A homemade MLPA assay detects known CTNS mutations and identifies a novel deletion in a previously unresolved cystinosis family.

Infantile nephropatic cystinosis is a rare, recessive, and genetically homogeneous disorder impairing renal function. It is caused by mutations in CTNS. Several large copy number aberrations have been identified but, for the majority of these, heterozygous patients and carriers can not easily be identified. We therefore developed a multiplex ligation-dependent probe amplification assay targeting eight of the twelve CTNS exons. We show that this assay is valid in detecting known deletions in both the homozygous and heterozygous state. The application to a family previously found mutation-negative by conventional screening revealed a novel large deletion which, as the first of its kind, does not involve the coding region. We conclude that our assay represents a valid tool for further completing the CTNS mutation spectrum and for simplified carrier testing in cystinosis families harboring copy number mutations. More generally, our study exemplifies the use of synthetic, homemade MLPA probesets as cheap, efficient, and rapidly available screening tools for small genes and/or very rare diseases.

TC II deficiency: avoidance of false-negative molecular genetics by RNA-based investigations.

Transcobalamin II (TC II) is a plasma transport protein for cobalamin. TC II deficiency can lead to infant megaloblastic anemia, failure to thrive and to neurological complications. This report describes the genetic work-up of three patients who presented in early infancy. Initially, genomic investigations did not reveal the definite genetic diagnosis in the two index patients. However, analysis of cDNA from skin fibroblasts revealed a homozygous deletion of exon 7 of the TC II gene caused by the mutation c.940+303_c.1106+746del2152insCTGG (r.941_1105del; p.fs326X) in one patient. The other patients were siblings and both affected by an insertion of 87 bp on the transcript which was caused by the homozygous mutation c.580+624A>T (r.580ins87; p.fs209X). Additional experiments showed that cDNA from lymphocytes could have been used also for the genetic work-up. This report shows that the use of cDNA from skin fibroblasts or peripheral lymphocytes facilitates genetic investigations of suspected TC II deficiency and helps to avoid false-negative DNA analysis.

Diversity of cystathionine beta-synthase haplotypes bearing the most common homocystinuria mutation c.833T>C: a possible role for gene conversion.

Homozygosity or compound heterozygosity for the c.833T>C transition (p.I278 T) in the cystathionine beta-synthase (CBS) gene represents the most common cause of pyridoxine-responsive homocystinuria in Western Eurasians. However, the frequency of the pathogenic c.833C allele, as observed in healthy newborns from several European countries (q(c.833C) approximately equals 3.3 x 10(-3)), is approximately 20-fold higher than expected on the basis of the observed number of symptomatic homocystinuria patients carrying this mutation (q(c.833C) approximately equals 0.18 x 10(-3)), implying clinical underascertainment. Intriguingly, the c.833C mutation is also present in combination with a 68-bp insertion, c.[833C; 844_845ins68], in a substantial proportion of chromosomes from nonhomocystinuric individuals worldwide. We have sought to study the relationship between the pathogenic and nonpathogenic c.833C-bearing chromosomes and to determine whether the pathogenic c.[833C; -] chromosomes are identical-by-descent or instead arose by recurrent mutation. Initial haplotype analysis of 780 randomly selected Czech and sub-Saharan African wild-type chromosomes, employing 12 intragenic markers, revealed 29 distinct CBS haplotypes, of which 10 carried the c.[833C; 844_845ins68] combination; none carried an isolated c.833C or c.844_845ins68 mutation. Subsequent examination of 69 pathogenic c.[833C; -] chromosomes, derived from homocystinuria patients of predominantly European origin, disclosed three unrelated haplotypes that differed from their wild-type counterparts by virtue of the presence of c.833C, thereby indicating that c.833T>C transition has occurred repeatedly and independently in the past. Since c.833T does not reside within an obvious mutational hotspot, we surmise that the three pathogenic and comparatively prevalent c.[833C; -] chromosomes may have originated by recurrent gene conversion employing the common nonpathogenic c.[833C; 844_845ins68] chromosomes as templates.

Paragliding accidents with spinal cord injury: 10 years' experience at a single institution.

