Analyses of the mitochondrial mutations in the Chinese patients with sporadic Creutzfeldt–Jakob disease

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause a variety of chronic diseases in central nervous system (CNS). However, the role of mtDNA mutations in sporadic Creutzfeldt–Jakob disease (sCJD) has still been unknown. In this study, we comparatively analyzed complete mtDNA sequences of 31 Chinese sCJD patients and 32 controls. Using MITOMASTER and PhyloTree, we characterized 520 variants in sCJD patients and 507 variants in control by haplogroup and allele frequencies. We classified the mtDNAs into 40 sub-haplogroups of 5 haplogroups, most of them being Asian-specific haplogroups. Haplogroup U, an European-specific haplogroups mtDNA, was found only in sCJD. The analysis to control region (CR) revealed a 31% increase in the frequency of mtDNA CR mutations in sCJD versus controls. In functional elements of the mtDNA CR, six CR mutations were in conserved sequence blocks I (CSBI) in sCJD, while only one in control (P<0.05). More mutants in transfer ribonucleic acid-Leu (tRNA-Leu) were detected in sCJD. The frequencies of two synonymous amino-acid changes, m.11467A>G, p.(=) in NADH dehydrogenase subunit 4 (ND4) and m.12372G>A, p.(=) in NADH dehydrogenase subunit 5 (ND5), in sCJD patients were higher than that of controls. Our study, for the first time, screened the variations of mtDNA of Chinese sCJD patients and identified some potential disease-related mutations for further investigations.     

MITOMASTER: a bioinformatics tool for the analysis of mitochondrial DNA sequences

We have developed a computer system, MITOMASTER, to make analysis of human mitochondrial DNA (mtDNA) sequences efficient, accurate, and easily available. From imported sequences, the system identifies nucleotide variants, determines the haplogroup, rules out possible pseudogene contamination, identifies novel DNA sequence variants, and evaluates the potential biological significance of each variant. This system should be beneficial for mtDNA analyses of biomedical physicians and investigators, population biologists and forensic scientists. MITOMASTER can be accessed at http://mammag.web.uci.edu/twiki/bin/view/Mitomaster.     

Functional consequences of mitochondrial tRNATrp and tRNAArg mutations causing combined OXPHOS defects

Combined oxidative phosphorylation (OXPHOS) system deficiencies are a group of mitochondrial disorders that are associated with a range of clinical phenotypes and genetic defects. They occur in approximately 30% of all OXPHOS disorders and around 4% are combined complex I, III and IV deficiencies. In this study we present two mutations in the mitochondrial tRNA^Trp (MT-TW) and tRNA^Arg (MT-TR) genes, m.5556G>A and m.10450A>G, respectively, which were detected in two unrelated patients showing combined OXPHOS complex I, III and IV deficiencies and progressive multisystemic diseases. Both mitochondrial tRNA mutations were almost homoplasmic in fibroblasts and muscle tissue of the two patients and not present in controls. Patient fibroblasts showed a general mitochondrial translation defect. The mutations resulted in lowered steady-state levels and altered conformations of the tRNAs. Cybrid cell lines showed similar tRNA defects and impairment of OXPHOS complex assembly as patient fibroblasts. Our results show that these tRNA^Trp and tRNA^Arg mutations cause the combined OXPHOS deficiencies in the patients, adding to the still expanding group of pathogenic mitochondrial tRNA mutations.     

Exaggerated status of "novel" and "pathogenic" mtDNA sequence variants due to inadequate database searches

Given its relative ease, screening the entire mitochondrial DNA (mtDNA) for heteroplasmic or novel homoplasmic mutations has become part of the routine diagnostic workup for the molecular geneticist confronted with a disease case exhibiting clinical and biochemical features of mitochondrial dysfunction. "Novelty" of a given mtDNA variant is most often equated with nonregistration in the extensive MITOMAP database (www.mitomap.org). This practice has led to a number of spurious findings and wrong conclusions concerning the pathogenic status of specific mtDNA mutations, especially in the absence of proper evaluation and pathogenicity scoring. We demonstrate by way of real cases targeting the mt-tRNA(Cys) (MT-TC) gene and a stretch within the MT-ND3 gene, that a straightforward Google search can identify twice as many previously observed mutations than any MITOMAP query could achieve. Further, we reassess the recent rediscovery of m.15287T>C by listing all known occurrences and, where possible, providing the haplogroup context, shedding new light on the potential pathogenicity status of m.15287T>C.     

