Real time PCR assays to detect common mutations in the biotinidase gene and application of mutational analysis to newborn screening for biotinidase deficiency

Biotinidase deficiency is an autosomal recessive disorder of biotin metabolism caused by defects in the biotinidase gene. Symptoms of biotinidase deficiency are resolved or prevented with oral biotin supplementation and as such newborn screening is performed to prospectively identify affected individuals prior to the onset of symptoms. Biotinidase deficiency is detected by determining the activity of the biotinidase enzyme utilizing the newborn dried blood spot and colorimetric end point analysis. While newborn screening by enzyme analysis is effective, external factors may compromise results of the enzyme analysis and difficulty is encountered in distinguishing between complete and partial enzyme deficiencies. In the United States, the four mutations most commonly associated with complete biotinidase deficiency are c98:d7i3, Q456H, R538C, and the double mutation D444H:A171T. Partial biotinidase deficiency is almost universally attributed to the D444H mutation. To more effectively distinguish between profound and partial biotinidase deficiency, a panel of assays utilizing real time PCR and melting curve analysis using Light Cycler technology was developed. Employing DNA extracted from the original dried blood specimens from newborns identified through prospective newborn screening as presumptive positive for biotinidase deficiency, the specimens were analyzed for the presence of the five common mutations. Using this approach it was possible to separate newborns with partial and complete deficiency from each other as well as from many of those with false positive results. In most cases it was also possible to correlate the genotype with the degree of residual enzyme activity present. In newborn screening for biotinidase deficiency, we have shown that the analysis of common mutations is useful in distinguishing between partial and complete enzyme deficiency as well as improving specificity. Combining biotinidase enzyme analysis with genotypic data also increases the sensitivity of screening for biotinidase deficiency and provides information useful to clinicians earlier than would otherwise be possible.     

Double mutation (A171T) and (D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States

Biotinidase deficiency is inherited as an autosomal recessive trait that, unless treated with pharmacologic doses of biotin, can result in neurologic and cutaneous symptoms. We have identified two new mutations in exon D of the biotinidase gene of children with profound biotinidase deficiency ascertained by newborn screening. Transition 511G→A near the 5′ end of exon D results in a substitution of threonine for alanine171 (A171T) and transversion 1330G→C occurs close to the 3′ end of exon D causing a substitution of histidine for aspartic acid 444 (D444H). The D444H mutation was detected in four individuals from our normal population whose mean serum biotinidase activity is 5.25 nmol/min/ml, which is significantly lower than the mean normal activity (7.1 nmol/min/ml). We calculated that this mutation causes a 52% loss of activity in the aberrant enzyme. Twenty-three individuals with the D444H mutation were found by allele specific oligonucleotide analysis of DNA from 296 randomly-selected, anonymous dried-blood spots. We estimate the frequency of this allele in the general population to be 0.039. In contrast, no individuals in 376 have the A171T mutation. Fourteen children (eleven probands and three siblings) out of 31 enzyme-deficient children have both the A171T and D444H mutations. Both mutations are inherited from a single parent as a double mutation allele. The nine families in which this allele was identified are of mostly European ancestry, although the mutation cannot be attributed to a specific nationality or ethnic group. The serum of a child who is homozygous for the double mutation allele has very little CRM and the aberrant enzyme has very low biotinyl-hydrolase activity and no biotinyl-transferase activity. This double mutation allele (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. Hum Mutat 11:410, 1998. © 1998 Wiley-Liss, Inc.     

