Identification Of The Causative Mutations For Two Families With Monogenic Diseases

Genetic disease is caused by one or more genetic problems of genome, which is often determined at birth (congenital), can also be an acquired disease. The pathogenesis are entirely determined by genetic factors, whose obvious symptoms often appears in several years or even decades later, Such as hereditary spastic paraplegia, hereditary pseudo progressive rheumatoid dysplasia, poly cystic kidney disease, etc.. Most genetic diseases are quite rare, probably, the incidence rate is one in several thousand or several million.Single-gene genetic disease is defined by a single gene mutation leading to disease. There have been more than 6500 kinds of single-gene disorders found up to now, which can be passed to offspring in several ways.Autosomal dominant genetic disease locates on autosomal alleles. Even one mutation can result in disorders, whose known amount has reached more than 17OO, such as familial multiple colon polyps. Its pedigree characteristics relates to gender, the equal incidence opportunities for men and women, a party of patients’parents always tend to be patient and successive generations suffering. Sometimes, mutant autosomal dominant genes don’t lead to diseases because of the environmental aspect (incomplete penetrance). The penetrance of autosomal dominant disease was 60% -90%.Autosomal recessive genetic diseases appear only when their causative genes are homozygous, which means that most of recessive genetic disease patients are the offspring of two carriers. About 1,200 kinds of these diseases have been identified, such as congenital deaf, albinism, phenylketonuria. Its pedigree characteristics are the equal incidence opportunities for men and women, discontinue generations suffering, greatly increasing morbidity of recessive genetic disease in inbreeding.Sex-linked genetic diseases include X-linked genetic diseases and Y-linked genetic diseases. X-linked recessive genetic disease is more common. The disease gene on the X chromosome, is recessive traits, and women are only carriers. X-linked dominant genetic disease is rare, with a high incidence and light condition on women. Y-linked genetic disease is very rare, which could only be passed to son.Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary kidney disease, whose main features are gradually increasing bilateral renal cysts leading to kidney failure ultimately. Its prevalence worldwide is between 1/400 to 1/1000, and accounted for 5%-10% of patients with advanced renal transplant patients. There will be some symptoms besides kidney, such as other epithelial organs, liver and pancreatic cysts. In addition, some ADPKD patients will exhibit some connective tissue defects, such as intracranial aneurysms.Spinocerebellar ataxia (SCAs) are a class includes a variety of subtypes, including neurodegenerative diseases with a high degree of genetic heterogeneity, whose onset often in adulthood. Common sites suffering from SCAs include major nervous system (cerebellum, brainstem, spinal cord, peripheral nerves, etc.) and the site out of nervous system (eyes, bones, heart, etc.). Clinical manifestations is complex and diverse. Shrinking cerebellar cortex are often found in CT.In our study, we collected two pedigrees with autosomal dominant inheritance of a single gene, in which molecular genetics research about both single-gene genetic diseases above-mentioned were performed to examine the genes involved in pathogenesis and identified the causative mutation. Our studies aim to lay a groundwork for looking into the physiological mechanisms of disorder above-mentioned. Available Genetic counseling and prenatal diagnosis are also our goal for this family members.Part 1 Disease-causing mutation analysis of a dominant polycystic kidney familyPolycystic kidney disease (PKD) is a serious life-threatening or even lethal single gene disorders. Its main clinical symptoms are many fluid-filled cyst in renal. Polycystic kidney disease patients showed progressive cyst formation and enlargement, kidney enlargement, eventually developing advanced kidney failure. The disease is divided into autosomal dominant (ADPKD) and autosomal recessive (ARPKD).Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary kidney disease whose main features are gradually increasing bilateral renal cysts leading to kidney failure ultimately, with an incidence between 1/500 to 1/1000. Its Clinical manifestations include the cysts of other epithelial organs, such as the liver, spleen. Patients also showed connective tissue defects, such as intracranial aneurysm, heart valve abnormalities, dissection, abdominal hernia. Hypertension is a very common symptom of ADPKD.Alport Syndrome (AS) is an inherited kidney disease, whose incidence is at least 1 in 50,000. It is often in childhood onset and showed microscopic hematuria, by age increasing macroscopic hematuria, proteinuria, renal dysfunction, and ultimately renal failure in adulthood. Some patients have symptoms besides the kidney, including gradual deafness, visual impairment. Alport syndrome is mainly caused by mutations in the X-linked COL4A5 gene (85%), which is a dominant pathogenic. The remaining 15% of Alport syndrome is attributed to autosomal recessive mutations.We found a pedigree with 3 generations family members, five known patients in nanfang hospital, whose polycystic kidney disease are preliminary diagnose with