Identification Of Novel Mutation Research Articles

Identification of novel mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the Greek population

42 Hereditary pancreatitis − managing patient clinical information and sequence variants in the CFTR, PRSS1 and SPINK1 genes A. Millson1, C. Miller2, T. Lewis1, G. Pont-Kingdon1, E. Lyon1,2,3. 1ARUP Institute for Clinical and Experimental Pathology, Research and Development, Salt Lake City, UT, United States; 2ARUP Laboratories, Salt Lake City, UT, United States; 3University of Utah, Pathology, Salt Lake City, UT, United States

Polyuria and polydipsia in a young child: diagnostic considerations and identification of novel mutation causing familial neurohypophyseal diabetes insipidus

A 3-year 5-month-old boy was seen for second opinion regarding polydipsia and polyuria. Previously, a diagnosis of primary polydipsia was made after normal urine concentration after overnight water deprivation testing. The boy's father, paternal grandfather, and paternal aunt had diabetes insipidus treated with desmopressin acetate. Based on this young boy's symptoms, ability to concentrate urine after informal overnight water deprivation, and family history of diabetes insipidus, we performed AVP gene mutation testing. Analysis of the AVP gene revealed a novel mutation G54E that changes a normal glycine to glutamic acid, caused by a guanine to adenine change at nucleotide g.1537 (exon 2) of the AVP gene. Commonly, patients with familial neurohypophyseal diabetes insipidus (FNHDI) present within the first 6 years of life with progressively worsening polyuria and compensatory polydipsia. Since these patients have progressive loss of arginine vasopressin (AVP), they may initially respond normally to water deprivation testing and have normal pituitary findings on brain MRI. Genetic testing may be helpful in these patients, as well as preemptively diagnosing those with a mutation, thereby avoiding unnecessary surveillance of those unaffected.

Abstract 239: Mutation of the inhibitory adaptor protein LNK drives potentiated JAK-STAT signaling in patients with JAK2 V617F-negative myeloproliferative neoplasms

Abstract Dysregulated JAK-STAT signaling via activating mutations in tyrosine kinases (e.g. JAK2 and MPL) is a hallmark of chronic myeloproliferative neoplasms (MPNs). Even in the absence of mutations in JAK2 or MPL, JAK-STAT activation can be demonstrated, suggesting that alterations of other regulatory elements in this pathway may contribute to MPN pathobiology. One regulator of JAK-STAT signaling is LNK (SH2B3), an adaptor protein that binds to MPL via it SH2 domain and co-localizes to the plasma membrane via its pleckstrin homology (PH) domain. Upon cytokine stimulation, LNK binds strongly to JAK2 and dampens or terminates downstream STAT activation. LNK−/- mice exhibit features consistent with an MPN phenotype, including splenomegaly, leukocytosis, and thrombocytosis. We therefore sequenced LNK in 34 JAK2 V617F-negative MPN patients, and report the identification of novel mutations in exon 2 of LNK in two patients. In a patient with primary myelofibrosis, a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains was identified. A second patient with essential thrombocythemia exhibited a missense mutation leading to an E208Q substitution in the PH domain. DNA isolated from cultured skin fibroblasts revealed wild-type (WT) sequence, confirming that these mutations were somatic. TPO-dependent BaF3-MPL cells were transduced with WT and mutant LNK. While WT LNK inhibited TPO-dependent growth and activation of JAK2-STAT3/5, the DEL mutation led to loss of these negative feedback properties, thereby permitting augmented and sustained JAK-STAT activation in response to TPO stimulation. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes. In peripheral blood samples obtained from MPN patients, stimulation with TPO or G-CSF revealed a unique phosphorylated STAT3/5 (pSTAT3+/5+) subpopulation that was increased in DEL compared with normal donor samples. A similar pSTAT3+/5+ subpopulation was seen with JAK2 V617F and MPL W515L-positive samples, suggesting that this may be a shared feature of MPNs. E208Q cells exhibited STAT3/5 phosphorylation in response to TPO, but not G-CSF, indicating that a partial loss of LNK function may generate differential STAT activation profiles in response to specific cytokines. The cytokine-responsive pSTAT3+/5+ cells from DEL were primarily CD34+, and the DEL mutation was detected in this subset, suggesting that LNK mutations arise in a hematopoietic stem or progenitor cell. Finally, the pSTAT3+/5+ response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Thus, mutations in LNK, the first reported in human disease, lead to loss of LNK negative feedback function and represent a novel mechanism of MPN pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 239.

