新疆少数民族苯丙酮尿症患者的突变基因分析

目的 探讨新疆少数民族苯丙酮尿症(PKU)患者的基因突变特征,为有针对性的防治策略提供科学依据.方法 采用单链构象多态性分析技术和PCR产物直接测序方法 ,检测12例少数民族PKU患者的苯丙氨酸羟化酶(PAH)基因突变.结果 从12例维吾尔族、回族和哈萨克族PKU患者(24个PAH等位基因)中,检出13种基因突变,包括错义突变8种、无义突变1种、剪切位点突变3种,其中突变频率最高的是EX6-96A＞G和P281L,EX6-96A＞G常见于国内和亚洲地区,P281L多见于欧洲各地.突变频率较高的R243Q、R111X、R176X和F161S 4种突变中,其中R243Q是我国北方地区居首位的突变,R111X处于第3位.而R176X和F161S在世界范围内都极为少见,尤其F161S是具有中国特色的基因突变,是第2次在中国人中发现.结论 新疆少数民族中的PAH突变基因不仅与亚洲黄种人和欧洲及拉美人种表现出密切的联系,而且也存在着明显的差异,形成了自身独立的遗传规律和特点,是我国一个十分特殊的PAH突变基因分布区域.     

Tetrahydrobiopterin responsiveness in patients with phenylketonuria

OBJECTIVES: To investigate the BH4 response in a group of patients with phenylketonuria (PKU) in order to offer this alternative treatment to the responsive patients. DESIGN AND METHODS: The 24-h-long Phe/BH4 loading test was performed on 64 PKU patients requiring dietary treatment. RESULTS: All patients with mild-PKU and 75% of patients with moderate-PKU were BH4 responsive, while only 11% of classic-PKU patients showed good/partial response (P < 0.0001). The percentages of Phe decrease after the BH4 loading test were significantly different in the three PKU phenotypes (mild PKU: 67.9 +/- 18.7; moderate PKU: 37.4 +/- 16.8; and classical PKU: 21.9 +/- 13.7; ANOVA with Bonferroni correction: P < 0.0001). We report four mutations (P147S, D222G, P275S, and P362T) not previously associated with BH4 responsiveness, all of them combined with mutations with zero predicted residual activity. CONCLUSION: Both the percentage of Phe decrease and the Phe value achieved 24 h after BH4 loading are valuable data in predicting a response. We report four mutations not previously associated with BH4 responsiveness.     

In vitro characterization of missense mutations associated with quantitative protein S deficiency

OBJECTIVE: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.     

Mutational spectrum of the phenylalanine hydroxylase gene in patients with phenylketonuria in the central region of China

Phenylketonuria (PKU, OMIM 261600) caused by phenylalanine hydroxylase (PAH) deficiency is an autosomal recessive disease that is characterized by abnormalities of phenylalanine metabolism. In this study, a total of 77 patients, originating from the central region of China and who were diagnosed with PAH deficiency at the third affiliated hospital of Zhengzhou University, were enrolled in this study. The 13 exons and 12 flanking introns of the PAH gene were analyzed by Sanger sequencing and next generation sequencing. The sequencing data were aligned to the hg19, PAHvdb and HGMD databases to characterize the genotypes of PKU patients, and genotype–phenotype correlations and BH4 responsiveness predictions were performed using BIOPKUdb. In total, 149 alleles were characterized among the 154 PKU alleles. These mutations were located in exons 2–13, and intron 12 of the PAH gene, with a relative frequency of ≥5%, for EX6-96A>G, p.R241C, p.R243Q, p.V399V and p.R53H. Additionally, a novel variant, p.D84G, was identified. The genotype correlated with clinical symptoms in 33.3–100% of the cases, depending on the disease severity, and BH4 responsiveness predictions show that only five patients with MHP-PKU and one patient with Mild-PKU were predicted to be BH4 responsive. In conclusion, we have characterized the mutational spectrum of PAH in the central region of China and have identified a novel mutation. The hotspot mutation information might be useful for screening, diagnosis and treatment of PKU.     

