Detection of Parkin (PARK2) and DJ1 (PARK7) mutations in early-onset Parkinson disease: Parkin mutation frequency depends on ethnic origin of patients

Mutations in the Parkin (PARK2) and the DJ1 (PARK7) gene cause early-onset Parkinson disease (EOPD). We tested 75 Serbian EOPD patients for mutations in both genes by conventional mutational screening (SSCP/dHPLC/sequencing) to detect small sequence alterations and by gene dosage studies (quantitative PCR) to reveal deletions or multiplications of one or more exons. A compound heterozygous Parkin mutation (exon deletion and point mutation; [c.836_972del]+[c.1411C>T]; +1 is first nucleotide of GenBank AB009973.1) was identified in a patient who showed a relatively benign course after a disease onset at 41 years. Another case had a heterozygous exon deletion in DJ1 ([c.253_322del]+[?]) and presented with an age at onset of 45 years and a rapid disease course. In conclusion, Parkin mutations are surprisingly rare in our Serbian EOPD sample, suggesting that the mutation rate depends on the ethnic origin of the patients. Although DJ1 mutations appear to be rare, we confirm their role in EOPD and demonstrate the importance of gene dosage studies.     

Germline mutations in MLH1, MSH2 and MSH6 in Korean hereditary non-polyposis colorectal cancer families

Hereditary non-polyposis colorectal cancer (HNPCC), the most common hereditary colon cancer syndrome, is a dominant disorder caused by germline defects in mismatch repair (MMR) genes. Identification of MMR gene mutations can have direct clinical implications in counseling and management of HNPCC families. We screened 44 HNPCC and 97 suspected HNPCC Korean families for germline mutations in three MMR genes: MLH1, MSH2 and MSH6. We identified twelve novel mutations: nine in MLH1(c.632_633insT, c.808_811delACTT, c.845C>G, c.1625A>C, c.1730+1delG, c.1907T>C, c.1918C>T, c.2104-2A>G and c.2170T>A), two in MSH2 (c.1886A>G, c.1316_1318delCCT) and one in MSH6 (c.3488A>T). In addition, two statically significant cSNPs in MLH1: c.1128T>C ( p=0.008 in HNPCC and p=0.037 in early-onset CRC) and c.2168C>A ( p<0.001 in HNPCC). Interestingly, the most frequent mutation, c.1757_1758insC in MLH1, was a founder mutation inherited from a common Korean ancestor.     

Association of PINK1 and DJ-1 confers digenic inheritance of early-onset Parkinson's disease

Mutations in genes encoding both DJ-1 and pten-induced kinase 1 (PINK1) are independently linked to autosomal recessive early-onset familial forms of Parkinson's disease (PD). We here report identification of a family with PD patients harboring novel heterozygous missense mutations in both PINK1 and DJ-1 genes encoding DJ-1A39S and PINK1P399L, respectively. In transfected cells, DJ-1 interacts with PINK1. PINK1P399L is less stable than the wild-type protein and is degraded via the ubiquitin-mediated proteasomal pathway. Expression of wild-type DJ-1 increased steady-state levels of PINK1, whereas expression of DJ-1A39S reduced steady-state levels of PINK1. Furthermore, co-expression of wild-type DJ-1 and PINK1 suppresses neurotoxin 1-methyl-4-phenylpyridinium (MPP(+))-induced death of dopaminergic SH-SY5Y cells. In contrast, co-expression of PD-associated DJ-1A39S and PINK1P399L significantly potentiated susceptibility of SH-SY5Y cells to MPP(+)-induced cell death. This study reports the first case of autosomal recessive PD with digenic inheritance and demonstrates that DJ-1 and PINK1 physically associate and collaborate to protect cells against stress via complex formation.     

Splice-site contribution in alternative splicing of PLP1 and DM20: molecular studies in oligodendrocytes

Mutations in the proteolipid protein 1 (PLP1) gene cause the X-linked dysmyelinating diseases Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia 2 (SPG2). We examined the severity of the following mutations that were suspected of affecting levels of PLP1 and DM20 RNA, the alternatively spliced products of PLP1: c.453G>A, c.453G>T, c.453G>C, c.453+2T>C, c.453+4A>G, c.347C>A, and c.453+28_+46del (the old nomenclature did not include the methionine codon: G450A, G450T, G450C, IVS3+2T>C, IVS3+4A>G, C344A, and IVS3+28-+46del). These mutations were evaluated by information theory-based analysis and compared with mRNA expression of the alternatively spliced products. The results are discussed relative to the clinical severity of disease. We conclude that the observed PLP1 and DM20 splicing patterns correlated well with predictions of information theory-based analysis, and that the relative strength of the PLP1 and DM20 donor splice sites plays an important role in PLP1 alternative splicing.     

