| Neurogenetic disorders are defined as clinical diseases caused by defects in one or more genes which affect the function of the nervous system. Neurological disorders include a considerable number of neurogenetic disorders:a relatively high proportion of Mendelian single gene inheritance compared with other systems. It is estimated that among the 5,000 known genetic disorders, as many as half of them have important neurologic involvement and more than 300 are primarily neurologic. Many of these neurogenetic disorders manifest themselves early in life leading to a lifelong disability with an enormous burden on affected individuals, their families, and society. The pathological mechanisms of most neurogenetic disorders are incompletely understood. In the past decade considerable progress has been made in cloning the mutation gene of many important neurogenetic disorders, gene diagnosis, and gene therapy.Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of conditions that are characterised by the presence of lower limb spasticity and weakness, which results from the selective degeneration of upper motor neurons (UMNs). The common pathological feature of these conditions is retrograde degeneration of the longest nerve fibers in the corticospinal tracts and posterior columns. Charcot-Marie-Tooth disease (CMT) comprises a group of clinically and genetically heterogeneous peripheral neuropathies. Pathological studies of peripheral nerves show evidence of either demyelinating neuropathy or axonal degeneration. There are evidences that HSP and CMT may share similar mechanisms in the pathogenesis of dying back neuropathies. By understanding the pathways together, we can combine our knowledge of mechanisms, and potentially also treatment strategies, from different axonal disorders.In this study, we performed clinical and genetic studies on two neurogenetic disorder kindreds and identified the causative genes. Through linkage study of a pedigree of HSP with autosomal-dominant inheritance, we mapped the causative gene to 3q24-q26. Screening of candidate genes revealed that the HSP is caused by a missense mutation in the gene for acetyl-CoA transporter(SLC33A1). After cloning of the mutation gene in the SPG42 pedigree, we successfully performed prenatal diagnosis for two family members. In our study, we also identified a novel mutation in GJB1 (c.110delT) in a large Han Chinese family with CMTX. The deletion mutation results in a frameshift beginning at amino acid 37 and also generates a premature stop codon at position 83. As more genes causing neurogenetic disorders are identified, we will have a better understanding of the molecular pathology of these diseases and find potential treatments.Part I Clinical and Genetic Features of a Large Chinese Pedigree with Hereditary Spastic ParaplegiaA family member from a large kindred with HSP contacted us for genetic counseling. The disease of the kindred clearly followed a pattern of autosomal dominant inheritance, though with incomplete penetrance. Of the 57 family members in the pedigree examined,20 individuals were diagnosed as definitely affected,3 as mildly affected and 1 asymptomatic carrier. Genomic DNA was extracted via standard techniques from blood samples.Clinical examinations of 57 subjects in the family were performed by an experienced neurologist. The typical patients presented with hyperreflexia, spasticity of the lower limbs, increased muscle tone, and Babinski sign. The age at onset and severity of the disease varied greatly among the affected patients. The symptoms of the patients progressed slowly, which might go unnoticed for years. No additional neurological symptoms were detected in this family. Two affected family members were further examined by magnet resonance imaging (MRI) and muscle biopsy, and no abnormalities were detected. Therefore, the patients in this pedigree are diagnosed as an uncomplicated form of HSP.Part II A New Locus for Autosomal Dominant Hereditary Spastic Paraplegia Maps to Chromosome 3q24-q26We first tested, by linkage analysis, whether the HSP in this family was caused by a mutation in any of the known HSP loci. Linkage to any of the known 11 AD HSP loci was excluded. Negative LOD scores, as well as obligates recombinants in patients, were observed for all tested loci. On the basis of our exclusion data, we considered this pedigree for a new locus for pure autosomal dominant HSP.After exclusion