A synonymous variant in MYO15A enriched in the Ashkenazi Jewish population causes autosomal recessive hearing loss due to abnormal splicing

Nonsyndromic hearing loss is genetically heterogeneous. Despite comprehensive genetic testing, many cases remain unsolved because the clinical significance of identified variants is uncertain or because biallelic pathogenic variants are not identified for presumed autosomal recessive cases. Common synonymous variants are often disregarded. Determining the pathogenicity of synonymous variants may improve genetic diagnosis. We report a synonymous variant c.9861 C > T/p.(Gly3287=) in MYO15A in homozygosity or compound heterozygosity with another pathogenic or likely pathogenic MYO15A variant in 10 unrelated families with nonsyndromic sensorineural hearing loss. Biallelic variants in MYO15A were identified in 21 affected and were absent in 22 unaffected siblings. A mini-gene assay confirms that the synonymous variant leads to abnormal splicing. The variant is enriched in the Ashkenazi Jewish population. Individuals carrying biallelic variants involving c.9861 C > T often exhibit progressive post-lingual hearing loss distinct from the congenital profound deafness typically associated with biallelic loss-of-function MYO15A variants. This study establishes the pathogenicity of the c.9861 C > T variant in MYO15A and expands the phenotypic spectrum of MYO15A-related hearing loss. Our work also highlights the importance of multicenter collaboration and data sharing to establish the pathogenicity of a relatively common synonymous variant for improved diagnosis and management of hearing loss.Subject terms: Genetic testing, Genetic testing     

Characterization of mtDNA variation in a cohort of South African paediatric patients with mitochondrial disease

Mitochondrial disease can be attributed to both mitochondrial and nuclear gene mutations. It has a heterogeneous clinical and biochemical profile, which is compounded by the diversity of the genetic background. Disease-based epidemiological information has expanded significantly in recent decades, but little information is known that clarifies the aetiology in African patients. The aim of this study was to investigate mitochondrial DNA variation and pathogenic mutations in the muscle of diagnosed paediatric patients from South Africa. A cohort of 71 South African paediatric patients was included and a high-throughput nucleotide sequencing approach was used to sequence full-length muscle mtDNA. The average coverage of the mtDNA genome was 81±26 per position. After assigning haplogroups, it was determined that although the nature of non-haplogroup-defining variants was similar in African and non-African haplogroup patients, the number of substitutions were significantly higher in African patients. We describe previously reported disease-associated and novel variants in this cohort. We observed a general lack of commonly reported syndrome-associated mutations, which supports clinical observations and confirms general observations in African patients when using single mutation screening strategies based on (predominantly non-African) mtDNA disease-based information. It is finally concluded that this first extensive report on muscle mtDNA sequences in African paediatric patients highlights the need for a full-length mtDNA sequencing strategy, which applies to all populations where specific mutations is not present. This, in addition to nuclear DNA gene mutation and pathogenicity evaluations, will be required to better unravel the aetiology of these disorders in African patients.     

Use of stereotypical mutational motifs to define resolution limits for the ultra-deep resequencing of mitochondrial DNA

Massively parallel resequencing of mitochondrial DNA (mtDNA) has led to significant advances in the study of heteroplasmic mtDNA variants in health and disease, but confident resolution of very low-level variants (<2% heteroplasmy) remains challenging due to the difficulty in distinguishing signal from noise at this depth. However, it is likely that such variants are precisely those of greatest interest in the study of somatic (acquired) mtDNA mutations. Previous approaches to this issue have included the use of controls such as phage DNA and mtDNA clones, both of which may not accurately recapitulate natural mtDNA. We have therefore explored a novel approach, taking advantage of mtDNA with a known stereotyped mutational motif (nAT>C, from patient with MNGIE, mitochondrial neurogastrointestinal encephalomyopathy) and comparing mutational pattern distribution with healthy mtDNA by ligation-mediated deep resequencing (Applied Biosystems SOLiD). We empirically derived mtDNA-mutant heteroplasmy detection limits, demonstrating that the presence of stereotypical mutational motif could be statistically validated for heteroplasmy thresholds ≥0.22% (P=0.034). We therefore provide empirical evidence from biological samples that very low-level mtDNA mutants can be meaningfully resolved by massively parallel resequencing, confirming the utility of the approach for studying somatic mtDNA mutation in health and disease. Our approach could also usefully be employed in other settings to derive platform-specific deep resequencing resolution limits.     

