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Abstract: Plasmodium vivax is the predominant species of the human malaria parasite present in the Indian subcontinent. There have been recent reports on Chloroquine (CQ) resistance and severe manifestations shown by P. vivax from different regions of the world including India. This study focuses on Bikaner, India where during the last few years there have been continuous reports of severe manifestations by both Plasmodium falciparum and P. vivax. This region has a widespread use of Chloroquine and Sulfadoxine–Pyrimethamine for the treatment of malaria, but the resistance profiles of these drugs are not available. We report here the profile of mutations in marker genes associated with Chloroquine and antifolate drug resistance among the P. vivax parasites obtained from patients with severe (n =30) and non-severe (n =48) manifestations from this region. Most isolates showed the wild type alleles for both the Chloroquine and antifolate resistance markers (P <0.0005). Except for one isolate showing Y976F mutation in the Pvmdr-1 gene, no reported mutation was observed in the Pvmdr-1 or Pvcrt gene. This is in accordance with the fact that till date no Chloroquine resistance has been reported from this region. However, the single isolate with a mutation in Pvmdr-1 may suggest the beginning of the trend towards decreased susceptibility to Chloroquine. The frequency of PvDHFR–PvDHPS two locus mutations was higher among the patients showing severe manifestations than the patient group with non-severe (uncomplicated) malaria (P <0.003). None of the parasites from patients with uncomplicated P. vivax malaria showed the mutant PvDHPS genotype. Novel mutations in PvDHFR (S117H) and PvDHPS (F365L, D459A and M601I) were observed only in the parasite population obtained from patients exhibiting severe complications. Preliminary homology modeling and molecular docking studies predicted that these mutations apparently do not have any effect on the binding of the drug molecule to the enzyme. However, the presence of novel mutations in the PvDHPS gene indicate a degree of polymorphism of this molecule which is in contrast to available published information. [Copyright &y& Elsevier]

Eyries M, Coulet F, Girerd B, Montani D, Humbert M, Lacombe P, Chinet T, Gouya L, Roume J, Axford MM, Pearson CE, Soubrier F. ACVRL1 germinal mosaic with two mutant alleles in hereditary hemorrhagic telangiectasia associated with pulmonary arterial hypertension. Germline mutations in genes encoding members of the transforming growth factor- β (TGF- β)/bone morphogenetic protein (BMP) superfamily are causal for two hereditary vascular disorders, hereditary hemorrhagic telangiectasia (HHT) and heritable pulmonary arterial hypertension (PAH). When the two diseases coexist, activin A receptor type II-like kinase-1 ( ACVRL1) gene mutations are usually identified. We report a remarkable ACVRL1 germinal and somatic mosaicism characterized by the presence of two distinct mutant alleles and a non-mutant ACVRL1 allele in a woman diagnosed with PAH at the age 40. She also met the Curaçao diagnostic criteria for HHT based on additional findings of telangiectases, epistaxis and arteriovenous malformations. Mutation analysis of ACVRL1 identified two adjacent heterozygous deleterious mutations within exon 10: c.1388del (p.Gly463fsX2) and c.1390del (p.Leu464X) in a region enriched by mutation-associated DNA motifs. The mother transmitted the c.1388del to one child and the c.1390del to two children confirming germinal mosaicism. Allele-specific polymerase chain reaction analysis showed that c.1388del is the predominant mutation in lymphocytes of the index case. Haplotype analysis revealed that both mutant alleles have a common chromosomal origin which is distinct from that of the mother's non-mutant ACVRL1 allele. These distinct mutant alleles in tissues and germline could have arisen by DNA structure-mediated events occurring in the early stages of the mother's embryogenesis, prior to the segregation of her germline, which ultimately led to the independent transmission of each allele. These highlight the complexity of genomic events occurring during early embryogenesis and the consequences of mutational mosaicism upon pathogenic variability. [ABSTRACT FROM AUTHOR]

Purpose: To identify underlying genetic defects in the carbohydrate sulfotransferase-6 (CHST6) gene in North Indian patients with macular corneal dystrophy (MCD). Methods: 30 clinically diagnosed MCD patients from 21 families and 50 healthy normal controls were recruited in the study. Detailed clinical evaluation in the patients was undertaken followed by histopathology and ultrastructural studies in corneal tissues. DNA from blood samples was amplified for the CHST6 coding and upstream region followed by direct sequencing and in silico analysis. Results: We identified pathogenic mutations in 17 patients from 11 families. Of these 4 were novel (p.Ser54Tyr, p.Gln58Arg, p.Leu59His and p.Leu293Phe), 2 were previously reported (Arg93His and Glu274Lys) homozygous, 1 heterozygous stop codon (p.Trp123X) and 2 compound heterozygous (p.Arg93His + p.Arg97Pro; p.Leu22Arg + p.Gln58X) mutations. A missense single-nucleotide polymorphism was also identified in 11 patients. The novel mutations were conserved as shown by in silico analysis. Thirteen patients did not show any pathogenic CHST6 changes. Conclusions: This is the first report on molecular analysis of MCD in North Indian patients. All cases could not be explained by mutations in CHST6, suggesting that MCD may result from other changes in the regulatory elements of CHST6 or from genetic heterogeneity. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Aims of the present study are: 1) to report an additional Sicilian patient with autoimmune polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED), whose genotypic features are original, in that she is the first APECED patient with three coexisting mutations of Autoimmune Regulator (AIRE) gene, two of which had never been reported to date; 2) to review the genotypic epidemiology of APECED in Italy, in order to underline the great genetic heterogeneity of this disorder in Italian patients. Both the novel mutations of our new patient, named S107C and Q108fs respectively, were detected on exon 3 and were inherited by the mother, whereas the third one (T16M) was inherited by the father. Phenotypically, the present case is quite characteristic, in that she exhibited a classical clinical picture, with no original features. Conclusions: a) this is the seventh Sicilian APECED patient identified to date, and the first one with no R203X mutation; b) our data confirm that distribution of mutations may considerably vary according to different geographical distribution, within the same country, thus suggesting a potential founder effect. [Copyright &y& Elsevier]

