNS gene. Currently, there is growing evidence that exonic variants in many diseases can affect pre-mRNA splicing. The impact of CTNS gene exonic variants on splicing regulation may be underestimated due to the lack of routine studies at the RNA level. Here, we analyzed 59 exonic variants in the CTNS gene using bioinformatics tools and identified candidate variants that may induce splicing alterations by minigene assays. We identified six exonic variants that induce splicing alterations by disrupting the ratio of exonic splicing enhancers/exonic splicing silencers (ESEs/ESSs) or by interfering with the recognition of classical splice sites, or both. Our results help in the correct molecular characterization of variants in cystinosis and inform emerging therapies. Furthermore, our work suggests that the combination of in silico and in vitro assays facilitates to assess the effects of DNA variants driving rare genetic diseases on splicing regulation and will enhance the clinical utility of variant functional annotation.     

The genomic region encompassing the nephropathic cystinosis gene (CTNS): complete sequencing of a 200-kb segment and discovery of a novel gene within the common cystinosis-causing deletion

Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations in CTNS are deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization of CTNS and to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassing CTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanning CTNS was constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of the CTNS exons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization of CTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboring CTNS (human ortholog of rat vanilloid receptor subtype 1 gene, CARKL, TIP-1, P2X5, and HUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, both CTNS and CARKL are absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion). [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF168787 and AF163573.]     

Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin.

Infantile nephropathic cystinosis is a rare, autosomal recessive disease caused by a defect in the transport of cystine across the lysosomal membrane and characterized by early onset of renal proximal tubular dysfunction. Late-onset cystinosis, a rarer form of the disorder, is characterized by onset of symptoms between 12 and 15 years of age. We previously characterized the cystinosis gene, CTNS, and identified pathogenic mutations in patients with infantile nephropathic cystinosis, including a common, approximately 65 kb deletion which encompasses exons 1-10. Structure predictions suggested that the gene product, cystinosin, is a novel integral lysosomal membrane protein. We now examine the predicted effect of mutations on this model of cystinosin. In this study, we screened patients with infantile nephropathic cystinosis, those with late-onset cystinosis and patients whose phenotype does not fit the classical definitions. We found 23 different mutations in CTNS; 14 are novel mutations. Out of 25 patients with infantile nephropathic cystinosis, 12 have two severely truncating mutations, which is consistent with a loss of functional protein, and 13 have missense or in-frame deletions, which would result in disruption of transmembrane domains and loss of protein function. Mutations found in two late-onset patients affect functionally unimportant regions of cystinosin, which accounts for their milder phenotype. For three patients, the age of onset of cystinosis was <7 years but the course of the disease was milder than the infantile nephropathic form. This suggests that the missense mutations found in these individuals allow production of functional protein and may also indicate regions of cystinosin which are not functionally important.     

Ultrastructure of the liver in a case of childhood cystinosis

Summary Ultrastructural findings in the liver in a case of childhood cystinosis are reported. Crystalline structures were found mainly in Kupffer cells. The presence of dark cells, with or without crystals, was the most striking feature observed. Such cells have already been noted within the kidney on one occasion when it was shown that the dark substance was L-cystine (Spear et al., 1971). In this case identical dark material was also found extracellularly.The data shows that free cystine can fill cell cytoplasm and extracellular spaces and the possibility that cystine overproduction may take place in the hyaloplasm should be considered. Extracellular location of cystine in the tubules might account for an increase in epithelial permeability and thus for the Fanconi syndrome.Chargé de Recherche à l'I.N.S.E.R.M.     

High cystine in platelets from patients with nephropathic cystinosis: a chemical, ultrastructural, and functional evaluation