Whole gene deletion and splicing mutations expand the PINK1 genotypic spectrum

Autosomal recessive parkinsonism is a genetic condition closely resembling Parkinson disease, the only distinguishing features being an earlier age at onset and a slower disease progression. Three causative genes have been identified so far. While exon rearrangements are frequently encountered in the Parkin gene, most PINK1 mutations are represented by single nucleotide changes. We report a sporadic parkinsonian patient carrying a deletion of the entire PINK1 gene and a splice site mutation (g.15445_15467del23) which produces several aberrant mRNAs. This report expands the genotypic spectrum of PINK1 mutations, with relevant implications for molecular analysis of this gene.     

Mutation analysis of the parkin and PINK1 genes in American Caucasian early-onset Parkinson disease families

Mutations in the parkin gene and the PTEN-induced putative kinase 1 gene (PINK1) have been identified as the most common causes of autosomal recessive early-onset Parkinson disease (EOPD). To investigate the presence of the parkin and PINK1 gene mutation(s) and to explore genotype-phenotype correlations in American Caucasian families with EOPD from North American, we screened these two genes in probands of six families by direct sequencing, semi-quantitative PCR and RT-PCR. No PINK1 gene mutation was found in any of the probands, but compound heterozygous mutations (EX 3 del and EX 3_4 del) in the parkin gene were identified in one family. Extended analysis of the parkin-positive family showed the phenotype of patients was that of classic autosomal recessive EOPD, characterized by early age at onset, slow progression, beneficial response to levodopa, and levodopa-related motor complications. Three heterozygous mutation carriers (EX 3 del or EX 3_4 del) were free of any neurological symptoms. None of 62 healthy controls harbored EX 3 del or EX 3_4 del mutation. Our data suggest that compound heterozygous mutations (EX 3 and EX 3_4 del) in the parkin gene were the cause of EOPD in one of six Caucasian families; heterozygous EX 3 del and heterozygous EX 3_4 del forms were insufficient to cause this disorder, consistent with a loss-of-function mechanism of the parkin mutations. The results may provide new insights into the cause and diagnosis of PD and have implications for genetic counseling.     

Three Korean patients with maple syrup urine disease: four novel mutations in the BCKDHA gene

Maple syrup urine disease (MSUD) is a rare, autosomal recessive disorder of branched-chain amino acid (BCAA) metabolism caused by dysfunction of the multienzyme branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex. Although a few cases of MSUD have been reported in the Korean population, the genetic background of MSUD is not well understood. In this study, we investigated three newborn males who were diagnosed with MSUD using a standard newborn screening test and amino acid analysis. We screened all coding regions of the BCKDHA, BCKDHB and DBT genes for abnormalities using direct sequencing. Changes in these genes are associated with MSUD. For one patient with complex deletion/duplication mutations, we also performed TOPO TA cloning sequencing. Amino acid analysis showed elevated levels of all branched chain amino acids (BCAAs) in all patients. Three patients were either homozygous or compound heterozygous for the BCKDHA mutations. Patient 1 was homozygous for c.1036C>T (p.R346C); patient 2 was heterozygous, with c.632C>T (p.T211M) and c.659C>T (p.A220V); and patient 3 had c.1204_1209dupAAACCC (p.L402_P403dup) and c.1280_1282delTGG (p.L427_A428delinsP). Among these mutations, c.1036C>T, c.632C>T, c.1204_1209dup and c.1280_1282del were novel. These patients had no mutations in either the BCKDHB or the DBT gene. Although this study included only three patients, the five different mutations in these patients may indicate mutational heterogeneity in Korean patients with MSUD. In addition, the BCHDHA gene may present a primary target for clinical genetic analysis. To the best of our knowledge, this is the first report of genetically confirmed MSUD in Korea.     

