Implementing Whole Gene Sequencing and Genetic Counseling Internationally for People with Parkinson’s Disease: The PD GENEration Experience (P3-5.007)

Objective To investigate the mutations in methyl-CpG-binding protein 2 gene(MECP2 gene) from typical sporadic Rett syndrome patients, explore the correlations between their genotype and phenotype, assist in genetic counseling. Methods Genomic DNA was extracted from peripheral blood leukocytes from 2 patients and their parents using standard protocols.Polymerase chain reaction and direct sequencing were performed using specific primers from 4 exons in MECP2 gene. Results No mutations were found in exon 1, 2, 3.Two different heterozygous missense mutations in exon 4 within MECP2 gene were identified from 2 patients.Their nuclear acid changes were: c.C473T and c. C397T, leading to amino acid change accordingly: p.T158M and p. R133C.There were no same mutations from their parents.Phenotype of patient with c. C397T was milder than patient with c. C473T. Conclusions Most of typical Rett syndrome patients had mutations in MECP2 gene.Gene test should be performed.Their biological parents should be detected accordingly if the patient had positive found to support genetic counseling. Key words: Rett syndrome; Methyl-CpG-binding protein 2; Gene mutation; Phenotype; Genetic counseling

Objective To summarize the clinical features of 22 probands diagnosed with congenital muscular dystrophy (CMD), and to provide genetic counseling and prenatal diagnosis for 23 fetuses of these pedigrees. Methods Data of 22 CMD patients who were treated in the Pediatric Department of Peking University First Hospital during October 2006 to March 2016 were analyzed. Informed written consents for participation in this study were obtained from the parents or guardians. Prenatal diagnosis was performed using DNA samples extracted from fetal villus cells of 12 cases at 11-13 gestational weeks and amniotic fluid of 11 cases at 18-22 gestational weeks. Direct DNA sequencing by polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) were used to detect CMD-related gene mutations. Linkage analysis of short tandem repeats (STRs) was used to identify maternal blood contamination and biological parents. Results Thirteen out of the 22 probands with CMD were diagnosed with congenital muscular dystrophy type 1A (MDC1A), and all of them carried compound heterozygous mutations in LAMA2 gene. Prenatal diagnosis of 13 fetuses from these pedigrees found that four fetuses were wild-type, seven were heterozygotes and two carried the same mutations as their proband. Three probands with LMNA-related congenital muscular dystrophy (L-CMD) carried de novo mutations in LMNA gene. In these pedigrees, two fetuses were wild-type and one whose mother was mosaicism carried the same mutations as the proband. One proband with Ullrich congenital muscular dystrophy carried compound heterozygous mutations in COL6A2 gene and the fetus of the same pedigree was wild-type. Five probands were diagnosed with α-dystroglycanopathies. And among them, two cases of muscle-eye-brain disease (MEB) carried compound heterozygous mutations in POMGnT1 gene and the fetuses of the two peidgrees were heterozygotes; one case of congenital muscular dystrophy type 1C (MDC1C) had compound heterozygous mutations in FKRP gene and the fetus carried the same mutations; one patient diagnosed with POMGnT1-related congenital muscular dystrophy with mental retardation (CMD-MR) carried compound heterozygous mutations in POMGnT1 gene, and the fetus was positive for the same mutations; one proband with POMT1-related CMD-MR was positive for compound heterozygous mutations in POMT1 gene and the results of prenatal diagnosis for two fetuses of this pedigree showed that the first fetus had the same mutations as the proband, while the second was heterozygote. Conclusions No effective therapeutic method is available for CMD. Therefore, accurate genetic counseling and prenatal diagnosis are necessary to prevent CMD child from birth. Key words: Muscular dystrophies; Prenatal diagnosis; Genetic testing