高分辨熔解曲线检测苯丙氨酸羟化酶基因突变的临床价值

目的 研究用HRM分析技术检测经典型PKU患者PAH基因突变的临床应用价值。方法采用HRM分析技术对17例经典型PKU患者PAH基因的13个外显子及外显子-内含子交界区域进行检测,并采用DNA测序验证HRM检测结果。同时评价HRM诊断PKU的敏感度和特异度。另外,对其中2个家系的风险胎儿进行产前诊断。结果采用HRM分析技术从17例经典型PKU患者中检出有2个致病突变的患者16例,检出有3个致病突变患者l例。同时检出194V(c.280A＞G)、IVS4nt-1G＞A(c.442-1G＞A)、R158Q(c.473G＞ A)、Q160X(c.478C＞ T)、W187X(c.561G＞ A)、E6nt-96A＞ G(c.611A＞ G)、G239D(c.716G＞ A)、R241C(c.721C＞ T)、R243Q(c.728G＞ A)、G247R (c.739C-＞ C)、G247V(c.740G＞ T)、R261X(c.781C ＞T)、R261Q(c.782G＞ A)、H264R(c.791A ＞G)、F302 fsX39(c.904delT)、E305K(c.913G＞ A)、G312V(c.935G＞ T)、Y356X(c.1068C＞A)、V399V (c.1197A ＞T)、R408Q(c.1223G＞ A)、T418P(c.1252A ＞C)、A434D(c.1301C＞ A) 22种致病突变;Q232Q(c.696G＞A)、V245V(c.735G＞ A)、L385L(c.1155C＞G)3种沉默突变;1种单核苷酸多态点rs2280615(c.402A＞ C)。其中194V(c.280A＞ G)、Q160X(c.478C＞ T)、H264R(c.791A ＞G)、G312V (c.935G＞T)和E305K(c.913G＞ A)为PAH基因新发现的突变。2例风险胎儿经产前诊断,1例未发现致病突变,1例诊断为携带者。HRM突变筛查经典型PKU的敏感度和特异度均为100％,其检测结果与DNA测序结果完全一致。结论HRM分析技术是一种简单、准确、快速、高通量、低成本的遗传学分析方法,可用于经典型PKU的PAH基因突变筛查,并可用于已知父母突变的经典型PKU家系快速产前诊断。     

苯丙酮尿症患儿苯丙氨酸羟化酶exon6基因、exon7基因突变的研究

目的 探讨山西省苯丙酮尿症(PKU)患儿的苯丙氨酸羟化酶(PAH)exon6 基因、exon7 基因的突变特征.方法 通过测序及序列比对的方法对56 例PKU 患儿和112 例健康儿童的336 个PAH exon6 基因、exon7 基因进行序列分析,以确定其突变位点、性质和频率.结果 通过序列分析,发现在PKU 患儿和健康儿童中均高频率的出现Q232Q(CAA→CAG)、V245V(GTG→GTA)两种同义突变,其中cDNA 696 位点的频率高达96.2%,cDNA 735 位点的频率高达76.1%.健康儿童的其他序列与Genbank 完全相同.而PKU 患儿的基因序列中还发现了9 种共计37 个突变基因,占全部PAH 突变基因的33.04%.exon 6 仅发现一种突变基因Y204C,突变频率达9.8%;exon 7 中R243Q 的突变率最高,占10.7%,其次为Ivs7 +2 T ＞A,占5.4%,其余的G247V、R252Q、L255S、R261Q、T278I 和E280K 分别占0.9%、0.9%、0.9%、0.9%、2.7%和0.9%.9 种突变都在之前文献中有过报道,其中有7 种错义突变和2 种剪接位点突变.结论 明确了PAH exon6 基因、exon7 基因的突变种类和分布等特征,表明exon6 基因、exon7 基因中Y204C 和R243Q属于山西人群中PAH 基因突变的热点.     