Double mutation (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening the the United States. Mutations in brief no. 128. Online

Biotinidase deficiency is inherited as an antosomal recessive trait that, unless treated with pharmacologic doses of biotin, can result in neurologic and cutaneous symptoms. We have identified two new mutations in exon D of the biotinidase gene of children with profound biotinidase deficiency ascertained by newborn screening. Transition 511G->A near the 5' end of exon D results in a substitution of threonine for alanine 171 (A171T) and transversion 1330G->C occurs close to the 3' end of exon D causing a substitution of histidine for aspartic acid 444 (D444H). The D444H mutation was detected in four individuals from our normal population whose mean serum biotinidase activity is 5.25 nmol/min/ml, which is significantly lower than the mean normal activity (7.1 nmol/min/ml). We calculated that this mutation causes a 52% loss of activity in the aberrant enzyme. Twenty-three individuals with the D444H mutation were found by allele specific oligonucleotide analysis of DNA from 296 randomly-selected, anonymous dried-blood spots. We estimate the frequency of this allele in the general population to be 0.039. In contrast, no individuals in 376 have the A171T mutation. Fourteen children (eleven probands and three siblings) out of the 31 enzyme-deficient children have both the A171T and D444H mutations. Both mutations are inherited from a single parent as a double mutation allele. The nine families in which this allele was identified are of mostly European ancestry, although the mutation cannot be attributed to a specific nationality or ethnic group. The serum of a child who is homozygous for the double mutation allele has very little CRM and the aberrant enzyme has very low biotinylhydrolase activity and no botinyl-transferase activity. This double mutation allele (A171T and D444H) is a common cause of profound biotinidase deficience in children ascertained by newborn screening in the United States.     

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.     

Analysis of mutations causing biotinidase deficiencya

Biotinidase deficiency is an inherited disorder in which the vitamin, biotin, is not recycled. Individuals with biotinidase deficiency can develop neurological and cutaneous symptoms if they are not treated with biotin. Biotinidase deficiency screening has been incorporated into essentially all newborn screening programs in the United States and in many countries. We now report 140 known mutations in the biotinidase gene (BTD) that cause biotinidase deficiency. All types of mutations have been found to cause biotinidase deficiency. Variants have been identified throughout the coding sequence. Essentially all the variants result in enzymatic activities with less than 10% of mean normal enzyme activity (profound biotinidase deficiency) with the exception of the c.1330G>C (p.D444H) mutation, which results in an enzyme having 50% of mean normal serum activity. The putative three‐dimensional structure of biotinidase has been predicted by homology to that of nitrilases/amidases. The effect of the various missense mutations can be predicted to affect various important sites within the structure of the enzyme. This compilation of variants causing biotinidase deficiency will be useful to clinical laboratories that are performing mutation analysis for confirmational testing when the enzymatic results are equivocal for children identified through newborn screening. Hum Mutat 31:983–991, 2010. © 2010 Wiley‐Liss, Inc.     

β-Glucocerebrosidase gene mutations in two cohorts of Greek patients with sporadic Parkinson's disease

An increasing number of clinical, neuropathological and experimental evidence linking Gaucher disease and a spectrum of synucleinopathies, including Parkinson's disease (PD) has emerged over the last decade. In particular, several studies, despite individual differences, have shown that mutations in the β-glucocerebrosidase gene (GBA) are a risk factor for PD. Recently a study from Northern Greece has shown a significant overrepresentation of such mutations only in patients with early onset PD. In the present study 8 different GBA mutations covering 87% of the mutations identified in Gaucher disease patients diagnosed in Greece were investigated in two ethnic Greek cohorts of patients with sporadic Parkinson's disease. Cohort A included patients residing and originating from Thessaly, Central Greece (n=100) and cohort B included patients residing and/or originating from the greater area of Athens (n=105). Age-gender-ethnicity matched healthy individuals from the same areas were included as controls (n=206). In patients of cohort A 11 carriers of GBA mutations were identified (5/11:N370S, 2/11:L444P, 2/11: D409H;H255Q, 1/11:H255Q, 1/11D409H) as opposed to 3 in the controls (n=105) (1/3:N370S, 1/3:H255Q, 1/3:Y108C) (p=0.021, OR 4.2, 95% CI=1.14-15.54). In patients of cohort B 10 carriers of GBA mutations were identified (4/10:L444P, 4/10:D409H;H255Q, 1/10:N370S, 1/10:IVS10-1G→A) as opposed to 4 in controls (n=101) (3/4:N370S, 1/4:L444P). However the difference was not statistically significant (p=0.113, OR 2.5, 95% CI=0.77-8.42). In both cohorts, patients with PD harboring a GBA mutation had an earlier onset of symptoms than non-carriers (p=0.034, p=0.004). The overall difference in the number of carriers identified in PD patients and controls was statistically significant (p=0.006; OR 3.24; 95% CI=1.35-7.81). The association was reinforced in the early onset PD patients (EOPD; n=28, p=0.000, OR 11.37; 95% CI=3.73-34.6). In conclusion GBA mutations were identified with increased frequency in both geographical cohorts of patients with sporadic PD studied compared to control individuals, with the difference being statistically significant only in cohort A. An impressive association with EOPD was found and one third of the EOPD patients examined harbored a GBA mutation. Qualitative differences regarding the type of mutations and/or their relative frequencies were observed between cohorts A and B of PD patients. Genetic and/or environmental factors may account for the observed differences.     