Objective To investigate the characteristics of the phenylalanine hydroxylase(PAH) gene mutations in patients with phenylketonuria(PKU) in Guangxi region, in order to provide clinical data for genetic counseling and prenatal gene diagnosis. Methods Thirty-seven children diagnosed as PKU in the Maternal and Children′s Hospital of Guangxi Zhuang Autonomous Region were enrolled in the study between January 2009 and December 2017. Venous blood was collected and the PAH gene sequence was determined by Sanger sequencing after amplification with the polymerase chain reaction technique. The new gene mutations were defined based on the national and international literature review and databases.Meanwhile, 100 healthy individuals were selected as the control group for gene sequencing to confirm whether the mutation was a new one. Results Thirty-seven cases of PKU were detected for 68 mutations, with the detection rate being 91.89% (68/74). Six mutations were identified in exon 7, which accounted for 31.08% of all, exon 12(18.92%), exon 8(10.81%) and exon 6 (10.81%) followed.A total of 25 different mutations were identified which including 14 missense mutations(56.00%), 7 nonsense mutations(28.00%), 3 splicing junction mutations(12.00%), and 1 deletion mutation(4.00%). The most common mutations included c. 1223G>A(p.R408Q), c.728G>A(p.R243Q) and c. 721C>T(p.R241C), accounting for 14.86%, 13.51%, and 10.81%, respectively.After querying international databases, including PAH mutation database and Human Gene Mutation Database and forecasting software, three kinds of mutations c. 314C> T (p.T105I), c.583A> G (p.K195E), c .851G> A(p.C284Y) were verified as novel PAH gene mutations. Conclusions The mutation spectrum of the PAH gene in Guangxi has been identified.And 3 kinds of mutations have been identified.This may accumulate valuable information for gene diagnosis and prenatal diagnosis of PKU in Guangxi region. Key words: Phenylketonuria; Phenylalanine hydroxylase; Gene mutation

Cancer is a genetic condition driven by a series of both inherited (germline) and tumour specific (somatic) mutations. The analysis of tumour to identify somatic alterations and generate a genetic profile has given rise to a personalised approach for treating oncology patients, guiding optimal therapy. Next generation sequencing (NGS) technology is now routinely utilised in oncology to screen for both genes known to be associated with improving treatment response, as well as, genes of uncertain significance to contribute to new findings in cancer treatment. In the development of genome wide mutation analysis of tumour tissue, germline DNA has also been routinely collected for comparative analysis in order to identity tumour specific mutations. As the identification of germline mutations is secondary to the main purpose of tumour testing for targeted treatment, the management of germline findings has been extensively debated, in particular, around if and when to communicate these findings to patients. The role of the genetic counsellor in this process has been pertinent to these discussions. Clinical practice, however, is shifting towards a preference of sequencing tumour tissue alone to characterise its molecular profile for reasons such as reducing cost and facilitating logistics of sample collection. This method raises the question of how the testing of tumour alone contributes to the identification of germline mutations. The laboratory at POLARIS, Singapore, has generated genomic data of 130 colon tumours using a panel of 84 genes, including 29 germline susceptibility genes associated with known inherited syndromes. The analysis of this data has given an insight into the proportion of mutations that are potentially germline, which in turn will impact how patients are consented for tumour profiling and the delivery of the results. In generating these findings, however, the analysis process has demonstrated a number of the complexities in interpreting the pathogenicity of the variants, as well as, determining the somatic or germline origin of the variant. The challenges in interpreting germline mutations from tumour next generation data, the importance of conveying a germline finding to the patient and the integration of the genetic counsellor in this process will be discussed.

