Study of Toll-like receptor gene loci in sarcoidosis.

Sarcoidosis is a multi-factorial systemic disease of granulomatous inflammation. Current concepts of the aetiology include interactions of unknown environmental triggers with an inherited susceptibility. Toll-like receptors (TLRs) are main components of innate immunity and therefore TLR genes are candidate susceptibility genes in sarcoidosis. Ten members of the human TLR gene family have been identified and mapped to seven chromosomal segments. The aim of this study was to investigate all known TLR gene loci for genetic linkage with sarcoidosis and to follow positive signals with different methods. We analysed linkage of TLR gene loci to sarcoidosis by use of closely flanking microsatellite markers in 83 families with 180 affected siblings. We found significant linkage between sarcoidosis and markers of the TLR4 gene locus on chromosome 9q (non-parametric linkage score 2.63, P = 0.0043). No linkage was found for the remaining TLR gene loci. We subsequently genotyped 1203 sarcoidosis patients from 997 families, 1084 relatives and 537 control subjects for four single nucleotide polymorphisms of TLR4, including Asp299Gly and Thr399Ile. This genotype data set was studied by case-control comparisons and transmission disequilibrium tests, but showed no significant results. In summary, TLR4 - w ith significant genetic linkage results - appears to be the most promising member of the TLR gene family for further investigation in sarcoidosis. However, our results do not confirm the TLR4 polymorphisms Asp299Gly and Thr399Ile as susceptibility markers. Our results rather point to another as yet unidentified variant within or close to TLR4 that might confer susceptibility to sarcoidosis.

Evidence for linkage of restless legs syndrome to chromosome 9p: are there two distinct loci?

BACKGROUND: Restless legs syndrome (RLS) is a common sensory-motor disorder characterized by paresthesias and an intense urge to move the legs with a considerable familial aggregation. To date, no gene mutation has been found, but five gene loci have been mapped in primary RLS to chromosomes 12q, 14q, 9p, 2q, and 20p (RLS1 through 5). PATIENTS/METHODS: We identified a four-generational German RLS family with 37 family members including 15 affected cases. We performed linkage analysis using microsatellite markers at the five known loci. Prompted by the identification of a potentially shared haplotype near the RLS3 locus, we expanded the investigated linkage region on chromosome 9p using additional DNA markers. RESULTS: Mode of inheritance in our RLS family was compatible with an autosomal dominant pattern, and disease onset was mainly in childhood or adolescence. We excluded linkage to the RLS1, RLS2, RLS4, and RLS5 loci. However, we identified a likely new RLS gene locus (RLS3*) on chromosome 9p with a maximum lod score of 3.60 generated by model-based multipoint linkage analysis. A haplotype flanked by D9S974 and D9S1118 in a 9.9-Mb region, centromeric to RLS3, was shared by all 12 investigated patients. In addition, 11 of them carried a common haplotype extending telomeric to D9S2189 that is located within RLS3. CONCLUSIONS: We demonstrate linkage to a locus on chromosome 9p that is probably distinct from RLS3. Our family with a rather homogeneous phenotype and very early disease onset represents a unique opportunity to further elucidate the genetic causes of the frequent restless leg syndrome.

Spinocerebellar ataxia type 4 (SCA4): Initial pathoanatomical study reveals widespread cerebellar and brainstem degeneration.

Spinocerebellar ataxia type 4 (SCA4), also known as 'hereditary ataxia with sensory neuropathy', represents a very rare, progressive and untreatable form of an autosomal dominant inherited cerebellar ataxia (ADCA). Due to a lack of autopsy cases, no neuropathological or clinicopathological studies had yet been performed in SCA4. In the present study, the first available cerebellar and brainstem tissue of a clinically diagnosed and genetically-confirmed German SCA4 patient was pathoanatomically studied using serial thick sections. During this systematic postmortem investigation, along with an obvious demyelinization of cerebellar and brainstem fiber tracts we observed widespread cerebellar and brainstem neurodegeneration with marked neuronal loss in the substantia nigra and ventral tegmental area, central raphe and pontine nuclei, all auditory brainstem nuclei, in the abducens, principal trigeminal, spinal trigeminal, facial, superior vestibular, medial vestibular, interstitial vestibular, dorsal motor vagal, hypoglossal, and prepositus hypoglossal nuclei, as well as in the nucleus raphe interpositus, all dorsal column nuclei, and in the principal and medial subnuclei of the inferior olive. Severe neuronal loss was seen in the Purkinje cell layer of the cerebellum, in the cerebellar fastigial nucleus, in the red, trochlear, lateral vestibular, and lateral reticular nuclei, the reticulotegmental nucleus of the pons, and the nucleus of Roller. In addition, immunocytochemical analysis using the anti-polyglutamine antibody 1C2 failed to detect any polyglutamine-related immunoreactivity in the central nervous regions of this SCA4 patient studied. In view of the known functional role of affected nuclei and related fiber tracts, the present findings not only offer explanations for the well-known disease symptoms of SCA4 patients (i.e. ataxic symptoms, dysarthria and somatosensory deficits), but for the first time help to explain why diplopia, gaze-evoked nystagmus, auditory impairments and pathologically altered brainstem auditory evoked potentials, saccadic smooth pursuits, impaired somatosensory functions in the face, and dysphagia may occur during the course of SCA4. Finally, the results of our immunocytochemical studies support the concept that SCA4 is not a member of the CAG-repeat or polyglutamine diseases.

