Utility of MLPA in deletion analysis of <Emphasis Type="Italic">GCH1</Emphasis> in dopa-responsive dystonia

We applied multiple ligation-dependent probe amplification (MLPA) to patients from three families with characteristic dopa-responsive dystonia (DRD) but no base change in the gene GCH1. We found a complete deletion of GCH1 in affected members of family 1, and partial deletions in affected individuals of family 2 (exons 4-6) and of family 3 (exons 2-6). The findings were confirmed by quantitative real-time PCR. Our investigations demonstrate the utility of MLPA for routine deletion analysis of GCH1 in DRD patients with no sequence changes in this gene.     

GCH1 mutation and clinical study of Chinese patients with dopa‐responsive dystonia

Dopa‐responsive dystonia (DRD) is typically caused by heterozygous mutations in GTP cyclohydrolase 1 gene (GCH1). Our aim was to investigate the clinical and genetic features of Chinese DRD patients. We analyzed a cohort of Chinese DRD patients' clinical data. Mutation of the GCH1 gene was screened by direct sequencing. Additionally, multiplex ligation‐dependent probe amplification (MLPA) assay targeting the GCH1 and the TH gene to evaluate large exon deletion or duplicate mutation of the genes were performed in point mutation‐negative patients. Ten sporadic DRD patients and two pedigrees including six patients were included in the study. The onset age ranged from 3 to 15 years old. All patients initially presented with walking problems due to lower limb dystonia. The delay between onset and diagnosis ranged from 1 to 42 years old. The symptoms were completely or near‐completely abolished with low dose levodopa treatment (dosages ranged from 25 mg to 400 mg/day). Direct sequencing in 14 patients found two known mutations (Gly203Arg in exon 5 in four unrelated patients and Met102Lys in exon 1 in one patient) and one new mutation (Thr186Ile mutation in exon 5 in two unrelated pedigrees). A heterozygous exon 2 deletion in the GCH1 gene was found in one of three point mutation‐negative patients by MLPA analysis. Our clinical findings in DRD patients were consistent with other studies. GCH1 gene mutations were quite common in Chinese patients. MPLA should be performed in routine deletion analysis of GCH1 in point mutation‐negative DRD patients. © 2010 Movement Disorder Society     

Novel non‐sense GCH1 mutation in a South African family diagnosed with dopa‐responsive dystonia

Background: Dopa‐responsive dystonia (DRD), a movement disorder characterized by onset in early childhood and a dramatic response to low doses of levodopa, has been shown to be caused by a number of different mutations in the GCH1 gene. Methods: We identified a South African family which presented with DRD in three family members. Polymerase chain reaction (PCR) primers were designed to span all six exons of GCH1 and the PCR products were screened for pathogenic mutations using direct sequencing. Results: A novel non‐sense mutation (c.233delT; p.I78fsX79) was identified in the DRD patients, which would produce a markedly truncated protein of only 78 amino acids. This mutation was also present in a number of asymptomatic family members. Conclusions: A novel non‐sense mutation in the GCH1 gene can be associated with DRD and reduced penetrance in South African patients.     

Novel GCH1 mutation in a Brazilian family with dopa‐responsive dystonia

Dopa responsive Dystonia (DRD) was first described in 1971 and typically begins at childhood with gait dysfunction caused by foot dystonia progressing to affect other extremities. There is marked diurnal fluctuation and sustained improvement of symptoms with low dose levodopa therapy. Heterozygous mutation of the gene GCH1 has been shown to cause DRD. We studied GCH1 in nine patients with DRD from six families of Federal University of Minas Gerais Movement Disorders Clinic. We identified three mutations; two affected siblings carried a novel T209P mutation and two siblings from another family were compound heterozygous carriers of Met211Val and Lys224Arg mutations. To our knowledge this is the first report of GCH1 mutations underlying DRD in patients from Brazil. © 2007 Movement Disorder Society     

Occurrence of GCH1 gene mutations in a group of Indian dystonia patients

The aim of this study is to examine the role of GCH1 among Indians affected with dopa responsive dystonia (DRD) and early onset Parkinson’s disease (EOPD). The patients (n?=?76 including 19 DRD and 36 EOPD) and controls (n?=?138) were screened for variants in GCH1 by PCR amplification of exons, splice junctions and 1?kb upstream region followed by SSCP and DNA sequencing. Four novel variants (p.Met1Val, p.Val204_205del, IVS3+68A>G, and IVS5?6T>G) were identified in 10 patients but not in the controls. In addition to two nonsynonymous changes, identified in four DRD patients in heterozygous condition, one intronic variant (IVS5?6T>G) could be linked to pathogenesis of the disease since it has the potential of altering the splice site as assessed by in silico analysis. Patients carrying different nonsynonymous variants had remarkable variation in clinical phenotype. Consistent with earlier reports, severity of clinical phenotype and the age of onset varied among family members harboring the same mutation. No mutation was detected in the EOPD patients. Three novel mutations in GCH1 gene have been found and are shown to be associated with variable clinical phenotypes mostly within the spectrum of DRD. The mutations identified represent 15.79% (3/19) of east Indian DRD patient cohort.     

Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction–restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.     

Dopa-responsive dystonia due to a large deletion in the GTP cyclohydrolase I gene

Although it is assumed that most patients with autosomal dominant dopa-responsive dystonia (DRD) have a GTP cyclohydrolase I dysfunction, conventional genomic DNA sequencing of the gene (GCH1) coding for this enzyme fails to reveal any mutations in about 40% of DRD patients, which makes molecular genetic diagnosis difficult. We found a large heterozygous GCH1 deletion, which cannot be detected by the usual genomic DNA sequence analysis, in a three-generation DRD family and conclude that a large genomic deletion in GCH1 may account for some "mutation-negative" patients with dominantly inherited DRD.     

A novel missense mutation in GTP cyclohydrolase I (GCH1) gene causes dopa‐responsive dystonia in Chinese Han population

Background: Dopa‐responsive dystonia has been shown to be caused by a number of different mutations in the GCH1 gene. Up to now, only several genetic studies of Chinese patients with Dopa‐responsive dystonia (DRD) have been reported. Methods: We performed a genetic analysis by amplifying the entire coding region of GCH1 gene and direct sequencing in four DRD families from mainland China. Results: A novel missense mutation, Gly155Ser, has been identified in a sporadic case from a consanguineous marriage family. Furthermore, two known mutations, Met137Arg and Gly203Arg, have also been detected in the other families. Conclusions: A novel missense mutation in the GCH1 gene can be associated with DRD. Our findings further expanded the mutational spectrum of GCH1 gene associated with DRD.     

多巴反应性肌张力障碍患者的GCH1基因突变检测

目的分析多巴反应性肌张力障碍(dopa responsive dystonia,DRD)患者的GCH1基因突变。方法我们抽取21例来自医院门诊及住院的散发型多巴反应性肌张力障碍患者的肘静脉血,并提取外周血全基因组DNA。Primer3设计GCH1基因6个外显子的引物,PCR扩增GCH1基因的外显子及周边部分内含子序列,并对PCR产物进行测序。测序结果与正常序列进行比对,发现碱基变异后进行序列分析以确定是否多态。结果成功扩增21位DRD患者GCH1基因的6个外显子。经过分析,GCH1基因外显子序列未发现基因突变。仅在4个患者的1号外显子发现1个单核苷酸多态(SNP)c.68CT,该SNP没有产生氨基酸的改变。结论本地区多巴反应性肌张力障碍患者未发现GCH1基因突变,DRD患者可能存在其他致病基因。GCH1基因突变检测目前仍不能作为早期诊断的依据。     

Are dopa-responsive dystonia and Parkinson’s disease related disorders? A case report

Objectivel-Dopa-responsive dystonia (DRD) is a hereditary dystonia characterized by an excellent response to low dosages of levodopa. DRD patients may also develop Parkinsonism which resembles idiopathic Parkinson’s disease. In classical DRD no changes in the dopaminergic uptake have been observed.MethodsA 65-year old woman presented with clinically remarkably slowly progressing Parkinson’s disease (PD) without any dystonic signs and excellent response to dopaminergic medications. We obtained a [¹²³I] FP-CIT-SPECT (DaTSCAN?) in order to elucidate a striatal dopaminergic deficit.ResultsWe found a reduced uptake in the [¹²³I] FP-CIT-SPECT (DaTSCAN?) contralateral to the more affected body side. Additionally, the patient showed a heterozygous deletion of the GHC1 gene.ConclusionsPatients with mild parkinsonian symptoms, excellent response to low dosages of dopaminergic drugs and a reduced dopamine-transporter uptake in [¹²³I] FP-CIT-SPECT might more commonly be GCH1 mutation carriers than has previously been supposed. PD patients with a positive family history of DRD and combination of these clinical symptoms should be offered genetic counselling and testing for GCH1.     