Objective To analyze the clinical characteristics, muscle pathological features and pathogenic gene mutation in 4 cases with LMNA-related congenital muscular dystrophy(L-CMD). Methods Clinical data of the probands and the parents were collected. Skeletal muscle specimens were biopsied from the probands for pathological analysis. Genomic DNA and RNA were extracted from peripheral blood leukocytes, and PCR, reverse transcription(RT)-PCR and DNA direct sequencing were employed to analyze the LMNA gene to determine the gene mutation and confirm the pathogenicity. Results Four patients had symptoms from fetal period to several months after birth. They presented with motor retardation, muscle weakness with prominent the proximal upper limbs, distal lower limbs and neck extensor, hypotonia, contractures, with mild to moderate elevation of CK level. The muscle biopsies showed muscular dystrophic and with inflammatory changes, and the abnormal nuclear morphology was observed with transmission electron microscopy. Genetic analysis of them detected 4 dominant de novo mutations. Three of them had unreported pathogenic mutations. The same sites of the LMNA gene were wild type in their parents. Conclusions Four cases of L-CMD are genetically identified. Genetic counseling of the family can be possible. The patients should be considered LMNA gene mutation of they present themselves with muscle weakness with the proximal upper limbs, distal lower limbs and neck extensor, hypotonia, contractures, mild to moderate elevation of CK level, and if the biopsies show muscular dystrophic changes but also with inflammatory changes should be considered LMNA gene mutation. Genetic analysis is the most reliable method for diagnosing L-CMD. Key words: LMNA related congenital muscular dystrophy; Inflammatory myopathy; LMNA gene; Mutation

Objective To investigate the spectrum of CYP21A2 gene mutation and the correlation between genotype and phenotype in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas. Methods Genomic DNA was extracted from the peripheral blood samples of the proband. Locus-specific PCR, direct sequencing of PCR amplification products, and multiplex ligation-dependent probe amplification were applied to detect pathogenic gene CYP21A2 and the relationship between genotypes and phenotypes was analyzed. Results (1) Of 35 patients with 21-hydroxylase deficiency, 25 were classified as salt-wasting phenotype and 10 were simple virilizing phenotype. (2) 69 mutant alleles were detected in a total of 70 alleles in 35 patients. Only one mutant allele was detected in one patient. Two mutant alleles were detected in all other patients, with the mutation detection rate 98.6%. (3) A total of 6 types of mutations were detected, of which c. 293-13C /A>G (I2G) was the most common, accounting for 57.1% (40/70), followed by 18.6% (13/70) for large gene deletion or conversion, and 14.3% (10/70) for p. I173N. In addition, a novel mutation, c. 949C>T (p.R317X), which has not been reported previously, was detected as a pathogenic mutation. (4) Correlation analysis of genotype and phenotype in 35 children showed that the phenotype predicted by genotype was consistent with the actual salt-wasting phenotype in 31 children, and those in three children were inconsistent with the actual clinical phenotype. Conclusion The mutation characteristics of CYP21A2 gene in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas are slightly different from those reported in other regions in China. A mutation c. 949C>T has not been reported, which enriches the mutation spectrum of CYP21A2 gene and provide the foundation for genetic counseling and prenatal diagnosis. Key words: 21-Hydroxylase deficiency; CYP21A2 gene; Mutation

Omenn syndrome is a rare autosomal recessive hereditary severe combined immunodeficiency. The purpose of this study was to understand clinical characteristics and genetic mutation type of Omenn syndrome and to improve the recognition of Omenn syndrome among pediatric clinicians. One suspected case of severe combined immunodeficiency was found to have pneumonia repeatedly, intractable diarrhea, poor antibiotic treatment effect, lymphadenopathy, hepatosplenomegaly and erythroderma. The patient was diagnosed as having Omenn syndrome by RT-PCR, and the expression of RAG1/RAG2 and gene analysis of RAG1/RAG2 were performed. The classification of lymphocyte was CD3(+) cells (35.3%), CD19(+) cells (0.4%), CD16(+) cells (57.6%). After stimulation with phytohemagglutinin (PHA), lymphocyte proliferation of the child was extremely low. Genetic studies showed RAG1 homozygous deletion mutation (2302 del T). He had detectable activated T-lymphocytes with low circulating B-lymphocytes and no evidence of maternal T-cell engrafment as indicated by the short tandem repeat (STR) analysis. Omenn syndrome is a severe combined immunodeficiency disease caused by mutations in the RAG1/RAG2 gene. The disease has been reported rarely in China. The clinical manifestations of the disease is early postnatal repeated infections and erythroderma. Mutation analysis of RAG1/RAG2 gene may help to confirm the diagnosis and may be useful in early immune reconstitution and genetic counseling.