Spinocerebellar ataxia type 4. Investigation of 34 candidate genes.

The spinocerebellar ataxias (SCAs) with autosomal dominant inheritance are a clinically and genetically heterogeneous group of neurodegenerative disorders. To date 24 different loci have been identified for these conditions. A locus at chromosome 16q22.1 co-segregates with the disease phenotype in families of Scandinavian, Japanese and German origin. The corresponding SCA4 locus was narrowed down to 7.94 Mb for the two European and to 1.25 Mb for Japanese pedigrees. Unfortunately, because of the phenotypic differences between patients from Japan and Europe it is not possible to decide if SCA families linked to chromosome 16q22.1 share a common disease genotype or not. To look for mutations in the German family we screened 34 candidate genes in a 3.69 cM region. With the exception of two cSNPs, no segregation of DNA variations with the disease phenotype was found.

Rational approach to genetic testing of cystic fibrosis (CF) in infertile men.

Male infertility as a result of isolated congenital bilateral absence of the vas deferens (CBAVD) is one primary genital form of cystic fibrosis (CF) and occurs in 1-2% of infertile men. Assisted fertilization in patients with CBAVD increases the risk of transmitting mutations in the CF gene. We developed a rational approach to genetic CF testing in infertile men. A total of 282 infertile male patients were screened for the most common CF mutations (DeltaF508, R117H, IVS8-5T). Clinical data including medical history, examination, semen analysis, sweat tests, karyotypes and hormonal values were analysed. We identified 23 patients carrying mutations in the CF gene (DeltaF508: 10 patients; R117H: six patients; IVS8-5T: 11 patients). Two patients were compound heterozygote for DeltaF508/R117H, two others for DeltaF508/IVS8-5T. Correlating these molecular analyses with the clinical data pertaining to serum follicle-stimulating hormone concentration, semen pH, sperm count and total testicular volume, we were able to develop a score with a high specificity (98.4) for the presence of a cystic fibrosis transmembrane conductance regulator (CFTR) mutation, but only with a low sensitivity (positive post-test likelihood: 62.5%; negative post-test likelihood: 6.3%). With regard to the low sensitivity and the high number of CFTR mutations found in this heterogeneous group of infertile men, we still recommend genetic CF testing before assisted fertilization.

Extension of the mutation spectrum in Friedreich's ataxia: detection of an exon deletion and novel missense mutations.

Friedreich's ataxia (FRDA), the most common autosomal recessively inherited ataxia, is due to a homozygous GAA triplet repeat expansion in the first intron of the FRDA gene in about 96% of patients. Approximately 4% of FRDA patients are compound heterozygotes with a GAA repeat expansion in one allele and a point mutation in the coding region of the second allele. To reinvestigate the mutation spectrum, we searched for mutations including exon deletions in six patients heterozygous for the GAA repeat expansion and found two unknown missense mutations, p.Asn146Lys and p.Leu186Arg, in trans to the expanded FRDA allele. Interestingly, we detected a heterozygous 2776 bp deletion including exon 5a in one of our patients. This deletion removes 50 of the 210 residues of the frataxin. Furthermore, since no FRDA case with two-point mutations is known, we screened eight patients with FRDA phenotype but GAA alleles within the normal range but did not reveal a mutation within the FRDA gene. In addition, DNA polymorphisms have been found in four out of 100 control individuals in this study.

Low-level gonosomal mosaicism in women undergoing ICSI cycles.

PURPOSE: Females with low-level gonosomal mosaicism (LLGM) scheduled for ICSI were retrospectively analyzed and compared to control groups. The prevalence of this mosaicism as well as its impact for an ICSI treatment were evaluated. METHODS: Routine cytogenetic analysis was done in 891 females scheduled for ICSI treatment. For comparison 294 females with recurrent abortions and 104 women with clinical or cytogenetical affected children, who also had routine chromosome analysis, were acquired. RESULTS: In the ICSI group the incidence of LLGM was significantly lower compared to the group of females with recurrent abortions (3.9% vs. 8.5%, p < 0.01). The incidence was similar between the groups of ICSI females and females with cytogenetical or clinically affected children (5.8%, p = 0.43). Neither anamnestic factors nor outcome parameters were different between females with and without LLGM undergoing ICSI. CONCLUSION: There is no higher incidence of LLGM in females undergoing ICSI. In those who show LLGM, fertility capacity is not reduced.