Compound heterozygous or homozygous truncating MYBPC3 mutations cause lethal cardiomyopathy with features of noncompaction and septal defects

Familial hypertrophic cardiomyopathy (HCM) is usually caused by autosomal dominant pathogenic mutations in genes encoding sarcomeric or sarcomere-associated cardiac muscle proteins. The disease mainly affects adults, although young children with severe HCM have also been reported. We describe four unrelated neonates with lethal cardiomyopathy, and performed molecular studies to identify the genetic defect. We also present a literature overview of reported patients with compound heterozygous or homozygous pathogenic MYBPC3 mutations and describe their clinical characteristics. All four children presented with feeding difficulties, failure to thrive, and dyspnea. They died from cardiac failure before age 13 weeks. Features of left ventricular noncompaction were diagnosed in three patients. In the fourth, hypertrabeculation was not a clear feature, but could not be excluded. All of them had septal defects. Two patients were compound heterozygotes for the pathogenic c.2373dup p.(Trp792fs) and c.2827C>T p.(Arg943*) mutations, and two were homozygous for the c.2373dup and c.2827C>T mutations. All patients with biallelic truncating pathogenic mutations in MYBPC3 reported so far (n=21) were diagnosed with severe cardiomyopathy and/or died within the first few months of life. In 62% (13/21), septal defects or a patent ductus arteriosus accompanied cardiomyopathy. In contrast to heterozygous pathogenic mutations, homozygous or compound heterozygous truncating pathogenic MYBPC3 mutations cause severe neonatal cardiomyopathy with features of left ventricular noncompaction and septal defects in approximately 60% of patients.     

Single‐fiber studies for assigning pathogenicity of eight mitochondrial DNA variants associated with mitochondrial diseases

Whole mitochondrial DNA (mtDNA) sequencing is now systematically used in clinical laboratories to screen patients with a phenotype suggestive of mitochondrial disease. Next Generation Sequencing (NGS) has significantly increased the number of identified pathogenic mtDNA variants. Simultaneously, the number of variants of unknown significance (VUS) has increased even more, thus challenging their interpretation. Correct classification of the variants' pathogenicity is essential for optimal patient management, including treatment and genetic counseling. Here, we used single muscle fiber studies to characterize eight heteroplasmic mtDNA variants, among which were three novel variants. By applying the pathogenicity scoring system, we classified four variants as “definitely pathogenic” (m.590A>G, m.9166T>C, m.12293G>A, and m.15958A>T). Two variants remain “possibly pathogenic” (m.4327T>C and m.5672T>C) but should these be reported in a different family, they would be reclassified as “definitely pathogenic.” We also illustrate the contribution of single‐fiber studies to the diagnostic approach in patients harboring pathogenic variants with low level heteroplasmy.     

Consolidating biallelic SDHD variants as a cause of mitochondrial complex II deficiency

Isolated mitochondrial complex II deficiency is a rare cause of mitochondrial respiratory chain disease. To date biallelic variants in three genes encoding mitochondrial complex II molecular components have been unequivocally associated with mitochondrial disease (SDHA/SDHB/SDHAF1). Additionally, variants in one further complex II component (SDHD) have been identified as a candidate cause of isolated mitochondrial complex II deficiency in just two unrelated affected individuals with clinical features consistent with mitochondrial disease, including progressive encephalomyopathy and lethal infantile cardiomyopathy. We present clinical and genomic investigations in four individuals from an extended Palestinian family with clinical features consistent with an autosomal recessive mitochondrial complex II deficiency, in which our genomic studies identified a homozygous NM_003002.3:c.[205 G > A];[205 G > A];p.[(Glu69Lys)];[(Glu69Lys)] SDHD variant as the likely cause. Reviewing previously published cases, these findings consolidate disruption of SDHD function as a cause of mitochondrial complex II deficiency and further define the phenotypic spectrum associated with SDHD gene variants.Subject terms: Disease genetics, Genetic counselling, Metabolic disorders, Genetics research     

Pathogenic mitochondrial mt-tRNAAla variants are uniquely associated with isolated myopathy