Clinical features,BTD gene mutations,and their functional studies of eight symptomatic patients with biotinidase deficiency from Southern China

Biotinidase (BTD) deficiency is a rare autosomal recessive metabolic disease, which develops neurological and cutaneous symptoms because of the impaired biotin recycling. Pathogenic mutations on BTD gene cause BTD deficiency. Clinical features and mutation analysis of Chinese children with BTD deficiency were rarely described. Herein, for the first time, we reported the clinical features, BTD gene mutations and their functional studies of eight symptomatic children with BTD deficiency from southern China. Fatigue, hypotonia, proximal muscular weakness, hearing deficits, rash and respiratory problems are common clinical phenotype of our patients. Seizures are observed only in patients with profound BTD deficiency. Five novel mutations were detected, among which c.637delC (H213TfsTer51) was found in 50% of our patients and might be considered as a common mutation. In vitro studies confirmed three mild mutations c.1368A>C (Q456H), c.1613G>A (R538H), and c.644T>A (L215H) which retained 10–30% of wild type enzyme activity, and six severe mutations c.235C>T (R79C), c.1271G>C (C424S), c.1412G>A (C471Y), c.637delC (H213TfsTer51), c.395T>G (M132W), c.464T>C (L155P), and c.1493dupT (L498FfsTer13) which retained <10% of wild type enzyme activity. c.1330G>C (D444H) decreased the protein expression but not activity of BTD enzyme, and H213TfsTer51 was structurally damaging while L498FfsTer13 was functionally damaging. These results will be helpful in establishing the definitive diagnosis of BTD deficiency at the gene level, offering appropriate genetic counseling, and providing clues to structure/function relationships of the enzyme.

     

Molecular analysis of northwestern Mexican patients with cystic fibrosis: screening of 10 known mutations. Mutations in brief no. 185. Online

We have analyzed 45 unrelated Northwestern Mexican patients with Cystic Fibrosis for 10 known CF mutations (DF508, G542X, G551D. R553X, W1282X, NI303K, R334W, R347H, S549R, and R1162X). Screening was performed on exons 7, 10, 11, 19, 20 and 21 using standard methods such as polymerase chain reactions, reverse dot blot hybridization (non-radioactive), and restriction enzyme digestion. The analysis for these ten mutations permitted the identification of only two mutations in 37.7% of CF chromosomes in this sample. The major mutation, delta F508, accounts for 34.4% of CF chromosomes. Of the 45 CF patients 9 (20.0%) were homozygous delta F508 deletion, 11 (24.4%) were heterozygous for the delta F508 mutation and an unknown mutation. One additional mutation G542X was also found in 3 chromosomes in our population (3.3%). Two patients were documented to be a compound heterozygote for DF508/G542X, and other one heterozygous for G542X and an unknown mutation. Therefore 62.2% of chromosomes remain uncharacterized.     