STUDY DESIGN: A retrospective analysis of 41 patients with spinal cord injury (SCI) after paragliding accidents. OBJECTIVE: To determine the lesioned pattern and prognostic radiologic factors for rehabilitation potential. SUMMARY OF BACKGROUND DATA: Paragliding accidents with SCI present a new injury pattern, dealt with in the current literature from a purely orthopedic, sports medicine, or insurance point of view. Few combinations of orthopedic and neurologic data are available. METHODS: Over a 10-year period, the case records of 41 patients with SCI caused by paragliding accidents were analyzed with regard to vertebral and other skeletal fractures, neurologic recovery (American Spine Injury Association score), and professional reintegration. RESULTS: Vertebral fractures peaked in the thoracolumbar region, with L1 most frequently (30%) affected. The levels of vertebral lesion and neurologic deficit differed in 32% of patients. Combination with lower-limb fractures was characteristic for paragliding SCI (P < 0.001); 93% of patients with initial bony occlusion of the spinal canal of <70% left the clinic ambulatory. CONCLUSION: Paragliding accidents with SCI show a characteristic injury pattern associated with a high recovery potential if the initial bony spinal canal occlusion is <70%. Half the patients will reintegrate in their former profession and place of employment.

Congenital glutamine deficiency with glutamine synthetase mutations.

Glutamine synthetase plays a major role in ammonia detoxification, interorgan nitrogen flux, acid-base homeostasis, and cell signaling. We report on two unrelated newborns who had congenital human glutamine synthetase deficiency with severe brain malformations resulting in multiorgan failure and neonatal death. Glutamine was largely absent from their serum, urine, and cerebrospinal fluid. Each infant had a homozygous mutation in the glutamine synthetase gene (R324C and R341C). Studies that used immortalized lymphocytes expressing R324C glutamine synthetase (R324C-GS) and COS7 cells expressing R341C-GS suggest that these mutations are associated with reduced glutamine synthetase activity.

Long-term carbimazole treatment of neonatal nonautoimmune hyperthyroidism due to a new activating TSH receptor gene mutation (Ala428Val).

BACKGROUND: Hereditary nonautoimmune hyperthyroidism is caused by activating germline mutations in the thyrotropin receptor gene. Antithyroid treatment failed to control hyperthyroidism in most cases, so that primary thyroid ablation or 131I therapy is advocated as the preferred treatment of choice. PATIENT/METHODS: We describe a case of neonatal nonautoimmune hyperthyroidism treated with carbimazole. Molecular analysis revealed a new heterozygous point mutation (A428V) in the TSH receptor (TSHR) gene. RESULT: Antithyroid treatment was successful in controlling hyperthyroidism for the first 5.9 years of age. CONCLUSION: We conclude that carbimazole therapy is effective in treating nonautoimmune hyperthyroidism. It may be an alternative to thyroidectomy or radioiodine treatment.

Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study.

Molecular typing of normal (n = 456) and small-colony-variant (SCV; n = 239) Staphylococcus aureus isolates cultured from the airways of 52 of 72 cystic fibrosis (CF) patients (72.2%) during a 6-year prospective study revealed a median long-term persistence of 37 months (range, 6 to 70). SCV persisted longer in the airways than the normal S. aureus (statistically not significant). Pulsed-field gel electrophoresis identified six prevalent clonal lineages, which were cultured from more than one patient (3 to 12 patients), and 39 individual clones, which were isolated only from single patients. The SCV phenotype was not restricted to a distinct clonal lineage but occurred in many different clones. Most patients (33 of 52, 63.46%) harbored single clones. This study provides a basis for improved understanding of S. aureus colonization and infection dynamics in CF patients.

Argininosuccinate lyase (ASL) deficiency: mutation analysis in 27 patients and a completed structure of the human ASL gene.

Argininosuccinic aciduria is an urea cycle disorder caused by argininosuccinate lyase (ASL) deficiency and is inherited as an autosomal-recessive trait. To date, mutation analysis has been limited because of incomplete sequence information about the human ASL gene. As a consequence, only 12 different mutations in 12 patients have been reported, so far. This study aimed at the completion of the structure and the sequence of the human ASL gene, the development of a genomic DNA-based system for mutation analysis and, finally, the characterisation of the molecular genetic background of ASL deficiency in 27 unrelated patients. This report provides transcript variants, the complete sequence of the human ASL gene and a complete ASL homologue on chromosome 22. The homologue was formerly thought to be a pseudogene but was found, in this study, to be correlated with an immunoglobulin-lambda-like mRNA. On the basis of the novel sequence data, a polymerase reaction chain system for mutation-screening in all 16 coding exons of the ASL gene was established and applied to the analysis of the ASL-deficient patients. We found mutations in all of the 54 investigated alleles and identified 23 (19 novel) different mutations. Some mutational hot-spots were identified (mainly in exons 4, 5, and 7) as were several predominant mutations: IVS5+1G-->A (15 alleles), c.532G-->A (7), c.346C-->T (6), c.1153C-->T (4). This study introduces a system for mutation analysis in the ASL gene, thereby elucidating the genetic background of ASL deficiency, which was found to be associated with considerable allelic heterogeneity.