Abstract: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease of small vessel caused by mutations in the NOTCH3 gene (NCBI Gene ID: 4854) located on chromosome 19p13.1. NOTCH3 consists of 33 exons which encode a protein of 2321 amino acids. Exons 3 and 4 were found to be mutation hotspots, containing more than 65% of all CADASIL mutations. We performed direct sequencing on an ABI 3130 Genetic Analyser to screen for mutations and polymorphisms on 300 patients who were clinically suspected to have CADASIL. First, exons 3 and 4 were screened in NOTCH3 and if there were no variations found, then extended CADASIL testing (exons 2, 11, 18 and 19) was offered to patients. Here we report two novel non-synonymous mutations identified in the NOTCH3 gene. The first mutation, located in exon 4 was found in a 49-year-old female and causes an alanine to valine amino acid change at position 202 (605C>T). The second mutation, located in exon 11, was found in a 66-year-old female and causes a cysteine to arginine amino acid change at position 579 (1735T>C). We also report a 46-year-old male with a known polymorphism Thr101Thr (rs3815188) and an unreported polymorphism NM_000435.2:c.679+60G>A observed in intron 4 of the NOTCH3 gene. Although Ala202Ala (rs1043994) is a common polymorphism in the NOTCH3 gene, our reported novel mutation (Ala202Val) causes an amino acid change at the same locus. Our other reported mutation (Cys579Arg) correlates well with other known mutations in NOTCH3, as the majority of the CADASIL-associated mutations in NOTCH3 generally occur in the EGF-like (epidermal growth factor-like) repeat domain, causing a change in the number of cysteine residues. The intronic polymorphism NM_000435.2:c.679+60G>A lies close to the intron–exon boundary and may affect the splicing mechanism in the NOTCH3 gene. [Copyright &y& Elsevier]

Abstract: Background: JAG1 is an evolutionarily conserved ligand for Notch receptor and functions in the cell fate decisions, cell–cell interactions throughout the development of heart especially right heart development. Tetralogy of Fallot (TOF) is essentially a right sided heart disease with characteristic features of ventricular septal defect, right ventricular outflow tract obstruction, aortic dextroposition and right ventricular hypertrophy. Hence, the present study was investigated to identify mutations of JAG1 gene in an Indian cohort of patients with TOF. Methods: The clinical data and blood samples from 84 unrelated subjects with TOF were collected and evaluated in comparison with 87 healthy individuals. PCR based single strand conformation polymorphism analysis and subsequent bidirectional DNA sequencing of conformers was carried in the exon 6 of JAG1 gene. Results: The DNA sequences aligned with NCBI-BLAST led to the identification of four novel variations including one nonsense 765 C>A, two missense 814 G>T, 834 G>T; and one silent alteration 816 G>T in TOF patients. The protein structure of JAG1 predicts that these variations effect first and second epidermal growth factor like repeat and might disturb ligand-receptor binding ability. The presence of similar variations was not observed in healthy controls. The software CLUSTAL-W showed the inter species conservation of altered amino acids in missense mutations. Conclusion: Disease-associating novel JAG1 gene variations were found in TOF patients, and seem to play an important role in the causation of the disease. [Copyright &y& Elsevier]

McDonald J, Damjanovich K, Millson A, Wooderchak W, Chibuk JM, Stevenson DA, Gedge F, Bayrak-Toydemir P. Molecular diagnosis in hereditary hemorrhagic telangiectasia: findings in a series tested simultaneously by sequencing and deletion/duplication analysis. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by a unique pattern of telangiectasia and arteriovenous malformations (AVMs). Mutations in one of two genes ( ENG and ACVRL1) cause approximately 85% of cases. Genetic testing impacts clinical management because genotype/phenotype correlations exist, and early preventive screening for internal AVMs is recommended in affected individuals prior to the age at which a diagnosis can typically be made based on clinical criteria. We report 383 consecutive cases in which sequencing and large deletion/duplication analysis were performed simultaneously for endoglin ( ENG) and activin-like receptor kinase 1 ( ACVRL1). We report the first case of mosaicism in an affected individual and 61 novel mutations. We discuss the potential benefits of a diagnostic testing approach for HHT whereby ENG and ACVRL1 are analyzed simultaneously by sequencing and a method which detects large deletion/duplications, rather than by a sequential or reflex testing protocol. We report a case in which a deletion would probably have been missed if large deletion/duplication analysis was performed only if a suspected pathogenic mutation was not first identified by sequencing. [ABSTRACT FROM AUTHOR]

Abstract: CHARGE syndrome is an autosomal dominant disorder characterized by features represented in its acronym: Coloboma, Heart defect, Atresia of the choanae, Retarded growth and development, Genital abnormalities, Ear anomalies/deafness. We report two patients with a diagnosis of typical CHARGE syndrome and one with atypical clinical diagnosis. All the three patients had uni- or bilateral choanal atresia and sensorineural hearing loss. The patients were screened for CHD7 gene mutations. Three novel occurring de novo heterozygous mutations were identified: a mutation in the donor splice site of intron 24, a missense mutation in exon 2 and a deletion in exon 11. [ABSTRACT FROM AUTHOR]

The article discusses novel mutations in two Japanese patients with campomelic dysplasia (CD) who showed long-term survival. A medical profile of these patients are presented together with the process of screening for genetic mutations by extracting genomic DNA from peripheral blood leukocytes. Results of DNA sequencing are discussed with emphasis on the creation of SOX9 mutation, with one leading to a truncated protein and the other to a formation of an extended protein.