Molecular analysis of Chanarin-Dorfman syndrome (CDS) patients: Identification of novel mutations in the ABHD5 gene

Chanarin-Dorfman syndrome (CDS) is an autosomal recessive metabolic disorder associated with congenital ichthyosis and a multisystemic accumulation of neutral lipids in various types of cells. Recently, mutations of the ABHD5 gene were identified as the cause of CDS. In this work, we carried out molecular analysis of the ABHD5 gene in 6 unrelated patients. We identified one previously reported mutation, N209X and two novel genetic alterations; a nonsense mutation (p.Y50X) and missense mutation (p.S73A).

Identification and functional characterization of a novel nonsense mutation in FGA accounting for congenital afibrinogenemia in six Egyptian patients

Congenital afibrinogenemia is a rare coagulation disorder attributed to over forty mutations found either in homozygosity or in compound heterozygosity, the majority localized in FGA encoding the fibrinogen Aalpha-chain. Despite the number of genetic analyses performed the study of additional patients still allows the identification of novel mutations and a better understanding of fibrinogen structure and function. Here we report the identification and functional analysis of a novel nonsense mutation in FGA exon 5: c.718C>T (CAG>TAG) p.Q240X (Q221X in the mature chain lacking the signal peptide), accounting for fibrinogen deficiency in six Egyptian patients. Expression of the mutant Aalpha-chain cDNA in combination with wild-type Bbeta-chain and gamma-chain cDNAs demonstrated that although the mutant chain could be detected in the cell media of transfected COS-7 cells it was less secreted in comparison to the wild-type Aalpha-chain. Our patients were all homozygous for p.Q240X(Q221X) yet their clinical spectrum varied considerably in their onset of presentation or severity, with bleeding ranging from moderate mucous membrane bleeds in adolescence to life threatening intracranial hemorrhage in infancy.

Identification of novel and known KRT5 and KRT14 mutations in 53 patients with epidermolysis bullosa simplex: correlation between genotype and phenotype

Basal epidermolysis bullosa simplex (EBS) is a hereditary skin blistering disorder resulting in most cases from missense mutations in the keratin 5 (KRT5) or keratin 14 (KRT14) genes. To identify the underlying mutations in different EBS subtypes and correlate genotype and phenotype. Mutation analysis was performed in 53 patients with EBS and their families by direct sequencing of the KRT5 and KRT14 genes. We identified 39 different mutations, of which 15 have not been published previously. Three novel deletion/insertion mutations, among them one in-frame duplication, were associated with the rare phenotype of EBS with mottled pigmentation. We identified for the first time a patient with compound heterozygosity for KRT5 mutations causing Dowling-Degos disease and EBS. Identification of novel mutations and genotype-phenotype correlations in EBS allow improved understanding of disease pathogenesis as well as better patient management.

Phenylalanine hydroxylase gene mutations in phenylketonuria patients from India: Identification of novel mutations that affect PAH RNA

Analysis of seven Indian phenylketonuria families has revealed four novel mutations in the phenylalanine hydroxylase gene; two affected consensus splice sequence and the 3′ UTR, respectively, while the other two were single base insertion and deletion mutations, respectively. A novel 3′ splice site mutation c.168-2A>G resulted in the activation of a cryptic 3′ splice site that generated a premature termination codon leading to very low levels of the mutant transcript, probably due to activation of the nonsense-mediated decay (NMD) pathway. This is probably the first report of PKU caused by the activation of NMD.

The identification of novel mutations in the biotinidase gene using denaturing high pressure liquid chromatography (dHPLC)

Biotinidase deficiency (BD) is an autosomal recessive disorder of biotin metabolism that causes incomplete recycling of free biotin. The resulting depletion of intracellular biotin leads to impaired activities of biotin-dependent carboxylases. The ensuing clinical phenotype includes progressive neurologic deterioration with epileptic seizures, muscular hypotonia as well as skin eczema. BD may be readily diagnosed by analysing enzyme activity in dried blood spots during newborn screening but typically requires molecular confirmation. More than 100 different mutations in the biotinidase gene have been reported to date. To simplify molecular testing we have developed a rapid and accurate denaturing high pressure liquid chromatography (dHPLC) method of the promoter, 3′UTR, all exons including exon/intron boundaries as a first line screen followed by direct sequencing of the respective PCR products. To validate this method we used DNA from 23 different, newly diagnosed patients with biochemically proven BD from Austria, India, Morocco and Spain. A total of 11 mutations, missense 7, frameshift 3 and 1 nonsense, were screened. Six mutations were novel to this study. All mutations revealed distinct dHPLC pattern thus enabling their accurate detection. This study revealed that dHPLC method is robust, automated, economical and above all highly sensitive for the molecular analysis of biotinidase gene and should be used as a pre-analytical tool followed by sequencing of aberrant heteroduplex forming amplicons.