AIM: To investigate the morphology and function of platelets in nephropathic cystinosis (NC).METHODS: Seven patients (mean age, 6.5 years; SD, 20 months) with NC were investigated. Their platelets were examined by transmission electron microscopy (TEM) and the characteristics of the dense granules (DGs) were determined by mepacrine labelling and the uranaffin reaction. Bleeding time, turbidometric aggregation, and luminescence aggregation were studied and intraplatelet cystine was measured. RESULTS: Increased intraplatelet cystine, primary and secondary aggregation defects, and the absence of ATP release were demonstrated. TEM revealed DGs of various shapes and sizes and lamellary or amorphous cytoplasmic inclusions. Viscous material had been released into the vacuolar spaces and enlarged open canalicular system. Mepacrine labelling revealed that the numbers of DGs/platelet were comparable between the patients and the controls (mean, 2.9 (SD, 0.22) v 3.32 (0.18); p = 0.34). The uranaffin reaction revealed that the numbers of type 1, 3, and 4 DGs were comparable between the patients and the controls, but that there were fewer type 2 DGs in the patients (mean, 8.5 (SD, 1.95) v 17.22 (1.58); p = 0.01). TEM for platelet aggregation revealed a lack of induction and/or defective execution and/or delayed transmission. The patients' intraplatelet cystine concentrations were higher than the controls (mean, 1.56 (SD, 0.84) v 0.08 (0.01) nmol/mg protein; p = 0.009). CONCLUSIONS: This is the first report to demonstrate raised intraplatelet cystine, abnormal platelet ultrastructural findings, and defective aggregation in NC.     

Independent origin of single and double mutations in the human glucose 6-phosphate dehydrogenase gene

The vast majority of both polymorphic and sporadic G6PD variants are due to single missense mutations. In the four polymorphic variants that have two point mutations, one of the mutations is always 376 A→G (126 Asn→Asp), which on its own gives rise to the nondeficient polymorphic variant, G6PD A. In a study of G6PD deficient patients who presented with clinical favism in Spain, we have found a new polymorphic variant that we have called G6PD Malaga, whose only abnormality is a 542 A→T (181 Asp→Val) mutation. This is the same mutation as previously found in association with the mutation of G6PD A in the double mutant, G6PD Santamaria. G6PD Malaga is associated with enzyme deficiency (class III), and the enzymic properties of G6PD Malaga and G6PD Santamaria are quite similar, indicating that in this case the effects of the two mutations are additive rather than synergistic. G6PD Santamaria might have been produced by recombination between G6PD A and G6PD Malaga; however haplotype analysis, including the use of a new silent polymorphism, suggests that the same 542 A→T mutation has taken place independently in a G6PD B gene to give G6PD Malaga and in a G6PD A gene to give G6PD Santamaria. These findings help to outline the relationship and evolution of mutations in the human G6PD locus. © 1996 Wiley-Liss, Inc.     

Molecular characterization of familial hypercholesterolemia in German and Greek patients

We used the denaturing gradient gel electrophoresis (DGGE) method to define mutations in the promoter region, the 18 exons, and their flanking intronic sequences of the low-density lipoprotein (LDL) receptor gene LDLR, causing familial hypercholesterolemia (FH) phenotype in 100 German and in 100 Greek hypercholesterolemic individuals. In addition, we tested all patients for the presence of mutations in codons 3456-3553 of the gene encoding apolipoprotein B-100 (APOB). Twenty-six aberrant DGGE patterns were identified and subsequently directly sequenced. In LDLR, two novel missense mutations (c.1957G>T/p.V653F, c.647 G>A/p.C216Y) and one novel homozygous base substitution c.1-156 C>T in the repeat 2 of the promoter region were identified among German FH patients; one novel splice site c.1060+10C>G was identified among Greek FH patients. One of the German FH patients was a carrier for the mutations c.1171G>A/p.A391T and p.V653F, and two of the Greek FH patients were compound heterozygotes for the mutations c.1150C>T/p.Q384X and c.1158C>G/p.D386E. Two German FH patients carried the mutation p.R3500Q within APOB. Comparing the mutations within the LDLR gene of the two European FH populations, the German population seems to be more heterogeneous than the Greek cohort. Further studies in progress are trying to elucidate the responsiveness to drug therapy in association with LDLR genotype and the nutritional habits of the two FH populations.     