Clin Microbiol Infect 2010; 16: 1454–1457 Fluoroquinolones are increasingly used for the treatment of infections caused by multidrug-resistant Mycobacterium tuberculosis. Our study was designed to determine the frequency of the emergence of ciprofloxacin-resistant isolates in a university hospital (Rabta University Hospital Tunis, Tunisia) and to characterize the mutations responsible for the resistance phenotype. A total of 495 clinical M. tuberculosis isolates obtained from January 2005 to July 2008 were investigated for their susceptibility to ciprofloxacin, using the standard proportion method, PCR and DNA sequencing. Four resistant isolates (0.8%) were identified. Among these, only two carried point mutations in gyrA leading to amino acid changes other than the phenotypically silent S95T substitution. No gyrB missense mutations were found in any of the clinical isolates. Although fluoroquinolone resistance is still rare in Tunisia, accurate surveillance is needed in order to control the possible emergence of resistance to fluoroquinolones, which are essential for the successful treatment of multidrug-resistant tuberculosis. [ABSTRACT FROM AUTHOR]

Autosomal recessive primary microcephaly (MCPH) is a rare neurological disorder, in which the patients exhibit reduced occipital frontal head circumference (>3 standard deviations) and mild-to-severe mental retardation. Autosomal recessive primary microcephaly is genetically heterogeneous and 7 loci have been reported to date. Mutations in ASPM (abnormal spindle-like, microcephaly associated) gene are the most common cause of autosomal recessive primary microcephaly in the majority of the reported families. In the current investigation, we have located and studied 21 families with autosomal recessive primary microcephaly. Genotyping using polymorphic microsatellite markers linked to 7 autosomal recessive primary microcephaly loci revealed linkage of 18 families to the MCPH5 locus. Sequence analysis of the ASPM gene in 18 linked families detected 2 novel nonsense mutations (c.2101C>T/p.Q701X; c.9492T>G/p.Y3164X) in 2 families and 2 novel deletion mutations (c.6686delGAAA/p.R2229TfsX9; c.77delG/p.G26AfsX41) in 2 other families. Three previously described mutations (c.3978G>A/p.W1326X; c.1260delTCAAGTC/ p.S420SfsX32; c.9159delA/p.K3053NfsX4) were also detected in 11 families. These identified mutations extended the body of evidence implicating the ASPM gene in the pathogenesis of human hereditary primary microcephaly. [ABSTRACT FROM AUTHOR]

β-Thalassemia (β-thal) is one of the most common autosomal recessive disorders in Iran, with more than 15,000 registered cases of thalassemia major in the country. Iran has a multiethnic society and knowledge of the mutation spectrum and regional distribution is an essential requirement for health planning and a prenatal diagnosis program. We have determined the spectrum of mutations in patients from the Isfahan region of Iran. A study of 190 chromosomes revealed 24 different mutations, including three novel ones: −41 (A>C), IVS-I-109 (–T) and codon 24 (–G). The most common mutation was IVS-II-1 (G>A) (20.5%), followed by IVS-I-5 (G>C) (11%). The findings for the Isfahan region confirm the extremely heterogeneous nature of the molecular basis of β-thal in Iran. The results show that a strategy of using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) for 14 of the most common mutations and DNA sequencing for the rare mutations can be used for prenatal diagnosis of β-thal in this region. [ABSTRACT FROM AUTHOR]

L-2-hydroxyglutaric aciduria (L-2-HGA, MIM 236792) is a neurometabolic disorder caused by the toxic accumulation of high concentration of L-2-hydroxyglutaric acid in plasma and cerebrospinal fluid. Distinct mutations on the L2HGDH gene have been associated with the clinical and biochemical phenotype. Here we present three novel mutations (Gln197X, Gly211Val and c.540+1 G>A), which increase the present deleterious collection of L2HGDH gene up to 35 mutations that we have compiled in this study. In addition, we used the haplotypic information based on polymorphic markers to demonstrate the common origin of Gly57Arg harboring chromosomes. [ABSTRACT FROM AUTHOR]

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disease caused by mutations in the adenomatous polyposis coli ( APC) gene. Massive formation of colorectal adenomas, of which some will inevitably develop into adenocarcinomas, is the hallmark of the disease. Characterization of causative APC mutations allows presymptomatic diagnosis, close follow-up and prophylactic intervention in families. To date more than 900 different germline mutations have been characterized worldwide demonstrating allelic heterogeneity. The germline mutation spectrum of APC identified in 69 apparently unrelated Norwegian FAP families are presented and discussed with reference to clinical phenotype and novel mutation rate. Different methods have been used over the years. However, all mutations were confirmed detectable by an implemented denaturing high-performance liquid chromatography screening approach. Multiplex ligation-dependent probe amplification analysis was employed for potential gross rearrangements. Fifty-three distinctive mutations were detected, of which 22 have been detected in Norway exclusively. Except for two major deletion mutations encompassing the entire APC, all mutations resulted in premature truncation of translation caused by non-sense (31%) or change in reading frame (69%). A high ratio of novel APC mutations continues to contribute to APC mutation heterogeneity causing FAP. This is the first comprehensive report of APC germline mutation spectrum in Norway. [ABSTRACT FROM AUTHOR]

