Four novel TMC1 (DFNB7/DFNB11) mutations in Turkish patients with congenital autosomal recessive nonsyndromic hearing loss.

Mutations in the transmembrane channel-like gene 1 (TMC1) cause prelingual autosomal recessive (DFNB7/11) and postlingual progressive autosomal dominant (DFNA36) nonsyndromic hearing loss. To determine the genetic causes of autosomal recessive nonsyndromic hearing loss (ARNSHL) in the northeast and east of Turkey, 65 unrelated families without mutations in the protein coding region of the GJB2 (GJB2-negative) were analyzed. A genomewide scan for homozygosity and linkage analysis in one of these families revealed a 13.2 cM critical region between D9S273 and D9S153 at chromosome 9p13.2-q21.31 with a maximum two-point lod score of 4.00 at theta=0.0 for marker D9S175. TMC1 is in this critical region. Homozygosity screening with intragenic markers for TMC1 in the remaining 64 families suggested involvement of this gene in three additional families. Subsequent sequencing of TMC1 in these four families revealed four novel homozygous mutations, c.776A>G [p.Tyr259Cys], c.821C>T [p.Pro274Leu], c.1334G>A [p.Arg445His], and c.1083_1087delCAGAT [p.Arg362ProfrX6]. Our results indicate that TMC1 mutations account for at least 6% (4/65) of ARNSHL in GJB2-negative Turkish families from the northeast and east of Turkey.

Molecular analysis of the coronavirus-receptor function of aminopeptidase N.

Aminopeptidase N (APN) is a major cell surface for coronaviruses of the serogroup I. By using chimeric APN proteins assembled from human, porcine and feline APN we have identified determinants which are critically involved in the coronavirus-APN interaction. Our results indicate that human coronavirus 229E (HCV 229E) is distinct from the other serogroup I coronaviruses in that determinants located within the N-terminal parts of the human and feline APN proteins mediate the infection of HCV 229E, whereas determinants located within the C-terminal parts of porcine, feline and canine APN mediate the infection of transmissible gastro-enteritis virus (TGEV), feline infectious peritonitis virus (FIPV) and canine coronavirus (CCV), respectively. A further analysis of the mapped amino acid segments by site directed mutagenesis revealed that a short stretch of 8 amino acids in the hAPN protein plays a decisive role in mediating HCV 229E reception.

Isolation and recombinant expression of an MHV-JHM neutralising monoclonal antibody.

The monoclonal antibody A1 (mab A1) efficiently neutralises the infection of susceptible cells by the murine hepatitis virus MHV-JHM in vitro and in vivo (Wege et al., 1984). The variable regions of mab A1 were amplified from mRNA of the respective hybridoma cell line by RT-PCR and integrated into different eukaryotic expression vectors. The biological function of the recombinant antibody constructs was verified by virus neutralisation assays. Whereas a complete recombinant antibody (mab A1rec.) expressed in transfected murine myeloma cells inhibited the MHV-JHM infection as well as the parental antibody, a single-chain Fv derived from mab A1 did not show any neutralising activity.

Rapid detection of point mutations and polymorphisms of the alpha-globin genes by DGGE and SSCA.

We report the application of DGGE and SSCA for the identification of point mutations causing alpha-thalassemia. The alpha-globin genes were amplified in three overlapping fragments of 250 bp (I), 540 bp (II), and 600 bp (III), respectively. Fragments II and III were analysed by DGGE, while fragments I and II were analysed by SSCA. A panel of seven previously identified mutations was employed to test the combined DGGE/SSCA strategy: 5/5 and 6/7 mutations were detected by SSCA and DGGE, respectively. The same approach has also led to the identification of eight disease-causing mutations in a sample of 18 presumed non-deletional alpha-thalassemia carriers. During this pilot study, two novel mutations as well as three new polymorphisms were found. The combined application of SSCA and DGGE allows the rapid identification of mutations responsible for alpha-thalassemia and abnormal globin chain variants. Moreover, it will prove extremely useful for pre- and postnatal diagnosis and in screening programs for non-deletional alpha-thalassemias.

Characterization of functional domains in the human coronavirus HCV 229E receptor.

