Mutation Detection In Chinese Patients With Glycogen Storge Diseases Multation Analysis Of The ELN Gene In A Child With Mild Congenital Cutis Laxa

Paper 1:Mutation detection in Chinese patients with glycogen storage diseasesGlycogen storage diseases (GSDs) are a group of inherited diseases caused by abnormalities of the enzymes that regulate the synthesis or degradation of glycogen. They are divided into 13 types from 0 to XII according to the differences in enzyme deficiency and the affected tissues. The biochemical deficiency of GSD is the excessive deposit of glycogen in liver, muscle and kidney. The overall GSD incidence is estimated to be 1 case per 20,000-43,000 live births and the most common types are type I, type III and type IX. Most types of GSDs are inherited in autosomal recessive patterns except for GSD IX which is an X-linked GSD and is caused by the deficiency of hepatic phosphorylase kinase activity. Our study focused on patients affected with GSD I and GSD III in Chinese population. These cases are suspected as GSD I or GSD III by pediatrician of the Peking Union Medical College Hospital on the basis of clinical and biochemical findings. The present study aimed at finding pathogenic mutations, providing definite diagnosis and prenatal diagnosis for patients with GSD I and GSD III.Part I:Mutation analysis of the AGL gene in Chinese patients with glycogen storage disease type IIIGlycogen storage disease type III (GSD III, MIM:232400) is a rare genetic disorder of glycogen metabolism inherited as an autosomal recessive trait. It is caused by the deficiency of the glycogen debranching enzyme (AGL), which has two independent catalytic activities:amylo-1,6-glucosidase (EC 3.2.1.33) and 4-alpha-glucanotransferase (EC 2.4.1.25). GSD III can be classified into four subtypes (a, b, c and d) according to the different affected organs and loss of different catalytic activities of the glycogen debranching enzyme. Approximately 85% of GSD III is GSDⅢa. All types of GSD III are caused by various mutations in the AGL gene (MIM:610860). Clinical manifestations of GSD III include hepatomegaly, fasting hypoglycemia, growth retardation, and, in many patients, progressive myopathy and cardiomyopathy. Hepatomegaly tends to resolve spontaneously with high frequency. In patients with GSDⅢa, cardiomyopathy may become predominant in adults.Human AGL gene is located on human chromosome 1p21 and consists of 35 exons spanning approximately 85kb of genomic DNA. The AGL gene encodes about 7kb mRNA and produces at least 6 isoforms by alternative splicing. The major mRNA isoform presenting in both muscle and liver encodes a protein of 1532 amino acid residues with a calculated molecular weight of 170kDa. To date, mutation analysis of the AGL gene in GSD III patients from different countries and populations have been described, including African, Asian (mainly in Japanese population), European and American, whereas only six cases with mutation screening have been reported in Chinese population.79 mutations identified in the AGL gene, including 10 missense mutations, 22 nonsense mutations,10 splicing site mutations,23 small deletions,11 small insertions, one complex deletion/insertion mutation, one gross deletion and one complex rearrangement mutation.In the present study, a cohort of 87 subjects clinically suspected as GSD III was enrolled for mutation screening. After informed consent was obtained from participating individuals, peripheral blood samples along with clinical data were collected. The ethical approval was obtained from the Peking Union Medical College Hospital Institutional Review Board. Polymerase chain reactions (PCR) of the AGL gene coding region were carried out with the template of AGL cDNA and genomic DNA. After purification of the PCR products and automatic sequencing, putative disease-causing mutations were identified in 52 patients (from 50 families) out of the 87 index cases. A total of 58 different mutations, including 51 novel ones, were identified, comprising 8 missense mutations (13.8%),13 nonsense mutations (22.4%),17 splice-site mutations (29.3%),14 small deletions mutations (24.1%), five small insertions mutations (8.6%) and one complex insertion/deletion mutations (1.7%). To confirm the pathogenicity of the novel missense mutations, restriction fragment length polymorphism (RFLP) analysis were conducted in 50 Chinese Han normal controls with none of the novel missense mutations detected in controls. The most frequently detected mutation we found is c.1735+1 G>T, which accounts for 14.0% of the 100 independent alleles in our patients. It is reported that c.1735+1G>T has the highest frequency (11.8%) in Korean and Japanese patients with GSD III. The mutations with the second high frequency were c.100C>T (p.Arg34X) and c.4234delC (p.Gly1413AlafsX2), and both were found in 4.0% of our patients with GSD III. These three prevalent mutations account for 22% of the 100 independent alleles. Based on the results of mutation analysis, we performed prenatal diagnosis for one fetus at high risk. The results showed that the fetus inherited one pathogenic mutation from the mother, while a normal allele from the father which defined the fetus as an unaffected carrier. Correlation between the genotype and phenotype was noticed as patients with missense mutations or deletions of a single amino acid showed normal or slightly elevated CK values, while patients with splicing or frameshift mutations showed apparently elevated CK values.In summary, by mutation