Abstract: The disorder trimethylaminuria (TMAu) often manifests itself in a body odor for individuals affected. TMAu is due to decreased metabolism of dietary-derived trimethylamine (TMA). In a healthy individual, 95% or more of TMA is converted by the flavin-containing monooxygenase 3 (FMO3, EC 1.14.13.8) to non-odorous trimethylamine N-oxide (TMA N-oxide). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAu. Herein, we report two novel mutations observed from phenotyping and genotyping two self-reporting individuals. Sequence analysis of the exon regions of the FMO3 gene of a young woman with severe TMAu revealed heterozygous mutations at positions 187 (V187A), 158 (E158K), 308 (E308G), and 305 (E305X). Familial genetic analysis showed that the E158K/V187A/E308G derived from the same allele from the mother, and the E305X was derived from the father. FMO3 variants V187A and V187A/E158K were characterized for oxygenation of several common FMO3 substrates (i.e., 5- and 8-DPT, mercaptoimidazole (MMI), TMA, and sulindac sulfide) and for its thermal stability. Our findings show that with the combination of V187A/E158K mutations in FMO3, the enzyme activity is severely affected and possibly contributes to the TMAu observed. In another study, genotyping analysis of a 17year old female revealed a mutation that caused a frame shift after K415 and resulted in a protein variant with only 486 amino acid residues that was associated with severe TMAu. [Copyright &y& Elsevier]

Juvenile polyposis syndrome (JPS), a rare polyposis syndrome in childhood, is characterized by multiple diffuse hamartomatous polyps in the gastrointestinal tract (GI) with an estimated incidence of one in 50,000 to 100,000 live births. Although a juvenile polyp is a non-cancerous lesion, affected individuals have an increased risk of GI malignancy. JPS is a disease related to loss-of-function mutations in BMPR1A and SMAD4 involved in the biological maintenance of cellular growth. Pathogenic mutations in those genes have been identified in approximately 50–60% of JPS cases. Our report describes two JPS cases with loss-of-function mutations of BMPR1A identified by a high-throughput nucleotide sequencing. Both cases had a similar clinical manifestation, symptomatic lower GI bleeding. Both patients underwent a total colectomy after histological confirmation. Consequently, Family members who had had genetic confirmation of these mutations were invited to be screened by enteroscopy. JPS is a hereditary disease caused by dominantly inherited loss-of-function of the BMPR1A and SMAD4 genes. Identifying loss-of-function mutations in particular genes allows early screening, diagnosis, and prevention of malignancy among the index cases and their families. High-throughput genome sequencing is a current molecular approach for improving the precision of mutation detection and minimizing time to diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In previous studies, we have identified novel mutations in Tunisian FH families. In this study, we have extended our investigation to additional families. Five unrelated probands were screened for mutations in the LDLR and APOB genes, using direct sequencing and enzymatic restriction. We identified two novel LDLR mutations: a missense mutation in exon 7: p.Gly343Cys (c.1027G>T), and a nonsense mutation in exon 17: p.Lys816X (c.2446A>T). Using the PolyPhen and SIFT prediction computer programs the p.Gly343Cys is predicted to have a deleterious effect on LDL receptor activity. The missense mutation we found in exon 3, p.Cys89Trp (c.267C>G), has previously been identified in patients from United Kingdom and Spain, and is reported here for the first time in the Tunisian population. Finally, the framshift mutation in exon 10, p.Ser493ArgfsX44, is reported here for the fourth and fifth time in Tunisian families. The latter is the most frequent FH-causing mutation in Tunisia. These LDLR gene mutations enrich the spectrum of mutations causing FH in the Tunisian population. The framshift mutation, p.Ser493ArgfsX44, seems to be a founder mutation in this population. [Copyright &y& Elsevier]

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia (CAH), an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease- causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. In this report, we report the characterisation of two novel CYP21A2 missense mutations (p.H119R and p.I194N) found in two unrelated Italian patients with nonclassic (NC) CAH clinical diagnosis. Functional in vitro assays for mutagenized CYP21 enzymes were performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained for p.H119R and p.I194N mutants allowed to classify them as NC-CAH associated mutations. These results correlate with the rate of severity of the patients' disease. Finally, the new p.H119R and p.I194N mutations should be included in the panel of those already listed for association with the NC form of 21-hydroxylase deficiency. © 2009 IUBMB IUBMB Life 61(3): 229–235, 2009 [ABSTRACT FROM AUTHOR]

Abstract: Mutations of the receptor tyrosine kinase (RTK) are frequently reported in acute myeloid leukemia (AML) with a normal karyotype. In this study, Southeast Asian AML patients with a favorable karyotype including t(8;21)/AML-ETO, inv(16)(CBFβ/SMMHC), and t(15;17)/PML-RARα were genotyped for KIT and FLT3 RTK mutations by PCR and sequencing. The combined frequency of KIT/FLT3 mutations in patients with t(8;21), inv(16) and t(15;17) was 35%, 18% and 41%. KIT mutations were mainly detected in patients with t(8;21) (23%) and undetectable in patients with t(15;17). Two novel KIT mutations were identified. FLT3 mutations were preferentially found in patients with t(15;17) (41%). Patients with inv(16) had a strikingly low frequency of both KIT and FLT3 mutations (9% each). KIT-mutated patients were older than FLT3-mutated patients and demonstrated a high expression of myeloid antigens and CD56 lymphoid antigen. FLT3 mutation was coexistent with PML-RARα with markedly low or no CD11c and HLA-DR expression. KIT and FLT3 mutations preferentially exist in distinct clinical and genetic AML subtypes, reflecting unique leukemogenetic mechanisms. Targeting therapy with specific RTK inhibitors should provide benefits for a subgroup of AML patients with favorable chromosomes who also carry selective types of RTK mutations. [Copyright &y& Elsevier]