Metabolic basis of sexual dimorphism in PKU mice after genome-targeted PAH gene therapy.

We have previously reported a transgene delivery system based on phiBT1 bacteriophage integrase that results in targeted insertion of transgenes into mammalian genomes, and its use in the delivery of murine phenylalanine hydroxylase (PAH) complementary DNA (cDNA) into the hepatocytes of male phenylketonuria (PKU) mice, leading to a complete and permanent correction of their hyperphenylalaninemic phenotype. In this study, we report only partial phenotypic correction in female PKU mice, even though hepatic PAH activities in both sexes after gene treatment were similar. Daily injections of tetrahydrobiopterin (BH4), an essential co-factor for phenylalanine hydroxylation, in the gene-treated females led to complete correction of their PKU phenotype. After gonadectomy, serum phenylalanine levels in the gene-treated females were reduced to normal, whereas those in the gene-treated males remained unchanged. The sterile gene-treated PKU mice were subjected to daily sex hormone injections. Whereas the estradiol-treated sterile males developed hyperphenylalaninemia, the dihydrotestosterone-treated sterile females remained normal phenylalaninemic. The results indicate that it is estrogen that suppresses the steady-state levels of BH4 in mouse hepatocytes that became limiting, which is the underlying mechanism for the observed sexual dimorphism in PKU mice after PAH gene treatment. Livers of the PAH gene-corrected PKU mice also appeared normal and without apparent pathologies.     

Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH). Over 500 disease-causing mutations have been identified in humans, most of which result in PAH protein misfolding and increased turnover in vivo. The use of pharmacological chaperones to stabilize or promote correct folding of mutant proteins represents a promising new direction in the treatment of misfolding diseases. We performed a high-throughput ligand screen of over 1,000 pharmacological agents and identified 4 compounds (I-IV) that enhanced the thermal stability of PAH and did not show substantial inhibition of PAH activity. In further studies, compounds III (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) and IV (5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno[2,3- d]pyrimidin-4(1H)-one) stabilized the functional tetrameric conformation of recombinant WT-PAH and PKU mutants. These compounds also significantly increased activity and steady-state PAH protein levels in cells transiently transfected with either WT-PAH or PKU mutants. Furthermore, PAH activity in mouse liver increased after a 12-day oral administration of low doses of compounds III and IV. Thus, we have identified 2 small molecules that may represent promising alternatives in the treatment of PKU.     

BH4 responsiveness associated to a PKU mutation with decreased binding affinity for the cofactor

BACKGROUND: Tetrahydrobiopterin (BH4), cofactor of phenylalanine hydroxylase, can be used to treat a subset of phenylketonuria (PKU) patients as it results in a reduction in blood phenylalanine levels. The molecular basis of the response appears to be multifactorial. METHOD: A standard BH4 loading test (20 mg/kg) was performed. Genotyping was performed by DGGE and sequencing analysis. Expression analysis of the D129G mutation was performed in E. coli (expression as fusion protein MBP-PAH) and in a human hepatoma cell line with an N-terminal FLAG epitope. RESULTS: We report the positive response and long-term treatment of a patient functionally hemizygous for the D129G mutation in the phenylalanine hydroxylase gene. Expression in the prokaryotic system revealed partial activity and a decreased binding affinity for BH4 of the mutant protein. In the eukaryotic system the mutant protein shows reduced stability. CONCLUSION: The D129G mutation which confers a BH4-responsive phenotype, has a decreased binding affinity for BH4.     