Human aminopeptidase N (hAPN or CD13) and porcine aminopeptidase N (pAPN) are functional receptors for human coronavirus (HCV) 229E and porcine transmissible gastroenteritis virus (TGEV), respectively. However, hAPN cannot function as a receptor for TGEV and pAPN cannot function as a receptor for HCV 229E. In this study, we constructed a series of chimeric hAPN/pAPN genes and expressed the corresponding proteins in transfected cells. Subsequently, we identified the chimeric proteins that can function as a receptor for HCV 229E. The results show that replacement of a small region of pAPN sequence (pAPN amino acids 255-348) with the corresponding hAPN sequence (hAPN amino acids 260-353) converts pAPN into a functional receptor for HCV 229E. The region of hAPN that we have defined in this way does not correspond to the region of pAPN that has been identified as essential for the TGEV-receptor interaction. We conclude that although both viruses use a homologous receptor protein, different regions of the protein are required to mediate susceptibility to infection with HCV 229E and TGEV.

alpha-Thalassemia in The Netherlands: a heterogeneous spectrum of both deletions and point mutations.

In this article we describe the molecular characterization of 104 independent alpha-thalassemia patients identified by hematological analysis and family studies. During the study, another six chromosomes were identified with rearrangements of the alpha-cluster or point mutations in the alpha 2-globin gene, not associated with alpha-thalassemia, in healthy relatives of the patients. The molecular defects were established by Southern blot analysis and, if no deletions could be identified, the alpha-globin genes were investigated by denaturing gradient gel electrophoresis and single strand conformation analysis for the presence of point mutations. Following this strategy, we were able to identify the molecular basis of 131 independent alpha-thalassemia chromosomes. In two individuals, the alpha-thalassemia determinant could not be demonstrated at the molecular level. We identified eight different deletion and five non-deletion alpha-thalassemias, three rearrangements in the alpha-cluster, two alpha-chain variants, and a silent mutation in the alpha 2-globin gene not associated with alpha-thalassemia. The large heterogeneity of alpha-thalassemia mutations seen in the Dutch population might be typical for northern European countries where, besides the more common mutations introduced by migration, a variety of sporadic mutations was also found in the autochthonous population. The screening strategy as described here, capable of identifying a wide spectrum of both deletions and point mutations, identified 98% of the alpha-thalassemia determinants present in 133 chromosomes.

The molecular spectrum of beta-thalassemia and abnormal hemoglobins in the allochthonous and autochthonous dutch population.

The prevalence at birth of hemoglobin defects in the autochthonous North-European population is low. However, the long immigration and colonial history of the Netherlands has resulted in a group of about 1-2 million 'autochthonous' inhabitants, with Asian, South-European or African ancestors, in whom a moderate birth prevalence of globin gene mutations can be expected. Furthermore, at least 10% of the Dutch population consists of recent immigrants from different countries with high birth prevalence of hemoglobinopathies. Because of the endogamous partner choice, which is prevalent in this population, the risk for homozygous progeny remains elevated. At least 100,000 carriers of hemoglobinopathies of recent allochthonous origin are present in the Netherlands, and the number of homozygous children is rising. Prevention by prenatal diagnosis requires a suitable protocol and knowledge about the molecular defects present in the country. Therefore we have analyzed a large number of patients and carriers, both at the hematological and at the DNA level. Our survey revealed 47 different beta-thalassemia determinants, characterized on 223 independent chromosomes from individuals of different ethnic origins. As expected, the most prevalent mutations were largely represented. The cd39 (C-->T) mutation was found in 70% of the immigrants from Morocco, Sardinia and other Central-West-Mediterranean regions while the IVS-I-110 (G-->A) was prevalent in the East-Mediterranean populations. The IVS-I-5 (G-->C) mutation was found in 45% of the patients of Indonesian origin. We also registered 308 independent chromosomes with common structural defects (HbS, HbC, HbE, Hb Lepore, Hb Constant Spring and HbD Punjab) and 33 chromosomes with 19 different, less frequent, rare or very rare mutants. Seven structural mutants were described for the first time and published separately. Furthermore, 139 independent chromosomes with deletional and nondeletional alpha-thalassemia defects were characterized.