A novel silent substitution (C8516T) in exon 9 of the human PROC gene.

Protein C is a vitamin K dependent serine protease zymogen, which has a regulatory influence over the coagulation cascade via the inhibition of factors Va and VIIIa. Hereditary protein C deficiency is associated with an increased risk of thromboembolic disease. A multitude of families displaying protein C (PROC) gene defects have been reported, and a number of DNA sequence polymorphisms are known to occur in the PROC gene. We have identified a previously undescribed silent substitution (C8516T) by direct DNA sequencing in a Korean patient with thrombosis and protein C deficiency. In addition, a rare T allelic frequency (0.016) was determined in 123 patients with acquired or hereditary protein C deficiency.     

Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India

We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH‐cytochrome b5 reductase (NADH‐CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice‐site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three‐dimensional (3D) structure of human erythrocyte NADH‐CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype–phenotype correlation in NADH‐CYB5R deficiency.     

Identification of 9 novel IDS gene mutations in 19 unrelated Hunter syndrome (Mucopolysaccharidosis type II) patients

Hunter syndrome is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS deficiency can be caused by several different types of mutations in the IDS gene. We have performed a molecular and mutation analysis of a total of 19 unrelated MPS II patients of different ethnic origin and identified 19 different IDS mutations, 9 of which were novel and unique. SSCP analysis followed by DNA sequencing revealed four novel missense mutations: S143F, associated with the 562C→T polymorphism, C184W, D269V and Y348H. Two novel nonsense mutations were found: Y103X (433C→A) and Y234X (826C→G). In two patients two novel minor insertions (421insA and 499insA) were identified. In one patient a complete IDS deletion was found, extending from locus DXS1185 to locus DXS466. © 1998 Wiley-Liss, Inc.     

Mutational screening of APP gene in patients with early-onset Alzheimer disease utilizing mismatched PCR-RFLP

To elucidate the frequency of mutations of the β/A4 amyloid protein precursor (APP) gene in early-onset Alzheimer disease, we designed a mismatched PCR-RFLP that can identify all kinds of missense mutations at codon 717 in addition to the seven kinds of known mutations at exon 17. When we screened mutations at exon 17 utilizing this method and the double missense mutations at exon 16 of the APP gene by PCR-RFLP, no cases revealed mutations of the APP gene among 13 familial and 54 sporadic cases, except one family (OS-1) that had previously been reported and used as a positive control of APP717(Val → Ile). Our results support the hypothesis that mutations in the APP gene are not major causes in early-onset Alzheimer disease.     

Characterization of the sialidase molecular defects in sialidosis patients suggests the structural organization of the lysosomal multienzyme complex

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation and hepatosplenomegaly (sialidosis type II). We have analyzed the genomic DNA from nine sialidosis patients of multiple ethnic origin in order to find mutations responsible for the enzyme deficiency. The activity of the identified variants was studied by transgenic expression. One patient had a frameshift mutation (G623delG deletion), which introduced a stop codon, truncating 113 amino acids. All others had missense mutations: G679G-->A (Gly227Arg), C893C-->T (Ala298Val), G203G-->T (Gly68Val), A544A-->G (Ser182Gly) C808C-->T (Leu270Phe) and G982G-->A (Gly328Ser). We have modeled the three-dimensional structure of sialidase based on the atomic coordinates of the homologous bacterial sialidases, located the positions of mutations and estimated their potential effect. This analysis showed that five mutations are clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We hypothesize that this region may be involved in the interface of sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in their high-molecular-weight complex required for the expression of sialidase activity in the lysosome.     

Molecular defects of the C7 gene in two patients with complement C7 deficiency

Different genetic mutations have been described in complement components resulting in total or subtotal deficiency states. In this work we report the genetic basis of C7 deficiency in a previously reported Spanish patient exhibiting a combined total deficiency of C7 and C4B associated with systemic lupus erythematosus. Exon-specific polymerase chain reaction and sequencing revealed a not previously described single base mutation in exon 10 (T1458A) leading to a stop codon that causes the premature truncation of the C7 protein (C464X). Additionally, a C to A transversion at position 1561 (exon 11) was found in the patient resulting in an amino acid change (R499S). This latter mutation has been previously reported in individuals with subtotal C7 deficiency or with combined subtotal C6/C7 deficiency from widely spaced geographical areas. Another novel mutation was found in a second patient with meningococcal meningitis of Bolivian and Czech origin; a 11-base pair deletion of nucleotides 631-641 in exon 6 leading to the generation of a downstream stop codon causing the premature truncation of the C7 protein product (T189 x 193). This patient was found to be a heterozygous compound for another mutation in C7; a two-base pair deletion of nucleotides 1922 and 1923, 1923 and 1924 or 1924 and 1925 in exon 14 (1922delAG/1923delGA/1924delAG), leading again to the generation of a downstream stop codon that provokes the truncation of the C7 protein (S620x630). This latter mutation has been recently reported by our group in another Spanish family. Our results provide more evidences for the heterogeneous molecular basis of C7 deficiency.     