Background. Congenital nephrotic syndrome (CNS) is de- fined as nephrotic syndrome that manifests at birth or within the first 3 months of life. Most patients develop end-stage renal disease (ESRD) within 2 to 3 years of life. CNS of the Finnish-type (CNF) features a rather specific renal histology and is caused by recessive mutations in the NPHS1 gene encoding nephrin, a major structural protein of the glomerular slit-diaphragm. So far, more than 80 different mutations of NPHS1 causing CNF have been published. [ABSTRACT FROM PUBLISHER]

The article presents a study on three novel mutations of the ectodysplasin A (EDA) gene in Chinese patients with X-linked hypohidrotic ectodermal dysplasia (XLHED). The researchers believe that a pathogenic mutation located in the introns or other regulatory regions might be responsible for the disease phenotype. Furthermore, the study revealed evidence for an unequal homologous recombination between two LINE-1 elements as the molecular mechanism in the pathogenesis of XLHED.

Factor XI (FXI) deficiency disorder is caused by defects in the F11 gene . The affected patients may suffer unexpected and major bleeding after trauma. Hence, the aim of this study was to identify the mutations underlying FXI deficiency in Iranian patients. The genetic basis of FXI deficiency was investigated in nine Iranian patients from unrelated families using conformation-sensitive gel electrophoresis (CSGE) and direct sequencing. Nine different mutations were detected among which seven changes were not previously reported. Among the novel mutations, one was a point mutation that interfered with normal splicing of the mRNA; the other six changes were missense mutations that resulted in amino acid substitutions. Five mutations out of nine were heterozygous and were found in moderately affected patients, whereas the other four changes were homozygous among severely affected patients. [ABSTRACT FROM AUTHOR]

Mutations in the TBX22 gene underlie an X-linked malformation syndrome with cleft palate (CP) and ankyloglossia. Its mutations also result in non-syndromic CP in some populations. To investigate whether mutations in TBX22 play a part in the formation of non-syndromic CP in the Thai population, we performed mutation analysis covering all the coding regions of the TBX22 gene in 53 unrelated Thai patients with non-syndromic CP. We identified four potentially pathogenic mutations, 359G→A (R120Q), 452G→T (R151L), 1166C→A (P389Q), and 1252delG in four different patients. All mutations were not detected in at least 112 unaffected ethnic-matched control chromosomes and had never been previously reported. R120Q and R151L, found in two sporadic cases, were located in the DNA binding T-box domain. P389Q and 1252delG, found in two familial cases, were at the carboxy-terminal region, which has never been described. Our study indicates that TBX22 mutations are responsible for a significant proportion of Thai non-syndromic CP cases confirming its importance as a frequent cause of non-syndromic CP across different populations. [ABSTRACT FROM AUTHOR]

Abstract: The Wiskott–Aldrich syndrome (WAS) is an X-linked disorder including microthrombocytopenia, eczema and immunodeficiency. A mild form is known as the X-linked thrombocytopenia (XLT). We screened 150 individuals or families based on a multiplex PCR method. We found 28 novel mutations (7 missense, 1 nonsense, 1 nonstop change, 5 splice site mutations and 14 deletions or insertions). The method relied on the co-synthesis of 5 amplicons and direct sequencing, optimizing the novel protocol proposed by Jones et al. [L.N. Jones, M.I. Lutskiy, J. Cooley, et al. A novel protocol to identify mutations in patients with Wiskott–Aldrich syndrome, Blood Cells Mol. Dis. 28 (2002) 392–398]. It was thus possible to identify faster and at a lower cost the mutations in newly diagnosed patients. The mutation distribution, according to the type, was in keeping with the distribution reported previously. No clear-cut genotype–phenotype correlation was observed. [Copyright &y& Elsevier]

Autosomal recessive limb-girdle muscular dystrophies (LGMD2s) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement of the proximal limb girdle muscles; the group includes at least 10 different genetic entities. The calpainopathies (LGMD2A), a subgroup of LGMD2s, are estimated to be the most common forms of LGMD2 in all populations so far investigated. LGMD2A is usually characterized by symmetrical and selective atrophy of pelvic, scapular and trunk muscles and a moderate to gross elevation of serum CK. However, the course is highly variable. It is caused by mutations in the CAPN3 gene, which encodes for the calpain-3 protein. Until now, 161 pathogenic mutations have been found in the CAPN3 gene. In the present study, through screening of 93 unrelated LGMD2 families, we identified 29 families with LGMD2A, 21 (22.6%) of which were identified as having CAPN3 gene mutations. We detected six novel (p.K211N, p.D230G, p.Y322H, p.R698S, p.Q738X, c.2257delGinsAA) and nine previously reported mutations (c.550delA, c.19_23del, c.1746-20C>G, p.R49H, p.R490Q, p.Y336N, p.A702V, p.Y537X, p.R541Q) in the CAPN3 gene. There may be a wide variety of mutations, but clustering of specific mutations (c.550delA: 40%, p.R490Q: 10%) could be used in the diagnostic scheme in Turkey. [ABSTRACT FROM AUTHOR]