The molecular basis of phenylketonuria in Latvia

Characterization of the molecular basis of phenylketonuria (PKU) in Latvia has been accomplished through the analysis of 96 unrelated chromosomes from 50 Latvian PKU patients. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed through a combined approach in which R158Q, R252W, R261Q, G272X, IVS10-11G>A and R408W mutations were first screened for by PCR or restriction generating PCR amplification of PAH gene exons 5, 7, 11 and 12 followed by digestion with the appropriate diagnostic enzyme. Subsequently 'broad range' denaturing gradient gel electrophoresis analysis of the 13 PAH gene exons has been used to study uncharacterized PKU chromosomes. A mutation detection rate of 98% was achieved. 12 different mutations were found, with the most frequent mutation, R408W, accounting for 76% of Latvian PKU alleles. Six mutations (R408W, E280K, R158Q, A104D, R261Q and P281L) represent 92% of PKU chromosomes. PAH VNTR and STR alleles have been also identified and minihaplotype associations with PKU mutations were also determined.     

Mutation spectrum in a large cohort of unrelated consecutive patients with hypertrophic cardiomyopathy

Defects in nine sarcomeric protein genes are known to cause hypertrophic cardiomyopathy (HCM). Mutation types and frequencies in large cohorts of consecutive and unrelated patients have not yet been determined. We, therefore, screened HCM patients for mutations in six sarcomeric genes: myosin-binding protein C3 (MYBPC3), MYH7, cardiac troponin T (TNNT2), alpha-tropomyosin (TPM1), cardiac troponin I (TNNI3), and cardiac troponin C (TNNC1). HCM was diagnosed in 108 consecutive patients by echocardiography (septum >15 mm, septal/posterior wall >1.3 mm), angiography, or based on a state after myectomy. Single-strand conformation polymorphism analysis was used for mutation screening, followed by DNA-sequencing. A total of 34 different mutations were identified in 108 patients: 18 mutations in MYBPC3 in 20 patients [intervening sequence (intron) 7 + 1G > A and Q1233X were found twice], 13 missense mutations in MYH7 in 14 patients (R807H was found twice), and one amino acid change in TPM1, TNNT2, and TNNI3, respectively. No disease-causing mutation was found in TNNC1. Cosegregation with the HCM phenotype could be demonstrated for 13 mutations (eight mutations in MYBPC3 and five mutations in MYH7). Twenty-eight of the 37 mutation carriers (76%) reported a positive family history with at least one affected first-grade relative; only eight mutations occurred sporadically (22%). MYBPC3 was the gene that most frequently caused HCM in our population. Systematic mutation screening in large samples of HCM patients leads to a genetic diagnosis in about 30% of unrelated index patients and in about 57% of patients with a positive family history.     

Fetal akinesia deformation sequence: a study of 30 consecutive in utero diagnoses

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. The H1069Q mutation in exon 14 of ATP7B is far the most frequent in Wilson patients of European origin. Mutations in exon 8 and 15 are also common among the over 150 described mutations in the WD gene. The aim was to investigate the frequency of these common WD gene mutations in Hungarian patients. A total of 42 patients with WD from 39 Hungarian families were examined. The H1069Q mutation was assessed by a seminested polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay, while mutations in exons 8, 13, 15, and 18 of WD gene were identified by sequencing. In addition, haplotype analysis was performed using three common microsatellite markers (D13S314, D13S301, D13S316). The H1069Q mutation was found in 27 patients (64.3%). Nine patients were H1069Q homozygous. Eighteen patients were H1069Q compound heterozygous, two of them had H1069Q/P969Q and one patient H1069Q/3400delC genotype. In two of the 15 H1069Q-negative patients a novel mutation in exon 13 (T977M) was detected. One H1069Q-negative patient had a mutation in exon 8 (G710S). None of the studied mutations was detected in 12 WD patients. H1069Q-positive patients from various European countries had the same haplotype pattern. The H1069Q point mutation is frequent in Hungarian patients with WD and appears to have originated from a single founder in Eastern Europe. In contrast, mutations in exons 8, 13, 15, and 18 are uncommon in Hungarian WD patients.     