Twelve novel JAG1 gene mutations in Polish Alagille syndrome patients

Alagille syndrome (AGS) is an autosomal dominant disorder with developmental abnormalities of the liver, heart, eyes, vertebrae, and face. Mutations in the JAG1 (Jagged 1) gene, coding a ligand in the evolutionarily conserved Notch signaling pathway, are responsible for AGS. Here we present sixteen different JAG1 gene mutations, among them twelve novel, not described previously. Seven frameshift: c. 172_178del7 (p.Ala58fs), c.509delT (p.Leu170fs), c.1197delG (p.Val399fs), c.1485_1486delCT (p.Pro495fs), c.1809_1810insTGGG (p.Lys604fs), c.2122_2125delCAGT (p.Gln708fs), c.2753delT (p.Ile918fs); five nonsense: c.383G>A (p.Trp128X), c.496C>T (p.Glu166X), c.841C>T (p.Gln281X), c.1207C>T (p.Gln403X), c.1603C>T (p.Gln535X); two splice site: c.388-1G>C, c.3048+1_3048+2insG and two missense mutations: c.359T>A (p.Ile120Asn), c.560G>A (p.Cys187Tyr) were found. Forty percent of the changes were identified in exons 2 and 4, the remaining mutations are distributed along the entire coding sequence of the gene. Seventy-five percent of the mutations lead to creation of premature termination codons. Family studies revealed that the specific mutations were inherited in 3 out of 11 investigated cases. No correlation between genotype and phenotype was observed.     

Exon dosage variations in Brazilian patients with Parkinson's disease: analysis of SNCA, PARKIN, PINK1 and DJ-1 genes

Parkinson’s disease is one of the most common neurodegenerative disorders associated with aging, reaching ∼ 2% of individuals over 65 years. Knowledge achieved in the last decade about the genetic basis of Parkinson’s disease clearly shows that genetic factors play an important role in the etiology of this disorder. Exon dosage variations account for a high proportion of Parkinson’s disease mutations, mainly for PARKIN gene. In the present study, we screened genomic rearrangements in SNCA, PARKIN, PINK1 and DJ-1 genes in 102 Brazilian Parkinson’s disease patients with early onset (age of onset ≤ 50 years), using the multiplex ligation-dependent probe amplification method. Family history was reported by 24 patients, while 78 were sporadic cases. Screening of exon dosage revealed PARKIN and PINK1 copy number variations, but no dosage alteration was found in SNCA and DJ-1 genes. Most of the carriers harbor heterozygous deletions or duplications in the PARKIN gene and only one patient was found to have a deletion in PINK1 exon 1. Data about dosage changes are scarce in the Brazilian population, which stresses the importance of including exon dosage analysis in Parkinson’s disease genetic studies.     

Neurofibromatosis type 1 (NF1): Identification of eight unreported mutations in NF1 gene in Italian patients [corrected

In the present study the entire NF1 coding region was analyzed for mutations in 132 unrelated Italian NF1 patients. Using PTT, SSCP, and DNA sequencing, we found 8 novel mutations. Clinical diagnosis of NF1 was established according to the NIH consensus criteria. We detected 59 truncated fragments, and 46 of them were characterized by SSCP and direct sequencing. Eight mutations represent novel changes that contribute to the germline mutational spectrum of the NF1 gene. In two patients, premature termination was due to substitutions at nucleotide c.3982C>T (Q1298X) and c.7411C>T (Q2471X), respectively. Two other mutations were caused by the deletions (1756delA, 4699delA), and two by the insertions (c.5266_5267insT, c.7464_7465insTCCA) of a small number of nucleotides. Lastly, we found 2 splice-site mutations (c.2252-2A>C, c.2251+1G>A).     

Identification of novel MLC1 mutations in Chinese patients with megalencephalic leukoencephalopathy with subcortical cysts (MLC)

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal, recessively inherited disease caused by mutations in the MLC1 gene. Most of the previously published studies have been carried out in ethnic populations other than the Chinese. In this study, the analysis of clinical features and MLC1 mutation screening were performed in 13 Chinese patients for the first time. A total of 10 MLC1 mutations were identified in these patients, including five novel missense mutations (c.65G>A, p.R22Q; c.95C>T, p.A32V; c.218G>A, p.G73E; c.823G>A, p.A275T; c.832T>C, p.Y278H), one novel splicing mutation (c.772-1G>C in IVS9-1), one novel small deletion (c.907_930del, p.V303_L310del), one known nonsense mutation (c.593delCTCA, p.Y198X) and two known missense mutations (c.206C>T, p.S69L; c.353C>T, p.T118M). Mutation c.772-1G>C in IVS9-1, accounting for 27.3% (3/11) of the total number of genetically confirmed patients found in this study, is thus a putative hot-spot mutation in the present study group. The existence of a unique MLC1 mutation spectrum in Chinese MLC patients was shown. A systemic study to assess the mutation spectra in different populations should be undertaken.     