Expression of 14 von Willebrand factor mutations identified in patients with type 1 von Willebrand disease from the MCMDM-1VWD study

Summary.  Background:  Candidate von Willebrand factor (VWF) mutations were identified in 70% of index cases in the European study 'Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease'. The majority of these were missense mutations. Objectives:  To assess whether 14 representative missense mutations are the cause of the phenotype observed in the patients and to examine their mode of pathogenicity. Methods:  Transfection experiments were performed with full-length wild-type or mutant VWF cDNA for these 14 missense mutations. VWF antigen levels were measured, and VWF multimer analysis was performed on secreted and intracellular VWF. Results:  For seven of the missense mutations (G160W, N166I, L2207P, C2257S, C2304Y, G2441C, and C2477Y), we found marked intracellular retention and impaired secretion of VWF, major loss of high molecular weight multimers in transfections of mutant constructs alone, and virtually normal multimers in cotransfections with wild-type VWF, establishing the pathogenicity of these mutations. Four of the mutations (R2287W, R2464C, G2518S, and Q2520P) were established as being very probably causative, on the basis of a mild reduction in the secreted VWF or on characteristic faster-running multimeric bands. For three candidate changes (G19R, P2063S, and R2313H), the transfection results were indistinguishable from wild-type recombinant VWF and we could not prove these changes to be pathogenic. Other mechanisms not explored using this in vitro expression system may be responsible for pathogenicity. Conclusions:  The pathogenic nature of 11 of 14 candidate missense mutations identified in patients with type 1 VWD was confirmed. Intracellular retention of mutant VWF is the predominant responsible mechanism.     

Identification of exonic deletions in the PAH gene causing phenylketonuria by MLPA analysis

BACKGROUND: Multiplex ligation probe amplification (MLPA) is a sensitive and efficient technique for molecular diagnosis of diseases involving deletions or duplications of large genomic regions. In phenylketonuria (PKU), most of the mutant alleles correspond to missense mutations and large deletions have been scarcely identified. In this study, we report for the first time the use of MLPA analysis on PKU patients to detect exonic deletions. METHOD: DNA from 22 unrelated PKU patients with an incomplete genetic diagnosis after standard mutation detection analysis were subjected to MLPA analysis. Deletions were confirmed by long-range PCR and sequence analysis. RESULTS: The technique identified two large genomic deletions in the phenylalanine hydroxylase (PAH) gene, of 6.6 kb and 1.8 kb, including exons 3 and 5, respectively. The chromosomal breakpoints were established by long-range PCR and chromosomal walking, confirming the involvement of repetitive sequences in the deletions. CONCLUSION: MLPA may complement routine mutation screening in PKU patients, although, in the sample studied, exonic deletions in the PAH gene do not appear to be a frequent cause of PKU.     

The metabolic basis of the hyperphenylalaninemias and tyrosinemia]

The hyperphenylalaninemias are caused by the defect of either phenylalanine hydroxylase (PAH) or tetrahydrobiopterin (BH4) cofactor. The former is diagnosed as phenylketonuria (PKU) or benign hyperphenylalaninemia, based on the serum phenylalanine values. The latter, so called malignant hyperphenylalaninemia, includes three enzyme defects, dihydropteridine reductase (DHPR), 6-pyruvoyl tetrahydropterin synthase (PT PS) and guanosine triphosphate cyclohydrolase (GTP-CH). Excess phenylalanine and its metabolites cause brain damage before 6 years of age. Deficiency of BH4 impairs two other hydroxylases (tyrosine and tryptophan), and severe neurological symptoms develop because of the lack of neurotransmitters. Tyrosinemia I, II, and III are different enzyme defects, fumarylacetoacetate hydrolyase (FAH), hepatic tyrosine aminotransferase (TAT), and 4-hydroxyphenylpyruvate acid oxidase, respectively. Tyrosinemia I is associated with severe involvement of the liver, kidney and central nervous system. Tyrosinemia II has mental retardation, palmar hyperkeratosis and corneal ulcers. Tyrosinemia III has mild mental retardation but no eye or skin manifestations.     