Fanconi anemia (FA) is a rare genetic syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. FA is usually inherited as an autosomal recessive condition. This disease is genetically heterogeneous and mutations in 16 different genes have been identified in FA patients to date. An accurate diagnosis needs detection of pathogenic variations in the FA genes along with positive results from chromosome breakage test. In this study, 48 families with at least 2 affected FA patients and positive chromosome breakage test were enrolled from the Iranian population. Molecular analysis of FA genes was performed using Next Generation Sequencing (NGS) method and Multiple Ligation Dependent Probe Amplification (MLPA). Causal mutations for 30 (63%) patients were identified in homozygous or compound heterozygous forms. FANCA had the highest mutation frequency rate (83%) followed by FANCG (10%), FANCD2 (3%) and FANCL (3%). A significant proportion (44%) of FANCA mutations were large rearrangements. Genetic testing for FA patients improves the accuracy of diagnosis and also will be essential for genetic counselling and prenatal diagnosis for future pregnancies in the family. Availability of NGS technology has made the screening of all known FA genes at once more practical and affordable.

Objective To investigate the clinical manifestations and biochemical characteristics of 3-hydroxy-3-methylglutaric aciduria, and discuss results of 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HMGCL) gene mutation. Methods The clinical data of a boy with 3-hydroxy-3-methylglutaric aciduria who was treated at Peking University First Hospital on December 7th 2014 was collected into this study. The admission examination, auxiliary examination, diagnosis and treatment, gene detection and follow-up of this boy were retrospectively analyzed. The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Peking University First Hospital. Informed consents were obtained from patient's parents. Results The boy complained of unexplained diarrhea, vomiting, repeated hematemesis, fever 2 d and coma 3 h. The admission examination and auxiliary examination results showed increased white blood cell count, liver damage, electrolyte imbalance, low blood sugar, blood clotting abnormalities, low-protein, high ammonia, metabolic acidosis. Cranial MRI result showed that abnormal signal could be found in bilateral lateral ventricle and double fronto-temporal parietal cortex. The final diagnosis of the boy was 3-hydroxy-3-methylglutaric aciduria. By limiting protein intake, intravenous infusion of glucose and L-carnitine treatment, the symptom alleviated gradually. Genetic analysis results showed that exon region of HMGCL was found two heterozygous mutation point: c. 509G> T and c. 348 + 1 G>C, where c. 509G> T was heterozygous mutation from the mother, c. 348 + 1 G>C emerged in child homozygous mutation, the mutation site of c. 348+ 1G>C splice site mutation caused amino acid changes p. Cys170Phe, splicing (cysteine phenylalanine, shear mutation). After discharge, the six months follow-up results showed as follow. Psychomotor developed slightly behind the health children, but no significant setback. The boy lived in good general condition, and biochemical markers and the level of urinary 3-hydroxy-3-methylpentyl reduce to normal range. Conclusions 3-hydroxy-3-methylglutaric aciduria is one of rare autosomal recessive genetic disease. Genetic gene detection and genetic analysis of HMGCL was helpful not only to diagnose the disease, but also crucial important to genetic counseling for family guidance and prenatal diagnosis. Key words: 3-Hydroxy-3-methylglutaric aciduria; 3-Hydroxy-3-methylglutaryl-coenzyme A lyase gene; Organic acid; Metabolic diseases; Child