Low frequency of GCH1 and TH mutations in Parkinson's disease

BackgroundThe causes of Parkinson's disease (PD) are unknown in the majority of patients. The GCH1 gene encodes GTP-cyclohydrolase I, an important enzyme in dopamine synthesis. Co-occurrence of dopa-responsive dystonia (DRD) and a PD phenotype has been reported in families with GCH1 mutations. Recently, rare coding variants in GCH1 were found to be enriched in PD patients, indicating a role for the enzyme in the neurodegenerative process.MethodsTo further elucidate the contribution of GCH1 mutations to sporadic PD, we examined its coding exons in a targeted deep sequencing study of 509 PD patients (mean age at onset 56.7 ± 12.0 years) and 230 controls. We further included the tyrosine hydroxylase gene TH, also known to cause DRD. Gene dose assessments were performed to screen for large copy number variants in a subset of 48 patients with early-onset PD.ResultsNo putatively pathogenic GCH1 mutations were found. The frequency of rare heterozygous variants in the TH gene was 0.69% (7/1018) in the patient group and 0.22% (1/460) in the control group (p = 0.45).ConclusionsPrevious studies have found that coding variants in the GCH1 gene may be considered a risk factor for PD. Our study indicates that mutations in GCH1 are rare in late-onset PD. Several patients carried heterozygous variants in the TH gene that may affect protein function. Our study was not designed to determine with certainty if any of these variants play a role as risk factors for late-onset PD.     

Heterozygous mutation in 5’-untranslated region of sepiapterin reductase gene<Emphasis Type="Italic"> (SPR)</Emphasis> in a patient with dopa-responsive dystonia

The search for mutations in genes coding for components of the biopterin pathway other than GTPCH1 revealed a mutation in the gene coding for sepiapterin reductase ( SPR) in 1 of 95 patients with GCH1-negative dopa-responsive dystonia (DRD). The mutation detected in SPR is a GA transition at position –13 of the untranslated region of the gene. This resulted in drastically reduced activity of sepiapterin reductase in the patients fibroblasts. The findings indicate that haploinsufficiency of SPR can be a rare cause of DRD.     

A new mutation of GCH1 in triplets family with dopa‐responsive dystonia

Background: Dopa‐responsive dystonia (DRD) is associated with mutations of the GCH1. We first report four female siblings with DRD from one family, including three monozygotic triplets patients clinically and genetically. Methods: We performed GCH1 analysis by direct sequencing of PCR product amplified with primers designed to cover the entire exons of GCH1 in those four patients and their mother. Results: In all four patients with DRD, a new frameshift mutation (c.729delG; p.A190fsX191) was identified in the exon 5 of GCH1. Conclusions: The frameshift mutation results in truncated GCH1 protein which is suspected to result in loss of function of the catalytic GTP‐cyclohydrol domain.     

Frequency of GCH1 deletions in Dopa-responsive dystonia

We performed a systematic study on the frequency of point mutations and deletions of the gene GCH1 in dopa-responsive dystonia (DRD). A total of 136 dystonia patients were studied. Fifty of these had a sustained response to oral L-Dopa therapy (group 1: definite diagnosis of DRD), whereas the response to L-Dopa was incomplete or not tested in 86 patients (group 2: possible diagnosis of DRD). We found a GCH1 point mutation in 27 patients of group 1 (54%) and in four patients of group 2 (5%). Of these, nine single and one double mutation have not been described before. GCH1 deletions were detected in four patients of group 1 (8%) and in one patient of group 2 (1%). Among GCH1 point-mutation-negative patients with a definite diagnosis of DRD (group 1), the frequency of GCH1 deletions was 17% (4/23). We conclude that GCH1 deletion analysis should be incorporated into the routine molecular diagnosis of all patients with DRD with a sustained response to L-Dopa.     

Identification of Alu elements mediating a partial PMP22 deletion

Hereditary neuropathy with liability to pressure palsies (HNPP) is most frequently caused by deletion of a 1.4-Mb region in chromosome 17p11.2-12 including the peripheral myelin protein 22 (PMP22) gene. Smaller deletions partially affecting the PMP22 gene are less frequently observed. We identified in a HNPP patient a deletion of the 5′ region of PMP22 including non-coding exon 1, coding exons 2 and 3, whereas, exons 4 and 5 were present. PMP22 exon 3- and 4-specific qPCR resulted in a deletion of one exon 3 allele but in the presence of 2 exon 4 alleles. SNP analysis revealed the presence of heterozygosity for PMP22 coding exons 4 and 5. Finally, MLPA specific for the CMT1A region defined this deletion for the entire 5′ region of PMP22 (exons 1, 2 and 3). These partial HNPP deletions may be missed by other techniques, e.g., STR marker analysis. Alu elements have been reported to mediate non-allelic recombination events. Bioinformatic analysis revealed 12 Alu elements flanking in close neighbourhood the estimated 40-kb deletion region as candidates for recombination events. PCR primers were designed to identify a breakpoint-spanning product including the respective Alu elements. PCR-driven identification of a junction fragment was successful with AluJo–AluSq and AluYb9–AluSq specific primer pairs comprising the same intronic region of PMP22. Sequence analysis of these breakpoint-overlapping PCR fragments revealed a 29-bp motif including a chi-like sequence (GCTGG) present both in the AluYb9 and the AluSq element. These data confirm that low-copy repeats (LCRs) mediate non-allelic homologous recombinations (NAHR).     