Identification of seven novel mutations of F8C by DHPLC

Hemophilia A is an X-linked recessive disorder resulting from deficiency of Factor VIII (F8C), an important protein in blood coagulation. A large number of disease producing mutations have been reported in the F8C gene. However, a comprehensive analysis of mutations is difficult to conduct due to the large gene size, its many scattered exons, and the high frequency of de novo mutations. In this study, we performed analysis using PCR, Conformation Sensitive Gel Electrophoresis (CSGE), Denaturing High Performance Liquid Chromatography (DHPLC) and direct sequencing. We found seven novel mutations causing severe, moderate and mild Hemophilia A: IVS14-1G>A, G458V, T1695S, L1758P, Q2311P, 1441delT, 1269-1271insA. At least four variants detected by DHPLC (IVS14-1G>A, Q2311P,_R698W and D1241Q) were not detectable by CSGE.     

一例P．Gly400Val和P．Arg532Ter导致的遗传性FXI缺陷症患者的临床表型CSCD

目的分析1例遗传性凝血因子XI（FXI）缺陷症患者的临床表型和基因突变特征。方法用凝固法检测先证者及家系成员活化部分凝血活酶时间（activated partial thromboplastin time, APTT）、凝血酶原时间（prothrombintime,PT）、凝血因子Ⅺ活性（FⅪactivity,FXI：C）,ELISA方法检测凝血因子Ⅺ抗原（FXI antigen,FXI：Ag）。对F11基因第1～15外显子及其侧翼序列进行PCR扩增、纯化和测序,寻找突变位点并用Pymol软件对突变进行分析。结果先证者APTT为70．3S,明显延长,FXI：C和FⅪ：Ag同时下降为6％和1．9％。先证者儿子FⅪ：C和FⅪ：Ag均下降为31％和39％。测序结果显示先证者携带F11基因第11外显子C．1296G〉T（P．Gly400Val）错义突变和第14外显子C．1691A〉T（P．Arg532Ter）无义突变;先证者儿子为C．1296G〉T（P．Gly400Val）杂合突变携带者。Pymol软件分析显示P．Gly400Val突变导致FⅪ蛋白氢键数量变化,使蛋白质二级结构改变。根据人类基因突变数据库（HGMD professional 2016．4）,F11 NM_13142C．1691A〉T（p．Arg532Ter）为未报道过的新突变,根据美国医学遗传学与基因组学学会（ACMG）2015年指南判断为功能缺失型突变。结论F11NM_13142 C．1296G〉T（p．Gly400Val）和F11 NM_13142C．1691A〉T（P．Arg532Ter）复合杂合突变是导致先证者遗传性FXI缺陷症的致病原因,引起FXI抗原和活性同时下降。     

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.     

Mechanisms of the interaction between two ADAMTS13 gene mutations leading to severe deficiency of enzymatic activity

The inherited deficiency of the von Willebrand factor-cleaving protease ADAMTS13 is associated with rare forms of thrombotic thrombocytopenic purpura (TTP). We investigated a woman with a family history of chronic recurrent TTP and undetectable plasma levels of ADAMTS13 activity. Genetic analysis revealed two missense mutations in the heterozygous state: p.Val88Met substitution in the metalloprotease domain and p.Gly1239Val substitution in the first CUB domain of ADAMTS13. To explore the mechanism of ADAMTS13 deficiency in this patient, the wild type (WT; ADAMT13(WT)) and each mutant construct (ADAMTS13(Val88Met), ADAMTS13(Gly1239Val)) were transiently expressed in HEK 293 and COS-7 cells. To recapitulate the compound heterozygous state of the patient, both mutant ADAMTS13 proteins were also expressed together. The p.Val88Met mutation led to a defect of secretion of the protease associated with a reduction of enzymatic activity, the p.Gly1239Val mutation led to a secretion defect causing intracellular accumulation of the protease. The mechanistic effects of the mutations were further explored by means of differential immunofluorescence, that demonstrated an homogeneous distribution of ADAMTS13(WT) in the Cis-Golgi and endoplasmic reticulum (ER) compartments, a reduction of ADAMTS13(Val88Met) in both compartments, while ADAMTS13(Gly1239Val) failed to reach the Cis-Golgi compartment and remained in the ER.     