Urea cycle disorders (UCDs) are a group of autosomal or X-linked, recessively inherited errors of metabolism that lead to severe neurological disease due to insufficient detoxification of excess nitrogen. The resulting hyperammonemia is the key feature of UCDs, but at the same time only a surrogate marker. In the majority of cases, a sole biochemical analysis is indicative but not diagnostic. Therefore, additional means are required and mutation analysis is the method of choice for con- firmation in most cases of UCD. In addition to confirming the diagnosis, mutation analysis enables genetic counseling and prenatal testing and contributes to new research approaches. All genes involved in any of the enzymatic (namely the genes for N-acetylglutamate synthase, NAGS; carbamoylphosphate synthetase, CPS1; ornithine transcarbamylase, OTC; argininosuccinate synthetase, ASS1; argininosuccinate lyase, ASL; arginase, ARG1) or transporter steps (namely the genes for the ornithine/ citrulline antiporter ORNT1, SLC25A15; glutamate/aspartate antiporter citrin, SLC25A13) of the urea cycle are known and thus accessible for genetic testing. In most situations, direct Sanger sequencing using DNA from peripheral blood cells can be done but there are exceptions to this such as in the case of the relatively large CPS1 gene which renders RNA based mutation analysis an attractive alternative. With this review, we provide an overview on the current methods used for mutation analysis of each UCD gene, we mention possible pitfalls and their solutions, and discuss alternative methods which may become standard in the future.

Objective To analyze the clinical characteristics and genetic variation of megalencephalic leukoencephalopathy with subcortical cysts(MCL), then to explore the genetic characteristics so as to help families by providing genetic counseling. Methods The clinical data of the children and their family members were collected, and the peripheral blood DNA of the children and family members were extracted.Then, the MLC1 gene mutation in the children was detected by using the target sequence capture high-throughput sequencing technology and Sanger sequencing technology. Results (1)MCL often presented abnormal head circumference in infants as the first symptom.The main clinical manifestations were hypoevolutism in motor development, retrogression of early school age, then the movement disorder progressed and finally paralyzed; epilepsy was common in early childhood; head magnetic resonance imaging showed white matter in bilateral cerebral hemisphere diffusing abnormal signal with temporal lobe cystic change in the early stage, and then showed brain atrophy.(2)The gene results showed that the 2 girls with MLC had both c. 368C>T (p.Thr123Ile) and c. 353C>T (p.Thr118Met) complex heterozygous variation, which existed in the MLC1 gene.The girls′ father and a sister carried c. 368C>T (p.Thr123Ile), while the mother carried c. 353C>T (p.Thr118Met) heterozygous variation, all of whom were normal phenotypes. Conclusions MCL is one cause of hypoevolutism in motor development in children and abnormal head circumference of infants is usually the first symptom.The MLC1 gene c. 368C>T(p.Thr123Ile) is a pathogenic mutation for MLC, and may be another new pa-thogenic mutation. Key words: Megalencephalic leukoencephalopathy with subcortical cysts; MLC1 gene; Mutation

To investigate the incidence and the gene mutation frequencies and patterns of β-thalassemia (β-Thal) in ethnic Han children in Chongqing city. A total of 1726 children were screened by using automatic hemocytic analyzer, cellulose acetate electrophoresis and fetal hemoglobin alkali denaturation test. Samples with mean corpuscular volume (MCV) < 80 fl, cell hemoglobin content (MCH) < 27 pg and hemoglobin A2 (HbA2) levels >3.3%, fetal hemoglobin (HbF) >2% for β-Thal screening indicators. The positive samples of screening indicators were detected and identified by PCR-reverse dot blot method for 18 common β-Thal mutations in Chinese populations, unknown mutations samples were subjected to DNA sequencing analysis of the β-globin gene. Twenty-five cases of β-Thal carriers were observed from the 1726 samples, with 24 cases of β-Thal heterozygote and one case of double heterozygote. Therefore, the β-Thal carrier rate was 1.51%. After 1726 peripheral venous blood samples analyzed by hematological parameters, 164 positive cases of β-Thal screening indicators were found, with the positive rate being 9.50% (164/1726). A total of 6 different gene mutations were detected, the four most common mutations were as the following: CD41-42, IVS-II-654, CD17 and beta E. These four mutations as the major types in this area accounted for 88.00% of all the mutations. In addition, one rare mutation of 5 'UTR; + (43 -40) was found, and one case of the hemoglobin variant of Hb Zurich was reported in Chinese people for the first time. Chongqing is a high risk region of the β-Thal. Epidemiological Data from the research was useul for the genetic counseling and the prevention of β-Thal major.