遗传性凝血因子Ⅶ缺陷症家系中R152Q及IVS6+1G→T突变的鉴定

目的检测1个遗传性凝血因子Ⅶ缺陷症家系中因子Ⅶ基因的突变。方法应用PCR结合直接测序的方法对先证者的因子Ⅶ基因9个外显子及其侧翼序列进行分析，鉴别其中可能存在的基因变异。应用PCR产物反向测序法或PCR限制性内切酶分析技术对突变位点进行确证。随机选取100名健康体检者作为正常对照。结果先证者FⅦ基因第6外显子和第6内含子分别存在R152Q和IVS6＋1G→T杂合突变，家系分析表明这2个突变是双重杂合子型，前者遗传自父亲，后者遗传自母亲。100名健康对照者均未发现R152Q错义突变。结论FⅦ基因第6外显子R152Q错义突变和第6内含子供体剪接位点IVS6＋1G→T突变可能是先证者因子Ⅶ先天性缺陷的原因。     

Mutation analysis of phenylketonuria patients from Morocco: High prevalence of mutation G352fsdelG and detection of a novel mutation p.K85X

Objective:The knowledge of the molecular basis of the Phenylketonuria (PKU, MIM# 261600) in different countries provides relevant information for undertaking specific and rational mutation detection strategies in each population and for the implementation of adequate dietary and cofactor treatment. There are no data available in Moroccan population.Design and methods:In this work we describe the genetic analysis by mutation scanning using denaturing gradient gel electrophoresis (DGGE) and subsequent direct sequencing of 20 different PKU families from Morocco. We have also included the study of 7 Moroccan PKU patients living in Spain detected by the Spanish newborn screening program.Results:The mutational spectrum in the first sample included eight different changes, one of them, p.K85X, was novel. The most common mutation was the frame shift change p.G352fsdelG identified in 62.5% of the mutant chromosomes studied. Other changes (p.R176X, IVS10nt-11 g > a, p.W120X, p.A165T, p.R243X and p.R243Q) were identified, respectively, in 2 or 3 mutant alleles. All detected mutations were severe according to the classical phenotype of the patients. In the 7 patients living in Spain we have detected 4 severe mutations (p.G352fs, p.R176X, Y198fs and Exon3del) and also milder changes such as p.A403V, p.S196T, p.D145V and p.R408Q detected in 3 mild hyperphenylalaninemia (MHP) patients and a novel p.L258P found in a mild PKU patient.Conclusion:The results provide important information on the distribution of PKU mutations in this Mediterranean area gaining insight into the genetic epidemiology of the disease.     

The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II

PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II-associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep.     

马凡综合征原纤蛋白质-1突变的蛋白质三维结构分析

目的 分析马凡综合征(Marfan syndrome,MFS)患者原纤蛋白质1（fibrillin 1, Fib-1）基因（FBN1）错义突变的蛋白质三维结构变化。 方法 应用Swiss-Model 软件分析福建地区已发现的4例MFS患者的FBN1错义突变c.5015G>C (p.C1672S)、c.5309G>A(p.C1770Y)、c.7241G>A(p.R2414Q)与c.7769G>A (p.C2590Y),模拟突变后的蛋白质三维结构变化。 结果 Swiss-Model 模拟蛋白质结构显示,4个错义突变中,3个发生在半胱氨酸位的错义突变(p.C1672S、p.C1770Y与p.C2590Y)蛋白质三维结构发生明显改变。 结论 用计算机模拟基因突变后的蛋白质三维结构结果有助于了解突变对FBN 1蛋白质三维结构的影响。     

Differential diagnosis and therapy of various forms of hyperphenylalaninemia: facts and fiction]