detection of the patients clinically diagnosed as GSD III, we found several novel pathogenic mutations of the AGL gene, extend its mutation spectrum and enrich its mutation database, discover the correlation between the genotype and phenotype and provide prenatal diagnosis for one fetus at high risk. These works expand our knowledge on molecular basis of GSD III patients and lay the foundation for genetic diagnosis in future in Chinese population. Part II:Mutation analysis of the G6PC and G6PT1 genes in Chinese patients with glycogen storage disease type IGlycogen storage disease type I (GSD I, von Gierke disease) comprises a group of autosomal recessively inherited disorders, characterized by impaired glucose homeostasis due to the deficiency of glucose-6-phosphatase (G6Pase) system. The combined frequency of the disease is approximately 1 in 100,000 live births. The type I GSDs are divided into four subtypes corresponding to defects in different enzyme activities:GSD la (MIM:232200), defect in the G6Pase catalyticunit (G6PC); GSD Ib (MIM:232220), defect in the G6P transporter (G6PT1); GSD Ic (MIM:232240), defect in a putative phosphate transporter, T2; and GSD Id, defect in a putative glucose transporter, T3.GSD la is the most prevalent form of GSD I, representing over 80% of GSD I cases. The typical clinical characteristics involve severe fasting hypoglycaemia, hepatomegaly, growth retardation and bleeding tendency, concomitant fasting lactic acidosis, hyperlipidaemia and hyperuricaemia. Long-term complications such as gout, osteoporosis, impaired platelet function, nephropathy, liver adenoma, and pancreatitis may emerge. GSD Ib has a similar clinical course, with additional findings of neutropenia and impaired neutrophil function resulting in recurrent bacterial infections. However, neutropenia may not appear in all the patients with GSD-Ib and some of the patients with GSD la can suffer from mild neutropenia as well. Therefore, a definite diagnosis may still require the mutation analysis of both G6PC and G6PT1 genes. GSD Ic and GSD Id were reported in only a few patients who have not been characterized at the molecular level. In clinical cases reported to represent GSD Ic and GSD Id, deleterious G6PT1 mutations were identified, so GSD I tends to be divided into two types:GSD la and GSD I non-a.Human G6PC gene (MIM:232200), a single-copy gene containing five exons, spans 12.5 kb of DNA on chromosome 17q21 and encodes an endoplasmic reticulum membrane associated protein containing 357 amino acids. GSD la is not restricted to any ethnic group. To date,89 separate G6PC gene mutations have been identified out of GSD-Ia patients from different populations. Taking all these patients together, the most frequently detected mutations are p.Arg83Cys (29.0%), c.648G>T(16.4%), p.Gln347X (14.3%) and p.Arg83His(3.9%). Some studies showed that in Chinese patients with GSD la, the most prevalent mutations are c.648G>T (54%) and p.Arg83His (26%); among the Japanese and Korean, c.648G>T accounts for 91% and 75% of the patients, respectively. Human G6PT1 gene (MIM:602671) consists of nine exons and spans 5.3kb in the chromosomal region 11q23. G6PT1 encodes a 46 kDa protein of 429 amino acids which has both G6P and inorganic phosphate translocase activities. To date, more than 150 GSD I non-a patients have been studied for the G6PT1 gene mutation. Most of them are GSD Ib patients, six are GSD Ic patients and one is GSD Id patient. From these patients,82 separate G6PT1 gene mutations have been identified with the major mutation type as missense mutation accounting for 42.7% of the mutations. As with the GSD la mutations, mutation frequency in GSD Ib patients also shows ethnic variability. In Caucasian patients, p.Leu348ValfsX53 (32%) is the most prevalent mutation. While in Japanese patients, p.Trpll8Arg is the prevalent mutation, accounting for 44%. Few cases in Chinese patients with GSD Ib have been reported.In the present study, using PCR amplification and automatic sequencing of G6PC and G6PT1 genes coding region, we studied a group of Chinese patients clinically suspected as GSD I, including 64 GSD Ia patients and 12 GSD Ib patients (from 10 families). With informed consent of all participating individuals and ethical approval of the Peking Union Medical College Hospital Institutional Review Board, peripheral blood samples were collected from the patients and their family members.As a result,14 different pathogenic mutations were identified in the G6PC gene from 27 suspected GSD la patients, including seven missense, four nonsense, two small deletions and one splicing mutation. Among them five are novel mutations. The c.648G>T mutation was most common, accounting for 24 (44.4%) of the 54 mutant alleles. The second common mutation was p.Arg83His, accounting for 9 (16.7%) of the 54 mutant alleles. The frequencies of these two mutations are slightly lower than those in other previous studies in Chinese patients.As the c.648G>T mutation in G6PC is the predominantly mutation in Chinese patients with GSD la, we performed high resolution melting (HRM) assay which is more adaptable for hotspot mutation screening to detect this mutation in 10 patients who have been verified to be GSD la by sequencing analysis. The HRM analysis results of mutation c.648G>T are completely consistent with those from sequencing analysis. So HRM analysis is proved to be a simple and fast technique for detecting frequent mutations in Chinese GSD la patients.We were able to identify both mutant alleles in