Abstract: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. We analysed the ARSA gene in eight unrelated Italian families with different clinical variants of MLD and identified three novel mutations: two Ser406Gly, (Glu329Ter) associated with late infantile MLD and one (Leu52Pro) with juvenile MLD. Only one family carried a pseudodeficiency allele (Asn350Ser). The IVS2+1G>A mutation occurred in four families. We also identified three polymorphisms, all in heterozygosis: Thr391Ser was present in five families, Trp193Cys in four families, and Ala210Ala in one family. We could identify 100% of the alleles causing MLD in the families, involving 12 different mutations, resulting in improved prognosis and genetic counselling. [Copyright &y& Elsevier]

Thyroglobulin abnormality is a rare cause of congenital hypothyroidism and only a limited number of mutations in the thyroglobulin gene have been reported. We analyzed the thyroglobulin gene in a patient with congenital goitrous hypothyroidism. This girl was identified with hyperthyrotropinemia in a neonatal mass-screening test. The patient had goiter, and her body weight gain was poor. Distal femoral epiphysis was absent on roentgenography. Her serum thyroxine level was low; however, her triiodothyronine level was high. Autoantibodies against triiodothyronine, thyroid peroxidase, and thyroglobulin were all negative. Her serum thyroglobulin level was undetectable. The thyroglobulin gene from the genomic DNA of the patient was analyzed by direct sequencing. Two novel heterozygous missense mutations, Cys1897Tyr (exon 31) and Arg2336Gln (exon 40), were found in the patient. The former mutation was derived from her mother, suggesting a compound heterozygous state. Normal triiodothyronine and low thyroxine concentrations are often observed in patients with thyroglobulin gene mutations. We considered that some patients with thyroglobulin abnormality might have high triiodothyronine levels. In cases of congenital goitrous hypothyroidism with normal-to-high triiodothyronine levels and low serum thyroglobulin levels, thyroglobulin abnormality should be considered. [ABSTRACT FROM AUTHOR]

The development of neutralising antibodies to factor VIII (FVIII) is a major complication of haemophilia A (HA) therapy. We aimed to construct an individual risk profile for the development of inhibitors in HA and started by screening for the causative mutation in our HA patient population. A total of 109 patients and 28 carriers were screened. The analysis revealed 38 different mutations in the FVIII gene, of which 13 have not been described on the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS). Twenty-five mutations have been reported previously and all except two had a similar phenotype to what has been described. Three novel mutations were associated with severe HA: one non-missense mutation, a small insertion in the A2 domain, and two missense mutations, a H256R mutation in the A1 domain and a L2025P substitution in the C1 domain. One novel mutation, Y156C, was associated with moderate HA. Nine novel mutations caused mild HA. The P130R, D167E and V278M mutations are located in the A1 domain. R439C, Y511H, A544G and Q645H in the A2 domain, L1758F in the A3 domain and a S2157R mutation in the C1 domain. In conclusion, the genotypic profile of our HA population was not different from others described and is suitable to study inhibitor formation. [ABSTRACT FROM AUTHOR]

Purpose: Usher syndrome is an autosomal recessive disease associating retinitis pigmentosa and neurosensory deafness. Three clinical types (USH1, USH2, USH3) and 11 mutated genes or loci have been described. Mutations in MYO7A and USH2A are responsible for about 40% and 60% of Usher syndromes type 1 and 2, respectively. These genes were screened in a series of patients suffering from Usher syndrome. Methods: We performed SSCP screening of MYO7A in 12 unrelated patients suffering from Usher syndrome type 1 (USH1) and USH2A in 28 unrelated patients affected by Usher syndrome type 2 (USH2). Results/conclusions: Six mutations in MYO7A were found in five patients, including two novel mutations c.397C>G (His133Asp) and 1244-2A>G (Glu459Stop), accounting for 42% of our USH1 patients. Twelve mutations in USH2A were found in 11 patients, including four new mutations c.850delGA, c.1841-2A >G, c.3129insT, and c.3920C>G (Ser1307Stop), accounting for 39% of our USH2 patients. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegia (HSP) is a group of neurodegenerative disorders mainly characterized by progressive spasticity of the lower limbs. The major features of HSP are a marked phenotypic variability both among and within families and an extended genetic heterogeneity. More than 20 HSP loci and 10 spastic paraplegia genes (SPG) have been identified to date, including the genes responsible for the two most frequent forms of autosomal dominant spastic paraplegia (AD-HSP), encoding spastin (SPG4) and atlastin (SPG3A), respectively. To date, only eight mutations have been described in the atlastin gene, which was reported to account for about 10% of all AD-HSP families. We investigated 15 German and French AD-HSP families, including the 3 large pedigrees that allowed the mapping and subsequent refinement of theSPG3Alocus. Three novel mutations were found in exons 4, 9, and 12 of the atlastin gene and the common R239C mutation located in exon 7 was confirmed in a 7th family of European origin. Overall, the comparison of the clinical data for all SPG3A-HSP families reported to date failed to reveal any genotype/phenotype correlation as demonstrated for other forms of AD-HSP. However, it confirmed the early onset of this form of HSP, which was observed in almost all affected individuals with a mutation in the atlastin gene. [ABSTRACT FROM AUTHOR]