应用多重连接依赖性探针扩增定量技术检测假肥大型肌营养不良重复突变及携带状态

目的 应用多重连接依赖性探针扩增(MLPA)技术对假肥大型肌营养不良(DMD及BMD)患者及其家系成员进行dystrophin基因分析,探讨MLPA定量技术在本病重复突变及携带者检测中的优势.方法 以355例DMD及BMD患者、46名缺失型患者之母和8名重复型患者之母为研究对象,应用MLPA技术对dystrophin基因全长外显子进行分析,对于单一外显子缺失的样本采用PCR及测序进行验证.结果 经MLPA分析,全部355例患者中190例为dystrophin基因缺失型患者,在其余非缺失型患者中检测出34例重复型突变.此外,在46名缺失型患者的母亲中发现了28名携带者,在8名重复型患者的母亲中发现了6名携带者,两组患者母亲携带者频率差异无统计学意义.经过测序验证,在1例单一外显子缺失的患者中发现17号外显子存在AGGGAACAGATCCTGGTAAAGCA小片段缺失.结论 与传统的定量方法相比,MLPA定量技术可对DMD及BMD患者全长外显子区域同时进行缺失、重复分析,并能对患者家系成员的携带状态进行判定.此外,MLPA检测结果受模板DNA的浓度及纯度影响较小.     

Genetic heterogeneity in 30 German patients with oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is due to short elongations of a polyalanine tract in the poly(A) binding protein nuclear 1 (PABPN1) gene. Originally GCG expansions in which (GCG)₆ is extended to (GCG)_7–13 were found. Subsequently five further genotypes with additional GCA– and GCG–trinucleotides were identified in single OPMD patients. This indicated larger genetic heterogeneity and showed that unequal crossing–over and not replication slippage must be the underlying mechanism of elongation.We performed sequencing of the PABPN1 gene in 30 German OPDM index patients to determine the exact genotype. The original GCG expansion ranging from (GCG)₈ to (GCG)₁₁ was found in 22 patients. In 8 patients, however, three different elongated alleles other than classical (GCG)_7–13 were observed. Two of these genotypes had already been identified in Japanese patients. One genotype was recently identified showing (GCG)₆ followed by inserted (GCA)₃GCG in four unrelated patients. This study further supports the theory of unequal crossing over as the molecular mechanism leading to elongation. It shows that other genotypes than classical (GCG)_7–13 are rather common in German OPMD patients. The data imply that there is no single founder effect in German OPMD patients. Drs. Müller and Deschauer contributed equally to this work.     

Phenocopies in a large GCH1 mutation positive family with dopa responsive dystonia: confusing the picture?

BACKGROUND: Dopa responsive dystonia (DRD) is a disorder characterised by childhood onset dystonia but a wide range of clinical presentations has now been described. OBJECTIVE: To study a large Canadian family with presumed DRD. METHODS: The clinical features of the family were collected before molecular genetic mutational analysis. RESULTS: All nine individuals in whom a clinical diagnosis of DRD was definite or probable were heterozygous for a GCH1 gene deletion. However, eight of nine possibly clinically affected members did not carry the GCH1 mutation. CONCLUSIONS: Great care must be taken in diagnosing DRD even in families with the classic phenotype, because of potential phenocopies of the disease.     

A new complex homozygous large rearrangement of the PINK1 gene in a Sudanese family with early onset Parkinson’s disease

PARK2 and PINK1 gene mutations are involved in recessive early onset Parkinson’s disease (EOPD). In order to determine the causative mutations in three affected sibs from a consanguineous Sudanese family with EOPD, multiplex ligation-dependent probe amplification was performed and revealed that the patients were homozygous for a deletion of PINK1 exons 4 to 8. Breakpoint analysis revealed a complex rearrangement combining a large deletion and the insertion of a sequence duplicated from the DDOST gene intron 2, located near the PINK1 gene. As breakpoint sequences displayed only three base pairs of homology, this rearrangement may result from Fork Stalling and Template Switching mechanism. This third large rearrangement of PINK1 enlarges the mutation spectrum and, together with recent published data in Tunisian patients with EOPD, points out that PINK1 gene analysis, including search for large rearrangement, should be considered in early onset recessive PD patients, particularly those from Arab origin.