Pathogenic mitochondrial DNA (mtDNA) point mutations are associated with a wide range of clinical phenotypes, often involving multiple organ systems. We report two patients with isolated myopathy owing to novel mt-tRNA^Ala variants. Muscle biopsy revealed extensive histopathological findings including cytochrome c oxidase (COX)-deficient fibres. Pyrosequencing confirmed mtDNA heteroplasmy for both mutations (m.5631G>A and m.5610G>A) whilst single-muscle fibre segregation studies (revealing statistically significant higher mutation loads in COX-deficient fibres than in COX-positive fibres), hierarchical mutation segregation within patient tissues and decreased steady-state mt-tRNA^Ala levels all provide compelling evidence of pathogenicity. Interestingly, both patients showed very high-mutation levels in all tissues, inferring that the threshold for impairment of oxidative phosphorylation, as evidenced by COX deficiency, appears to be extremely high for these mt-tRNA^Ala variants. Previously described mt-tRNA^Ala mutations are also associated with a pure myopathic phenotype and demonstrate very high mtDNA heteroplasmy thresholds, inferring at least some genotype:phenotype correlation for mutations within this particular mt-tRNA gene.     

An integrated approach for classifying mitochondrial DNA variants: one clinical diagnostic laboratory’s experience

PurposeThe mitochondrial genome is highly polymorphic. A unique feature of deleterious mitochondrial DNA (mtDNA) mutations is heteroplasmy. Genetic background and variable penetrance also play roles in the pathogenicity for a mtDNA variant. Clinicians are increasingly interested in requesting mtDNA testing. However, interpretation of uncharacterized mtDNA variants is a great challenge. We suggest a stepwise interpretation procedure for clinical service.MethodsWe describe the algorithms used to interpret novel and rare mtDNA variants. mtDNA databases and in silico predictive algorithms are used to evaluate the pathogenic potential of novel and/or rare mtDNA variants.ResultsmtDNA variants can be classified into three categories: benign variants, unclassified variants, and deleterious mutations based on database search and in silico prediction. Targeted DNA sequence analysis of matrilineal relatives, heteroplasmy quantification, and functional studies are useful to classify mtDNA variants.ConclusionClinical significance of a novel or rare variant is critical in the diagnosis of the disease and counseling of the family. Based on the results from clinical, biochemical, and molecular genetic studies of multiple family members, proper interpretation of mtDNA variants is important for clinical laboratories and for patient care.     

Prenatal diagnosis of myopathy, encephalopathy, lactic acidosis, and stroke-like syndrome: contribution to understanding mitochondrial DNA segregation during human embryofetal development

Introduction

Myopathy, encephalopathy, lactic acidosis, and stroke‐like (MELAS) syndrome, a maternally inherited disorder that is among the most common mitochondrial DNA (mtDNA) diseases, is usually associated with the m.3242A>G mutation of the mitochondrial tRNA^leu gene. Very few data are available with respect to prenatal diagnosis of this serious disease. The rate of mutant versus wild‐type mtDNA (heteroplasmy) in fetal DNA is indeed considered to be a poor indicator of postnatal outcome.

Materials and methods

Taking advantage of a novel semi‐quantitative polymerase chain reaction test for m.3243A>G mutant load assessment, we carried out nine prenatal diagnoses in five unrelated women, using two different fetal tissues (chorionic villi v amniocytes) sampled at two or three different stages of pregnancy.

Results

Two of the five women, although not carrying m.3243A>G in blood or extra‐blood tissues, were, however, considered at risk for transmission of the mutation, as they were closely related to MELAS‐affected individuals. The absence of 3243A>G in the blood of first degree relatives was associated with no mutated mtDNA in the cardiovascular system (CVS) or amniocytes, and their three children are healthy, with a follow‐up of 3 months–3 years. Among the six fetuses from the three carrier women, three were shown to be homoplasmic (0% mutant load), the remaining three being heteroplasmic, with a mutant load ranging from 23% to 63%. The fetal mutant load was fairly stable at two or three different stages of pregnancy in CVS and amniocytes. Although pregnancy was terminated in the case of the fetus with a 63% mutant load, all other children are healthy with a follow‐up of 3 months–6 years.