Effects of prenatal stress on motor performance and anxiety behavior in Swiss mice

Stressor presence during the last weeks of gestation has been associated with behavioral disorders in later life. In this study we support further research on the long term effects of prenatal stress on Swiss mice descendant's behavior. Prenatal stress procedure consisted on restraining the dams under bright light for 45 min, three times per day from the 15th day of pregnancy, until birth. After weaning, offspring's motor performance and spontaneous exploratory behavior were measured by the tight-rope and T-maze tests, respectively. We also evaluated anxiety behavior using elevated plus maze test. We found that maternal stress improves the performance of the animals in the tight rope test and that this effect was sex and age dependent: prenatal stressed males obtained the best scores during the first month of life, while in females the same was achieved at the second month. Spontaneous exploratory behavior analysis revealed that it was elevated in prenatal stressed males and that this effect persisted on time. However, we did not find significant differences on this behavioral response among both females groups. Finally, differences on anxiety behavior were found only in females: prenatally stressed animals showed a higher proportion of entries into the open arms of a plus maze (reduced anxiety) compared to the control group. Our results show that prenatal stress modifies the normal behavior of the progeny: prenatal stressed animals have a better performance in the carried out test. These notably results suggest the existence of an adaptive response to prenatal stress.     

(ATT) trinucleotide repeats in the antithrombin gene and their use in determining the origin of repeated mutations

Two (ATT) trinucleotide repeat polymorphisms have been identified in the tails of Alu repeat elements in intron 5 of the antithrombin gene. The frequency and distribution of allele sizes for the Alu 5 and Alu 8 tail polymorphisms have been defined in a sample Caucasian population. The Alu 5 polymorphism has two alleles while that of Alu 8 has 10 alleles with a heterozygosity of 0.83. These polymorphisms have been used in combination with four previously described polymorphisms within the antithrombin gene to construct antithrombin gene haplotypes in the sample Caucasian population. Twenty-two different haplotypes were observed, with the Alu 8 polymorphism being particularly useful in subdividing the core haplotype based on the previously identified polymorphisms. The haplotype data were used to investigate the origin of repeat mutations within the antithrombin locus. We compared the haplotypes associated the mutant antithrombin genes in five families with the mutation 2759C→T (L99F) and five families with the mutation 5381C→T (R129Stop). The mutation 2759C→T (L99F), which occurs within a non-CpG dinucleotide, was carried on a gene associated with an identical haplotype in each of the five families. The mutation 5381C→T (R129Stop), a single base substitution within a CpG dinucleotide, was associated with at least two different haplotypes. The findings suggest a founder effect in the five families sharing the 2759C→T (L99F) and at least two independent origins for the CpG dinucleotide mutation 5381C→T (Rl29Stop). © 1994 Wiley-Liss, Inc.     

Molecular epidemiology of the nasal colonization by methicillin-susceptible Staphylococcus aureus in Swiss children

Nasal carriage of Staphylococcus aureus contributes to an increased risk of developing an infection with the same bacterial strain. Genetic regulatory elements and toxin-expressing genes are virulence factors associated with the pathogenic potential of S. aureus. We undertook an extensive molecular characterization of methicillin-susceptible S. aureus (MSSA) carried by children. MSSA were recovered from the nostrils of children. The presence of Panton-Valentine leukocidin (PVL), exfoliatins A and B (exfoA and exfoB), and the toxic-shock staphylococcal toxin (TSST-1) and agr group typing were determined by quantitative PCR. A multiple-locus variable-number of tandem repeat analysis (MLVA) assay was also performed for genotyping. Five hundred and seventy-two strains of MSSA were analysed. Overall, 30% were positive for toxin-expressing genes: 29% contained one toxin and 1.6% two toxins. The most commonly detected toxin gene was tst, which was present in 145 (25%) strains. The TSST-1 gene was significantly associated with the agr group 3 (OR 56.8, 95% CI 32.0–100.8). MLVA analysis revealed a large diversity of genetic content and no clonal relationship was demonstrated among the analysed MSSA strains. Multilocus sequence typing confirmed this observation of diversity and identified ST45 as a frequent colonizer. This broad diversity in MSSA carriage strains suggests a limited selection pressure in our geographical area.     