We have identified 16 different mutations of the lowdensity lipoprotein receptor (LDLR) gene in 25 unrelated Korean patients with heterozygous familial hypercholesterolemia (FH), including five novel mutations, C83Y, 661del17, 1705insCTAG, C675X, and 941-1G>A. The 1705insCTAG mutation in which the four 3'-terminal nucleotides of exon 11 are duplicated was found to prevent splicing of exon 11 and would therefore generate a truncated polypeptide. The in-frame 36-bp deletion (1591del36) in exon 11, which had been reported only in one Korean FH patient, was also found. We showed that this change affects transport of the LDL receptor from the endoplasmic reticulum to the cell surface. In addition, we found 8 mutations (-136C>T, E119K, E207K, E207X, F382L, R574Q, 1846- 1G>A, and P664L) that had been described in other ethnic groups but not in Koreans, and 2 mutations (R94H and D200N) that had been described in Koreans as well as other ethnic groups. 5 mutations (1591del36, E119K, E207X, E207K, and P664L) were found more than once in the Korean FH samples. Identification of the novel and recurring LDLR mutations in Korean FH patients should facilitate prenatal and early diagnosis in families at high risk of FH. [ABSTRACT FROM AUTHOR]

Autosomal recessive limb-girdle muscular dystrophy linked to 19q13.3 (LGMD2I) was recently related to mutations in the fukutin-related protein gene (FKRP) gene. Pathogenic changes in the same gene were detected in congenital muscular dystrophy patients (MDC1C), a severe disorder. We have screened 86 LGMD genealogies to assess the frequency and distribution of mutations in the FKRP gene in Brazilian LGMD patients. We found 13 Brazilian genealogies, including 20 individuals with mutations in the FKRP gene, and identified nine novel pathogenic changes. The commonest C826A European mutation was found in 30% (9/26) of the mutated LGMD2I alleles. One affected patient homozygous for the FKRP (C826A) mutation also carries a missense R125H change in one allele of the caveolin-3 gene (responsible for LGMD1C muscular dystrophy). Two of her normal sibs were found to be double heterozygotes. In two unrelated LGMD2I families, homozygous for novel missense mutations, we identified four asymptomatic carriers, all older than 20 years. Genotype-phenotype correlation studies in the present study as well as in patients from different populations suggests that the spectrum of variability associated with mutations in the FKRP gene seems to be wider than in other forms of LGMD. It also reinforces the observations that pathogenic mutations are not always determinant of an abnormal phenotype, suggesting the possibility of other mechanisms modulating the severity of the phenotype that opens new avenues for therapeutic approaches.European Journal of Human Genetics (2003) 11, 923-930. doi:10.1038/sj.ejhg.5201066 [ABSTRACT FROM AUTHOR]

A 39-year-old Japanese women who has been jaundiced since childhood and indicative of Dubin–Johnson syndrome (DJS) underwent MRP2/ABCC2 mutation analysis. The results of the analysis revealed two novel mutations, C298T and C3928T, and two single nucleotide polymorphisms (SNPs), C3972T and C-24T. One of the two mutations (C298T) is in the transmembrane domain. The other mutation (C3928T) and the SNP (C3972T) are in the cytoplasmaic domain which are concentrated in the second adenosine triphosphate (ATP)-binding cassette. Both of the mutations, C298T and C3928T, are immature stop codons. C298T, C-24T and C3972T (originating from the mother) and C3928T (from the father) were observed to be heterozygous in this patient. Light-microscopy examination of the liver biopsy specimens revealed the accumulation of dark pigment granules in most of the hepatocytes. In the immunohistochemistry using pAb recognizing the C-terminal of the human MRP2, no staining of MRP2 in the canalicular membrane domain was observed in the liver sections from this patient. In this patient, the mutations with immature stop codons presumably resulted in instability of MRP2 mRNA and/or defective synthesis of the truncated protein, which may underlie the loss of the expression of functional MRP2 protein. [Copyright &y& Elsevier]

We have analyzed Twinkle, the causative gene for autosomal dominant progressive external ophthalmoplegia (adPEO) on chromosome 10, in 11 Australian autosomal dominant progressive external ophthalmoplegia families of Caucasian origin, and investigated whether there are distinct molecular and clinical features associated with mutations in this gene. We found two new mutations in Twinkle, in 3 of the 11 pedigrees examined. One resides in the linker region of this gene while the other is in the primase domain. Both regions are highly conserved between species. Multiple deletions in the mtDNA from muscle are not always prominent and there are significant variations in the clinical presentation within and between families with mutations in the Twinkle gene. Therefore, genotype/phenotype predictions are difficult. No mutations were found in adenine nucleotide translocator 1 (ANT1), another known adPEO causative gene, in four of the seven remaining families investigated. Thus, Twinkle appears to be the most common gene associated with adPEO in Australian families. [Copyright &y& Elsevier]