Identification of three novel mutations of the palmitoyl-protein thioesterase-1 (PPT1) gene in children with neuronal ceroid-lipofuscinosis

Eight unrelated children with progressive neurological deterioration and granular osmiophilic deposits (GROD) due to an underlying palmitoyl-protein thioesterase deficiency were analyzed for mutations in the PPT1 gene. Three novel mutations (G118D, Q291X and F84del) were identified. The novel Q291X mutation was observed in an African-American child. The G118D and Q291X mutations occurred in infantile-onset subjects. These two mutations would be predicted to have severe effects on enzyme activity. The novel F84del mutation involves an invariant phenylalanine residue. A missense mutation, Q177E, occurred in three subjects from two families with late-infantile NCL, confirming an association of the Q177E mutation with a late-infantile phenotype. Other previously described mutations were R151X (5/16 alleles), T75P (3/16 alleles), R164X (1/16 alleles), and V181M (1/16 alleles). The current study expands the spectrum of mutations in PPT1 deficiency and further confirms the broad range of age of onset of symptoms resulting from an enzyme deficiency previously associated only with infantile NCL.     

X-连锁的肾上腺脑白质营养不良患者ABCD1基因突变分析

目的　分析我国 X-连锁的肾上腺脑白质营养不良 (X- linked adrenoleukodystrophy,X- AL D)患者 ABCD1基因突变。方法　提取 2 5例 AL D患者外周血基因组 DNA,用 PCR扩增和 DNA直接测序的方法 ,分析 AL D患者 ABCD1基因突变。结果　 2 5例患者中发现 18例有 17种不同的 ABCD1基因突变 ,突变存在于除 4、9和 10以外的每一个外显子 ,错义突变为最常见的类型。10例患者有 10种不同的错义突变 ,其中4种错义突变是国际首次报道 ;3例患者有 3种不同的无义突变 ;1例患者有碱基缺失导致移码突变 ;1例患者有剪切部位的碱基缺失 ;2例患者同时具有两个相同的同义突变。结论　我国 AL D患者大部分存在ABCD1基因突变 ,无突变热点。不同个体常有不同的突变点 ,检测到的突变率约为 70 %。突变类型和表型之间无特殊的相关关系。     

Fabry disease: 20 novel GLA mutations in 35 families

Thirty two mutations have been found in 35 unrelated patients of European origin with Fabry disease, including 8 females. Twenty of the mutations are novel and comprise of 13 missense: H46Y, W47G, R49P, C94S, F113S, G258R, P259R, Q279H, Q280H, R363H, A377D, P409A, P409T; 1 nonsense: L294X; 5 small deletions: 154delT, 520delT, 909‐918del10, 1152‐1153delCA, 1235‐1236delCT and 1 splice site mutation: IVS5+2t→c. The remaining 12 mutations have all been reported previously. All patients with deletions had the classic form of the disease but it was not possible to predict the phenotype from the missense mutations. © Wiley‐Liss, Inc.     

Common mutations of ATP7B in Wilson disease patients from Hungary

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. The H1069Q mutation in exon 14 of ATP7B is far the most frequent in Wilson patients of European origin. Mutations in exon 8 and 15 are also common among the over 150 described mutations in the WD gene. The aim was to investigate the frequency of these common WD gene mutations in Hungarian patients. A total of 42 patients with WD from 39 Hungarian families were examined. The H1069Q mutation was assessed by a seminested polymerase chain reaction (PCR)‐based restriction fragment length polymorphism (RFLP) assay, while mutations in exons 8, 13, 15, and 18 of WD gene were identified by sequencing. In addition, haplotype analysis was performed using three common microsatellite markers (D13S314, D13S301, D13S316). The H1069Q mutation was found in 27 patients (64.3%). Nine patients were H1069Q homozygous. Eighteen patients were H1069Q compound heterozygous, two of them had H1069Q/P969Q and one patient H1069Q/3400delC genotype. In two of the 15 H1069Q‐negative patients a novel mutation in exon 13 (T977M) was detected. One H1069Q‐negative patient had a mutation in exon 8 (G710S). None of the studied mutations was detected in 12 WD patients. H1069Q‐positive patients from various European countries had the same haplotype pattern. The H1069Q point mutation is frequent in Hungarian patients with WD and appears to have originated from a single founder in Eastern Europe. In contrast, mutations in exons 8, 13, 15, and 18 are uncommon in Hungarian WD patients. © 2002 Wiley‐Liss, Inc.     

Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease

Wilson disease (WD) is an autosomal recessive disorder of copper transport. WD patients are presenting with a wide range of heterogeneous clinical syndromes including hepatic, neurological, or psychiatric presentations. The disease is caused by mutations in the ATP7B gene. This study presents the results of comprehensive mutation analysis in 227 WD patients from 200 unrelated families (173 from Czech Republic and 27 from Slovakia). More than 80% of all mutant alleles were identified, using a combination of PCR/RFLP, DGGE, TTGE, DHPLC, and sequencing. A total of 40 different mutations and 18 polymorphisms were detected on 400 independent mutant chromosomes. The most common molecular defect was H1069Q (57% of all 400 studied alleles). Each of the other 39 mutations was present in no more than 4% of WD alleles and 23 mutations were found in only one WD allele each (0.25%). Thirteen novel mutations were identified, including seven missense mutations (L641S, T737R, D918E, T1033S, G1111D, D1271N, and G1355C), four small deletions (19_20delCA, 1518_1522del5, 3140delA, and 3794_3803del10), and two splice-site mutations (2446-2A>G, 2865+1G>A). We did not find a significant correlation between H1069Q homozygosity and age of onset, and clinical and biochemical manifestation. Our data provide evidence that the H1069Q mutation-the most common molecular defect of the ATP7B gene in the Caucasian population-originates from Central/Eastern Europe. Screening of five prevalent mutations is predicted to reveal 70% of all mutant alleles presented in WD patients. This will provide a good starting point for early clinical classification of WD in our population.     

Characterization of cystathionine beta-synthase gene mutations in homocystinuric Venezuelan patients: identification of one novel mutation in exon 6

This study describes for the first time the cystathionine beta-synthase (CBS) gene mutations in Venezuelan patients. A total of five disease-causing mutations were identified in 9 out of 10 independent chromosomes. Four of the mutations have been previously described (G85R, T191M, D234N, and D444N) and a novel mutation was found (Q243X). Two common polymorphisms (699C/T and 1080C/T) were found in the CBS gene. Mutation analysis was performed using a combined screening approach for CBS mutations: restriction analysis, single-strand conformational polymorphism (SSCP) scanning, and sequencing. All the mutations were detected in homozygous state, except for Q243X, detected in three heterozygous siblings. Each one of the patients studied presented a different mutation. All mutations and polymorphisms detected involved hypermutable CpG sites, except for the novel mutation Q243X. The most common mutations I278T and G307S were not found in any of the patients. The CBS mutations present in each country differ from each other depending on the demographic profile; therefore, specific mutations scanning must be performed in each population for diagnosis and prognosis purposes.     

The molecular basis of cystathionine beta-synthase (CBS) deficiency in UK and US patients with homocystinuria

The molecular basis of cystathionine beta-synthase (CBS) deficiency has been studied in 536 patient alleles with 130 different mutations described. To date, no study has reported on the incidence of any of the reported mutations in patients from the UK and the US. We developed a new antisense oligonucleotide (ASO) PCR/hybridization method to screen for 12 of the most frequent CBS mutations in 14 unrelated patients from the UK and 38 unrelated patients from the US, a total of 104 independent alleles. We determined 16/28 (57%) and 28/76 (37%) of the affected alleles in the UK and US patients, respectively. Four different mutations were identified in the UK patients (c.374G>A, R125Q; c.430G>A, E144K; c.833T>C, I278T; c.919G>A, G307S) and 8 mutations identified in the patients from the US (c.341C>T, A114V; c.374G>A, R125Q; c.785C>T, T262M; c.797G>A, R266K; c.833T>C, I278T; c.919G>A, G307S; g.13217A>C (del ex 12); c.1330G>A, D444N). The I278T was the predominant mutation in both populations, present in 8 (29%) of 28 independent alleles from the UK and in 14 (18%) of 76 independent alleles from the US. The incidence of the G307S mutation was 21% in the UK patients and 8% in the US patients. The spectrum of mutations observed in the patients from the UK and US is closer to that which is observed in Northern Europe and bears less resemblance to that observed in Ireland.     