Screening for point mutations in the LDL receptor gene in Bulgarian patients with severe hypercholesterolemia

Familial hypercholesterolemia (FH) is a common, autosomal dominant disorder of lipid metabolism, caused by defects in the receptor-mediated uptake of LDL (low-density lipoproteins) due to mutations in the LDL receptor gene (LDLR). Mutations underlying FH in Bulgaria are largely unknown. The aim of the present study was to provide information about the spectrum of point mutations in LDLR in a sample of 45 Bulgarian patients with severe hypercholesterolemia. Exons 3, 4, 6, 8, 9, and 14, previously shown to be mutational hot spots in LDLR, were screened using PCR-single-strand conformation polymorphism (SSCP). Samples with abnormal SSCP patterns were sequenced. Three different, hitherto undescribed point mutations (367T>A, 377T>A, 917C>A) and two previously described mutations (858C>A and 1301C>T) in eight unrelated patients were identified; four of the detected point mutations being missense mutations and one, a nonsense mutation. One of the newly described point mutations (917C>A) is a base substitution at a nucleotide position, at which two other different base substitutions have already been reported. Thus, all three possible base substitutions at this nucleotide position have been detected, making it a hot spot for point mutations causing FH. This is the first such mutational hot spot described in exon 6 of LDLR.     

Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene

Achromatopsia (ACHM) is a hereditary cone photoreceptor disorder characterized by the inability to discriminate colors, nystagmus, photophobia, and low‐visual acuity. Six genes have been associated with this rare autosomal recessively inherited disease, including the GNAT2 gene encoding the catalytic α‐subunit of the G‐protein transducin which is expressed in the cone photoreceptor outer segment. Out of a cohort of 1,116 independent families diagnosed with a primary clinical diagnosis of ACHM, we identified 23 patients with ACHM from 19 independent families with likely causative mutations in GNAT2, representing 1.7% of our large ACHM cohort. In total 22 different potentially disease‐causing variants, of which 12 are novel, were identified. The mutation spectrum also includes a novel copy number variation, a heterozygous duplication of exon 4, of which the breakpoint matches exactly that of the previously reported exon 4 deletion. Two patients carry just a single heterozygous variant. In addition to our previous study on GNAT2‐ACHM, we also present detailed clinical data of these patients.     

Mutations of theTyrosinase gene in three Korean patients with Type I oculocutaneous albinism

Summary Oculocutaneous albinism (OCA) is an inherited disorder of the melanin pigmentary system, characterized by a decrease or an absence of melanin in the skin, hair, and eyes. Type I (tyrosinase-deficient) OCA results from mutations of thetyrosinase (TYR) gene encoding tyrosinase, the enzyme that catalyzes at least the first two steps of melanin biosynthesis. We have analyzed theTYR gene in three Korean patients with severe type I OCA. Two patients were compound heterozygotes for the Arg (CGG) to Gln (CAG) mutation at position 77 and a C insertion mutation at position 310. The other was a compound heterozygote for a C insertion mutation at position 310 and the Asp (GAT) to Asn (AAT) mutation at position 383. These mutations were easily detected by restriction enzyme digestion or by SSCP analysis. Such methods of mutation analysis thus provide a basis for a screening system for theTYR gene mutations in Korean patients with type I OCA.     

Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF

Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disorder in Caucasians, and is associated with at least one mutation on each CF transmembrane conductance regulator (CFTR) allele. Some patients, however, with only one identifiable point mutation carry on the other allele, a large deletion that is not detected by conventional screening methods. The overall frequency of large deletions in patients with CF is estimated to be 1-3%. Using the CFTR Multiplex Ligation dependent Probe Amplification Kit (MRC-Holland, Amsterdam, Netherlands) that allows the exact detection of copy numbers from all 27 exons in the CFTR gene, we screened 50 patients with only one identified mutation for large deletions in the CFTR gene. Each detected deletion was confirmed using our real-time polymerase chain reaction (PCR) assay and deletion-specific PCR reactions using junction fragment primers. We detected large deletions in eight patients (16%). These eight CF alleles belong to four different deletion types (CFTRindel2, CFTRdele14b-17b, CFTRdele17a-17b and CFTRdele 2-9) whereof the last is novel. Comparing detailed clinical data of all these patients with CF and the molecular genetic findings, we were able to elaborate criteria for deletion screenings and possible genotype-phenotype associations. In conclusion, we agree with other authors that deletion screenings should be implemented in routine genetic diagnostics of CF.