of the known AD HSP loci, we performed a genome-wide scan using 205 microsatellite markers spaced, on average,15-20 cM apart on 22 autosomes. Samples from 24 family members (14 definitely affected,2 mildly afeected,1 carrier and 7 unaffected spouses) were used for genome-wide scan.Among the 205 markers genotyped, six markers produced positive LOD scores, of which two mapped to chromosome 3q and the other four were on chromosome 4 (D4S2431),6 (D6S1959),7 (D7S2201), and 20 (D20S162). Additional microsatellite markers at a higher density were then used to analyze the regions surrounding these loci. For the regions on chromosomes 4,6,7, and 20, all additional markers analyzed generated LOD scores less than -2. In contrast, seven additional markers genotyped on chromosome 3q all gave LOD scores> 3, with a peak of 5.085 at 0=0 for D3S1746, showing strong evidence of linkage between the chromosomal region and the disease. Multipoint linkage analysis with the use of 11 markers on chromosome 3q reached a peak LOD score between D3S2326 and D3S3053. Haplotype analysis indicated that all of the markers flanked by D3S2326 and D3S3053 co-segregated with the disease. This 22 cM interval flanked by D3S2326 and D3S3053 corresponds to a physical distance of 27.54 Mb. These results clearly establish the existence of a locus for AD HSP within chromosome 3q24-q26. The locus was named as SPG42 by the HUGO nomenclature.PARTâ…¢A Missense Mutation in SLC33A1 Causes SPG42After we mapped the locus for the SPG42 pedigree to chromosome 3q24-q26, we performed candidate cloning for the disease gene. More than 130 genes have been mapped to the 22cM interval region. A group of genes were selected, on the basis of their expression pattern in neuron and their possible gene function in relevance to the disease, for mutation analysis. No noticeable mutation was found in the first five genes sequenced PFN2, SCHIP1, SLITRK3, MYNN, and CLDN11. A heterozygous c.339 T>G transversion in exon 1 of SLC33A1 gene was found by sequencing analysis. The c.339T>G transversion in exon 1 resulted in a missense mutation that changed serine to arginine at the codon 113 (p. S113R). We used an allele-specific tetra-primer PCR assay and sequencing analysis to determine whether the mutant allele cosegregated with the disease. The mutant allele was present in all of the definitely affected patients tested but not in any of the unaffected family members. And the mutatnt allele was excluded as a SNP, as it was not detected in a panel of 200 healthy controls. SLC33A1 encodes the acetyl-CoA transporter, which consists of 549 amino acids and contains multiple transmembrance domains, with a leucine zipper domain. In silico topology prediction, with SOSUI software, it is predicted that the S113R mutation would dislodge the second transmembrane domain, leading to its expulsion from the membrane bilayer. Furthermore, it would also cause all domains starting from 113R to be placed in an opposite orientation across the membrane compared with the normal transporter. We confirmed that the subcellular localization of SLC33A1 didn't change by the S113R mutation by immunofluorescence colocalization. Both the wild type and mutant SLC33A1 protein localized to the endoplasmic reticulum (ER), as evidenced by their co-localization with the ER protein CALNEXIN. Cross-species alignment of the amino acid sequences for SLC33A1 also shows that the serine at position 113 is highly conserved among vertebrates, emphasizing its important role in the function of the protein. Recent research papers demonstrated SLC33A1 is the ER membrane acetyl-CoA transporter, regulating the acetylation status of ER transiting proteins. Our study illustrated a new AD HSP (SPG42) gene SLC33A1. And for the first time, SLC33A1 was discovered as causative gene of a disorder. The findings have provided key insight into underlying causes of other neurodegeneration disorders such as Parkinson's disease and Alzheimer disease and inspired further research aimed at prevention and therapy.Part IV Prenatal Diagnosis of Autosomal Dominant Hereditary Spastic Paraplegia (SPG42) in a Chinese KindredHereditary spastic paraplegias are a highly heterogeneous group of neurodegenerative disorders. The age of symptom onset and degree of severity may vary widely within a given family and between families with the same genetic type of HSP. The diagnosis of hereditary spastic paraplegia (HSP) is typically