Mutation in TNXB gene causes moderate to severe Ehlers-Danlos syndrome

We report a 28-year-old female who presented with severe joint pain, chronic muscle weakness, Raynaud’s phenomenon, and hypermobility. She was found to have a 6074A > T nucleotide transition in the TNXB gene causing an amino acid protein change at Asp2025Val classified as likely pathogenic. We add this clinical report to the literature and classical human disease gene catalogs to identify this specific mutation as disease-causing. This gene variant was reported previously in a different 36-year-old patient who shared our patient’s symptoms of joint hypermobility, skeletal and joint pain, skin elasticity and musculoskeletal problems, thereby causing a more severe presentation than seen in the hypermobility type of Ehlers-Danlos syndrome (EDS). At the time of writing, a few mutations in the TNXB gene have been recognized as pathogenic causing EDS due to tenascin-X deficiency, but the variant identified in our patient has not been recognized as pathogenic in online genetic databases. Our case study in combination with peer-reviewed literature suggests that the 6074A > T nucleotide transition in the TNXB gene may be classified as disease-causing for EDS due to tenascin-X deficiency.     

Clinical, immunological and molecular characteristics of 37 Iranian patients with X-linked agammaglobulinemia

BACKGROUND: X-linked agammaglobulinemia (XLA) is a hereditary immunodeficiency characterized by an early onset of recurrent bacterial infections, a profound deficiency of all immunoglobulin isotypes and a markedly reduced number of peripheral B lymphocytes. Eighty-five percent of the patients with this phenotype have mutations in Bruton's tyrosine kinase (BTK) gene. METHODS: To provide an informative outlook of clinical and immunological manifestations of XLA in Iran, 37 Iranian male patients with an age range of 1-34 years, followed over a period of 25 years, were studied. Twenty-four of the 37 patients were screened for BTK gene mutation using PCR-SSCP followed by direct sequencing. BTK protein expression assay was done by flow cytometry in 9 families. RESULTS: All patients first presented with infectious diseases, the most common of which were respiratory tract infections. Eighteen different mutations were identified, 13 of which were novel: IVS1+5G>C, 1896G>A, 349delA, 1618C>T, 1783T>C, 2084A>G, 1346delT, 1351delGAG, 587A>G, IVS14-1G>A, IVS3+2T>C, 1482G>A, 1975C>A. CONCLUSION: The fact that we found a great number of novel mutations in a relatively limited number of patients underlines the heterogeneity of BTK mutations in the Iranian population. The large number of new mutations indicates that extended studies in this region would be rewarding.     

Mutations in BTD causing biotinidase deficiency

Biotinidase (BTD) is the only enzyme that can cleave biocytin, a product of the proteolytic digestion of holocarboxylases. Profound BTD deficiency (less than 10% mean normal activity in serum) is an autosomal recessive disorder that can result in neurological and cutaneous abnormalities. Both the cDNA and the genomic DNA of normal BTD gene have been isolated and characterized. The BTD gene is localized to chromosome 3p25. Thus far 61 mutations in three of the four exons of the BTD and one mutation in an intron gene that cause profound BTD deficiency have been reported. Mutations occur at different frequencies in symptomatic children than they do in children ascertained by newborn screening. Two mutations, 98‐104del7ins3 and R538C, were present in 52% or 31 of 60 alleles found in symptomatic patients. Three other mutations, A755G, Q456H, and 511 G>A; 1330G>C (double mutation), accounted for 52% of the alleles detected by newborn screening in the United States. Two asymptomatic adults, parents of children with profound BTD deficiency detected by newborn screening, have been described. Additional different mutations have been found in Turkish, Saudi Arabian, and Japanese children with profound BTD deficiency. Partial BTD deficiency (10–30% of mean normal serum activity) is predominantly caused by the single 1330G>C mutation that results in D444H on one allele in combination with one of the mutations causing profound deficiency on the other allele. Four intragenic polymorphisms, three neutral and one amino acid change, have also been found. Although a preponderance of mutations causing the production of truncated BTD protein occurs in symptomatic children with profound deficiency, preliminary studies fail to demonstrate clear genotype–phenotype correlations. Hum Mutat 18:375–381, 2001. © 2001 Wiley‐Liss, Inc.     

Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK‐deficient patients from 35 unrelated families. Twenty‐nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in‐frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N‐terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three‐dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme‐linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype‐to‐phenotype correlation in PK deficiency as well as the enzyme's structure and function. Hum Mutat 0, 1–8, 2008. © 2008 Wiley‐Liss, Inc.     

