Evaluation of locus heterogeneity and EXT1 mutations in 34 families with hereditary multiple exostoses

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by growth of benign bone tumors. Three chromosomal loci have been implicated in this genetically heterogeneous disease: EXT1 at 8q24, EXT2 at 11p13, and EXT3 on 19p. EXT1 and EXT2 were recently cloned. We evaluated 34 families with EXT to estimate the proportion of disease attributable to EXT1, EXT2, and EXT3 and to investigate the spectrum of EXT1 mutations. Linkage analyses combined with heterogeneity testing provides strong evidence in favor of linkage of disease to both chromosomes 8 and 11, but does not support evidence of linkage to chromosome 19 in this data set. The 11 EXT1 exons were PCR-amplified and sequenced in all 11 isolated cases and in 20 of the 23 familial cases. Twelve different novel EXT1 mutations were detected, including 5 frame-shift deletions or insertions, 1 codon deletion, and 6 single base-pair substitutions distributed across 8 of the exons. Only 2 of the mutations were detected in more than one family. Three mutations affect sites in which alterations were previously reported. Nonchain-terminating missense mutations were identified in codons 280 and 340, both coding for conserved arginine residues. These residues may be crucial to the function of this protein. Although the prevalence of EXT has been estimated to be approximately 1/50,000 individuals, the disease has been reported to occur much more frequently in the Chamorro natives on Guam. Our detection of an EXT1 mutation in one Chamorro subject will allow investigation of a possible founder effect in this population. Combined mutational and heterogeneity analyses in this set of families with multiple exostoses suggest that 66% of our total sample, including 45% of isolated and 77% of familial cases, are attributable to abnormalities in EXT1. Hum Mutat 11:231–239, 1998. © 1998 Wiley-Liss, Inc.     

经典型苯丙酮尿症基因全长外显子的突变检测和分析

目的探讨中国PKU患者PAH基因突变特征。方法运用PCR-SSCP及PCR-DNA直接测序检测40例经典型PKU患者和30例正常对照的PAH基因。结果在PAH基因上8个外显子共发现11种突变和3种多态,其中R243Q和Y204C为两个高频突变位点,突变率分别为27.5%和10%,280 insT、M276K、M276R、IVS10nt+32T→A、IVS4nt+46C→T、H290R是首次发现的新突变。结论中国PKU患者基因外显子突变是以两个突变热点和罕见突变并存为特征。     

Haplotypes at the DRD2 locus and severe alcoholism

Association studies of the minor TaqI A allele of the D(2) dopamine receptor (DRD2) gene with alcoholism have produced conflicting findings. Failure to assess alcoholics for severity of their disorder and to screen controls for substance use have been proposed as causes for the discrepant results. In the present study, five diallelic sites spanning the DRD2 gene were determined in combined Caucasian (non-Hispanic) studies of more severe alcoholics (n = 92) and controls screened for substance use (n = 85). The frequency of the minor alleles at the 3'-untranslated site (TaqI A) and two intronic sites (TaqI B and intron 6) of the DRD2 gene were each strongly associated with alcoholism. Moreover, the alcoholics compared with the controls at these three sites had a significantly higher frequency of the minor/major allele heterozygote haplotype combination (A1/A2 B1/B2 T/G) than the major allele homozygote haplotype combination (A2/A2 B2/B2 G/G). However, exon 7 and promoter alleles were not associated with alcoholism. In neither the alcoholics nor in the controls were there departures from Hardy-Weinberg equilibrium at any of the five sites examined. The most significant diallelic composite genotypic disequilibria were found when comparisons were made between TaqI A and TaqI B, TaqI A and intron 6, and TaqI B and intron 6 sites. Weaker but still significant disequilibria were observed when TaqI A and exon 7, TaqI B and exon 7, intron 6 and exon 7, and promoter and exon 7 sites were compared. However, no significant disequilibria were noted when TaqI A and promoter, TaqI B and promoter, and intron 6 and promoter sites were compared. In sum, the study found significant evidence for association of the minor alleles in the untranslated sites of the DRD2 gene and their haplotypes with the more severe alcoholic phenotype.     