Genetic heterogeneity in ten families with myoclonus-dystonia.

BACKGROUND: Myoclonus-dystonia (M-D) is a movement disorder with autosomal dominant inheritance and reduced penetrance but may also occur sporadically. Recently, mutations in the epsilon-sarcoglycan gene (SGCE) were shown to cause M-D. Furthermore, single variants in the dopamine D2 receptor (DRD2) and DYT1 genes were found in combination with SGCE mutations in two M-D families, and another M-D locus was recently mapped to chromosome 18p11 in one family. METHODS: The authors clinically and genetically characterised ten consecutive cases with myoclonus-dystonia; seven familial and three sporadic. Twenty nine M-D patients and 40 unaffected family members underwent a standardised clinical examination by a movement disorder specialist. Index cases were screened for mutations in the SGCE, DYT1, and DRD2 genes and for deletions of the SGCE gene. Suitable mutation negative families were tested for linkage to the SGCE region and to chromosome 18p11. RESULTS: Two SGCE mutations were detected among the seven familial but no mutation in the sporadic cases. Haplotype analysis at the new M-D locus was compatible with linkage in two families and excluded in another family, suggesting at least one additional M-D gene. There were no obvious clinical differences between M-D families with and without detected mutations. CONCLUSION: M-D is genetically heterogeneous with SGCE mutations accounting for the disease in only part of the clinically typical cases.

DJ-1 (PARK7) mutations are less frequent than Parkin (PARK2) mutations in early-onset Parkinson disease.

BACKGROUND: Mutations in the Parkin gene (PARK2) are the most commonly identified cause of recessively inherited early-onset Parkinson disease (EOPD) but account for only a portion of cases. DJ-1 (PARK7) was recently reported as a second gene associated with recessively inherited PD with a homozygous exon deletion and a homozygous point mutation in two families. METHODS: To investigate the frequency of DJ-1 mutations, the authors performed mutational analysis of all six coding exons of DJ-1 in 100 EOPD patients. For the detection of exon rearrangements, the authors developed a quantitative duplex PCR assay. Denaturing high performance liquid chromatography analysis was used to screen for point mutations and small deletions. Further, Parkin analysis was performed as previously described. RESULTS: The authors identified two carriers of single heterozygous loss-of-function DJ-1 mutations, including a heterozygous deletion of exons 5 to 7 and an 11-base pair deletion, removing the invariant donor splice site in intron 5. Interestingly, both DJ-1 mutations identified in this study were found in the heterozygous state only. The authors also detected a polymorphism (R98Q) in 1.5% of the chromosomes in both the patient and control group. In the same patient sample, 17 cases were detected with mutations in the Parkin gene. CONCLUSIONS: Mutations in DJ-1 are less frequent than mutations in Parkin in EOPD patients but should be considered as a possible cause of EOPD. The effect of single heterozygous mutations in DJ-1 on the nigrostriatal system, as described for heterozygous changes in Parkin and PARK6, remains to be elucidated.

Spinocerebellar ataxia type 5: clinical and molecular genetic features of a German kindred.

The authors report a German family with autosomal dominant cerebellar ataxia tightly linked to the spinocerebellar ataxia type 5 (SCA5) locus (multipoint lod score 5.76). The phenotype is characterized by a purely cerebellar syndrome with a downbeat nystagmus occurring prior to the development of other features. Imaging studies demonstrated cortical cerebellar atrophy. Progression is slow even in patients with a disease onset during the second decade. The age at onset varies from 15 to 50 years.

CARD15 gene mutations in sarcoidosis.

Sarcoidosis, Blau syndrome and Crohn's disease are complex disorders, characterised by granulomatous inflammation affecting a variety of organs. Mutations of the CARD15 gene, on chromosome 16, have been shown to contribute significantly to Crohn's disease and to cause Blau syndrome. These factors prompted the current authors to study CARD15 mutations in sarcoidosis. A total of 138 families were genotyped, including 302 patients with sarcoidosis and 127 patients without a family history of sarcoidosis (together with their parents), for four main coding CARD15 polymorphisms associated with increased risk of Crohn's disease. Furthermore, the gene segment that harbours Blau syndrome mutations was sequenced in 39 selected patients from 39 families with affected siblings identical for one or two parental chromosomes 16s and in eight patients from multi-case families. None of the reported Blau syndrome mutations and no new sequence alterations were found. There was an increased frequency of transmission of the rare allele of the polymorphic sites 802C>T (SNP5) and 2722G>C (SNP12) in at least one of the two study groups. In conclusion, CARD15 mutations, which are important in Crohn's disease and Blau syndrome, play no major role in sarcoidosis in this study population. However, these mutations could be of limited importance, especially in patients without a family history of sarcoidosis.