Hypodysfibrinogenaemia due to production of mutant fibrinogen alpha-chains lacking fibrinopeptide A and polymerisation knob ‘A’

Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia) or both (hypodysfibrinogenaemia). Extensive allelic heterogeneity has been found for all these disorders: in congenital afibrinogenaemia for example more than 40 mutations, the majority in FGA , have been identified in homozygosity or in compound heterozygosity. Numerous mutations have also been identified in patients with hypofibrinogenaemia, many of these patients are in fact heterozygous carriers of afibrinogenaemia mutations. Despite the number of genetic analyses performed, the study of additional patients still allows the identification of novel mutations. Here we describe the characterization of a novel FGA intron 2 donor splice-site mutation (Fibrinogen Montpellier II) identified in three siblings with hypodysfibrinogenaemia. Functional analysis of RNA produced by the mutant minigene in COS-7 cells revealed that the mutation led to the in-frame skipping of exon 2. Western blot analysis of COS-7 cells expressing an exon 2 deleted FGA cDNA revealed that an alpha-chain lacking exon 2, which codes in particular for fibrinopeptide A and polymerisation knob 'A', has the potential to be assembled into a hexamer and secreted. Analysis of precipitated fibrinogen from patient plasma showed that the defect leads to the presence in the circulation of alpha-chains lacking knob 'A' which is essential for the early stages of fibrin polymerisation. Fibrin made from purified patient fibrinogen clotted with thrombin displayed thinner fibers with frequent ends and large pores.

Hereditary predominantly motor neuropathies

We review recent advances in Charcot-Marie-Tooth disease (CMT), the most frequent inherited neuromuscular disorder. During the last year further progresses have occurred in this field and concerned identification of novel mutations in recently identified genes, allowing better definition of associated phenotypes; increased knowledge on pathophysiologic mechanisms of the different CMT types, with the contribution of cellular and animal model studies; studies on the natural history of CMT and attempts at developing appropriate outcome measures to assess disease course and intervention efficacy; trials with ascorbic acid in CMT type 1A; and studies on new possible therapeutic strategies. Such advances have implications on clinical management of CMT and are modifying the clinical approach to CMT, by improving diagnostic tools, allowing better definition of prognosis, and increasing the hope for future effective treatments. Research on CMT is important as is shedding light on important pathways that regulates the normal function of axonal transport, vesicular trafficking, and also revealing new aspects of intracellular organelles' function and interactions.

Functional Complementation in Yeast Allows Molecular Characterization of Missense Argininosuccinate Lyase Mutations

Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL(null) strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, "compound heterozygous" yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.

Systematic molecular genetic analysis of congenital sideroblastic anemia: Evidence for genetic heterogeneity and identification of novel mutations

Sideroblastic anemias are heterogeneous congenital and acquired bone marrow disorders characterized by pathologic iron deposits in mitochondria of erythroid precursors. Among the congenital sideroblastic anemias (CSAs), the most common form is X-linked sideroblastic anemia, due to mutations in 5-aminolevulinate synthase (ALAS2). A novel autosomal recessive CSA, caused by mutations in the erythroid specific mitochondrial transporter SLC25A38, was recently defined. Other known etiologies include mutations in genes encoding the thiamine transporter SLC19A2, the RNA-modifying enzyme pseudouridine synthase 1 (PUS1), a mitochondrial ATP-binding cassette transporter (ABCB7), glutaredoxin 5 (GLRX5), as well as mitochondrial DNA deletions. Despite these known diverse causes, in a substantial portion of CSA cases a presumed genetic defect remains unknown. In the context of the recent discovery of SLC25A38 as a major novel cause, we systematically analyzed a large cohort of previously unreported CSA patients. Sixty CSA probands (28 females, 32 males) were examined for ALAS2, SLC25A38, PUS1, GLRX5, and ABCB7 mutations. SLC19A2 and mitochondrial DNA were only analyzed if characteristic syndromic features were apparent. Twelve probands had biallelic mutations in SLC25A38. Seven ALAS2 mutations were detected in eight sporadic CSA cases, two being novel. We also identified a novel homozygous null PUS1 mutation and novel mitochondrial DNA deletions in two patients with Pearson syndrome. No mutations were encountered in GLRX5, ABCB7, or SLC19A2. The remaining undefined probands (43%) can be grouped according to gender, family, and clinical characteristics, suggesting novel X-linked and autosomal recessive forms of CSA.