Impact of PKU on the reproductive patterns in collaborative study families

Factors that relate to reproductive patterns in 129 families after the birth of a child with phenylketonuria (PKU) include birth order of the index child, age of the parents at the birth of the index child, and expressed intentions of the parents whether or not to have additional children. Factors that do not correlate with reproductive histories include knowledge of the genetic and metabolic nature of PKU, the relationship of PKU to mental retardation and special diet, parental upset about the diagnosis, sex of the affected child, parental IQ, religion, education, and social class. Correlations found related to the question, "Is PKU the reason you don't want more children?" include stress factors in family functioning, mother's upset with the diagnosis, father's concerns about being a carrier, sex of the child with PKU, and degree of knowledge about PKU. Many of the Collaborative Study clinics tend to be more concerned about the consequences of PKU on the family than on society, and feel that families should receive genetic counseling to determine their reproductive risks and future plans. Upon self-report, many clinics declare their counseling to be either "completely nondirective" or making a "conscious effort to be nondirective."     

Identification of three novel TECTA mutations in Iranian families with autosomal recessive nonsyndromic hearing impairment at the DFNB21 locus

Forty-five consanguineous Iranian families segregating autosomal recessive nonsyndromic hearing loss (ARNSHL) and negative for mutations at the DFNB1 locus were screened for allele segregation consistent with homozygosity by descent (HBD) at the DFNB21 locus. In three families demonstrating HBD at this locus, mutation screening of TECTA led to the identification of three novel homozygous mutations: one frameshift mutation (266delT), a transversion of a cytosine to an adenine (5,211C > A) leading to a stop codon, and a 9.6 kb deletion removing exon 10. In total, six mutations in TECTA have now been described in families segregating ARNSHL. All of these mutations are inactivating and produce a similar phenotype that is characterized by moderate-to-severe hearing loss across frequencies with a mid frequency dip. The truncating nature of these mutations is consistent with loss-of-function, and therefore the existing TECTA knockout mouse mutant represents a good model in which to study DFNB21-related deafness.     

Neu Laxova syndrome in two Egyptian families

We report on 2 Egyptian girls from 2 families with 8 affected relatives, 4 in each family. The 2 propositae had unusual facial appearance, severe microcephaly, generalized edema, contractures of limbs, and generalized ichthyotic skin lesions. Findings in the present 2 patients were compared with those in previously reported cases of Neu Laxova syndrome. In our cases, consanguinity, affected sibs, and affected cousins were noted. Autosomal recessive inheritance and lethality are emphasized. A high frequency of the Neu Laxova syndrome in Egyptians is suspected as a result of the increased consanguinity rate.     

PAH gene mutations in the Sicilian population: association with minihaplotypes and expression analysis.

The molecular basis of PAH deficiency in the Sicilian population is characterized by a marked heterogeneity, with 44 mutations at a single locus identified by a "gene-scanning" approach and accounting for a detection rate of 91%. The remaining 9% of PAH alleles does not bear mutations in any of the 13 exons and 24 exon/intron junctions. Three mutations IVS10nt-11 G > A, R261Q, and A300S accounted for 30.5%, whereas the remaining mutations were found at relative frequencies of less than 5% and 20 mutations were observed once only. Five mutations have been detected only in Sicilians so far. By studying the association of mutations with intragenic STR-VNTR haplotypes ("minihaplotypes"), "identity by descent" has been established for 24 mutations also detected in other populations. This finding supports the hypothesis of a multipolar origin for a large proportion of PAH mutant alleles currently detected in Sicilians. In order to improve our understanding of the clinical heterogeneity of PAH deficiency in this population, we have for the first time analyzed three missense mutations L41F, T92I, and P211T in vitro by the pCDNA3/COS-7 eukaryotic expression system and found an activity of 10, 76, and 72%, respectively, compared to normal PAH. In two HPA patients with mild PKU and mild hyperphenylalaninemia (MHP), harboring respectively L41F/R261Q and T92I/P281L genotypes, the predicted biochemical effect of these genotypes appeared to be consistent with the metabolic phenotypes. In contrast, discordant metabolic phenotypes (mild PKU and MHP) were observed in two unrelated patients bearing the same R261Q/P211T genotype, a finding which underscores the complex relationship linking genotype to phenotype in PAH deficiency. Hypotheses on the possible mechanisms responsible for the observed discordance are discussed. The spectrum of PAH gene mutations in Sicily reflects the complex demographic history of this island at the crossroad of prehistoric and historical migrations in the Mediterranean sea. The data presented in this study also add to the present knowledge on the relationship between PAH genotypes and HPA phenotype and are expected to improve PAH genotyping among individuals with hyperphenylalaninemia.     