autosomal dominant disease. We made a clinical analysis of the family and related tests, collected DNA samples of family members, and get the first certificate of kidney tissue samples. Through the probands of next-generation sequencing we get the possible pathogenic PKD1 mutation, this mutation to shift mutation and patients with family in the separation. At the same time in the family found a were annotated for the pathogenicity of the COL4A5 mutations G953V rs78972735, this gene mutation will lead to more serious alports syndrome.Through the comparison of the PKD1 gene encoding protein PC1 first differences syndrome and normal tissue, the expression of patients with advanced renal failure found in PC1 increased by about 1.7 times; immunohistochemistry also showed that in the patients with dilated tubules have significantly increased the expression of Ki67 positive cells. At the same time to improve and increase the proportion of pS6/S6 tips the patients in the tubular high value-added state; but TUNEL test showed that the apoptosis of renal cells of patients also increased significantly this indicates that patients with kidney tissue in a high and high proliferation apoptosis remodeling. Anastomotic fibrosis increased expression of some genes related to the mechanism of reconstruction of anti and advanced kidney the clinical manifestations of COL4A5 carriers. Through analyst system, we found that the COL4A5 mutation in the family did not cause Alport syndrome, and A bioinformatic prediction contradictions; analysis report of this mutation literature we found only one case of the COL4A5 separate mutations in patients, patients than the usual point mutation in patients with more serious and in the survey did not detect the gene rearrangement. So, I think separate COL4A5 G953 V mutation may not pathogenic.The discovery of a new PKD1 mutation in the family provides convenience for the future genetic counseling and prenatal diagnosis of the family.Part 2 Identification of the Causative Mutation for a Spinocerehenar ataxia FamilieSpinocerehenar ataxia(SCAs), is a kind of neurodegenerative disease including many subtypes. SCAs is a disease of complicated and vrious clinical manifestations;its lesions includes from major nervous system (cerebellum, brainstem, spinal cord, peripheral nerves, etc.) to outside the nervous system (eyes, bones, heart, etc.). The main clinical features of SCAs are:early insidious onset, walking gait disturbance, limb shaking, slow movement and deteriorated accuracy response; in the mild,it progressively develops into ambiguous pronunciation when speaking, weak tone control, irregular eye movements, images tend to overlap, worse muscle incongruity, unable to write, sometimes feel difficulty swallowing; when the late, very unclearly speaking not even language, limb weakness, can not stand, to rely on a wheelchair, the gradual decline in the ability to understand, finally lost consciousness, lethargy. Imaging mainly for shrinking cerebellar cortex.SCAs is a disease of complicated and vrious clinical manifestations, and the only exact and effective method of classification is genotyping by the mode of inheritance, SCAs can be divided into autosomal dominant, autosomal recessive and X-linked recessive. Among the most common —autosomal dominant, so far, has been located out about 35 sites, and was classified into three major types:(1) the coding region of CAG trinucleotide repeats cause an increase in poly glutamine ataxia, including SCA3, SCA6 and SCA7; (2) an increase in non-coding regions repeat cause of ataxia; (3) conventional mutation (frameshift, missense mutations, nonsense mutations, cut mutation, etc.) and copy number variation caused by ataxia. Autosomal recessive SCAs is relatively rare,and the mainly of which —Freidreich disease is relatively common in the West,while the domestic is rare.But even found so many disease-causing genes, there are still about 40% of patients with SCAs can not be found the cause of the disease, indicating the presence of disease-causing genes are still undiscovered, and almost every year new virulence genes were found.We have found a SCAs family in the ZhuJiang Hospital. There are four patients with obvious dominant inheritance in the family. The age of onset is about 40 years old of patients with typical symptoms of spinocerebellar ataxia in the family.Walking proband initially unexplained instability when walking and standing, the bilateral body down, walk slowly, difficulty standing, followed by worsening symptoms. Lower limbs superficial sensory loss, upper limbs radial periosteum, biceps, triceps reflex. Lower limbs knee, Achilles tendon reflex. MRI showed:bilateral cerebellar atrophy, inflammation around the waist 3/44/5 lumbar intervertebral disc endplates.After studying the results of the patient’s symptoms and SCAs epidemiological investigation in China, we have decided to give priority screening mutations of SCA3, SCA1, SCA3, SCA6, SCA7 in the family. In subsequent electrophoresis and capillary electrophoresis, we have found that the CAG copy number of patients exceeds the normal threshold— Ⅲ1:27/68 times; Ⅲ3:27/68 times, while the similar age of family members don’t. Meanwhile, we have found three potential patients who have not yet reached the age of onset:Ⅲ4:14/68 times; Ⅳ8:14/68 times; Ⅳ10:29/71 times. Our findings have laid the foundation for genetic counseling and prenatal diagnosis of the family members later.