SCA17 caused by homozygous repeat expansion in TBP due to partial isodisomy 6.

An expanded polyglutamine domain in the TATA-binding protein (TBP) has been described in patients with spinocerebellar ataxia type 17 (SCA17) characterized by cerebellar ataxia associated with dementia. TBP is a general transcription initiation factor that regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. SCA17, as an autosomal dominantly inherited progressive neurodegenerative disorder, is caused by heterozygous expansion of a CAG repeat coding for glutamine. Alleles with 27 to a maximum of 44 glutamine residues were found as the normal range, whereas expansions above 45 repeat units were considered pathological. Here, we present a patient with a very severe phenotype with a late onset but rapidly progressing ataxia associated with dementia and homozygous 47 glutamine residues caused by an apparent partial isodisomy 6. This extraordinary case has important implications for the insights of TBP and SCA17. The expanded polyglutamine domain in both TBP copies is not correlated with embryonic death indicating that the normal function of the protein is not disrupted by this kind of mutation but may account for the dementia seen in this patient.

Refinement of the spinocerebellar ataxia type 4 locus in a large German family and exclusion of CAG repeat expansions in this region.

Spinocerebellar ataxia type 4 (SCA4) is an autosomal dominant disorder mapped to chromosome 16q22.1 in a large Utah kindred. The clinical phenotype is characterized by cerebellar ataxia with sensory neuropathy. We describe a five-generation family from northern Germany with similar clinical findings linked to the same locus. Haplotype analyses refined the gene locus to a 3.69 cM interval between D16S3019 and D16S512. Analysis of nine CAG/CTG tracts in this region revealed no evidence for a repeat expansion.

Exon deletions in the GCHI gene in two of four Turkish families with dopa-responsive dystonia.

Most cases of dopa-responsive dystonia (DRD) are thought to be caused by mutations in the GCHI gene; however, by sequencing, mutations are found in only 40% to 60%. Recently, a single report identified, via Southern blot analysis, a large genomic GCHI deletion in a "mutation-negative" case. This report describes four families with DRD, two of which carry large deletions, thus confirming that deletions are an important subtype of GCHI mutations. These deletions were detected by quantitative duplex PCR that is amenable to DNA diagnostics.

Complete cDNA sequence, expression, alternative splicing, and genomic organization of the mouse Nfat5 gene.

Members of the NFAT (nuclear factors of activated T cells) gene family have been investigated in numerous organisms, including man and mouse. All NFATs may be synthesized in several isoforms differing in amino or carboxy termini due to 5' and 3' alternative splicing of the corresponding mRNA. Recently, we mapped the murine Nfat5 gene to chromosome 8D. In the present paper we describe for the first time the complete sequence and primary structure of murine Nfat5, two new spliced isoforms, and the expression of murine Nfat5 in embryonic and adult mouse tissues.

Evaluation of 50 probands with early-onset Parkinson's disease for Parkin mutations.

BACKGROUND: Early onset PD has been associated with different mutations in the Parkin gene, including exon deletions and duplications. METHODS: The authors performed an extensive mutational analysis on 50 probands with onset of PD at younger than 50 years of age. Thirteen probands were ascertained from a registry of familial PD and 37 probands by age at onset at younger than 50 years, blind to family history. Mutational analysis was undertaken on the probands and available family members and included conventional techniques (single strand conformation polymorphism analysis and sequencing) and a newly developed method of quantitative duplex PCR to detect alterations of gene dosage (exon deletions and duplications) in PARKIN: RESULTS: Using this new technique, the authors detected eight alterations of gene dosage in the probands, whereas 12 mutations were found by conventional methods among the probands and another different mutation in an affected family member. In total, the authors identified compound heterozygous mutations in 14%, heterozygous mutations in 12%, and no Parkin mutation in 74% of the 50 probands. We expanded the occurrence of Parkin mutations to another ethnic group (African-American). CONCLUSION: The authors systematically screened all 12 Parkin exons by quantitative PCR and conventional methods in 50 probands. Eight mutations were newly reported, 2 of which are localized in exon 1, and 38% of the mutations were gene dosage alterations. These results underline the need to screen all exons and to undertake gene dosage studies. Furthermore, this study reveals a frequency of heterozygous mutation carriers that may signify a unique mode of inheritance and expression of the Parkin gene.