all 12 GSD Ib patients. Altogether,10 different mutations, six of which were novel, were identified in the G6PT1 gene, including six missense, two splicing, one nonsense, and one small deletion mutation. The most prevalent mutation in our patients is p.Pro191Leu, which accounts for 7 (35%) of the 20 mutant alleles. Because of the small number of the samples in our study, we can’t arbitrarily conclude that this mutation is the most frequent mutation in Chinese GSD Ib patients.In summary, our findings expand the spectrum of mutation in the G6PC and G6PT1 genes. We found two frequent mutations in our GSD la patients and one frequent mutation in our GSD Ib patients. These findings helped us better understand the molecular basis of GSD I patients. Paper 2:Mutation analysis of the ELN gene in a child with mild congenital cutis laxaCutis laxa (CL) comprises a rare and heterogeneous group of disorders characterized by lax inelastic skin with or without internal organ manifestations. They can be divided into congenital and acquired forms. Congenital cutis laxa include at least three forms of the inheritance:autosomal dominant, autosomal recessive, and X-linked form. Autosomal dominant CL is associated with a more normal lifespan and internal organ abnormalities are less frequent. Compared with autosomal dominant CL, autosomal recessive CL are known to date less frequent but more severe, and they often involve vital organs and are often lethal. CL may also be acquired following trauma or febrile exanthema.To data, several genes mapped on different human chromosomes involved in cutis laxa have been reported, such as FBLN4, FBLN5, ELN, ATP7A and ATP6V0A2. Mutation in ELN and FBLN5 can cause either autosomal dominant or autosomal recessive cutis laxa. The 45 kb human elastin gene (ELN, MIM:130160) maps to 7q11.2 and comprises 34 exons. Elastin is secreted from the cell as a soluble monomer called tropoelastin. The tropoelastin molecule is characterized by a series of tandem repeats, each include a lysine-containing cross-linking region followed by a hydrophobic motif. The hydrophobic domains are responsible for the elastic properties of elastin, whereas the lysine residues form covalent crosslinks between molecules. In CL patients nine mutations in the ELN gene have been described so far. These mutations include four small deletions, two missense mutations, one gross deletion, one partial tandem duplication, and one splicing mutations. One missense mutation was found in an acquired cutis laxa patient who developed CL following a Toxocara canis parasitism.We now present one patient with mild CL who was an 18-month-old girl born to nonconsanguineous and phenotypically normal parents. She presented with lax and wrinkled skin all over the body at birth, and gave an appearance of premature aging with normal wound healing. She had a hoarse voice, down-slanting palpebral, large ears, and drooping cheeks. No pulmonary or heart manifestations were observed. Based on these clinical manifestations, she was diagnosed as congenital cutis laxa.After informed consent was obtained, genomic DNA from the patient and her parents were isolated from peripheral blood leukocytes. We performed DNA mutation analysis only in the 3’end exons (from exon 28 to 34) of the ELN gene, because this is the region where mutations most likely causing these disorders have been reported. After bi-directional sequencing of PCR products from the patient, we found two novel nucleotides changes in exon 28 and intron 33 respectively:c.1819G>C (p.Gly607Arg) and c.2132-7C>A. The c.1819G>C mutation was inherited from her father, while the c.2132-7C>A is a de novo mutation. To confirm the pathogenicity of the novel mutations, restriction fragment length polymorphism (RFLP) analysis were performed in 120 Chinese Han normal controls. The results showed that both of these two novel mutations were not detected in control individuals. To test whether the nucleotide change occurred in intron 33 can cause aberrant splicing, we performed cDNA level studies. Total RNA was isolated from the cultured skin fibroblasts of the patient and one normal control individual. Reverse transcriptase PCR was performed. As expected, c.2132-7C>A created a novel splice donor site and introduced 5 bp nucleotides insertion, leading to a frameshift in exon 34 of the elastin gene which is predicted to replace the 14 amino acids at the C-terminus of elastin by 41 novel amino acids. Multiple sequence alignment of elastin polypeptides from different species showed that p.607G1y and the 14 amino acids of C termini amino acids were highly conserved among different species. All these facts support the pathogenicity of the two novel mutations. Immunostaining of cultured fibroblasts from patient and healthy control with monoclonal antibody against human elastin showed no obvious differences in deposition or abundance of elastin. These results indicate that the two mutations may not affect the synthesis and secretion of elastin. Her father had the same missense mutation and showed normal phenotype which suggests that the inheritance in this family is autosomal recessive.In summary, we reported two novel compound heterozygous mutations in ELN causing mild autosomal recessive cutis laxa. These results enrich the spectrum of mutations in the ELN gene. To our knowledge, this is the second report of a mild CL phenotype caused by ELN mutation with an autosomal recessive form of inheritance and expands our knowledge on genetic heterogeneity of congenital cutis laxa.