Conclusion

These data suggest that a prenatal diagnosis for MELAS syndrome might be helpful for at‐risk families.Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Most of these defects result in a progressive disabling neurological syndrome with premature death. Among them, the myopathy, encephalopathy, lactic acidosis, and stroke‐like (MELAS) syndrome (OMIM: 540 000) is one of the most common and serious conditions. MELAS syndrome is mainly caused by the m.3243A>G mutation in the mitochondrial tRNALeu gene¹ (Genbank NC001 807), which produces a generalised dysfunction of the mitochondrial respiratory chain. The clinical features are highly variable, not only in terms of age at onset and severity of symptoms but also in relation to the specific organs associated.² Many patients present with mellitus diabetes, deafness, cardiomyopathy, external ophthalmoplegia and skeletal myopathy, variously associated in different degrees.³ The interfamilial and intrafamilial variability of the clinical phenotype is classically related to the properties of mtDNA segregation: heteroplasmy, “threshold” effect, mitochondrial “bottleneck” and variation in the tissue distribution⁴.Owing to the severity of the disease, the high risk of recurrence in siblings and the absence of efficient treatment, couples at risk of transmitting the m.3243A>G mutation often ask for prenatal diagnosis. However, very few data are so far available with respect to the prenatal diagnosis of MELAS syndrome.^4,5 Thus, whether prenatal assessment of the fetal mutant load (rate of mutant v wild‐type mtDNA) is a good predictive marker for the postnatal clinical outcome remains a matter of debate. In the few available reports, imprecise assessment of the fetal mutant load, most often studied in a single fetal tissue, hampers consideration of this issue. We report here a sensitive novel technical approach, designed to quantify as accurately as possible the mutant load in a given tissue. The results of prenatal analyses in a series of nine cases are detailed.     

Fine Time Scaling of Purifying Selection on Human Nonsynonymous mtDNA Mutations Based on the Worldwide Population Tree and Mother–Child Pairs

A high‐resolution mtDNA phylogenetic tree allowed us to look backward in time to investigate purifying selection. Purifying selection was very strong in the last 2,500 years, continuously eliminating pathogenic mutations back until the end of the Younger Dryas (～11,000 years ago), when a large population expansion likely relaxed selection pressure. This was preceded by a phase of stable selection until another relaxation occurred in the out‐of‐Africa migration. Demography and selection are closely related: expansions led to relaxation of selection and higher pathogenicity mutations significantly decreased the growth of descendants. The only detectible positive selection was the recurrence of highly pathogenic nonsynonymous mutations (m.3394T>C‐m.3397A>G‐m.3398T>C) at interior branches of the tree, preventing the formation of a dinucleotide STR (TATATA) in the MT‐ND1 gene. At the most recent time scale in 124 mother–children transmissions, purifying selection was detectable through the loss of mtDNA variants with high predicted pathogenicity. A few haplogroup‐defining sites were also heteroplasmic, agreeing with a significant propensity in 349 positions in the phylogenetic tree to revert back to the ancestral variant. This nonrandom mutation property explains the observation of heteroplasmic mutations at some haplogroup‐defining sites in sequencing datasets, which may not indicate poor quality as has been claimed.     

A novel mutation in the mitochondrial tRNAPro gene associated with late-onset ataxia, retinitis pigmentosa, deafness, leukoencephalopathy and complex I deficiency

We present a patient with ataxia, retinitis pigmentosa, dysarthria, neurosensorial deafness, nystagmus and leukoencephalopathy. A novel heteroplasmic G to A transition at nucleotide 15 975 was found, affecting the T arm of the mitochondrial (mt) tRNA^Pro gene. A biochemical analysis of respiratory chain enzymes in muscle revealed isolated complex I deficiency. This is the fourth pathogenic tRNA^Pro point mutation to be associated with an mt disorder. The result highlights the importance of molecular dissection of mtDNA in patients with defined mt disorder and confirms the clinical and biochemical heterogeneity associated with tRNA^Pro mutations.     

The role of mitochondrial genome in essential hypertension in a Chinese Han population

Earlier genetic studies of essential hypertension have focused on nuclear genes or family-based mitochondrial screening in Caucasian and African-American pedigrees. The role of mitochondria in sporadic Chinese hypertensives is unknown. We sequenced mitochondrial genomes in 306 age- and gender-balanced Chinese Han hypertensives and controls. In 153 hypertensives, putative functional changes included 4 changes in rRNA genes, 11 changes in tRNA genes and 25 amino-acid substitutions. The remaining variants were synonymous changes or non-coding regions. In the 153 controls, 2 base changes in the tRNA genes and 13 amino-acid substitutions were found. A8701G in ATP6 gene (belongs to haplogroup M; P=0.0001) and C8414T in ATP8 gene (belongs to haplogroup D; P=0.01) were detected significantly different in the cases and controls. Interestingly, the cases were more likely to have two or more amino-acid changes and RNA variants compared with the controls (57.43 versus 23.81%, P=0.0001). In addition, several variants we found were highly conserved and/or specifically located at the 3′ end adjacent to the anticodon, which may contribute to the stabilization of structure, and thus lead to the decrease of tRNA metabolism. In conclusion, mitochondrial SNPs (mtSNPs) may affect the course of hypertension in sporadic Chinese hypertensives. Some specific mtSNP within mitochondria may have potential role in the Chinese hypertensives due to their function. Synergetic interaction between mitochondrial mtSNPs and/or haplogroups is needed to be investigated in the future.     