High prevalence of Arginine to Glutamine Substitution at 98, 141 and 162 positions in Troponin I (TNNI3) associated with hypertrophic cardiomyopathy among Indians

ABSTRACT: BACKGROUND: Troponin I (TNNI3) is the inhibitory subunit of the thin filament regulatory complex Troponin, which confers calcium-sensitivity to striated muscle actomyosin ATPase activity. Mutations (2-7%) in this gene had been reported in HCM. However, the frequencies of mutations and associated clinical presentation have not been established in cardiomyopathy patients of Indian origin, hence we have undertaken this study. METHODS: We have sequenced all the exons, including the exon-intron boundaries of TNNI3 gene in 101 hypertrophic cardiomyopathy patients (HCM), along with 160 healthy controls, inhabited in the same geographical region of southern India. RESULTS: Our study revealed a total of 16 mutations. Interestingly, we have observed Arginine to Glutamine (R to Q) mutation at 3 positions 98, 141 and 162, exclusively in HCM patients with family history of sudden cardiac death. The novel R98Q was observed in a severe hypertrophic obstructive cardiomyopathy patient (HOCM). The R141Q mutation was observed in two familial cases of severe asymmetric septal hypertrophy (ASH++). The R162Q mutation was observed in a ASH++ patient with mean septal thickness of 29 mm, and have also consists of allelic heterogeneity by means of having one more synonymous (E179E) mutation at g.4797: G [RIGHTWARDS ARROW] A: in the same exon 7, which replaces the very frequent codon (GAG: 85%) with rare codon (GAA: 14%). Screening for R162Q mutation in all the available family members revealed its presence in 9 individuals, including 7 with allelic heterogeneity (R162Q and E179E) of which 4 were severely affected. We also found 2 novel SNPs, (g.2653; G [RIGHTWARDS ARROW] A and g.4003 C [RIGHTWARDS ARROW] T) exclusively in HCM, and in silico analysis of these SNPs have predicted to cause defect in recognition/binding sites for proteins responsible for proper splicing. CONCLUSION: Our study has provided valuable information regarding the prevalence of TNNI3 mutations in Indian HCM patients and its risk assessment, these will help in genetic counseling and to adopt appropriate treatment strategies.     

Preconception and prenatal cystic fibrosis carrier screening of African Americans reveals unanticipated frequencies for specific mutations.

PURPOSE: It is recommended that cystic fibrosis (CF) carrier screening be made available to African Americans who are either pregnant or planning a pregnancy. We analyzed the carrier and mutant allele frequencies for African Americans undergoing CF carrier screening in our laboratories. METHODS: Between December 2001 and September 2003, we performed carrier screening for 2189 African Americans, testing for at least the 25 recommended mutations. RESULTS: A total of 33 CF carriers were identified. The most common mutations detected were deltaF508, G622D, R117H/7T, and G551D. The G622D allele frequency among African Americans was 0.18%. We did not detect any 3120 + 1G --> A carriers, although 4 were expected (P < 0.05). CONCLUSIONS: When considering only the 25 recommended CF mutations, 1 in 75 African Americans screened in our laboratories were carriers (within the expected range, given a 69% mutation detection rate). The addition of 2 mutations, G622D and Q98R (incidentally identified while screening for ACOG/ACMG mutations), increased the observed carrier frequency to 1 in 66, which is not significantly different from the known African American carrier frequency of 1 in 65. The frequencies of several specific mutations detected were unanticipated, as was the absence of 3120 + 1G --> A carriers. Further studies on African American patients with classic CF are needed to examine the incidence of CF mutations that are not part of the current panel, such as G622D.