based upon symptoms, a careful physical examination, family history, and various specialized tests such as MRI, EMG, and muscle biopsy. The diagnosis is often difficult, especially for patients with mild symptoms and asymptomatic carriers. Gene diagnosis is gold standard in diagnosis of HSP.Autosomal dominant HSP is the most prevalent form and represents around 70% of cases. That means each affected person usually has one affected parent. There is a 50% chance that a child will inherit the mutated gene. HSP sometimes have reduced penetrance, which means not all individuals who inherit the mutation present symptoms. In genetic counseling, asymptomatic carrier detection is very important for HSP. Gene diagnosis allows precise prenatal diagnosis and carrier detection of HSP. After cloned the mutation gene in the kindred, we carried out gene diagnosis for all the individuals in the family. Then, we performed prenatal gene diagnosis for two at-risk pregnancies in the SPG42 kindred. The sequencing results of the two at-risk pregnancies showed that neither of the two fetuses inherited the S113R mutation of SLC33A1 from their affected parents. Linkage analysis confirmed our results that the fetuses of the two at-risk pregnancies are not carriers of S113R mutation. By the combined use of linkage analysis and direct sequencing, we successfully carried out prenatal diagnosis of two pregnancies in the SPG42 kindred. The linkage analysis we performed ruled out possible contamination of fetus DNA by the maternal genomic DNA. Our study illustrates the feasibility of preventing HSP by prenatal diagnosis.Partâ…¤Identification of the Mutation for X-linked Charcot-Marie-Tooth Disease in a Large Chinese FamilyCharcot-Marie-Tooth disease (CMT) comprises a group of clinically and genetically heterogeneous peripheral neuropathies diseases. It is characterized by progressive distal muscle atrophy and weakness, sensory disturbance, hyporeflexia, and foot deformities. Most patients displayed phenotypes before 20 years old.CMT can be subdivided into two main types based on electrophysiological and pathological findings:CMT1, the demyelinating form, exhibits moderately to severely reduced motor nerve conduction velocities(the median nerves MNCV<38 m/s), and onion bulb formation on the nerve biopsy; CMT2, the axonal form, shows normal or mildly reduced MNCVs(the median nerves MNCV>38 m/s), and axonal loss on the nerve biopsies. CMT can be transmitted in an autosomal dominant, autosomal recessive, or X-linked pattern. Autosomal dominant CMT1A, which accounts for 40-50% of CMT cases, is caused by a duplication or point mutation of PMP22 gene. X-linked Charcot-Marie-Tooth disease (CMTX), the second most common form, accounts for about 10-20% of all CMT cases. CMTX1 is caused by mutations in the GJB1 gene that encodes CONNEXIN32, accounting for about 90% of all reported cases of CMTX. CX32, a 32-kDa gap junction protein, is required for the transfer of ions and small molecules between cells.A family with a clinical diagnosis of CMT was found in Jining, Shandong China. The family consists of 5 living affected males and 4 mildly affected females. On the basis of clinical and electrophysiological features, CMTX was highly suspected. After carefully clinical examinations and linkage analysis and mutation analysis, we identified a novel deletion mutation in GJB1 in the family. The deletion mutation results in a frameshift beginning at amino acid 37 and generates a premature stop codon at position 83. The mutation should cause a loss of function of CX32. With the isolation of the gene, genetic counseling and precise diagnosis is now possible by molecular analysis.In summary, we identified two mutant genes responsible for neurogenetic disorders with demyelinating and axonal degeneration in this study. These findings provide the first direct evidence that SLC33A1 mutation is the cause of autosomal dominant hereditary spastic paraplegia. In this work, we also provided prenatal diagnosis for individuals in the kindred by analyzing the mutation in the SLC33A1 gene. We identified a novel deletion mutation in GJB1 gene which results in a frameshift beginning at amino acid 37 and generates a premature stop codon at position 83 in a Charcot-Marie-Tooth disease family. These results advanced our understanding of the etiology of HSP and CMT and the pathological mechanisms of neurogenetic motor disorders. |
PDF Full Text Request