The principal PINK1 and Parkin cellular events triggered in response to dissipation of mitochondrial membrane potential occur in primary neurons

PINK1 and PARKIN are causal genes for hereditary Parkinsonism. Recent studies have shown that PINK1 and Parkin play a pivotal role in the quality control of mitochondria, and dysfunction of either protein likely results in the accumulation of low‐quality mitochondria that triggers early‐onset familial Parkinsonism. As neurons are destined to degenerate in PINK1/Parkin‐associated Parkinsonism, it is imperative to investigate the function of PINK1 and Parkin in neurons. However, most studies investigating PINK1/Parkin have used non‐neuronal cell lines. Here we show that the principal PINK1 and Parkin cellular events that have been documented in non‐neuronal lines in response to mitochondrial damage also occur in primary neurons. We found that dissipation of the mitochondrial membrane potential triggers phosphorylation of both PINK1 and Parkin and that, in response, Parkin translocates to depolarized mitochondria. Furthermore, Parkin's E3 activity is re‐established concomitant with ubiquitin–ester formation at Cys431 of Parkin. As a result, mitochondrial substrates in neurons become ubiquitylated. These results underscore the relevance of the PINK1/Parkin‐mediated mitochondrial quality control pathway in primary neurons and shed further light on the underlying mechanisms of the PINK1 and Parkin pathogenic mutations that predispose Parkinsonism in vivo.     

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.     

Clinical characteristics and molecular analysis of 21 Chinese children with congenital agammaglobulinemia

Congenital agammaglobulinemia is a humoral primary immunodeficiency and affected patients have extremely low levels of peripheral B cells and profound deficiency of all immunoglobulin isotypes. Mutations of the Bruton's tyrosine kinase (BTK) gene are responsible for most of the congenital agammaglobulinemia. In this study, the phenotypes of congenital agammaglobulinemia were investigated in 21 male children from 21 unrelated Chinese families. Sixteen different mutations of BTK gene were identified in 18 patients, and three patients did not have BTK gene mutations. Nine mutations had been reported previously including one gross deletion (c.722_2041del), one missense mutation (c.1764G>T), three non-sense mutations (c.194C>A, c.895C>T and c.1821G>A) and four invariant splice-site mutations (c.971+2T>C, c.1481+2T>A, c.1482-2A>G, c.1699-2A>G). Seven novel mutations were identified (c.373_441del, c. 504delG, c.537delC, c.851delA, c.1637G>A, c.1879T>C and c. 1482_1882 del). Ten of the eighteen mutations of BTK gene were located in the TK domain, four in the PH domain, three in the SH3 domain and one spanned the TH, SH3, SH2 and TK domain. Candidate genes of autosomal-recessive agammaglobulinemia, including IGHM, CD79a, CD79b and IGLL1, were screened in three patients without mutations in the BTK gene. A compound heterozygosity mutation in the IGHM gene (c.1956G>A, c.175_176insC) was identified in one patient. The results of our study further support that molecular genetic testing represents an important tool for early confirmed diagnosis of congenital agammaglobulinemia and may allow accurate carrier detection and prenatal diagnosis.     

Kallmann syndrome: 14 novel mutations in KAL1 and FGFR1 (KAL2)

Kallmann syndrome (KAL) combines hypogonadotropic hypogonadism and anosmia. Hypogonadism is due to Gonadotropin Releasing Hormone (GnRH) deficiency and anosmia is related to hypoplasia of the olfactory bulbs. Occasional symptoms include renal agenesis, bimanual synkinesia, cleft lip palate, dental agenesis. KAL is genetically heterogeneous and two genes have so far been identified, namely KAL1 (Xp22.3) and FGFR1/KAL2 (8p12), which underlie the X chromosome‐linked form and an autosomal dominant form of the disease, respectively. We studied a cohort of 98 unrelated Caucasian KAL patients. We identified KAL1 mutations in 14 patients, of which 7 (c.3G>A (p.M1?), g.IVS1+1G>T, c.570_571insA (p.R191fsX14), c.784G>C (p.R262P), c.958G>T (p.E320X), c.1651_1654delinsAGCT (p.P551_E552delinsSX), c.1711T>A (p.W571R)) have not been previously reported. In addition, we found FGFR1 mutations in 7 patients, namely c.303G>A (p.V102I), C.385A>C (p.D129A), c.810G>A (p.V273M), c.1093_1094delAG (p.R365fsX41), c.1561G>A (p.A520T), c.1836_1837insT (p.Y613fsX42), c.2190C>G (p.Y730X), all of which were novel mutations. In this study, unilateral renal agenesis and bimanual synkinesia were exclusively found associated with KAL1mutations, cleft palate and dental agenesia with FGFR1mutations. © 2004 Wiley‐Liss, Inc.     