Novel pathogenic mutations in the glucocerebrosidase locus

To determine the frequency of mutations responsible for Gaucher's disease, we systematically sequenced the GBA1 gene as part of a molecular characterization of 73 adult patients in the United Kingdom. Five hitherto unknown pathogenic variants were identified, one of which is a splice site change; the others are novel missense mutations. Given that GBA1 gene mutations are an important risk factor for the development of Parkinson's disease, we contend that a complete analysis and molecular characterization of both the known and novel GBA1 variants will be needed before the biochemical processes underlying this genetic association can be fully understood.     

Kinetic and stability analysis of PKU mutations identified in BH4-responsive patients

From all the different molecular mechanisms put forward to explain the basis of BH4 responsiveness in PKU patients, a clear picture is now emerging based on the results from expression studies performed with a number of missense mutations identified in patients with a positive response in BH4 loading tests. Two of the proposed mechanisms, namely decreased binding affinity of the mutant proteins for the natural cofactor and stabilization effect of BH4, have been confirmed for several PKU mutations and the results are reviewed here. The actual view supports a multifactorial basis of the response, highlighting the necessity of detailed in vitro characterization of each mutant PAH protein. Several of the confirmed molecular mechanisms may be operating simultaneously, as exemplified in the data presented, and this may result in different degrees of BH4 responsiveness.     

苯丙氨酸羟化酶基因的六种新型突变

目的　检测中国苯丙酮尿症 ( phenylketonuria ,PKU )患者苯丙氨酸羟化酶 (phenylalaninehy droxylase ,PAH)基因新的突变位点。 方法　应用聚合酶链反应 单链构象多态性及DNA直接测序检测 40例经典型PKU患者和 3 0名正常对照的PAH基因。结果　在PAH基因上共发现 11种突变和 3种多态 ,3 0名正常对照者PAH基因未发现异常。结论　经与国际PAH基因突变数据库比较 ,确认M 2 76K、M 2 76R、2 80insT、IVS10nt 3 2T→A、IVS4nt 4 7C→T是国际上首次发现的突变 ,H 2 90R是中国PKU患者基因上首次发现的新型突变。     

SERPING1 mutations in 59 families with hereditary angioedema

Hereditary angioedema due to C1 Inhibitor (C1Inh) deficiency (HAE types I and II) is a rare, life-threatening disease causing spontaneous edema of the submucosal layers. A cohort of 127 individuals with symptoms of recurrent familial angioedema from 59 non-related families was studied. All the patients included fulfilled the diagnostic and biochemical criteria of HAE, including low C1Inh function and/or concentration. Genetic studies were carried out by PCR and sequencing of the C1NH locus followed, in the negative cases, by MLPA, long-range PCR and restriction enzyme analysis of genomic DNA to detect potential large rearrangements. Mutations located in consensus splicing sequences or nearby positions were studied by RT-PCR. The study identified 52 different mutations (25 missense, 15 frameshift, 7 splicing defects and 5 large deletions) responsible for the disease in 56 HAE families. In the remaining three families no molecular alteration could be detected. Twenty-seven of the mutations in this cohort are novel and 10 are confirmed de novo cases. The pathologic effect of the 5 splicing defects first reported here was assessed at the RNA and protein levels. Large deletions affecting exons 4 and 7, ranging from approximately 1500 to 2500 bp, were partially characterized by their altered restriction patterns upon long-range amplification. These results highlight the heterogeneity of mutations in the C1NH gene causing C1Inh deficiency and HAE. An approach to the molecular effects associated to each of the mutations reported here was made when possible based on the available data of pathological variants of serpins.     

Four mutations of the spastin gene in Japanese families with spastic paraplegia

Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower limbs. HSP is caused by failure of development or selective degeneration of the corticospinal tracts, which contain the longest axons in humans. The most common form of HSP is caused by mutations of the spastin gene (SPAST), located on chromosome 2p21-p22, which encodes spastin, one of the ATPases associated with diverse cellular activities (AAA). In this study, we detected four causative mutations of SPAST among 14 unrelated patients with spastic paraplegia. Two missense mutations (1447A→G, 1207C→G) and two deletion mutations (1465delT, 1475-1476delAA) were located in the AAA cassette region. Three of these four mutations were novel. Previous reports and our results suggest that the frequency of SPAST mutations is higher among Japanese patients with autosomal dominant HSP, although SPAST mutations are also observed in patients with sporadic spastic paraplegia.Rehana Basri, Ichiro Yabe and Hiroyuki Soma have contributed equally to this work.     