Genetic testing in familial AD and FTD: Mutation and phenotype spectrum in a Danish cohort

Autosomal dominantly transmitted Alzheimer's disease (AD) and frontotemporal dementia (FTD) are genetically heterogeneous disorders. To date, three genes have been identified in which mutations cause early-onset autosomal dominant inherited AD: APP, PSEN1, and PSEN2. Mutations in two genes on chromosome 17, the MAPT and the PGRN genes, are associated with autosomal dominant inherited FTD. The aim of this study was to characterize the mutation spectrum and describe genotype-phenotype correlations in families with inherited dementia. The identification of novel mutations and/or atypical genotype-phenotype correlations contributes to further characterizing the disorders. DNA-samples from the 90 index cases from a Danish referral-based cohort representing families with presumed autosomal dominant inherited AD or FTD were screened for mutations in the known genes with sequencing, denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA) techniques. Seven presumed pathogenic mutations (two PSEN1, one PSEN2, one APP, one MAPT, and two PGRN) were identified, including a novel PSEN2 mutation (V393M). No dosage aberrations were identified.

Identification of novel mutations and sequence variation in the Zellweger syndrome spectrum of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBD) are a heterogeneous group of autosomal recessive neurodegenerative disorders that affect multiple organ systems. Approximately 80% of PBD patients are classified in the Zellweger syndrome spectrum (PBD-ZSS). Mutations in the PEX1, PEX6, PEX10, PEX12, or PEX26 genes are found in approximately 90% of PBD-ZSS patients. Here, we sequenced the coding regions and splice junctions of these five genes in 58 PBD-ZSS cases previously subjected to targeted sequencing of a limited number of PEX gene exons. In our cohort, 71 unique sequence variants were identified, including 18 novel mutations predicted to disrupt protein function and 2 novel silent variants. We identified 4 patients who had two deleterious mutations in one PEX gene and a third deleterious mutation in a second PEX gene. For two such patients, we conducted cell fusion complementation analyses to identify the defective gene responsible for aberrant peroxisome assembly. Overall, we provide empirical data to estimate the relative fraction of disease-causing alleles that occur in the coding and splice junction sequences of these five PEX genes and the frequency of cases where mutations occur in multiple PEX genes. This information is beneficial for efforts aimed at establishing rapid and sensitive clinical diagnostics for PBD-ZSS patients and interpreting the results from these genetic tests.

Identification of novel mutations in theSLC25A15gene in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome: A clinical, molecular, and functional study

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder of the urea cycle. With the exception of the French-Canadian founder effect, no common mutation has been detected in other populations. In this study, we collected 16 additional HHH cases and expanded the spectrum of SLC25A15/ORC1 mutations. Eleven novel mutations were identified including six new missense and one microrearrangement. We also measured the transport properties of the recombinant purified proteins in reconstituted liposomes for four new and two previously reported missense mutations and proved that the transport activities of these mutant forms of ORC1 were reduced as compared with the wild-type protein; residual activity ranged between 4% and 19%. Furthermore, we designed three-dimensional (3D)-modeling of mutant ORC1 proteins. While modeling the changes in silico allowed us to obtain new information on the pathomechanisms underlying HHH syndrome, we found no clear-cut genotype-phenotype correlations. Although patient metabolic alterations responded well to low-protein therapy, predictions concerning the long-term evolution of HHH syndrome remain uncertain. The preference for a hepatic rather than a neurological presentation at onset also continues, largely, to elude us. Neither modifications in oxidative metabolism-related energy, such as those expected in different mtDNA haplogroups, nor sequence variants in SLC25A2/ORC2 seem to be crucial. Other factors, including protein stability and function, and ORC1-ORC2 structural interactions should be further investigated.

