David I. Hoar

Molecular detection of a translocation (Y;15) in a 45,X male.

SummaryA 45,X male individual was shown to have a translocation of Y-chromosome material to the short arm or proximal long arm of chromosome 15. This translocation was detected by genomic DNA blotting and in situ hybridization with Y-chromosome-specific DNA probes.

Prenatal diagnosis of hypophosphatasia.

Hypophosphatasia is a rare, recessively inherited metabolic disorder characterized by low serum and tissue alkaline phosphatase, the presence of phosphoethanolamine in the urine1 , 2 and osseous ch...

Assessment of the sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y-bodies*†

The effectiveness of sephadex sperm filtration on altering the sex ratio of human sperm was tested using three different methods. For each ejaculate the ratio of X- and Y-bearing sperm was analysed before and after sephadex filtration using three different methodologies: sperm chromosome analysis after fusion of human sperm with hamster oocytes, deoxyribonucleic acid analysis using the Y-preferential probe pS4 and the fluorescent Y-body test. In 182 sephadex-treated sperm analysed by sperm chromosomes the sex ratio (55.5%X) did not differ significantly from 226 untreated sperm (54.7%X) from the same semen samples. The DNA and Y-body tests demonstrated variability in both degree and pattern of enrichment for replicates but no consistent enrichment for X-chromosome-bearing sperm was observed for any one fraction after sephadex gel filtration.

Prenatal identification of a deleted Y chromosome by cytogenetics and a Y-specific repetitive DNA probe

SummaryA very small sex chromosome was identified prenatally as a Y chromosome by using molecular hybridization in conjunction with conventional cytogenetics techniques. The combination of R-banding, Q-banding, distamycin-DAPI staining suggested that the chromosome might be a de novo deletion of the Y chromosome as the fathers Y chromosome was normal. Restriction enzyme analysis of amniotic fluid cell DNA using a Y chromosome repetitive probe confirmed the origin of this chromosome.

Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis

SummaryThe molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5′ TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3′ TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.

Severe combined immune deficiency presenting with cyclic hematopoiesis

At age 2 months a male infant presented with a cyclic clinical syndrome every 14–21 days that included pharyngeal aphthous ulcers, high fever, lymphadenopathy, pallor, and malaise. Serial blood studies indicated cycling of all blood cell elements, compatible with a diagnosis of cyclic hematopoiesis (CH). He also manifested a progressively severe immune deficiency, not described before in human CH. When first studied at age 5 months, he was hypogammaglobulinaemic with normal B lymphocyte numbers. By 6.5 months, he was agammaglobulinaemic. At age 8 months, he developed severe pneumocystis carinii pneumonia, and studies showed a state of severe combined immune deficiency. The patient received a bone marrow transplant from his HLA-identical sister with no preconditioning therapy. Subsequently, normal immune function developed and the cyclic hematopoiesis resolved. The majority of lymphocytes is of donor origin. Persistence of erythrocytes and neutrophils of recipient origin suggests that the hematopoietic stem cells were not abnormal. We speculate that this patient had a primary deficiency of a differentiation factor affecting maturation of lymphoid and myeloid progenitor cells.

Ethnic-Specific Allelic Variation within the D1Z2 Locus

DNAs from 122 individuals representing 5 ethnic groups (Black, Chinese, Japanese, Caucasian and Melanesian) were analyzed for restriction fragment length polymorphisms (RFLPs) with a hypervariable repeated sequence located uniquely on chromosome 1 (hMF No.1; is a component of the D1Z2 locus). When human genomic DNA is digested with a variety of enzymes (TaqI, EcoRI, SinI, PstI, HaeIII) the hMF No.1 probe reveals multiple RFLPs. Ethnic group differences were found in the frequencies of specific EcoRI bands. The most striking ethnic group variation was the presence of a unique fragment amongst the Japanese.