Identification Of The Causative Mutation For Two Families With Hereditary Neuromuscular Rare Diseases

Neurogenetic disorders are defined as clinical diseases caused by defects in the numbers, the structure or the function of germ cells or oosperm mainly resulting in neural dysfunction of the developing individual. Neurogenetic disorders, which constitute an important part of neurological disorders and medical genetics, are the most vital type in all of the human genetic diseases. More than a half of found genetic diseases involve in nervous system. There are a great variety of neurogenetic disorders, most of which are familial and livelong, but the pathogenesis and mechanism of many of them is never clearly explored. Neurogenetic disorders may manifest themselves at any time of life but many of these do before30. Currently, many neurogenetic disorders have never obtained affective therapy, a high proportion of which leading to teratogeny, disability or death, and therefore, the outcome is adverse. In the past decade or more, considerable progress has been made in cloning the mutation gene of many important neurogenetic disorders, gene or gene-product diagnosis, and gene therapy and will make the research in neurogenetic disorders better.Charcot-Marie-Tooth disease (CMT) comprises a group of genetically heterogeneous peripheral neuropathies. CMT is the most common type of genetically peripheral neuropathies with the incidence of1/2500. CMT is a type of neurogenetic disorders and myelin sheath and the pathologic changes are mainly the demyelination and degeneration of neural axis. Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neuropathies with the main presence of lower limb spasticity, weakness and spastic gait. The incidence is2-10/100000. HSPs are similar with CMT and both are neurogenetic disorders affecting neural axis. Differently, HSPs mainly affect neural axis of upper motor neurons but CMT mainly affect ones of lower motor neurons and sensory nerve. There are increasing evidences that HSP and CMT may share similar mechanisms in the pathogenesis. It is meanwhile to study the mechanism of the two axonal disorders for better understanding the maintenance of the normal function of neural axons.In the study, we conducted a research on molecular genetics of the two collected families from Shandong province, and detected genes associated with diseases and identified the mutation. The causative gene identification will lay solid foundations for revealing the mechanism of the above two diseases. And, we can offer prenatal diagnosis for the other members of the two families in order to prevent the birth of the unhealthy babies and improve the quality of the population.Part I Identifying the causative mutation responsible for an X-linked Charcot-Marie-Tooth familyCharcot-Marie-Tooth disease (CMT) comprises a group of highly genetically heterogeneous peripheral neuropathies. The characters include progressive distal muscles atrophy and weakness of limbs, especially apt to affect lower limbs more worse; the formation of claw foot, equinovarus and perpendicular foot; the worse abnormality of the patients may be like Crane legs or champagne bottle; sensory disturbance; hyporeflexia. CMT is the most common type of genetically peripheral neuropathies with the incidence of1/2500. The inheritance pattern includes autosomal dominant (AD), autosomal recessive (AR) and X-linked. The onset age of the affected is early and most affected displayed phenotypes before20years old.According to electrophysiological and pathological findings, CMT can be subdivided into two main types:CMT1(the demyelinating form) and CMT2(the axonal form). CMT1(the demyelinating form) exhibits moderately to severely reduced motor nerve conduction velocities (the median nerves MNCV<38m/s), and onion bulb formation on the nerve biopsy; CMT2(the axonal form) shows normal or mildly reduced MNCVs (the median nerves MNCV>38m/s), and axonal loss on the nerve biopsies. Other studies showed there was another form-CMTDI, which shows the median nerves25m/s<MNCV<45m/s.CMT1A, which accounts for40-50%of CMT cases, is the most common type of autosomal dominant CMT and caused by a duplication mutation, the most common mutation, or point mutation of PMP22gene. The second most common form: X-linked Charcot-Marie-Tooth disease (CMTX), accounts for about10-20%of all CMT cases.90%of CMTX, CMTX1, is caused by mutations in the GJB1gene, lying on Xq13.1, which encodes CONNEXIN32(CX32). CX32, a32-kDa gap junction protein, is required for the transfer of ions, small