The problem of novel FVIII missense mutations for haemophilia A genetic counseling

Molecular genetic testing for factor VIII (FVIII) mutations is indicated in haemophilia A since determination of FVIII activity cannot reliably identify female carriers. Given the large number of FVIII mutations the identification of novel mutations is not uncommon. Since amino acid polymorphisms of FVIII are rare, missense mutations in patients with haemophilia A which are not found in the normal population are considered as causative in general practice when no other mutation can be detected by complete FVIII gene sequencing. We report a novel rare missense variant (P2311S) in a haemophilia A family that was mistakenly considered as pathogenic leading to amniocentesis, prenatal diagnosis and influenced the peripartal management of the putatively affected child. Subsequently, we identified the novel causative mutation V197G in the family's index case which could be detected neither in the neonate nor in his mother. This case emphasizes the necessity to establish the molecular diagnosis in the family's index case and to perform expression studies of novel mutations to prove their causative nature.

Identification of novel mutations of the WFS1 gene in Brazilian patients with Wolfram syndrome

Wolfram syndrome (WS) is a rare, progressive, neurodegenerative disorder with an autosomal recessive pattern of inheritance. The gene for WS, WFS1, was identified on chromosome 4p16 and most WS patients carry mutations in this gene. However, some studies have provided evidence for genetic heterogeneity and the genotype-phenotype relationships are not clear. Our aim was to ascertain the spectrum of WFS1 mutations in Brazilian patients with WS and to examine the phenotype-genotype relationships in these patients. Clinical characterization and analyses of the WFS1 gene were performed in 27 Brazilian patients with WS from 19 families. We identified 15 different mutations in the WFS1 gene in 26 patients, among which nine are novel. All mutations occurred in exon 8, except for one missense mutation which was located in exon 5. Although we did not find any clear phenotype-genotype relationship in patients with mutations in exon 8, the homozygous missense mutation in exon 5 was associated with a mild phenotype: onset of diabetes mellitus and optic atrophy during adulthood with good metabolic control being achieved with low doses of sulfonylurea. Our data show that WFS1 is the major gene involved in WS in Brazilian patients and most mutations are concentrated in exon 8. Also, our study increases the spectrum of WFS1 mutations. Although no clear phenotype-genotype relationship was found for mutations in exon 8, a mild phenotype was associated with a homozygous missense mutation in exon 5.

Identification of novel mutations in WISP3 gene in two unrelated Chinese families with progressive pseudorheumatoid dysplasia

IntroductionProgressive pseudorheumatoid dysplasia (PPD) is an autosomal recessive genetic disease and it has been reported that PPD is caused by mutations of the Wnt1-inducible signaling pathway protein 3 (WISP3) gene which is located on chromosome 6q22. Up to date, 16 different mutations in the WISP3 have been identified in patients with PPD in different countries previously, but only two mutations in exon 5 were previously identified from Asian origin. Our study aimed to characterize the clinical manifestations and features of PPD and screen the mutations of the disease causing WISP3, and try to elucidate the molecular pathogenesis of PPD. Materials and methodsAltogether, 153 persons, including 4 affected individuals, 49 unaffected individuals from two unrelated Chinese families, and 100 healthy donors were recruited and genomic DNA was extracted. PPD was diagnosed based on the clinical manifestations, physical examination, characteristics of their bones on X-ray and laboratory results. All 5 exons and their exon–intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR) and sequenced directly. ResultsIn family 1, we identified that the proband (IV4) carried a novel non-sense mutation (G46X) which consisted of a homozygous C to T transition at c.8004 in exon 3. This mutation changed codon CAG to TAG and resulted in a subsequent change of the glutamine codon to stop codon and truncation at p. 46. In family 2, a novel missense mutation (C114Y) was found in the three patients (IV6, IV7, IV8), namely, a homozygous G to A transition at c.8209 in exon 3, which resulted in a cysteine (TGT) to tyrosine (TAT) substitution at p.114. Neither G46X nor C114Y was found in 100 normal controls. Meanwhile, we found that these patients had some different phenotypes, compared with the affected individuals with PPD from cases reported previously. ConclusionsOur study suggests that the novel G46X and C114Y mutations in exon 3 in WISP3 gene are responsible for PPD in Chinese patients. Furthermore, many heterozygous carriers (c.8004C>T and c.8209G>A) are found in the two families, suggesting the existence of a founder effect in the locality where they live, respectively.