P gene mutations associated with oculocutaneous albinism type II (OCA2)

Oculocutaneous albinism type II (OCA2) is the most common form of albinism in humans. OCA2 has been previously associated with mutations of the P gene, the human homologue to the murine pink-eyed dilution gene. The P gene encodes a 110 kDa protein containing 12 potential membrane spanning domains and is associated with melanosomal membranes. The specific function of the P protein is currently unknown but is thought to be involved in tyrosinase processing and transport. We report nine novel mutations in the P gene associated with OCA2. These include two missense mutations, c.1938A>C (p.Ile646Val) and c.1556T>C (p.Val519Ala); one nonsense mutation c.612G>A (p.Trp204X); five frameshift mutations: c.2372_2373delTC, c.1555delG, c.1938_1939insC, c.2050delT, and c.1045_1046delAT; and a splice site mutation c.1951+1G>A. We also report 12 novel polymorphisms including one amino acid substitution, c.2365_2366GC>CA (p.Ala789Glu). At present, there is no functional assay to determine if a mutation is truly pathogenic. The presence of numerous polymorphisms of the P gene in the coding region, several of which result in amino acid substitutions, makes molecular diagnosis problematic. To ensure accurate molecular diagnosis, further mutational analysis will be necessary to produce a comprehensive list of mutations associated with OCA2. This information will also help define the critical functional domains of the P protein. Mutations associated with OCA2 can be found in the Albinism Database (http://albinismdb.med.umn.edu).     

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.     

Evidence for founder effect of the glu104asp substitution and identification of new mutations in triosephosphate isomerase deficiency

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder of glycolysis characterized by multisystem disease and lethality in early childhood. Among seven unrelated Northern European kindreds with clinical TPI deficiency studied, a single missense mutation at codon 104 (GAG;Glu→GAC;Asp) predominated, accounting for 11/14 (79%) mutant alleles. In three families molecular analysis revealed compound heterozygosity for Glu104Asp and novel missense mutations. In two cases the second mutation was a Cys to Tyr substitution at codon 41(TGT→TAT) and in one an Ile to Val substitution at codon 170(ATT→GTT). The origin of the Glu104Asp mutation was defined by haplotype analysis using a novel G/A polymorphism at nucleotide 2898 of the TPI gene. Cosegregation of the low frequency 2898A allele with the G→C base change at nucleotide 315 supports a single origin for the Glu104Asp mutation in a common ancestor. Hum Mutat 10:290–294, 1997. © 1997 Wiley-Liss, Inc.     

Structural insights on pathogenic effects of novel mutations causing pyruvate carboxylase deficiency

Pyruvate carboxylase (PC), a key enzyme for gluconeogenesis and anaplerotic pathways, consists of four domains, namely, biotin carboxylase (BC), carboxyltransferase (CT), pyruvate carboxylase tetramerization (PT), and biotin carboxyl carrier protein (BCCP). PC deficiency is a rare metabolic disorder inherited in an autosomal recessive way. The most severe form (form B) is characterized by neonatal lethal lactic acidosis, whereas patients with form A suffer chronic lactic acidosis with psychomotor retardation. Diagnosis of PC deficiency relies on enzymatic assay and identification of the PC gene mutations. To date, six mutations of the PC gene have been identified. We report nine novel mutations of the PC gene, in five unrelated patients: three being affected with form B, and the others with form A. Three of them were frameshift mutations predicted to introduce a premature termination codon, the remaining ones being five nucleotide substitutions and one in frame deletion. Impact of these mutations on mRNA was assessed by RT‐PCR. Evidence for a deleterious effect of the missense mutations was achieved using protein alignments and three‐dimensional structural prediction, thanks to our modeling of the human PC structure. Altogether, our data and those previously reported indicate that form B is consistently associated with at least one truncating mutation, mostly lying in CT (C‐terminal part) or BCCP domains, whereas form A always results from association of two missense mutations located in BC or CT (N‐terminal part) domains. Finally, although most PC mutations are suggested to interfere with biotin metabolism, none of the PC‐deficient patients was biotin‐responsive. Hum Mutat 0:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Rapid and simple determination of hereditary haemochromatosis mutations by multiplex PCR–SSCP: detection of a new polymorphic mutation

Hereditary haemochromatosis is a common inherited disorder leading to excessive accumulation of iron in various organs. Two missense substitutions at the HFE-gene have recently been associated with the disease, 187C G and 845G → A (mutations H63D and C282Y, respectively). We present a simple, rapid PCR–SSCP multiplex screening method allowing the simultaneous detection of both substitutions. Furthermore, testing the method on 420 Danish blood donors revealed the presence of a hitherto undetected third substitution in 13 individuals. The new substitution, a 193A → T transversion, affects codon 65 changing the code for serine to that of cysteine (S65C). It may thus have functional consequences for the HLA class protein encoded by the HFE-gene. The allele frequencies observed were: H63D 14.8%, C282Y 6.2% and S65C 1.5%, which for the two former alleles are in agreement with frequencies reported for other North European population samples.