Stability of the m.8993T->G mtDNA mutation load during human embryofetal development has implications for the feasibility of prenatal diagnosis in NARP syndrome

Background

Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Because of the high transmission risk and the absence of therapy in these disorders, at‐risk couples often ask for prenatal diagnosis (PND). However, because heteroplasmy load (coexistence of mutant and wild‐type mtDNA) may vary among tissues and with time, the possibility that a single fetal sample may not reflect the whole neonate impedes prenatal diagnosis of mtDNA diseases.

Methods

We performed 13 prenatal diagnoses for the NARP (neurogenic weakness, ataxia, retinitis pigmentosa) m.8993T→G mtDNA mutation (p.Leu156Arg) in the ATP synthase subunit 6 gene. Analyses were performed on chorionic villous (CVS) and/or amniocyte samples carried out at various stages of pregnancy, using a method enabling quantification of low DNA amounts.

Results

Maternal mutant loads ranged from 0 to 75% in blood and had no predictive value for the fetus status, except for women with no detectable mutant DNA, whose fetuses were consistently mutation‐free. In 8/13 PND, mutant load was <30%. These children are healthy at 2–7 years of age. In 5/13 PND, mutant load ranged from 65 to 100%, and parents preferred to terminate the pregnancies (15–22 weeks of gestation). Single‐cell analysis of 20 trophoblastic cells and 21 amniocytes isolated from two affected fetuses found an average mutant load close to the overall CVS and amniocyte mutant load, despite striking intercellular variation. The m.8993T→G mutant loads, assessed in 7, 17, 11, and 5 different tissues from 4 terminations, respectively, were identical in all tissues from a given individual (mean (SD) 78 (1.2)%, 91 (0.7)%, 74 (2)%, and 63 (1.6)% for the 4 fetuses, respectively).

Conclusions

Our results indicate that the placental/amniotic mutant loads do reflect the NARP mutant mtDNA load in the whole fetus, even when the sample amount is small, and suggest that heteroplasmy level remains stable during pregnancy, at least after 10 weeks of gestation. Although these data establish the feasibility of PND for this mutation, assessing more precisely the correlation between mutant load and disease severity should further help in interpreting PND results.     

Identification and in silico analysis of 14 novel GJB1, MPZ and PMP22 gene mutations

Duplication within the chromosome 17p11.2 (CMT1Adup), peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) and gap junction β1-protein (GJB1) gene mutations are frequent causes of the Charcot-Marie-Tooth disease (CMT). A large number of mutations in these genes are listed in databases. Sequence variants identified in patients are frequently reported as mutations without further evaluation. We analyzed 250 consecutively recruited unrelated Austrian CMT patients for CMT1Adup by microsatellite marker typing, real-time PCR or MLPA, and found 79 duplications (31.6%). The coding regions of the PMP22, MPZ and GJB1 genes were analyzed by direct sequencing in the remaining patients; 28 patients showed mutations, 14 of which were novel. We scored the pathogenicity of novel missense mutations by segregation studies and by their exclusion in control samples. Our comprehensive literature study found that up to 60% of the reported mutations in these genes had not been evaluated regarding their pathogenicity, and the PANTHER bioinformatics tool was used to score novel and published missense variants. The PANTHER program scored known polymorphisms as such, but scored ∼82–88% only of the published and novel mutations as most likely deleterious. Mutations associated with axonal CMT were less likely to be classified as deleterious, and the PMP22 S72L mutation repeatedly associated with severe CMT was classified as a polymorphism using default parameters. Our data suggest that this in silico analysis tool could be useful for assessing the functional impact of DNA variations only as a complementary approach. The CMT1Adup, GJB1, MPZ and PMP22 mutation frequencies were in the range of those described in other CMT patient collectives with different ethnical backgrounds.