Systematic Analysis of Known Candidate Genes in 58 Probands with Previously Uncharacterized Congenital Sideroblastic Anemia: Evidence for Genetic Heterogeneity and Identification of Novel Mutations in ALAS2 and PUS1

Sideroblastic anemias are a heterogeneous group of congenital and acquired bone marrow disorders characterized by pathologic iron accumulation in the mitochondria of erythroid precursors. Whereas the molecular ontology of acquired sideroblastic anemia is largely obscure, the genetic etiology is known for many patients with congenital sideroblastic anemia (CSA). Mutations in the heme biosynthetic enzyme 5-aminolevulinate synthase 2 gene (ALAS2)—associated with the classic, X-linked form of the disease—or mitochondrial DNA mutations/deletions (as in Pearson syndrome) have been reported in many cases. Rare cases are associated with mutations in the genes encoding a thiamine transporter (SLC11A2), pseudouridine synthase 1 (PUS1), a mitochondrial ATP-binding cassette transporter (ABCB7), and glutaredoxin 5 (GLRX5). Nonetheless, the molecular defect in a majority of cases of CSA remains unknown and there appears to be a significant bias in underreporting those cases that go genetically uncharacterized. Given this, we undertook a systematic genetic investigation of a large series of previously unreported CSA patients.METHODS AND RESULTS: 58 probands (26 female, 32 male) with CSA were studied. This cohort was comprised of 49 singleton patients and 10 families, including 8 families with at least 2 affected full siblings (1 set of monozygotic male twins, 1 each male or female sibling pairs, 2 male/female sibling pairs, 1 set each of 3 female or 3 male siblings, and one set of 3 siblings of unknown sex), and 2 families in which there was a history of CSA in a prior generation. In the great majority, CSA was the only clinical phenotype. We employed a sequential sequencing strategy to identify variants in ALAS2, PUS1, and GLRX5; ABCB7, SLC19A2, and mitochondrial DNA variants were not included in our analysis due to the absence of associated syndromic phenotypes in the cohort. To begin, ALAS2 was characterized by sequencing the coding region, intron-exon boundaries, proximal promoter, and intron 8, which contains an erythroid-specific enhancer, in all subjects. We detected 7 different missense mutations in 8 of the 58 probands, each of which was a singleton patient. No ALAS2 mutations were found in the promoter or intron 8. Two of the ALAS2 mutations were novel: V301A in a male patient and R517G in a female patient with a skewed X chromosome inactivation pattern in peripheral blood leukocytes as assessed by the human androgen receptor assay (HUMARA). The coding regions and intron-exon boundaries of PUS1 and GLRX5 were sequenced in all probands lacking ALAS2 mutations. Among these, a novel homozygous null (Q154X) PUS1 mutation was found in one familial case; only known polymorphisms in GLRX5 were detected. In 48 probands, including the 9 remaining familial cases, we did not identify a disease-causing mutation. However linkage studies in three families with affected males only were consistent with linkage to the ALAS2 locus in each case. CONCLUSIONS: These results demonstrate that:ALAS2 mutations are commonly associated with CSA,ALAS2 mutations and non-random X inactivation in affected females underscore the importance of ALAS2 analysis in women as well as in men with CSA,many potentially X-linked cases of CSA may have either ALAS2 mutations not detected by conventional sequencing approaches or mutations in other X-linked genes,PUS1 and GLRX5 variants are unusual causes of CSA, andthere is strong genetic evidence that other, autosomal recessive forms of CSA exist.

Identification of novel mutations in five patients with mitochondrial encephalomyopathy

MELAS, MERRF, LHON and NARP, are well-established mitochondrial syndromes associated with specific point mutations of mitochondrial DNA (mtDNA). However, these recurrent mtDNA mutations account for only a minority of mitochondrial disease cases. To evaluate the impact of novel mtDNA mutations, we performed mtDNA sequence analysis in muscle and other tissues of 240 patients with different mitochondrial neuromuscular syndromes. We identified a total of 33 subjects with novel, private or uncommon mutations. Among these, five novel mutations were found in both paediatric and adult cases. We here report on the clinical description of these patients, as well as the biochemical and molecular genetic characterization of the corresponding mutations. Patients 1 and 2 showed changes in ND genes, patient 3 carried a heteroplasmic deletion in the COI gene, patients 4 and 5 carried heteroplasmic mutations in tRNATrp and tRNAPhe, respectively. Altogether, these data indicate that mtDNA analysis must become part of the routine screening for mitochondrial disorders.