Spectrum of novel mutations in the human PKLR gene in pyruvate kinase-deficient Indian patients with heterogeneous clinical phenotypes

Eighteen unrelated pyruvate kinase (PK)-deficient Indian patients were identified in the past 4 years with varied clinical phenotypes ranging from a mild chronic haemolytic anaemia to a severe transfusion-dependent disorder. We identified 17 different mutations in the PKLR gene among the 36 mutated alleles. Ten novel mutations were identified: 427G>A, 499C>A, 1072G>A, 1180G>T, 1216G>A, 1220A>G, 644delG, IVS5 (+20) C>A, IVS9 (+44) C>T, and IVS9 (+93) A>C. A severe syndrome was commonly associated with some mutations, 992A>G, 1436G>A, 1220A>G, 644delG and IVS9 (+93) A>C, in the PKLR gene. Molecular graphics analysis of human red blood cell PK (RPK), based on the crystal structure of human PK, shows that mutations located near the substrate or fructose 1,6-diphosphate binding site may change the conformation of the active site, resulting in very low PK activity and severe clinical symptoms. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. In particular, the 1216G>A and 1219G>A mutations significantly affect the interdomain interaction because they are located near the catalytic site in the A/B interface domains. The most frequent mutations in the Indian population appear to be 1436G>A (19.44%), followed by 1456C>T (16.66%) and 992A>G (16.66%). This is the first study to correlate the clinical profile with the molecular defects causing PK deficiency from India where 10 novel mutations that produce non-spherocytic haemolytic anaemia were identified.     

Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site

Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron-exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3'UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3'UTR for mutations in a large series of BRCA-mutation negative, population and clinic-based breast cancer cases, and controls. Fifteen novel BRCA1 3'UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.* 528G>C, c.* 718A>G, and c.* 1271T>C and four found in controls, c.* 309T>C, c.* 379G>A, c.* 823C>T, and c.* 264C>T, reduced 3'UTR activity (P < 0.02), whereas two variants found in cases, c.* 291C>T and c.* 1139G>T, increased 3'UTR activity (P < 0.01). Three case variants, c.* 718A>G, c.* 800T>C, and c.* 1340_1342delTGT, were predicted to create new miRNA binding sites and c.* 1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3'UTR reporter activity when coexpressed with the predicted targeting miRNA, miR-103. This is the most comprehensive identification and analysis of BRCA1 3'UTR variants published to date.     

McArdle disease: the mutation spectrum of PYGM in a large Italian cohort

Deficiency of the muscle isozyme of glycogen phosphorylase is causative of McArdle disease or Glycogen storage disease type V (GSD-V), the most common autosomal recessive disorder of glycogen metabolism. The typical clinical presentation is characterized by exercise intolerance with cramps, and recurrent myoglobinuria. To date, 46 mutations in the PYGM gene have been detected in GSD-V patients. We report the mutational spectrum in 68 Italian patients. We identified 30 different mutations in the PYGM gene, including 19 mutations that have not been reported previously. The novel mutations include: eight missense mutations (c.475G>A, p.G159R; c.689C>G, p.P230R; c.1094C>T, p.A365E; c.1151C>A, p.A384D; c.1182C>T, p.R428C; c.1471C>T, p.R491C; c.2444A>C, p.D815A; c.2477G>C, p.W826S), two nonsense mutations (c.1475G>A, p.W492X; c.1627A>T, p.K543X), five splice site mutations (c.855 +1G>C; c.1092 +1G>A; c. 1093-1G>T; c.1239 +1G>A; c.2380 +1G>A), and four deletions (c.715_717delGTC, p.V239del; c.304delA, p.N102DfsX4; c.1970_2177del, p.V657_G726; c.2113_2114delGG, p.G705RfsX16). Whereas we confirmed lack of direct correlation between the clinical phenotype and the genotype, we also found that the so-called 'common mutation' (p.R50X) accounted for about 43% of alleles in our cohort and that no population-related mutations are clearly identified in Italian patients.