Objective To explore the prevalence and spectrum of β-thalassemia mutations in Fujian province,and to provide a reference for prenatal diagnosis and genetic counseling in this population.Methods Two thousand three hundred and one blood samples were randomly selected from 9 different areas of Fujian province from May 2008 to December 2010.PCR and reverse dot blot hybridization (RDB) were adopted for detection of the 17 common types of mutation,and the frequency of each genotype of β-thalassemia mutations was calculated.The β-globin gene of unknown positive samples were analyzed directly with DNA sequencing.Results Three hundred and fifty-nine cases were detected with β-thalassemia mutations of the 2301 copy blood samples submitted,and the detection rate was 15.60％ (359/2301).Of the mutated genes,12 different mutations were identified,namely IVS-2-654(C→T),CD41-42(-TCTT),CD17(A→T),-28(A→G),CD27-28(+C),CD26(G→A),CD71-72(+A),IVS-1-1(G→T),CD43(G→T),-29(A→G),initiation codon ATG→AGG and CD36(-C).Mutation frequencies were 46.54％ (175/376),33.24％ (125/376),9.31％ (35/376),5.05％ (19/376),2.13％(8/376),1.33％(5/376),0.80％(3/376),0.27％(1/376),0.27％(1/376),0.27％(1/376),0.53％(2/376),and 0.27％(1/376),respectively.The most common mutations were IVS-2-654 (C→T) and CD41-42 (-TCTT),which accounted for 79.78％(300/376) of total genetic mutations.In addition,a novel β-globin gene mutation CD36 (-C) allele was detected for the first time,the deletion of a nucleotide C at code 36 within exon 2 lead to a frameshift mutation that could result in a premature termination at code 60.Conclusions β-thalassemia mutations in Fujian province are complex with significant genetic heterogeneity.We present for the first time the detection of a new β-thalassemia mutation in the population:CD36(-C),which provides valuable information for genetic counseling and prenatal diagnosis in Fujian province. Key words: 3-thalassemia; Genotype; Reverse dot blot hybridization

Educating individuals who seek information about genetic testing for cancer susceptibility is a complex task. Because of the psychological and social risks to patients of being identified as susceptible to a genetic disorder, it is important that education take place before a patient undergoes gene testing. However, it is not known who will provide such education. Since primary care physicians have limited knowledge, time, and confidence with genetic counseling, they are not ideal educators. Genetic counselors are well trained to provide the service, but many patients lack access to them. Other specialists are scare. Interactive computers hold much promise as a supplemental or alternative modality for education. The authors describe their experience with developing an interactive CD-ROM on gene testing and breast cancer, and respond to anticipated criticisms of this technology.

To explore the clinical and genetic characteristics of a family affected by genetic cholestasis. Clinical data of the patient was collected. Targeted exome sequencing was carried out to detect the pathogenic mutations. The results were confirmed by Sanger sequencing. The patient, a 5-year-old boy, presented with severe cholestatic cirrhosis. Genetic analysis revealed that he has carried compound heterozygous mutations c.1006-2A>G and c.3580C>T (p.R1194X) of the ABCB4 gene, which were inherited from his father and mother, respectively. By structural prediction, the mutation c.3580C>T can give rise to a truncated multi-drug resistance protein 3 (MDR3). The patient was diagnosed with progressive familial intrahepatic cholestasis type 3 (PFIC-3) based on clinical and molecular findings. Detection of novel mutations of the ABCB4 gene has provided valuable clues for the diagnosis and genetic counseling.