The various conditions which can lead to elevated blood phenylalanine (PHE) levels must be differentiated promptly in the neonatal period so that the correct treatment can be implemented as soon as possible. In order to exclude the rare tetrahydrobiopterin (BH4) deficiency, it is advisable to perform a BH4 loading test and to determine the renal excretion of pterins, as well as the dihydropteridine reductase activity in erythrocytes. The practical consequence of differentiating the various types of PHE hydroxylase deficiency is that with both phenylketonuria (PKU; PHE greater than 20 mg/dl) and hyperphenylalaninemia (HPA) with PHE levels above 15 mg/dl a diet restricted in PHE is initiated, whereas HPA infants with PHE levels below 8 mg/dl are fed normally. In the case of PHE concentrations between 8 and 15 mg/dl standardized protein intake can be used to decide whether a diet restricted in PHE or only a restriction of protein intake has to be instituted. Optionally a protein challenge can be performed at the age of six months in order to evaluate the individual PHE tolerance. Psychometric investigations of PKU patients after diet discontinuation at different ages, as well as animal studies are in favour of a diet-for-life. The diet in PKU patients is known to lead to some side effects such as bony changes and amino acid imbalance, as well as deficiency of selenium and carnitine. Finally, great efforts have to be made in order to avoid the increasing danger of PHE embryofetopathy in the offspring of PKU mothers (maternal PKU).     

Development of a skin-based metabolic sink for phenylalanine by overexpression of phenylalanine hydroxylase and GTP cyclohydrolase in primary human keratinocytes

Phenylketonuria, PKU, is caused by deficiency of phenylalanine hydroxylase (PAH) resulting in increased levels of phenylalanine in body fluids. PAH requires the non-protein cofactor BH4 and the rate-limiting step in the synthesis of BH4 is GTP cyclohydrolase I (GTP-CH). Here we show that overexpression of the two enzymes PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH4 supplementation. Integration of multiple PAH and GTP-CH transgenes were achieved after optimized retroviral transduction. Phenylalanine clearance was measured ex vivo in primary human keratinocytes cotransduced with PAH and GTP-CH (more than 370 nmol/24 h/106 cells), a level exceeding that of a human liver cell line (HepG2 cells). Cells overexpressing either one of the enzymes alone did not clear significant amounts of phenylalanine. Transfer of the two genes into the same cell was not necessary, since cocultivation of cells transduced separately with PAH and GTP-CH also resulted in phenylalanine clearance. Thus the experiments indicate metabolic cooperation between cells overexpressing PAH and cells overexpressing GTP-CH, possibly due to intercellular transport of synthesized BH4.     

Diversity of Ribosomal Mutations Conferring Resistance to Macrolides, Clindamycin, Streptogramin, and Telithromycin in Streptococcus pneumoniae

Mechanisms of resistance were studied in 22 macrolide-resistant mutants selected in vitro from 5 parental strains of macrolide-susceptible Streptococcus pneumoniae by serial passage in various macrolides (T. A. Davies, B. E. Dewasse, M. R. Jacobs, and P. C. Appelbaum, Antimicrob. Agents Chemother., 44:414-417, 2000). Portions of genes encoding ribosomal proteins L22 and L4 and 23S rRNA (domains II and V) were amplified by PCR and analyzed by single-strand conformational polymorphism analysis to screen for mutations. The DNA sequences of amplicons from mutants that differed from those of parental strains by their electrophoretic migration profiles were determined. In six mutants, point mutations were detected in the L22 gene (G95D, P99Q, A93E, P91S, and G83E). The only mutant selected by telithromycin (for which the MIC increased from 0.008 to 0.25 microg/ml) contained a combination of three mutations in the L22 gene (A93E, P91S, and G83E). L22 mutations were combined with an L4 mutation (G71R) in one strain and with a 23S rRNA mutation (C2611A) in another strain. Nine other strains selected by various macrolides had A2058G (n = 1), A2058U (n = 2), A2059G (n = 1), C2610U (n = 1), and C2611U (n = 4) mutations (Escherichia coli numbering) in domain V of 23S rRNA. One mutant selected by clarithromycin and resistant to all macrolides tested (MIC, >32 microg/ml) and telithromycin (MIC, 4 microg/ml) had a single base deletion (A752) in domain II. In six remaining mutants, no mutations in L22, L4, or 23S rRNA could be detected.