Background: Congenital adrenal hyperplasia arising from 21-hydroxylase deficiency is associated with mutations in the CYP21 gene on chromosome 6p. This is the first report on the mutational spectrum of the CYP21 gene in Singapore. Methods: To catalogue the mutations, ten exons of the CYP21 gene from 28 Singaporean patients were analyzed by PCR amplification and direct sequencing. Results: Common mutations in descending order were the intron 2 splice site mutation (32.7% of the alleles), the I172N mutation (23.1% of the alleles), and the R356W mutation (19.2% of the alleles). Two potentially novel mutations were discovered: (1) duplication of 111 bp from codon 21 to codon 57 (exon 1) and (2) missense mutation (L261P, exon 7). There was generally a good genotype-phenotype correlation, allowing accurate prediction of the disease severity.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Fabry disease is a X-linked recessively inherited metabolic disorder, which results from the deficient activity of the lysosomal hydrolase α-galactosidase A leading to the systemic deposition of glycosphingolipids with terminal α-galactosyl moieties. Single-strand conformation polymorphism (SSCP) analysis was performed, followed by DNA sequencing of PCR amplified exons of the human α-galactosidase A gene in 5 unrelated Korean patients with classic Fabry disease. Five different mutations were identified; two nonsense mutations (Y86X and R342X), one missense mutation (D266N), and two small deletions (296del2 and 802del4). Except for R342X mutation, four were novel mutations (Y86X, D266N, 296del2, 802del4). A T to G transversion at nucleotide position 5157 in exon 2 caused a tyrosine-to-stop substitution at codon 86. A G to A transition at position 10 287 in exon 5 substituted an asparagine for an aspartate at codon 266. Mutation 296del2 in exon 2 resulted in a frame shift with a stop signal at the 22th codon downstream from the mutation, whereas mutation 802del4 resulted in a stop codon at the site of 4 bp deletion. In addition, the 802del4 was found to be a de novo mutation. This is the first report on mutation analysis of the human α-galactosidase A gene in Korean patients with Fabry disease. [ABSTRACT FROM AUTHOR]

DNA screening for mutations in the α-l-iduronidase (IDUA) gene was performed in a Chinese mucopolysaccharidosis type IH/S patient. The patient had two different mutations: the maternal allele has L346R (t–g transversion in codon 346) and the paternal allele has 388-3c–g (c–g transversion at position -3 of the 3′ splice site of intron 2). In transfected COS-7 cells, L346R showed no appreciable IDUA activity (0.4% of normal activity), although it did not cause an apparent reduction in IDUA mRNA or protein level. The 388-3c–g mutation profoundly affects normal splicing leading to a very unstable mRNA. Expression of the IDUA cDNA containing the mutated acceptor splice site showed trace amounts of enzyme activity (1.6% of normal activity). The results provide further support for the importance of cytosine at the -3 position in RNA processing. [ABSTRACT FROM AUTHOR]

This study has investigated a total of 51 Acinetobacter baumannii isolates for the prevalence of resistant determinants in tigecycline susceptible and non-susceptible clinical isolates of A. baumannii. Antimicrobial susceptibility testing revealed 74% of isolates were tigecycline resistant. Mutations in RND-efflux pump regulatory genes and the expression of efflux pump genes were measured in tigecycline resistant isolates. There was a strong co-relation between the blaNDM-1 and armA wherein majority of the isolates that are positive for blaNDM-1 have also harbored armA. Compared with TSAB (tigecycline susceptible A. baumannii), TNAB (tigecycline non-susceptible A. baumannii) isolates show increased distribution of blaNDM-1 (P = 0.048), blaIMP-1 (P< 0.0001) and blaOXA-51 (P = 0.0029) carbapenemase genes. The variants of RND-efflux pump regulatory genes due to amino-acid mutations in adeS (F12S, K84E, W61R, N268H and Q299R) and adeL (G21R and Q262R) were identified in tigecycline resistant isolates as well as ISAba1 mediated disruption of adeN were observed causing overexpression of adeIJK efflux pump. Additionally, mutations in adeRS were also associated with increased expression of adeABC efflux pump. Besides, TNAB isolates showed significantly (P< 0.0001) higher ability of biofilm formation as compared to TSAB isolates. The tigecycline resistance due to mutations in contemporary A. baumannii isolates having a higher ability to form biofilm may pose therapeutic difficulties., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

To describe clinical and genetic characteristics in a series of Chinese patients with non-syndromic retinitis pigmentosa, a total of 20 unrelated Chinese pedigrees with non-syndromic retinitis pigmentosa were evaluated. Complete ophthalmic examinations data including the Humphrey visual field, spectral domain-optical coherence tomography, full-field electroretinography, and fundus fluorescence were collected and analyzed. Targeted exome sequencing was utilized to investigate variations in 260 known genes of inherited retinal disease, including the 90 known causative retinitis pigmentosa genes. We initially identified the potential candidate variants in the pedigrees, then validated the variants using the Sanger sequencing and performed segregation analysis to verify that the variants constituted disease-causing mutations in these pedigrees. We detected three novel (likely) pathogenic and eight previously reported (likely) pathogenic variations in nine genes reported to be related to non-syndromic retinitis pigmentosa in nine of the pedigrees. We report clinical characteristics of Chinese patients with retinitis pigmentosa and novel mutations responsible for non-syndromic retinitis pigmentosa in Chinese pedigrees, expanding the number of gene mutations associated with this disorder and clarifying its genetic basis in the Chinese population. These data will help with rapid and efficient molecular diagnosis and the study of targeted treatment for retinitis pigmentosa in this population.

This review collates information on the onset of COVID-19, SARS-CoV-2 genome architecture, emergence of novel viral lineages that drove multiple waves of infection around the world and standard and fast track development of vaccines. With the passage of time, the continuously evolving SARS-CoV-2 has acquired an expanded mutational landscape. The functional characterization of spike protein mutations, the primary target of diagnostics, therapeutics and vaccines has revealed increased transmission, pathogenesis and immune escape potential in the variant lineages of the virus. The incurred mutations have also resulted in substantial viral neutralization escape to vaccines, monoclonal, polyclonal and convalescent antibodies presently in use. The present situation suggests the need for development of precise next-generation vaccines and therapeutics by targeting the more conservative genomic viral regions for providing adequate protection., (© 2022 Future Medicine Ltd.)