Detection of mosaic RB1 mutations in families with retinoblastoma

The RB1 gene mutation detection rate in 1,020 retinoblastoma families was increased by the use of highly sensitive allele specific‐PCR (AS‐PCR) to detect low‐level mosaicism for 11 recurrent RB1 CGA>TGA nonsense mutations. For bilaterally affected probands, AS‐PCR increased the RB1 mutation detection sensitivity from 92.6% to 94.8%. Both RB1 oncogenic changes were detected in 92.7% of sporadic unilateral tumors (357/385); 14.6% (52/357) of unilateral probands with both tumor mutations identified carried one of the tumor mutations in blood. Mosaicism was evident in 5.5% of bilateral probands (23 of 421), in 3.8% of unilateral probands (22 of 572), and in one unaffected mother of a unilateral proband. Half of the mosaic mutations were only detectable by AS‐PCR for the 11 recurrent CGA>TGA mutations, and not by standard sequencing. This suggests that significant numbers of low‐level mosaics with other classes of RB1 mutations remain unidentified by current technology. We show that the use of linkage analysis in a two‐generation retinoblastoma family resulted in the erroneous conclusion that a child carried the parental mutation, because the founder parent was mosaic for the RB1 mutation. Of 142 unaffected parental pairs tested, only one unaffected parent of a proband (0.7%) showed somatic mosaicism for the proband's mutation, in contrast to an overall 4.5% somatic mosaicism rate for retinoblastoma probands, suggesting that mosaicism for an RB1 mutation is highly likely to manifest as retinoblastoma. Hum Mutat 0, 1–10, 2009. © 2009 Wiley‐Liss, Inc.     

Comprehensive molecular analysis of 61 Egyptian families with hereditary nonsyndromic hearing loss

Nonsyndromic hearing loss is an extremely heterogeneous disorder. Thus, clinical diagnostics is challenging, in particular due to differences in the etiology of hearing loss between populations. With this study, we wanted to elucidate the genetic basis of hearing loss in 61 consanguineous Egyptian families. In 25 families, linkage analysis was used as a prescreening to identify regions for targeted sequencing of candidate genes. Initially, the coding regions of 12 and later of 94 genes associated with hearing loss were enriched and subjected to massively parallel sequencing (MPS) with diagnostic yields of 36% and 75%, respectively. Causative variants were identified in 48 families (79%). They were found in 23 different genes with the majority being located in MYO15A (15.3%), SLC26A4 (9.7%), GJB2 (8.3%), and MYO7A (6.4%). As many as 32 variants were novel ones at the time of detection. Five variants were shared by two, three, or even four families. Our study provides a first survey of the mutational spectrum of deaf patients in Egypt revealing less GJB2 variants than in many European populations. It underlines the value of targeted enrichment of well-selected deafness genes in combination with MPS in the diagnostics of this frequent and genetically heterogeneous disorder.     

New mutations in the GLA gene in Brazilian families with Fabry disease

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the alpha-galactosidase A (GLA) gene. Evaluating the enzymatic activity in male individuals usually performs the diagnosis of the disease, but in female carriers the diagnosis based only on enzyme assays is often inconclusive. In this work, we analyzed 568 individuals from 102 families with suspect of FD. Overall, 51 families presented 38 alterations in the GLA gene, among which 19 were not previously reported in literature. The alterations included 17 missense mutations, 7 nonsense mutations, 7 deletions, 6 insertions and 1 in the splice site. Six alterations (R112C, R118C, R220X, R227X, R342Q and R356W) occurred at CpG dinucleotides. Five mutations not previously described in the literature (A156D, K237X, A292V, I317S, c.1177_1178insG) were correlated with low GLA enzyme activity and with prediction of molecular damages. From the 13 deletions and insertions, 7 occurred in exons 6 or 7 (54%) and 11 led to the formation of a stop codon. The present study highlights the detection of new genomic alterations in the GLA gene in the Brazilian population, facilitating the selection of patients for recombinant enzyme-replacement trials and offering the possibility to perform prenatal diagnosis.