Saul Surrey

Connexin-26 mutations in sporadic and inherited sensorineural deafness

BACKGROUNDnHearing impairment affects one infant in 1000 and 4% of people aged younger than 45 years. Congenital deafness is inherited or apparently sporadic. We have shown previously that DFNB1 on chromosome 13 is a major locus for recessive deafness in about 80% of Mediterranean families and that the connexin-26 gene gap junction protein beta2 (GJB2) is mutated in DFNB1 families. We investigated mutations in the GJB2 gene in familial and sporadic cases of deafness.nnnMETHODSnWe obtained DNA samples from 82 families from Italy and Spain with recessive non-syndromic deafness and from 54 unrelated participants with apparently sporadic congenital deafness. We analysed the coding region of the GJB2 gene for mutations. We also tested 280 unrelated people from the general populations of Italy and Spain for the frameshift mutation 35delG.nnnFINDINGSn49% of participants with recessive deafness and 37% of sporadic cases had mutations in the GJB2 gene. The 35delG mutation accounted for 85% of GJB2 mutations, six other mutations accounted for 6% of alleles, and no changes in the coding region of GJB2 were detected in 9% of DFNB1 alleles. The carrier frequency of mutation 35delG among people from the general population was one in 31 (95% CI one in 19 to one in 87).nnnINTERPRETATIONnMutations in the GJB2 gene are a major cause of inherited and apparently sporadic congenital deafness. Mutation 35delG is the most common mutation for sensorineural deafness. Identification of 35delG and other mutations in the GJB2 gene should facilitate diagnosis and counselling for the most common genetic form of deafness.

Rapid sizing of polymorphic microsatellite markers by capillary array electrophoresis.

Genetic mapping and DNA sequencing projects could potentially be completed more rapidly by using capillary array electrophoresis (CAE) systems running 48-96 capillaries simultaneously. Currently, multiplex polymerase chain reaction (PCR) and multicolor fluorescent dye-labeling strategies are used to generate DNA profiles containing 18-24 genotypes per sample. By using 4-color fluorescence detection and these multiplex PCR strategies, a CAE system has the capacity to generate up to 5.5 million genotypes per year. CAE offers extremely fast, high-resolution separation of DNA and more automated sample processing than conventional systems because the labor-intensive slab-gel pouring and sample-loading steps are eliminated. We used a prototype CAE system in an ongoing linkage analysis study of inherited deafness in Mediterranean families. CA-repeat markers linked to deafness susceptibility genes on chromosomes 7, 11 and 13 were analyzed and DNA profiles generated which contain 6 markers per color. Fragment sizes of over 28,000 short tandem repeat alleles and 3200 CA-repeat alleles have been determined by CAE. An average sizing precision of +/- 0.12 base pairs (bp) for fragments up to 350 bp was realized in 1-h runs. In addition, a versatile non-denaturing matrix was used to separate DNA sizing standards, restriction digests, and multiplex PCR samples. Application of this matrix to Duchenne muscular dystrophy exon deletion screening is also described. These CAE approaches should facilitate rapid genotyping of microsatellite markers and subsequent identification of disease-causing mutations.

Identification of the Nucleotide Change (CAC→CGC) Responsible for Hb P-Galveston [β117(G19)His→Arg]

Hb P-Galveston is a P-globin variant first identified in 1957 by Schneider and Haggard (1). The properties of this hemoglobin (Hb) variant were examined by Schneider et al in 1969 (2) and Di Iorio et a1 in 1975 (3). Hb P Galveston is associated with minimal hematologic abnormalities: target cells, hypochromia and anisopoikilocytosis. The molecule shows only a minimally increased oxygen affinity and normal stability (3). Until now, the nucleotide change responsible for this substitution was only presumed. We now demonstrate that the single nucleotide change of A+G at the second position of codon 1 17 is responsible for this variant. A child was referred after newborn screening identified an abnormal variant by high performance liquid chromatography (HPLC). Hematologic parameters at 8 months of age were as follows: Hb 9.9 g/dL, MCV 79 fL, MCHC 26.0 g/dL, reticulocytes 2.1%, RDW 12.9%. Genomic DNA was isolated from the child’s peripheral blood, and the P-globin gene was amplified by polymerase chain reaction (PCR); the third exon was examined by automated fluorescence-based DNA sequence analysis. Our results show heterozygosity for an