molecules and signal molecules between cells and expresses both in Schwann cells and oligodendrocytes.We found a large CMT family with5generations in Linyi, Shandong province and obtained blood of14members. There are12affected male members without male-male transmission, and female ones with the mutation have no symptom. On the basis of clinical and electrophysiological features, CMTX was highly suspected. After carefully clinical examinations, assistant examination and mutation detection of the coding sequence of GJB1, we identified a missense mutation in GJB1in the family. The missense mutation (c.614A>G) changed asparagine to serine at the205th amino acid. So, we considered that the mutation should results in the dysfunction of CX32. With the identification of the causative mutation of the gene, genetic counseling and precise diagnosis for the family members is now possible by molecular genetic analysis. Part II Identifying a novel causative mutation responsible for an AD-HSP familyWe collected a large hereditary spastic paraplegia family with four generations in Weihai, Shandong province. There were4patients who had typical symptom, and the onset age was about45. There were affected members of both genders and the members of female patients were more than the male ones. According to the analysis of the pedigree, we presumed the inheritable pattern of the HSP family was autosomal dominant, the most common pattern.On the basis of clinical examinations, the characteristics of the HSP family were as follows:①he onset age of the affected members was about45;②) The symptoms of the patients were progressive, which might go unnoticed for years, and the severity of the disease varied greatly among the affected patients;③The typical patients presented with hyperreflexia, spasticity of the lower limbs, increased muscle tone, and Babinski sign;④No other abnormalities were detected when the patients were examined by magnet resonance imaging (MRI) and muscle biopsy and others. From the above, we can conclude that the pattern of this pedigree was a pure form of HSP.We tested a known causative gene, SPASTIN gene which is the most common causative gene, as a candidate one by linkage analysis. We selected two microsatellite markers (short tandem repeat, STR)-D2S2351(lying in the upstream0.2Mb) and D2S2347(in the downstream0.9Mb of SPAST)-both of which had high heterozygosity in the population according to Marshfield genetic maps. And then, we performed the genotyping and calculated logarithm of odds (LOD) score in order to exclude or confirm the candidate gene. The family members had no polymorphism at the site of D2S2351. The family members available for linkage analysis were so fewer that we were unable to get a high LOD score at D2S2347. Although we could not confirm that SPAST was the disease gene, we could also not exclude it as a candidate causative gene.We performed the amplification of the entire exonic sequences of SPASTJN gene by Polymerase Chain Reaction (PCR) and direct sequencing. Result revealed a novel14-bp heterozygous deletion (c.1630-1643delTACTCAGGAAGTGA) in exon15of SPASTIN gene, which led to a frameshift mutation and premature termination of translation (p.tyr544profsX28). The mutation is co-segregated with the phenotype in the family. The14-bp heterozygous deletion was precisely defined by subcloning PCR products in E. coli and sequencing.Mutation (p.tyr544profsX28) between amino acids544and571, with a premature termination is predicted to disrupt the highly conserved functional domain (the AAA cassette), which includes the predicted ATP binding and hydrolysis sites. The likely pathogenic mechanism for the frameshift mutation was haploinsufficiency, which appears to lead to unstable mRNA levels and proteins absent or expressed at low levels.In summary, we reported two causative mutations resulting in two neurogenetic disorders in this study:GJBI mutation (c.614A>G) is the cause of the Charcot-Marie-Tooth disease in the CMTX family; a novel14-bp heterozygous deletion (c.1630-1643delTACTCAGGAAGTGA) in exon15of SPASTIN gene is the causative mutation of the hereditary spastic paraplegia family, which led to a frameshift mutation and premature termination of translation (p.tyr544profsX28). We can offer genetic counseling and prenatal diagnosis for the two family members in order to prevent the birth of unhealthy babies. And all of the results made it better for us to understand the diagnosis, the pathology and the mechanism of CMT and HSP and provided important reference value for other neurogenetic diseases.